Chitin and Chitosan - Proceedings of The Second International Conference

Chitin and
Chitosan
Wiley Series
in
Renewable Resources
Series Editor:
Christian V. Stevens, Faculty of Bioscience Engineering, Ghent University, Belgium
Titles in the Series:

Wood Modification: Chemical, Thermal and Other Processes
Callum A. S. Hill
Renewables‐Based Technology: Sustainability Assessment

Jo Dewulf, Herman Van Langenhove
Biofuels
Wim Soetaert, Erik Vandamme
Handbook of Natural Colorants

Thomas Bechtold, Rita Mussak
Surfactants from Renewable Resources

Mikael Kjellin, Ingegärd Johansson
Industrial Applications of Natural Fibres: Structure, Properties and Technical Applications

Jörg Müssig
Thermochemical Processing of Biomass: Conversion into Fuels, Chemicals and Power

Robert C. Brown
Biorefinery Co‐Products: Phytochemicals, Primary Metabolites and Value‐Added Biomass

Processing
Chantal Bergeron, Danielle Julie Carrier, Shri Ramaswamy
Aqueous Pretreatment of Plant Biomass for Biological and Chemical Conversion to Fuels and
Chemicals
Charles E. Wyman
Bio‐Based Plastics: Materials and Applications

Stephan Kabasci
Introduction to Wood and Natural Fiber Composites

Douglas D. Stokke, Qinglin Wu, Guangping Han
Cellulosic Energy Cropping Systems

Douglas L. Karlen
Introduction to Chemicals from Biomass, 2nd Edition

James H. Clark, Fabien Deswarte
Lignin and Lignans as Renewable Raw Materials: Chemistry, Technology and Applications
Francisco G. Calvo‐Flores, Jose A. Dobado, Joaquín Isac‐García, Francisco J. Martín‐Martínez
Sustainability Assessment of Renewables‐Based Products: Methods and Case Studies
Jo Dewulf, Steven De Meester, Rodrigo A. F. Alvarenga
Cellulose Nanocrystals: Properties, Production and Applications

Wadood Hamad
Fuels, Chemicals and Materials from the Oceans and Aquatic Sources
Francesca M. Kerton, Ning Yan
Bio‐Based Solvents
François Jérôme and Rafael Luque
Nanoporous Catalysts for Biomass Conversion

Feng-Shou Xiao and Liang Wang
Thermochemical Processing of Biomass: Conversion into Fuels, Chemicals and Power,

2nd Edition
Robert C. Brown
Forthcoming Titles:
The Chemical Biology of Plant Biostimulants
Danny Geelen, Lin Xu
Biorefinery of Inorganics: Recovering Mineral Nutrients from Biomass and Organic Waste
Erik Meers, Gerard Velthof
Waste Valorization: Waste Streams in a Circular Economy

Sze Ki Lin, Chong Li, Guneet Kaur, Xiaofeng Yang
Process Systems Engineering for Biofuels Development

Adrián Bonilla-Petriciolet, Gade Pandu Rangaiah
Biobased Packaging: Material, Environmental and Economic Aspects

Mohd Sapuan Salit, Rushdan Ahmad Ilyas
Chitin and Chitosan:
Properties and
Applications
Edited by
LAMBERTUS A.M. VAN DEN BROEK

Wageningen Food & Biobased Research
Wageningen
The Netherlands
CARMEN G. BOERIU
Wageningen Food & Biobased Research
Wageningen
The Netherlands
This edition first published 2020
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Contents
List of Contributors xvii

Series Preface xxi
Prefacexxiii
1 Sources of Chitin and Chitosan and their Isolation 1

Leen Bastiaens, Lise Soetemans, Els D’Hondt, and Kathy Elst
1.1 Chitin and Chitosan 2
1.1.1 Chemical Structure 2
1.1.2 Different Crystalline Forms of Chitin 2
1.2 Sources of Chitin and Chitosan 5
1.2.1 Sources of Chitin 5
1.2.2 Sources for Chitosan 10
1.3 Isolation of Chitin 11
1.3.1 Technology Principles 11
1.3.2 Isolation of Chitin from Crustaceans 13
1.3.3 Isolation of Chitin from Insects 16
1.3.4 Isolation of Chitin from Other Biomass Types 16
1.4 Production of Chitosan 19
1.4.1 Conversion of Chitin to Chitosan 19
1.4.2 Chitosan Extracted from Fungi 24
1.5 Towards Commercial Applications 25
1.6 Outlook 28
References28
2 Methods of Isolating Chitin from Sponges (Porifera) 35

Sonia Ż ółtowska, Christine Klinger, Iaroslav Petrenko, Marcin Wysokowski,
Yvonne Joseph, Teofil Jesionowski, and Hermann Ehrlich
2.1 Introduction 35
2.2 Brief Overview of Classical Methods of Isolating Chitin from
Invertebrates38
viii Contents
2.3 The Modern Approach to Chitin Isolation from Sponges 40

2.3.1 Methods of Isolating Chitin from Glass
Sponges (Hexactinellida) 41
2.3.2 Methods of Isolating Chitin from
Demosponges (Demospongiae) 43
2.4 Prospective Applications of Poriferan Chitin 49
2.4.1 Poriferan Chitin and Modern Bioinspired Materials Science 49
2.4.2 Chitinous 3D Scaffolds of Sponge Origin for
Tissue Engineering 51
2.5 Outlook 54
Acknowledgment54
References54
3 Physicochemical Properties of Chitosan and its Degradation Products 61

Karolina Gzyra‐Jagieła, Bożenna Pęczek, Maria Wis ́niewska‐Wrona, and
Natalia Gutowska
3.1 Physicochemical Properties of Chitosan 62
3.1.1 Determination of Molar Mass 62
3.1.2 Determination of the Deacetylation Degree 67
3.1.3 Determination of Dynamic Viscosity 70
3.1.4 Determination of Nitrogen 70
3.1.5 Determination of Ash Content 71
3.1.6 Determination of Heavy Metal Content 71
3.1.7 Determination of Water Retention Value (WRV) 72
3.1.8 Determination of Solubility in Hydrochloric Acid 72
3.1.9 Determination of Water Content 72
3.1.10 Determination of Protein Content 73
3.1.11 Quantitative Determination of Chitosan by Ninhydrin 73
3.2 Products of Degradation and their Application 74
3.3 Outlook 77
References77
4 New Developments in the Analysis of Partially Acetylated Chitosan

Polymers and Oligomers81
Stefan Cord‐Landwehr, Anna Niehues, Jasper Wattjes, and
Bruno M. Moerschbacher
4.1 Introduction 82
4.2 Chitosan Oligomers 83
4.2.1 Degree of Polymerisation (DP), Fraction and Pattern of
Acetylation (FA and PA)83
4.3 Chitosan Polymers 86
4.3.1 Molecular Weight (MW) / Degree of Polymerisation (DP)
and its Dispersity (ÐMW / ÐDP)86
4.3.2 Fraction of Acetylation (FA) and its Dispersity (ÐFA)87
4.3.3 Pattern of Acetylation (PA)89
4.4 Outlook 91
References92
Contents ix
5 Chitosan‐Based Hydrogels 97
Zhengke Wang, Ling Yang, and Wen Fang
5.1 Introduction 97
5.2 Chitosan‐Based Multilayered Hydrogels 98
5.2.1 Periodic Precipitation 99
5.2.2 Alternating Process 100
5.2.3 Induced by Electrical Signals 100
5.2.4 Layer‐by‐Layer (LbL) Assembly 101
5.2.5 Sequential Curing 101
5.3 Chitin/Chitosan Physical Hydrogels Based on Alkali/Urea Solvent System 103
5.3.1 Chitin Hydrogels Based on Alkali/Urea Solvent System 104
5.3.2 Chitosan Hydrogels Based on Alkali/Urea Solvent System 104
5.4 Chitosan‐Based Injectable Hydrogels 108
5.4.1 Physical Association Networks 108
5.4.2 Chemical Association Networks 110
5.4.3 Double‐Network Hydrogels 116
5.5 Chitosan‐Based Self‐Healing Hydrogels 119
5.5.1 Physical Interactions 119
5.5.2 Dynamic Chemical Bonds 121
5.6 Chitosan‐Based Shape Memory Hydrogels 125
5.6.1 Water‐/Solvent‐Triggered Shape Recovery 126
5.6.2 pH‐triggered Shape Recovery 126
5.6.3 Ultrasound Triggered Shape Recovery 126
5.6.4 Self‐Actuated Shape Memory Hydrogels 127
5.6.5 Chitosan‐Based Hydrogels with Triple Shape Memory Effect 127
5.7 Superabsorbent Chitosan‐Based Hydrogels 131
5.7.1 Cross‐Linked Chitosan‐Based Hydrogels 132
5.7.2 Hydrogels by Graft Copolymerization 133
5.7.3 Chitosan‐Based Composite Hydrogels 134
5.7.4 Pure Chitosan‐Based Materials 135
5.8 Outlook 136
References136
6 Beneficial Health Effects of Chitin and Chitosan 145

Liyou Dong, Harry J. Wichers, and Coen Govers
6.1 Immunomodulatory Effects of Chitin and Chitosan as Demonstrated
with In Vitro Studies 146
6.2 Beneficial Health Effects Mediated by Chitin and Chitosan as
Demonstrated with Animal Studies 149
6.2.1 Immune Modulation 149
6.2.2 Anti‐Pathogenic Effects 155
6.2.3 Anti‐Tumour Effects 157
6.3 Beneficial Health Effects Mediated by Chitin and Chitosan as
Demonstrated with Clinical Trials 158
6.3.1 Cholesterol Reduction and CVD Preventive Effects 158
6.3.2 Other Health Effects 160
6.4 Requirements to forward the Field of Study Towards the Beneficial
Health Effects of Chitin and Chitosan 163
x Contents
6.5 Outlook 164
Acknowledgement164
References164
7 Antimicrobial Properties of Chitin and Chitosan 169

Magdalena Kucharska, Monika Sikora, Kinga Brzoza‐Malczewska, and
Monika Owczarek
7.1 Microbiological Activity of Chitosan – The Mechanism of its Antibacterial
and Antifungal Activity 169
7.2 The use of Chitin/Chitosan’s Microbiological Activity in Medicine
and Pharmacy171
7.3 Microbiological Activity of Chitosan in the Food Industry 174
7.4 Microbiological Activity of Chitosan in Paper and Textile Industries 176
7.5 Microbiological Activity of Chitosan in Agriculture 177
7.6 Outlook 181
References181
8 Enzymes for Modification of Chitin and Chitosan 189

Gustav Vaaje‐Kolstad, Tina Rise Tuveng, Sophanit Mekasha, and
Vincent G.H. Eijsink
8.1 CAZymes in Chitin Degradation and Modification 190
8.1.1 Chitinases 191
8.1.2 β‐N‐acetylhexosaminidases195
8.1.3 Exo‐β‐glucosaminidases195
8.1.4 Chitosanases 197
8.1.5 Lytic Polysaccharide Monooxygenases 199
8.1.6 Carbohydrate Esterases 200
8.1.7 Carbohydrate‐Binding Modules 204
8.2 Modular Diversity in Chitinases, Chitosanases and LPMOs204
8.3 Biological Roles of Chitin‐Active Enzymes 205
8.4 Microbial Degradation and Utilisation of Chitin 208
8.4.1 Chitin Degradation by Serratia marcescens209
8.4.2 Chitin Degradation by Bacteria in the Bacteroidetes
Phylum211
8.4.3 Chitin Degradation by Thermococcus Kodakarensis211
8.4.4 Chitin Degradation by Fungi 212
8.5 Biotechnological Perspectives 213
8.6 Biorefining of Chitin‐Rich Biomass 214
8.7 Outlook 216
References216
9 Chitin and Chitosan as Sources of Bio‐Based Building Blocks

and Chemicals229
Malgorzata Kaisler, Lambertus A.M. van den Broek, and
Carmen G. Boeriu
9.1 Introduction 230
9.2 Chitin Conversion into Chitosan, Chitooligosaccharides and
Monosaccharides232
Contents xi
9.2.1 Chitosan Production 232

9.2.2 Production of Chitooligosaccharides 234
9.2.3 Production of GlcNAc and GlcN from Chitin 235
9.3 Building Blocks for Polymers from Chitin and its Derivatives 238
9.3.1 Furan‐Based Monomers 238
9.3.2 Amino Alcohol and Amino Acid Building Blocks 239
9.4 Outlook 239
Acknowledgement240
References240
10 Chemical and Enzymatic Modification of Chitosan to Produce New

Functional Materials with Improved Properties 245
Carmen G. Boeriu and Lambertus A.M. van den Broek
10.2 Functional Chitosan Derivatives by Chemical and Enzymatic
Modification246
10.2.1 Anionic Chitosan Derivatives 248
10.2.2 Hydroxyalkylchitosans 250
10.2.3 Quaternised and Highly Cationic Chitosan Derivatives 250
10.2.4 Hydroxyaryl Chitosan Derivatives 250
10.2.5 Carbohydrate‐Modified Chitosan 251
10.3 Graft Co‐Polymers of Chitosan 251
10.4 Cross‐Linked Chitosan and Chitosan Polymer Networks 254
10.5 Outlook 254
References255
11 Chitosan‐Based Drug Delivery Systems 259

Cristian Peptu, Andra Cristina Humelnicu, Razvan Rotaru,
Maria Emiliana Fortuna, Xenia Patras, Mirela Teodorescu,
Bogdan Ionel Tamba, and Valeria Harabagiu
11.2 Beneficial Effects of Chitosan 261
11.2.1 Interaction with Anionic Drugs 263
11.2.2 Mucoadhesive Properties 263
11.2.3 Transfection Activity 263
11.2.4 Efflux Pump Inhibitory Properties 265
11.2.5 Permeation‐Enhancing Properties 265
11.3 Chitosan—an Active Polymer for Bypassing Biological Barriers 265
11.3.1 Skin Barrier 266
11.3.2 Mucosa Barrier 267
11.3.3 Ophthalmic Barrier 269
11.3.4 Blood–Brain Barrier 270
11.4 Chitosan‐Based DDS Formulations 271
11.4.1 Hydrogels 275
11.4.2 Micro/NPs 275
11.4.3 Nanofibers 275
11.4.4 Scaffolds and Membranes 275
xii Contents
11.5 Outlook 276
Acknowledgment276
References276
12 The Application of Chitin and its Derivatives for the Design of Advanced

Medical Devices 291
Marcin H. Struszczyk, Longina Madej‐Kiełbik, and Dorota Zielińska
12.1 Selection of the Raw Sources: Safety Criteria 291
12.1.1 Aspect of Animal Tissue‐Originated Derivatives 292
12.1.2 General Requirements for Chitinous Biopolymers Applied
in Designing Medical Devices 292
12.1.3 Characterisation of the Biopolymer for Application in Wound
Dressing Designing293
12.1.4 Aspect of the Sterilization of the Final Wound Dressing 295
12.2 Types of Wound Dressings Consisting of Chitin‐Derived Biopolymers
Available in the Market297
12.3 Performance and Safety Assessment 297
12.4 New Ideas and Concepts 301
12.5 Risk Acceptance and Design Process Aspects 306
12.6 Outlook 308
Acknowledgements308
References308
13 Food Applications of Chitosan and its Derivatives 315

Suse Botelho da Silva, Daiana de Souza, and Liziane Dantas Lacerda
13.2 Chitosan and its Derivatives as Food Additive 316
13.2.1 Antioxidant 318
13.2.2 Antimicrobial 319
13.2.3 Stabilizer and Thickener 319
13.3 Functional Ingredient and Health Beneficial Effects 320
13.4 Active Packaging 321
13.5 Enzyme Immobilization 331
13.6 Encapsulation and Delivering of Bioactive Ingredients 332
13.7 Adsorption and Chelation of Toxic and Undesirable Compounds 334
13.8 Outlook 339
References340
14 Potential of Chitosans in the Development of Edible Food Packaging 349

Véronique Coma and Artur Bartkowiak
14.1 Potential Limitations for Real Introduction into the Market 350
14.1.1 Generally Recognized as Safe (GRAS)351
14.1.2 Solubility 351
14.1.3 Source—Origin 352
14.1.4 Structure Variability 352
14.2 Films and Coatings for Food Preservation 353
Contents xiii
14.2.1 Definitions and Interests 353

14.2.2 Main Relevant Chitosan‐Based Material Properties 353
14.3 Specific Case of Chitosan Nanoparticles (CSNPs)357
14.3.1 CSNPs 357
14.3.2 CSNPs in Various Edible Films 358
14.3.3 Antimicrobial Activities of CSNPs in Edible Films 359
14.3.4 Toxicity Studies of CSNPs360
14.4 Applications to Sensitive Real Food Products 360
14.4.1 Fruits and Vegetables 361
14.4.2 Meat and Meat Products 362
14.4.3 Fish and Seafood Products 362
14.5 Conclusions 364
References364
15 The Use of Chitosan‐Based Nanoformulations for Controlling Fungi

During Storage of Horticultural Commodities 371
Silvia Bautista‐Baños, Zormy Nacary Correa‐Pacheco, and Rosa Isela
Ventura‐Aguilar
15.2 Importance of Fruits and Vegetables 372
15.3 Storage Disorders and Diseases of Horticultural Products 374
15.4 Plant Fungi Inhibition by Chitosan Application 375
15.5 Chitosan Integrated with Other Alternative Methods for Controlling
Postharvest Fungi 376
15.6 Chitosan‐Based Formulations 376
15.7 Physiological Response and Quality Retention of Horticultural
Commodities to Chitosan Coating Application 376
15.8 Influence of Chitosan Coatings on the Shelf Life of
Horticultural Products 378
15.9 Effects of Chitosan Coatings with Additional Compounds on Quality
and Microorganisms Development 379
15.10 Integration of Chitosan Nanoparticles into Coating Formulations
and their Effects on the Quality of Horticultural Commodities
and Development of Microorganisms 384
15.11 Outlook 387
Acknowledgments387
References387
16 Chitosan Application in Textile Processing and Fabric Coating 395

Thomas Hahn, Leonie Bossog, Tom Hager, Werner Wunderlich, Rudi Breier,
Thomas Stegmaier, and Susanne Zibek
16.1 Chitosan in the Textile Industry 396
16.2 Textile Production 398
16.3 General Test Methods 400
16.4 Fibres and Yarns from Chitin and Chitosan 401
16.4.1 Chitin and Chitosan Solubilisation for Spinning Purposes 402
xiv Contents
16.4.2 Chitosan Spinning Processes 402

16.4.3 Mechanical Properties of Chitosan Fibres/Yarns 404
16.5 Sizing with Chitosan 406
16.5.1 Miscibility of Chitosan with Other Sizing Agents 407
16.5.2 Viscosity of Chitosan‐Containing Sizing Agents 408
16.5.3 Adhesion and Wetting 410
16.5.4 Mechanical–Physical Properties of Chitosan Films 411
16.5.5 Removal and Processing of Chitosan Sizing after Weaving 412
16.6 Chitosan as a Finishing Agent or Coating 414
16.6.1 Chitosan as a Carrier and Linker 415
16.6.2 Formation of a Durable Finish with Chitosan 416
16.6.3 Chitosan as an Active Agent 417
16.7 Outlook 419
Nomenclature420
References421
17 Chitin and Chitosan for Water Purification 429

Petrisor Samoila, Andra Cristina Humelnicu, Maria Ignat,
Corneliu Cojocaru, and Valeria Harabagiu
17.2 Wastewater Treatment by Adsorption 432
17.2.1 Principle of the Adsorption Process 432
17.2.2 Adsorption of Organic Compounds 434
17.2.3 Adsorption of Heavy Metals 437
17.3 Wastewater Treatment by Coagulation/Flocculation 440
17.4 Wastewater Treatment by Membrane Separation 446
17.4.1 Principle of Ultrafiltration Process 446
17.4.2 Fabrication of Ultrafiltration Blend Membranes 448
17.4.3 Chitosan‐Enhanced Ultrafiltration 450
17.5 Outlook 452
Acknowledgement452
References453
18 Chitosan for Sensors and Electrochemical Applications 461

Suse Botelho da Silva, Guilherme Lopes Batista, and
Cristiane Krause Santin
18.2 Chitosan: A Biopolymer with Unique Properties 462
18.3 Modification and Preparation of Chitosan‐Based Materials
for Electrochemical Applications 463
18.4 The Proton Conductivity of Chitosan 465
18.5 Selected Applications 467
18.5.1 Electrochemical Sensors 467
18.5.2 Spectroscopic Sensors 470
18.5.3 Other Electrochemical Devices 471
18.6 Outlook 472
References473
Contents xv
19 Marketing and Regulations of Chitin and Chitosan from Insects 477

Nathalie Berezina and Antoine Hubert
19.1 Historical Outline 477
19.2 Natural Origins of Chitin 478
19.3 Specificities of Chitin Biopolymer 479
19.4 Differences Among Chitins from Insects and Other Sources 479
19.4.1 Differences of Chemical Compositions of the Cuticles 479
19.4.2 Differences of Physical Assemblies of Chains and Molecules 480
19.5 Extraction and Purification Specificities of Chitins from Insects 480
19.5.1 Different Cuticle Structures and Contents of Insects 480
19.5.2 Chemical Extraction 480
19.5.3 Biological Extraction 481
19.5.4 Characterization and Transformation into Chitosan 481
19.6 Market Opportunities and its Regulations 482
19.6.1 Agriculture Applications 482
19.6.2 Water Treatment Applications 483
19.6.3 Material Applications 483
19.6.4 Biomedical Applications 484
19.7 Outlook 485
References485
Index491
List of Contributors
Artur Bartkowiak Center of Bioimmobilisation and Innovative Packaging Materials,

Faculty of Food Sciences and Fisheries, West Pomeranian University of Technology,
Szczecin, Poland
Leen Bastiaens VITO (Flemish Institute for Technological Research), Mol, Belgium
Silvia Bautista‐Baños Centro de Desarrollo de Productos Bióticos (CEPROBI), Instituto
Politécnico Nacional (IPN), Yautepec, Morelos, Mexico
Nathalie Berezina Ynsect, Évry, France
Carmen G. Boeriu Wageningen Food & Biobased Research, Wageningen, The
Netherlands
Leonie Bossog Textilchemie Dr. Petry GmbH, Reutlingen, Germany
Suse Botelho da Silva Food and Chemical Engineering, Polytechnic School, Unisinos
University, São Leopoldo, RS, Brazil
Rudi Breier Textilchemie Dr. Petry GmbH, Reutlingen, Germany
Lambertus A.M. van den Broek Wageningen Food & Biobased Research, Wageningen,
The Netherlands
Kinga Brzoza‐Malczewska Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Corneliu Cojocaru ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian
Academy, Iași, Romania
Véronique Coma University of Bordeaux, LCPO, UMR 5629, Centre National de la
Recherche Scientifique (CNRS), Pessac, France
Stefan Cord‐Landwehr University of Münster, Institute for Biology and Biotechnology
of Plants, Münster, Germany
Zormy Nacary Correa‐Pacheco CONACYT-CEPROBI, Instituto Politécnico Nacional,
Yautepec, Morelos, Mexico
xviii List of Contributors
Els D’Hondt VITO (Flemish Institute for Technological Research), Mol, Belgium
Liyou Dong Food & Health Research, Wageningen Food & Biobased Research,
Wageningen, The Netherlands; Food Chemistry, Wageningen University, Wageningen, The
Netherlands
Hermann Ehrlich Institute of Electronics and Sensor Materials, TU Bergakademie‐
Freiberg, Freiberg, Germany
Vincent G.H. Eijsink Faculty of Chemistry, Biotechnology, and Food Science, The
Norwegian University of Life Sciences (NMBU), Ås, Norway
Kathy Elst VITO (Flemish Institute for Technological Research), Mol, Belgium
Wen Fang Institute of Biomedical Macromolecules, Department of Polymer Science
and Engineering, Zhejiang University, Hangzhou, China
Maria Emiliana Fortuna ‘Petru Poni’ Institute of Macromolecular Chemistry,
Romanian Academy, Iași, Romania
Coen Govers Food & Health Research, Wageningen Food & Biobased Research,
Wageningen, The Netherlands
Natalia Gutowska Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Karolina Gzyra‐Jagieła Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Tom Hager German Institutes of Textile and Fiber Research, Denkendorf, Germany
Thomas Hahn Fraunhofer Institute of Interfacial Engineering and Biotechnology,
Stuttgart, Germany
Valeria Harabagiu ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian
Antoine Hubert Ynsect, Évry, France
Andra Cristina Humelnicu ‘Petru Poni’ Institute of Macromolecular Chemistry,
Romanian Academy, Iași, Romania
Maria Ignat ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian Academy,
Iași, Romania
Teofil Jesionowski Institute of Chemical Technology and Engineering, Faculty of
Chemical Technology, Poznan University of Technology, Poznan, Poland
Yvonne Joseph Institute of Electronics and Sensor Materials, TU Bergakademie‐
Freiberg, Freiberg, Germany
Malgorzata Kaisler Bioprocess Engineering Group, Wageningen University,
Wageningen, The Netherlands; Wageningen Food & Biobased Research, Wageningen, The
Netherlands
Christine Klinger Institute of Physical Chemistry, TU Bergakademie‐Freiberg,
Freiberg, Germany
List of Contributors xix
Cristiane Krause Santin Food and Chemical Engineering, Polytechnic School, Unisinos
University, São Leopoldo, RS, Brazil; itt CHIP – Unisinos Semiconductor Institute, São
Leopoldo, RS, Brazil
Magdalena Kucharska Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Liziane Dantas Lacerda Food and Chemical Engineering, Polytechnic School, Unisinos
Guilherme Lopes Batista itt CHIP – Unisinos Semiconductor Institute, São Leopoldo,
RS, Brazil
Longina Madej‐Kiełbik The Institute of Security Technologies “MORATEX”, Lodz,
Poland
Sophanit Mekasha Faculty of Chemistry, Biotechnology, and Food Science, The
Bruno M. Moerschbacher University of Münster, Institute for Biology and
Biotechnology of Plants, Münster, Germany
Anna Niehues University of Münster, Institute for Biology and Biotechnology of Plants,
Münster, Germany
Monika Owczarek Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Xenia Patras ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian Academy,
Iași, Romania
Bożenna Pe ̨czek Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Cristian Peptu ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian Academy,
Iași, Romania
Iaroslav Petrenko Institute of Experimental Physics, TU Bergakademie‐Freiberg,
Freiberg, Germany
Razvan Rotaru ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian
Petrisor Samoila ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian
Monika Sikora Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Lise Soetemans VITO (Flemish Institute for Technological Research), Mol, Belgium
Daiana de Souza Food and Chemical Engineering, Polytechnic School, Unisinos
Thomas Stegmaier German Institutes of Textile and Fiber Research, Denkendorf,
Germany
Marcin H. Struszczyk The Institute of Security Technologies “MORATEX”, Lodz,
Poland
xx List of Contributors
Bogdan Ionel Tamba A&B Pharm Corporation, Roman, Neamt ̦, Romania

Mirela Teodorescu ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian
Tina Rise Tuveng Faculty of Chemistry, Biotechnology, and Food Science, The
Gustav Vaaje‐Kolstad Faculty of Chemistry, Biotechnology, and Food Science, The
Rosa Isela Ventura‐Aguilar CONACYT-CEPROBI, Instituto Politécnico Nacional,
Zhengke Wang Institute of Biomedical Macromolecules, Department of Polymer
Science and Engineering, Zhejiang University, Hangzhou, China
Jasper Wattjes University of Münster, Institute for Biology and Biotechnology of
Plants, Münster, Germany
Harry J. Wichers Food & Health Research, Wageningen Food & Biobased Research,
Wageningen, The Netherlands; Food Chemistry, Wageningen University, Wageningen, The
Netherlands
Maria Wis ń iewska‐Wrona Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Werner Wunderlich German Institutes of Textile and Fiber Research, Denkendorf,
Germany
Marcin Wysokowski Institute of Chemical Technology and Engineering, Faculty of
Chemical Technology, Poznan University of Technology, Poznan, Poland; Institute of
Electronics and Sensor Materials, TU Bergakademie‐Freiberg, Freiberg, Germany
Ling Yang Institute of Biomedical Macromolecules, Department of Polymer Science
and Engineering, Zhejiang University, Hangzhou, China
Susanne Zibek Fraunhofer Institute of Interfacial Engineering and Biotechnology,
Stuttgart, Germany
Dorota Zielińska The Institute of Security Technologies “MORATEX”, Lodz, Poland
Sonia Ż ółtowska Institute of Chemical Technology and Engineering, Faculty of
Chemical Technology, Poznan University of Technology, Poznan, Poland; Institute of
Electronics and Sensor Materials, TU Bergakademie‐Freiberg, Freiberg, Germany
Series Preface
Renewable resources, their use and modification are involved in a multitude of important
processes with a major influence on our everyday lives. Applications can be found in the
energy sector; paints and coatings; and the chemical, pharmaceutical, and textile industry,
to name but a few.
The area interconnects several scientific disciplines (agriculture, biochemistry, chemis-
try, technology, environmental sciences, forestry), which makes it very difficult to have an
expert view on the complicated interaction. Therefore, the idea to create a series of scien-
tific books, focusing on specific topics concerning renewable resources, has been very
opportune and can help to clarify some of the underlying connections in this area.
In a very fast‐changing world, trends are not only characteristic of fashion and political
standpoints; science too is not free from hypes and buzzwords. The use of renewable
resources is again more important nowadays; however, it is not part of a hype or a fashion.
As the lively discussions among scientists continue about how many years we will still be
able to use fossil fuels – opinions ranging from 50 to 500 years – they do agree that the
reserve is limited, and that it is essential not only to search for new energy carriers but also
for new material sources.
In this respect, the field of renewable resources is a crucial area in the search for alterna-
tives for fossil‐based raw materials and energy. In the field of energy supply, biomass‐ and
renewables‐based resources will be part of the solution alongside other alternatives such as
solar energy, wind energy, hydraulic power, hydrogen technology and nuclear energy. In
the field of material sciences, the impact of renewable resources will probably be even big-
ger. Integral utilisation of crops and the use of waste streams in certain industries will grow
in importance, leading to a more sustainable way of producing materials. Although our
society was much more (almost exclusively) based on renewable resources centuries ago,
this disappeared in the Western world in the nineteenth century. Now it is time to focus
again on this field of research. However, it should not mean a ‘retour à la nature’, but
should be a multidisciplinary effort on a highly technological level to perform research
towards new opportunities, to develop new crops and products from renewable resources.
This will be essential to guarantee an acceptable level of comfort for the growing number
of people living on our planet. It is ‘the’ challenge for the coming generations of scientists
to develop more sustainable ways to create prosperity and to fight poverty and hunger in
the world. A global approach is certainly favoured.
xxii Series Preface
This challenge can only be dealt with if scientists are attracted to this area and are recog-
nised for their efforts in this interdisciplinary field. It is, therefore, also essential that con-
sumers recognise the fate of renewable resources in a number of products.
Furthermore, scientists do need to communicate and discuss the relevance of their work.
The use and modification of renewable resources may not follow the path of the genetic
engineering concept in view of consumer acceptance in Europe. Related to this aspect, the
series will certainly help to increase the visibility of the importance of renewable resources.
Being convinced of the value of the renewables approach for the industrial world, as well
as for developing countries, I was myself delighted to collaborate on this series of books
focusing on the different aspects of renewable resources. I hope that readers become aware
of the complexity, the interaction and interconnections, and the challenges of this field, and
that they will help to communicate on the importance of renewable resources.
I certainly want to thank the people of Wiley’s Chichester office, especially David
Hughes, Jenny Cossham and Lyn Roberts, in seeing the need for such a series of books on
renewable resources, for initiating and supporting it, and for helping to carry the project to
the end.
Last, but not least, I want to thank my family, especially my wife Hilde and children
Paulien and Pieter‐Jan, for their patience, and for giving me the time to work on the series
when other activities seemed to be more inviting.
Christian V. Stevens,
Faculty of Bioscience Engineering
Ghent University, Belgium
Series Editor, ‘Renewable Resources’
June 2005
Preface
Chitin was reported for the first time about 200 years ago, in extracts of mushrooms and
insects. About 40 years later, chitosan was obtained from chitin by acid treatment. These
polysaccharides are among the most abundant natural biopolymers in the world. They are,
for example, present in crustaceans, insects and fungi. Just before World War II, there was
a huge interest in the applications of these polysaccharides as a bioplastic. However, the
simultaneous upcoming of synthetic polymers and the exponential increase in high‐
performance synthetic polymers, which outperformed their natural counterparts, resulted
in a decrease of interest in chitin/chitosan materials. In the 1970s, large‐scale production
of chitin and chitosan from the shells of marine organisms started, owing to the develop-
ment of aquaculture and the enactment of severe environmental regulations to decrease
the amount of shellfish dumping in the oceans. Nowadays there is a need to be less
dependent on fossil resources. The transition to a biobased economy and the increasing
societal demand for more green and environmentally friendly products urge us to look for
chemicals, materials and fuels based on renewable resources. The enormous potential of
chitin and chitosan on account of their abundance, unique properties and numerous appli-
cations makes them interesting biomass resources. This book, Chitin and Chitosan:
Properties and Applications, shows the state‐of‐the‐art and future perspectives of chitin
and chitosan materials and applications. The book presents the most recent developments
in the science and technology of all related fields, from extraction and characterisation to
modification, material synthesis and end‐user applications. This book comprises 19 chapters
that deal with most topics related to chitin and chitosan polymers and materials.
In Chapters 1–4, the sources of chitin and chitosan are described and how these biopoly-
mers can be isolated. Next to the isolation, the analysis of the biopolymers is described.
The different sources and/or isolation methods can result in different structures and proper-
ties. In Chapter 5–7, hydrogels, health effects and the anti‐microbial effects of chitin and
chitosan are discussed. To improve or to modify the properties, enzymes and chemical
reactions can be applied to customise these biopolymers, as shown in Chapters 8–10. The
applications of chitin and chitosan in drug delivery, medical devices, agriculture, food,
packaging, horticulture, textile, water purification and sensors are discussed in more detail
in Chapters 11–18. And finally, Chapter 19 is devoted to the market and regulation of chitin
and chitosan.
xxiv Preface
These topics have never been addressed previously in a single book. Books, book chap-
ters and reviews have been dedicated to the specific fields of application of chitin and chi-
tosan materials. This book presents an overview of the latest scientific and technological
advances in almost all areas of application, and show the great potential of chitin and chi-
tosan as materials of the future. We hope that the reader will be inspired by the examples
given of these biopolymers in different areas. We are confident that chitin and chitosan will
become major renewable resources in the biobased circular economy.
This book should be useful for scholars and those in academia, such as undergraduate
and postgraduate students in the areas of agriculture, polymer and material sciences,
biobased economy and life sciences. In addition, we hope this book will aid researchers
and specialists from industry in the field of (bio)polymers, packaging, biomedical applica-
tions, water treatment, textiles, sensors, and agriculture and food – as well as regional and
national policy‐makers.
The input is from well‐known experts from all over the world. We would like to express
our great gratitude to all chapter authors of this book, who have made excellent contribu-
tions. In addition, we would like to thank Sarah Higginbotham, Emma Strickland and
Lesley Jebaraj from Wiley for all their help.
Lambertus A.M. van den Broek and Carmen G. Boeriu

Wageningen 2019
1
Sources of Chitin and Chitosan
and their Isolation
Leen Bastiaens, Lise Soetemans, Els D’Hondt, and Kathy Elst
VITO – (Flemish Institute for Technological Research), Mol, Belgium
Chitin is a natural biomolecule that was reported for the first time in 1811 by the French
professor Henri Braconnot as fungine [1] and in 1823 by Antoine Odier as chitin. Chitin
consists of large, crystalline nitrogen‐containing polysaccharides made of chains of a mod-
ified glucose monosaccharide, being N‐acetylglucosamine. It is ubiquitously present in the
world and has even been reported to be one of the most abundant biomolecules on earth,
with an estimated annual production of 1011–1014 tons [2, 3]. Chitin serves as template for
biomineralization such as calcification and silicification, providing preferential sites for
nucleation, and controlling the location and orientation of mineral phases [4, 5]. This phe-
nomenon explains the presence of chitin in solid structures in a variety of biomass such as
cell walls of fungi and diatoms and in exoskeletons of Crustaceans. Chitin is present in
diverse structures in at least 19 animal phyla besides its presence in bacteria, fungi, and
algae [5].
Chitosan is mainly known as a partially deacetylated derivative of chitin that is more
water soluble than chitin, and as such is easier to process. For this reason, chitosan—and,
in some cases, even more preferably, the relatively small sized (1–10 kDa) chitosan
oligomers—are the molecules that are envisioned for multiple applications such as agricul-
ture; water and wastewater treatment; food and beverages; chemicals; feed; cosmetics; and
personal care [6, 7]. In addition, chitosan oligomers have been reported as being bioactive
[8], offering potential for application in, for instance, wound dressing and cosmetics.
Although chitin and chitosan are versatile and promising biomaterials [9], the extraction
Chitin and Chitosan: Properties and Applications, First Edition.

Edited by Lambertus A.M. van den Broek and Carmen G. Boeriu.
© 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.
2 Chitin and Chitosan: Properties and Applications
and purification of chitin and its conversion to chitosan (oligomers) require several process
steps, and these have been mentioned as bottlenecks that hinder the wider use of the under-
spent chitin in the world.
This chapter intends to provide more information related to (1) the structure of chitin, (2)
sources of chitin and chitosan, and (3) their extraction and purification, as well as (4) the
conversion of chitin into chitosan. The further conversion of chitosan to chitosan oligomers
is the subject of Chapter 3.
1.1 Chitin and Chitosan

1.1.1 Chemical Structure
Chitin, and its derivate chitosan, are natural polysaccharides consisting of 2 monosaccha-
rides, N‐acetyl‐D‐glucosamine and D‐glucosamine, connected by β‐1,4‐ glycoside bonds.
Depending on the frequency of the latter monosaccharides, the molecule is defined as
chitin or chitosan. Chitin contains mainly N‐acetyl‐D‐glucosamine and can be transformed
to chitosan by partial deacetylation of the monomer N‐acetyl‐D‐glucosamine to D‐glucosa-
mine (see Figure 1.1) [7]. Diverse definitions of chitin and chitosan circulate in literature.
Most sources mention a deacetylation degree of at least 50% [7, 10] as a criterion to define
the molecule as chitosan. Others report a deacetylation degree of at least 60% or 75% for
chitosan, implying that, respectively, more than 60% or 75% of the monosaccharides are
D‐glucosamine moieties [11–13]. Chitin in its natural appearance is usually already a het-
eropolymer, with a deacetylation degree ranging between 5% and 20% [14]. The structure
of chitin is very similar to that of cellulose and shares generally the same function of pro-
viding structure integrity and protection of the organism.
1.1.2 Different Crystalline Forms of Chitin

Chitin usually functions as a supporting material and is composed of layers of polysaccha-
ride sheets. Each individual sheet consists of multiple parallel‐positioned chitin chains
[17], as schematically presented in Figure 1.2. Highly crystalline fibers are formed when
the polymer sheets are placed next to each other and form interactions [12]. Depending on
their orientation, three crystalline forms have been reported (α, β, and γ).
The most abundant form is α‐chitin, which is present in almost all crustaceans, insects,
fungi, and yeast cell walls [7]. In this formation, the chitin sheets (three sheets as example
in Figure 1.2a), consisting of parallel chitin chains (for each sheet, two chains are pre-
sented in Figure 1.2a), are positioned in an anti‐parallel way, allowing a maximum forma-
tion of hydrogen bonding. More specifically, two intramolecular and two intermolecular
bondings are formed: a first intermolecular bonding with a vertical neighbor chain (in the
same sheet), and another with a horizontal neighbor chain form a different sheet [15].
These hydrogen bounds create a remarkably high crystallinity, resulting in a more stiff
and stable material. Therefore, α‐chitin is characterized as a non‐reactive and insoluble
product [16]. Since this form is the most common polymorphic, α‐chitin has been exten-
sively studied [12].
On the other hand, in β‐chitin, the chitin sheets are ordered in parallel (Figure 1.2b)
with weaker intermolecular forces. This results in a softer molecule with a higher affinity
Insects
Algae Crustaceans
Chitin
OH OH OH OH
H O H O H H O H H O H
H O H H O H
H H H H
OH OH OH OH
O H
O O
H HN H H NH H NH
NH2
O O O
H3C H3C H3C
n
Deacetylation
Chitosan
Fungi OH OH Mollusks
OH OH
H O H O H H O H H O H
H O H H O H
H H H H
OH OH OH OH
O H
O O
H NH2 H H NH H NH2
NH2
O
H 3C
n
Figure 1.1 Chemical structure of chitin and chitosan and some examples of species that contain chitin.
(a) (b)
Sheet 1
H C
H C H C
H C
H C H C O
H C O O
H C
HO O O
H C O O
H C HO
O O O O
O O
OH O
O NH OCH
O
O NH OCH NH OCH O
O O O HO
O OH
HO O NH CH
OH NH OCH NH CH HO
NH OCH O NH CH
O H C NH HO HO
HO
OH NH CH HO HO OH
NH CH H CO NH O OH OH
HO OH
HO OH
OH O
O OH OH
O OH O
OH
O O
O O O
O O O
OH O O O O
O O O
O O O
O NH OCH
O
OH NH OCH NH OCH O
OH NH OCH O O HO
NH OCH H C O HO NH CH
O NH HO NH CH HO
OH NH CH NH CH
HO NH CH O HO
H CO NH HO OH
HO OH HO
HO O OH OH
OH O OH
O O OH OH OH
OH O
O O O O O
O
OH O
O
O O O O
O O O O
O OH O O
O OH NH OCH O NH OCH
NH OCH O NH OCH NH OCH O
O H C NH HO O HO
OH NH CH HO O NH CH
HO NH O NH CH HO
CH H CO NH NH CH
HO HO
HO OH HO OH
OH O
O OH HO
O
OH
OH OH
OH OH
O O OH O
O O
OH O O
O O O
O OH O O O O
O OH O O O
O NH OCH O O
O NH OCH H C NH HO NH OCH
OH NH OCH O
HO NH CH O O NH OCH HO
NH CH H CO NH O HO NH
HO HO CH
HO OH NH CH NH CH
OH O HO
O HO OH
O OH OH HO
OH HO
OH
HO O OH
CH OH OH HO
HO HO
HO CH HO
Chain 1 n HO HO
HO
n
n n
Figure 1.2 Schematic representation of (a) α‐form and (b) β‐form of chitin.
Sources of Chitin and Chitosan and their Isolation 5
for solvents and a higher reactivity. It is proven to be soluble in formic acid and can be
swollen in water [15]. This chitin form is present in the squid pen, in the tubes of pogo-
nophoran and vestimentiferan worms, and in monocrystalline spines excreted by diatoms
such as Thalassiosira fluviatilis [7]. Although squid and tubes of Tevnia jerichonana both
contain β‐chitin, their crystallinity differs. This implies that the crystallinity also depends
on the source. Chitin obtained from squid pens is semi‐crystalline, and chitin from T. jeri-
chonana is almost complete crystalline [7, 8, 16].
The third formation, γ‐chitin, is less common. It is considered to be a mixture or inter-
mediate form of α‐ and β‐chitin with both parallel and antiparallel arrangements [16]. More
specifically, every third chitin chain has the opposite direction to the two preceding chitin
sheets [13, 15]. Very few studies have been carried out on γ‐chitin, and it has been s uggested
that γ‐chitin may be a distorted version of the other two instead of a true third polymorphic
form.
1.2 Sources of Chitin and Chitosan

1.2.1 Sources of Chitin
For more than a century, scientists reported chitin to be present in a variety of organ-
isms. Initially, zoologists named all hard yellow–brownish structures chitin, without
chemical analysis, sometimes generating misleading data. Later on, it was accepted that
the presence of chitin could only be demonstrated after chemical tests. Hymann (1958),
for instance, used an iodine‐based color test to study the presence of chitin in different
sea animals. Later on, more sophisticated techniques such as Fourier transform infrared
spectroscopy (FTIR), nuclear magnetic resonance (NMR), mass spectroscopy (MS),
X‐ray diffraction (XRD), and Raman spectroscopy were used [18]. Quantification of
chitin is challenging and only reported in more recent publications. Currently, quantita-
tive data on chitin contents are still incomplete, and available numbers need to be inter-
preted with care. Not only are different quantification methods used, but also varying
parts of the biomass are considered (whole organism versus chitin‐rich part of the
organisms).
Nowadays, it is estimated that a large portion of chitin produced in the biosphere is
present in the oceans [19, 20]. It can be found in aquatic species belonging to phyla
such as Cnidaria (corals [21, 22]), Entoprocta [23], Phoronida (horseshoe worms [18]),
Ectoprocta [18], Brachiopoda (lamp shells [18]), Bryozoa [19], Porifera (sponges [5,
24]), and Mollusca (squid [8, 23], cuttlefish [26], and clams [8]). Further, chitin has
also been detected in fungi (mushrooms and yeasts [1]), algae (diatoms [27], coralline
algae [28], green algae [29, 30]), Onychophora (velvet worms), and protozoa [31]. The
most easily accessible sources of chitin, however, are the exoskeletons of Arthropoda,
which includes insects [32–35], arachnids (spiders [36] and scorpions [37]), myria-
pods (millipedes and centipedes [38]), as well as Crustaceans (shrimp, krill, crab, and
lobster [8, 9, 18, 37]).
Table 1.1 lists examples of chitin‐containing sources, along with available compositional
data. The amount of chitin varies with species type, the biomass part considered, and even
with seasons and growth stages [40]. Values ranging from <1% to 72% (w/w) on dry matter
basis on the biomass type have been reported.
Table 1.1 Sources of chitin.
% (w/w) chitin in biomass

Origin Species Biomass type N (%) CaCO3 (%) % protein type of dry weight Ref.
Crustaceans (Arthropoda phylum)
Crab cuticle 40–50 15–30* [8]
Blue swimming crab (male) Portunus pelagicus Shells 68.87 10.33 20.8 [26]
Blue swimming crab (female) Portunus pelagicus Shells 65.5 14.36 20.14 [26]
Crabs Shells 66.58 16.68 16.73 [25]
Marbled crab Grapsus marmoratus 10 [41]
Red crab Portunus puber 10 [41]
Spider crab Maja squinado 16 [41]
Cancer crab Cuticula 72.1 [40]
Carcinus crab Whole body 64.2 [40]
King crab Paralithodes Whole body 35 [40]
Shrimp cuticle 20–30 30–40* [8]
Jinga shrimp Metapenaeus affinis Shells 45.66 37.59 16.75 [26]
Brown shrimp Penaeus aztecus Shells 48.97 29.5 21.53 [25]
Pink shrimp Penaeus duorarum Shells 42.26 34.02 23.72 [25]
Shrimp Palaemon Fabricius 22 [41]
Shrimp Penaeus monodon Shells 5.74 10 [33]
Grooved tiger prawn Penaeus semisulcatus Shells 52.03 28.84 19.13 [26]
Scyllarid lobster Thenus orientalis Shells 61.81 16.93 21.26 [26]
Locust lobster Scyllarus arctus 25 [41]
Spiny lobster Palinurus vulgaris 32 [41]
European lobster Homarus vulgaris 17 [41]
Crayfish Procambarus clarkii Shells 63.94 15.56 20.6 [25]
Crayfish Astacus fluviatilis 36 [41]
Krill cuticle 20–25 20–30* [8]
Barnacle Lepas anatifera 7 [41]
Squilla Squilla mantis 24 [41]
Isopoda Oniscus asellus Dried adult 4.7 6–7* [32]
0004461239.INDD 6 10/26/2019 1:16:41 PM

Insects (Arthropoda phylum)
Honey bees Apis mellifera Exoskeletons 5.56 2.5 [33]
Grasshopper Aiolopus simulatrix Fully dried adult 5.3* [42]
Aiolopus strepens Fully dried adult 7.4* [42]
Duroniella fracta Fully dried adult 5.7* [42]
Duroniella laticornis Fully dried adult 6.5* [42]
Oedipoda miniata Fully dried adult 8.1* [42]
Oedipoda caerulescens 8.9* [42]
Pyrgomorpha cognata 6.6* [42]
Schistocerca gregaria Exoskeletons 2.92 12.2 [33]
Black soldier fly Hermetia illucens Fully dried prepupae 2.7–19.7 2 37.7–40.7 5.6–6.7 [34]
Hermetia illucens Fully dried larvae 17.5 2.10 3 [43]
Hermetia illucens Whole larvae 6.4 Own work
Lesser mealworm Alphitobius diaperinus Whole worm 5.6 Own work
Beetle Melolontha melolontha Fully dried adult 6.72 13–14* [35]
Beetle Calosoma rugosa Exoskeletons 5 [33]
Cockroach Blattella 18.4 ^ [40]
Cockroach Periplaneta Cuticle 54.8^ [40]
Blatta lateralis Fully dried nymphs 19.0 0.67 3 [43]
Silkworm Bombyx 44.2^ [40]
Bombyx mori L. Cuticle 23–52 36–62 [44]
Waxworm Galleria 33.7^ [40]
Tebo worms Chilecomadia moorei Fully dried larvae 15.5 1.11 3 [43]
Tobacco hornworm Manduca sexta Exoskeleton of the 60 20 [21]
adult (organic part)
Ladybug Coleoptera 27–35^ [40]
Shield bug Palomena prasina Fully dried adult 10.8* [45]
Butterfly Pieris 2 [40]
Housefly Musca domestica Fully dried adults 19.7 1.19 3 [43]
Mollusks (Mollusca phylum)
Squid (Cephalopoda) Pen Negligible 20–40* [8]
Pen 4.74 46.23 49 [25]
Loligo vulgaris 40 [41]
(Continued )
0004461239.INDD 7 10/26/2019 1:16:41 PM

Table 1.1 (Continued)
% (w/w) chitin in biomass

Origin Species Biomass type N (%) CaCO3 (%) % protein type of dry weight Ref.
Cuttlefish (Cephalopoda) Sepia spp. Shells 91.25 1.35 7.4 [26]

Pens 88.48 6.12 5.4 [25]
Sepia officinalis 20 [41]
Clam/oyster (Bivalvia) Shell 85–90 3–6* [8]
Other animals
Bryozoa Plumatella repens Dried 13.3* [32]
Black coral (Cnidaria) Antipathella fiordensis Skeleton (organic 70 10 [21]
part)
Horseshoe worms Tube [18]
(Phoronida)
Sponges (Porifera) [28]
Spiders (Arachnids) Geolycosa vultuosa 6.42 8–8.5 [36]
Hogna radiata 6.41 5.5–7 [36]
Fungi
Basidiomycota (yeast) Fomes fomentarius 2.4* [45]
Lactarius vellereus 19 [40]
Full biomass 11 [46]
Basidiomycota (mushroom) Agaricus bisporus Cell wall 43.8 [47]
Zygomycota Mucor rouxii Cell wall 50.1 [48]
44.5 [40]
Rhizopus oryzae Full biomass 14.6 [49]
Ascomycota (yeast) Aspergillus niger Cell wall 42 [40, 48]
Penicillium chrysogenum Cell wall 20.1 [40]
Penicillium notatum Cell wall 18.5 [40]
Saccharomyces cerevisiae Cell wall 2.9 [40]
Algae
Diatoms Thalassiosira fluviatilis Ropes [50]
Green algae Pithophora oedogonia Cell wall [30]
Chlorella vulgaris Cell wall [29]
Note: *not provided how it is measured; ^based on the weight of the organic cuticle, others were measured based on weight differences of the raw materials and that of the sample
obtained after acid and alkaline treatments2, crude ash3 based on acid detergent fiber, minus present amino acids.
0004461239.INDD 8 10/26/2019 1:16:41 PM

1.2.1.1 Crustaceans (Part of Arthropoda)

Chitin is located in the exoskeletons of Arthropoda. The skeleton is a tough and hard mate-
rial designed for mechanical support to the body and functions as an armor against preda-
tors. These characteristics can be dedicated to the presence of highly crystalline α‐chitin
that, combined with proteins, forms a hybrid material with high stiffness (at least 150 GPa).
These chitin–protein complexes, together with minerals for strength, form a fibrous struc-
ture that is among the most resistant organic materials [38, 52]. The shells of crustaceans
mainly contain chitin (20–30%), proteins (20–40%), minerals (30–60%), pigments, and
sometimes also lipids (0–14%) [8, 38]. Based on Table 1.1, it can be said that the chitin
content ranges from 6% to 72% in crustacean shells. This large variation can be explained
by the origin of the biomass (e.g., differences in species such as gray shrimp versus pink
shrimp, or in growth phase), the part of the biomass considered for chitin analysis (e.g.,
shells as such or stripped of remaining flesh), or the varying pretreatment or analysis
method used. Crabs and shrimp are mostly used at an industrial level and contain 10–72%
chitin. As mentioned previously, the differences in species impacts the chitin content—for
example, the Cancer crab contains 72% chitin as compared to 64% chitin in the Carcinus
crab and 35% chitin in the king crab.
1.2.1.2 Insects (Part of Arthropoda)

Chitin is found in the exoskeleton of insects, but also in internal structures such as the inner
cuticular linings of the alimentary canal and the tracheal system. In contrast with the exo-
skeletons of crustaceans, insect cuticles contain also catecholamines besides chitin, pro-
teins, lipids, and minerals. The catecholamines are cross‐linked by o‐quinones with proteins
and possibly also with chitin [14]. α‐Chitin in the exoskeleton of insects serves the same
function as for crustaceans. It increases the strength of the skeleton, gives structure, pre-
vents physical and chemical damages, and protects against infectious diseases [14]. The
chitin content differs significantly between different insect species. In addition, Kaya et al.
found that the chitin content is also significantly dependent on the life stage of the insect.
The larvae of Vespa crabro (wasp) had a chitin content of 2.2% dry weight (DW) in com-
parison with 6.2% for pupa and 10.3% of the adult. This phenomenon can be explained by
the different chitin functions in different body parts during different stages [52]. Similar
results were obtained by Kaya et al. with the larvae of the potato beetle (7% chitin) and the
adult beetle (20% chitin) [53].
1.2.1.3 Other Sources

Spiders (also part of the Arthropoda) contain α‐chitin (5–8.5%) with a high acetylation
degree. Kaya et al. characterized physicochemically the chitin structure of two spider spe-
cies (Geolycosa vultuosa and Hogna radiata), for which acetylation degrees of 97% and
99% were found, respectively [36]. Within the Mollusca, squids receive major attention
because they are the prototype for β‐chitin. In addition, high chitin content (up to 49%) in
the pen have been reported [25], which may be the basis to conclude that squid pen can
become increasingly common as another potentially important chitin source [54]. However,
it should be kept in mind that these high percentages are related to the composition of the
chitin‐rich pen that represents a very minor fraction of the whole squid. Chitin may be
involved in the formation of skeletons in calcifying marine sponges [28]. Sponges are
described more in detail in Chapter 4. Within the Cnidarian taxa, skeletons often contain,
besides chitin, calcium‐based minerals. Black corals form an exception and have a unique
halogenated scleroprotein named antipathin associated with chitin [22]. Lophophorates
(marine and freshwater Octopoda, Phoronida, Brachiopoda) have exoskeletons, named
tubes, that consists of chitin [18]. Chitin is the most important ultrastructural compound of
fungal cell walls, where it is embedded in the amorphous matrix and provides the frame-
work of the cell wall morphology [55]. It exists in the spores, mycelia, and stalks, and has
only been detected as α‐chitin [55]. Its amount ranges from 2% to 50% (w/w) on dry cell
wall base, whereby the lowest value corresponds to yeasts [56] and the highest to
Euascomycetes [55]. Depending on the class of fungi, the cell wall can also contain glu-
cans, mannans, as well as chitosan. As the cell wall is only a part of the fungal biomass, the
overall chitin (plus chitosan) yield is lower, and values have been reported (glucosamine on
dry matter base) of 8–16% (w/w) for Aspergillus niger and Mucor rouxii mycelia [48] and
12% (w/w) for Agaricus bisporus stalks [47].
In the case of algae, since 1965, diatoms such as Thalassiosira were reported to secrete
β‐chitin ropes that span between two recently divided daughter cells to keep them together,
creating flexible cell chains that float in the water [27, 50]. However, chitin has also been
shown to be present in diatoms in other forms—for instance, in the siliceous shell. In calci-
fied coralline algae such as Clathromorphum compactum, chitin has been reported to be
present that strengthens the skeleton and protects the algae from ocean acidification and
grazing in shallow waters [28]. The presence of chitin was also demonstrated in the cell
walls of the green algae Pithophora oedogonia [30] and Chlorella vulgaris [29]. Quantitative
data on the chitin content in the algae, however, are scarce. The fact that chitin in algae is
plant‐based can be an advantage for some applications.
1.2.2 Sources for Chitosan

Chitosan is mainly known as a partially deacetylated derivative of chitin, but has also
been found to be naturally present in some types of biomass. Some fungi contain chi-
tosan as an important constituent of their cell wall at various stages their life cycle. The
class of Zygomycetes (e.g., the Mucor, Absidia, Benjaminiella, Cunninghamella,
Gongronella, and Rhizopus genera) has especially been recognized as a valuable source
of chitosan [59, 60]. Chitosan content of 1–10% on dry biomass base have been found
with a reported degree of deacetylation of 83–94%. Chitosan is not directly synthesized,
but is rather the result of an efficient conversion of chitin to chitosan by the presence of
a deacetylase enzyme [57]. The deacetylation enzymes are thought to be in close proxim-
ity to the regions where the chitin transverses the plasma membrane [59]. As chitin is
synthesized, the deacetylase enzyme converts it to chitosan [57]. Since chitosan can be
isolated with less extreme procedures, fungi may become an interesting source of chi-
tosan in the future [26].
In addition to fungi, bacteria too have been reported to be able to convert chitin into
chitosan using enzymatic deacetylation. Kaur et al. isolated, from soil, bacteria (Bacillus
sp. and Serritia sp.) that produce chitin deacetylase and release chitosan. Although the
efficiency of this process is limited due the insolubility of chitin [13], it contributes to the
degradation of chitin in nature. Further, these fast‐growing bacteria, or the isolated enzymes,
offer a tool for biological chitosan production [3] (see Section 1.4).
1.3 Isolation of Chitin

Although large quantities of chitin are available and future applications look promising,
chitin is still an underutilized resource. Limited attention has been given to chitin, in con-
trast to the extensive studies on cellulose, because of the relatively laborious isolation pro-
cess. Chitin present in biomass is strongly connected to other biomolecules such as proteins
and minerals. These impurities need to be removed to generate a high‐purity product for
application development [60]. Traditional extraction processes are long, involve high con-
sumption of hazardous chemicals, are energy consuming, and are environmentally pollut-
ing due to the need for high amounts of mineral acids and alkali [8, 40, 62].
1.3.1 Technology Principles

The process steps that have been reported to isolate and purify chitin from biomass are
summarized in Figure 1.3, and explained in more detail in the subsequent subsections. It
concerns biomass pre‐treatment, deproteination, demineralization, decoloration, and post‐
treatment processes. Depending on the biomass type, the individual steps can change order,
or may even not be required. The degree of crystallinity and the deacetylation degree of
chitin depend on the source, and may also alter during the purification process, as was
shown for crustaceans [62].
Chitin
Pre- Post-
containing treatment Deproteination Demineralisation Decoloration treatment Chitin
biomass
Figure 1.3 Processes involved in the isolation and purification of chitin from biomass.
1.3.1.1 Pre‐Treatment
The pretreatment step groups all activities that are required to prepare the biomass for chi-
tin extraction. This may comprise the removal of soft tissues by scraping, boiling, and
pressing. For other organisms, boiling can be a part of a hygienization step. The biomass is
mostly dried and reduced in size. However, for fermentation purposes, for example, drying
is not necessary. Examples of pre‐treatments are given in Table 1.2.
1.3.1.2 Demineralization
When a high amount of minerals is present, demineralization is advisable. For example, the
exoskeleton of crustaceans can contain more than 50% (w/w) of CaCO3 to enhance its
strength [38, 52]. Two approaches have been reported—chemical and biological deminer-
alization. Chemical demineralization uses acids such as HCl, HNO3, H2SO4, CH3COOH,
and HCOOH [60]. Among these acids, HCl is the most commonly used reagent for remov-
ing the mineral constituents [40]. Biological demineralization is based on acid‐producing
biological processes using bacteria [3, 39, 40] or enzymes such as Alcalase® [40]. The acids
Table 1.2 Examples of pre‐treatment steps.
Origin Biomass type Pretreatment step(s) Ref.

Crustaceans Shells Washed with tap water or boiled; drying, mechanical [63]
crushing to reduce size; sieving aiming at 60–120 μm
fraction
Shrimp shells Pressing and washing to remove residual meat and [64]
connective tissue
Shrimp waste Shells were dried at 50°C for 24 h and homogenized in [65]
a laboratory mixer
Shrimp waste Washed with tap water, dehydrated at room [66]
temperature, and shred into pieces smaller than 2 mm
Shrimp shells Scraped to remove loose tissue, washed and dried, and [33]
finally grounded to pass through a 250 μm sieve
Insects Grasshopper Cleaned by washing with distilled water, dried at room [42]
temperature and pulverization
Crickets, waxworms, Starved for 24 hours to clear the gastrointestinal tract of [67]
mealworm, any residual food, killed by freezing
silkworms, beetles
Beetles Starved for 48 hours to clear the gastrointestinal tract of [68]
any residual food, washed with water, and killed by
freezing. Thawed at room temperature and air‐dried at
50°C for 2 days. The dried beetles were milled to a
powder to pass through a 20‐mesh screen.
Mollusks Squid pen The squid pens were washed with water, dried, and cut [69]
in small pieces (sieved from 2 to 5 mm).
Squid pen Samples were ground into about 18 mesh (ASTM) size [70]
formed (such as lactic acid) react with calcium carbonate, resulting in a precipitate that can
be removed by washing. Khanafari et al. stated that fermentation is less likely to change the
physicochemical characteristics of the chitin chain [65]. Table 1.3 summarizes some dem-
ineralization procedures as examples.
1.3.1.3 Deproteination
Chitin is traditionally viewed as a chain that is embedded in a protein‐matrix. During the
purification step, complete removal of proteins is advisable when applications in medical,
food, or feed are envisioned since proteins can be allergenic [10]. The proteins are bound
by multiple hydrogen bridges, and the ‘free’ amino groups of the chitin may covalently be
bound to the proteins [8, 51]. Therefore, extreme process parameters are often required for
the deproteination. Chemical deproteination is commonly performed with sodium hydrox-
ide as a preferential reagent. The effectiveness of alkali deproteination depends on the
process temperature, the alkali concentration, as well as the alkali/biomass ratio [26].
During deproteination, chitin may undergo some changes such as partial deacetylation and
hydrolysis of the polymer [10]. Also, biological deproteination using (1) proteases (such as
pepsin, papain, and trypsin) secreted by proteolytic bacteria in the fermentation medium or
(2) isolated proteases (crude or purified) has been reported [39, 40]. As the conditions are
less harsh as compared to chemical deproteination, small peptides and amino acids remain
attached to the chitin chain after enzymatic hydrolysis [10], and the deacetylation degree is
altered less (less increase) [72]. Some examples are summarized in Table 1.4.
Table 1.3 Examples of demineralization steps.
Origin Biomass type Demineralization approach (scale) Ref.

Crustaceans Crustacean shell Chemical: 0.2–2 M HCl; 1–48 hours; 0–100°C [39]
waste
Prawn shells Chemical: 0.5–1% (v/v) HCl; 24 hours; 25°C; 4:1 or 10:1 (v/w) [63]
Shrimp shells Chemical: 0.25 M HCl; 0.25 hours; 25°C; 40:1 (v/w); 2–5 [60]
repeats
Shrimp waste Biological: Lactobacillus plantarum (PTTC 1058), Lactobacillus [65]
acidophilus (PTTC 1643), and Lactobacillus rhamnosus (PTCC
1637) were added (OD600 =0.8–1) to 50 mL water + 5 g of
shrimp waste. Incubation for 7 days at 30°C and 5% CO2
Shrimp shells Biological: Lactobacillus plantarum PTCC 1058—5% w/v [40]
shrimp shell powder—addition of peptone, yeast extract,
meat extract, K2HPO4
Crustaceans/ Barnacles, crab, Chemical: 0.5–2 M HCl; 0.5–1.5 hours; 4–50°C; 10:1 (v/w): [71]
mollusks lobster, crayfish, 1–3 repeats
shrimp, cuttlefish,
squid
Prawn, shrimp, crab, Chemical: 0.25 M HCl; 0.25–3 hours; 40 mL/g [26]
lobster, cuttlefish
Marine shell waste Chemical: 0.55 M HCl; 2 hours; 25°C; 11.25: 1 (v/w) [41]
Crustaceans/ Shrimp shells, Desert Chemical: 1 M HCl; 25°C; 15 mL/g [33]
insects locus, honey bee,
beetles
Insects Palomena prasina Chemical: 2 M HCl; 2 hours; 100°C [45]
Grasshopper Chemical: 4 M HCl; 1 hour; 75°C [42]
Beetle Chemical: 1 M HCl; 30 min; 100°C [68]
1.3.1.4 Decoloration and Other Post‐Treatment Processes

When desired, a decoloration step can be introduced to remove pigments (such as pink
colors for crustaceans). A mild oxidizing treatment with, for example, hydrogen peroxide
[74] or potassium permanganate [75], or an extraction with solvents such as acetone [63],
ethanol, and chloroform [42], have been reported. Finally, some post‐treatment steps such
as neutralization, drying, and milling may be required to finalize the chitin production
process (Table 1.5).
1.3.2 Isolation of Chitin from Crustaceans

Isolation of chitin from crustaceans is well studied and documented. Generally, chitin is
produced from crustacean waste (shells) through washing and crushing of the raw material
in a pretreatment step (Table 1.2). The shells are washed with tap water or boiled before
drying [64]. After drying, the shells are mechanically crushed. Different particle sizes are
obtained and reported, such as, for example, 60–120 μm [63], less than 250 μm [26],
0.5–1 mm [76], or 0.5–10 mm [77].
Acid calcination is selected most often as a demineralization step (Table 1.3), with HCl
as the preferred reagent [40]. A concentration below 1 M is usually applied at room tem-
perature. For crustaceans, the mineral content differs significantly, resulting in the need for
different treatments [25]. As a result, the time and number of repeats can differ, depending
on the biomass. Percot et al. found that, for shrimp shells, the demineralization was
Table 1.4 Examples of deproteination steps.
Origin Biomass type Deproteination approach (scale) Ref.

Crustaceans Marine shell waste Chemical: 30–50% (w/v) NaOH; 1–72 hours; 0–100°C [40]
Shell waste Chemical: 1 M NaOH; 1–72 hours; 65–100°C [39]
Shrimp shells Chemical: 1% (w/v) NaOH; 24 hours; 70°C [60]
Chemical: 0.3 M NaOH; 1 hour; 80–85°C; repeat till [41]
absence of color
Shrimp shells Biological: Protease production of Bacillus cereus SV1 [72]
was used, shells with water (1:2 w/v) were cooked for
20 min and protease was added, incubation for
3 hours at 40°C
Shrimp shells Biological: Bacillus mojavensis A21 and Bacillus subtilis [71]
A26 were used to produce proteases, 15 g shrimp
shells + 45 mL water was mixed with the protease
Crustaceans/ Shrimp waste Chemical: 0.5–1.25 M NaOH; 0.5–24 hours; 60–100°C; [66]
Mollusca 1:10 (w/v)Room temperature
Fish scales Chemical: 0.5 M NaOH; 18 hours; 25°C; repeated till [73]
absence of color
Crustaceans/ Shrimp shells, squid Chemical: 1 M NaOH; 105–110°C; repeated till absence [25]
Mollusca pens, crab shells, of color
lobster shells
Prawns shells Chemical: 5% (w/v) NaOH; 2 hours; 60°C; 8:1 (v/w) [63]
Bryozoan, Chemical: 2 M NaOH; 20 hours; 140°C; [45]
insects,
fungal
Marine shell waste Chemical: 0.3 M NaOH; 1 hour; 80–85°C; 2–7 repeats [74]
Insects Grasshopper Chemical: 2 M NaOH; 18 hours; 175°C [42]
Beetle Chemical: 1 M NaOH; 24 hours; 80°C [68]
c omplete within 15 min at ambient temperature [60]. Younes et al. performed a two‐level
fractional factorial design and concluded that the number of batches and the concentration
of HCl are the most influential parameters for the demineralization efficiency. As tempera-
ture was not found to be a significant parameter, lower temperatures are preferred to reduce
chain degradation and chitin deacetylation. The best demineralization for shrimp was
found when using lower temperatures and longer reaction times [71]. Tolaimate et al. con-
firmed that the number of repeats (a multi process) had a positive influence, but did not find
a relation between higher acid concentration and longer reaction time in their settings [74].
Biological demineralization by the use of fermentation, for instance, solid‐state fermenta-
tion with organic acid–producing bacteria, received more attention recently as being a
more eco‐friendly, safer, and more technologically flexible alternative to chemical demin-
eralization. Arbia et al. and Kaur et al. gave an overview of the most common bacteria
utilized [39, 40]. Lactic acid–producing bacteria were usually selected, for example,
Lactobacillus spp. B2 or L. plantorum A3. Other acid‐producing microorganisms that were
tested include Bacillus subtilis, Pseudomonas aeruginosa K‐187, and Serratia marcescens
FS‐3. Since the acid also lowered the pH, which activated proteases [10], demineralization
and deproteination occurred partially simultaneously. Khanafari et al. proved that deminer-
alization using fermentation could be evenly effective if not better than chemical deminer-
alization. They also stated that fermentation was less likely to change the physicochemical
characteristics of the chitin chain [65].
Table 1.5 Examples of decoloration and post‐treatment steps.
Origin Biomass type Post treatments approach Ref.

Crustaceans Shrimp waste Sample filtrated under vacuum and washed with [66]
deionized water
Decoloration: hydrogen peroxide (35% (v/v)) overnight
at room temperature
Post‐treatment: filtered, washed, and dried at 50°C for 3
hours
Shrimp waste Post‐treatment: filtered, washed to neutrality, freeze‐ [72]
dried
Shrimp shells, Sample washed with deionized water to neutrality and [25]
crab shells, lobster dried
shells Decoloration: KMnO4 + oxalic acid + H2SO4 or refluxing
in ethanol for 6 hours
Post‐treatment: filtered, washed, and dried at 50°C for 3
hours
Prawn, shrimp, crab, Post‐treatment: washed to neutrality and washed with [26]
lobster, cuttlefish hot ethanol (10 mL/g), and later boiled in acetone to
remove any impurities; purified chitin then dried
Bryozoa, Decoloration: refluxed with distilled water, methanol, [45]
insect, and chloroform (4:2:1), respectively, room
fungi temperature, 2 hours
Post‐treatment: neutralized and dried (3 days, 50°C)
Insect Grasshopper Decoloration: chloroform, methanol, and distilled water [42]
(1:2:4), rinsed with distilled water
Post‐treatment: dried (60°C, 24 hours)
Beetle Decoloration: 1% (w/v) potassium permanganate [68]
solution, 1 hour, rinsed with distilled water
Post‐treatment: dried (50°C)
In respect to deproteination (Table 1.4), a chemical approach with sodium hydroxide is

often used, even if reaction conditions are very variable. The concentration of NaOH ranges
from 0.1 to 5 M, and the temperature can increase up to 160°C. Chang and Tsai found that
2.5 M NaOH at 75°C and a minimal solution‐to‐solid ratio of 5 mL/g was the most effec-
tive to clean shrimp shells [78]. In contrast, Tokatli and Demirdoven did a Box–Behnken
experimental design and found that 0.95 M NaOH for 75.6 min at 60.5°C were the optimal
conditions to deproteinate a similar biomass [66]. Both studies did not assess possible
changes on the physicochemical properties of the polymer. During deproteination, partial
deacetylation of chitin has often been reported [10]. Nevertheless, Percot et al. found that
temperature and time had no influence on hydrolysis and deacetylation with NaOH con-
centrations below 1 M and temperatures below 70°C [60]. In summary, literature reported
divided conclusions concerning optimal process parameters, which can be attributed to the
different sources (species) used or the different pretreatment applied. For example, Younes
et al. also found that the particle size obtained in the pretreatment step impacted the effi-
ciency of the purification of chitin [71]. Biological deproteination with proteolytic enzymes
and protease‐producing bacteria is an alternative to remove proteins during chitin isolation
[39, 40]. In comparison to chemical deproteination, the efficiency of the enzymatic method
is lower, leaving approximately 5–11% residual protein [10, 72]. In addition, the use of
crude proteolytic extracts obtained from different microorganisms or even from fish v iscera
have been studied [61]. Although the deproteination levels achieved in such cases are gen-
erally lower than those obtained using alkaline treatments, this alternative has the advan-
tage to produce nutritionally valuable protein hydrolysates in addition to chitin.
In conclusion, the intensity of the demineralization and deproteination steps depends on
the source [74]. It is generally accepted that these steps significantly change the physico-
chemical properties of chitin, for example, the molecular weight and acetylation degree
[40, 60]. Both the degree of crystallinity and the acetylation degree of chitin depend on the
source and the method of purification [62]. Most researchers favor performing the demin-
eralization first, followed by deproteination [74]. However, it is considered that the order
of these two phases in interchangeable depending on the source [61, 79].
1.3.3 Isolation of Chitin from Insects

In recent years, the production of proteins from agri‐waste using insects has increased
rapidly, and numerous startup companies have emerged. This has led to a new chitin‐rich
side‐stream composed of spent prepupae shell, residue after molting, and dead insects,
which offers a tremendous potential for chitin and chitosan production as a by‐product.
The procedure to isolate chitin from insect cuticles is very similar to the one used in crus-
taceans. Nevertheless, insects have lower levels of inorganic material, allowing a more
gentle demineralization step [2]. Some examples of chitin extractions from insects are
given in Table 1.6. Only a few articles could be found, indicating that insect chitin is a
relatively new field. For this reason, no standard or engineered procedure for insects was
found.
1.3.4 Isolation of Chitin from Other Biomass Types

As mentioned in Section 1.2.1.3, fungi can contain considerable amounts of chitin, reach-
ing up to 50% of the cell wall on a dry matter base. They are, however, one of the very few
biomass sources in which chitosan can naturally be present in significant amounts, and
many studies target the isolation of chitosan rather than that of chitin (see Section 1.4).
Nevertheless, the isolation of (crude) chitin is regularly targeted on a lab scale, mostly to
investigate its properties [29, 48, 63]. As the characteristics of the fungal biomass differ
from the ones of the traditional and commercial source of chitin (i.e., crustaceans), the
chitin isolation procedure deviates in some aspects from process steps as outlined in
Figure 1.2. During the pretreatment, the filtered and washed fungal biomass is typically
dried, which requires a more extensive dewatering/drying step, given the wet nature of the
biomass. No demineralization step is performed, since the fungal biomass does not contain
appreciable quantities of calcium carbonate and other mineral salts [88]. After pretreat-
ment, a base extraction is applied, which not only aims to remove the proteins but also the
alkali‐soluble polysaccharides, leaving the alkali‐insoluble cell wall behind. This chitin‐
rich cell fraction is characterized and used [89, 90] as such or further purified to remove the
remaining glucans, for instance, by selective enzymatic hydrolysis [91] (or separate chi-
tosan, if present).
Mollusks are generally processed following the standard procedure elaborated for crus-
taceans. Nevertheless, some differences are reported. For instance, it was noticed that the
CO2 emission was stronger in case of cuttlefish than other (crustacean) species [26].
Table 1.6 Examples of chitin extraction procedures applied on insects.
Insect Insect stage Pretreatment Demineralization Deproteination Post‐treatment Ref.

Colorado potato Larvae/adult Drained in 70% (v/v) 20 g + 100 mL 2 M 50 mL 2 M NaOH Filtered and washed, drained in [53]
beetle beetle alcohol, washed, dried at HCl at 65–75°C for at 80–90°C for chloroform (1), methanol (2), and
room temperature, 2 hours 16 hours water (4) for 1 hour, filtered and
grounded, dried at 65°C washed, dried at 60°C
for 3 hours
Crickets: Gryllus Dried crickets, grounded, (3) Oxalic acid, (1) 40 g + 400 mL (2) Drained in ammonium persulfate [80]
bimaculatus washed, and dried at 60°C 3 hours at room 1 M NaOH 95°C solution (50% (w/v)) at 50°C for
temperature for 6 hours 6 hours
(4) Dried at 60°C overnight
Calliptamus Grounded, dried at 70°C for 2 g + 100 mL 1 M HCl 1 M NaOH at Washed and dried at 55°C, drained [81]
barbarus and 30 min at 100°C for 30 min 80–90°C for in chloroform (1), methanol (2),
Otopharynx 21 hours and water (4) for 1 hour
decorus
House fly: Musca Fourth instar Washed with 15% (w/v) (3) 10 mg/mL oxalic (1) 10 g + 100 mL (2) 10 mg/mL potassium [82]
domestica larvae aqueous NaCl, freeze‐ acid aqueous for 1 M NaOH at permanganate aqueous solution
dried, grounded 3 hours 95°C for 6 hours for 4 hours
(4) Neutralized and freeze‐dried
Silkworm Chrysalides Dried by lyophilization for 1 M HCl at 100°C for 1 M NaOH at 80°C Washed with Na2CO3 0.4% (w/v), [83]
12 hours 20 min (10 mL for 24 hours dried at 80°C
HCl/g) (10 mL NaOH/g)
Blowfly: Larvae Washed with NaCl, dried at (3) Oxalic acid (10 mg/ (1) 10 g + 100 mL (2) Decolored with NaCl (0.5%, [84]
Chrysomya 50°C, grounded mL) for 3 hours NaOH 1 M at w/v) for 3 hours
megacephala 95°C for 6 hours (4) Washed to neutral with distilled
water and then freeze‐dried in
vacuum
Black soldier fly: Pupal exuviae Cleaned, dried, grounded 1 M HCl for 1 hour 1 M NaOH at 80°C 1% KMnO4 and excess of KMnO4 [85]
Hermetia and dead for 24 hours was removed by 4% (w/v) oxalic
illucens imago acid
House cricket: 8 weeks old Starved for 48 hours, (2) 200 mL 10 M (1) 20 g + 200 mL 200 mL 1% NaCl (1%, w/v), at room [86]
Brachytrupes frozen‐washed with water, oxalic acid at room 1 M NaOH at temperature for 3 hours, filtered,
portentosus dried at 60°C for 48 hours temperature for 95°C for 6 hours washed to neutral pH, dried at
3 hours 60°C overnight
(Continued)
0004461239.INDD 17 10/26/2019 1:16:43 PM

Insect Insect stage Pretreatment Demineralization Deproteination Post‐treatment Ref.

Beetle: Holotrichia Adult beetles Starved for 48 hours, 5 g + 250 mL 1 M HCl 1 M NaOH at 80°C Washed till neutral pH, 1% (w/v) [68]
parallela washed, frozen, defrosted, at 100°C for 30 min for 24 hours potassium permanganate for
dried at 50°C for 48 hours, 1 hour, washed and dried at 5°C
grounded
7 Orthoptera Grasshoppers Washed, dried at room 2 g + 100 mL 4 M HCl 50 mL 2 M NaOH Filtered, washed, drained in [42]
species temperature, grounded at 75°C for 1 hour at 175°C for chloroform (1), methanol (2), and
18 hours water (4), washed with distilled
water, dried at 60°C for 24 hours
Desert locus, Exoskeletons Scraped free from loose 1 M HCl at room 1 M NaOH at Filtered, washed till neutral pH, [33]
beetles, honey tissue, washed, dried, temperature 100°C for washed with hot ethanol and
bee grounded (15 mL/g) 8 hours, repeated boiled in acetone, dried in
several times vacuum oven at 50°C
Melolontha Adults Killed using a killing agent, 2 g + 50 mL 4 M HCl 4 M NaOH at Filtered, washed till neutral pH, [35]
melolontha washed, dried at 60°C for at 75°C for 2 hours 150°C for drained in chloroform (1),
24 hours, grounded 18 hours methanol (2), and water (4) for
20 min, washed, filtered, dried at
60°C for 24 hours
0004461239.INDD 18 10/26/2019 1:16:43 PM

1.4 Production of Chitosan

Due to the high crystallinity, chitin is insoluble in many common solvents. Even if there are
some exceptions, however, these solvents are usually toxic, corrosive, or degradative, and
thus cannot be used in scaling up procedures for pharmaceutical applications [92]. Due to
this insoluble nature of chitin, attention has been given to convert chitin into more soluble
derivatives. Among the various reactions that can disrupt the intra‐ and intermolecular
bonds causing the high crystallinity, N‐deacetylation is the simplest modification, trans-
forming chitin to chitosan [79]. Chitosan is generally insoluble in organic solvents and
water, but soluble in diluted acid solutions with a pH below 6. This is because the chitosan
amino groups have pKa values around 6.3, implying that, at low pH, the amines will be
protonated and become positively charged. There is a positive correlation between the
degree of deacetylation (DDA) and the number of positive charges potentially present in
the chitosan chain. The charge density, as well as the exact pKa value, and in turn also the
solubility of chitosan, are highly dependent on DDA [7, 87]. Other factors affecting the
charge density of chitosan, such as the ionic strength and the distribution of acetyl groups
along the chain, also affect the solubility of chitosan [7]. Chitosan is chitin’s most impor-
tant derivate in terms of application [7], since the amino group on chitosan makes easy
chemical modification possible [11] and gives the molecule some specific functionalities.
The production of chitosan can be achieved via two approaches that start from a different
biomass type (Figure 1.4). The most common approach is converting extracted and purified
chitin into chitosan via a deacetylation step that may be linked to some pre‐treatment (to
decrease crystallinity) and post‐treatment steps. A second option is to start from chitosan‐
containing fungi biomass and separate it from other biomass components via a cleaning
and chitosan extraction step, potentially linked to some pre‐treatment (drying, etc.) and
post‐treatment steps (drying, milling, etc.). Both approaches are detailed in the following
sections.
Chitin Pretreatment Deacetylation Post-treatment
Chitosan
Fungi Pre- Chitosan Post-

treatment Cleaning step
biomass extraction treatment
Figure 1.4 Processes involved in the production of chitosan.
1.4.1 Conversion of Chitin to Chitosan

1.4.1.1 Deacetylation Reaction Mechanism
Deacetylation is a two‐step nucleophilic substitution reaction (Figure 1.5). The first step
consists of a nucleophilic addition of an hydroxide on the carboxy groups [74], while, in
the second step, an amine is formed (=chitosan) when acetic acid is split off. The reaction
OH OH OH OH OH OH
OH OH
H O H O H H O H H O H H O H O H
H O H H O H
H O H H O H H O H O
H H H
OH H H H H H H H
H OH OH OH OH OH OH OH
O O O H
O O O
H HN H H NH H NH H NH NH NH
NH H NH H H
O O O O
H C H C H C H C
n n
CH3
R1
O O– O
O
OH
NH
R NH + OH– ⇋ R ⇋ R OH
+ NHR1–
O
NH O HO
1
R OH O
1 HO O
R
NH
OH
O
O CH3
+ n
NH2R1 R
R O–
Figure 1.5 Reaction mechanism of the deacetylation step.

follows a pseudo‐first‐order kinetic during the initial period (first hour) when the concen-
tration of alkaline is high [76, 93].
1.4.1.2 Chemical Deacetylation Process

Deacetylation is traditionally performed in an alkaline solution using sodium or potassium
hydroxide at concentrations of 30–50 w% [13]. A two‐step homogeneous deacetylation
process can be realized with water‐soluble chitin, referring to chitin with a DDA between
45% and 55% that is soluble in neutral water [16]. Chitin can be made water‐soluble by
treating it with 10% (w/v) NaOH at room temperature for 70 hours (step 1). The subse-
quent deacetylation with soluble chitin is considered to take place randomly along the main
chain and occurs under moderate alkaline conditions—for instance, with 13 w% alkali for
12–24 hours at 25–40°C (step 2) [13]. A heterogeneous deacetylation can be realized in a
one‐step process with solid chitin. Under these conditions, mainly the amorphous zones are
impacted, leading to a heterogeneous, more block‐like deacetylation pattern [13, 16].
Heterogeneous deacetylation is commonly performed with higher aqueous alkali concen-
trations (40–50% (w/v)) and at higher temperatures (100–160° C), and is preferred for
industrial purposes [40]. Some examples of deacetylation conditions and linked pre‐ and
post‐treatment are summarized in Table 1.7 and Table 1.8, respectively.
Table 1.7 Examples of chitin deacetylation.
Origin Biomass type Deacetylation approach (DDA‐reached) Ref.

Not specified Not specified 13 w% alkali for 12–24 hours at 25–40°C [13]
(homogeneous deacetylation)
Crustaceans Crabs, lobsters, crayfish, 50 w/w% KOH, 25 w/w% 96° (v/v) ethanol, [74]
and shrimp, cuttlefish, squid 25 w/w% monoethylene glycol process,
Mollusca 0.5 g scale
Crabs, lobsters, crayfish, 50 w/w% NaOH, 80°C, 60 mL/g, under [74]
shrimp, cuttlefish, squid nitrogen atmosphere (Kurita process), 0.5 g
scale
Crustaceans Prawn shells 25–50% NaOH; 80–100°C; 2, 5, 10 hours; [63]
solid/liquid 1:5
Lithodes antarcticus, 50 w% NaOH; 110°C; 4 hours; 10 mL/g, [76]
Paralomis granulosa, under N2 atmosphere
Palinurus vulgaris
Shrimp waste 50 w% NaOH; 105°C; 1–4 hours; 20 mL/g [94]
Shrimp waste 421 g/L NaOH; 130°C; 16.5 g/L [95]
Shrimp shells 40–50 w% NaOH; 90°C; 1.5–2 hours [70]
Insects Larvae and adult beetles of 50% NaOH (w/v 1:20) at 100°C for 3 hours [53]
the potato beetle
Calliptamus. barbarus and 50% NaOH (w/v 1:15) at 130°C for 2 hours [81]
Otopharynx. decorus
Larvae of the blowfly 670 g/L NaOH at 90°C for 9 hours (with the [84]
renewal of alkali every 3 hours)
Mollusca Squid (β‐chitin) 40–50% NaOH; 60–90°C; 2–4–6 hours; [70]
solid/liquid 1:20; up to three treatment
steps
Squid (β‐chitin) 20–60 w% NaOH; 60–110°C; 5–80 min; [96]
solid/liquid 5:100
Table 1.8 Examples of pre‐ and post‐treatment steps linked to chitin deacetylation.
Origin Biomass type Deacetylation approach (scale) Ref.

Not specified Not specified Pre: pre‐swelling of chitin to increase the number of [13]
amorphous zones
Crustaceans Krill shells Pre: mechanical disintegration; repeated freezing and [97]
thawing of alkaline chitin solutions (13–24% (w/v)
NaOH), increase the number of amorphous zones
Lithodes antarcticus, Pre: milling and sieving (0.5–1 mm fraction) [76]
Paralomis Post: washed with distilled water until pH 7
granulosa,
Palinurus vulgaris
Crustaceans Crabs, lobsters, Post: wash step with water to neutral pH, wash step [74]
and Mollusca crayfish, shrimp, with methanol, wash step with acetone, drying
cuttlefish, squid 50°C for 12 hours
Two traditional deacetylation procedures described by Broussignac [98] and Kurita et al.
[99] were compared by Tolaimate et al. using α‐chitin and β‐chitin from different sources.
The Broussignac procedure is based on an anhydrous reaction medium composed of 50
w/w% potassium hydroxide, 25 w/w% 96% (v/v) ethanol, and 25 w/w% monoethylene
glycol. For the Kurita procedure, an aqueous sodium hydroxide solution is used. Tolaimate
et al. showed a benefit of the Kurita approach for the deacetylation of β‐chitin, but not for
α‐chitin. For α‐chitin, the method of Broussignac proved to be more efficient, explained by
the higher solubility of NaOH in aqueous media as compared to the solubility of KOH in
the anhydrous media. The dielectric constant of NaOH in aqueous solution is much higher
and perhaps favors the development of charges (during the deacetylation reaction). They
also found that α‐chitin treated with the Kurita medium required a higher reaction time for
reaching the same DDA as Broussignac, and this happened at the expense of the molecular
weight of chitosan and its quality. They suggested that the difference between α and β chi-
tin can be due to morphological changes over time leading to more accessible amine
groups. For α‐chitin, these changes may be quicker in time for the Broussignac media, and
may thus lead to more efficient deacetylation [74]. Despite these results, the Kurita proce-
dure is considered the most common process, as indicated in Table 1.7. In general, deacety-
lation is performed at high NaOH concentrations (30–60 w%), high temperatures
(80–150°C), and with long reaction times (1–80 hours) [8, 13, 70, 100, 101].
1.4.1.3 Tailoring Deacetylation Degrees of Chitosan

The specific characteristics (DDA and molecular weight) of the resulting chitosan are not
only influenced by the deacetylation process parameters (reagent type, reagent concentra-
tion, temperature, reaction time, atmosphere, repeating steps), but also by the nature of the
chitin source, such as its physical structure (e.g., particle size) and the isolation process
applied [10, 74, 102]. For example, the time needed to obtain a similar DDA with the
method of Broussignac differed significantly. As such, a general procedure to obtain chi-
tosan with specific characteristics does not exist.
• Example 1: The deacetylation procedure took 1 hour for squid chitin, 2 hours for grey
shrimp and Squilla chitin, 3–4 hours for pink shrimp and prawn chitin, and 6–7 hours
for lobster or red crab chitin [74]. The intensity of the deproteination caused differences
in partial deacetylation (5–20%) [16].
• Example 2: The optimal process parameters for α‐chitin extracted from shrimp waste
were 100°C for 12 hours with 50 w% NaOH in the study by Tokali et al., where a DDA
of 80.06% was obtained [66]. For β‐chitin, 50 w% NaOH at 90°C for 6 hours proved to
be best and a DDA of 95.6% was achieved [70].
• Example 3: Younes et al. investigated the impact of seven factors on the deacetylation of
shrimp chitin. A clear significant effect of temperature and the alkali reagent was
detected (NaOH was much more efficient than KOH). Also, the DDA was significantly
affected by the use of repeats, the reaction time, and the concentration of alkali. The
atmospheric (nitrogen or air) condition and the use of reducing agents (NaBH4), how-
ever, did not have a significant effect on the DDA, but influenced the molecular weight
of chitosan.
1.4.1.4 Deacetylation Drawbacks and Alternative Methods

During the alkaline treatments, chain degradation occurs. Although the mechanism is not
yet fully known, the high concentration and prolonged reaction time required to obtain a
complete deacetylation seem to be disadvantageous [41]. Galed et al. studied the degrada-
tion during deacetylation for three different sea animals. Even when a nitrogen environ-
ment was applied, chain degradation occurred (±1000 kDa to ±500 kDa after 250 min at
110°C, 50 w% NaOH) [76]. Tolaimate et al. found that β‐chitin, deacetylated by 40%
(w/v) NaOH at 80°C for 6 hours, had a larger molecular weight than the one treated at the
same conditions for 9 hours [69]. Methacanon et al. reported that a twofold increase in
time and a 1.25‐fold increase in temperature resulted in a chitosan molecular weight
decrease of 20–30% and 60%, respectively [94]. A nitrogen environment or the presence
of a reducing agent (such as NaBH4 or thiophenol) could reduce the degradation of chi-
tosan [10, 40].
From the preceding text, it is clear that chemical deacetylation results in high amounts
of alkaline wastewater [61]. Therefore, alternative deacetylation procedures are studied to
replace the environmental‐unfriendly method of Kurita and Broussignac or to decrease the
degrading effect.
• Acid hydrolysis was not considered to be suitable, since the reaction was found to be
limited due to steric effects, and it resulted in even more extensive hydrolysis of the
backbone [54].
• Tsai et al. described a process of steam explosion for deacetylation. Flash treatment was
also reported to give highly deacetylated chitosan over a shorter time [103].
• The use of enzymes such as deacetylases has been reported. The deacetylase was found
to recognize a sequence of four N‐acetyl glucosamine units, whereby one underwent
deacetylation. The produced chitosan showed a more regular deacetylation pattern as
compared to chemical deacetylation. However, it was concluded that deacetylase was
not effective, as the DDA remained low (<10%) on natural, insoluble, crystalline chitin
[10]. Pre‐treatment to obtain better access to the acetyl groups was considered to be
necessary. In this case, when chitin was soluble and more amorphous, deacetylase was
able to perform deacetylation [104]. For example, Kim et al. were able to prepare
c hitosan with a DDA of 71–88% with deacetylase [105]. Besides the necessary pre‐
treatment, deacetylase has a high production cost and requires a long reaction time. This
makes the enzymatic approach less ideal for commercial applications [100].
1.4.2 Chitosan Extracted from Fungi

Many fungal species have been investigated for the production of chitosan [47, 57, 106–
109]. Typically, waste fungal biomass from the biotech industry as well as dedicated fungi
are considered. More than 60% of the biotech industries use fungi in different processes
such as brewing and baking, and for food, antibiotics, pharmaceuticals, organic acid, and
enzyme production. For instance, citric acid, the most widely used organic acid in a wide
array of food, beverage, and pharmaceutical applications, is produced with Aspergillus
niger, which results in an annual waste of ~80 kTon mycelium [58]. As another example,
the cultivation of Agaricus bisporus, the most popular mushroom worldwide, results in a
waste of 5–20% in the form of stalks and mushrooms with irregular dimensions and shapes.
This already corresponds to an annual mushroom waste of ~50 kTon in the United States
[48]. These specific biotech wastes, among others, have been investigated for the extraction
of chitin and chitosan. As a second approach, the direct fermentation of fungi for chitinous
material production is also considered. Mucor rouxii has been the most commonly investi-
gated [109–112] as the content of chitosan can reach 35% in the cell wall dry weight [112].
These fungi are usually harvested at their late exponential growth phase to obtain maximal
yield [113].
As compared to the recovery of chitin from marine sources, differences can be noted in
the isolation and production process. Whereas the isolation of chitin from marine sources
is performed at an industrial scale, the recovery from fungi is still in its infancy, whereby
procedures are applied that are more common to a lab environment [114]. The overall pro-
cess can be separated into a pretreatment, an alkali‐based “cleaning” step, an acid extrac-
tion step, followed by a posttreatment for chitosan separation, and purification.
1.4.2.1 Pretreatment
Fungi are a wet biomass and are typically extensively washed, dried, and refined before
processing. Their low mineral content, on the other hand, does not require a demineraliza-
tion step that is needed in the chitin production from marine resources. Moreover, as some
fungi of particular interest are rich in the deacetylated chitosan, often the isolation of this
particular compound is targeted, leaving the (crude) chitin behind. This not only alters the
details of the extraction step to allow the specific isolation of chitosan, but also avoids the
need for a post‐deacetylation processing step.
1.4.2.2 Alkali‐Based “Cleaning”

Even if the details of the extraction method vary among the different fungal sources/wastes
used, the extraction of the fungal chitinous material always starts with a dilute alkaline treat-
ment to remove proteins, glycoproteins, and alkali‐soluble polysaccharides. This p rocess
resembles the alkaline process also performed to produce chitin from marine wastes. It is
typically performed at low substrate loading (~1:30–1:40 w/v), high alkali concentration
(1–4 M), high temperature (80–121°C), and short contact times (15 min–3 hours) [48, 59,
60, 89, 106]. The alkali‐insoluble residue, containing the chitin, chitosan, and beta‐glucan,
is separated and extensively washed till neutral pH before further processing.
1.4.2.3 Chitosan Extraction and Post‐Treatment

In a next step, the residue is typically extracted with an acidic solution to dissolve the chi-
tosan, leaving behind chitin and beta‐glucan as insoluble residues. Acids such as HCl,
H2SO4, and mostly acetic acid are used at a concentration of 0.5–2% (v/v), and the extrac-
tion is typically performed at a high temperature (60–95°C) over 6–14 hours [57]. The
residual crude chitin is typically associated to the beta glucans through covalent bonds, and
is difficult to recover in a chemical approach without degradation. The chitosan‐loaded
acid solution is recovered and brought to pH 10 to precipitate the chitosan. The precipitate
is extensively washed, usually with a combination of water, ethanol, and acetone, and
finally dried.
In a more industrial approach, recently described in patent literature, some modifications
were suggested to improve the procedures described earlier. Rather than drying the bio-
mass first, the alkali extraction was immediately performed on the wet biomass. Moreover,
by optimizing the alkaline extraction to increase deacetylation of the chitin or, alterna-
tively, to enzymatically hydrolyze the beta‐glucans with specific enzymes to purify the
crude chitin, increased extraction of chitosan/chitin was targeted [112, 113]. Some exam-
ples of chitosan extraction procedures from fungi are summarized in Table 1.9.
1.5 Towards Commercial Applications

Currently, the main sources for commercial chitin and chitosan production are crustaceans
[58]. The annual worldwide production of crustacean shells amounts 1.2 million tons [118],
and approximately 80000 tons of chitin is obtained from marine by‐products [58]. Shells
of crustaceans such as crabs and shrimp are mostly used to extract commercial chitin, for
the simple reason that they are conveniently available as waste from seafood‐processing
industries [16]. About 35% of the captured marine organisms are handled as by‐products
and waste. This includes the skeletons, heads, and the viscera. This waste is mostly dumped
back into the sea, causing environmental pollution [61]. Kurita noted that the most likely
promising source in the future for chitin extraction will be krill (20–30%) [16]. Nevertheless,
other sources such as fungi, squids, oyster, cuttlefish, and insects also offer potential [2].
Rhazi (2000) indicated that squid pen can become increasingly common as another poten-
tially important chitin source [54]. An overview of advantage and disadvantages for three
chitin sources is given in Table 1.10, being crustaceans, fungi, and insects.
The advantages associated with crustaceans are the existence of full‐scale industrial pro-
cesses, the availability of significant amounts of seafood waste, and extended data sets on
chitin extraction and processing. The disadvantages reported comprise discontinuous sup-
ply due to seasonal variations of marine sources, heterogeneity of the biomass (different
species), the high mineral contents that require long processing times and more stringent
process conditions, and allergens.
Fungi have recently received increased attention as a source of chitin due some specific
advantages as compared to crustaceans. The fungal mycelium can be obtained by convenient
Table 1.9 Examples chitosan extraction procedures from fungi.
Origin Biomass type Extraction approach (scale) Ref.

Fungi Absidia coerulea Alkali “cleaning” with 1 N NaOH/121°C/30 min [106]
(Zygomycota) Chitosan extraction with 2% (v/v) HCl/95°C/12 h
Fungi Mucor rouxii Washing of mycelia and drying [115,
(Zygomycota) Rhizopus oryzae Alkali “cleaning”: 1 N NaOH/121°C/15 min/substrate 116]
ratio 1:30 w/v
Recovery of insoluble fraction and washing till
neutral pH
Chitosan extraction: 2% (v/v) acetic acid/95°C/8 hours/
substrate ratio 1:40 w/v
Recovery of soluble fraction and precipitation at pH 10
Recovery of chitosan precipitate and washing with
water, ethanol, and acetone, and drying
Fungi Rhizopus pusillus Concentrated mycelium [117]
(Zygomycota) Alkali “cleaning”: 0.5 N NaOH/90°C/120 min/
Recovery of insoluble fraction and washing till
neutral pH
Chitosan extraction: 1% (v/v) sulfuric acid/121°C/20
min/substrate ratio 1:100 w/v
water
Scale: < 1 kg mycelium
Fungi Various Washing of mycelia and drying [87]
(Basidiomycota) Basidiomycetes Alkali “cleaning”: 1 N NaOH/40°C/overnight/substrate
ratio 1:33 w/v
Recovery of insoluble fraction and water wash at 95°C
Chitosan extraction: 5% (v/v) acetic acid/90°C/3 hours
Recovery of soluble fraction and precipitation with 1 N
NaOH
Recovery of chitosan precipitate and water wash till
neutral pH
Scale: 3 g
Fungi Mucor rouxii Freeze‐dried mycelia [48]
(Zygomycota) Alkali “cleaning”: 1 N NaOH/95°C/30 min/substrate
ratio 1:40 w/v
Recovery of insoluble fraction and washing with water
and ethanol
Chitosan extraction: 2% (v/v) acetic acid/95°C/6 hours/
water till neutral pH and drying
fermentation, and its availability does not experience geographic or seasonal limitations.
The better controllable fermentation guarantees a chitin/chitosan final product with more
consistent properties [119]. Fungal mycelium also has some compositional advantages as
compared to crustacean wastes. It contains less inorganic compounds, avoiding the need
for a demineralization treatment in the chitin production [120]. Moreover, whereas c hitosan
Table 1.10 Advantages and disadvantages of three chitin sources (partially based on [58]).
Crustaceans Insects Fungi

Source Main source for industrial Only lab scale [124] Only lab scale [19]
purposes nowadays [19,
124]
Side‐stream of seafood Side‐stream of breeding Side‐stream from
industry [39], which is (cocoons, sheddings) and production of
widely available, other industries (e.g., the mushrooms [124]
renewable, and zero cost silk industry) [84]
[124] Possibly, a side‐stream when Controlled production all
proteins are extracted for year round that results
food/feed industries in better reproducibility
Side‐stream when insects are [47]
caught in the prevention of
pest outbreak [35, 53]
Seasonal supply [19] Can be bred on cheap bio‐ Can be bred on cheap
waste [84], easy to bio‐waste [124]
massively rear [84],
tremendous potential as a
natural resource because
of high biodiversity of
insects [68]
Purification Long processing times, high Lower level of inorganic No demineralization
of chitin temperatures, and high material as compared to needed and mild
concentration of acids and crustaceans [124] extraction parameters
alkalis are necessary to [19]
purify [10, 19]
Production Well‐established method for Limited literature data; same No chemical
of chitosan industrial production of procedure as crustaceans deacetylation needed.
chitosan is mostly applied Fermentation leads to a
more reproducible
product by better
controlled process
parameters [57]
High cost and laborious
process, harsh process
parameters, high quantity
of waste water [19]
Enzymatic deacetylation
needs pretreatment [104],
high cost of enzymatic
deacetylation, and long
reaction time [100]
End product High molecular weight [19] No literature data found Medium–low MW [19], a
more narrow molecular
mass distribution than
the chitosan produced
from seafood [13]
Animal protein contamination More controlled
with possible allergenic molecular weight DDA
properties, which limits the and distribution of
use in biomedicine and deacetylated groups [9]
various other sectors [19]
Heterogeneous biomass leads Physical properties of
to inconsistent levels of extracted chitosan
DDA and high MW [19, notably related to the
125] growth substrate
composition [47]
is not native to animal resources, it is present as structural components in the cell wall of
fungi from the Zygomycetes class, such as Mucor, Absidia, and Rhizopus [55]. In addition,
recent observations suggested that non‐Zygomycetes plant and insect pathogenic fungi
also have high proportions of chitosan in the cell walls [121]. These types of fungi allow
the direct production of chitosan, avoiding the need for a deacetylation step to transform
chitin to chitosan. Additionally, fungal chitosan is free of allergenic shrimp protein and has
been reported to have beneficial properties in terms of homogeneous polymer length and
solubility over the current marine source [58]. However, the processes of chitosan produc-
tion from fungi are not scaled up to industrial level [106].
As insect farms are currently being established in Europe with focus on selected insect
species, insects become a potential alternative source for chitin production. Compared to
crustaceans, the insect‐based source (exoskeletons) is more homogeneous (mostly one spe-
cies) and is not seasonally dependent. As with fungi, the mineral content is lower, which
justifies reducing the demineralization step to a minimum. On the other hand, the chitin
production process from insects is less studied, and mostly procedures elaborated for crus-
taceans are applied.
Although commercial facilities for chitin and chitosan production exist (for Crustaceans),
the examples mentioned in the previous sections were mainly performed at lab scale (<1–
40 g). Upscaling to pilot and full scale usually requires modifications to the protocols.
Lopez et al. estimated, based on lab date, the environmental impact of chitin production
processes that involve strong acids and bases at high temperature in multiple process steps
[122]. The chemical extraction of chitin would require, per ton chitin, 72.7 m3 of water,
12231 kWh, 727 kg of NaOH, 2319 kg HCl, and 354 kg NaClO, and would produce 1.8
tons of CO2 and 21.8 tons of wastewater. Switching to enzymatic deproteintion and water
recovery would generate only half of the chitin from the same biomass, but could reduce
the environmental impact per ton to 0.9 m3 of water, 516 kWh, 46 kg of NaOH, 339 kg HCl,
84 kg NaClO, and 3.7 kg of enzymes, and would produce 0.08 tons of CO2 and 0.2 tons of
wastewater. Both approaches were predicted to be profitable. Gómez‐Rios (2017) also
reported on profitable chitosan production from shrimp shell waste, with low volumetric
yields and high water requirements as main drawbacks. Re‐use of water, including enzy-
matic processes and co‐valorization of other compounds, would increase the sustainability
of the process [123].
1.6 Outlook
Chitin is a natural polysaccharide that is omnipresent in nature. Despite the diverse poten-
tial applications of chitin, the compound is underspent. Upscaling of the multistep extrac-
tion process and the production of reproducible chitin and chitosan products in a more
sustainable way are challenges that need to be overcome to increase the commercial pro-
duction activities of crustaceans and extend them to other source such as fungi and insects.
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2
Methods of Isolating Chitin
from Sponges (Porifera)
Sonia Ż ółtowska1,2, Christine Klinger4, Iaroslav Petrenko3,
Marcin Wysokowski1,2, Yvonne Joseph2, Teofil Jesionowski1,
and Hermann Ehrlich2
1
Institute of Chemical Technology and Engineering, Faculty of Chemical Technology,
Poznan University of Technology, Poznan, Poland
2
Institute of Electronics and Sensor Materials, TU Bergakademie‐Freiberg, Freiberg, Germany
3
Institute of Experimental Physics, TU Bergakademie‐Freiberg, Freiberg, Germany
4
Institute of Physical Chemistry, TU Bergakademie‐Freiberg, Freiberg, Germany
Chitin is recognized as an evolutionarily ancient and fundamental skeletal construct, com-

monly found in diverse uni‐ and multicellular (mostly invertebrate) organisms across the
globe. The chapter first discusses the occurrence and structural peculiarities of chitin from
sponges (Porifera), as well as methods for isolating this structural aminopolysaccharide
from these organisms. It then describes trends for the application of this unique, naturally
pre‐fabricated, three‐dimensional (3D) chitin towards biomedical (tissue engineering) and
technological (biomaterials‐inspired science) goals.
2.1 Introduction
Chitin is known to be one of the most abundant biopolymers in nature. It occurs in vari-
ous contexts across a broad range of species, including fungi, yeast, marine protists,
foraminifera [1, 2], diatoms [3, 4], hydroids, coelenterates, brachiopods, polychaetes,
Pogonophora [5], molluscs [6, 9], and crustaceans [10–15], and even in the epidermal
cuticles of fish [16] and the scales of salmon [17]. Interestingly, the existence of chitin in

sponges (phylum Porifera) was poorly studied until 2007. Previously, it was stated that
the presence of chitin in several sponges might be related to ‘contamination’ by a variety
of chitin‐containing micro‐invertebrates harboured by the sponges [18], such as poly-
chaete worms (Haplosyllis basticola) colonising the Ianthella basta demosponge. Later,
the presence of chitin in sponges was considered to be limited only to the gemmules of
freshwater Spongillidae sponges. However, in 2007, for the first time, a team led by
Hermann Ehrlich described the discovery of chitin in the skeletal fibres of the demos-
ponge Verongula gigantea. They showed that chitin isolated from V. gigantea is very
similar in structure to the α‐chitin found in other invertebrates, including arthropods [19].
The authors suggested that the system of chitin production might be an ancestral feature
in Metazoa. This hypothesis indicates that the synthesis of chitin may have evolved in the
Precambrian, much earlier than previously described. In the same year, Ehrlich et al. [20]
published the first report of the presence of chitin in endogenous material within the
skeletal frameworks of the glass sponge Farrea occa. They put forward the hypothesis
that chitin molecules are part of a very old organic template system involved in the
biosilicification process, which was established a long time before the origin of glass
sponges [21].
In 2009, Ehrlich’s team continued systematic studies on the isolation of chitin from
diverse demosponges of the order Verongiida. Their results indicate that the obtained chi-
tin scaffolds closely resemble the shape and morphology of native sponge skeleton for all
tested species [22–24]. The presence of chitin in Mexican Pacific species of the genus
Aplysina and in the sponge Aplysina fistularis was confirmed in 2012 [25] and 2013 [26],
respectively. In 2013, an important discovery was reported by Ehrlich’s team, who
described, for the first time, the isolation of chitin from skeletons of the freshwater dem-
osponges Lubomirskia baicalensis [27] and Spongilla lacustris [28]. The authors sug-
gested that the incorporation of chitin into the proteinaceous skeletons of S. lacustris
appears to be selectively favoured, because the resulting material becomes more rigid.
They also suggested that chitin identified in the holdfast of L. baicalensis plays an impor-
tant role in the adhesion of sponges to the rocky substrate. The presence of chitin in the
heavily mineralised skeleton of the Verongiida demosponge Suberea clavata was con-
firmed in 2017 [29]. In that study, the authors proved that chitin can be an organic tem-
plate for both magnesium‐bearing calcite and amorphous silica, which were shown to be
intercalated in a chitinous matrix. These results imply that chitin is involved in the phe-
nomenon of multiphase biomineralization in Verongiida demosponges (see [21] for
details). Recently, a 3D fibrous chitin scaffold was isolated from another representative of
the order Verongiida, Pseudoceratina purpurea [30]. Intriguingly, in 2018, chitin has also
been discovered in non‐Verongiida marine demosponges from the family Poecilosclerida:
Acarnus wolffgangi, Echinoclathria gibbosa [31], and Mycale euplectellioides [32]. In
contrast to the Verongiida, these demosponges have siliceous spicules in their chitinous
skeletons.
Considering the evolutionary aspect, chitin is hypothesised to have been a major organic
component of prehistoric invertebrates. Interestingly, the oldest chitin found in nature was
also discovered in a fossil of sponge origin. Ehrlich and his co‐authors described the pres-
ence of chitin in 505‐million‐year‐old (Middle Cambrian) Vauxia gracilenta sponge fos-
sils [33]. To date, the chitin found in this sponge is the oldest known chitin, which is
Methods of Isolating Chitin from Sponges (Porifera) 37
exceptionally well preserved in comparison with previously reported chitin‐containing

fossils [34].
It should be noted that chitin‐based structures have never been isolated from calcareous
sponges (class Calcarea), although chitin synthase genes have been identified in two
species [35]. Additionally, there are no reports to date concerning the presence of chitin in
skeletons of sponges of the class Homoscleromorpha (Figure 2.1).
The chitin isolated from demosponges has a 3D, tubular and fibrous morphology, with
numerous channels and chambers having the ability to swell. These tube‐like constructs,
up to 50 μm in diameter, can operate like capillaries (Figure 2.2) [36, 37]. These unique
morphological and physicochemical properties of chitin‐based skeletons make them
promising ready‐to‐use scaffolding materials for practical applications in biomedicine
and tissue engineering [24, 38–40]. Furthermore, due to its thermal stability up to 400°C,
sponge chitin may be superior to the chitin of lower thermal stability (up to 248°C)
derived from certain fungi [41]. This high thermal stability of Poriferan chitin has
recently been successfully exploited in the book titled Extreme Biomimetics [42], by
Hermann Ehrlich, for the development of diverse metal oxide–based functional 3D com-
posites [37, 42–52].
Sycon ciliatum
Marine species
Spongilla lacustris
Calcarea Lubomirskia baicalensis
Freshwater species
Hexactinellida CHITIN IN PORIFERA Demospongia
Marine species Marine species

Euplectella aspergillum Homoscleromorpha Verongula gigantea
Farrea occa Aplysina gerardogreeni
Rossella fibulata Not examined Aplysina aerophoba
Aplysina revillagigedi
Aplysina cauliformis
Aplysina clathrata
Aplysina fulva
Aplysina fistularis
Aiolochoria crassa
Ianthella basta
Mycale euplectellioides
Acarnus wolffgangi
Echinoclathria gibbosa
Pseudoceratina purpurea
Suberea clavata
Figure 2.1 Presence of chitin in diverse representatives of Porifera.

(a) (b) 1 cm (e)
1 cm
(c) (d)
on
ti
ac
C a p ill a r y
1 cm 10 μm
Figure 2.2 From sponge to chitinous scaffold. Demineralisation of dried Ianthella sp. demosponge (a)
leads to a flat 3D‐structured skeleton (b). Treatment of this skeleton with 5% (w/v) NaOH at 37°C leads
to the isolation of a translucent, pigment‐free chitinous scaffold (c) that resembles the square‐like mesh
architecture of the skeleton. Such meshes (d) are made of tube‐like fibres with diameter around 50 μm
(SEM image e) and exhibit capillary action (arrows) when placed in liquids. Due to this capillary action,
the whole construct can swell and become well visible even in air (c).
2.2 Brief Overview of Classical Methods of Isolating Chitin

from Invertebrates
Chitin, the second‐most abundant biopolymer after cellulose, is the single‐largest waste
resource from the fish processing industry having high technological potential, especially
as a source for chitosan production (see [53–55] for review). The main industrial sources
of raw material for the production of chitin are the naturally mineralised cuticles of certain
crustaceans, principally crabs and shrimps. Nevertheless, the first step in chitin extraction
is the selection of shells. In the case of lobsters and crabs, for example, such selection has
a great influence on the quality of the final isolated material. Ideally, shells of the same
species and similar size are chosen. In the case of shrimps, the shell wall is thinner, thus the
isolation of chitin is easier than from other types of shells. The selected shells are cleaned,
dried, and ground into small pieces [56, 57]. By this procedure, chitin can finally be
obtained only in the form of microparticles and powders. Such a consistency is suitable for
subsequent chitosan production – but not, for example, for the production of 3D scaffolds
for tissue engineering.
Because chitin in marine arthropods is found as a constituent of a complex network, with
proteins and calcium carbonate deposits located within the rigid shells, the main industrial
methods of chitin isolation in this case are based on chemical treatments which enable the
hydrolysis of proteins, depigmentation of pigments [58] and demineralisation of inorganic
matter [59–64]. Demineralisation can be achieved using various acids, most commonly
diluted hydrochloric acid, acetic acid or sulphuric acid (see [57, 65, 66] for examples). To
decompose all of the inorganic salts, the acid intake should be greater than the stoichiometric
quantity of minerals [67, 68]. Due to the heterogeneity of the solid matrix and difficulties
in removing calcium debris, larger volumes or more concentrated acid solutions are
commonly used [56, 65]. Longer demineralisation times – even up to several days – may
cause degradation of the polymer matrix [69]. It has also been reported that the use of high
temperature for demineralisation stimulates the reaction by promoting the penetration of
the solvent into the chitin matrix. Therefore, some attempts have been made to carry out
demineralisation at higher temperatures [70]. It has further been shown that the penetration
of solvent into the chitin matrix is strongly dependent on the particle size. Thus, Marquis‐
Duval reported that the decisive factor in demineralisation is related to the contact area
between the chitin matrix and the solvent [71]. However, it has been shown that high
temperatures, longer incubation times and high acid concentrations affect the final
properties of the isolated chitin. Recently, Kaya et al. proposed to use sodium hypochlorite
(NaClO) pre‐treatment prior to traditional methods of acid‐based demineralisation [72], a
method that appears to be promising in terms of time and energy savings. Also,
ethylenediaminetetraacetic acid (EDTA) is recognised as a useful reagent for the
decalcification of chitin‐based crustacean shells [73].
Industrial deproteinisation is carried out using a base solution such as NaOH or KOH
(see [74, 75] for review). The effectiveness of deproteinisation depends on the ratio of shell
to solution, the temperature and duration of the process, and the concentration of the base.
Industrial methods also include a decolourisation step, which improves the colour of chitin.
For this purpose, hydrogen peroxide or sodium hypochlorite solutions are used [57]. In the
case of fungi which contain no mineral phases within their skeletal structures, only
deproteinisation is necessary [76]. For example, chitin from the cultivated mushroom
Ganoderma lucidum was obtained when the biomass was sonicated and washed with
ethanol, deproteinised with 4 M NaOH at 100°C for 2 h, and then bleached using potassium
permanganate and oxalic acid [41].
However, deproteinisation and demineralisation can also be carried out using
microorganisms and corresponding enzymatic treatment. A comparative study of chemical
and biological methods of chitin extraction was carried out by Khanafari et al. [77]. They
showed that biological treatment produced better results than the chemical method, because
it preserved the structure of chitin. Similarly, a study by Bustos and Healy [78] demonstrated
that chitin obtained by the deproteinisation of shrimp shells with various proteolytic
microorganisms has higher molecular weights than chemically prepared shellfish chitin.
The biological extraction of chitin offers high reproducibility in a shorter time, simpler
manipulation, lower solvent consumption and lower energy input. Diverse alternative
procedures to the chemical extraction of chitin have recently been reported, using such
bacteria as Bacillus subtilis and Lactobacillus plantarum [79], lactic acid fermentation
with Pseudomonas aeruginosa [80] and L. plantarum DSM 20174 [81], Bacillus mojavensis
A21 and Balistes capriscus [82], B. subtilis A26, B. mojavensis A21, Bacillus pumilus A1,
Bacillus amyloliquefaciens An6, Bacillus licheniformis NH1, Bacillus cereus BG1 [83],
Streptomyces griseus [84], Bacillus invictae [85] and Bacillus safensis S406 [86] proteases.
Interestingly, chitin from Portunus segnis and Penaeus kerathurus shells has been isolated
by means of crude digestive alkaline proteases from the viscera of P. segnis [87]. There is
Shell
Chemical Washing Washing Biological
treatment Grinding Drying treatment
Crushing
Demineralisation Penetrated shell waste
HCl Organic acid
(lactic acid)-producing bacteria
Minerals
Minerals
(CaCO3, Ca3(PO4)2)
(CaCO3, Ca3(PO4)2)
Deproteinisation Demineralised shell waste
Base Protease-producing bacteria
Proteins Proteins
Pigments
Bleaching and decolouration Deproteinised shell waste
H2O2 Additional chemical
deproteinization
Pigments Proteins
Chitin
Deacetylation
(NaOH/KOH 30–50%)
80–150 °C
Washing and drying
Chitosan
Figure 2.3 Industrial methods of chitin extraction from crustacean shells.
even a report of pilot‐scale chitin extraction from shrimp shell waste with bacterial enrich-
ment cultures [88].
A schematic overview of chemical and biological methods of chitin isolation is pre-
sented in Figure 2.3. Owing to differences in the ultrastructure of the initial material, all
such treatments must be adapted to the specific chitin source, to produce high‐quality chi-
tin, and then chitosan.
2.3 The Modern Approach to Chitin Isolation from Sponges

Poriferan chitin, as a linear polysaccharide, is composed similarly to α‐chitin from crusta-
ceans, from 1,4‐N‐acetyl‐D‐glucosamine residues, including the acetamide group at the
C‐2 position of glucosamine, the secondary hydroxyl group at C‐3 and the primary hydroxyl
group at C‐6, and exhibiting along the chain a repeating unit 10.3 Å in length [89]. Chitin
oligomers of more than 10 monomers are hardly soluble in water and spontaneously assem-
ble into fibres [90]. This poor solubility is a consequence of the close packing of chains and
the strong inter‐ and intramolecular hydrogen bonds between acetamide groups, hydroxyl
groups and carbonyl groups. Furthermore, sponge chitin is found in the form of silica–chitin
(glass sponges) or calcium carbonate–chitin–silica (demosponges) biocomposites that
resemble the inner morphology of the corresponding sponge skeletons. Consequently,
in contrast to the case of crustacean shells, both decalcification and desilicification
procedures must be carried out for chitin isolated from sponges.
2.3.1 Methods of Isolating Chitin from Glass Sponges (Hexactinellida)

Sponges of the class Hexactinellida, with their biosilica‐based skeletal structures (spicules,
networks), may be the oldest metazoan taxon in Earth’s history [91]. The spicules of these
sponges are characterised by high flexibility and toughness, this being related to the
presence of an organic matrix of proteinaceous (i.e. collagen) or polysaccharide (i.e. chitin)
origin (see [92] for review).
The first attempt to isolate the organic component from glass sponges was made by
Kölliker in 1864. He used HF solution for the demineralisation of Hyalonema spicules
[93]. In 1888, Sollas described a desilicification method based not only on HF, but also on
boiling solutions of KOH [94]. The procedure of HF‐etching of siliceous spicules for their
microscopic study was developed by Vosmaer and Wijsmann in 1902 [95], and is still used
today. An analogous method was used by Schmidt in 1926 to study the organic and
inorganic composition within spicules of Hyalonema and Monorhaphis species [96]. The
use of HF solutions for the dissolution of silica was applied for the visualisation of axial
filaments of glass sponges, but this method is satisfactory only for determining their gross
morphology, because HF partially masks the fine details of organic axial filaments [97].
The cathepsin‐like proteins called silicateins were successfully isolated from the siliceous
spicules of Tethya aurantia by treatment with HF/NH4F solutions [98]. Travis et al. used a
method based on purging the sample with gaseous HF in vacuum [99], and reported the
presence of parallel‐oriented cellulose‐like filaments with a considerable quantity of
hexosamine. However, the HF‐based silica dissolution procedure was criticised as being
rather aggressive and capable of significantly changing the structure of the organic matrix
[99–101]. Another desilicification method, based on treatment with Na2CO3 or 0.5 M
NaOH with subsequent heating at 85°C, was investigated, but this method is also not
acceptable because of the destruction of the organic matrix [101].
To solve this problem, Ehrlich’s team developed a novel slow‐etching method, using a
2.5 M NaOH solution and carrying out demineralisation at 37°C for a long period – 14 days
or more – without stirring of the mixture [101–104]. A schematic representation of this
approach is given in Figure 2.4.
This type of demineralisation is based principally on alkali–silica reactions. In essence,
the hydroxyl ions attack the stronger siloxane bridge near the surface of the reactive
siliceous component and break it down. The negative charge created by this breakdown is
balanced by the positively charged alkali ions. As the reaction proceeds, alkaline hydroxide
penetrates into the siliceous particle, thus loosening the lattice structure. This type of
biosilica dissolution is practically impossible in well‐crystallised silica such as quartz, but
is much more effective in amorphous silica because of the larger surface area and the
disordered, open lattice structure [104].
Additionally, study of the presence of chitin within the skeleton of Demospongiae [19]
has led to the hypothesis that chitin, if present within the glass sponges, must occur as a
structural component of their skeletons. Therefore, in order to dissolve the siliceous exo-
skeleton, the slow‐etching method using a solution of NaOH was applied, to obtain a
chitin matrix from the representatives of Hexactinellida – from Farrea occa (Figure 2.5)
[20] and from spicules of E. aspergillum [105]. The same approach was successfully used
in 2008 to examine the presence of chitin in another Hexactinellida sponge, Rossella fibu-
lata [106]. Chitin, in contrast to many pigments, lipids and proteins, remains stable after
I. Washing with deionised water,

removal of soluble salts
24 h, 25°C
II. Removal of organic residues

using 10% H2O2
15 min, 25°C
III. Removal of collagen 24 h, 15°C IV. Digestion of exogenous chitin

contamination using collagenase by treatment with chitinase enzyme
enzyme
48 h, 25°C
14 DAYS, 37°C
V. Alkali etching using
2.5 M NaOH solution, CHITIN MATRIX
without shaking
Figure 2.4 Schematic representation of the slow‐etching approach for chitin isolation from glass sponges.
200 μm
Figure 2.5 Wide field fluorescence microscopy image providing strong evidence for the presence of
chitin (in blue) after Calcofluor‐white staining of an alkali‐resistant siliceous skeleton of Farrea occa glass
sponge.
treatment in both alkali‐based solution (e.g. 5% (w/v) NaOH) and HF up to a concentra-

tion of 40% at temperatures between 25°C and 40°C. This feature is critical for the extrac-
tion and isolation of chitin in purified form from diverse representatives of the phylum
Porifera [31].
It is important to note that Hexactinellida sponges may have evolved from cellular
sponges, and the syncytial tissues are not a primitive trait of the Metazoa. However, due to
their favoured deep‐sea habitat, the Hexactinellida are still poorly investigated as regards
their biology and the nature of the organic matrix forming their skeleton. The discovery of
chitin in the skeleton of glass sponges suggests that it is silica–chitin composites that play
an important role as a template for skeleton formation, rather than silica–protein scaffolds.
This leads to the hypothesis that chitin molecules are involved in a very old biomineralisation
motif, which was established a long time before the origin of Hexactinellida sponges and
collagen as a structural protein. Therefore, the identification of chitin as a main component
of axial filaments is also especially important from the point of view of evolution and
systematics [106]. It should be noted that the NaOH concentration commonly used for
chitin isolation (2.5 M corresponds to 4% v/v) is well below the critical concentration of
25–30% v/v at which the transformation of β‐chitin into α‐chitin starts to take place [107].
Therefore, the crystal structure of the chitin‐based scaffolds is not influenced by the
extraction procedure, which means that purified chitin scaffolds are obtained without
pigments, proteins, lipids and silica residues and without changing the morphology and
chemistry of the organic matrix [22, 23].
2.3.2 Methods of Isolating Chitin from Demosponges (Demospongiae)

The class Demospongiae includes over 7000 species. In Systema Porifera, this class was
divided into 13 orders, as a result of morphological studies by international researchers
[108]. Based on the latest molecular phylogenetic studies, this classification has been
revised. The class Demospongiae is now considered to contain 22 orders [109]. In 2007,
chitin was isolated from demosponges for the first time, and since that time further
investigations have been performed to analyse its distribution in this class of sponges and
to gain knowledge regarding its function and properties [19, 23, 24, 26–28]. Examination
of pure isolated chitin skeletons from Aplysina cauliformis, Aplysina sp., V. gigantea and
Aplysina aerophoba provides an insight into the fibrous architecture [23]. Each single fibre
consists of multi‐layered concentric layers. By cross‐linking, these fibres form a 2D or 3D
hierarchical organised structure [22–24, 110] (Figure 2.2).
Demineralisation is an essential step to obtain more knowledge about chitin as a template
for biomineralisation and to reveal new potential applications in the field of biomimetic
materials [21]. It is advantageous to apply mild conditions so as to retain the unique
structure provided by Verongiida sponges while dissolving all other unwanted organic
components, minerals, pigments and typical sponge metabolites [111]. This approach has
given rise to a suitable method for chitin isolation.
2.3.2.1 Demineralisation as a Crucial Step in Chitin Isolation from Demosponges

The standard method for chitin isolation from sponges was established in 2007 [19]. The
following diagram shows this process schematically (Figure 2.6).
This places importance on maintaining the architecture of the sponge chitin and providing
reliable purification for further applications, for example, as a scaffold enhancing cell
growth in biomedical applications [39].
The first step in the isolation of chitin from marine demosponges involves rinsing with
deionised water. The sponge sample is placed in deionised water for 24 hours at 37°C.
Water‐soluble components are dissolved, and the sponge’s cells are lysed by osmosis (see
Figure 2.2b).
Repeated until complete demineralisation
Deionised 20% acetic 2.5 M NaOH,

H2O, 37°C acid, 37°C 37°C
Decalcification Desilicification
Figure 2.6 Scheme of the standard method for chitin isolation from demosponges.
The second step is the decalcification of calcium carbonates, achieved by applying 20%
(v/v) acetic acid for 24 hours at 37°C. In addition to the degradation of calcium carbonates,
further pigments and proteins are dissolved. The decalcification of biominerals was
described in detail by Ehrlich’s team in 2009 [112]. The following equation describes the
reaction of degradation using acetic acid:
CaCO3 CH 3COOH Ca 2 CH 3COO H 2 O CO2 (1)

2
After the treatment, the sample has to be neutralised by rinsing with deionised water.
Then the third step, desilicification, can be carried out. As previously noted, beside the
crystalline calcium carbonate, the appearance of amorphous silica in Verongiida sponges
has been detected [113]. For the degradation of this silica, 2.5 M NaOH is applied for
24 hours at 37°C. The alkaline extraction reaction can be described as follows:
SiO2 2 NaOH SiO32 2 Na H 2 O (2)
As the reaction equation shows, the silica is dissolved by the alkali to form silicic acid.
Furthermore, other alkali‐soluble proteins, lipids and pigments are dissolved. After the
alkaline extraction, the sponge sample is neutralised by rinsing with deionised water. The
alternating treatment, using low‐concentration acetic acid and alkaline solution, is repeated
until the chitin matrix is completely cleaned, demineralised and nearly transparent. Then
the final step is the application of 35% (v/v) hydrogen peroxide to remove the remaining
pigments from the chitin matrix. In the chitin isolation process, the number of alternating
treatments depends on the morphology of the treated sponge species. For example, some
determining factors are the degree of mineralisation and the type of minerals which are
present. Thus, treatment times of up to 4 days are reported [29].
According to the latest findings on chitin isolation from the demosponges Suberea clav-
ata, Pseudoceratina purpurea, Acarnus wolffgangi, Echinoclathria gibbosa and Mycale
euplectellioides, where silica residues as spicules or other incorporated silica particles were
not completely degraded by alkaline extraction, a further step is necessary [29–32]. As is
established for the less mild demineralisation of glass sponges, desilicification is performed
(a) (b)
100 μm 100 μm
Figure 2.7 The skeleton of Mycale euplectellioides before (a) and after (b) HF treatment. The siliceous
spicules present in the chitinous skeleton of M. euplectellioides are marked with arrows.
applying low‐concentration (up to 10% (v/v)) hydrofluoric acid for 6–12 hours at room
temperature (Figure 2.7).
2.3.2.2 Ultrasonic Treatment in the Isolation of Chitin from Demosponges

Ultrasonic waves have frequencies higher than 20 kHz [114]. These waves consist of
mechanical vibrations, which make them an excellent tool for extraction treatments or for
the degradation of solids in solution. Ultrasound treatment leads to cavitation bubbles.
The collapse of these bubbles excites two mechanical effects: microjets and microstreams.
Both microjets and microstreams stress the solid surface mechanically, inducing ruptures.
These ruptures enhance the entry of the solvent. Thus, the diffusion of particles is also
enhanced. Cavitation is also known to increase temperature and pressure [115]. As a con-
sequence, the proteins are denaturated. With regard to the complex composition of the
skeleton of Verongiida sponges, there are a variety of effects which probably increase the
degradation of silica and calcium carbonate, deproteinisation, the dissolution of pigments
and lipids, and the lysis of sponge cells. It was recently reported that ultrasound improves
the extraction of polysaccharides from fungi [116]. As is well known, one typical polysac-
charide present in fungi is chitin. Unfortunately, the composition of the extracted polysac-
charides was not analysed. In another context, ultrasonic treatment is often described as
one possibility for the deacetylation of chitin to chitosan and for its depolymerisation
[117, 118]. Regarding the isolation of chitin from sponges, an ultrasound‐assisted method
was developed to optimise the treatment with respect to time and the volume of chemicals.
The method was tested using fragments of Aplysina fistularis, Aplysina archeri and
Verongula rigida from St. Vincent (Caribbean). All three species belong to the order
Verongiida, but successful chitin isolation was reported only in the case of A. fistularis
[26]. Complete demineralisation and deproteinisation are achieved by applying ultrasonic
sound during the alternating treatment with 2.5 M NaOH and 20% (v/v) acetic acid. The
optimum temperature was found to be 70°C. Depending on the morphology of the sponges
studied, there is an appreciable difference in the time of treatment and the chemicals used.
For chitin isolation from V. rigida and A. archeri, treatment with acetic acid while apply-
ing ultrasound is essential to obtain a completely demineralised and transparent chitin
matrix (Figure 2.8).
For chitin isolation from A. fistularis, ultrasound‐assisted treatment with alkaline solu-
tion for 1.5 hours is sufficient for complete demineralisation and deproteinisation. Further
acidic extraction applying ultrasound causes damage to the typical chitin fibres. The dam-
age is obvious when the chitin skeleton is viewed with the naked eye, as shown in the fol-
lowing photographs (Figure 2.9).
Altogether, the treatment time is reduced to a maximum of 4 hours and the quantity of
applied chemicals is significantly reduced when the standard method for chitin isolation is
combined with ultrasound. Given the variety of chitin deacetylation methods using ultra-
sound‐assisted alkaline treatment, verification of the obtained chitin is necessary.
(a) (b)
200 μm 200 μm
Figure 2.8 Skeleton isolated from Aplysina archeri marine demosponge (a) with the standard method
and (b) with the ultrasound‐assisted method. The direct comparison shows the ‘defibrillation’ of the chitin
fibres by ultrasound.
(a) (b)
1 cm 1 cm
Figure 2.9 The skeleton isolated from Aplysina fistularis (a) after the first isolation step applying 2.5 M
NaOH and after treatment with 20% (v/v) acetic acid (b). The uncompleted demineralisation at the inter-
mediate stage is visible because of the presence of pigmentation.
2.3.2.3 Microwave‐Assisted Methods for Chitin Isolation from Demosponges

Microwaves are electromagnetic waves and are a type of non‐ionising radiation. Their
frequencies range from 300 MHz up to 300 GHz. Microwaves generate an oscillating
electromagnetic field. In this field, the polar molecules align due to rotation displace-
ment, which leads to temperature increase. In this process, superheating conditions
appear that cause extreme high temperatures at certain local spots. Additionally, the
effect of rotation displacement is coupled with the conductivity mechanism. This relates
to the electrophoretic transfer of ions and electrons due to the electric field. Both mech-
anisms cause internal heating [119]. The heat facilitates both damage and diffusion. For
example, microwaves are used in mineral treatment processes. In the separation of dif-
ferent minerals, the impact of the conductivity mechanism on various minerals is
observed. However, the impact of microwaves on silica and calcium carbonates has
been shown to be minimal [120]. Furthermore, microwaves are used in food science and
technology, for example, in heating and extraction processes [121]. A recent report con-
cerns chitin isolated through microwave‐assisted dissolution using ionic liquids, as a
biopolymer of high molecular weight that can be manufactured into materials with
diverse architectures [122]. The most commonly mentioned field of application for
microwaves is the deacetylation of chitin. The deacetylation of crab chitin is performed
with 45% (w/v) NaOH solution under microwaves for 5.5 minutes at 900‐watt power
[123]. A more comprehensive study investigated the impact of the concentration of the
alkali solution on the degree of deacetylation, using 30%, 40% and 50% (w/v) NaOH
for 10 min and 1400‐watt power. The resulting degree of deacetylation was found to
increase proportionally with the concentration of the alkali solution [124]. Additionally,
the time‐saving potential of microwave‐assisted treatment was well documented by the
deacetylation of chitin flakes from shrimp shells. A 60% (w/v) alkaline solution and
microwaves were applied for 3 hours under N2 atmosphere, and resulted in 95% dea-
cetylation and seven times shorter treatment time [125]. Another possibility is to apply
microwaves for chitin deproteinisation in combination with hot 6% (w/v) NaOH at
100°C, with 300‐watt power. A significantly shortened treatment time was also reported
in this context [126]. For β‐chitin, it is reported that microwave treatment causes pre‐
swelling, which leads to disruption of the native crystalline structure [127]. To date,
however, there is no method for applying alkaline solution of low concentration in the
treatment of α‐chitin. Therefore, in the context of isolation of chitin from sponge skel-
etons, the positive impact of microwaves on the decalcification, desilicification and
deproteinisation processes is considered.
The microwave‐assisted method combines the established method of chitin isolation
from demosponges [19, 22–24] with exposure to microwaves. The selected Verongiida
sponge samples must be pre‐treated with 2.5 M NaOH for 24 hours to extract organic
compounds, including pigments (bromotyrosines). The resulting sponge skeleton is treated
alternately with alkaline solution and acetic acid in a microwave oven. We developed this
method using fragments from A. aerophoba. Applying microwaves, in contrast to traditional
methods, it was possible to reduce the treatment time from 3–7 days to a maximum of just
30 minutes. Even including the pre‐treatment time of 24 hours, this is only a third of the
standard treatment time. After the treatment, the Verongiida sponge skeletons were fully
demineralised and transparent (Figure 2.10).
(a) (b)
1 cm 1 cm
Figure 2.10 (a) Cell‐free but pigmented skeleton of Aplysina aerophoba and (b) the 3D chitinous scaffold
obtained from the same skeleton after complete alkaline and acidic extraction assisted by m icrowaves
(750 W).
(a) (b)
200 μm 10 μm
Figure 2.11 Deformed chitin fibres isolated by the microwave‐assisted method from the skeleton of
Aplysina aerophoba sponge, under different magnifications.
As regards the power applied, it was shown that a power of 400 W provides a milder
procedure in terms of the destructive effect on the structure. As the SEM images show,
microwave‐assisted chitin isolation promotes the breakdown of the characteristic multi‐
layered structure known for A. aerophoba. The isolated chitin scaffolds (Figure 2.11)
promote the typical chitin fibres and flattened chitin layers, which leads to surface
enlargement. Considering the advantages of large surfaces, there arise new potential fields
of application. Despite the breakdown of a great part of the chitin fibres, the isolated matrix
is still capable of liquid adsorption.
During the development of the microwave‐assisted method for chitin isolation, tem-
perature measurements were also made. Superheating led to temperatures of up to
118°C. Due to the reported deacetylation of chitin to chitosan at high temperatures, it
was necessary to analyse the chitin using Raman and FTIR spectra, chitinase digestion
and Calcofluor‐white staining. The spectra of the microwave‐assisted isolated chitin
were found to be in agreement with the spectra of chitin isolated using the standard
method. In comparison with a chitosan standard, significant differences were present in
the spectra. Therefore, no deacetylation to chitosan takes place on exposure to micro-
waves during alkaline and acidic extraction. Overall, the microwave‐assisted method
enables chitin isolation in 99.3% of the treatment time of the standard method.
Furthermore, it reduces the quantity of solvents required and offers a new shape of iso-
lated chitin fibres.
2.4 Prospective Applications of Poriferan Chitin

In contrast to the powdered chitin that is the final product of chitin extraction from marine
crustaceans, Poriferan chitin offers unique morphological properties, especially due to its
naturally prefabricated centimetre‐sized 3D scaffolds. This unique architecture enables a
wide variety of possible applications.
2.4.1 Poriferan Chitin and Modern Bioinspired Materials Science

Modern bioinspired materials science uses a biomimetic approach. This means that it
imitates natural processes to create new technologies or methodologies. Given the function
of Poriferan chitin as a scaffold and template for biomineralisation, it is a suitable substrate
for the development of novel bioinspired materials [36, 47, 128]. Thermogravimetric
analysis has shown that the thermal stability of chitin depends on its origin, and it may
remain stable up to 360°C [129]. This property opens the way to the use of well‐known
solvothermal and hydrothermal synthesis reactions established as common methods for the
creation of novel nanostructured inorganic–chitin composites [52]. In hydrothermal
synthesis, the reactants – also called precursors – may be added as solutions, gels,
suspensions and colloids. One advantage of multi‐layered tubes such as the chitin fibres
forming the sponge skeleton is their ability to retain water. The capillary action of the chitin
skeleton is advantageous with regard to the pre‐treatment with liquid precursor.
Comprehensive studies since 2013 have confirmed the methods of hydrothermal and
solvothermal synthesis for the creation of novel composites. The first hydrothermal
synthesis was performed with modified Stöber conditions using sponge chitin. A solution
of tetraethyl orthosilicate and a chitin skeleton from I. basta were used for hydrothermal
synthesis at 120°C [45]. This yielded a chitin–silica composite, and led to understanding of
the dependency of the rate of silicification on the hydrolysis of the precursor. Hydrothermal
synthesis was also applied for zirconia deposition on chitin isolated from A. aerophoba
[37]. In that case, the chitin scaffold was soaked in a solution of ammonium zirconium (IV)
carbonate as precursor. After heating of the swollen scaffold at 150°C for 48 hours, a new
zirconia‐based composite was formed. It was suggested that the zirconia binds to the chitin
framework via hydrogen bonds. The new composite may find applications in the field of
biosensors and biocatalysts [37].
20 μm 2 μm
Figure 2.12 SEM images of naturally prefabricated 3D chitin–GeO2 composite under different
magnifications.
In 2015, applying extreme biomimetic conditions (pH 1.5, 80°C), a monolithic silica–
chitin composite was obtained using chitin from A. cauliformis [44]. It was shown that
silica was deposited exclusively in the inside of the tube‐like chitin. Due to the excellent
biocompatibility of both chitin and silica, this is a promising material in the field of tissue
engineering. Furthermore, in the same year, the synthesis of a unique 3D germanium
oxide–chitin composite was reported [47]. The following images (Figure 2.12) show the
germanium oxide nanocrystals on the surface of a fibre of chitin isolated from A. cauli-
formis, viewed by means of scattering electron microscopy with 2000‐ and 6000‐fold
magnification.
After soaking in a solution of germanium tetraethoxide (TEOG), used as the precursor
of germanium oxide, the chitin scaffold was hydrothermally treated at 120°C and pH 14.
The resulting new 3D GeO2–chitin composite exhibited interesting fluorescent and lumi-
nescent properties [47].
The hydrothermal synthesis of a chitin–hematite composite at pH 1.5 and 90°C for 48 hours
is another prime example of the application of sponge chitin in Extreme Biomimetics.
Wysokowski and his team [49, 50] were able to demonstrate the function of chitin as a
carrier and the nucleation of hematite crystals at the surface of chitin nanofibres. This
material was proposed as a promising composite for research on sensors and biocatalysts.
The second of the aforementioned methods for transforming chitin scaffolds of Poriferan
origin into new biocomposites is solvothermal synthesis [52]. The chitin scaffold is treated
together with the reactant at high temperature. This method has been used for the deposi-
tion of polyhedral oligomeric silsesquioxanes (POSS) on the chitin skeleton isolated from
A. cauliformis [46] (Figure 2.13). The deposition increased the hydrophobicity and oleo-
philicity of the material.
Another relatively new approach for the creation of novel bioinspired materials is
bioelectrometallurgy [130]. Because neither the isolated sponge skeleton nor the isolated
chitin scaffolds are electro‐conductive, pre‐treatment of the biopolymers with silver nitrate
and ammonium iron (II) sulphate is an indispensable step for bioelectrometallurgy. The
latter substance reduces the quantity of silver ions deposited on the cell‐free skeleton. The
resulting electro‐conductive matrix is applied as a cathode in a copper sulphate solution.
The electrochemical reaction causes the deposition of copper on the surface of this chitin‐
based cathode [131]. In Figure 2.14, a metallised chitin scaffold isolated from the I. basta
(a)
5 mm
(b) (c)
20 μm 2 μm
Figure 2.13 Chitinous scaffold isolated from the demosponge Aplysina cauliformis (a and SEM image b)
was used as a template for the solvothermal synthesis of POSS microcrystals in chloroform at 100°C (SEM
image c).
demosponge (see Figure 2.2) is shown. The large amount of copper and copper oxide
deposited on the chitin scaffold can be clearly seen without further tools. Using scattering
electron microscopy, the complete metallisation of the chitin scaffold is also verified on a
micrometric scale.
2.4.2 Chitinous 3D Scaffolds of Sponge Origin for Tissue Engineering

Every day, destroyed tissues are repaired or replaced in thousands of surgical interventions
worldwide. This is the main goal of tissue engineering, which aims to regenerate damaged
tissues by combining cells from the body with highly porous scaffold biomaterials, which
act as templates for the regeneration and growth of new tissue. Scaffolds, cells and growth‐
stimulating signals are generally referred to as the key components of engineered tissues.
Scaffolds, typically made of polymeric biomaterials, provide the structural support for cell
attachment and growth. Researchers have an enormous variety of options when selecting
(a) (c)
1 mm
(b)
200 μm 1 μm
Figure 2.14 The electrochemical reduction of copper leads to a chitin scaffold completely metallised
with Cu/Cu2O (stereo microscopy images a and b). Nanocrystals of the metal phases (SEM image c)
become tightly covered with nanofibres of sponge chitin and seem to be intercalated within this organic
construct. These investigations resulted in an electro‐conductive material with a number of possible
applications – for example, as a catalyst.
scaffolds for tissue engineering, but these scaffolds have to meet various standards relating
to biocompatibility, biodegradability, appropriate mechanical properties compatible with
the tissue where they will be applied, as well as appropriate architecture. Scaffolds should
possess an interconnected pore structure with high porosity to ensure cellular penetration
and adequate diffusion of nutrients to cells within the construct and to the extra‐cellular
matrix formed by those cells. Another key feature is the pore size of the scaffolds and the
presence of ligands on their surface. The pores should be large enough to allow cells to
migrate into the structure, where they eventually become bound to the ligands within the
scaffold, but also small enough to establish a sufficiently high specific surface area, leading
to a minimal ligand density to allow efficient binding of cells to the scaffold. Other
important factors include the cost of manufacture and the availability of the selected
carriers. Typically, three different groups of biomaterials are used in the fabrication of
scaffolds for tissue engineering: inorganic materials, synthetic polymers and natural poly-
mers [132, 133] (Figure 2.15).
Each of these groups has specific advantages and disadvantages. Therefore, great atten-
tion is paid to the modification of existing carriers as well as the search for and production
of new types of carriers [132].
Particular attention is paid to sponges of the order Verongiida, whose 3D chitinous skel-
etons meet the standards for fabrication of tissue engineering scaffolds [38]. A first attempt
to use 3D chitin scaffolds from Aplysina cavernicola, A. cauliformis, A. fulva and
Aiolochroia crassa as templates for chondrocyte culture in an in vivo environment was
reported by Ehrlich et al. in 2010. The results show that the deposition of proteoglycan‐rich
extracellular matrix and the presence of cartilage‐specific collagen type II on the chitinous
Collagen
Gelatin
Proteins Fibrin Hyaluronic acid
Silk fibroin Alginate
Natural polymers Polysaccharides Chitin
Chitosan
Natural composites Chondroitin sulphate
Poly(hydroxyacid)s
Scaffolds of Aliphatic polyesters
Synthetic polymers Hydrogels
tissue engineering Polyurethanes
Polyphosphazenes
Chitin scaffold
Inorganic materials Calcium phosphate
Figure 2.15 Categorisation of scaffolds for tissue engineering based on their source.
(a) (b)
100 μm
100 μm
Figure 2.16 Chitinous scaffold from Ianthella basta as carrier after 2 days of incubation with hMSCs (a).
The distribution of hMSCs within the pores of the chitin carrier is well visible even on day 21 (b).
matrix is similar to what is found in other cartilage tissue engineering constructs.

Interestingly, the authors showed that the chitin scaffolds also supported the deposition of
a proteoglycan‐rich extracellular matrix of chondrocyte‐seeded bioconstructs [24]. The
positive results of these tests have encouraged the scientific community to continue research
using chitin of Verongiida origin. The chitinous scaffolds derived from I. basta were used
to investigate the distribution, metabolic activity and capacity for directed differentiation of
human adipose tissue–derived mesenchymal stromal cells (hMSCs). More detailed study
of the chitinous scaffolds of I. basta origin as a carrier for hMSCs clearly indicates that the
cells were attached to the walls and flattened on them, obtaining a fibroblast‐like morphol-
ogy. Seven days after the seeding of the culture, there was a gradual filling of scaffold pores
with cells from the periphery to the centre. After 21 days of culture, the formed cell sheets
entirely filled the carrier pore lumens (Figure 2.16).
The successful study with I. basta encouraged Mutsenko’s team to test the possibility of
using a 3D chitin scaffold isolated from the A. aerophoba demosponge as a carrier for
hMSCs in vitro [39, 40]. The results showed that the chitin isolated from A. aerophoba
supports in vitro adhesion, growth and proliferation of these cells. Differentiation studies
suggest that the chitin scaffold may support the multilineage differentiation of hMSCs after
culture in differentiation media, indicating their cytocompatibility. These results indicate
that scaffolds prepared by simple demineralisation of sponge skeletons may be a novel,

renewable source of supports for tissue engineering purposes [39, 40].
Thus, there is no doubt that Poriferan chitin, as a ready‐to‐use naturally prefabricated 3D
biomaterial, is very promising for use in tissue engineering and regenerative medicine,
particularly in view of its nontoxicity, biodegradability as well as centimetre‐size
dimensions.
2.5 Outlook
The newly established methods for the isolation of chitin from sponge skeletons, using
ultrasound or microwaves during alkaline and acidic extraction, offer significant economic
advantages. Even including the pre‐treatment step for extraction of the pharmaceutically
interesting bromotyrosines, which takes one additional day, the ultrasound‐assisted method
of chitin isolation from sponges requires not more than a third of the treatment time of the
standard method. Furthermore, it is possible to reduce the quantities of alkaline solution
and acetic acid used by at least 40%. For A. fistularis, it was possible to isolate pure chitin
without the application of acetic acid; hence, in this case, it is possible to eliminate the
acidic extraction completely when ultrasound is applied. The microwave‐assisted method
of isolation of chitin from sponges also enables a time saving of up to a third of the treatment
time of the standard method, as well as a reduction in the quantity of chemicals used.
These findings open the door to new methods for an industrial approach to chitin
isolation. It is not without significance that marine sponges can be easily farmed in marine
ranching conditions, providing a renewable source of 3D, naturally prefabricated chitin.
Consequently, these methods offer a wide range of opportunities to integrate sponges as a
renewable source for pharmacy and tissue engineering on an industrial scale. Nevertheless,
the fundamental question of the possibility of applying such methods to other chitin sources
remains open. There is a lack of knowledge concerning the mechanism of chitin isolation
using ultrasound or microwaves; such knowledge may help establish the optimum
conditions for the isolation process and avoid the destruction of 3D chitin scaffolds.
Additionally, no research has been done on the possibility of isolating chitin from non‐
Verongiida sponges using treatments of this kind. The potential use of ultrasound and
microwaves in chitin isolation from spiculated sponges also remains unexplored. It seems
that now is the time to develop new and less time‐consuming methods of isolation of both
chitin and secondary metabolites from sponges, to realise the full potential of these
promising marine invertebrates.
Acknowledgment
This work was partially supported by DFG Project HE 394/3‐2 (Germany) and PUT
Research Grant no. 03/32/DSMK/0810 (Poland). M.W. is grateful for financial support
from the Foundation for Polish Science – START 097.2017, and S.Ż .A. for support from
the DAAD and Erasmus Plus programmes.
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3
Physicochemical Properties
of Chitosan and its Degradation
Products
Karolina Gzyra‐Jagieła, Bożenna Pe ̨czek, Maria Wisń iewska‐Wrona,
and Natalia Gutowska
Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Unique properties such as biodegradability, bioactivity, biocompatibility, non‐toxicity,

spin‐ability, ability to form film, and miscibility with other polymers make chitosan suita-
ble for use in many different sectors such as medicine, pharmacy, veterinary, tissue engi-
neering, agriculture, environment protection, food and packaging industry [1–7]. Some of
chitosan properties largely depend upon its molecular structure. For example, the reduction
of its molecular weight is desired for a variety of applications. Low‐molecular fractions of
chitosan, called chitooligomers, obtained by degradation of the polymer, gained particular
attention due to high biological activity [8]. A strict correlation exists between
physicochemical and structural properties of the polymer and its practical use. The term
chitooligomers applies to products with a polymerization degree below 20 and an average
molecular mass of up to 3900 Da. Chitooligomers with polymerization degree below 6 are
water‐soluble, and thus well suited to many applications such as crop‐spraying in
agriculture. They also reveal beneficial biomedical properties bearing antibacterial and
antifungal activity [9–11]. Chitooligomers show anticancer action, stimulate the immunity
system, control blood pressure, prevent heart diseases and constipation, reduce the level of
urea in the blood, reduce low‐density lipoprotein (LDL) and increase high‐density
lipoprotein (HDL) in cholesterol [8,10–12].

3.1 Physicochemical Properties of Chitosan

The physicochemical characteristics (Figure 3.1) determine the relationship that exists
between the chemical structure of chitosan compounds and their potential uses in many
branches of science and industry [13, 14]. Deacetylation degree (DD) and average molar
mass are the most frequently assessed properties, regardless of the intended application of
a given compound. Often analyzed are other features such as solubility; crystallinity;
viscosity; content of water, nitrogen, and ash; and water retention value. The contents of
heavy metals and protein and the level of endotoxins in chitosan are key factors in
determining its use as a biomaterial [15].
The following text presents the main physicochemical methods prepared at the Institute
of Biopolymers and Chemical Fibres (IBWCH), Łódź, Poland, based on professional lit-
erature and standardization documents. Some of the methods were modified or adapted to
the needs of the research in chitosan. For many years, the Institute has been one of the
most important centers engaged in R&D on the use of chitosan and its degradation
products.
3.1.1 Determination of Molar Mass

Estimation of molar mass counts as the most important analyses in the assessment of
chitosan since the parameter determines the biological activity of the polymer. Extensive
investigation has shown that chitosan activity increases with decreasing molar mass and
DD [16–18]. The determination of molar mass is of particular importance in medical
chitosan where the relationship between molecular structure and biological activity appears
crucial. It enables the examination of chitosan‐induced biochemical processes in the cells
[17–19].
Solubility Viscosity
Crystallinity Endotoxin levels

Degree of
deacetylation
Water content Protein content
Average
molar mass
Water retention Heavy metal
values content
Ash content Nitrogen content
Figure 3.1 Physicochemical characteristics of chitosan.

Physicochemical Properties of Chitosan and its Degradation Products 63
3.1.1.1 Determination of Molar Mass by GPC/SEC

Gel permeation chromatography/size exclusion chromatography (GPC/SEC) is considered
among the most important and modern analytical methods used to examine polymers.
Offering information on the molecular structure of examined polymers, it is particularly
suitable in industry and research [20]. In addition to the polymer itself, attention is increas-
ingly being focused on the degradation products of chitosan. Numerous investigations have
confirmed their specific properties, which are most valuable in medical application. The
anticancer and antioxidant activities of chitosan oligomers are intriguing. A precise deter-
mination of the biological activity of the antitumor compounds calls for the examination of
their molecular characteristics [21]. For many years, IBWCH has conducted chitosan
investigation using the GPC/SEC technique, equipped with a refractometric detector (RI).
The use of the detector enables obtaining values relative to universal calibration by using
polymeric standards and the Mark–Houwink–Sakurada equation. GPC/SEC measurements
characterize the polymers on the molecular level, estimating important molecular parame-
ters: number average molar mass (Mn), weight average molar mass (Mw), polydispersity
(Mw/Mn), and, most importantly, the function of molar mass distribution (MMD). The
principle of GPC analysis is the separation of polymer molecules with regard to their
hydrodynamic volume, which is proportional to their molar masses. The separation process
occurs inside an SEC column which is packed with porous gel of different size. The sepa-
rated molecules, after leaving the column, are detected by the RI detector, which produces
a signal registered and transformed by computer with suitable software. If only the RI
detector is used, the GPC method requires calibration by means of a polymer standard with
known molar mass. To obtain the function of molar mass distribution, a raw chromatogram
must be calculated in relation to the calibration curve on the base of the universal calibra-
tion, using parameters values a and k from the Mark–Houwink–Sakurada equation:
kM a
Here, Ƞ = intrinsic viscosity; M = molar mass; and a and k are Mark‐Houwink‐Sakurada

constants
The molar mass of chitosan is calculated on the basis of a universal calibration method
using the parameters k = 74×10−5 mL/g and a = 0.760 for chitosan, and for standards,
poly(ethylene oxide) (PEO) and poly(ethylene glycol) (PEG), k = 62×10−5 mL/g, a = 0.625
[22]. Using the GPC/SEC method the effect of different chitosan degradation methods on
chitosan’s molecular parameters, average molar mass, polydispersity, and function of
molecular mass distribution, were studied. Two different grades of chitosan were used with
varied DD and water retention value (WRV): chitosan A (DD=97.7%, WRV=60%) and
chitosan B (DD=74.4%, WRV=113%). Assessed was also the impact of degradation time
on the properties of the obtained degradation products.
Two degradation processes were employed to degrade the microcrystalline chitosan A:
• Thermal
• Thermal–mechanical
The aim of the research was to evaluate the effectiveness of the two methods. The thermal
degradation was carried out in a steam autoclave at 121°C for 2 hours.
The thermal–mechanical degradation combines two treatments: 2 hours in a steam autoclave
at 121°C, and homogenization for 20 minutes at room temperature. For the GPC analysis,
Table 3.1 GPC/SEC analysis of chitosan A degradation products.
Sample Mn, g/mol Mw, g/mol Mw/Mn

Virgin chitosan 38 670 97 650 2.5
After thermal degradation 17 720 61 640 3.5
After thermal–mechanical degradation 17 720 68 110 3.8
Molar Mass Distribution
1
Differential weight fraction
0.8
(1/log(g/mol))
0.6
0.4
0.2
0
3 3.5 4 4.5 5 5.5 6
logM g/mol
Virgin Thermal degradation Thermal–mechanical degradation
Figure 3.2 Distribution of average molar mass (MMD) of microcrystalline chitosan samples after different
degradation processes.
Table 3.2 Results of GPC/SEC analysis of chitosan with varied DD.
Sample Time of degradation (h) Mn, g/mol Mw, g/mol Decrease of Mw, % Mw/Mn
Chitosan A (DD = 97.7%) Virgin 38 670 97 650 ‐ 2.5
0.5 26 650 105 740 ‐ 4.0
1 23 760 103 400 ‐ 4.4
Chitosan B (DD = 74.4%) Virgin 38 600 120 130 ‐ 3.1
0.5 37 480 115 830 3.6 3.1
1 32 370 108 250 9.89 3.1
two columns were used: PLaquagel–OH MIXED and PLaquagel–OH 40, 300 mm long,
made by Agilent Co. The results of the analysis are shown in Table 3.1 and Figure 3.2.
The thermal degradation results in a distinct drop in the average molar mass. Combining
of the two processes, thermal and mechanical degradation, does not cause any considerable
decrease in the average molar masses in comparison to the thermal treatment. The impact
of the thermal treatment time upon the structure of polymers with varied DD was also
examined. The results are shown in Table 3.2, and Figures 3.3 and 3.4.
It may be noted that the average molar mass dropped in chitosan B with lower DD and
increased for the high‐DD chitosan A; this is probably caused by cross‐linking of the
polymer. Chitosan B has a lower DD, marking a higher content of acetyl groups prone to
the forming of hydrogen bonds and, as a consequence, has cross‐linked polymers. It was
earlier incubated in strong alkaline medium at high temperature for deacetylation.
1
0.8
(1/log(g/mol))
0.6
0.4
0.2
0
3 3.5 4 4.5 5 5.5 6
logM g/mol
Virgin After 0.5 h After 1 h
Figure 3.3 Distribution of average molar mass of degraded microcrystalline chitosan A with high DD
(97.7%).
1
0.8
(1/log(g/mol))
0.6
0.4
0.2
0
3 3.5 4 4.5 5 5.5 6
logM g/mol
Virgin After 0.5 h After 1 h
Figure 3.4 Distribution of average molar mass of degraded microcrystalline chitosan B with low DD
(74.4%).
Solutions of degraded chitosan A (DD=97.7%) and chitosan B (DD=74.4%) were also

GPC/SEC‐analyzed. Thermal degradation was carried out at 0.5 h and 1.0 h. Water was
removed from the post‐filtration solid by:
• Drying in a vacuum dryer at 35°C to complete water removal.
• Evaporation in IKA RV 10 vacuum evaporator at 50°C to complete evaporation, fol-
lowed by additional drying in the vacuum dryer for 2 h at 35°C.
The samples were analyzed in acetate buffer (pH 4.5) in a system with a series of chro-
matographic columns (PLaquagel–OH MIXED, PLaquagel–OH 40, and PLaquagel–OH
30, all 300 mm long, made by Agilent Co.). The results are shown in Table 3.3. The set of
standards PEO and PEG was used for universal calibration.
Table 3.3 Analytical results of solutions after the degradation of two chitosan grades (universal
calibration).
Sample Time of degradation, h Method of drying Mn, g/mol Mw, g/mol Mw/Mn
Solution chitosan A 0.5 Dryer 1 410 1 430 1.0
(DD 97.7%) Vacuum evaporator 1 350 1 360 1.0
1 Dryer 1 340 1 360 1.0
Vacuum evaporator 1 330 1 330 1.0
Solution chitosan B 0.5 Dryer 1 650 1 870 1.1
(DD 74.4%) Vacuum evaporator 1 720 2 040 1.2
1 Dryer 2 070 2 730 1.3
Vacuum evaporator 2 170 3 070 1.4
The degradation of chitosan resulted in oligomers with average molar mass in the
range of 1400–2000 g/mol. The obtained results do not show any distinct impact of the
drying method. Oligomers with higher molar mass were achieved for chitosan with a
lower DD, that is, chitosan B. Prolonged degradation time caused a distinct increase of
the molar mass only for chitosan B. The preceding is an example of the practical use of
the GPC/SEC method. It is an important method to follow—that is, not only the degrada-
tion of the polymer, but also its molar mass distribution after other treatments and
processing.
3.1.1.2 Determination of the Viscometric Average Molar Mass

The viscometric average molar mass is calculated on the basis of the limiting viscosity
number [η], determined at 25°C. An aqueous solution of 0.2 mol/dm3 acetic acid, 0.1 mol/dm3
sodium chloride, and 4 mol/dm3 urea is used as the solvent.
A dilution viscometer is employed in the testing. Viscosity is measured by passing down
a solution of chitosan (with defined concentration) and four of its consecutive dilutions
through a capillary of the viscometer. The value of reduced viscosity ηred is calculated for
each of the solution concentration from the formula:
t n to
red
c to
Here, tn = flow time of a solution with given concentration, s; t0 = flow time of the
pure solvent, s; and c = amount of chitosan in 100 cm3 of the solution considering the
dilution, g.
The limiting viscosity number is estimated in a coordinate system of reduced viscosity
and concentration of chitosan in solution by extrapolating to zero concentration. The limit-
ing viscosity number is expressed by the formula:
lim red
c 0
The average viscometric molar mass is calculated from the Mark–Houwink equation:
k Mv
Here, [η] = limiting viscosity number; k, α = Mark–Houwink constants; and Mv = average

viscometric molar mass.
3.1.2 Determination of the Deacetylation Degree

The deacetylation degree (DD) is defined as the ratio of the number of glucosamine groups
to the overall number of N‐acetylglucosamine (GlcNAc) and glucosamine (GlcN) groups.
The level of DD value determines whether the polymer is chitin or chitosan. The polymer
is considered a chitosan if its DD is higher than 60% [23]. Many analytical methods are
used to assess the degree of deacetylation—for example, infrared spectroscopy (FTIR),
near‐infrared spectroscopy (NIR), UV spectroscopy, potentiometric titration, magnetic
resonance (NMR), etc. [24, 25].
3.1.2.1 Estimation of DD by Nuclear Magnetic Resonance (NMR) Method

Among the available measuring techniques, NMR is considered to be a perfect tool for the
estimation of DD. Literature recommends two techniques: 1H NMR and 13C NMR. For
proton NMR, just dissolve 5–10 mg of chitosan and measure your sample. The DD can be
calculated according to Hirai et al or Vårum et al. [26, 27]. Here, we describe the 13C NMR
method, in which the spectra are standardized against signals derived from the carbon C‐1.
DD is estimated from the equation:
DD 1 A CH3 / A C1
Here, ACH3 = intensity of the CH3 band, and AC1 = intensity of the C1 band.
3.1.2.2 Estimation of DD by Fourier‐Transform Infrared Spectroscopy (FTIR)

Infrared spectroscopy occupies a special place in literature concerning research in chitosan
deacetylation. Interpretation of the spectrum is particularly divergent; moreover, the way
the background is removed of the peaks is problematic (Figure 3.5). The multitude of ways
by which the spectra are interpreted stems from the lack of a well‐defined procedure which
would lead toward reproducible results. Based on literature, practical methods to determine
DD were applied, producing quite divergent results [28].
In Table 3.4, results are shown of DD estimation in three chitosan samples depending on
the method of peak interpretation and the type of selected bands. The results reveal
considerable differences, depending on the selected bands and interpretation method
(choice between surface area or height of the peak). From among all possible interpretation
methods, the selected method was that one that produced results comparable to those
obtained with the 13C NMR technique. The band selected for this method is the intensity of
the peak at wave number 1650 cm‐1, which is characteristic of amide I, and as reference
band the intensity of the peak at wave number 3450 cm‐1, which is characteristic of the OH
group. The intensity of the bands was based on height with subtraction of the background
for the single peaks. Unfortunately, analyzing the IR bands of chitosan raises difficulties
due to the complex structure of the polymer with the presence of hydrogen bonds origi-
nated from OH, C=O, and NH groups.
80,0
75
70
65
60 2551 cm–1
55
50
45
%T
40
35
30
25 2907 cm–1
20
1290 cm–1
15
1429 cm–1
10 1023 cm–1
3451 cm–1 1322 cm–1
5 1626 cm–1
1553 cm–1
3323 cm–1 1072 cm–1
0,0
4000,0 3000 2000 1500 1000 500 400,0
cm–1
Figure 3.5 Background removal from absorption peaks in an IR spectrum of chitosan [28].
Table 3.4 Results of DD estimation of different chitosan samples.
DD, %
Sample Proportion band IR Surface area of peak Height of peak

Chitosan no 1 1650/3450 95.1 77.2
1320/3450 97.2 57.5
1320/1420 76.8 68.5
Chitosan no 2 1650/3450 95.9 80.7
1320/3450 98.1 61.2
1320/1420 77.5 71.0
Chitosan no 3 1650/3450 97.4 93.7
1320/3450 97.3 77.8
1320/1420 88.7 67.7
Divergence in the assessment of DD occurs in the selected method too, depending on the
way in which the tested material is prepared. It is therefore recommended to prepare the
tested material for comparison always by the same technique—for example, in KBr tablets
or in film. No comparison should be made between results of DD obtained from FT‐IR
tests employing different methods of sample preparation.
3.1.2.3 Determination of DD by Potentiometric Titration Method

Potentiometric titration generally proceeds in a non‐aqueous medium. Content of water up
to 10% (v/v) is acceptable, since it does not affect the accuracy of the experiment. The
measuring system is composed of a glass and a calomel electrode looped by an electrolytic
bridge and connected to an electromotive force (emf) meter. The chitosan sample is first
dissolved in a small amount of 1% (v/v) acetic acid, and then anhydrous acetic acid is
added to have a water content below 10% (v/v) in the solution. The tested chitosan solution
and 1,4‐dioxan are put to a measuring vessel. Peracetic acid with concentration of 70%
(v/v) serves as the titrant. A diagram is drawn up of the relationship emf/volume of the
perchloric acid solution, and the volume is read, which responds to the neutralization of the
NH3 groups. The amount of HClO4 solution needed to obtain neutralization, titer of the
solution, chitosan sample weight, and volume of chitosan solution are all taken into account
in the calculation of DD. The procedure was prepared according to the OECD guidelines
[29]. The potentiometric titration produces reproducible results comparable to those from
the NMR measurements. The drawback of the method is its application, which is limited to
easy dissolvable preparations.
3.1.2.4 Determination of DD by a Linear Potentiometric Titration Method

The titration proceeds in an aqueous medium. Chitosan is dissolved in 0.1 mol/dm3 solution
of acetic acid and titrated with a solution of 0.01 mol/dm3 sodium hydroxide. A pH value
close to 2 is considered as starting point of the titration, and the titration is continued to pH 3.
Next, the value of function f(x) responding to the volume of added NaOH is calculated
from the formula:
v0 v
f x H OH
NB
Here, vo = volume of chitosan solution (cm3); v = volume of added NaOH solution
(cm3); NB= concentration of NaOH solution (mol/dm3); [H+] = concentration of ions [H+]
(M); and [OH–] = concentration of ions [OH–].
The curve of the linear potentiometric titration is obtained by calculating the relationship
of the f(x) value from the volume of added NaOH solution (0.01 mol/dm3). The volume v
of NaOH at the final titration point is determined by linear extrapolation of the titration
curve to the x axis. DD is calculated from the equation:
DD % W 161 / 204 100
Here, ϕ = (NAVA – NBVe)/1000; NA= concentration of HCl solution (mol/dm3); VA = volume

of HCl solution (cm3); NB= concentration of NaOH solution (mol/dm3); Ve = volume of
NaOH solution at the final titration point; and W = mass of chitosan sample used in the
analysis.
The low cost of the method is an advantage. The analysis is time‐ consuming due to long
duration required for the chitosan to dissolve. The results in the method differ somehow
from those obtained by 13C NMR measurements.
3.1.2.5 Determination of DD of the First Derivative of the UV Spectrum Method

The method consists of the preparation of the first derivative spectrum for a chitosan
solution in 0.01 mol/dm3 acetic acid in the wavelength range of 195–250 nm, and the
determination of the distance from the zero line to the point corresponding to the minimum
of the first derivative. The results are compared with the calibration curve for the solution
of N‐acetyl‐D‐glucosamine in 0.01 mol/dm3 acetic acid in the concentration range of

0.006–0.048 mg/cm3. For each of the calibration GlcNAc solutions, the distance (H, mm)
is defined from the zero point to the minimum of the first derivative. Then, a calibration
curve is drawn of the dependence of the GlcN concentration on the height of value H. The
content of D‐glucosamine must be taken into account in the calibration curve; it may exert
an influence on the increase of the H‐value for GlcNAc.
The three methods—potentiometric titration, linear potentiometric titration, and first
derivative of UV spectrum—were compared in respect of their practical use. The results of
the comparison are shown in Table 3.5.
Comparison of results obtained by different methods is not advisable, and only the
results from the same DD testing method should be compared. The correlation between the
preceding DD testing methods and the 13C NMR technique was examined. The results,
expressed as R2 correlation coefficient, is as follows: potentiometric titration: 0.978; linear
titration: 0.852; and first derivative: 0.971. The method of first derivative appears best
suited for its good correlation with 13C NMR and for technical and economic reasons.
In Table 3.6, an overview is shown of the advantages and disadvantages of the various
methods of DD determination.
3.1.3 Determination of Dynamic Viscosity

Dynamic viscosity is an important quality factor in the industrial processing of poly-
mers. It is estimated by means of the rotation viscometer Brookfield at 25°C ± 1°C. The
viscometer measures the force of resistance to rotation stemming from the viscosity of
the tested medium. The dynamic viscosity of chitosan solutions is calculated from the
reading of the equipment taking into account a constant from a table for the given rota-
tional speed and specific spindle used. The measured parameter is expressed in cen-
tipoises [cP] [30].
3.1.4 Determination of Nitrogen

The content of nitrogen in chitosan can be assessed by means of the automatic elemental
analyzer Vario Macro Cube. Oxidizing high‐temperature combustion of the tested sample
is the main principle in quantitative CHNS analysis. Gaseous products of the combustion
Table 3.5 Comparison of DD measurement methods
Deacetylation degree, %
Potentiometric Linear potentiometric First derivative of UV

No. of chitosan sample titration titration spectrum
1 82.4 90.6 83.1
2 70.9 82.9 74.0
3 77.5 87.5 79.7
4 76.6 86.0 78.1
5 82.6 88.3 84.2
6 75.3 90.6 80.6
Table 3.6 Comparison of DD determination methods for chitosan.
Advantages Methods Disadvantages

Absolute values NMR Special, expensive equipment, difficult to
access
Easily accessible equipment First derivative of theDifficult preparation of the calibration curve
Simple, quick, sensitive method UV spectrum
Reproducible method
Contamination in the tested
material do not affect the results
Small sample
Easy available equipment and Potentiometric titration Method for well‐soluble chitosan only
reagents
Results well comparable with
those from NMR
Reproducibility
Availability of equipment FTIR Method leads toward divergent results
depending on interpretation of the
spectrum
Simple and cheap method Linear potentiometric Time‐consuming
Harmless reagents titration Inflated results
Large sample
are purified, separated to single components, and detected in a measuring cell of the ther-
mal conduction detector (TCD). The applied method enables computer‐controlled optimi-
zation of the combustion, selective separation of the gaseous components in absorption
columns, and a sequential detection of signals in the TCD of high selectivity. Solid sam-
ples are mostly closed in a tin foil and then compacted to tablets in a press. The samples
are burnt in the oven at 1150°C [31]. Other methods that can be used are the Kjeldahl and
Dumas.
3.1.5 Determination of Ash Content

The determination of ash content is particularly important when chitosan is intended for
medical applications. The applied method employs burning of the tested material and
weighing of the residual ash. The sample is first burnt on an electric heater or at a gas
burner and then in a muffle furnace at 800°C ± 10°C. Combustion is performed till a
constant weight is obtained for the residual ash. The procedure for ash estimation was
performed at IBWCH according to the standard ISO 3451‐1:1997 [32].
3.1.6 Determination of Heavy Metal Content

The content of heavy metals—cadmium, chromium (sum of all oxidation states), lead,
zinc, and mercury—in chitosan can be determined by atomic absorption spectroscopy. The
flame method ASA (FAAS) is suitable for determining cadmium, chromium, lead, and
zinc, while the cold vapor generation method ASA (CVAAS) is used in measurements of
mercury content (own method).
3.1.7 Determination of Water Retention Value (WRV)

WRV is an important parameter whenever chitosan is in the form of powder or flakes and
used as sorption material in medical devices such as wound dressings. The method estimates
the water content that the tested material can accumulate. The sample is first put into water
for 20 hours at room temperature and then centrifuged to remove excessive water. The
material is next dried at 105°C to constant weight, resulting in the complete removal of
water. The difference between the mass of the polymer after centrifuging and drying gives
the water retention value [33].
3.1.8 Determination of Solubility in Hydrochloric Acid

Solubility is another important feature of the polymer. The applied method consists of the
estimation of the content of substance insoluble in 0.5% (v/v) hydrochloric acid after 2
hours of incubation with dynamic stirring at room temperature. The un‐dissolved portion is
separated on a G‐5 Schott funnel and dried to constant weight at 105°C. The percentage of
the fraction insoluble in 0.5% (v/v) HCl is determined by weighing.
3.1.9 Determination of Water Content

The simple method of weighing after incubation in a furnace at 105°C for a defined time is
commonly used. The coulometric method using the Karl Fischer reaction is presently
recommended for determination of water content:
ROH SO2 3 RN I 2 H2O (RNH) SO 4 R 2 (RNH)I
Iodine is electrochemically generated by the anode oxidation of iodide in a coulometric

cell according to the half‐reaction:
2I I2 2e
Iodine is generated on the electrode in the glass titration cell. The generating cell is situ-
ated close to a measurement electrode—a double‐needle platinum electrode—which, by a
voltmeter, monitors the potential of the sample solution in the course of the coulometric
titration. A classical coulometric cell is composed of two parts: an anode chamber and
cathode chamber separated by a diaphragm. The anode chamber contains an anolyte, that
is, a Karl Fischer electrolyte, which makes the oxidation process run. Iodine is generated in
the anolyte as soon as voltage is connected to the electrode. The anolyte contains sulfur
dioxide, imidazole, and iodine salts. Methanol or ethanol is used as solvent. The cathode
holds a catholyte, which is a reagent that enables the entire electrochemical reaction. The
amount of water titrated in the coulometric technique is proportional to the electric charge
(in coulombs) used to generate iodine. Once the entire water content has reacted with
iodine, free iodine emerges in the titrated solution/anolyte. The titration is stopped as soon
as free iodine has been detected in the titrated cell. The apparatus is equipped with an oven
for keeping the sample at suitable temperature. Streaming nitrogen connected to the
apparatus carries the delivered water molecules to the measuring cell. The method is highly
accurate, producing measurement results on the ppm level [34].
3.1.10 Determination of Protein Content

Derived by deacetylation from the natural polymer chitin, chitosan may contain small
amounts of other substances, such as protein. The content of soluble protein can be esti-
mated by the Lowry method. Two features of protein are exploited in the method: the occur-
rence of peptide bonds and the presence of aromatic amino acids. Two steps can be
distinguished in the method: the first involves a biuret reaction featured by the formation of
colored complexes by the peptide bonds and copper ions in alkaline medium. In the second
step, the biuret reaction is intensified by the reaction with Folin–Ciocalteu reagent, in which
the phosphomolybdic and phosphotungstic acids are reduced to oxides in the presence of
mainly tyrosine and tryptophan. Concentration of the colored (violet) complex can be meas-
ured in the range of visible spectrum at wavelengths of 600–700 nm. As in other colorimet-
ric methods, the concentration of protein is calculated from a calibration curve. Chitosan
samples are incubated for 24 hours in 0.5% (w/v) NaOH at 20°C under dynamic conditions.
The extraction is repeated three to four times in 4 hours to release all proteins in the solution
in order to estimate the entire content of protein in the filtrate. The final result is the total
content of protein in all solutions after extraction. The content of protein in the alkaline solu-
tions for the chitosan samples is calculated from the formula, taking into account the color
developed due to the presence of amino sugars (e.g. D‐glucosamine) present in the extract:
X d·R K mg /cm 3
Here, d = dilution; R = protein concentration from the calibration curve for bovine serum
albumin; and K = correction coefficient of amino sugars (concentration of amino sugars
determined from the analytical curve prepared for D‐glucosamine)
The protein content in the starting chitosan sample is calculated from the formula:
n
i 1
Xi Vi n
Z 1000 ppm
m
Here, Xi = content of protein in the extract (mg/cm3); Vi = volume of the extract (cm3);
and m = mass of chitosan sample (g).
3.1.11 Quantitative Determination of Chitosan by Ninhydrin

Thanks to its biological properties—that is, bioavailability, and antineoplastic‐,
immunogenic‐, and antibacterial activity—chitosan is very often used as a component
in polymer composites. The content of chitosan in the composite should therefore
be determined. A colorimetric method is used here. The reaction of ninhydrin with a pri-
mary amino group will form a colored reaction product—diketohydrindylidene–
diketohydrindamine—also called Ruhemann’s purple (Figure 3.6).
Only the 2‐amino‐2‐deoxy‐β‐D‐glucopyranose (GlcN) units of chitosan yield colored
products with ninhydrin. The reaction of chitosan with ninhydrin is sensitive and reproduc-
ible. After the reaction between ninhydrin and chitosan in 0.5% (v/v) hydrochloric acid, the
absorbance at the wavelength of 570 nm is measured by a UV‐VIS spectrophotometer
against water used as blank. In the same way, standards of chitosan solutions are prepared.
A calibration curve is used for estimating the concentration [35, 36].
R
O H2N C COO– O O
H
OH
2 N
OH
CO2, 3 H2O –
O O O
O
C
H R
Figure 3.6 Ninhydrin reaction.
3.2 Products of Degradation and their Application

New, effective methods are sought to degrade chitosan that would lead to the genera-
tion of oligomeric products with biological activity surpassing that of high‐ and low‐
molecular chitosan. Well known are chemical, physical, and biochemical chitosan
degradation methods including radiation, oxidizing, hydrolysis, and the mechanical‐,
thermal‐, and enzymatic treatment that induce changes in the structural and properties
of chitosan [37–40]. The degradation process is a result of broken glycoside bonds in
the chitosan chain. The chemical degradation of chitosan proceeds in the presence of
various acids and free radicals. Degradation processes with acids (acidic hydrolysis)
are non‐specific, yielding a variety of oligomers and massive amounts of the monomer
D‐glucosamine. In contrast, the enzymatic depolymerization of chitosan is a specific
method that most effectively runs in the presence of specific and expensive enzymes of
the chitinase and chitosanase family [41]. It may, however, also be accomplished with
the more common and cheap cellulase enzymes. Cellulolytic enzymes with high hydro-
lytic activity are produced by mildew fungi of the species Trichoderma, Penicillium,
Aspergillus, and Fusarium [42, 43]. Most of the microorganisms produce cellulolytic
enzymes in the form of a complex that combines the activity of various enzymes, exert-
ing a diversified impact on cellulose—for example, endo‐1,4‐β‐glucanases, exo‐1,4‐β‐
glucosidases, cellobiohydrolases, and β‐glucosidases. Endoglucanases randomly cut
the 1,4‐β‐glycoside bonds in the chain’s interior, primarily in the amorphous regions,
leading to a statistical fragmentation of the cellulose chain. Cellobiohydrolases uncou-
ple cellobiose particles from the non‐reductive end of the chain, both in the amorphous
and crystalline regions, while β‐glucosidases hydrolyze cellobiose and short oligosac-
charides to glucose. Similar phenomena may be expected with the said enzymes
applied to chitosan degradation. As chitosan and cellulose structures are quite close to
each other, this could lead to the conclusion that enzymes would more or less similarly
affect their substrate.
Proper selection of the cellulolytic complex allows the preparation of the chitosan oli-
gomers in a controlled manner with the preferred molecular structure. Chitosan also under-
goes biodegradation by the action of lysozyme (N‐acetylmuramine glucanohydrolase),
which appears in practically all human and animal body fluids and tissues [44].
Partly hydrolyzed chitosan exerts the ability to inhibit activity and expression of metal-
loproteinase‐2 (MMP‐2) in fibroblasts of human skin; the process proceeds by reducing
gene expression. In consequence, chitosan reduces the intensity of MMP‐2‐induced

hydrolysis of collagen type IV, which constitutes an important portion of basement
membranes.
Experience gained so far in projects carried out at IBWCh concerns the degradation of
various forms of chitosan (virgin, microcrystalline, salts) by biotechnological methods
using properly selected enzymes of the cellulase, xylanase, and lipase group. Varlamov
et al. proposed a method to prepare a low‐molecular chitosan by the use of a Streptomyces
kurssanovii–derived chitinolytic complex. Low‐molecular chitosan with average molar
mass of 4–8 kDa was obtained by the hydrolysis of chitosan salts in a sodium acetate
solution by using a suspension of cells from the culture of S. kurssanovii as enzymes [45].
Other enzymes active toward chitosan include the lipase derived from Rhizopus japonicus
[46] and the commercial beta‐glucosidase separated from bitter almonds [47]. Muzzarelli
et al. [48] investigated chitosan degradation using a preparation of lipase derived from
wheat germs. Various forms of chitosan were put to enzymatic degradation: chitosan
lactate, chitosan methyl pyrrolidone, and N‐carboxymethyl‐chitosan by using lipase. Due
to this enzyme, the chitosan preparation’s viscosity was dramatically reduced in only a few
minutes, and the molar mass from about 700 kDa was degraded to 13 kDa, whereby N‐
carboxymethyl‐chitosan appeared the most susceptible to the treatment. The enzymatic
degradation was studied, and the concentration of the substrate, enzyme/substrate
proportion, temperature, and pH were taken into account. The degradation was accomplished
by the use of such enzyme preparations as neutral cellulase, Cellulase‐Kinase CE (from AB
Enzymes Oy, Finland), and xylanase Pulpzyme HA (from Novo Industri A/S, Denmark).
All forms of chitosan were, to varying degrees, susceptible to enzymatic degradation, with
virgin chitosan being the least prone (which makes it difficult for the enzyme to penetrate
the structure). The degradation with xylanase and acidic cellulase had, under the adopted
conditions, a statistical character; a reduction could be observed of the average molar mass
by up to 80% at saccharification degree not exceeding 3%. The degradation induced by the
neutral cellulase was, except of the significant decrease of the average molar mass, accom-
panied by a massive formation of soluble amino‐oligosaccharides.
Worth to note, the enzymatic degradation process can be carried out in a controlled way
that leads to oligomeric products with defined molecular‐ and supermolecular structure.
Oligomers of chitosan are obtained as a mixture of products with varied degrees of
polymerization, identification of which is difficult due to the lack of proper standards at
that time. Enzymes in the method must be recovered, and the oligomeric products carefully
cleaned of the residual enzymes by ultrafiltration or ion‐exchange chromatography. The
proteins were removed from the oligomeric mixture by an ultrafiltration apparatus equipped
with a polyethersulfone membrane (Vivaflow 200) with a molecular weight cut‐off of
5000. Another way to remove proteins is the use of anion exchange DEAE –cellulose,
cation exchange CM‐cellulose, and organic‐synthetic ion exchangers such as (ion‐exchange
resins)‐derivatives of polystyrene. The latter are more costly and time‐consuming
operations.
The characteristics of the molecular structure of the degradation products (oligomers) of
chitosan is essential both for the understanding of the degradation mechanisms and the
impact of process parameters on the structure of the obtained oligomers and their
applicability. Such knowledge provides the chance of controlling the process run and
obtaining oligomers with the preferred structure. The assessment of chitosan molecular
structure actually calls for the solution of two analytical problems:
• Separation of the oligomeric mixture to separate oligomers. The only applicable method
is liquid chromatography HPLC.
• Identification of the oligomers by the offline use of NMR measurements or mass
spectroscopy–type ESI‐MS or MALDI‐TOF MS after the separation of the oligomeric
mixture to more homogeneous fractions.
Due to the cationic nature of the chitosan oligomeric molecules, three HPLC methods
can be employed for the chromatographic separation:
• Partition chromatography with reverse phases: The difference of the chemical affinity
of oligomer molecules to the column filling and eluent (mobile phase) is exploited.
• Ion‐exchange chromatography: Separation is based on the varied cation potential of
oligomers with different polymerization degrees.
• Gel permeation chromatography: The separation proceeds as result of different
hydrodynamic volumes of the oligomers with different polymerization degrees.
It is known from literature, and our own experience, that the partition chromatography
with reverse phases by the use of the aminopropyl column LC‐NH2 (Supelco) gives
difficulties, and does not produce satisfying result. This is due to the partial adsorption of
oligomers on the column filling reflected in non‐reproducible chromatograms with a
changeable profile and ever poorer intensity [49–52].
Heterophase degradation of microcrystalline chitosan (MCCh) in the presence of
cellulolytic enzymes yielded a product with partly reduced molar mass and a blend of
water‐soluble oligomers. It was found that MCCh can be degraded with high yield (ca.
99%), while neutral cellulase proved most effective for the material. Wide‐angle X‐ray
scattering (WAXS) examination of water‐soluble degradation products showed the presence
of barely discernible crystallinity peaks characteristic of chitin and a peak unrelated with
elementary chitin cells, originated from a different type of order. The size of the crystallites
is much smaller than of those in virgin and partly degraded chitosan. A study to assess the
impact of chitosan biodegradation products on the stimulation of the germination power of
the tested plants—radish and lettuce (number of germinated seeds, mass of green germs,
and their length in comparison to reference‐water)—showed that oligomers and partly
degraded MCCh stimulate the germination at a lower concentration (0.001% (w/v)) as
compared to virgin chitosan. D (+) glucosamine hydrochloride used for comparison
revealed phytotoxic action at a concentration of 0.01% (w/v). Among the tested preparations,
partly degraded MCCh and water‐soluble oligomers showed bacteriostatic action against
Escherichia coli and poor bacteriostatic activity against Staphylococcus aureus.
Investigation of the activity against plant viruses and bacteria carried out in an agriculture
research center revealed that chitosan oligomers quite effectively (100%) inhibited the
growth of Lucerne mosaic virus (AIMV) and, to a lesser degree, the growth of tobacco
mosaic virus (TMV). The soluble products of chitosan degradation inhibit the growth of
plant bacteria Clavibacter michiganensis subsp. michganensis, Erwinia carotovora subsp.
carotovora, and E. coli at concentrations of up to 0.5% (w/v). It was found that chitosan
and its degradation products showed activity against pathogens of mushroom plants:
Rhizoctonia solani and Myrothecium roridum. Investigation in the use of chitosan
degradation products in agriculture indicate that it would be appropriate to continue works

in that direction, mainly in the protection of plants against viruses and bacteria. Water‐
solubility of chitosan oligomers is a much desired property, offering an easy use of the
material. Lack of effective environment‐friendly preparations for the protection and growth
stimulation of plants prompts further research in the area [53–57].
Medical investigation for the possible use of selected chitosan preparations in cancer
treatment were conducted at the Institute of Medical Biochemistry, Collegium Medicum of
the Jagiellonian University, Kraków. The examination revealed that chitosan preparations
compared with a reference had no cytotoxic action against the cells of both Ehrlich tumor
and regular cells of mice nipple cuticle. In the presence of the preparations with oligomers
obtained after enzymatic treatment, a slight reduction could be observed of the proliferation
of cancer cells (mitotic division). In trials with MCCh and oligomers after thermal
degradation, a decrease was observed of the level of pyruvate kinase isoenzyme M2 of
tumor cells; the effect was particularly pronounced with MCCh after 72 hours. Oligomers
prepared by enzymatic degradation did not display any influence on the parameter. None
of the tested preparations affected the metabolism of regular cells M1. It was found that
oligomers prepared by methods other than enzymatic degradation, mainly those soluble in
water, are more effective in their ability to inhibit the proliferation of tumor cells than other
forms of chitosan. It may be supposed that chitosan oligomers bearing a positive charge
would display a higher ability to inhibit the proliferation of tumor cells due to a better
solubility in body liquids and more effective action on the negatively charged surface of the
tumor cell membrane [58, 59].
3.3 Outlook
The physicochemical properties of chitosan and its degradation products (molar mass, DD,
etc.) are crucial for their application, making testing of the properties a critical part of the
investigation in that sector. A precise and reliable analysis is a must when it comes to
medical applications bearing hazard to human health and life. Thanks to the development
of new testing techniques, instrumental analysis plays an ever‐growing role in
physicochemical examinations, though experience remains a key factor.
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4
New Developments in the Analysis
of Partially Acetylated Chitosan
Polymers and Oligomers*
Stefan Cord‐Landwehr#, Anna Niehues#, Jasper Wattjes#, and
Bruno M. Moerschbacher
University of Münster, Institute for Biology and Biotechnology of Plants, Münster, Germany
A detailed structural analysis of partially acetylated chitosans is a prerequisite for the

establishment of molecular structure–function relationships and also for the development
of reliable applications for chitosan‐based products. Today’s second‐generation chitosans
are well‐defined in terms of their degree of polymerisation (DP) and fraction of acetylation
(FA), but less well in terms of their dispersity in DP (ÐDP), and typically not at all regarding
their dispersity in FA (ÐFA). Moreover, all chitosans of today have a more or less random
pattern of acetylation (PA), but enzymatic modification may yield chitosans with non‐
random PA. Novel methods based on capillary electrophoresis (CE) for ÐFA and enzymatic /
mass spectrometric (MS) fingerprinting for PA are currently being developed, possibly pav-
ing the way for third‐generation chitosans which will be even better defined and less dis-
perse. In this chapter, we will briefly present the established gold standards for the analysis
of DP, FA and ÐDP, and describe in more detail the emerging techniques for ÐFA and PA
analysis of chitosan polymers. We will also briefly present the established methods, and
* This review is dedicated to our friend and teacher Dr. Nour Eddine ’Nouri’ El Gueddari who sadly passed
away in February 2018.
# These authors contributed equally.

describe in more detail the newly emerging mass spectrometric tools for the structural
analysis of partially acetylated chitosan oligomers (paCOS). Quantitative MS sequencing
allows the full structural description of paCOS mixtures, and it is also a prerequisite for
enzymatic / MS fingerprinting analyses of chitosan polymers.
4.1 Introduction
The functional biopolymer chitosan has long been, and far too often still is, hailed as a
cure‐all wonder drug. Not surprisingly, this has raised some unrealistic expectations lead-
ing to disappointment upon failure, or scepticism leading to blunt disbelief and rejection.
However, research done over the past two decades has changed this picture by increasingly
elucidating structure–function relationships of partially acetylated chitosans, in terms of
both their physico‐chemical properties and biological functionalities. Different chitosans
behave differently in aqueous solutions and exhibit different bioactivities. There is not one
cure‐all chitosan; instead, there are many different chitosans, and choosing the right one for
a given task is key to reliable success. Paramount to this transition from the ill‐defined
first‐generation chitosan to well‐defined second‐generation chitosans has been the devel-
opment of reliable methods for the structural characterisation of chitosans. These in turn
have enabled the development of reliable processes for the reproducible production of chi-
tosans with known structural properties and low batch‐to‐batch variabilities. These are now
becoming available in the market in industrially relevant quantities from different suppliers
worldwide, allowing the development of reliable agricultural or biomedical products with
known functionalities.
The structural characteristics which have been the focus of development of second‐
generation chitosans have been DP and FA. For both parameters, a number of analytical
methods have been developed with different advantages and disadvantages, and gold
standards have been established. A number of reviews have been published in the past
describing these, so short summaries will suffice here. It is important to realise that both
DP and FA values are invariably average values as any chitosan sample will be a mixture of
molecules differing in their DP and FA. As a consequence, description of a chitosan sample
should ideally not only indicate these average values, but also their variation within the
sample. While polymer chemistry offers different methods for analysing the ÐDP (‘D
stroke’), describing the FA variability ÐFA is still in its infancy.
A third parameter, namely PA, that is, the distribution of acetylated GlcNAc and deacety-
lated GlcN residues within the chitosan chains, has been discussed controversially. It had
initially been thought that chitosan produced by heterogeneous partial de‐N‐acetylation of
chitin would possess non‐random, block‐wise PA, while chitosans produced by homogene-
ous partial de‐N‐acetylation of chitin or by homogeneous partial re‐N‐acetylation of poly-
glucosamine would possess random PA. Reliably random PA was regarded as a quality
criterion, with some authorities even going as far as defining chitosan as a copolymer of
randomly distributed GlcNAc and GlcN residues. Later, more detailed investigations,
however, suggested that all chitosans available in the market appear to be characterised by
random PA. As a consequence, the possible influence of PA on physico‐chemical properties
and/or biological functionalities is unknown at present. However, recent results showed
that enzymatic rather than chemical modifications of the FA of chitosans can lead to non‐
random PAs. Interestingly, chitosans with either more block‐wise or more regular than
New Developments in the Analysis of Partially Acetylated Chitosan Polymers and Oligomers 83
random PA can be produced using different chitin deacetylases. Functional analyses of first
such chitosans suggest that these structurally novel chitosans are also functionally differ-
ent from conventional chitosans with random PA. PA, thus, emerges as an additional struc-
tural parameter of significant relevance for chitosans’ functionalities. This emerging
insight has been strongly supported by novel analytical methods to characterise the PA of
chitosan polymers.
In parallel to this development, and partially as a consequence of it, the focus of chitosan
research has lately shifted somewhat from polymers to oligomers. This shift was motivated
by the realisation that at least some of the biological activities of chitosan polymers may in
fact be conveyed by their oligomeric breakdown products generated by chitosan‐hydrolysing
enzymes such as chitinases or chitosanases present in the target tissues. In addition, the
shift was clearly favoured by the fact that the structural analysis of chitosan oligomers is
much more straightforward than that of chitosan polymers. It needs to be noted that there
is no clearly defined boundary between chitosan oligomers and chitosan polymers.
Chitosans of DP <10 are clearly oligomers and chitosans of DP >100 are clearly polymers,
and everything in between may be considered large oligomers or, rather, small polymers. A
practical boundary may be a DP of around 20, coinciding cum grano salis with the bound-
ary of analytical techniques best suited for their analysis.
The structural analysis of paCOS is more advanced than that of chitosan polymers,
particularly with the recent developments in mass spectrometry. At the same time, paCOS
analysis is also an integral component of the recently developed enzymatic / MS
fingerprinting analyses of chitosan polymers. Therefore, we will first discuss the structural
analysis of chitosan oligomers followed by that of chitosan polymers.
4.2 Chitosan Oligomers

4.2.1 Degree of Polymerisation (DP), Fraction and Pattern of Acetylation (FA and PA)
The DP of paCOS is rather easily determined using thin‐layer chromatography (TLC),
size‐exclusion chromatography (SEC), gel electrophoresis (GE), nuclear magnetic
resonance (NMR), or preferably mass spectrometry (MS) [1, 2]. While NMR can only give
an average value for the DP of a chitosan oligomer sample, TLC, SEC, GE and much more
precisely MS will give direct insight into the DP diversity of the sample. Similarly, FA of
paCOS can be determined by different methods, with NMR again giving average values
only, while MS can give accurate insight into their FA diversity [3]. In contrast to DP and FA
analysis, the investigation of the PA in paCOS is more demanding and challenging. NMR
can partly reveal the PA of paCOS by analysing the occurrence of GlcN and GlcNAc
residues at the reducing and non‐reducing ends (and to some extent at the penultimate
positions) [2]. This means that the power of NMR increases with increasing purity and
decreases with increasing size of the paCOS analysed, and the single paCOS of very low
DP may even be fully sequenced [4]. Sequencing of larger paCOS can also be achieved by
stepwise enzymatic degradation using specific glycosidases removing GlcN or GlcNAc
residues from the non‐reducing ends, but only if single paCOS are available [5, 6]. In
contrast, MS allows PA determination by sequencing of paCOS even in mixtures as long as
they are not too complex. For paCOS mixtures of low DP, that is, up to DP 6, even
quantification of the components in the mixture is possible using MS of isotopically
labelled oligomers [7].
All commercially available chitosan oligomers and most of the paCOS used for research
are mixtures of oligomers, usually differing in all the three parameters described earlier,
namely DP, FA and PA, rarely in FA and PA only [8]. Therefore, the identification, analysis
and quantification of the paCOS within one sample taking into account DP, FA and PA is the
crucial step to correlate the biological functions of paCOS to their structure. Furthermore,
a detailed analysis of the sample is important to allow batch‐to‐batch comparisons, a con-
ditio sine qua non for reproducible results. To this end, MS is currently seen as the most
suitable method, the gold standard, for a large number of biomolecules. Even in complex
samples, paCOS with different DP and FA can easily be identified due to their different
masses, and PA can be analysed with multiple‐stage mass spectrometry (MSn) experiments.
In these experiments, the paCOS are fragmented in the MS system, and the mass‐to‐charge
ratios of the fragments then allow the deciphering of the sequence of GlcN and GlcNAc
units in the paCOS [3, 9]. A major drawback in MS analysis of paCOS and other com-
pounds is that it yields qualitative but not quantitative data. This situation arises from mul-
tiple problems, for example, that DP, FA and PA are all influencing the ionization efficiency,
that different adducts are formed during ionization, that ion suppression may occur, or that
in‐source fragmentation leads to over‐representation of small and under‐representation of
large paCOS. Furthermore, the distribution of GlcN and GlcNAc units in the oligomer
influences the fragmentation of the paCOS, which is required to elucidate their sequence in
the oligomer, as the glycosidic linkages within the four possible chitosan diads (GlcNAc–
GlcNAc, GlcNAc–GlcN, GlcN–GlcNAc, GlcN–GlcN) are chemically not identical, so that
some fragmentation events will occur more frequently, while some fragments will be
under‐represented [10]. As a consequence, only isolated paCOS can be sequenced without
further precautions. In a mixture of just two paCOS having the same DP and FA, a quantita-
tive statement about the sample composition is not possible using simple MSn, and if too
many paCOS with different PA are in the sample, or if the paCOS are too large, not even the
identification of the PA is possible in this way.
To overcome these drawbacks, several improvements in the analytical process had to be
developed. In a first step, the MS detection was combined with liquid chromatography
(LC) to at least partially separate the paCOS before they are analysed by MS. This
separation can be achieved using hydrophobic chromatography, for example, on a C18
column, or using hydrophilic interaction chromatography (HILIC), for example, on an
amide column [11]. HILIC has been confirmed as the most suitable method for the
separation of complex paCOS mixtures containing oligomers of both low and high DP and
FA [9]. These are separated according to their hydrophilicity, a parameter that is influenced
by both the DP and FA, but very little by the PA. The advantage of a partial or complete
paCOS separation before they are analysed by MS is that problems like ion suppression or
in‐source fragmentation can be easily overcome. Importantly, a complete separation of all
compounds in the LC step is not required.
A further parameter complicating quantitative paCOS analysis is the effect of
their N‐acetylation on ionization and fragmentation. Therefore, quantitative chemical
re‐N‐acetylation of the paCOS mixture using isotopically labelled acetic anhydride is per-
formed before the sample is analysed by MS. The acetic anhydride reacts with the free
amino group of GlcN, resulting in fully acetylated paCOS – in fact, chitin oligomers. To
distinguish between ‘old’, originally present acetyl groups and ‘new’, chemically attached
acetyl groups, deuterated [2H6] acetic anhydride is used in this process, so that the ‘new’
GlcNAc units are 3 Da heavier than the ‘old’ ones, allowing them to be distinguished in MS
[12]. A further benefit of the re‐N‐acetylation is that the chemical complexity of the sample
is strongly reduced, as all chitosan oligomers are converted to chitin oligomers. As a con-
sequence, HILIC gives access to well‐separated fractions containing chitin oligomers of a
single DP each, but, of course, with different amounts and distributions of isotopically
labelled and non‐labelled acetyl groups within each fraction. Therefore, only a few internal,
double isotopically labelled standards, one for each DP, are sufficient to enable quantitative
analysis, as only the effect of DP on quantification remains, while those of FA and PA are
eliminated. These standards are produced from a series of fully deacetylated GlcN
oligomers with defined DP by chemical re‐N‐acetylation using acetic anhydride, which has
six deuterium atoms and four 13C carbon atoms, leading to GlcNAc units which are 5 Da
heavier than the non‐labelled ones. In the next step, the [2H3]-re‐N‐acetylated paCOS mix-
ture and known amounts of the [13C2;2H3]-labelled internal standards are mixed and ana-
lysed by HILIC‐MS. HILIC separation leads to co‐elution of oligomers having the same
DP, including the internal standard. MS detection is used to characterise different paCOS
in the mixture concerning their DP and FA based on their mass, and the intensity relative to
the internal standards is used for quantification. This process eliminates the influence of
DP and FA on ionization efficiency and fully resolves potential problems of ion suppression
and in‐source fragmentation [7].
Still, the third structural parameter, PA, has to be included in the quantitative analysis. To
this end, the [2H3]‐modified paCOS samples are analysed by MSn, where the compounds
are fragmented in the MS detector and the fragments are used to sequence the oligomers.
Re‐N‐acetylation of the sample eliminates the effect of FA on fragmentation, but a second
modification is required to distinguish between fragments coming from the reducing versus
non‐reducing end of the oligomer. Reduction of the reducing end, coupling the reducing
end to 2‐aminoacridone (AMAC) or exchanging the oxygen in the hydroxyl group of the
reducing end against 18O oxygen are commonly used methods for this step [12–14]. This
change in mass of the reducing end sugar unit allows for the unambiguous allocation of
each fragment to either the reducing or non‐reducing end, a prerequisite for sequencing.
Finally, the quantitative data obtained during MS of the deuterated paCOS mixture
informing about DP and FA of the oligomers is combined with the quantitative pattern
analysis obtained during MSn of the deuterated and 18O‐labelled paCOS mixture. This
method allows to fully and quantitatively analyse complex paCOS mixtures regarding their
DP, FA and PA. One remaining disadvantage of the method is that it is restricted to the
analysis of paCOS with a DP <8 as larger chitin oligomers are not soluble in aqueous
media. This limitation does not appear to significantly impede the analysis of paCOS
mixtures obtained by chemical or enzymatic depolymerisation of chitosan polymers, which
in most cases contain mainly small oligomers [15, 16]. But if a chitosan polymer of low FA
is digested by a chitinase requiring consecutive GlcNAc units for cleavage, or if a chitosan
polymer of high FA is digested by a chitosanase requiring consecutive GlcN units for cleav-
age, large fragments will also occur. As such mixtures might be of special interest when it
comes to their biological activities, overcoming this size limitation is an important task for
future method development.
An alternative or complementing method for PA analysis has recently been described
with the combination of mass spectrometry and infrared (IR) spectroscopy [17]. When
analysing carbohydrates, IR spectroscopy has the advantage of giving information on, for
example, the monosaccharide content, regio‐ and isomer conformations, and the linkage
type between the carbohydrate monomers [18]. Additionally, IR spectroscopy is quantitative,
if a reference standard for each compound is available [19]. By performing IR spectroscopy
inside the MS detector, the advantages of both analytical methods can be combined. MS
detection gives the m/z ratio and enables fragmentation of the compounds, and the IR spec-
tra of the compounds and, even more importantly, of their fragments are used to further
characterise and quantify them. Applying IR spectroscopy to the fragment ions reduces the
number of reference IR spectra needed to identify the compounds to just a few, as all larger
oligomers can be fragmented into smaller ones. In future, it should become possible to use
this method for the analysis of larger oligomers and possibly even polymers that cannot be
analysed by LC–MS alone.
4.3 Chitosan Polymers

4.3.1 Molecular Weight (MW) / Degree of Polymerisation (DP)
and its Dispersity (ÐMW / ÐDP)
The first and rather simple structural parameter governing physico‐chemical properties and
biological functionalities of chitosans is the DP, that is, the number of monomeric units
linked by β‐1,4‐glycosidic bonds. DP is the major parameter determining the MW of a
chitosan, and methods to determine DP therefore rely on measuring MW, then calculating
DP by taking into account FA, as GlcN and GlcNAc monomers significantly differ in their
MW. There are different methods for determining the MW of chitosan polymer samples,
and several excellent reviews exist describing them [8, 20, 21]. They mainly include visco-
simetry giving an average value, and SEC giving additionally insight into the MW disper-
sity of the sample. Depending on the method used, SEC can give relative values (when
compared to the elution volume of standards) or absolute values (when refractive index
(RI) combined with multi‐angle laser light scattering (MALLS) are used as detectors, and
the dn/dc value for the polymer to be analysed under the given solution conditions is
known) for the MW of a polymer.
Polymer chemistry distinguishes between different average values for the MW / DP,
namely the number‐average MW / DP (MWn / DPn) and the weight‐average MW/DP
(MWw / DPw), and in principle also the viscosity‐average MW / DP (MWv / DPv) and
z‐average MW/DP (MWZ / DPZ), depending on the method used for its determination.
MWn / DPn is the normal arithmetic mean of the molecular masses of the individual
polymer molecules in the sample; each molecule contributes equally to this mean. In
contrast, the larger polymer molecules contribute more to MWw / DPw than the smaller
polymers in the sample. While this appears counter‐intuitive at first, it has its reason
because some polymer characteristics are more strongly influenced by the larger mole-
cules than by the smaller ones. High‐performance SEC‐RI‐MALLS allows to determine
both MWn and MWw, where MWn is typically smaller than MWw. The ratio of both
values – the dispersity ĐMW = MWw/MWn (and, accordingly, ĐDP = DPw/DPn) – is an
indication of the width of the MW distribution of the sample: the narrower the distribu-
tion, the smaller the Đ value; a (theoretical) sample in which all polymers would have
the identical size would have Đ = 1. Chitosan polymer samples with Đ <2 are typically
considered to be of good quality.
Đ is rarely given in publications reporting biological activities of chitosans (all too often,
not even average MW or DP and average FA values are reported, though such work should
not even be considered for publication in a reputed journal). However, without knowing the
dispersity, it is impossible to judge the relevance of conclusions concerning a possible
influence of MW / DP on a given biological activity. In an extreme (but not at all rare) case,
a sample with a low average MW / DP might consist of a mixture of small oligomers and
some large polymers, in which case clearly the average MW / DP would be strongly
misleading! This problem is probably one of the reasons for the controversy concerning the
antimicrobial activities of chitosan ‘oligomers’ (as is the ambiguous definition of
‘oligomers’).
4.3.2 Fraction of Acetylation (FA) and its Dispersity (ÐFA)

Next to MW / DP, the fraction of acetylation is the second rather well‐researched structural
parameter of chitosans, and the one that is now known to have the strongest influence on a
range of bioactivities. Analysis of FA, therefore, is done on a routine basis by all serious
suppliers, and is indicated in publications by all serious research groups. A number of dif-
ferent approaches have been described in literature during the last decades that range from
ultra violet/visible (UV/VIS) spectroscopy, IR spectroscopy, diverse titration techniques,
acetate determination, monomer analysis and CHN elemental analysis, to X‐ray diffrac-
tion and others. These have been extensively reviewed [22–24]. The methods most widely
used and accepted are based on NMR spectroscopy. Due to the presence of 1H, 13C and 15N
in the chitosan monomers GlcN and GlcNAc, 1H‐NMR, 13C‐NMR and 15N‐NMR can be
used for analysis. Because of the high abundance of 1H, 1H‐NMR is the most commonly
used NMR technique. Irrespective of the element being investigated, the ratios of signals
representing acetylated units and de‐N‐acetylated units are used to calculate the average FA
of the sample.
A disadvantage of most of these methods for FA determination is the relatively high
amount of sample required for analysis as well as the often elaborate sample preparation
and high measurement time. However, high‐throughput analysis of many samples in a short
time might be key for a better understanding of structural properties as well as structure–
function relationships of chitosans and chitosan‐modifying enzymes. Moreover, sample
amounts are often limited when working with experimental chitosans on a lab scale.
Modern MS is probably the most sensitive and accurate technique for the analysis of low‐
MW substances, and, at the same time, it allows high‐throughput measurements. As
described earlier, MS‐based analysis of chitin and chitosan oligomers is already highly
advanced, and the advantages of modern MS can also be used for the analysis of chitosan
polymers.
One of the more recent approaches for FA determination of chitosan polymers that can
include MS analysis is the use of enzymatic fingerprinting. The general idea of fingerprinting
techniques used for biopolymer analysis is to first degrade the polymeric samples to
oligomers which can be analysed afterwards with methods that are not easily available for
polymeric material, such as MS. In principle, degradation of the polymer can be carried out
physically, chemically or enzymatically. Of course, an important requirement is that the
parameter to be investigated is not affected by the treatment. For the depolymerisation
of chitin and chitosan, different methods have been described, including acid hydrolysis,
nitrous acid depolymerisation, sonication, plasma‐assisted processes or enzymatic treat-

ments. A major advantage of enzymatic treatments compared to mechanical or chemical
treatments are the rather mild reaction conditions as well as higher specificity of the reac-
tion, in particular avoiding the risk of concomitant de‐acetylation. As a consequence, chi-
tosanolytic enzymes can be used to produce unique oligomeric fingerprints that are
dependent on the parameters of the starting material. Niehues and Wattjes et al. [25] showed
that, in the case of chitosan, fingerprints produced by a recently described [26], novel chi-
tosanolytic enzyme, chitinosanase, are specific for distinct FAs. This fact can be used to
accurately determine the FA of a sample based on the fingerprint of the enzymatic treat-
ment. Using in silico simulation techniques as well as multivariate predictive models, they
were able to establish an FA determination method with accuracy similar to that of NMR,
but requiring only microgram amounts of sample. Another advantage of enzymatic / MS
fingerprinting over NMR analyses is that the latter is sensitive to free acetate, which is
often present in chitosan samples, while the former is not. As described, the technique uses
the unique chitinosanase isolated from the fungus Alternaria alternata, which is currently
not available commercially, thereby severely limiting its applicability. However, the method
should in principle work with other chitosanolytic enzymes as well. The unique advantage
of chitinosanase with its absolute subsite specificities only becomes visible when it is used
for the determination of PA, as described later in text.
In contrast to the enzymatic hydrolysis carried out by Niehues et al. [25], Han et al. [27]
used nitrous acid depolymerisation to degrade polymeric chitosan to oligomers, followed
by MS and UV measurements for FA determination. This reaction specifically targets the
glucosamine units in chitosan, which are deaminated in the process, leading to the formation
of 2,5‐anhydro‐mannose residues at the reducing ends of the oligomers formed. If the
reaction is carried out to the endpoint, no GlcN units are left in the sample since all of them
are converted to 2,5‐anhydro‐mannose units. In their study, Han and co‐workers showed
that 1‐phenyl‐3‐methyl‐5‐pyrazolone (PMP) can be used to label these reducing ends and
developed a method to determine the FA of chitosan samples by high performance liquid
chromatography‐(HPLC)‐UV detection, verifying the method using MS measurements
and oligomeric standards. PMP derivatization tremendously lowers the detection limit of
the oligomers, and the presence of a single 2,5‐anhydro‐mannose unit per oligomeric or
monomeric product of nitrous acid depolymerisation allows a simple calculation of the
ratio between GlcN and GlcNAc and, thus, determination of FA. The reliability of the
method was verified using titration and NMR experiments that were used for reference FA
values.
Another recent study used a Quadrupole Time‐of‐Flight (Q‐TOF) mass spectrometer for
the direct determination of the FA of chitosan polymers [28]. They observed dissociation of
the glycosidic linkage and dehydration processes that took place during ionization which is
part of the MS analysis. Apparently, the capillary voltage of the electrospray ionization
(ESI) source was the key factor responsible for dissociation of the glycosidic linkage. A
detailed analysis of the mass spectra obtained and comparison with conventional methods
revealed that this method has the potential to investigate the FA of polymeric chitosan
samples and possibly also their PA.
As mentioned earlier, MW / DP and FA values of chitosan polymer samples are always
average values. However, while methods for determining the dispersity ÐMW / ÐDP are well
established, though little used, this is not the case for ÐFA analysis. But there is good reason,
though little evidence, to believe that chitosan polymer samples can vary strongly in this
respect. It is rather reasonable to assume, as in the case of ÐMW / ÐDP, that ÐFA will have a
strong influence on the physico‐chemical properties and biological functionalities of
chitosan polymer samples. It is, therefore, necessary to develop methods that can reliably
distinguish between intra‐chain PA and inter‐chain ĐFA of chitosan polymers.
As early as 1997, Roberts [29] suggested that the differences observed between the bulk
properties of chitosans produced by different production routes might not be explained by
different intramolecular distributions of acetylated units along the chitosan chains (PA), but
rather by an intermolecular variation in FA between chitosan chains (ĐFA). He showed that
a chitosan sample produced by partial deacetylation under homogeneous conditions had a
low variation in FA, whereas a sample produced under heterogeneous deacetylation
conditions had a broad distribution of FA when separated by a pH‐solubility fractionation
technique. He therefore proposed to characterise chitosan samples in terms of their
dispersity in FA, since this is likely to influence the polymers’ physico‐chemical as well as
biological properties, just like ÐMW / ÐDP.
Later, Ngyuen et al. [30] studied the MW and FA of chitosans after fractionation by semi‐
preparative SEC. For a low‐MW chitosan fraction (prepared by partial nitrous acid
depolymerisation of a commercial high‐molecular‐weight chitosan, whose method of
preparation is not documented), they found intermolecular heterogeneity in FA where the FA
increased with increasing molecular weight (from FA 0.03 at MWn 4 kDa to FA 0.13 at MWn
31 kDa). This dispersity was thought to be caused by recovering the material after
depolymerisation through alkaline precipitation and centrifugation as the solubility of
chitosans depends on both MW and FA.
Recently, the ĐFA of chitosan polymers was investigated by Mnatsakanyan et al. [31]
and by Thevarajah et al. [32]. They used free solution CE and UV absorption detection to
study the electrophoretic mobility distribution of different chitosan samples. They found a
correlation between FA and weight‐average electrophoretic mobility. However, aggrega-
tion of polymers was a problem when using this method. Another challenge for studying
the effect of ĐFA on the electrophoretic mobility of the polymer is the dependence on refer-
ence chitosans with a narrow FA distribution. Such chitosans can, however, be produced by
chemical re‐N‐acetylation of polyglucosamine under homogeneous conditions [29]. The
electrophoretic mobility of chitosan might also be influenced by its PA, but this has not
been investigated, for obvious reasons.
4.3.3 Pattern of Acetylation (PA)

In contrast to PA analysis of paCOS, the analysis of the PA in chitosan polymers is far
more challenging. The first studies addressing the distribution of GlcN and GlcNAc units
in chitosan polymers were carried out in the last decade of the twentieth century. As pio-
neers in the field, Vårum and co‐workers used 1H‐NMR [2] as well as 13C‐NMR [33] to
study the distribution of GlcN and GlcNAc units within chitosan polymers. The fact that,
in NMR, different chemical environments can be studied since they are responsible for
differences in the chemical shift of certain hydrogen or carbon atoms was used to inves-
tigate the influence of different neighbouring units on the occurrence of a certain C or H
signal within the spectrum. NMR methods can give average values for monad, diad or
even triad frequencies. Vårum et al. compared diad (GlcNAc–GlcNAc, GlcNAc–GlcN,
GlcN–GlcNAc, GlcN–GlcN) frequencies obtained by 1H‐NMR and 13C‐NMR to those of

theoretical values based on random (Bernoullian) statistics. It is important to note that if the
diad frequencies of a chitosan polymer mixture correspond to the expected frequencies for
random distribution, this only allows the conclusion that the PA can be random, but not that
it actually is random. Reversely, if the diad frequencies observed differ from the ones
expected for random, PA is certainly non‐random. In other words, observing the expected
diad frequencies for random distribution is a necessary, but not a sufficient condition for
assuming random PA.
Based on this work, Weinhold and Kumirska et al. [34, 35] later systematically
investigated the four diad frequencies of chitosans obtained by different chemical
production routes using the C‐5 resonance region. Based on a formula reported by Bovey
and Mirau [36], they proposed a single parameter calculated from the diad frequencies to
describe the pattern of acetylation relative to a random pattern. This value was referred to
as a ‘PA‐value’ by Weinhold and Kumirska. In order to avoid confusion with the abbrevia-
tion for the pattern of acetylation, we prefer the designation PΣ as originally used by Bovey
and Mirau. It equals 1 for random distribution of acetyl groups, while tending towards 0 for
perfectly block‐wise and towards 2 for perfectly alternating distributions. However, the
same limitation as indicated in the preceding text applies: PΣ ≠ 1 is a safe indication of
non‐random PA, while PΣ = 1 is no proof of random PA. The authors found that, irrespective
of the kind of chitosan and its production route – homogeneous or heterogeneous partial
de‐N‐acetylation of chitin or homogeneous partial N‐acetylation of polyglucosamine – all
samples had a PΣ ≈ 1 (ranging from ca. 0.5 to 1.1), indicating, but not proving, more or less
random PA. Interestingly, a systematic deviation from PΣ = 1 was found for all chitosans
with a low FA, which invariably had PΣ <1 (and chitosans with high FA had PΣ >1), but this
observation was assumed to be most likely an artefact caused by inaccuracies of the NMR
diad frequency analysis.
In analogy to some pectin methylesterases which can de‐esterify pectin to yield par-
tially esterified pectin with block‐wise pattern of esterification, it has been proposed that
chitin deacetylases may yield partially acetylated chitosans with block PA [37]. However,
few purified or recombinant chitin deacetylases were available at the time, and these were
almost inactive on fully acetylated, crystalline chitin polymers. In an earlier study, how-
ever, Martinou et al. [38] had shown that treating a high‐FA chitosan with a chitin deacety-
lase originating from the fungus Mucor rouxii indeed seemed to yield low FA chitosan
with a distribution of acetylated and de‐N‐acetylated groups deviating from a random
distribution, as indicated by the NMR diad frequency analysis. The inability to act on
chitin polymers as a substrate was unfortunately confirmed for all chitin deacetylases
characterised since, even though the presence of chitin binding domains in some chitin
deacetylases appears to slightly improve their activity on insoluble chitin polymers [39].
Some recombinant chitin deacetylases were tested on chitosan polymers with different FA
(and known random PA due to their production route using partial chemical N‐acetylation
of polyglucosamine) as substrates (e.g., FA = 0.3 and FA = 0.6), invariably yielding prod-
ucts with lower FA (e.g., FA = 0.1 and FA = 0.3, respectively), but failing to fully deacety-
late the polymers [39, 40]. A similar observation, that is, that chitin deacetylases tend not
to fully deacetylate their substrates, had previously been shown repeatedly when acting
on chitin oligomers [41]. As neither addition of fresh enzyme nor removing potentially
inhibitory acetate allowed the enzymes to continue the deacetylation process, this obser-
vation would appear to suggest that the enzymatic deacetylation had created low‐FA chi-
tosan polymers with a non‐random PA.
In a more systematic study, Niehues and Wattjes et al. [42] recently compared the PA
produced by three different fungal chitin deacetylases originating from Podospora anse-
rina, Pestalotiopsis sp. and Puccinia graminis f. sp. tritici [39, 40, 43]. Using highly acety-
lated chitosan with FA = 0.6 (and random PA, as in the preceding text), they showed that all
enzymes de‐N‐acetylated the substrate to a product with FA = 0.3, but with differences in
PA. These differences were evident both in traditional 13C‐NMR diad frequency analysis
and in newly developed enzymatic fingerprinting approaches. Especially for PA analysis of
chitosan polymers, enzymatic fingerprinting turned out to be a highly valuable tool, able
to overcome some of the drawbacks of 13C‐NMR analysis. The main disadvantage of
NMR, besides the high sample amount required for a single sample and the rather long
measurement time of up to several hours, is that the diad (and even triad) frequencies will
always inform about acetyl distributions on a very short length frame of two (to three)
monosaccharide units. In contrast, MS‐based fingerprinting analysis allows to look at a
high number of different oligomers with a DP of up to 6 if performed quantitatively (and
even higher if only semi‐quantitatively), thus giving better insight into the dispersity within
the sample.
As already mentioned earlier, enzymatic / MS fingerprinting can, in principle, be per-
formed with any chitosanolytic enzyme, as all of them appear to possess some degree of
subsite specificities or preferences. Clearly, the higher the specificity, the more revealing is
the analysis. In this respect, chitinosanase [26], with its absolute specificity for the diad
GlcN–GlcNAc at subsites (‐2) and (‐1), is of particular interest. As already suggested in the
original publication, this specificity leads to the production of oligomeric products that
represent one full GlcNAc‐block of the substrate followed by a full GlcN‐block.
Chitinosanase fingerprinting, thus, allows the determination of GlcNAc‐ and GlcN‐block
size frequencies. Since chitinosanase‐catalysed depolymerisation can be simulated in silico
[25], it is possible to compare the experimentally determined block size frequencies to
theoretical frequencies based on random statistics. In future, the insight into PA gained by
this method will allow studying the mode‐of‐action of chitin deacetylases on polymeric
substrates in more detail. Also, it will open the door towards analysing the influence of PA
on physico‐chemical properties and biological functionalities of partially acetylated
chitosan polymers.
4.4 Outlook
In the past decade, advances in analytical techniques have paved the way for the transition
from yesterday’s ill‐defined first‐generation chitosan (singular) to today’s well‐defined
second‐generation chitosans (plural). The term ‘well-defined’ refers to the two most prom-
inent structural parameters of partially acetylated chitosans, namely DP and FA. These chi-
tosans, which were first available on a small lab scale only, have been used extensively to
elucidate the structure–function relationships of chitosans, in terms of both their physico‐
chemical properties and biological functionalities. As a consequence, second‐generation
chitosans are not only structurally well defined, but their biological activities are also at
least partially known. Such chitosans are now available in the market in industrial quantities
and reliable qualities, thanks to the implementation of strict quality management measures
by some chitosan‐producing companies routinely making use of these analytical tools. This
availability is beginning to be a game changer for the chitosan markets. For the first time,
global market demand exceeds global production of high‐quality chitosans, leading to
increasing prices. And, products based on second‐generation chitosans are appearing in
different markets.
We believe that the new developments described in this chapter, most prominently MS‐
based sequencing and fingerprinting tools, will induce a similar development towards
third‐generation chitosans of the future. These will be well-defined also in terms of their
ĐDP and ĐFA, and their different, random or non‐random PA. Once available on lab scale,
they will be used not only to improve our understanding of molecular structure–function
relationships, but also to help elucidating cellular modes of action. This development is
already ongoing, using fully defined paCOS. We trust that, eventually, selected third‐gen-
eration chitosans will also be mass‐produced, most likely using biotechnological in vitro
biorefinery or in vivo cell factory processes, making them available for new chitosan‐based
products that will be successful in the bio‐economy markets of the future.
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5
Chitosan‐Based Hydrogels
Zhengke Wang, Ling Yang, and Wen Fang
Institute of Biomedical Macromolecules, Department of Polymer Science and Engineering,
Zhejiang University, Hangzhou, China
Hydrogels are cross‐linked polymeric networks that can retain a moisture environment and
mimic human soft tissues. Chitosan, a linear polysaccharide with pH‐responsive self‐
assembling properties, is an attractive candidate for hydrogel construction owing to the
existence of intrinsic intermolecular and intramolecular hydrogen bonds and versatile reac-
tive amino groups. Recently, a variety of chitosan‐based hydrogels have been developed
via diverse approaches, which could be generally categorized as multilayered hydrogels,
hydrogels based on alkali/urea solvent system, injectable hydrogels, self‐healing hydro-
gels, shape memory hydrogels, and superabsorbent hydrogels. Following a brief introduc-
tion of the chitosan hydrogels, this chapter introduces recent advances in chitosan‐based
hydrogels with emphasis on their preparation, characterization, properties, and possible
applications. In addition, current challenges and future perspectives in this field are also
discussed to suggest a new developing direction.
5.1 Introduction
Hydrogels are chemically or/and physically cross‐linked polymeric networks with hydro-
philic groups that can absorb and retain plenty of liquids in an aqueous environment [1].
They are similar to the human soft tissues (e.g. skin, cartilage, muscle, etc.) due to their
high water content and softness, which enable them to become the best candidates for arti-
ficial tissues [2]. Till date, numerous natural polymers, especially polysaccharides, have
been used to design and prepare eco‐friendly hydrogels owing to their abundant resources,
renewability, low production cost, and biodegradability [3]. Chitosan, the main derivative

of chitin with a higher degree of deacetylation, is an attractive linear amino polysaccharide,

composed primarily of the repeating units of β‐(1‐4)‐2‐amino‐2‐deoxy‐D‐glucose
(D‐glucosamine) [4]. It contains excellent biocompatibility, easy biodegradation, and
nontoxic and good antibacterial properties. Chitosan‐based hydrogels have been widely
studied and applied in the biomedical fields, such as controlled drug delivery [5], wound
healing, and tissue engineering [6].
Chitosan is a pH‐responsive self‐assembling polysaccharide as a consequence of its
reactive amino groups, which are protonated in acid media, making chitosan transform into
a water‐soluble cationic polyelectrolyte. Under neutral and basic ambient environments,
the deprotonation of amino groups enables the formation of intermolecular and intramo-
lecular hydrogen bonds and concomitant self‐assembly of polymer chains, leading to the
construction of chitosan hydrogels and poor water solubility [7]. Thus, a series of water‐
soluble derivatives of chitosan (such as carboxymethyl chitosan, chitosan quaternary
ammonium salt, etc.) and novel solvent systems such as the alkali/urea system have
emerged. Moreover, large quantities of reactive hydroxyl/amino groups and the excellent
intrinsic pH sensitivity enable chitosan to become a multifunctional material after chemical
modification and reaction.
This chapter aims to highlight the recent advances in chitosan‐based hydrogels with
emphasis on their production, characterization, properties, and possible applications. We
will first introduce the multilayered chitosan‐based hydrogels formed by chitosan in
acidic solution and chitosan/chitin physical hydrogels based on alkali/urea solvent sys-
tem. Then, smart chitosan‐based hydrogels with specific functions including injectable,
self‐healing, shape memory, and superabsorbent properties are discussed. Finally, the
remaining problems, challenges, and prospects in the formation of chitosan‐based hydro-
gels with unique structures and distinctive functions are briefly summarized at the end of
this chapter.
5.2 Chitosan‐Based Multilayered Hydrogels

Generally, hydrogels are homogeneous materials with amorphous structures. However,
human tissues usually possess three‐dimensional (3D) ordered structures, such as mul-
tilayered or orientated structures, which are necessary for highly elaborate functions of
living organisms [8]. Moreover, inspiration from nature has attracted extensive interests
for the development of materials with complicated and organized structures. For exam-
ple, multilayered structures are often observed in natural substances, such as onions,
nacre shells, and the annual rings of tree trunks. As mentioned earlier, chitosan is a
pH‐sensitive self‐assembling material, and its gelation significantly depends on the
external cues which are able to trigger sol–gel transitions, such as sodium hydroxide
(NaOH) [9]. The formed shape and structures of chitosan hydrogels can be easily
“erased” and regenerated by adjusting the ambient pH [7]. In addition to their native wet
state, these hydrogels can be converted to aerogel, xerogel, or other functional dry mate-
rials with different forms by the removal of water. In this section, we present some cur-
rently typical technologies for fabricating multilayered chitosan‐based hydrogels. We
will introduce these technical approaches briefly while mainly exhibiting the unique
character and classical applications of the resultant structured hydrogels and their cor-
responding dry products.
Chitosan‐Based Hydrogels 99
5.2.1 Periodic Precipitation

Periodic precipitation occurs when a gelation system forms a multilayered hydrogel spon-
taneously. The formation of layers can be explained by the phenomenon named Liesegang
rings [10], which are caused by a special diffusion–precipitation process in the supporting
medium by the encounter of an inner and an outer electrolyte (Figure 5.1a,b).
Hu et al. [11] first prepared multilayered chitosan rods with concentric structures and
great mechanical performances by in situ periodic precipitation. In this work, chitosan in
acidic solution was filled in a semipermeable membrane cylindrical mold and immersed in
a coagulation bath (5 wt% NaOH solution). Then, hydroxyl ions (OH−) diffused into the
inner chitosan system gradually and induced the gelation process. Nie et al. [12] obtained
various hydrogels with different shapes by similar components and procedures. They fur-
ther studied the morphology, mechanism, and design principle of the multilayered products
with oriented structures. Subsequently, Nie et al. [13] also discussed the influence of intro-
ducing certain metal ions (e.g. Cu2+) into chitosan hydrogels and found that the strong
affinity between chitosan and metal ions led to structural transition from orientation to
multilayered structure.
In order to identify the microstructure of physical chitosan hydrogels formed by periodic
precipitation, Sereni et al. [15] prepared thick gels by placing the glass cells containing
chitosan solutions in a NaOH coagulation bath. The results showed that the microstructure
of gels was continuously evolving from the top to the bottom, with mainly two structural
transition zones separating three types of hydrogels (Figure 5.1c). Zone I, close to the sur-
face of the hydrogel, was compact and homogeneous, while the subsequent zone II con-
tained perpendicularly aligned, tubular capillaries. The deepest zone III exhibited small
(micrometer‐range) oriented microstructures that gradually replaced the capillary
morphology.
Some cross‐linking agent was added to regulate the structures and improve the proper-
ties of the prepared chitosan materials. Novel biomimetic multilayered chitosan‐based
materials were developed by the incorporation of a popular ionic cross‐linking agent, trip-
olyphosphate (TPPS) [14]. Ma et al. [16] synthesized a multilayered honeycomb chitosan
hydrogel bead via breath‐figure templating in combination with in situ precipitation
(Figure 5.1d).
(a) (b) (c) (d)

I II-III
II III
30 μm
Figure 5.1 Liesegang ring phenomenon and microstructures of multilayered chitosan‐based hydrogels
formed by periodic precipitation spontaneously. (a) A concentric multilayered hydrogel formed by chi-
tosan acidic solution [12]. (b) Liesegang ring phenomenon occurred in chitosan solution [14]. (c) Confocal
laser scanning microscopy (CLSM) micrographs of a physical chitosan hydrogel obtained at different dis-
tances from the top of the gel [15]. (d) Scanning electron microscope (SEM) images of chitosan honey-
combs formed spontaneously by in situ coprecipitation [16].
5.2.2 Alternating Process

It is well known that chitosan hydrogels with multilayered structures are conveniently
obtained with proper concentration of chitosan and diffusion rate of OH− via periodic pre-
cipitation [12]. Nevertheless, the width and number of layers are not controllable by the
spontaneous process. In recent years, people found that the formation of multilayers can
also be achieved by an alternating process through the spatiotemporal sequence of cue,
which is capable of triggering self‐assembly. Compared with periodic precipitation, the
alternating process is remarkably dependent on manual operations rather than the nonlinear
dynamics of sol–gel transition, resulting in more sophisticated and diversified structures.
Chitosan was utilized to generate physical multilayered hydrogels with “onion‐like”
structures by alternate soaking in a NaOH coagulation bath and water for several times
[17]. The separated concentric multilayers formed due to the fluctuation of hydroxide ions
concentration when the neutralization was interrupted, while the thickness of each layer
was controlled by the base concentration and immersion times. The ordered chitosan
hydrogels were not only applied in drug uploading [18] for their large porosity and equilib-
rium swelling ratio, but also beneficial for cell cultures [17]. Furthermore, the hydrogels
could serve as a template for the generation of hard inorganic particles with complex inter-
nal structures [19].
Besides construction from outside to inside, the multilayered chitosan hydrogels were
also prepared from the core to the periphery with arbitrary shapes by tailoring the gel‐
core template [20]. To form the first gel membrane, the chemical cross‐linkers (e.g. tere-
phthalaldehyde) were loaded onto the chitosan gel‐core and were soaked in a chitosan
solution, and then the sample was ripened in a cross‐linker solution to load chemical
cross‐linkers for the following layer. A multilayered hydrogel could be formed by repeat-
ing these steps.
5.2.3 Induced by Electrical Signals

Many researchers found that the neutralization mechanism allowed chitosan hydrogels to
be electrodeposited at a cathode in which the high cathodic pH induced the sol–gel transi-
tion [21]. Therefore, electrical signals have been applied in the formation of multilayered
chitosan‐based hydrogels, which can be arbitrarily sequenced with high temporal and
quantitative control. The inputs of electrical signals induce the formation of hydrogel lay-
ers from inside to outside, while short interruptions create interfaces between each layer.
Thus, the number of multilayers is controlled by the number of interruptions, while the
thickness of each layer is modulated by the charge transfer during the individual deposition
step (Figure 5.2) [22].
Yan et al. [24] developed an agarose (thermosensitive) and chitosan (pH‐sensitive) dual
hydrogel network. The hybrid polysaccharide hydrogel could undergo reversible sol–gel
transitions in response to imposed thermal or pH cues, which could be controlled by elec-
trical signals. During the erasing and reprogramming experiment, the organized hydrogel
became a solution without structure information after heating and acid treatment. Upon
recooling to 25°C, the hydrogel regenerated owing to the thermal‐responsive agarose. Then
a reprogrammed multilayered structure was formed by inserting a wire electrode and
imputing on–off current signals.
(a) (b)
OH OH Electrical Structural
On
O O pKa O O input output
HO O Off
HO O Optical image
~6.3 +
NH2 n NH3 n
High pH Low pH Structural
e– pH Front output
50 μm
H+ SEM image
Cathode
Hydrogel Soluble chitosan chain

+ + + pH Gradient
+ + +
Chitosan/Agarose
pH Gradient Hydrogel 200 μm
Figure 5.2 Programmable fabrication of chitosan hydrogels with multilayered structures by using on–off
electrical inputs to cue self‐assembly. (a) Neutralization mechanism of the cathodic electrodeposition of
chitosan [23]. (b) Complex self‐assembled structures (chitosan multilayers) within the agarose hydrogel
medium induced by electrical signals and optical/SEM images of the resultant hydrogels [24].
It has been demonstrated that short sequences (minutes) of low‐power (about 1 V) elec-
trical inputs can guide self‐assembly to create complicated soft matters [23]. The multi-
layer formation induced by electrical signals is similar to the hydrogels formed through the
alternating process. Imposing and regulating electrical inputs are much simpler and con-
venient as compared to transferring products among different solutions. Nevertheless, the
shape of the resulting hydrogels in this approach is limited to some extent.
5.2.4 Layer‐by‐Layer (LbL) Assembly

LbL assembly [25] has been explored as a route for the preparation of multilayered chi-
tosan materials, which is dependent on molecular physical interactions, such as electro-
static interactions or hydrogen bonds. The LbL method is helpful for the incorporation of
different drugs, cells, or bioactive molecules in multilayered structures, which show great
potential in biomedical applications. Yoo et al. [26] prepared a novel capsosome with a
cationic liposomal core, which was coated with multilayered polyelectrolyte by sequential
adsorption of negatively charged sodium hyaluronate and positively charged chitosan via
LbL layer deposition. Further, smaller‐sized liposomes were incorporated to the capso-
some for cell mimicking (Figure 5.3). Based on the diversity in the release kinetics between
absorbed liposomes, this capsosome could function as a multidrug delivery system and
shows specific response to the ambient conditions.
5.2.5 Sequential Curing

Sequential curing methods are similar to alternating process methods, which are character-
ized by the relative independence of layers by using solutions with different compositions.
Han et al. [27] developed a gelatin–chitosan (Gtn–CS) hybrid hydrogel with a biomimetic
multilayered structure for osteochondral defect repair via quickly sequential photo‐cross‐
linking (Figure 5.4). The solutions with designed compositions from deep layer to
O– Na+ OH OH OH
O
O O O O O
O HO O
HO
OH
O
NH
HO HO
NH
+
NH2
O n O n +
Chitosan + +
Sodium hyaluronate –
– +
+ – +
+ +
+
+ – +
+ – +
– +
Cationic + +
liposome +
Cationic
liposome core Multilayered liposome
– –
– – Hyaluronate
– – – shell
– + –
–
+ –
– – Chitosan
+ shell
–
–
– –
+ +–
– Liposome
– –
– – +
– – –
–
–
(Liposome adsorption) Liposome core
capsosome
Figure 5.3 A liposomal core coated with alternately multilayered sodium hyaluronate and chitosan mate-
rials via layer‐by‐layer (LbL) assembly [26].
superficial layer were rapidly transferred into cylindrical molds and exposed under UV
light to gelate. The inner pore size, swelling ratio, as well as compressive mechanical prop-
erties of this hydrogel exhibited a graded transition from top to bottom.
Unprecedented multilayered hydrogels exhibit sophisticated internal structures and tun-
able properties, which show great potential in various biorelated fields, especially involv-
ing cell bioreactors [28], drug codelivery [29], tissue engineering [14], etc. Nevertheless,
the design and formation of multilayered chitosan‐based hydrogels still remain great chal-
lenges: (i) the relationship between microstructures with microscopic properties is unclear,
especially the tribological properties such as wear resistance [30], which is vital for some
specific biomedical applications such as joint replacement; (ii) the studies of multilayered
hydrogels mainly focused on their preparation, structure, and physical properties, while
lacking feasibility analysis and experimental tests for certain applications; (iii) most
researches fabricated multilayered hydrogels with the same component in each layer [29],
which may cause unalterable structures and the boundary disappearance of two layers
under long treatment duration; and (iv) the mechanical strength of synthesized structural
hydrogels usually cannot be as good as natural tissues. Recently, the 3D printing technique
[31] has shown great advantages in the preparation of soft materials with more complex
patterns and sophisticated capabilities. Thus, the sustained development of novel tech-
niques will promote the construction of multilayered hydrogels to a great extent.
Interface
T
50 μm
100 μm
UV
T
S: G1/CS/PEGDMA
T: G2/CS/PEGDMA
D: G3/CS/PEGDMA/OV-POSS
50 μm
D T
Interface
D
50 μm 100 μm
Figure 5.4 SEM micrographs of the multilayered Gtn–CS hydrogel with graded compositions [27]. G1,
G2, and G3 are referred to the methacrylatedgelatin with 22, 39, and 43% of methacrylation degrees,
respectively. S, T, and D are referred to as superficial, transitional, and deep layers, respectively.
5.3 Chitin/Chitosan Physical Hydrogels Based on Alkali/Urea

Solvent System
Chitin, a linear polysaccharide composed of β‐(1‐4)‐linked 2‐acetamino‐2‐deoxy‐D‐
glucose units, possesses excellent biocompatibility, biodegradability, and biofunctionality,
with extensive applications as chelating agents, drug carriers, membranes, water treatment
additives, etc. However, its poor solubility in dilute aqueous solvents and common organic
solvents greatly restricts its processing and preparation for macroscopic materials. Chitosan
is obtained by alkaline deacetylation of chitin with better water solubility in acidic media,
but the poor solubility in neutral and alkaline pH region limits its widespread use.
Approaches to acquire homogenous chitosan solution should focus on the breakage of the
hydrogen bonds that exist within the chitosan system via either the protonation of –NH2 in
the acidic aqueous solution or hydrogen formation with other small additives.
Inspired by an efficient cellulose solvent composed of urea and NaOH [32], a novel
NaOH/urea aqueous solution was proposed by Hu et al. for the first time to dissolve chi-
tosan [33]. Subsequently, Fan et al. put forward a prominent solvent system containing 4.8
wt% LiOH/8 wt% urea to dissolve chitosan through a freeze–thawing process. Inclusion
compound (IC) of urea, LiOH, and chitosan was formed with urea as the host, and the
LiOH–chitosan complex as the guest. It was concluded that the added LiOH and urea
played vital roles in the breakage of intramolecular and intermolecular hydrogen bonds in
chitosan, and the freezing treatment was crucial [34–36]. In this section, chitin and chitosan
physical hydrogels based on novel alkali/urea solvent system differing from traditional
acidic solvent system are introduced and discussed.
The dissolution process of chitin in NaOH/urea solvents was illustrated in four main
steps: (a) swelling of chitin immersed in NaOH/urea aqueous solution at ambient tempera-
ture; (b) entry of water molecules into the chitin molecular chain assisted by NaOH;
(c) breakage of the inter‐hydrogen and intra‐hydrogen bonds caused by the freezing and
expansion of water at the freezing point; and (d) solubility promotion of chitin chains
(Figure 5.5a–d) [33]. For the purpose of figuring out the conformation of chitin in NaOH/
urea aqueous solution, a variety of measurements were conducted to study the intramolecu-
lar and intermolecular interactions of chitin [37]. The results revealed that chitin presented
an extended worm‐like chain conformation in the NaOH/urea aqueous solution, with a
sheath‐like structure consisting of NaOH and chitin forming a hydrogen‐bonded complex
surrounded by the urea hydrates (Figure 5.5e).
5.3.1 Chitin Hydrogels Based on Alkali/Urea Solvent System

It was pointed out by Hu et al. that chitin in NaOH/urea aqueous solution possessed thermal‐
induced gelation property, and that the gelation speed was positively correlated with the
environmental temperature and concentration [38]. The mechanism for this thermosensitive
sol–gel transition was supposed as follows: (a) at a low temperature, the chitin solution was
stabilized by surrounding small molecules such as urea and NaOH, the combination of
which acted as an “overcoat” to break the strong intramolecular and intermolecular hydro-
gen bonds; and (b) when elevating the temperature, the overcoat layer was broken owing to
the rapid thermal escape motion of small molecules, causing the exposure of the hydroxyl
groups on the chitin chain and subsequent gelation. In addition to this, the sol–gel transition
could be reversibly conducted via temperature regulation. The thermal‐sensitive properties
of chitin provided a facile approach for fabricating chitin physical hydrogels.
A high‐efficiency and energy‐saving solvent composed of 3.5 M KOH/0.6 M urea for
chitin dissolution was proposed by Cai et al., endowing the continuous dissolution of chitin
within 10 min at ‐30°C without the necessity of repeating a freeze–thawing process [39–
41]. Notably, chitin double‐cross‐linked (DC) hydrogels with high flexibility and high
toughness were achieved by sequential chemical and physical cross‐links [39]. Specifically,
the α‐chitin, dissolved in 0.25 M KOH/0.067 M urea aqueous solution, was first chemically
cross‐linked by epichlorohydrin (ECH) at 5°C for 40 h, and then physically cross‐linked by
immersion in aqueous ethanol to form hydrogen bonding, hydrophobic interactions, and
crystalline hydrates. It was noteworthy that the formed DC hydrogels with both high
strength and high toughness could be realized through very simple approaches without the
addition of fillers. They could even be knotted into a “love knot” and wrapped like a braid
without rupture (Figure 5.6).
5.3.2 Chitosan Hydrogels Based on Alkali/Urea Solvent System

High‐strength chitosan physical hydrogels based on alkali/urea solvent system were pre-
pared by Sun et al. [42]. Briefly speaking, after dissolving in 4 wt% LiOH/6 wt% urea
solvent system via the freeze–thawing treatment, the chitosan solution was then poured
(a) (b) (e)
+ + +
+
+
+ + +
(c) (d)
+ +
+ +
+ + +
+
+ + + + +
+ +
+ +
NaOH hydrate Urea hydrate
Chitin chain
Hydrogen bond Water
Figure 5.5 The diagrammatic illustration of the dissolution process of chitin, including: (a) chitin soaked in 8 wt% NaOH/4 wt% urea aqueous solution at ambi-
ent temperature; (b) water molecules entering the chitin molecular chain facilitated by NaOH; (c) water molecules freezing and expanding at the freezing tem-
perature, breaking the inter‐hydrogen and intra‐hydrogen bonds; (d) promoting solubility of the chitin [33]; and (e) a model description of a chitin complex chain
in NaOH/urea aqueous solution [37].
(a)
Step 1
+ +
Chitin solution Chitin chemically cross-linked hydrogel
Step 2
Chemically cross-linked domain Physically cross-linked domain

CH2OH NHCOCH3 CH2OH NHCOCH3
O O
OH O OH O OH O OH O
O O
NHCOCH3 CH2O NHCOCH3 CH2OH +
HO CH2
CH2O
CH2 OH
NHCOCH3 CH2OH NHCOCH3 Chemically cross-linked domains
O O
OH O OH O OH O OH O
NHCOCH3 CH2OH
O O
NHCOCH3 CH2OH
Physically cross-linked domains
Chitin double-cross-linked hydrogel
(b) (c) (d) (e)
Figure 5.6 Preparation and gross mechanical characterization of double‐cross‐linked (DC) chitin hydrogels. (a) Preparation of the DC chitin hydrogels. Step 1:
the chitin chains form covalent cross‐links induced by ECH that produce chemically cross‐linked domains; step 2: physically cross‐linked domains formed through
hydrogen bonding, hydrophobic interactions, and the formation of crystalline hydrates of α‐chitin are intertwined with the chemically cross‐linked domains to
create the DC chitin hydrogels. The red and blue parts represent the chemically and physically cross‐linked domains, respectively. Photographs of the DC chitin
hydrogels under (b) stretching, (c) compression, (d) knotting, and (e) wrapping. The inset shows a traditional Chinese knot [39].
into the mold and immersed in an aqueous solution of H2SO4/ethanol at 40–50°C for 24 h
to neutralize the alkali completely. Finally, the resultant chitosan hydrogels were thor-
oughly washed with tridistilled water to remove any residues. Compared with chitosan
hydrogels formed via the acidic system, chitosan hydrogels via the alkaline system exhib-
ited better mechanical properties, reflected in the high ultimate strength and energy absorp-
tion before breaking [43].
For in‐depth knowledge on the gelation mechanism, Wang et al. introduced a novel,
visualized approach to elaborate the mechanism of the gelation process. Tetraphenylethylene
(TPE), a typical aggregation‐induced emission fluorogen (AIE gens), was employed to
directly observe the transformation of network, since stronger fluorescent emission was
deemed to tighten network formation [44]. It was noted that the formation of chitosan
hydrogel via LiOH/urea aqueous system was unique, with two distinct but integrated
stages: the thermal gelation stage and rinse stage (Figure 5.7). The thermal gelation stage
was driven by the elevated temperature, forming the embryonic structure and crystalline
regions, while the rinse stage was driven by the continuous change of system components,
greatly increasing intermolecular/intramolecular hydrogen bonds.
Various functional hydrogels were fabricated based on the LiOH/urea solvent system
[45–47]. Inspired by the bilayer structures of plant organs, Duan et al. constructed bio‐
hydrogel actuators from chitosan and cellulose/carboxymethylcellulose (CMC) solution in
an alkali/urea aqueous system utilizing ECH as a cross‐linker [46]. Strong electrostatic
attraction and chemical cross‐linking endowed tight adhesion between two layers and
excellent mechanical properties for carrying out rapid, reversible, and repeated self‐rolling
deformation actuated by pH‐triggered swelling/deswelling. The significant difference in
the swelling behavior between the positively charged chitosan and the negatively charged
cellulose/CMC layers generated enough force to actuate the performance of the hydrogels
as soft grippers, smart encapsulators, and bioinspired lenses, revealing potential applica-
tions in biorelated fields.
(a) (b) (e) (f)
250 μm 250 μm
(c) (d) (g) (h)
250 μm 25 μm
Figure 5.7 Confocal laser scanning fluorescence microscope images of the gelation process of tetraphe-
nylethylene‐chitosan (TPE‐CS). (a) Solution; (b) gel after thermal gelation; (c, d) hydrogel after rinse
procedure—schematic illustration of the formation of junction points of chitosan hydrogel in LiOH–urea
solution; (e) well‐dissolved solution; (f) formation of intermolecular/intramolecular hydrogen bonds of
chitosan, induced by heating; (g) formation of crystalline regions in chitosan gel; and (h) further formation
of hydrogen bonds of chitosan, induced by the removal of LiOH and urea [44].
5.4 Chitosan‐Based Injectable Hydrogels

Injectable hydrogels formed by in situ chemical polymerization or by the sol–gel phase
transition have recently been paid much attention. These material systems are flowable
precursor solutions before administration, but, once injected, rapidly gel under physiologi-
cal conditions, which can be easily applied through a syringe to undergo a rapid sol–gel
transition at the target site. They can readily take the shape of a cavity, providing a good fit
and interface between the hydrogel and tissue [48]. The gel formation after injection brings
about some advantages: an injectable matrix can be implanted in the human body with
minimal surgical wounds, and bioactive molecules or cells can be incorporated simply by
mixing before injection. Following gelation, these matrices become drug delivery deposits
in pharmaceutics or cell‐growing depots for tissue regeneration [49, 50].
Injectable hydrogels can be classified in two main groups according to the nature of their
in situ cross‐linking mechanism, namely, chemically and physically cross‐linked hydrogels.
The gelation of chitosan‐based injectable hydrogels is less invasive and can be initiated by
either physical stimulus or chemical reaction. By controlling the chemical composition, a
number of chitosan‐based injectable hydrogels have been studied for drug delivery, tissue
engineering, and cancer treatment. Furthermore, it is important that they have the ability to
deliver cells, drugs, and/or other bioactive molecules [51]. The construction mechanism of
chitosan‐based injectable hydrogels is either physically associated or chemically cross‐
linked to form the hydrogel. Our discussion in the following text will mainly focus on the
different preparation approaches and the corresponding biomedical applications for various
chitosan‐based injectable hydrogels. The classification is mainly based on the in situ cross‐
linking mechanisms for injectable hydrogel constructions [51, 52].
5.4.1 Physical Association Networks

5.4.1.1 Ionic Interactions
Ionic interactions are constructed via the reversible electrostatic interactions among oppo-
site‐charged ions. The amino group of chitosan would be protonated in acidic environ-
ments, leading to easy complexion with negatively charged polymers (e.g. alginate, heparin,
and polyglutamate) to form hydrogels in situ via the ionic interactions [53–56]. In addition,
the abundant ─OH and ─NH2 functional groups on the native biopolymer chitosan endow
it coordination capacity with a variety of metal ions (e.g. Fe3+, Co2+, Ni2+, Cu2+, Zn2+, and
Ag+) to produce a series of supramolecular hydrogels.
Heparin is clinically used as an antithrombotic agent, and periodate‐oxidized (IO4−) hep-
arin, a negatively charged heparin derivative without a specific pentasaccharide structure,
exhibits lowered anticoagulant activity as compared to native heparin. Fujita et al. reported
a preparation method for the construction of chitosan–heparin‐based injectable hydrogels
via the ionic interactions between these two polyelectrolytes [53]. The viscous lactose‐
modified chitosan solution easily gelled upon mixing with the periodate‐oxidized (IO4−)
heparin, resulting in an injectable hydrogel.
In view of the strong and reversible coordinate interactions between catechol groups and
Fe (III) ions, Yavvari et al. constructed an injectable hydrogel system, combining aqueous
solutions of catechol‐appended chitosan and Fe (III) ions (CAT‐Gel). The optimal Fe (III)–
catechol molar ratio as 1:3 could generate the hydrogel within few minutes (Figure 5.8a).
(a) Fe(III)-catechol
(b)
hydrogel
(coordinative
crosslinking)
Hydrogel
CAT-CHIT Drug loaded hydrogel injection
Low solubility Strong gel

Heating
Sequential and
sustained drug
release Cooling
Tumor bearing
mice
Healthy cell
Localized treatment Weak gel
Reduced toxicity Fe(III) Cancerous cell High solubility
High tumor suppression Dexorubicin Dead cell
Docetaxal Blood vasculature Chitosan PNIPAAm
Figure 5.8 (a) Schematic representation of the study showing the coordinatively cross‐linked catechol–
derived chitosan hydrogel induced by Fe (III)–catechol interactions for localized delivery of a combination
of drugs as effective anticancer therapy [55]; (b) schematic description on solubility and gelation behavior
of chitosan‐g‐PNIPAAm: copolymers with long side chains are viscous and less soluble at low tempera-
tures, but can form strong hydrogels at high temperatures, whereas copolymers with short side chains are
more soluble at low temperatures. However, they cannot form strong hydrogels at high temperatures [58].
Two different populations of catechols exist in the CAT‐Gel, where a major population
formed monocomplexes with Fe (III) ions and a minor population formed complexes of
higher coordination number with Fe (III) [55]. This combined population of the complexes
contributed to the requisite cohesive interactions needed for in situ gelation, and the revers-
ible nature of coordinate cross‐linking imparts favorable injectability nature to the resulting
CAT‐Gel.
5.4.1.2 Hydrogen Bonds

Hydrogen bonds are a kind of physical interactions that occur between hydrogen atoms and
highly electronegative atoms (e.g. O, N, or F), contributing to reversible cross‐links formed
by electrostatic interactions. Theoretically speaking, chitosan is a highly hydrophilic poly-
mer chain with various ─OH and ─NH2 groups grafted as side chains. Nevertheless, the
compact intramolecular and intermolecular hydrogen bonds substantially restrain the solu-
bility of native chitosan. It seems that the reconstruction of the intramolecular and intermo-
lecular hydrogen bonds of chitosan in aqueous solution would be a promising method for
hydrogel formation.
A classical injectable chitosan hydrogel system with thermoresponsive properties was
first proposed by Chenite et al. [57]. In this system, chitosan was first dissolved in acidic
environments via protonation of ─NH2. Then, the pH value of the solution was regulated to
approximately 6.2 while maintaining the solution statement. With the addition of polyol
salts bearing a single anionic head, such as glycerol‐, sorbitol‐, fructose‐, or glucose‐
phosphate salts (polyol‐phosphate or sugar‐phosphate), the cationic polysaccharide solu-
tion was transformed into thermally sensitive pH‐dependent gel‐forming aqueous solution.
The mixture solution of chitosan and polyol salts remained liquid at ambient environment,
and turned into gel when injected in vivo or heated at body temperature. Upon heating, the
water sheaths around chitosan chains are removed and the heat‐induced transfer of protons
from protonated amino groups to glycerol phosphate occurs, and consequently the forma-
tion of hydrogen bonds and hydrophobic interaction among chitosan chains is facilitated.
5.4.1.3 Hydrophobic Associations

The hydrophobic association appears with the aggregation of nonpolar hydrophobic sur-
faces or hydrophobes in aqueous media. The hydrophobic association has been regarded as
a driving force for a variety of biochemical processes, including conformational changes of
biopolymers, cell membrane and vehicle formation, and protein folding. Furthermore, it
has been utilized to trigger the self‐assembly of amphiphilic molecules to fabricate micelles,
liposomes, and physically cross‐linking hydrogels.
Considering the hydrophilic nature of chitosan polymer chains, one efficient way to
import hydrophobic association might be the introduction of hydrophobic sequence onto
the chitosan chains. Various trials have been done to synthesize amphiphilic chitosan‐
derivate polymers for hydrogel construction [59–62], and the interactions between poly-
mer hydrophobes would lead to a transient macromolecular network, thereby imparting
significant viscoelasticity to the solution. Among them, poly(N‐isopropylacrylamide)
(PNIPAAm) and Pluronic F127 are the two types of well‐known thermal polymer chains
with their lower critical solution temperature (LCST) near the body temperature.
Considering the biological activity of chitosan, there is a tendency of incorporating
PNIPAAm [58, 63, 64] or Pluronic F127 [65, 66] chain into water‐soluble chitosan chains
physically or chemically to acquire temperature‐responsive chitosan‐based hydrogels.
Mellati et al. synthesized chitosan‐g‐poly(N‐isopropylacrylamide) (CS‐g‐PNIPAAm)
and found that the resulting copolymers with a small amount of long PNIPAAm side chains
exhibited poor solubility at low temperatures, but can form stronger hydrogels (almost
fivefold) at high temperatures. Whereas copolymers with a high amount of short PNIPAAm
side chains are more soluble at low temperatures, they cannot form strong hydrogels at high
temperatures (Figure 5.8b) [58]. In a physiological pH, an optimized balance between the
solubility (as the prerequirement for cell dispersion and injectability) of copolymers at
ambient temperature and enhanced gel mechanical strength (as the essential parameter of
stem cell microenvironments) at body temperature could be achieved through controlled
reaction conditions. CS‐g‐PNIPAAm was proved to have excellent biocompatibility, bio-
degradability, and rapid phase transition after injection, suitable to serve as cell carriers or
implanted scaffolds. However, weak mechanical properties significantly limit its potential
for biomedical fields.
5.4.2 Chemical Association Networks

Formation of injectable hydrogels can also be done by means of a variety of in situ chemi-
cal reactions, including radical polymerization (induced by heat or UV light), oxidation,
Schiff base reaction, phenylboronate ester bond, click chemistry (e.g. Michael type addi-
tion, Diels–Alder (DA) cycloaddition, and amino‐yne click reaction), and enzyme‐mediated
cross‐linking. Efficient cross‐linking is important for minimizing toxicity associated with
reactive chemical species or leachable small molecules. The abundant amino groups on the
chitosan polymer chains endow chitosan with tremendous potential to construct injectable
hydrogels via the chemically cross‐linking approaches.
5.4.2.1 Schiff Base Reaction

Schiff base reaction can be used to achieve in situ cross‐linking of functional amino and
aldehyde groups in natural biomaterials without the need for any extraneous chemical
cross‐linking agents. This process could be conducted under physiological condition
and without cytotoxic effects as compared to other commonly used cross‐linking
approaches. The abundant amino groups on chitosan polymer chain could react with
aldehyde‐functionalized polymers to generate chitosan‐based injectable hydrogels with
rapid and pH‐responsive gelation properties.
Several polysaccharides such as hyaluronic acid [68–71], alginate [72–74], dextran
[75–77], chondroitin sulfate [78], and xanthan [79] are partially oxidized and the generated
aldehyde groups could be employed for hydrogel preparation via Schiff base reaction with
chitosan. Tan et al. utilized oxidized aldehyde hyaluronic acid (A‐HA) to react with substi-
tuted N‐succinyl‐chitosan (S‐CS) by Schiff base reaction to fabricate a biocompatible and
biodegradable composite hydrogel that has injectability [68]. The gelation time for these
composite hydrogels was within 1–4 min. The ratio of modified HA and chitosan had
strong impact on the gelation time, pore size, and mechanical properties. The A‐HA–S‐CS
hydrogels exhibited slow hydrolysis rate under physiological conditions and could remain
stable in phosphate‐buffered‐saline (PBS) for 4 weeks. Analogically, other kinds of water‐
soluble chitosan were synthesized to incorporate with oxidized HA to prepare similar
injectable hydrogels based on the Schiff base reaction, manifesting prospective application
on postoperative adhesion prevention [69], drug delivery [71], tissue regeneration [80, 81],
and cell encapsulation [70].
Apart from oxidized polysaccharides, telechelic dialdehyde‐functionalized poly(ethylene
glycol) (DF‐PEG) was also widely employed to react with the amino groups of water‐soluble
chitosan [67, 82]. Tseng et al. prepared an injectable and self‐healing hydrogel with intrin-
sic healing by the dynamic Schiff base linkages between benzaldehyde groups at DF‐PEG
chain ends and NH2 groups on glycol chitosan (GC) [67]. The mixture aqueous solution of
DF‐PEG and GC was able to gel rapidly at ambient environment and possessed a gelation
time as approximately 220 s at 25°C, shorter than 100 s at 37°C. The different gelation
times and curing properties of this gel responding to different temperatures gave it the body
injectability for further drug and cell encapsulation (Figure 5.9). Moreover, these DF‐
PEG–GC hydrogels with proper modulus (≈1.5 kPa) were chosen to repair the central nerv-
ous system, triggering the twice faster neurosphere‐like progenitor growth and remarkable
healing effect on neural development (≈81% recovery). For the purpose of mechanical
enhancement and gelation time contraction, multi‐benzaldehyde‐functionalized PEG
analogues [2] and benzaldehyde‐terminated [83] multiarm PEG were used to form more
efficient Schiff base linkages with soluble chitosan.
5.4.2.2 Phenylboronate Ester Bond

Boronic acid derivatives have been extensively explored as a promising candidate for the
designing of glucose detection system in view of their well‐known property of binding to
diol moieties with high affinity through reversible boronate ester formation. In particular,
the introduction of phenylboronic acid (PBA) moieties into polymers sheds light on the
design of glucose‐responsive systems, due to their selective glucose‐responsiveness in
(a) O
OH (c) Self-healing chitosan hydrogel
O O
O O
O OH
O 1000
H HO
n
O
90-92 O
Low viscosity
NH2
100 Gʹ
Glycol chitosan DF-PEG Gʺ
10
Gʹ, Gʺ (Pa)
25ºC
1 ~220 s
OH OH 0.1
O O Plenty of time for
O O 0.01 operation at 25ºC
O OH
HO O HO
H
N x NH2 y 0.001
0 200 400 600 800 1000
x+y=n
*
Time (s)
10000 Gʹ
~1.5 kPa Gʺ
1000 Dynamic
(b) Self-healing hydrogel (injectable) 100 self-healing
Gʹ, Gʺ (Pa)
37ºC
10
1
0.1 Rapid gelation in 37ºC
26-gauge needle 0.01
1 cm
0 500 1000 1500
Time (s)
Figure 5.9 Preparation of DF‐PEG–GC injectable hydrogels. (a) Benzaldehydes at both ends of difunc-
tionalized PEG (DF‐PEG) cross‐linking with glycol chitosan to form injectable hydrogel; (b) the injectable
hydrogel can pass through a 26‐gauge (260 μm) needle without clogging; (c) the gelation time and modu-
lus (G’ and G”) of the injectable hydrogels at 25 and 37°C [67].
aqueous milieu and their advantages of greater stability, longer‐term storability, and lower
cytotoxicity, as compared to protein‐based components such as glucose oxidase and conca-
navalin A.
A series pH and glucose dual‐responsive injectable hydrogels were developed by Li
et al. through the cross‐linking of Schiff’s base and phenylboronate ester using phenylbo-
ronic‐modified chitosan (CSPBA) and oxidized dextran (Oxd), and water was the only
by‐product of the two cross‐linking reactions [84]. CSPBA and Oxd were used as starting
materials in this work, they were able to form covalently cross‐linked hydrogels through
imine bond and phenylboronate ester, and encapsulate drugs and cells with pH changing
from slightly acidic to physiological condition (Figure 5.10a). Oxd with aldehyde groups
and vicinal diol were selected as macromolecular cross‐linkers to form hydrogels with
CSPBA containing amino groups and PBAs. Hydrogels can form by the in situ cross‐
linking reaction between CSPBA and Oxd through two kinds of dynamic covalent bonds,
that is, the pH‐responsive imine bond by the reaction of amino between aldehydes, and the
glucose‐responsive phenylboronate ester by the combination of PBA with polyol. The rapid
gelation and biocompatible cross‐linking chemistry are appropriate for the incorporation of
drug molecules and cells by in situ gel formation.
5.4.2.3 Click Reaction

Recently, click chemistry has received much attention in hydrogel synthesis and prepara-
tion owing to its significant features, such as high chemoselectivity, high reaction efficiency,
(a)
HO
HO O O
O OH
HO
O
O OH
O NH2 CH2 CH COO O CO CHO
OH + + OHC CH2 CH2
O NH2
OH n n
NH
CO CSPBA PVA OHC-PEO-CHO
B(OH)2
H+/glucose OH–
–
Low pH lable Glucose responsive
covalent linker moiety
– OH
– OH
– N=C C=N
H H O
HO HO H2N –
– NH2 O
B B OH
– O
– OH
O N=C C=N
HO H H O
– –
– – B O
HO O B
OH OH
O N=C C=N
– H
H HO
HO NH2 H2N
Drug – Cell
(b)
OR OR
O O
O O
HO NH n HO NH2 m H O
O N O
R = H or CH2COONa
CM-Chitosan (CMC)
O
O
Oz
O
DA-PEG Hydrogel
(c)
H+ OH– H+ OH–
5 cycles
H O H+ O H+ O
N O N O NH2 O
OH– OH– OHC
Enamine Imine Amine + Aldehyde
Figure 5.10 (a) Illustrative formation of the CSPBA/PVA/OHC–PEO–CHO hydrogel [85]; (b) schematic
representation of DA‐PEG/CMC hydrogels by cross‐linking CMC with DA‐PEG; and (c) pH sensitivity of
the DA‐PEG/CMC hydrogel (upper layer) and its mechanism (lower layer) [85].
and mild reaction conditions. Several click reactions, including azide–alkyne

cycloadditions, thiol–ene/yne conjugation reactions, Diels–Alder cycloadditions, strain‐
promoted azide–alkyne cycloadditions, tetrazine–norbornene reactions, etc., have been
employed in drug delivery system and tissue engineering communities. Latest developed
metal‐free click chemistry on chitosan‐based hydrogels would bring good news for
clinical applications [86–89].
Notably, Huang et al. have recently reported an approach to construct injectable and
degradable pH‐responsive hydrogels via metal‐free amino‐yne click reaction [86].
Carboxymethyl chitosan (CMC) was mixed with telechelic electron‐deficient dipropiolate
ester of polyethylene glycol (DA‐PEG) via the spontaneous amino‐yne click reaction with-
out using any initiator or catalyst under physiological conditions (Figure 5.10b,c). The
gelation time varied from 7 to 20 min with adjustable CMC concentrations and stoichio-
metric ratios. DA‐PEG/CMC hydrogels exhibited tunable mechanical properties, which
could be optimized to match the host tissues ranging from 100 to 10 000 Pa. The formation
of enamine bonds was sensitive to acidic stimulus and the balance between enamine and
amino plus aldehyde groups led to pH‐induced sol–gel transitions for the obtained DA‐
PEG/CMC hydrogels.
5.4.2.4 Light‐Triggered Radical Cross‐Linking

Photopolymerization is initiated by free radicals produced by photoinitiators upon UV or
visible light irradiation. The produced radical species attack the double bond of monomers
and propagate to form cross‐linked polymer networks. Photopolymerization to form hydro-
gels has attracted considerable interest in the field of tissue engineering and drug delivery
for the reason that the constructed hydrogels have similar water contents to the extracellu-
lar matrix, allowing for efficient nutrient transport. In tissue engineering applications, pho-
topolymerization is regarded as a less invasive manner for rapid entrapment of cells and
versatile geometries. Effective cell attachment and promoted proliferation could be
achieved by judicious choice of the photopolymerizable macromonomer and triggering
environment.
Various attempts have been made to fabricate double‐bond modified chitosan for pho-
topolymerization under the excitation of UV and appropriate initiators [90–92]. For
instance, carboxymethyl‐chitosan‐grafted‐poly(N‐isopropyl acrylamide)‐glycidyl meth-
acrylate (CMCS‐PNIPAAm‐GMA) was synthesized and cross‐linked by UV to develop
cytocompatible pH‐responsive/thermoresponsive injectable hydrogels for localized drug
delivery [93]. The introduction of PNIPAAm endowed the hydrogels with thermosensitive
properties, while a large amount of carboxyl groups and amino groups on the chitosan
chain led to the pH‐responsiveness for the hydrogels (Figure 5.11a).
Nevertheless, excessive exposure to UV may lead to thermogenic effect and also cause
damage to cells and tissues. Additionally, the limited transmitting depth through tissues of
UV would largely restrict its application in vivo. One feasible solution proposed by Hu
et al. [94] was illustrated as the introduction of visible light photoinitiating system for pho-
topolymerization. Camphorquinone (CQ), fluorescein, and riboflavin are the three blue
light initiators, which could be employed to in situ cross‐link methacrylated glycol chi-
tosan (MeGC). The gelation behavior and biological performance were investigated for
hydrogels that were cross‐linked by these three initiating systems, the consequences of
(a)
O
CH3
CH2 CH C NH CH
CH2OCH2COOH CH3
H O O (NIPAm)
OH H
APS, 70, 3h
H
H NH2 n
(CMCS-PNIPAm)
(CMCS) O O
Ring opening
O reaction
(GMA)
Photo-irradiation
I2959
(b)
VBL curing Riboflavin (RF)
O
N
NH
RF
N N O
OH
MeGC HO
OH
OH
MeGC
+
pH neutralization
Col II
MeGC/Col II-L MeGC/Col II-H
+
– – –
– + – +
+ +– +–
–
–
CS +
MeGC/CS-L MeGC/CS-H
Figure 5.11 (a) Schematic illustration for synthetic route of CMCS‐PNIPAAm‐GMA hydrogels [93].
(b) Design of visible blue light cross‐linkable chitosan/ECM composite hydrogels. See text for details [95].
which revealing that photopolymerized MeGC hydrogels initiated with riboflavin possessed
the most favorable cell viability and mechanical properties for tissue engineering applica-
tions. This visible light initiating system was further utilized to generate injectable chitosan
hydrogels incorporated with Col II, manifesting increased chondrogenesis for cartilage
repair (Figure 5.11b) [95]. Apart from this, another blue‐light‐mediated cross‐linking
approach based on ruthenium (Ru)‐catalyzed photochemical cross‐linking process, which
could form covalent dityrosine bonds between phenolic groups by blue‐light illumination,
was also applicable for construction of chitosan‐based injectable hydrogels [96].
5.4.2.5 Enzyme‐Mediated Gelation

Recently, a considerable amount of research interest has been placed on developing hydro-
gels that undergo cross‐linking mediated by enzymes (both endogenous and exogenous
enzymes). Enzymatic reactions are catalyzed by most enzymes at neutral pH, in an aqueous
milieu and at moderate temperatures, implying their potential to develop in situ forming
hydrogels. Additionally, these reactions are usually very specific and thus avoid side reac-
tions and limit concerns of toxicity.
For chitosan, the most commonly employed enzymatic system is the horseradish peroxi-
dase (HRP) with hydrogen peroxide (H2O2) as substrate. The mechanism for the in situ
gelation is based on the conjugation of phenol and aniline derivatives catalyzed by HRP in
the presence of H2O2. In detail, the HRP can promptly combine with hydrogen peroxide
and the formed complex can oxidize hydroxyphenyl groups ulteriorly [97]. In view of this,
the primary step for building chitosan‐based hydrogels is to incorporate phenolic or aniline
groups into the chitosan chains [98–100]. Jin et al. grafted phloretic acid (PA) onto the
water‐soluble chitosan derivatives (chitosan‐grafted‐glycolic acid, CS‐GA) and the gela-
tion occurred fast with the aid of HRP/H2O2 [98]. Gelation time varied from 4 min to 10 s
by elevating the concentration of polymer, and these hydrogels could be readily degraded
by lysozyme. However, biocompatibility of the HRP‐cross‐linked hydrogel system remains
a concern due to the plant origin of HRP.
Tyrosinase, also known as phenoloxidase and monophenol monooxygenase, is a cop-
per‐containing enzyme that catalyzes the oxidation of phenols, such as in tyrosine residues
and dopamine, into activated quinones, in the presence of O2. Activated quinones can react
with a hydroxyl group or amino group mainly via a Michael‐type addition reaction [101,
102]. Jin et al. fabricated the enzyme‐mediated fast injectable hydrogels based on chitosan‐
glycolic acid/tyrosine (CS‐GA/Tyr) conjugates gelled by tyrosinase under physiological
conditions [99]. The gelation time of the CS‐GA/Tyr hydrogels was tuned from seconds to
minutes by modulating the concentration of the enzymes/conjugates and degree of substi-
tution of Tyr residue. Compared to the HRP‐cross‐linked hydrogels, the tyrosinase‐cross‐
linked hydrogels possessed lower cytotoxicity and better biocompatibility, manifesting to
be a more compatible enzymatic system for tissue engineering.
5.4.3 Double‐Network Hydrogels

Although there are various positive attributes for single network injectable hydrogels such
as structural utility, bioactivity, and cell‐laden possibility, some problems still exist for
hydrogels formed by a single network. For instance, physical cross‐linking frequently
results in low stability, weak mechanical property, and fast degradation of the gelation
system, while covalently cross‐linked hydrogels are typically embedded with poor tough-
ness and unfavorable cytotoxicity. Thus, each individually fails to recapitulate biological
tissues’ resilience toward repeated loading. Addressing these limitations, double‐network
(DN) hydrogels, a subset of specifically structured interpenetrating networks (IPNs) that
exhibit resistance to mechanical failure, have evolved to produce desirable mechanical
properties for biomedical applications.
A variety of attempts have been made to construct chitosan‐based DN hydrogels [103–
112]. Typically, dynamic covalent bond, integrating both the stability of covalent bond
and the reversibility of noncovalent bond in one system, was employed to build injectable
CEC‐I‐OSA‐I‐ADH DN hydrogels composed of N‐carboxyethyl chitosan (CEC) with
medium molecular weight, adipic acid dihydrazide (ADH), oxidized sodium alginate
(OSA), and CEC with high molecular weight (Figure 5.12a,b) [103]. Imine bonds and
acylhydrazone bonds were two main dynamic covalent bonds that could undergo dynamic
reaction under physiological conditions, and the addition of high‐molecular‐weight CEC
could largely enhance the mechanical properties of the hydrogels. The homogeneously
mixed solution was able to form hydrogel within 20 s at room temperature, and it pos-
sessed the capability to be injected after gelation, since the broken hydrogel pieces
squeezed from the needle can self‐assemble and self‐heal into an integral hydrogel at the
target site, with more uniform distributions of cargos and more controllable placement of
hydrogels in vivo.
Additionally, an efficient approach to construct DN hydrogels is the selective and
sequential combination of physical, chemical, and enzymatic cross‐linking methods
with good compatibility. For instance, bioinspired ultratough hydrogels composed of 3,4‐
dihydroxiphenylalanine amino acid (DOPA)‐functionalized chitosan (DOPA‐CHT) were
generated via the covalent cross‐linking using genipin (GP) as a cytocompatible chemical
cross‐linker along with the noncovalent cross‐linking through coordination bonds among
the catechol groups present in DOPA‐CHT and Fe3+ ions (Figure 5.12c). And the DN
hydrogels were further produced by incorporating a small portion of medium‐molecular‐
weight CHT (MMW‐CHT) in the hydrogel precursor solution containing DOPA‐CHT, GP,
and Fe3+ ions. Except for the potential injectability, the introduction of dynamic ionic
interaction conferred an increased toughness, mechanical properties, and self‐healing
capacity, allowing for the recovery of initial mechanical properties in few minutes [104].
Injectable hydrogels are an important class of materials in light of the drive in the clinic
toward minimally invasive procedures. A variety of approaches have been utilized to
develop chitosan‐based injectable hydrogels, providing opportunities for more versatile
and safe means for constructing the hydrogels with varied and useful properties. Although
a number of chitosan‐based injectable hydrogel systems have proved to be promising can-
didates for biomedical applications, several significant challenges for future work remain
as follows: (i) efficient control of the suitable gelation time and stable gelation behaviors
for different applications; (ii) avoidance of the poor mechanical properties and inferior bulk
stability; (iii) minimization on the cytotoxicity induced by unreacted small molecules,
unfriendly fabrication process, and severe cross‐linking simulation; (iv) maintenance of the
balance between the bulk stability and appropriate degradation behaviors; and (v) explora-
tion on hydrogels with multifunctionality such as self‐healing properties, electroconducti-
bility, magnetism, and shape memory capacities. Therefore, further improvements in
(a) (c)
DN DN
OSA CEC-l-OSA-l-ADH DC DC
CEC+ADH Hydrogel
OH OH
O O OH
O NH
RT, < 20s HO NH HO NH
O
n OH
(PBS 7.0) H N
(b) DOPA-CHT
O
HO OH
O O
O Fe O
OH OH
HO OH
i) O O
H O NH
N
O HH N
GP ii) H OH
H O
CH HO
O O H HH
H
O O HO
– – NH
Cl Cl NH
H H HO
(CEC) Cl3Fe.6H2O H
Fe H HN
O
Cl
–
O
iii) H O
H H
H H HO H N
O
(OSA) HO
OH OH
OH OH H
O O PDMS NH
NH
N
O O iv)
(ADH) HO NH x HO NH y
O
H
NH
NH
C O
(Imine) (Acylhydrazone) H C MMW-CHT
Figure 5.12 Synthesis scheme of the CEC‐I‐OSA‐I‐ADH hydrogels. (a) The photographs of before and after gelation of CEC‐I‐OSA‐I‐ADH hydrogel (R = 0.5) in
PBS (pH 7). (b) Chemical structures of the CEC‐I‐OSA‐I‐ADH hydrogel obtained by condensation reaction of the aldehyde groups (from OSA) with the amino
groups (from CEC) and hydrazides (from ADH), resulting in dynamic imine and acylhydrazone bonds, respectively [103]. (c) Schematic representation of the
method developed to fabricate the DC and DN hydrogels. Besides the DOPA‐CHT, GP, and Fe3+ ions, DN hydrogels are also composed of MMW‐CHT. The inset
depicts a picture of the obtained hydrogels (DC and DN showed a similar appearance). Two cross‐linking processes were employed to produce the hydrogels,
namely, a covalent cross‐linking using GP and a physical cross‐linking through coordination bonds in presence of Fe3+ ions. Therefore, DC hydrogels can be
composed of bis‐ and tris‐complexes Fe: (i) DOPA‐CHT or/and covalent bonds between (ii) two DOPA‐CHT chains. Additionally, DN hydrogel can also establish
covalent bonds (iii) between a chain of DOPA‐CHT and a chain of MMW‐CHT or/and (iv) between two chains of MMW‐CHT [104]. More details are presented in
the text.
material design to obtain enhanced mechanical properties and multifunctionality, stability,

biocompatibility, as well as tunable gelation and biodegradability are required for wide-
spread utility of chitosan‐based injectable hydrogels.
5.5 Chitosan‐Based Self‐Healing Hydrogels

Natural soft tissues usually exhibit excellent mechanical strength and some smart proper-
ties, such as self‐healing, self‐sensing, and stimuli‐responsive performance [113]. However,
conventional hydrogels are weak and brittle, showing intrinsic poor mechanical perfor-
mances. Moreover, the traditional gels are vulnerable to stress‐induced formation of cracks,
and then propagation of these cracks may largely influence the integrity of their network
structures [114]. Therefore, inspired by the biological systems where damage triggers a
healing process automatically, hydrogels, which can heal themselves visually after fracture
and recover original material functions to improve their lifetime and safety, have been
extensively studied and applied [115]. Till date, various strategies have been devised to
engineering self‐healing hydrogels through physical interactions or dynamic chemical
linkages [116]. Self‐healing mechanisms are reliant on the dynamic equilibrium of the
interactions or cross‐links utilized in the fabrication of hydrogel networks [117].
In this section, we will mainly introduce the current advances in the preparation and
applications of self‐healing chitosan‐based hydrogels. The classification is mainly based
on the cross‐links or interactions which are responsible for self‐healing property of
products.
5.5.1 Physical Interactions

5.5.1.1 Ionic Interactions
One of the common strategies to prepare hydrogels via ionic interactions is the incorpora-
tion of metal ions, especially involving ferric ions. Skin‐inspired multifunctional chitosan
hydrogels were developed based on physically and chemically cross‐linked double net-
works through a two‐step synthesis [118] (Figure 5.13). First, polypyrrole (PPy) was
grafted to chitosan units decorated by methacrylic anhydride (DCh). Next, free radical
polymerization of acrylic acid (AA) monomers happened in the presence of FeCl3 and
DCh‐PPy. Therefore, dynamic ionic interactions between the carboxylic groups of
poly(acrylic acid) and ferric ions endowed an eye‐catching autonomous intrinsic self‐healing
property and 100% mechanical recovery in 2 min, while the covalent cross‐linking was
used to support the bulk mechanical structure of the hydrogel.
(Before cut) (Cut) (2 halves attached) (30 S) (60 S)
(90 S) (120 S) (150 S) (180 S) (Stretching after 180 S)
Figure 5.13 The self‐healing process of a skin‐inspired chitosan hydrogel after being cut into two halves
and then brought together again with intervals of 30 s [118].
Additionally, catechol and ferric ions have also been widely applied in the formation of
self‐healing. Due to the high grafting ratio of catechol, these chitosan derivatives could
form gels even in acidic medium upon the addition of Fe(III) and Cu(II) ions as cross‐
linkers [119]. A novel catechol‐modified N‐(furfural) chitosan‐based hydrogel was formed
by Diels–Alder click reaction and coordination bonds [120], which retains great
mechanical properties as well as excellent self‐healing performance. A mussel‐inspired
DN was synthesized by incorporating a small fraction of chitosan in the former hydrogel
precursor solution containing DOPA‐CHT (a chitosan derivative functionalized with cat-
echol groups), genipin (GP), and Fe3+ ions [104]. The prepared hydrogel was cleaved in
half and then immediately brought into contact. Five minutes after this operation, the DN
product had completely recovered without signs of a cut.
5.5.1.2 Hydrogen Bonds

He et al. [31] formed a chitosan‐based hydrogel that could be triggered to reversibly switch
cross‐linking mechanisms. Under acidic medium, the chitosan chains were electrostati-
cally cross‐linked by sodium dodecyl sulfate (SDS) micelles. However, chitosan became
neutral in basic solvent, where a self‐assembled crystalline network generated owing to a
lot of hydrogen bonds. The two physical cross‐linking mechanisms could switch in time
and space repeatedly to create soft matters with self‐healing, programmable, and reconfig-
urable properties.
To enhance the self‐healing property of hydrogels prepared via hydrogen bonds, peculiar
dual‐network cross‐linked gels were fabricated (Figure 5.14) [121]. First, chitosan acidic
solution played the role of a template, and then AA and ferric ions were added. After homo-
geneous mixing, free radical polymerization happened in the presence of potassium sulfite
(KPS) as an initiator. The hydrogen bonds between poly(acrylic acid) and chitosan and the
coordination of Fe3+ with carboxyl or amino groups cooperatively formed the dual‐network.
The hydrogels showed electrical conductivity and remarkable self‐healing efficiency
Cut
Chitosan Fe3+ KPS
Free radical
polymerization
Acrylic acid Blade Healed
Self-healing
–NH2 –COOH Fe3+
Figure 5.14 Photographs demonstrating the electrical conductivity and self‐healing property of the dual‐
network cross‐linked CS‐PAA‐Fe(III) hydrogel [121].
ithout external stimulus, which had potential applications in the field of artificial skin or
w
wearable devices.
5.5.1.3 Other Physical Interactions

Host–guest interactions are another type of physical bonds formed by a unique structural
recognition, such that one moiety (the guest) is physically inserted and included inside
another moiety (the host). Host–guest interactions are reversible and can be employed as a
self‐healing mechanism. For example, HA modified by adamantane and β‐cyclodextrin,
respectively, was utilized to develop self‐healing hydrogels through the host–guest interac-
tions [122].
Polymer chains with hydrophobic groups easily form a cross‐linked network and quickly
rearrange via hydrophobic interactions when disturbed, which is the basis of a self‐healing
mechanism [123]. It was reported that self‐healing polyacrylamide (PAAm) [124] and
poly(acrylic acid) hydrogels [125] had been formed by hydrophobic associations. Although
there are few articles about self‐healing chitosan‐based hydrogels prepared by host–guest
or hydrophobic interactions at present, their developments are necessary and inevitable.
5.5.2 Dynamic Chemical Bonds

5.5.2.1 Imine Bonds
Imine bonds, also known as Schiff base linkages, have two main advantages: (i) the dynamic
interactions between amino groups and aldehyde groups are easily available; and (ii) the
reversible nature of imine bonds is displayed under neutral pH, which is suitable for the
preparation and application of self‐healing hydrogels in vivo [126].
A series of self‐healing chitosan‐based hydrogels were facilely synthesized upon mixing
DF‐PEG solution with chitosan or its derivatives solution [67, 127–129]. For example,
after being injected through a 21‐gauge needle, the broken hydrogel pieces regenerated a
completely homogeneous hydrogel within 30 min (Figure 5.15a) [128]. Moreover, the fac-
titious hole in the gels closed up gradually and finally disappeared within 15 min
(Figure 5.15b) [127]. The CS‐PEG hydrogel system could be used for controlled release of
(a) (b)
A-1) Injected A-2) 0h 0.75 h 2h
United gel
30 min A-3) A-4)

later Gelatin gel
Figure 5.15 Self‐healing phenomena of the CS‐PEG hydrogel system. (a) Self‐healing process after injec-
tion (blue: self‐healing hydrogel, red: gelatin hydrogel) [128]. (b) Appearance of self‐healing hydrogels
versus time (5 wt% gelatin gel as control) [127].
small bioactive molecules [127], or drugs [129]. Additionally, the gels showed great advan-
tages in 3D cell culture and injection cell therapy [130], which were an excellent material
to encapsulate neural stem cells (NSCs) for rescuing the central nervous system [67].
Further, inorganic particles (e.g. Fe3O4 and SiO2) [131, 132] had been incorporated into this
system to obtain better functions.
Water‐soluble derivatives of chitosan have also been developed to form self‐healing
hydrogels [83, 133]. Further, the addition of natural nontoxic cross‐linking agents to gelate
chitosan became a safer and environmentally friendly way [134, 135]. Chen et al. [74]
formed a bioadhesive and self‐healing hydrogel based on N‐carboxyethyl chitosan and
sodium alginate dialdehyde, with the introduction of carbon quantum dots (C‐dots), which
performed the dual role of bioimaging and reactive oxygen species (ROS) scavenging.
Recently, zinc phthalocyanine tetra‐aldehyde (ZnPcTa), a good photosensitizer and gelator,
has been used to develop self‐healing chitosan‐based hydrogels with porous nanostructures
(Figure 5.16) [136]. The crack (width, 1 mm) of hydrogels made by a razor blade gradually
healed after 10 min and completely disappeared after 15 min. Besides, imine bonds broke
quickly due to the more acidic tumor environment (pH 6.5–6.8), leading to the rapid release
of ZnPcTa for localized photodynamic treatment [137]. The self‐healing chitosan‐based
hydrogels could also be applied as hemostatic materials [138], or utilized in magnetic reso-
nance imaging (MRI) in vivo [139].
5.5.2.2 Acylhydrazone Bonds

Acylhydrazone bonds, which are formed via the reaction of aldehyde and hydrazine groups,
are similar to dynamic imine bonds, but are much more stable. Thus, the acylhydrazone
bonds have been utilized to construct self‐healing hydrogels with robust mechanical prop-
erties [140]. A novel biocompatible polysaccharide‐based self‐healing hydrogel was
reported by Wei et al. [103]. The hydrogel contained dynamic acylhydrazone bonds as well
as reversible imine bonds, which showed excellent self‐healing ability with a high healing
efficiency of up to 90 ± 2.7% after 48 h at 25°C. In addition, the healing efficiency of mate-
rial can reach as high as 95 ± 2.2%, when the healing temperature increased to 37°C.
Therefore, the hydrogel could be promising to be applied as a cell or drug delivery carrier
with great self‐healing property and cytocompatibility.
5.5.2.3 Other Dynamic Covalent Bonds

Enamine bonds, which belong to the family of dynamic imine bonds, can be formed by the
reaction of amines with acetoacetates. A novel polysaccharide hydrogel was built by sim-
ply mixing chitosan acidic solution and cellulose acetoacetate (CAA) aqueous media [141].
The hydrogel pieces could heal autonomously without external stimuli. Boronic acids have
been extensively studied as a consequence of their ability to react reversibly with certain
diols to develop boronate esters [142]. The dynamic nature of boronate ester bonds has
enabled the development of various self‐healing biomaterials for the lack of apparent toxic-
ity [143]. Disulfide bonds, made by thiol/disulfide exchange reaction, appear as outstand-
ing candidates to endow final products with self‐healing functionality under mild conditions
while retaining a reasonable level of bond strength [144]. DA reaction has been gradually
developed for the preparation of self‐healing hydrogels for its abundant advantages, such
(a) (b)
OH OH
OH CN OH
CN O
HO OHO O
n HO
+ N NH2 NH2
1 2 O
CHO
O H NO2 O
N
(cut)
N N
N Zn N
O N N
N
O
O CN O
3 OHC
H CN OHC H2N N
H2N OHO
OH O
CHO OH O
n
O HO HO
MW O
HO
10 min
N
N N
N Zn N
N N OH OH OH
O O O O O
N HO
O HO HO HO
NH2 NH2 NH2
O n
OHC
CHO 20 min 30 min
Figure 5.16 Novel self‐healing chitosan hydrogel cross‐linked by zinc phthalocyanine tetra‐aldehyde (ZnPcTa). (a) Chemical structure of in situ formation of
ZnPcTa‐conjugated chitosan through Schiff base reaction [137]. (b) Digital photographs of flexibility and self‐healing process of this hydrogel nanocomposite
[136].
(a) (b)
OCOOH OH OH
O O O O O O O
HO HO HO
N NH2 NH
O
Self-healing
O
O
O
N O
O
OH
O HN O
n
OH
2C
OH
2 H
O
OH C
OH
O HN
O
OH
OH
O OH O
N
O
O O
O
O Acid-resistant
N H2N HN
OH OH OH
O O O O O O O
HO HO HOOCO
Figure 5.17 Chitosan hydrogel with double cross‐linked networks (DN) by combining reversible imine bonds with Diels–Alder (DA) reaction [146].
(a) Chemical structure of the DN hydrogel. (b) Gelation behavior, acid resistance, and self‐healing properties of the DN hydrogel.
as high efficiency, high selectivity, no side reactions, no need of additives (catalysts or

coupling reagents), etc. [145]. Due to the higher stability of these dynamic chemical bonds,
they often fabricate self‐healing chitosan‐based hydrogels with double cross‐linked net-
works (DN) in company with reversible imine bonds (Figure 5.17) [146].
Compared with traditional hydrogels, self‐healing hydrogels can restore pristine struc-
tures and functions after damage and exhibit various advantages such as the enhanced
durability, prolonged lifetime, and improved mechanical strength. Though great advances
have been made in the field of self‐healing chitosan‐based hydrogels, their extensive appli-
cations still face some challenges, namely: (i) the self‐healing mechanisms of chitosan‐
based hydrogels are mostly attributed to the dynamic exchange of imine bonds, remarkably
limiting their developments and applications; (ii) the self‐healing processes of gels should
be quick, autonomous, and complete, meaning that the mechanical functions and other key
properties, such as biocompatibility of the healed samples, should be similar to the original
hydrogels, which is hard to achieve; and (iii) good mechanical properties are essential for
artificial tissues fabrication, which should be comparable to articular cartilage (fracture
energy of about 1000 J m−2 and tensile strength of up to 30 MPa). Self‐healing hydrogels
are liable to achieve enhanced mechanical strength by reinforcing the cross‐links, but
resulting in poor healable functions.
5.6 Chitosan‐Based Shape Memory Hydrogels

Being one of the most important stimuli‐responsive materials, shape memory polymers
(SMPs) are polymeric materials that are capable of fixing one (dual shape memory effect)
or more (multi shape memory effect) temporary shapes and subsequently recovering to the
permanent shape in response to external stimuli (e.g. heat, light, magnetism, or chemicals).
The existence of physical or chemical cross‐linking points is required for the shape mem-
ory effect (SME) to recover the initial shape after deformation and fixation. SMP contains
a network architecture consisting of netpoints that are connected with stimuli‐sensitive
macromolecular chains. The netpoints determine the permanent shape, and the segment
chains are used as a kind of molecular switch. The switchable segments give flexibility for
the deformation and offer stiffness for shape fixation under different stimuli [40]. In this
section, typical chitosan‐based shape memory hydrogels and their mechanisms would be
introduced in the following text (Figure 5.18).
(a) Shape recovery (b)

ractions
inte
ular ds
Metal
coo
c bo
n rdi
n
n
ole
at
ge i
A
Shape fixation
o
am
Deformation
on
dr
M
D
Hy
bo
A
Supr
shape mem
nds
A
ar
D
or
l
pramolecu
y
H o s t- g u e s t i n
hydrogels
b on d s
SSMHs
Su
ase
N
nds
fb
ter
h if
a
bo
c ti
Sc
ns
o
nt
Bo d s
le
ro n a a
te ester bon
ov
ic c
Dy n a m
Permanent crosslinks Reversible interactions
Figure 5.18 (a) Mechanism of shape memory hydrogels. (b) Strategies employed to construct shape
memory hydrogels (SMHs) based on reversible interactions [40].
0004461243.INDD 125 10/26/2019 1:22:33 PM

5.6.1 Water‐/Solvent‐Triggered Shape Recovery

Water‐ or solvent‐driven shape recovery effects have been used as switching transition in
SMPs having glass transitions. This type of polymer could absorb a large amount of water,
which affects its mechanical and physical properties by disrupting intramolecular hydro-
gen bonds. Water acts as a plasticizer to reduce the glass transition temperature and hence
effectively allowing for room temperature actuation. Various attempts have been made to
fabricate chitosan‐based solvent‐triggered shape memory hydrogels [147–151].
Correia et al. demonstrated that chitosan‐based porous scaffolds can present a shape
memory effect triggered by hydration. Scaffolds of genipin‐cross‐linked chitosan (CS 1)
and non‐cross‐linked chitosan (CS 0) were immersed in various compositions of water–
ethanol mixtures [147, 149, 151]. With higher contents of ethanol, the dehydrated scaffolds
with vitreous‐like nature could fix their temporary shapes under compression, while the
presence of water was able to disrupt intermolecular hydrogen bonds and permit large‐
scale segmental mobility of the chitosan chains, thus leading to the recovery of the perma-
nent shape of a pre‐deformed scaffold. Compared to CS 0, CS 1 exhibited higher recovery
ratio and better tolerance to large deforming strains, and this phenomenon could be
explained as that the chemical cross‐linking via genipin provided extra‐anchorage points
for improvement of the recovery capability of the scaffolds.
5.6.2 pH‐triggered Shape Recovery

Chitosan can form stable physically cross‐linked hydrogels on the basis of abundant hydro-
gen bonds and hydrophobic interactions among chitosan polymer chains. However, these
physical associations are vulnerable to concentration of H+ and possess reversible deforma-
tion tuned by the pH value [24, 152, 153]. Considering this attribute, bioinspired, nacre‐
like chitosan/graphene oxide (CS/GO) nanocomposite hydrogel films were fabricated
through a simple water evaporation induced self‐assembly method followed by physical
cross‐linking in alkaline solution [152]. The strong and tough hydrogel films showed
unique pH‐driven shape memory behaviors. After immersing in pH 3 aqueous solution for
5 min, the straight samples became soft and easily deformed into “U”‐like shape by exter-
nal stress followed by shape fixation at pH 12 aqueous solution for 10 min. The deformed
samples were transferred to pH 3 aqueous solution again and gradually recovered to their
original shape within 9 min. The incorporation of GO layer was proved to endow a better
shape memory effect than pure chitosan, attributing to the additional physically cross‐link-
ing points of GO.
5.6.3 Ultrasound Triggered Shape Recovery

Bao et al. explored an ultrasound‐modulated shape memory and payload release effects in
a biodegradable cylindrical rod based on chitosan‐functionalized poly(lactic‐co‐glycolic
acid) microspheres (CTS‐PLGA) [154]. Herein, the incorporation of chitosan via elec-
tronic interaction was intended to modify the biocompatibility and biodegradability of the
PLGA microspheres, and was further verified to enhance the thermal stability, elevate the
critical temperature (Ttrans, from 45 to 50°C), and strengthen the compressive modulus of
PLGA microspheres and sintered cylindrical rods. It is noted that the shape recovery ratio
0004461243.INDD 126 10/26/2019 1:22:33 PM

reached the maximum of 97.6% when the PLGA/chitosan ratio was 2.5:1. Besides,
the remote high‐intensity focused ultrasound (HIFU) activation could be utilized to
simultaneously control the shape recovery process and the drug release behavior of
chitosan‐functionalized lysozyme‐loaded PLGA microspheres via variation in irradiation
power and duration time (Figure 5.19). A retarded degradation was observed from the com-
posite microspheres in contrast to the pristine PLGA due to the effective acid‐neutralizing
effect of chitosan on acid metabolites of PLGA. The underlying mechanism for synchro-
nous effects by this indirect trigger was illustrated as that the absorbed ultrasound energy
transformed into heat energy, leading to a rapid rise in temperature above the Ttrans, and thus
enhancing the diffusion coefficient of the SMP, resulting in rapid release of the entrapped
drug molecules.
5.6.4 Self‐Actuated Shape Memory Hydrogels

The temporary shapes of traditional shape memory polymers are often created as well as
defined by an external force. Considering that the possible complex shape or the operation
environment is inappropriate for direct contact, the commonly used deformation method
will be limited, greatly restricting the potential applications of shape memory polymers.
Therefore, a novel shape‐generating method developed by Chen et al. removed this con-
straint via introducing an actuating system into the shape memory polymers to endow them
with various temporary shapes and noncontact self‐deformation behavior.
Specifically, a bilayer self‐deformed shape memory hydrogel, with a thermoresponsive
poly(N‐isopropylacrylamide) (PNIPAAm) layer as the actuating layer and a pH‐responsive
poly(acrylamide)‐chitosan (PAAm‐CS) layer as the memorizing layer, was fabricated
through photopolymerization techniques with a tight junction layer, ensuring the integral-
ity during the bending–recovering process [155]. With a temperature higher than the LCST
of the PNIPAM network, the actuating layer of the bilayer hydrogel would shrink to deform
as a result of the hydrophilic–hydrophobic transition, avoiding inaccuracy and inconven-
ience of traditional shape memory hydrogel. As shown in Figure 5.20, a straight strip of
hydrogel turns into a circle and reaches equilibrium in about 8 min after the temperature
increases to 45°C. Then the deformed bilayer hydrogel was immersed into alkaline solu-
tion for 20 min, the chitosan chains in the PAAm‐CS network would become hydrophobic
due to the deprotonation effects, and the transparent PAAm‐CS layer turns opaque gradu-
ally, manifesting the formation of microcrystallization of chitosan chains and fixation of
the deformed shape of the bilayer hydrogel. When decreasing the pH to 2, the semicircle
hydrogel was able to become straight again due to the disentanglement of the chitosan
chains.
5.6.5 Chitosan‐Based Hydrogels with Triple Shape Memory Effect

Xiao et al. reported a novel poly acrylamide/chitosan‐oxidized dextran (PAAm/CS‐Oxd)
hydrogel with multiresponsive and triple shape memory properties, involving two reversi-
ble interactions such as Schiff base bonds and metal coordination interactions [156].
Chitosan and oxidized dextran (Oxd) endowed it multiresponsive shape memory properties
contributing to the versatile Schiff base bonds among the amino groups of chitosan
and aldehyde groups of Oxd. In addition, PAAm was designed to provide a chemically
0004461243.INDD 127 10/26/2019 1:22:34 PM

(a)
Lyz
CTS 0.5% PVA aqueous
Lysozyme solution
aqueous solution
PLGA solution
+CTS
T>Tg
Hot-pressing
T<Tg T>Tg T>Tg
Ultrasound Ultrasound
Drug release
Shape recovery and drug release
(b)
0s
Initial shape
30 s 60 s
90 s 120 s
Figure 5.19 Preparation procedures and shape recovery effect of chitosan‐functionalized poly(lactic‐
co‐glycolic acid) (PLGA) microspheres. (a) Schematic illustration of the experimental procedures for
chitosan‐functionalized PLGA microspheres with ultrasound‐modulated shape memory and payload
release effects. (b) Photographs showing the shape recovery effect during four cycles of HIFU irradiation
at 30 s per cycle in panel (b); the sample was immersed in the water bath of the HIFU reactor at 37°C
throughout the examination [154].
0004461243.INDD 128 10/26/2019 1:22:35 PM

(a)
Hand deformation Shape memory
Actuating layer
Self-deformation Shape memory
Memorizing layer
O NH O N O N O N O N
O NH H H H
H
H H H H
HN O N O N O
OH–
HN O N O N O
T>LCST
+ + + +
+ + + +
+ + + + + +
OH NH2 OH NH2
n O O HO O O HO O
= + = HO
O
O O O n
NH3+ NH
O NH2 OH
O
OH
n
= NH = microcrystal = H2O
O
(b)
Initial shape Deformed shape Memorized shape Recovered shape
ºC ºC ºC ºC
NaOH HCl
(c) (d) (e)

3.0 Self-deformation Shape memory in NaOH Shape recovery in HCl
2.0 0.5
2.5
1.6 0.4
Curvature
2.0
Curvature
Curvature
1.5 1.2 0.3

1.0
0.8 0.2
0.5
0.4 0.1
0.0
0 4 8 12 16 0 40 80 120 160 0 40 80 120 160
Time (min) Time (min) Time (min)
Figure 5.20 Mechanism and shape recovery behavior of self‐deformed hydrogel. (a) Schematic illustra-
tion and mechanism for novel self‐deformed shape memory process. (b) The self‐deformed hydrogel
reversibly self‐deformed between 20 and 45°C. The deformed shape of the hydrogel is partially maintained
in NaOH solution and completely recovered in HCl solution. (c–e) The curvature of a bilayer strip against
time in 45°C KNO3 aqueous solution, 20°C NaOH solution, and 20°C HCl solution [155].
cross‐linked and irreversible network via the free radical polymerization of acrylamide
(AAm). Owning to the excellent chelating capacity of chitosan with various metal cations
such as Fe3+, Ag+, Ni2+, etc., the coordination effects could also stabilize the temporary
shape and mold the hydrogels.
The PAAm/CS‐Oxd hydrogel was authenticated to possess triple shape memory behav-
ior, as shown in Figure 5.21. A hydrogel strip dyed in pink and green was first immersed in
the alkaline buffer solutions (pH 8) to fix temporary shape I based on the formation of
Schiff base bonds (Figure 5.21c). Then the hydrogel strip was deformed to temporary
0004461243.INDD 129 10/26/2019 1:22:36 PM

(a) (c)
Oxalate or H2SO4
EDTA Ag+ OH–

Ag+
Fe3+ Co2+ OH– Ag+
H+ EDTA
Cu2+ Zn2+ Original shape Temporary Temporary
shape I shape II
Ni2+
OH– Ag+
H+ EDTA
(b)
Oxalate or H2SO4
Ox O
ala Ag+ SO
4 HO
HN
HO H 2N
te H2 O OH O OH
O O O
O OH O O OH O OH NH2 OH NH2
H 2N
HO
H2N HO
n
O
O HO O O HO O PAAm
O O HO O
HO O
NH2 NH n
OH O OH
OH OH
– O
– /Br
ED O O HO HO
H 2N C N
Cl TA HO HN OH
O
O O O O OH O O O
OH O OH O O OH O
O O n
H 2N HO H2N HO
Figure 5.21 The process and mechanism of triple shape memory and shape recovery processes. (a) The shape memory behavior based on the coordination of
chitosan with various metal ions. All the dashed lines indicate that the temporary shape hydrogel will be recovered in the EDTA aqueous solution. (b) Shape
memory behavior based on CS–Ag+ coordination. (c) The process and mechanism of the triple shape memory and shape recovery; the triple shape recovery could
be realized step by step or in one step [156].
shape II and transferred into the AgNO3 aqueous solution to fix temporary shape II via
metal coordination interaction. Finally, an “N”‐shaped hydrogel strip was obtained. After
being sequentially transferred into the aqueous solutions of Ethylenediaminetetraacetic
acid (EDTA) and acetic acid–sodium acetate buffer solutions (pH 3), the complex “N”
temporary shape would be gradually recovered to temporary shape I and then to the origi-
nal straight shape. Moreover, the complex “N” temporary shape could also be directly and
quickly recovered to the starting straight shape when using oxalate as the external stimulus.
This phenomenon was termed as triple shape memory capability ascribed to the dual attrib-
utes of oxalate acid both serving as a moderately strong acid and a good binding agent to
Ag+, causing simultaneous destruction on the two reversible interactions. The programma-
ble triple shape memory effect triggered by two different stimuli step by step, or a single
stimulus in one step, largely expanded the potential applications of reversible interaction‐
induced SMPs.
So far, various chitosan‐based hydrogels with fast shape memory/recovery rates or good
shape fixity/recovery ratios have been constructed. However, it is still challenging to com-
bine all these advantages into one single hydrogel system. Most of the previously reported
SMHs have poor mechanical properties, thus limiting their applications. Besides, most
investigations in this field are focused on dual SMHs. In order to meet the increasing
demands for versatile materials, it is of urgent need to develop more shape memory hydro-
gels with elegant triple/multi shape memory properties. Even though great efforts have
been primarily devoted to the preparation of novel SMHs in the last decades, less attention
has been given to the practical applications of SMHs, especially in the fields of biomedical
applications, such as drug delivery systems, tissue engineering, biosensing, etc. Considering
the excellent attributes of chitosan, SMHs with excellent biocompatibility can be con-
structed and be employed for in vivo studies. It is also quite promising to develop new types
of SMHs with multifunctionality, such as magnetism, luminescence, electrical conductiv-
ity, expansion–contraction, self‐healing properties, etc. Such supramolecular hydrogels
with various functions are expected to be the next generation of smart soft materials that
can function more closely with natural or biological systems.
5.7 Superabsorbent Chitosan‐Based Hydrogels

Typically, superabsorbent hydrogels (SH) are 3D, hydrophilic networks with the ability to
absorb much more water than its mass, ranging from hundreds to thousands of times [157].
Further, SH can keep their networks stable even in a swollen state without being dissolved
and retain a large fraction of water even under load (Figure 5.22). Currently, worldwide
products of SH are generally synthetic polymers, such as polyacrylamide, poly(acrylic
acid), and other polyacrylates. Nevertheless, their worse biodegradability has caused heavy
environment pollution and their utilization in the field of biomedicine has been limited due
to the poor biocompatibility and their toxicity. Therefore, numerous natural polymers,
especially the polysaccharides, have been used to replace (or be combined with) those
synthetic polymers [158].
Superabsorbent chitosan‐based hydrogels have drawn significant attention. In this sec-
tion, we aim to introduce the recent progress in the chitosan‐based superabsorbent hydro-
gels (CSSH), providing a brief overview of their preparation methods, capacity (e.g. water
absorbency, water retention, and salt tolerance), and promising applications. We classify
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(a) (b)
1 cm 1 cm
Figure 5.22 Photographs of a superabsorbent hydrogel in (a) dry state and (b) the swollen state [159].
these CSSH into four types according to the components of synthetic systems: (i) cross‐
linked chitosan (or their derivatives) hydrogels without synthetic polymers; (ii) chitosan
hydrogels prepared through graft copolymerization of vinyl monomers, such as AA or
acrylamide (AAm) on the polymer chains; (iii) chitosan‐based composite hydrogels
obtained via the incorporation of inorganic or organic substances; and (iv) pure chitosan
materials fabricated without any additives, such as initiators, vinyl monomers, or
cross‐linkers.
5.7.1 Cross‐Linked Chitosan‐Based Hydrogels

Commonly, chitosan is soluble in acid solvents and therefore cannot form hydrogels. To
overcome this problem, chemical or physical cross‐linking throughout covalent or physical
interactions has been introduced. Glutaraldehyde, a normal cross‐linker for chitosan‐based
system, has been used to form superabsorbent hydrogels. A superabsorbent chitosan poly-
mer was provided via carboxymethylation of chitosan, followed by cross‐linking with glu-
taraldehyde and freeze‐drying. The water uptake capacities of the SH measured after
submersion in distilled water, 0.9 wt% NaCl solution, synthetic urine, and artificial blood
for 10 min were 104, 33, 30, and 57 g/g, respectively [160]. Additionally, some different
organic cross‐linkers have also been applied. A novel hydrogel was synthesized success-
fully through the cross‐linking of chitosan chains with the EDTA–urea adduct. The product
exhibited a much higher water uptake capacity (570 g/g) than a commercial diaper material
(238 g/g) [161].
To eliminate the toxicity, other biological cross‐linkers might be used. A new full‐
polysaccharide hydrogel was prepared by cross‐linking modified chitosan and periodate‐
oxidized sucrose [162]. Narayanan et al. [163] prepared the hydrogels by using renewable
sources such as chitosan, urea, and citric acid. The product was found to absorb a maximum
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of around 1250 g/g of distilled water and 210 g/g of 0.1% (w/v) sodium chloride solution.
The new material with rapid and high water uptake could play a vital role in applications
that demand the quick absorption and slow release of water, such as agriculture.
The advantage of a chemical approach (cross‐linking through covalent bonds) is that the
hydrogel matrix remains stable over time. However, the agents used in the chemical cross‐
linking are often toxic and the residual cross‐linkers must be removed before using in
biomedical applications. Thus, physical cross‐linking is an efficient route to prepare hydro-
gels without using toxic chemical agents. A novel gellan gum–chitosan superabsorbent
hydrogel was constructed via the ionic bonds between chitosan and gellan gum [164]. The
best water absorption capacity (more than 218 g/g) was obtained and the fertilizer‐release
test showed that almost complete release occurred in approximately 8 h. Additionally, the
hydrogel showed excellent ability of water retention through the water evaporation analy-
sis. Therefore, this material could be applied in environment‐friendly agriculture success-
fully for controlling soil humidity and releasing the fertilizer.
5.7.2 Hydrogels by Graft Copolymerization

Several studies have concluded that the graft polymerization of hydrophilic monomers
(such as AA and AAm) onto chitosan can enhance the water uptake capacity of the back-
bone and hydrophilicity, making it an efficient way to acquire superabsorbents with differ-
ent functional properties. Furthermore, the effect of polymerization variables, including the
content of cross‐linker, initiator and additives, the weight ratio of monomer to chitosan, the
reaction temperature, time, and drying method, on water absorbency had been studied in
various researches.
A novel superabsorbent polymer was prepared through graft polymerization of AA onto
the chain of carboxymethyl chitosan and subsequent cross‐linking [165]. The polymer syn-
thesized under the best conditions is able to absorb over 1180 g/g of distilled water, over
162 g/g normal saline, over 116 g/g artificial blood, and over 108 g/g artificial urine. In
addition, acrylic acid could also be grafted onto chitosan chains under microwave irradia-
tion with shortened fabrication period [166].
AAm monomer was also used to modify chitosan chains to develop superabsorbent
hydrogels with higher salt‐resistance property. Water‐soluble chitosan was first reacted
with glycidyl methacrylate (GMA) and then AAm monomers were added. The highest
water‐swelling percentage (about 1900%) was obtained under particular experimental
reaction conditions. Superabsorbent hydrogels were successfully synthesized from AA,
AAm, and quaternary ammonium chitosan with high substitution degree [159]. The AAm
(the content was 10 wt%) could increase the water absorbency in 0.9 wt% NaCl solution
from about 40 to 88 g/g. When the content of AAm was 10%, the maximal synergistic
effect and water absorbency (about 760 g/g) were obtained.
A novel chitosan‐based superabsorbent hydrogel was prepared through ceric‐initiated
graft polymerization of acrylonitrile onto chitosan in a homogenous medium and followed
by saponification [168]. The newly synthesized hydrogel exhibited low salt sensitivity
when the degree of graft was low. Moreover, the synthesis and study of polyampholyte
superabsorbent polymers had drawn significant attentions, whose cross‐linked networks
contained both positively and negatively charged repeating units. Polyampholyte superab-
sorbents were prepared through the graft copolymerization of AA and dimethyl diallyl
0004461243.INDD 133 10/26/2019 1:22:48 PM

1000
Repulsive
force
800
Repulsive
force
600
Q (g/g)
400
200
0 2 4 6 8 10 12 14
pH
Figure 5.23 Effects of pH of the solution on water absorption capacity of the polyampholyte superabsor-
bents [167].
ammonium chloride (DMDAAC) onto the chain of carboxymethyl chitosan [167]. The
highest swelling ratios in distilled water and normal saline water could be as high as 612
and 81 g/g, respectively. The swelling and shrinking of the products exhibited unique pH‐
dependent properties and showed peaks in swelling capacity at both acidic condition (pH
3.5) and basic condition (pH 9.5) (Figure 5.23), which made them good candidates as
potential drug carriers.
5.7.3 Chitosan‐Based Composite Hydrogels

Recently, the preparation of superabsorbent composites has attracted considerable atten-
tion because of their relatively low production cost, improved swelling properties, and
enhanced gel strength [169]. Natural aluminosilicate inorganic compounds (e.g. montmo-
rillonite, kaolin, and attapulgite) are often applied due to their hydrophilic nature.
Additionally, organic substances were common additives as well.
5.7.3.1 Inorganic‐Chitosan Composites

A novel chitosan‐g‐poly(acrylic acid)/montmorillonite superabsorbent nanocomposite was
prepared by in situ intercalative polymerization, with water absorbency of 160.1 g/g in
distilled water and 46.6 g/g in 0.9 wt% NaCl solution. The introduced montmorillonite
could form a loose and porous surface and improve the water absorbency of this hydrogel.
Kaolin is very suitable to use as a reactive additive owing to its hydrophilic nature. A
superabsorbent composite based on chitosan was prepared by graft copolymerization of
AA in the presence of kaolin powder. The maximum water absorbency (421 g/g) was
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obtained at the optimum condition. Attapulgite contained a lot of cations and they were
easily ionized and dispersed into the superabsorbent network, which enhanced hydrophi-
licity of the composite and made it swell more. Zhang et al. [170] prepared a chitosan‐g‐
poly(acrylic acid)/attapulgite composite hydrogel with water absorbency of 159.6 g/g in
distilled water and 42.3 g/g in 0.9 wt% NaCl solution.
Wang et al. have applied several inorganic clay materials to form superabsorbent chi-
tosan‐based composites. A novel chitosan‐g‐poly(acrylic acid)/vermiculite composite was
prepared with a high water absorbency (585.3 g/g) and fast swelling rate [171]. Another
composite material was synthesized by replacing vermiculite with rectorite. It was found
that introducing 10 wt% of rectorite could enhance water absorbency and water retention
in the same time. The maximum water uptake capacity (415 g/g) was obtained under opti-
mum reaction condition.
5.7.3.2 Organic‐Chitosan Composites

A novel superabsorbent hydrogel composite, based on acrylic monomers grafted onto chi-
tosan and the incorporation of an industrial waste, rice husk ash (RHA), was developed
[172]. Hydrogels filled with RHA previously calcinated at 900°C (RHA900) showed better
water uptake (225 g/g) than those with husk calcinated at 400°C (RHA400) (198 g/g) due
to the higher purity and crystallinity. The utilization of wasted RHA materials was an envi-
ronmentally friendly alternative. Liu et al. [173] made a chitosan‐g‐poly(acrylic acid)/
sodium humate superabsorbent in the presence of sodium humate. The introduced sodium
humate could enhance water absorbency, and the content of 10 wt% sodium humate gave
the best absorption (183 g/g in distilled water and 41 g/g in 0.9 wt% NaCl solution, respec-
tively). Superabsorbent hydrogel composites made of cellulose nanofibers and chitosan‐
grafted‐poly(acrylic acid) copolymer were developed by Spagnol et al. [174]. The hydrogel
composite presented higher water absorption capability (486 g/g) than the chitosan‐grafted‐
poly(acrylic acid) hydrogel (381 g/g). The composites showed responsive behavior in rela-
tion to pH and salt solution. Such characteristics made these smart materials suitable for
several technological applications as devices for controlled release.
5.7.4 Pure Chitosan‐Based Materials

Generally, unmodified chitosan does not have enough water absorbency capacity to be used
as a superabsorbent material. However, a pure chitosan film with high water uptake perfor-
mance was fabricated without any additives, such as initiators, vinyl monomers, or cross‐
linkers, simply by dissolving chitosan in a ternary solvent system composed of acetic acid,
dioxane, and dimethyl solfoxide (DMSO), and subsequently freeze‐drying [175]. The
water uptake capacity of this chitosan film prepared under the optimal process parameters
was 896 g/g. The water absorbency of products tested between 10 and 40°C was higher
than 500 g/g, indicating excellent thermal stability.
In addition to the preceding examples of superabsorbent chitosan‐based hydrogels, some
chitosan derivatives modified with functional groups can be used as macromolecular cross‐
linkers to form hydrogels. A high‐swelling superabsorbent was synthesized with biode-
gradable N‐maleyl chitosan as a cross‐linker and AA and AM as the monomers, by means
of aqueous solution polymerization. The water absorbencies of superabsorbents under the
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optimal conditions were 1812 and 92.7 g/g in distilled water and 0.9 wt% NaCl solution,
respectively [176].
Nowadays, superabsorbent hydrogels formed by various materials and methods have
been applied in extensive technological applications, such as waste‐water treatment, per-
sonal hygiene, and agricultural and biomedical fields. However, some challenges still exist
as follows: (i) various reaction parameters have been investigated in order to form high
performance superabsorbent hydrogels. However, most studies have just drawn qualitative
conclusions, or their quantitative conclusions are not widely applicable. There is still a lack
of understanding regarding the relationship between the network structure and properties
of superabsorbent hydrogels; (ii) hydrogels with superabsorbent characteristics and excel-
lent mechanical properties at the same time are difficult to achieve; (iii) most of the supera-
bsorbent hydrogels absorb much less water in salt medium than in distilled water, which
exhibits poor salt‐resistance; (iv) hydrogels prepared through graft copolymerization of
vinyl monomers show excellent water uptake capacity, but the worse biodegradation prop-
erty; and (v) lowering the final cost production remains as a challenge for applications of
chitosan‐based superabsorbent hydrogels, because chitosan raw materials show higher cost
than other natural polysaccharides (e.g. cellulose and starch). More strategies should be
developed for the formation of environment‐friendly chitosan‐based superabsorbent poly-
mers with other functional properties, such as good mechanical strength, self‐healing, and
responsive property, which have potentials to become substitutes for synthetic commercial
superabsorbent polymers.
5.8 Outlook
Chitosan is chemically similar to glycosaminoglycans, and regarded as an excellent candi-
date matrix for hydrogels, since it is nontoxic, stable, biodegradable, biocompatible, and
can be sterilized. Moreover, the free amino and hydroxyl groups in the polymer chains
endow chitosan with covalent binding sites for versatile small molecules and macromole-
cules. Two completely opposite solvent systems in nature, acidic solution and alkali/urea
solvent, have been utilized to prepare chitosan‐based hydrogels with unique structures and
enhanced mechanical performance. Based on their intrinsic pH‐sensitivity, chitosan‐based
hydrogels that exhibit multifunctional properties, such as injectability, self‐healing, shape
memory, and superabsorbent performance, have been widely synthesized and discussed,
showing great potential in various fields, especially involving biomedical applications.
Further progresses are needed to obtain chitosan‐based hydrogels with other excellent
functions such as being conductive, stimuli‐responsive, luminescent, ultratough, and highly
stretchable.
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6
Beneficial Health Effects of Chitin
and Chitosan
Liyou Dong1,2, Harry J. Wichers1,2, and Coen Govers1
1
Food & Health Research, Wageningen Food & Biobased Research, Wageningen,
The Netherlands
2
Food Chemistry, Wageningen University, Wageningen, The Netherlands
Chitin and chitosan have been recognised for their beneficial health effects since the 1980s
[1]. Over the past few decades, numerous studies and several clinical trials have been per-
formed which demonstrated that these compounds can reduce body weight and cardiovas-
cular disease (CVD), improve wound healing, but can also modulate the immune system
and demonstrate antifungal and antibacterial activity. In particular, weight reduction and
improvement of cardiovascular status are interesting targets as the prevalence of obesity
and CVD are increasing. Both diseases are associated with various pathological disorders,
including diabetes and hypertension and put a strain on healthcare costs and capacity. In
general, lifestyle‐based interventions such as the oral intake of chitin and/or chitosan are
becoming increasingly popular as these are easily integrated into current treatments and
improve the self‐assertiveness of patients. Of interest, but beyond the scope of this chapter,
is the use of chitin‐glucans as supplements in cosmetics [2, 3]. Several studies have dem-
onstrated that chitin‐glucan reduced wrinkling and skin-ageing, suggesting an interaction
between the chitin‐glucan and cells of the epidermis. Although these effects may not be
attributed to chitin alone, this does demonstrate the strong biological potential of chitin on
cells of the human body. In this chapter, scientific literature has been reviewed that demon-
strated the beneficial health effects of chitin and chitosan from an immunomodulatory
point-of-view. First, we provide an overview of in vitro studies that offer in‐depth mecha-
nistic insights, followed by describing preclinical animal studies. Finally, we list various

human intervention trials that most clearly demonstrate the beneficial health effects of
chitin and chitosan. Furthermore, we purposely discriminate between data on chitin and
chitosan as they are chemically distinct and therefore possibly demonstrate unique effects
on health.
6.1 Immunomodulatory Effects of Chitin and Chitosan

as Demonstrated with In Vitro Studies
Chitin and chitosan are not endogenous to mammals. In contrast, they can be found in vari-
ous pathogens, such as Candida albicans, Brugia malayi and Plasmodium [4–6], and hence
may function as pathogen‐associated molecular patterns (PAMPs). The immune system
has developed specific mechanisms and receptors, that is, pattern recognition receptors
(PRRs) which are recognised and activated by such PAMPs. A number of PRRs have been
identified that bind to chitin and chitosan such as FIBCD1, NKR‐P1, RegIIIγ, galactin‐3,
mannose receptor, dectin‐1 and various toll‐like receptors (TLRs) which are listed in
Box 6.1 [7].
The recognition and binding of chitin and chitosan by cell surface receptors results in
downstream signalling, transcription factor activation, gene transcription and finally
cellular activation. Cellular activation encompasses a number of biological responses, of
which the production and secretion of signalling molecules such as cytokines and
chemokines are often investigated. These molecules can influence the direct environment
and induce, for instance, an inflammatory condition in response to pathogenic recognition.
Most studies that investigate immune responses upon recognition of chitin and chitosan
have used innate‐type immune cells and analysed cytokine secretion.
Although epithelial cells are not officially classified as immune cells, they are the first
cells to come into contact with foreign material such as chitin and chitosan and therefore
are the first cells to initiate cellular responses. In relation to a fungal lung inflammation in
mice, the responses of lung epithelial cells to chitin was studied [19]. This demonstrated
that the epithelial cells respond to chitin with the secretion of cytokines and chemokines
such as interleukin‐25 (IL‐25), IL‐33 and thymic stromal lymphopoietin (TSLP). These
signalling molecules in turn activated lung‐resident immune cells (i.e. innate lymphoid
type 2 cells) to secrete IL‐5 and IL‐13, which induced the migration of other immune cells
(i.e. eosinophils and macrophages) to the site of chitin contact. The influx of eosinophils
was corroborated in another lung infection model where increased exposure to chitin
particles resulted in the expression of eosinophil chemoattractant CCL11 and in addition
revealed a Th2 shift in T cell activation [20]. In another study focussing on fungal lung
infections, chitin was also shown to induce Th2 skewing with increased production of IL‐5,
IL‐13 and CCL5 [21]. The strength of Th2 responses were linked to Chit1chitinase activity,
but not to AMCase, suggesting that smaller chitin fragments have an increased stimulatory
effect. In addition, although dendritic cells were demonstrated to play an important role in
Th2 skewing, the initiating signal appears to originate from epithelial cells in the form of
TSLP production.
The epithelial cell layer in our body is also lined with immune cells for protection. A
typical innate‐type immune cell is the macrophage which expresses a number of receptors
that can directly bind to chitin (see Box 6.1). Chitin induced secretion of the anti‐
inflammatory cytokine IL‐10 by macrophages. In contrast, chitin also induced the secretion
Beneficial Health Effects of Chitin and Chitosan 147
Box 6.1 Receptors that have been demonstrated to bind to chitin or chitosan.
TLRs: Toll‐like receptors are expressed on the surface of multiple cells, including that of
immune cells such as macrophages and dendritic cells. TLR2 and TLR4 are shown to bind to
chitin or chitosan, and subsequently activate an innate immune response [8, 9]. In addition to
TLRs expressed on the cell surface, TLR9, which is expressed in the endosomal compart-
ments, was identified to be involved in chitin and chitosan‐mediated production of the anti‐
inflammatory cytokine IL‐10 [10].
Dectin‐1: This is a member of the C‐type lectin receptors that is mainly involved in the
recognition of fungal pathogens. It is also important for the immune response towards bacteria,
viruses and parasites [11, 12] and reported to be able to recognise chitin.
Mannose receptor (MR): MR is an endocytic receptor that also belongs to the C‐type lectin
family. It is predominately expressed on the surface of macrophages and dendritic cells. Chitin
can bind to the mannose receptor and activate the phagocytic process of immune cells [13].
NKR‐P1: This again is a C‐type lectin receptor that is primarily expressed on natural killer
(NK) cells and T cells. This receptor was shown to possess a chitin oligomer binding domain
which leads to the activation of NK cells [14].
FIBCD1: This is a transmembrane endocytic receptor that is expressed primarily by intesti-
nal epithelial cells. It serves as an acetyl binding receptor to recognise chitin [15].
RegIIIγ: This is a secreted C‐type lectin receptor which is expressed by intestinal epithelial
cells. It can recognise N‐acetyl‐glucosamine oligomers [16].
Galectin‐3: This is a member of the galectin family that is abundantly expressed on mac-
rophages. This receptor was shown to have an N‐acetyl‐glucosamine binding domain [17].
NOD2: NOD2 is an intracellular chitin recognition receptor which is primarily expressed
by macrophages and epithelial cells. It was reported to mediate chitin‐induced IL‐10
production [18].
Abbreviations: FIBCD1: Fibrinogen C Domain Containing 1; NKR‐P1: Killer cell lectin‐
like receptor subfamily B, member 1; RegIIIγ: regenerating islet‐derived protein 3 gamma;
TLR: toll‐like receptor; NOD2: nucleotide‐binding oligomerization domain‐like receptors.
of the pro‐inflammatory cytokines IL‐6 and TNFα by peripheral blood mononuclear cells
(PBMCs) which contain a large variety of immune cells dominated by lymphocytes (i.e. T
and B cells) [10]. A similar chitin preparation with a size of 1–10 μm was shown to induce
IFNγ, IL‐12 and TNFα secretion by an immune cell mixture isolated from the spleen [22].
Despite the differences in immune cell responses to chitin, both studies demonstrated that
the responses were dependent on chitin interaction with the mannose receptor, NOD2 and
TLR9, and also that immune responses can be chitin‐concentration‐dependent. Low
concentrations of chitin induced secretion of low levels of TNFα, whereas this exponentially
increased upon stimulation with higher concentrations of chitin. In contrast, all chitin
concentrations induced similar concentrations of IL‐10.
Next to concentration, the size of chitin too was demonstrated to affect immune
responses. Upon exposure to chitin fractions of <40 μm, 40–70 μm or 70–100 μm,
macrophages showed different responses in cytokine production, receptor binding and
downstream signalling pathway activation [23]. The smallest fraction induced both TNFα
and IL‐10 secretion, whereas the intermediate‐sized chitin induced only TNFα secretion
and the large particles did not induce any cytokine production. The varying dependency of
cytokine production on receptor binding adds to the complexity of immune responses and
downstream signalling. TNFα secretion in response to small‐sized chitin proved to be
dependent on dectin‐1 recognition with optional TLR2 and mannose receptor binding, and
relied on downstream NF‐κB and partial Syk signalling. In contrast, TNFα secretion in
response to intermediate‐sized chitin was dectin‐1 and NF‐κB dependent, but also fully
TLR2 dependent and mannose receptor independent. The intermediate‐sized chitin
fragments were also subject to a more in‐depth study which demonstrated that these also
induced IL‐17, IL‐12 and IL‐23 production in a TLR2‐ and MyD88‐dependent manner
[24]. Interestingly, IL‐12, IL‐23 and TNFα production all depended on the initial IL‐17
production. Furthermore, intermediate‐sized chitin also induced, independently of IL‐17,
CCL5 and CXCL2.
Similar to chitin, chitosan has also been shown to exert pro‐ and anti‐inflammatory
effects on immune cells. Chitosan oligomers with a limited polymerisation degree of 3–8
units were shown to activate macrophages [9]. This particular macrophage cell‐line (RAW
264.7 macrophages) demonstrated increased proliferation and production of pro‐inflam-
matory mediators TNFα and nitric oxide. TLR4 was shown to be important for these
responses as an anti‐TLR4 antibody appeared to block the direct interaction between chi-
tosan and macrophages and nitric oxide production. Small fragments of chitosan were
also shown to be most potent in enhancing intra‐epithelial lymphocyte (IEL) cytotoxicity
against YAC‐1 cells ex vivo [25]. As YAC‐1 cells are particularly sensitive to NK cell–
mediated cytotoxicity, these results suggest that chitosan specifically enhanced NK cell
activity in the IEL population. In addition to the direct immunomodulatory effects of
chitosan, a number of studies investigated the modulatory effects of chitosan upon pri-
mary lipopolysaccharides (LPS)‐stimulation. LPS‐stimulated macrophages demonstrated
an increase in IL‐1β production when co‐stimulated with chitosan, but not chitin [26].
This increase in IL‐1β production is both concentration‐ and size‐dependent, with higher‐
concentration and smaller‐sized chitosan inducing a more potent IL‐1β production.
Indeed, chitosan <20 μm proved most potent and >100 μm almost functionally inert,
which is reminiscent of other reported chitin‐size‐dependent effects. In this study, the
authors also demonstrate that chitosan depends on the NLRP3 inflammasome for intracel-
lular signalling.
In a similar study, treatment of LPS‐stimulated macrophages with chitosan reduced the
TNFα, IL‐6 and NO production [27]. Unfortunately, it is unclear whether these experiments
were performed with similar small‐sized chitosan. Another study, in which the production
of pro‐inflammatory mediators following LPS‐stimulation of macrophages was again
measured, demonstrated that chitosan negatively affected the production of nitric oxide,
PGE2, TNFα, IL‐6 and also IL‐1β [28]. Here, the authors again investigated the effect of
chitosan size and demonstrated that the inhibiting effects on these mediators were stronger
for smaller particles. Still, a direct comparison between studies remains difficult as the size
of chitosan in this study is not described in degrees of polymerisation or μm but in kDa.
Also, the authors demonstrated that the inhibiting effects of chitosan are mediated by
reducing intracellular MAP‐kinase activation. Interestingly, Li and colleagues revealed that
LPS‐mediated cytokine production is regulated by the transcription factor NF‐κB, which in
turn is activated by MAP‐kinases [29]. Therefore, it appears that chitosan is inhibiting
LPS‐mediated pro‐inflammatory macrophage responses by reducing intracellular MAP‐
kinase activation, potentially via activation of TLR4.
Initial insights in cellular responses towards chitin and chitosan were acquired using in
vitro studies and some ex vivo studies. These studies have demonstrated that there are a
number of receptors that can recognise chitin and chitosan, which leads to modulating
responses of a number of cell types. It is important to note that the immunomodulatory
effects of chitin and chitosan are highly concentration‐ and size‐dependent. The variation
of chitin and chitosan concentration and size leads to different immune responses. In in
vitro studies, chitin concentration has a positive correlation with its immunomodulatory
activity. In contrast, chitin or chitosan size was inversely linked with its immune activity,
and particles with a size bigger than 100 μm were considered as immunomodulatory inert.
These in vitro studies were in part related to the analysis of in vivo effects of chitin or
chitosan towards fungal lung infection and tumour growth. The in vitro results corroborated
the in vivo data which was encouraging for the potential of translational research to estimate
the potential health effects of chitin and chitosan, which is described in more detail in the
following paragraphs. However, it should be noted that, in in vitro studies, LPS contamination
is commonly present and can strongly interfere with proper interpretation of the
immunomodulatory effects of chitin or chitosan. The chitosan studies especially
demonstrate clearly that the presence of LPS can strongly affect the net result of immune
responses. Nevertheless, many studies did not determine the LPS content during the
preparation of chitin or chitosan products [24, 30]. The presence of LPS in chitin or chitosan
samples does not interfere with readouts when using in vivo models, which is another
advantage beyond testing the full complexity of the body. Therefore, we will focus on the
beneficial health effects and validation of immunomodulatory effects of chitin or chitosan
in in vivo studies following oral intake while also focussing on the used particle sizes and
doses.
6.2 Beneficial Health Effects Mediated by Chitin and Chitosan

as Demonstrated with Animal Studies
The next step in the analysis of immunomodulatory effects of chitin and chitosan is to
translate and validate in vitro findings to animal studies. In the following text, we have
summarised animal studies in which chitin and chitosan demonstrated beneficial health
effects. We have divided the various studies into their reported immune modulation
(Table 6.1), anti‐pathogenic (Table 6.2) and anti‐tumour effects (Table 6.3).
6.2.1 Immune Modulation

Cellular studies on the modulation of inflammation by chitin and chitosan have been
discussed in the preceding section. The described inflammation modulatory properties are
essential for the prevention or treatment of some inflammation‐related diseases. A mouse
model of ragweed allergy nicely demonstrates the potential of chitin to modulate immune
responses [31]. In this model, the mice undergo an 11‐day treatment regime to induce a
Th2‐dominated allergic response. Mice are sensitised to ragweed by two intraperitoneal
(i.p.) injections containing alum as adjuvant and a subsequent intratracheal challenge with
ragweed, and then assessed for allergic parameters on day 13. Starting 3 days before
initiating treatments and during the 13 days of the trial, an oral intervention is performed
with saline solution as control or small particulate chitin as treatment (1–10 μm diameter),
Table 6.1 Immune‐modulatory effects of chitin and chitosan.
No. of subjects
(experimental arm/control);
Products Origin Size/MW DD Product administration subject status Summary of effects References
Chitin microparticles n.d. 1–10 μm n.d. 6.5 mg/day for 16 days; 6 or 7/6 or 7; allergic mice ↓IL‐4, IL‐5, IL‐10 (p<0.05); [31]
oral administration IgE levels (p<0.0005)
↑IFNγ (p<0.05); IgG2a levels
(p<0.05)
LM‐chitosan n.d. <1 kDa 98.5% 16.5 mg/kg twice a day for 14 10–16/10–16; asthmatic ↓TNFα, IL‐4, IL‐5, IL‐13 [32]
days; oral administration mice (p<0.001)
Chitin microparticles n.d. 1000 kDa n.d. 0.2% wt chitin contained diet for 8/8; peanut allergic mice ↓IL‐5, IL‐10, IL‐13 (p<0.05) [33]
6 weeks; oral administration
Chitosan microparticles n.d. 70 kDa 91% 0.2% wt chitin contained diet for 8/8; peanut allergic mice ↓IL‐5, IL‐10, IL‐13 (p<0.05)
6 weeks; oral administration ↓serum IgE (p<0.05) [33]
Chitin nanofibrils Crab 200 μm n.d. 1% (w/v) chitin for 7 days; oral 5/5; IBD mice ↓NF‐κB staining area (p<0.05) [34]
administration ↓MCP‐1 (p<0.05)
Abbreviations: DD: degree of de‐acetylation; IBD: inflammatory bowel disease; IFN: interferon; Ig: immunoglobulin; IL: interleukin; LM: low molecular; MCP‐1: monocyte
chemotactic protein‐1; MW, molecular weight; NF‐κB: nuclear factor kappa light chain enhancer of activated B cells; n.d.: not described; NK, natural killer.
0004461244.INDD 150 10/26/2019 11:43:27 AM

Table 6.2 Antipathogenic effects of chitin and chitosan.
No. of subjects
Products Origin Size/MW DD Product administration subjects status Summary of effects References
Chitin n.d. n.d. n.d. 50 mg/kg per day for 6 18 (i.p.) or 20 (i.v.)/18 or ↑Serum polymorphonuclear [38]
days at a 2‐day 20; C. albicans infected leucocytes (p<0.01)
interval; intravenous mice ↑Survival time
or intraperitoneal
administration
Chitosan n.d. n.d. n.d. 50 mg/kg per day for 6 18 (i.p.) or 20 (i.v.)/18 or ↑Active oxygen generating cells [38]
days at a 2‐day 20; C. albicans infected (p<0.01)
interval; intravenous mice ↑Survival time
or intraperitoneal
administration
Chitosan oligosaccharides Crab 208 kDa 70–100% 0.5 or 1 mg per day for 5/5; S. aureus infected mice ↑The number of monocytes (p<0.05) [39]
5 and 10 days; ↑IFNγ, IL‐6 (p<0.01)
intraperitoneal ↑Survival time
injection
0.5, 1, 2 mg per day
for 7 days; oral
administration
Chitin Crab n.d. n.d. 4 μg/g or 6 μg/g for 10/10; V. alginolyticus ↑THC, respiratory burst (p<0.05) [40]
once; injection infected shrimp ↑Phagocytosis ability
↑Survival time
Chitosan Crab n.d. n.d. 2 μg/g or 4 μg/g for 10/10; V. alginolyticus ↑THC, respiratory burst (p<0.05) [40]
once; injection infected shrimp ↑Phagocytosis ability
↑Survival time
Chitin Prawn n.d. 26.7% 1% (wt) chitin 50/50; A. invadans infected ↑WBC, RBC, haematocrit, [41]
contained diet for 15 fish lymphocytes, monocytes,
days; oral neutrophils (p<0.05)
administration ↑Phagocytosis activity
↓Mortality
(Continued)
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No. of subjects
Products Origin Size/MW DD Product administration subjects status Summary of effects References
Chitosan Prawn n.d. 58.5% 1% (wt) chitin 50/50; A. invadans infected ↑WBC, RBC, haematocrit, [41]
contained diet for 15 fish lymphocytes, monocytes,
days; oral neutrophils (p<0.05)
administration ↑Phagocytosis activity,
↓Mortality
Chitin microparticles n.d. <40 μm n.d. 100 μg/μL for 2 weeks 5/5; L. major infected mice ↓Parasitic load (p<0.05) [42]
at a 2‐day interval; ↓Lesion formation (p=0.021)
injected at the ↑TNFα, IL‐10 (p<0.001)
infected site
Chitosan microparticles n.d. <40 μm n.d. 100 μg/μL for 2 weeks 5/5; L. major infected mice ↓Parasitic load (p< 0.05) [42]
at a 2‐day interval; ↓Lesion formation (p=0.039)
injected at the ↑TNFα, IL‐10 (n.s.)
infected site
Chitin microparticles n.d. <44 μm n.d. 100 μg/μL for 2 weeks 6/6; L. major infected mice ↓Lesion formation (p=0.023) [43]
at a 2‐day interval; ↓TNFα (p=0.026)
injected at the ↑IFNγ/IL‐5 (p=0.023)
infected site ↑IL‐10
Chitosan n.d. n.d. n.d. 250 mg/kg once a day 8/8; E. papillata infected ↓The number of parasites in the [45]
for 5 days; oral mice developmental stage (p<0.01)
administration ↓oocyst in faeces (p<0.01)
↓NO production; TNFα, TGFβ gene
expression (p<0.001)
↑IL‐4, IL‐10 gene expression
(p<0.001)
Chitosan particles n.d. n.d. 88.5% 500 μg once a week 10/10; H. nana infected ↓Gene expression of iNOS, IFNα, [46]
for 4 weeks; oral mice IFNγ, IL‐9 and TNFα (p< 0.001)
administration ↑The number of mucosal mast cells;
gene expression of IL‐4 (p<0.001)
Abbreviations: DD: degree of de‐acetylation; IFN: interferon; IL: interleukin; iNOS: inducible nitric oxide synthase; i.p.: intraperitoneal injection; i.v.: intravenous injection; MW: molecular
weight; NF‐κB: nuclear factor kappa light chain enhancer of activated B cells; n.d.: not described; NK: natural killer; NO: nitric oxide; n.s.: not significant; RBC: red blood cells; THC:
haemocyte count; TNF: tumour necrosis factor; TGF: transforming growth factor; WBC: white blood cells.
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on average 6.5 micrograms per mouse per day. The preventive treatment with chitin signifi-
cantly (p<0.0005) reduced the serum IgE levels and ragweed‐specific IgE levels. In con-
trast, the ragweed‐specific IgG2a levels are significantly (p<0.05) increased. By using
bronchoalveolar lavage (BAL) to harvest cells and cytokines from the lung, it became
evident that oral chitin intervention significantly reduced (p<0.05) the migration of eosino-
phils and lymphocytes to the lung and drastically (p<0.05) altered cytokine profiles from
an IL‐4‐ to an IFNγ‐dominated type. Strikingly, not only did a 13‐day preventive adminis-
tration of chitin drastically alter a Th2‐dominated immune response to a Th1‐dominated
type, but also a curative setting in which chitin was provided on days 12 and 13 signifi-
cantly (p<0.01) decreased total serum IgE levels and immune cell lung infiltrates (p<0.05).
A similar curative allergy model was used to study the immunomodulatory effects of low
molecular (LM) chitosan [32]. In this study, mice were sensitised to ovalbumin (OVA) with
a 12‐day protocol using three i.p. injections with alum as adjuvant, after which allergic
responses were induced by intranasal administration of OVA. The intranasal administration
was performed from day 13 to 27 and coincided with daily oral administration of LM‐chitosan
(16.5 mg/kg) or saline solution as control. Analysis of the allergic responses were focussed
on cytokine production. BAL fluids of mice treated with LM‐chitosan demonstrated a
significant (p<0.001) reduction in IL‐4, IL‐13 and TNFα levels. Furthermore, lung tissue
also revealed a significant reduction of mRNA levels of IL‐4, IL‐5, IL‐13 and TNFα.
Interestingly, no change in IFNγ mRNA levels were observed. In comparison to the previ-
ous study, no chitosan‐mediated skewing of the immune response was observed (i.e. Th2
towards Th1), but LM‐chitosan did induce a strong reduction in allergic responses.
Chitosan and chitin were directly compared as preventive treatment in a peanut allergy
model [33]. This model is, in essence, similar to the earlier described models, but with a
different timeline and location of sensitisation and challenge. The mice were provided with
α‐chitin, β‐chitin or β‐chitosan mixed in food pellets at a concentration of 0.2% (w/w). The
interventions resulted in anaphylaxis reduction when the sensitised mice were challenged
with peanut. To investigate whether the chitin or chitosan indeed affected immune cell
responses, splenocytes were isolated and ex vivo stimulated with peanut extracts. This
revealed that splenocytes from mice treated with α‐chitin, β‐chitin or β‐chitosan were less
sensitive to the peanut extract and produced significantly (p<0.05) less IL‐5, IL‐13 and
IL‐10. Authors also investigated serum peanut–specific IgE levels which were lowered by
α‐chitin and β‐chitosan, but not β‐chitin. Within the group of β‐chitin‐treated mice, there
was a strong division in mice with low and high IgE levels, indicating that β‐chitin has the
potency to lower allergic responses, but is dependent on a parameter beyond the scope of
this study.
Instead of redirecting immune responses towards a Th1 type response, chitin has also
demonstrated the potency to reduce the strength of immune responses. To investigate the
potential of chitin to reduce the inflammation in inflammatory bowel disease (IBD), a
mouse model was used in which ulcerative colitis was induced using dextran sulphate
sodium (DSS) [34]. Regular chitin with an average diameter of 200 μm or specifically
generated chitin nanofibrils with average lengths of 4 nm at a concentration of 0.1% (w/v)
in tap water was provided as a 7‐day intervention before the DSS‐mediated induction of
ulcerative colitis. Analysis of colonic epithelium demonstrated a significant (p<0.05)
reduction in activation of the NF‐κB transcription factor, and serum analysis revealed sig-
nificantly (p<0.05) reduced levels of monocyte chemotactic protein‐1 (MCP‐1), which is
Table 6.3 Anti‐tumour effects of chitin and chitosan.
No. of subjects
(experimental arm/
Products Origin Size/MW DD Product administration control); subjects status Summary of effects References
Chitin and chitosan Crab n.d. n.d. 1, 2, 4% (wt) chitin or 8/8; Colon‐26 tumour‐ ↓Tumour weight (p<0.05) [47]
oligomers chitosan contained diet bearing mice ↑IFNγ, IL‐12p70 (p<0.01)
for 28 days; oral
administration
LM‐chitosan n.d. 21, 46 and 130 kDa n.d. 100 mg/kg or 300 mg/kg 10/10; Sarcoma 180‐ ↓Tumour weight and growth [25]
twice daily for 20 days; bearing mice (p<0.05)
oral administration ↑NK cells activity
LM‐chitosan Shrimp 30 kDa 95% 100 mg/kg or 200 mg/kg per 10/10; Liver tumour ↓Tumour weight (100 mg/kg) [48]
day for 14 days; oral H‐22 bearing mice (p<0.05)
administration ↓Tumour weight (200 mg/kg)
(p<0.01)
↑Leucocytes, monocytes,
neutrophils and lymphocytes
(p<0.05)
↑The number of CD3/CD4 T‐
cells, NK cells (p<0.05)
↑TNFα, IL‐12 (p<0.05)
Abbreviations: CD3: cluster of differentiation 3; CD4: cluster of differentiation 4; DD: degree of de‐acetylation; IFN: interferon; IL: interleukin; MW, molecular weight; n.d.: not
described; NK, natural killer; TNF: tumour necrosis factor.
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the key chemokine for the migration and infiltration of monocytes and macrophages. These
effects, however, were only observed following preventive treatment with chitin nanofibrils
and not with regular chitin. Based on the knowledge gained from in vitro analysis, it appears
that the chitin length in in vivo situations as well might be a crucial parameter to reduce
intestinal inflammation.
Chitosan and, in particular, chitin have long been associated with allergenic response.
Recent studies indicate that these correlations are mainly attributable to various proteins
[35], and clinical trials have demonstrated the safety of using chitosan in bandages [36].
The general observation that chitin and chitosan prevent allergenic responses in the lung
and shift Th2 responses towards Th1 substantiates this. In addition, chitin also demonstrated
lowering of intestinal immune responses. Both applications could potentially improve the
quality of life for many patients.
6.2.2 Anti‐Pathogenic Effects

Although chitin and chitosan have already been used as antimicrobial agents for decades,
the underlying mechanism remains unclear, but it has been proposed to involve cell lysis,
penetration of cytoplasmic membranes and chelation of cations [37]. These mechanisms
might also result in fungicidal and bactericidal effects. Furthermore, chitin and chitosan
also appear to possess anti‐parasitic activity, which can be correlated to improved mac-
rophage phagocytosis capacity as demonstrated in in vitro experiments and overall improve
innate and adaptive immune responses.
A study performed already over three decades ago demonstrated the potential of chitin
and chitosan to reduce infections in mice with the yeast Candida albicans [38]. In this
study, mice were pretreated with i.p. injections of chitin or chitosan 6 days, 4 days and 2
days before i.p. C. albicans injections. The pretreatment with chitin, but even more so with
chitosan, increased the survival rates of mice. These beneficial effects coincided with
increased levels of polymorphonuclear leucocytes in blood (chitin) and peritoneal immune
cells (chitosan). These immune isolates from chitin or chitosan treated mice demonstrated
to possess significantly increased reactive oxygen species production and candidacidal
activity. Similar results were obtained when chitosan was orally provided in a preventive
setting to treat Staphylococcus aureus infections [39]. After a 7‐day pretreatment period, S.
aureus was injected i.p. and the survival of mice was monitored over a 6‐day period. With
the preventive strategy, the survival of mice increased from 10% to 70–100% depending on
the chitosan concentration.
This preventive strategy also proved to be efficient when treating shrimp or fish with
microbial infections. Shrimp were treated with an injection of chitin or chitosan and chal-
lenged the next day with a Vibrio alginolyticus injection [40]. The pretreatment signifi-
cantly (p<0.05) enhanced shrimp survival during the 6‐day follow‐up. In addition, the
immediate and significant (p<0.05) increase of leucocyte responses such as respiratory
burst activity and phagocytic activity proved to be transient in nature and subsided after 4
days. Of note, again chitosan appeared to be more potent regarding the antimicrobial and
immunestimulation than chitin. Also, a similar increase in leucocyte activity was observed
when carp‐derived leucocytes were directly ex vivo incubated with small chitin particles
and demonstrated increased phagocytic activity [41]. This increase in phagocytic capacity
was unfortunately not measured when carp were provided with diets enriched in chitosan
or chitin (1% (w/w)) to treat Aphanomyces invadans infection. Interestingly, in this study,
pretreatment and continued additions of either chitin or chitosan reduced mortality in fish
from 70% to almost the levels of uninfected fish 4 weeks post‐infection. Similar as for
shrimp, chitosan was slightly more effective in reducing the mortality in fish which was
accompanied by an increased level of red and white blood cell counts, among which were
monocytes, lymphocytes and neutrophils, which was already apparent at the first moment
of measuring 1 week after infection.
Studies to investigate anti‐parasitic activity of chitin and chitosan were mostly performed
in a curative setting. In a model to test the effects against Leishmania major infections,
mice were subcutaneously injected with the parasite and treated with local injections of
small‐sized chitin or chitosan (<40 μm) for 2 weeks with 2‐day intervals [42]. The onset of
lesions in the treated groups was delayed and ulcer diameter of the lesions was significantly
smaller (p<0.05). Twelve weeks after infection, the mice were sacrificed and analysis
revealed a significantly (p<0.05) reduced parasitic load in the draining lymph nodes. In
relation to reducing infections, the authors investigated the potential of chitin and chitosan
to induce production of cytokines by lymph node‐derived immune cells. Only chitin
incubation resulted in a significant (p<0.001) increase in both IL‐10 and TNFα production.
Although the production of these cytokines appears contradictive, an activation of Th1
response which is stimulated by TNFα enhanced macrophage activity and increased IL‐10
production critically limited the harmful side effects of inflammatory responses. In a
previous L. major infection mice trial performed by the same group, chitin was shown to
also increase IFNγ and reduce IL‐5 production [43]. These results were in line with an in
vitro study, also performed by this group, which showed that chitin induced the increase of
TNFα production in peritoneal macrophages after L. major infection [44]. Although these
ex vivo cytokine productions do not directly correlate to a lowered burden of infection, it
does appear to substantiate that chitin enhances the immune system’s ability to combat L.
major.
The treatment of the intestinal parasite Eimeria papillata was only tested with chitosan.
Mice were intestinally infected with E. papillata and subsequently treated with a daily dose
of chitosan for 5 days. The infectious grade was determined on day 5 by the oocyte count
in the faeces of the mice and was found to be significantly reduced (p<0.01) because of the
chitosan treatment when compared to non‐treated mice [45]. In line with this finding, the
authors observed a significantly reduced number of parasites per crypt (p<0.01) and a
reversion of the decline in goblet cells in histological sections following chitosan treatment.
Chitosan treatment also reversed other infection‐related immunological changes such as an
increase in the level of oxidative stress, neutrophil infiltration and inflammatory cytokine
gene transcription, and a decrease in anti‐inflammatory cytokine gene transcription. In
contrast, E. papillata infection also reduced the levels of T cells in the villi and IgA levels
in sera and the intestine, but treatment with chitosan did not prevent this.
Interestingly, when compared head‐to‐head based on a reduction of the burden of infec-
tion and accompanying histological and immunological changes, it appears that chitosan is
more effective in reducing the burden of microbial infection whereas chitin appears more
effective against parasites. After all these studies, it remains unclear, however, whether
these chitin‐ and chitosan‐mediated effects are instigated by potentiating the immune sys-
tem or reducing the virulence of microbes and parasites and thereby lowering immune
responses.
A single study has demonstrated effects of chitosan beyond restoring immune parame-
ters towards non‐infected controls. To treat the infection of an intestinal tapeworm
(Hymenolepis nana) mice were pretreated with a weekly dose of 500 μg chitosan for 4
weeks [46]. As observed in the earlier described studies, most parameters, histological and
immune‐related, that were affected by the H. nana infection were restored to or towards the
levels found in non‐treated mice by the pretreatment with chitosan (p<0.01). Similar to the
earlier described study, the authors also observed a reduced load in H. nana adults and
eggs, which altogether again suggests that chitosan reduces the virulence of H. nana.
However, in contrast to the earlier described study, mast cell concentrations were investi-
gated and these were not affected by the H. nana infection but significantly increased by
chitosan beyond levels found in both non‐treated and infected mice. It, therefore, appears
that chitosan pretreatment increases mast cell numbers and, potentially, their activity which
allows a quick and efficient anti‐H. nana response and thereby prevents a full‐scale immune
response resulting in histological damage and production of inflammatory cytokines.
So far, most studies using various microbes or parasites infecting either the skin or
intestine demonstrate that chitin and chitosan can indeed reduce these infections although
the mechanism behind this remains elusive. By reducing the extent of infection, chitin and
chitosan most likely also reduce the inflammatory response, which explains the lowered
cytokine levels and immune cell recruitment. Mast cells appear to play a critical role as
their presence is selectively increased by chitosan in the intestinal infection by H. nana. In
contrast, the production of IL‐5, which is related to mast cell activity in allergenic responses,
is reduced by chitin in ex vivo immune cell stimulations, which demonstrates the necessity
for further research into the anti‐parasitic potential of chitin and chitosan.
6.2.3 Anti‐Tumour Effects

The anti‐tumour effects of chitin and chitosan are strongly related to their immunomodulatory
effects. The final anti‐tumour activity is performed by cytotoxic T‐cells. The activation of
these T‐cells is mediated by antigen‐presenting cells such as macrophages and dendritic
cells, which are strongly responsive to chitin and chitosan (see Box 6.1). So far, however,
animal studies have focussed more on the effects of tumour growth and less on the
underlying immunomodulatory effects. For instance, fully or partially de‐acetylated
chitosan has been tested for its anti‐tumour property in a colon‐26 tumour‐bearing murine
model [47]. Mice were provided with a diet containing 1%, 2% and 4% (w/w) of chitosan
for 28 days before the tumour inoculation, which was continued for another 14 days
thereafter. This diet significantly (p<0.05) reduced tumour growth which correlated with
increased apoptosis in the tumour cells. The authors also observed a significant increase
(p<0.01) in serum IFNγ and IL‐12p70 levels which could have activated macrophages
leading to an anti‐tumour effect.
Similarly, low‐MW, water‐soluble chitosan has also been tested for its anti‐tumour effect
in a sarcoma 180‐bearing mice model [25]. Chitosan with various MW (21 kDa, 46 kDa,
130 kDa) was orally administered to mice twice daily at 100 mg/kg or 300 mg/kg for 20
days, starting 12 hours after the transplantation of tumour cells. The results showed that
chitosan significantly (p<0.05) decreases tumour weight and also inhibits tumour growth
in a size‐dependent manner. The lowest MW chitosan demonstrated the strongest effects
whereas the largest MW chitosan proved ineffective in reducing tumour growth. Of interest,
chitosan could not be detected in blood or spleen but was detected in the small intestine.
Coincidently, IELs from mice that were orally administered low molecular chitosan, but
again not high molecular chitosan, demonstrate significantly (p<0.05) enhanced ex vivo
cytotoxicity against YAC‐1 cells in a size‐dependent manner. Recently, a group from China
confirmed that low MW water‐soluble chitosan contains anti‐tumour activity and did so in
an H22 liver tumour mice model [48]. The chitosan was orally administrated to mice at 100
or 200 mg/kg for 14 days, starting 12 hours after the inoculation of the tumour cells. The
treatment revealed significant concentration‐dependent (p<0.05) reduction in tumour
growth and spleen weight compared to non‐treated animals. At a cellular level, the number
of leucocytes, lymphocytes, monocytes and also neutrophils were significantly (p<0.05)
increased in the chitosan intervention group in comparison to the untreated group. In par-
ticular, the CD4+ T‐cells, but not CD8+ T‐cells, and NK cell concentrations were signifi-
cantly (p<0.05) increased after chitosan intervention. Finally, also TNFα and IL‐2 serum
levels, cytokines that are critical in anti‐tumour response, were significantly (p<0.05)
increased upon chitosan treatment [48, 49].
These papers demonstrate that low MW chitosan can modulate the immune system and
induce pro‐inflammatory anti‐tumour activities in different tumour‐bearing murine models.
In these studies it is more plausible that the observed immune cell counts and activities are
a direct chitosan‐derived effect, in contrast to the potential physical interference in anti‐
pathogen studies. Therefore, low MW chitosan may be a promising adjuvant for anti‐
tumour therapies.
6.3 Beneficial Health Effects Mediated by Chitin and Chitosan

as Demonstrated with Clinical Trials
Extensive in vitro and animal studies indicate the potential beneficial health effects medi-
ated by chitin and chitosan. It is crucial to validate such observations in human clinical
trials. Part of this concerns the development of treatment regimens, as especially the dose
and duration may be completely different from that in animal studies. So far, human clinical
studies investigating the beneficial health effects of chitin and chitosan have been limited
in number and type of investigations. Studies have largely focussed on the effect of
cholesterol reduction, weight loss and glucose control.
6.3.1 Cholesterol Reduction and CVD Preventive Effects

Chitin has been investigated in clinical trials for its hypocholesterolaemic effects in a
mixture with glucan (chitin‐glucan; Table 6.4). This insoluble chitin‐glucan was tested in a
single‐blind pilot study for its effect on obesity in hypocholesterolaemic subjects. The
subjects orally ingested 4.5 g/day of chitin‐glucan before every main meal for a period of
4 weeks. After this intervention, the results showed a decrease in oxidised low‐density
lipoprotein (LDL) levels in blood, which reduces the risk for CVD [50]. Another study with
the same chitin‐glucan was performed in a fully randomised, double‐blind and placebo‐
controlled setting with a longer follow‐up and a larger study population (n=68) [51].
This phase IV clinical study revealed similar results as the pilot study and demonstrated
that chitin‐glucan significantly lowers oxidised LDL blood levels compared to the
placebo group.
Table 6.4 Clinical trials demonstrating cholesterol reduction and CVD preventive effects by chitin and chitosan.
No. of subjects (experimental arm/

Products Origin Size DD Product administration Study design placebo); subjects status Summary of effects References
Chitin‐glucan Fungi n.d. n.d. 4.5 g/day for 4 weeks; Single‐blind pilot 20/10; slightly overweight and ↓Oxidised LDL [50]
orally ingested hypercholesteraemic male ↓Oxidised glutathione
↑Anti‐oxidant enzyme
activity
Chitin‐glucan Fungi n.d. n.d. 4.5 g/day for 6 weeks; Randomised, 33/35; healthy subjects ↓Oxidised LDL [51]
orally ingested double‐blind, (p=0.035)
placebo‐controlled
Chitosan Fungi n.d. n.d. 125 mg/day for 4 A pilot study 28/n.a.; hypertriglyceridaemia ↓Total cholesterol [52]
months; orally patients (p=0.019)
ingested ↓LDL cholesterol
(p>0.05)
↓TG levels (p<0.001)
↑HDL cholesterol
(p=0.016)
↑LDL‐2 particles
(p<0.0001)
Chitosan Crab <1 kDa 90% 1 g/day for 6 weeks; Open trial 19/n.a.; healthy male smokers and ↓Total cholesterol [53]
orally ingested non‐smokers (p<0.01)
↓LDL cholesterol
(p<0.05)
Abbreviations: DD: degree of de‐acetylation; HDL: high‐density lipoprotein; LDL: low‐density lipoprotein; n.a.: not applicable; n.d.: not described; TG: triglyceride
0004461244.INDD 159 10/26/2019 11:43:27 AM

Chitosan has also been tested in clinical trials for its hypocholesterolaemic potency. A
pilot study was conducted by Rizzo and colleagues to investigate the effect of fungal chi-
tosan on regulation of plasma lipid and lipoprotein in hypertriglyceridaemia patients [52].
Subjects were orally administered 125 mg/day of chitosan tablets for a period of 4 months.
This intervention resulted in a significant decrease in total cholesterol (p=0.019) and tri-
glyceride (TG) levels (p<0.001) and a concomitant significant increase in high‐density
lipoprotein (HDL) cholesterol (p=0.016) and LDL‐2 particles (p<0.0001), a less athero-
genic LDL particle. Total LDL levels did decrease although not significantly (p>0.05). The
observed increase in HDL and lowering of LDL constitutes a beneficial change in plasma
lipid composition and lowering the risk of CVD. The potential of chitosan to reduce the
risk of CVD has also been tested in smokers who are at higher risk of developing this dis-
ease [53]. The study setup included a higher dose of chitosan (1 g/day) for a shorter period
(6 weeks), which also resulted in a significant decrease in total cholesterol (p<0.01) and
LDL cholesterol (p<0.05) levels.
These results for chitosan have led to a European Food Safety Authority (EFSA) claim
on cholesterol lowering, and chitosan is available as a commercial product. So far, how-
ever, the mechanism behind chitosan’s effect remains to be elucidated. Chitosan might bind
bile acids in the intestine, preventing their reuptake and forcing enhanced cholesterol
metabolism to generate bile acids [54]. Other potential mechanisms are the increased
expression of LDL receptors in the liver, which would result in enhanced uptake and break-
down of serum LDL or the inhibition of pancreatic lipase activity to reduce the digestion
and uptake of dietary fats [55]. Depending on the mechanism, and whether the acetylation
level is involved in this, a similar EFSA claim might be possible for chitin, which clearly
demonstrated its potency in clinical trials.
6.3.2 Other Health Effects

Next to reducing CVD risk, chitosan was also tested for its potential to address other ill-
nesses such as reducing oxidative stress (Table 6.5). Here, water‐soluble chitosan with a
MW of 20 kDa was administered to young and healthy subjects at a daily dose of 540 mg
for 4 weeks in an open‐label study [56]. In line with the studies described earlier, again a
significant increase in HDL (p<0.05) and (non‐significant) reduction in LDL levels were
observed along with a significant decrease in the atherogenic index (p<0.05; ratio between
triglycerides and HDL‐C levels). Oxidative stress levels in blood were determined by
measuring the oxidised albumin ratio and total plasma antioxidant capacity. Both param-
eters were significantly reduced (p<0.05), which suggested that chitosan has antioxidant
properties and reduces oxidant stress. Perhaps unexpectedly, the authors also observed a
significant (p<0.05) reduction in blood glucose levels following chitosan intervention.
This finding has been the primary read‐out parameter of another clinical trial that focussed
on the effect of chitosan on glucose control [57]. In this randomised, double‐blind,
placebo‐controlled study, pre‐diabetic patients were administered a daily dose of 1.5 g
chitosan for 12 weeks, which is a considerably higher dose for a longer period compared
to the previous study. Again, a significant lowering of serum glucose levels (p=0.03) was
observed. Also, the glycated haemoglobin levels (i.e. HbA1c) that are indicative of blood
sugar levels over a prolonged period were significantly (p=0.021) reduced in the chitosan
treated patients when compared to the placebo group. Surprisingly, no change in blood
Table 6.5 Clinical trials demonstrating beneficial health effects of chitin and chitosan.
No. of subjects
(experimental arm/
Products Origin MW DD Product administration Study design placebo); subject status Summary of effects References
WSC n.d. 20 kDa 95% 540 mg/day for 4 weeks; Open label 10/n.a.; healthy subjects ↓Plasma glucose (p<0.05) [56]
orally ingested ↓Atherogenic index (p<0.05)
↑HDL cholesterol (p<0.05)
Chitosan n.d. n.d. n.d. 1.5 g/day for 12 weeks; Randomised, 25/26; pre‐diabetic ↓Serum glucose control [57]
orally ingested double‐blind, participants (p=0.03);
placebo‐controlled ↓Plasma level of
haemoglobin level
(p=0.023)
↑Plasma adiponectin
(p=0.013)
Chitosan n.d. n.d. n.d. 2.25 g/day for 3 months; Randomised, 6/6; obese subjects ↓Body weight (p=0.027) [58]
orally ingested double‐blind ↓TG level (p=0.028)
Chitosan Fungi n.d. n.d. 2.5 g/day for 90 days; Single‐blind, 64/32; obese subjects ↓Body weight (p<0.0001) [59]
orally ingested placebo‐controlled, ↓HbA1c level (p=0.0343)
randomised
Water‐soluble Crab 3–5 kDa 70% 37.5 or 150 mg/day for Randomised, 12/n.a.; healthy subjects ↓Dental plaque formation (S. [62]
chitosan 6 weeks; rinsed with double‐blind, mutans) (p=0.014)
mouth rinse solution placebo‐controlled
LMW‐WSC n.d. 5 kDa n.d. 20 mg chitosan solution Randomised, 40/n.a.; denture ↓Formation of erythematous [61]
four times a day for 2 single‐blind stomatitis patients surface area (p<0.001)
weeks; immerse in ↓Number of blastopores and
chitosan solution mycelia of C. albicans
(n.s.)
Chitosan n.d. n.d. n.d. 2% w/v solubility in Randomised, 50/n.a.; healthy subjects ↓Amount of bacteria [63]
saliva; chewed eight double‐blind, (Streptococci) (p<0.05)
gums in total placebo‐controlled
Chitin Prawn 1–20 μm n.d. 2 mg in total; intranasal Randomised, 14/n.a.; healthy ↓IL‐4; IL‐6 [64]
administration double‐blind, participants ↑Leukotriene B4
placebo‐controlled
Abbreviations: DD: degree of de‐acetylation; HbA1c: haemoglobin A1c; HDL: high‐density lipoprotein; IL: interleukin; LDL: low‐density lipoprotein; LMW‐WSC: low‐molecular‐
weight water‐soluble chitosan; MW: molecular weight; n.a.: not applicable; n.d.: not described; n.s.: not significant; TG: triglyceride.
0004461244.INDD 161 10/26/2019 11:43:27 AM

insulin levels were observed, which might have been expected with lowered blood glucose
levels. Another study also designed as randomised, double‐blind and placebo‐controlled
analysed insulin sensitivity, rather than insulin levels, in obese subjects following chitosan
intervention [58]. Subjects orally ingested 2.25 g of chitosan (750 mg before every main
meal) for a period of 3 months, which are conditions similar to the previous study, which
did not yield a change in blood insulin level. As hypothesised by the authors, chitosan
significantly increased insulin sensitivity (p=0.43). At the same time, subjects also lost a
significant amount of body weight through the intake of chitosan (p=0.027), which was
accompanied by a significant decrease in body mass index (BMI) (p=0.028) and waist
circumference (p=0.028). These changes might relate again to the reduction in uptake of
cholesterol and bile acids as described earlier since triglycerides levels were found to be
significantly reduced (p=0.028). This potential relationship was used as the hypothesis to
investigate the effect of chitosan on body weight reduction [59]. Again similar study con-
ditions were used with a 90‐day intervention period and daily 2.5 g intake of chitosan and
a randomised, double‐blind, placebo‐controlled setting. Of note, this study included a
larger amount of subjects (i.e. 96 instead of 12), which increased the significance and
validity of the study. Again in this study, chitosan proved to significantly (p<0.0001)
reduce body weight when compared to the placebo group and again corresponding meas-
urements such as BMI, body fat and visceral fat were also significantly reduced (p<0.001).
Taken together, these clinical studies demonstrate that chitosan has a clear effect on
metabolism that is reflected in cholesterol regulation, body weight, antioxidant capacity
and insulin sensitivity.
The cationic interactions that underlie the binding of bile acids by chitosan might also
make chitosan a suitable bactericidal and fungicidal component [60], in addition to the
other putative mechanisms as described earlier. The positive charge of the amino group can
interact with anionic cell surface components, such as lipopolysaccharides of gram‐
negative bacteria. This interaction was shown to destabilise the integrity of the outer mem-
brane which might sensitise bacteria but cannot directly be described as bactericidal
activity. This aspect of chitosan was utilised in a number of studies investigating the benefi-
cial effect on oral health [61–63]. Chitosan was added to mouthwash or chewing gum and
indeed reduced the plaque index and related oral bacterial levels and even local
inflammation.
In contrast to chitosan, only a single clinical trial was performed with chitin focussing on
non‐CVD end‐points. This study (Table 6.5) did include the analysis of interleukin produc-
tion, which are key mediators of immune cells. This study was designed as a randomised,
double‐blind, placebo‐controlled, crossover study with a 2‐week washout period in between
each intervention [64]. Healthy participants were randomised into two groups and received
either intranasal chitin microparticles (1–20 μm) or placebo. Chitin microparticles demon-
strated a significant reduction in local IL‐4 and IL‐6 levels but an increase in leukotriene
B4 levels and total white blood cell counts. These results are somewhat contradicting as
IL‐6 is also an important pro‐inflammatory marker, preventing a general conclusion on
how chitin microparticles affect the intranasal immune system.
Taken together, it is not possible to draw firm conclusions on chitin‐mediated beneficial
health effects because of the limited number of clinical studies. Although the potential of
chitin in CVD treatment has been demonstrated, it has only been tested in healthy subjects
with relatively low oxidised LDL. More studies are required to potentially acquire an EFSA
status similar to chitosan. The clinical studies towards the effect of chitosan on cholesterol
reduction, glucose control and weight loss were more comprehensive and clearly demon-
strated beneficial health effects.
6.4 Requirements to forward the Field of Study Towards the Beneficial

Health Effects of Chitin and Chitosan
Modulation of immune response as induced by chitin or chitosan is dependent on their
physical and chemical properties. Variations in the physical properties such as size and
concentration, and purification methods and chemical properties including degree of de‐
acetylation (DD) levels and sources, may induce different immune responses [65]. This has
been illustrated by various in vitro studies.
Size‐mediated variations of immune responses may result from different mechanisms of
interaction and downstream signalling pathways. Specifically, small‐sized chitin (<10 μm)
could be taken up by phagocytosis, leading to the induction of immune responses in a
phagocytosis‐dependent manner [22]. In contrast, cells are physically limited in absorbing
particles bigger than 10 μm. These larger particles most likely affect cellular responses via
receptor clustering. Such differences in interaction with cells likely result in various down-
stream signalling pathways, which consequently leads to different cellular responses.
Next to this, the DD has been shown to be a critical factor to determine cellular responses
to chitin and chitosan. Julianne and colleagues found that chitin with different DD can
induce distinct cytokine production [66]. Fully acetylated chitin (100%) only induces
TNFα production, but chitin with a DD of 39% induces both TNFα and IL‐1β production
by THP‐1 macrophages. Moreover, chitin with a DD of 85% also induces the production of
TNFα and IL‐1β by THP‐1 macrophages, but at a lower level. Similarly, acetylation levels
were also demonstrated to affect the amount of accumulated leucocytes at the sites of
inflammation [67].
Additionally, chitin or chitosan isolated from different sources can lead to distinct
results. Human PBMCs exposed to chitin from crustaceans were found to produce TNFα
and IL‐6 [68]. Chitin of similar size from fungi induces the production of IL‐10 when pre-
sented to human PBMCs [10]. In fact, chitin isolates from the same kingdom sources but
different genus also shows different immunogenicity. Chitin derived from Aspergillus
fumigatus induces six times higher IL‐6 production than C. albicans–derived chitin in
human PBMCs [65].
Chitin or chitosan may also induce different responses when they have been alternatively
isolated from the same source, as purification methods during isolation may differ in dis-
tinct studies. Interaction of residual proteins, fibres or secondary metabolites during extrac-
tion of chitin and chitosan can affect immune responses. Chitin extracted from shrimps and
crabs are commonly commercially used; however, the purification steps during the extraction
process can be different [22, 23, 30]. For instance, it has been demonstrated that β‐glucan
may largely remain in chitin extracts if not specifically removed [65]. β‐glucan is a well‐
known and well‐described biological response modifier and capable of influencing immune
cell phenotypes, production of cytokines and recruitment of immune cells [69].
Surprisingly, many of these key characteristics have not been described in many of the
animal studies and clinical trials (see Tables 6.1–6.5). Although the impact of these charac-
teristics has only more recently become evident, this does not take away from the fact that
study compound details should have been described. This, however, is a challenge of the
growing field of studies towards beneficial health effects of food compounds or orally
administered supplements such as protein, dietary fibres, etc. The lack of detailed infor-
mation prevents a direct comparison of results and therefore hampers the analysis of
contradictory results and accumulation of knowledge to unlock the potential of chitin
and chitosan.
6.5 Outlook
The terms ‘chitin’ and ‘chitosan’ cover a wide range of molecules with varying MW, acety-
lation levels and sources and purification methods. All these variables are demonstrated to
impact immune responses. There is a large body of work describing the immunomodula-
tory effects of chitin and chitosan, but often lacks a detailed description of the structural
characteristics of the preparations under investigation, which hampers clear‐cut conclu-
sions of the potential of chitin and chitosan. As for other areas of food and nutrition
research, we are only scraping the surface knowledge of interaction with the immune sys-
tem. Most studies focus on epithelial and macrophage responses which represent important
parts of the innate immune system. Studies towards the adaptive immune system are more
complicated and arguably more receptive to minor differences in chitin and chitosan struc-
ture. To ensure that work benefits the current body of knowledge on immunomodulatory
effects of chitin and chitosan, it is crucial that all parameters as indicated earlier are ana-
lysed and described.
Currently, the beneficial health effects of chitin and chitosan have already been com-
mercialised in various products (e.g. Chitosan®, xtendlifeTM). The main functions of these
products are weight loss and cholesterol reduction. The clinical trials and animal studies
performed to demonstrate other beneficial health effects demonstrate the potency of chitin
and chitosan to be more broadly applied to improve health.
Acknowledgement
This work was supported by the Dutch Ministry of Economic Affairs and Climate Policy
(KB‐23‐001‐015).
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7
Antimicrobial Properties of Chitin
and Chitosan
Magdalena Kucharska, Monika Sikora, Kinga Brzoza‐Malczewska,
and Monika Owczarek
Institute of Biopolymers and Chemical Fibres, Lodz, Poland
This review describes possible applications of broad‐spectrum antimicrobial activity of

chitin and chitosan. The antimicrobial activity of chitosan against Gram‐negative and
Gram‐positive bacteria as well as filamentous fungi and yeasts is presented. The mecha-
nism of antibacterial and antifungal action of chitosan on the cell wall of microorganisms
and the influence of physicochemical parameters of the polymer on its biological activity
are discussed. In addition, the relationship between chitosan antimicrobial activity and the
cell wall structure of bacteria and fungi is elucidated, and the influence of environmental
parameters (pH and temperature) as well as the age of the bacterial cell on the antimicrobial
activity of the polymer are characterised. The second part includes the possibilities of using
the antimicrobial activity of chitin and chitosan in medicine, pharmacy, agriculture and in
food, paper and textile industries.
7.1 Microbiological Activity of Chitosan – The Mechanism of its

Antibacterial and Antifungal Activity
The antimicrobial activity of chitosan is influenced, among others, by temperature and pH
value of the environment where the reaction takes place, the growth phase of the bacterial
cell as well as the presence of salts able to complexate with chitosan. According to the
literature, and supported by research, the antimicrobial and antifungal activity of chitosan
is attributed, among others, to positively charged amino groups that react with negatively

charged groups of lipopolysaccharides and proteins on the surface of the microbial cells,
which results in disintegration of the cell membrane and damage of the bacterial cell wall.
The pH values below the pKa of chitosan (< 6.3) result in the formation of a polycationic
compound capable of interacting with negatively charged groups on the surface of micro-
bial cells. Chitosan at pH above 7 loses its antimicrobial properties, due to the lack of
protonated amino groups and low solubility. Therefore, in an acidic environment, chitosan
is characterised by the highest solubility and the ability to react with the negatively charged
surface of bacterial cells. All changes associated with the permeability of the cell mem-
brane cause leakage of intracellular substances as well as inactivation and lysis of the cell.
The conducted research also reveals that the complexes of chitosan with some metals
(mainly chitosan‐Ag, chitosan‐Zn and chitosan‐Cu) show a broad antibacterial spectrum
and act more effectively on some Gram‐negative bacteria including Escherichia coli.
Literature reports indicate that the antibacterial activity of chitosan is also influenced by
the ‘age’ of the bacterial cell (its growth phase). Additionally, it is associated with changes
in the membrane potential on the surface of the cells, which increases in the logarithmic
phase of cell growth and gradually decreases in the stationary phase. Therefore, the most
susceptible to the antibacterial action of chitosan are ‘young’ cells (in the early and late
exponential phase), and the least susceptible are the cells already entering the stationary
phase. The mechanism of this bactericidal action is also associated with inactivation of
bacterial enzymes, substitution of metal ions and interaction with teichoic acid on the sur-
face of the bacterial cell [1–3].
The other parameter, temperature, also affects bacterium–chitosan interactions, being
the most optimal in the range of 4–37 °C. Lower or higher temperatures may cause changes
in the cell surface structure and affect cell electronegativity, thus, reducing the number of
free sites for binding with chitosan. The presence of sodium, calcium or magnesium salts
can adversely affect the antimicrobial activity of chitosan, by forming complexes and
reducing the amount of free chitosan and, thus, preventing the interaction between chitosan
and cell wall.
The antibacterial effect of chitosan varies also with its degree of deacetylation and
molecular weight. High‐molecular‐weight chitosan is believed to be capable of forming a
polymeric film with a microbial cell wall, which, among others, prevents the delivery of
nutrients and, hence, the death of bacterial cells. On the other hand, low‐molecular‐weight
chitosan can penetrate into the cells and bind with negatively charged intracellular compo-
nents, for example, phosphate residues of DNA molecules, which results in blocking reac-
tions on the transcriptional level and synthesis of mRNA. The antibacterial effect of
chitosan is associated with the structure of the microbial cell wall. In Gram‐positive bacte-
ria, due to the greater thickness of the cell wall, high‐molecular‐weight chitosan shows
better bactericidal effect, forming a coating encapsulating bacterial cells. Gram‐negative
bacteria have much thinner cell walls, and that is why low‐molecular‐weight chitosan acts
most effectively, penetrating into cells and disintegrating their genetic material [2, 4].
Chitosan is active against both Gram‐negative and Gram‐positive bacteria as well as fila-
mentous fungi and yeasts. Its antifungal activity mainly consists of the inhibition of sporu-
lation and germination of spores. Chitosan oligomers diffuse inside the fungal hyphae and
interfere with the activity of the growth‐promoting enzymes. It is active against a whole
range of microorganisms such as Aspergillus niger, Candida albicans, Candida glabrata,
Fusarium solani (fungi), Staphylococcus aureus, E. coli, Salmonella typhimurium and
Antimicrobial Properties of Chitin and Chitosan 171
Pseudomonas aeruginosa (bacteria). Chitosan inhibits bacterial growth, both in logarith-

mic and stationary phases, but in the case of the latter one, this is much less effective due
to changes in membrane potential of the cells [5–8].
According to literature data, commonly used indicators of fungicidal or bactericidal
activity of chitosan preparations are MIC (minimum inhibitory concentration), MBC (min-
imum bactericidal concentration) and MFC (minimum fungicidal concentration). MIC is
the lowest concentration of the microbiocide that will inhibit microbial growth, expressed
in mg/L. MIC indicates at what concentration the active substance inhibits the growth of
bacteria or fungi. MBC is the lowest concentration of bactericide at which 99.9% of bacte-
ria are killed, expressed in mg/L. MBC indicates at what concentration the preparation
shows bactericidal activity, directly killing vegetative forms of bacteria. MFC is the lowest
concentration of fungicide at which 99.9% fungi are killed, expressed in mg/L. All three
parameters are determined in vitro for a given type and species of bacteria or fungi [8].
Chitosan, being a non‐toxic, biodegradable and biocompatible natural polymer with a
bactericidal and fungicidal activity, is the subject of interest for many scientists. Its antimi-
crobial properties may be applied in many areas such as medicine, agriculture as well as
food, textile and paper industries [9].
7.2 The use of Chitin/Chitosan’s Microbiological Activity in Medicine

and Pharmacy
Chitosan, due to its high microbiological activity as well as its unique biological features
such as stimulation of wound healing and haemostatic properties has been applied in the
construction of various types of (wound) dressings. Currently, a wide range of dressings is
available on the medical market, based on various forms of chitosan, which, besides the
ability to quickly stop bleeding, are also characterised by high effectiveness in reducing
bacterial infection in wounds. These types of dressings include HemCon Bandage™
(HemCon), Clo‐Sur P.A.D.™ and Clo‐SurPLUS P.A.D.™ (SCION Cardio‐Vascular, Inc.) as
well as Chitodine® dressing (IMS) based on chitosan with the addition of iodine [10–13].
The first Polish dressing based on chitosan is TROMBOGUARD (Tricomed SA), devel-
oped as a result of the cooperation between the Institute of Biopolymers and Chemical
Fibers (IBWCh), Lodz, Poland and the Institute of Safety Technology MORATEX, Lodz,
Poland. An active layer of this innovative dressing is a mixture of chitosan and sodium/
calcium alginate with the addition of silver salts [14]. The dressing has haemostatic and
antibacterial properties and is intended for the treatment of post‐traumatic and post‐opera-
tive wounds. Its immediate antihaemorrhagic and antibacterial effectiveness was confirmed
by in vitro and in vivo studies in animals as well as in clinical testing.
At IBWCh, innovative nanoforms of chitosan as well as a chitosan–sodium/calcium
alginate complex were also developed, which served as modifiers of cellulose carriers
intended for medical and cosmetic purposes. Modified fibrous products with nanochitosan
or nanocomplex exhibit very high antibacterial activity against E. coli and S. aureus as well
as antifungal activity against A. niger and C. albicans. The activity of the developed materi-
als did not change after 12 months, which was confirmed by aging tests [15, 16]. Among
the results of research conducted at IBWCh are also some composite materials which con-
sist of chitosan antibacterial fibres and/or various forms of chitosan (microcrystalline chi-
tosan, chitosan gel form). Composite materials based on various forms of chitosan were
produced in the form of a sponge for its utilisation in dressing purposes. According to the
assumption, these dressings can be used in the initial period of wound healing accompa-
nied by inflammation. The obtained wound dressings are characterised by antibacterial
activity against E. coli and S. aureus [17].
Helicobacter pylori is a kind of bacteria that may lead to the development of gastric and
duodenal ulcers, chronic gastritis, gastric cancer, possibly also ischemic heart disease, and
atherosclerotic vascular disease. According to literature, chitosan is highly active against
this bacterium. The research focussed on the determination of the antibacterial activity of
chitosan ascorbate. Chitosan with 60% deacetylation degree prepared from krill chitin was
used to produce this form of polymer. Chitosan ascorbate activity was determined against
17 H. pylori strains. The strains were isolated from the periodontal pockets (7 strains),
atherosclerotic plaque from the carotid and femoral arteries (10 strains). The studies
showed that the flagella of H. pylori strains isolated from the periodontal pockets showed
higher susceptibility to chitosan ascorbate than the strains isolated from atherosclerotic
plaque [18].
In stomatology and treatment of oral cavity diseases, chitosan is used as a viscous, bio-
adhesive antimicrobial, prolonging release of the drug in the treatment of periodontal dis-
ease, as well as for reduction of plaque formation. According to literature, chitosan
ascorbate also shows activity against bacteria isolated from pathological gum pockets, root
canals and abscesses that cause infections in the oral cavity. The experiments included 70
clinical isolates and 4 standard strains. The following bacterial strains were tested:
Aggregatibacter (22 strains), Corynebacterium (3 strains), Capnocytophaga (5 strains),
Campylobacter (19 strains), Eikenella (17 strains), Rothia (4 strains) and 4 standard strains:
E. coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), S. aureus (ATCC 25923) and
P. aeruginosa (ATCC 27853). Chitosan ascorbate showed good activity against microaero-
philic bacteria. The most sensitive to the polymer were strains of Corynebacterium matru-
chotii (100% sensitive) and Aggregatibacter actinomycetemcomitans (72% sensitive). The
Gram‐negative and Gram‐positive bacteria showed slightly less sensitivity (48% and 43%,
respectively), while the least sensitivity exhibited strains were Capnocytophaga gingivalis
and Rothia dentocariosa [19].
A. actinomycetemcomitans plays a key role in the pathogenesis of periodontal diseases:
periodontitis and gingivitis. Kochańska et al. reported that in vitro ascorbic salt of chitosan
shows good activity against these bacteria. The MIC for 77% of A. actinomycetemcomitans
strains is in the range of ≤ 0.06–0.5 mg/mL [20].
An antimicrobial formulation in the form of a thermally sensitive mucoadhesive gel has
been developed for the treatment of oral mucositis. It is a mixture of chitosan trimethylsilyl
or chitosan methyl pyrrolidinone with glycerophosphate [21].
Antibacterial properties of chitosan have also been used to develop a preparation for the
treatment of chronic periodontitis. Chitosan gel with or without metronidazole, as an active
substance, was used to treat this disease. The evaluation of the effectiveness of the devel-
oped product was carried out in clinical tests. They studied the evaluation of the gel’s
impact on clinical parameters such as depth of a gum pocket, clinical attachment level,
gingival recession, plaque index, gingival index and gingival bleeding time index. The
research shows that chitosan alone, as well as in combination with metronidazole, is an
effective preparation for the treatment of periodontitis. Throughout the whole study, no
complications associated with the use of chitosan in patients were observed [22].
In addition to the antibacterial action of chitosan on microorganisms in the oral cavity, it

also affects the adhesion of microorganisms to the surface of the teeth. This adhesion plays
a key role in the formation of plaque and development of caries. Chitosan has the ability to
prevent the adhesion of Streptococcus mutans to the tooth enamel. Mechanism of this
action consists of stimulation and regeneration of soft tissues in the oral cavity by chitosan,
thereby preventing the harmful effects of organic acids and impairing the bacteria. The
mode of interaction between chitosan and external membranes of bacterial cells appears to
be related to the chitosan concentration. At low concentrations (< 0.2 mg/mL), chitosan is
probably attached to negatively charged bacterial surfaces, causing agglutination of bacte-
ria. At higher chitosan concentrations (>2 mg/mL), resulting in a large number of positive
charges, allows bacteria to maintain in suspension [23].
Tooth decay is the most common oral disease. In the development of this disease, various
types of bacteria are involved such as S. mutans, Streptococcus sanguinis, Streptococcus
salivarius and Lactobacillus casei. Synthetic antimicrobials are used against tooth decay,
but many of these substances cause side effects. An alternative to that type of agents may
be a lacquer made on the basis of propolis and chitosan. Studies concerning the effective-
ness of the lacquer showed its high ability to inhibit all pathogenic microorganisms that
cause caries [24]. As part of the work carried out at IBWCh, in vitro tests were conducted
to assess the effect of chitosan on the growth of plaque bacteria such as S. mutans and
Lactobacillus sp. as well as E. coli. The creation of favourable conditions for the permanent
binding of filling materials with tooth tissues, that is, enamel and dentin through the bind-
ing system, is important for limiting the penetration of chemicals and microorganisms
harmful to the pulp of the tooth. This is related to complications such as dentin hypersen-
sitivity and secondary caries [25].
Chitosan has also been used to treat surgical site infections – the complications occur-
ring, for example, in treatment of hernia with the use of surgical mesh. According to litera-
ture, a surgical mesh has been developed, which has been surface‐modified by a chitosan
gel with the addition of an antibacterial substance – triclosan. In vivo studies in rats infected
with S. aureus have shown that the developed implant has a satisfactory prophylactic action
against infection caused by these bacteria [26].
Chitosan has also the potential to be used in ophthalmology. Antibacterial activity of the
polymer as well as good wetting properties are required in the treatment of dry eye syn-
drome [27]. Chitosan has all the features needed to produce perfect contact lenses, due to
its ability to form films characterised by adequate gas permeability, especially oxygen, and
having antimicrobial and hydrophilic properties as well as optical clarity [28].
The emergence of antibiotic‐resistant microorganisms is a serious health risk which
implies the urgent need to develop alternative antibiotics. It has been proven that chitosan
microparticles can potentially be used as an alternative antimicrobial to limit the develop-
ment of E. coli in a model involving cows with uterine diseases. In the study, treatment with
chitosan microparticles effectively reduced the secretion of intrauterine pathogenic bacte-
ria as compared to antibiotic treatment. Treatment with chitosan microparticles did
not induce bacteriophages or Shiga toxins in E. coli O157: H7, suggesting that chitosan
microparticles could be a potential candidate for the treatment of infections caused by this
pathogen [29].
The evaluation of the antifungal activity of chitosan against the clinical form of the fun-
gus from the Candida family is also reported. The most susceptible to infection with this
type of pathogen are patients with diabetes and endocrinopathies. The clinical form of
Candida is often associated with a predisposing factor, such as AIDS, neutropenia, cancer,
radiotherapy as well as destructive periodontal disease, gingivitis and caries. It is known
that the growth of Candida depends on the availability of sugars, especially glucose.
Ascorbic salt of chitosan was selected for the activity tests against fungi of the Candida
family. The antifungal effect of this form of chitosan on different strains such as C. albi-
cans, C. tropicalis, C. guilliermondii, C. parapsilosis, C. kefyr, C. krusei and the standard
PCM1409 strain has been assessed. Conducted studies demonstrated that in experimental
conditions, chitosan ascorbate showed different antifungal activity. The most sensitive to
the preparation was C. tropicalis strain (MIC ≤ 1 mg/mL, 33% strains), and the least C. guil-
liermondi and C. parapsilosis strains (MIC ≥ 2 mg/mL, 100% strains). Strains belonging to
C. albicans, the dominant species in the study, were less susceptible to chitosan ascorbate
(at a concentration of 0.12–0.5 mg/mL 25% of strains inhibited). It was also found that 31%
of the tested Candida strains were not sensitive to chitosan ascorbate at the concentration
tested (MIC > 2 mg/mL) [30].
The potential biomedical use of chitin includes surgical sutures and wound healing
materials. Chitin has also been tested for its antibacterial and tissue regenerating properties,
which make it useful in the wound healing process and in preparation of materials for
wound treatment, mainly adhesive plasters and dressings. Researchers, after trials on ani-
mals, place great hope in the use of such dressings in wound treatment using chitin with a
molecular weight in the range of 10–50 kDa [31].
7.3 Microbiological Activity of Chitosan in the Food Industry

In the food industry, chitosan is used, among others, for clarification and deacidification
of beverages as well as colour stabilisation. It can also be used as a thickening and stabilis-
ing agent, texture‐controlling agent as well as an antioxidant and preservative [32]. There
is an increasing interest in the use of natural antimicrobial materials to prevent spoilage of
food. Chitosan films are considered as biofunctional materials, which, due to their proper-
ties, are very well tolerated by living tissues and have the specific use as edible films and
coatings. Their role is to extend the shelf life and preserve the quality and freshness of
food products [33].
Chitin and chitosan have many features attributed to packaging materials, among others,
they have the ability to form films and show resistance to heating. These compounds are
also characterised by good permeability to oxygen and carbon dioxide, have good mechan-
ical properties, and thanks to their biodegradability, are environmentally friendly. The dis-
advantage of chitosan packaging is their low moisture resistance, due to the hygroscopic
nature of chitosan [34]. Therefore, research is conducted to develop the optimal composi-
tion of chitosan film in order to reduce its sensitivity to water, and, at the same time, not
changing its antibacterial properties. Previous studies on the composition of chitosan films
provide information on the effectiveness of chitooligosaccharides (COS) in neutralised or
heat‐treated chitosan films. The addition of COS decreases the film solubility and increases
antibacterial activity, by strengthening the action of functional groups, and do not reduce
the water vapour permeability [33]. In studies on stored fresh fish, the good bactericidal
effect had coatings based on chitosan with the addition of herbal extracts, for example,
thyme. Fresh eel fillets covered with a mixture of chitosan and thyme oil showed extended
shelf life, and maintained freshness by inhibiting the growth of microorganisms responsible
for food spoilage without altering the taste. In addition, antioxidant properties of thyme
extract inhibited lipid peroxidation in fillets. A similar effect may be demonstrated by a
combination of chitosan with essential oils (cinnamon, oregano, tangerine) [35, 36], as well
as chitosan with bamboo vinegar, used to prolong the shelf life of pork [37].
Thanks to modern methods of electrospinning or encapsulation it is possible to exploit
the ability to form nanofibers and nanoparticles (e.g., nanoemulsions) by many natural
polymers (including chitosan) and to use them in the food industry to produce modern,
biologically active and biodegradable food packaging, microbiologically active coatings
and natural preservatives [36, 38]. Studies conducted so far have proved the applicability
of chitosan membranes for coating fresh fruits and vegetables (mainly strawberries, blue-
berries and grapes, fresh cucumbers, tomatoes and carrots), and the addition of herbal
extracts (e.g., rosemary), garlic, nisin, phenolics and flavonoids, or lysozymes and makes
it possible to use these mixtures as antioxidants and natural preservatives protecting against
the development of food spoilage by bacteria and fungi [34, 39, 40]. The use of edible
chitosan films as a matrix for introducing bioactive components could effectively prolong
the shelf life of food products and enrich them with active compounds with a positive effect
on health and food. Actively acting nanoemulsions have a strong antibacterial and antifun-
gal effect, limiting food spoilage [41]. Other literature data indicate positive effect of chi-
tosan on colloidal stabilisation of light beer and wines. Due to its ability to actively
counteract protein coagulation, chitosan can be a natural clarification agent for beers and
wines, and some chitosan derivatives are able to absorb heavy metals from them [42, 32].
The antioxidant properties of chitosan were used, among others, for food preservation,
mainly as an addition to meat and fish. Chitosan acts as a preservative in combination with
other natural antioxidant compounds, for example, with lycopene (a health‐promoting
compound naturally found in tomatoes). It prevents the oxidation of compounds present in
the meat product, limiting its spoiling without changing the taste and nutritional values
and, in addition, it is neutral for the body. In the food industry, the chitosan application
techniques used are usually dipping, spray coating and vacuum packaging of food products
[43, 44].
Chitosan is a strong chelating agent and easily forms complexes with metals. This prop-
erty can be used in design of chitosan–metal nanocomposites with antifungal and antibac-
terial activity. Studies carried out so far prove that chitosan–copper and chitosan–zinc
nanocomposites in vitro strongly inhibit growth of S. aureus, P. aeruginosa and Salmonella
enterica. This feature can be used to limit the growth of these pathogens in the food as well
as cosmetic and textile industries [45].
In the era of propagation of many civilisation diseases, which is often associated with an
improper diet, special attention has to be paid to the consumption of fresh fruits and vegeta-
bles and the beneficial polyunsaturated fatty acids, whose best sources are fresh fish and
seafood. The problem of fresh fish storage is associated with its high content of moisture
and nutrients that enable development of bacterial microflora, which accelerates decompo-
sition of the product, changing its organoleptic and pro‐health properties. Chitosan oligo-
saccharides, which are low‐molecular‐weight, biodegradable products of chitosan, can
prevent these processes. They are a mixture of oligomers that consist of D‐glucosamine
residues connected by β‐1,4 bonds. Due to their low molecular weight, low viscosity and
good solubility in water, they can be applied in food industry as an additive to fish and
seafood. It has been shown that chitosan oligosaccharides have neuroprotective, anticancer,
immunity‐strengthening and cholesterol‐reducing effects. Besides health‐promoting
action, the addition of natural antioxidants and preservatives based on chitosan reduces
economic losses caused by generation of large volume of food waste [46, 47].
7.4 Microbiological Activity of Chitosan in Paper and Textile Industries

The development of functional materials in the paper industry is increasingly focused on
the search for new solutions, beneficial not only to the consumers but also to the environ-
ment. Demand for paper‐making antibacterial materials is still growing, which is mainly
associated with social and economic development. For the production of antibacterial
paper, carboxymethyl chitosan can be used, which can be obtained by introducing carboxy-
methyl quaternary amine groups at the chitosan chain. The use of carboxymethyl chitosan–
silver nanocomposites as paper coating or as pulp additive has positive effect on the
physicochemical properties of paper, and it induces antifungal and antibacterial activity
[48]. In addition, the molecular structure of chitosan, similar to cellulose, enables strong
bonding, increasing mechanical strength of paper. The improvement of antibacterial and
physicochemical properties of paper can be applied, among others, in production of anti-
bacterial packaging materials used in food, medical and healthcare industries [49].
In the textile industry, there is a growing demand for new medical textiles and textiles
related to health care. The development of research in this field was largely due to the
events associated with the rapid propagation of dangerous pathogens that may cause epi-
demics (e.g., severe acute respiratory syndrome (SARS) and H1N1 virus). Until now, many
chemicals have been used in antimicrobial textiles such as phenols and thiophenols, antibi-
otics, heterocyclic compounds with anionic groups, nitro compounds, urea, formaldehyde
and amine derivatives, which show bactericidal effect, but in most cases are toxic to humans
and harmful to the environment. That is why the textile industry is looking for new, eco‐
friendly solutions that are non‐toxic to people and are safe for the environment. Chitosan is
a good material for use in the textile industry due to its antimicrobial and biodegradable
properties.
Chitosan derivatives with an increased number of functional groups can be obtained by
modifying the chemical structure of chitosan, which results in an increase in its antimicro-
bial activity. Quaternary ammonium salts not only increase the antibacterial effect but also
the solubility of chitosan in water. Chitosan derivatives were employed to finish cotton
fabrics using citric acid as a cross‐linking agent. In vitro modified fabrics showed inhibi-
tory effects on the growth of pathogens such as E. coli and S. aureus, which makes it pos-
sible to use the antibacterial potential of chitosan in production of new microbiologically
active fabrics. Using the technique of encapsulation of active substances, such as medicines
or antimicrobial agents, depolymerised chitosan lysosomal emulsions can be obtained. The
use of chitosan in this form may be an unconventional method for impregnation of cotton
fabrics with active compounds [50, 51]. Fabrics treated with nanoparticles of chitosan‐ZnO
complex showed increased resistance to the pathogenic strains of E. coli, S. aureus and
E. faecalis [8].
Chitosan also has applications in textile industry for fixing dyes, as a thickener and
binder in dyeing of cellulose fabrics. It also increases mechanical strength of dyed polyes-
ter–cotton textiles [52]. Tests carried out on cotton and polyester fabrics coated with
modified chitosan proved that they were resistant to high temperatures, humidity and
mechanical washing. Application of modern methods for the production of antibacterial
fabrics for medicine or health care may be a breakthrough in the struggle against spreading
of pathogenic microorganisms, and thus protect society against epidemics threats [49].
7.5 Microbiological Activity of Chitosan in Agriculture

Nowadays, in developed countries, there is a tendency to limit the use of chemical plant
protection agents in agriculture and horticulture, due to their potential harmfulness [53–
55]. So far, the dominant forms of plant protection against pathogens (75–80%) are chemi-
cal methods. Excessive use of synthetic fertilizers and pesticides in cultivation of plants
contributes not only to significant increase in productivity and crop yield, but also causes
environment pollution, destroys pests’ natural enemies, adversely affects biocenotic diver-
sity and natural ecosystems and, above all, negatively affects consumers’ health, leading to
development of antibiotic‐resistant pathogenic strains [54, 56, 57].
Natural products are a great alternative to synthetic pesticides in reducing negative health
and ecological aspects and in the development of good practice principles in the use of
plant protection products, eliminating all adverse effects of pesticides and promoting
organic farming [56].
In agriculture, fungi, bacteria and viruses are the cause of many serious diseases of crops
and ornamental plants and are responsible for huge losses in harvest and crop quality
around the world. In addition, several species of pathogenic fungi can produce harmful
toxins and metabolites during the infection process, which is a potential threat to the safety
of agricultural products and, as a consequence, to the health and life of people and animals.
The final result of infection of plants by pathogenic fungi and bacteria is reduction of plant
growth rate, lower yields, poorer crop quality and huge economic losses. Therefore, crop
infections must be strictly controlled and promptly eliminated in order to maintain the
quality and safety of agricultural products [57–59].
The first reported attempts to use natural organic biological agents in plant protection
were carried out in 19th century by Metchnikoff (1880) and Krassilstschik (1888), who
used Metarhizium anisopliae insecticide fungi obtained in mass‐culture to control cereal
and beet agrophages [55]. Currently, many preparations based on extracts or substances of
natural origin are available on the market, which play a very important role in effective
protection of plants against pests [55, 60]. These include various forms of chitosan such as
chitosan oligomers, chitosan salts, for example, chitosan lactate, microcrystalline chitosan
(MKCh), polymeric compositions, for example, chitosan–lignin, chitosan gels, as well as
chitosan‐based nanomaterials (NM). As part of the previous studies, the usefulness of vari-
ous chitosans differentiated in terms of the degree of polymerisation and the degree of
deacetylation for protection of plants against soil pathogens (fungi) as well as bacteria and
viruses was assessed. Laboratory results and practical tests confirmed the bioactivity of
chitosan in the inhibition of plant pathogens [61, 62].
Chitosan‐based NM can penetrate plant tissues by overcoming size limits (about
1–1000 nm), for example, through cuticles, trichomes, stomata, wound sites, hydatodes and
root systems. Small‐size NM can easily pass through plant surfaces and cause uniformly
strong effect, compared to larger and more complex NM. High surface charge (zeta poten-
tial) of chitosan NM gives higher affinity to biological membranes and causes stronger
biological response to etiological agents in the form of bacteria (Xanthomonas sp.,

Euphorbia pulcherrima, Ralstonia solanacearum, Acidovorax citrulli, Pseudomonas
syringae and others), fungi (Fusarium graminearum, Fusarium oxysporum, Alternaria
alternata, Macrophomina phaseolin, Rhizoctonia solani, Pyricularia grisea and others)
and viruses (alfalfa mosaic virus (AMV), tobacco necrosis virus (TNV), tobacco mosaic
virus (TMV), peanut stunt virus (PSV), cucumber mosaic virus (CMV), potato virus X
(PVX), potato spindle tuber viroid (PSTV) and others [54]. Induction of natural defence
response in plants, enhanced by the presence of chitosan nanoparticles consists of increased
accumulation of phenolic compounds, cell wall synthesis and lignification as well as pro-
duction of reactive oxygen species [53, 54]. Chandra et al. [63] reported that solutions
(0.001% (w/v)) containing spherical chitosan nanoparticles (with a diameter of 42–180 nm)
increase the activity of plant defence enzymes (Camellia sinensis leaves) even fourfold,
after 24 hours. In addition, chitosan nanoparticles also enhanced the level of antioxidant
enzymes and secondary metabolites such as superoxide dismutase, catalase and phenol,
which limit the invasion of fungi on plant crops [53, 54].
Some studies have shown that chitosan oligomers (pentamers and heptamers) show
better antifungal activity than chitosan with a higher degree of polymerisation [64]. In
other cases, the antimicrobial activity increases with the increase of the molecular mass
of chitosan [65], and the rate is faster in the case of fungi and algae than in the case of
bacteria [66].
As a result of the work carried out, among others, at IBWCh, the influence of molecular
and supermolecular structure of chitosan and microcrystalline chitosan on bioactivity was
determined, and the possibility of creating this bioactivity by physicochemical modifica-
tion was examined [54, 67, 68]. IBWCh in cooperation with other research groups has
carried out a series of application works related to the use of chitosan in agriculture as a
biopreparation for the protection of plants against fungal and bacterial diseases. Study on
various usable forms of chitosan revealed that chitosan, depending on its form, apart from
direct interaction with pathogens, is characterised by the ability to induce natural immunity
that protects the plants. This phenomenon is strongly influenced by the presence of oligo-
aminosaccharides in the preparation or the possibility of their formation as a result of plant
enzymes’ action on chitosan [69, 70]. Their biological activity is manifested by the inhibi-
tion of in vitro growth of phytopathogenic fungi and bacteria, which is characteristic to
classical plant protection agents, but mainly they modify plant metabolism so as to be
hostile to the development of harmful etiological factors, which is manifested by the inhibi-
tion of infection and disease development in arable crops. Conducted research has shown
that the interaction of these polymers with plant metabolic factors is unfavourable for path-
ogens; still, it does not harm the plant and, if used in proper concentration, stimulates plant
growth [71–79]. It was also shown that the effectiveness of oligomeric chitosan fractions is
higher in the case of bacterial infections of plants and such form of chitosan can be included
in elicitors, that is, new generation of preparations, activating defence mechanisms, as a
result of which the immunity of the whole plant is induced to a number of diseases caused
by pathogens [80–83]. Other researchers assume that elicitors, in addition to inducing a
number of immune mechanisms, can also directly affect pathogens. Sun et al. [84] have
tested the antioxidant activity of several chitosan oligomers with different molecular
weights and determined the IC 50 of their ‘scavenging ability’ against the superoxide anion
and hydroxyl radicals. The lowest ‘scavenging’ activity against the superoxide anion and
hydroxyl radicals was noted for the oligomer with the highest molecular weight. At the
same time, the lower‐molecular‐weight chitosan oligomers showed better antioxidant
activity [84, 85].
So far, a number of compounds is known that induce plant resistance – elicitors natu-
rally occurring in nature as well as synthesised chemical substances. The natural elicitors
are some components of cell membranes of phytopathogenic fungi and plants, such as
glucan, pectin, as well as chitin and chitosan. Large‐scale production of elicitors (induc-
ers) of fungal origin is technically impossible; that is why natural polymers with proper-
ties similar to elicitors from fungi and bacteria and easily available in large quantities are
sought. It should also be noted that, in plant protection against pathogens, the condition
of plants is very important, especially in the initial stages of development. Plants with
stimulated rapid growth easily leave the development stage in which they are very sus-
ceptible to diseases. This effect will be particularly strong if it is supported by the stimu-
lation of immune processes in the plant. Chitosan as a polymer having a positive charge
when applied to the plant from the outside reacts with negatively charged molecules on
the surface of the fungal cell and reacts with specific active areas, mutually chemically
compatible, which causes significant changes in the composition of the membrane func-
tion. Properly selected chitosan also directly affects fungi by inhibiting their growth [68].
Based on the research, it was found that the use of chitosan pentamers on the surface of
soy and maize leaves, affected the net coefficient of photosynthesis one day after appli-
cation [86], which correlated with an increase in stomatal conductance and transpiration
rate. The use of chitosan formulations on leaves had no effect on the concentration of
intercellular CO2 which is necessary for the process of photosynthesis. The foliar appli-
cation of these oligomers also did not negatively affect the height of the corn or soy, the
length of the roots, the surface of the leaves and the total amount of dry matter. Bittelli
et al. suggested that chitosan may be an effective measure to prevent excessive transpira-
tion in order to reduce the need for huge water amounts for irrigation of crops. Researchers
studied the possibility to use chitosan oligomers on the leaf surface for the transpiration
of pepper plants in greenhouse and fields conditions. In both cases, water consumption
was monitored directly and indirectly. Plant biomass and crop yield were also checked to
calculate biomass/water ratio, and the differences in water vapour resistance were ana-
lysed, comparing control and chitosan‐treated plants. Using scanning electron micros-
copy and histochemical analyses, it was shown that the stomata in the leaves treated with
the preparation containing oligomers became closed, which reduces the transpiration
process, providing the leaves with the optimal turgor. The reduction in water consump-
tion for pepper plants after chitosan treatment was estimated at 26–43% compared to the
control, with no changes in the production efficiency and biomass yield [87]. Iriti et al.
revealed some aspects by which chitosan was able to reduce transpiration in bean plants
after application as a foliar spray. The authors have shown that this activity probably
occurred due to the increase in the content of abscisic acid (ABA) in the treated leaves.
Using scanning electron microscopy and histocytochemistry techniques, the authors
showed that after applying the chitosan preparations and increasing the ABA content,
partial closure of stomata occurred, which led to a decrease in the loss of water from the
treated plants [88]. Faoro et al. [58] showed that the use of chitosan as a spray on barley,
locally and systemically reduced infection with the pathogen of powdery mildew
Blumeria graminis f. sp. hordei [89].
As a result of physicochemical modification of the initial chitosan by continuous or

periodic methods, researchers from IBWCh have obtained new valuable usable chitosan
forms, that is, microcrystalline chitosan (MKCh) and chitosan gel form with improved
sorptive and chelating properties, increased susceptibility to enzymatic degradation and
biodegradation in water and soil conditions and, above all, increased biological activity. In
particular, the latter advantage makes such forms of chitosan particularly well suited for
agricultural applications [90]. Niekraszewicz et al. showed in their research that relative to
the control the highest ability to stimulate germination of radish seeds was observed after
the treatment of the seeds with MKCh with a molecular weight of 371 kDa. In the selected
range of concentrations (from 0.005 to 0.1% (w/v)), the increase in sprout mass relative to
the control was 128.8–136.9% and the length of sprouts was 137.1–177.1%. It was also
found that with the decrease in molecular weight of MKCh (201 kDa, 113 kDa and 24 kDa),
the germination stimulation effect of the treated seeds was slightly lower with respect to the
MKCh with a molecular weight of 371 kDa. Research was also carried out on the effect of
chitosan–lignin preparations obtained by agglomerating chitosan from a solution in lactic
acid using sodium lignin sulphonate. The highest sprout‐stimulating properties of treated
lettuce seeds were found in compositions obtained from MKCh with a 371 kDa molecular
weight both not washed and washed with buffered water at pH 7.01 in order to remove free
lignin [91].
As a result of the conducted research, the technology was developed and the pilot pro-
duction of a preparation Biochikol 020 PC (currently, a commercial preparation under the
trade name Beta‐Chikol) in the form of buffered chitosan lactate was started at IBWCh.
This biopreparation is used to protect crops, and vegetables and ornamental plants against
bacterial and fungal diseases. The developed preparation induces the natural immunity of
plants, and acts like an autovaccine, triggering intensive self‐defence processes in plant and
protecting against the penetration of fungal, bacterial and viral plant pathogens. Biochikol
020 PC is also used for treating seeds, tubers and bulbs and if applied in a spray form in
vegetative reproduction, it contributes to faster formation of the extended root system of
the plant and its faster growth. Kurzawińska et al. showed in their research that the
Biochikol spraying on potato (Solanum tuberosum) cuttings resulted in less disease symp-
toms on potato leaves caused by a fungus of genus Alternaria spp. [71]. Also, Włodarek
et al. proved that the result of using that preparation as a foliar fertilizer was 85% effective
in protection of celery (Apium graveolens) cultivation from the leaf spot caused by Septoria
apiicola [60].
Niekraszewicz et al. showed in their studies that chitosan lactate solutions of different
pH stimulate the germination of dressed radish seeds, contributing mainly to the increase
in mass of sprouts and their length. Chitosan lactate, with a 5% (w/v) addition of galacto-
glucomannans in the pH range of 6.56–6.63, was the best among the tested preparations.
After radish seed treatment, a visible increase in the length of shoots was observed, exceed-
ing 100% already at a concentration of 0.005% (w/v) [92].
Many scientific research groups are focusing on various forms of chitosan that may
eventually be used in agriculture. Some examples of research are mentioned in the follow-
ing text. It was shown that the addition of chitosan to the growing medium of Eustoma
grandiflorum intensified the growth of these plants. Plants that were grown on the substrate
with 1% (v/v) chitosan entered the flowering phase 15 days earlier in comparison to the
control plants [93]. Cho et al. in their research proved that soaking of sunflower seeds
(Helianthus annuus L.) in 0.5% (w/v) chitosan solution for 18 hours can increase the mass
of sunflower shoots by 12.9%. It was also found that the emergence of seeds soaked in a
chitosan solution was 16.0% higher in relation to the control seeds soaked in water [94].
Foliar application of chitosan compounds was used in cultivation of tobacco (Nicotiana
tabacum L.), and tobacco seeds were encapsulated in chitosan solution to protect plants
against fungi. Researchers showed that both these ways of using chitosan strengthened the
immune reactions, making them more resistant to Phytophthora parasitica nicotianae [95].
Orlikowski et al. showed that soaking tulip bulbs (Tulipa sp.) in chitosan solution within
30 minutes caused inhibition of fungus F. oxysporum f. sp. tulipae [96, 97]. Chitosan used
in the cultivation of multiflora beans and head lettuce in the form of seed dressing solution
(concentration of 1% (w/v)) effectively protects beans and lettuce against fungal diseases
caused by Bortytis cinerea, F. solani, F. oxysporum f. sp. phaseoli, Fusarium culmorum,
Pythium irregulare and R. solani. Plants grown from dressed seeds were healthier com-
pared to those that were grown from seeds not treated before sowing [98, 99].
7.6 Outlook
The preceding studies show that chitosan is an environmentally safe polymer that stimu-
lates growth and development of certain plant species. It can be used not only for treating
plants by spraying but also for so‐called soil activation. Soil activation is associated with
limiting the number of harmful organisms, but above all with the stimulation of the increase
in the number of chitinolitic microorganisms that inhibit the growth of fungal soil patho-
gens. The results of numerous field trials also confirm that chitosan compounds can be used
to stimulate immune reactions in crop plants. They can be used during cultivation, but also
to treat fruits and vegetables after harvesting in order to improve their health and quality.
The efficacy of chitosan as a preservative during cultivation and storage of fruits and veg-
etables is slightly weaker than fungicides, but the advantages of chitosan such as natural
origin, high biological activity and harmlessness to humans and the environment, makes it
frequently recommended for use in agriculture and horticulture, especially in organic farm-
ing and in the production of healthy food.
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8
Enzymes for Modification of
Chitin and Chitosan
Gustav Vaaje‐Kolstad, Tina Rise Tuveng, Sophanit Mekasha, and
Vincent G.H. Eijsink
Faculty of Chemistry, Biotechnology, and Food Science, The Norwegian University of Life Sciences
(NMBU), Ås, Norway
Nature is abundant in carbohydrates in monomeric, oligomeric and polymeric forms, and

different enzymes have evolved to synthesize, modify or degrade complex carbohydrate
structures. Carbohydrate‐active enzymes (CAZymes) are organised and classified in the
CAZy database, where they are divided into different classes, according to function, and
families, according to amino acid sequence similarity [1]. Currently, the CAZy database
contains five enzyme classes and one class of non‐catalytic modules that are associated
with CAZymes. The glycoside hydrolase (GH) class contains enzymes that hydrolyse gly-
cosidic linkages. Some GHs also have transglycosylating activity, where a sugar (instead of
water, as in hydrolysis) acts as an acceptor resulting in the formation of a new glycosidic
bond [2]. Glycosyl transferases (GT) synthesize glycosidic linkages using activated sugars,
while polysaccharide lyases (PL) perform non‐hydrolytic cleavage of glycosidic bonds.
Carbohydrate esterases (CE) remove ester modifications on carbohydrates [2]. The fifth
class is referred to as auxiliary activities (AA) and contains a variety of redox enzymes
acting in conjunction with other CAZymes [3]. The AA class includes the so‐called lytic
polysaccharide monoxygenases (LPMOs; see Section 8.1.5) that were discovered in 2010
[4] and play a pivotal role in polysaccharide degradation. The AA class also contains redox
enzymes acting on lignin, since lignin is found together with polysaccharides in plant cell
walls. Next to these five classes of catalytic domains, the carbohydrate‐binding module

(CBM) class contains proteins with no enzymatic activity. CBMs are normally covalently
attached to enzymes and their primary function is to promote substrate binding [2].
8.1 CAZymes in Chitin Degradation and Modification

A variety of enzyme families in the CAZy database are involved in the modification and
depolymerisation of chitin, including chitinases, chitosanases, carbohydrate esterases, β‐N‐
hexosaminidases, exo‐β‐glucosaminidases and lytic polysaccharide monooxygenases
(Figure 8.1). The roles of these enzyme families in chitin degradation are described in the
sub‐chapters that follow.
Chitin
1 2 3
LPMOs Chitinases Deacetylases
AA10 GH18 CE4
AA11 GH19 CE14
AA15
Lactone Aldonic acid Chitobiose Chitosan*

β-N-acetylhexosaminidases Chitosanases Chitinases
GH3 GH5 GH46 GH18
GH20 GH7 GH75 GH19
GH84 GH8
Chitinases +
β-N-acetylhexosaminidases
OR
GlcNAc GlcN-GlcNAc (GlcN)2
exo-β-glucosaminidases
GH2
GH9
GH35
GlcNAc-GlcNAc1A
OR
GlcNAc GlcN
Figure 8.1 Schematic illustration of enzymatic pathways for degradation of chitin. The dominant CAZy
families responsible for performing each step in the chitin degradation pathways are indicated next to the
arrows. Chitin‐active lytic polysaccharide monooxygenases (LPMOs) cleave glycosidic bonds in crystal-
line regions of chitin using an oxidative mechanism, producing oxidised CHOS (lactones that are in
equilibrium with their aldonic acids; pathway 1). Chitinases and β‐N‐hexosaminidases catalyse the con-
version of the oxidised products to GlcNAc and the aldonic acid GlcNAc–GlcNAc1A (chitobionic acid;
this compound is relatively resistant to hydrolysis by β‐N‐hexosaminidases [5]). The grey colour of the
aldonic acid product symbolises the low amount of this product compared to native GlcNAc. Chitinases
convert polymeric and oligomeric chitin chains to GlcNAc–GlcNAc (chitobiose), which will be con-
verted to GlcNAc by β‐N‐hexosaminidases (pathway 2). The modification of chitin through deacetylation
of the C‐2 carbon is catalysed by deacetylases and produces chitosan (which may vary in terms of the
degree of deacetylation). Chitosan will then be degraded to mixed dimeric products containing acety-
lated and deacetylated sugars (GlcN–GlcN, GlcN–GlcNAc or GlcNAc–GlcN). Further degradation of
these dimers to either GlcN and/or GlcNAc is catalysed by glucosamine specific exo‐β‐glucosaminidases.
Note that the separation of chitinases and chitosanases is not absolute; many chitinases will also cleave
a wide range of chitosans (e.g. [6]).
Enzymes for Modification of Chitin and Chitosan 191
8.1.1 Chitinases
Chitinases, enzymes that hydrolyse the β-1,4 glycosidic bonds in chitin chains, are found
in GH families 18 and 19 [7]. GH18s have a (β/α)8‐barrel fold [8, 9], while GH19 chitinases
mainly comprise α‐helices [10]. GH18 chitinases occur in a wide variety of organisms and
have been studied more extensively than GH19 chitinases, which were originally discov-
ered in plants, but also occur in actinomycetes and other bacteria. The two enzyme types
use different catalytic mechanisms. GH18s use a substrate‐assisted mechanism that is
retaining [11], the latter meaning that the anomeric configuration is retained. GH19s use a
classical inverting mechanism, that is, the anomeric configuration is inverted [12]. Bond
cleavage by inverting enzymes proceeds through a single displacement mechanism, con-
trary to the double displacement mechanism in retaining enzymes. Both mechanisms use
general acid catalysis and require a pair of carboxylic acids. One carboxylic acid acts as the
catalytic acid, whereas the other acts as a water‐activating base in the inverting mechanism
or as a nucleophile in the retaining mechanism. The retaining mechanism entails the forma-
tion of an intermediate that is hydrolysed by water that is activated by the deprotonated
catalytic acid. Both reactions proceed through oxacarbenium‐ion‐like transition states [13].
GH18 family chitinases use a special version of the retaining mechanism, namely a
substrate‐assisted mechanism (Figure 8.2), where the oxygen of the N‐acetyl group in the
substrate acts as the nucleophile, leading to the formation of an oxazolinium ion intermedi-
ate. In GH18 enzymes, the catalytic acid is a glutamate, which is located in a diagnostic
DXDXE sequence motif that occurs in all active GH18 chitinases [14–16].
Chitinases degrade chitin chains from one of the chain ends (exo‐mechanism) or from a
random point on the chain (endo‐mechanism). In addition, the endo‐ or exo‐activity can be
combined with processivity, meaning that the enzyme stays attached to the substrate after
a successful cleavage. The enzyme thus slides along the chitin chain, making several suc-
cessive cleavages before it detaches from the substrate [12, 17]. Processive and non‐processive
enzymes have been studied in detail, revealing both sequence and structural differences
that underlay processivity and its direction [17–19]. Importantly, variation in these features
is most prominent, and has been studied most extensively, in the GH18 chitinases, which
are more involved in microbial chitin turnover in nature and show more structural variation
compared to GH19 enzymes.
During growth on chitin, the well‐known chitinolytic Gram‐negative bacterium Serratia
marcescens produces mainly three chitinases (SmChiA, SmChiB, and SmChiC), of which
SmChiA and SmChiB are processive exo‐enzymes, while SmChiC is a non‐processive
endo‐enzyme. The structures (Figure 8.3) of these proteins show that SmChiA and SmChiB
have deep active site clefts, contrary to the shallow and open active site in SmChiC. The
deep clefts of the two processive enzymes are defined by several loops, and a small sub‐
domain that occurs only in a subset of GH18 enzymes (including SmChiA and SmChiB, but
not SmChiC) and that has been named the α+β domain [8]. Aromatic residues lining the
surface of the active site cleft form another characteristic feature of processive enzymes.
One of these aromatic amino acids is next to a highly conserved SXGG sequence motif,
which is followed by a Trp in processive enzymes [19]. These aromatic residues likely help
the enzyme to stay attached to the substrate as it moves along the chain. Horn et al. [6] and
Zakariassen et al. [18] mutated several of the aromatic residues in SmChiB and SmChiA,
respectively, to non‐aromatic residues and showed that some of these mutations almost
O O
Asp215 NH
O O
HO
NH
O HO O O
O O
O HO OH
HO O H
NH O O
HO
O O
HO O Tyr214
O O
N H O
Asp140 Asp140
O H O O O H
CH3
O O H OH O H
Asp142 O H
O
Asp142 O
Glu144 Glu144
NH
HO O
Asp215 HO
O
HO
O OH
O O O
OH
HO NH H H
O
O O
O H
O
HO
Tyr214
O
O
Asp140 N H O
O H O O H
Asp140 O CH3
O O H
Asp142 O H Asp142 O
O
O O
Glu144 Glu144
Figure 8.2 The substrate‐assisted mechanism used by GH18 chitinases. Binding of the substrate leads to
distortion of the sugar bound in subsite –1 towards a boat conformation. Simultaneously, Asp142 rotates
and forms hydrogen bonds with the catalytic Glu144 and the acetamido group of the sugar in the –1
subsite. At this point, Glu144 acts as a general acid by protonating the glycosidic oxygen, which supports
leaving group departure (i.e. cleavage of the glycosidic bond) that is further promoted by nucleophilic
attack of the acetamido group on the anomeric carbon, which leads to formation of an oxazolinium ion
intermediate. Glu144 then acts as a general base, activating an incoming water molecule that hydrolyses
the oxazolinium ion. The product is released from the active site and Asp142 rotates back to its original
conformation. Notably, if the water molecule hydrolysing the oxazolinium ion is outcompeted by another
acceptor, transglycosylation will occur instead of hydrolysis [23, 24]. Amino acid numbering is based on
chitinase B (SmChiB) from Serratia marcescens. The figure was obtained from [16] and permission was
granted through International Association of Scientific, Technical & Medical Publishers (STM).
(a)
(b)
(c)
Figure 8.3 Structures of Serratia marcescens chitinases. The left figures show (a) SmChiA (PDB id 1CTN),
(b) SmChiB (PDB id 1E15) and (c) SmChiC (PDB id 4AXN) in cartoon representation. The α+β domain
present in SmChiA and SmChiB is shown in pink. Extra domains that promote substrate binding in SmChiA
(FnIII) and SmChiB (CBM5) are shown in green, as is a small β‐hairpin domain in SmChiC possibly aiding
substrate binding. Note that SmChiC has an FnIII and a CBM5/12 domain, but structural data for these
domains are not available. The catalytic Glu is shown as sticks with blue carbons. The figures in the middle
show the chitinases in surface representation, with aromatic residues lining the active site highlighted in
orange and the catalytic Glu in blue. The right figures show the differences in depth of the active site clefts.
The figure was made using PyMol.
abolished processivity while maintaining or even improving catalytic activity towards solu-
ble substrates. Of note, this large and functionally relevant structural variation among the
catalytic domains of GH18 chitinases, is not observed for GH19 chitinases, although some
minor variation in the substrate binding cleft has been observed, which is due to variation
in the lengths of certain loops [20–22] (Figure 8.4).
Glu70
Figure 8.4 The structure of the GH19 chitinase BcChi‐A from Bryum coronatum. Chitotetraose, shown in
stick representation with green‐coloured carbon atoms, can be observed binding to the −2 to +2 subsites.
The catalytic base (Glu70) is shown in stick representation and is coloured with yellow carbon atoms. The
catalytic acid (Glu61) is mutated to alanine in this structure and therefore not shown. A deeper analysis of
the structure of this enzyme and its putative catalytic mechanism can be found in [22].
It is worth noting that chitinases in families GH18 and GH19 also act on chitosan, as
documented in various studies [6, 18, 25–27]. Expectedly, the efficiency of the enzymes
decreases as the degree of acetylation (FA) decreases. Furthermore, due to the substrate‐
assisted nature of the catalytic mechanism, GH18 enzymes only cleave the chitin chain
when an acetylated monosaccharide is placed in the –1 subsite.
Experimental determination of the processivity and the endo‐ or exo‐nature of a chi-
tinase is challenging. For GH18 chitinases, insight can be obtained from studies with highly
acetylated chitosan as shown [17]. When using water‐soluble chitosans, processive SmChiA
and SmChiB mainly produce even‐numbered oligosaccharides, while the endochitinase
SmChiC produces equal amounts of even‐ and odd‐numbered chitooligosaccharides
(CHOS) [17, 26, 28]. The production of even‐numbered oligosaccharides by processive
chitinases is expected, as an N‐acetyl group in subsite –1 is essential for catalysis and the
repetitive unit in chitin and chitosan is a dimer [16]. Initial productive binding of the sub-
strate to the enzyme will yield products of any length. If the enzyme is processive, it will
move by two monosaccharide moieties at a time, until a new productive complex is formed,
meaning that any further products resulting from the same initial enzyme‐substrate asso-
ciation will be even‐numbered. Non‐processive enzymes will detach and rebind in between
each reaction, thus yielding a continuum of product lengths. The exo‐ or endo‐activity of
a chitinase can be determined by measuring the reduction of viscosity during reactions
with chitosan. An endo‐enzyme cutting randomly along the chitosan chain will lead to fast
reduction of viscosity [29]. On the contrary, an exo‐enzyme cutting from the chain ends
will lead to slow reduction of viscosity [28]. Further considerations concerning the analysis
of processivity are provided in [29].
8.1.2 β‐N‐acetylhexosaminidases
The most dominant product arising from chitin degradation by GH18 and GH19 chitinases
is (GlcNAc)2. To further convert (GlcNAc)2 to GlcNAc, most chitinolytic enzyme systems
contain a GH20 β‐N‐acetylhexosaminidase (also known as chitobiase), but other enzymes
performing similar reactions are also known (e.g. in families GH3 and GH84). The chito-
biase cleaves off the non‐reducing end sugar of CHOS using a catalytic mechanism similar
to the substrate‐assisted mechanism used by chitinases [11, 30]. Since most chitinases
yield (GlcNAc)2 as their primary product, this also represents the primary substrate for
chitobiases. However, it is well‐known that chitobiases are capable of efficiently removing
GlcNAc residues from the non‐reducing end of longer CHOS [30].
The structure of the chitobiase from S. marcescens (Figure 8.5) reveals the presence of
four domains: I, II, III and IV. Domain III accommodates the catalytic site whereas the
roles of domains I, II and IV are not known. Domain I shares some features with a CBM,
but the chitobiase binds only weakly to chitin. Domains II and IV lack similarity to any
domain/enzyme with a known function [16, 31].
Interestingly, like the GH18s, the catalytic domain of chitobiase (domain III) has a (β/α)8
fold, and catalysis is retaining and substrate‐assisted. Still, the catalytic site is different in
that the catalytic acid (Glu540) is directly adjacent to the aspartate that is co‐responsible
for positioning and activating the N‐acetyl group of the sugar bound in the –1 subsite
(Asp539) (Figure 8.5). Despite these differences, the determination of the complex struc-
ture as depicted in Figure 8.5, showing a substrate spanning the active site [31] was instru-
mental in unravelling a similar mechanism in the GH18 chitinases [11]. The substrate‐binding
site of chitobiase contains aromatic and charged amino acids that are responsible for sub-
strate binding (Figure 8.5).
8.1.3 Exo‐β‐glucosaminidases
Glucosamine‐specific exo‐β‐glucosaminidases (EC 3.2.1.165) are found in families GH2,
GH9 and GH35 [32] and could play a role in the chitosan pathway for chitin degradation
(Figure 8.1, pathway 3). Family GH2 and GH35 enzymes exploit retaining catalytic
mechanisms while family GH9 enzymes use the inverting mechanism. Only a few
exo‐β‐glucosaminidases have been characterised. Among these are bacterial GH2 exo‐β‐
glucosaminidases from Amycolatopsis orientalis named CsxA [33], and a bacterial GH9,
from Photobacterium profundum named PpGlcNase [34], which have been characterised
both functionally and structurally. The structures of these exo‐β‐glucosaminidases show a
narrow substrate‐binding pocket that is shaped by negatively charged residues that are
assumed to interact with the amine group of the substrate. The GH2 exo‐β‐glucosaminidases
consist of an incomplete (β/α)8 barrel and a domain with immunoglobulin‐like topology
consisting of four β‐sandwiches. The GH9 exo‐β‐glucosaminidases consist of an N‐terminal
domain with an FnIII‐type fold and unknown function and a catalytic C‐terminal (α/α)6
barrel. The structures of these enzymes in complex with GlcN revealed distinct interactions
IV
E739 D671
R349 W685
Y669
II
+1
–1
W737
W616
III W639
D539 E540
Figure 8.5 Structure of the chitobiase from Serratia marcescens in complex with (GlcNAc)2. The overall
structure of the chitobiase (shown on the left) shows the presence of four domains; I, II, III and IV. Domain
I, II and III are associated to one another whereas domain IV protrudes relative to the other three domains.
The upper right panel shows the substrate binding pocket (shown as a light grey surface) and (GlcNAc)2
(magenta‐coloured sticks); the sugar bound in the –1 subsite, that is the non‐reducing end of the chitobi-
ose substrate, is buried deep in the protein, whereas the more visible sugar bound in the +1 subsite is
exposed to the solvent. The lower right panel shows details of the interaction between (GlcNAc)2 and cata-
lytic site residues. The (GlcNAc)2 is shown as magenta‐coloured sticks and residues from the chitobiase
are shown as light grey sticks. The catalytic acid (E540) is shown in yellow in all figures. The figure was
obtained from [16] and permission was granted through STM.
of active site residues with the substrate. CsxA (Figure 8.6a) displays a dominance of acidic
residues interacting with the hydroxyl groups and the amino group of the GlcN moiety. In
PpGlcNase, interactions involving aromatic residues seem to play a more prominent role
(Figure 8.6b).
In addition, a fungal GH2 exo‐β‐glucosaminidase from Trichoderma reesei and two
GH35 exo‐β‐glucosaminidases from the hyper‐thermophilic archaea Pyrococcus horiko-
shii and Thermococcus kodakarensis have been functionally characterised. The two
archaeal GH35 enzymes are homodimeric enzymes. All reports on the functionality of
exo‐β‐glucosaminidases belonging to families GH2, GH9 and GH35 conclude that the
enzymes act at the non‐reducing end of CHOS [33–37].
Interestingly, extensive studies of various enzymes from T. kodakarensis have led to the
conclusion that this microbe indeed uses the ’chitosan’ pathway for chitin saccharification
(see Section 8.4.3). In short, the organism degrades chitin to GlcNAc–GlcNAc using a
chitinase and the non‐reducing terminal sugar is then specifically deacetylated by a
(a)
D203 E394
O
O H D469
O
W642 O
H
NH O OH
O
NH3
OH
H O
N O
O O O
W204 E541
D469
E394
E541
(b) E555
Q448 (acid)
Y445 N524
D143
(base)
W446
H150 W557
F231 D139
Q161
Y147
Figure 8.6 Structure of the active sites of CsxA (a) and PpGlcNase (b) in complex with GlcN. Panel A
shows the structure of CsxA in a complex with GlcN. The left figure is a schematic illustration of how the
enzyme interacts with GlcN; H‐bonds are indicated by dashed lines. The right‐side figure shows the pocket
accommodating the GlcN; the side chains of three acidic residues are shown in yellow and the grey
dashed lines represent H‐bonds with distances shown in angstrom. Asp469 is the catalytic acid/base and
Glu541 is the catalytic nucleophile. Figure source: [33], permission of use granted by the publisher. Panel
B shows the superimposed structures of PpGlcNase in a complex with GlcN complex (green) and of ligand
free PpGlcNase (magenta). H‐bonds are indicated by dashed lines. Glu555 acts as the catalytic acid (pos-
sibly via a water molecule), and Asp143, with help from Asp139, acts as the catalytic base. The figure has
been obtained from [34] and permission is granted through STM.
d eacetylase, producing GlcN–GlcNAc. The product is then hydrolysed by the exo‐β‐

glucosaminidase to mono‐sugars GlcN and GlcNAc [36, 38].
8.1.4 Chitosanases
Chitosanases are found in the GH5, GH7, GH8, GH46, GH75 and GH80 families of the
CAZy database [32, 39]. Among these, families GH5, 7 and 8 contain a few chitosanases,
while families GH 46, 75 and 80 exclusively contain chitosanases [39]. GH 5 and 7 chi-
tosanases utilise retaining catalytic mechanisms, while enzymes belonging to families
(a) (b)
Asp55 Asp40 Glu309
Glu122
Glu22
Glu37
Figure 8.7 Structure of family GH46 chitosanases. Panel A shows the structures of GH46 enzymes from
Bacillus sp. MHK1 (left) and from Streptomyces sp. N174 (right); the arrows indicate the substrate binding
clefts. The extra loop protruding from the ’upper’ domain of the MHK1 chitosanase is coloured yellow. See
the text and [46] for more details. Panel B shows the structure of a GH8 chitosanase from Bacillus sp. K17.
This structure comprises a double‐(α/α)6 barrel. The side chains of the catalytic residues are shown in stick
representation and labelled.
GH 8, 46, 75 and 80 use inverting mechanisms. Regarding taxonomical classification,

families GH5 (the most diverse of the GH families) and GH75 contain chitosanases from
bacteria and eukaryotes, GH7 only contains enzymes from eukaryotes, while families GH8
and GH80 only contain bacterial chitosanases. The majority of characterised chitosanases
belong to the family GH46, and members of this family come from a wide variety of
sources, including archaea, bacteria, eukaryota and viruses [32, 40].
Bacterial chitosanases have been described for a wide variety of species, including
Serratia sp. [41], Acinetobacter sp. [42], Bacillus sp. and Streptomyces sp. Among these,
chitosanases from Bacillus and Streptomyces species, which are members of the GH5,
GH8 and GH46 families, have been studied most extensively, addressing both functional
and structural properties [43–46]. Based on their substrate cleavage‐site specificities, chi-
tosanases are classified into three subclasses. Subclass I includes a relatively well‐studied
GH46 chitosanase from Streptomyces sp. N174 and contains enzymes cleaving both GlcN–
GlcN and GlcNAc–GlcN bonds [47]. Subclass II includes the GH8 chitosanase from
Bacillus sp. K17, which is specific for GlcN–GlcN linkages [48]. Subclass III includes the
GH46 chitosanase from Bacillus sp. MHK1 and enzymes in this class split both GlcN–
GlcN and GlcN–GlcNAc [49]. Notably, this classification is not ’perfect’. The family
GH46 chitosanase from Streptomyces coelicolor, ScCsn46A, was characterised in detail by
[50], who showed that the enzyme prefers GlcN–GlcN bonds but can also cleave GlcN–
GlcNAc and GlcNAc–GlcN bonds. Similar observations have been made for a GH46 chi-
tosanase from Bacillus [51].
Structural characterisation of the GH46 chitosanases from Streptomyces sp. N174 and
Bacillus sp. MHK1 showed similar overall structures mainly composed of α‐helices
(Figure 8.7). The substrate‐binding site of the enzymes occurs at the interface of two
structural domains named the upper and lower domain. Figure 8.7 shows that, despite
similarities, the substrate‐binding site clefts and catalytic centres of the two enzymes
show distinct differences. The MHK1 enzyme has an additional loop in the upper domain
that makes the substrate‐binding site less open compared to the N174 enzyme. The sub-
strate binding clefts of both enzymes contain carboxylic amino acid side chains which can
interact with the positively charged amino‐groups of the substrate. The catalytic acid and
base for both enzymes are a Glu and an Asp, respectively, where the distance between
these residues is about 10 Å, which is a typical distance for enzymes with an inverting
mechanism [45, 46, 52, 53].
The crystal structure of a family GH8 chitosanase from Bacillus sp. K17 shows an (α/α)6
double barrel and long loops containing small β‐strands and 310 helices that protrude from
the helices and form a substrate binding cleft and catalytic centre on top of the barrel.
Despite structural differences between the GH46 and GH8 enzymes, the distance between
the catalytic residues is similar in all these chitosanases. Similar to the N174 and MK1
chitosanases, the substrate‐binding cleft of the K17 chitosanase contains acidic residues
that are likely to interact with positive charges on the substrate. Aromatic residues with the
potential to interact with the substrate are also present in the active cleft [43].
8.1.5 Lytic Polysaccharide Monooxygenases

In 2005, Vaaje‐Kolstad and colleagues showed that a chitin‐binding protein produced by S.
marcescens, named CBP21 (also known as SmLPMO10A, the name used in the rest of the
text) and originally classified as a CBM33 contributed to chitin degradation by strongly
boosting chitin solubilisation by chitinases [54]. At the time, SmLPMO10A was assumed
to have no catalytic activity, but rather acts as a ’helper protein’ (e.g. ’substrate‐disrupting
protein’) for chitinases in the chitin degradation process. However, in 2010, it was shown
that SmLPMO10A was an enzyme capable of cleaving chitin chains by an oxidative mech-
anism [4]. The reaction products of these enzymes were found to contain a single oxygen
derived from dioxygen [4], and, therefore, the enzymes were named LPMO [55].
The most remarkable feature of these oxidative enzymes is their ability to cleave poly-
saccharide chains that are embedded in a crystalline environment, something that is both
sterically and energetically difficult for canonical hydrolytic enzymes such as chitinases
and cellulases. By making nicks on the surface of the polysaccharide crystals, LPMOs
likely disrupt the crystal surfaces and provide attachment points for the hydrolytic enzymes,
which explains the synergistic effects that are observed when combining LPMOs and
hydrolytic enzymes [4, 56–58]. LPMOs cannot use the catalytic power of substrate distor-
tion inflicted by the extended binding clefts of hydrolytic enzymes, as in the example of
GH18 chitinases (see Section 8.1.1), but use instead powerful redox chemistry facilitated
by a catalytic centre that contains a copper ion coordinated by two fully conserved histidine
residues and the N‐terminal amino group of one of these histidines (Figure 8.8).
The discovery of LPMOs led the CAZy team to create the auxiliary activity class, which
today contains 15 families. LPMOs are categorised in AA families 9, 10, 11, 13, 14 and 15
and are found in several organisms, including bacteria, fungi, viruses and higher eukary-
otes like insects [3]. Chitin‐active LPMOs are found in AA families 10, 11 and 15. So far,
chitin‐active fungal LPMOs have only been described for the AA family 11. Family AA15
only contains LPMOs from insects and viruses.
LPMO activity was originally discovered for chitin but it was immediately obvious that
similar enzymes acting on cellulose would exist, in particular, enzymes that were at the
time erroneously classified as GH61 [4]. Since the discovery of the first chitin‐active
LPMOs, LPMOs acting on cellulose have gained much attention due to their industrial
relevance [59–63]. LPMOs show different oxidative regioselectivities and these differ
between chitin‐active and cellulose‐active LPMOs (Figure 8.8). While only C1 oxidising
chitin‐active LPMOs have been described, cellulose‐active LPMOs can be strictly C1
OH Lactone
O
(a)
O C1-oxidation R
OH H2 O 2 HO
NH
or O
O NH
R HO R O2 + 2H+ + 2e– O
HO O
NH O LPMO O
AA10
OH
O AA11 NH
C4-oxidation ? HO R
AA15
O
O
(b) OH
Figure 8.8 Catalytic centre and reaction mechanism of LPMOs. (a) Reaction mechanism of chitin‐active
LPMOs showing possible C1 and C4 oxidised products (see text for details; C4 oxidation of chitin has not
been observed for chitin‐active LPMOs, but has been documented for other substrates with a β‐1,4 glucose
backbone, such as cellulose). (b) The catalytic centre in LPMOs exemplified by BaLPMO10A from Bacillus
amyloliquefaciens (PDB id 2YOX) showing the T‐shaped coordination (called the histidine brace; [59]) of
the Cu‐ion (brown sphere). The two conserved histidines, coordinating with the Cu‐ion, are shown as
sticks with pink‐coloured carbon atoms. One of these histidines is the N‐terminal residue of the protein.
oxidising, strictly C4 oxidising, or be able to oxidise both the C1 and C4 position [64, 65].
Independent of regioselectivity, LPMOs depend on copper‐reducing equivalents [59, 61] to
reduce this copper and dissolved molecular dioxygen in order to perform catalysis.
However, in a recent publication, Bissaro et al. (2017) [66] questioned the validity of diox-
ygen being the LPMO co‐substrate and provided compelling evidence indicating that
hydrogen peroxide (H2O2) is the true co‐substrate of LPMOs. Notably, LPMOs can them-
selves generate H2O2 from O2 [67].
8.1.6 Carbohydrate Esterases

CEs are enzymes catalysing the O‐ or N‐deacetylation of substituted saccharides and of the
16 CE families known to date (September 2019). CEs deacetylating chitin and its deriva-
tives are only found in CE families 4 and 14 [32]. The CE14 family only contains a few
characterised chitin deacetylases, all being archaeal, deacetylating the non‐reducing end of
(GlcNAc)2 as part of the chitinolytic pathway of the organism [see Section 8.4.3 [38, 68]].
The CE4 family comprises several bacterial and eukaryotic esterases that deacetylate
GlcNAc units in peptidoglycan, chitin, chitosan and oligomers of chitin and chitosan
His417 His417 His417
O HN N+ O HN N+ H O HN N R
H R
– HN – R – H2N
HN
O O O
Asp391 C HN Asp391 HN Asp391 CH3 HN
O
H3 H3C O
Tyr367 Tyr367 Tyr367
Asp275 H OH Asp275 OH Asp275 OH O
O OH OH
–
O O O
H2N Zn H 2N + Zn H2N Zn
+ His326 +
NH2 Asp276 NH2 Asp276 His326 NH2 Asp276 His326
HN Arg364 His330 HN Arg364 His330 HN Arg364 His330
Figure 8.9 Reaction mechanism of CE4 deacetylases. The figure shows a proposed acid/base reaction
mechanism for CE4 deacetylases that remove N‐acetyl groups. The amino acid numbering is based on
SpPgdA. See the main text for a detailed description of the catalytic mechanism. The figure was obtained
from [76]. Copyright (2005) National Academy of Sciences, U.S.A.
(CHOS). The CE4 family also contains enzymes capable of removing O‐linked acetyl
groups from acetyl xylan [69] and several family members are known to act both on xylan
(O‐deacetylation) and chitin (N‐deacetylation [70–73]). The activity and structural features
of CE4 deacetylases are discussed in more detail in the following text.
The first deacetylase acting on chitin was found in extracts from the fungus Mucor rouxii
[74]. However, a few years earlier, Araki and co‐workers described an enzyme from
Bacillus cereus that deacetylates GlcNAc units in peptidoglycan [75]. Both these enzymes
are today classified in the carbohydrate esterase family 4. CE4 deacetylases removing the
N‐acetyl group from GlcNAc units share five conserved sequence motifs: motif 1, T(F/x)
DD; motif 2, H(S/T)xxH; motif 3, R(P/x)PY; motif 4, (Dxx)D(W/Y); motif 5, LxH [76].
Blair et al. (2005) [76] first proposed a catalytic mechanism for family CE4 deacetylases,
which, notably, depends on a bound metal ion, preferably zinc or cobalt [76–78]. They sug-
gested a general acid/base reaction mechanism based on an extensive structural analysis
(Figure 8.9). In this catalytic cycle, the catalytic base (the first Asp in motif 1) activates a
metal‐bound water molecule, which subsequently performs a nucleophilic attack on carbon
in the scissile C‐N bond creating a tetrahedral oxyanion intermediate. The metal ion and
backbone nitrogen of tyrosine in motif 3 stabilises the negative charge on the carbonyl
oxygen. The catalytic acid (His in motif 5) protonates the nitrogen in the substrate generat-
ing a free amine on the deacetylated product and leads to release of acetate [76, 78].
Motif 1, 2, and 5 are highly conserved between different deacetylases, while motif 3 and
4 display more sequence variation (Figure 8.10). The Asp‐His‐His metal binding triad is
located in motif 1 and 2 (Figure 8.10 and Figure 8.11, panel A). Motif 3 and 4 form one side
of the active site groove each (Figure 8.11, panel A [76, 78]).
As shown in Figure 8.10, some CE4 members have big insertions, representing loops
that are located close to the active site (Figure 8.11, panel B). Andrés et al. (2014) [78]
proposed a ’subsite capping model’ involving six loops (indicated in Figure 8.10) that each
cap parts of the active site cleft. Such loops would contribute to substrate specificity and
could endorse the deacetylase with sequence specificity because they could define which
substrates bind to the enzymes and which GlcNAc unit in the substrate becomes deacety-
lated. The family CE4 representative, VcCDA, from Vibrio cholera, is special in that these
loops are particularly long and form a buried active site (Figure 8.11, panel B). It was fur-
ther suggested that these loops may rearrange depending on the length of the substrate [78].
Figure 8.10 Structure‐based sequence alignment of CE4 enzymes. The five conserved sequence motifs
are indicated with dark purple background. The yellow asterisks indicate the metal binding triad, while a
red triangle and circle indicate the catalytic base and acid, respectively. The deacetylases included in the
alignment are: SpPgdA, peptidoglycan deacetylase from Streptococcus pneumoniae [77]; AnCDA, chitin
deacetylase from Aspergillus nidulans [73]; SlCE4, acetyl xylan deacetylase from Streptomyces lividans
[77]; BsPdaA, peptidoglycan deacetylase from Bacillus subtilis [84]; ClCDA, chitin deacetylase from
Colletotrichum lindemuthianum [80]; VcCDA, chitin deacetylase from Vibrio cholerae [78]. Loop number-
ing and colouring was taken from [78]. The alignment was prepared using PyMod 1.0 [85].
Indeed, VcCDA is a highly specific enzyme that is restricted to deacetylate the GlcNAc
next to the non‐reducing end in CHOS. It is important to note that most other deacetylases
in this family, including the other structurally characterised ones (Figure 8.10), have shorter
loops and, hence, more open active sites (Figure 8.11, panel C). These enzymes generally
show less specificity compared to VcCDA, deacetylating a variety of substrates at several
positions [73, 76, 79–81]. Hence, CE4 deacetylases are generally considered to have broad
substrate specificity [70, 73]. For example, a deacetylase from Aspergillus nidulans
(AnCDA), having an open active site, is able to deacetylate all GlcNAc units in a chitohexa-
ose and also shows activity towards acetyl xylan [73].
The GlcNAc residue to be deacetylated binds in subsite 0, and apart from the interac-
tions between the enzyme and substrate in subsite 0, little experimental data for enzyme‐
substrate interactions of other subsites exist. The crystal structure of VcCDA in complex
with (GlcNAc)2 and (GlcNAc)3 was determined by Andrés et al. (2014) [78]. The dimer
occupies subsite 0 and –1, while the trimer occupies subsite –1 to +1, with the non‐reducing
end in subsite –1 in both cases. VcCDA makes several interactions with the sugar in subsite –1,
and binding of a sugar in subsite +1 requires rearrangement of several loops to allow its
binding. As pointed out above and illustrated in Figure 8.11, the structure of VcCDA is very
(a) (b)
MT3
MT4 MT2
MT5
MT1
(c) (d)
W148
H173
D32
D33 Y123
H82
H86
Figure 8.11 Structure of CE4 deacetylases. (a) ClCDA in cartoon representation zooming in on the
active site. The side chains of the most important residues from the five catalytically important sequence
motifs are shown as sticks. The metal ion (blue sphere) is coordinated in an octahedral fashion (black
dashed lines) by the metal binding triad, a water molecule (red sphere), and an acetate ion (sticks with
pink carbons). (b) Surface representation of VcCDA (PDB id 4NY2) highlighting the loops that are pro-
posed to be involved in the substrate‐capping model. (c) Surface representation of ClCDA (PDB id
2IW0), showing the open active site. The loops are coloured according to the colour scheme used in
Figure 8.10: loop 1 in yellow, loop 2 in blue, loop 3 in red, loop 4 in orange, loop 5 in green and loop 6
in black. The metal ion is shown as a grey sphere. (d) The active site of ArCE4A in cartoon representation
with the GlcNAc2 substrate (yellow‐coloured carbon) bound in the active site. The active site residues
involved in substrate binding and catalysis are shown in stick representation with grey‐coloured carbon
atoms. Black dashed lines indicate hydrogen bonds. The figure was made using PyMol [86].
different compared to other CE4 enzymes with a known structure. Blair et al. (2006) [80]
performed a docking of (GlcNAc)3 bound in subsite –1 to +1 in ClCDA, which indicated
that there are no interactions between the enzyme and sugar bound in subsite –1.
This is different from what Andrés et al. (2014) [78] found for VcCDA. Recently, the
structure of ArCE4A was determined in complex with (GlcNAc)2 occupying subsite
0 and +1 (Figure 8.11D [82]). Interestingly, the authors used (GlcNAc)4 as substrate in the
crystallisation, however, no interactions between the enzyme and substrate was observed
beyond subsite 0 and +1, similar to the findings in [83].
From an applied point of view, utilisation of deacetylases for production of chitosans
and CHOS with a defined degree of polymerisation (PA) is of great interest since the PA
together with the FA influences the biochemical properties of chitosan and CHOS [87, 88].
In this context, VcCDA and NodB from Rhizobium sp. GRH2 have been utilised to pro-
duce CHOS that are deacetylated specifically at the non‐reducing end and the neighbour-
ing sugar unit [88]. Recently, a fungal deacetylase from Puccinia graminis was shown to
deacetylate all GlcNAc units in different CHOS, except the two at the non‐reducing end
[89], thus providing another type of specificity. Importantly, Hembach et al. have recently
demonstrated the production of a series of CHOS with defined sequences by using com-
binations of deacetylases with varying specificities [81]. Notably, the use of deacetylases
to directly convert non‐soluble polymeric chitin into soluble polymeric chitosan has been
regularly presented as an attractive biocatalytic scenario, but has not yet been
accomplished.
8.1.7 Carbohydrate‐Binding Modules

In recalcitrant polysaccharides such as chitin and cellulose, the substrate is often difficult
to access for CAZymes. Many CAZymes have solved this problem by including one or
several non‐catalytic CBMs that promote association of the enzyme to polysaccharide and
which may also contribute to correct (’productive’) positioning of the catalytic module
[90]. A recent study showed that the beneficial effect of CBMs on enzyme efficiency is
reduced at higher substrate concentrations, underpinning their role in substrate binding
[91]. Currently (September 2019), 85 different CBM families exist, and chitin‐binding
CBMs are found in families 1, 2, 3, 5, 12, 14, 18, 19, 37, 50, 54, 55 and 73 [32]. CBM fami-
lies 5 and 12 are distantly related and often referred to as CBM5/12. Of the different CBM
families, the CBM50 family contains the most number of entries. CBM50 proteins are also
known as LysM domains, which bind to various GlcNAc containing carbohydrates such as
peptidoglycan, chitin and CHOS [92, 93].
In addition to the CAZy classification, CBMs have been divided into three types based
on structural and functional similarities: type A, surface‐binding CBMs; type B, glycan‐
chain‐binding CBMs; type C, small‐sugar‐binding CBMs [90]. The binding of CBMs to
crystalline surfaces involves aromatic residues on the binding surface of the module, and
several papers have demonstrated the importance of these aromates for the activity of the
appended catalytic domains towards crystalline substrates (e.g. [94–97]). CBMs are not
usually considered to be important for enzymatic efficiency towards oligomeric substrates
and, indeed, studies have shown that the aromatic residues in the binding surface of a CBM
are not important for the activity of the appended catalytic domain towards such short (and
soluble) substrates [94, 96, 98].
8.2 Modular Diversity in Chitinases, Chitosanases and LPMOs

The protein family (Pfam) database is a valuable tool for obtaining information on protein
families and protein domain structures based on evolutionary relationships between
sequences [99]. Many chitinases, chitosanases and LPMOs contain one or more additional
domains and the various domain compositions can be extracted from Pfam.
The most common domains appended to these enzyme types are non‐catalytic domains
known as CBMs. CBMs contribute to substrate binding and make sure that the enzyme
does not wander too far off from its substrate [90, 100, 100]. The positive impact of CBMs
on overall enzyme efficiency is well documented in literature.
Other domains often found include the fibronectin type III (FnIII) domain, an evolution-
ary conserved domain found in many extracellular enzymes [102, 103]. This domain is
found in chitinases, chitosanases, LPMOs and many other GHs (Figure 8.12), but its role
is not always well established and may relate both to substrate‐binding and positioning of
the other domains [98, 99, 104].
Interestingly, a wide variety of non‐catalytic and catalytic domains may be appended to
chitinases, chitosanases and LPMOs and the function of some of these domains is unclear.
For example, the RicinB lectin occurring in some chitosanases was originally found in
seeds of castor bean, Ricinus communis. RicinB lectin domains are found in a wide variety
of CAZymes and also in proteases and are known to bind mono‐sugars, lactose and galac-
tose [105–107]. The F5/F8 type domains seen in some chitosanases, also known as discoi-
din domains, are found in many blood coagulation factors [108]. DUF 2272 and 4157 are
domains of unknown function.
The domain architectures shown in Figure 8.12 also demonstrate that several GH
domains may occur in the same protein, which is not uncommon. Only a few such enzymes
have been studied, but the general impression is that such multi‐catalytic enzymes may be
quite effective [109, 110]. Furthermore, Figure 8.12 shows that GH domains may also be
coupled to other catalytic domains that are not GHs or LPMOs. The PD1 (Polysaccharide
deacetylase 1) domain is a family CE4 CAZyme containing the NodB domain and likely
responsible for deacetylation of CHOS. The GT2 (glycosyltransferase family 2) domain
likely synthesizes glycosidic bonds and family GT2 members include cellulose, chitin and
CHOS synthases [111]. PGB (peptidoglycan binding) domains occur in many enzymes
involved in bacterial cell‐wall degradation [112, 113]. CHAP (cysteine, histidine‐dependent
amidohydrolases/peptidases) domains are found in several amidase families and are
assumed to hydrolyse peptidoglycans [114, 115]. Most of the enzymes having complex
domain architectures discussed in this section have not been biochemically characterised,
which implies that enzyme diversity available for chitin conversion and modification still
remains a large untapped resource with a great future potential.
8.3 Biological Roles of Chitin‐Active Enzymes

Chitin‐active enzymes are important for all organisms that contain or metabolise chitin.
From a biotechnological and biorefining point of view, the conversion of chitin to soluble
building blocks for subsequent chemical or fermentative processes is perhaps the most
central. In addition to the metabolic (degradative) function of chitin‐active enzymes, a
multitude of other functions have been described. Chitin‐active enzymes are widespread in
nature, found in all domains of life and the biological roles of these proteins vary.
Humans, although devoid of chitin, have two GH18 chitinases encoded in the genome:
human chitotriosidase [116] and acidic mammalian chitinase [117]. The human chitotriosi-
dase plays a role in the innate immune system against chitin‐containing pathogens [118],
(a)
(b)
(c)
(d)
Figure 8.12 Schematic illustration of modular architectures of chitinolytic enzymes. GH18 chitinases
(red; a), GH19 chitinases (orange; b), GH8, GH46 and GH75 chitosanases (black; c) and AA10 LPMOs
(green; d). The figure was made by gathering data from the Pfam database. Pfam accessions numbers:
GH18, PF00704; GH8, PF01270; GH46, PF01374, GH75, PF07335, AA10 and PF03067. The numbers
shown in parenthesis indicate the number of sequences found in the Pfam database with the shown modu-
lar architecture. See text for further details.
while the acidic mammalian chitinase has gained attention due to its possible link to the
pathophysiology of asthma [119]. In fungi, chitinases are postulated to have a wide variety
of functions including degradation of exogenous chitin for nutrition, remodelling of the
(own) fungal cell wall (which contains chitin), and defence against other fungi and arthro-
pods [120]. Fungal chitinases have also been proposed to act as virulence factors in insect
pathogenic fungi [121]. The number of chitinases encoded by fungi varies from 1 to over
30, making it an extensive task to determine the exact role of each chitinase [122, 123]. In
plants, chitinases, primarily of the GH19 type, are important in the defence against fungal
attacks [124, 125]. Bacteria generally use chitinases to degrade chitin for utilising it as a
nutrient source [126, 127]. However, there are indications that bacterial chitinases have
additional roles, based on putative activities on non‐chitin substrates such as glycoproteins
[128, 129]. Of note, the Listeria monocytogenes chitinase, ChiA, has been shown to con-
tribute to suppression of host innate immunity [130].
Chitobiases have several biological roles, dependent on the organism and even the cell
type. In addition to participating in chitin catabolism, several additional biological func-
tions of bacterial chitobiases have been proposed. For example, a chitobiase in Escherichia
coli contributes to cell wall recycling by hydrolysing the β‐1,4‐linkage between GlcNAc
and anhydro‐N‐acetylmuramic acid [131]. In the biofilm‐forming bacterium
Actinobacillus actinomycetemcomitans, a chitobiase is important for detachment of cells
from the biofilm in order to enable spreading of the biofilm to other surfaces [132]. In
fungi, chitobiases have several biological roles, especially in controlling the composition
of chitin in the cell wall. Chitobiase activity is also important for nutrient release during
the saprophytic and mycoparasitic growth phases of fungi and may be involved in insect
pathogenesis [132].
Chitosanases are found in bacteria, fungi and plants, having different biological func-
tions [133]. Some organisms are thought to secrete chitosanases to degrade chitosan and
utilise this as a nutrition source [134]. Saito et al. (2009) [135] demonstrated that a GH46
chitosanase from Amycolatopsis sp. CsO‐2 has antifungal activity against Rhizopus oryzae
and could thus be part of a defence system. Chitosanases may also have a role in protection
against the well‐known antimicrobial activity of chitosan and CHOS [136].
Chitin‐active LPMOs are mostly involved in the process of chitin catabolism (i.e.
making crystalline chitin more amenable to enzymatic depolymerisation), but some are
(also) suggested to act as virulence factors. Listeria monocytogenes possesses two
chitinases and an LPMO is possibly involved in the pathogenesis of this bacterium
[57, 137]. In V. cholerae, an LPMO termed GbpA binds mucin, and thereby enhances
bacterial colonisation of the intestine [138]. Mucin consists of glycoproteins, which are
glycosylated with different carbohydrates including GlcNAc [139]; it is not yet known
whether the LPMO domain of GbpA acts on the mucins, but its activity on chitin has
been proven [5]. Another example of an LPMO potentially involved in bacterial viru-
lence is SmLPMO10A from S. marcescens. Kawada et al. (2008) [140] showed that
knocking out this protein significantly decreased adhesion of the bacterium to colonic
epithelial cells, and suggested that SmLPMO10A and similar proteins are involved in
bacterial adhesion to such cells.
Deacetylases in the CE4 family are believed to have a role in pathogenesis of Gram‐positive
bacteria, since they, by modifying the peptidoglycan layer, make the bacteria less sus-
ceptible to the host innate immune system [141, 142]. The CE4 peptidoglycan deacetylase
of S. pneumonia (SpPgdA) acts as a virulence factor by deacetylating GlcNAc residues in

peptidoglycan, thereby obstructing lysozyme activity of the host. Knocking out the PgdA
gene made the bacterium lysozyme sensitive [143, 144]. In fungi, CE4 deacetylases are
thought to have a similar role in pathogenesis by deacetylating the chitin in the fungal cell
wall, creating a poorer substrate for chitinases secreted by the organisms that they are
attacking, such as plants [145, 146]. It has also been shown that deacetylases are required
for yeast spore wall formation [147, 148], which indicates that remodelling of chitin plays
a role in this process. The heterodimer GlcNAc–GlcN, produced by highly specific chitin
deacetylases in Gram‐negative Vibrio species, induces the production of chitinases by
functioning as a signal molecule in the catabolic chitin cascade [149]. Deacetylases in
Vibrios and Photobacteria produce CHOS with a deacetylated sugar next to the penulti-
mate GlcNAc, and the resulting products, GlcNAc–GlcN–(GlcNAc)n [150], resemble those
produced by NodB, another specific CE4 deacetylase producing GlcN–(GlcNAc)3‐4 [142].
The products from NodB are intermediates in the biosynthesis of Nod factors, which are
important in the communication between symbiotic nitrogen fixing bacteria and plants
[142]. This suggests that the products produced by deacetylases in Vibrios and Photobacteria
could be important in cellular signalling [142, 150]. Due to the earlier mentioned biological
functions of CE4 deacetylases, they are interesting targets for biological control of
pathogenesis.
In addition to the important role of CBMs in promoting substrate binding when associ-
ated with catalytic domains, many other biological roles of CBMs have been suggested,
including roles in virulence which relate to the ability of CBMs to bind to carbohydrate
structures in the host [151]. Although CBMs often are associated with catalytic domains
hydrolysing different polysaccharides, they can also exist as single domains or with cata-
lytic domains targeting non‐polysaccharide substrates. For example, the plant pathogenic
fungus Cladosporium fulvum secretes Av4, a CBM14 protein, which binds specifically to
chitin in fungal cell walls, protecting the fungi from host chitinases during infection [152,
153]. In the same fungus, a LysM domain (CBM50) mediates virulence through perturba-
tion of chitin‐triggered host immunity [154].
8.4 Microbial Degradation and Utilisation of Chitin

Chitinolytic microbes are widespread in nature and are able to degrade chitin under both
aerobic and anaerobic conditions. In general, chitin catabolism is achieved by microorgan-
isms through secretion of a set of chitinolytic enzymes that attack the insoluble chitin
generating shorter soluble CHOS that are imported into the cell and processed further by
other enzymes for utilisation as a nutrient source. The strategy used by the microbe seems
to depend on the environmental conditions in which the microbe dwells. For example, in a
comparative study of chitinolytic proteins encoded by bacterial genomes performed by Bai
et al. (2016) [155], substantial differences were found when comparing aquatic and ter-
restrial bacteria. The modular composition of chitinases differed substantially between the
two habitats and terrestrial bacteria seemed to be more adapted to various chitin sources,
having a higher number of chitinase genes, higher diversity of associated CBMs and a
higher number of LPMOs [155]. Different microorganisms have developed different sys-
tems for chitin modification and utilisation, and a few of such systems are discussed in
detail in the following text.
–1 +1
Chitobiase
Chi
–1 B ChiA
+1 +1
–1
CBM
5/12 CBP21 Fnlll
NR R
–1 +1
GlcNAc
ChiC
GlcNAcA
Figure 8.13 The chitinolytic machinery of Serratia marcescens. The chitinases SmChiA, SmChiB, and
SmChiC hydrolyse glycosidic bonds in different fashions. SmLPMO10A (named CBP21 in the figure) is an
LPMO making oxidative cuts in the crystalline parts of the chitin. The chitobiase degrades soluble products
generated by the chitinases and LPMO into monomers. Note that SmChiC has an FnIII and a CBM5/12
domain and that the chitobiase is a four‐domain protein; see text for details. Figure is obtained from [16]
and permission is granted through STM.
8.4.1 Chitin Degradation by Serratia marcescens

S. marcescens is a Gram‐negative bacterium and one of the most studied chitinolytic bac-
teria. The SmLPMO10A protein from S. marcescens BJL200, originally classified as
CBM33, was the first of its kind to be identified as a lytic polysaccharide monooxygenase
[4, 54]. The chitinolytic machinery of S. marcescens (Figure 8.13) consists of three chi-
tinases (SmChiA, SmChiB and SmChiC), SmLPMO10A and a chitobiase. SmChiA and
SmChiB are predominantly processive exo‐acting enzymes degrading the chitin from the
reducing and non‐reducing end, respectively [8, 156–158]. Contrary to SmChiA and
SmChiB, SmChiC is a non‐processive endo‐acting enzyme [17, 159]. As early as in 2005
[54], it was shown that SmLPMO10A promotes chitinase activity on chitin, and since 2010
[4], we know that SmLPMO10A is an LPMO that makes oxidative cleavages in crystalline
regions of the substrate, creating new ends for the chitinases to bind to [16]. The main
product produced by the chitinases is (GlcNAc)2, which is cleaved into monomers by a
GH20 chitobiase (Figure 8.13).
As described in Section 8.2, catalytic domains degrading crystalline polysaccharides
often have CBMs to aid in substrate binding. Accordingly, SmChiA has an N‐terminal FnIII
module that contributes to substrate binding and improves catalytic activity [8, 98], while
SmChiB has a C‐terminal CBM5/12 module [156]. SmChiC has two C‐terminal domains,
one FnIII and one CBM5/12 module. The culture supernatant of S. marcescens grown on
chitin commonly contains two versions of SmChiC that are commonly found, C1 and C2.
The C1 version contains the catalytic domain and two C‐terminal domains, while the C2
version consists of the catalytic domain only [126, 160].
When grown on chitin, S. marcescens secretes all the three chitinases, the chitobiase and
SmLPMO10A [161–164]. Interestingly, SmChiB and SmChiC lack a conventional signal
peptide. Hamilton et al. (2014) [164] showed that secretion of the chitinolytic machinery in
S. marcescens Db10/11 is dependent on a holin‐like protein and an endopeptidase. However,
despite these findings, the exact secretion mechanism and the role of the holin/endopepti-
dase system remain unclear. Interestingly, the genes encoding SmLPMO10A and SmChiB
are localised in a gene locus dedicated to chitin metabolism. This gene locus also contains
the holin and endopeptidase genes, a LysR transcriptional regulator involved in regulation
of chitinase expression and two spanin genes [164, 165]. The chitobiase, whose expression
is induced by the presence of (GlcNAc)2, is located in the periplasm [166], although small
amounts of the enzyme have been detected in culture supernatants [167]. Figure 8.14 shows
a proposed model for the chitin utilisation pathway of S. marcescens. Generally, the
Chitin
O
OH
NH H
H O
O HO O
HO O
O
NH
OH
O n
SmChi18A, B- & C
Extracellular
SmLPMO10A
O
OH OH
NH O
O
HO HO OH Chitobiase HO
HO O HO OH
O NH
NH
OH
O O
Putative chitoporin Putative chitoporin
O OH
OH
O
Periplasm
NH
O
HO OH Chitobiase HO
HO OH
HO
HO O NH
O
NH
OH O
O
PTS permease PTS permease

OH
HO O OH
P
O
putative
HO O
O phoshpo- OH P
O NH chitobiase O O
HO OH HO
HO
Cytoplasm
HO O HO OH O
O NH
NH HO
HO O
OH NH
O O
O
GlcNAc
kinase
Aminosugar
catabolism
Figure 8.14 Catabolic pathway for chitin utilisation in Serratia marcescens. Dashed arrows indicate
minor pathways. It should be noted that the oxidised CHOS formed by the activity of the LPMO and
SmLPMO10A are not shown in this figure. The catabolic fate of oxidised CHOS (i.e. aldonic acids) is not
known. For a detailed discussion of chitin catabolism by S. marcescens, see [167].
c atabolic pathway in S. marcescens is similar to the chitinolytic machineries described for

other Gram‐negative [168] and Gram‐positive bacteria [169].
8.4.2 Chitin Degradation by Bacteria in the Bacteroidetes Phylum

The Bacteroidetes phylum contains Gram‐negative bacteria that are generally recognised
for effectively degrading carbohydrates. Although most studied, Bacteroidetes are anaer-
obes related to the gut microbiota of mammals. Bacteroidetes can be found in both anaer-
obic and aerobic environments [170]. The genes encoding the proteins needed for
degrading a specific carbohydrate are often organised in gene clusters called polysaccha-
ride utilisation loci (PULs [171]). The first described PUL is the starch utilisation system
of the anaerobic bacterium Bacteroides thetaiotaomicron, which, next to enzymes con-
tains a so‐called SusC/D pair, i.e. an outer membrane porin and a carbohydrate‐binding
protein, respectively [172, 173]. SusC/D‐like pairs are now used as identifiers for PULs in
different organisms [174].
Recently, Larsbrink et al. (2016) [110] described the first chitin utilisation locus, which
was found in the aerobic Bacteroidetes Flavobacterium johnsoniae. This PUL comprises
11 genes, encoding four enzymes, a predicted two‐component sensor/regulator system, and
two SusC/D‐like pairs (Figure 8.15). The four enzymes are a secreted multidomain GH18
chitinase (ChiA), an outer‐membrane anchored GH18 chitinase (ChiB), a periplasmic chi-
tobiase (GH20) and a glucosamine‐6‐phosphate deaminase (NagB). Interestingly, to date,
no LPMOs have been discovered in Bacteroidetes [110, 175]. The absence of LPMOs may
be related to the fact that most Bacteroidetes live in anaerobic conditions. It is worth noting,
however, that, as mentioned in Section 8.1.5, recent findings suggest that molecular oxygen
is not crucial for LPMO activity [66]. Another explanation could be that the PULs in them-
selves are so powerful when it comes to polysaccharide degradation that the need for
LPMOs is relieved [110].
8.4.3 Chitin Degradation by Thermococcus Kodakarensis

Tanaka and co‐workers have studied the rather peculiar chitinolytic pathway of the hyper-
thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1[36, 38, 176, 177].
The chitin utilisation system of this archaeon is organised in a gene cluster encoding a
multimodular family GH18 chitinase (Tk‐ChiA), a family CE14 deacetylase (Tk‐Dac), a
family GH35 glucosaminidase (Tk‐GlmA), an ABC transporter system, and several hypo-
thetical proteins. The chitinase has two catalytic GH18 domains and three CBMs. The cata-
lytic GH18 domains show different activities. The first domain is an exo‐type chitinase
while the second domain is an endo‐type chitinase [177]. Two of the CBMs belong to
family 2 and can bind both chitin and cellulose, while the last CBM belongs to family 5 and
binds to chitin [32, 176,178]. Tk‐Dac is a highly specific deacetylase that only deacetylates
the non‐reducing end of fully acetylated CHOS as well as GlcNAc [38]. Tk‐GlmA is active
against a range of fully deacetylated CHOS, although the dimer GlcN–GlcNAc is consid-
ered as the physiological substrate [36, 38]. In the proposed pathway for chitin conversion
by T. kodakarensis KOD1 (Figure 8.16), Tk‐ChiA produces (GlcNAc)2 that is imported into
the cells by the ABC transporter. The intracellular deacetylase, Tk‐Dac, specifically dea-
cetylates the non‐reducing end of (GlcNAc)2 after which the glucosaminidase, Tk‐GlmA,
ChiA
ChiB
CusD
CusC
GH20
TonB- CusS
ExbBD IM
complex transp.
NagA NagB CusR
P P P
NagK
GlcNAc GlcNAc-6P GlcN-6P Fru-6P
Figure 8.15 Proposed pathway for chitin utilisation in Flavobacterium johnsoniae. The secreted multi‐
domain ChiA converts chitin to CHOS. ChiB can also degrade CHOS and chitin, although not as efficiently
as ChiA. The SusC/D‐like pairs (here called CusC/D) capture and import CHOS to the periplasm. The
chitobiase (GH20) converts CHOS to monomers before translocation of the monomers to the cytoplasm
where they are converted to fructose‐6‐phosphate by NagK, A and B. Proteins not encoded by the PUL are
coloured grey. This figure has been obtained from [110], an original BioMed Central (BMC) publication.
hydrolyses the GlcN–GlcNAc dimer. Finally, Tk‐Dac deacetylates the GlcNAc monomer
to generate GlcN [38].
8.4.4 Chitin Degradation by Fungi

Fungal genomes often encode several chitinases; it is not exceptional to find 10‐25 differ-
ent chitinases, which, all belong to family GH18 [120, 122]. For example, the genome of
the well‐studied entomopathogen Beauveria bassiana encodes 20 GH18 chitinases possi-
bly involved in pathogenesis by aiding the penetration of the chitin‐containing exoskeleton
of the host [179, 180]. Trichoderma species, which are known for biomass degradation as
well as chitinase‐mediated biocontrol functionalities, contain various numbers of GH18
encoding genes, and as many as 36 GH18s are encoded by Trichoderma virens [122, 181].
O O
OH OH
H O HO NH O H Tk-ChiA O HO NH
O HO OH
O O
HO HO
NH O O
NH
OH n OH
O O
Tk-Dac
O O
OH OH
O NH O NH
HO OH Tk-Glm HO
HO + HO OH
HO OH HO O
O HO
NH2 NH2 O
OH OH
Tk-Dac
Figure 8.16 The chitinolytic pathway of Thermococcus kodakarensis. Tk‐ChiA converts chitin to
(GlcNAc)2, which is deacetylated on the non‐reducing end by Tk‐Dac. Tk‐GlmA then hydrolyses the
GlcN–GlcNAc dimer and Tk‐Dac finally deacetylates GlcNAc to produce GlcN.
Chitinases in fungi are sometimes divided into three subgroups, A, B and C, based on their
modular structures. Group A contains chitinases without extra domains, group B contains
chitinases linked to CBM1s, while group C comprises chitinases with CBM18s and LysM
(CBM 50) domains [120, 181]. Of note, chitin is an important part of the fungal cell wall,
which also contains β‐1,3‐glycan and a manno‐protein layer [182].
As mentioned in Section 8.3, fungal chitinases are involved in cell wall remodelling and
in degradation of exogenous chitin, and an often raised question is how the fungi discrimi-
nate between self and non‐self chitin. It has been proposed that different chitinases are
specialised to do different jobs. Gruber and Seidl‐Seiboth (2012) [122] hypothesised that
differentiation between self and non‐self is regulated by substrate accessibility [120, 152].
The high number of chitinases makes genetic assessment of chitinase functionality a chal-
lenging task. It has been shown that double or multiple knock‐outs are needed to get a
phenotypic effect indicating possible redundancy among these many fungal chitinases
[123, 183].
Some fungi contain chitin‐active LPMOs [184], which likely play a role in chitin conver-
sion, but whose roles in chitin degradation and interplay with chitinases have not yet been
assessed. Several fungal chitin deacetylases have also been characterised (see Section 8.1.6)
and are believed to be involved in cell wall remodelling during growth [185] or to protect
hyphae during host invasion (plant pathogenic fungi [186]).
8.5 Biotechnological Perspectives

In nature, chitin is usually part of a complex co‐polymeric recalcitrant matrix, which,
together with its insolubility, limits applications. Despite this limitation, chitin is currently
used in a variety of sectors, but primarily after being chemically converted to chitosan.
Chitin and chitosan are of interest in a variety of applications due to their biodegradability,
non‐toxicity, biocompatibility and potential to be modified. In the medical field, chitosan

is used in wound‐dressing materials [187, 188] as well as in tissue engineering [189, 190].
In the hygiene sector, chitosan is added in some toothpastes and other dental care products
[191]. In the cosmetic industries, the polycationic properties of chitosan are very useful;
chitosan retains moisture and becomes viscous and neutral when dissolved in diluted acid.
For this reason, several products for hair and skin care contain chitosan [192]. Chitosan is
also applied in wastewater treatments for removal of heavy metals and as a flocculating
agent [192–194]. In the food industries, chitosan‐based materials are used as wrapping and
packaging materials with a positive impact on quality preservation [192, 195, 196]. GlcN
and GlcNAc, the mono‐sugars of chitosan and chitin, respectively, are used as dietary sup-
plements [197, 198]. In agriculture, chitosan is used as a plant protecting agent due to
chitosan’s antimicrobial and antifungal properties [199, 200]. Production of bioethanol
from GlcNAc has been reported [201]. In the chemical industries, GlcNAc is used as a
starting molecule for the production of different chemicals [202].
The preceding examples indicate the potential and value of chitin and chitosan. However,
the full potential of this bioresource seems not yet fully exploited, and more research is
required to unleash its additional potential. Importantly, whereas most commercial efforts
to valorise cellulose today are based on enzyme technology, most chitin applications still
rely on classical chemical methods for purification of chitin, production of chitosan and the
depolymerisation of chitin or chitosan to oligomers and monomeric sugars. There is con-
siderable potential for the implementation of biotechnological methods, including (1)
enzymatic methods for purifying chitin from co‐polymeric chitin‐rich feedstocks, (2) enzy-
matic methods to convert chitin to chitosan, and (3) enzymatic methods to depolymerise
chitin or chitosan, both to mono‐sugars and oligosaccharides. Section 8.6 provides a fur-
ther discussion of enzymatic methods for chitin valorisation.
The biotechnological potential of chitinases is diverse, ranging from medical applications
to biocontrol. Oranusi and Trinci (1985) [203] demonstrated that an antifungal agent (ampho-
tericin B) was potentiated by chitinases, indicating the possibility of improving existing treat-
ments for fungal infections. Furthermore, van Eijk et al. (2005) [118] showed that the human
chitinase ’chiotriosidase’ inhibits the growth of the fungal pathogen Cryptococcus neofor-
mans, suggesting that chitinases have potential as an antifungal agent in humans. Based on
the affinity of chitinases for the fungal cell wall, Vega and Kalkum (2012) [204] suggested
that these enzymes may be used to detect fungal infections in humans. In agriculture, several
studies have reported that transgenic plants over‐expressing one or more chitinases display an
increased ability to resist fungal infections (see reviews in [205] and [206]). A multitude of
chitinases have been evaluated for their antifungal/ anti‐insect properties, especially in the
context of use as pesticides (see e.g. [207]). However, to the best of our knowledge, there are
currently no major commercially available pesticide products containing chitinases. The only
exception is the indirect application of chitinolytic enzymes through biocontrol agents such
as the entomopathogen Bacillus thuringiensis and Baculoviruses.
8.6 Biorefining of Chitin‐Rich Biomass

The recalcitrant nature of chitin makes efficient in vitro enzymatic depolymerisation chal-
lenging. Furthermore, despite the large interest in utilising chitin‐containing co‐products in
a biorefining context, little research has been conducted on developing efficient chitinolytic
cocktails tailored for industrial use, which could entail a need for using non‐natural pro-
cessing conditions, such as high temperature, low pH and a high dry matter content.
All recalcitrant polysaccharide‐containing biomasses require pretreatment in order to
increase the surface area of the biomass and thereby obtain efficient enzymatic saccharifi-
cation. Chitin‐containing biomass is usually deproteinised and demineralised in order to
isolate the chitin (Aye and Stevens, [208] 2004). Depending on the application and particu-
larly if the goal is to produce chitosan, an extra alkaline pretreatment step is needed [195].
Mechanical size‐reduction of the chitin particles has proven to be successful for further
improvement of degradability [56].
Subsequent enzymatic hydrolysis of the purified chitin or chitosan will generate the
desirable oligomeric or monomeric products. So far, enzymatic saccharification of chitin
and chitosan has received little attention and has been mainly focused on bacterial enzymes.
There are no commercial products available for efficient saccharification of chitin or chi-
tosan. In order to obtain efficient and low‐cost enzymatic conversion of chitin, optimisation
of enzyme mixtures and characterisation of more chitin‐targeting enzymes seems to be
required, as shown and discussed by Mekasha et al. (2017) [209].
It is important to note that low‐molecular‐weight chitin and chitosan, that is, CHOS,
have received attention in different fields due to reported bioactivities. CHOS with distinct
physiochemical properties such as molecular weight, FA and acetylation pattern have poten-
tial in different applications in medicine and agriculture [87]. Further research on the enzy-
matic conversion of chitin‐rich biomass should consider the production of CHOS and full
saccharification will not always be the key target of a chitin conversion process. The direct
production of well‐defined CHOS from chitin by a combination of enzymatic deacetyla-
tion and (controlled) hydrolysis is an attractive scenario for valorisation of chitin‐rich bio-
mass [87, 210], which, however, has not yet been achieved.
Enzymatic conversion of chitin has not been explored in the same scale as cellulose. In
order to put the efforts of chitin biorefining in perspective, it is useful to briefly review the
status of the cellulose biorefining field, which resembles chitin biorefining in many ways
(abundant feedstock, recalcitrant and insoluble, and valuable for industrial use, with one
bottleneck being the efficiency and cost of enzyme cocktails).
In recent years, efficient processes for the conversion of lignocellulosic biomass have
been developed, and these are currently being used commercially. Industrial conversion of
lignocellulosic biomass to streams of mono‐sugars (primarily cellulose‐derived glucose)
involves a substrate pretreatment step that alters structural features of lignocellulose, breaks
the ‘lignin‐barrier’ and promotes substrate accessibility for subsequent enzymatic hydroly-
sis. The pretreated material is then hydrolysed using either cell‐free enzyme systems or
microbes. The resulting sugars are used in different applications, one of the best known
being the production of ethanol [211–213]. The term ’consolidated bioprocessing’ is used
when enzymatic depolymerisation and fermentation of the solubilised sugars are performed
simultaneously, meaning that the pretreated feedstock is directly converted to the final
product, for example ethanol, in a one‐pot reaction.
Enzymatic hydrolysis of lignocellulosic biomass is commonly performed using complex
enzyme mixtures, which may be crude or composed of purified enzymes. Cellulolytic
enzymes from fungal strains such as T. reesei show high hydrolytic potential on ligno-
cellulosic biomass and are among the best known cellulases, which were prominent in
early commercial enzyme cocktails [214]. Today’s enzyme cocktails are so efficient that
commercial production of lignocellulosic ethanol is feasible, with one cause being the
inclusion of LPMOs [215–218].
An important lesson to be learned from the field of cellulose biorefining is that fungal
enzymes dominate. Essentially all commercially available enzyme cocktails developed for
degradation of cellulose‐containing biomass are based on fungal enzymes. Interestingly,
few fungal chitin‐degrading enzymes (and chitin‐degrading fungi) have been characterised
in detail. If fungal chitin‐degrading enzymes show characteristics similar to fungal cellu-
lose degrading enzymes, they may represent a great potential for future chitin biorefining.
It is important to note the potential of chitin‐degrading enzymes from thermophilic
archae, as reviewed by Egorova and Antranikian (2005) [219] and described in more detail
earlier, for T. kodakarensis (Section 8.3). Such enzymatic systems have superior properties
due to the extreme stability of enzymes that allow chitin conversion at temperatures where
potentially contaminating bacteria do not grow. Furthermore, enzymes from thermophiles
are often also tolerant to extreme pressure and pH, which may be beneficial for industrial
processes.
8.7 Outlook
In conclusion, while today we know a lot about enzymes for the modification of chitin and
chitosan, the industrial use of these enzymes for biorefining of chitin‐rich biomass is still
in its infancy. Importantly, next to the development of efficient enzymatic processes for
controlled conversion of chitin or chitosan to monomeric sugars or CHOS, truly ’green’
processes for chitin biorefining would also require the development of enzymatic or micro-
bial pretreatment techniques. Such techniques should replace the current chemical tech-
nologies that are needed to convert the original complex feedstock into reasonably clean
chitin or chitosan, which can then be used for further enzymatic valorisation.
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9
Chitin and Chitosan as Sources
of Bio‐Based Building Blocks
and Chemicals
Malgorzata Kaisler1,2, Lambertus A.M. van den Broek2, and
Carmen G. Boeriu2
Bioprocess Engineering Group, Wageningen University, Wageningen, The Netherlands
1
2
Wageningen Food & Biobased Research, Wageningen, The Netherlands
Chitin and chitosan polymers are a valuable source of functional chemicals and materials.
Chemical and/or enzymatic depolymerisation processes have been developed for the
production of chitooligosaccharides (COS), N‐acetylglucosamine (GlcNAc) and
glucosamine (GlcN), which have a wide variety of applications. New technologies are now
emerging to convert chitin and its derivatives into platform chemicals. Chemical liquefaction
of chitin can lead to bulk chemicals like acetic acid and platform chemicals like
hydroxymethylfurfural (HMF) and amine‐containing monomers for polymers, in low
yield. The monomers GlcNAc and GlcN can be converted into N‐containing HMF
derivatives, opening a pathway for furan‐based monomers for polyamides. Selective
catalytic oxidation of GlcN results in the production of D‐glucosaminic acid (DGA). This
acid is a valuable building block for the synthesis of various amino acids for biomedical
applications and bio‐based chiral polyamides. Further technological improvements are
necessary to increase the selectivity and efficiency of reactions, particularly for the
conversion of polymeric chitin and chitosan into building blocks.

9.1 Introduction
In the past decades, significant technological developments have been achieved in
the utilisation of lignocellulosic biomass as an alternative source to fossil fuels for the
production of chemicals and materials for the food, feed and chemical industry. The
production of building blocks for materials and chemicals from renewable biological
resources, such as plants, microorganisms and animals creates the opportunity to reduce
the emission of greenhouse gas and reduce our dependence on coal, oil and gas, and
constitutes as such the fundamental concept of bio‐based economy [1, 2]. Recent devel-
opments and regulations significantly enlarge the scope of bio‐based economy towards
a zero‐waste circular economy that extends the biomass source towards industrial
side streams and promotes the recycling of plastics and biodegradable materials in the
process cycle.
In this context, biomass side stream (biowastes) generated by different industries are
considered an attractive renewable biological resource for chemicals, building blocks and
materials. The recovery of valuable components such as biopolymers, fats and fine
chemicals, pigments and pharmaceuticals from biowastes decreases landfilling of wastes
and protects the environment. Moreover, conversion of cheap biowastes to added‐value
products can bring additional economic benefit [3]. Recovered biopolymers like proteins,
lignin, starch and chitin are of high importance, since they can be used directly as feed or
food or can be applied as a resource of building blocks for the production of non‐food bio‐
based products.
Chitin, one of the most abundant biopolymers on earth, is generated in huge amounts as
a by‐product (biowaste) from the food and chemical industry. Industrial sources of chitin
and chitosan are the side streams of the shell food industry, insect farms, mushroom
cultivation and the fungal fermentation industry (Figure 9.1).
So far, the seafood industry generates the biggest amounts of chitin‐containing biomass
(6–8 million tons/year produced globally) comprising the shells from crustaceans (e.g.
prawns, shrimps, crabs, lobsters) [4]. This biomass is often considered as a waste and is just
dumped in the sea or landfilled without any processing [5]. However, such a landfilling
leads to serious environmental pollution of coastal areas. Thus, in the 1970s, the USA
introduced strict environmental regulations and banned the dumping of waste shells without
any processing [6], which directly resulted in a significant increase of chitin utilisation. In
some countries, for example, in Australia, disposal of crustacean shells is costly and can be
up to €130 per tonne [5]. Crustacean shells from the food industry are processed by drying
and desizing (i.e. crushing and milling) and sold as a low‐value fertiliser at a much lower
price (€44/tonne) as compared to the price of dried crustacean shells (€85–102/tonne) [5].
However, chitin‐containing biomass has a much higher potential than only being a low‐
value fertiliser. A small part of chitin‐rich streams is used though for the extraction and
valorisation of chitin and chitosan, chitin’s most valuable derivative.
Besides chitin, chitin‐containing biomass comprises proteins, fats, glucans and pigments
(e.g. astaxanthin) (Figure 9.1). All the components can be separated in a biorefinery concept
using chemical and/or enzymatic extraction methods. Apart from food, the refined
components can be used as animal feed (e.g. proteins) or can be further converted into
valuable chemicals or materials. Lipids can be used in the production of high‐value oil,
biodiesel and animal feed [5]. Calcium carbonate (CaCO3) is applied in the pharmaceutical,
Cephalopods Crustaceans Insects Fungi
Industrial Meat Meat • Silk production • Organic acid production

main stream • Meat • Brewing and baking
• Mushroom cultivation
• Enzymes production
• Antibiotic production
Industrial Beaks and cuttlebones Shells Cuticle Fungal mycelia, 80 ktons/year
side stream 6 – 8 million tons/year 65 ktons/year Mushroom waste, 50 ktons/year
Biomass 15– 40% d.w. chitin

26–50% d.w. chitin up to 60% d.w. chitin 14–16% d.w. chitin
composition 20 –40% d.w. proteins up to 50% d.w. proteins 15– 35% d.w. glucans
40–64% d.w.
Proteins 0.4% d.w. pigments up to 5% d.w. lipids 2–3% d.w. protein
20 –50% d.w. CaCO3
Process Chemical or enzymatic extraction of components (biorefinery)
Glucans food, medicine, pharmacy

Pigment (Astaxanthin) food/feed, cosmetics
Conversion
Chitin chitosan, COS, GlcNAc, GlcN, functional materials
and
application Proteins animal feed, fertilisers
Lipids high-value oil, biodiesel, animal feed
Calcium carbonate minerals for agriculture, pharmaceutical, construction and paper industries
Figure 9.1 Valorisation of chitin‐containing biomass within the bio‐based economy.

agricultural, construction and paper industries [5]. Astaxanthin, a carotenoid with antioxi-
dant properties, is widely used in aquaculture feeds, food, cosmetic, pharmaceutical and
medical applications [7]. Glucans recovered from fungi have potential application in food
and feed, medicine and pharmacy [8].
The first step of chitin valorisation is its extraction. Traditionally, chitin is extracted by
the removal of proteins (deproteinisation) with alkali treatment (e.g. with NaOH) and
removal of CaCO3 (demineralisation) with acidic treatment (e.g. HCl) [9, 10]. After
extraction, chitin is often decoloured with acetone or an organic acid solvent mixture [11].
This conventional fractionation process for chitin extraction is, however, destructive and
has a high environmental impact due to the use of high amounts of corrosive acids and
alkaline reagents and large wastewater streams. In the past years, research focused on the
development of more efficient and greener alternative routes based on solvent extraction
with ionic liquids (ILs) [12–14] and biotechnological conversion using microorganisms or
isolated proteolytic enzymes [10, 15].
The production of chitin is less than 100,000 metric tons per year [16], but reliable data
are difficult to be found in open literature. Almost all commercial chitin is produced from
crustacean shells, but in the past years new chitins from insects, fungal mycelia and
mushrooms have emerged in the market. Due to its intrinsic functional properties, chitin
has many industrial applications: in agriculture, for controlling plant diseases and as plant
growth regulators, in the food and drink industry for clarification of fruit juices, in water
treatment, as well as in the cosmetic, pharmaceutical and biomedical industry [10, 17].
However, an important application of chitin is as a source for the production of
glucosamine (GlcN), N‐acetylglucosamine (GlcNAc), chitosan and COS. In the past years,
novel promising routes have been demonstrated on a laboratory scale for the conversion of
chitin into bulk chemicals like acetic acid, platform chemicals like 5‐HMF and levulinic
acid (LA), and amine‐containing monomers for polymers. An overview of the chemicals
that can be produced from chitin and chitin’s monomers GlcN and GlcNAc is shown in
Figure 9.2.
Many reviews have been dedicated in the past years to the use of chitin as a renewable
source of chemicals and materials, promoting the concepts of ’shell biorefinery’ as a major
technology platform of the Blue Biotechnology and Circular Bioeconomy roadmaps [1, 3,
5, 15, 18–21]. In this chapter, we will discuss only the latest development in the production
of high‐value carbohydrates and bio‐based building blocks derived from chitin.
9.2 Chitin Conversion into Chitosan, Chitooligosaccharides

and Monosaccharides
9.2.1 Chitosan Production
Chitosan represents a family of linear co‐polymers of 2‐acetamido‐2‐deoxy‐β‐D‐glucose
(GlcNAc) and 2‐amino‐2‐deoxy‐β‐D‐glucose (GlcN) moieties that are obtained through
the deacetylation of chitin in varying degrees. Generally, polymers with a degree of
acetylation (DA) between 0–50 mol% are classified as chitosan [22], and differ from chitin
by the higher positive charge density and increased solubility in acidic aqueous solutions.
Industrially, chitosan is obtained in a thermocatalytic process where acetamido groups are
hydrolysed under alkaline conditions with a concentrated NaOH solution [23]. The
H3C O
C OH
NH O OH OH O
HO O
OH HO O HO
O O
NH2 OH
O OH OH NH2
n
HAc Chitosan, DDA > 60% DGA
OH
Chemical or O OR OR O
HO
Enzymatic
Polymer
HO OH H2 N OH
NH2
OR OR
GlcN
H C O
Chemical or
C OH Enzymatic
s
O
NH H C O
HO O O
O
C
Chemical or H2N OH
O O HO
NH
NH
HO O Enzymatic OH
OH
n O
O O
HO HO
COS, 2<n<20 OH n
HO OH
NH2 O
OH
Chitin O
O HO
GlcNAc HN
O
O
O O
Pyrolysis OH
OH HO
O Hydrogenolysis 3,6-anhydro-GNF O
Liquefaction Hydrogenation O
O HN
5-HMF Chemical HO
LA O OH
O HO
3,6-anhydro-MNF
O O N OH OH
O O H
O OH OH
HO 5-FMF H2N OH
HN
O ADS OH NH2
AS
3A5AF
s
Polymer
Figure 9.2 A schematic representation of chitin conversion into chitosan, chitooligosaccharides (COS), N‐acetylglucosamine (GlcNAc), glucosamine (GlcN)
and various platform chemicals and building blocks for polymers. 3,6‐anhydro‐GNF is 2‐acetamido‐3,6‐anhydro‐2‐deoxy‐D‐glucofuranose; 3,6‐anhydro‐MNF is
3,6‐anhydro‐2‐deoxy‐D‐mannofuranose; 3A5AF is 3‐acetamido‐5‐acetylfuran; 5‐FMF is 5‐[(formyloxy)methyl]furfural; 5‐HMF is hydroxymethylfurfural; ADS is
2‐acetamido‐2‐deoxy‐D‐sorbitol; AS is 2‐amino‐2‐deoxy‐D‐sorbitol; DGA is D‐glucosaminic acid; HAc is acetic acid; and LA is levulinic acid.
c oncentration of hydroxide, temperature and reaction time strongly influence the molecular
properties of the chitosan product, in particular the DA and the molecular weight (Mw)
[24]. Although effective, chemical deacetylation has a number of drawbacks, of which high
energy consumption, use of corrosive chemicals, large streams of polluting wastes and
large variation in product properties are most relevant. Enzymatic chitin deacetylation
using chitin deacetylases of fungal [22, 25–28], bacterial [29, 30] and insect origin [24, 31]
has been proposed as a green and environment‐friendly alternative for chitosan production
that offers the possibility to obtain well‐defined chitosan through a controlled, mild and
non‐destructive process [24]. However, the efficiency of all tested chitin deacetylases on
polymeric, crystalline chitin is very low, and less than 10% of the N‐acetylglucosamine
residues are deacetylated rapidly [22, 25, 26, 28–30, 32]. Based on these results, it was
concluded that pretreatment of chitin is required in order to increase the accessibility of the
acetyl groups for deacetylation. Indeed, low‐Mw, partly deacetylated chitin/chitosan are
rapidly deacetylated by chitin deacetylases from Mucor rouxii [22], Absidia coerulea [32]
and Podospora anserina [33] to degrees of deacetylation of up to 90–97 mol%.
Next to chitosan, large amounts of acetic acid (HAc in Figure 9.1), a valuable bulk
chemical, are also produced, irrespective of the method used for chitin deacetylation.
There are three characteristics, which make chitosan an interesting industrial
polysaccharide: (i) it behaves like a polyelectrolyte with positive charge density at low pH,
(ii) is the only known high‐Mw cationic natural biopolymer with unique and exceptional
properties and (iii) it is and is often claimed to be GRAS (generally recognised as safe)
[34]. In addition, chitosan is biocompatible, bioabsorbable and nontoxic, and has a plethora
of bioactive properties, like antioxidative and antimicrobial activities, among others. These
characteristics enable chitosan to have applications in various fields. For example, chitosan
is used in medicine (as an antibacterial lining for bandages and wound dressings),
agriculture (for coating seeds to enhance disease resistance), food industry (as food
preservative, active edible packaging, dietary fibre), in water treatment as a bioflocculant,
in textiles and bioprinting, in cosmetics and the pharmaceutical industry for drug delivery
[10, 11, 35]. Chitosan is also used as a dietary supplement for weight loss since it was
shown to bind fat and hence should prevent fat digestion in the human body [34].
Chitosan prices depend on the quality of the product given mainly by chitosan’s degree
of deacetylation (DDA), Mw, water solubility, viscosity and residual protein, pigment and
mineral [36]. The demand for chitosan is increasing every year. In 2012, the global market
for chitosan was estimated at 21,400 metric tons, while in 2015, 0.2 million tons were
produced. The global chitosan market size is expected to reach €2.2 million by 2022 from
€1.1 million in 2015, with a CAGR of 18% (https://www.alliedmarketresearch.com).
9.2.2 Production of Chitooligosaccharides

Depolymerisation of chitin and chitosan can result in the formation of COS. Chitosans with
a degree of polymerisation (DP) <20 and average Mw less than 3,900 Da are defined as
COS [37]. On the other hand, Jeon and Kim divided COS into low‐, medium‐ and high‐Mw
COS, that have a Mw of <0.6, 1.5–6 and 7–24 kDa, respectively [38]. COS can have, for
example, antimicrobial, antitumour, antioxidant, immunostimulatory and anti‐inflammatory
activities. The activities depend on the DP, DA, pattern of acetylation (PA) and Mw. The
structural characteristics can be measured as described by Cord‐Landwher et al. [39]. The
Chitin and Chitosan as Sources of Bio‐Based Building Blocks and Chemicals 235
low viscosity, low Mw, short chain lengths and water solubility make COS more suitable
for some industrial applications than chitin and chitosan [11, 40]. Due to their biological
activities, COS have high potential to be used on an industrial scale [40–42], and, at the
moment, different companies produce COS on a commercial scale [40].
COS can be prepared by chemical, physical and enzymatic treatments. Chemical
methods consist mainly of acid hydrolysis although oxidative reductive agents also can be
used. The most commonly applied chemical hydrolysis is the use of concentrated HCl at
80°C for 1–2 h. Other acids that can be used are nitrous acid, hydrofluoric acid and
phosphoric acid. As oxidative reductive agents, hydrogen peroxide, ozone and persulphate
can be used [42, 43]. A drawback of chemical hydrolysis is low yield, (detrimental)
secondary products can be formed and in general small size COS with a DP of 1–4 are
produced [41]. The mechanism of chemical hydrolysis is described in detail in [37].
Physical hydrolysis of chitin and chitosan can be performed by sonication, microwave
and irradiation. These high‐energy‐impact treatments result in cleavage of the O‐glycosidic
linkages [42]. Sonication can be performed in combination with acids to obtain COS [44,
45]. If sonication was used without acid, then only degradation of chitosan into lower Mw
was observed [46, 47]. Gamma and electron‐beam irradiation also resulted only in
degradation of chitosan into lower Mw [48]. In addition, gamma irradiation is more
effective in aqueous solution than in the solid state [49]. Production of COS from chitin and
chitosan in electron‐beam plasma gases has been reported [50]. Microwave irradiation is
performed in the presence of chemicals like acid or salts to obtain COS [51, 52].
Enzymatic methods are another option to produce COS in a more controlled way. Chitin
and/or chitosan can be converted to COS with glycoside hydrolases like chitinases or
chitosanases [53]. Depending on the structure of the starting material (DP, Mw, PA and
DA) enzymes can cleave the glycosidic linkages or modify the starting material (e.g.
deacetylation). Depending on the specificity of the enzymes, specific oligosaccharides can
be released. The characteristics of these enzymes are described in more detail in [54]. As
mentioned earlier, different methods or combinations hereof can be used to obtain COS. In
general, chemical and enzymatic treatments are used. Acetic acid is the main by‐product of
COS production processes.
9.2.3 Production of GlcNAc and GlcN from Chitin

Both chitin and chitosan can be hydrolysed into their constitutive monomers, that is,
GlcNAc and GlcN. These monomers are widely used for the treatment of joint diseases
(e.g. osteoarthritis) and inflammatory bowel disease in children [55]. GlcNAc was shown
to have a potential effect on the cartilage metabolism at a lower dose (500–1,000 mg/day)
as compared to GlcN (1,500 mg/day) [56]. In cosmetics, GlcNAc is a valuable ingredient
for improving skin quality [57, 58]. In the food industry, GlcNAc is used as an additive in
beer, milk and wine [59–61]. Recently, GlcNAc was proposed as a biological C6 source for
bioethanol production through fermentation [62]. Other promising valorisation of
monomers is their application as platform chemicals for synthesis of high‐value chemicals
like 5‐hydroxymethylfurfural (5‐HMF) among others (Figure 9.1), as we will discuss in the
following text. 5‐HMF is an important platform chemical as it can be converted into
numerous sustainable chemicals such as alkoxymethylfurfurals, levulinic acid, adipic acid,
furan‐based monomers, etc., which can be used to make different bio‐based polymers [63].
Hydrolysis is traditionally performed with chemical methods using a strong acid as cata-
lyst (e.g. HCl) which breaks the glycosidic bonds in chitin and chitosan polymers [11].
Depending on process conditions, either GlcN or GlcNAc are produced. GlcN is usually
obtained from chitin with concentrated HCl (20–37%) at 100°C [64]. GlcNAc is obtained
by acid hydrolysis at lower temperature, for example, 40–80°C [65] or by re‐acetylation of
GlcN with acetic anhydride [66]. The chemical production of GlcNAc from chitin is esti-
mated to be economically feasible, but there are some drawbacks, including low yield
(below 65%), low specificity of the chemical catalyst, high operational costs and genera-
tion of large streams of acidic wastes [18].
Mechanocatalytic conversion of chitin to GlcNAc has recently been reported [67]. This
process consists of two steps: (i) impregnation of chitin with catalytic amounts of H2SO4,
and (ii) ball‐milling of acid‐impregnated chitin for 6 h, at 500 rpm, under solvent‐free con-
ditions, which results in total dissolution of chitin and the formation of low‐Mw chitin
oligosaccharides, with DP2 to DP5 and some longer chitin oligomers. Further hydrolysis
of the mixture at high temperature for a short time results in 53% GlcNAc, while
methanolysis produces the 1‐methyl ether derivative of GlcNAc, in 70% yield [67]. The
1‐O‐methyl‐N‐acetylglucosamine formed is a functional building block for biodegradable
polyesters and polyamides [67].
Following long and intensive research, enzymatic methods are now confirmed to be a
viable green alternative to acid depolymerisation for the production of GlcNAc and
GlcN from chitin and/or chitosan. Using chitin‐ and chitosan‐specific enzymes of fun-
gal and/or bacterial origin, chitin and chitosan can both be converted to GlcN, while
only chitin could be used to produce GlcNAc as a major product, in a selective way.
Figure 9.3 illustrates two major enzymatic pathways occurring in nature, which have
also been partly translated into laboratory or industrial processes for the production of
GlcNAc.
The chitinolytic pathway (route 1 in Figure 9.3) is based on the synergistic action of
three groups of chitin‐hydrolysing enzymes: chitinases, N‐acetylglucosaminidases
(NAGase) and lytic polysaccharide monooxygenases (LPMOs). Chitinases cleave
β‐1,4‐glycosidic linkages within chitin polymer chains and intermediate chitin oligo-
saccharides to generate chitobiose, (GlcNAc)2, which is finally degraded to GlcNAc by
Chitinase Chitinase GlcNAc

(GlcNAc)n, (GlcNAc)2
LPMO n>2 NAGase
(1) Deacetylase
Chitin GlcN
GlcNase
(2)
Chitin Chitosanase/ (GlcN)2

Chitosan GlcN-GlcNAc, GlcNAc-GlcN
deacetylase Chitinase*
Figure 9.3 Enzymatic degradation of chitin via chitinolytic pathway (1) and chitosanolytic pathway (2).
LPMO, lytic polysaccharide monooxygenase; NAGase, N‐acetylglucosaminidase; GlcNase, glucosami-
nidase. *Some chitinases are able to degrade chitosan.
N‐acetylglucosaminidase (NAGase) [68, 69]. LPMOs cleave glycosidic bonds in the

crystalline regions of chitin via an oxidative reaction [70] generating oxidised COS with
an N‐acetylaminogluconic acid terminal residue. LPMOs can be considered as ’process-
ing aids’, since, through their action, they enhance the hydrolytic efficiency of chi-
tinases by providing them with less crystalline, better accessible substrates. The final
product, GlcNAc, can be enzymatically deacetylated by N‐acetylglucosamine deacety-
lase to GlcN.
The second pathway (route 2 in Figure 9.3) involves the deacetylation of chitin by chitin
deacetylases, producing chitosan with different DDA, which is subsequently hydrolysed by
chitosanases and exo‐1,4‐β‐D‐glucosaminidase to produce GlcN. Formation of other
products such as GlcNAc and/or heterodimers composed of GlcN and GlcNAc is determined
by the DDA and PA of chitosan and the specificity of the enzymes. Some chitinases are
also able to degrade chitosan, and their activity is influenced by the DDA and PA of chitosan
[71–74].
In the past years, enzymes involved in chitin and chitosan depolymerisation pathways
have been obtained, isolated and characterised, and their efficiency in producing GlcNAc
and GlcN, respectively, has been estimated in in vitro studies. Due to the higher availability
and lower price, chitin is the preferred substrate for the production of monomers. However,
the insolubility and crystalline nature of chitin hamper its interaction with enzymes and
decreases the efficiency of enzymatic hydrolysis of the polymer chain, significantly slowing
down the process. Degradation experiments of crystalline chitin with crude chitinolytic
enzyme preparations from bacteria and fungi, for example, Aeromonas hydrophila H‐2330,
Bacillus licheniformis SK‐1, Burkholderia cepacia TU09 and Aspergillus sp., typically
took a few days, and the yield of GlcNAc was still low (41–85%) [75, 76]. The problem of
chitin inaccessibility has been solved by development of pretreatment methods like acid
treatment [77], dissolution in ILs [78], ultrasonication and steam explosion [79], gamma‐
and microwave‐irradiation [80, 81], high‐pressure homogenisation [82], mechanochemical
grinding [83], dry ball milling [84], and subcritical and supercritical water treatment [85],
which considerably increased the hydrolytic efficiency of enzymes and decreased the
reaction time.
Recently, we have reported an efficient two‐step process for chitin depolymerisation
consisting of a mechanochemical pretreatment of chitin and subsequent depolymerisation
with an enzyme cocktail containing two thermostable enzymes, Chitinase Chi1 and a beta‐
N‐acetylglucosaminidase MthNAG, both from the fungus Myceliophthora thermophila C1,
that work in synergy [74, 86]. After 6 h of incubation at 50°C and pH 5 with an optimised
cocktail composition and enzyme load, the amorphous chitin was converted into GlcNAc
with a yield of 72.6% and a productivity of 6 g L‐1 h‐1.
An elegant fully enzymatic cascade process for production of GlcNAc from commercial
acid–pretreated chitin from shrimp and crab, using a mixture of five mono‐component
chitinolytic enzymes from the chitin‐degrading bacterium Serratia marcescens, was
reported by Eijsink and his co‐workers [87]. The enzyme cocktail contained an endo‐acting
chitinase, SmChiA; two exo‐acting chitinases, SmChiB and SmChiC; a lytic polysaccharide
monooxygenase SmLPMO10A; and a β‐N‐acetylhexosaminidase, SmCHB. The authors
report a GlcNAc yield of 70–75% when pretreated chitin was reacted with an optimised
cocktail composition [87].
9.3 Building Blocks for Polymers from Chitin and its Derivatives

9.3.1 Furan‐Based Monomers
Furan‐based monomers, like 2,5‐furan dicarboxylic acid (FDCA), 2,5‐bishydroxymethyl
furan (BHMF), 5‐hydroxymethyl‐furan‐2‐carboxylic acid (HMFA) and other bifunctional
furan derivatives including N‐containing furans, are now established building blocks for
polyesters, polyamides and polyurethanes. Furan monomers are obtained from 5‐HMF, an
important building block derived from glycans and lignocellulosic biomass. The use of
chitin, chitosan and their lower‐Mw derivatives, that is, oligosaccharides, GlcN and
GlcNAc, for the production of 5‐HMF has been thoroughly investigated in the past years.
One advantage of chitin versus cellulose is that it is also possible to obtain N‐containing
HMF derivatives, thereby opening a simpler pathway of aminated furan‐based monomers
for polyamides.
Chitin conversion into 5‐chloromethylfurfural (5‐CMF), a precursor of 5‐HMF, in a mild
acid hydrolysis with concentrated HCl and LiCl, at 100°C and with CH2Cl2 as solvent, was
first described by Mascal and Nikitin [88]. The yield of 5‐CMF was 45%, significantly
lower than the yield obtained from cellulose under similar conditions, which was about
84% [88]. 5‐HMF can easily be obtained from 5‐CMF in high yields by mild acid hydrolysis.
Despite the low efficiency, production of 5‐HMF from chitin via the 5‐CMF route is
attractive, but the process requires optimisation to become a technological and economic
viable process.
Milder direct routes to 5‐HMF that avoid the use of strong acid catalysts and chlorinated
organic solvents are currently being studied. Conversion of chitosan to 5‐HMF in water
using hydrated SnCl4 or SnO2/HCl as catalyst was achieved with a product yield of 13.2%
that has been reported [89]. Analysis of reaction intermediates showed the formation of
GlcN that was further deaminated and dehydrated to give 5‐HMF. It was also shown that,
with the process conditions, further dehydration of 5‐HMF into LA occurs [89].
Hydrothermal conversion of chitosan at low temperature, with diluted H2SO4 [90] or
acidic IL catalysts [91], produced 5‐HMF in moderate yield. Temperature, reaction time,
pH and type of acidic catalyst influenced the product yield significantly. The highest
5‐HMF yield, 29.5%, was obtained with 4 wt% N‐methylimidazolium hydrogen sulphate
([MIM]HSO4) aqueous solution under hydrothermal conditions, at 180°C and a reaction
time of 5 h [91].
The production of 5‐HMF in moderate yield is possible by conversion of GlcN, GlcNAc
and various chitosan polymers in concentrated ZnCl2 aqueous solution [92]. The highest
yield of 5‐HMF, 21.9%, was obtained from GlcN, in 67% wt% ZnCl2 aqueous solution, at
120°C without co‐catalyst. Under these conditions, GlcN was fully converted into 5‐HMF
and humin. GlcNAc and the chitosan polymers tested gave lower yields of 5‐HMF at
similar conditions [92].
Recently, the production of 3‐acetamido‐5‐acetylfuran (3A5AF), a N‐containing furan‐
type monomer, has been achieved from GlcNAc and chitin as substrates [93–95]. 3A5AF
was obtained in over 60% yield by conversion of GlcN in both ILs [94] and organic sol-
vents [95] using dispersed boric acid as a catalyst. Conversion of native chitin by using
boric acid and alkaline chlorides as additives and NMP as a solvent produced 3A5AF in
low yield, for example, 7.5 wt%, next to levoglucosenone, 4‐(acetylamino)‐1,3‐benzenediol,
acetic acid and chitin‐humins as side products [93]. Disruption of crystalline regions
of chitin by dry ball mill grinding prior to reaction significantly increased the yield of
3A5AF to 28.5 wt% [84].
9.3.2 Amino Alcohol and Amino Acid Building Blocks

Selective catalytic oxidation of GlcN results in the production of DGA, which is a valuable
building block for the synthesis of various amino acids for biomedical applications and for
bio‐based chiral polyamides [96]. DGA has been obtained in high yield and with high
selectivity by oxidation with air or oxygen using Au, Pt or Pd catalysts in aqueous solution
[97, 98]. Using Au‐catalysed oxidation, N‐acetyl‐D‐glucosaminic acid has been obtained
from GlcNAc [97]. A very attractive alternative to chemical synthesis of DGA with noble
metal catalysts is the enzymatic oxidation of GlcN with glucose oxidase from Aspergillus
niger, a robust industrial enzyme, that selectively produces DGA at 80% yield at very mild
conditions [99]. Microbial production of DGA at 80% yield by oxidative fermentation of
GlcN by strains of lactic acid bacteria has also been reported [100].
Catalytic conversion of chitin and GlcNAc can be driven to produce amino alcohols,
another class of functional and reactive building blocks. Hydrolytic hydrogenation of
GlcNAc by activated carbon‐supported metal nanoparticles, like Ru/C, Rh/C, Pd/C and
Pt/C, to the corresponding aminopolyols was reported [101]. Total conversion of GlcNAc
was achieved with Ru/C catalyst, at 80°C, and the 2‐acetamido‐2‐deoxy‐D‐sorbitol (ADS)
was obtained at 98% yield. The direct conversion of chitin to ADS in a one‐pot two‐step
reaction using mechanocatalysis with H2SO4 pretreatment and subsequent hydrolytic
hydrogenation with H2SO4 and Ru/C, with an overall yield up to 52% was recently
reported [102].
Low yields of 2‐acetamido‐3,6‐anhydro‐2‐deoxy‐D‐glucofuranose (3,6‐anhydro‐GNF)
and 2‐acetamido‐3,6‐anhydro‐2‐deoxy‐D‐mannofuranose (3,6‐anhydro‐MNF) have been
obtained by noncatalytic conversion of GlcNAc at high temperature in water [103]. These
compounds are rigid N‐containing diols belonging to the class of dianhydroalditols of
which the most known representative is isosorbide, an important rigid bio‐based building
block for polyesters. The utility of 3,6‐anhydro‐GNF and 3,6‐anhydro‐MNF for polymer
synthesis is still to be demonstrated.
9.4 Outlook
Valorisation of chitin and its derivatives is an important aspect of the bio‐based circular
economy in general and of the blue bioeconomy in particular, which recognises the need to
maximise the enormous economic potential presented by the ocean while preserving it.
Production of chitin, chitosan and chitin monomers is already demonstrated and applied on
an industrial scale, and chitin‐derived products are largely utilised. Recent studies
demonstrated the feasibility of converting chitin, chitosan and their monomers, that is,
glucosamine and N‐acetylglucosamine, into platform chemicals of high relevance for the
chemical and polymer industry. Further technological improvements are necessary to
increase the selectivity and efficiency of the reactions, particularly for the conversion of
polymeric chitin and chitosan into building blocks. Also, a better understanding of the
relationship between structural and molecular properties of chitin and chitosan (e.g.
crystallinity, Mw, DA, PA, etc.) and their reactivity and process efficiency is necessary. The
technological and economic feasibility of chitin conversion into functional N‐containing

chemicals is a fact, but this still has to be demonstrated for the production of non‐N‐
containing chemicals, like 5‐HMF and levulinic acid, which can be easily obtained from
C6 and C5 sugars from first‐ and second‐generation biorefineries.
Acknowledgement
Malgorzata Kaisler received funding from the Netherlands Organisation for Scientific
Research (NWO) in the framework of the TASC Technology Area BIOMASS.
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10
Chemical and Enzymatic
Modification of Chitosan
to Produce New Functional
Materials with Improved
Properties
Carmen G. Boeriu and Lambertus A.M. van den Broek
Wageningen Food & Biobased Research, Wageningen, The Netherlands
Chitin and chitosan are natural biopolymers with applications in different areas. However,
to broaden its application area, research is needed to impart new functionality, develop new
and better materials and to explore the whole potential of chitin/chitosan derivatives. These
derivatives can be made by chemical and enzymatic synthesis. Here, the main classes of
chitosan derivatives obtained by using green and sustainable conversion routes and as
much as possible using green and/or bio‐based chemicals for modification and their poten-
tial areas for application are discussed.
10.1 Introduction
Chitin and chitosan are natural biopolymers with unique structures and properties. Both
polymers are aminated linear polysaccharides with N‐acetylamide and free amino groups
at the C2 position of the β‐glucose unit, that are connected through rigid β‐1,4‐glycosidic
linkages. The β‐1,4‐configuration of the N‐acetylglucosamine (GlcNAc) and glucosamine
(GlcN) building blocks results in a rigid configuration of the linear polymer chains that are

associated through intra‐ and inter‐molecular hydrogen bonds into linear aggregates with
high crystallinity [1]. Chitin and chitosan differ from each other by the content of acety-
lated and free amino groups, expressed as degree of acetylation (DA) and the distribution
of the acetyl group along the polymer chain, referred to as pattern of acetylation (PA), both
having a strong influence on the polymers’ properties. The distribution of the GlcNAc and
GlcN residues in the chitosan chain can be block‐wise or random, depending on the condi-
tions of the deacetylation process [1]. In nature, chitin is most probably fully acetylated,
but during the extraction process some deacetylation of the GlcNAc units occurs and the
isolated (commercial) chitin is partly deacetylated. It has been shown that even low dea-
cetylation of chitin, for example, 10–15% free amino groups, can induce bioactivities and
increase solubility and reactivity. Therefore, technologies have been developed to deacety-
late chitin to obtain chitosan, a family of deacetylated chitin derivatives with degree of
deacetylation (DDA) ranging from 50% to 99.8% and molecular weight between 5 × 104
and 2.5 × 106 g/mol [2, 3]. The high density of glucosamine residues in chitosan has tre-
mendous effects on its properties. The polymer is positively charged in acidic solution, due
to the protonation of the free amine groups (pKa~ 6.3), which increases chitosan’s solubil-
ity by increasing both the polarity and extent of electrostatic repulsion. Water‐soluble salts
of chitosan with formate, acetate, lactate, malate, citrate, tartrate, glycolate and other weak
organic acids are used for chitosan processing and the production of biomedical devices.
The cationic sites along the polymer chain are responsible for biological activity, for exam-
ple, antibacterial and antifungal [4, 5], and hydrogel formation through electrostatic asso-
ciation with anionic biopolymers and synthetic polymers [6]. This makes chitosan a
valuable component in food, feed, agrochemical, cosmetics, biomedical and pharmaceuti-
cal applications. Chitosan also shows pH‐dependent metal chelating properties that form
the basis for its utilisation in the removal of heavy metals from polluted water [7, 8] or as
support for metal catalysts in heterogeneous synthetic reactions [9] and antimicrobial coat-
ings and fibres [10–13], for example. This unique assembly of multiple and versatile func-
tional activities associated with biocompatibility, biodegradability and lack of toxicity
distinguishes chitosan from all other commercial natural and synthetic polymers.
The potential for application of native chitosan in different areas has been proven, but
research has also shown the need to enhance its properties, impart new functionality,
develop new and better materials and explore the whole potential of the compounds. There
is a huge interest in modification of chitosan, illustrated by the high number of research
papers published each year: a search in the web of science (https://login.webofknwoledge.
com/) for ’chitosan AND modification’ gave 598 hits in 2018. Numerous comprehensive
reviews, books and book chapters addressing chitosan modification are available. In
Figure 10.1, we show an overview of the most important modification strategies applied for
chitosan derivatisation and application areas.
In this chapter, we discuss the main classes of chitosan derivatives obtained by using
green and sustainable conversion routes and as much as possible using green and/or bio‐
based chemicals for modification and their potential areas for application.
10.2 Functional Chitosan Derivatives by Chemical and Enzymatic

Modification
A large diversity of chitosan derivatives with various functional substituents have been
obtained by direct modification of the reactive amino and hydroxyl groups using a limited
OH
O OH
O
HO O
NH2 O Cosmetics Pharma
HO
NH O Wound healing
O
Biomedical
Functional group modification

Biosensors
Modified
- O/N Carboxymethylation Chain elongation and branching chitosan
- Acylation Packaging
- Hydroxyalkylation - Graft polymerisation
- Sulphation - Cross-linking Textiles
- Quaternisation
- Oxidation - Polymer networks
- Condensation Agriculture
Water treatment
Figure 10.1 Derivatisation of chitosan and fields of application of the modified biopolymers. Dashed arrows indicate the most reactive groups prone to
modification.
number of chemical reactions (Figure 10.2). Highly cationic and quaternised chitosan, ani-
onic chitosan with carboxyl or sulphate groups, arylated, nonpolar or hydrophobic chitosan
derivatives have all been obtained by chemocatalytic or mild and selective enzymatic con-
version. Some examples are given in Figure 10.2. Recent developments will be discussed
in the following text.
10.2.1 Anionic Chitosan Derivatives

Anionic chitosan derivatives with acidic groups on the polymer backbone are ampho-
teric polyelectrolytes containing both cationic and anionic charges, with increased solu-
bility at neutral and alkaline pH and a pH‐dependent behaviour, that can be controlled
by the charge density and degree of substitution. Carboxyalkyl chitosan derivatives,
both N‐ and O‐substituted, are usually synthesised by carboxymethylation with monoh-
alocarboxylic acids at controlled conditions to attain N/O selectivity [3, 14]. With this
method, N/O and O‐carboxymethyl chitosan [15], carboxypropyl chitosan and carboxy-
butyl chitosan derivatives [16] with antibacterial properties have been obtained.
Carboxyalkyl chitosan derivatives with glycine and carboxybenzyl pending groups have
been obtained by reductive amination with glyoxylic acid [17] and 2‐carboxybenzalde-
hyde [18], respectively. N‐carboxymethyl chitosan is soluble in water, has high viscos-
ity, large hydrodynamic volume and film and gel forming properties and is suitable for
application in food and cosmetics [3]. The N‐carboxybenzyl chitosan is formed by
cross‐linking with glutaraldehyde, a pH‐responsive hydrogel for colon‐specific delivery
of drugs [19].
Succinyl‐ and glutaryl‐chitosan derivatives with anticoagulant and antiplatelet activity
have been obtained by N/O acylation with succinic and glutaric anhydride, respectively
[19, 20].
An elegant and mild chemoenzymatic route for the synthesis of carboxyl chitosan deriv-
atives by selective oxidation of the C6‐hydroxyl group has been recently developed by our
group [21]. A range of oxidised chitosan derivatives with different degrees of oxidation and
properties were obtained by controlling the reaction conditions. These derivatives showed
increased solubility and decreased viscosity in solution. If chitosan is dissolved in diluted
hydrochloric acid prior to TEMPO‐laccase oxidation, a cross‐linked chitosan derivative
was produced, which was able to form a self‐assembling pH‐responsive hydrogel [21]. The
pH‐responsive chitosan derivative obtained showed a sol–gel transition approximately
around physiological pH (7.4) and had a potential application as a platform for delivery
drugs directed to the stomach.
Chitosan sulphates are an important class of anionic chitosan derivatives with strong
anticoagulation and haemagglutination inhibiting properties that can be utilised as an
alternative to heparin [3, 22–24]. Chitosan sulphates also exhibit high sorption capacity
and can be used for metal ion recovery and water and soil bioremediation. Chitosan sul-
phates are commonly obtained by reacting with chlorosulphonic acid in homogeneous or
heterogeneous conditions. The reaction is not selective and mixtures of N‐, O‐monosub-
stituted and N‐, O‐disubstituted derivatives are obtained. Selective synthesis of N‐ or
O‐monosulphate chitosan derivatives can be achieved by starting from N‐ or O‐ protected
chitosans [25, 26].
OH O
O O R
O
HO
NH O O
HO
OH O R NH2
O O R N-acyl chitosan O-acyl chitosan
HO OH OH O
NH O O +
O O N
R + O O
O HO
R HO NH
N-, O O
NH2 O-A HO
Hydroxyalkyl chitosan cyla O
on
tion + NH2
N
lati
HO OH
lky
O OH N-betaine chitosan O-betaine chitosan

O
O-A
O O O
O O
HO O HO
O NH
N-,
O NH2
HO O Chitosan
NH2 O Re
HO d
O-carboxymethyl-chitosan N-carboxymethyl-chitosan
ion am uct
at ina ive
ulf
OH
Arylatio
S tio
n O
n
OSO3Na O O
tio
HO
NH
ida
O O HO
n
OH
Ox
HO
NH2 O OH HO OH
HO
Chitosan sulfate O O
COOH
HO O Sugar-linked chitosan
O NH n
O
HO R
OH O
6-Carboxychitosan HO
OH
Arylchitosan
Figure 10.2 Functional chitosan derivatives obtained by chemical or enzymatic modification of amine or primary hydroxyl groups.
10.2.2 Hydroxyalkylchitosans
Hydroxyalkylchitosans – that is hydroxyethyl chitosan, hydroxypropyl chitosan and glycol
chitosan – are obtained by reacting with alkyl epoxides (e.g. ethylene oxide, propylene
oxide, butylene oxide) and glycidol, which is a common procedure for modification of
polysaccharides, including cellulose and starch. Selective synthesis of N‐hydroxyalkyl or
O‐hydroxyalkyl derivatives can be controlled by the reaction conditions, particularly the
process temperature and catalyst used. Hydroxyalkylchitosans are water soluble and show
antimicrobial properties and have potential application as a temperature‐sensitive injecta-
ble carrier for cells [27] and as self‐assembled nanoparticles for drug delivery [28–30].
10.2.3 Quaternised and Highly Cationic Chitosan Derivatives

The cationic character of chitosan is essential for its biological activities and applications,
therefore highly cationic chitosans and permanently charged quaternary chitosan deriva-
tives have been synthesised, characterised and tested. Cationic chitosans are usually pre-
pared by aminoalkylation with dialkylaminoalkyl chloride, glycidyl‐trimethylammonium
chloride (GTAC) or glycidyltrimethylammonium oxide at alkaline conditions. The highly
cationic chitosan derivatives are water soluble and find applications in cosmetics for hair
and skin protection [1, 3, 31]. Chitosan derivatives with N‐aminoethyl, N‐dimethylaminoe-
thyl, N‐diethylaminoethyl, N‐dimethylaminoisopropyl display cytotoxic activities and
might have antitumour effects, eliciting dose‐dependent inhibitory effects on the prolifera-
tion of tumour cell lines [32, 33].
Quaternised chitosan derivatives like N,N,N‐trimethylchitosan chloride (TMC), N‐
propyl‐N,N‐dimethyl chitosan chloride are obtained by methylation of amino groups in
chitosan using well‐established chemical procedures. Quaternised chitosans show antibac-
terial and antifungal properties, hydroxyl radical scavenging activity and have improved
mucoadhesive properties as compared to native chitosan [3].
Betaine‐chitosan derivatives represent a greener class of quaternary chitosans, consist-
ing only of natural and nontoxic components. Fully bio‐based quaternary chitosan deriva-
tives have been obtained by N‐acylation of chitosan with glycine betaine, a natural
quaternary amino acid found in sugar beet and many other plants [34, 35]. Chitosan betain-
ates showed a uniform structure and exhibited biological activities like enhanced cellular
uptake and cytotoxicity [35].
10.2.4 Hydroxyaryl Chitosan Derivatives

Chitosan adducts with phenols and phenolic acids with increased water solubility under
alkaline conditions, which has been achieved by enzymatic oxidative addition reactions
using tyrosinase, laccase or peroxidase as catalysts [36–39]. All three enzymes have broad
substrate specificity and can convert a wide variety of phenolic substrates in aqueous solu-
tion and mild reaction conditions. With laccase and tyrosinase as catalysts, hexyloxyphenol
[38], chlorogenic acid [39], 4‐hydroxyphenyl‐3‐propionic acid [37] and gallic acid [40]
have been coupled to chitosan via an o‐quinone intermediate to yield either Schiff‐bases or
Michael‐type adducts. Adducts of dodecyl gallate with chitosan have been obtained using
peroxidase [41]. All these phenolic‐chitosan adducts have increased water solubility under
Chemical and Enzymatic Modification of Chitosan to Produce New Functional Materials 251
(a)
C C C C C C C C C C C C
C
C +
C
C
(b)
C C C C C C C C C C
(c)
C C C C C C C C C C C C C C C
Figure 10.3 Schematic illustration of (a) grafting through, (b) grafting to, and (c) grafting from. Encircled
C is a chitosan monomer (e.g. N‐acetyl glucosamine, glucosamine or chitosan molecule); X is the initiating
functionality; tail is a co‐polymer; star is another co‐polymer added to the tail.
both acid and alkaline conditions, even at low substitution degrees. These phenolic chi-
tosans show antioxidant and antimicrobial activity, and have a rheological behaviour char-
acteristic of associating water‐soluble polymers [3].
10.2.5 Carbohydrate‐Modified Chitosan

Another class of biologically active chitosan derivatives is represented by carbohydrate‐
modified chitosans. Sugar‐linked chitosan derivatives are obtained by reductive amination
(N‐alkylation) of chitosan with various aldehydes, monosaccharides, disaccharides and oli-
gosaccharides [42–45]. Carbohydrate‐bound chitosan derivatives with D‐ and L‐fructose,
lactose, galactose and mannose showed specific interaction with cells and showed high
cellular recognition ability.
Cyclodextrin‐bound chitosan derivatives have shown useful properties for application as
a matrix for drug delivery in cosmetics and analytical chemistry [3].
10.3 Graft Co‐Polymers of Chitosan

Graft co‐polymers of chitosan consist of a linear chitosan backbone and randomly distrib-
uted branches of co‐polymers. The properties of graft co‐polymers strongly depend on the
type, length and number of chains attached to the chitosan backbone. In general, three
approaches for graft synthesis can be applied: ’grafting through’, ’grafting to’ and ’grafting
from’. ’Grafting‐through’ is the cross‐linking of monomers (e.g. N‐acetyl glucosamine,
glucosamine or chitosan molecule) with other monomers with a defined co‐polymer
attached to it (Figure 10.3a). For the production of graft co‐polymers of chitosan only the
other two methods are used. In the case of ’grafting to’, end‐group functionalised co‐polymers
react with complementary functional groups on the chitosan chain (Figure 10.3b).
Telechelic polymers, polyethylene glycol and polydimethylsiloxane are some examples
that are usually utilised for grafting to chitosan. ’Grafting from’ is often based on the intro-
duction of a reactive group, usually referred to as initiator, on the chitosan backbone medi-
ated by the ’grafting to’ method. The initiator, that can be an acrylate or vinyl, is used to
covalently link co‐polymers to the chitosan chains (Figure 10.3c.) [46]. The efficiency and
percentage of grafting is dependent on the type of monomer and initiator concentration and
also reaction temperature and time.
Techniques that can be used for grafting are chemical [47–52] including grafting through
living polymerisation [48, 53], irradiation [47, 48, 51, 52], photochemical [48], plasma‐
induced [48, 50], and enzymatic synthesis [47, 48, 50, 51, 54]. Chemical grafting can be
performed by free radical and ionic approach. Free radicals are produced from initiators
and transferred to the chitosan backbone which will form the graft co‐polymer. Examples
of initiators are ammonium persulphate, potassium persulphate, ceric ammonium nitrate,
thiocarbonate–potassium bromate, potassium diperiodatocuprate (III), 2,2’‐azobisisobu-
tyronitrile and ferrous ammonium sulphate (FAS) [51]. Ionic mode grafting can be per-
formed by cationic and anionic mechanisms using alkali metal suspensions in a Lewis base
liquid, organometallic compounds, sodium naphthalenide and sodium‐ammonia as initia-
tors [48]. A drawback of the free radicals and ionic approach is that the polymerisation
process is less controlled, resulting in high polydispersity of the grafted co‐polymers.
’Living’ polymerisation, a process defined by Szwarc [55], results in grafted co‐polymers
with regulated molecular weights and low polydispersity. In case of living polymerisation,
controlled free radical polymerisation can be performed with atom transfer radical
polymerisation (ATRP). The advantage of ATRP is twofold due to the following principles.
First, the initiation reaction is fast enough in order to have a continuous concentration of
growing polymer chains. Second, the graft co‐polymers are able to grow due to the equilibrium
between the radicals formed and graft co‐polymers synthesised [56].
In addition, click chemistry can be used to combine/conjugate different polymeric chains
to synthesise grafted co‐polymers with different structures such as linear, branched, comb‐
shaped and star‐shaped co‐polymers [57].
Photochemical grafting is initiated by chromophores that absorb light and as a conse-
quence dissociate into reactive radicals. Photosensitisers can be used to accelerate the pro-
cess. Irradiation‐induced grafting can be performed by microwave and γ‐irradiation such as
60
Co γ‐irradiation. Plasma‐induced grafting depends on electron‐induced excitation, ioni-
sation and dissociation; the released energy, like in irradiation, is able to form radicals.
Enzymatic conjugation is another possibility to graft chitosan to biomacromolecules like
peptides and proteins. Tyrosinase, transglutaminase, laccase and horseradish peroxidase
have been used for the synthesis of chitosan‐protein bioconjugates with a wide range of
proteins and peptides, such as, for example, gelatin, sericin‐peptides and collagen peptides,
among others [58–62]. These chitosan‐protein and chitosan‐peptide conjugates offer inter-
esting mechanical and rheological properties, comparable with that of natural glycopro-
teins, and are suitable for biomedical and industrial applications. The grafted chitosan
co‐polymers may form hydrogels and also display various biological properties. Some
examples of graft co‐polymers of chitosan and their potential application are listed in
Table 10.1.
10.4 Cross‐Linked Chitosan and Chitosan Polymer Networks

Homopolymeric networks consisting of cross‐linked only‐chitosan derivatives are obtained
using bifunctional reactive cross‐linkers such as (i) aliphatic or aromatic dialdehydes and
(ii) diepoxides, through intermolecular bridges between chitosan chains. Most common
cross‐linkers are glutaraldehyde and ethylene glycol diglycidyl ether (DGDE), which link
the chitosan chains via imine (Schiff‐base) or ether bond formation, respectively. Cross‐
linked chitosan derivatives have the ability to form hydrogels and show various functional
properties that can be enhanced by the cross‐linker. For example, a range of multifunctional
cross‐linked chitosan derivatives have been obtained using aromatic dialdehyde and dike-
tones as cross‐linking agents [76]. These derivatives are able to form water‐insoluble
hydrogels, with high metal‐chelating capacity and antimicrobial activity, due to the phe-
nolic groups of the cross‐linker, and could be applied in many areas such as metal remov-
ing, water removing, and biological applications [76].
Chitosan‐based interpenetrating polymer networks (IPNs) have been obtained with a
number of natural and synthetic polymers. Some examples are chitosan IPN hydrogels
with gelatine and cellulose ([77], methyl cellulose [78], hyaluronic acid [79], polyvinyl
alcohol ([80, 81], polyacrylonitrile [82] and polythylene oxide/poly‐N‐isopropylacryla-
mide [83]. IPNs are attractive materials since they exhibit synergistic properties from the
component polymers with tuneable hydrophilic‐hydrophobic balance, temperature and pH
responsive properties [79, 83–85] and electric conductivity [82, 86]. Chitosan‐based IPNs
have been shown to be excellent materials for immobilisation and delivery of drugs,
for immobilisation and stabilisation of enzymes, and as a scaffold for cartilage tissue
engineering [1, 77, 87].
10.5 Outlook
Decades of research on chitosan and its modified derivatives have shown, beyond any
doubt, the huge application potential for use in various fields including medical, pharma-
ceutical, cosmetics, bio‐related science and technology, food industry, agriculture, and
environmental protection. Viable technologies and processing routes have been developed
for the synthesis of chitosan derivatives and chitosan‐based materials, and a number of
them are already reaching the consumer in various end‐use products. Now, when the
world is facing the transition from the petrochemical economy to a sustainable bio‐based
circular economy, there are many opportunities for chitosan‐derived chemicals and mate-
rials to be recognised as a major bioresource for bioactives and biomaterials and to expand
their market share. Chitosan and its derivatives are very useful since they can be made
available in a variety of morphologies including fibres, films, hydrogels, membranes,
nanoparticles, microparticles and with various functional properties and bioactivities.
Nevertheless, scientific and technological developments are still needed to develop
greener and more sustainable routes for chitosan production and modification, addressing
not only process efficiency but also the source of substrates, catalysts and solvents, the
carbon and energy balance, the environmental impacts and the process and product costs.
Standardisation of methods for chemical, structural and functional characterisation of chi-
tosan and chitosan derivatives is needed to allow objective tools and criteria to compare
different materials.
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85. Kim, S.J. et al., Water and temperature response of Semi‐IPN hydrogels composed of chitosan and
polyacrylonitrile. J. Appl. Polym. Sci., 2003. 88(12): p. 2721–2724.
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p. 365–374.
11
Chitosan‐Based Drug
Delivery Systems
Cristian Peptu1, Andra Cristina Humelnicu1, Razvan Rotaru1, Maria
Emiliana Fortuna1, Xenia Patras1, Mirela Teodorescu1, Bogdan Ionel
Tamba2, and Valeria Harabagiu1
1
‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian Academy,
Iași, Romania
2
A&B Pharm Corporation, Roman, Neamț, Romania
Drug delivery systems (DDSs) represent one of the most active research topics with a
history as long as more than 50 years, and it is an increasing actual interest focused on
sustained/controlled and targeted pharmaceutical formulations able to provide increased
compliance of the patients, personalized, and more efficient therapeutic effects. DDSs of
different shapes ranging from microparticles to nanoparticles (NPs), from hydrogels to
scaffolds, and membranes containing the drugs embedded into organic, inorganic,
polymeric matrices or combinations between them were investigated, a few of them being
already commercialized.
Chitosan and chitosan‐modified polymers are one of the most studied materials of natural
origin in their use as matrices for the preparation of DDSs. This chapter describes the unique
properties of chitosan/modified chitosan as cationic polymers in providing increased bioa-
vailability and higher therapeutic efficiency of various anionic drugs/genetic materials or of
hydrophobic/hydrophilic drugs as a result of their higher bioadhesion to different tissues/
mucosa, transfection properties, efflux pump inhibition or permeation across biological bar-
riers (oral, nasal, and vaginal mucosa or skin, ophthalmic, gastrointestinal, and blood–brain
barriers (BBB)). Recent and representative results on the preparation and applications of
chitosan/chitosan derivatives–based DDSs are also reviewed.

11.1 Introduction
Drug delivery systems (DDSs) could be defined as engineered devices aimed to provide
sustained, controlled, and/or targeted release of therapeutic agents. These systems are
composed of the therapeutic agent embedded into a matrix that could be an organic/
inorganic compound or a polymer. The main advantages of controlled‐release systems are
(i) the reduction of the drug side effects by maintaining their concentration in the therapeutic
range between the minimum effective and the toxic concentrations, (ii) increased
compliance of the patients by diminishing the number of administrations, and (iii) reduced
amounts of therapeutic agents. However, one should also mention the drawbacks of such
systems linked to the low biocompatibility and toxicity of the matrix or of its biodegradation
compounds, any discomfort of the patient due to the administration method (e.g., surgery
for implantation) or a higher cost as compared to the traditional drug administration as
solutions and ointments.
Three steps in the development of DDSs were identified by Park 2014. Thus, during a
30‐year period, in the second part of the twentieth century (1950–1980), the first generation
of pharmaceutical formulations of sustained release was commercialized for oral and
transdermal delivery systems, the physicochemical properties of controlled DDSs were
established, and the mechanisms of drug release (dissolution, diffusion, osmosis, and ion‐
exchange) were identified as a function of drug/matrix nature and of the administration
procedure. That time, macroscopic and microscopic polymeric DDSs were mainly
developed.
The second generation of DDSs (1980–2010) started with the development of the zero‐
order release kinetics formulations, considered to be the only systems able to provide a
steady blood concentration of the drug. However, the thinking “flatter is better” [1] was
proved to be inappropriate, as an efficient but variable drug concentration between the
lower and higher limits of the therapeutic range is registered for many delivery systems, on
one hand, and zero‐order release kinetic does not always warrant a constant blood
concentration. This is especially the case of the oral drug administration since a decrease
of the drug absorption was observed when DDS moves from upper small intestine to the
colon. Moreover, for some drugs—nitroglycerin, pituitary gland hormones, and insulin—a
constant blood concentration is even not desired. Thus, other release kinetics was proposed
for both biodegradable and nonbiodegradable matrices [2]. Drug conditioning into
cyclodextrin macrocycles and sustained drug release from the obtained inclusion complexes
were also investigated [3–9]. An important research effort was done to elaborate stimuli‐
responsive [10–14] and targeted DDSs [15], part of them being prepared by using
nanotechnology concepts, but only a few formulations achieved the clinical application
maturity due to the difficulties in overcoming the biological barriers, a challenging issue to
be solved during the next period.
Although a pioneering report on NPs designed for drug delivery appeared many years
ago [16], the miniaturization of DDSs at nanoscale (the third generation) was boosted by
introducing the nanoscience and nanotechnology program in USA, the beginning of 1990s,
since a growing interest in nanomedicine was registered everywhere in the world, generating
valuable results on liposomal formulations, polymer–drug conjugates [17–21], or
poly(ethylene glycol) ((PEG)ylated) NPs [22, 23] showing long circulation in the body
[24]. The enhanced permeation and retention effect by the conjugates accumulated in
Chitosan‐Based Drug Delivery Systems 261
tumor due to the “leaky” vasculature [25] was developed and the effects of passive
(determined by NP physicochemical properties, dimension, and shape) and active targeting
(generated by affinity ligands linked to NP surface) were compared [26].
A recent paper presenting very interesting views of medical specialists from Harvard
Medical School [27] considers the evolution of the DDSs from macroscopic (1960s) to
microscopic (1980s) and nanoscopic formulations (1990s). The advantages and
disadvantages of each type of drug delivery formulations are described, and the classical
and new mechanisms and mathematical models for the drug release are also compared.
Several macro‐ and microscaled DDSs that were already commercialized and of targeted
nanoscaled‐drug delivery formulations which are in the phases of preclinical and clinical
testing are presented in Table 11.1 (entrees 1–8, 9–11, and 12–18, respectively). More
information on commercialized DDSs was published by Kearney and Mooney (2013) [28].
Many research efforts were focused on the development of preparation methods, such
as ProLease technique to obtain injectable microparticles with prolonged release [29],
nanoprecipitation [30, 31] and nanoemulsion [32, 33] or microfluidics [34] to allow the
nanoentrapment of hydrophobic and hydrophilic drugs, the last mentioned technique
offering the advantages of a better control of NP dimensions and a higher content of
encapsulated drug. Drug delivery NPs containing two drugs dispersed in polymeric layers
of different structures were also prepared through layer‐by‐layer deposition, a method that
allows the release rate control of each drug [35].
When choosing a polymer to be used as a matrix in the preparation of DDSs, one should
consider its physicochemical (chemical structure/functionality, molecular weight,
solubility, degradability, etc.) and biological properties as well as the drug properties and
mechanism of action. This chapter concentrates on chitosan‐based DDSs considering the
biological properties of this biopolymer that generate unique end‐use properties of
pharmaceutical formulations containing either pristine chitosan, chemically/physically
modified chitosan or combinations of chitosan with other polymers (Figure 11.1). As very
rich information focusing on chitosan‐based DDSs is available in review articles [56–64],
only the most recent and representative results on different formulations shapes (micro/
NPs, gels, membranes, nanofibers, etc.) and their specific applications are discussed here.
11.2 Beneficial Effects of Chitosan

Chitosan—characterized by biological properties such as hemostatic, bacteriostatic,
anticholesteremic, anticarcinogenic, and fungistatic properties [59]—is considered as one
of the most suitable candidates for biomedical applications, including drug delivery. Being
a natural biopolymer, its biocompatibility and lack of toxicity are generally accepted.
Moreover, chitosan was already approved by the Food and Drug Administration as the
HemCon dressing in wound treatment [65]. Low‐molecular‐weight and high‐molecular‐
weight chitosan are metabolically and enzymatically degraded [66], respectively, in the
body, providing an easy elimination by renal clearance. The rate of degradation is also
depending on the degree of deacetylation. Chitosan is soluble in acid media, and becomes
insoluble at physiologic (7.4) and basic pH. Moreover, having two hydroxyl groups on
each structural unit and a content of primary amine groups depending on the degree of
deacetylation, chitosan is a versatile candidate for the preparation of materials with
modulated properties through chemical modification and physical interaction with organic/
Table 11.1 Drug delivery systems on different technological maturity levels.
No. DDSs type: release mechanism Matrix* Active principle Reference Commercial trademark: therapeutic
application
1 Reservoir: diffusion Silicone Anesthetic gases [36] First report on macroscaled DDS
2 Implant: diffusion Progestin levonorgestrel [37] Norplant: contraceptive drug
Progesterone
3 Implant: reservoir diffusion PEVA Pilocarpine [38] Ocusert: intraocular antiglaucoma drug
4 Implant: reservoir diffusion Progesterone [38, 39] Progestasert: intrauterine contraceptive drug
5 Skin patch: zero‐order kinetics Scopolamine [40] Transderm‐Scop: drug treating motion sickness
6 Oral delivery DDSs: pulsatile Polymeric capsules with drilled Salbutamol, nifedipine, [41] Different commercialized drugs
release, osmotic control surface membrane hydromorphone, verapamil,
paliperidone
7 Polymeric tablet: swelling, HPMC Diclofenac, molsidomine, [42] Geomatrix: platform for different drugs
gelling, erosion zileuton, nisoldipine, etc.
8 Disk‐like brain implant Poly(carboxyphenoxy Bis(2‐chloroethyl) nitrosourea [43] Gliadel: treatment of brain cancer
propane)—sebacic acid
9 Depot microparticles PLGA Luteinizing hormone‐releasing [44] Decapentyl LP or Lupron Depot: treatment of
hormone prostate cancer
10 Injectable microspheres PLGA Risperidone [45] Risperdal Consta: antipsychotic drug
11 Microparticles Poly(butylene terephthalate) Interferon‐α2b [46] Locteron: (treatment of hepatitis C
12 Micelle formulation PEG–PLA Paclitaxel (treatment of different [47] Genexol‐PM: antitumor
tumors)
13 Drug‐loaded NPs Poly(L‐glutamic acid) Paclitaxel [15] Xyotax: ovarian and lung cancer
14 The systems are in different Cyclodextrin‐ polymer conjugate Camptothecin [48–50] CRLX‐101 or IT‐101
15 phases of preclinical and PEG–PLA Docetaxel [51] BIND‐014: prostate, non‐small lung, cervical,
clinical studies. head and neck cancers
16 Adamantine‐PEG and siRNA [52] CALAA‐01: siRNA delivery
adamantine‐PEG conjugated
with human transferrin
17 PLGA and PEG–PLA Nicotine antigen, T‐helper cell [53, 54] SEL‐068: antismoking vaccine (first NP for
peptide, and adjuvant vaccine formulation)
Under clinical trials at SELECTABIO: www.
selectabio.com
18 Poly(isohexyl cyanoacrylate) Doxorubicin [55] Livatag: treatment of hepatocellular carcinoma
Note: *PEVA = poly(ethylene‐co‐vinyl acetate); HPMC = hydroxypropyl methylcellulose; PLGA = poly(lactic acid‐co‐glycolic acid) copolymers; PEG-PLA = poly(ethylene glycol -co- lactic acid)
copolymers; and NPs = nanoparticles; DDSs = drug delivery systems.
0004461249.INDD 262 10/26/2019 11:59:03 AM

inorganic compounds, metallic or metal oxide NPs, or other polymers [58, 59, 67]. Chitosan
used in the formulation of DDSs is mainly based on its cationic nature that provides useful
properties such as interactions with anionic drugs, mucoadhesion, increased permeation,
and transfection or inhibition of the pump efflux to the system [68].
11.2.1 Interaction with Anionic Drugs

Chitosan is a cationic polymer suitable for sustained release DDLs through its electrostatic
interaction with anionic or polyanionic drugs such as naproxen [69] and enoxaparin [70],
respectively. The release profile of chitosan‐based DDSs could be manipulated by
complexing chitosan with anionic polymers, [71] tripolyphosphates, or sulfates [72].
11.2.2 Mucoadhesive Properties

The mucoadhesion of chitosan is based on its hydration with water molecules attracted
from the mucus through hydrogen bonding and electrostatic interaction of positively
charged quaternized groups of chitosan with negatively charged functionalities of the
mucus gel [73]. Having the cationic amino groups linked in the less accessible third position
of the glucopyranose ring, chitosan is characterized by quite weak cohesion between its
macromolecular chains and mucoadhesion to the anionic mucus substructures as compared
to other polymers [74].
The mucoadhesion properties of chitosan were strongly improved by increasing its hydro-
philicity through the quaternization of the primary amino groups and PEGylation [75]. The
thiolation of chitosan was proved to be even a more powerful way to increase both the
mucoadhesion and the chitosan cohesion, since disulfide units could be formed not only
with the glycoproteins of the mucus substrate but also between the polymer chains [76].
Mucoadhesive and mucopenetration properties of different anionic and cationic polymer‐
based DDSs were recently reviewed as a function of their chemical structure and molecular
characteristics (molecular weight, functional groups nature/content) [77]. The influence of
the molecular weight, the degree of deacetylation, and the solvent nature on the hydrody-
namic stability and mucoadhesion of electrosprayed chitosan microparticles was also
deeply analyzed in view of their use as oral DDSs [78].
11.2.3 Transfection Activity

Transfection properties of a vector refer to the transport of large nucleic acid molecules or
antisense oligonucleotide (ASO) into targeted cells by surpassing different anatomical and
physiological barriers, such as epithelial and endothelial cell linings and endonucleases
from extracellular matrix that could degrade DNA, phagocytic Kupffer cells, and
macrophages from the circulation that determine a rapid clearance of ASO. Although well
known for its ability to complex with negatively charged macromolecules, such as nucleic
acids, and to protect them against the degradation by serum nucleases, the properties of
chitosan as a candidate for gene therapy are under continuous investigation [79–82].
The interactions between transfection vectors and nucleic acids yield complex
supramolecular structures (polyplexes). Polyplex NPs with dimensions lower than 100 nm
and positive zeta potential were found to undergo endocytosis and to behave as nonviral
CHITIN
CH3
OH O
Crustaceans O HO NH
O O O
HO
O
NH
O
Squid Deacetylation OH
CH3
CH3
OH O
O HO NH
O O
Fungi O
HO O
NH2
Insects OH
BIOLOGICAL CHITOSAN END-USE PROPERTIES OF

PROPERTIES (CS) CS/MODIFIED CS
Biodegradable Cationic properties

Biocompatible Water solubility
Cytocompatible Cell targeting
CHITOSAN
Mucoadhesive Increased mucoadhesion
MODIFICATIONS
Antimicrobial Interaction with anionic drugs
Hemostatic Chemical modification and genetic material
Non-antigenic (chitosan derivatives) In situ gelling properties
Non-toxic Grafting Transfection enhancing
Antioxidant Permeation enhancing
Crosslinking
Anti-inflammatory Blending Efflux pump inhibitory activity
DRUG DELIVERY
SYSTEMS
MICRO/ MEMBRANES THIN

HYDROGELS BEADS NANOFIBERS
NANOPARTICLES AND SCAFFOLDS FILMS
Sustained release
(antibiotics; anti-inflammatory Modulated release Targeted release
Gene therapy
drugs, growth factors, (insulin) (tumoral cells)
steroids, diuretics)
Figure 11.1 Properties of chitosan/modified chitosan and their applications in the preparation of drug
delivery systems.
vectors [83]. However, the transport capacity of chitosan is lower as compared to the viral
vectors. Branched structures [84, 85], thiolated chitosan [86, 87], thiolated/PEGylated chi-
tosan able to stabilize the supramolecular complexes [88], poly(L‐lysine)‐grafted chitosan
[89], and chitosan/cyclodextrin NPs [90] were found to possess improved transfection
properties.
11.2.4 Efflux Pump Inhibitory Properties

Drug‐specific or multidrug efflux systems represent one of the major causes of the
resistance to chemotherapeutics and antibiotics. Thus, intense research efforts were directed
in identifying both low‐molecular‐weight and polymeric efflux pump inhibitors. The
polymeric inhibitors were found to show lower systemic toxicity since they are less
absorbed into the gastrointestinal tissues due to their higher molecular weight, while
thiolated‐chitosan derivatives were proved to provide improved oral delivery for different
P‐glycoproteins substrates, such as saquinavir, paclitaxel, and rhodamine 123 [91–93].
Thiolated chitosan with a molecular weight of 150 kDa was also demonstrated to inhibit the
multidrug‐resistance activity of P‐glycoprotein in the excised small intestine [94].
11.2.5 Permeation‐Enhancing Properties

Depending on the method of administration, a drug has to cross different epithelial and
mucosal membranes. The permeation enhancers are compounds able to temporarily disrupt
such membranes to allow the transport of the drug into blood or lymph streams. Chitosan
and its N‐trimethylated derivative were found to increase the permeation of the large
hydrophilic drugs through the intestinal epithelial tight junction [95, 96]. The permeation‐
enhancing properties are higher for chitosan of increased molecular weight and degree of
deacetylation as proved by ganciclovir–chitosan coadministration when a two‐fold
improved oral bioavailability of the drug was registered [97]. Thiolated chitosan [98] and,
more recently, succinyl‐derivatized chitosan [99] were also found to enhance the intranasal
absorption of isosorbide dinitrate and the buccal absorption of peptides, respectively. A
permeation‐enhancing effect manifested on the entire duodenum was found for NPs of
chitosan combined with cyclodextrins, as compared to simple chitosan NPs that have
limited effect only on the first segment of the duodenum [100]. Synergic effects were
observed for combinations or mixtures of permeation enhancers [101].
As the effectiveness of a DDS is strongly dependent on its ability to interact with the
biological environment, to protect the drug and to release it at a targeted site, the next
paragraph describes the performances of chitosan‐based carriers in bypassing different
biological barriers.
11.3 Chitosan—an Active Polymer for Bypassing Biological Barriers

The development of DDSs has to respond to the challenge of proposing effective formula-
tions or devices that allow the transport of a suitable amount of the desired substance with
therapeutic effect to the site of action in the body in a timely controlled manner. Usually,
such a task has to take into account the presence of the biological barriers. These barriers
are, in fact, differentiated tissues that form an interface between different environments and
perform various functions such as physical–chemical protection while insuring and facili-
tating certain transport/exchange phenomena between the delimitated biological environ-
ments. Such barriers may be found at the cellular level (e.g., cellular membrane) and at the
tissue level (various number of cellular layers—e.g., skin tissue; intestinal tissue, etc.).
Thus, some of these membranes are fulfilling a physiological function by allowing the
selective passage of various entities and, in the same time, they may perform a protection
function against allogeneic entities, for example, bacteria, viruses, drugs, etc. Thus, in
order to achieve drug delivery, the strategies employed must take into account defeating the
natural protection of certain biological barriers. The intracellular delivery of the drug
molecules must defeat the cellular membrane and ensure transcellular transport, while the
passage through a tissue may occur either by defeating the cellular membrane or by pas-
sage through the intercellular junctions (paracellular transport). Some drugs may penetrate
the biological membranes by passive diffusion and without energy consumption, while
other drugs require an energetically activated mechanism. The drug delivery devices are
aimed to facilitate the drug penetration and, among these devices, the nanoparticulate car-
riers are proved to be a very effective tool for various types of biological membranes.
Chitosan‐based nanocarriers raised an enormous interest due to the above-mentioned
outstanding properties promoting their use for the preparation of DDSs across various
biological membranes.
11.3.1 Skin Barrier

The skin barrier represents the external protective layer of the human body and consists of
three sub‐layers: the epidermis; the dermis; and the hypodermis. The outer layer, the epi-
dermis, is actually performing the barrier function of the skin due to the stratum corneum,
10–20 μm thick, composed of corneocyte cells tightly connected in a “brick‐and‐mortar”
like structure. These cells are embedded in lipid bilayers which are inducing an overall
hydrophobic character and increased connectivity. The presence of these lipids in the skin
barrier structures blocks the passage of the hydrophilic compounds while the hydrophobic
ones are allowed to pass through. However, the second barrier, the stratum granulosum, is
built from tightly packed keratinocyte cells [102]. The stratum corneum presents some
interruptions allowing the passage of hair follicles and excretion channels of the sweat
glands. Such spaces may be actually exploited in order to achieve penetration of the skin
barrier by NP carriers. Thus, depending on the NPs dimensions, their fate when crossing
the skin barrier may greatly differ. For example, the particles with dimensions higher than
100 nm may not pass, while particles with diameters lower than 20 nm tend to accumulate
in the hair follicles [103]. However, according to various types of skin diseases, such
requirements may significantly change. Thus, other authors [104] established that dimen-
sions between 300 and 600 nm are ideal for the transfollicular passage.
The percutaneous transit of the drug molecules must defeat mainly the corneocyte cell
layer, and among the employed strategies NPs having appropriate size were proved to be
successful. The nanoparticulate devices may pass the barrier together with the drug to
reach systemic circulation or they may be trapped in the stratum corneum junctions or at
the skin surface and release the drug load locally.
Chitosan, as a constituent polymer for transdermal delivery devices, was demonstrated
to enhance the transdermal drug passage due to the electrostatic bonds between the
chitosan’s positive charges located at the amino group’s level and the negatively charged
phospholipids from the cellular membranes of the corneocytes cells [105]. Such interactions
disrupt the barrier function of the cells, thus facilitating the transdermal delivery. Also, it
has been shown that chitosan can disturb, on one hand, the tight junctions between the
epithelial cells by extracting the proteins responsible for the formation of the intercellular
connections from the cellular membrane [106] and, on the other hand, can disturb the over-
all ultrastructure of rat epidermis [107].
The effect of chitosan as penetration enhancer for transdermal drug delivery has been
demonstrated [108] by preparing chitosan‐conjugated, pluronic‐based nanocarrier as a
r eservoir for hydrophilic proteins. The addition of chitosan increased the average NPs diam-
eter from approx. 60 to 70 nm. However, fluorescence analysis of the penetration efficiency
clearly demonstrated that chitosan‐conjugated nanocarriers were superior to bare nanocar-
rier, in spite of the size difference. The hydrophilic nature of chitosan is limiting its use as a
carrier for hydrophobic drugs. Recent studies [109] focused on the use of cyclodextrin–
warfarin complexes in combination with chitosan NPs for transdermal delivery. Thus, it was
demonstrated that cyclodextrin increases the drug amount to be loaded in the chitosan NPs
and the overall transdermal delivery of warfarin. Moreover, cyclodextrins were proved to act
as skin penetration enhancers [110]. Tacrolimus (FK506), a potent macrolide immunosup-
pressive agent, was delivered using NPs (average size of 110 nm) based on a combination of
chitosan and nicotinamide, a water‐soluble derivative of vitamin B3, proved to enhance the
solubility of some poorly water‐soluble molecules [111]. The abovementioned studies
revealed that the drug‐complex loaded particles were more effective than neat drug‐complex
in delivery at the level of both superficial and deeper layers of the skin. Recent studies, [112]
by using confocal laser scanning microscopy, demonstrated that the curcumin‐loaded
chitosan NPs, having different average diameters ranging from 70 to 190 nm, get accumu-
lated in the hair follicles where they act as reservoirs for the drug delivery.
11.3.2 Mucosa Barrier

Mucosal barriers consist of cell tissues covering the body cavities, which are in contact
with the external environment. A mucosal tissue may be also encountered at the level of the
digestive tract. The epithelial tissues belonging to the mucosa barrier have a similar
structure to the skin although the stratum corneum is missing. Mucosal membranes are
covered by a mucus layer with a gel structure which consists of mucin proteins having the
bioadhesion function and insuring the epithelium moisture. The mucus layer is continuously
secreted by the adjacent epithelium and has a decreased viscosity in contact with the air,
thus insuring a lubricating effect that helps in dissipating the eventual mechanical stress
and protection of the deeper mucus layers. The mucus layer has a mesh‐like structure
having the dimensions ranging between 30 and 100 nm, and is resistant to blending, which
imposes a diffusion‐limited drug delivery. The physical interactions of the penetrating
entities through the mucus barrier condition the diffusion process. The hydrophilic
substances are easily diffusing through the mucus layer, while the hydrophobic ones may
have a delayed diffusion due to interactions with the hydrophobic mucus components. The
local pH also influences the mesh density of the mucus; the acidic conditions are leading
to a decreased mesh size, while an alkaline environment induces an opposite effect. The
nanoparticulate drug delivery devices aimed for passing through the mucus barrier are
conditioned by their surface electrical charge. Thus, particles with neutrally charged surface
will diffuse according to their size, while the positively charged particles strongly interact
with the mucus and disrupt its ultrastructure, thus diminishing the barrier properties.
11.3.2.1 Nasal Mucosa

The nasal mucosa functions as a barrier at three levels. First, there is a physical barrier
consisting of mucus and ciliated epithelium; second, the mucus flow which is responsible
for permanent local clearance; and third, a chemical barrier formed by the enzymes present
in the mucus. The chitosan‐based nanoparticulate systems were proved particularly
effective in drug delivery via the nasal mucosal barrier. The successful nasal delivery of
insulin‐mediated by PEG‐graft‐chitosan nanogels with average diameters from 175 to
280 nm showed that, besides the size of the nanocarriers, the surface charge is also very
important for successful drug delivery [113]. Chitosan particulate carriers were also found
effective for delivery of vaccines via the nasal pathway, as previously reviewed [114]. The
modifications of chitosan, as in N‐trimethyl chitosan (TMC) or mono‐N‐carboxymethyl
chitosan (MCC), were tested in view of the performance in nasal delivery of tetanus toxoid
antigen [115]. The TMC‐based particles had positive surface charge, while MCC had
negatively charged surface. The delivery performance of the tetanus toxoid antigen was
found slightly better in the case of TMC NPs characterized by smaller average diameters,
a feature that compensates for the lack in positive charges for the enhancement of the
delivery through the nasal mucosa. The performance of chitosan NPs can be further
enhanced by combination with other delivery strategies. In a recent study, chitosan was
conjugated with monophosphoryl lipid A as a novel formulation for use as a mucosal
adjuvant to generate protective immune responses against Mycobacterium tuberculosis
infection [116].
11.3.2.2 Barriers Encountered in Oral Delivery

The delivery of drugs by oral administration may target local tissues along the digestive
tract or the delivery of active principles in the systemic circulation. The passage of the
drugs from oral cavity to the intestinal tube encounters different biological barriers, having
as goal the processing of the nutrients while ensuring protection against the toxins or
pathogenic agents. The first encountered obstacle is the environment from the oral cavity
where drugs may pass into the blood stream via the oral mucosa.
The surface of oral mucosa is partially covered by nonkeratinized (30% of the surface)
and keratinized epithelium (50% of the surface). The saliva, which is continuously secreted
by the salivary glands, creates a permanent fluid at the surface of the mucosa which
influences the passage of any pharmaceutical product. Thus, the barrier function at the
buccal level consists of selectivity induced by saliva‐dependent factors such as pH, fluid
amount, enzymatic activity, and, finally, the permeability of the epithelial tissue. However,
the delivery through oral mucosa should be also regarded in the context of salivary clearance
which brings into discussion the delivery via epithelial tissue covering the next segments of
the digestive tube. The intestinal barrier presents also a mucus layer, which is continuously
secreted, and an epithelial tissue including cellular tight junctions. The mucus layer is
designed to allow the passage of water and nutrients, whereas bacteria and pathogens
cannot pass. The overall electrostatic charge in the mucus layer is supposed to be negative.
The particular properties of the intestinal barrier (epithelial tissue) impose the passage of
lipidic substances up to 700 Da, while the tight junctions have an even lower cut‐off value
of 200 Da. Another component of the digestive barrier has a chemical nature and consists
of different pH values (1.2–3 in the stomach and 6.5–8 in the thin intestines) and also the
strong enzymatic activity (pepsin in the stomach, pancreatic enzymes in the luminal area,
cytosolic proteases bound to the enterocyte membrane).
The requirements for a drug delivery device aimed to overcome the digestive barrier
have to take into account the complex chemical, physical, and biological barriers. Thus,
NP‐based DDSs may address these problems by their protective capacity towards chemical
attacks, good contact with the intestinal epithelium due to high surface/volume ratio, and
appropriate size to favor the trans‐ and paracellular passage. The chitosan‐based particles
were proven to be able to disrupt the epithelial tight junctions due to its positively charged
nature. However, the low pH in the stomach induces changes of the chitosan nanoparticulate
carriers, and, as a consequence, chitosan has been modified or used in combination with
other polymers. Thus, a succinic anhydride derivative (2‐dodecenyl succinic anhydride)
was used to modify the chitosan amino groups [117]. The obtained product, chitosan with
increased hydrophobicity, was further used to prepare insulin‐loaded NPs, and the authors
observed that the release at intestinal pH is slightly reduced as compared with unmodified
chitosan but the protection against acid pH was significantly improved. In a recent study,
polymeric NPs based on quaternary ammonium‐chitosan and S‐protected thiolated
derivative were prepared by cross‐linking low‐molecular‐weight hyaluronic acid [118]. It
was shown that S‐protected thiolated chitosan derivative has better mucoadhesion, probably
because of the cleavage of the S–S bonds and exchange reactions with the thiol components
of the mucus. The increased adhesion was considered responsible for prolonging the drug
residence (a model drug for insulin) at the absorption site, and enhancing the drug
bioavailability. In another study, chitosan was combined with cyclodextrins in order to
enhance the intestinal delivery of peptide glutathione [100]. Transport studies performed in
the frog intestine model confirmed that both types of NPs could induce permeabilization of
the intestinal epithelia; however, the cyclodextrin‐based NPs exhibited absorption‐
enhancing properties in all segments of the duodenum, whereas chitosan‐only NPs effect
was restricted to the first segment of the duodenum.
11.3.2.3 Vaginal Mucosa

Vaginal mucosa represents a frequently used pathway for the delivery of drugs with local
or systemic effect. The main barriers against drug absorption are actually the natural
barriers against infections and comprising chemical barriers (the vaginal fluid having a low
pH) and physical barriers (the vaginal epithelium and mucus layer). Chitosan‐based NPs
loaded with a model anti‐HIV microbicide (tenofovir) were demonstrated efficient in terms
of mucoadhesivity and vaginal drug delivery [119]. NPs prepared from poly(D,L‐lactide‐
co‐glycolide) and surface coated with chitosan, when used as vaginal delivery system, were
showed to increase the antifungal activity of Clotrimazole by fourfold against Candida
albicans based on increased mucoadhesive properties of the NPs [120]. S‐protected
chitosan was also proved as a mucoadhesive excipient for the release of metronidazole
from vaginal tablets [121].
11.3.3 Ophthalmic Barrier

Another area of interest for the local administration of therapeutic agents is the eye, and the
main target of ophthalmologic treatments is to bypass the biological barriers without
damaging the sensitive healthy tissue. A first barrier encountered in eye drug administration
is the muco‐aqueous layer that protects the anterior surface of the eye followed by corneal
epithelium, iris blood vessels, nonpigmented layer of the ciliary epithelium, retinal pigment
epithelium, and the epithelium of the retinal vessels. The different strategies for the drug
administration must consider particular requirements for each area of interest. Generally,
the drugs may be delivered to the anterior or to the posterior sides of the eye. The noninvasive
drug delivery via the anterior side of the eye, topical instillation, is the most commonly
used pathway, [122] and must defeat the lacrimal clearance. The posterior eye treatment
may use the periocular pathway which must pass several barriers (ocular tissues)—
episclera, sclera, choroid, Bruch’s membrane, and retinal pigment epithelium—to reach the
retina and vitreous humor [123]. Besides the tissues bypass, there is also a dynamic barrier
consisting of blood flow, lymphatic drainage, fluid flow from the intraocular drainage
systems, and enzymatic degradation. A more direct drug delivery pathway may be used
when large amounts of drugs are needed in the posterior eye segment, and that is the
intravitreal injection. However, potential side effects such as retinal detachment, cataract,
endophthalmitis, etc., may prevent its usage. Nevertheless, intravitreal administration is the
preferred drug delivery route in treating diseases of the posterior segment, achieving the
highest intraocular bioavailability [124].
Topical administration of drugs using chitosan NPs (around 400 nm diameter) suggests
that they may penetrate into the corneal and conjunctival epithelia as demonstrated by
confocal laser scanning microscopy studies [125]. The prepared chitosan NPs were able to
interact and remain associated to the ocular mucosa for extended periods of time, thus
enabling the local drug delivery. Chitosan was also combined with cyclodextrins in order
to improve the drug delivery capacity. Thus, the naringenin‐loaded sulfobutylether‐β‐
cyclodextrin/chitosan NPs were shown to have better ability to prolong the ocular mucosa
residence time than the naringenin suspension [126]. Also, chitosan was used together with
alginate in the preparation of NPs by polyanion–polycation complexation. Thiolated
chitosan–alginate NPs were showed to have improved mucoadhesive properties when
tested on corneal tissue [127]. Hyaluronic‐acid‐modified lipid–polymer hybrid NPs were
designed to improve the ocular bioavailability of the strongly hydrophilic moxifloxacin
hydrochloride [128]. The proposed ocular drug delivery platform could effectively cross
the ocular tissues through both para‐ and transcellular routes. The authors propose that the
internal core of the lipid–polymer hybrid NPs could transiently open the intercellular tight
junctions by altering the levels of junction protein. Next, the external lipid layer possessed
biomimetic advantages and could significantly prolong precorneal retention. Furthermore,
the surface‐modified hyaluronic acid in the structure could readily target the CD44
receptors on epithelial cells and promote endocytosis.
The chitosan‐based NP systems were not only tested for topical delivery of adrenaline
[129] but also for intravitreal injection administration of a model drug, cefuroxime.
Chitosan–gelatin nanocapsules prepared by double cross‐linking in double emulsion were
tested for intraocular drug delivery via intravitreal injection [130]. This experimental study
demonstrated the ability of fluorescent chitosan‐based nanocarriers to penetrate ocular
tissues close to the administration site (intravitreal injection), and especially their tendency
to migrate deeply in the retina at time intervals of 72 hours. Moreover, after initial burst
release, the NPs continue to release the drug up to 96 hours, while the drug administered as
the solution is removed rapidly by the intravitreal clearance system.
11.3.4 Blood–Brain Barrier

This barrier represents an interface between the blood systemic circulation and the central
nervous system (CNS), insuring its protection against undesired active principles. The BBB
is located at the level of the blood vessels irrigating the CNS, and it blocks the passage of
most of the drugs. It is built of endothelial cells and makes part of the capillary walls. In
normal capillary vessels, the endothelial cells are connected by more loose junctions but in
the case of the BBB capillaries, the endothelial cells are connected through tight junctions
which are regulating the diffusion processes from the blood to the brain. The delivery of
therapeutic agents to the brain may be performed by invasive and noninvasive methods.
The invasive methods are consisting of direct injection of drugs or drug delivery devices at
CNS level or by temporary physical disruption of BBB using ultrasounds, osmotic shocks,
etc. The noninvasive methods consist of using chemical BBB penetration enhancers (e.g.,
lipid nanocarriers or lipid conjugation of drugs), biological enhancers (drug conjugation
with biologically active ligands that insure the transport across BBB), and the delivery via
nasal mucosa (the olfactory and trigeminal nervous tract shortcut). The delivery via nasal
mucosa pathway was previously detailed and, in the following text, only the delivery across
BBB using chitosan colloidal carriers will be discussed. The mechanisms of breaching
through BBB for CNS‐targeted drug delivery may consider several strategies [131]. One
possibility is that the NPs may be retained in the BBB capillaries and from there they may
create a local concentration gradient that might enhance the drug transport across the
barrier. Also, a certain disruption of the BBB by affecting the epithelial cells membrane of
tight junctions may enhance the NPs permeation through the BBB. Another strategy may
consist of surface masking of the NPs in order to be transported through the BBB cell via
activated transport mechanisms.
The studies of Yemisci et al. (2015) [132] employed the avidin–biotin coupling technol-
ogy to design chitosan nanospheres conjugated with PEG and bearing the OX26 monoclonal
antibody whose affinity for the transferrin receptor may trigger receptor‐mediated transport
across the BBB. The NPs having a diameter of approximately 200 nm were observed in the
lumen of the blood vessels and also in the brain interstitium, thus demonstrating the success
of the delivery strategy. Other studies showed that the results obtained by this strategy are
highly dependent on the degree of surface functionalization [133]. Thus, NPs having too
many interacting sites may remain bound to BBB and have reduced drug delivery efficiency.
A similar approach used nanoprobes comprising an iron oxide NP coated with a PEGylated
chitosan‐branched copolymer, to which a targeting ligand, chlorotoxin, and a near‐infrared
fluorophore, Cy.5.5, were conjugated [134]. In this design, chitosan was used as a linker to
anchor the polymer to the iron oxide surface through electrostatic interaction and physical
adsorption. Also, the chitosan’s role was to stabilize the nanostructure by alleviating the
need for cross‐linking agents and to provide sites for subsequent conjugation of ligands
without the need for further chemical modification. Moreover, the bound PEGylated chi-
tosan also acts as a sterically stabilizing corona, preventing particle aggregation under physi-
ologic conditions. More recent studies employing the inhibition of BBB cellular uptake by
chlorpromazine clearly demonstrated that the transferrin receptor antibody‐conjugated NPs
were preferentially crossing BBB via the receptor‐mediated endocytosis pathway [135].
11.4 Chitosan‐Based DDS Formulations

As a result of their unique physicochemical properties resulting in beneficial end‐use
effects, chitosan and chitosan derivatives were used as carriers for DDSs under differently
shaped formulations such as microspheres, beads, tablets, NPs, nanofibers, films,
hydrogels, conjugates, or nanocomposites [136]. Table 11.2 summarizes some of the
recent results.
Table 11.2 Composition and applications of chitosan‐based formulations for drug delivery.
No. Formulation type Matrix composition* Released drug Applications Ref.

1. Hydrogel Chitosan/catechol Lidocaine Drug administration via buccal [155]
mucosa
2. β‐cyclodextrin‐grafted carboxymethyl Acetylsalicylic acid Controlled DDS [156]
chitosan
3. Chitosan/catechol Sulfasalazine Rectal drug delivery vessels to treat [157]
ulcerative colitis
4. Chitosan/gelatin/glycerol phosphate Latanoprost Eye drop formulation for glaucoma [158]
treatment
5. Chitosan/gelatin/glycerol‐2‐phosphate Stromal cell‐derived Corneal epithelium regeneration [159]
disodium salt factor‐1 alpha
6. Chitosan/glycerol‐2‐phosphate Liposomal NPs Local inner ear application [160]
7. Chitosan/alginate/gelatin Tetracycline hydrochloride Wound healing [161]
8. Chitosan/sodium alginate/ Doxorubicin Liver cancer [152]
hydroxyapatite
9. Imino–chitosan 5‐fluorouracil Local anticancer therapy [144]
10. Hydrogel beads Chitosan/silver Ibuprofen Controlled DDS [162]
11. Chitosan/ZnO Ibuprofen Controlled DDS [163]
12. Powder Chitosan‐g‐poly(acrylamide)/Zn Ofloxacin Drug release [164]
13. Microspheres embedded Glutaraldehyde cross‐linked chitosan Salicylic acid Sustained drug delivery [150]
in thermosensitive
hydrogel
14. Thermosensitive Carboxymethyl chitosan‐graft‐poly 5‐fluorouracil Injectable DDS (anticancer and [165]
hydrogels (N‐isopropyl acrylamide)‐glycidyl anti‐inflammatory)
methacrylate
15. Chitosan cross‐linked with glycerol Adipose‐derived Acute kidney injury [166]
phosphate mesenchymal stem cells
16. Chitosan/glutaraldehyde/polyvinyl Paclitaxel Intratumoral delivery [148]
alcohol
17. Chitosan/glucose 1‐phosphate Eosin Y disodium salt Drug delivery [147]
Diclofenac potassium
18. Chitosan/hydroxypropyl Bovine serum albumin Delivery system [149]
methylcellulose/glycerol
19. Magnetic thermosensitive Chitosan/β‐glycerol phosphate/Fe3O4 Bacillus Calmette–Guérin Bladder cancer [146]
hydrogel
20. Injectable hydrogel Quaternized chitosan/gelatin Dopamine metronidazole Parkinson’s disease [153]
0004461249.INDD 272 10/26/2019 11:59:04 AM

21. Gel‐forming tablets Chitosan/xanthan gum chitosan/ Sodium valproate/valproic Oral drug delivery [154]
carrageenan acid
Chitosan/sodium carboxymethyl
cellulose chitosan/sodium alginate
22. NPs Chitosan/iron oxide Doxorubicin Targeted delivery applications on [167]
MCF‐7 breast cancer cells
Chitosan/hyaluronic acid 5‐fluorouracil Antitumor‐targeted drug delivery via [168]
CD44.
23. NPs Chitosan diacetate and chitosan Doxorubicin Delivery systems for anticancer [169]
triacetate drugs
24. Chitosan/carboxymethyl‐ Silver sulfadiazine Burn wounds [170]
β‐cyclodextrin
25. Beads Carboxymethyl Diclofenac sodium DDSs [171]
chitosan/κ‐carrageenan
26. NPs/fibers Chitosan/tripolyphosphate/Spanish Vancomycin Antibacterial wound dressing [172]
broom fibers
27. Membranes Chitosan/poly(vinyl alcohol) (S)‐ibuprofen Wound healing [173]
28. Scaffold Chitosan/poly(2‐hydroxyethyl acrylate) Levofloxacin Wound infections management [174]
29. Implantable nanofibrous Chitosan/PEO Chlorhexidine Silver Active antibacterial dressing for [175]
membranes local delivery
30. Films Chitosan Ibuprofen Oral mucosal drug delivery [176]
31. Chitosan/PEG‐block‐poly(propylene Metformin Oral antidiabetic DDS [177]
glycol)‐block‐PEG
32. Magnetic NPs Chitosan/carboxymethyl‐β‐ 5‐fluorouracil Anticancer drug delivery [178]
cyclodextrin/Fe3O4
33. Nanocomposite Chitosan/PEG/iron oxide Methotrexate Cancer therapy [179]
34. Chitosan/sodium tripolyphosphate Insulin Oral drug delivery [180]
35. NPs Carboxymethyl chitosan Resveratrol Oral drug delivery [181]
36. NPs Chitosan/tripolyphosphate/mannitol/ Isoniazid rifampicin Treatment of tuberculosis [182]
leucine
37. Chitosan/hydroxyapatite/polymer Candesartan cilexetil Oral drug delivery [183]
micelle
38. Chitosan/folic acid Doxorubicin hydrochloride Tumor‐targeted drug delivery [184]
39. Chitosan/silica Carvedilol Oral drug delivery [185]
40. Chitosan/sodium tripolyphosphate Interferon alpha Oral drug delivery [186]
(Continued)
0004461249.INDD 273 10/26/2019 11:59:04 AM


41. Chitosan/dextran sulfate Protein/siRNA Drug delivery [187]
42. Magnetic NPs Chitosan/PEG/polyvinylpyrrolidone/ Curcumin Anticancer drug delivery [188]
iron oxide
43. Composite NPs Chitosan/ZnO Gentamicin Wound care [189]
44. Cross‐linked chitosan with Doxorubicin Tumor therapy [190]
bis(acryloyl)cystamine /mesoporous
silica
45. Microparticles Chitosan/sodium tripolyphosphate Isoniazid Pulmonary delivery [191]
46. Composite nanofibers Chitosan Temozolomide Glioblastoma cancer treatment [192]
47. Blend nanofibers Chitosan/polycaprolactone 5‐Fluorouracil Anticancer drug delivery [193]
48. Composite nanofibers Chitosan/graphene oxide Curcumin Cancer therapy [194]
49. Nanofibers Chitosan/phospholipids Curcumin, diclofenac, and Transdermal drug delivery [195]
vitamin B12
50. Chitosan/xanthan gum Curcumin Hydrophobic bioactive delivery [196]
51. Nanocomposite Chitosan/PEO/graphene oxide Doxorubicin Lung cancer treatment [197]
nanofibrous scaffolds
52. Scaffold Chitosan/collagen Curcumin Diabetic wounds [198]
53. Injectable scaffold Chitosan particles embedded in p‐DNA and bone Alveolar bone repair and drug [199]
chitosan hydrogel morphogenetic protein release
54. Nanocomposite scaffold Chitosan‐graft‐poly(acrylic acid‐co‐ Celecoxib Bone engineering and drug release [200]
acrylamide)/hydroxyapatite
55. Chitosan/hydroxyapatite microtubes Gentamicin sulfate Bone engineering and drug delivery [201]
56. Microspheres Chitosan–chondroitin sulfate Bovine serum albumin Cartilage tissue engineering [202]
Scaffold
57. Nanofibrous scaffold Chitosan–polycaprolactone Ferulic acid and resveratrol Wound dressing/therapeutics [203]
58. Nanocomposite Chitosan/(ureasil–PEO hybrid) Pramoxine Drug delivery [204]
59. membranes Chitosan/PEO–PPO–PEO/Mesoporous Metformin Antidiabetic drug delivery [177]
silica
60. Membranes Chitosan Ibuprofen Oral mucosal drug delivery [176]
61. Chitosan/terpenes Ondansetron Transdermal delivery [205]
hydrochloride
62. Chitosan/PEG/polyvinyl pyrrolidone/ Tetracycline hydrochloride Wound healing [206]
cotton
63. Poly(vinyl alcohol)/chitosan Ibuprofen Wound healing and drug release [173]
Note: PPO = poly(propylene oxide); PEO = poly(ethylene oxide).

*
0004461249.INDD 274 10/26/2019 11:59:04 AM

11.4.1 Hydrogels
Hydrogels are cross‐linked, three‐dimensional polymeric materials prepared by chemical
and physical methods [137]. They were intensively studied as carriers for bioactive
compounds (low molecular drugs, nucleic acids, proteins) or cells. In situ gelling can be
realized by solvent exchange, UV‐irradiation, ionic cross‐linking, pH and temperature
changes [138–142], by using the reaction of thiolated chitosan with oxidizing agents, [143]
or the dynamic imino–chitosan chemistry [144, 145].
One of the most studied systems is represented by thermosensitive hydrogels [146–151].
Recent studies have described the sustained release of doxorubicin [152] and dopamine/
metronidazole [153] from injectable hydrogels in the treatment of liver cancer and
Parkinson’s disease, respectively. In another study, different hydrophilic gel‐forming
matrix tablets based on chitosan and anionic polymers for sustained oral release of sodium
valproate and valproic acid were evaluated [154].
11.4.2 Micro/NPs
Chitosan‐based micro/nanoparticulate systems were extensively investigated as DDSs.
Therefore, for the development of micro/NP forms, researchers used various methods such
as emulsion cross‐linking [207], spray drying [182], precipitation [178, 179], coacervation
[208], ionic gelation [180], sieving [183], or reverse micellar technique [209]. Among
drug‐loading methods, another important factor to be considered is the size of the particles,
which has to be in accordance with the administration route [186, 210].
Recent papers demonstrated significantly enhanced drug delivery from chitosan NPs
after conjugation with folic acid [184], grafting with silica [185], obtaining ZnO‐based
nanocomposites [189] or introducing the magnetic properties of Fe3O4 [188].
11.4.3 Nanofibers
Based on the large values of their surface/volume ratio and porosity, the nanofibrous mate-
rials obtained by electrospinning of chitosan, chitosan derivatives, or combinations with
other polymers [211–214] were shown to possess higher drug‐loading capacity as com-
pared to similar materials but of other morphologies, enhanced drug transport properties as
well as cell attachment properties [215]. To improve the ability of chitosan nanofibers for
drug and gene delivery, their functionalization [216] or the preparation of composite
nanofibers were also proposed [175, 217, 218].
11.4.4 Scaffolds and Membranes

Chitosan and its derivatives were intensively used to prepare multi‐stimuli‐responsive scaf-
folds and membranes as DDSs [205, 219], but usually they were employed both as DDSs
and tissue engineering or wound healing devices [220, 221]. Chitosan‐based scaffolds were
obtained by different methods like—lyophilization, electrolytic techniques, or precipita-
tion and electrospinning of physically and chemically cross‐linked materials [222]. They
were used as freestanding three‐dimensional final shapes or as stimuli‐sensitive hydrogels
by in vivo gelling. Apart of their cell proliferation external matrix role, depending on
specific uses, they were charged with antibiotics, anti‐inflammatory, analgesic drugs,
proteins, growth factors, nucleic acids, or cells [110, 223, 224].
11.5 Outlook
As a result of high impact on human health and well‐being, DDSs represent one of the most
studied topics at frontiers between chemistry, biology, medicine, and bioengineering.
During more than 50 years of fundamental and applied research, DDSs preparation
procedures, as well as their shapes and administration routes, were continuously diversified.
Based on fundamental insights into the release mechanisms correlated with the biological
mechanisms of drug action, the performance of DDSs evolved from sustained release and
long in vivo circulation to controlled and targeted delivery.
Chitosan is one of the most studied biopolymers for application as a carrier in DDSs. The
interest in this biopolymer resides in its physicochemical characteristics (easily to be modulated
by changing the molecular weight, the degree of deacetylation or by chemical and physical
modifications of chitosan with organic/inorganic compounds or other biopolymers) and benefi-
cial biological properties mainly based on its cationic chemical structure (biodegradation, inter-
action with anionic drugs/tissue components, adhesion to different mucosa, inhibition of pump
efflux or promoting increased permeation of different drugs and transfection of nucleic acids
and proteins). Moreover, chitosan and its metabolic degradation products are non‐toxic. Thus,
hydrogels, micro/NPs, nanofibers or scaffolds, and membranes containing chitosan‐based
carriers for DDSs addressed to various diseases and able to surpass skin, mucosa, gastrointesti-
nal, ophthalmic, and blood–brain biological barriers were prepared and tested.
However, there are still challenges in the development and commercialization of efficient
DDSs related to analytical methods to evaluate in vivo the biocompatibility/toxicity of
complex drug formulations or to mathematical models meant to correlate the in vitro
release to the in vivo pharmacokinetic profile of specific drugs. With the increasing
incidence of diabetic and oncological diseases, patients and medical doctors are expecting
innovative solutions from researchers and bioengineers for strict “on–off” release
mechanism, for modulated insulin delivery, efficient tumor‐targeting systems, and long‐
term depot formulations for proteins and peptides without important burst effect.
Acknowledgment
The financial support of European Social Fund for Regional Development, Competitiveness
Operational Programme Axis 1—Project “Petru Poni Institute of Macromolecular
Chemistry—Interdisciplinary Pol for Smart Specialization through Research and
Innovation and Technology Transfer in Bio(nano)polymeric Materials and (Eco)
Technology,” InoMatPol (ID P_36_570, Contract 142/10.10.2016, cod MySMIS: 107464),
is gratefully acknowledged.
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12
The Application of Chitin and its
Derivatives for the Design
of Advanced Medical Devices
Marcin H. Struszczyk, Longina Madej‐Kiełbik, and Dorota Zielińska
The Institute of Security Technologies “MORATEX”, Lodz, Poland
Chitin is a copolymer of β‐1,4‐N‐acetylglucosamine (GlcNAc) with an amount of glu-

cosamine (GlcN) lower than 50% that is very abundant in the animal world, although it is
not synthesised by humans. Chitin is a renewable biopolymer originating from natural
sources and is biodegradable, nontoxic and insoluble in water and many organic solvents.
It is mostly known as a component of the exoskeleton of arthropods and the cell walls of
fungi [1], but it is also common in cephalopods and has been found in one species of fish
[2]. In recent years, there has been an increase in research on chitin due to its potential use
in medical fields (tissue regeneration, wound healing, drug delivery, gene therapy, immu-
nology), antimicrobial properties (food technology, agriculture) and designing of bioma-
terials, among others [3]. Many studies correlate the exposure of animal cells to chitin
with a pro‐inflammatory immune response [4–17] although, a few reports have shown
immunosuppressant effects [18–20].
12.1 Selection of the Raw Sources: Safety Criteria

Chitin and its derivatives, including the one often used for designing medical devices –
chitosan – are promising materials for the development of new, innovative solutions in med-
icine, both in designing medical devices as well as medicinal products. However, there
are several factors affecting the safe application of those biopolymers in medical areas.

The two most important European Union (EU) regulations that specify the possibility of
chitin and chitosan medical application are as follows:
• Medical Devices Directive (MDD) – Directive 93/42/EEC (MEDDEV);
• EU Regulation 2017/745 on medical devices, amending Directive 2001/83/EC,
Regulation (EC) No 178/2002 and Regulation (EC) No 1223/2009, and repealing
Council Directives 90/385/EEC and 93/42/EEC, which will be implementing a new
regulation of the medical devices in 2020.
12.1.1 Aspect of Animal Tissue‐Originated Derivatives

Chitin and its derivatives mainly originate from animals: crustacean, insects, etc. Due to
rule 17 of MEDDEV, all medical devices containing substances derived from animals are
incorporated in the highest class of risk – class III. For the determination of the safety of
the final wound dressing containing chitin derivatives, several verification stages are
followed:
• Strict determination of the nature of animal tissue and/or characterisation of the animal
origin (species, country of origin/fishing area);
• Characterisation of the origin of animal materials (method of the removal of tissues/
organs and precautions adopted to limit cross‐contaminations during the collection,
hygiene requirements adopted for each stage of the biopolymer separation phase,
method of testing/verification of the traceability of the chitinous raw materials);
• Screening the historical data of chitinous raw materials clinical application, data of the
viral/microbiological assessment in aspect of their use in the manufacturing medicinal
products or medical devices already Conformité Européenne (CE) marked or Food and
Drug Administration (FDA) registered;
• Completion of the entire process of biopolymer separation from collection phase to the
final quality control, as well as identification of all subcontractors participating in the
manufacture of the biopolymer;
• Detailed identification of the manufacturing process of the biopolymer separation with
detailed definition of the condition/factors of the process affecting the elimination of the
pathogens (such as temperature, pH, concentration of the processing aids, etc.);
• Determination of the maximal biopolymer yield (output);
• Description of the quality control tools applied to ensure the traceability and reproduci-
bility of each biopolymer batch;
• Documented, scientific data of the efficiency of the viral/pathogens removal/inactiva-
tion tools applied in the process;
• Validation of the viral/pathogens removal/inactivation process [21].
12.1.2 General Requirements for Chitinous Biopolymers Applied

in Designing Medical Devices
Designing of medical devices, including wound dressing, is limited by the obligation to
meet the essential requirements taking into account the published standards, clinical and
scientific data (literature) and validated test results. The process of selecting the suitable
The Application of Chitin and its Derivatives for the Design of Advanced Medical Devices 293
chitinous raw materials consists of several verification and validation steps. The general
limitations for selecting the most proper raw sources are:
• Detailed characterisation of raw materials (chemical, physical verification) for the deter-
mination of the biopolymers degree of purity as well as determination of the characteristic
of the initial raw sources as an input for ensuring the reproducibility of the manufacture;
• Verification of their biocompatibility regarding the future application and design attrib-
utes of the medical device;
• Determination of the biodegradation susceptibility or selection of the biopolymers
characteristic related to the required biodegradation period;
• Verification of the extent and effect of leakage of the substances (such as low‐molecular‐
weight fractions, proteins, minerals, etc.);
• Determination of the effect of the fabrication process including the sterilization with
suitable selection of the optimal sterilization agent (irradiation, steam or ethylene oxide
(EO)) on the biopolymer characteristic (degradation, chemical modification, changes in
crystallinity, etc.) and performance (changes in insolubility or solubility);
• Determination of the possible effects of the wound dressing performance due to the
interaction of the biopolymer and other constituents, processing aids, etc.;
• Verification of the wound dressing biocompatibility and determination of the constitu-
ent effects, risk of cross‐contamination, as well as manufacture process (including
sterilization);
• Verification of the microbiological and particulate contamination levels, both of the
raw materials and the final wound dressing (before sterilization) due to the risk of
pyrogenicity [21].
The crucial limitations in applying chitin and its derivatives for designing the wound
dressing are:
• Acquisition of reproducible sources of raw chitin (animals) as a starting point for the
separation of high‐quality biopolymer;
• Absence of the European (EN) standards describing the essential requirements for
chitin‐originated raw materials applicable for medical devices;
• Restricted access for the reproducible, ultra‐pure biopolymer manufactured in controlled
environments (reduction of the microbiological contamination) with verifiable
purification steps;
• Obligation to meet requirements of EN ISO 22442 standards regarding the documented
validation of the elimination of animal originated pathogens as well as measurable
determination of the risk of pathogens transfer to human in consideration to the process
of biopolymer separation as well as main process of the wound dressing manufacture;
• Necessity to use sterile wound dressing in relation to risk of modification of its perfor-
mance and safety by action of the sterilization agent (mainly EO or irradiation) [21,22].
12.1.3 Characterisation of the Biopolymer for Application in Wound

Dressing Designing
There are several general standards/guidelines in EU identifying the essential require-
ments for chemical and structural characterisation of the chitinous biopolymers to be
applied in wound dressings. They include European Pharmacopoeia as well as European
Standards: EN‐ISO10993‐18 (Biological evaluation of medical devices. Chemical

characterization of materials) and EN‐ISO 10993‐19 (Biological evaluation of medi-
cal devices. Physico‐chemical, morphological and topographical characterization of
materials).
Additionally, the Standard: EN‐ISO 10993‐12 (Biological evaluation of medical devices.
Sample preparation and reference materials) describes a methodology of extracting
leachable substances from materials; the method could be easily adopted for purity
determination of the chitinous biopolymers applied in medical devices. Due to the role
played by medical devices, conditions of the extraction, selection criteria for the extraction
conditions as well as selection of the references are given in details in the above‐mentioned
standardisation document.
The document gives only a general pathway for the proper identification and selection of
testing tools and methodologies related to the determination of the main parameters
characterising chitin and its derivatives [23].
More detailed requirements are included in the ASTM Standards:
• ASTM F2103‐18 (Guide for Characterization and Testing of Chitosan Salts as Starting
Materials Intended for Use in Biomedical and Tissue‐Engineered Medical Product
Applications);
• ASTM F2260‐03 (Test Method for Determining Degree of Deacetylation in Chitosan
Salts by Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy);
• ASTM F2602‐13 (Test Method for Determining the Molar Mass of Chitosan and
Chitosan Salts by Size Exclusion Chromatography with Multi‐angle Light Scattering
Detection (SEC‐MALS)).
The most applicable analytical tests to verify the quality of chitosan that is most often used
for designing the wound dressings are focused on (but not limited to) the determination of:
• Moisture;
• Residual substances (ash, protein, dyes, heavy metal) content;
• Degree of insolubility;
• Solution turbidity;
• Viscosity;
• Degree of deacetylation (DD);
• Crystallinity;
• Molecular mass and degree of polymerization.
The quality control should consider the identification of the usable form of the biopoly-
mer (powder, flakes, etc.) and colour [22].
Moreover, the crucial aspect for the verification of the safety of chitinous biopolymers is
the determination of microbiological contamination (bioburden ‐ aerobic bacteria and
fungi content) affecting the potential risk of the pyrogenicity of the final wound dressing.
In some critical applications, such as implants, the endotoxin level should also be
determined using the selected methodology, since chitosan as a polyaminosaccharide
induces an increase of the endotoxins level. An outline (flow chart) of the chitosan purity
determination process is shown in detail in [23].
12.1.4 Aspect of the Sterilization of the Final Wound Dressing

It is well known that all materials intended for contact with a wound or body fluids must be sterile
[24,25]. Sterilization aims to kill or inactivate any form of microbial contaminant. Many com-
mon sterilization techniques can induce changes in the properties of chitin and chitosan. Changes
to these properties may have potential effects on the performance of chitin as a biomaterial.
Therefore, the sterilization method used for medical devices should not affect their properties
and intended functions. After careful investigation of the possible effects of the sterilization
method on the material, a specific sterilization technique must be chosen. Before a final choice
of the method for sterilization of medical devices containing chitosan is made, its effects on the
properties and the conclusive performance of the polymer will have to be documented.
Chitin and its derivatives are widely used for developing wound dressings. Any wound
dressing used in the clinic should be sterile according to appropriate standards. The
sterilization of wound dressings, prior to use on a patient, is important to ensure the safety
of the patient. As it can be seen from the data presented in the literature, there are many
methods of sterilization of chitin and its derivatives [26]. Despite various studies involving
the use of chitin and its derivatives as biomaterials, information about sterilization methods
and their effects on the properties of the materials is limited.
The most important aspect of the sterilization of final wound dressing is the packaging
of the product. Each sterilization method has advantages and disadvantages in terms of the
required types of packaging, cost and impact on the properties of the dressing throughout
its shelf life. This requirement to penetrate the packaging material in order to ensure steril-
ity limits the type of packaging that can be used (i.e. the packaging must be permeable to
the sterilizing agent).
The sterilization process is most critical due to the safety as well as remaining perfor-
mance of the wound dressing, especially when a biopolymer of chitin origin is used for a
design. The aim of the sterilization of medical devices is to cleanse these devices of the
microbiological contamination that originate from the raw materials and conditions of the
manufacture process. Microbial survival probability in a sterilized medical device should
be ≤1 in 1 million (10−6) [21].
It should also be noted that the process of sterilization eliminates living microorganisms,
but does not eliminate the hazards of the presence of pyrogenic substances from bacterial
cell walls. Thus, the limitation of the bioburden on the raw materials is crucial for the safety
of the final medical device.
The sterilization process commonly applied in the medical devices area utilises three
types of the sterilization agents:
a. Exposure to dry heat – sterilization using dry heat led to the reduction of chitosan dis-
solution potential in aqueous solution [24]. In some cases, this process resulted in
totally insoluble biopolymer losing its most important behaviour – affinity for hydrogel
formation. The phenomenon can be explained by the interchain cross‐link involving the
amine groups in chitosan.
b. Saturated steam at various temperatures (126°C or 121°C) – the steam sterilization of
chitosan films caused mechanical behaviour deterioration as well as increase in the rate
and extent of the thermal treatment. Chitosan films lost their solubility behaviour with
extensive reduction in tensile strength [24] and molecular mass [27]. Moreover, steam
sterilization yielded great darkening of the colour of chitosan powder with the colora-
tion deepening (yellow to brown) by an increase in the temperature and prolongation of
heat exposure [24]. Steam sterilization of chitosan flakes dispersed in water remained
the molecular weight of the biopolymer [28]. However, the changes induced by dry
steam did not significantly affect sterilized chitosan under anoxic conditions [24].
c. EO – EO sterilization of chitinous biopolymers or wound dressing (in the usable form
of a film) containing these biopolymers produces by‐products and cross‐linking result-
ing in reduction of mechanical strength [26]. Additionally, EO is irritating to human
body and is mutagenic, has fetotoxic and teratogenic behaviour, can adversely affect
testicular function and shows systemic toxicity. After exposure to EO, several adverse
systemic effects were detected, such as the induction of various types of neoplastic
changes including leukaemia, brain tumours and mammary tumours [29]. The risk of
the adverse accumulation of EO (after sterilization) increases with the porosity of the
materials, and affects cytotoxicity, irritation, mutagenicity and genotoxicity potential.
According to Standard EN‐ISO 10993‐7 (Biological evaluation of medical devices – Part
7: Ethylene oxide sterilization residuals), the EO residue reacts easily with water (such
as hydrogels), resulting in ethylene glycol formation. In some cases, ethylene chloro-
hydrin is also a co‐product of the reaction of EO residues. It shows high irritation affin-
ity, acute toxicity as well as weak mutagenic and carcinogenic potential. The study by
França et al. [30] shows that EO exposure on three types of chitosan with different
degree of N‐deacetylation, and carboxyethyl chitosan resulted in unexpected changes
in the chemical structure of the biopolymers, nonetheless without any negative effect
on the biocompatibility and toxicity. Additionally, EO exposure led to a minor change
in the crystallinity and network structure of chitosan powder. EO sterilization of medi-
cal devices based on chitinous biopolymers is limited to the dry state due to the possible
derivatisation of chitinous biopolymers if the wet state underwent sterilization [28].
This research underlined the risks associated with toxicity, flammability and aspects of
material contamination with EO residues.
d. Irradiation (γ‐irradiation or accelerated electrons) – γ radiation is a rapid, residue‐free
method that can be monitored by dosimetry. However, γ radiation cannot be used to
sterilize some types of polymers because of their inherent sensitivities or susceptibilities.
Sterilization of chitosan in the film form by γ‐irradiation with irradiation dose higher
than 10 kGy resulted in biopolymer chain degradation, deterioration of the mechanical
properties as well as decrease in the susceptibility to swell. On the other hand, realisa-
tion of the process under anoxic conditions significantly reduces negative alteration,
indicating that the irradiation process formed free radicals and accelerated the process of
biopolymer chains degradation. The standard γ irradiation sterilization dose of 25 kGy is
used in the industry for most medical devices that come in contact with skin or tissue.
This dose is considered sufficient for gaining a minimum of 10−6 sterility assurance level
(1 in 1,000,000 of medical devices can be unsterile after the sterilization process).
Each sterilization agent induces changes in the biopolymer structure, and initiates deg-
radation, cross‐linking and/or chemical modification affecting the performance of the final
product, as well as its safety. The determination of the effect of sterilization on biopolymers
from the possible interaction of the constituents and processing aids is the main aspect of
the process of proper selection of the biopolymer parameters, as well as in designing the
wound dressing.
12.2 Types of Wound Dressings Consisting of Chitin‐Derived Biopolymers

Available in the Market
Wound dressings containing chitin and its derivatives are available in various forms such as
nonwoven, sponge/foam, gel or powder. Most of them are used primarily as bleeding
control products, especially during battle warp where the cause of 80% deaths is
haemorrhage. However, only a small part of wound dressings consisting of chitin is
available nowadays on market (Table 12.1).
There are only a few companies offering chitosan wound dressing despite a lot of scien-
tific publications about chitin and its derivatives as components of dressings. This is due to
the fact that market clearance of a medical device requires several verifications and valida-
tion stages, clinical trials, certification and standardisation, which takes time and money.
12.3 Performance and Safety Assessment

Chitin is a valuable natural polymer indicating excellent bioactive properties. Chitin prod-
ucts are antibacterial, antiviral, antifungal, nontoxic and nonallergic. Chitin as well as chi-
tosan are widely used for the design of medical devices (Table 12.1). These polymers have
many useful and advantageous biological properties in the application as a wound dressing,
namely biocompatibility, biodegradability, haemostatic activity, anti‐infection activity and
capability of wound healing acceleration [43].
For medical devices and medicinal products design, chitin and chitosan need to be highly
purified, since residual proteins and pigments can cause side effects [44]. When chitosan is
to be used for applications inside the body, as a wound dressing or other medical devices,
strict requirements of manufacture, characterisation and purity need to be met for the
biomaterial, as discussed in the preceding text.
According to the essential requirements for medical devices and in order to meet the
safety rules, medical devices should be designed and manufactured in a way that they
perform according to the intention of the manufacturer, compromising neither the safety of
patients nor the safety or health of other persons, provided they are used under conditions
and for the purposes as intended.
Medical devices based on chitin and its derivatives should be designed and manufactured
in such a way that, during normal conditions of use, they are suitable for their intended
purpose and achieve the performance intended by the manufacturer. Therefore, in the
selection of materials to be used in medical device manufacturing, special attention should
be paid to the properties of the materials, which include particularly chemical, toxicological,
physical, morphological and mechanical properties. The most important factors that should
be taken into account for the estimation of chitosan and chitin safety are:
• Chitin sources (Chitin is industrially extracted from crab and shrimp shells obtained as
by‐products in the seafood industry. The production of chitin and chitosan from fungal
sources has gained increased attention in recent years.)
• Purity and impurity of the biomaterial (relates to the presence of extraneous substances
and materials in the biopolymer). The major concern of impurities include, but are not
limited to, the following:
∘∘ Endotoxin content
∘∘ Protein content (residual protein in chitosan could cause allergic reactions such as
hypersensitivity)
Table 12.1 Examples of wound dressings consisting of chitin‐derived biopolymers available on the market [31–42].
Wound dressing Manufacturer Main constituent Usable form and main performance
Celox™ Gauze Medtrade Products Ltd. Chitosan • 5’ Z‐fold gauze or gauze 10′ roll impregnated by haemostatic granules
• Bleeding control
• Chosen by multiple NATO forces
• CE‐certified
Celox RAPID Gauze • 5’ Z‐Fold gauze impregnated by haemostatic granules
• Works with just 60 seconds compression
• CE‐certified
Celox™ Haemostatic • Granules are available in a 15 g sachet
Granules • Bleeding control
• Moulding to the shape of the wound
• Registered as class III CE‐marked medical device
Celox™‐A • A unique applicator pre‐packed with 6 g of Celox™ granules
• CE‐certified
HemConChitoGauze® Tricol Biomedical, Inc. Chitosan • 10 cm × 3.5 m Z‐fold gauze coated by chitosan
PRO • Antibacterial properties
• Stops severe bleeding
• Reduces blood loss
• CE‐certified
ChitoGauze® XR PRO • 7.6 cm × 3.7 m Z‐fold gauze coated by chitosan with x‐ray detectable element
• Antibacterial properties
• Stops severe bleeding
• Reduces blood loss
• CE‐certified
HemCon Patch® PRO • 2.5 cm × 7.5 cm haemostatic patch
• Severe bleeding control
ChitoDot® • ∅1,9 cm a double‐sided haemostatic dressing
HemCon® Strip PRO • Haemostatic bandage
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ChitoSAM™ 100 Sam Medical Chitosan • Nonwoven chitosan dressing spun directly from chitosan, available in two
forms: Z‐fold gauze and smaller single ply
• Stops lethal bleeding rapidly
• An innovative package which allows for quick access
• CE‐certified
Tromboguard® Tricomed Chitosan and alginates • Three layers: contact layer (composition of chitosan and alginates),
absorptive layer (high‐absorption polyurethane foam), protection layer
(water‐resistant polyurethane membrane)
• Quick haemostatic action
Axiostat® Axio Chitosan • Very flexible, both sides active sponge, 5 cm × 5 cm, 8 cm × 5 cm
• Temporary control of wound bleeding
• CE‐certified
Axiostat® Military • Very flexible, both sides active sponge, 8 cm × 8 cm
variant MIL88 • Temporary control of wound bleeding
• CE‐certified
• Packed in camouflaged, rugged metal pouch
Axiostat® Vascular • Very flexible, both sides active sponge, 5 cm × 5 cm
• Stops bleeding during vascular procedures
• CE‐certified
Axiostat® Dental • Very flexible, both sides active sponge, 1 cm × 1 cm
• CE‐certified
• Severe bleeding control during dental procedures
SyvekExcel® Marine Polymer Poly‐N‐acetyl • 3 cm × 4 cm lyophilised pad of poly‐N‐acetylglucosamine with foam backing
Technologies, Inc. glucosamine • CE‐certified
• FDA market clearance
SyvekNT® • 3 cm × 3 cm nonwoven pad of poly‐N‐acetylglucosamine fibres isolated from
microalgae
• CE‐certified
• FDA market clearance
Clo‐Sur PAD Scion BioMedical Chitosan • 4 cm × 4 cm nonwoven hydrophilic wound dressing
• CE‐certified
Clo‐Sur PLUS
PAD • 4 cm × 4 cm nonwoven hydrophilic wound dressing
• Antimicrobial barrier for up to 6 days
• CE‐certified
(Continued )
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Chitoderm® Plus Trusetal Chitosan • Foam, 10 cm × 12 cm
Verbandstoffwerk • Super absorber with antibacterial action
GmbH • Promotes wound healing
• Inhibits the growth of bacteria
• Rapid reduction of pathogenic organisms
ExcelArrest® XT Hemostasis, LLC. Carboxymethylated • Foam in three various sizes: 5 cm × 5 cm, 5 cm × 10 cm, 10 cm × 10 cm
chitosan • Moderate to severe bleeding control
• CE‐certified
• FDA‐cleared
KytoCel® Aspen Medical Chitosan • Gelling fibre dressing available in six sizes: 5 cm × 5 cm, 10 cm × 10 cm,
15 cm × 15 cm, 4 cm × 10 cm, 4 cm × 20 cm, 4 cm × 30 cm
• Minor bleeding control
HEMO‐ Coreleader Biotech Chitosan • Woven into a strong haemostatic gauze in three various sizes:
FiberKompresse Co. Ltd. 7.5 cm × 300 cm, 10 cm × 200 cm, 10 cm × 300 cm
• Control of severe bleeding
• Biocompatibility
• High tensile strength
• CE‐certified
Note: *NATO = North Atlantic Treaty Organization
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∘∘ Heavy metals (Marine sources of chitosan can contain heavy metal contaminants
such as lead and mercury, and these should be analysed. Mercury has been desig-
nated as a hazardous material that can damage the central nervous system, kidney and
liver. Therefore, properties of chitosan should be evaluated using test methods that
are appropriate to ensure safety and efficacy.)
∘∘ Microbiological burden (bacteria, yeast and mould; the presence of bacteria may also
contribute to the presence of endotoxins)
• Reproducibility in the manufacture of the compound
• Compatibility with the essential requirements for medical devices and the European
Standards (e.g. EN ISO 22442‐1/2/3 Standards)
• Sterilization of medical devices containing chitin or its derivatives – most critical pro-
cesses may affect their biocompatibility [21].
The safety of chitosan in biomedical applications should be determined according to
current guidelines, such as ISO 10993 and ASTM F748 standards.
The performance assessment should review analytical performance data and, where
appropriate, clinical performance data in the form of any:
• Literature
• Performance study reports
• Experience gained by routine diagnostic testing.
This is to establish that medical devices achieve their intended performance under normal
conditions of use, and that known and foreseeable risks, and any undesirable effects, are
minimised and acceptable when weighed against the benefits of the intended performance.
12.4 New Ideas and Concepts

Chitin and chitosan can be easily processed into different forms such as membranes, sponges,
gels, scaffolds, microparticles, nanoparticles and nanofibres for a variety of biomedical appli-
cations such as drug delivery, gene therapy, tissue engineering and wound healing [45,46].
The adhesive nature of chitin and chitosan, together with their antifungal and bacteri-
cidal action and their permeability to oxygen, has very important advantages associated
with the treatment of wounds and burns. Efforts have been made during the last few years
towards the development of new wound dressings based on chitin and its derivatives, which
will meet the requirements necessary for the treatment of major skin wounds. Over the last
years, recent trends have come up in the form of composite membranes, where a textile
material is coated with the polymer solution.
Due to its aforementioned remarkable properties, chitosan appears as a relevant candi-
date for the preparation of such biomaterials, which could be a substitute for missing or
damaged tissue and organ [47]. The potential applications of chitin and its derivatives result
from the possibilities of processing them into different forms (hydrogel, beads, films, nano-
gel, nanofibres, micro/nanoparticles, scaffolds/bandages, membranes/spongers) [46].
The reports published in the literature [48] suggest that chitin and its derivatives may be
used as:
• Biomedical 3D scaffolds for tissue engineering in the form of chitosan hydrogels and foams
• 2D scaffolds as films and fibres for wound healing application.
In recent years, growing scientific attention to wound dressing based on chitin and its
derivatives, including chitosan, is observed. The Scopus database shows how many
publications (keywords: ‘chitosan’ and ‘dressing’) appeared since 2010 (Figure 12.1).
Since 2010, the number of documents has doubled. This shows that a large number of
research groups are dedicated to producing new and improved wound dressings. Most
papers are connected with materials science and engineering (Figure 12.2).
Some new ideas and concepts of dressing based on chitosan and its derivatives are
discussed in the following text.
The gel‐e® [49] products are made from modified chitosan with natural fatty acids (corn
and sunflower oil). It is capable of rapidly self‐assembling into robust physical seals
designed to stop bleeding. It is available in four forms: Vascular gel‐e®, Nuisance bleed
gel‐e®, Chronic wound gel‐e® (films, bandages, gels) and Surgical gel‐e® (gels, foams,
sponges). All these products are not yet commercially available, but at the stage of
preclinical and clinical testing. Only Vascular gel‐e® has been cleared by the FDA for use
in vascular access procedures.
Sarhan et al. developed biocompatible, antimicrobial, nanofibrous wound dressing based
on fabricated honey, poly(vinyl alcohol) and chitosan nanofibres. A preliminary in vivo
study proved the biocompatibility and showed that developed nanofibre mats enhanced the
wound healing process [50].
Lu et al. incorporated Ag/ZnO nanocomposites into a chitosan sponge, and then it was
exposed to a lyophilisation process [51]. The results showed high porosity and swelling, as
well as enhanced blood clotting and antibacterial activity. Cytocompatibility test evaluated
in vitro demonstrated a very low toxic nature of the composite dressing, and in vivo
evaluation in mice showed that the developed composite dressing enhances wound healing
200
180 180
160 163
147
140
Published documents
120
107
100
96
81 83
80
72
60
40
20
0
2010 2011 2012 2013 2014 2015 2016 2017
Figure 12.1 Published documents on the application of chitin and its derivatives, including chitosan, for
designing wound dressing, as listed in the Scopus database (Scopus; 2018.02.15).
Environmental Other Immunology and

Physics and Science 5% Microbiology
Astronomy 1% 1%
5%
Pharmacology,
Toxicology and
Pharmaceutics
7%
Materials Science
23%
Medicine
10%
Engineering
Chemical 15%
Engineering
10%
Chemistry
Biochemistry, 12%
Genetics and
Molecular Biology
11%
Figure 12.2 Published documents on the application of chitin and its derivatives as listed in the Scopus
database, divided into science areas (Scopus; 2018.02.15).
and promotes re‐epithelialisation and collagen deposition. Authors suggested that there
was a possibility of using this novel chitosan–AgZnO composite dressing for wound care
application.
According to the literature [52], chitosan dressing with silver can be used as a burn
dressing. It offered an optimal environment for moist wound management. Moreover, it did
not demonstrate shrinkage or disintegration as compared to the widely used hydrofibre
dressings. Studies by Alemdaroğlu et al. [53], related to the development of chitosan gel
formulation containing epidermal growth factor, suggested a potential opportunity to use
that material for healing second‐degree burn wounds (the studies were carried out on rats).
Chilarski et al. [54] suggested a potential opportunity to use the chitin derivative
dibutyrylchitin for the treatment of burn wounds, postoperative/posttraumatic wounds and
various other conditions causing skin/epidermis loss.
Chitosan–sulphadiazine membrane has been developed by Dragostin et al. [55]. The
authors prepared six chitosan–sulphonamide derivatives. The results demonstrated an
improved healing effect and enhanced epithelialisation of chitosan–sulphonamide
derivatives as compared to untreated chitosan.
N,N,N‐trimethyl chitosan was deposited onto polypropylene and polylactide nonwovens
[56]. The results showed that modified material exhibited good antibacterial properties
against Gram‐positive bacteria Staphylococcus aureus.
The research carried out by Zhou et al. indicated that N,N,N‐trimethyl chitosan fibres
have the potential to be used as wound dressing materials [57]. The results showed that the
obtained fibres had higher absorption capacity, higher antibacterial performance compared
to chitosan fibres, and were non‐toxic to mouse embryo fibroblasts.
Chitosan was used by Dumont et al. for coating alginate fibres [58]. The obtained fibres
contained about 10% (v/v) of chitosan by an external coating. The result showed that
chitosan‐coated alginate fibres presented an antibacterial activity against Gram‐negative
Escherichia coli sp., and more interestingly against Gram‐positive Staphylococcus genus.
Moreover, it was demonstrated that antibacterial properties did not change after steriliza-
tion process by beta‐irradiation.
Alginate fibres with chitosan sheath were prepared by Chang et al. with the electrospinning
method [59]. Obtained fibres exhibited a core–sheath structure, and the average diameter
of the fibres ranged from 600 to 900 nm, depending on the electrospinning parameters.
They suggested that that core–sheath electrospun fibre had a potential for biomedical
applications.
The research carried out by Chen et al. [60] was focused on electrospun nanofibre mats
of pectinate, alginate and chitosan. They showed that all nanofibre mats had comparable
mechanical strength and vapour permeability. However, pectinate nanofibre mats could
absorb more exudates in less time and suppress the growth of bacteria more effectively, so
the authors suggested that pectinate nanofibre mats might be a superior wound dressing as
compared to the alginate and chitosan nanofibre mats.
Nanofibre mats were also developed by Kohsari et al. [61]. The chitosan–PEO nanofibre
mats were prepared by the electrospinning technique. Silver nanoparticles were added in
two different weight percentages. The results showed that the obtained 21anofibers
exhibited 100% antibacterial performance against both the Gram‐positive S. aureus and
Gram‐negative E. coli bacteria.
In another study [62], chitosan membranes were prepared as potential coating for tita-
nium implants. A surface roughness and fibronectin adsorption increase was observed with
increased DD. The authors concluded that there was a possibility of preparing chitosan
membranes by selecting from chitosan types with a wide range of DD, thereby displaying
that different bioactivity would be a useful advancement in the coating of titanium for
orthopaedic applications.
Composite wound dressing based on the chitosan‐oxidised Bletilla striata polysaccharide
and chitosan‐Ag, both cross‐linked with genipin, were successfully fabricated for wound
repair applications by Ding et al. [63]. The in vivo studies indicated that wound dressing
significantly accelerated the healing rate of cutaneous wounds in mice, and it had great
potential in wound dressing applications.
The aim of the research of Saporito et al. was to develop sponge‐like dressings based on
chitosan and chondroitin sulphate or hyaluronic acid [64]. It was prepared by lyophilisation
and loaded with tranexamic acid. The in vitro and ex vivo studies showed that dressings
were biocompatible and able to sustain cells. The result suggested that these dressings are
effective tools to enhance haemostasis and healing in bleeding wounds.
The surface of nonwoven chitosan was modified by the succinyl groups, carboxymethyl
groups and quaternary ammonium groups by Yan et al. [65]. The results demonstrated that
the modified nonwoven exhibited better haemostatic property than gauze and initial
nonwoven, especially, the nonwoven with succinyl groups had an excellent haemostatic
effect, compared to other nonwoven samples.
The research carried by Zielińska et al. [66] described two usable forms of a topical
haemostatic agent: chitosan/alginate lyophilised foam and chitosan/alginate impregnated
gauze. The presented forms of the topical haemostatic agent, being the main part of the
modified combat gauze, were fabricated using a chitosan/alginate complex. A comparison

of the properties of the developed usable forms with the commercially available haemostatic
dressings showed the supremacy of the former. Newly designed usable forms were
characterised by high absorption under free soaking that indirectly supports the process of
the haemostasis and moisture vapour transmission rate. After the accelerated ageing test, a
non‐significant decrease of approximately 7% was found in absorption under free soaking
in the lyophilised foam. In the second‐tested usable form, the absence of significant change
was also detected in the above‐mentioned parameter. The results of the test confirmed the
stability of the tested parameters of newly developed usable forms of the haemostatic
agents. Moreover, the accelerated ageing study allowed for estimating the performance
stability of the developed topical agents during the storing and transportation.
Kamel et al. [67] incorporated banana peels with chitosan as wound dressing. Glycerol
was added as plasticiser and cross‐linker to the membranes. The results showed that the
addition of banana peels decreased the degree of swelling of chitosan matrix; the obtained
membranes had antimicrobial properties with the highest activity at 10 wt%. The most
sensitive microorganism recorded for these membranes was Candida albicans.
The aim of the research of Kang et al. was to establish a modified procedure for preparing
chitosan dressings [68]. The chitosan matrix after freezing and lyophilisation was treated
with sodium hydroxide and/or sodium tripolyphosphate, subsequently washed and
lyophilised again to produce dressings. In vitro blood clotting tests showed that the obtained
dressing absorbed blood quickly and accelerated blood clotting. Meanwhile, the
commercially available dressing Clo‐Sur required more time to completely absorb the
same amount of blood.
In vivo studies of nonwoven chitosan gauze impregnated with PolySTAT have been
examined by Chan et al. [69]. The results showed that no appreciable differences were
observed in fibre size, morphology or pore size between obtained PolySTAT/chitosan
nonwoven and a commercially available chitosan‐containing gauze (Celox® Rapid). The
PolySTAT/chitosan nonwoven rapidly absorbed blood, reduced blood loss and improved
survival rate.
Hydrogels are interesting biomaterials as their high water content makes them compat-
ible with a majority of living tissues. Moreover, they are soft and bendable, which mini-
mises the damage to the surrounding tissue during and after implantation in the patient
[70]. Chitosan foams, for example, chitosan [71], chitosan/tricalcium phosphate (TCP)
[72] and chitosan/collagen sponges [73], are mainly used as wound healing materials as
they can soak up the wound exudates while helping the tissues to regenerate. Chitosan
sponges also find application in bone tissue engineering as a filling material [72].
2D‐shaped scaffolds such as films and porous membranes are candidates to tackle the
challenges addressed in skin repair [48]. Types of chitosan films are as follows:
• Phosphorylated chitosan films with higher ionic conductivity than normal chitosan films
• Films based on chitosan and minocycline hydrochloride, accelerate the healing of burns
• Chitosan/poly(vinyl alcohol)/alginate films, to create a moist environment within the
framework of wound healing
• Non‐adherent film of β‐glucan and chitosan.
Kumar et al. reported the suitability of nanohydroxyapatite‐incorporated α‐ and β‐chitin
hydrogel and chitosan hydrogel scaffolds for bone tissue engineering applications [74,75].
Subsequent researches have shown that chitosan derivatives might be used in the preparation
of influenza vaccines [76,77]. The research carried by Frohbergh et al. [78] described
nanofibrous scaffolds from chitosan for bone tissue engineering applications. Scientists
have developed a one‐step technology to generate electrospun and hydroxyapatite‐
containing fibrous chitosan scaffolds that are subsequently cross‐linked with genipin as
potential substitutes for periosteum. The authors proposed that electrospun cross‐linked
hydroxyapatite‐containing chitosan nanofibrous scaffolds subsequently cross‐linked with
genipin were potential candidates for non‐weight‐bearing bone tissue engineering – that is,
for cranial and maxillofacial reconstruction.
The smartness of chitin nanofibril/chitosan glycolate‐based preparations, a spray
(Chit‐A), a gel (Chit‐B) and a gauze (Chit‐C), in healing cutaneous lesions was assessed
macroscopically and by light microscopy immunohistochemistry [79]. Those evaluations
were compared to the results obtained using a laser co‐treatment. The wound repair pro-
vided by those preparations was clearly evident even without the synergistic effect of the
laser co‐treatment.
Chitosan derivatives with quaternary ammonium groups showed very good performance
against bacteria and fungi. The explanation that the cytoplasmic membrane of bacterial
cells is the target of these cationic polymers is generally accepted [80]. The photo‐cross‐
linked electrospun mats containing quaternary chitosan (QCS) were capable of inhibiting
the growth of both Gram‐positive and Gram‐negative bacteria [81]. The results indicate
that the cross‐linked QCS/poly(vinyl alcohol) (PVA) electrospun mats are advantageous
material applicable for wound dressing. Likewise, the photo‐cross‐linked electrospun
nanofibrous QCS/PVA products exhibited optimal bactericidal activity towards the Gram‐
negative E. coli and Gram‐positive S. aureus bacteria [81].
In vivo studies of a bioactive dressing containing silver sulphadiazine (AgSD) as anti‐
infective drug and alpha tocopherol (αTph) as antioxidant agent, and intended to be loaded
with autologous platelet lysate in the treatment of chronic skin wounds, were performed by
Bonferoni et al. [82]. As both AgSD and αTph are hardly soluble, nanocarriers such as
polymeric micelles (AgSD‐Mic) and nanoemulsions (αTphNE), based on an amphiphilic
salt of chitosan with oleic acid, were previously developed and characterised to improve
their dispersion in aqueous environment. The results confirmed the positive encapsulation
of poorly soluble hydrophobic molecules in chitosan oleate. The authors suggested that the
bioactive wound dressing can be a suitable and versatile support of the haemoderivative for
the treatment of wounds. Moreover, the effect on wound healing was dose‐dependent, mak-
ing it a flexible tool in wound treatment.
12.5 Risk Acceptance and Design Process Aspects

The application of chitin and its derivatives originating from animals carries risks in terms
of the uncontrolled pathogen transmission to humans, as well as reproducibility in terms of
the quality of the raw materials used for the manufacture of an innovative wound dressing.
The main tool helpful for the proper planning of the design process is risk analysis.
The risk analysis for the wound dressings should be performed according to the guide
given in EN ISO 14971 (Medical devices. Application of risk management to medical
devices) (specifying the main guide for the risk analysis for medical device) and
selected parts of EN ISO 22442‐1 (Medical devices utilizing animal tissues and their
derivatives. Application of risk management) (describing the aspect of biological risks

including the animal‐originated pathogen transmission to humans). The most optimal
technique for the risk analysis management is the Failure Mode and Effect Analysis
(FMEA) method [83].
The application of chitinous biopolymers is limited by the following risks: the presence
of parasites and unclassified pathogenic factors; qualitative and quantitative microbiologi-
cal contamination with bacteria, fungi and yeast; virus presence with consideration to the
requirements of EN ISO 22442‐2 (Medical devices utilizing animal tissues and their deriv-
atives. Controls on sourcing, collection and handling) and EN ISO 22442‐3 (Medical
devices utilizing animal tissues and their derivatives. Validation of the elimination and/or
inactivation of viruses and transmissible spongiform encephalopathy (TSE) agents); pyro-
genicity; immunology; as well as both local and systemic toxicity [23, 84].
Additionally, risk analysis covers the estimation and identification of biological hazards;
environmental hazards; hazards originating from leachable substances; hazards related to
non‐proper use; and hazards related to production, quality control, ageing and clinical
adverse events, etc. [85].
The risk reduction procedure is performed by the identification as well as estimation of
the influence and probability of each risk related to the hazardous situation, effect(s) of the
risk occurring in the area of performance (defined functionality) and, finally, the application
of risk steering identification tools for reducing the risk to acceptable levels [85].
The design of a wound dressing consists of several verification and validation phases,
estimation of the effectiveness yielded from an appropriate testing methodology and
research programme development. The application of chitinous biopolymers, especially
into new usable structures that are the effects of the design synergistically supported by a
risk analysis management, will result in improvements in the performance as well as in
maintaining the required safety. The design of the usable forms of the biopolymer aims at
maximising the performance of developed wound dressing regarding the anatomical
localisation and contact period [66].
The biocompatibility of the biopolymers as well as the final medical devices comprising
chitin or its derivatives should be evaluated according to the selected biocompatibility test
specified in EN ISO 10993‐1 (Biological evaluation of medical devices. Evaluation and
testing), where medical devices are classified based on the clinical anatomical localisation
and the period of the exposure.
Moreover, an estimation of the shell life should be performed, preferably based on the
accelerated studies guided and adopted with use of ASTM 1980F (Standard Guide for
Accelerated Aging of Sterile Barrier Systems for Medical Devices) [83]. Specifications for
tests of the medical packaging can be adopted for designing the conditions of the stability
testing of the newly developed wound dressing themselves [85].
Evaluation of the performance of wound dressings should take into account the
methodology described in the EN 13726 standard, where guidelines are provided for the
testing methodologies of fluids transport, adsorption under free soaking, dispersion
characteristics, moisture vapour transmission (in contact with water vapour or with fluid
simulated exudate), water tightness and conformability/handling. The above‐mentioned
tests should be selected rationally, considering the nature of the designed wound dressing.
For each testing area, the sterile wound dressing should be used to evaluate the medical
devices in the clinically applied form.
The design of the new usable form of the chitinous biopolymers applicable for innova-
tive wound dressing is supported by initiation activity focused on understanding the prob-
lem to be solved with strict interconnection of the identified problems with the hazards in
the risk analysis (the risk identification).
The initiation design phase is helpful at defining and making assumptions of the concept
to be developed, by the ideation of the models and prototypes that would be verified and
validated during the testing phase of the design process. Creating the design of a usable
form of the biopolymers included three phases: initialisation, main design process and the
post‐design processes (collecting end‐user feedback) [66].
The initialisation is related to the problem identification by the design team, mostly by
deeply empathising with the end‐users (practitioners and patients). As a result of this phase,
initial assumptions for the raw materials, technology (to be developed) and design of the
final wound dressing should be defined. The main document – the research programme
based on the initial risk analysis – is also the main output of the initialisation phase. As a
minimum, the research programme should cover testing methods for risk reduction to an
acceptable level, as well as define the optimal level of the parameters describing each
functionality of the wound dressing to be designed [66].
The development phase consists of the verification and validation stages of the designed
models and the resulting prototypes. During the main development activities, the models
and prototypes of the usable forms of the topical haemostatic agents are verified and
validated. The primary verification activities are usually focused on testing the usable
properties that define the functionalities of the designed medical devices, and then
verification of the biocompatibility, stability as well as validation stages, including process
validation, sterilization validation and, finally, the clinical study [66].
12.6 Outlook
Chitin and its derivatives, including chitosan – the chitinous polymer most commonly used
for designing wound dressings – are promising biopolymers in this kind of applications due
to the documented wide‐range bioactivity and biocompatibility. The haemostatic, wound‐
healing acceleration, high gelling affinity and various usable forms are the main indicators
for selecting chitinous biopolymers for medical applications. However, the critical disad-
vantage of the above‐mentioned polymers is the absence of reproducibility of the initial
parameters because of the specificity of the natural origins. The elimination of this aspect
should allow for enhancing the application of chitinous biopolymers in medical fields.
Acknowledgements
This research was supported by the National Science Centre for Research and Development
under the research project No. DOB‐BIO6/19/98/2014 ‘The dressing set for protecting the
injuries which occur at the duty activities of uniformed forces’.
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13
Food Applications of Chitosan
and its Derivatives
Suse Botelho da Silva, Daiana de Souza, and Liziane Dantas Lacerda
Food and Chemical Engineering, Polytechnic School, Unisinos University, São Leopoldo, RS, Brazil
This chapter presents the current and potential applications of chitosan and its derivatives
in food processing. Chitosan is a polysaccharide obtained from partial or full deacetylation
of chitin. Having highly reactive amino and hydroxyl groups, its structure imparts
antioxidant, antimicrobial, and chelating properties to this polymer. These characteristics
are associated with good film‐forming ability, biodegradability, and nontoxicity of chitosan,
and have allowed the development of a wide range of food applications. In this chapter,
recent works are presented as examples of different ways in which chitosan can be applied,
focusing on uses as a food additive and functional ingredient, material for active packaging,
enzyme immobilization, encapsulation of probiotics and bioactive compounds, and for
removal of toxic substances from food. Research on new chitosan derivatives, combination
with other materials, and the advances made by nanotechnology are reflected in these
applications and have allowed for improved materials and high‐performance processes.
13.1 Introduction
Chitosan is a polymer of D‐glucosamine and N‐acetyl‐glucosamine linked through β‐(1–4)
glycosidic linkages, obtained from partial or full deacetylation of chitin with a molecular
weight typically ranging between 16 and 1,000 kDa or even more [1]. Oligomers obtained
from the depolymerization of chitosan, having a degree of polymerization less than 20
(MW < 4 kDa), are recognized as chitooligosaccharides (COS) [2, 3]. Except for cellulose,
chitin is the most abundant polysaccharide in nature, being present in the exoskeleton of

crustaceans and insects, and also in mollusks, annelids, fungi, algae, and protozoa.
Commercially available chitosan is obtained from crustacean chitin processing waste from the
food industry in countries such as USA, Japan, India, and Brazil [4, 5]. The industrial produc-
tion of chitosan contributes, therefore, to minimizing waste generation and to produce a
high‐value added material for the food industry itself [2].
Biodegradability, biocompatibility, and nontoxicity are characteristics attributed to
chitosan; in addition, this polysaccharide has a GRAS (generally recognized as safe) status
by the FDA (Food and Drug Administration) [6, 7]. Chitosan is also approved by the
European authorities and by the International Organization of Vine and Wine to be used as
a fining agent and antimicrobial in wines [8]. Recently, the toxicity of shrimp and fungal
chitosan and nanostructures (5–13 nm) obtained hereof was assessed by brine shrimp and
rat bioassays [9]. The brine shrimp bioassay technique showed that no toxic compounds
were detected for both chitosan and nano‐chitosan tested at concentrations up to 15,000 ppm.
Adding chitosan and nano‐chitosan in the rat diet (100 and 200 mg kg−1 BW rat) showed
no significant changes in blood biochemical profile, oxidative stress parameters, and in the
histopathology of liver, kidney, and stomach, when compared with the control group.
Chitosan has a good film‐forming ability and exhibits a significant pH‐sensitive behavior
due to the presence of amino groups in its structure. At pH lower than 6.2, the protonation
of amino groups occurs, imparting a cationic character to chitosan, that allows its solubility
[4]. Apart from highly reactive amino groups in C‐2, chitosan also has hydroxyl groups
(primary hydroxyl at C‐6 and secondary hydroxyl at C‐3) that are also easily modified [5].
This peculiar structure makes chitosan a remarkable material to be used as an antioxidant,
antimicrobial, or adsorbent agent, and for encapsulation or immobilization of bioactive
compounds in food processing and packaging applications. Even so, in recent years, modi-
fied derivatives from chitosan such as grafted and cross‐linked structures have been devel-
oped for improving its properties [5, 6, 10].
This chapter will present the current and potential applications of chitosan and its
derivatives in the food processing and food packaging area. These applications involve the
use of chitosan as an antioxidant, antimicrobial, or stabilizing agent in product formulations
and in active packaging; as a nutritional ingredient in functional foods; as a chelating or
adsorbent material for metals or toxic substances; and also as a material for immobilization
of enzymes and for encapsulating of microorganisms and other bioactive compounds.
13.2 Chitosan and its Derivatives as Food Additive

Lipid oxidation, enzymatic activity, microbial growth, Maillard reaction, and emulsion
destabilization are some of the most significant deterioration reactions that occur in food.
Some food additives are capable to prevent or retard these reactions extending the shelf life
of products. The remarkable structure of chitosan allows a diversified use as a food additive
with different aims and in different food matrices; however, the poor solubility of chitosan
is a limiting factor for some of its applications.
Here, we present some applications of chitosan and its derivatives as antioxidant,
antimicrobial, stabilizer and thickener additive, including information about their
mechanism of action which allows maintenance of the safety and quality of food products
throughout the shelf life. Table 13.1 shows recent works involving the application of
chitosan as a food additive in some food matrices.
Table 13.1 Recent studies of chitosan as a food additive.
Target food
Chitosan or chitosan derivative Concentration Food deterioration reaction Main result Reference
Chitooligosaccharides (COS) 0.5% (w/w) Fish, meat, Maillard reaction Inhibition of the formation of AGEs in the [21]
added with transglutaminase sausage (harmful advanced product. Cross‐linking of proteins and
(TGase) glycation end glycosylation of glutamine induced by TGase
TGase/COS 1:1 products—AGEsa) and COS resulted in steric hindrance that
inhibited glycation.
Chitosan (Mw 650.00 kDa, 1 g/100 g total weight Bread Microbial growth Chitosan and COS showed an inhibitory effect [22]
DD 30.8%) basis against Bacillus cereus and Rhizopus sp.
COS (Mw 13 KDa, DD 54.8%) growth and delayed ropiness and fruity odor
in bread stored under 30°C for 4 days. Better
results were obtained with COS, resulting in a
shelf life extension of the bread in comparison
with the control.
Chitosan (Mw 32 kDa, DD 1% (w/w) chitosan + Minced Microbial growth Chitosan could control Listeria monocytogenes [23]
90.4%) from Aspergillus LBE at percentages catfish (pathogenic bacteria) and lipid oxidation in fish mince, and its
niger and Goji berry (Lycium of 0.2 and 0.4% and lipid oxidation efficacy was higher when LBE was added at
barbarum) extract (LBE) (w/w) 0.2 and 0.4% (w/w). The sensorial assessment
of treated minces indicated better acceptance
of samples with chitosan and LBE.
Chitosan (not defined Mw 0.2 g/L Wine Microbial growth Chitosan induced a reduction of viability of [8]
and DD) (acetic acid bacteria) acetic acid bacteria immediately after its
addition, showing the similar effectiveness of
SO2 in wine preservation.
Chitosans (MW 310 kDa, DD 100 mg chitosan/kg Mayonnaise Lipid oxidation Retarding of lipid oxidation of the mayonnaise [24]
77.7%) and (MW 123 kDa, product for 63 days under accelerated storage.
DD = 83.2%) Improvement of the sensorial attributes (taste
and odor) compared to the control.
Chitosan (Mw 160 kDa, 95% 0.5% (w/v) chitosan, Stewed pork Microbial growth, lipid Reduce the values of pH, lipid oxidation markers [25]
DD) and aqueous extract 5% (v/v) extract (dipping oxidation, (TVB‐N, peroxide value, TBA value) and total
GOG (ginger, onion, and GOG solution) discoloration, and bacterial count during 12 days of refrigerated
garlic) flavor deterioration storage. Reduction of off‐flavors and better
acceptance than control.
Note: aAGEs: advanced glycation end products; COS: chitooligosaccharides; DD: degree of deacetylation; LBE: Lycium barbarum extract (Goji berry) extract; TBA: 2‐thiobarbituric acid;
TGase: transglutaminase; TVB‐N: total volatile basic nitrogen.
0004461251.INDD 317 10/26/2019 12:14:51 PM

13.2.1 Antioxidant
Antioxidants are compounds capable of retarding or inhibiting oxidative processes in
food, and thereby they can stabilize the color and flavor of food and protect sensitive
nutrients from oxidation. Most antioxidants currently used in the food industry are low‐
molecular‐weight compounds, which may eventually have their efficiency reduced by
processes such as heating or leaching [11]. Antioxidant polymers such as chitosan, on the
other hand, tend to be more resistant to this type of degradation or loss [11], although
they are not considered a practical antioxidant ingredient due to their typical low
solubility [10].
The antioxidant property of chitosan is attributed to its ability to chelate metal ions and
to act on free radicals [12]. In both, this activity is due to the presence of amino group and
hydroxyl groups in the chitosan structure that imparts a strong hydrogen‐donating ability
of this polymer [13, 14]. The chelating activity involves binding of metal ions with the
hydroxyl group at the C6 and with the amino at the C2 of chitosan [13, 15] preventing
metal ions from initiating lipid peroxidation. Similarly, it occurs with the free radicals
which react with active hydrogen atoms of the hydroxyl or amino groups of chitosan to
form a very stable macromolecular radical and, consequently, block the advance of the
oxidative reactions [14]. The antioxidant activity of chitosan is therefore related to the
structural characteristics of this polymer, including its molecular weight, deacetylation
degree, and origin [6].
Antioxidant activity increases with the deacetylation degree and decreases with the
molecular weight, in both cases due to the increase of its solubility [13]. The lower the
molecular weight, the more flexible the chain, so that amino and hydroxyl groups have less
impediment to react with free radicals or to chelate metals, and chitosan exhibits a higher
antioxidant activity [6]. The number of free amino groups (deacetylation degree) itself also
exerts great importance on the antioxidant activity of chitosan, as shown by Yen et al. [13]
working with crab chitosan with different deacetylation degrees (DD). These authors
showed that chitosan with a higher DD had more amino groups to inhibit the oxidation both
via radicals scavenging and metal chelating.
In order to improve the antioxidant activity, especially for chitosan of medium and
high molecular weights, several chemical and enzymatic modification can be per-
formed. These modifications aim to increase the solubility of chitosan and/or improve
the free radical scavenging activity of the structure and conjugation of small antioxi-
dant molecules [6]. Grafting chitosan with flavonoids and phenolic acids using oxida-
tive enzymes is a nontoxic and eco‐friendly alternative for functionalization chitosan
to improve its antioxidant activity [10, 11, 16–18]. For instance, using this approach,
Božič et al. [10] functionalized chitosan with caffeic acid (CA) and gallic acid (GA)
using laccase from Trametes versicolor. In this reaction, the oxidative enzyme con-
verted phenols in reactive o‐quinones which reacted with primary amino groups of
chitosan producing phenolic acid chitosan derivatives with higher antioxidant activity
compared to untreated chitosan. Since the solubility and the protonation of amino
groups of chitosan are dependent on pH, derivatives produced under different pHs
undergo different functionalization mechanisms and intensity of antioxidant activity,
as shown by the authors. The maximum activity was found for chitosan modified at
pH 4.5 [10].
Food Applications of Chitosan and its Derivatives 319
13.2.2 Antimicrobial
The electrostatic interaction between the polycationic structure of chitosan and the predomi-
nantly anionic components (lipopolysaccharides, peptidoglycan, and teichoic acid) of the bac-
terial surface is the main factor responsible for its antimicrobial activity [6]. Chitosan
antimicrobial activity is, therefore, influenced by the deacetylation degree, and also by origin
and degree of polymerization of the polymer, and by the environmental conditions, especially
pH [6, 19, 20]. The higher the degree of deacetylation and the lower the pH, the greater the
number of amino groups likely to be positively charged to interact with the cell surface of the
microorganisms [6, 20]. The degree of polymerization of chitosan affects the antioxidant activ-
ity of different ways, depending on whether the bacteria are Gram‐positive or ‐negative [19].
Recently, Verlee et al. [1] clarified some aspects regarding the antioxidant activity of
chitosan and highlighted the type of microorganism also as an important factor to understand
the mechanism behind the antimicrobial activity of chitosan. According to these authors,
there are four types of microorganisms regarding its susceptibility towards chitosan and,
consequently, four different mechanisms of action: Gram‐positive bacteria, Gram‐negative
bacteria, chitosan‐sensitive fungi, and chitosan‐resistant fungi.
In Gram‐positive bacteria, the main mechanism of action of chitosan is through
electrostatic interaction with teichoic acids in the peptidoglycan layer, leading to disruption
of different functions and resulting in cell death [1]. In this case, the antimicrobial activity
tends to enhance on increasing the molecular weight of chitosan, suggesting that high‐
molecular‐weight chitosan forms films around the cell that inhibit the absorption of
nutrients [1, 19]. In Gram‐negative bacteria, two different mechanisms of action of chitosan
are suggested: chelation with divalent cations under acidic pH decreasing the stability of
the membrane and the nutrient uptake; and, electrostatic interactions with the
lipopolysaccharide at the outer membrane enabling chitosan to penetrate towards the inner
membrane, resulting in cell leakage [1]. In this case, low‐molecular‐weight chitosan
penetrates more easily into Gram‐negative bacteria, binding to DNA, preventing
transcription and translation, and causing disturbances in cellular metabolism [19]. In
chitosan‐sensitive fungi, chitosan affects the cell membrane via electrostatic interactions
with the negatively charged phospholipids, causing the disruption of the membrane, and
further penetrates in the cell and inhibits DNA/RNA synthesis [1]. For chitosan‐resistant
fungi, chitosan is unable to permeabilize the cell membrane which has a high amount of
unsaturated fatty acids and remains at the outer surface [1].
The chitosan modifications that increase amino groups and decrease the molecular
weight frequently enhance its antimicrobial activity [6]. Grafting chitosan with groups with
antimicrobial properties, such as phenolic compounds, also enhance the antimicrobial
activity of the polymer as demonstrated by Božič et al. [10]. These authors functionalized
chitosan with CA and GA and reported an increase in activity against Escherichia coli and
Listeria monocytogenes compared to untreated chitosan.
13.2.3 Stabilizer and Thickener

Polysaccharides and proteins are hydrocolloids and frequently used as natural food
structuring and stabilizer additives. They can be applied to gel, aqueous solutions,
stabilizing foams, emulsions, and dispersions; inhibit the formation of ice and sugar
crystals; and control the release of flavors [26]. Among them, chitosan is considered as an
excellent emulsifier capable of stabilizing oil‐in‐water (O/W) emulsions, which is very
useful in the development of products as dairy desserts, ice creams, and sauces [27]. This
property results from the protonation of the amino groups of chitosan in acidic medium,
which confers an amphiphilic character to the molecule, allowing the adsorption at the oil/
water interfaces and the formation of the emulsion by the decrease of the interfacial tension
[6]. In this sense, the emulsifying ability of chitosan is highly dependent on its molecular
weight and degree of deacetylation [6].
Souza Soares et al. [27] evaluated the performance of chitosan as a thickener and
emulsion stabilizer. O/W emulsions were prepared from 0.1% (m/v) chitosan dispersions
in lactic acid solutions (pH 3.0, 3.5, or 4.0) with Tween 20 (1% v/v) and sunflower oil (9:1).
Emulsions of lactic acid solutions with Tween 20 and sunflower oil was used as a control.
The results showed chitosan as an effective thickener agent even at only 0.1% (m/v),
improving both the size distribution and average size of droplets in emulsions. The surfaces
of oil droplets in emulsions containing chitosan showed highly positive ζ‐potential values,
which is important to sustain the emulsion stability. The average diameter and the ζ‐
potential values did not show significant differences after storage at 25°C for 7 days [27].
Another important aspect of this work is that chitosan has been dispersed in lactic acid
rather than commonly used acetic acid. This new approach would allow producing chitosan
emulsions without the off‐flavor characteristic of acetic acid.
13.3 Functional Ingredient and Health Beneficial Effects

Chitin, chitosan, and COS have been reported for their beneficial health effects on humans
and animals, and they have been proposed as functional ingredients for food and feed
products [2, 3, 28, 29]. Both chitosan and COS are considered as sources of dietary fiber
since they resist intestinal enzymes and acidic gastric conditions [2]. They also demonstrated
anti‐obesity and satiety effects when given in the diet [29, 30].
Some works have reported COS as a prebiotic ingredient [31, 32]. The term “prebiotics”
is defined as “a substrate that is selectively utilized by host microorganisms conferring a
health benefit” [7]. This recent consensus includes as prebiotics, besides nondigestible
food ingredient, other substances that beneficially affects the host by selectively stimulating
health‐promoting bacteria in the colon [7, 33]. Health effects due to prebiotics range from
benefits for the gastrointestinal tract (inhibition of pathogens and immune stimulation) to
cardiometabolism (reduction in blood lipid levels, effects on insulin resistance), mental
health (brain function, energy, and cognition), and bone (mineral bioavailability) [7].
Besides the selective stimulation of health‐promoting bacteria in the colon, other health
beneficial effects of COS and chitosan have also been reported as lowering blood cholesterol
and blood pressure, scavenging of reactive oxygen species (ROS), protective effects against
infections, controlling arthritis, improvement of calcium uptake, and enhancing antitumor
properties [3, 14]. The antitumor activity of COS seems to be related to the protonation of
amino groups and to the low molecular weight [3, 12]. Free radicals or ROS are also
generated in vivo as the oxidation products from biological reactions or induced by
exogenous factors [14]. These species are able to react with biomolecules, attacking
proteins, and impairing its functioning inducing lipid peroxidation of cellular membranes
and oxidizing and damaging DNA [14]. Human metabolism is generally capable to protect
cells and organs against these deleterious effects; however, an imbalance between the
antioxidant protection system and ROS leads to oxidative stress, which is associated with
various chronic diseases [34].
Halder et al. [32] evaluated the bioactivity of COS from fermented shrimp shell hydro-
lysates and chitosan (60% deacetylated) in male albino rats, regarding hypocholester-
olemic, antioxidant, and prebiotic effects. The levels of triglycerides, total cholesterol
(TC), low‐density lipoprotein cholesterol (LDL cholesterol), and atherogenic index (TC/
high‐density lipoprotein cholesterol) were significantly lower in animals fed on chitosan or
COS compared to animals that received the same cholesterol‐rich feeding but did not
receive these chitosan supplements. The antioxidant activity and the resistance to lipid
peroxidation were higher due to COS than to chitosan feeding. COS also selectively stimu-
lated the growth of health beneficial microbes (Bifidobacterium spp. and Lactobacillus
spp.) and inhibited pathogenic bacteria, demonstrating, therefore, prebiotic effect. The
same observation was not found for chitosan.
Long et al. [35] performed a study where orally COS (500 mg kg−1) was given to normal
mice and colitis‐treated mice. The results showed that COS could act as prebiotics by
increasing the levels of Bacteroidetes and Actinobacteria phyla, and common health‐
promoting bacteria as Lactobacillus spp. and Bifidobacterium spp. and inhibiting the
growth of Firmicutes and Proteobacteria phyla, and potential pathogens in both normal and
colitic mice. Additionally, the oral intake of COS induced the enhancement of the
concentrations of short‐chain fatty acids (SCFAs) in the colon. These findings suggest that
COS administration have beneficial effects on the intestinal tract and can protect against
the development of colitis [35].
Regarding chitosan health claims, the European Food Safety Authority (EFSA) Panel on
Dietetic Products, Nutrition and Allergies provided a scientific opinion in 2011 [36].
According to them, there is sufficient evidence between cause and effect to attribute to
chitosan the claim of Maintenance of normal blood LDL‐cholesterol concentrations,
informing that 3 g of chitosan should be consumed to obtain the claimed effect. Nevertheless,
for claims of reduction in body weight, reduction of intestinal transit time, and reduction of
inflammation, the Panel concluded that a cause‐and‐effect relationship has not been
established respecting to the consumption of chitosan.
13.4 Active Packaging

Nowadays, many efforts have been made on searching for new alternatives in food pack-
aging due to the consumer demand for healthier and minimally processed foods, but with
extended shelf life and ready‐to‐eat concept [19]. Beyond the primary functions of
conventional food packaging (containment, protection, communication, and conveni-
ence), active packaging systems are capable of interacting with the product and enhance
its quality and/or increase its shelf life [37]. Active packaging is intentionally designed to
incorporate components that would release (antimicrobial or antioxidant agents) or absorb
(oxygen, ethylene, or water vapor) material into or from the product or the environment
[38]. The active packaging technology has been developed for several decades, but it is a
very dynamic field with continuous advancements [37].
Additionally, searching for eco‐friendly packaging is also increasing, due to the serious
environmental problem related to the accumulation of synthetic plastics, mainly from food
packaging. In this scenario, there is a growing research effort in the development of

biodegradable films and coatings. The biopolymers used as raw material to prepare
biodegradable films should be renewable, abundant, low‐cost, and can be obtained from
wastes, in some cases [39]. Furthermore, biopolymers with film‐forming ability can be
used to produce edibles films and coatings, which represents a stimulating route for creating
new packaging materials. This is because edible films and coatings are available with a
wide range of properties that can help to alleviate many problems encountered with foods
[40]. The most studied materials to develop biodegradable packaging films and coatings
are polysaccharides. They are able to form films and coatings with good barrier properties
against the transport of gases such as oxygen and carbon dioxide [39]. Many studies in
material sciences are focused on the structure–properties relationship to predict and control
the functional film properties. Currently, special attention has been given to chitosan,
which is a suitable material for active film development and has numerous other favorable
properties such as biodegradability, antimicrobial property, and excellent film‐forming
ability [19, 41–44]. Chitosan also can form transparent films, which may find application
in a variety of packaging needs [45].
The main method applied for production of chitosan and other biopolymers films at lab
scale is the casting method. Using this technique, raw materials are first dissolved or
dispersed in a solvent such as water, alcohol, a mixture of water and alcohol, or a mixture
of other solvents. Plasticizers, antimicrobial agents, coloring or flavoring agents can be
added during this step. Adjusting the pH and/or heating the solutions may be necessary to
facilitate the solubility of some biopolymers. Then, the film‐forming solution is cast and
dried under controlled temperature and relative humidity conditions to obtain free‐standing
films. As a coating, film‐forming solutions could be applied to food by several methods
including dipping, spraying, and brushing followed by drying [40].
For chitosan film production, the biopolymer is typically dispersed in acetic acid
solutions (1.0%, v/v) in concentrations ranging from 1 to 4%, and generally glycerol is
used as plasticizer in proportions ranging from 0 to 75% (w/w) based on polymer
concentration (the most applied glycerol concentration is 30% w/w of chitosan).
Antimicrobial or antioxidant agents are also incorporated at this moment. Tween 80 is used
when it is necessary to incorporate some compound by emulsion dispersion. After the
dissolution of chitosan under stirring generally at room temperature (24–48 h), the solution
is filtered to remove particulate matter. The solution is degassed under vacuum to remove
air bubbles. Finally, the chitosan solutions are cast over the Petri dishes for 24–48 h at
25–50°C. After drying, the chitosan films are carefully peeled and stored in a humidity
chamber at 25°C for 48 h for characterization studies [46–59].
The barrier and mechanical properties of chitosan films are strongly affected by the DD,
pH, and the type of acid used for dissolution [60]. The lower the degree of chitosan
deacetylation, the lower the water vapor permeability (WVP), and higher tensile strengths
(TS) of the films produced. Films obtained from acetic and propionic acid provide greater
water vapor barrier and higher TS than films produced with lactic acid. As pH increased
from 3 to 5, WVP of chitosan films tended to increase while TS decreased significantly.
The mechanical properties, gas and water vapor permeability of the polysaccharide films
can limit their use. The TS values showed by polysaccharide‐based films greatly vary from
each other, but some of them exhibit similar values to those observed in synthetic polymers
values. For example, TS values of films based on high amylose starch or chitosan are
comparable to those values found in high‐density polyethylene films. The values of the
percentage of elongation are the main concern, which are far from the desirable values
found for synthetic polymers [39].
Blending chitosan with other polymers had been a strategy to obtain chitosan‐based
films and coatings with enhanced mechanical and physical properties [42]. For blending
chitosan, some methods are available such as solvent blending, extrusion blending, and
reactive extrusion blending, and are reviewed in detail by van den Broek et al. [61]. These
authors emphasize that for increasing the antimicrobial effect of chitosan and improving
the mechanical properties of the material at the same time, a good interfacial adhesion
between chitosan and the polymer matrix is needed. In this sense, the reactive extrusion
blending at which the chitosan and polymer matrix will be covalently connected is a better
option of the blending method. Another advantage of the reactive extrusion is that it is
possible to direct the orientation of functional groups of chitosan to the outside of the
polymeric matrix, improving in this way the antimicrobial activity of the film [61].
The study of combinations of polysaccharides with other materials to improve the barrier
and mechanical properties is a field with continuous advancements and may allow
biopolymer matrices to replace synthetic polymers [39]. Indeed, the high hydrophilicity of
the polysaccharides is one of their major limitations, because as soon as the films enter in
contact with water, they undergo changes in their physical aspect and properties, which
also compromise their acceptance. Moreover, the disintegration of the films could also hap-
pen. For example, the incorporation of the hydrophobic poly(ε‐caprolactone) to the chi-
tosan matrix and the use of glacial acetic acid may be useful tools to prevent undesirable
swelling in contact with food [62].
Recently, the application of nanocomposite technology has been proven as a promising
option to improve the mechanical properties of chitosan films [44, 63, 64]. With the aim
focused on the development of a composite‐improved film, chitosan was incorporated with
montmorillonite (MMT). Film thickness, water vapor permeability, and mechanical and
optical properties were investigated. MMT could enhance water vapor permeability and
mechanical properties of the chitosan‐based films [63]. In another study [64], chitosan/
nanoclay nanocomposite active films containing three different levels of sodium MMT
(1, 3, and 5% w/w based on chitosan) and Silybum marianum L. extract (SME) (0.5, 1, and
1.5% v/v) were prepared. The results showed that water vapor permeability and solubility
of films reduced significantly by incorporation of MMT and SME. The tensile properties
of the prepared nanocomposites indicated an improvement in the mechanical properties in
comparison to control chitosan film, which can be explained by the formation of exfoliated
structure. Improved elastic modulus clearly demonstrates the reinforcing effects of the
nanoparticles on the films, which could occur as a result of strong hydrogen bonding
between MMT and chitosan [64].
A sodium lactate–loaded chitosan–polyvinyl alcohol/montmorillonite (NaL‐CS/PVA/
MMT) barrier film with antibacterial activity was developed by the coating method [65].
An intercalated structure was achieved for the CS/PVA/MMT film and the interfacial
interactions among CS, PVA, and MMT were intermolecular hydrogen bonds. An
appropriate increase of MMT contents (15 wt% and below) could achieve a remarkable
enhancement in TS and Young’s modulus, meanwhile, the water vapor, oxygen, and carbon
dioxide barrier properties of the films were also significantly improved. The NaL‐CS/PVA/
MMT film exhibited good antibacterial activity against E. coli and well‐controlled release
of NaL [65]. To improve the mechanical properties of polyvinyl alcohol/chitosan (PVA/

CS) biodegradable films, a study was developed for addition of SiO2 in situ to enhance
PVA/CS biodegradable films via hydrolysis of sodium metasilicate in the presence of PVA
and chitosan solution. The TS of PVA/CS biodegradable films was improved by 45% when
0.6 wt% SiO2 was incorporated into the films. According to the authors, the Si‐O group of
in situ‐synthesized silica allowed the formation of hydrogen bonds between silica/PVA and
silica/CS, which contributed to the enhanced mechanical properties. The in situ SiO2‐
enhanced PVA/CS biodegradable films possessed not only excellent mechanical properties
but also a barrier of oxygen and water for food packaging to extend the shelf life [44].
Despite the widely studied intrinsic antimicrobial potential of chitosan, several recent
studies have sought to investigate the effect of the incorporation of many (mostly natural)
antioxidants and antimicrobial compounds in the chitosan film. The aim of these studies is
to produce films or coatings with enhanced properties capable of acting as active food
packaging. Due to the good compatibility of chitosan with other active compounds, both
antimicrobials and antioxidants, these chitosan films have shown great promise for their
application in food preservation [19, 37]. However, the addition of bioactive compounds
may influence the physicochemical and mechanical properties of the films [66]. Table 13.2
shows some recent studies related to the development of antioxidants/antimicrobial
chitosan films incorporated with different kind of molecules. It is possible to note that the
antioxidant activity is enhanced in the incorporated films, and the films showed antimicrobial
activities (in vitro) against various microorganisms, increasing food safety.
The interactions that occur between food and films related to the migration rates of the
active compounds are dependent on the storage conditions, and they may interfere with
the results of the application. Therefore, to perform the study of film application or coat-
ing on foods, simulating the real conditions of use is very important. Several studies have
recently been carried out, applying films or coatings of chitosan to perishable foods,
evaluating their effect on the shelf life of the food. Some studies are shown in Table 13.3.
These studies have obtained very promising results in producing active packaging using
chitosan as a base polymer with different antimicrobial compounds and/or natural
antioxidants.
The study of the application of chitosan in the production of edible and biodegradable
films and coatings has been extensively carried out in recent years. The use of different
active molecules incorporated into chitosan films allows obtaining a material with a higher
potential to act as active packaging for food. Additionally, the results obtained in the
application of chitosan‐based biofilms or coatings in various perishable foods have shown
how promising is this polymer. Issues related to the release and migration rates of
compounds for food should always be considered in order to qualify the packaging or
coating for a specific application as an active food packaging.
The main constraints for the commercialization of antimicrobial packaging systems,
although extensive research has been carried out in the development of packaging solutions,
are mainly regulatory issues and also technical limitations that need to be solved. So, the
successful implementation of antimicrobial packaging solutions in the market needs a
multidisciplinary approach involving researchers from different disciplines working
together with the packaging industry [37]. Additionally, it is important to have low costs for
these packaging solutions, since they should be as low as possible to obtain an economi-
cally viable product [61].
Table 13.2 Recent studies on antioxidant/antimicrobial chitosan films incorporated with different molecules.
Film‐forming Molecules or
solution extracts tested Concentrations Antioxidant activity Antimicrobial activity against Effects on film properties Reference
Chitosan Eucalyptus globulus 0, 1, 2, 3, and Significantly enhanced Escherichia coli, Staphylococcus Significantly decreases water [67]
(2% w/v) (EG) essential oil 4% (v/v) with increasing EG aureus, Pseudomonas solubility
essential oil aeruginosa, Candida albicans,
concentration and Candida parapsilosis;
enhanced with increasing EG
essential oil concentration
Chitosan Spirulina extract (SE) 0, 2.5, 5, 10, 15 Significantly enhanced E. coli, S. aureus, P. aeruginosa, The incorporation of SE into [59]
(2% w/v) and 20% (w/v) with increasing SE L. monocytogenes, crab chitosan film led to
concentration Salmonella typhimurium, considerable improvement
Bacillus subtilis, B. cereus; in mechanical and barrier
films exhibited concentration‐ properties of the resulting
dependent antimicrobial film.
activities against bacteria.
Chitosan Turmeric ethanol 67% (v/v) ‐ TEE incorporated chitosan film The addition of turmeric to [68]
(2% w/v) extract (TEE) reduced the counts of S. chitosan film significantly
aureus and Salmonella sp. increased the TS of the film
significantly, compared to and improved the
native chitosan film during ultraviolet‐visible light
the 3 h exposure period. barrier of the film.
Chitosan Epigallocatechin 1% (w/v) EGCG grafted‐chitosan The antibacterial activity of the EGCG‐grafted chitosan [69]
(1% w/v) gallate (EGCG) showed higher EGCG‐grafted chitosan was showed higher antioxidant
antioxidant activity increased compared to pure activity than blank
than native chitosan. EGCG or native chitosan chitosan.
against S. aureus and
Pseudomonas sp.
Chitosan Ziziphora 0 and 1% v/w, The highest antioxidant S. aureus, B. subtilis, B. cereus, ZEO and GSE reduces [70]
(2% clinopodioides separately and activity was found in L. monocytogenes, S. swelling index, TS,
w/v)— essential oil (ZEO) in chitosan enriched with typhimurium, E. coli puncture force, and
Gelatin Ethanolic grape seed combination 1% (v/w) ZEO in O157:H7 puncture deformation of
(3% w/v) extract (GSE) combination with 1% chitosan and gelatin films.
(v/w) GSE
(Continued )
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Chitosan Thinned young 0.25, 0.50, 0.75, The antioxidant activity YAP‐chitosan films have A significant increase in the [52]
(2% w/v) apple polyphenols and 1.0% of 1.0% (w/v) YAP‐ concentration‐dependent thickness, density, swelling
(YAP) (w/v) chitosan films antimicrobial activities against degree, solubility, and
increased nearly bacteria (E. coli, L. opacity of chitosan film,
threefold compared to monocytogenes, and S. aureus) but the water content,
the control films. and molds (Colletotrichum water vapor permeability,
fructicola, Botryosphaerial and mechanical properties
dothidea, and Alternaria of the film were decreased.
tenuissima) but no activity
against yeasts (Saccharomyces
cerevisiae, baker’s yeast, and
tropical candida)
Chitosan Ellagic acid (EA) 0.5, 1.0, 2.5 and Concerning the chitosan/ Chitosan/EA films totally These set of films display high [56]
(1% w/v) 5.0% (weight EA films, DPPH a
inhibited the growth of both mechanical properties,
percentage of scavenging activity Gram‐positive (S. aureus) and thermal stability up to 215
chitosan) increased from 0.0 to Gram‐negative (P. aeruginosa) and 220°C, UVA‐ and
about 28% with the food pathogenic bacteria due UVB‐barrier properties,
increase of EA content to chitosan antimicrobial moderate water vapor
from 0.5 to 5.0 wt% effectiveness. permeability.
Chitosan Honeysuckle flower 5, 10, 20, and The incorporation of HFE Film solution created a larger Compared with the chitosan [51]
(4% w/v) extract (HFE) 30% (weight enhanced the inhibitory zone (127.36 mm ) 2
control film, the chitosan/
percentage of antioxidant activity of against E. coli compared with HFE films had darker
chitosan) the films. 36.77 mm without HFE.
2
appearance, higher water
solubility, and lower TS
and elongation at break.
Chitosan Apple peel (APP) 0.25, 0.50, 0.75, Incorporating of APP E. coli, B. cereus, S. aureus, and Thickness, density, swelling [46]
(2% w/v) and 1.0% significantly improved S. typhimurium; films degree, solubility, and
(w/v) the antioxidant activity exhibited concentration‐ opacity of film were
of chitosan‐based films, dependent antimicrobial significantly increased, but
and the antioxidant activities against bacteria. the water content and
activity was enhanced vapor permeability were
with increased decreased.
concentration of APP.
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Chitosan Procyanidin (PC) 0.25, 0.50, 0.75, Films showed high Solutions possessed The mechanical properties [71]
(1% w/v)— and 1.00 mg/ antioxidant activity antibacterial activity against (TSb and EABc) of films
Fish gelatin mL S. aureus and E. coli strains were significantly
(3% w/v) due to the presence of improved, these being
chitosan. more resistant and
PC content did not impact on extensible. Results denoted
activity. that PC acted like
plasticizer in prepared
films, providing better
mobility.
Chitosan Methanol extracts of 1.7% (v/v) of The addition of methanol Proteus microbilis, Proteus The elasticity of chitosan‐ [49]
(1% w/v) stem, leaf, and each extract extracts from P. vulgaris, P. aeruginosa, and E. seed and chitosan‐stem
seed obtained terebinthus’ stem, leaf, coli. Chitosan‐seed film films was improved.
from Pistacia and seed notably indicated the greatest
terebinthus improved the antimicrobial activity than the
antioxidant properties other blended films
of the blend films.
Note: aDPPH: 1,1‐diphenyl‐2‐picrylhydrazyl radical; bTS: is tensile strengths; cEAB: elongations at break.
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Table 13.3 Recent studies of the application of chitosan films or coatings to perishable food, evaluating their effect on the shelf life.
Application
Film‐forming solution Food tested technique Molecules or extracts tested Concentration Main result Reference
Chitosan (1.5% w/v) Fresh red drum Coating Grape seed extract (GS) tea 0.2% w/v of each Both treatments extend the shelf [72]
(Sciaenops polyphenols (TP) extract, separately life of fish by 6–8 days compared
ocellatus) fillets with the control group.
Chitosan (1% w/v) Eggs Coating Lysozyme (L) 0, 10, 20, and 60% 20% w/v L–chitosan coating [73]
(dry weight maintained the internal quality
lysozyme/dry of eggs 3 weeks longer than
weight chitosan) observed for uncoated control
Chitosan (2% w/v) Lean pork slices Coating Clove oil (CO)— 0.05% v/v (CO) Chitosan + CO and [74]
ethylenediaminetetraacetate 10 mM (E) Chitosan + CO + E coatings have
(E) a good potential for
antimicrobial purposes in
refrigerated pork as active
packaging materials.
Chitosan (1.5% w/v) Strawberry Coating Nisin (NS) natamycin (NT) 1% w/v of each, Antimicrobial agents incorporated [75]
pomegranate extract (PE) separately in chitosan coating improved the
grape seed extract quality and extended the shelf
life of fresh strawberry up to 30
days
Chitosan (1% w/v), Shrimp Coating ‐ ‐ A shelf life of <14 days was [76]
gelatin (3% w/v), observed for the coated shrimp,
70:30 as compared to only <8 days for
the control.
Chitosan (1% w/v) Barracuda fish Film Ginger essential oil (GEO) 0.1, 0.2, 0.3% v/v Sensorially, chitosan‐GEO film [41]
wrapping wrapped sample was acceptable
till the end of storage for 20 days
as compared to 12 days for
unwrapped control and fish
steak packed in EVOH film.
Chitosan (2% w/v) Shrimp Film Z. clinopodioides essential oil 1% v/v (ZEO and The group treated with chitosan-ZEO [48]
wrapping (ZEO) pomegranate peel PPE); 1% w/v 1% + PPE 1% + CN 1% had the
extract (PPE) cellulose (CN), separately best antibacterial effectiveness
nanoparticle (CN) and in and also the highest organoleptic
combination scores after 11 days.
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Application
Chitosan (1.5% w/v) Butter cake Film Potassium sorbate (PS) vanillin 0.2 g g−1 chitosanChitosan film can delay mold [77]
wrapping (V) (PS); 0.3 g g−1 growth on butter cake and
chitosan (V), double the shelf life of butter
separately cake. Further, addition of
potassium sorbate and vanillin in
the film can reinforce the
inhibitory effect of chitosan film
and almost triple the shelf life of
butter cake.
Chitosan (1.5% w/v) Whole milk Immersion Cinnamaldehyde ‐ Although the release of the agent [78]
with covalent (inoculated with of film in caused a perceptible cinnamon
immobilization of L. monocytogenes) the aroma in milk, the sensory panel
cinnamaldehyde via sample considered this effect as positive,
Schiff base treated milk being preferred to
the control sample.
Chitosan/polyvinyl “Superior seedless” Coating Ascorbic acid (AA) 2.8, 5.6, and 8.2 CS/PVA‐AA 8.2 mM coating fruit [79]
alcohol (CS/PVA) grapes mM cluster of superior seedless
grapes exhibited a slower rate of
deterioration, as compared to
uncoated fruit clusters and CS/
PVA only.
Chitosan/cassava Mangaba fruits Coating Myrcia ovata Cambessedes 0.5, 1.25, 2.0, 2.5% The edible coating containing [80]
starch essential oil (EO) w/v 0.5% cassava starch, 0.5%
chitosan, 1.25% EO reduced the
natural microbiota and inhibited
B. cereus (artificially
contaminated) in Mangaba fruits
stored at 10°C for 12 days
Chitosan (1% w/v) Beef (inoculated Film Liposome‐encapsulated phage ‐ The chitosan film embedded with [58]
with E. coli wrapping liposome‐encapsulated phage
O157:H7) exhibited high antibacterial
activity against E. coli O157:H7,
without the impact on the
sensory properties of beef.
(Continued)
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Application
Chitosan (1.25% w/v) Salmon (Salmo Film Grape seed extract and With and without This study reports the beneficial [81]
salar) wrapping carvacrol microcapsules GSECM effects of the chitosan film with
(GSECM) GSECM on salmon samples
stored at 5°C, with the samples
remaining acceptable between
the fourth and seventh days of
storage.
Chitosan (1% w/v) Minced beef Film Kombucha tea (KT) 1, 2, and 3% (KT) The shelf life of stored minced beef [55]
wrapping (w/w) packaged in chitosan/KT can be
extended up to 6 days. The
Staphylococcus growth counts
were also inhibited by packaging
in chitosan containing KT.
Chitosan (2% w/v) Cherry tomato fruit Film Titanium dioxide (TiO2) 1% The quality changes of climacteric [50]
wrapping nanopowders (T) fruit are known to be triggered
and regulated by ethylene;
hence, the chitosan‐T
nanocomposite film which
exhibited ethylene
photodegradation could delay
the ripening process and extend
the storage life of the tomato
fruit
Nanomontmorillonite‐ Minced camel’s Film Z. clinopodioides essential oil 0.5, 1, and 2% w/v An increase in the shelf life of [47]
chitosan (MMT meat wrapping (ZEO) Ficus carica extract (ZEO) alone or in minced camel meat by active
0.1% w/v chitosan (FCE) combination of Chitosan‐MMT films was
2% w/v) 1% v/v (FCE) observed.
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13.5 Enzyme Immobilization

Enzyme immobilization is a relatively simple methodology and offers many benefits on
biocatalysis such as efficient reuse of the enzyme, continuous operation, enhanced stability,
under both storage and operational conditions, easy separation from the medium reaction,
thereby minimizing or eliminating protein contamination of the product, low or no aller-
genicity, among others [82]. According to the type of interaction between enzymes and
support carriers, enzyme immobilization methods are classified into physical and chemical
methods. Adsorption and entrapment are considered as physical methods, in which there
are no covalent interactions between enzymes and support carriers, while covalent attach-
ment and cross‐linking are related to chemical methods. In some cases, multiple enzyme–
support interactions are exploited for enzymes immobilization [83]. Different materials can
be used as enzyme supports, consisting of organic or inorganic materials such as polysac-
charides, proteins, polymers, activated carbon, and metals. Advantage of chitosan‐based
materials is related to their biodegradability, antibacterial activity, hydrophilic property, as
well as presence of polar groups which are able to interact also with other polymers (‐OH
and ‐NH2 groups involved in hydrogen bonds and the N‐acetyl groups in hydrophobic inter-
actions) [2, 84].
Ricard et al. [85] prepared inorganic–organic composite material based on silica/chi-
tosan using the sol–gel method, and a silica material, which was organofunctionalized
with 3‐aminopropyltrimethoxysilane for comparison. These materials were applied to
the immobilization of β‐galactosidase from Kluyveromyces lactis. The results aligned
the enzyme stabilization properties of chitosan with the mechanical resistance of silica,
demonstrating compatibility for food processing. For α‐galactosidase immobilization,
chitosan and Sepabeads EC‐EA (polymethacrylate‐based synthetic material with hexa-
methyl ethylene diamino groups) were functionalized with aminophenyl boronic acid by
using glutaraldehyde. Under optimized immobilization conditions, chitosan and
Sepabeads EC‐EA immobilized enzymes exhibited activity yield of 89.5 and 72%,
respectively [86].
A simple embedding method was developed to synthesize a new kind of chitosan‐
enriched magnetic composites (MCCs) to immobilize α‐glucosidase (α‐Glu) [87]. By add-
ing the mixture of Fe3O4 nanoparticles and chitosan solution to sodium hydroxide solution,
MCCs were prepared. Then, by cross‐linking with glutaraldehyde, α‐Glu was covalently
cross‐linked on MCCs. The immobilized α‐Glu showed a broader working pH and tem-
perature range than the free enzyme [87]. Immobilization also enhanced protection against
high temperatures for dextransucrase from Leuconostoc mesenteroides covalently immobi-
lized on glutaraldehyde‐activated chitosan particles [88]. In this case, the immobilization
was carried out in two steps: first, the hydrolysis of the dextran layer around dextransucrase
molecules was performed using dextranase; then, the immobilization of the enzyme pro-
ceeded on glutaraldehyde‐activated chitosan particles. The results suggest the use of the
immobilized dextransucrase on chitosan particles as a promising novel biocatalyst to pro-
duce dextran and oligosaccharides.
In the fruit juice industry, the presence of polysaccharides in the form of the disrupted
vegetal cell wall can lead to an increase in turbidity, color intensification, and the formation
of sediments in the product during storage. The breakdown of such biological
macromolecules including pectin and cellulose is vital for beverage processing. Results
showed that chitosan is considered an excellent supporting matrix for the immobilization
of pectinolytic enzymes such as polygalacturonase pectin lyase, and pectin methylesterase
[89]. Khoshnevisan et al. [90] provided a detailed overview of the applications and recent
advances of immobilization of cellulase onto different functionalized magnetic nanoparticles
(MNPs). For instance, the synthesis of chitosan‐MNPs was carried out by adsorption or
covalent binding after activating the hydroxyl groups of chitosan with carbodiimide,
methanediimine, or cyanuric chloride.
Some recent studies focus on using carboxypeptidase A (CPA) insoluble derivatives for
producing hydrolysates of food proteins mainly from milk and by‐products for obtaining
food preparations with low allergenicity and standardized for phenylketonuric consumers
[91]. Manzo et al. [91] developed strategies for effective multipoint covalent immobilization
of CPA metalloenzyme on chitosan beads by N‐alkylation. Urrutia et al. [92] reported a
complete analysis of the use of hydrophobic chitosan as a support for the immobilization
of lipases and their further application in the selective hydrolysis of fish oil. According to
these authors, the length of alkyl chains linked to chitosan is a critical factor in the final
behavior of immobilized lipases, mainly affecting biocatalyst activity and stability.
Chitosan and modified chitosan have a strong affinity towards immobilization of biomol-
ecules, which have demonstrated also a huge potential in providing a platform for the fabri-
cation of biosensors. A biosensor is an analytical device that uses specific biochemical
reactions mediated by a biological recognition element (an enzyme) immobilized onto a
signal transducer. Quantification limit (LOQ), low limit of detection (LOD), response time,
repeatability, reproducibility, and accuracy are other factors responsible for the characteriza-
tion of biosensor performance [93, 94]. Neusakumar et al. [95] reported a lactic acid biosen-
sor by immobilizing lactate dehydrogenase (LDH) with chitosan onto ZnO nanorods. The
one‐dimensional ZnO nanorods exhibit high surface area and act as an immobilization
matrix for LDH. This lactate detecting biosensor was shown to have an LOD of 4.73 nmol L−1,
LOQ of 15.75 nmol L−1, the response time of < 1 s, sensitivity of 1.832 μA μmol−1 L, and
linearity of 0.2–0.8 μmol L−1. This sensor was stable up to 23 days under dry conditions at
room temperature.
13.6 Encapsulation and Delivering of Bioactive Ingredients

Encapsulation may be defined as a process to entrap one active agent (core) within another
material structure (wall) for protection of the “core” from drastic conditions of the
environment [96]. Bioactive compounds (vitamins, phenolic compounds, and bioactive
peptides) and probiotic microorganisms are widely recognized as health‐promoting
ingredients, nevertheless, they are frequently sensitive to food processing and gastrointestinal
tract conditions [96, 97]. Encapsulating materials should provide adequate protection
against these issues, showing nontoxicity. The encapsulating systems can offer additional
other desirable characteristics to the core in food matrices, such as solubility, improvement
of rheological and sensorial properties, and controlled releasing of the incorporated
materials [96, 98].
Chitin and chitosan are materials normally used as coating agents because they produce
a very good encapsulation system for food compounds and controlled release since they
are nontoxic, highly biocompatible, and mechanically strong [98, 99]. Additionally,
chitosan and some derivatives exhibit a pH‐responsiveness behavior due to the presence
of amine groups on D‐glucosamine units [4, 5]. This characteristic leads to an interesting
property to chitosan, which is dependent on the protonation of amino groups and results
in an excellent mechanism which can be used to release the encapsulated active com-
pounds as pH changes [4, 99].
Several works are found in the literature describing the preparation of encapsulating
systems made only or in combination with chitosan. These systems are obtained using
primarily mechanical techniques as emulsification, spray drying, spray chilling, fluidized‐
bed coating, centrifugal extrusion, electrospinning/electrospraying, pressure extrusion, and
hot‐melt extrusion, or essentially chemical techniques as ionic gelation, simple or complex
coacervation, and coating with solvent evaporation [97–101]. The more advanced
procedures involve the addition of a cross‐linker (tripolyphosphate, transglutaminase, or
genipin) during the last step of coacervation [96]. Even using merely mechanical techniques
to produce the beads, chemical modifications of the polymer may eventually be induced, or
further steps may be added for that purpose. This is especially true for charged polymers or
having chemically active functional groups.
In ionic gelation, a hydrocolloid with a bioactive compound are dripped or atomized into
an ionic solution under constant stirring, obtaining spherical gel structures [99]. For
chitosan‐based systems, typically chitosan is dissolved in diluted acetic (or other) acid
solution in order to induce the protonation of amino groups and exhibit its cationic character
followed by dripping in an anionic solution. In coacervation, the formation of complex
structures results from liquid–liquid phase separation from an initial suspension followed
by deposition and solidification of the dense coacervate phase surrounding the core material
[26, 100]. The simple coacervation result from the presence of only one polymer or
hydrocolloid in the initial suspension, while for complex coacervation it is necessary to
have two or even more hydrocolloids and polymers in an initial suspension mixture [96].
The formation of the coacervate phase is due to changes in the physical parameters
(temperature, pH, or composition) of the initial suspension leading to the separation of
incompatible polymer [100].
Chitosan is an extremely useful material in a chemical encapsulation process due to its
hydroxyl and amino‐reactive groups which can be used for modification of this polymer
and for obtaining complex structures [5, 102]. In particular, the protonation of the amino
groups of chitosan in low pH imparts a polycationic character to the polymer and allows
the formation of polyelectrolyte complexes of chitosan (PEC) prepared with natural,
naturally modified or synthetic polyanions [103]. In ionic gelation, the electrostatic
interaction occurs between the amine group of chitosan and a negatively charged group of
polyanion such as tripolyphosphate [104]. In these approaches, not only electrostatic
interactions between chitosan and anionic polymers are involved but also hydrogen bond-
ing and hydrophobic interactions [6].
Among the bioactive compounds which exhibit huge potential for use in nutraceutical
encapsulation systems are polyphenols and vitamins. Polyphenols are beneficial to human
health, however, they are highly unstable to conventional food process conditions and
frequently have an unpleasant taste [105]. So, encapsulation is an effective tool for
preserving the stability and ensuring bioactivity and bioavailability to these compounds, as
demonstrated by several authors [98, 105–110]. Vitamins, especially those that have
antioxidant potential as vitamin C or vitamin E, are susceptible to degradation and can have
their bioavailability limited by low absorption and degradation during delivery [111].
Liposoluble vitamins are not readily soluble in water, which also restricts their use in some
food matrices. Encapsulating systems have been investigated and proposed to improve the
solubility, stability, and bioavailability of vitamins, but also to enhance proprieties as
antioxidant activity [112]. Several works involving chitosan‐based systems to encapsulate
polyphenols and vitamins have been published in recent years, showing the emergence of
this research area. Some examples of these works are shown in Table 13.4.
Even more than for bioactive chemicals, encapsulation systems are crucial for probiotics,
in order to ensure their viability during food processing and storage, as well for delivering
the microorganisms through the gastrointestinal tract [113, 114]. Probiotics are defined as
live microorganisms that when administered in adequate amounts in food confer a health
benefit on the host [115]. Thus, metabolically stable and active cells and ensured high
viability rates are the requirements to consider microorganisms as functional probiotics
[100]. In this sense, encapsulating systems capable to protect microorganisms in acid
gastric condition and their release under intestine conditions are particularly desired.
Several works have demonstrated the ability of chitosan in increasing the viability of
encapsulating microorganisms in food matrices under storage or under gastrointestinal
conditions [116–122]. Frequently in these works, the microorganism is initially encapsulated
using alginate, pectin, or another hydrocolloid, followed by application of chitosan as a
coating. Bepeyeva et al. [119] prepared gel beads by extrusion of amidated pectin in cal-
cium chloride solution, coated with or without chitosan. The authors reported the protec-
tive effect of chitosan in this encapsulating system. Capsules with chitosan provided
improved protection to Lactobacillus casei in simulated gastric juice resulting in high lev-
els of viable bacteria released in simulated intestinal juice [119]. Brinques and Ayub [116]
encapsulated Lactobacillus plantarum BL011 in beads of sodium alginate coated with chi-
tosan and verified that the viability of the encapsulated cells under refrigerated storage was
greatly enhanced compared to unencapsulated microorganisms. However, the viability was
drastically reduced in the simulated gastric medium [116]. These results demonstrate that
the effectiveness of encapsulation and the viability of microorganism depend on the encap-
sulation materials used beyond the selected strain [101]. Details of encapsulating tech-
nique, materials, capsule dimension, and main results for these works are shown in
Table 13.5.
13.7 Adsorption and Chelation of Toxic and Undesirable Compounds

The occurrence of chemical contaminants in food as pesticide residues, mycotoxin, or
heavy metals represents a serious public health problem worldwide. The use of agents
capable to remove, bind, or inactivate such contaminants and toxic compounds in food has
been studied extensively, being chitosan considered as one of the most promising materials
for this purpose [124]. At pH < 6.2, chitosan exhibits a positive surface charge due to
protonation of the amine groups (‐NH3+) [4], which makes this polymer an excellent mate-
rial to be used as an adsorbent or chelating agent [125]. Moreover, the modification of
chitosan molecule with grafting (insert functional groups) or cross‐linking reactions leads
to the formation of chitosan derivatives with superior properties, enhancement of adsorp-
tion capacity and resistance in extreme media conditions, respectively [126].
Among the toxic compound that can contaminate food, mycotoxins represent a special
concern. Mycotoxins are secondary metabolites produced by mycotoxigenic fungi which
Table 13.4 Recent works using chitosan‐based systems to encapsulate polyphenols and vitamins.
Bioactive Encapsulation Capsule

ingredients Encapsulating materials techniques diameter Main results Reference
Eugenol Chitosan, sodium Ionic gelation 80–100 nm Improved thermal stability of eugenol by [106]
tripolyphosphate and mediated by encapsulation. Beads with strong antibacterial
Tween20 ultrasonic‐ activity against S. aureus and E. coli O157:H7, P.
emulsification aeruginosa, and Salmonella.
Catechin and Chitosan and sodium Ionic gelation 180.4 ± 3.1 nm In vitro sustainable release. Stronger or comparable [107]
quercetin tripolyphosphate and followed by potency in scavenging DPPH, ABTS•+, •OH,
subsequent modification cross‐linking and O2−• radicalsa, and in suppressing growing
by genipin. of E. coli, S. aureus, and B. subtilis as compared
to native catechin and quercetin.
β‐carotene Amphiphilic chitosan Complex 97 ± 4 nm to High efficiency in retaining β‐carotene in the core [112]
(conjugated with polylactic coacervation 144 ± 6 nm structure, and enhancement of the antioxidant
acid) and nucleic acid activity.
Propolis (great Heat‐denatured zein, Complex 130–304 nm Encapsulated propolis exhibited control pH‐ [109]
polyphenol carboxymethyl chitosan coacervation dependent release, improved water solubility
content) and Ca2+ followed by and antioxidant activity
cross‐linking
Epigallocatechin Carboxymethyl chitosan, Ionic gelation 50–300 nm Zein coating greatly improved the controlled [110]
gallate (EGCG) chitosan hydrochloride, release of EGCG and the antioxidant activity.
and zein
Propolis (great Cross‐linked chitosan by Membrane 1650 ± 20 μm Propolis–chitosan beads inhibit the growth of S. [108]
polyphenol genipin emulsification aureus and other food spoilage and/or pathogen
content) followed by microorganisms in the liquid culture medium.
cross‐linking
Note: aABTS•+ is 2,2’‐azino‐bis‐(3‐ethylbenzothiazoline‐6‐sulfonate) radical cation; DPPH is 1,1-diphenyl-2-picrylhydrazyl radical.
0004461251.INDD 335 10/26/2019 12:14:52 PM

Table 13.5 Recent works using chitosan‐based systems for the encapsulation of probiotic microorganisms.
Probiotic Encapsulating Encapsulation Capsule diameter Food Matrix Main Results Reference
microorganisms materials techniques
Lactobacillus casei Calcium alginate, Ionic gelation 71.5 ± 4.9 μm for uncoated Ice cream Chitosan and poly L‐lysine coatings [118]
ATCC 39392 and wheat, rice, and mediated by capsules, 94.58 ± 2.28 μm significantly improved the
Bifidobacterium high‐amylose corn emulsion and 95.6 ± 3.2 μm for encapsulated probiotic survival
adolescentis ATCC (Hylon VII) starches, followed by chitosan and poly L‐lysine in ice cream after 100 days of
15703 chitosan, and poly coating coated capsules, storage at ‐30°C.Encapsulated
L‐lysine respectively probiotic showed no significant
effect on typical sensorial
characteristics of ice cream.
L. casei NCIMB Pectin and chitosan Ionic gelation 2.57 ± 0.02 to 3.50 ± 0.05 mm No food Coating with chitosan effectively [119]
30185 (PXN37) mediated by for uncoated capsules and matrix protected the bacterial cells in
extrusion 0.236 ± 0.061 mm for tested pectin capsules from the acid in
followed by coating thickness the simulating gastric juice, while
coating capsules without chitosan
coating had limited protection.
Lactobacillus Chitosan, Ionic gelation Microparticles (by nozzle‐ No food The capsules were stable at the [120]
rhamnosus GG carboxymethyl‐ mediated by spraying): 5 μm matrix gastric pH conditions (pH 2.4)
LMG 18243 cellulose, and extrusion (syringe macroparticles (obtained tested and swell considerably at higher
genipin or nozzle sprayer) with a syringe): 2 mm pHs, close to intestine pH
followed by (pH 7.4).
coating
Lactobacillus Sodium alginate, Ionic gelation 1,302–1,335 μm No food Survival of encapsulated [121]
plantarum LAB12 xanthan gum, and mediated by matrix microorganism at pH 1.8 with
chitosanβ‐ extrusion tested facilitated release at pH
cyclodextrin as a followed by 6.8.Minimum loss of the bacterial
supplement coating viability at 75 and 90°C and
survival over cold storage (4°C)
for 4 weeks.Cholesterol‐lowering
effects of β‐cyclodextrin‐
supplemented beads were
demonstrated and
cytocompatibility was assessed
in vitro, confirmed nontoxicity.
0004461251.INDD 336 10/26/2019 12:14:52 PM

L. plantarum TN8 Sodium alginate, Ionic gelation 22–23 mm Corn– When administrated in broiler diet, [122, 123]
xanthan gum, mediated by soybean the probiotic preparation caused
fructose, maltose, extrusion and meal (for a decrease in total cholesterol,
MRS broth, coating broilers) HDL‐cholesterol, LDL‐
glycerol, and cholesterola, and triglycerides,
chitosan and improved the performance
(body gain weight, feed
consumption, and feed
conversion).
L. rhamnosus GG Alginate, inulin, and Ionic gelation 1.40 ± 0.08 and 1.39 ± 0.06 Apple juice Probiotic encapsulation improved [117]
chitosan mediated by mm in beads encapsulated bacterial survival in apple juice
extrusion and with and without inulin, during storage at 4 and 25°C for
coating respectively 90 days, and no adverse sensorial
modifications were observed.
Encapsulation improved probiotic
survival under simulated
gastrointestinal conditions.Better
results were found when inulin
was added to the encapsulating
system.
L. plantarum BL011 Alginate, low‐ Emulsion and Microcapsules (size not Yogurt Chitosan increased the stability of [116]
methoxyl citric coating specified) microorganism in the
pectin, and encapsulating systems added in
chitosan yogurt under cold storage.
Note: aHDL‐cholesterol: high‐density lipoprotein cholesterol; LDL‐cholesterol: low‐density lipoprotein cholesterol.
0004461251.INDD 337 10/26/2019 12:14:52 PM

are strongly toxic to humans and animals. These toxins can contaminate cereal grains and
other feedstuffs generating global concern [124]. When animals ingest contaminated feed,
mycotoxins are metabolized and transferred to food products, such as milk or meat, thereby
becoming a risk to human health [127]. There are more than 300 mycotoxins identified
worldwide, and the most monitored have been aflatoxin, ochratoxins, zearalenone,
trichothecenes, and fumonisins [124].
Some studies report the use of chitosan as an adsorbent to sequester mycotoxins.
Experimental data are frequently tested to fit Langmuir, Freundlich, and Hill models.
Quintela et al. [128] reported that chitosan could efficiently bind ochratoxin in wine and
improve wine safety. Zhao et al. [124] investigated the capability of chitosan salts and
cross‐linked chitosan beads for the adsorption of multiple mycotoxins including aflatoxin
B1 (AFB1), ochratoxin A (OTA), zearalenone, fumonisin B1 (FB1), deoxynivalenol
(DON), and T‐2 (T2) toxin. Among the tested adsorbents, cross‐linked chitosan–
glutaraldehyde complex presented the highest adsorption capability for AFB1 (73%),
OTA (97%), ZEN (94%), and FB1 (99%), but no obvious adsorption for DON and T2
(< 30%). Luo et al. [129] prepared a magnetic chitosan adsorbent combining Fe3O4 par-
ticles and chitosan using Triton X‐100 as an emulsifier and evaluated the effective
absorption of patulin, a mycotoxin produced by certain fungi on harvested fruits. These
authors reported patulin adsorption in kiwi fruit juice without any impact on the flavor
and quality of treated juice [129].
Besides mycotoxins, heavy metals are also of great concern for food safety. Lead (Pb) is
one of the most toxic heavy metals which is the main source of contamination for humans
being found in water and food. Abou El Fadl [130] studied the use of natural alginate and
modified alginate/chitosan beads synthesized by graft copolymerization with acrylic acid
by gamma radiation as sorbents. The graft copolymer showed a greater ability to absorb Pb
ions compared to ungrafted beads.
In addition to the removal of toxic compounds in foods, some studies have focused on
the use of chitosan as a chelating agent for compounds associated with food spoilage and/
or have nutritional or health concerns, and also as a technological adjunct. The International
Union of Pure and Applied Chemistry (IUPAC) provides a detailed description of chela-
tion: it involves the creation or even the presence of two or more separate coordinate
bonds between a polydentate (multiple bonded) ligand and a single central atom. In the
majority of cases, the ligands are organic‐based compounds (namely chelants, chelators,
chelating agents)[126]. Examples of chitosan as chelating agent are discussed in the
following text.
Since ferrous ions are the most effective prooxidants in the food system, the high ferrous
ion chelating abilities of chitosan would be beneficial if they were formulated into food.
Fungal chitosans were prepared by alkaline N‐deacetylation of crude chitin, which was
obtained from air‐dried shiitake stripes. At 1.0 mg/mL, chelating abilities of chitosan on
ferrous ions were superior to 85% [131]. Zhu et al. [132] evaluated the antimicrobial, anti-
oxidant, and darkening inhibitory activities of Maillard Reaction Products (MRPs) pre-
pared from chitosan and xylose, and their effects on the preservation and quality of
semi‐dried noodles. The chitosan–xylose mixture in this study showed better antimicrobial
and antioxidant activities than chitosan alone. Additionally, MRPs exhibited copper‐
chelating property and good inhibitory effect on polyphenol oxidase, which is recognized
as the main factor leading to the darkening of Chinese and Japanese noodles [132].
Lamas et al. [133] employed four different strategies to obtain egg‐derived products in form of
sticks with a reduced cholesterol content (use of β‐cyclodextrins, extraction of egg‐yolk granules,
bioconversion of cholesterol by cholesterol oxidase, and use of chitosan as a chelating agent).
Considering all the results obtained, egg‐derived yolk that was chelated by chitosan contained the
best balance out of the four methodologies employed, considering price, the ease of industrial
application, and the minor changes in color and consumer acceptability. The final reduction of
cholesterol content using this method was about 50%. Do Amaral [134] investigated the fat
absorption capacity of chitosan in low‐fat fresh pork sausage. The reduction of fat content to
levels of 5% was positively achieved with the incorporation of chitosan. The sensorial analysis
showed that panelists did not detect any significant difference in taste and unfavorable effect on
the sausage appearance as a consequence of chitosan addition and variation of fat [134].
Chitosan can also be used as an effective coagulating agent of suspended particles in
juices since the amino groups became protonated under acidic conditions, and chitosan
exhibits a typical behavior of a polyelectrolyte [135]. In this sense, clarification conditions
of apple juice were optimized using response surface methodology and also compared to
the traditional method of clarification during storage. Clarification of apple juice by using
chitosan (191.58 mg/100 mL) was suitable and easily applicable compared to the traditional
clarification method. Nevertheless, the high cost of chitosan may seem like a disadvantage
as compared to the traditional clarification agents [135].
13.8 Outlook
The development of applications for chitosan and its derivatives in the food area has been
extensively carried out in recent years, motivated by the investigation of new routes for
chitosan modification and preparation of blends, and also for the development of
nanotechnology. Although chitosan already exhibits widely recognized antimicrobial and
antioxidant properties, mainly due to the presence of reactive amino and hydroxyl groups,
these same groups have been the starting point for modification reactions, blending
processes, and obtaining complex structures with enhanced functional properties.
The encapsulation systems for bioactive compounds and active packaging have shown
great growth in recent years. Chitosan is a polymer that presents great compatibility with
several antimicrobial and antioxidant agents and can act as support for these compounds.
The application of nanotechnology to the production of chitosan nanocomposites is a very
promising area, which can solve some bottlenecks still present in packaging technology,
related to the mechanical and barrier properties of chitosan‐based films. In vitro assays for
evaluation of microbial or antioxidant properties or to test the efficiency of encapsulation
and delivering systems are very useful, but they need to be complemented with test
applications in food processing and under storage conditions. When applicable, delivering
through the gastrointestinal tract should be evaluated, as well as the bioactivity and
bioavailability of encapsulated compounds, even in simulated conditions.
The wide dissemination of chitosan applications by the food industry depends, in many
cases, on safety assessment and regulation. In addition, it is important to keep in mind that
the adoption of new technologies in substitution as conventional, needs besides the
comparable or better efficiency and environmental safety, also of economic viability. To
this end, the study and development of alternative routes and the optimization of processes
are increasingly necessary.
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14
Potential of Chitosans in the
Development of Edible
Food Packaging
Véronique Coma1 and Artur Bartkowiak2
University of Bordeaux, LCPO, UMR 5629, Centre National de la Recherche Scientifique (CNRS),
1
Pessac, France
2
Center of Bioimmobilisation and Innovative Packaging Materials, Faculty of Food Sciences and
Fisheries, West Pomeranian University of Technology, Szczecin, Poland
The development of bio‐based and biodegradable packaging materials is currently a crucial

challenge regarding environmental impact and human health issues. The majority of
materials used in the packaging industry are produced from fossil fuels and are non
degradable. For this reason, packaging materials represent a serious global environmental
problem [1]. Knowing that polysaccharides are the most abundant of the four major classes
of biomolecules, which also include proteins, lipids, and nucleic acids, the creation of
polysaccharide‐based materials is, thus, an active research area. In parallel, food safety has
been recognized by many national governments as a major social cost, threatening consumer
health, and creating trade barriers across the global food web [2]. Mild food processing
methods—processing technologies that apply a mild temperature (<40°C) to destroy
microbial food contaminants—have been developed. Thus, the development of antimicrobial
polysaccharide‐based edible coatings can be included in this field, in addition to physical
treatments such as high‐pressure processing, UV light, and so on. Although the use of films
and edible coatings in food quality preservation is not a new concept, research in this field
has recently been intensified. As a result, the increasing interest and research activity in

OH
NH2
O
O HO O
HO O
NH O
C
H3C O OH
y
n
Figure 14.1 Repeating units of partially acetylated chitosan characterized by an average degree of
acetylation.
edible bioactive packaging have been motivated by both increasing consumer demand for
safe, convenient, and stable foods and also awareness of the negative environmental impacts
of nonbiodegradable packaging waste. Chitosans, therefore, have potential in a large
domain of applications, especially due to their strong film‐ and gel‐forming properties.
Chitosan is a family of polysaccharides with many different structures. It is a high‐
molecular‐weight linear basic polycationic heteropolysaccharide, consisting of two mono
saccharides, N‐acetyl‐D‐glucosamine and D‐glucosamine, linked together by β (1→4)
glycosidic bonds (Figure 14.1).
Due to variable relative amounts of both monosaccharides, chitosans are not a uniquely
defined compound. This diversity in structure leads to diversity in biological activities.
Much of the commercial interest in chitosans and their derivatives arises from the fact that
they combine several favorable biological characteristics, including nontoxicity and poten
tial antimicrobial activity. Regarding the latter, numerous publications are now available
showing not only the wide range of chitosan activities toward human pathogens as well as
foodborne organisms but also the influence of different factors on this activity (i.e., molec
ular weight, deacetylation degree, pH). There is still a lack of knowledge about the mecha
nism of this antimicrobial action, but on bacteria, the site of action is more probably the
microbial cell surface, such as the majority of cationic antibacterial agents.
The use of chitosans in the formulation of edible films or coatings can, thus, lead to new
active food packaging materials. This is a modern strategy seeking to promote an additional
active role to assist in maintaining product quality. The focal point of this chapter is to
provide an overview of bioactive chitosan edible coatings that have the potential to enhance
the preservation of packaged products with a particular emphasis on (i) criteria limiting
their market introduction, (ii) studies based on real food applications and emerging new
technologies that can improve their future commercialization. Only non‐chemically‐
modified chitosans are targeted.
14.1 Potential Limitations for Real Introduction into the Market

Chitosans are really fascinating biopolymers, and their applications can be found in edible
packaging, and particularly in active materials, but some limitations exist due to the
polymer. Sustainable biopolymer‐based packaging materials with new and active
functionalities may help to introduce new products onto the market with a higher value
Potential of Chitosans in the Development of Edible Food Packaging 351
than traditional products. However, we have seen many scientific publications during the
last decades with no real market introduction. In this regard, the main aim of the current
Actinpack COST FP1405 (active and intelligent fiber‐based packaging—innovation and
market introduction) is to develop a knowledge‐based network around sustainable, active,
and intelligent bio‐based packaging in order to overcome current technological, industrial,
and social limitations that hinder the wide deployment of existing and newly developed
solutions in market applications. In the following text, only the limitations connected to
chitosans will be discussed.
14.1.1 Generally Recognized as Safe (GRAS)

To be accepted, an edible film should be generally recognized as safe (GRAS) and used
within any limitations specified by the U.S. Food and Drug Administration (FDA). Its oral
LD50 in mice was found to be in excess of 16 g/day/kg body weight, which is higher than
that of sucrose [3, 4]. Chitosans have not been officially proclaimed GRAS by the FDA but
as specified by Fouad (2008) [4], one Norwegian company (Primex Ingredients ASA),
which manufactures shrimp‐derived chitosans, announced in 2001 that its purified chitosan
product (ChitoClear) had achieved a self‐affirmed GRAS status in the US market. In any
case, to date, the use of chitosans is not allowed by the European regulation for organic
production.
14.1.2 Solubility
Chitosans have a great advantage over chitin regarding solubility but this is still a point that
limits its real application. Chitin is insoluble in most organic solvents, while chitosans are
readily soluble in dilute acidic solutions. The presence of the amino groups indicates that
pH substantially alters the charged state and properties of chitosans, and the soluble–
insoluble transition occurs at their pKa value around pH values between 6 and 6.5 ([5],
Figure 14.2). In practice, chitosan film‐forming solutions were prepared at low pH, in
OH OH
pH>PKa
O O O O
OH OH
O pH<PKa O
NH2 NH3+
Water solubility
Potential
antimicrobial
action
Figure 14.2 Impact of pH on the protonation of the amino group of the glucosamine monomer of
chitosans.
general close to 3–5, and even if an adjustment can be made before applying to the food
surface, the pH of edible chitosan solutions is generally close to 5. This insolubility at pH
higher than 6.5 could be a disadvantage for many industrial applications such as edible
coatings for pH‐sensitive food. For food coatings produced from unmodified chitosans,
different dilute acid solutions can be used and the frequent counter ions found in the
literature are acetate, lactate, citrate.
14.1.3 Source—Origin
The question is “Which chitosan is best for a specific application such as edible packag
ing?” Because a large amount of the crustacean exoskeleton is readily available as a by‐
product of the seafood processing industry, chitosans from crab shell or the fishing
industry are the most frequent one on the market. However, the marine origin of chi
tosans is sometimes considered as problematic. Chitosans are also found in nature, for
example, in the cell walls of fungi of Zygomycetes as well as in insect cuticles. Recent
advances in fermentation technology suggest that the cultivation of fungi (Aspergillus
niger) can provide an alternative source of chitosan. In addition, Greenaltech in Spain
has recently discovered the presence of natural chitosans in certain green microalgae
species (Derek Latil). The microalgal chitosans do not undergo any chemical
modifications.
One important point is that various chitosan sources can be responsible for structural
differences [6]. Some authors have shown that whereas the acetyl groups in chitosan
produced from crustacean chitin are uniformly distributed along the polymer chain, a
chitosan of similar degree of deacetylation (DD) isolated from fungal cell walls would
possess acetyl residues that are grouped into clusters [4, 7]. This variability in relation to
the source or origin of chitosans can be a key point for the industrial development of
chitosan‐based edible coatings and films.
14.1.4 Structure Variability
As already mentioned, chitosan structure may differ regarding not only the sources but
also the methods used to transform chitin into chitosans. Today, commercially available
chitosans are often produced from fungal strains or shrimp shell chitin. The main reason
for using the latter is the current context of waste by‐product valorization, particularly
waste from fish‐ and sea‐food factories. Variations in chemical structure could be due to
batch‐to‐batch differences, posing a problem of reproducibility. In addition, the end
product sometimes does not offer the required purity needed for food applications. The
problem is that in order to be able to explore the benefits of chitosans in depth, they
need to have a defined structure. One idea could be to start from materials readily avail
able from natural resources and then transform them through chemical reactions into
monomers designed to self‐condense in a well‐defined manner when reacting in the
presence of an engineered enzyme. In the same way, Dr. Moerschbacher’s team from
the University of Munster in Germany is working on the production of well‐defined
chitosans with known structures and functionalities through biotechnological
approaches. The tools required for this approach come from enzymes such as chitin
synthases, which produce chitin from small sugar molecules, and chitin deacetylases,
which convert chitin into chitosan. The recombinant enzymes were then characterized
and used for the biotechnological conversion of chitin into chitosans. However, and
perhaps interestingly, the chitosans obtained differ in their fine structure from all cur
rently available chitosans, which are invariably produced from chitin using chemical
methods. This could be the first step toward “third generation chitosans,” with poten
tially different biological activities.
In any event, chitosans from marine organisms are currently the most studied ones.
Despite the limitations raised previously, many scientific papers related to chitosan‐based
materials for food applications are published every year, showing that these biopolymers
have great potential and could be developed and used in the future because of some specific
properties discussed below.
14.2 Films and Coatings for Food Preservation

The need for biodegradable polymers for direct packaging, including edible films, has
fostered the development of novel, biodegradable polymeric materials from natural sources,
as an alternative to reduce the amount of waste and environmental impacts. Use of edible
film is a popular method, which has many functional properties such as protecting foods
from the adverse effects of the surrounding environment and carrying active ingredients.
14.2.1 Definitions and Interests

Although the terms “edible films” and “edible coatings” are sometimes referred to as syn
onymous, a distinction must be made between them. Films are preformed separately and
then applied to the food surface, whereas coatings are formed directly onto food surfaces
[8, 9]. In other words, a film is a thin skin, which has been formed, for example, by casting
a biopolymer solution separately from the food and then applying it to the food. By con
trast, a coating is a suspension or an emulsion, which is applied directly to the food surface,
and later becomes transformed into a film [10].
Important is that the formulation of films and coatings must contain at least one
component capable of forming a cohesive structural matrix [11]. In order to form a three‐
dimensional structural matrix, the key material, a polymer, is required. In addition, it is
well known that cationic chitosans possess good film‐forming properties after solubilization
in acidic conditions.
14.2.2 Main Relevant Chitosan‐Based Material Properties

14.2.2.1 Surface Properties/Adhesion Properties for Coatings
Chitosans exhibit great surface properties leading to good adhesion on the surface of
various food products. Thus, a uniform distribution of polar groups throughout the polymer
chain increases the ability of a coating material to form hydrogen bonds and participate in
ionic interactions [12, 13]. Adhesion between the final dried film and the food is extremely
important; a layer of the dried, solid film should attach to the food, particularly in surface
regions where there may be discontinuities. Wettability is then an important criterion, given
that optimum wettability requires the greatest possible area for contact between a solution
and the surface to be coated, thus, avoiding the disruption of air between the solution and
the surface. As discussed by Lin and Zhao (2007) [14] and Arnon‐Rips et al. (2018) [15],
the viscosity, density, and surface tension of the coating formulation should be adjusted to
the surface tension and roughness of a specific food product.
14.2.2.2 Possibility of Using the Layer‐by‐Layer Technique (LbL)

At its core, layer‐by‐layer (LbL) is a solution deposition technique in which layers of
cationic and anionic materials are built up via electrostatic attractions in an alternating
fashion. Due to their cationic charges after solubilization in dilute acid environments,
chitosans could be an excellent option. In the literature, when electrostatic interaction is
targeted, chitosans can be associated with anionic molecules such as alginate, pectin,
gelatin, or carboxymethyl cellulose. Progress in the development of active edible coatings,
by using LbL, has been summarized in a very recent review [15].
14.2.2.3 Mechanical and Barrier Properties

Good mechanical resistance can be a key point when applying edible coatings to brittle or
fragile foods, such as minimally processed fruits and vegetables. Poverenov et al. (2014)
[16] showed that a bilayer coating from a chitosan–alginate combination can slow down
tissue texture degradation in fresh‐cut melon. Chitosans have also been applied to maintain
the firmness of fresh‐cut papaya [17]. In general, the mechanical properties of coatings are
investigated on “model film templates” prepared by a solution casting method. Chitosan‐
derived materials generally exhibit poor mechanical properties in terms of strength and
deformation, especially for very thin matrices. In contrast, relatively high Young’s moduli
were obtained. For films prepared with 1–2% (w/v) chitosan, the tensile strength values
ranged from 22.2 to 79 MPa, with variations that could be due to several factors, including
chitosan composition, chitosan source, film preparation, and storage [18]. The addition of
plasticizers such as glycerol (the most commonly used) is one strategy followed to
overcome this problem [19, 20].
To know more precisely if an edible coating can maintain the integrity of the target food
because of its suitable mechanical properties, a study on real coated food is needed.
Unfortunately, real applications are not commonly found in scientific papers.
Because of their hydrophilic character, it is well known that chitosan films or coatings
have poor water barrier properties but relatively high oxygen barrier properties in dry
environments. The barrier properties of chitosans compared to other polysaccharide‐based
materials were proposed in a recent review by Yousuf et al. (2017) [21]. Although film
properties are frequently studied, edible coating properties are not, despite this criterion
being of great interest for edible coatings. This is due to technical problems in characterizing
thin films, and we can generally have an idea of such properties by comparing coated food
to an uncoated one directly. To improve the barrier properties of chitosan coatings, the most
frequent strategies are based on the use of additives, while others look at the process to
create the coatings. The use of lower drying temperatures, for example, can have a positive
effect on the water vapor permeability (WVP) of chitosan films, while the use of higher
drying temperatures (40°C) can enhance some mechanical properties such as the tensile
strength [22].
14.2.2.4 Inherent Antimicrobial Properties

One of the most interesting properties of chitosans for edible coatings is their antimicrobial
activities on both fungal and bacterial strains. A recent review of the antimicrobial activity
of chitosans has now been published [23]. They can prevent bacteria from growing and
then can be used in food to prevent food spoilage.
Regarding antimicrobial properties, two main categories of antimicrobial matrices
potentially used in edible packaging applications have to be considered (Figure 14.3).
These are based on the following processes [24]:
i. Utilization of polymers, which are chemically modified in order to produce bioactive
properties or use inherently antimicrobial polymers exhibiting film‐forming properties
such as chitosans.
ii. Direct incorporation of the antimicrobial agent into an edible coating, which acts as a
carrier for the antimicrobial agent. In this category, the active agent can generally
release the antimicrobial agents onto the surface upon which antimicrobial action is
needed. The system is more efficient than a direct application of the antimicrobial
agent onto selected surfaces, because it slows the migration of the agents away from
the surface, and thus, helps to maintain high concentrations where they are needed.
As a result, chitosans can be used as an inherent active polymer and also as a carrier for
other active agents previously incorporated into edible chitosan‐based coatings.
There is still a great controversy regarding the phenomenology and mechanisms of the
antimicrobial properties of chitosans and their derivatives (Figure 14.4). Even if the
mechanism is not yet fully understood, it is known that it involves cell lysis, breakdown of
the cytoplasmic membrane barrier, and selective chelation of trace metal cations that could
be necessary for microorganism growth. Raafat et al. (2008) [25] showed that there might
not be a single classical target that would explain chitosan antimicrobial action. After
studying the impact of chitosans on Staphylococcus aureus, they speculated that binding
chitosans to teichoic acids, coupled with a potential extraction of membrane lipids
(predominantly lipoteichoic acid), results in a sequence of events, ultimately leading to
bacterial death.
Chitosan as
intrinsically active
polymer
Food
Edible
coating
Chitosan as a
carrier of
bioactive agents
Bioactive agent Desorption, and
or active diffusion
functional groups
Figure 14.3 Concepts of antimicrobial coatings for food preservation.

Direct antimicrobial mechanisms

NH2
HO O
O O
OH y
Adsorption to negatively
Protonation charged cell membrane
Decrease in osmotic stability
pH < PKa of the cell
Leakage of intracellular
constituants
Indirect antimicrobial
mechanisms Protonation Formation of an impermeable
coat on the microbial surface
pH < PKa
Interaction with exoenzymes
Low molecular Diffusion through the cell wall

Interaction with substances
Chelation of trace metals in the cell, interaction
weight fragments
with DNA
Sensitization of
microorganisms
to antimicrobial agents
Figure 14.4 Suggested mechanisms of antimicrobial activity of chitosans.
14.2.2.5 Carrier Properties
The incorporation of additives or extracts from natural sources into edible chitosan films
and coatings is one of the methods being explored to increase the shelf life of foods and
then to provide high‐quality products (fresh/safe). Chitosans can be used as a simple carrier
of various molecules such as antimicrobials, antioxidants, vitamins, flavors, and prebiotics
[26–29]. Interestingly, the use of chitosan as a carrier can lead to adequately dispersed
agents. Chitosans can increase the solubility of some hydrophobic molecules in water‐
based formulations.
Regarding studies of the release of active agents, the process responsible for mass
transfer within a system is diffusion, and diffusion coefficients have a much wider range of
values in solids compared to liquids and gases, differing by a factor of more than 1010 as
specified by Flores‐Martinez [30]. When an additive is expected to diffuse through a
material, this corresponds to diffusion in a solid system. A fairly new concept in interactive
packaging is to control this release of additives [31]. Diffusion in polymers is relatively
complex and depends strongly on the concentration and degree of polymer swelling. This
degree of swelling also depends on the free volume in the materials, that is, space not
occupied by polymeric chains or molecule segments. An increase in the amount of free
volume leads to an increased diffusion rate. As a result, external factors such as temperature
greatly affect the amount of free volume (Flores‐Martinez et al. 2015) [30]. As a result, in
terms of practical importance, active agent diffusivity values are essential in predicting the
shelf life of coated food products under changing storage conditions. Regarding the
literature, there is clearly a lack of studies on the diffusivity of active agents. A better
understanding of the mechanism of preservative transport in chitosan‐based edible films is,
thus, needed.
Direct incorporation into the film‐forming solution is used in numerous studies. However,
because of some disadvantages with such blends, other studies are considering the
development of multilayer systems, leading to more sophisticated processes. According to
Suppalkul (2015) [32] and Han (1996) [33], a multilayer structure for bioactive‐release
packaging systems can be organized comprising an outer bioactive barrier layer (a barrier
layer to prevent the loss of bioactive agents), an active‐containing matrix layer (provides a
very rapid diffusion rate of the bioactive agent), a release‐control layer (controls the initial
lag period and the subsequent flux of the substance), and food. Chitosans could be good
options to create this multilayer design, which has the advantage of the control layer to alter
the diffusivity of bioactive substances.
14.3 Specific Case of Chitosan Nanoparticles (CSNPs)

14.3.1 CSNPs
Nanoscience is an emerging area of science that has the potential to generate radical new
products and processes. Nanotechnology deals with the application, production, and
processing of materials with sizes less than 1000 nm. Advances in processes of production
of nanostructured materials coupled with appropriate formulation strategies have made
possible the production and stabilization of NPs that have potential applications in the food
and related industries.
Relatively poor mechanical and water vapor barrier properties of edible films result in a
major limitation of their industrial use. Over recent years, in order to overcome these
drawbacks of biodegradable films, different types of nanofillers have been introduced. By
adding appropriate NPs, it is possible to produce materials with improved mechanical
reinforcement, higher thermal stability and barrier properties, and lower moisture sensitivity
[34]. Moreover, implementation of edible film and nanoencapsulation technologies for the
incorporation of various active and functional materials into a food product has been, in
recent years, the main topic of many studies. Immobilized active compounds such as
antioxidant substances, flavors, antimicrobial agents, and texture enhancers can be
incorporated into the formulations of edible films and coatings in order to increase the shelf
life of foods [35, 36].
Polymeric NPs may be produced with different polymers, including poly (ε‐caprolactone)
(PCL), carrageenan, chitosan, polylactic acid, gelatin, polyglycolic acid, alginate, and zein
[37, 38].
Chitosan microparticles and NPs can be prepared by various methods (Table 14.1) such
as emulsion cross‐linking, coacervation/precipitation, spray‐drying, ionic gelation in TPP
using standard precipitation and membrane process, combined spray‐drying and ionic
gelation, and by use of a reverse micellar and sieving method [39, 40].
The most common systems are chitosan–TPP NPs, which have been synthesized and
mainly used as drug carrier as reported for more than a decade in many studies [41, 42].
Parameters such as TPP concentration, pH, and chitosan concentration and molar mass
Table 14.1 Methods for chitosan nanoparticle formation.
No. Method Additional substances References

1. Emulsification solvent diffusion Vanillin as cross‐linker; formethylene [48]
chloride/acetone
2. Coacervation/precipitation Sodium hydroxide (NaOH), methanol, or [40]
ethanediamine
3. Spray‐drying ‐ [49]
4. Ionic gelation in tripolyphosphate (TPP) TPP [50]
using standard precipitation
5. Ionic gelation in TPP using membrane TPP [51]
process
6. Combined spray‐drying and ionic TPP [52]
gelation
7. Reverse micellar and sieving method Surfactant (sodium bis(ethylhexyl) [53]
sulfosuccinate), n‐hexane, Tris–HCl buffer
were found to significantly affect the formation of the NPs. It was postulated [43] that the
pH of TPP affects the electronegative potential of the molecule in its reaction with free
amine groups in chitosan. At lower pH values, TPP becomes less reactive for chemical
interactions with chitosan because it is buffered by more positive ions in solution (H3O+ and
H+). TPP, therefore, reacts with fewer amino groups of chitosan (−NH3+), leading to the
formation of smaller‐sized NPs that are more monodisperse. The interactive effect of
molecular weight (MW) and pH on the formation of CSNPs showed that optimal particle
size was obtained when low‐MW CS at a pH of 4.6 was used [44].
Due to their polymeric cationic characteristics, CSNPs can interact with negatively
charged molecules and polymers. CSNPs have been successfully used as fillers to improve
mechanical and barrier properties as well as the thermo‐stability of films, decrease solubility
and produce more compact and dense materials [45–47].
The use of NPs in food packaging has been proposed on the basis that it could improve
the protection of foods by reducing the permeation of gases, minimizing odor loss, and
increasing mechanical strength and thermal stability. The typical process of edible
nanocomposite film formation is divided into two stages: the first is the synthesis of CSNPs,
and the second is the production of the films containing NPs.
14.3.2 CSNPs in Various Edible Films

CSNPs have been incorporated in various eatable film materials such as methylcellulose,
hydroxypropyl methylcellulose, chitosan, gelatin, tara gum (TG), and pectin. De Moura
et al. (2008) [46] described the effects of addition that NPs made, using chitosan and
poly(methacrylic acid) (PMAA), on the mechanical properties, water vapor, and oxygen
permeability of hydroxypropyl methylcellulose films used in food packaging.
The mechanical, thermal, and water barrier properties of edible films based on
hydroxypropyl methylcellulose (HPMC) with papaya and guava [54, 55] purees were
improved by CSNPs. It was observed that when the NPs were added to HPMC and guava
puree films, they decreased the films’ solubility and permeability. The films prepared with
guava puree, HPMC, and NPs are easy to handle and visibly homogeneous, in addition to
the fact that the color and aroma of the films remained for about a year.
Ultrasound‐assisted preparation of size‐controlled CSNPs plus characterization and fab

rication of transparent chitosan biofilm were described by Souza et al. (2013) [56].
Irrespective of the degree of DD, the application of longer sonication times reduced the
viscoelasticity of the solutions and yielded NPs with lower size (and molecular mass). The
results showed a significant decrease in the permeability with decreasing the molecular
mass. However, the mechanical properties were adversely affected. These findings may not
only be useful for the future design of bioplastics with improved properties, but also for the
development of biocompatible NPs with tunable size and molecular mass.
Bio‐nanocomposite films based on fish gelatin (FG) and chitosan/sodium–TPP NPs
have been successfully developed [57]. The particles were spherical in shape with a size
range of 40–80 nm and were evenly dispersed in the FG matrix at lower‐loading levels.
They improved FG film’s water vapor barrier as well as tensile strength and elastic
modulus.
Glycerol‐plasticized TG films were successfully produced with the inclusion of bulk
chitosan or CSNPs at various concentrations [58]. CSNPs improved mechanical,
physicochemical, and barrier properties. Tensile strength was increased by 35.73 MPa,
while the elongation was decreased by 7.21%. Water solubility and WVP were reduced by
74.3 and 22.7%, respectively. The compact structure of the CSNPs reduced the free volume
of the polymer matrix more than bulk chitosan by obstructing the diffusion of water and,
thereby, decreasing the moisture content of the films. CSNPs have been reported to
reinforce banana‐based edible films [59]. Nanocomposite pectin films, comprising two
pectin types (high‐methyl (HDM) and low‐methyl (LDM)), were successfully produced as
homogeneous, compact, and continuous CSNPs based materials [60]. The addition of
CSNPs improved the mechanical properties, being tensile strength the most affected
mechanical attribute (increased from 30.81 to 46.95 MPa and from 26.07 to 58.51 MPa for
HDM pectin/CSNP and LDM pectin nanocomposite film, respectively). Water permeability
was affected only in the LDM pectin films, mainly due to larger interaction and solubility
in water of the pectin chains, since pectin is consisted of carbohydrate molecules having
greater interactions with water molecules. These results show that the produced pectin
nanocomposites had improved mechanical properties when compared with control pectin
films, making these novel materials promising for food packaging production.
14.3.3 Antimicrobial Activities of CSNPs in Edible Films

The broad antimicrobial properties of CSNPs have been reported and has been attributed to
their high surface area and charge density [61].
Paomephan et al. (2018) [62] investigated the influence of MW and particle size
characteristics on the antibacterial property of CSNPs for application as a vegetable wash
disinfectant. A time‐dependent antibacterial assay against Escherichia coli was used as a
model and showed that CSNPs with smaller size produced from either low‐ or high‐MW
chitosan were effective antibacterial agents, leading to an approximate 2 log reduction in
the number of bacteria within 12 h. Once demonstrated to have good antibacterial activity,
all CSNPs were formulated as vegetable wash disinfectants in citric acid and evaluated
using an in vitro inactivation assay with E. coli and a pathogenic bacterium (Salmonella
typhimurium), known to be possible contaminants on fresh vegetables. The wash solution
containing CSNPs was found to be the most effective in killing more than a 1 log reduction
within 15 min using of both inoculated E. coli and S. typhimurium populations, suggesting
their potential use as an effective disinfectant in washing fresh vegetables.
Incorporation of CSNPs produced TG films less effective against E. coli compared to S.
aureus than films with bulk chitosan, and their antimicrobial activity was reduced at high
concentrations of CSNPs probably due to agglomeration [58]. It has been shown that
antibacterial activity of CSNPs could be significantly enhanced by metal ion loaded,
especially for Cu2+, and Zn2+, compared to those of CSNPs and related metal ions, where
Gram‐negative bacteria were more sensitive than Gram‐positive bacteria. It was found that
antibacterial activity was directly proportional to the zeta potential [63].
New active packaging films based on chitosan printed with chitosan–TPP NPs loaded
with thymol using inkjet printing were evaluated by Caro et al. (2016) [64]. The printed
films had enhanced barrier properties compared to the control. The efficiency of thymol
incorporation by inkjet printing was dependent on the number of printed layers, the contact
angle, the amount of glycerol added to the dispersion, and the film type. The printed films
exhibited higher antimicrobial activity against Listeria innocua, S. aureus, S. typhimurium,
Enterobacter aerogenes, Pseudomonas aeruginosa, and E. coli than both non‐printed and
control films.
Recently, several chitosan/sodium–TPP NPs embedded with Torreya grandis aril
essential oils (TEOs) were synthesized using an emulsion‐ionic gelation technique and in
order to replace acetic acid, an ionic liquid (IL) was employed to dissolve chitosan [65].
The obtained results revealed that TEO‐loaded NPs synthesized in acid and IL–aqueous
systems have stronger antibacterial activities against S. aureus than CSNPs alone.
14.3.4 Toxicity Studies of CSNPs

The considerable potential for use of chitosan particles in the area of edible films needs
assessment of their toxicity and any possible risks they may pose to human health and the
environment.
The genotoxicity of different polymeric CS/PMAA NPs synthesized by polymeriza
tion of methacrylic acid in chitosan solution (size in range 60–111 nm) was evaluated at
different concentration levels [66], using the Allium cepa chromosome damage test as
well as cytogenetic tests employing human lymphocyte cultures. Results showed no evi
dence of DNA damage caused by the NPs; however, the 82 and 111 nm NPs reduced
mitotic index values at the highest concentration tested (180 mg/L), indicating that the
NPs were toxic to the cells used at this relatively high concentration. In the case of the
60 nm CS/PMAA NPs, no significant changes in the mitotic index were observed at all
concentration levels tested, indicating that these particles were not toxic. The obtained
results suggest that smaller particles can be used safely at higher concentrations than
large particles.
14.4 Applications to Sensitive Real Food Products

Knowledge of surface properties is essential for understanding film adhesion and optimiz
ing performance characteristics such as water permeability. Good surface wettability does
not always correspond to good adhesion because wettability is necessary but not sufficient
for proper adhesion [13].
14.4.1 Fruits and Vegetables

A lot of work has been done on studies connected to the preservation of very sensitive fruits
such as strawberries, because postharvest rot is still a major problem in such fruit production,
leading to low stability under commercial conditions. In addition, due to this high sensitivity
and degradation rates, strawberries are the perfect food model for scientists trying to
develop active coatings, because its short shelf life is compatible with the majority of
scientific projects. For example, to extend the shelf life of fresh strawberry, Duran et al.
(2016) [67] studied a chitosan coating associated with nisin, natamycin, pomegranate and
grape seed extracts. By using this coating, they showed significant improvement in the
stability of pH, total soluble solid content and texture of fruits, and regarding the
development of aerobic microbial strains. Perdones et al. (2016) [68] used chitosan–lemon
essential oils as strawberry coatings. They showed that coatings affected the metabolic
pathways and volatile profile of the fruits. Interestingly, no effect of chitosan coatings was
perceived sensorially, and the addition of lemon oil led to effective coatings able to control
fruit fungal decay at 20°C for 7 days. According to Valenzuela et al. (2015) [69], chitosans
can be combined with quinoa protein to create an edible coating, thus, improving the global
quality of strawberries by prolonging the storage period for 7 days at a temperature of 20°C
and relative humidity of 53%, and slowing down their senescence process compared to
uncoated strawberries. In addition, the addition of beeswax as a separate layer or as a
component in the composite coating showed a beneficial effect.
Xu et al. (2018) [70] tried to improve the shelf life of tangerine fruits by chitosan‐based
coatings. The results demonstrated that combining chitosans with nanoclay (montmorillonite)
clearly enhanced stability and provided a longer shelf life. Cinnamaldehyde–chitosan
coating could also be used to extend the storage time and maintain the quality of citrus
fruit. Gao et al. (2018) [71] observed that this association significantly reduced the decay
rate, weight loss, and global senescence of navel orange fruits during 120 days of storage
at 10°C and 80–90% relative humidity. Interestingly, Mannozzi et al. (2018) [72] created
an edible coating based on chitosan from mushrooms enriched with procyanidins extracted
from grape seeds. They demonstrated the efficacy of such a coating to maintain the overall
quality of fresh blueberries during 14 days of storage at 4°C.
The effects of chitosan‐based NPs on microbiological quality, color, polyphenol oxidase
(PPO) and peroxidase activity (POD) and firmness of fresh‐cut “Gala” apple slices during
storage at 5°C for 10 days were elaborated by Pilon et al. (2015) [73]. There was an increase
in chroma and a proportional decrease in hue angle and lightness. Browning of the slices
coated with conventional chitosan and control was slightly intense than those coated with
CSNPs of 110 and 300 nm. The PPO and PDO activities increased with time for all samples,
with irrelevant difference among the treatments. Flesh firmness did not change for any
treatment and period. Coatings with CSNPs of 110 nm showed higher antimicrobial activity
against molds, yeasts, and mesophilic and psychrotrophic bacteria than the other treatments.
No Salmonella, and total and fecal coliforms were detected. This investigation supports the
potential use of CSNPs as edible coatings in controlling microbial activity in fresh‐cut
apples.
In the study by Lustriane et al. (2018) [74], the effect of different concentrations of
chitosan and CSNPs as an edible coating in extending shelf life and maintaining the quality
of banana fruits (Musa acuminata AAA group) was evaluated. The fruit treated with 1.15%
(w/v) chitosan, 1.25% (w/v) chitosan, and CSNPs were stored at ambient temperature
(25 ± 1°C). The shelf life of banana, starch content, weight loss, pulp to peel ratio, total
soluble solid, surface morphology of banana peel, and sensory evaluation were analyzed.
Molecular analysis on the effect of chitosan was also conducted. Based on the results of
Lustriane et al. (2018) [74], it was confirmed that CSNPs and chitosan can provide good
effects on postharvest quality of banana including shelf life, starch content, weight loss,
pulp to peel ratio, total soluble solids, and sensory quality.
14.4.2 Meat and Meat Products

Another food product frequently used as a target food in research studies is meat. Recent
reviews regarding the packaging concepts for meat are now available [75, 76]. By applying
a chitosan film infused with essential oils extracted from thyme, Quesada et al. (2016) [77]
showed a good control of the growth of yeasts in a refrigerated ready‐to‐eat meat product.
They also observed reduced water condensation inside the pack. Essential oils or extracts
are, thus, frequently associated with chitosan to improve antimicrobial and antioxidant
properties of the edible coating potentially used for meat preservation [78–81]. A 2% (w/v)
chitosan solution coating pork loins showed antioxidant and antimicrobial properties, but
the addition of 0.2% (w/v) gallic acid further enhanced these properties under high‐oxygen‐
modified atmosphere packaging storage at 4°C [78]. Vargas et al. (2011) [82] developed
chitosan coatings combining a high‐molecular‐weight aminopolysaccharide with sunflower
oil for pork meat used in hamburgers. These authors mentioned that it could, however, be
necessary to modulate the oxygen permeability of films in such applications to complete
the food protection. As a result, chitosan‐based coatings could be very useful in decreasing
the development of spoilage and pathogen strains. It is also worth noting, as specified and
studied by Alnoman et al. (2017) [83], that the food industry is really looking for novel
bacterial spore‐inactivation strategies, in which chitosan coatings could also be effective.
In the study by Ozvural and Huang (2018) [84], different formulations of chitosan and
chitosan/sodium–TPP matrix solutions including β‐carotene were used as additives and
edible coatings in hamburger patties, and the treatments were compared to control in terms
of quality, oxidative and microbiological features of the patties. The comparison of different
treatments including the solutions of chitosan or chitosan/sodium–TPP matrix containing
β‐carotene showed that incorporation of the solution as an edible coating was more effective
in lipid oxidation and microbial growth than its utilization as an additive on quality of
patties according to the results on the last day of storage.
14.4.3 Fish and Seafood Products

It is well documented that the quality preservation and shelf life of Atlantic cod, herring
fresh fillets, rainbow trout, oysters, shrimp, salmon, and sea bass can be improved by using
chitosan as edible films or coatings.
The effects of chitosan coating on the quality and shelf life of silver carp during
refrigerated storage has been investigated [85]. In a study, 3% (w/v) chitosan solutions
incorporating 10% fish oil (w/w chitosan, containing 91.2% EPA and DHA), with or
without the addition of 0.8% vitamin E, were used as coating solution of fresh lingcod
(Ophiodon elongates) fillets and vacuum‐impregnated at 100 mmHg for 10 min followed
by atmospheric restoration for 15 min [86]. The chitosan–fish oil coating reduced the lipid
oxidation process in both fresh and frozen samples, and also decreased the drip loss of
frozen samples by 14.1–27.6%. Chitosan coatings resulted in 0.37–1.19 and 0.27–
1.55 log CFU/g reductions in total plate and psychrotrophic counts in cold‐stored and
frozen‐stored samples, respectively. The positive effect of chitosan coating on the shelf life
extension of salmon (Salmo salar) fillets during storage at 0°C was demonstrated by Souza
et al. (2010b) [87].
The results of Ojagh et al. (2010) [88] indicated that the chitosan coating enriched with
cinnamon oil enabled the good quality characteristics of rainbow trout (Oncorhynchus
mykiss) to be retained longer and helped extend the shelf life during refrigerated storage.
The possibility of improving the shelf life of fresh sea bass (Dicentrarchus labrax) fillets
by using vacuum packaging and wrapping with chitosan‐based edible films during cold
storage at 4°C was assessed by Günlü and Koyun (2013) [89]. The results showed that the
shelf life of the control and vacuum‐packaged groups ended within 5 days, whereas that of
vacuum‐packaged, chitosan‐film‐wrapped samples ended at 25–30 days. Therefore, the
shelf life of sea bass fillets wrapped in chitosan was prolonged by about 20 days.
The potential of CSNPs produced via ionotropic gelation with sodium TPP as a glazing
material for shrimp was investigated by Solval et al. (2014) [90]. Glazing containing
CSNPs reduced the lipid oxidation in shrimp during frozen storage at −20°C. CSNP glazing
had not affected the weight of frozen shrimp as compared to the controls. In addition,
CSNPs reduced total aerobic counts, yeast, and molds without affecting the color and
texture properties of frozen shrimp during 30 days of storage at −20°C.
Recent results of Wang et al. (2015) [91] demonstrated that CSNP coating exhibited a
better effect on the quality maintenance of shrimp as compared to carboxymethyl–chitosan
coating. On day 10 of storage at 4°C, the highest hardness and springiness and lowest total
viable counts of microorganisms were obtained in case of coating with CSNP and followed
by carboxymethyl–chitosan coating. Lower thiobarbituric acid and higher inosinic acid
content were obtained in NP‐coated sample than those of the control on day 10. Similarly,
Wang and Li (2011) [92] proved that CSNPs also improved fat composition and inosinic
acid content of tilapia (Oreochromis niloticus) as compared to normal chitosan materials.
A chitosan/sodium TPP NP solution was developed and applied to shrimp through
vacuum tumbling, and the quality characteristics during frozen storage were evaluated
[93]. Chitosan and CSNP treatments could preserve the color, texture, and moisture content
of shrimp throughout 120 days of frozen storage at −20°C. In addition, chitosan‐ and
CSNP‐treated shrimp had the highest reduction in lipid oxidation as compared to other
treatments at 120 days of storage at −20°C. This study showed that vacuum tumbling with
a chitosan or CSNP solution can be effective at reducing aerobic plate counts and lipid
oxidation in shrimp during frozen storage while maintaining desired physicochemical
properties.
The responsive intelligent film based on poly(vinyl alcohol) (PVA), CSNPs, and
mulberry extracts (MBEs) were successfully prepared and characterized by Ma et al.
(2018) [94]. CSNPs were prepared by using ionotropic with sodium TPP gelation method
to enhance the mechanical properties of PVA‐based films. The film with 6% (w/v) CSNPs
had the highest tensile strength (∼73.43 MPa). The film containing 20% (w/w) MBE had
the highest tensile strength and showed visible color responses to variations across pH
1–13. This film changed from red to green during the process of fish spoilage due to the
change of pH, indicating that it can detect quality changes in fish. Hence, CSNP coating
might represent a potentially efficient and beneficial natural preservative and freshness
indicator for seafood quality, although the further safety evaluation and elucidation of
biological mechanisms are still required for the transition from laboratory systems to
commercial applications in the seafood industry.
14.5 Conclusions
Although chitosans are bio‐based polymers, their environmental impact must be investigated
further, from the extraction of raw materials to the finished product. One point to keep in
mind is that applied chemical methods to produce chitosans from animal chitin require
large amounts of chemicals and produce a lot of wastewater, which has a harsh impact on
the environment. Life cycle assessments have to be conducted more regularly, especially
with the data from the real chitosan producers.
As a conclusion, taking into account that one of the greatest challenges of the food
industry is microbial contamination, chitosan‐based materials including NPs will certainly
be developed considerably during the next decade.
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15
The Use of Chitosan‐Based
Nanoformulations for Controlling
Fungi During Storage of
Horticultural Commodities
Silvia Bautista‐Baños1, Zormy Nacary Correa‐Pacheco2,
and Rosa Isela Ventura‐Aguilar2
1
Centro de Desarrollo de Productos Bióticos (CEPROBI), Instituto Politécnico Nacional (IPN),
2
CONACYT-CEPROBI, Instituto Politécnico Nacional, Yautepec, Morelos, Mexico
Biodegradable antimicrobial compounds, such as chitosan, integrated with essential oils,

plant extracts, and propolis, have been incorporated into a diverse range of formulations.
Likewise, there are various microorganisms that cause serious economic damage,
principally during storage of horticultural commodities. Coatings have become increasingly
more important as a means to deliver fresh horticultural commodities to the consumer, in
safe conditions. Furthermore, studies have confirmed that their applications on agricultural
products have a remarkable potential to inhibit microorganisms as compared to the uncoated
products. In addition, generally there have been no reports of adverse or side effects on the
ripening process of the horticultural products. Currently, the application of nanotechnology
has shown great benefits in different fields, such as medicine, energy, the environment, and
food. In the latter, the study of food conservation and control of microorganisms that cause
decay and disease, through the designing of new materials (nanostructured coatings, among
others) has been noteworthy. Regarding this subject, the research group of the Postharvest

Technology Laboratory of Agricultural Products of the Center for the Development of

Biotic Products (CEPROBI‐IPN) has considered the design and application of nanomateri-
als as the main theme of their research, based on nontoxic and naturally occurring com-
pounds, focusing on the control of the principal postharvest microorganisms.
15.1 Introduction
Interest in the use of chitosan has increased significantly in comparison to synthetic fun-
gicides. Chitosan can be considered a bioactive natural product, since it possesses impor-
tant attributes such as antimicrobial, low‐toxicity, biocompatibility, and biodegradability
characteristics. Chitosan (poly‐D‐glucosamine) is provided by an extensive deacetylation
of the polymer chitin (poly‐N‐acetyl‐glucosamine) and can be found in fungal cell walls
of Zygomycetes, some insects and algae, and from various marine origin exoskeletons,
including crabs, shrimps, and shells, among others [1]. This biopolymer provides a wide
range of biological functions due to its cationic characteristics (free amino groups present
in its structure). Its interactions with negatively charged structures like those of patho-
genic fungal cell walls are of great interest. Such interactions provide a fungicidal activity
of paramount importance for controlling many species of microorganisms [2, 3]. In addi-
tion, there is plenty of evidence that chitosan is a reliable elicitor, inducing resistance
mechanisms through a systemic induced resistance (SAR) in the affected horticultural
commodity ([4]).
Chitosan can be dissolved in diluted aqueous acidic solvents allowing the formation of
structures, such as edible films and coatings, that combined with some natural antimicrobials
(essential oils and plant extracts, among others) and one or two bioactive products result in
active films/coatings that protect the horticultural commodity against fast moisture and
oxygen losses and from external environmental factors, including temperature, relative
humidity, heat, and microorganisms [5, 6]. At the present time, the application of
nanotechnology has shown great benefits in different fields, such as medicine, energy, the
environment, and food preservation. In the latter, the study of food conservation and control
of microorganisms that cause decay and disease during the postharvest stage through the
designing of new materials (nanostructured coatings) has been noteworthy [6, 7]. The
application of nanotechnology using nanostructured coatings on agricultural products
based on botanical and animal derivatives, such as chitosan, has a remarkable potential to
inhibit microorganisms as compared to the uncoated products. Its application has no
adverse effects on the ripening process of the product [8].
This chapter discusses the current studies and future research on chitosan coating,
formulated alone or with new materials, including nanoemulsions, for preserving fresh
fruits and vegetables during storage, emphasising their effects on the fruit quality and the
control of different microorganisms.
15.2 Importance of Fruits and Vegetables

Fresh fruits and vegetables serve as key sources of nutrition for many human inhabitants
of the world, and can prevent chronic and serious diseases [9]. In several areas, fruit crops
are the main dietary components and serve as a clean source of essential ingredients [10].
According to Kader [11] fruit, nuts, and vegetables contribute about 91% of vitamin C,
48% of vitamin A, 27% of vitamin B6, 17% of thiamine, and 15% of niacin. They also
The Use of Chitosan-Based Nanoformulations for Controlling Fungi During Storage 373
Table 15.1 Response of various fungal pathogens causing plant and fruit diseases to chitosan
application.
Stage of fungal
Fungal disease/microorganism development affected Range of tested doses References
Black rot, leaf, and fruit spot/Alternaria Mycelium, sporulation, 0.1–2.5% (w/v) [27–29]
alternata conidial germination
Aspergillus ear, kernel rot/Aspergillus Mycelium 0.1–5.0 g L−1 [30]
flavus
Black mould/Aspergillus niger Mycelium, conidia [31]
Brown spot/Bipolaris oryzae Germ tube, conidial 300–1000 mg L−1 [32]
germination
Gray mould/Botrytis cinerea Mycelium 0.75–6.0 mg mL−1; [17]
0.01–1.0% (w/v) [33]
Root rot/Cylindrocarpon destructans/; Mycelium 0.5–20 mg mL−1 [34]
damping‐off, root rot, foliage
blight/Cylindrocladium floridanum
Scab of cucurbits/Cladosporium Mycelium 50–1000 μg mL−1 [35]
cucumerinum
Anthracnose/Colletotrichum Mycelium, conidial 0.10–2.5% (w/v) [23, 24, 36]
gloeosporioides sporulation,
germination
Anthracnose/Colletotrichum musae Mycelium 10–2.0 % (w/v) [26]
Damping off/Fusarium acuminatum Mycelium 0.5–2.0 mg mL−1 [34]
Vascular wilt, Fusarium wilt/Fusarium Mycelium 0.5–2.0 mg mL−1; [34]
oxysporum 50–1000 μg mL−1; [35] [37]
0.30–4.5 g L−1
Fusarium wilt/Fusarium oxysporum f.sp. Mycelium, spore 0.6 mg mL−1; 0.5–2.0 [38, 39]
radicis‐lycopersici germination mg mL−1
Root and fruit rot/Fusarium solani Mycelium, sporulation, 1–5 g L−1; 0.05–0.20% [40, 41]
spore germination (w/v)
Ear rot, kernel rot/Fusarium verticillioides Mycelium 1.0–4.0 g L−1 [42]
Take all/Gaeumannomyces graminis var. trici Mycelium 0.5–2.0 mg mL−1 [39]
Basal canker/Leptographium procerum Mycelium 0.05 and 0.075% [43]
Damping off, seedling blight, and root Mycelium 1–5 g L−1 [40]
rot/Macrophomina phaseolina
Brown rot/Monilinia fructicola Mycelium 50–1000 μg mL−1 [35]
Green mould/Penicillium digitatum Mycelium 0.5–3.0% (w/v) [44]
Damping off, basal rot/Pythium sp. Mycelium 0.5–2.0% (w/v) [45]
Damping off, root rot/Pythium ultimum Mycelium 0.5–2.0% (w/v); [39, 45]
0.5–2.0 mg mL−1
Root rot, late blight/Phythophthora Mycelium 0.1–2.0 mg mL−1 [46]
botryosa
Rice blast/Pyricularia grisea Mycelium 5 g L−1 [47]
Collar rot, root rot, wire stem/Rhizoctonia Mycelium 1–5 g L−1 [40]
solani
Soft rot/Rhizopus stolonifer Mycelium 0.75–6.0 mg mL−1; [17] [28]
100–1000 μg L−1; [48]
2–6 g L−1
White mould/Sclerotinia sclerotiorum Mycelium 1–5 g L−1 [40]
Maize head smut, sorghum head Mycelium, sporidia, 10–10 000 μg L−1 [49]
smut/Sphacelotheca reiliana teliospore
Anthracnose, scab /Sphaceloma pelinum Mycelium 1000–100 000 ppm [50]
Diplodia tip blight/Sphaeropsis sapinea Mycelium 0.05 and 0.075% (w/v) [43]
Corn smut/Ustilago maydis Mycelium 10–10 000 μg L−1 [49]
Verticillium wilt/Verticillium dahliae Mycelium 0.5–2.0 mg mL−1 [39]
Note: Modified from Bautista‐Baños et al. [3].

Table 15.2 Volatile compounds as molecules responsible for strawberry aroma of —control‐

and chitosan‐coated (FEC) strawberries stored at 5°C for 17 days.
Control FEC
Volatile compounds 0 17 0 17
Ethyl acetate + nd + +
Butanoic acid methyl ester + nd + +
Butanoic acid, ethyl ester + nd + +
Hexanoic acid, methyl ester + nd + +
Hexanoic acid, ethyl ester + nd + +
3‐Hexen‐1‐ol, (Z) + nd + +
Acetic acid, phenyl methyl ester + nd + +
2,4‐Di‐tert‐butylphenol + nd + +
p‐Mentha‐1(7),8(10)‐dien‐9‐ol + nd + nd
2‐Octyn‐1‐ol nd nd nd +
Decanoic acid, 10‐bromo‐methyl ester nd nd nd +
N‐Methyl‐D‐glucamine nd nd nd +
E‐9‐Tetradecenoic acid nd nd nd +
1‐Butanol, 3‐methyl, acetate + nd + +
4‐Penten‐1‐ol, 3‐methyl, acetate nd nd nd +
2‐Hexen‐1‐ol, acetate, (E) nd nd nd +
3(2H)‐Furanone, 4‐methoxy‐2,5‐dimethyl nd nd nd +
(+)‐3‐Carene nd nd nd +
Alpha‐terpineol nd nd nd +
Camphene nd nd nd +
Beta‐pinene nd nd nd +
Acetic acid pentyl ester nd nd nd +
4‐Hexen‐1‐ol, acetate nd nd nd +
Note: nd: data not available; + flavour component present; FEC: non‐inoculated fruit with chitosan coating.
s upply 16% of magnesium, 19% of iron, and 9% of total calories. Legume vegetables,
such as potatoes, provide about 5% of proteins, which contain high quality essential
amino acids. Other important nutrients supplied by these agricultural commodities include
folacin, riboflavin, zinc, calcium, potassium, and phosphorus. Thus, some human dis-
eases can be closely related to the lack of appropriate intake of nutrients. The most signifi-
cant nutrition‐related disease is chronic undernutrition; other diseases such as beriberi,
scurvy, and anaemia are associated with the lack of vitamin B3, vitamin C, and iron,
respectively, all principally present in fruits and vegetables (https://www.britannica.com/
science/nutritional‐disease).
15.3 Storage Disorders and Diseases of Horticultural Products

Fruits and vegetables are highly perishable products, especially during the postharvest
phase, when considerable losses due to physical damage (mechanical injuries), physiologi-
cal disorders (low and high temperature disorders and mineral deficiencies), and microbio-
logical diseases (fungi and bacteria) can occur [12]. The extent of postharvest losses due to
microorganisms has not been properly documented; however, they are estimated to be
between 10 and 30%, and in crops from tropical latitudes estimates can reach up to 50%
[13]. According to Coates and Johnson [14], apart from direct economic considerations, an
additional risk due to pathogenic infection is the mycotoxin contamination, which can
occur when the agricultural commodity has undergone further processing. Fungal genera
such as Penicillium, Alternaria, and Fusarium are known to produce mycotoxins under
certain conditions. The main fungi causing postharvest diseases belong to the following
genera: Alternaria, Colletotrichum, Monilinia, Penicillium, Aspergillus, Rhizopus, Botrytis,
Fusarium, and Thielaviopsis [13].
15.4 Plant Fungi Inhibition by Chitosan Application

Chitosan is a compound that presents biofunctional characteristics, which make it suitable
to replace traditional methods for the control of microorganisms. In addition, it can be
safely used to produce edible coatings [15]. A coating is formed on the surface of the fruit
or vegetable, which acts as a mechanical barrier that protects it from infections caused by
fungi, thereby helping to decrease the disease level during storage. At present, there is a
vast literature reporting that the antimicrobial activity of this compound depends on factors
strongly associated with the intrinsic physicochemical characteristics of this polymer,
including molecular weight, degree of deacetylation, and origin. Other factors, such as
chitosan concentration (the fungal growth inhibition increased as the concentration of
chitosan was increased) and fungal strain, can also be considered [2, 6]. A number of
studies have also confirmed the fungicidal effect of chitosan. Fungi causing serious plant
pathogenic diseases were in vitro inhibited by the application of this polymer (Table 15.1).
Those studies confirmed that the level of inhibition of fungi is mostly associated with
chitosan concentration [2]. However, other authors have mentioned that the degree of dea-
cetylation of this polymer is the factor having the highest influence on the antimicrobial
activity of chitosan, since the amount of free amino groups in the chitosan molecule has
been related to this activity [16].
For in situ evaluations, various edible chitosan‐based coatings have been developed
till date. For example, antimicrobial coatings with chitosan have been the subject of
experimentation since 1992 by El Ghaouth et al. [17]. In those studies, a concentration of
15 mg mL−1 inhibited the growth of Botrytis cinerea and Rhizopus stolonifer in strawber-
ries (Fragaria × ananassa). Also, the typical infection signs of these fungi appeared after
5 days of being stored at 13°C, while in the control the symptoms were visible on the first
day. By the end of the storage period, the infection revealed an over 60% decrease in
affected strawberries treated with chitosan. In further studies, significant chitosan fungicidal
activity was reported by Romanazzi et al. [18] when it was applied as a coating to grapes (Vitis
vinifera) and strawberries against B. cinerea. Also, Jiang et al. [19] reported that chitosan
at a concentration of 2% (w/v) was able to inhibit the appearance of microorganisms in
litchi fruit (Litchi chinensis) by the end of the evaluation, after being incubated for 12 h.
However, higher chitosan concentrations did not significantly increase the beneficial effects
of chitosan in the control of microorganisms, according to Zhang and Quantick [20].
Remarkable control has been observed in various species of the genus Colletotrichum
with the application of chitosan coatings in papaya (Carica papaya), tomato (Lycopersicon
esculentum), and grape with a concomitant reduction in lesion diameter [21–23]. In addi-
tional studies [24], anthracnose incidence was significantly lower in the 1% (w/v) chitosan‐
treated fruit. It was reported that the anthracnose development in soursop (Anonna
muricata) was inhibited by 80%, while in mango (Magnifera indica) and banana (Musa sp.)
the inhibition was 100% as compared to the untreated fruit, in which little or no disease
control was observed. With regard to this fruit, Kyu Kyu Win et al. [25] mention that in the
cv. Kluai Hom Thong treated with 1% (w/v) chitosan, there was a considerable reduction
of up to 75% in the development of crown rot after five weeks of storage, while Jinasena
et al. [26] reported very similar results with the cv. Embul, since in this case the anthrac-
nose decreased by up to 90% during 14 days of storage.
15.5 Chitosan Integrated with Other Alternative Methods for Controlling

Postharvest Fungi
There is a large amount of data indicating that the chitosan may interact with other posthar-
vest treatments, which, in turn, can improve its effects for preventing fungal decay of hor-
ticultural commodities during storage of crops. The synergistic effects among chitosan
formulations combined with physical means including heat and UV irradiation exposures,
and modified atmosphere and hypobaric storage, are undeniable. In addition, chitosan inte-
grated with natural products, plant derivatives such as plant extracts and essential oil,
organic salts and acids, and antagonistic microorganisms, including yeast and bacteria, can
be very effective for reducing fungal postharvest rots. Synthetic fungicides and other poly-
mers may also potentiate the synergistic effects [5, 6].
15.6 Chitosan‐Based Formulations

To overcome contamination problems due to the disposal of plastic synthetic packaging,
new alternatives, such as the use of natural polymers, have been sought. As previously
stated, chitosan represents a good alternative. Chitosan‐based edible films and coatings
have been used as packaging materials to extend the shelf life of fresh and minimally
processed fruits and vegetables. Chitosan films exhibit low permeability to O2 and CO2 and
good mechanical and barrier properties. However, its water barrier property is poor, due to
chitosan’s hydrophilic nature, and this is why other compounds, such as lipids or essential
oils, are incorporated into formulations. Chitosan coatings influence the sensorial and qual-
ity attributes of postharvest fruits and vegetables by delaying the rate of respiration and
decreasing the weight loss [51, 52]. There are two main ways of applying a coating to a
fruit or vegetable: dipping and spraying. In the dipping procedure, the food product is intro-
duced into a coating formulation, and in the spraying process, an aerosol is used to cover
the fruit or vegetable with the solution. However, application conditions affect the coating
thickness [53]. Some studies have demonstrated that lower microbial counts can be found
for dipping than for spraying, due to the higher thickness of the dip coating. On the other
hand, firmness and texture are better preserved for sprayed samples [54].
15.7 Physiological Response and Quality Retention of Horticultural

Commodities to Chitosan Coating Application
Worldwide, horticultural products are marketed and consumed based on their quality. Thus,
attention to the quality concept is important. It can be analysed from two main different
perspectives, according to Espejel et al. [55]. The first is the quality objective, which refers
to the technical, measurable, and verifiable nature of products. While the second is the
subjective or perceived quality that allows the consumer to evaluate the products through
their intrinsic attributes related to the physical aspects of a product, such as colour, fla-
vour, shape, and appearance, and also from extrinsic attributes which are related to the
nature of the product, such as brand name, stamp of quality, price, country of origin, stor-
age, packaging, and production information, in response to rising concerns of safety,
health, convenience, ethical factors, and nutritional composition [56]. Thus, the quality of
fruits and vegetables will depend on their sensory and functional features, before and after
storage. Hence, postharvest technologies for preserving the product’s quality are limited
to the use of nontoxic compounds to ensure its acceptability by consumers. In this regard,
chitosan is widely used in different areas. For example, the United States Environmental
Protection Agency (EPA) has approved it as a plant growth enhancer, and as a substance
that boosts the ability of plants to trigger certain defence mechanisms against fungal
infections [57]. In addition, Shields et al. [58] reported the use of this polymer as a human
dietary supplement for weight loss and cholesterol reduction. Particularly in the food sec-
tor, the use of chitosan and chitin is regulated by international standards organisations,
such as the Codex Alimentarius Commission and the Japanese Ministry of Health and
Welfare [59, 60].
At present, more than one hundred research papers are published every year on the physi-
ological, biochemical, and physical features of fruits and vegetables treated with chitosan
coatings alone, or enriched with bioactive compounds as a technology for improving their
quality and safety. As outlined previously, horticultural products are highly perishable due
to their composition. Many agricultural commodities have particular compositions that
make them susceptible to deterioration. This includes a volume of water up to 90% of the
total mass, and other compounds that promote microbial growth, including (a) carbohy-
drates, representing 50–80% of the total dry weight, (b) proteins, which represent less than
1% of the fresh mass with the exception of leguminous seeds that are rich in protein, con-
taining up to 15–30%, (c) fats that are usually below fresh 1%, with exceptions in products,
such as avocado (35–70%), olive (30–70%), grapes (0.2%), banana (0.1%), and apple
(0.06%), and (d) vitamins and antioxidants, such as vitamin C (30–150 mg per 100 g fresh
weight) [61]. Consequently, a wide variety of experiments have been conducted on chitosan
coatings, since they preserve different quality parameters of numerous agricultural com-
modities, such as weight. Fruits and vegetables are basically formed by water, and it repre-
sents an economic value during their marketing. The water interchange that occurs between
the film and fruit during storage has been very well explained by Lin et al. [62]. They indi-
cated that, during the interaction between the agricultural commodity and the chitosan coat-
ing, one side, exposed to air, presents a smooth surface, being the chitosan molecular chains
in an orderly arrangement, and the other side, in contact with the pericarp, presents an
irregular face, being the chitosan molecular chains randomly arranged, due to the high con-
tent of moisture. It was observed that the greater roughness resulted in the higher water
vapour permeability (WVP), which meant that the moist surface facilitates the transport of
water. On the contrary, the air‐dried surface prevents the transfer of water, due to its higher
uniform and compact molecular chains (–NH2 and –OH groups of water inside).
Consequently, the firmness and volatile compounds of aroma are preserved, too.
Another important feature is that chitosan coatings neither modify the appearance (firm-
ness, colour, and absence of visual defects) nor the taste of the treated commodities after
application, even if some additives are also incorporated. According to Arnon‐Rips and
Poverenov [63] the advantage of using this coating is that the firmness and texture of
horticultural products can be improved through the addition of Ca2+ ions and other addi-
tives, thus reducing the water vapour permeability of the product by utilising water‐insoluble
or poorly soluble components in the coating formulation, and, therefore, avoiding the
incidence of microorganisms that cause softening as a consequence of the damage in the
cell wall of the products. In addition, the colour of the horticultural products can be
maintained because chitosan is a polymer without colour and it is able to adhere to their
surface. It is possible that the NH3 group with its positive charge is able to form bonds
with the compounds present on the surface. On the other hand, the coatings may limit the
diffusion rate of oxygen; consequently, the oxidation reactions and enzymatic browning,
which cause undesired colour changes, can occur slowly. Additionally, the shape, size,
and brightness of the products are improved by using chitosan coatings, because they
prevent the loss of water and dehydration. Consequently, the deformation of products
is reduced and the gloss on the external surface is perceivable. Also, skin blemishes
are caused by handling, and microbial and mechanical damages are reduced, since the
coatings act as packaging [63, 64].
Sweetness, sourness, and bitterness of the fruit treated with chitosan coatings can be
slightly modified in the first few days after application, but this abnormality disappears
during storage, as reported by Devlieghere et al. [65]. Chitosan solution is normally
adjusted to a pH value below 6, but this value can be modified by interacting with the
constituents of the product until reaching its pH of the product. Regarding aroma, our
working group trials have shown an increase in volatile compounds after 17 days in 0.4%
(w/v) chitosan‐treated strawberries to which an aqueous extract of Roselle calyces (1%
w/v) and cinnamon essential oil (0.025% w/v) were added. Results indicated that at the end
of the storage, terpenes and esters were synthesised. These are compounds that are
recognised for providing pleasant and fruity aromas. Esters are synthesised by esterases
that show considerable activity during the maturation stage of the fruit. Similar compounds
of aroma were reported by Ayala‐Zavala et al. [66]. Thus, it is confirmed that the chitosan
coatings do not cause changes in the aroma of the product after prolonged storage
(Table 15.2). This confirms the fact that chitosan coatings can be a suitable technology for
quality retention including turgidity, colour, aroma, and total soluble solids (TSS) content,
among others.
15.8 Influence of Chitosan Coatings on the Shelf Life of Horticultural

Products
The beneficial effects of chitosan coatings on the shelf‐life maintenance of various horti-
cultural products have been subject to a great deal of study. On this subject, Khaliq et al.
[67] used 1% (w/v) chitosan on the mango fruit at 13°C and obtained a firmer fruit but with
a lower content of soluble solids than the control group. Nevertheless, the CO2 and ethylene
contents between the control group and treated mangoes were similar, which indicates that
the ripening process was not affected by chitosan. In the same way, plums (Prunus domes-
tica) treated with chitosan coatings showed a delay in the rate of ripening that was observed
through firmness retention and significantly less weight loss, change of colour, and reduc-
tion in ethylene evolution rate during storage for 35 days [68].
For mango, Cosme et al. [69] applied chitosan coatings with different concentrations
(1%, 2%, and 3% w/v). The chitosan coating delayed the climacteric peak, water loss,
and firmness. No significant changes were observed for the other evaluated variables, such
as solid soluble content, titratable acidity, pH (pulp), and sugar content. However, starch
degradation decreased. Therefore, the chitosan coating was considered to be effective in
preserving the quality attributes and starch degradation rate for mango.
Additionally, Pagno et al. [71] reported that chitosan coating at 1.5% (w/v) influenced
the nutraceutical quality of tomatoes, delaying the peak of accumulation of β‐carotene and
lycopene, slowing down the loss of caffeic and ferulic acids during storage, and although
transiently, increasing the levels of efficient antioxidants, such as chlorogenic acid (135%),
caffeic acid (121%), and quercetin (115%), until the final day of storage.
Also, an effect of chitosan coatings on the oxidative stress on fruit has been reported
through changes of enzymatic and nonenzymatic antioxidants, and malondialdehyde
(MDA). In this regard, Li et al. [72] studied the oxidative stress of yali pears (Pyrus bretsch-
neideri) and observed that chitosan treatment of pears, before and after being bruised, not
only decreased the MDA content as an indicator of the breakage of cell membranes, but also
enhanced the activities of antioxidant enzymes in this fruit. Based on these results, it can be
hypothesised that chitosan might delay the senescence of damaged pears by means of regu-
lating the antioxidant enzymes system.
Batista et al. [74] evaluated the physicochemical and antioxidant activities of chitosan‐
coated guava using the same coatings. Cosme et al. [69] reported that chitosan coating sup-
pressed the respiratory rate, weight loss, firmness, and skin colour, and the antioxidant
process was reduced for concentrations of 2% and 3%, thus maintaining fruit quality and
delaying ripening during storage for 96 h. On the other hand, Kaya et al. [70] used chitosan
coating to preserve the shelf life of red kiwifruit (Actinidia deliciosa) berries kept at room
temperature (20 ± 2°C) for a period of 26 days. The coating was effective for the first 12
days of storage. The coated berries preserved higher amounts of biologically active com-
pounds (phenolic and ascorbic acid) at the end of the storage period. The decomposition
rate was lower for the coated samples, as evidenced by the changes in soluble solids.
Therefore, chitosan coating was effective in preserving the short shelf life of the berry fruit.
15.9 Effects of Chitosan Coatings with Additional Compounds on Quality

and Microorganisms Development
Incorporation of antimicrobial, antioxidant, and barrier components into formulations is an
effective method of microorganism control, rather than the addition of the component to
the food, because it can gradually migrate from the film or coating onto the food surface
[73]. For example, for blueberries, Vieira et al. [75] studied the effects of a chitosan–aloe
vera coating on the fruit quality during storage at 5°C. The antifungal and antioxidant
properties were evaluated. From the results, microbiological growth (fruit inoculated with
B. cinerea) and water loss were reduced by 50% and 42%, respectively, in coated blueberries
after the storage of 25 days. In general, chitosan coating extended the shelf life of the fruit
for 5 days. In further studies, Mannozzi et al. [76] evaluated the efficacy of a chitosan
edible coating loaded with a flavonoid (procyanidin) from grape seeds on fresh blueberries
during the storage of 14 days at 4°C. They found that the chitosan‐based coated samples
showed an important decrease in yeast and mould growth compared to the uncoated
samples. Also, the coating maintained firmness, increased the antioxidant activity in the
blueberries, and maintained the quality during storage.
Another study related to mango was undertaken by Jongsri et al. [77] in which the fruit
was coated with chitosan combined with spermidine and evaluated after storage for 9 days
at 25 ± 2°C. The effect of the coating on anthracnose disease of the mango fruit after
inoculation with Colletotrichum gloeosporioides was studied. Combination of both
components in the formulation induced the fruit’s defence mechanism against anthracnose.
The mango softening was reduced due to the decrease of soluble pectin during the ripening
stage. The coating was effective for improving firmness and delaying the deterioration of
postharvest mangoes.
Shen and Yang [78] elaborated the three coating formulations based on chitosan, chitosan
and salicylic acid, and chitosan‐g‐salicylic acid, studying the effects on decay incidence,
quality, and shelf life during the cold storage of grape fruit. The chitosan‐g‐salicylic acid
enhanced the activity of diverse enzymes (lyase, chitinase, and glucanase), also promoting
the accumulation of phenolic compounds with antifungal activity against B. cinerea. Other
benefits were the decrease of respiration rate, decay incidence, and weight loss. Also, TSS,
titratable acidity, and sensory properties were improved due to the coating during storage at
0°C for 42 days. Pomegranate arils were coated with a formulation containing chitosan and
ascorbic acid [79], and stored at 5 ± 1°C for 28 days. During storage, bacterial and fungal
growth was inhibited. The shelf life of the coated arils was extended up to the storage of 21
days, in contrast with noncoated products with a shelf life of less than 10 days.
Awad et al. [80] evaluated the behaviour of bananas coated with a mixture of 2% (w/v)
chitosan and 1% (w/v) gallic acid. The inclusion of the gallic acid compound into the
chitosan matrix improved the antioxidant properties of the fruit. Moreover, the chitosan–
gallic acid coating delayed the fruit ripening as reflected by a higher firmness, lower TSS
and TSS/acid ratio, and slightly higher vitamin C (0.064 g kg−1) during storage at 20 ± 2°C
as compared to the control group.
Regarding the incorporation of essential oils into chitosan‐based formulations, Shahbazi
[81] applied a carboxymethyl cellulose‐chitosan coating containing mentha (Mentha
spicata) essential oil to fresh strawberries to study the antimicrobial effect and the
physicochemical and sensory properties after 12 days of shelf life extension. It was tested
against Listeria monocytogenes, being the coating effective as an antimicrobial agent. Also,
positive effects for weight loss, titratable acidity, pH, water vapour resistance, and
respiration rate were observed for the coating used as an active packaging.
Yuan et al. [82] prepared chitosan coatings, and the in vitro and in vivo activities of
essential oils as antioxidants, antibacterials, and antifungals were evaluated. Their use in
food composites was also considered. The efficacy of essential oils as antioxidants, anti-
bacterials, and antifungals was increased for the in vitro test. Also, the coating extended the
shelf life of fish and meat products.
Perdones et al. [83] studied the volatile profile of strawberries coated with chitosan‐
lemon essential oil during storage at 20°C for 7 days. They found that the coating was
effective for controlling the fruit’s fungal decay, affecting the metabolic pathways and
volatile profile of the strawberries, and also enhancing the perception of aroma. Coatings
based on chitosan and M. piperita or M. villosa essential oils were tested on cherry tomatoes
by Dantas et al. [84] to prevent postharvest mould infections caused by Aspergillus niger,
B. cinerea, Penicillium expansum, and R. stolonifer, and to maintain the quality of the fruit.
The physicochemical and sensory properties were tested for two storage conditions: at
room temperature (25°C for 12 days) and low temperature (12°C for 24 days), measuring
the minimum inhibitory concentration (MIC) for the different fungi. The results showed
mycelial growth and spore germination inhibition for concentrations of 4 mg mL−1
(chitosan) and 2.5 or 1.25 μL mL−1 for the essential oils for both tested temperatures. Also,
physicochemical and sensory properties such as weight loss, firmness, titratable acidity,
and soluble solids were not affected.
Antimicrobial activity of chitosan coatings and films against L. monocytogenes on
black radish was assessed by Jovanović et al. [85]. Chitosan‐gelatin coatings were evalu-
ated on shredded black radish samples stored for 7 days at 4°C. The highest antibacterial
activity was observed for chitosan prepared with acetic acid, and for films with higher
chitosan concentration and essential oil incorporation. Especially films with 1% (w/v)
chitosan and 0.2% (w/v) thyme essential oil were most effective. However, these films
had a negative impact on the consumers due to the flavour and aroma of coated radish.
Therefore, mint essential oil, also tested, but having lower antibacterial activity, was
selected, because it had the least negative impact on the sensory properties mentioned
earlier.
With regard to the interaction between chitosan and essential oils, Devlieghere et al. [65]
showed that, during this interaction, the chitosan is positioned at the outside of the emulsion
drops, due to the interaction between the positively charged chitosan and the negatively
charged free fatty acids. In this way, the chitosan formed larger positively charged drops
that still maintained their antimicrobial activity. Additionally, when the essential oil is used
in combination with chitosan, the antimicrobial effect can be enhanced. This is possible
because essential oils are complex mixtures of volatile metabolites that can be isolated by
pressing and distillation from a whole plant or plant part. The main compounds contained
in the essential oils are derived from three biosynthetic pathways: 1) the mevalonate
pathway leading to sesquiterpenes, 2) the methyl‐erythritol pathway leading to mono and
diterpenes, and 3) the shikimic acid pathway en route to phenylpropenes. Each one has a
specific mode of action on the microorganism. Dorman and Deans [86] explained that the
antimicrobial activity can be enhanced by the effect of the functional groups present in the
essential oil. There are three examples of this: 1) in phenols, an alkyl substitution occurs in
the nucleus and this chemical operates to affect gram‐negative bacterial growth, 2) the
bacteriostatic and fungistatic actions of terpenoids are increased when they are carbonylated,
i.e., when a carbon atom is bonded to an oxygen atom by a double bond, and 3) an aldehyde
group conjugated to a carbon‐to‐carbon double bond is a highly electronegative arrangement
that can interfere in biological processes involving electron transfer and react with vital
nitrogen components, e.g. proteins and nucleic acids. Therefore, it is able to inhibit the
growth of the microorganisms, among others.
Other bioactive compounds with antimicrobial and antioxidant properties, such as plant
extracts, have been incorporated into chitosan‐based formulations. Plant extracts used for
edible coatings are obtained from fruits and herbs, thus adding medicinal benefits to the
coating. Edible coatings incorporated with plant extracts can replace non‐biodegradable
polyethylene plastic films used for the packaging of fruits and vegetables allowing product
cooling, among other benefits [63].
On this subject, Sneha et al. [87] studied the effect of chitosan‐alginate‐based coatings
enriched with pomegranate peel extract to extend the shelf life of guava. The formulation
composition was 1% (w/v) chitosan, 2% (w/v) alginate, and 1% (w/v) of the plant extract.
After the storage of 20 days at 10°C, changes in respiration, ripening, and colour were
observed. The guavas coated with the pomegranate plant extract maintained their quality
compared to the uncoated ones. The visual attributes and nutritional properties of the guava
were improved, retarding respiration rate and delaying senescence. Likewise, Duran et al.
[88] observed that the strawberries treated with chitosan 1.5% (w/v) and pomegranate seed
extract 1% (w/v) showed a reduction of mesophilic bacteria count, but the surface colour
and texture profile analysis were not improved.
Chitosans incorporated with different concentrations of phenolic compound from
blueberry leaf‐and‐fruit extracts were tested by Yang et al. [87] against Staphylococcus
aureus, L. monocytogenes, Salmonella typhimurium, and Escherichia coli to preserve the
postharvest quality of fresh blueberries. Fruits were stored at 2 ± 1°C for 35 days and then
for 3 days at room temperature. Five concentrations were used containing 2% (w/v)
chitosan and 4–12% (w/v) plant extracts. Also, for the 12% (w/v) plant extract, a modified
atmosphere packaging (3 kPa O2 + 12 kPa CO2) was used. Proper antimicrobial activity
against tested microorganisms was observed, for a minimum inhibition concentration from
25 to 50 g L−1. More effective control on fruit decay was obtained for the modified
packaging atmosphere with 12% (w/v) leaf plant extract. All treated samples maintained
higher nutritional properties, total phenolic content, and radical scavenging activity as
compared to the control.
Breda et al. [90] did not find a clear significant difference of colour, firmness, or
weight loss in tomatoes coated with chitosan alone or in combination with the extract
of pequi (4:1) (Caryocar brasiliense), and stored at 22 ± 2°C. However, chitosan coat-
ings and extracts of pequi showed benefits by means of maintaining 70% lower levels
of mould and yeast counts during all storage periods of tomatoes than the control group.
Sun et al. [91] reported that carvacrol at ≥0.1% (w/v) or trans‐cinnamaldehyde at ≥0.2%
(w/v) incorporated with chitosan coatings improved the antimicrobial activity against
E. coli and Penicillium digitatum, and retarded the loss of firmness of blueberries dur-
ing storage. The results suggest that the antimicrobial properties of the compounds
added to chitosan, and their effect on quality will depend on their concentration and
structure.
Studies on propolis or bee glue, a resinous mixture produced by honey bees, have dem-
onstrated effective antimicrobial characteristics; therefore, its incorporation into chitosan
coatings has also been the subject of experimentation. For example, Migliori et al. [92]
tested a propolis coating on “Vesuviano” tomatoes at room temperature for 120 days. The
coating formulation was compared with the effect of thyme essential oil and chitosan on
the postharvest fruit quality and organoleptic properties, and carotenoids and phenol con-
tents were evaluated. Thyme essential oil decreased the number of rotten fruits after stor-
age of 40 days. However, propolis and chitosan had the same effect after 80 days.
Moreover, chitosan retarded the senescence for the whole storage period. The postharvest
quality of the fruit was not affected by the different formulations. However, total soluble
sugar (for chitosan and thyme), total carotenoids and flavonols (for chitosan), and total
organic acids and terpenes (for propolis) were better retained during the postharvest
period. As a conclusion, the best result as a fungicide was obtained for chitosan, which
most effectively reduced the fruit’s senescence maintaining tomato quality for a long
period of time.
In Tables 15.3 and 15.4, the different coatings, bioactive components, and their effects
on the horticultural commodity are summarised.
Table 15.3 Chitosan edible coatings on preserving the quality and nutraceutical compounds of fruits
and vegetables.
Topics References
• Potential applications and modes of action of chitosan and its derivatives as [93]
antimicrobials: a review
• Chitosan as a novel edible coating for fresh fruits: a review [94]
• Influence of chitosan coating on loquat’s nutritional quality during cold storage [95]
• Chitosan coating and storage effect on food storage chamber in banana stored at 25°C [96]
• Chitosan coating at concentrations from 0.7 to 2 g L−1 on physicochemical, nutritional, [97]
and microbiological parameters of fresh‐cut “Rocha” pear
• Chitosan coating on the enzymatic antioxidant system and the membrane integrity [98]
maintenance of sweet cherry during cold storage
• Effect of chitosan/nanosilica on enhancing chilling tolerance in loquat fruit during cold [99]
storage
• Edible coatings combined with repetitively pulsed light treatment on microbiological [100]
stability, quality, and physicochemical changes of fresh‐cut cantaloupes during storage
at 4°C
• Application of Cu‐nanoparticles, introduced in chitosan–polyvinyl alcohol hydrogels, [101]
on the growth of jalapeño pepper plants, the antioxidant content in fruits, and their
postharvest characteristics
• Improvement in the fruit storability of Punica granatum using prestorage chitosan [102]
coating technique
• Chitosan coating added with rosemary extracts, chitosan with ascorbic acid on the [103]
browning characteristics, and the physicochemical and sensory properties of fresh‐cut pears
Table 15.4 Chitosan coating/bioactive component and its effects on fruits/vegetables.
Coating/bioactive
component Effects Fruit References
Chitosan/procyanidin Decreased yeast and mould growth, intact Blueberry [76]
firmness, and increased antioxidant activity
Chitosan/aloe vera Microbial growth and water loss were reduced Blueberry [75]
Chitosan Climacteric peak, water loss, and firmness were Mango [69]
delayed
Chitosan/spermidine Defence mechanism against anthracnose was Mango [77]
induced. Firmness was improved and
deterioration of fruit was delayed.
Chitosan Respiratory rate, weight loss, firmness, and Guava [74]
antioxidant process were reduced. Ripening
was delayed.
Chitosan Biologically active compounds (phenolics and Red kiwifruit [70]
ascorbic acid) were preserved. Lower berries
decomposition rate.
Chitosan, chitosan/ Activities of lyase, chitinase, and glucanase Grape [78]
salicylic acid, and enzymes were enhanced. Decrease on
chitosan‐g‐salicylic respiration rate, decay incidence, and weight
acid loss.
Chitosan/ascorbic acid Bacterial and fungal growth was inhibited. Pomegranate [79]
arils
Chitosan‐ carboxymethyl The coating effective as antimicrobial agent. Strawberries [81]
cellulose/Mentha Positive effects for weight loss, titratable acidity,
spicata essential oil pH, water vapour resistance, and respiration rate.
(Continued)
Coating/bioactive
component Effects Fruit References
Chitosan/lemon essential Fruit fungal decay was controlled. Aroma Strawberries [81]
oil perception was enhanced.
Chitosan/Mentha piperita Inhibition of mycelial growth and spore Cherry [84]
L. or Mentha × villosa germination. Physicochemical and sensory tomato
Huds. essential oils properties did not change during storage.
Chitosan‐gelatin/thyme Higher antibacterial activity, higher flavour and Black radish [85]
or mint essential oil aroma (thyme), and better sensory properties
with low antibacterial activity (mint).
Chitosan‐alginate/ Visual attributes and nutritional properties were Guava [87]
pomegranate peel improved. Respiration rate and senescence
extract were delayed.
Chitosan/phenolic Good antimicrobial activity. Effective control of Blueberry [89]
compound from fruit decay.
blueberry
Chitosan vs. propolis vs. Total soluble sugar (for chitosan and thyme), total Tomato [92]
thyme essential oil carotenoids and flavonols (for chitosan), and
total organic acids and terpenes (for propolis)
were retained. The best result as fungicide was
obtained for chitosan.
15.10 Integration of Chitosan Nanoparticles into Coating Formulations

and their Effects on the Quality of Horticultural Commodities
and Development of Microorganisms
Nowadays, nanotechnology is defined as the “technology at the nanoscale,” considering the
nanoscale in a range from 1 to 100 nm. Many structures have been developed at nanolevel,
including nanoparticles, nanofibers, nanowhiskers, nanorods, and nanotubes, among others.
The good performance of nanomaterials lies in the surface‐to‐volume ratio, which increases
their reactivity [104]. There are many methods available to elaborate chitosan nanoparticles.
The most frequently used are: nanoprecipitation and ionotropic gelation, microemulsion,
emulsification solvent diffusion, and polyelectrolyte complex method [105].
Chitosans have been incorporated as nanoparticles in diverse formulations, alone or with
bioactive compounds. Divya et al. [106] studied the antifungal and antioxidant activities of
chitosan nanoparticles (1–5% w/v) prepared by ionic gelation. The mechanism of action
and antimicrobial activity were evaluated against Rhizoctonia solani, Fusarium oxyspo-
rum, C. acutatum, and Phytophthora infestans. The coating was tested in vivo on tomato,
chilli, and eggplant. Good antifungal activity was found, which was better than that of
Amphotericin (a commercially available antifungal). A significant antioxidant activity was
also observed. Reduced weight loss was obtained for the coated vegetables. The in vitro
antimicrobial activity of gel‐based chitosan nanoparticles on the physicochemical and sen-
sory properties of table grapes was assessed by Ferrão et al. [107]. The minimum inhibitory
and antibacterial concentrations against foodborne pathogens (Salmonella spp., E. coli,
S. aureus, Pseudomonas aeruginosa, L. monocytogenes) were calculated. The edible chi-
tosan nanoparticle coating was effective at delaying the ripening of the fruit. Weight loss,
soluble solids, and sugar contents were reduced. Moisture retention was increased and
titratable acidity was intact. In conclusion, the postharvest quality of the grapes was pre-
served with the use of the edible coating.
Polyphenols of strawberry extract were encapsulated in chitosan nanoparticles and the
release kinetics was studied by Pulicharla et al. [108]. Their final results showed that the
encapsulation increased the bioavailability and release effectivity of the phytochemical.
The encapsulation efficiency was 58% of polyphenols. With regard to the incorporation of
essential oils into chitosan nanoparticles, Sotelo‐Boyás et al. [109] also studied the release
kinetics and inhibitory effect of thyme essential oil‐loaded chitosan nanoparticles and
nanocapsules against six foodborne bacteria (S. aureus, L. monocytogenes, Bacillus cereus,
S. typhi, Shigella dysenteriae, and E. coli). The results showed 68% of encapsulation
efficiency. The release from thyme was faster for nanoparticles than for nanocapsules. The
highest inhibitory effect was observed against S. aureus for the nanoparticles.
The antimicrobial activity and stability of cinnamon (Cinnamomum zeylanicum)
essential oil‐loaded chitosan nanoparticles against Phytophthora drechsleri as a coating on
cucumber were studied by Mohammadi et al. [110] during 7 days of storage at 4°C and 2–3
additional days at 20°C. The physicochemical properties were evaluated. Encapsulation
efficiency was about 2–17%. Encapsulated oil decreased the disease severity and incidence
more efficiently in Phytophthora‐inoculated cucumbers. The shelf life of cucumbers coated
with cinnamon essential oil‐loaded chitosan nanoparticles was extended up to 21 days at 10
± 1°C, while it was 15 days for the uncoated vegetable. As a conclusion, the coating
improved the physicochemical quality of the cucumbers, preserving firmness, and main-
taining colour and water content. Lower microbial counts were found for the coated fruit.
Mohammadi et al. [111] studied the in vitro and in vivo antifungal activities of satar (Zataria
multiflora) essential oil encapsulated in chitosan nanoparticles on strawberries. The encap-
sulation efficiency was 45%. The coating was effective in controlling the incidence and
severity of B. cinerea in inoculated strawberries during the storage for 7 days at 4°C fol-
lowed by 2–3 more days at 20°C. The same essential oil was tested on cucumber by
Mohammadi et al. [112], who studied the antioxidant activity on the shelf life extension of
nano‐chitosan‐based coating with the addition of this essential oil for a 21‐day storage
period. The coating improved the quality of cucumber and protected the bioactive
compounds during storage. Firmness, respiration rate, and 2,2‐diphenyl‐1‐picrylhydrazyl
(DPPH) radical scavenging capacity were best kept when the coating was used. The coating
was effective in inhibiting the growth of total aerobic bacteria, yeasts, and mould.
On the other hand, the antibacterial activity of chitosan nanoparticles and nanocapsules
incorporated with lime essential oil was assessed for S. aureus, L. monocytogenes, S. dys-
enteriae, and E. coli by Sotelo‐Boyás et al. [113]. It was found that the antibacterial activity
of the nanoparticles was higher than that for nanocapsules, and the highest inhibition halo
was found for S. dysenteriae for 40 mL of minimum inhibitory volume.
The in vitro and in vivo activities of the coating of chitosan nanoparticles and chitosan‐
thyme (Thymus sp.) essential oil nanoparticles against C. gloeosporioides on avocado cv.
“Hass” and its fruit quality were evaluated by Correa‐Pacheco et al. [8] after 6 days of stor-
age. The fungal activity and fruit quality were evaluated. In vitro results showed that the
growth of C. gloeosporioides was completely controlled with the incorporation of thyme
essential oil into the nanoparticles at thyme essential oil concentrations of 3% (w/v) and 5%
(w/v). A coating composed of 55% of chitosan‐thyme essential oil nanoparticles resulted in
a reduction of up to 60% of anthracnose disease without affecting the quality of the fruit. In
addition, firmness was better maintained for the coated fruit in comparison with the uncoated
one. On the other hand, the use of plant extracts incorporated into chitosan nanoparticles
was assessed by Barrera‐Necha et al. [114]. In this study, the antifungal activity of chitosan
nanoparticles loaded with ethanolic extracts of nanche (Byrsonima crassifolia) and blue-
berry (Vaccinium corymbosum) was evaluated on A. alternata isolated from fig (Ficus car-
ica) and rosemary (Rosmarinus officinalis) and from C. gloeosporioides isolated from
papaya and soursop. The ethanolic extract of nanche was effective on C. gloeosporioides
showing the mycelial growth inhibition of 79% and 82% for the fungi isolated from papaya
and soursop, respectively. Germination inhibition was 100%. Furthermore, the ethanolic
extract of blueberry inhibited the mycelial growth of A. alternata by 83% and 76% for fig
and rosemary, respectively, and germination inhibition was 75% for rosemary.
Tables 15.5 and 15.6 summarise the chitosan nanoparticle–based coatings and the effects
of pathogenic microorganism development on fruits and vegetables.
Table 15.5 Level of control of chitosan nanoparticles on fungi.
Fungi Inhibition (%) References

A. flavus 94 [115]
R. solani 67
A. alternata 78
Rhizopus 98 [116]
Colletotrichum capsici 98
C. gloeosporioides 98
A. niger 95
A. alternata 82 [117]
M. phaseolina 88
R. solani 34
A. flavus 100 [118]
C. gloeosporioides 100 [119]
Source: Sotelo et al. [120].
Table 15.6 Effects of chitosan nanoparticle–based coating/bioactive component applied on fruits/

vegetables.
Coating/bioactive Fruits/
component Effects of the coating vegetables References
Chitosan nanoparticles Good antifungal and antioxidant activities. Tomato, [106]
(NPs) Reduction of weight loss. chilly, and
eggplant
Chitosan Good antibacterial activity. Fruit ripening was Grape [107]
delayed. Reduction on weight loss, soluble
solids, and sugar contents. Moisture retention
was increased. Titratable acidity was preserved.
Chitosan/Cinnamomum Disease severity and incidence were decreased. Cucumber [110]
zeylanicum essential oil Firmness, colour, and water content were intact.
Chitosan/Zataria multiflora Firmness, respiration rate, and DPPH radical Strawberry [111]
essential oil scavenging capacity were preserved. Bacteria,
yeast, and mould growth were inhibited.
Chitosan/thyme essential Good fungal inhibition. Incidence of fungi was Avocado [8]
oil reduced and firmness was maintained.
15.11 Outlook
Horticultural products have proven to be an excellent source of minerals, vitamins, and
functional compounds for human consumption, resulting in food production evolving from
a local to a globalised environment. Contamination of fruits and vegetables by fungal and
bacterial pathogens that causes serious economic losses to producers, traders, and consum-
ers can occur any time from early growth to harvest and during handling and final market-
ing operations.
Controlling practices include chemical and nonchemical methods. Within the last few
years, coatings have become increasingly more important as a means to deliver fresh hor-
ticultural commodities to the consumer in safe conditions. Currently, there is a growing
trend to design and evaluate formulations based on nontoxic, naturally occurring com-
pounds. In this respect, chitosan‐based coatings can be a good alternative as an active food
packaging allowing the shelf life extension and quality preservation of fruits and vegeta-
bles by means of controlling the growth of microorganisms. Development of coatings
based on versatile biopolymers like chitosan will have an important role in promising hor-
ticultural applications, including those for food preservation. The incorporation and release
of bioactive compounds, such as antimicrobials, antioxidants, and nutrients, among others,
will produce deliverable solutions for protection against pathogens, enhancing at the same
time with sensory and functional properties. Certainly, this will depend on the interactions
among the coating, the bioactive ingredients, and the fruit or vegetable at issue.
We firmly believe that the use of nanotechnology will enable some drawbacks of the
existing edible coatings to be solved and better functional properties obtained. However,
ethical, security, and legislative problems regarding the use of nanotechnology, and scaling
from the laboratory to industry are issues that must be resolved. At present, there is a need
for further research related to the use of chitosan or chitosan nanobased formulations for
the control of microorganisms during the storage of fruits and vegetables, in particular
emphasising the incorporation of nontoxic compounds.
Acknowledgments
The authors wish to thank Mexico’s National Science and Technology Council for support-
ing professorships of Rosa Isela Ventura‐Aguilar (No. 772) and Zormy Correa‐Pacheco
(No. 1549).
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16
Chitosan Application in Textile
Processing and Fabric Coating
Thomas Hahn1, Leonie Bossog2, Tom Hager3, Werner Wunderlich3,
Rudi Breier2, Thomas Stegmaier3, and Susanne Zibek1
1
Fraunhofer Institute of Interfacial Engineering and Biotechnology, Stuttgart, Germany
2
Textilchemie Dr. Petry GmbH, Reutlingen, Germany
3
German Institutes of Textile and Fiber Research, Denkendorf, Germany
Textile manufacturers and related industries are undergoing a change due to legal regula-
tions becoming effective soon, as well as their commitment for social responsibility and the
increased ecological awareness of customers. Turning from established fossil‐based value
chains towards more sustainable bio‐based products is one of the key factors to achieve
these goals. This means to substitute petrol‐based agents with reactants and products
derived from renewable resources, like cellulose and chitosan. Cellulose is, as the main
content of cotton, an omnipresent and abundant material already known in the textile
industry for centuries. By comparison, the utilisation of chitosan in textile processing is
relatively new but not less promising. The benefits of chitosan include biodegradability,
antibacterial effects and further functionalisations that enable a broad range of applications
within the textile industry. Together with a previous view on the textile processing value
chain in general, the most promising application fields of chitosan are elucidated within
this review. A summary of the commonly applied physical–mechanical or application‐
specific test methods for textile fabrics is also part of the chapter in order to assess chi-
tosan performance. A brief overview about chitin fibre production introduces the different
methods used to manufacture chitosan fibres, revealing that wet spinning and electrospin-
ning are techniques of choice. The physical–mechanical properties are discussed in more

detail whereby chitosan fibres show low to medium tensile properties. A further application
field being considered is the utilisation of chitosan as a sizing agent that efficiently protects
yarns during weaving. Chitosan films exhibit a suitable value range of significant proper-
ties, such as viscosity, surface tension, adhesion power and mechanical properties. Removal
of chitosan from the yarn or fabric as well as recycling after weaving is explained in differ-
ent sections. A further part of the article deals with the application of native or modified
chitosan as a permanent coating or finishing agent on a textile fabric meant to be non‐
removable. Due to the amine functionality of chitosan, which offers a reactive coupling
site, the polysaccharide could be modified with versatile reagents to gain various desired
properties. Furthermore, a cross‐linking is desired in order to increase the washing fastness
required for a marketable product. Besides antibacterial activity, which has already resulted
in the development of commercial chitosan‐containing formulations, further beneficial
properties that have commercial viability are anticipated. The removal of dyes and heavy
metals with chitosan‐grafted fabrics, the increased dyeing ability of fabrics, and advanta-
geous crease recovery properties are presented in the last section of this chapter.
16.1 Chitosan in the Textile Industry

Clothing is one of the basic human needs besides food and shelter. It is accepted that
humans started to wear apparels 100,000–500,000 years ago, enabling them to resist unfa-
vourable weather conditions [1]. In the Mesolithic period, Cro‐Magnons and Neanderthals
used bone tools with rounded ends for leather softening and eying needles to sew animal
skins together, as per evidence from 40,000‐year‐old historical sites [2, 3]. The first textiles
were animal-based. In contrast to that, the processing of renewable resources, such as flax
and hemp, emerged later as one of the fundamental technologies. With the help of findings
at the Dzudzuana Cave in Georgia, scientists proved that flax fibres were woven or sewn to
produce baskets, ropes, etc., more than 30,000 years ago [4]. Hints on the cultivation of flax
date back to 8,000 BC [5]. Hand spinning, commonly applied to the manufacture of plant
material fabrics for thousands of years, was replaced by wheel spinning, invented in India
during 500–1,000 AD [6]. Through such history, textile trade developed into a highly tech-
nical industrial sector. A milestone in history was the machine manufacturing of cotton‐
based textiles initiating the Industrial Revolution in the nineteenth century. At the same
time, experiments were carried out to modify cotton and cellulose to improve their original
properties. The first truly synthetic fibre, called nylon, was commercialised in the first half
of the twentieth century [7]. Chemical modification of yarns and fabrics as well as the
application of synthetic fibres for textile production is common nowadays. What has
changed though is the increased consumer demand in the application and production of
more eco‐friendly and sustainable agents for textile products fostered by governmental
legislative initiatives concerning the application of chemicals with adverse health effects.
This does not only concern the fibre itself but also the manufacturing process, modification
reagents and post‐use [8]. Coincidentally, the functionality and quality of materials should
be at least of equal value.
Searching for materials and reagents that could contribute to functionality and sustain-
ability, researchers identified chitin and its derivative chitosan as the agents of choice.
Chitin is the second most abundant biopolymer on earth and can be obtained from fishery
waste, as crab and shrimp shells, but also from fungi and insects. When compared to
Chitosan Application in Textile Processing and Fabric Coating 397
polysaccharides from first‐generation biomass, such as starch, chitin production does

not need arable land and is not competitive to food production. The long‐chain chitin
consists of 5,000–8,000 ß‐1,4‐glycosidic‐linked N‐acetyl glucosamine and a minor
amount of glucosamine units. The glucosamine units confer a positive charge to the
polysaccharide at lower pH values, providing a unique characteristic in nature with its
particular negatively charged and neutral biopolymers. Deacetylation of chitin results in
conversion to chitosan (per definition a deacetylation degree of >50%). Chitin is insolu-
ble in most solvents, whereby chitosan can be solubilised in organic and mineral acids,
providing a valuable benefit for successive processing. Several investigations indicated
that chitosan displays anticholesterolaemic, haemostatic, analgesic, antitumour and
antioxidative activities besides biodegradability, biocompatibility, non‐toxicity as well
as antibacterial effects [9, 10]. These properties are worthwhile for pharmaceutical and
medical purposes. The benefits of using chitosan in textile production are provided in
the following section:
1. Chitosan is biocompatible and biodegradable: The textile industry currently uses syn-
thetic auxiliaries and coatings that are mainly based on fossil fuels, for example, poly-
vinyl chloride, polyvinyl alcohols, acrylic polymers or polyurethanes, that act as a
predominant matrix for coatings [11]. In the field of technical textiles, the application
of perfluorinated compounds is omnipresent, since they provide a unique combination
of water‐ and dirt‐repellent properties. Because of recently passed laws, the regulation
within Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
and the increased environmental awareness and health issues, scientists are eager to
substitute toxic synthetic compounds with non‐toxic greener agents. The current pro-
duction of chitosan suffers from extensive application of caustic soda and requires high
temperatures. However, it can be assumed that the intensive research performed nowa-
days will result in a process aiming to reduce the carbon footprint of the production
process as well as the final product [12, 13]. The biodegradability and biocompatibility
of chitosan already meets customer demands today.
2. Chitosan has free amino groups: Textile production requires the application of various
agents with properties depending on the desired effect on the fabrics. Non‐flammable
textiles need a coating that greatly differs from hydrophobic finishing. Nitrogen in
chitosan is bound as a free amino group. This provides a strong nucleophile‐enabled
grafting with a reactant bearing an electrophilic function. Besides this, the primary
alcoholic group at C6 and residual hydroxyl groups enable the functionalisation at
these sites, too. The versatility of reactions, grafting and modifications performed with
chitosan was already demonstrated in numerous studies [14]. This polysaccharide can
be grafted, for example, to obtain a more hydrophobic functionalisation [15] or to bear
anionic charges [16]. Another modification is cross‐linking [17].
3. Chitosan is easy to handle: Chitosan in its native form is a polymer that can be solubi-
lised in cold diluted acids with pH values of ≤5 at low concentrations. This pH can be
obtained with organic acids such as acetic acid, formic acid or lactic acid provided by
biotechnological production processes. Since no caustic and corrosive mineral acids
are required for pH adjustment, endangerment of the operating staff is substantially
reduced. Furthermore, there is no great investment to adapt already existing process
steps if using chitosan.
4. Chitosan has antibacterial activity: Medical fabrics, for example, wound dressings, are
commonly applied in order to support wound healing after surgery or to treat harmless
injuries. The predominant requirement of medical fabrics is sterility in order to avoid
bacterial and viral infections. Thus, the most common commercial biocidal textile fab-
rics include metals and metal salts, triclosan, polyhexamethylene biguanide (PHMB)
and quaternary ammonium compounds [18]. Chitosan provides the opportunity to
incorporate a bio‐based bacteriostatic and hence an anti‐odour property to fabrics. This
activity is based on the interaction of the polycationic molecule with the negative
charges of the microorganism’s cell membranes [19].
Because of these features, there are several possible chitosan application fields in the
textile industry. Consequently, the polysaccharide can be potentially applied – in textile
manufacturing or in processes directly linked to fabric production – in the native or modi-
fied form as [20]:
• A sizing agent to protect the yarn during weaving [21, 22]
• An adsorbent for the removal of dyes to purify textile effluents [23, 24]
• An auxiliary in the textile dyeing process in order to increase the uptake of anionic dyes
[25, 26]
• A carrier and linker of flame retardation agents [27]
• A thickener in the cotton print process [28]
• A fibre material or mixed/blended fibres with natural and/or synthetic polymers [29]
• A finishing agent for anti‐creasing properties or enhanced crease recovery of textile
fabrics [30, 31]
• An antibacterial finishing or anti‐odour agent [32, 33]
• A superhydrophobic coating for cotton fabrics [34, 35]
• A shell material for microencapsulation processes [36]
The list emphasises the versatility of chitosan in the textile industry, although it has to be
stated that chitosan is frequently used as an auxiliary rather than as an agent with the
desired functionality. This is due to the amine groups providing a suitable site for anchor-
ing and grafting. However, several applications seem to be more promising than others,
giving rise to the first technical chitosan products on the market, described in the course of
this review. The most important applications indicated by increased research activities of
the scientific community will be discussed in the following pages. Since most readers are
probably not familiar with the processes and relevant measurements taking place during
textile manufacture, the section will start with a brief overview for a comprehensive under-
standing. This is required to evaluate chitosan performance in textile processes described
for fibres, sizing and finishing agents.
16.2 Textile Production

Manufacturing of textiles, such as jeans, canvas or tarpaulins, is a complex system of dif-
ferent process steps. This requires several mechanical, physical and chemical operations
within a common textile value chain from fibre production to ready textile. For illustration
purposes, the textile manufacturing process is graphically shown in Figure 16.1, starting
from petrol‐ or bio‐based commodities.
• Dissolving cellulose
• Transfer into the
spinning mass
Petroleum Plant, animal
Gas, hydrocarbons Cellulose, cotton, wool, silk
• Production of the mono- • Polycondensation, • Washing

meric building blocks polymerisation or
(several reaction steps) polyaddition
Man-made fibres Natural fibres
• Finishing • Finishing
• Oiling
Yarn
Spun fibres
• Sizing • Sizing
• Weaving preparation • Pretreatment
• Dyeing • Dyeing
Textile fabric
Weaving, knit fabric, fleece
• Pretreatment • Pretreatment
• Dyeing • Dyeing
• Finishing • Finishing
Everyday textiles
Clothing, home textiles, technical textiles
Figure 16.1 Textile value chains from the basic commodity to the textile. The figure was reprinted and
modified according to [37] and permission was obtained by the Fund of the chemical industry and
TEGEWA e. V.
The backbone of textile fibres commonly consists of linear macromolecules besides a

minor amount of other compounds. Man‐made fibres, on one hand, can be divided into
natural (e.g. cellulosic) fibres, such as viscose, or synthetic chemical fibres, for example
polyester, polyamide and polyacrylate. The extraction of raw materials for natural fibre
production begins with the shearing of sheep for wool production or the harvesting of
plants to produce cotton fibres. Cellulosic chemical fibres are based on the physicochemi-
cal treatment of wood. Synthetic fibres derived from crude oil or gases are monomeric
molecules linked to polymers for higher molecular weight.
The process step of obtaining yarn is called ‘spinning’ and can be divided into primary
and secondary spinning. Within primary spinning, cellulosic or synthetic polymer granules
are melted or dissolved in solvents and subsequently extruded with a spinneret to obtain
filament fibres with almost infinite length. In contrast, natural fibres from wool or cotton
are of finite length. Secondary spinning is the process of achieving staple fibre yarns by
drawing and twisting both yarn types, natural and synthetic fibres. To obtain optimised
properties, blend yarns are produced by mixing natural and chemical fibres. To increase the
efficiency of weaving processes due to the high mechanical load, as well as to conserve the
quality of the fabric, warp yarns get a protective coating before weaving [21]. After the
weaving process, the surface structure of the fabric is pretreated for dyeing, then dyed and
printed. Following this procedure, if a desired property is not an inherent characteristic of
the fibre, the fabric obtains a finishing, such as making shirts or blouses easier for ironing
or making seat covers and carpets in aircraft or train compartments flame retardant [37].
The last processing step converts the fabric into the designed textile ready for further use.
Considering the value chain of natural fibres, it should be noted that the number of chemi-
cal processing steps and the amount of chemicals consumed (textile auxiliaries) is as high as
for the production of man‐made fibres. This is due to the requirements that textiles must
meet. Many properties required by customers (e.g. water‐ and dirt‐repellence, moisture
absorption, water resistance, elasticity, the feel of the fabric on the skin, and resistance to
heat, cold, bacteria, etc.) are adjusted by applying thin coatings within the textile finishing
process. These chemicals end up in wastewater and could pollute the environment. This
confirms the need to integrate a higher content of environment‐friendly raw materials, such
as renewable resources. For this reason, there is a trend towards using bio‐based materials
and products. At the same time, customers expect equal or even better performance and
properties substituting fossil‐based compounds. Chitosan has, as an abundant natural poly-
mer, the potential to substitute several petrol‐based agents while providing new properties to
the fabrics. To evaluate chitosan properties and to guarantee quality criteria for commercial
textiles, specific measurement methods are necessary to confirm the required performance.
16.3 General Test Methods

Precursor products (e.g. fibres, yarns) and textile fabrics undergo extensive checks to meet
the requirements and desired properties. The test methods in the textile industry are versa-
tile and adapted to the application of the final product. The same criteria need to be applied
for chitosan‐containing or chitosan‐treated fibres, yarns and fabrics. Table 16.1 summa-
rises the basic test methods used to check qualitative and quantitative parameters in the
production of a textile.
The majority of physical–chemical investigations is based on standardised test norms.
Not only do scientists and researchers use these methods to evaluate their products, but
textile manufacturers too are committed to performing these measurements routinely. The
methods are a decisive factor for the quality assurance system and are commonly per-
formed under controlled conditions, keeping environmental conditions constant to obtain
expressive data. Table 16.1 illustrates that tensile properties are important measurements
for fibres, yarns and fabrics, resulting in expressive values for breaking force, elongation
and tenacity. The values indicate the mechanical stress that textile products can withstand
in processing or successive applications. Flame‐retardation and dirt‐repellence investiga-
tions are unlisted, since these test methods are either more country‐specific, strongly
dependent on the application itself and/or greatly different in the operation procedure.
Furthermore, numerous tests exist with regard to a specific product group, especially inves-
tigating technical or functional textiles. In addition to commonly existing internal facilities to
evaluate quality, a rising number of external textile‐specific laboratories offer their services.
Table 16.1 A selection of test methods applied in the textile industry dependent on the material.
Test material Commonly applied measurements Test standard/norm Common units

Fibre (natural or synthetic) Composition of a blend ISO 1833‐1 %
Weight‐related fineness ISO 1973 dtex
Length DIN 53808‐1 mm/μm
Tensile Breaking force ISO 5079 cN
properties Elongation %
Tenacity cN/dtex
Yarn Fineness ISO 2060 dtex
Unevenness ISO 16549 U%
Tensile Breaking force ISO 2062 cN
Tenacity cN/dtex
Twist ISO 2061 turns/m
Fabric (dyed or Abrasion resistance ISO 12947 Martindale
functionalised) Contact angle and surface energy DIN 55660‐2 °, mN/m
Oil repellence AATCC 118‐2002 Numerical value
Tensile Breaking force ISO 13934‐1 cN
Tenacity cN/dtex
Water repellence 3M test method Numerical value
Abbreviations: ISO: International Organization for Standardization; DIN: German Industry Norm ; AATCC: American Norm.
They do not only perform textile‐ and clothing‐relevant measurements of fibres, yarns and
threads of woven, knitted, braid, and laid textile fabrics, but also carry out health‐relevant
investigations of textile medicinal products. This is especially important since the predomi-
nant property of chitosan – that is, being relevant for commercial use – is the result of
antimicrobial activity. On the one hand, antibacterial activity can be implemented by
finishing/coating the textile fabric at the end of the production chain. On the other hand, the
application of chitosan fibres and yarns offers the opportunity to implement these proper-
ties at the initial step of the value chain.
16.4 Fibres and Yarns from Chitin and Chitosan

Fibres are the basic module of every yarn and thus of each textile. According to Figure 16.1,
fibres can be either fossil‐ or natural‐based. Within the spinning process of natural fibres,
fibres are aligned (carding), stapled, twisted and subsequently manufactured into yarns
suitable for mechanical weaving or knitted into cloth. Man‐made fibres are processed by
another method. The first step is to solubilise or disperse the polymer to obtain the spinning
fluid. This also applies for chitin (see Section 16.4.1) and chitosan. The succeeding extru-
sion of the solution results in the formation of synthetic fibres. Different spinning processes
for chitosan fibre production have been described in literature and are summarised in
Section 16.4.2, whereas assessment with regard to the mechanical properties of fibres fol-
lows in Section 16.4.3. Lack of scientific data results particularly from the insolubility of
chitin in most solvents, and the mechanical properties thus cannot be discussed in this
chapter.
16.4.1 Chitin and Chitosan Solubilisation for Spinning Purposes

Spinning is the formation of fibres with polymer solutions. The first attempts to form chitin
fibres with the latter process were performed in the 1920s in order to obtain artificial silk
[38]. The need for previous solubilisation is mandatory but adverse since chitin is not solu-
ble in solvents that can be applied for cellulose dissolution such as Schweizer’s reagent or
cadoxen [39]. Chitin is soluble in solvents such as hexafluoroacetone and hexafluoroiso-
propanol (HFIP) [40]. Recent investigations revealed that chitin could be successfully solu-
bilised by the application of dimethylacetamide, N‐methyl‐2‐pyrrolidone or blends of both
solvents in the presence of LiCl too [41]. This fostered the development of a prospective
economically viable process, however the low number of related current research activities
shows that it is actually of little interest. The application of ionic liquids to dissolve poly-
saccharides not soluble in aqueous solutions enables the wet spinning of chitin, avoiding
the utilisation of toxic organic solvents but requires the application of these normally
expensive organic salts [42]. A further opportunity is to use organic or mineral acids at high
concentrations in conjunction with chloroalcohols to dissolute the chitin. It has to be stated
that dissolution in that case is always accompanied with degradation [20].
Obviously, chitin dissolution is the constraining factor for an economic spinning of
fibres since the suitable solvents are corrosive (strong mineral acids), toxic (dimethylaceta-
mide) or expensive (ionic liquids). Previous derivatisation of chitin, for example, with
perchloric acid and butyric anhydride to obtain dibutyryl chitin, is an alternative enabling
the solubilisation in more common solvents such as acetone [43, 44]. The derivatisation
provides a further processing step, needs additional time, produces waste streams and
consumes energy, and is thus not advantageous.
Due to the facts given earlier, chitin solubilisation and spinning is challenging. In con-
trast, chitosan spinning is the subject of several comprehensive research studies, although
chitin conversion to achieve chitosan also requires high concentrations of alkali and high
temperatures. This is owing to the functionality provided by the higher amount of primary
amine groups for chitosan, which is one of the major advantages of chitosan towards chitin.
Another reason is the solubility in diluted organic acids, such as lactic, formic or acetic
acid, or mineral acids at low concentrations. However, the alternative solvents used for
chitosan solubilisation are trifluoroacetic acid (TFA) and HFIP, since the usage of these
solvents results in uniform chitosan fibres after spinning. Blending aqueous chitosan solu-
tions with coadjutant polymers is one of the promising alternatives to increase solubility.
This avoids the application of harsh solvents and improves the spinnability of chitosan,
independent of the spinning process used, as explained in Section 16.4.2 [45].
16.4.2 Chitosan Spinning Processes

Several spinning techniques were already investigated and declared unfit or excluded in
advance, for example, melt spinning and dry spinning. Melt spinning comprises the melt-
ing of polymers, pressing it out through a nozzle and the resulting fibre formed by cooling.
It has the advantage of avoiding the application of solvents. Since there are strong interac-
tions between hydroxyl, acetamido and amino groups, decomposition of chitosan (and chi-
tin) occurs before reaching the melting point [46], excluding the application of melt
spinning [38]. The dry spinning technique is commonly used for polymers, which cannot
be melt spun. It utilises the extrusion of a polymer‐containing solvent with a spinneret,

whereas the liquid evaporates while impinging on a stream of hot air. Few investigations
are known with regard to the application of this technique for the production of chitosan
fibres. Notin et al. described a method called pseudo‐dry‐spinning, whereby the chitosan
solution streams in gaseous ammonia for coagulation. They neither used a cross‐linking
agent nor an organic solvent, as is commonly performed for chitosan spinning [47].
Besides these techniques, two other chitosan spinning processes with higher relevance
are described in the literature: electrospinning and wet spinning to obtain a pure fibre or a
blend with other compounds [20]. Wet spinning as a common and known method for chi-
tosan fibre production also involves dissolution in 1–10% (v/v) acetic acid; the application
of other acids is also possible. The solution is used to forward extrusion with a nozzle
submerged in an alkaline bath to precipitate chitosan in fibres [48]. The factors relevant
for the quality of the resulting chitosan fibres are: the coagulation bath concentration, the
kind of base and organic solvent additives, the spin‐stretch ratio, the type of organic acid
used for dissolution and the drying rate [49–51]. The most used technique for the forma-
tion of man‐made fibres is electrospinning, utilising electrostatic forces to obtain fine
fibres with diameters in the nano‐ and micro‐scale. This method utilises a high‐voltage
supplier forming an electrically charged polymer solution jet towards a collector which is
connected to an electrode of opposite charge or merely grounded. The solvent evaporates
before the jet impinges on the collector and forms the fibre [52]. An excellent overview
about the method, relevant parameters and applications can be obtained from Bhardwaj
and Kundu but is not part of this chapter [53]. Repulsive forces between the ionic groups
of chitosan hamper the formation of continuous and homogeneous fibres resulting in the
formation of beads [54] and there were several obstacles in order to obtain chitosan fibres.
A harsh acid is thus needed to form stable salts and to disturb the strong repulsive forces
between the amine groups. The first time that electrospinning was successfully applied
aiming to achieve pure chitosan fibres (see Figure 16.2) was in the year 2004, using trif-
luoroacetic acid and a small portion of dichloromethane as solvent [55]. Since that date,
researchers have focused on the use of electrospinning for chitosan fibre production,
whereas a combined electro‐wet‐spinning technique was developed, too, applying a volt-
age and coagulation bath [56].
Several investigations followed, setting a framework of relevant conditions and param-
eters to electrospin chitosan. Exceeding a certain viscosity and concentration is mandatory
for the formation of a Taylor cone at the tip of the spinneret and satisfying fibre quality [57,
58]. Chitosan solutions satisfy this requirement even at low concentrations. Otherwise,
high viscosities of chitosan solutions provide a challenge since they affect processibility
during extrusion requiring high voltages [59]. In order to circumvent the described draw-
backs, scientists further grafted chitosan or chitosan derivatives to improve spinnability.
Suitable derivatives include hexanoyl, PEGylated, and quaternized carboxymethyl chi-
tosan. For the same reason, several researchers investigated the application of chitosan
mixtures. The resulting blends with non‐ionic or anionic polymers, such as polyvinyl alco-
hol (PVA), polyethylene oxide (PEO), collagen, polyacrylamide, zein (a protein isolated
from maize), agarose and nylon [59, 60], increase the processibility. It can also be con-
cluded that blending chitosan with other materials is suitable to overcome much more
drawbacks [61]. Investigations indicated that pure chitosan fibres reveal some adverse
effects such as high moisture sensitivity and moderate mechanical properties.
(a) (b)
6 μm 6 μm
(c) (d)
6 μm 3 μm
Figure 16.2 Scanning electron microscope (SEM) figures of chitosan fibres obtained by solubilisation of
the polysaccharide in different solvent mixtures; (a) 90:10 v/v TFA/dichloromethane (DCM), (b) 80:20
v/v TFA/DCM, (c) 70:30 v/v TFA/DCM (5000x magnified), (d) 70:30 v/v TFA/DCM (10,000x magnified).
Reprinted from Ohkawa et al.[55] with permission from Wiley.
16.4.3 Mechanical Properties of Chitosan Fibres/Yarns

The mechanical properties of fibres are of high relevance for the application of the ready
textile as well as the load applied during processing. As one of the first process steps, fibres
must form a linear continuous strand, the yarn. Monofilament or multifilament yarns are
manufactured by endless man‐made fibres, commonly exhibiting high durability and
strength. Except silk, natural raw materials such as cotton or wool provide fibres spun into
a staple yarn, showing characteristics that depend on the spinning process and its parame-
ters. The properties greatly differ with the geometrical and physical arrangement of fibres
along the yarn and the cross‐section. Hence, mechanical properties are affected by the
chemical nature or structure of the underlying polymer, but are moreover determined by the
spinning conditions [62]. However, different polymers, whether man‐made or natural,
reveal typical value ranges of the mechanical properties of fibres and yarns. Electrospun or
wet‐spun chitosan fibres and yarns thereof give a range of characteristic values as dis-
cussed in the following sections.
In general, chitosan fibres have similar characteristics to that of viscose fibres but with
slightly lower tenacity values and higher moisture regain [63]. The high moisture regain
(16.2%) indicated a strong hygroscopic behaviour as for wool, resulting in an increased

dye adsorption and improved wearing comfort of the textile. However, humidity of the
fibre also affects the physical and mechanical properties such as tenacity and elongation.
Fineness is one of the most important fibre characteristics determining how many fibres are
in the cross‐section of a yarn at a given thickness. Lee described fineness values for chi-
tosan fibres of 1.4–1.7 dtex, which is in the range of cotton, silk or casein, representing fine
to medium fibres [64]. However, fineness is a property which can be easily adjusted by
spinning conditions. Tenacity is a further relevant fibre property, which is related to the
fineness of the fibre and depends on the proportion of crystalline or amorphous regions.
Typical chitosan fibres have tenacities of about 1–2 cN/dtex, whereas dry fibres exhibit
higher tenacities than wet ones. Young (initial) moduli of chitosan fibres are described to
be in the range of 30–84 cN/dtex [65, 66]. This means that high stress needs to be applied
to deform the fibre in comparison to nylon with values of 9–35 cN/dtex [67]. This also
applies for the breaking extension reaching values of ~10% varying with the moisture con-
tent of the fibre [64]. Besides spinning conditions, influencing parameters such as molecu-
lar weight or DA are mainly responsible for the uneven data obtained from literature.
Increasing DA results in higher fibre strength due to enhanced intermolecular forces [38,
65]. The increase is based on decreased repelling forces and higher crystallinity as it is for
chitin fibres [41]. Increasing molecular weight results in improved tenacity values [50]. In
contrast to this assumption, chitin fibres exhibit comparable or even lower values, espe-
cially in the wet state [68]. The moderate values regarding the mechanical properties of
chitosan fibres impeded a broad application and led to further research activities to circum-
vent this drawback. Various approaches could or have been taken:
1. Modification of chitosan prior to or during spinning: It can be assumed that the graft-
ing, especially at the free primary amine groups, affects the properties, since they are
particularly responsible for chitosan characteristics. There are only a few studies per-
formed regarding the spinning of derivatised chitosan, but the majority of the investiga-
tions concentrate on electron microscopy pictures or medicinal or biological tests rather
than investigation of the tensile properties [69, 70]. Electrospinning of PEGylated or
carboxymethyl chitosan is reported to be challenging and could only be performed in
the presence of auxiliaries [71, 72].
2. Adaption of spinning conditions: There are various tuning parameters to adjust either
wet or dry spinning in order to obtain chitosan fibres with optimised properties.
However, comprehensive studies focusing on the systematic optimisation of spinning
parameters have not been carried out yet. Using ionic liquids rather than acetic acid as
the chitosan solvent increases the tenacity by about four times, emphasising the poten-
tial of adapting the spinning conditions [73]. Post‐spinning drying is also considered as
a relevant process step to influence the mechanical properties of chitosan fibres.
Researchers confirmed that drying with methanol, based on the increase in the degree
of crystallinity, improves tensile strength and biodegradation resistance [74].
3. Post‐modification of chitosan fibres: Cross‐linking is an effective way to modulate the
properties. Glyoxal or glutardialdehyde application led to an improvement of tenacity
from 1 to 2 cN/dtex [75, 76]. Treatment with acyl anhydrides is an alternative method
to hydrophobise chitosan fibres. This revealed decreased strength but increased elonga-
tion value with increasing chain length of the acyl chain [77]. Post‐modification does
not necessarily result in altered properties. Constant or even worse properties were
reported with regard to breaking elongation and tenacity after derivatising chitosan
fibres with acrylic acid [78].
4. Admixture of other polymers in the spinning dope (co‐spinning): Different polymers
could be solubilised together in a suitable solvent, enabling the production of fibres
with properties completely different from the fibres derived from pure compounds.
Zheng et al. solubilised PVA and chitosan in an acetic acid solution containing urea.
The dry and wet tensile strengths of PVA/chitosan fibres were 10–40% higher than that
of pure chitosan [79]. There are other known studies that utilise the wet‐spinning tech-
nique to process chitosan with molecules such as heparin or cellulose. This has led to
the formation of bio‐filaments, providing a holistic renewable approach. Yan et al.
observed that a spike in chitin nanocrystals in the chitosan spinning dope raised tenac-
ity and young modulus by 54% and 85%, respectively [80].
5. Applying chitosan fibres with other polymer fibres to gain blended yarns: The merits
pointed out to blend two polymers as stated earlier apply here in the same way. Blending
chitosan with polyacrylonitrile fibres, while providing a non‐sustainable fibre, showed
superior yarn performance and minimised wastage [81]. Lam et al. investigated the pro-
duction of chitosan/cotton blend yarns and evaluated their mechanical properties. They
stated that the mechanical properties of the resulting yarns can be optimised by applying
chitosan fibres of shorter length and blending them with the sliver technique [82].
As can be obtained from the quoted examples, mechanical properties of chitosan fibres
can be affected and improved by several techniques. Nevertheless, the tensile strengths of
chitosan fibres or fibre blends with a major content of chitosan are at a disadvantage as
compared to other man‐made fibres, although the potential for improvement has not yet
been fully exploited and should be part of prospective research projects. Based on this
reason, few chitosan fibres or blends thereof are currently commercialised, for example
Crabyon (Swicofil), Sunray (Hismer Bio‐technology Co. Ltd) and Ultex (Shanghai
Nantec), and predominantly offered for special applications. To strengthen the turnover, the
following challenge needs to be overcome: developing an economic process and material
with higher mechanical properties.
16.5 Sizing with Chitosan

Besides the spinning process, tensile properties play a big role within the weaving process.
Here, providing a mechanical load stress in combination with high abrasion results in
yarn breakage. To avoid this, a sizing agent must be applied. This topic will be described
in this chapter.
To protect the yarn during the automated weaving process from abrasion, breakage,
increased hairiness and clinging tendency, a sizing agent is applied. Sizing agents protect
the yarn due to their ability to form a film. Using a sizing agent makes the warp threads
more resistant to mechanical loads such as thread–thread friction, thread–metal friction,
cyclical tensile stress and bending. The application of the sizing agent provides surface
protection, enabling the spun yarns to resist mechanical loads by adhering to protruding
fibres and/or smoothing yarns. In the case of filament yarns, the main aim is to tie the fila-
ments together [40]. Besides film‐forming macromolecules, the size potentially contains
viscosity regulators, sizing fats, wetting agents, defoamers and preservatives [83].
The majority of sizing agents contain fossil‐based polymers like PVA, PAC and PES or
biopolymer‐like starch‐based compounds. Starch and its derivatives are the predominant
sizing agents of earlier times, but their manufacturing competes with food production.
Furthermore, they are not very effective in the current applied high‐speed weaving looms.
Hence, chitosan as a polysaccharide from renewable resources exhibiting film‐forming and
biodegradation properties could be an alternative. An advantage is that chitosan has limited
valuable use in the food or feed industry. The competitive pressure within the textile indus-
try forces, besides the textile material, the use of cheap auxiliaries and reagents. However,
at the same time, customers demand more sustainable products. Sustainability of chitosan
is proven but the costs are nowadays higher as compared to conventional sizing agents.
Thus, it should show superior characteristics while, as with all other sizing agents, meeting
the following criteria [83, 84]:
• The liquid should have high wetting ability and appropriate surface tension with regard
to the applied yarn.
• The ability to form a homogeneous and closed film on the warp thread unaffected by
different fibre finishes is mandatory. Additionally, the film‐forming property should not
be influenced by atmospheric conditions.
• The resulting film should have a certain elasticity so that no breakage of the film occurs
during the various stretching and bending stresses of the weaving process.
• A high adhesive power is required to stick the protruding fibre ends to the yarn surface,
resulting in the prevention of friction during the weaving process.
• A significant reduction of the warp thread elongation by the sizing agent is not
required.
Chitosan‐specific requirements can be summarised in five statements as follows:
1. For economic reasons, chitosan should be miscible with conventional, inexpensive siz-
ing agents and additives. It should be possible to produce homogeneous solutions.
2. To ensure good workability in standard sizing boxes, the sizing liquid should have, at
maximum, a viscosity of 200 mPa·s (at a shear rate of 500 s−1).
3. In order to achieve good solubility and film formation as well as suitable viscosity in
the solution, the DDA should be at least 80%.
4. Easy removal of chitosan after weaving is highly demanded. This is negligible if chi-
tosan does not affect further processing of the fabric.
5. Ash content should be lower than 1% (w/w) of the solid in order to achieve high adhe-
sive power.
The general and specific requirements for chitosan will be discussed in detail in the fol-
lowing text.
16.5.1 Miscibility of Chitosan with Other Sizing Agents

Usually, a sizing agent is a formulation of different compounds and contains a couple of
different chemicals. All of them need to be miscible and show suitable film‐forming prop-
erties. To achieve a significant market share, on the other hand, chitosan should improve
the functionalities and reduce sizing costs. One aim could be lowering chitosan concentra-
tion within sizing formulation while providing a robust size liquid avoiding the formation
of precipitates, the phase separation during long‐time storage. Since chitosan is a polymer
whose application is demonstrated as a coagulation/flocculation auxiliary in wastewater
treatment, it readily forms precipitates, especially with anionic compounds. Renault et al.
gives an excellent overview about the chitosan coagulation/flocculation application with a
variety of compounds stating the effective interaction between them [85]. However, inves-
tigations in blending pure starch and chitosan revealed miscibility over the entire range
measured [86]. Sudhakar et al. indicated positive interactions only up to 40% starch content
in the blend but stated a significant temperature effect [87]. A German research project
investigated the miscibility of chitosan with different sizing agents [88]. Studies performed
by the authors indicated that there are incompatibilities with carboxymethylated starch,
carboxymethyl cellulose, PVA and polyacrylate. In combination with other selected modi-
fied starch solutions, chitosan application is possible. Nevertheless, preliminary miscibility
investigations in a long‐term test are a prerequisite to evaluate the performance and exclude
undesired effects while sizing. Another fact to be considered while applying different com-
pounds in an approach is that the molecular interaction could result in high‐viscosity solu-
tions aggravating the application. Besides, viscosity is also affected by several other factors,
as described in the following chapter.
16.5.2 Viscosity of Chitosan‐Containing Sizing Agents

Viscosity of the liquid plays a major role for application in the sizing box as well as in the
wetting behaviour of the sizing liquid. The processing of highly viscous liquids is not prac-
ticable and requires increased pumping pressures. The high viscosity of the sizing liquid
results in a robust film and protection around the yarn. The sizing agent adheres the fibres
together since the liquid cannot penetrate the fibres [89]. In contrast, low viscosity decreases
jacket film thickness [90] and allows the size to penetrate into the core of the yarn, not
forming the necessary protection layer on the surface. Furthermore, there is an increased
likelihood that yarn breakage occurs during sizing due to hydrodynamic forces in the gap
of the squeezing rollers used to wring out the liquid after sizing. As a consequence, the
viscosity of the sizing solution has to be adapted to the properties of the yarn. The param-
eters influencing the viscosity of chitosan solutions are:
• Molecular weight: The higher the molecular weight of chitosan, the higher the viscosity
(see Figure 16.3a). In a common conversion process, chitosan is produced from chitin by
using high concentrations of caustic soda. This leads to low molecular weight (LMW).
• Acetylation degree: This is a parameter strongly determining the viscosity of chitosan,
and the viscosity increases with a higher degree of acetylation. The opposite applies in
the presence of high salt concentrations [91].
• Acetic acid concentration: Increasing acetic acid volume at low concentrations decreases
the viscosity of chitosan. In contrast, increasing acetic acid volume at high concentra-
tions increases viscosity [39].
• Concentration: A higher chitosan content leads to higher viscosity with non‐Newtonian
behaviour (see Figure 16.3a). The viscosity at a given shear rate increases exponentially
with increasing concentration. According to the graph, the manageable concentration of
commercially available chitosans with average molar masses is 2–3% (w/v) at
maximum.
(a) (b)
400 400
350 350
LMW Chitosan
300 HMW Chitosan 300
Viscosity [mPa·s]
Viscosity [mPa·s]
250 250
Threshold value
200 200
3%
150 150
2.5%
100 100 2%
50 50 1.5%
1%
0 0
0.5 1.0 1.5 2.0 2.5 3.0 500 1000 1500 2000 2500 3000
Concentration [%] Shear rate [s–1]
(c) (d)
90 80
80
70
70
Viscosity [mPa·s]
Viscosity [mPa·s]
60
60
50
40 50
30
40
20
10
30 40 50 60 70 80 90 0 1 2 3 4 5
Temperature [°C] Week
Figure 16.3 (a) Viscosity of low‐molecular‐weight (LMW) and high‐molecular‐weight (HMW) crab shell chitosan in 2% acetic acid at different concentrations
(% w/v) and at a constant shear rate of 500 s−1; (b) viscosity of LMW chitosan in 2% acetic acid at different chitosan concentrations as a function of the shear
rate; (c) viscosity of 2% LMW chitosan in 2% acetic acid as a function of the temperature (at 500 s−1); and (d) viscosity of 2% LMW chitosan solution in 2%
acetic acid at 500 s−1 as a function of the storing time. LMW chitosan: deacetylation degree (DDA) = 90%; HMW chitosan: DDA = 85%, measured with cone‐
plate rheometer.
• Shear rate: Chitosan displays a pseudoplastic (shear‐thinning) behaviour, which is illus-

trated in Figure 16.3b. The viscosity decreases with increased shear rate. This applies all
the more for higher‐concentration chitosan solutions than for lower‐concentration ones.
High shear stress at high weaving rates thus leads to a lower film thickness and
reorientation of the chitosan molecules.
• Temperature: The viscosity of chitosan solutions decrease with increasing temperature
as shown in Figure 16.3c. This is due to the distortion of intermolecular interactions, and
is important, since sizing agents are commonly applied at 85–95°C.
• Storing time: Studies from Nguyen et al. proved that the viscosity of chitosan decreases
with ongoing storage time. This was confirmed by our investigations (see Figure 16.3d).
The decrease is based on a first‐order rate hydrolysis of chitosan chains, whereas acid
concentration has no significant effect [92].
As with several other gel‐forming polysaccharides, chitosan exhibits significant viscos-
ity even at low concentrations. The viscosity of chitosan‐containing solutions can be sig-
nificantly modified by the application of LMW chitosan rather than HMW chitosan. Ageing
of chitosan in solution even reduces the viscosity values. The authors assume that there is
optimum molecular weight providing suitable viscosity while maintaining (especially) the
film‐forming properties. The film formation itself solely depends on the molecular forces
between chitosan chains, and the magnitude of interaction to the yarn material is deter-
mined by the adhesive strength.
16.5.3 Adhesion and Wetting

A high adhesion strength of the sizing agent with different fibres is necessary. If the adhe-
sion of the film to the fibre is too low, increased abrasion occurs when the yarn is subjected
to load by deflection or weft thread insertion. In general, good adhesives exhibit properties
such as elevated viscosity and elasticity of the resulting films, as well as high adhesive
power and strong cohesive forces at the same time.
Proper wetting of the fibre surface by the sizing agent is a prerequisite for film formation
and tight adhesion. In general, to achieve suitable wetting surface tension of the sizing
agent, it should be lower than the surface free energy of the fibre material. A technique
commonly used for the determination of the wetting ability is the contact angle measure-
ment. A study revealed that the contact angles of water drops on chitosan films ranged from
50° to 100° in a given time span, depending on the nature of chitosan. The decrease of the
contact angle with increasing DDA emphasises that hydrophilicity is enhanced with higher
amounts of available primary amine groups, as well as with the mounting protonation of
these functionalities [93]. The wettability of chitosan‐containing sizes is also dependent on
external impurities and other additives of the size liquid. Alternatively, additives, for exam-
ple surfactants, emulsions of paraffin and polyethylene waxes or other surface‐active com-
pounds, could counteract poor wetting, leading to low contact angles [94]. The application
of additives decreases the surface tension of the liquid below the surface free energy of the
fibre surface if required for the spreading of the sizing agent.
The surface tension of the liquid and surface free energy of the substrate have a decisive
influence on wetting as well as on the strength of adhesion. A good adhesion distinguishes
itself by the contributions of polar and dispersive forces that are in a comparable range to
the polar and dispersive proportions of the material’s surface free energy. A low difference
between the surface free energy of the solid and surface tension of the liquid is, in general,
beneficial. Concerning the surface free energy of chitosan films, literature offers opposing
data. On the one hand, there are investigations reporting surface free energy of the films
from 36 to 41 mN·m−1. Based on this value, chitosan has a suitable adhesion to surfaces
with low surface free energy [95, 96]. Other studies revealed high surface tension of chi-
tosan solutions (65–75 mN·m−1) [97, 98]. The differences are likely related to various
measurement methods, water content of the films, concentration or the physical–chemical
properties of the polysaccharide applied. With regard to these inconsistencies, Cunha et al.
agreed that nonpolar impurities greatly affect measurement and result in high errors, con-
cluding that a high purification degree is essential for performance. Examining a film com-
posed of pure chitosan resulted in a surface free energy of 60 mN·m−1 [99]. As is the case
with other polymer solutions, the surface tension of chitosan solutions also increase with
increasing concentration and molecular weight of the chitosan used [100]. Chitosan is a
known, experimentally proven, well‐performing adhesive due to its positively charged
amino groups that can interact with negative surface charges via electrostatic forces exhib-
iting a high amount of polar forces. The contribution of hydrogen bonds and van der Waals
forces to the adhesive strength was also described, together with strong cohesive interac-
tions in acidic conditions [101].
The aforementioned properties resulted in the commercialisation of chitosan adhe-
sives for biomedical applications, as reported by Mati‐Baouche et al. [102]. In contrast,
only a few commercial sizing agents contain chitosan, although there are major advan-
tages as compared to conventional starch sizing, for example, of cotton. It can be assumed
that the adhesion of chitosan to cotton is probably due to the structural similarity with
cellulose and/or the opposite charge of the cotton derived by residual pectins or waxes.
However, high adhesion power in comparison to conventional PVA is also confirmed by
own investigations. Furthermore, the tensile strength of the yarn is also improved by the
increased adhesion of individual fibres to each other. The extent of improvement and
protection efficiency depends on the mechanical properties of the films formed in the
sizing process.
16.5.4 Mechanical–Physical Properties of Chitosan Films

Elasticity, tensile strength, and swelling behaviour are relevant film parameters and have a
great impact on the overall protection efficiency of the film on the yarn. Poor mechanical
properties lead to cracks and fissures in the film during the many stretching and bending
processes, thus leading to reduced protection efficiency and increased abrasion of the yarn.
Chitosan films were focused on in several studies concerning their physical–mechanical
properties, albeit not with regard to textile processing. The high cohesive strength of chi-
tosan films, which is a requirement for a well‐performing adhesive and sizing agent (see
Section 16.5.3), is proven by high shear modulus [103]. Furthermore, field literature
reported chitosan films revealing elongation values from 2.5 to 100% until breakage occurs,
whereas tensile strength is in the range of 2.5–27 MPa [104–107]. According to the values
given, both parameters are in a very broad range and can thus not be compared to films
produced by other biopolymers. It can be assumed that the variety depends on the quantity
of further compounds contained in the chitosan film. Souza et al. measured superior
mechanical properties since it has very high tensile modulus, high tensile strength and low
elongation values at low moisture content (10–15%). An improvement in the mechanical
properties can also be achieved by the application of plasticizers, such as, for example,
glycols, glycerol, sorbitol, sucrose and citrate [108]. At higher moisture content, modulus
and tensile strength decrease, whereas elongation until breakage greatly increases [109].
The same obviously applies for the organic acid concentration in the films previously
applied for acidification of the solution in order to solubilise chitosan. The properties are
altered in an order of the power of ten [110]. Hence, the protectability of the sizing agent
cannot necessarily be determined by the mechanical properties of chitosan films, but is
rather based on application‐specific trials, whereas the sizing agent demonstrated to be
eligible for this purpose. This also holds true for the water uptake behaviour with swelling
ratios measured in a range from 190 to 500% [104, 107], which is a crucial property for the
removal of the film after weaving (desizing) [111].
16.5.5 Removal and Processing of Chitosan Sizing after Weaving

Sizing is exclusively used to protect the warp yarn from mechanical loads during the weav-
ing process, and is commonly removed immediately after this. A washout of the sizing
agent is required to ensure that the subsequent finishing or dyeing process will not be dis-
turbed. Another reason for the removal is that the size has usually a significant effect on the
handle and affects the textile character of the product. Synthetic sizes require removal by
using hot water, whereas starch‐based desizing is carried out with enzymes, such as amyl-
ases [112]. The enzymatic desizing of starch‐sized cotton is a success story and part of
many investigations [113]. Besides, there are still further techniques that are applied: rot,
alkali or acid steeping (hydrolytic methods); and bromide, chlorite or ammonium persul-
phate desizing (oxidative methods).
Chitosan removal efficiency depends on the fibre material sized and the adhesion forces
between these. The desizing of synthetic fibres takes only a few minutes with slightly acidic
water. The chitosan layer around the synthetic fibre swells, solubilises in the acid, is removed
and the washing solvent reused. In contrast, the removal of chitosan from cellulose fibres
requires much more effort. In general, a total desizing is not feasible within a few washing steps.
Hebeish et al. confirmed the requirement to use 90°C hot water, resulting in removal efficiencies
of 75–100% for hydrolysed and carboxymethyl chitosan from cotton [22]. The need for high
temperatures is attributed to the formation of specific hydrogen bonds and undefined ion–dipole
interactions between the compounds, since anionic charges on the cotton surface exist [114,
115]. The anionic charges are based on fatty acids, hemicelluloses or pectins, which are also
contained in the cotton network or due to the oxidation of cellulose [116]. Furthermore,
there are other non‐ionic mechanisms present, since studies revealed that chitosan adsorp-
tion is maximised by pH values of ≥7, resulting in the deprotonation of chitosan [39].
Investigations confirmed that the application of chitosan‐degrading enzymes is favourable
for the removal of chitosan. These enzymes hydrolyse the adsorbed chitosan, resulting in deg-
radation to oligosaccharides or even monosaccharides and removal of the majority of chitosan.
However, a minor part of the chitosan remains on the textile. Figure 16.4 illustrates these points.
It requires extensive studies to elucidate the impact of residual adsorbed chitosan in the
following process steps. The effects of residual chitosan on the surface of the fabric in the
subsequent process steps were as follows:
• Impact on bleaching: Investigations revealed that bleaching subsequent to desizing
affords the same degree of whiteness independent of whether residual chitosan is present
After desizing After enzymatic

Without sizing After sizing desizing
with acidic water
Polyester
Cotton
Figure 16.4 Fabrics treated with ninhydrin, a chitosan‐specific dye. In the original state, ninhydrin gives
only a slight colour. After sizing with 1% (w/v) chitosan, the dark green colour is recognisable, indicating
the presence of adsorbed chitosan. Desizing the polyester fabric with acidic water results in the complete
removal of chitosan, whereas cotton still exhibits an excessive colouration. In comparison, enzymatic
desizing of chitosan‐sized cotton revealed a slightly lower colouration, indicating the presence of residual
chitosan on the fabric.
on the cotton surface or not. However, it must be stated that optical brighteners were not
used for the trials, since their anionic character could result in a strong electrostatic
interaction with the positively charged chitosan.
• Impact on dyeing: After desizing, it was possible to dye the polyester content of the
mixed fabric (polyester–cotton) without any problems. The single yarns of the mixed
fabric exhibited a comparable colouration. Chitosan‐sized cotton showed a slightly
deeper colouration after dyeing with direct dyes. Concerning reactive dyes, there were
no differences between chitosan‐sized cotton and cotton sized with usually applied
agents, for example, starch. The chitosan hardly affected the fastness of the dyeing.
• Impact on the fabric handle: No significant differences were determined concerning the
handle between fabrics sized with chitosan or with conventional sizing liquid after
desizing.
• Impact on the finishing process: Chitosan‐sized fabrics subjected to high‐grade finish-
ing (easy‐care finishing) exhibited slightly lower tensile strength and wrinkle recovery
angle. The impact of residual chitosan on fluorocarbon finishing was marginal.
As can be seen from the preceding text, the influence of chitosan on the subsequent pro-
cessing is low in general, even if chitosan washout was not completely performed. On the
other hand, if dyeing with acidic agents, chitosan should be completely separated to avoid
interaction. Further investigations must be carried out for this purpose. To the best of the
authors’ knowledge, comprehensive studies on performing a total removal of chitosan from
a cotton surface via enzymes are lacking. If not treated with enzymes, the removal solution
could theoretically be recycled to obtain a closed sizing loop.
Chitosan in its native form is a biodegradable, non‐toxic polysaccharide. However,
uncontrolled release into the wastewater system or the environment needs to be avoided,
since it is still expensive as compared to other polymers, and adverse effects on the
icrobiota cannot be excluded since it provides antibacterial effects. Furthermore, desizing

m
effluents, for example, those used to remove starch from the yarn, are responsible for
approximately 50% of the organic load in wastewater from wet processing in textile com-
panies, representing a substantial load for the sewage plants [117]. The recovery of chitosan
is thus more promising and the most economical solution rather than wasting chitosan with
the effluents. This obviously demands the application of non‐degrading or non‐modifying
techniques that enable the desizing and the subsequent recovery.
16.6 Chitosan as a Finishing Agent or Coating

Knitted, woven or non‐woven fabrics are subjected to the final steps of the textile value
chain – that is, pretreatment (washing, bleaching), colouration (dyeing, printing) and fin-
ishing. Although the term ’finishing’ is also used for all processes as a whole after the
fabric leaves the loom or knitting machine, we solely refer to the finishing process as the
very last step to produce the ready‐made textile [118]. Finishing gives the textile the desired
properties. The process can be carried out by four different techniques: physical, mechani-
cal, enzymatic and chemical processes. Commercial enzyme finishing is confined to
cellulase application to obtain a ’stonewashed’ look. Further studies investigated the modi-
fication of polyethylene terephthalate with cutinases, the scouring of vegetable and bast
fibres with pectinases, the treatment of wool and leather with transglutaminases and the
application of nitrilases or nitrile hydratases for polyacrylonitrile cross‐linking [119, 120].
Physical and mechanical finishing steps (also referred to as ‘dry finishing’), such as cutting,
brushing, heating or pressing, are commonly combined with and increase the efficiency of
the chemical processes (wet finishing).
The term ’finishing’ is often confused with ’coating’, and is thus unfortunately used
inconsistently in scientific literature, but precision is necessary and possible. The chemical
textile finishing differs from the so‐called coating. Finishing changes the physical proper-
ties, but does not change the visual impression of the carrier material. The finishing com-
pound covers only the individual fibres, whereby the pores of the textiles are usually not
closed as it is in the case for coating. Textile coating is the application of a visible plastic
layer to a textile substrate (carrier material). Basically, pastes or foams consisting of poly-
mers are applied for coating. In contrast, the parent process to apply a wet finish is the
pad–dry–cure process. The textile passes a bath containing the finishing agent, is then dried
and afterwards cured with heat or pressure to fix the chemicals or to start a cross‐linking
reaction. Up to now, it has largely been state of the art, primarily due to the traditional
production of clothing textiles, to process non‐functionalised yarns and only to produce the
desired functionalities in textile fabrics by suitable finishing and coating processes.
However, this approach reaches technical and economic limits if more complex structures
or combinations of functions are to be achieved. The processing of functionalised yarns,
which already carry important and desired functions in the final product, can overcome
these limits of textile functionalisation. The application methods of yarn functionalisation
are manifold and have developed further in recent years.
In the following, we refer to both finishing and coating treatments, without providing the
process details, since it is the chemical reaction from a molecular point of view we would
like to focus on. The chemical reaction forms the basis of the process, whereas the applica-
tion is the desired outcome. During a chemical finishing process, the chemical compound
is linked, whether covalently, ionic or via non‐ionic interactions, to the textile, conferring
the following properties [121, 122]:
• Wrinkle resistance or resiliency for cellulosic materials (durable‐press finish)
• Soil, oil and water repellence
• Chemical resistance
• Durable cleanability (soil release)
• Self‐cleaning ability
• Shrink‐proofing of wool and cellulose
• Pilling, abrasion and wear resistance
• Antistatic properties
• Resistance to UV light, heat and pollutants
• Minimising ironing or pressing (easy‐care finish)
• Resistance towards microorganisms and insects (especially wool fibres)
• Flame retardancy
• Thermal conductivity
The increasing trend towards the functionalisation of textile surfaces also sometimes
requires that textiles possess several such functionalities. The functional coating of techni-
cal textiles is usually based on the use of synthetic chemical auxiliaries based on polyure-
thanes, polyacrylates, polyvinyl chloride, silicone elastomers and fluoropolymers, which
are dispersed in water. However, Holme already stated, in the 1990s, that prospective com-
pounds should meet the demand for green finishing agents, specifically for ease of applica-
tion on automated machinery [123]. Since some of the properties listed are high‐tech
applications, the use of natural heterogeneous materials is not suitable for all of these func-
tionalities. Nevertheless, chitosan was already investigated for use in finishing formula-
tions, and revealed the most promising results concerning antibacterial or wrinkle‐free
finishes, but is not restricted to these [124]. As follows, chitosan is contained in several
coating or finishing formulations in order to modify the surface of textiles. Two options for
using chitosan in surface modification formulations are conceivable: on the one hand, the
application as an active agent conferring the desired property to the textile; and, on the
other hand, as a compound supporting the efficiency, respectively, of the release of active
agents or of performing as a carrier material. A sole use as one or the other is normally not
possible independently of each other, but the definition of a focus is. Both application pur-
poses will be explained in detail in the text that follows.
16.6.1 Chitosan as a Carrier and Linker

The incorporation of bioactive ingredients in microcapsules for application on textiles is to
control release or increase stability. Chitosan‐based capsules are already known and cur-
rently under investigation. The capsules are commonly prepared by the coacervation tech-
nique, dispersing the core material in the coating material, followed by rigidisation of the
coating material due to immiscibility in a certain phase. Different agents were incorporated
into chitosan‐containing microcapsules. Limonene essential oil is an antibacterial ingredi-
ent investigated for controlled release by chitosan microcapsules after impregnation of
cotton. The authors reported that chitosan capsules enabled the controlled release of oil,
and identified volatility as a parameter which can be adapted by the formation conditions [36].
Roy et al. highlighted the tuning of the active ingredient release by the layer‐by‐layer for-
mation of chitosan/carboxymethyl cellulose microcapsules and the succeeding application
of glutardialdehyde [125]. The impregnation of the textile with the capsules commonly
results in weak interactions. Thus, researchers grafted chitosan/gelatine microcapsules
onto a cotton fabric with the help of dimethylol dihydroxy ethylene urea. Herein, it was
demonstrated that patchouli oil in the core of the microcapsules also develops a significant
antibacterial effect, even after several wash cycles [126]. Ionic gelation with sodium trip-
olyphosphate (TPP) is a method to produce chitosan‐containing nanocapsules. The authors
described the incorporation of rose fragrance in the core of these particles as enhancing the
permanence on cotton fabrics [127]. Another study has focused on the use of microcapsules
with shell material based on TPP and chitosan formed by ionic gelation and grafting.
Incorporated ingredients such as rosehip oil and ceramide greatly increased the antibacte-
rial activity of the cotton fabric [128]. The same applies for Ag/ZnO particles embedded in
a chitosan matrix, resulting in a highly active antibacterial finishing agent for a cotton/
polyester blend [129].
In conclusion, mainly oils were investigated for incorporation into the capsules. The
release of active ingredients by chitosan capsules can be steered by the encapsulation pro-
cess itself derived by adaption to the conditions. A slow release is often desired, whereas
some applications require permanent immobilisation of the functional molecule. Chitosan
itself has a high affinity to, for example, cotton, resulting in tight adsorption (see
Section 16.5.5), but covalent bonds are usually necessary to implement a permanent effect
on the textile.
16.6.2 Formation of a Durable Finish with Chitosan

Chitosan is a multifunctional molecule with primary amino groups offering suitable bind-
ing sites to act as a carrier material by linkage to the agent with the desired properties. For
stable immobilisation and high washing stability of chitosan on the fabric, a chemical cova-
lent bond or permanent linkage is required. Here, a chemical reaction partner needs to be
available on the molecules within the fabric or yarn. Hence, derivatisation of the chitosan
is carried out to overcome these challenges [130]. The possible agents used for the fixation
or permanent finishing of chitosan include cyanuric chloride. Investigations proved the
antibacterial activity after reaction, but indicated the loss of bioactivity with succeeding
washing steps. Potassium periodate treatment of cellulose fibres under acidic conditions
forms aldehyde groups that can react with the amino groups of chitosan to Schiff bases
[131]. 1,2,3,4‐butanetetracarboxylic acid is an agent that enables both the coupling to cel-
lulose and the intramolecular cross‐linking of chitosan [132]. Citric acid, as tricarboxylic
acid, is one of the natural cross‐linking agents, substituting formaldehyde or glutardialdehyde
cross‐linkers to prevent chitosan leaching during several laundering cycles by covalent link-
ages to cotton [133]. Moreover, citric acid is proven to exhibit antibacterial activity alone,
supporting chitosan properties [134]. The majority of chitosan applications concerning the
treatment of cotton in particular is described in literature. Few other investigations focus on the
treatment of other raw materials such as wool or silk. For example, Pascual and Julià studied
the finishing of oxidised wool. They stated that the previous hydrogen peroxide treatment
generates anionic groups which result in a strong ionic interaction with chitosan [135].
A further promising grafting technique of wool with a bio‐based linker is described by

Ranjbar‐Mohammadi et al. [136]. They applied succinic anhydride to link chitosan to the
wool. The relatively slow reaction could be accelerated by the application of ultrasonic
waves, providing a technique which uses the efficiency‐enhancing effect of physical meth-
ods to covalently link biological products [137].
16.6.3 Chitosan as an Active Agent

The activity of an agent is strongly related to its concentration on the surface of the fabric.
Also, the formation of a covalent linkage between chitosan and the fabric alters the effec-
tiveness of the compound. The amino functions which are commonly used for the linkage
to resins are mainly responsible for the properties of chitosan. Therefore, a compromise
must be found between the degree of cross‐linking and free amino bonds in order to imple-
ment the properties permanently on the textile. The positive properties of chitosan applied
as refinement or finishing are explained in the following text.
16.6.3.1 Antibacterial Coating

Chitosan as an active coating or finishing agent particularly focuses on the antibacterial
activity. The antibacterial effect of chitosan is not well understood, but has been proven in
numerous studies and is part of several reviews [138, 139]. Interestingly, only a few chi-
tosan‐containing formulations are currently on the market to render a textile antibacterial.
This is partially owing to the pH‐sensitivity of chitosan revealing lower antibacteriality
at higher pH values [140]. Common commercially available antibacterial textiles utilise
metals, metal salts, quaternary ammonium compounds, PHMB and triclosan [141].
However, there are a few products already utilising chitosan or chitosan‐containing coat-
ings, especially for fibres and fabrics with application in the medical field or in imple-
menting skin‐beneficial properties. The Austrian company Lenzing has brought out a fibre
to the market, called Tencel® C, that leads to improved moisture retention, skin elasticity
and cell renewal [142]. The chitosan‐containing finishing agent Eosy® is produced and
distributed by Unitika. The application is said to confer antimicrobial and anti‐odour
effects to a fabric treated with the compound [143]. A good overview about further chi-
tosan‐containing commercialised wound dressings exploiting antibacterial and even hae-
mostatic effects of the polysaccharide was summarised by Kardas et al. [144]. Although
the given commercial products are promising towards the prospective coating or finishing
of fabrics or fibres with chitosan, research is not limited to these fields, and researchers
have identified a few other intended uses.
16.6.3.2 Removal of Heavy Metals and Dyes

Fabrics coated or finished with chitosan can be applied to purify polluted or contaminated
water from the textile industry. Authors reported that chitosan‐treated cotton gauze showed
suitable results concerning the removal of telon blue, reactive blue 4 and direct red 81
from synthetic wastewater. They applied an alkaline solution to desorb the dyes and
regenerate the material [145]. A study by Ali et al. investigated the removal of toxic dyes
by chitosan‐coated cotton. The working group exploited immobilised copper nanoparti-

cles on the resin to produce a fabric which showed efficient dye removal [146]. The high
affinity of chitosan towards copper ions is also utilised in another approach to remove
metal ions from wastewater. The immersion of a cotton textile in chitosan solution resulted
in the incorporation of copper and lead adsorption capacities with a performance compa-
rable to commercially available ion‐exchange textiles [147]. Hg (II) and Cr (VI) are other
heavy metal ions which could be removed by the application of chitosan‐coated cotton
[148, 149].
16.6.3.3 Increased Dyeability of Textiles

In contrast to the example cited earlier, the efficient adsorption of dyes is sometimes
required. A further application utilises the properties of chitosan to increase the dyeability
of fabrics. Cotton, for example, suffers from a non‐homogeneous colouration and exhibits
coloured or white spots after dyeing. Chitosan pretreatment is said to transform cotton into
a ’wool‐like fibre’, resulting in a more uniform colouration while reducing the amount of
electrolyte required [26]. Several authors have demonstrated increased uniform colouration
and dyeability of cotton after treatment with chitosan [150, 151]. Acidic dyes, according to
the oppositely charged chitosan with its cationic sites, were focused on in the majority of
studies, for example in the case of cotton as a fibre material. However, the colouration‐
enhancing effect of chitosan was also utilised to dye other fibres. Periolatto et al. con-
firmed, for example, that UV‐cured chitosan provides higher wool affinity towards acidic
dyes besides the increased antibacterial activity and the felting reduction [152]. Increased
dyeability is also determined for chitosan‐coated silk and cotton [153].
16.6.3.4 Improved Crease Resistance, Anti‐Felting Properties and Crease

Recovery Angles
Customers demand improved crease resistance and anti‐felting properties. Chitosan cou-
pled to cotton via a citric acid linker is proven to have a significant beneficial effect on crease
resistance. The authors stated that the effect is highly dependent on the concentration of
chitosan and citric acid, as well as the curing temperature applied [133]. In contrast, Roberts
and Wood demonstrated that there are no significant correlations between crease resistance
and molecular weight or deacetylation degree of chitosan measured for wool treatment. An
acylation of chitosan even increases the effectivity due to higher compatibility to the wool
surface [154]. Polyurethane is a widespread agent to confer crease resistance to a textile.
Authors linked chitosan to a polyurethane in order to treat cotton fabrics. They determined
an increased crease recovery angle for printed fabrics as compared to untreated ones
regardless of whether the warp yarn or weft yarn was considered. In the case of dyed fab-
rics, the results depended very much on the weft or weaving yarn. At the same time, both
fabrics exhibited higher stiffness after treatment, which impairs wearing comfort [155]. A
polyurethane/chitosan blend, not covalently linked, also works for the anti‐felting finishing
of wool, resulting in an overall reduction in the amount of polyurethane applied [156]. A
modified water‐soluble chitosan, carboxymethyl chitosan, also increases the crease recov-
ery angle and simultaneously provides increased practicability [157].
16.7 Outlook
In the course of human history, textile production has developed from manual time‐intensive
production of individual garments to highly automated production with high unit num-
bers. As a result, products with a higher degree of modification and quality were also
produced, which were either based on petrochemically produced raw materials or at least
treated with synthetic agents in the course of production. Today, due to customer demand
and ecological necessity, the trend is towards more sustainable and environmentally
friendly textile products. According to the results shown earlier, chitosan could contribute
to this. Chitosan can be implemented as a fibre material at the very first process step of the
textile value chain. Unfortunately, the spinning of chitosan is challenging and results in
fibres with poor mechanical properties. Adaption of spinning conditions, derivatisation or
blending can be applied to unfold a strengthening effect in order to resist the mechanical
loads in further processing. Weaving is one of these processes which cause high stress to
the fibres requiring sizing that protects the fibre. It turned out that chitosan is a sizing
agent providing a suitable protection film around natural and man‐made fibres, enabling
them to withstand stress. Prospective works should focus on the complete removal of
chitosan from the cotton fibre after sizing. However, residual chitosan adsorbed at the
fibre has no or marginal influence on the following process steps. A permanent fixation of
chitosan on the fabric is even desired in the case of coating or finishing. On the one hand,
chitosan can act as a carrier material or linker, transferring the beneficial properties of
another encapsulated or grafted active agent to the fabric. For a durable chitosan coating
or finishing, several synthetic or bio‐based cross‐linkers are suitable. On the other hand,
chitosan could act as the active agent itself, conferring antibacterial activity to the textiles,
which already led to commercial biomedical applications. A few more research studies
were promising and could lead to the development of marketable products, such as the
removal of dyes or metal ions from textile wastewater, the increased dyeability of textiles
or the crease resistance.
In contrast to the beneficial properties described, the production process requires much
more research and improvement to satisfy the demands of a holistic sustainable approach.
The positive is the substantial progress based on intensive research in the past, resulting in
lower consumption of chemicals and the reduction in energy input during the conversion of
chitin into chitosan [158]. Nevertheless, the development of biotechnological or at least
milder methods to produce chitosan is mandatory. As a consequence, the relatively high
cost of chitosan (US$10–100/kg) [159, 160] would considerably decrease and make chi-
tosan more competitive to other synthetic agents used. A price cut can also be expected
from a rise in further commercialised chitin sources, such as fungi or insects. As part of the
arthropods, insects do not only contain proteins in their exoskeleton but also high amounts
of chitin. Cultivation of insects and the products thereof are currently a ‘hot topic’, impli-
cating that a larger quantity of insect‐based chitosan will be available in the future. Due to
the more controlled conditions in the breeding of insects and the cultivation of fungi, alter-
ing external conditions do not influence the homogeneity and thus the quality of chitin and
chitosan, respectively, as is the case for crab shells. The application of defined and homo-
geneous chitosan is highly desirable for the substitution of synthetic polymers in the textile
value chain. However, chitosan is already showing promising results in its current form.
Now the existing potential must be fully exploited through intensive research work and the
optimisation of production processes in order to implement chitosan in commercial textile

processes and increase sustainability.
Nomenclature
Carding The process of preparing the fibres of cotton, wool, etc., for
spinning.
Dyes:
Direct Water‐soluble azo compounds for the direct adsorptive dyeing
of cellulose or cotton in alkaline or neutral solution.
Reactive Water‐soluble anionic compounds for the dyeing of fibres via
covalent bonds at alkaline conditions exhibiting high washing
and light fastness.
Fabric Artificial network of yarns and threads of fibres that has been
woven or knitted.
Colour fastness Describes the property of a material to maintain the colouration
by external influences.
Finishing The last process step to improve the textile’s aesthetic or func-
tional properties.
Easy‐care Cross‐linking of cellulose or cotton fibres with aldehyde‐
containing compounds to reduce swelling and improve wrinkle
recovery.
Knitting Knitted fabric consists of consecutive loops called stitches. As
each row progresses, a new loop is pulled through an existing
loop. The active stitches are held on a needle until another loop
can be passed through them.
Sizing process The process of applying an adhesive protective coating on the
warp yarns’ surface is called sizing. Sizing is done during weav-
ing preparation for getting a good weaving behaviour.
Sizing agent A compound applied to protect the warp yarn during the weav-
ing process.
Tenacity/specific strength The tenacity of a material is the mass stress at break. It is defined
as the specific stress corresponding to the maximum force on a
force/extension curve.
Spinning:
Primary Cellulosic or synthetic polymers are converted into fibres by
extrusion of a polymer melt or solution.
Secondary Processing of fibres into staple yarn.
Tensile strength The tensile strength of a material is the maximum amount of
tensile stress it can take before failure, such as breaking or per-
manent deformation. Tensile strength specifies the point when a
material goes from elastic to plastic deformation.
Wash fastness The resistance of a material to change in any of its colour char-
acteristics; when subjected to washing, is called colour fastness
to washing.
Weaving Weaving is the process of making cloth with two components, a

warp and a weft, and can be done by very simple techniques on
a complicated loom.
Weft thread/yarns The horizontal yarns in the weaving process are called weft
yarns.
Warp thread/yarns Warp yarns in the weaving process run vertically along the
length of the fabric.
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17
Chitin and Chitosan for Water
Purification
Petrisor Samoila, Andra Cristina Humelnicu, Maria Ignat,
Corneliu Cojocaru, and Valeria Harabagiu
‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian Academy, Iași, Romania
The interest in developing new and more efficient materials and technologies for the
removal of different pollutants from wastewater is constantly growing. To this end, chitin
and chitosan have attracted the attention of many researchers because of their interesting
removal capacities for certain hazardous pollutants such as water‐soluble organics (phe-
nols, dyes, pesticides, herbicides, etc.) and heavy metal ions. Moreover, these biocompat-
ible and biodegradable carbohydrate polymers fall in the category of low‐cost sorbents that
are abundant and renewable resources. Nevertheless, the limited solubility of chitin and the
high sensitivity to pH of chitosan generate some feasibility issues. In this respect, many
studies suggest the chemical modification of chitin and chitosan, in order to develop deriv-
atives and composites with improved properties for water purification purposes. In this
chapter, chitin and chitosan, together with their derivatives and composites, are reviewed
from the perspective of water purification applications, underlining their advantages for the
removal of representative organic pollutants and heavy metals from aqueous solutions.
Likewise, the principles and mechanisms governing the purification processes such as
adsorption, coagulation/flocculation and ultrafiltration are detailed.

17.1 Introduction
Water contamination by a wide range of toxic compounds, particularly organic pollutants
and heavy metals, has become a serious environmental concern because of the negative
impact on the quality of drinking water for generations to come. Under these circum-
stances, the interest on developing new and more efficient materials and technologies for
removal of polluting substances from wastewaters is in constant growth. Depending on the
processes involved, wastewater treatments can be classified as: physical, chemical and bio-
logical. The selection of the appropriate treatment is made by considering the pollutant
nature and concentration, as well as the investment and operational costs. Because of their
versatility and wide range of properties, polymers (either synthetic or natural) and polymer‐
based composites are playing a key role in wastewater treatments such as: adsorption [1],
coagulation/flocculation [2] or membrane separation by (micro‐/ultra‐/nano‐/hyper‐) filtra-
tion processes [3].
In comparison with synthetic polymers, natural polymers gained the attention of
researchers in order to develop cheaper and more effective adsorbents. Biopolymers repre-
sent a real alternative to synthetic polymers owing to their distinguished structure, physico-
chemical characteristics, high reactivity and chemical stability. The presence of different
functional groups in the chemical structure (hydroxyl, carboxyl, amino or acetamido) per-
mits the synthesis of new bioadsorbents with high selectivity to aromatic compounds and
metals [4]. Among these, polysaccharides such as chitin [5], chitosan [6], starch [7], cel-
lulose [8] and their derivatives are known for their eco‐friendly properties such as biodeg-
radability, non‐toxicity and biocompatibility [9]. Likewise, their ability to bind with a large
variety of molecules by physical and chemical interactions is of great interest. Therefore,
their efficient adsorption property for toxic compounds made them ideal candidates for
water treatment (removal of pollutants) [10] and for separation processes (recovery of
important metals) [11].
Chitin and chitosan were extensively studied and proved to have attractive removal
capacities for certain pollutants such as water‐soluble organics and metal ions. Moreover,
these carbohydrate polymers (chitin and chitosan) fall in the category of low‐cost sorbents,
if compared to commercial activated carbons or synthetic ion exchange materials.
Industrially, chitin – known as the second most abundant natural polymer on a global
scale – is extracted mainly from crustaceans (crab, krill, crayfish) by a three‐step procedure
as follows: (i) calcium carbonate dissolution by means of an acid treatment; (ii) protein
solubilisation by alkaline extraction; and (iii) pigment removal by decolourisation.
Consequently, chitosan – the most important chitin derivative – is usually obtained by par-
tial deacetylation in alkaline media [12]. In spite of the beneficial properties of these natu-
ral polymers, such as being environment‐friendly, abundant, biodegradable and renewable
resources, the limited solubility of chitin and the high sensitivity to pH of chitosan impose
some feasibility issues [13, 14]. In this respect, many studies have been published on the
chemical modification of chitosan. According to these studies, the chemical modification
of chitosan yielded derivatives and composites with improved properties for wastewater
treatment purposes [14].
To obtain chitosan‐based sorbents with adequate physical–chemical properties, cross‐
linking agents are often employed. Such cross‐linking agents imply: glutaraldehyde [12, 15],
genipine [16], glyoxal [17], epichlorohydrin [18] or ethylene glycol diglycidyl ether [19].
Some of the commonly used cross‐linking agents are detailed in Figure 17.1.
Chitin and Chitosan for Water Purification 431
CHEMICAL CROSS-LINKING
CS OH OH
CS H
CS OH CS N
H-C=O N=C-H H OH
NH2 NH2 O
+ + H
NH2 NH2
N=C-H O NH
H-C=O H
CS CS O OCH3 CS
CS
Glutaraldehyde Genipine
NH2 OH
OH NH2 NH2
OH OH
CS CS
CS CS CS
CS O CS
O O
N O O O
NH2 NH2
+ + OH sau
NH2 sau NH2 OH
O CS CI O O
CS O N
O
OH CS CS CS
OH CS
CS
OH NH2 NH2
OH
NH2
Glyoxal Epichlorohydrin
OH OH
CS CS
OH
NH OH
NH
O=C=N O
CS O NH CS HO
NH2 NH2 O O
+
NH2 +
NH2
O O
CS O=C=N CS
O NH OH
O
OH NH NH
OH
CS CS
OH OH
4,4ʹ-Methylenebis (phenyl isocyanate) Ethylene glycol diglycidyl ether
PHYSICAL CROSS-LINKING
OH
CS
OH
O– Na+ O– NH3+
CS Na O P = O
+ –
Na O– P = O
+
O O
NH2
+ Na+ O– P = O Na+ O– P = O
NH2
O O
CS Na+ O– P = O Na+ O–
P= O
+
O– Na+ O– NH3
OH
CS
OH
Pentasodium triphosphate
Figure 17.1 Types of chitosan cross‐linking and cross‐linking reagents used.

Chitosan‐based composites used in wastewater treatments involve various types of inor-

ganic substances such as activated clay, montmorillonite, bentonite or oxide particles with
magnetic properties [14, 20].
Considering the survey of literature on chitosan‐based materials used for wastewater
treatment, this chapter is focused on three directions: adsorption, coagulation/flocculation
and ultrafiltration purification processes. These processes are outlined because of the fol-
lowing reasons: adsorption – due to its high efficiency, ample availability, low cost and easy
handling [21]; coagulation/flocculation [22] – as one of the simplest and most efficient tech-
niques used to achieve solid–liquid separation; and by membrane ultrafiltration – a rela-
tively less studied approach as compared to adsorption.
Likewise, in this chapter, chitin and chitosan are discussed in detail from the perspective
of environmental applications, underlining their advantages for the removal of organic pol-
lutants and heavy metals from aqueous solutions. The main objective is to provide recent
information about the most important characteristics and advantages of these biopolymers
used for wastewater treatment and water purification.
17.2 Wastewater Treatment by Adsorption

The application of chitin and chitosan as adsorbents has attracted great attention for the
treatment of wastewater loaded with organics and heavy metals. Chitin is a natural polymer
presenting a large number of hydroxyl and acetamido groups. Chitosan (a chitin derivative)
is a hydrophilic polymer that contains mostly hydroxyl and amino groups. The adsorption
mechanism (implying chitin and chitosan) is based on electrostatic attraction (for anionic
organic pollutants) and dative coordination (for heavy metals cations). The involved moie-
ties in such a mechanism are acetamido and amino groups from chitin and chitosan, respec-
tively [14, 23].
17.2.1 Principle of the Adsorption Process

As a surface phenomenon, adsorption is defined as the augmentation in concentration of a
specific compound at the surface or at the interface of two phases. The component adhering
onto a solid surface is known as an adsorbate, and the surface itself is called an adsorbent.
The nature of adsorbent and adsorbate, together with the experimental conditions (tem-
perature, pH, contact time, pollutant concentration and contact surface area), are the main
factors influencing the adsorption process [24].
Adsorption represents a widely used technique to remove certain pollutants from waste-
water, particularly those that are not readily biodegradable, such as the metal cations and
refractory organic molecules dissolved in water [25].
Depending on requirements, two adsorption systems can be used for wastewater treat-
ments: batch and continuous adsorption. Batch technique is used in order to establish the
maximum adsorption capacities and adsorption rates, and also to investigate the thermody-
namic parameters and interactions between the adsorbent and sorbate [26]. Continuous
adsorption is used for large‐scale applications. The continuous process is carried out in
packed‐bed columns. However, the removal efficiency yielded in continuous mode is gen-
erally lower than that achieved by batch processes [27].
Regarding solute affinity, it can be distinguished into three types of adsorption: physical,
ion exchange and chemical adsorption. Physical adsorption (or physisorption) is explained
mainly by Van der Waalls attraction complemented by electrostatic forces between the
adsorbate and the surface of the adsorbent [28]. The second type of adsorption relies mostly
on electrostatic forces occurring between ions retained on the surface, and is often called
ion exchange adsorption [29]. Chemical adsorption (or chemisorption) is the result of the
chemical interaction between the solid and the adsorbed substance. Chemisorption takes
place when the substance is linked to the solid surface by covalent bonds. Usually, physical
adsorption may occur on all surfaces, provided the pressure and temperature conditions are
favourable. In contrast, chemisorption is highly more selective. Also, it is possible only
between specific adsorptive and adsorbent species, especially if the active sites are not
blocked by previously adsorbed molecules [30].
The adsorption process is dependent on several factors, from the adsorbent nature and
structure to the physical and chemical properties of the adsorbate and the reaction medium.
Therefore, the most important factors that affect absorption capacity are: properties of the
sorbate (functional groups, polarity, hydrophobic/hydrophilic character, size and molecular
weight), medium condition (pH and temperature of solution, sorbent concentration and ionic
strength) and the type of sorbent (surface area, porosity, functional groups distribution) [31].
The adsorption process is based on the solute’s removal from a solution and changing the
surface concentration until an equilibrium between the solution and adsorbent is attained.
The adsorption amount at equilibrium is usually determined in accordance with the follow-
ing equation [32]:
C0 C V
q Eq. 17.1
m 1000
where q denotes the adsorption capacity (mg·g−1); C0 and C are, respectively, the initial and
final concentrations of pollutant in solution (mg·L−1); V is the volume of the solution (mL);
and m represents the mass of the adsorbent (g).
Frequently, adsorption isotherms are used to describe the equilibrium of adsorption of a
material on a surface (more generally, on a boundary surface) at a constant temperature.
Literature reports often provide isotherm models, such as Freundlich, Langmuir, Redlich–
Peterson, Sips and Dubinin–Radushkevich [33]. The conventional adsorption isotherm
equations are Langmuir and Freundlich isotherms, since both can be transformed into lin-
ear forms, and so the main parameters of the adsorption process are easily calculated either
by linear regression or by graphical means [34]. In addition, the Dubinin–Radushkevich
model is often used to determine the adsorption process type (physical, ion exchange or
chemisorption). The mean free energy of sorption ES (kJ·mol−1) is the main parameter of
this model that indicates the nature of adsorption. Generally, the process is considered to be
based on physisorption if ES <8 (kJ·mol−1). In turn, the sorption process relies on ion
exchange if ES ranges from 8 to 16 kJ·mol−1 [35]. For higher values of mean free energy,
the adsorption mechanism might be attributed to chemical interactions (chemisorptions).
In addition, thermodynamic parameters such as Gibbs free energy of adsorption (ΔGad) can
also indicate the nature of adsorption. For example, ΔGad ranges of 0 to −20 kJ·mol−1 and
−80 to −400 kJ·mol−1 can be attributed to physisorption and chemisorption, respectively

[36]. Following this line, it can be supposed that the in‐between interval (–20 to −80
kJ·mol−1) might be connected to the ion exchange phenomena.
17.2.2 Adsorption of Organic Compounds

The presence of reactive groups in the structure of chitin and chitosan offers different
adsorption interfaces with numerous organic pollutants such as phenols [37], pesticides
[38], herbicides [39], dyes [40], etc.
Phenol, a typical organic pollutant, is considered one of the most toxic and mutagenic
pollutants even at low concentrations and is known to be extremely harmful to living organ-
isms. Pigatto et al. [37] investigated phenol removal using commercial chitin as a bioadsor-
bent. They studied the influence of several parameters over adsorption capacity, such as: pH
ranging from 2 to 10, temperature ranging from 15 to 500°C, initial phenol concentration
ranging from 10.4 to 90.8 mg·L−1, etc. The maximum phenol adsorption capacity of 12.7
mg·g−1 was achieved at 25°C, pH 2 and 90.8 mg·L−1 initial pollutant concentration. The
increase in temperature up to 25°C was found to be beneficial for the adsorption capacity,
but detrimental at higher values, suggesting the appearance of thermo‐inactivation equilib-
rium. These authors are proposing an intermediate mechanism between the monolayer and
multilayer models based on the Langmuir, Freundlich and Temkin isotherm. Moreover, the
adsorption of phenol onto commercial chitin was explained mainly by the formation of
hydrogen bonds between the amide groups of chitin and phenol. Other authors [41] reported
a higher value of maximum phenol adsorption capacity (21.5 mg·g−1) working at 40°C, pH 1
and 300 mg·L−1 initial phenol concentration. According to the calculated thermodynamic
parameters (∆G = –19.4 kJ·mol−1; ∆H = 10.2 kJ·mol−1; ∆S = 0.093 kJ·mol−1·K−1), the adsorp-
tion of phenol onto chitin surface is spontaneous and endothermic.
The capacity of chitosan in its unmodified forms to remove phenols from wastewater is
known to be very low [42]. The wide majority of researchers are proposing the chemical
modification of chitosan and/or its combination with inorganic materials to obtain compos-
ites with enhanced performances. For example, Zhou et al. [43] modified chitosan with
salicylaldehyde and β‐cyclodextrin and used the obtained materials as adsorbent for phenol
and various chlorophenols. The adsorption capacities were ranging from 59.74 mg·g−1 for
phenol to 375.94 mg·g−1 for 2,4,6‐trichlorophenol. The higher maximum capacities
observed for chlorophenol were explained by the formation of hydrogen bonds and π–π
interactions. Moreover, the sorbents were easily regenerated by washing with alcohol. In
another study, Soni et al. [42] designed chitosan/activated carbon nanocomposites. They
found that the maximum adsorption capacity of their material was equal to 409 mg·g−1.
Contamination of both surface and ground waters with herbicides and pesticides are
among the most concerning environmental global problems. Because of their inherent toxic-
ity and high solubility, the removal of this kind of pollutants from wastewater represents a
great challenge for scientists. A previous study reported by Youshizuka et al. [38] dealt with
chitosan microparticles prepared by cross‐linking with glutaraldehyde and epichlorohydrin
for the adsorption of methyl parathion – a typical pesticide. They observed that the material
cross‐linked with glutaraldehyde showed superior adsorption performances over the epichlo-
rohydrin‐cross‐linked sample. Recently, Shankar et al. [44] have developed bioadsorbents
with improved adsorption performances for the removal of the pentachlorophenol pesticide
from synthetic waters by chemically modifying chitosan. In this respect, chitosan was sub-
jected to a cross‐linking process using 2‐hydroxy‐1‐naphthaldehyde. Further, the function-
alised biopolymer was modified by grafting with copper(II) chloride. The maximum
adsorption capacities were very sensitive to the modification of chitosan. The cross‐linked
chitosan exhibited the highest pesticide adsorption (39.1 mg·g−1) followed by the grafted
material (35.4 mg·g−1) and the unmodified polysaccharide (24.4 mg·g−1). These observations
shed light on the beneficial effect of chemical modification over the capacity of chitosan to
adsorb organic pollutants.
In their seminal paper, El Harmoudi et al. [45] recovered chitin by extraction technique
from shellfish waste and produced chitosan by the deacetylation of chitin. Further, they
used the obtained biopolymers for the removal of one of the most common organic herbi-
cides: 2,4‐dichlorophenoxyacetate (2,4‐D). During the adsorption tests, the researchers
studied the influence of some factors such as pH, the contact time and initial pollutant
concentration over the maximum adsorption capacity. The adsorption of the herbicide onto
chitosan and chitin surface was optimal at pH 3.7. According to their findings, at this pH
value, the 2,4‐D was in anionic form, and the functional groups (hydroxyl, amine and
amide groups) of the polysaccharides were protonated, favouring electrostatic attraction of
the system components. The adsorption capacities estimated from Langmuir equation were
equal to 11 mg·g−1 for chitosan and 6 mg·g−1 for chitin.
Other authors [39] dealt with montmorillonite–chitosan bionanocomposites applied as
adsorbents for 3,6‐dichloropyridine‐2‐carboxylic acid (clopyralid) as a representative mol-
ecule for anionic herbicides. They conducted their study by simulating waters and soil/
water suspensions polluted with clopyralid in order to determine the potential of their mate-
rials to prevent and remediate both water and soil contaminated with herbicides. The results
reported in this work underlined the main role of pH over adsorption performances. The
montmorillonite–chitosan composites showed good adsorption capacities at pH values that
enabled the protonated form of chitosan and anionic form of clopyralid, suggesting an ion
exchange adsorption mechanism. Moreover, the addition of bionanocomposites to acidic
soil significantly increased the pollutant adsorption, whereas negligible adsorption perfor-
mances were observed for alkaline soils. These observations recommended the immobilisa-
tion of montmorillonite–chitosan materials in certain soils to prevent the pollution of ground
waters. In another study, Ding et al. [46] designed the same kind of bionanocomposites for
removing the herbicide 3,7‐dichloroquinoline‐8‐carboxylic acid (quinclorac) from aqueous
solution. This study highlighted once again the influence of pH over adsorption perfor-
mances of montmorillonite–chitosan materials. These authors observed that the increase in
pH from 2.9 to 5.0 positively affected the adsorption capacity. In contrast, the pH increase
from 5.0 to 10.3 was detrimental for the maximum adsorption capacity. Furthermore, the
authors conducted desorption studies in order to elucidate the adsorption mechanism. The
results indicated a very high desorption rate in alkaline media as compared to distilled
water, suggesting that adsorption of quinclorac onto montmorillonite–chitosan biocompos-
ites occurred mainly by electrostatic attraction.
One of the most concerning environmental problem regarding wastewaters is caused by
the textile industry. The coloured effluents that result from textile manufacturing are gener-
ally considered to be highly toxic, since dyes are reported to cause allergy, skin irritation,
dermatitis and even cancer. Thus, the removal of dyes from effluents prior to their mixing
with clean natural water bodies is of crucial importance. Adsorption via natural biopolymers
such as chitin and chitosan was proved to be an effective treatment for discolouration of
textile dyes from effluents. Chemical modifications of these polysaccharides can induce
changes on their properties by promoting new materials with more appropriate characteris-
tics for the removal of various dyes.
The literature survey has indicated that chitosan is very effective for the adsorption of
anionic dyes (reactive and acid dyes), especially in an acidic environment, following the
ion exchange mechanism illustrated in Figure 17.2.
According to the adsorption mechanism proposed by Tan et al. [47], the amino groups of
chitosan are protonated in the presence of high concentrations of protons. Also, in acidic
conditions, anionic dye molecules are dissociated, favouring the electrostatic attraction
between anionic dye ions and protonated amino group of chitosan.
Recently, Humelnicu et al. [33] have reported on the adsorptive removal of anionic dyes
by rare‐earth‐doped cobalt spinel ferrite functionalised with chitosan cross‐linked with
epichlorohydrine. The selection of the cross‐linking agent was made in order to preserve
the amino groups’ number from the pristine polysaccharide (chitosan). The developed
composite material (CoFe1.98Sm0.02O4@Chitosan‐cL‐Epichlorohydrine) was used as sorb-
ent for Orange II removal in batch conditions. The maximal adsorption capacity was found
to be 182 mg·g−1. The model‐based optimisation was performed for the adsorption process
to evaluate the effects of pH and sorbent dosage over adsorption performances. The model‐
based optimisation performed by stochastic simulation using the Monte‐Carlo method sug-
gested that the optimal values of factors were pH 5.2 and sorbent dosage 1.60 g·L−1.
Furthermore, the energy values (ES) determined from Dubinin–Radushkevich isotherm
ranged from 9.85–11.78 kJ mol−1 depending on the temperature. These values were
included into the interval 8–16 kJ mol−1, suggesting that the mechanism predominant for
this adsorption process was ion exchange mechanism.
Nevertheless, chitosan‐based materials were also used for the removal of some cationic
dyes. Kamal et al. [48] investigated a new type of titanium oxide/chitosan nanocomposite
as an adsorbent for thymol violet dye removal. Both composite material and pristine chi-
tosan were adhered on the cellulose microfibre mat by using the doctor‐blade method. The
adsorption properties were evaluated as a function of pH, adsorbent dosage and contact
time. As expected, the optimum pH value for the adsorption process was found to be 7.96.
These authors argued that the superior values of adsorption capacities could be due to the
increasing number of negatively charged sites on the adsorbent. However, no details were
H+
CS NH2 CS NH3+
Protonation
H2O
Dye SO3Na Dye SO3– + Na+
Dissociation
CS NH3+ Dye SO3– CS NH3 + –O3S Dye

Electrostatic
attraction
Figure 17.2 Schematic representation of the adsorption mechanism of anionic dyes onto the chitosan
surface; adapted from [47].
given about the nature of these negatively charged sites onto the adsorbent surface.
Likewise, adsorption of the studied dye and adsorbent dosage were into a directly propor-
tional relation. Kinetic studies revealed that the composite adsorbed the dye rapidly and
equilibrium was achieved in 90 min, comparing with pure chitosan coated with cellulose.
In addition, the adsorption process involved particle diffusion mechanism and followed
pseudo‐second order kinetics. The calculated maximum adsorption capacities for the
adsorbents were equal to 84.32 mg·g−1 (chitosan–cellulose microfibre mat) and 97.51
mg·g−1 (TiO2/chitosan–cellulose microfibre mat), respectively.
Composite hydrogel beads based on chitosan and halloysite nanotubes were used as
absorbents for the removal of both methylene blue and malachite green from aqueous solu-
tions by Peng et al. [49]. The adsorbent materials were prepared by the dropping and pH‐
precipitation method. Adsorption data were fitted using Langmuir and Freundlich isotherm
models. It was observed that the addition of clay mineral into chitosan beads led to a sig-
nificant increase in the adsorption rate for both pollutants. Therefore, the maximum absorp-
tion capacity achieved for methylene blue was 270.27 mg·g−1 and for malachite green was
303.03 mg·g−1, attributed mainly to the numerous active sites of halloysite nanotubes.
Moreover, a regeneration experiment was done and showed that the adsorption capacity of
all hydrogel beads could be restored, especially for the case of methylene blue uptake.
Ultimately, it was inferred that chitosan/halloysite composite showed great potential as
reusable adsorbents for dye removal.
Table 17.1 summarises the performances of some adsorption processes dealing with the
uptake of organic dyes by chitosan‐based materials along with the adopted conditions of
experimentation. As one may notice from Table 17.1, most studies were focused on anionic
dyes (but not exclusively) and conducted in acidic environments.
17.2.3 Adsorption of Heavy Metals

The wastewaters loaded with heavy metals are considered to be very toxic because of the
long biological half‐lives and the non‐biodegradable nature of these kinds of pollutants.
The metals usually accumulate in different parts of the human body causing serious dis-
eases and disorders. In this regard, it is extremely important to control and eliminate the
hazardous waste generated by the industries responsible for heavy metals pollution [50].
A recent literature review on heavy‐metal ions adsorption by chitin has been reported by
Anastopoulos et al. [23]. The authors provided a relevant list of chitin‐based adsorbents
mentioning the best kinetic and isotherm fitting models, as well as the maximum adsorp-
tion capacity values. A special attention was given to the papers suggesting possible adsorp-
tion mechanisms. In most of the cases, the interaction mechanism between chitin and metal
occurs by coordination bonds involving the nitrogen atoms of the acetylamino groups of
the biopolymer. Generally, the nitrogen atom acts as a donor of lone‐pair electrons, whereas
the metal cation acts as the electron acceptor. Note that, the hybridisation of d‐orbitals of
the heavy metal cation (Mn+) creates vacancies able to accommodate lone‐pair electrons
from other atoms (e.g. N, O). In these lines, some studies (given in the review report by
Anastopoulos et al. [23]) also suggested the implications of oxygen atoms (O) from
hydroxyl groups of chitosan as the donor of lone‐pair electrons.
Figure 17.3 illustrates a possible mechanism for metal cations adsorption onto chitin and
chitosan materials by means of coordination bonds. The dative bond is supposed to be
Table 17.1 Application of chitosan (pristine and modified) as adsorbent for different types of dyes.
No. Chitosan‐based sorbent Form Dye Qmax (mg·g−1, mmol·g−1) pH T (°C) Reference
1 Chitosan Microparticles Acid Green 25 525 3 [51]
2 Chitosan Powder Acid Blue 9 210 3 25 [52]
Food Yellow 3 295
3 Chitosan Flakes Acid Green 25 645.1 4 25 [53]
Acid Orange 10 922.9
Acid Orange 12 973.3
Acid Red 18 693.2
Acid Red 73 728.2
4 Chitosan/Ethylenediamine Nanoparticles Acid Orange 7 3.47 4 25 [54]
Acid Orange 10 2.25 3
5 Chitosan/graphene Nano‐composite Acid Orange 7 42.7 3 25 [55]
6 Chitosan/TPP Nanoparticles Acid Green 27 1051.8 5 25 [56]
7 Chitosan/4‐formyl‐benzene‐1,3‐disulphonic acid Powder Basic Blue 3 166.5 3 25 [57]
8 Chitosan‐graft‐l‐monoguluronic acid Powder Congo Red 459.75 5 30 [58]
9 Chitosan‐g‐poly (acrylic acid) Microparticles Methylene Blue 1873 5 30 [59]
Chitosan‐g‐attapulgite 1848
10 Chitosan/Al2O3/Fe2O3 Nanoparticles Methyl Orange 417 25 [60]
11 Chitosan/Epichlorohydrine Beads Metanil Yellow 1334 3 30 [61]
Reactive Blue 15 722
12 Chitosan/montmorillonite Hydrogel Nitrazine Yellow 177 5 25 [62]
13 Chitosan/Epichlorohydrine Microparticles Remazol Black 2941 3 30 [63]
14 Chitosan‐graft‐poly(methylmethacrylate) Powder Procion Yellow MX 250 7 20–30 [64]
Remazol Brilliant Violet 357
Reactive Blue H5G 178
15 Chitosan‐graft‐poly(acrylamide) Powder Remazol Violet 1428 4 30 [65]
Procion Yellow 1000
16 Chitosan‐g‐poly(acrylamide) Powder Remazol Yellow Gelb 3RS 1211 2 25 [66]
Basic Yellow 37 528
17 Chitosan‐N‐lauryl/Fe2O3 Particles Remazol Red 198 267 25 [67]
18 Chitosan/Epichlorohydrine Beads Reactive Red 189 1642–1936 3 30 [68]
19 Chitosan/Sodium tripolyphosphate/Epichlorohydrine Beads Reactive Red 189 1802–1840 3 30 [69]
20 Chitosan‐g‐poly(methyl methacrylate) Powder Orange‐G 435 7 [70]
0004461255.INDD 438 10/26/2019 1:30:56 PM

(a) (b)
CT CS
NHAc NH2
Mn+ Mn+
NHAc NH2
CT CS
Figure 17.3 Schematic representation of the adsorption mechanism of heavy metals onto (a) chitin and
(b) chitosan materials.
formed involving the pair of electrons shared by the nitrogen atoms of biopolymers (donor)
with the metal cations (acceptor).
Chitin/chitosan are reported as efficient adsorbents for a very wide range of heavy met-
als, starting with elements usually present in the living organisms in low/moderate quantity,
but toxic at higher concentrations (Fe, Zn, Co, Cu, Mn, etc.), and finishing with Cd, As, Pb,
Hg or Cr, which are extremely hazardous even at lower concentrations.
For example, Forutan et al. [71] studied the adsorption of Pb(II) from synthetic wastewa-
ters by the chitin extracted from shells of pink shrimps. In order to evaluate the influence of
pH, sorbent dosage and initial concentration of lead, they conducted experiments varying
exclusively from the studied parameter. They reported a maximum adsorption capacity of
7.003 mg·g−1 in the following experimental conditions: initial concentration of lead equal to
7.99 ppm, absorbent dosage of 5 g·L−1, temperature 30°C and pH 9. In addition, Karthik and
Meenaksh [72] utilised chitin grafted with polypyrol for Pb(II) and Cd(II) ions removal
from aqueous solutions. They reported maximum adsorption capacities of 9.14 mg·g−1 for
lead and 6.49 mg·g−1 for cadmium, at 50°C. The adsorption mechanism suggested by these
authors involved electrostatic attraction followed by a final complexation step.
Singh and Nagendran [73] compared the adsorption efficiencies of chitin and chitosan
for chromium (III) from synthetic wastewaters. The maximum adsorption values estimated
from the Langmuir isotherm were very sensitive to the biopolymer nature. According to
their results, the deacetylated polysaccharide is far more suitable for Cr3+ removal. For
instance, the maximum adsorption capacity values were equal to 51.12 mg·g−1 for Cr3+/
chitosan, and only 7.738 mg·g−1 for the Cr3+/chitin adsorption system.
Ariff, Hanafiah and Ngah [74] prepared cross‐linked chitosan‐coated bentonite beads to
investigate their adsorption efficiency regarding Cu (II) removal form aqueous solution.
The beads were sieved in order to retain the microparticles greater than 200 μm. Several
factors impaired the absorption capacity. Therefore, the effects of temperature, stirring time
and Cu(II) concentrations were assessed. Adsorption kinetics was evaluated to determine
the optimum stirring time necessary for Cu(II) adsorption. The experimental data were
well‐fitted to the pseudo‐second‐order kinetic model suggesting that chemical adsorption

was the rate controlling mechanism. According to isotherm data, the Langmuir isotherm
model showed that the maximum monolayer adsorption of Cu(II) on chitosan‐coated ben-
tonite beads occurred at 27°C and was equal to 114.94 mg·g−1.
Recently, Ignat et al. [20] have reported a new synthesis strategy for zinc ferrite nanopar-
ticles coated with chitosan as sorbents for heavy metals. In their study, magnetic chitosan–
ZnFe2O4 composites were efficiently used as adsorbents for Ni(II) and Cr(III) cations
because of their appropriate physical and chemical properties and easy separation from an
aqueous environment under an external magnetic field. The single and competitive adsorp-
tions of nickel and chromium cations from synthetic wastewaters onto composite nanopar-
ticles were also studied. For the single cation sorption experiments, it was observed that the
adsorption capacity for Cr3+ (6.625 mg·g−1) was about twofold higher as compared to the
uptake of Ni2+ (3.096 mg·g−1). For the competitive system, the adsorption capacities of the
studied nanocomposites were equal to 0.788 mg·g−1 and 5.577 mg·g−1, for nickel cation and
chromium cations, respectively. Therefore, it was inferred that the chitosan–ZnFe2O4 nano-
composite adsorbed Cr(III) almost selectively in the presence of Ni(II).
Table 17.2 summarises the performances of various forms of chitosan and chitosan‐
based materials used for the adsorption of heavy metal ions from aqueous solutions. One
may observe that chitosan, chemically modified chitosan and chitosan composites were
successfully employed for a wide range of metal cations adsorption. In most of the cases,
the adsorption occurred in acidic conditions (pH<7) and at low temperatures (near to room
temperature).
17.3 Wastewater Treatment by Coagulation/Flocculation

Regardless of their origin, wastewaters contain dissolved solids (diameter <10−9 m), col-
loidal solids (10−9 m < diameter <10−6 m) and suspended solids (diameter >10−6 m). In order
to separate the suspended solids in wastewaters, coagulation and flocculation processes
are used.
Generally, the particles that are found in wastewaters could be of various origins, of dif-
ferent sizes and shapes, may vary in charge or may have different densities. Therefore, these
parameters play a crucial role in particle separation using coagulation/flocculation pro-
cesses. It is noteworthy to mention that many suspended particles in wastewater are nega-
tively charged. Therefore, such particles in solution repel each other due to the same surface
charge, and remain suspended in solution. As well, the charge of other particles may vary
with the pH of wastes, depending on its origin.
Flocculation/coagulation is a physicochemical process which implies the agglomeration
of colloids or suspended solids that cannot be separated by natural sedimentation alone, or
which form aggregates even if they sedimentate during a very long detention period. Such
a process was applied for many years to remove turbidity of water [75–77] and reduce the
inorganic [78] and organic matter content from wastewaters [79, 80]. Actually, during the
coagulation process, coagulant molecules, particles or species are added to wastes where
they incorporate suspended particles helping them to precipitate in the form of flocs. This
process is known since 1762 as a chemical precipitation process that consists of coagula-
tion of suspended, colloidal and dissolved constituents from sewage effluents [81]. The
coagulation was used to make the sedimentation of dirt more rapid and efficient. At that
Table 17.2 Application of chitosan (pristine and modified) as adsorbent for different heavy metals.
No. Chitosan‐based sorbent Form Heavy Metal Qmax (mg·g−1, mmol·g−1) pH T (°C) Reference
1 Chitosan Powder/solid Pb2+
141.10 4.5 20 [85]
Ni2+ 52.81 5.5
2 Chitosan Nanofibres Cu2+ 485.44 7 25 [86]
Pb2+ 263.15
3 Chitosan Particles Hg2+ 1127.1 6 25 [87]
4 Chitosan/Epichlorohydrine Powder Cr2+ 78 5 [88]
Cu2+ 80 3–5
5 Chitosan/glutaraldehyde Aerogel Cu2+ 35.08 25 [89]
6 Chitosan/graphene oxide Nanofibres Cr6+ 310.4 3 45 [90]
Cu2+ 423.8 6
Pb2+ 461.3 6
7 Chitosan/Fe3O4 Nanoparticles Cu2+ 21.5 2–5 [91]
8 Chitosan/phenylthiourea Beads Hg2+ 135 ± 3 5 30 [92]
Cd2+ 120 ± 1
Zn2+ 52 ± 1
9 Chitosan/clinoptilolite Beads Cu2+ 11.32 5 25 [93]
Co2+ 7.94
Ni2+ 4.209
10 Chitosan/(polyvinyl alcohol)/zeolite Nanofibres Cr6+ 0.17 [94]
Fe3+ 0.11
Ni2+ 0.03
11 Chitosan/thioglyceraldehyde Beads Hg2+ 85.33 5 [95]
Cu2+ 54.76
Zn2+ 35.12
12 Magnetic chitosan/glutaraldehyde Microsphere Zn2+ 32.16 5 [96]
13 Chitosan/poly(allylamine hydrochloride)–modified E. coli Fibres Pt4+ 290.98 2 40 [97]
14 Chitosan/glutaraldehyde Beads Co2+ 73.98 5 25 [98]
Cd2+ 68.52
Pb2+ 78.67
(Continued)
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No. Chitosan‐based sorbent Form Heavy Metal Qmax (mg·g−1, mmol·g−1) pH T (°C) Reference
15 Chitosan/montmorillonite Particles Pb2+
49.33 5 25–50 [99]
Cu2+ 25.28
Cd2+ 20.61
16 Chitosan/Fe3O4/thiourea Microspheres Hg2+ 625.2 28 [100]
Cu2+ 66.7
Ni2+ 15.3
17 Chitosan/glutaraldehyde Membrane Hg2+ 891.4 6 25 [101]
Spheres 647.9 ± 6
18 Chitosan/methyl iodide/EDTA dianhydride Powder Cu2+
0.698 4.5 25 [102]
Co2+ 1.125 4.5
Ni2+ 0.725 7.5
Cr6+ 1.910 2
19 Chitosan‐g‐Polyaniline Powder Pb2+ 16.07 6 50 [103]
Cd2+ 14.33
20 Chitosan/fly ash Particles Cd2+ 87.72 8 35 [104]
0004461255.INDD 442 10/26/2019 1:30:57 PM

time, chemical precipitation was done using inorganic coagulants such as lime alone or in
mixture with metal salts, magnesium chloride or calcium chloride. Later, other chemicals
were studied to be used as coagulants, namely ferric chloride, aluminium sulphate and cati-
onic polymers (e.g. polydiallyldimethyl ammonium chloride) [82–84]. Because of its very
large surface‐to‐volume ratio, a colloidal solution does not agglomerate naturally. Thus, the
small particles in such a stable solution, due to various factors (e.g. adsorption of ions onto
the particle surface, imperfection in crystal structure, ionisation of surface sites), can gain
net charges in aqueous solutions. This effect depends on the pH of the solution (at
pH>pHiso, the particles in solution are negatively charged; at pH<pHiso, the particles in
water are positively charged). The charge neutralisation, adsorption and precipitation are
often exploited to destabilise the colloidal particles.
Thus, over time, researchers found that coagulation using such organic/inorganic coagu-
lants may follow different mechanisms (Table 17.3). At low coagulant dosage and low pH
value, the coagulant neutralises the electrical charge of the suspended particles in wastes
Table 17.3 Mechanisms of coagulation; adapted from [105].
No. Mechanism Scheme

1 Inter‐particles bridging
2 Superficial charge neutralisation and

adsorption
3 Sweep coagulation
4 Double layer compression
5 Superficial charge neutralisation and

precipitation
Table 17.4 List of common inorganic coagulants.
Name of coagulant Hydrolysis reactions

Iron‐based coagulants [108] Fe(OH)3↔ Fe3+ + 3OH−
Fe2(SO4)3 Fe3+ + 2H2O ↔ Fe(OH)2+ + H3O+
FeCl3 Fe(OH)2+ + H3O+↔ Fe(OH)2+ + H3O+
2Fe3+ + 4H2O ↔ Fe2(OH)24+ + 2H3O+
Fe(OH)3 + OH−↔ Fe(OH)4−
Aluminium‐based coagulants [109] Al3+ + H2O ↔ AlOH2+ + H+
Al2(SO4)3 AlOH2+ + H2O ↔ Al(OH)2+ + H+
Polyaluminium chloride Al(OH)2+ + H2O ↔ Al(OH)3 + H+
Aluminium chlorohydrate Al(OH)3 + H2O ↔ Al(OH)4− + H+
causing the clumping effect, which is a predominant phenomenon. On the other hand, at high
coagulant dosage and pH value, the particles are just adsorbed on the coagulant surface.
In general, the most used coagulants are of inorganic nature (Table 17.4), acting as
charge neutralisers. Being added to water, this type of metal‐based coagulant dissociates
and releases metal ions in the solution. The released metal ions react with hydroxyl ions
(OH−), resulting in a hydrolysed species [106]. The formed hydroxides produce polymer‐
like chains allowing microfloc formation and growth to larger flocs.
One may observe that the most commonly used inorganic coagulants are based on alu-
minium and iron(III) salts. Nevertheless, these chemicals show some disadvantages in their
usage because they are effective only in a certain pH range. As a consequence, a good floc-
culation may not be achieved in some wastewaters, yielding an effluent with poor quality.
Despite these disadvantages, these kinds of coagulants can be found as residuals in treated
waters that are returned to nature. As they are not biodegradable, these residuals can cause
illness when the water is intended for human consumption. For example, Alzheimer’s dis-
ease is linked to aluminium accumulated from water sludge that causes problems of dis-
posal and treatment [107].
Consequently, researchers had to look for new solutions. In this context, polymers,
especially derived from certain kinds of plants and animal life, were found to be work-
able alternatives to inorganic coagulants. The natural coagulants were proven to be non‐
toxic flocculants in treatment of organic polluted wastewaters [110] and as a chelator of
toxic (heavy and radioactive) metals [111]. Due to their properties (availability, biocom-
patibility, biodegradability, polyelectrolisity, non‐toxicity and flocculating ability), pol-
ysaccharides can be used in multiple and various fields. For a few decades, these natural
additives attracted the attention of many researchers dealing with environmental protec-
tion. Hence, natural additives were employed as a flocculant/coagulant for wastewater
treatment [112].
The most challenging natural coagulants, over other polysaccharides, are chitin/chitosan
and their derivatives [113]. They have a chemical structure that contain very reactive amino
(–NH2) and hydroxyl (–OH) groups, which recommend them to be used as adsorbent mate-
rial. Also, their chemical structures allow designing chitin/chitosan macromolecules for a
particular application. Owing to the presence of reactive groups, chitin/chitosan is able to
form composites with various compounds (zeolites, magnetite, polyurethane, etc.) [114].
Recently, Dhananasekaran, Palanivel and Pappu [115] have found that α‐chitin
Table 17.5 Coagulant comparison; adapted from [119].
Characteristic Chitosan Al2(SO4)3·xH2O FeCl3·6H2O

Chemical Natural biopolymer Inorganic salt Inorganic salt
Optimum dose 8 30 30
(mg·L−1)
Optimum pH 5.5 6.0 5.5
Colour removal Most effective for removing Somewhat effective Increases colour due to
colour residual iron
Total Coliform and Effective and consistent with a Effective but inconsistent Most effective
Escherichia coli clear point of diminishing
removal return
Hardness removal Some removal at optimum dose Little to none Little to none
and pH
Settled water Somewhat effective; less Most effective; consistent Effective; consistent
turbidity removal effective with increase in pH; removal with increase in with increase in pH
consistent removals with pH and an increase in and an increase in
increase in algae count algae count algae count
n anoparticles, of 49 nm in size, 96.8% degree of acetylation (DA) and 83.73% crystallinity,

are promising materials for the purification of water dye stuff contamination. Such chitin
nanoparticles proved to adsorb dyes in a very short time in laboratory conditions. Chitin
can be also applied as a coagulant aid. For instance, Saritha, Srinivas and Srikanth Vuppala
[116] demonstrated that the turbidity of wastewaters can be removed up to 99% by both
chitin and aluminium sulphate, in an optimum dosage of 0.1–0.3 g L−1 at all pH values.
Dotto et al. [117] evaluated the efficiency of chitin as a coagulant. They reported that chitin
has an efficiency of 70% in suspended solid reduction (SSR) in comparison with alumin-
ium sulphate, which exhibited 90% of SSR. However, other authors [118] demonstrated
that chitosan can yield 95% of SSR.
The deacetylation degree of chitosan plays a crucial role in the removal of heavy metals
from wastewaters [50]. Chitosan is the single cationic biopolymer able to neutralise and
flocculate the anionic colloidal particles. This natural biopolymer acts as destabilising
agent and help flocs to grow. At high dosages, chitosan neutralises the charged particle,
then precipitates them, thus the coagulation process occurs by a charge neutralisation
mechanism. At low dosages, chitosan just adsorbs colloidal particles through the mecha-
nism of electrostatic charges [21].
Frederick [119] compared chitosan with inorganic coagulants and showed that chi-
tosan had the ability to work as an efficient flocculant in lower dosages (Table 17.5).
The coagulation and flocculation properties of chitosan are given by the positive charges
that are responsible for the removal of negatively charged colloidal particles [120].
Generally, the use of polymers has advantages over inorganic coagulants, such as: lower
coagulant dose requirements, a smaller volume of sludge, a reduction in the ionic load
of the treated water, avoidance of the presence of metal ions, and cost savings up to
25–30% [121].
Chitosan particles in various sizes and shapes were also applied as a flocculant in col-
loidal systems containing algae [122], bacteria [123] and emulsions [124].
17.4 Wastewater Treatment by Membrane Separation

Chitosan is a polycationic biopolymer with promising potential for applications related to
the membrane ultrafiltration process, which is an important water purification technology
designed for production of high‐purity water. In this respect, chitosan derivatives can be
employed as hydrophilic components for the fabrication of blend ultrafiltration mem-
branes. Likewise, chitosan can be applied as a binding agent to enhance the performance of
the ultrafiltration process. These aspects are outlined in the following subsections.
17.4.1 Principle of Ultrafiltration Process

Ultrafiltration (UF) represents a pressure‐driven filtration of liquids (through a porous
membrane), which is frequently applied as a separation process in chemical environmental
engineering, food industry and biotechnology. The removal mechanism by UF is based on
size exclusion [125–127]. According to this, water, ions and low‐molecular‐weight species
permeate a porous membrane. Instead, the tiny particles, bacteria, colloids, macromole-
cules and large organic molecules are retained by the membrane. Generally, a UF mem-
brane involves pores of size ranging from 0.002 to 0.1 μm. Note that the conventional
microfiltration process (MF), applied for the retaining of suspended solids, involves sym-
metrical microporous membranes (0.1–10 μm pore size) operated at a pressure of 0.5–3
bar. In turn, the ultrafiltration process is carried out on microporous‐asymmetric mem-
branes (0.002–0.1 μm pore size) subjected to a pressure of 1–10 bar [125].
Often, the pore sizes of the membrane are expressed indirectly as molecular weight cut‐
off (MWCO). This descriptor (MWCO) is measured by filtering surrogate molecules with
known molecular weights. Particularly, MWCO refers to the lowest‐molecular‐weight sol-
ute (in Daltons) in which 90% of the solute is retained by the porous membrane. The values
of MWCO range from 1,000 Da to 300,000 Da (i.e., 1–300 kDa) for the case of UF
membranes.
Some basic aspects of membrane processes are outlined in the following. Figure 17.4
shows the most important terms used in membrane separations along with two different
modes of running the ultrafiltration processes (i.e., dead‐end and cross‐flow filtration)
[125–127]. According to Figure 17.4, the feed side is referred to the bulk solution that
(a) (b)
Feed
Driving
force
Retentate Feed Retentate
(ΔP)
Membrane Membrane
Flux Flux
Permeate Permeate
Figure 17.4 Principle sketch of the pressure‐driven membrane processes: (a) dead‐end (in‐line) filtration
mode; (b) cross‐flow filtration mode.
undergoes ultrafiltration. Components of the bulk solution that are retained by the mem-
brane represent the retentate (concentrated solution). As the driving force acts, a higher‐
quality flux crosses through the membrane from the feed side to the permeate side. The
driving force of the filtration process is the transmembrane pressure (ΔP) [128, 129],
which can be calculated by Eq. 17.2 and Eq. 17.3 for dead‐end and cross‐flow filtration,
respectively.
Dead‐end filtration:
P Pfeed Ppermeate Pfeed Eq. 17.2

Cross‐flow filtration:
Pfeed Pretentate Pfeed Pretentate

P Ppermeate Eq. 17.3
2 2
Note that the pressure on permeate side (Ppermeate) is always much smaller than the pres-
sures on feed (Pfeed) and retentate (Pretentate) sides, that is, Pfeed>>Ppermeate.
The liquid going through the membrane with a certain velocity is designated as the per-
meate flux, and it is often reported in the units of L/(m2⋅h). Hence, the permeate flux is
calculated with respect to the membrane surface area, as given by [125, 127]:
V P
J Eq. 17.4
A t RM RCP RF

Here, A denotes the membrane area, and ΔV is the filtrate volume produced within time
interval Δt. On the other hand, the permeate flux is directly proportional to the transmem-
brane pressure (ΔP), and inversely proportional with the filtrate viscosity (μ) and total flow
resistance. The latter term involves three components of resistances against the solvent
transport, such as the resistance to flow provided by the membrane (RM); the resistance
caused by concentration polarization effect (RCP); and the resistance caused by membrane
fouling (RF) due to cake formation and pore blocking. Both phenomena, concentration
polarization (reversible) and fouling (irreversible), help achieve flux decline, thereby
reducing the filtration performance. Likewise, the quality of the permeate solution can be
evaluated by determining the rejection efficiency (Y, %), which can be expressed as:
Cp
Y 1 100 Eq. 17.5
Cb

where Cb is the solute concentration in the bulk solution from the feed side, and Cp is the
solute concentration in the permeate solution.
The greater the values of both responses (i.e., rejection efficiency, Y, and permeate flux,
J), the better is the performance of the UF process. Therefore, it is important to design
membranes with good selectivity and improved hydrophilicity. The latter characteristic
(hydrophilicity) helps in diminishing the concentration polarisation and fouling effects.
17.4.2 Fabrication of Ultrafiltration Blend Membranes

UF polymeric membranes are mainly produced by means of the phase inversion method.
The phase inversion is a demixing process by which the initially homogeneous polymer
solution is transformed, in a controlled fashion, from liquid to a solid state [126, 127, 130].
The phase inversion process is often realised by the immersion precipitation technique.
According to this, a polymer solution is cast first on a proper support (glass or nonwoven).
Then, it is immersed in a coagulation bath containing a non‐solvent. As a result, the pre-
cipitation (membrane formation) occurs owing to the exchange between the solvent and
non‐solvent. In this case, the phase inversion leads to the formation of asymmetric porous
membranes.
Typically, UF membranes are prepared from casting solutions with initial polymer
concentrations of 10–20% w/w [126]. The best solvents employed to prepare casting solu-
tions deals with aprotic solvents that lack acidic hydrogen (without labile H+), namely,
dimethylsulphoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC),
N‐methylpyrrolidone (NMP), tetrahydrofuran (THF), and others [126, 127]. Regarding the
coagulation bath, water is the most‐used precipitation medium (non‐solvent), although,
alcohols like methanol, ethanol and isopropanol can also be used as precipitation media,
leading to denser and lower‐flux membranes when compared to the ones precipitated in
water [127]. To enhance the filtration properties of membranes, the pore‐former agents
(porogens) are often used, such as LiCl, polyvinyl pyrrolidone (PVP) and polyethylene
glycol (PEG) [130]. The basic polymer choice is important for the fabrication of the UF
membrane, because it limits the solvents and non‐solvents that can be applied in the phase
inversion process. The common polymers used for the fabrication of UF membranes are
polysulphone, poly(ether sulphone), poly(vinylidene fluoride), polyacrylonitrile, polyim-
ide, and poly(ether imide) [126, 127, 130, 131]. Most of these polymeric materials are
hydrophobic in nature; therefore, they stimulate the concentration polarization effect.
To diminish the concentration polarization phenomena, and to improve the permeate
flux, additional hydrophilic polymers can be added during membrane manufacture.
This technique is known as blending with hydrophilic additives, and it has received great
attention because no supplementary steps are required [132]. In this regard, chitosan is a
hydrophilic material having many hydroxyl groups [133]. Therefore, chitosan can be added
to the composition of an UF membrane to induce anti‐fouling properties. However, there is
a limitation related to the fact that chitosan is insoluble in common organic aprotic sol-
vents. To resolve this issue, chitosan can be chemically modified to produce organosoluble
chitosan derivatives that can be further used in the blending process.
For example, Ghiggi et al. [132] produced a polyethersulphone/N‐phthaloyl‐chitosan
blend membrane for UF purposes. To this end, the chitosan was modified with phthalic
anhydride in a solution mixture of DMF:water (95:5 v/v) at 130°C under nitrogen atmos-
phere for 5 h reaction time [132–134]. Figure 17.5 illustrates the scheme of the reaction
for chemical modification of chitosan with phthalic anhydride. The resulted derivative
(N‐phthaloyl‐chitosan) was blended with polyethersulphone (PES) in a DMAC solvent,
and the asymmetric porous membranes were produced by phase inversion using water as a
non‐solvent [132].
The resulted blend membranes PES/N‐phthaloyl‐chitosan showed a low water contact
angle (WCA=56 ± 5) and improved permeate fluxes in protein filtration tests. Hence,
O
OH O O OH
DMF / H2O
O O O O
+
HO 130 °C/N2 HO
N
NH2 O O
n n
Figure 17.5 Schematic representation of chitosan modification with phthalic anhydride; adapted from
[132–134].
Ghiggi and collaborators demonstrated that the blend membranes (made of PES and N‐
phthaloyl‐chitosan) could significantly diminish the concentration polarisation effect and
fouling resistance, indicating their antifouling properties [132].
Other authors [135–137] reported the chemical modification of chitosan with succinic
anhydride, in mild thermal conditions (35–40°C), yielding N‐succinyl chitosan (NSCS)
derivative. For instance, Kumar, Isloor and Ismail [136] produced blend membranes made
of polysulphone/N‐succinyl chitosan (PSf/NSCS) that were applied for the UF of proteins.
Owing to the low solubility of NSCS in organic solvents, these authors proposed a new
method for blending of NSCS with PSf. According to that method, the maximum blending
of NSCS with PSf was achieved for the ratio of 80:20 (PSf:NSCS). The produced blend
membrane PSf/NSCS (80:20) showed a low contact angle equal to 60.9°, confirming the
improvement of membrane hydrophilicity. All PSf/NSCS membranes disclosed higher per-
meate fluxes during protein UF than the pristine PSf membrane. Hence, by blending NSCS
with PSf, both permeation flux and antifouling properties of membranes were enhanced
significantly. Finally, the authors concluded that N‐succinyl chitosan derivative (NSCS)
acted as a promising substitute for the hydrophilic polymeric additives like PVP, PEG or
polyvinyl alcohol (PVA).
The same authors [137] reported in another paper the synthesis of a different chitosan
derivative as an additive for polysulphone membrane. The water‐soluble chitosan deriva-
tive was designated as N‐propylphosphonic chitosan (NPPCS) having a terminal phospho-
nic acid group, and this derivative was synthesised by reacting chitosan with
hydroxybenzotriazole (HOBt) and propylphosphonic anhydride via a one‐pot reaction. The
produced blend membranes (polysulphone/N‐propylphosphonic chitosan) disclosed
improved antifouling properties when compared to pristine polysulphone membranes.
In their seminal paper, Zhang et al. [138] prepared a chitosan nanofiltration membrane
on polyacrylonitrile (PAN) UF membrane which was used as support. To improve the acid
resistance of the composite membrane, chitosan was cross‐linked by glutaraldehyde
through hydroxyl groups by protecting amine groups using copper ion chelation. The
cross‐linked chitosan/PAN membrane disclosed good rejection efficiency (95%) of γ‐
aminobutyric acid from aqueous solutions at pH 4.69.
A previous study reported by Xue et al. [139] dealt with chitosan‐functionalised gra-
phene oxide applied to improve the permeability and antifouling properties of UF mem-
branes. In their approach, the chitosan‐functionalised graphene oxide (CS‐GO) was
synthesised and incorporated into a polyvinylidene fluoride (PVDF) matrix. The produced
CS‐GO/PVDF blend membranes showed higher permeate flux and rejection efficiency,
when compared to PVDF, CS/PVDF, and GO/PVDF membranes. The optimal content of
CS‐GO into the PVDF matrix was found to be 0.6% wt%. This composite membrane
showed a reduced WCA (64.2°) and an improved antifouling property. Finally, these
authors concluded that the CS‐GO/PVDF composite membrane could be of great potential
for water treatment applications.
In another study, Hamzah et al. [140] designed chitosan/polysulphone (CS/PSf) self‐
assembly membranes to mitigate fouling and enhance the performance of the UF process.
To this end, the native PSf membranes were immersed into the chitosan solution (0.1%
wt%, in acetic acid pH 5) for 30–120 min to deposit chitosan macromolecules onto the PSf
membrane surface. This study highlighted that the integration of hydrophobic polysul-
phone and hydrophilic chitosan successfully upgraded the membrane UF performance by
minimising the fouling phenomena.
Preliminary work on the removal of chromium from wastewater by a cellulose acetate/
chitosan UF membrane was undertaken by Vinodhini and Sudha [141]. In their study, nan-
oparticles of chitosan were first prepared by sol‐gel method using sodium tripolyphosphate
(TPP) as a cross‐linker. Next, the UF membrane was prepared by blending cellulose acetate
(CA) with nanoparticles of chitosan (NCS) and PEG at 1:2:2 ratios. DMF was used as an
aprotic solvent to prepare casting solutions. These authors inferred that the produced blend
membrane CA/NCS/PEG could remove chromium from tannery effluents in an effective
way by the UF process.
17.4.3 Chitosan‐Enhanced Ultrafiltration

UF is less efficient for the removal of ions and small organic compounds from contami-
nated waters because these species normally pass through the membrane. However, the UF
process can be enhanced by adding water‐soluble polymers with functional groups able to
bind chemical species of low molecular weight. Such improved separation process is
known in literature under various titles, such as polymer‐enhanced ultrafiltration (PEUF),
complexation–ultrafiltration, adsorption–ultrafiltration, seeded UF or polymer‐assisted
ultrafiltration (PAUF) [142–146].
In PEUF, the water‐soluble macromolecules interact with ionic species (solutes) by
complexation, resulting in the formation of supra‐molecular aggregates that can be retained
by a porous asymmetric membrane (see Figure 17.6). The selection of a binding polymer
(complexing agent) depends on the nature of the ionic species (solute/contaminant).
Generally, the binding polymer has to fulfil particular requirements – that is, high molecu-
lar weight, good water solubility, ability of selective binding with ions and molecules, sta-
bility of complexes, and being non‐toxic and low‐cost [142]. Typical binding polymers are
PEG, polyethylene imine (PEI) and polyacrylic acid (PAA). Such binding agents have been
extensively applied for the removal of heavy metal cations and water‐soluble organic mol-
ecules (e.g. synthetic dyes) [129, 142–146].
Recently, low‐cost natural biopolymers have attracted attention as potential complexing
agents for PEUF. For example, Baharuddin, Sulaiman and Aroua [147–149] utilised starch
as a binding polymer in PEUF to remove heavy metal cations (Zn2+, Pb2+, Cr(VI), Cr3+)
from aqueous solutions.
(a) (b)
Feed Feed
Solvent (water)
Solute (pollutant)
Binding polymer
Driving
force
(∆P)
Membrane
Permeate Permeate
Figure 17.6 Schematic representation of ultrafiltration process: (a) ultrafiltration in the absence of a binding
polymer; (b) polymer‐enhanced ultrafiltration (PEUF).
A comparison between carboxymethyl cellulose (CMC) and chitosan as binding poly-

mers for UF was reported by Lam et al. [150]. They found that both polymers (CMC and
chitosan) showed similar performance for the UF of Ni2+ cations at natural conditions of pH
value (4 < pH <8). However, chitosan was a better option for basic (pH >8) or stronger acid
(pH <4) conditions. Interestingly, these authors applied chitosan‐enhanced UF to remove
heavy metal ions (Al3+, Co2+, Ni2+, Fe3+, Zn2+) from real wastewaters derived from the sur-
face treatment industry. Their results disclosed an improvement of metal rejections in the
presence of chitosan when compared to simple UF without adding chitosan.
A brief literature review on heavy metal ion removal by chitosan using PEUF processes
has been recently reported by Crini et al. [151]. Generally, the interaction mechanism (chi-
tosan–metal) can be explained by complexation [144, 151] that might occur via the forma-
tion of donor–acceptor bonds between the metal cation and nitrogen/oxygen atoms from
chitosan.
Recently, a relevant study has been reported by Déon et al. [152] regarding the remedia-
tion of aqueous solutions containing oxyanions of selenium using PEUF in the presence of
chitosan. These authors demonstrated that chitosan addition induced a positive influence
on selenium rejection on ceramic UF membranes with large pore sizes.
In a previous work, Dasgupta et al. [153] compared two cationic chelating polymers,
namely PEI and chitosan, for complexation and retention of Reactive Red 120 (RR
120 – anionic dye) by PEUF. The complexation–UF of RR 120 dye revealed that PEI‐
enhanced UF could provide a high rejection efficiency (>99%) and permeate flux of 148 L/
m2·h. In turn, the complexation–UF of RR 120 in the presence of chitosan unveiled a rea-
sonable rejection efficiency (>88%) and permeate flux (120 L/m2·h). The weaker interac-
tion between chitosan and RR 120 dye was explained by the presence of dye‐repelling
hydroxyl moieties in chitosan. Ultimately, these authors claimed that the successful appli-
cation of the futuristic PEUF process for water reclamation should imply a proper selection
of the chelating polyelectrolyte based on the ionic and chemical–structural properties of
the target solute molecule.
17.5 Outlook
Chitin and chitosan are known for their abundance, biodegradability, non‐toxicity, biocom-
patibility, renewable nature, low cost, versatility and ease of chemical modification. Based
on their properties, these polysaccharides are strong candidates for applications in the field
of wastewater treatment. This review was focused on the most relevant methods for water
purification involving chitin/chitosan materials, such as: adsorption, coagulation/floccula-
tion and UF purification processes. In spite of some rather satisfactory performances, the
solubility problems of chitin do limit the utilisation of this biopolymer in the development
of new materials dedicated to water purification treatments. In contrast, chitosan is easily
modified by cross‐linking, grafting, chemical functionalisation and/or forming composites,
thereby extending the range of applications or clearly improving its performance.
The use of chitin/chitosan (pristine and modified forms) for organic pollutant adsorption
is widely reported in literature. The majority of research works suggested that the adsorp-
tion occurred mainly in acidic medium and was realised via an ion exchange mechanism
based on electrostatic attraction between the protonated form of the biopolymer and organic
pollutant molecules frequently in their negatively charged form. Future work should be
dedicated to the application of chitin/chitosan‐based materials as adsorbents in real waste-
waters in order to determine their performances under real environmental conditions.
The performance of chitin/chitosan, both pristine and modified forms, for heavy metal
ion removal from wastewaters is reported by many authors. Nevertheless, the mechanism
governing metal cation adsorption onto adsorbent surface still needs to be detailed for each
particular application.
Many types of wastewaters can also be treated with chitin/chitosan‐based materials in order
to reduce the total solids, suspended solids and turbidity. In these lines, chitin/chitosan‐based
materials may act as coagulants/flocculants in the separation process. Such a process occurs
by a charge neutralisation mechanism or by the mechanism of electrostatic charges, depend-
ing on the coagulant concentration. Biomaterials (chitin/chitosan) showed improved coagu-
lant properties when compared to inorganic coagulants, even if they were used individually or
along with other organic/inorganic materials to treat wastewaters of different origins.
The literature survey has indicated that chitosan‐based materials are also of interest in
the field of membrane separations by UF. In this regard, chitosan derivatives were success-
fully employed to produce blend membranes with improved hydrophilic properties that can
diminish concentration polarisation and fouling phenomena.
The binding capacity of chitosan for metal cations, oxyanions and water‐soluble dyes
proves that this biopolymer is a good candidate substitute for conventional binding polymers
used in complexation–UF processes. Other advantages that make chitosan attractive for the
membrane separation processes are related to its non‐toxicity, recyclability and low cost.
Hence, it turns out that chitosan and its derivatives are prospective materials for environ-
mental applications related to water purification.
Acknowledgement
The authors Petrisor Samoila, Corneliu Cojocaru and Maria Ignat are grateful for the finan-
cial support of the Romanian Ministry of Research and Innovation, CNCS – UEFISCDI,
project number PN‐III‐P1‐1.1‐TE‐2016‐0805, within PNCDI III. Also, the authors Andra
Cristina Humelnicu and Valeria Harabagiu are grateful for the financial support of the
Romanian Ministry of Research and Innovation, CCCDI‐UEFISCDI, project number PN‐

III‐P1‐1.2‐PCCDI‐2017‐0194/25PCCDI/2018, within PNCDI III.
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18
Chitosan for Sensors and
Electrochemical Applications
Suse Botelho da Silva1, Guilherme Lopes Batista2, and
Cristiane Krause Santin1,2
1
Food and Chemical Engineering, Polytechnic School, Unisinos University, São Leopoldo, RS, Brazil
2
itt CHIP ‐ Unisinos Semiconductor Institute, São Leopoldo, RS, Brazil
This chapter discusses the use of chitosan for sensors and electrochemical applications.
The development of specific sensors and electrochemical devices is among the most inno-
vative applications of chitosan and its composites and derivatives. These materials present
characteristic chemical and electrical features, along with their interesting mechanical and
biological properties (which have been extensively exploited before), making them unique
materials for the referred applications. Although chitosan may present useful characteris-
tics alone, many applications explore its use through chemical modifications or in compos-
ites, leading to materials that may present mixed characteristics or, in some cases, better
performance due to synergic effects. This chapter will present how the structure of chi-
tosan, whether alone or modified, as well as the composite materials containing this poly-
mer, can present very interesting properties for its use in sensing platforms, solid‐state
batteries, and other electronic devices.
18.1 Introduction
In the last decades, there has been a huge demand for biocompatible, biodegradable, and/
or compostable materials in several areas, including in chemical, cosmetic, pharmaceutical,
and medical industries [1]. More recently, the use of “green materials” has also been shown
as a new trend in microelectronics, for applications as electronic devices, solid‐state

batteries, and sensors [2, 3]. In this sense, chitosan appears as a promising substitute for
synthetic polymers for use in electrochemical devices due to many peculiar properties and
characteristics. Chitosan, in pure form or combined with other compounds, can offer robust
and homogeneous materials in many different forms, such as layered films, hydrogels, or
other three‐dimensional materials. Its structure, containing both hydroxyl and amino
groups, is very versatile for chemical modifications for production of sensors and other
electrochemical devices, and also presents good interaction with different ions as
ionophores. Additionally, many chitosan‐based materials exhibit electrical conductivity
due to their proton binding sites that allow proton mobility. All these properties allow the
development of a wide range of applications. This chapter does not intend to cover
comprehensively all the applications of chitosan that are presented in literature, but rather
present some of the many different approaches in which chitosan can be used, making use
of its unique properties for different sensor technologies and other electrochemical
applications. For better understanding, some key aspects regarding these applications, such
as chitosan structure and relevant properties, the proton conductivity mechanism, and
methods for preparation of chitosan‐based materials, will be also presented in this chapter.
18.2 Chitosan: A Biopolymer with Unique Properties

Chitosan is recognized as a nontoxic, biodegradable, biocompatible, and amine‐rich
polymer with a good film‐forming ability [4, 5]. It is a random copolymer of D‐glucosa-
mine and N‐acetyl‐glucosamine (Figure 18.1), obtained from full or partial deacetylation
of chitin [5, 6]. Unlike chitosan, chitin is formed mainly by N‐acetyl‐glucosamine units.
When the deacetylation degree is greater than 50%, the protonation of the amino groups in
acidic pH causes the solubilization of the molecule [1]. The presence of amino groups in
the chitosan structure imparts peculiar properties to this polymer [7]. At pH below 6.2, the
protonation of the amine groups occurs (−NH3+), and chitosan is charged with a positive
surface charge on its D‐glucosamine units [1]. As a result of protonation, chitosan exhibits
a cationic character and can interact with negatively charged molecules [5]. Apart from
highly reactive amino groups at C‐2, chitosan also has hydroxyl groups (primary hydroxyl
at C‐6 and secondary hydroxyl at C‐3) that are easily modified by several organic reactions
[5, 8]. At the same time, these amino and hydroxyl groups tend to engage in intramolecular
and intermolecular hydrogen bonds resulting in the formation of linear aggregates with
extensive crystallinity [9].
The remarkable structure of chitosan with many amino and hydroxyl reactive groups in
a polysaccharide backbone allows biodegradability and biocompatibility, in addition to
chelating, complexing, antimicrobial, antioxidant, healing, and film‐forming properties.
O
H3C
OH
6
NH
3
2
1
O
4
5 O
HO
O 5
HO 2
4 O 3 1
6
NH2
OH
Figure 18.1 Chitosan structure.

Chitosan for Sensors and Electrochemical Applications 463
Due to all these properties, chitosan has been intensively studied, and several industrial and
technological applications have been developed in the last years, including uses in water
treatment, agriculture, food, pharmaceutical, biomedical, cosmetics, textile, and electronic
areas [5, 10–13]. Among the many properties exhibited by this polysaccharide, chelating
activity and electrical conductivity are of particular relevance in the development of
applications for sensors and other electrochemical devices. The hydrogen‐donating ability
of chitosan is the key factor for many properties exhibited by this polymer, including
electrical conductivity, which is supported by the hydrogen‐bond network along the
chitosan structure.
In solid state, chitosan is a semi‐crystalline polymer that is insoluble in water and most
organic solvents; however, it is soluble in dilute acid solutions [14]. Acetic acid is often
used for solubilization of chitosan, but other diluted organic acids (formic acid, propionic
acid, lactic acid) and inorganic acids (hydrochloric acid and nitric acid) have also been used
[15]. Chitosan is considered as a base which has primary amino groups of glucosamine
with a pKa value of 6.3 [16]. When chitosan is dissolved in acidic medium, it becomes a
cationic polyelectrolyte due to protonation of amino groups [17], according to the equation:
Chit NH 2 H 3O  Chit NH 3 H 2 O (1)
The degree of protonation is dependent on the concentration (pH) and the type (pKa) of
acid used [17]. Likewise, as the pH increases above the pKa, the amino groups deprotonate,
and the polymer loses its charge and becomes insoluble. The soluble–insoluble transition
occurs at its pKa value between pH 6 and 6.5 [16].
As the solubility of chitosan in the acidic medium depends on the ionic concentration, a
salting‐out effect can be observed in excess of hydrochloric acid (>1 M), making it possible
to isolate the hydrochloride form of chitosan [17]. On the other hand, as reported by
Rinaudo et al. [17], an excess of acetic acid or other weak acid increases the chitosan
solubility and no salting‐out was observed, even in high concentrations of acetic acid
(lower than 16.5 M), due to its small dissociation degree. When the hydrochloride and
acetate forms of chitosan are isolated, they are directly soluble in water giving an acidic
solution with pKa = 6.0 [14]. Chitosan salts can also be obtained by water evaporating from
an acid diluted solution [18].
18.3 Modification and Preparation of Chitosan‐Based Materials

for Electrochemical Applications
Despite the distinguished properties naturally provided by the chitosan structure, modifica-
tions in this polymer can still be performed to obtain a material with improved performance
or more suitable for a specific application. With these purposes, chitosan can be partially
depolymerized and amino and hydroxyl groups can be assessed using a physical, chemical,
and/or enzymatic approaches [5, 11, 13]. Quaternization, thiolation, glycation, tosylation,
alkylation, carboxylation, sulfonation, formation of Schiff’s base, and organocatalytic oxi-
dation are the main modifications of chitosan [5, 19]. These reactions occur and are induced
during polymer transformation processes, such as blending, grafting, and cross‐linking,
allowing the subsequent preparation of films, fibers, hydrogels, and other chitosan‐based
materials.
In blending, chitosan is combined with another polymer leading to a polymer blend with
better properties as compared to those obtained from the polymers alone [20]. Blends can
be homogenous or heterogeneous mixtures showing different morphologies, which depend
mainly on the type of process used to combine polymers (solvent blending, extrusion
blending, or reactive extrusion blending) [21]. In grafting, functional groups (and even
polymers) are inserted onto the backbone of chitosan via a chemical reaction or modification
of amino and hydroxyl groups. Functional groups such as carboxylic acid and hydroxyl
groups may be grafting on chitosan to increase the number of hydrogen bonds or to provide
more reactive groups for subsequent modifications [13].
The presence of amino and hydroxyls groups in the chitosan backbone also represents a
great potential for cross‐linking reactions. Dialdehydes can be used to interact with amino
groups of chitosan via covalent imine bonds (Schiff’s base), producing a cross‐linked
structure [9, 22]. Glutaraldehyde and glyoxal are the most commonly used cross‐linker
agents, but also genipin, diethyl squarate, and oxalic acid can be used [22]. Nevertheless,
cross‐linked chitosan can also be produced without an external cross‐linker, being formed
by direct interaction between polymeric chitosan chains through complexation with another
polymer or with a modified chitosan [7, 22]. A self‐cross‐linked amphiphilic structure was
obtained by (2,2,6,6‐tetramethylpiperidin‐1‐yl)oxyl (TEMPO)‐laccase oxidation of chi-
tosan, which added aldehyde groups to the structure from the oxidation of primary hydrox-
yls, allowing the formation of covalent imine bonds directly with the amino groups [19].
In addition to the covalent and ionic bonds, secondary interactions such as hydrogen
bonds and hydrophobic interactions are present in the three‐dimensional network formed
by the cross‐linked chitosan structures [23]. Three‐dimensional hydrophilic polymeric
networks, which absorb and retain water in their structure, are typically called hydrogels
[24]. Hydrogels can take up considerable amounts of water (up to thousands of times its
dry weight) without dissolving [24, 25]. They can occur as a colloid gel as well, in which
water is the dispersion medium [13].
Hydrogels, as well as films, are among the most used forms of chitosan in the development
of applications involving electrochemical devices and sensors. In some applications,
chitosan films or hydrogels can be used as a carrier or as a substrate for other compounds
or nanoparticles, resulting in composite materials with enhanced properties. It is especially
true for sensing applications. In this area, considerable advances have occurred in recent
years, mainly driven by the development of nanotechnology. Chitosan‐based nanomaterials
tend to exhibit superior physical and chemical properties as compared to conventional
chitosan [13]. Tensile strength, conductivity, and photoluminescence are among the
properties that are improved as a result of the typical high surface area of the nanomaterials
[13]. All these properties are particularly important for the manufacturing of films to be
used in sensor devices.
Chitosan films are usually prepared by the casting method [5]. Following this method,
first chitosan is dispersed in a diluted acid solution, typically in acetic acid. This dispersion
is kept under continuous stirring, generally at room temperature, until complete
solubilization. Then, the film‐forming solution is casted over a surface for drying under
controlled temperature and relative humidity [15, 26, 27]. The complete solubilization of
chitosan in a diluted acid solution may take some hours. After solubilization, plasticizers or
other agents can be added to improve the properties of films that will be formed [2, 20].
Likewise, chitosan solution can be filtered, degassed under vacuum, or centrifuged to
remove particulate or air bubbles [15, 28] As a polyelectrolyte, chitosan can also be
employed for the preparation of multilayered films, using electrostatic layer‐by‐layer
techniques based on the alternated adsorption of materials bearing opposite charges [29].
Recently, other methods of obtaining chitosan films, other than casting, have been
reported. Thin films have been prepared by spin‐coating [28] and electrochemical
deposition methods [30]. Even more in these cases, obtaining a filmogenic solution with
adequate viscosity and homogeneity is a necessary requirement. By spin‐coating, a dilute
acid chitosan solution is spread over a planar surface under high rotation speed [28]. While
the solution spins over the surface, the solvent is evaporated, being film thickness dependent
on polymer concentration, solvent volatility, and angular speed of spinning [31]. Controlling
these parameters is possible to prepare uniform ultrathin films [28, 31].
By an electrochemical deposition process, two electrodes (cathode and anode) are
dipped into the chitosan solution subjected to constant voltage [30]. The deposition of
chitosan on the cathode (negatively polarized electrode) is caused by the evolution of
hydrogen (H2) from the electrolysis of water, which consumes protons (H+) and generates
excess hydroxide ions (OH)− in the vicinity of this electrode. Thus, the protonated amino
groups of the chitosan are attracted electrostatically to the cathode, leading to the
insolubilization of the polymer next to the electrode. The continuous flow of electrons and
removal of protons by the cathode lead to the formation of a thin chitosan film on the
surface of the cathode [32]. To avoid the evolution of hydrogen gas and produce more
uniform coatings during the modification, a proton‐consuming reagent such as benzoquinone
or chloramphenicol can be added to the plating solution [33]. According to Suginta [32],
the electrodeposition of chitosan is particularly important in the development of
electrochemical biosensor devices, and in the selective placement of polymer deposits on
the active sites of microelectrode and nanoelectrode arrays that would not be easily
produced with conventional film‐forming procedures.
Apart from the method used to obtain chitosan films, the drying process exerts great
importance in the definition of the conductivity of film, especially in the conductivity–
temperature relationship behavior. Films dried in temperatures below Tg (glass temperature)
have higher conductivity than films processed over Tg. This difference is related to the
water content that remains adsorbed on the films, especially which are dehydrated at low
temperatures. The adsorbed water molecules form inter- and intra-form hydrogen bonds
with side groups of chitosan backbone and contribute for the enhancement of the conduc-
tivity of these films [26]. The mechanism behind this phenomenon will be explained in
more detail in section 18.4.
18.4 The Proton Conductivity of Chitosan

Although the use of chitosan associated with several other compounds has been widely
studied, the discussions presented here are mainly related to chitosan used alone, for
clarification purposes. However, the fundamental principles shown here can be extended to
chitosan derivatives and composite materials and constitute important insights for more
complex chitosan structures.
Two mechanisms explain the conductivity in chitosan films supported by proton mobility
(proton‐hopping), the “Grotthuss mechanism,” and the “packed‐acid mechanism” [26, 34, 35].
The first occurs in wet films, while the latter is observed in media with low humidity.
In dry state, chitosan films exhibit a very low electrical conductivity, less than 10−8 S
cm−1 [8]. However, if these films are hydrated or solvated with saline solutions, they
strongly enhance their conductivity [8, 23, 26]. Chitosan acetate films showed conductivity
as high as 10−4 S cm−1 after 1 h of hydration [8]. As shown by the authors [8], the conductiv-
ity increased greatly within 1 h but remained in the same order of magnitude for a longer
hydration time. This shows that the higher conductivity observed in wet films is due to the
adsorption of water, and gives insight that this may be related to conformational changes in
the chitosan chain. As discussed in section 18.2, the dissolution of chitosan in an acidic
solution causes the protonation of amino groups in the backbone structure, which remains
in chitosan films. In the same way, when dry chitosan films are hydrated, the amino groups,
which are weak alkaline groups, are partially protonated and some hydroxide ions are
formed [26]:
Chit NH 2 H 2 O  NH 3 OH (2)
Nevertheless, the increase on the conductivity of wet chitosan films is not due to ionic
conductivity (OH− ion conductivity) [26, 34], otherwise it is due to the “Grotthuss
mechanism” (proton‐hopping mechanism), as discussed by Prokhorov et al. [26]. When
water molecules are adsorbed by the chitosan chain, hydrogen bonds are formed
between water and protonated amino groups and other polar groups [34]. Several intra‐
hydrogen and inter‐hydrogen bonds in the chitosan structure create a kind of network
through which the protons (H+) can “jump” moving along the hydrogen bond network,
following a process of reorientation with cleavage/formation bonds with a local molec-
ular rearrangement [26, 34]. This proton mobility explains the difference in the conduc-
tivity of wet and dried chitosan films. It also supports the prevalent nonlinearity between
conductivity and temperature, described by Vogel–Fulcher–Tammann (VFT) model, in
heating experiments of wet films in the range of 25–70°C [26]. The VFT behavior is
related to the α‐relaxation process including the rotation of hydroxyl and amino groups
and breaking of hydrogen bonds due to water desorption for wet films obtained below
their Tg [26, 36]. These were confirmed by dielectric relaxation measurements [26].
Since the deacetylation degree of chitosan affects the water uptake, the proton
conductivity will also be affected, with highly deacetylated chitosan exhibiting higher
conductivity [23].
The conductivity exhibited by dried chitosan films can be explained by Grotthuss
“packed‐acid mechanism” [26, 36]. “Packed‐acid mechanism” is a kind of proton‐hopping
mechanism in which the conduction occurs by acid–acid interactions without the movement
of water [35, 37]. This mechanism was proposed by Ogawa et al. [37], and it occurs under
low humidity conditions and is typically observed in materials with a high acid concentration
[35]. A packed‐acid structure has a large number of proton donors, which increases the
concentration of protons in a packed‐acid structure, sufficiently to construct a weak
hydrogen‐bond network among the proton‐donors whereby protons move. [37]. Self‐
assembled polymer electrolytes can construct a similar structure as packed acids [37], dried
chitosan films produced by previous acid dissolution would be an example. In acetate
chitosan films, protons will be transferred through neighboring acid groups supported by
the formation of new bonds between −NH3+ groups and acetate ions [26]. Use of different
acids must give rise to different results in proton conductivity, since different acids induce
different protonation degrees and generate different counterions, producing a different

hydrogen bonding network.
Since the conductivity of chitosan is closely related to its structure, it is logical to expect
that structural modifications may lead to improvements in its conductive properties. The
modification of the amino or hydroxyl groups by grafting donor or electron acceptor groups
can alter the polymer charge and electrical properties, allowing fine‐tuning of the
electrostatic interaction between the chitosan and negatively charged molecules and/or
electrochemical mediators [32]. The introduction of bulky substituents can also control this
interaction by stereochemical impediment [32]. Khiar et al. [38] reported that the addition
of a proton donor leads to an increase in electrical conductivity in chitosan films, considering
a chitosan–NH4CF3SO3 system. The authors showed that the conductivity of films is a
function of the temperature and of salt concentration.
Du et al. [39] investigated the structural and electrical properties of chitosan electrolytes
doped by three different ammonium salts CH3COONH4, NH4Cl, and (NH4)2SO4, all
recognized as good proton donors. The results showed that the addition of ammonium salts
leads to complexation between ammonium salts, and the chitosan matrix enhancing the
amorphous phase of chitosan electrolyte and improving the conductivity. The improvement
in conductivity was attributed both to the increase in the number of proton donors and to
the enhancement in the amorphous nature of chitosan. As highlighted by authors, the
conduction of polymer electrolytes is dominated by the amorphous phase rather than the
crystalline phase. Regarding three types of ammonium salt‐doped chitosans as proton
donors, if the interaction between the ammonium salt’s anion and chitosan matrix is overall
strong, it does not support an H‐bond network for proton jumps, resulting in low
conductivity. In this sense, CH3COONH4–chitosan electrolyte exhibited the best electric
properties [39].
There are many other examples of materials obtained from chitosan, which have
great potential for the fabrication of sensors and other electrochemical devices. In section
18.5, some applications are presented.
18.5 Selected Applications

Chitosan has been proposed in a wide variety of electrochemical and sensing applications.
They benefit from its favorable properties for physical and chemical modification, high
hydrophilicity, and film‐forming properties. The most common approach, thus, is to
perform a chemical modification and/or make composite materials that enable its usage. It
will be clear, however, that in many cases chitosan presents unique chemical properties
beyond the already mentioned ones, and its use permits to develop some applications that
would not be possible by other means, or using other polymers usually applied for
composites. This section will illustrate how versatile these materials can be, as well as how
chitosan and its derivatives play their unique roles in these applications.
18.5.1 Electrochemical Sensors

Electrochemical sensors are materials that interact with molecules by charge transfer and/
or electrical field interactions. Typically, these materials are good electrical conductors and
their surface has specific or catalytic interactions with the molecule (or group of molecules)
to be measured. There are several approaches for electrochemical sensors and chitosan
derivatives, and composites have been used in many ways. As chitosan usually does not
present good electrical conductivity, it is rather common to make composites with more
conductive materials for making electrodes and sensors. Usually, the materials that have
been used more frequently are carbon‐based, especially carbon paste, graphite, graphene,
and carbon nanotubes. Chitosan composites can make very homogeneous dispersions,
retaining the conductive properties of these fillers and the chemical and catalytic properties
of chitosan and other composite materials.
The amino groups that are present on the chitosan molecules are binding sites for
electrostatic interactions. When in the protonated form, these groups interact well with
anions, and this property has been applied for selective potentiometric sensing of uranyl
ions (UO2−) [40] and monovalent anions [41]. In this type of sensor, the electric potential
of the membrane changes depending on the ion concentration, which is measured relative
to a reference electrode (with known electric potential) at zero current.
Although the surface of chitosan itself may be used as a sensing material for potentiometric
sensors, the surface can be further modified for the determination of different species. The
modification of sensors with enzymes has been extensively used for the fabrication of
analytical devices, and chitosan‐based materials are considered very suitable for this
application [42]. Enzymes have excellent catalytic activity and exhibit high selectivity
toward the target substrate, acting as excellent recognition molecules in sensors [43].
The modification of chitosan‐based materials with enzymes may happen in different
forms, that we can classify broadly as physical and/or chemical, and more than one
mechanism can occur simultaneously. For its modification, the methods may be classified
broadly into four groups: solvent evaporation method, in which a solution of chitosan in an
organic acid is casted and dried over a surface, typically an electrode (for this application,
it may be referred as drop‐casting); neutralization method, in which small particles of
chitosan are formed upon the titration of an acidic chitosan solution by a strong alkali;
cross‐linking method, using chemical agents to cross‐link chitosan molecules; and
coacervation, which precipitates chitosan on acidic solutions with anionic polyelectrolytes,
usually premodified with enzymes. A broad range of applications has been presented in a
review [42]. One example of enzymatic modification for a potentiometric approach used
glucose oxidase on a composite of chitosan with polyvinyl alcohol (PVA) by drop‐casting,
and the hydrogen peroxide formed in the reaction with glucose was sensed on platinized
paper. Although the enzyme is selective towards glucose the platinum surface was modified
with Nafion in order to enhance its selectivity on the potentiometric measurements,
avoiding anions to make contact with the platinum electrode [44].
Other biological molecules that have been incorporated to chitosan composites are
antibodies, which allow specific interaction with more complex molecules. Ma et al. [45]
developed a disposable amperometric immunosensor for determination of the Aflatoxin B1
(AFB1) in wheat based on chitosan and anti‐AFB1 antibody. Electrochemical immunosensors
are sensors based on solid‐state devices in which immunochemical reactions are coupled to
a transducer surface to generate the output as electrochemical signal [46]. Ma et al. [45]
reported that pH can interfere in the current measurements in an AFB1 sensor, therefore,
this effect must be understood and considered in the development of sensors using chitosan.
The modification of chitosan with inorganic materials may also render materials with
very interesting properties. For example, the composite of chitosan with graphite powder
and montmorillonite can form a structured three‐dimensional material when the amount of
chitosan is below the cationic exchange capacity (CEC) of the clay. However, when
chitosan is at a concentration higher than CEC, it forms a bidimensional nanostructured
material composed of a bilayer of chitosan intercalated with the interlayer region of the
clay, and the amino groups of the chitosan act as ionic exchange sites. This material was
used as a potentiometric sensor specific for monovalent ions, with more sensitivity for the
detection of nitrite ions [41].
Besides potentiometric measurements, many other modifications have been made to use
chitosan composites for sensor materials. One of the most common approaches for
electrochemical sensors is the measurement of an electric current on a working electrode
(which is a conductive surface) due to an electrochemical reaction, which is driven by a
difference of electrical potential applied between the sample and the electrode. These
modifications improve the sensor selectivity and sensitivity toward selected molecules or
groups of compounds [47].
A novel method for analysis of hydroxymethanesulfinate (HMS) was developed using
chitosan modified with carbon nanotubes and cetyltrimethylammonium (CTAB) [48]. In
bare glassy carbon electrodes, the signal of HMS is superimposed on the sulfite signal. The
modification, however, allows performing the analysis of both compounds simultaneously
due to the interaction of the CTAB in the interface with the sample.
More impressively, the incorporation of chiral compounds on chitosan composites may
allow the selective determination of chiral compounds. For example, an electrode composed
of chitosan modified with 3,4,9,10‐perylenetetracarboxylic acid (PTCA) for the
enantioselective determination of tryptophan presented a synergic effect that increased
substantially the selectivity of the determination of D‐tryptophan (selectivity factor of 2.6),
even comparing with glassy carbon electrode with PTCA or with chitosan alone (selectivity
factors of 1.1 and 1.2) [49]. Another interesting example of chiral determination is the
determination of atenolol using chitosan and chitosan succinamide with cyclodextrin
composites [50]. Although each enantiomer did not present a large difference in signals
between R‐atenolol and S‐atenolol, the enantiomeric determination could be accomplished
by the use of an electrode array with the different modifications combined to the use of
chemometric tools (such as principal component analysis) to evaluate the signals.
Another emerging approach for highly selective electrochemical analysis is by the use of
molecularly imprinted polymers (MIPs). These polymers are synthesized with known
cavity sizes for recognition of specific molecules, and the interaction of this polymer with
the target molecule will render an analytical signal. To build an electrically conductive MIP
on a stable surface, a carbon dots–chitosan composite was prepared and 3‐
aminobenzeneboronic acid was electrodeposited at its surface in the presence of glucose
molecules to build the cavities [51]. The system was tested to determine glucose on human
serum with excellent selectivity and fairly good detection limit (0.09 μM).
Other modifications that have been extensively studied are the incorporation or synthesis
of nanoparticles in chitosan composites. In many cases, the chitosan‐based materials
stabilize the nanoparticles of the composite, avoiding agglomeration and immobilizing on
the surface for sensor fabrication. Chitosan has been extensively used in combination with
carbon nanoparticles and derivatives. The simultaneous determination of mercury, copper,
lead, and cadmium was made by a fullerene (C60)–chitosan composite [52]. Besides
forming a stable and uniform film for the sensing surface, the positively charged chitosan
changes the electrical cloud distribution of C60. In another approach, a highly specific and
sensitive sensor for copper (II) was developed using amino‐functionalized graphene
combined with chitosan [53].
Besides being carbon‐based, much work has been done using metal nanoparticles on
chitosan composites. In many cases, they have catalytic properties on electrochemical
reactions, enhancing sensitivity and selectivity. The detection of ochratoxin A, a mycotoxin
that can be present in grape juice, could be achieved by amperometric detection using a
blend of graphene, multiwalled carbon nanotubes (MWCNT), ionic liquid, chitosan,
collagen, and nickel oxide nanoparticles [54]. While the carbon compounds enhance the
electrical conductivity and surface area of the sample, the nanoparticles increase sensitivity
and selectivity toward ochratoxin A, collagen can stabilize these nanoparticles, and chitosan
provides a film‐forming property to the mixture, as well as mechanical strength. In a
different approach, functionalized MWCNT and cobalt were dispersed on chitosan to the
determination of paracetamol. In this case, chitosan can disperse the f‐MWCNT
homogeneously and prevent its agglomeration. This sensor allowed the determination of
paracetamol in commercial tablets and human serum samples [55].
Glucose‐sensing could be accomplished by amperometry using chitosan modified with
copper (II) oxide [56]. This modification was made through a hydrothermal method, in
which the copper is oxidized after being coordinated with chitosan. This interaction
promotes differences in the homogeneity of the mixture and in the growth of copper oxide.
Besides being nonenzymatic, this method has high sensibility and low detection limit
(LD = 11 μM).
The selective determination of Cr3+ and Cr6+ could be accomplished using a dual
electrochemical detector using gold nanoparticle‐decorated carbon fiber chitosan‐modified
glassy carbon electrode [57]. This electrode presents the catalytic properties for chromium
electrochemical reactions on mild acidic pH and made the speciation of these species
possible with no prior chemical separation.
18.5.2 Spectroscopic Sensors

Different sensor technologies also benefit from these composites and properties that were
discussed above. Optical sensors have also been developed, taking advantage of the ease of
formulating composites and performing chemical modifications, as well as to the chelating
properties of chitosan and its derivatives.
The use of Cu2+/cysteine complexes and N‐(aminobutyl)‐N‐(ethylisoluminol) (ABEI)‐
functionalized gold nanoparticles combined with chitosan (Cu2+–Cys–ABEI–GNPs–CS)
made the determination of early acute myocardial infarction biomarker copeptin possible
by electro‐chemiluminescence (ECL). The use of chitosan on this composite can stabilize
electrogenerated intermediates, leading to a more sensitive determination with no need for
any coreactant [58].
Another very interesting application is the use of a composite of chitosan and titanium
oxide nanoparticles on a fluoride‐doped tin oxide conductive glass in order to perform ECL
imaging. While TiO2 increases the signal‐to‐noise ratio by strengthening the ECL signal in
luminol, chitosan was able to immobilize the cell and to increase the distance between the
cells and the electrode on the measurement, thus allowing more ECL reagent to be
in‐between the cell and electrode. With this, the system was sensitive enough to detect
H2O2 released at a single cell level [59].
The use of modified chitosan made the development of a wound‐healing membrane
possible, sensitive to the redox potential of the surface. For this, anthraquinone molecules
attached to gold nanoshells were loaded on the surface of a chitosan wound‐dressing
membrane, in order to monitor the redox potential of the material by surface‐enhanced
Raman scattering (SERS). This probe allows in situ measurement of redox potentials in a
noninvasive manner, making it possible to follow the wound‐healing process in a
noninvasive way [60].
Fluorescent detectors have also been implemented using chitosan composites. A
fluorophore, synthesized by cross‐linking chitosan using glutaraldehyde, was able to moni-
tor the concentration of Cr6+ selectively in the environment, by the oxidation of C═N bonds
of the composite, promoting a quenching effect on the fluorescence of the material [61].
A phosphorescent composite was obtained by the stabilization of trinuclear gold(I)
pyrazolate (Au3Pz3) on chitosan [62]. The polysaccharide, in this composite, has been used
as a template for Au3Pz3 synthesis, having an important role in the formation of gold(I)
pyrazolate complexes. The probe is especially sensitive to silver (I) ions in solution,
presenting zero interference from 15 other metals tested.
18.5.3 Other Electrochemical Devices

Chitosan composites have also been employed on different types of electronic devices,
especially in novel applications that demand very unique and sophisticated materials. In
some sense, these composites are very good candidates as protagonists for new technologies
that demand novel materials, as many of its properties are favorable to its applications.
The biocompatibility and some mechanical properties of chitosan and its derivatives
inspire the composition of materials for wound healing in many contexts [63]. More
recently, conductive composites have been proposed for nerve tissue wound healing and
regeneration. A hydrogel composed of sodium alginate, carboxymethyl chitosan doped
with polypyrrole and cross‐linked with calcium ions has been successfully tested on cell
cultures [64]. The composite conductivity was adjusted by the concentration of polypyrrole,
and proper electrical stimulation could promote differential growth on cell cultures. In
another very interesting investigation, a novel hydrogel composed of chitosan, poly(acrylic
acid) (PAA), and iron (III) ions presented self‐healing properties [65]. The PAA and
chitosan are linked to each other dynamically via hydrogen bonds and iron‐ion coordination.
Once the material possesses a crack or a notch, new cross‐linking points are formed due to
the dual network property of this hydrogel. The presence of iron ions also made the material
more conductive.
Other innovative materials that have been extensively searched for are polymer
electrolytes. These electrolytes are being tested especially for applications on energy
storage devices, as ionic conductors that perform electrolytic contact between anode and
cathode in battery systems. The high ionic conductivity of chitosan, its derivatives, and its
composites has inspired numerous formulations of polymer electrolytes, which has been
proposed mainly for fuel cell applications. Further information about its compositions and
specific applications can be accessed elsewhere [52, 66].
Another promising energy storage solution which has drawn much research efforts are
the supercapacitors [67]. They are based mainly on two phenomena: electrochemical dou-
ble layer capacitance (EDLC) and pseudocapacitance. In the EDLC, the surface of the
electrode presents a charge separation between the capacitor electrolytes, thus a large sur-
face area is desired. The pseudocapacitance is referred to fast and reversible faradaic reac-
tions between the electrode surface and the electrolyte [67]. The ability of chitosan
composites to form self‐assembling and cross‐linking structures with graphene oxide
results in a high‐porosity and low‐density material which has been proposed for superca-
pacitors [68]. Besides these interesting properties, the material presents exceptional
mechanical properties, such as compressive strength and resilience.
Although the chitosan composites can be used to enhance the surface area of electrodes
(making EDLC higher), its capacitance can benefit from its chemistry as well. Chitosan
composites with activated carbon present higher pseudocapacitance as a modified activated
carbon, due to its nitrogen containing groups, whose stability also favors its applicability
on supercapacitors [69].
In rechargeable batteries, the variation of volume on electrodes demands special
materials. In this context, a chitosan–glutaraldehyde composite linked by cyano bridges,
which behaves as a double network hydrogel, has been used with Sn–Fe nanoparticles
resulting on a 3D structure, confining the alloy particles in a hierarchical carbon framework.
As a result, the material exhibits long life cycles and high rate capability [70]. Chitosan was
also proposed in composition with natural rubber to form an elastic composite for Si anode
Li‐ion batteries [71]. High cyclic performance was achieved once natural rubber made the
material elastic in a reversible manner, while chitosan could anchor Si particles through
hydrogen bonding.
The synaptic transistor is a new type of device that presents nonvolatile memory and
history‐dependent analog‐like states, which simulate the behavior of a neuron and are
recently being proposed for new computing architectures [72]. A novel topology for this
kind of transistor is proposed using chitosan dispersed in acetic acid as a material for the
drain of the transistor, and indium tin oxide (ITO) for electrode materials on a polyethylene
terephthalate (PET) substrate [73], yielding a biocompatible and flexible synaptic
transistor.
18.6 Outlook
Chitosan has been widely investigated as a new alternative material for a broad range of
applications, and it presents unique characteristics that are very suitable in electrochem-
ical devices. Its structure allows physical dispersion and chemical modification, com-
bining conductive and functional materials to build very selective sensors and intelligent
materials. These dispersions can be tailored to synthetize and/or maintain nanoparticles
with reduced agglomeration effects, favoring the use of nanosized materials. Moreover,
the chemical structure of chitosan itself appears to be a convenient and selective mate-
rial for sensing applications. Other applications, moreover, use its proton conduction
property to assemble energy storage and other electrochemical devices. However, the
use of chitosan is still very recent in this area, and much novel applications are surely
about to come.
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19
Marketing and Regulations
of Chitin and Chitosan
from Insects
Nathalie Berezina and Antoine Hubert
Ynsect, Évry, France
Chitin, the second most abundant biopolymer worldwide, and chitosan, its deacetylated
derivative, are considered as very promising products in the market. Until now, chitin was
mainly sourced as a by‐product of marine arthropods, but with the emergence of the insect
industry this state is about to change. The aim of this chapter is to understand whether and
how this may have an incidence on chitin‐ and chitosan‐derived products, and examine the
main regulation constraints for their arrival in the market.
19.1 Historical Outline

A. Hatchett, an English scientist, is most probably the first who discovered chitin (in 1799),
and he described it as a “material particularly resistant to usual chemicals”; however, as he
did not push his investigations any further, the discovery of chitin is generally attributed to
H. Braconnot, a French naturalist who identified this biopolymer in mushroom extracts in
1811 [1]. Therefore, the first name ever given to chitin was “fungine,” and it was only in
1823 that A. Ogier, another French scientist, identified chitin in insects’ exoskeleton and
gave it the current name, derived from the Greek word “chiton,” meaning “tunic” [2].
Chitin and cellulose have very similar behaviors, serving as a protection from external
aggressions for some animals and plants. Therefore, studies were initiated by Payen on the

CH3
O CH3
OH O
NH
HO O NH
O HO Chitin
O O O
O HO O
NH
OH
O
CH3 OH
Deacetylation
CH3
OH O
NH2
HO O NH
O HO
O O O
O HO O Chitosan
NH2
OH
OH
Figure 19.1 Structure of chitin and chitosan.
differentiation between these two biopolymers, and, when exploring the exoskeleton of silk
worm, Bombyx mori [2], in 1843, Lassaigne noticed that, in contrast to cellulose, chitin
contained nitrogen in its intimate structure. Further studies on the chitin structure were
conducted, among others, by Ledderhose in 1879 and Gilson in 1894; finally, Purchase and
Braun established the structural information of chitin in 1946, declaring it to be a biopoly-
mer with repeating N‐acetyl‐glucosamine units (Figure 19.1) [2].
Chitosan is the deacetylated version of chitin (Figure 19.1). It is almost absent in its
actual form in nature and was first obtained by C. Rouget by boiling the chitin in a concen-
trated alkali solution [3]. Due to its natural protective function, chitin is extremely resistant
and almost insoluble; therefore, it is difficult to find any direct applications for it, whereas
once deacetylated to give chitosan, the latter becomes soluble in slightly acidic conditions,
allowing a number of applications. Therefore, the main market applications and the related
regulations discussed in this chapter concern chitosan rather than chitin.
19.2 Natural Origins of Chitin

As mentioned in the preceding text, chitosan is mainly obtained by the deacetylation of
chitin, which is widely found in nature. Indeed, there are many natural sources of chitin,
which is considered to be the second most abundant biopolymer worldwide after cellulose,
its plant‐based counterpart [4]. Many sources of chitin have been reported, such as irido-
phores and certain fishes, but the main sources remain arthropods, for example, crustaceans
and insects, and, to a lesser extent, cephalopods, mushrooms, and fungi [4]. The chitin
content usually ranges between 3 and 40% of cuticle dry mass [5–7] however, considera-
ble variations are found in content percentage among species, for example, 8–16% in
mushrooms [5], 23–32% in honey bees [5], and up to 49% in squid pens [6]. Also, it is
Marketing and Regulations of Chitin and Chitosan from Insects 479
α-chitin β-chitin γ-chitin
Figure 19.2 Different chitin assemblies according to Bouligand [10].
important to notice that the determination of the chitin content depends on the methods
used. Different techniques such as infrared or diffractive analysis remain only qualitative,
and the main quantitative method used is the extraction in basic medium, which strongly
depends on the matrix subjected to extraction and the operating conditions [8, 9]. Therefore,
contradictory results were found by different research groups concerning chitin content in
cuttlefish or shrimp [8, 9].
19.3 Specificities of Chitin Biopolymer

Chitin is a very strong, hardly soluble compound, as its main function in nature is to protect
the organism from external aggressions. However, its arrangement in nature might have
different forms and shapes. Thus, three main stereotypic arrangements were described by
Bouligand in 1965 (Figure 19.2). Those three types are called α, β, and γ. The α‐type,
exhibiting an antiparallel arrangement of chitin molecules, is the most robust and the most
frequently found in nature; the β‐type, exhibiting a parallel arrangement, is the weakest,
and therefore the easiest one for purification purposes; and finally, the γ‐type, exhibiting
both parallel and antiparallel arrangements, is the rarest type [10, 11]. The macroscopic
arrangement of chitin layers and the protein scaffolds surrounding them on a cholesteric
helix was also described [12]. A peculiar twisted plywood‐like structure was thus found in
a lobster, Homarus americanus [13], and in a crab, Loxhorhynchus grandis [14], and it is
also believed to be responsible for the iridescence of the scarab beetle [15].
19.4 Differences Among Chitins from Insects and Other Sources

19.4.1 Differences of Chemical Compositions of the Cuticles
Although there are many different sources of chitin, that from shellfish and other crustaceans
has mainly been used until now. The main reason is the development of aquaculture since
the 1970s, and the subsequent increase in the availability of crustaceans’ exoskeletons as
the main by‐product of this industry. However, the uniqueness of these marine arthropods
is actually their marine way of life, which is required for their protection—that is, the
reason for the high mineral content of their cuticles [16]. Insects, on the contrary, rarely live
in the same conditions; therefore, they do not have significant amounts of minerals in their
cuticles. The terrestrial life of insects confers another distinction to their cuticles—these
are usually covered by waxes, and these specific long‐chain alkanes are extremely
hydrophobic, helping preserve the insects from the penetration of water during rains and
other humid climatic events [11, 17].
19.4.2 Differences of Physical Assemblies of Chains and Molecules

Another important difference between the cuticles of crustaceans and insects is the physical
assembly of chitin. Although Bouligand structures are the same for crustaceans and insects,
with an abundance of α‐type structures, some other differences have been reported. Indeed,
mainly due to their marine life, crustaceans require strong and permanent resistance to the
pressure difference; therefore, their chitin exoskeletons are mainly organized in a network
of nodules [16]. On the contrary, the insects require more aerodynamism to help their
motion; therefore, their exoskeletons are mainly formed by fibers. These fibers are
extremely characteristic and specific, as their section is at the nanometric scale, whereas
their length is at the micrometric scale [18–20]. All these differences have important
impacts on both the extraction–purification processes and the market opportunities and
applications discussed in the following text.
19.5 Extraction and Purification Specificities of Chitins from Insects

19.5.1 Different Cuticle Structures and Contents of Insects
Insects are the most abundant eukaryotes worldwide; there are more than a million of
known insect species till date, and therefore it would be inaccurate to discuss about insects
in general, as important differences exist among their behaviors, feeding substrates, and
general environment. One of the possible distinctions among the insects could be their
motion behavior. We can thus find three main groups: flying insects (e.g. flies, butterflies,
etc.), jumping insects (e.g. crickets, grasshoppers, etc.), and others. Flying insects require
lots of energy for their motion; therefore, they possess a high content of fats, often greater
than 50%, in their pulp as well as in their cuticles [18–20]. On the contrary, jumping insects
make use of their muscles, that is, their protein content is much higher, close to 80%
[18–20]. The other insects are generally somewhere in between those two extremes. These
aspects are of main importance for the extraction and purification procedures of chitin
enclosed in the cuticle. Indeed, the fats are not covalently linked to the chitin and therefore
can be easily removed by either alkali or acidic treatments, whereas the proteins require
much stronger conditions for their removal; the chitin purification in this case is therefore
more complicated, and a less pure product is usually obtained [18–20].
19.5.2 Chemical Extraction

The chemical extraction of chitin is usually composed of two steps: acidic and base treat-
ments [16, 21, 22]. Acidic treatment, which used to be necessary for the demineralization
of chitin from marine arthropods, is sometimes used for the elimination of fats, when
applied to the purification of chitin from insects [23]; however, sometimes this step can
also be avoided when applied to insects [18–20]. The base treatment is required for protein
elimination and is somehow more difficult to adjust, as strong base treatment can damage
intimate chitin structure and/or drive to deacetylation, giving only chitosan and no longer
the chitin at the end of the process [16, 24, 25]. Although these treatments were subjected
to many optimizations, they call for considerable energy consumption, for example, heating
up to 60–90°C for 24–48 h, and remain environmentally unfriendly [5, 26]. Also, acidic
and base treatments are rarely enough for obtaining perfectly pure and white chitin; there-
fore, bleaching steps are often applied at the end of the processes [16].
19.5.3 Biological Extraction

To circumvent the energy consumption and environmental drawbacks of chemical
extractions, biological extractions were attempted [16]. They were imagined with whole‐
cell microorganisms [27] and purified enzymes [28, 29]. Similar enzymes, as compared to
marine arthropods, were mainly used for the purification of chitin from insects, that is,
proteases, and in some cases mixtures of proteases and lipases [18–20]. It is also worth
noticing that these techniques remain less efficient as compared to the ones used for
chemical extraction; therefore, the biological extraction is often coupled with some
complementary acidic, base, or bleaching chemical steps [16].
19.5.4 Characterization and Transformation into Chitosan

The characterization of chitin and its subsequent transformation into chitosan is not specific
to its origin, that is, we are giving here only a few reminders of these general aspects,
common to chitins of any origin.
For any utilization of chitin, first it needs to be extracted from its natural matrix, and then
the purity of the obtained polymer is analyzed. First, analytical issues are considered, since,
for now and to the best of our knowledge, no completely effective method for the determi-
nation of this purity exists. Usual techniques such as Fourier‐transform infrared (FT‐IR)
spectroscopy or X‐ray analysis are only qualitative [30], whereas some other techniques
such as liquid nuclear magnetic resonance (NMR) or chromatography are impossible due
to the high insolubility of the polymer. Therefore, the previously described “alkaline extrac-
tion” method is often applied; however, as mentioned earlier, this is also not completely
reliable [8, 9, 30]. Another approach is based on the specific analysis of known impurities
such as proteins, lipids, and ashes and the measurement of the so‐called “purity by differ-
ence” [18–20]; however, here again some limitations remain, such as the accuracy of the
analysis of each specific impurity, namely, proteins and lipids are subjected to improve-
ments; and the unknown or more specific impurities are not taken into consideration.
Also, as already mentioned in the preceding text, chitin is rarely used due to its
specificities, and needs to be transformed into chitosan for more widespread applications.
This transformation is usually performed in strong alkaline medium at high temperature
and consists of the deacetylation of chitin (Figure 19.1); the obtained product can be
subjected to NMR, size exclusion chromatography (SEC), and other more standard
analyses. Further modifications of chitosan have also been reported and applied for some
specific purposes [24]. Here again, even if some influence of the chitin origin can be
noticed while characterizing chitin itself, for example, MW or Bouligand’s arrangement, no
incidences of the origin have been reported when it comes to chitosan [8]. We can, however,
notice that, for some applications where high MW chitosan is suitable, the insect matrix is
of particular interest, as coming from fibers even in the case of α‐type arrangement, it
allows longer chains of final product; therefore, the specific utilization of the rare β‐chitin
containing sources is no longer mandatory [18–20].
19.6 Market Opportunities and its Regulations

Chitin is the second most abundant polymer worldwide after cellulose, and its extraction
and transformation into chitosan have been extensively studied since the 1980s, when the
aquaculture industry gained in maturity and significantly increased the production of
crustaceans and other marine arthropods. Several applications have been studied using its
properties such as biocompatibility [31] and antimicrobial [32], antioxidant [33], and
scavenging [34, 35] behaviors. Their applications in several fields from biomedical to
materials and agriculture were thus anticipated [36–39]. Biodegradability was also often
claimed, but as for now we are unable to find any reliable study on the biodegradation of
chitin or chitosan as required by the “ok compost” regulation following the EN 13432
norm, which combines the biofragmentation, bioassimilation, and ecotoxicity studies.
It is also worthwhile to notice that not all of these applications have found their way to
industry. Different reasons may explain that, namely the overoptimistic approach of some
studies, the actual price, the waste‐management constraints due to the production of chitin
and chitosan, and also the lack of reliable products in the market [4, 40]. Indeed, until now,
chitin and chitosan were mainly sourced from marine arthropods with huge variations in
quality due to the seasonability, mixture of different matrixes, etc.; hence, the emerging
insect industry can bring the advantage of more standardized production. Here, some of the
most promising market opportunities and the inherent regulations are discussed.
19.6.1 Agriculture Applications

Agriculture might be the only field where chitin as well as chitosan can be applied.
However, the applications of these two compounds are very different. Chitin is mainly
applied in seed protection, allowing the growth of microbial pesticides, such as
Trichoderma harzianium P1, active against foliar disease. Actually, the addition of chitin
was reported to have a double effect by promoting the growth of T. harzanium, and at the
same time inhibiting the growth of the pathogenic Rhizoctonia solani [41]. Chitosan, on
the contrary, is active as an elicitor for different plants. Several studies showed that the
introduction of chitosan in the growth medium increases the natural production of chitino-
lytic enzymes of several plants, thus enhancing their resistance against several pathogens,
mainly from fungal origin [42, 43]. Recent or anticipated regulations on pesticide control,
such as the restriction on using glyphosate and similar active principles, as well as the reti-
cence of several countries, such as Russia and those in Europe, about the utilization of
pest‐resistant genetically modified organisms (GMOs), are about to encourage the utiliza-
tion of natural protections of plants such as elicitors. Therefore, the utilization of chitin
and chitosan for these applications seems promising in the coming years. However, some
difficulties on the regulation level should also be mentioned. One of the main issues is the
lack of a general legal framework for biostimulants. In Europe, the current regulation N°
1107/2009 on plant protection products (PPPs) is the most reliable. However, the process
to obtain such a certificate is very long and costly; therefore, several companies are
tempted by the obtention of a “fertilizer” certificate at a country level, as the enhance-
ment of plant growth could be considered as a fertilizing effect by most country regu-
lations, whereas the European regulation N° 2003/2003 excludes biostimulants from
the scope of fertilizers, as it specifies that fertilizers should provide nutrients to the plants,
which the biostimulants clearly do not [44].
19.6.2 Water Treatment Applications

The purification of water remains a big challenge worldwide, either for purifying natural
sources for use by different populations or for the treatment of polluted effluents to protect
the environment. Different techniques are usually employed for water purification purpose,
such as scavenging, adsorption, flocculation, and biological treatments. Chitosan was
shown to be suitable for some of them, such as adsorption [45, 46] and flocculation [47].
However, even if chitosan was shown to improve the quality of water in a much better way,
as compared to commercially used polyaluminium chloride (PAC) by reducing the chemical
oxygen demand (COD) by 80% versus 40–45% for PAC, and turbidity by 85% versus
55–60% for PAC, its cost remains an obstacle for field applications, with PAC satisfying
the current regulation requirements [47].
19.6.3 Material Applications

19.6.3.1 Food Packaging
The antimicrobial properties of chitosan have made it a material of choice for food pack-
aging for a long time [48]. It has been shown to be active against postharvest strawberry
pathogens such as Botrytis cinerea and Rhizopus stolonifera [49], and also Listeria
monocytogenes—the “enemy no. 1” in the food ambit [50]. Recently, emerging nanosci-
ence has also been applied to food packaging purposes. Therefore, new specific biocom-
posites based on chitosan were synthesized and have shown outstanding physical
properties, particularly due to their antimicrobial effect [51] and nanoporous structure at
the same time [52].
These smart materials are, however, subject to different regulations [53, 54]. Here again,
there is no common legal framework between the United States and Europe. In the United
States, the smart packaging could have been concerned by the regulation on “food additives”
under the Federal Food, Drug, and Cosmetic Act. However, “food additive” is defined in
section 201 as a substance that is reasonably expected to become food under intended
conditions, which obviously does not cover the possible migration of the substance from
the packaging to its content; therefore, the smart packaging is not directly concerned by
this regulation [53]. In Europe, the situation is clearly different, and the regulation is still
evolving. So far, two main regulations are to be considered: the 1935/2004, which gives a
general framework, and the 450/2009, which is specific to active and intelligent materials,
and where the overall migration limits (OMLs) and specific migration limits (SMLs) are
considered [53].
Also, as mentioned earlier, nanomaterials containing or made of chitosan are about to
be introduced to the food packaging market. The regulatory organizations have been
warned about the potential damage to human health due to nanoparticles; however, no
specific regulation is available for now, as more specific studies on this topic remain to be
conducted [52].
19.6.3.2 Other Materials Applications

Other material applications of chitosan or its derivatives have also been reported [55–57].
One of the most interesting emerging topics seems to be the utilization of chitosan as a
catalyst support. This application combines innovative approaches in catalyst preparation,
such as freeze‐drying [58], utilization of supercritical CO2 (scCO2) [59], or ionic liquids
[60, 61]. Therefore, even if there is no particular regulation on this specific topic, it promotes
several among the green chemistry requirements: catalytic rather than stoichiometric
reactions, limitation of solvent use, and carbon preservation by the utilization of renewable
materials [4].
19.6.4 Biomedical Applications

19.6.4.1 Human Health
Chitosan has been used in wound healing since the 1990s [40]. This application combines
two among the most promising properties of chitosan—that is, biocompatibility and
antimicrobial behavior. Other biomedical applications have been demonstrated as well—
for example, as medical devices, namely bone substitutes [40, 62] and implants [63].
Chitosan has also been tested for blood interactions and as anti‐inflammatory [64, 65],
antihypertensive [66], and anticancer [67] drugs. Although chitosan has been considered
for a long time as an interesting candidate for drug delivery systems [40, 52], an emerging
topic advocates the use of its nanocomposites for the encapsulation of different active
molecules, mainly for anticancer [68], antiviral [68], and nucleic‐acid‐based [69] drugs.
This application combines the biocompatible properties of chitosan and the latest
developments in material nanoscience.
The utilization of chitosan for human health purposes requires compliance with very
severe and specific regulations in Europe, as well as in the United States and other parts of
the world. One of the main difficulties here concerns the purification and characterization
of the impurities of the biopolymer. Indeed, as with many other biopolymers, such as
natural rubber [70] or poly(hydroxyalkanoates) (PHAs) [71], the complete purification
from the natural matrix is the main issue. The tiny residues of proteins on natural rubber are
responsible for the so‐called “rubber allergies” and for the underutilization of PHAs in
biomedical applications.
19.6.4.2 Animal Health

Chitosan has also been reported for some applications in animal health. It was thus reported
to be active against cryptosporidiosis in goats by reducing the excretion of oocysts of
Cryptosporidium parvum, thus reducing the diarrhea in newborns and contributing to the
rapid weight gain of young animals [72].
Chitosan and, in some applications, chitin, contained in feed formulations, showed an
activity for the stimulation of the innate resistance of some organisms. Thus, the kelp
grouper species, Epinephelus bruneus, has been found to be resistant to the protozoan
parasite Philasterides dicentrarchi after ingestion of these biopolymers [73]. Similarly, the
supplementation of chitin in broiler diet inhibited the growth of foodborne pathogens such
as Escherichia coli or Salmonella spp [73].
To the best of our knowledge, no specific regulations have yet been issued for controlling
the addition of these substrates to animal diets. It is anticipated that, in addition to the
application of elicitors in agriculture for the replacement of pesticides, these applications
for the enhancement of the innate immune systems of animals will be highly appreciated,
as they have the potential to reduce the preventive antibiotic treatments of livestock.
19.7 Outlook
Chitin and chitosan are very promising molecules for several applications. In case of chi-
tosan, the initial source of the biopolymer is no longer of interest, as the extraction, purifi-
cation, and deacetylation of chitin tend to uniformize the obtained product, and the applied
process has more influence on the final product and its properties than the original
organism.
Among the different market opportunities reviewed, chitin is mainly concerned with a
few applications in seed protection in agriculture and improvement of the innate immune
system in animals, whereas chitosan has been found to present great possibilities in fields
such as water treatment and biomedical applications.
Bringing a new product into the market is always a costly and long process, as safety
concerns need to be addressed. We can regret that, in many fields, such as active packaging
and elicitors, no unified regulation frameworks are available, and sometimes the same
product can fall under several different categories with specific requirements to be
addressed.
However, we also have reasons to be optimistic, as chitin and chitosan exhibit actual,
diverse, and very interesting properties, with many valuable applications in several fields
where they can replace more drastic or harmful compounds. Also, the emergence of the
insect industry, even if they do not modify the intrinsic properties of these biopolymers,
allows their more consistent production; therefore, in the coming years, an increase in the
market share of chitin‐ and chitosan‐derived products can be anticipated.
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Index
Abscisic acid, 179 Acidic

Absorption capacity, 303, 339, 433, 437, 439 extraction, 46
Accelerated electrons, 296 groups, 248
3‐Acetamido‐5‐acetylfuran, 238 treatment, 232, 480
2‐Acetamido‐3,6‐anhydro‐2‐deoxy‐D‐ Acrylic acid, 119, 133, 135, 338
glucofuranose, 239 Acrylonitrile, 133
2‐Acetamido‐3,6‐anhydro‐2‐deoxy‐D‐ Activated transport mechanisms, 271
mannofuranose, 239 Active site, 191, 201–202
2‐Acetamido‐2‐deoxy‐D‐sorbitol, 239 Acylhydrazone bonds, 117, 122
Acetamido groups, 40, 232, 402 Added‐value products, 230
Acetic acid, 19, 44, 135, 229, 232, 234–235, 320, Administration, 108, 260, 265, 269–270, 275–276
322, 402–403, 408, 463 Adsorption, 432–440
Acetic anhydride, 84–85, 236 chemisorption, 433–434
N‐Acetylaminogluconic acid, 237 ion exchange adsorption, 433, 435–436
Acetylation physisorption, 433–434
degree, 9, 16, 232, 246, 408 Agarose, 100
fraction of, 81, 83, 87 Agricultural
pattern, 81, 83, 89–90, 215, 234, 246 commodities, 377
re‐, 82, 84–85, 236 products, 177, 371–372
N‐Acetyl‐D‐glucosaminic acid, 239 Agriculture, 77, 133, 177–178, 214, 482, 485
N‐Acetyl glucosamine (GlcNAc), 1–2, 23, 67, Ag/ZnO nanocomposites, 302
147, 197, 214, 229, 239, 245, 315, 350, Aldehyde groups, 111, 121–122, 381, 416
462, 478 Algae, 5, 10
N‐Acetylglucosamine deacetylase, 237 Alginate, 171, 270, 304, 334
N‐Acetylglucosaminidase (NAGase), Alkali, 12, 21, 24–25, 44
236–237 Alkaline
Acetyl group, 19, 23, 64, 84–85, 90, 191, conditions, 21, 420
194–195, 201, 246, 331, 352 extraction, 44, 481
Acid/base reaction, 201 solutions, 21
Acid catalysis, 191 treatment, 23

492 Index
Alkali/Urea solvent system, 98, 103–104 Astaxanthin, 232

N‐Alkylation, 251, 332 Asthma, 207
Alkyl epoxides, 250 ASTM Standards, 294
Allergic response, 153, 297 Atherogenic index, 160, 321
Allergy model, 149, 153 Atherosclerotic vascular disease, 172
Alternating process, 100–101 Atom transfer radical polymerisation (ATRP), 252
Amine group, 246, 333–334, 405, 462 Auxiliary activity (AA) family, 199
Amino Azide‐alkyne cycloadditions, 114
acid, 73, 191, 195, 198, 239
sequence, 189 Bacteria, 1, 10–12, 14, 39, 76, 162, 172–173,
alcohols, 239 208, 321
groups, 12, 19, 98, 111, 114, 263, 316, 318, 333, Bacterial
351, 397, 436, 462–464, 466 cell, 169–170, 173, 306
sugar, 73 cell wall, 170, 295
Ammonium persulphate, 252, 412 death, 170, 355
Amorphous, 10, 41, 74, 98 growth, 171, 381
Amphiphilic molecules, 110 infection, 171, 398
Amphoteric polyelectrolytes, 248 surface, 173, 319
2,5‐Anhydro‐mannose, 88 Bactericidal activity, 171
Animal Bacteriostatic, 76, 381
health, 484–485 Bacteroidetes, 211
studies, 149, 157–158, 163–164 Ball milling, 236
tissue, 292 Bandages, 234, 301–302
Anion exchange, 75 (α/α)6 Barrel, 195, 199
Anionic (β/α)8 Barrel, 191, 195
charges, 412 Beads, 333–334, 338, 403, 437, 439
chitosan derivatives, 248 Beer, 175, 235
drug, 263, 276 Benzaldehyde, 111
dyes, 398, 436 Beverages, 174
polymer, 275, 333, 403 Bile acids, 160, 162
Anomeric configuration, 191 Binding cleft, 193, 198–199
Antibacterial Bioactive
activity, 170–171, 175, 359–360, 381, 398 compounds, 175, 275, 324, 332–333, 357,
effect, 170, 417 381–382, 385, 387
material, 176 molecules, 108
textiles, 417 packaging, 350
Antibiotic resistant, 173, 177 properties, 297, 355
Antibiotics, 173, 265 Bioactivity, 177, 321
Antifungal activity, 169–171, 173–176 Bioadhesion, 259, 267
Antioxidant, 175, 318, 321, 379 Bioavailability, 259, 265, 269–270, 333–334, 385
Anti‐parasitic, 155–157 Bio‐based
Anti‐pathogenic, 149, 155 building block, 232, 239
Antisense oligonucleotide (ASO), 263 chemicals, 245–246
Anti‐tumour, 149, 157–158 circular economy, 239, 254
Aplysina, 36, 43, 45, 52 economy, 230
Aquaculture, 479, 482 materials, 400
Aqueous acidic solvent, 372 packaging material, 349, 351
Aromatic residues, 191, 196, 199, 204 products, 230, 395
Arthropoda, 5, 9 Bioburden, 294–295
Aspergillus niger, 10, 24, 170–171, 239 Biocompatibility, 116, 125, 131, 293, 302, 307
Index 493
Biocomposites, 40, 50, 435, 483 Bone tissue, 305–306

Biodegradable Boronate ester, 111, 122
films, 322, 324, 357 Boronic acid, 111, 122
polymers, 171, 236, 353 Bronchoalveolar lavage, 153
Biodegradation, 74, 136, 293, 482 Building block, 205, 229–230, 236, 238–239, 245
Bioelectrometallurgy, 50 Bulk chemicals, 229, 232, 234
Bioethanol, 214, 235 Burn dressing, 303
Biofilm, 207, 324, 359 Burns, 301, 305
Bioinspired material, 49–50
Biological Calcium carbonate, 12, 16, 38, 40, 45, 230, 430
activity, 62–63, 74, 83, 85, 87, 110, 178, 180, Cancer treatment, 77
235, 246, 250–251 Candida albicans, 155, 269, 305
barriers, 259–260, 265–266, 268–269 Capillaries, 37
function, 84, 86, 89, 91, 207 Carbohydrate, 189
response, 146, 163, 178 active enzymes (CAZymes), 189–190
Biomass, 1, 5, 9–11, 16, 19, 24–25, 39, 179, binding module (CBM), 189–190, 204
215–216, 230 bound chitosan, 251
Biomaterial, 51–52, 54, 62, 111, 122, 295, 297, esterase (CE), 189–190, 200–201
301, 305, 452 family
Biomedical application, 43, 102, 108, 131, 174, 4, 200–203, 205, 207–208
239, 301, 419, 484–485 14, 200, 211
Biomimetic, 49–50, 99, 101, 270 modified chitosan, 251
Biomineralization, 1, 36, 43, 49 structure, 189
Biopolymer, 47, 230, 276, 293, 372, 478 Carboxyl groups, 114
analysis, 87 Carboxylic acid, 191, 464
chain degradation, 296 Carotenoid, 232
characteristics, 293 Cartilage
chitinous, 293–294, 296, 307–308 repair, 116
chitosan, 82, 108 tissue, 53, 254
degree of purity, 293 Catabolism, 207–208
insoluble, 295 Catalyst, 114, 125, 236, 238–239, 246, 250
separation, 292 Catalytic
Biorefinery, 92, 230, 232 acid, 191, 195, 198, 201
Biosensor, 332, 465 activity, 193, 199, 209, 468
Biosilicification, 36 base, 201
Biotechnological methods, 75, 214 centre, 198–199
Biowaste, 230 domains, 189, 193, 195, 204–205, 208–209, 211
Bleeding, 171, 297, 302, 304 mechanism, 191, 194–195, 197, 201
Block‐wise, 21, 82, 90–91, 246 module, 204
Blood oxidation, 229, 239
brain barriers (BBB), 259, 270, 276 site, 195
capillaries, 271 Cationic
clotting, 302, 305 characteristics, 358, 372
stream, 265, 268 charges, 248, 354
systemic circulation, 270 interactions, 162
vessels, 270–271 polyelectrolyte, 98, 463
Blue bioeconomy, 239 polymers, 443, 445, 451
Body polysaccharide, 109, 259, 263
mass index, 162 Cavitation, 45
weight, 162, 321, 351 CAZy database, 189–190, 197
494 Index
Cell biomedical use, 174

attachment, 51, 114, 275 chain, 2, 5, 12, 191, 199
wall, 10, 28, 169–170, 207–208, 213, 295, 331, chemical structure, 2
352, 372 commercial, 25–28
remodeling, 207, 213 crystalline, 2
Cells, 10, 43, 51, 53, 77, 108, 114, 145–148, deacetylase, 90, 200, 234
156–158, 163, 170, 250, 263, 266, deacetylation
271, 306 chemical, 19–24
Cellular enzymatic, 200–201, 234
responses, 146, 149, 163 decolouration, 13, 232
signaling, 208 degradation, 208–213
Cellulase, 74–76, 215 demineralization, 11–12, 39, 43–45
Cellulose, 2, 74, 199, 204, 211, 214–216, 395, depolymerisation, 236–237
412, 420, 478 deproteination, 12, 39
binding domain, 195, 204–205, 208–209, enzymatic hydrolysis, 214–215, 236–237
211, 213 extraction
glycan‐chain‐binding, 204 biological, 481
non‐catalytic, 204–205 chemical, 480–481
small‐sugar‐binding, 204 fibres, 48, 402, 405
surface‐binding, 204 fungal, 24–25
fabrics, 176 glycol, 111, 250
fibres, 412, 416 health effects, 158–163
nanofibers, 135 hydrogel, 104
solvent, 103 immunomodulatory effects, 146, 149, 153,
Central nervous system, 111, 270–271, 301 155, 163
Cephalopods, 291, 478 isolation, 11–16, 24–25, 38–43, 45–49, 430
Chelating agent, 129, 175, 246, 254, 318, 338 oligomers, 85
Chemical(s), 46, 173, 176, 214, 229–230, 232, poriferan, 49–51
235, 364, 396, 400, 407, 414, 443–444 pre‐treatment, 11
bio‐based, 245–246 production, 232
bulk, 229, 232 scaffold, 43, 50–54
extraction, 28, 39, 480–481 sources, 5, 9–10, 478
high‐value, 235 sponges, 36–37
hydrolysis, 235 Chitinases, 83, 190–191, 193–195, 199, 205,
liquefaction, 229 207–209, 212–214, 236–237
methods, 177, 214, 235–236, 331 Chitin‐glucan, 145, 158
platform, 229, 232, 235, 239 Chitinolytic pathway, 200, 211, 236
polymerization, 108 Chitinous material, 24, 292–293
reactions, 110, 248, 352, 414, 464 Chitiobiose, 236
Chitin Chitobiase, 195, 207, 209–211
α‐, 2, 22, 479 Chiton, 477
β‐, 2, 5, 22, 479 Chitooligosaccharides, 61, 174, 194–196, 202,
γ‐, 5, 479 204–205, 207–208, 211, 215–216, 229,
adsorbent, 432, 437 234–235, 237, 315, 320–321
active enzymes, 205–208 even‐numbered, 194
active LPMOs, 199–200 odd‐numbered, 194
anti‐pathogenic effects, 155–157 Chitosan, 1, 98, 171, 315, 350, 462
anti‐tumour effects, 157–158 activity, 62
binding CBMs, 204 adsorbent, 338, 436, 439–440, 445
binding domain, 90, 199 alginate gauze, 304
Index 495
amino group, 19, 397, 462 gelatin

animal health, 484–485 coatings, 381
anionic derivatives, 248 microcapsules, 416
antibacterial activity, 170, 172, 398, 417 nanocapsules, 270
antimicrobial activity, 169, 319, 355, 359–360 gel formulation, 303
antioxidant properties, 160, 175, 179, health effect, 149, 176, 320–321, 484
318–319 high molecular weight, 170, 261
anti‐parasitic activity, 155 hydrogel, 97–100, 107–109, 116–117, 119–122,
anti‐tumour, 157–158 125–126, 131–133, 135–136
ascorbate, 172, 174 hydrolysing enzymes, 83
backbone, 251–252, 464–465 hydrophobic, 248, 332
betaine, 250 hydroxyalkyl, 250
biodegradation, 482 hydroxyaryl, 250
capsules, 415–416 immunomodulatory effect, 149, 153, 157
carboxyalkyl, 248 interpenetrating polymer networks, 254
N‐carboxybenzyl, 248 lactate, 180
carboxyethyl, 117, 122, 296 matrix, 305, 323, 380, 416, 467
carboxyl, 248 membrane, 304
carboxymethyl, 75, 114, 133–134, 176, 248, microcrystalline, 76, 178, 180
268, 363, 405, 418, 451 microparticles, 173, 357
cationic derivatives, 250 modification, 246, 319, 339
chain, 19, 74, 82, 89, 109, 133, 176, 194, molecular mass, 63
252, 410 molecular weight, 23, 86, 319
chelating properties, 246, 339 mucoadhesion, 263
coacervation, 275, 333, 357, 415, 468 nanocarriers, 266–267, 270–271
coatings, 324, 354, 361–362–363, 376–382, 417 nanofibre, 275, 304
composites, 134–135, 440, 468–472 nanomaterials, 177
cross‐linked, 254 nanoparticles, 178, 265, 267–268, 270,
cyclodextrin‐bound, 251 357–360, 384–386
deacetylated, 24 toxicity, 360
deacetylation degree, 22, 67, 237, 261, 296 native, 109, 246, 250, 397–398, 413
definition, 1 low‐molecular weight, 75, 153, 170, 261
degree of polymerisation (DP), 86, 91 oligomers, 1, 63, 74, 76–77, 83, 87, 148, 170,
determination 175–176, 178–179
ninhydrin, 73 oxidised, 248
diads, 84 packaging, 174
dressing, 303, 305 partially acetylated, 81, 91
drug delivery system, 261, 263 particles, 331
electrolyte, 467 pathway, 195–196
emulsifying ability, 320 pattern of acetylation (PA), 83, 89, 91, 237
encapsulation, 333–334 pKa, 170
extraction, 25 poly(vinyl alcohol)/alginate films, 305
fibre, 401–406 prices, 234
film, 135, 174, 295–296, 305, 322–324, 354, pristine, 261, 436, 452
362, 376, 411–412, 464–466 production
edible, 358–360 biological, 11, 235
flocculant, 445 chemical, 214, 232
fraction of acetylation (FA), 82–83, 87–91 crustacean, 25
fungicidal activity, 375 fungal, 24, 28
graft co‐polymers, 251–252 properties, 61
496 Index
Chitosan (cont’d) Co‐polymers

quaternary, 248, 250, 306 comb‐shaped, 252
derivatives, 250, 263 graft, 251–252
scaffolds, 275, 305 linear, 232
sized cotton, 413 star shaped, 252
sizing, 406–407 Copper ion, 73, 199, 418, 449
solubility, 19, 246, 351–352, 463 Corneal epithelium, 269–270
sorbent, 430 Corneocyte cells, 266
source, 10, 38, 230 Cosmetic industries, 175, 214
spinning, 402–403, 405 Cosmetics, 235, 250–251
sponges, 305 Crab, 9, 25, 38, 163, 237, 297, 372
succinyl, 111, 248, 265, 449 Cranial and maxillofacial reconstruction, 306
sulphadiazine membrane, 303 Cross‐contaminations, 292
sulphates, 248 Cross‐linking, 43, 110–112, 116–117, 248, 251,
TEMPO‐laccase oxidation of, 464 296, 334, 396, 405, 414, 464
thiolated, 265, 275 agent, 99, 111, 122, 176, 254, 271, 416,
N,N,N‐trimethyl, 303 430, 436
viscosity, 408, 410 chemical, 107, 126, 132–133, 468
wound dressing, 297, 471 enzyme‐mediated, 110
Chitosanase, 83, 85, 190, 197–199, 205, 207, mechanism, 108, 120
235, 237 physical, 126, 132–133
5‐Chloromethylfurfural, 238 points, 125–126, 471
Chondroitin sulphate, 304 Crustaceans, 5, 9, 13, 25, 28, 430
Chromophores, 252 chitin, 13, 163
Clarification agent, 175, 339 cuticles, 38, 480
Click chemistry, 110, 112, 114, 252 exoskeleton, 1, 9, 11, 479
Clinical shells, 9, 230
application, 114, 260–261, 292, 307 Crystalline
trials, 145, 155, 158, 160, 162–163, 171–172, environment, 199
297, 302, 308 forms, 2
Coastal areas, 230 network, 120
Colloidal particles, 443, 445 polysaccharides, 1, 209
Colon, 248, 260, 320–321 regions, 107, 209, 237–238, 405
Colour, 174, 296 structure, 47, 74
Compatibility, 50, 116, 125, 260–261, 293, 296, 301, surface, 204
307–308, 324, 331, 339, 397, 418, 471 Crystallinity, 2, 5, 19, 246, 294, 296, 405, 462
Compliance, 260, 484 degree of, 11, 16, 405
Composite material, 171, 331, 436 Crystal structure, 43, 199, 202
Confocal laser scanning microscopy, 267, 270 Cyclodextrins, 265, 267, 269–270, 434
N‐Containing diols, 239 Cytocompatibility, 53, 302
Controlled release, 121, 135, 260, 276, 323, 332, Cytoplasmic membrane, 155, 306, 355
413, 415 Cytotoxicity, 77, 111–112, 116–117, 148, 250
Conversion
biotechnological, 232, 353 3D
catalytic, 239 biomaterial, 54
chemocatalytic, 248 composites, 37, 50
enzymatic, 215, 248 network, 131
hydrothermal, 238 printing technique, 102
mechanocatalytic, 236 scaffolds, 36, 38, 49, 51–54, 301
mild, 248 skeleton, 52
Index 497
Deacetylation Dioxygen, 199–200

chemical, 21–23, 234 Disease, 177–181, 232, 266, 270, 276, 371–372,
chitin, 22, 47, 246 374–375, 437, 444, 482
degree of, 2, 11, 19, 62, 163, 177, 232, 246, 261, Dispersity, 81, 86–89, 91
296, 318–319, 322, 352, 375, 397, 445, DNA, 170, 263, 319–320, 360
462, 466 Domain
determination, 67–70 catalytic, 189, 193, 195, 204–205, 208–209, 211
tailoring, 22–23 chitin binding, 90, 147
enzymatic, 10, 90–91, 200, 215, 234 non‐catalytic, 205
heterogeneous, 21, 89 Double displacement, 191
microwave‐assisted methods, 47 Downstream signalling, 146–148, 163
NaOH, 15 Dressings, 171–172, 174, 303, 305
partial, 89, 430 Drug
pattern, 23 absorption, 260, 269
procedure, 23 administration, 260, 265, 269
reaction, 19, 21–23, 47 anionic, 263
Decalcification, 39–40, 44, 47 delivery, 108, 114, 234, 248, 250–251,
Decolorization, 39, 232, 430 260, 267
Degree of acetylation, 9, 16, 194, 232, encapsulated, 111–112, 261
246, 408 hydrophilic, 261, 265
Degree of polymerization, 81–83, 86–87, 177–178, hydrophobic, 261, 267
204, 234 in situ gel formation, 112
Degree of substitution, 116, 248 loading capacity, 275
Demineralization, 11–14, 16, 39, 41, 43–46, 480 polyanionic, 263
Demosponges, 36–37, 40, 43–45, 47, 51, 53 release, 260–261
Dendritic cells, 146–147, 157 side effects, 260
Depolymerization specific efflux system, 265
acid, 87, 236 uploading, 100
chemical, 229 Drug delivery systems, 108, 114, 259–261,
enzymatically, 74, 85, 88, 190, 207, 214–215, 266, 276
229, 237 anionic polymer based, 263, 265
nitrous acid, 88–89 cationic polymer based, 263
physically, 87 chitosan based, 261, 263, 275–276
plasma‐assisted, 88 micro/nanoparticulate, 275
sonification, 88 colon‐specific, 248
ultrasonic, 45 formulation, 261, 276
Deproteinization, 39, 45–46 hydrogel, 122, 275
Desizing, 230, 412–414 macroscopic, 260
Diabetes, 145, 174 microscopic, 260
Diad frequencies, 89–91 oral, 260, 263, 265, 268
Dialdehydes, 254, 464 stimuli‐responsive, 260
Diepoxides, 254 targeted, 260, 271
Dietary transdermal, 260
fiber, 164, 234, 320 Dry heat, 295
supplement, 214, 234, 377 Drying, 66, 72, 230, 322, 354, 405, 464
Digestive tract, 267–268 Duodenum, 265, 269
Diketones, 254 Dye, 176, 396, 398, 405, 413, 417–420, 435–437,
Dimer, 194, 202, 211–212 445, 451–452
Dimethyl diallyl ammonium chloride, 133–134 Dynamic imino–chitosan chemistry, 275
Dimethyl sulfoxide, 135, 448 Dynamic viscosity, 70
498 Index
Efflux pump, 263, 265, 276 modification, 82, 246, 468

inhibition, 259, 265 oxidative addition, 250
Electrochemical polymerization, 229
devices, 461–462, 471 Enzyme(s)
double layer capacitance, 472 antioxidant, 379
reaction, 50–51, 72, 469–470 bacterial, 170, 215
sensor, 467–469 cellulase, 74–76, 215–216
Electrochemically, 72 chitinases, 74, 83, 87, 191, 205, 208,
Electro‐conductive, 50, 117 236–237, 482
Electrode, 72, 100, 465, 468–470 chitosanases, 74, 83, 87–88, 236, 412
Electrolyte, 467, 471–472 deacetylases, 23
Electromagnetic exo, 191, 209
field, 47 fungal, 216
waves, 47 growth‐promoting, 170
Electron‐beam, 235 hydrolytic, 199
Electronic devices, 461–464, 471 immobilization, 331–332
Electron microscopy, 50, 179, 405 mediated
Electrons, 47, 296, 437, 465 cross‐linking, 110
Electrophoretic mobility, 89 gelation, 116
Electrospinning, 175, 275, 304, 395, 403 non‐processive, 191, 194
Electrospray ionization, 88 pectinolytic, 332
Electrostatic interactions, 101, 108–109, 246, 263, processive, 191
319, 333, 354, 413, 432, 435, 439, 452, proteases, 11, 15
467–468 redox, 189
Elemental analysis, 70, 87 substrate association, 194, 202
Elicitors, 178–179, 482, 485 technology, 214
Emulsion, 275, 316, 320, 353, 357 Eosinophils, 146, 153
Enamine bonds, 114, 122 Epichlorohydrin, 104, 430, 434, 436
Endo‐activity, 191, 194, 209, 211 Epidermis, 145, 266
Endochitinase, 194, 211 Epithelial
Endocytosis, 263, 270–271 cells, 146–147, 207, 263, 265–266
Endoglucanases, 74 tissues, 267–268
Endo‐mechanism, 191 Escherichia coli, 76, 170–173, 176, 304, 319,
Endopeptidase, 209–210 359–360, 382, 385
Endothelial cells, 263, 271 Essential oils, 175, 360–362, 371–372, 376,
Endotoxins, 62, 294, 301 380–381
Encapsulation, 175–176, 306, 316, 332–334, Ethanol, 13, 214–216, 235, 448
385 Ether bond formation, 254
Environmental Ethylene, 378
friendly, 122, 135, 419 Ethylene glycol, 296
impact, 28, 232, 364 Ethylene glycol diglycidyl ether (DGDE),
pollution, 11, 25, 131, 177, 230 254, 480
Enzymatic Ethylene oxide, 296
degradation, 75, 77, 83, 180, 270 Ethylenediaminetetraacetic acid (EDTA), 39, 131
depolymerization, 74, 85, 215, 229 Etiological agent, 178
deproteination, 28 Eukaryotes, 198–199, 480
extraction, 230 European Food Safety Authority (EFSA), 160, 321
fingerprinting, 87, 91 European Pharmacopoeia, 293
hydrolysis, 12, 215, 237 European standards, 293–294, 301
methods, 214, 236 European Union regulations, 292
Index 499
Exo‐activity, 191, 194, 209, 211 packaging, 175, 321, 324, 350, 358–359, 483
Exo‐enzyme, 195 preservation, 175, 234, 324, 353, 362, 371–372
Exo‐β‐glucosaminidases, 190, 195–197, 237 products, 174–175, 316, 353, 362
Exoskeleton, 1, 5, 9–10, 212, 291, 315, 352, 372, quality, 174, 349
477–478, 480 shelf life, 174, 324, 357
Eye, 269–270 spoilage, 174–175, 338, 355
waste, 176
Fabrics, 176–177, 396, 398, 400, 413–414, Food and Drug Administration (FDA), 261, 292,
417–418 316, 351
cellulose, 176 Formulations
cotton, 176, 398, 416, 418 chitosan, 179, 376, 380–381, 417
polyester, 176 drug delivery, 261
textile, 395, 400–401, 414 feed, 484
Failure Mode and Effect Analysis (FMEA), 307 macroscopic, 261
Fats, 160, 406, 480 microscopic, 261
Fatty acids, 175, 302, 319, 321, 381, 412 nanoscopic, 261
Feed, 12, 230, 338 pharmaceutical, 259–261
formulation, 484 Fossil fuels, 230, 349, 397
side, 446–447 Fourier‐transform infrared (FT‐IR) spectroscopy,
Ferric ions, 119–120 5, 67, 481
Fertilizer, 230, 483 Fractionation, 89, 232
foliar, 180 Fraction of acetylation, 81, 83, 87, 91
release, 133 Fragmentation, 84–86
synthetic, 177 Free radicals, 74, 114, 252, 296, 318, 320
Fibers, 2, 40, 43, 48, 303–304, 395–396, 398–406, Functional material, 176, 357, 472
410, 412, 419–420, 480 Fungal
Filling material, 173, 305 biomass, 10, 16, 24, 26
Film, 61, 305, 322–323, 353–354, 357–359, 362, cell wall, 10, 207–208, 352, 372
407–408, 410–412, 464–465 lung inflammation, 146, 149
antimicrobial properties, 170, 174, 296, 319, mycelium, 25–26
323, 359–360, 381 pathogens, 147, 177–180, 207, 214
biodegradable, 322, 324, 357 Fungi, 5, 10, 25, 28, 39, 169, 177–179, 207–208,
edible, 174–175, 322, 349–353, 357–360, 213, 375, 419
363, 372 chitosan extraction, 24–25
forming property, 406–407, 410, 462, 465, 470 filamentous, 169–170
gas permeability, 173 Fungicidal activity, 155, 171, 372, 375
hydrophilic properties, 173 Fungine, 1, 477
Fingerprinting, 87, 92 Furan‐based monomers, 229, 238
First derivative of UV spectrum, 69–70 2,5‐Furan dicarboxylic acid (FDCA), 238
Fish, 35, 155–156, 174–175, 291, 352, 362–364, 380
Flame retardation, 398, 400, 415 Gallic acid, 250, 318, 362, 380
Flocculant, 214, 444–445 Gastrointestinal
Flocculation, 408, 429–430, 432, 440, 444–445 tissues, 265
Foam, 301–302, 305, 414 tract, 320, 332, 334, 339
Food, 174–175, 230, 315–316, 318, 322, 324, Gel, 99, 108–109, 111–112, 119–122, 125, 134,
334, 338 172, 275, 297, 410
additive, 315–316, 483 colloid, 464
freshness, 174 core, 100
industry, 174–175, 214, 230, 232, 235, 316, cross‐linked, 120
318, 357 formation, 108, 112
500 Index
Gel (cont’d) Glycoside

mechanical strength, 110 bonds, 2, 74, 86, 189, 191, 205, 236–237, 350
membrane, 100 hydrolase (GH), 189, 235
mucoadhesive, 172 family, 19
porous, 63 2, 195–196
structure, 267, 333 3, 195
Gelatin, 101, 254, 354, 357, 359 5, 197
Gelation, 98, 104, 108–109, 111–112, 114, 7, 197
116–117, 119, 333, 363, 384 8, 197
ionic, 275, 333, 357, 360, 384, 416 9, 195–196
ionotropic, 363, 384 18, 191, 193–195, 199, 205, 211–212
mechanism, 107 19, 191, 193–195, 207
process, 99, 107 20, 195, 209, 211
speed, 104 35, 195–196, 211
stage, 107 46, 197, 207
system, 99, 117 61, 199
time, 111, 114, 116 75, 197
Gel permeation chromatography, 63, 76 80, 197
Gene transcription, 146, 156 84, 195
Generally Recognized as Safe (GRAS), 234, linkages, 84, 88, 189, 235–236, 245, 315, 397
316, 351 Glycosyl transferase, 189, 205
Gene Therapy, 263 Graft copolymerization, 132–134, 136, 338
Genetic materials, 170, 259 Graft co‐polymers, 251–252
Genipin, 126, 304, 306, 464 Grafting
Genotoxicity, 296, 360 chemical, 252
Germination, 76, 170, 180, 381, 386 enzymatic synthesis, 252
Glass sponges, 36, 41, 43–44 from, 251–252
Glass temperature, 126 ionic mode, 252
Global market, 92, 234 irradiation, 252
β‐Glucans, 25, 163, 232 living polymerization, 252
D‐Glucosamine, 2, 10, 40, 67, 74, 76, 88, 98, photochemical, 252
229, 232, 239, 245–246, 291, 315, plasma‐induced, 252
350, 397, 462 through, 251
D‐Glucosaminic acid, 229 to, 251–252
Glucose, 468–469 Graft synthesis, 251
control, 158, 160, 163 Gram‐negative bacteria, 162, 169–170, 172,
detection system, 111 191, 208–209, 211, 304, 306, 319,
dual–responsive injectable hydrogel, 112 360, 381
oxidase, 239, 468 Graphene oxide, 126, 449, 472
responsive phenylboronate ester, 112 Green and sustainable conversion routes, 245–246
responsive system, 111 Green chemistry, 484
Glutaraldehyde, 132, 248, 254, 331, 430, 434, 449, Greenhouse gas, 230
464, 471 Grotthuss mechanism, 465–466
Glycan, 213, 238 Growth factors, 276, 303
Glycerol, 305, 322, 354 Growth phase, 9, 169–170, 207
Glycidol, 250 exponential phase
Glycidyl methacrylate, 133 early, 170
Glycine betaine, 250 late, 24, 170
Glycoproteins, 207, 252, 263, 265 logarithmic phase, 170
Glycosidases, 83 stationary phase, 170–171
Index 501
Haemostatic double‐cross‐linked, 104

activity, 297, 397 double–network, 117, 119–120, 125, 472
agent, 304–305, 308 dual network
dressing, 305 pH sensitive, 100
effects, 417 thermosensitive, 100
properties, 171, 304 eco‐friendly, 97
Hair and skin, 214, 250 formation
Hair follicles, 266–267 electrostatic association, 246
Hazards, 295, 307–308 gelatin‐chitosan hybrid, 101
biological, 307 graft copolymerization, 133
HCl horseradish peroxidase/hydrogen peroxide, 116
demineralization, 11, 13 injectable, 97–98, 108, 110–111, 116–117
Health effects, 145, 149, 158, 160, 164, 320 glucose dual‐responsive, 112
Heavy metals, 62, 301, 429–430 pH responsive, 111–112, 114
absorption, 175, 338 thermo responsive, 114
adsorption, 437 memory, 127, 131
determination, 71 multilayered, 97, 99–102
ions, 451 physical, 100
pollution, 437, 439–440 pH responsive, 248
removal, 214, 246, 396, 417–418, 432, 445, 450 poly(acrylic acid), 121, 129
ultrafiltration, 450–451 polymeric network
α‐Helices, 191, 198 cross‐linked, 97
Helicobacter pylori, 172 photopolymerization of, 114, 116
Heparin, 108, 248 physically cross‐linked, 108, 110, 119, 126
Herbal extracts, 174–175 self‐healing, 97–98, 111, 119–122, 125
Hexactinellida, 41–43 shape memory, 97–98, 125–127, 131–132
biosilica‐based skeletal structure, 41 bilayer self‐deformed, 127
spicules, 41, 44 self‐actuated shape, 127
β‐N‐Hexosaminidases, 190 stimuli‐sensitive, 275
High‐density lipoprotein, 61, 160, 321 superabsorbent, 97–98, 131–132, 135–136
Histological damage, 157 supramolecular, 108, 131
Holin‐like protein, 209–210 thermosensitive, 275
Homopolymeric networks, 254 tyrosinase, 116
Horseradish peroxidase, 116, 252 Hydrogen bonds, 40, 64, 67, 97, 101, 109, 120,
Horticultural commodity, 371–372, 376, 382, 384 126, 331, 353, 434, 464, 466
Horticulture, 177, 181 Hydrogen peroxide, 39, 44, 116, 416, 468
Host‐guest interaction, 121 Hydrolytic hydrogenation, 239
Human body, 108, 145, 234, 266, 296, 437 Hydrophilic
Human intervention trials, 146 compounds, 266, 448
Hyaluronic acid, 111, 269–270 groups, 97
Hydrodynamic volume, 63, 76, 248 hydrophobic transition, 127
Hydrogel, 275, 305, 464 interaction chromatography, 84
alternating process, 100–101 nature, 110, 134, 267, 354, 376
bioinspired, 117 polymer, 109, 432
chemically cross‐linked, 108, 110, 119 Hydrophobic
chitosan‐based, 97–100, 116–122, 125, associations, 110, 121, 126
127, 131 chromatography, 84
composite, 134, 437 groups, 121
cross‐linked, 132 interactions, 104, 110, 121, 126, 333, 464
chitosan/graphene oxide nanocomposite, 126 molecules, 306, 356
502 Index
Hydrothermal Interferon (IFNγ), 147, 153, 156–157

conversion, 238 Interleukin (IL)
synthesis, 49–50 IL‐2, 158
Hydroxyapatite, 306 IL‐3, 146
Hydroxyl groups, 40, 85, 104, 116, 136, 246, 248, IL‐4, 153, 162
261, 316, 318, 332, 397, 432, 437, 444, IL‐5, 146, 156–157
448, 462, 464 IL‐6, 147, 162, 163
Hydroxyl radical, 178–179 IL‐10, 156, 163
scavenging activity, 250 IL‐12, 147–148
5‐Hydroxymethylfurfural (5‐HMF), 229, IL‐13, 153
232, 235, 237, 240 IL‐17, 148
Hypocholesterolaemic effects, 158, IL‐23, 148
160, 321 Intermolecular
bonds, 2
IgE levels, 153 hydrogen bond, 40, 98, 103–104, 107, 109, 126,
Imine bonds, 117, 121–122, 125, 464 246, 323, 462
Immune interaction, 410
cells, 146–148, 153, 155–158, 162–163 variation, 89
mechanisms, 178 Interpenetrating networks, 117, 254
modulation, 149 Invasive method, 271
response, 146–149, 153, 155–157, Inverting mechanism, 191, 195, 198–199
163–164, 268 Iodine, 72, 171
system, 146, 156, 158, 162, 164, 205 Ion‐exchange
Immunomodulatory effects, 145–146, 148–149, adsorption, 433
153, 157, 164 chromatography, 75–76
Inflammatory materials, 430
bowel disease, 153, 235 mechanism, 435–436, 452
condition, 146 textiles, 418
cytokines, 156–157 Ionic
responses, 156–157 interactions, 108, 117, 119, 353, 416
Initiator, 114, 120, 132, 252 liquids, 47, 232, 237–238, 402, 405
Innate immune Ionization efficiency, 84–85
response, 147 Irradiation, 296
system, 164, 205, 207, 485 electron‐beam, 235
Insects, 9 gamma, 235, 252, 296
chitin isolation, 16, 480–481 induced grafting, 252
cuticles, 352, 480 microwave, 133, 235
exoskeleton, 477 UV, 376
farms, 28 visible light, 114
industry, 482, 485 Isosorbide, 239
pathogenic fungi, 207
Insulin sensitivity, 162 Joint
Intramolecular diseases, 235
bonds, 2 replacement, 102
hydrogen bond, 40, 98, 103–104, 107, 109,
126, 462 Karl Fischer reaction, 72
variation, 89 Knock‐out, 207–208, 213
Intranasal administration, 153 KOH
Intraperitoneal injections (i.p), 149, 153, 155 deacetylation, 22–23
Intravitreal injection, 270 deproteination, 39
Index 503
Laccase, 250, 252, 318 Lymphocytes, 147–148, 153, 156, 158, 360
Lacrimal clearance, 270 Lymphopoietin, 146
Lactic acid, 320 Lyophilization, 302, 304–305
bacteria, 14 Lysis
biosensor, 332 cell, 155, 170, 355
fermentation, 39, 239 sponge cells, 45
Layer‐by‐layer Lysozyme, 74, 116, 127
assembly, 101 activity, 208
deposition, 261 Lytic polysaccharide monoxygenase (LMPOs),
formation, 416 189–190, 199–200, 204–205, 207–209,
technique, 354, 465 211, 213, 216, 236–237
Lead (Pb), 71, 301, 338, 418, 439, 469
Leakage Macromolecular
cell, 319 chain, 125, 263
cell membrane, 170 cross‐linkers, 112, 135
Leucocyte, 155, 158, 163 network, 110
activity, 155 radical, 318
responses, 155 Macrophage, 146–148, 155–157, 263
Levulinic acid, 232, 235, 238, 240 Maillard reaction, 316, 338
Lewis base liquid, 252 Mannose receptor, 147–148
Liesegang rings, 99 Manufacturing
Lignin, 180, 189, 230 film, 464
Lignocellulosic biomass, 215, 230, 238 medicinal products, 292
Linear process, 292–293, 295
aggregates, 246, 462 textile, 396, 398, 435
backbone, 251 Mark–Houwink equation, 66–67
macromolecules, 399 Mark–Houwink–Sakurada equation, 63
polysaccharide, 40, 98, 103, 232 Mass spectrometric tools, 76, 81
potentiometric titration, 69–70 Mass spectrometry, 83–85
Lipase, 75, 332, 481 Mast cell, 157
Lipid, 230, 266, 355, 376 Mathematical models, 261, 276
bilayers, 266 Matrix assisted laser desorption time of flight mass
composition, 160 spectrometry, 76
layer, 270 Meat, 362
nanocarriers, 271 Mechanical
oxidation, 316, 362–363 properties, 107, 110, 120, 125, 136, 322, 354,
peroxidation, 175, 318, 320–321 358, 472
Lipopolysaccharides, 148–149, 162, 170, 319 fibers/yarns, 404–406
Lobster, 23, 38, 479 films, 411–412
Loops, 191, 193, 199, 201–202, 420 strength, 102, 110, 119, 125, 176, 304
Low‐density lipoprotein (LDL), 61, 158, 160, Medical
162, 321 applications, 71, 171, 214, 292, 308
Lower critical solution temperature, 110 devices, 72, 291–297, 301, 306–308, 484
Low molecular fabrics, 398
chitosan, 75, 153, 158, 170, 215, 261, 319 textiles, 176
weight, 175, 265, 269, 320, 408, 446, 450 Membrane, 259, 276, 305, 447
Lowry method, 73 biological, 177, 266
Lymph blend, 448–450, 452
node‐, 156 cell, 170, 179, 271, 319, 398
stream, 265 cellular, 265–266, 320
504 Index
Membrane (cont’d) probiotic, 332, 334

chitosan, 175, 304 proteolytic, 39
composite, 301, 450 sterilisation, 295
cytoplasmic, 155, 306, 355, 379 Microparticles, 38, 162, 173, 259, 261, 357,
epithelial, 265 434, 439
gel, 100 Microspheres, 126–127, 271
mucosal, 265, 267 Microwaves, 47–49, 54
multi‐stimuli‐responsive, 275 Minimum bactericidal concentration, 171, 384
porous, 446, 448, 450 Minimum fungicidal concentration, 171
potential, 170–171 Minimum inhibitory concentration, 171–172, 174,
separation, 446, 452 381–382
ultrafiltration, 432, 446, 448–452 Moisture
Mercury, 71, 301, 469 resistance, 172
Metal, 170, 175, 398, 430, 437 retention, 385
catalyst, 239, 246 sensitivity, 357, 403
cation, 129, 355, 432, 437, 439–440, 452 vapour transmission, 305, 307
chelating properties, 246, 254, 318 Molar mass, 62–64, 75
coordination interaction, 127, 131 determination, 62–63
ion, 99, 108, 119, 201, 318, 360, 418–419, 430, distribution, 63, 66
444–445 number average (Mn), 63
recovery, 248 viscometric average, 66–67
removal, 254, 451 weight average (Mw), 63
suspension, 252 Molecular
Methacrylic acid, 119, 360 mass, 178, 359
1‐O‐Methyl‐N‐acetylglucosamine, 236 distribution, 63
Metronidazole, 172, 269, 275 parameter, 63
Mice, 146, 149, 153, 155–158, 321, 351 structure, 61–63, 75, 178
Michael‐type function relationship, 81, 92
addition, 110, 116 weight, 16, 22–23, 39, 86, 89, 170, 174, 215,
adducts, 110, 250 234, 276, 296, 318–319, 405, 408, 446
Microbes, 157, 208, 215, 321 Molecular weight cut‐off (MWCO), 446
Microbial Mollusks, 16
cell, 170 Monocytes, 155–156, 158
chitin degradation, 208 Montmorillonite, 134, 323, 361, 432, 469
growth, 171, 362, 377 chitosan material, 435
infection, 155–156 Mouse model, 149, 153
Microbiological Mucin, 207
burden, 301 Mucoadhesive
contamination, 293–295, 307 gel, 172
Microcrystalline, 76, 127, 178, 180 properties, 259, 263, 269–270
Microorganisms Mucor rouxii, 10, 24, 90, 201, 234
acid producing, 14 Mucosa, 259, 268–270, 276
antibiotic‐resistant, 173 Multi‐angle laser light scattering, 86, 294
cell surface, 319, 398 Multidrug
chitin catabolism, 208 delivery system, 101
encapsulation, 316, 334 efflux system, 265
growth, 355 resistance activity, 265
controlling, 371–372, 375, 379 Mushrooms, 24, 232, 361
inhibition, 175, 177, 371–372, 375, 381 Mutagenic, 296, 434
pathogenic, 173, 386 Mycotoxin, 334, 338, 375, 470
Index 505
Nanocarriers, 266–268, 270, 306 Obesity, 145, 158, 162

Nanocomposite, 126, 134, 175–176, 302, 304, Ocular
323, 358–359, 434, 436, 440, 484 drug delivery, 270
Nanoemulsions, 175, 261, 306, 357, 372 mucosa, 270
Nanofibers, 50, 135, 175, 275, 302 tissues, 270
Nanofibrils, 153, 155 Oligomers, 74, 76, 83, 85, 175
Nanomaterial, 177, 384, 464, 483 Oligosaccharides, 74, 194, 214, 235, 251, 331
Nanoparticles, 175–176, 178, 250, 259, 275, Organic
357, 384–386, 418, 440, 445, 450, acids, 14, 173, 232, 246, 397, 402–403, 412,
469–470, 483 463, 468
Nanostructures, 122, 316 compound, 260, 434, 450
Nanotechnology, 260, 339, 372, 384, 387 dyes, 437
NaOH farming, 177, 181
deacetylation, 22–23, 47 pollutants, 429–430, 432, 434–435, 452
deproteination, 15, 39, 45 solvent, 19, 103, 232, 238, 246, 291, 351,
titration method, 69 402–403, 463
urea solution, 103–104 Ophthalmologic treatment, 269
Natural Ophthalmology, 173
immunity, 178, 180 Oral
product, 177, 372, 376 administration, 153, 157–158, 160, 260,
Negatively charged, 440, 443 263, 268
colloidal particles, 445 cavity disease, 172–173
groups, 170 health, 162
molecules, 179, 358, 462, 467 intake, 145, 321
polymers, 108 release, 275
residues, 195 Oxacarbenium‐ion‐like transition states, 191
surface, 77, 170, 173, 268 Oxazolinium ion intermediate, 191
Neutrophils, 156, 158 Oxidative
Ninhydrin, 73 cleavage, 209
Nitric oxide, 148 enzymes, 199, 318
Non‐catalytic fermentation, 239
domains, 205 mechanism, 199
modules, 189 reaction, 237, 250, 318
Noninvasive method, 270–271, 471 reductive agents, 235
Non‐reducing end, 83, 85, 195–196, 200, 202, regioselectivities, 199
204, 209, 211 stress, 156, 160, 316, 321, 379
Nontoxic, 122, 318, 332, 372, 377, 387, 444 Oxygen, 85, 173–174, 191, 199, 211, 239, 301,
Nonwoven, 297, 303–305 322, 324, 354, 358, 378
Nuclear magnetic resonance, 5, 67, 69, 76, 83, atom, 381, 437, 451
294, 481
1
H NMR, 67, 87, 89–90, 294 Packaging material, 174, 176, 214, 295, 322,
13
C NMR, 67, 69–70, 87, 89–91 349–350, 376
15
N‐NMR, 87 Pancreatic lipase activity, 160
Nucleic acid, 263, 275–276, 349, 381 Paper industry, 176
Nucleophile, 191 Paracellular passage, 266, 269
Nucleophilic Parasite, 156–157, 307, 484
addition, 19 Particle size, 13, 15, 39, 149, 359
attack, 201 Pathogen‐associated molecular patterns
substitution, 19 (PAMPs), 146
Nylon, 396, 403, 405 Pathogenesis, 172, 207–208, 212
506 Index
Pathogenic endotoxin levels, 62, 294

agents, 268 heavy metal content, 71, 294
recognition, 146 molar mass, 62–64, 294, 408
transmission, 306–307, 321 nitrogen content, 70–71
Pathogens, 76, 146, 173–181, 205, 207–208, 214, protein content, 70, 73, 297
268, 292–293, 350, 359, 362, 384, 386, solubility, 72
482–483, 485 viscosity, 66–67, 70
Pattern of acetylation, 81, 83–85, 89–91, 234, 246 water content, 72
Penetration enhancers, 266–267, 271 water retention values, 63, 72
Periodic precipitation, 99–100 Pigments, 13, 38, 44, 230, 297
Periodontal diseases, 172, 174 pKa value, 19, 170, 351
Pesticides, 177, 214, 434, 482, 485 Placebo group, 158, 160, 162
Peptides, 12, 252, 265, 276 Plant
Peptidoglycan, 200–201, 204–205, 207–208, 319 growth rate, 177–178, 232, 377, 482
Permeability, 174, 268, 358–360 protection, 177, 179
cell membrane, 170 agents, 177–178
gas, 173 products, 482
oxygen, 301, 362, 376 Plasticizer, 126, 305, 322, 354, 412, 464
Permeation, 259–260, 263, 265, 271, 276, Plastics, 230, 321, 376, 420
358, 449 Platform chemicals, 229, 232, 235, 239
Peroxidase, 250 Pluronic F127, 110
activity, 361 Polarity, 246
Phagocytic activity, 155, 163 Pollutant, 429–430, 432, 434
Pharmaceutical formulations, 259–261 Polluted water, 246, 417, 435, 444
Pharmacokinetic profile, 276 Poly acrylamide chitosan, 127, 131
Phenolic groups, 116, 254 Polyacrylonitrile, 254, 448–449
Phenols, 116, 176, 178, 250, 318, 381–382, 434 cross‐linking, 414
Phenylboronate ester bond, 110–112 fiber, 406
Phenylboronic acid, 111–112 Polyamides, 229, 236, 238
Phospholipids, 266, 319 chiral, 229, 239
Phosphorylated chitosan films, 305 Polyanion–polycation complexation, 270
Photo‐cross linking, 101, 306 Polycationic
Photoinitiators, 114 compound, 170, 398
Photopolymerization, 114, 127 heteropolysaccharide, 350, 446
pH‐responsive properties, 214, 333
behavior, 332 structure, 319
gelation properties, 111 Polydispersity, 63, 252
hydrogel, 114, 248 Polyelectrolyte, 98, 101, 108, 234, 248, 333, 339,
imine bond, 112 384, 451, 463, 465, 468
self‐assembling properties, 97–98 Polyesters, 236, 238–239, 399, 413, 416
Physical fabric, 176
adsorption, 271, 433 Poly(ethylene glycol), 63, 111, 114, 252,
bonds, 121 260, 448
disruption, 271, 472 Poly(ethylene glycol)ylation, 260, 263–264
hydrolysis, 235 Polyhedral oligomeric silsesquioxanes, 50
seal, 302 Poly(N‐isopropylacrylamide), 110, 127, 254
Physicochemical properties, 82, 86, 89, 294 Polymer, 61, 63, 97, 108, 131, 175, 178–179, 235,
ash content, 71, 407 246, 263, 323, 333, 355–358, 376, 402,
average molar mass, 62–64, 66, 75, 408 404, 406, 414, 430, 444–445, 448,
crystallinity, 62, 76 450–451, 464, 467
degree of acetylation, 67–70 drug conjugates, 260
Index 507
Polyol, 109, 112 polymerization, 110, 119–120,

Polyplex, 263 129, 252
Polypyrrole, 119, 471 scavenging, 250, 318, 382, 385
Polysaccharide, 97, 111, 131, 204, 250, 315, 319, Ragweed, 149, 153
322–323, 349, 397–398, 430, 452 Random distribution, 82, 90, 251
based coating, 349 Re‐N‐acetylation, 82, 84–85, 89
lyase, 189 Reactive oxygen species (ROS), 122,
Polyurethanes, 238, 397, 415, 418, 444 155, 178, 320
Polyvinyl alcohol, 254, 397, 403, 449, 468 Recalcitrant, 204, 213–215
Porifera, 35 Receptor, 146, 149
Porosity, 52, 100, 275, 296, 302, 433, 472 binding, 147
Posterior eye segment, 270 cell surface, 146
Potentiometric C‐type lectin, 147
measurements, 468–469 dectin‐1, 146–148
sensor, 468–469 endocytic, 147
titration, 67–70 FIBCD1, 146–147
Prebiotic, 320–321, 356 galectin‐3, 147
Preservatives, 174–176, 181, 364, 406 intracellular chitin recognition, 147
Pretreatment, 11, 15–16, 24, 155–157, 215, 234, mannose, 146–148
237, 414 NKR‐P1, 146–147
Probiotic, 315, 332, 334 NOD2, 147
Processivity, 191, 193–195 pattern recognition, 146
Pro‐inflammatory immune response, 291 RegIIIγ, 146–147
Propolis, 173, 371, 382 toll‐like receptors, 146–147
Proteases, 12, 14–15, 39, 205, 481 transmembrane endocytic, 147
Protein, 9, 12, 16, 38, 44, 73, 75, 155, 205, 211, Recycling, 230, 396
252, 297, 319, 331, 430, 480–481 Redox chemistry, 199
family, 204 Reducing end, 85, 209
Pseudocapacitance, 472 Reductive amination, 248, 251
Pyrogenicity, 293–294, 307 Refractive index, 86
Pyrogenic substances, 295 Refractometric detector, 63
Regioselectivity, 199–200
Quadrupole Time‐of‐Flight, 88 Renewable biological resources, 230
Quality control, 292, 294, 307 Reproducibility, 39, 292–293, 301, 306,
Quaternary amine acid, 176 308, 332, 352
Quaternary amino acid, 250 Respiratory burst activity, 155
Quaternary ammonium Retaining, 122, 446, 468
chitosan, 133, 269 mechanism, 191, 195, 197
compound, 398, 417 Retina, 270
group, 304, 306 Reverse micellar technique, 275
salt, 98, 176 Reverse phase, 76
Quaternary chitosan, 250, 306 Risk, 301, 306–308, 338, 375
Quaternized chitosan, 248, 250 adverse accumulation ethylene oxide, 296
Quaternized groups, 263 analysis, 306–308
o‐Quinone intermediate, 250 cardio vascular disease, 158–160
Quinones, 9, 116, 318 class III, 292
contamination, 88, 293
Radiation, 47, 74 health, 173, 360
Radical, 74, 114, 252, 296, 318 pathogen transfer, 293
cross‐linking, 114 pyrogenicity, 293–294
hydroxyl, 178–179, 250 Ruhemann’s purple, 73–74
508 Index
Saccharification, 75, 196, 215 Sol‐gel

Safety, 119, 155, 177, 292–297, 301, 307, 316, method, 331, 450
338–339 transition, 98, 100, 108, 248
Saliva, 268 pH induced, 114
Salt‐resistance property, 133, 136 thermosensitive, 104
Scaffold, 43, 51–54, 126, 275–276, 306 Solubility, 72, 110, 170, 351
chitin, 36, 43, 48–50, 53 chitin, 103, 430, 452
2D, 301, 305 oligomer, 40, 77, 175–176, 235, 246
3D, 38, 49, 52–53, 301 chitosan, 19, 89, 232, 248, 250, 295, 316, 318,
Scanning electron microscopy, 179 402, 463
Scattering electron microscopy, 50–51 Sonication, 235, 359
Schiff base, 416 Sorption
adduct, 250 capacity, 248
linkages, 111, 121, 127, 129, 254 process, 433
reaction, 110–111 Spectroscopy
Seafood, 25, 175, 230, 297, 352, 362, 364 atomic absorption, 71
Selective oxidation, 248 FTIR, 5, 67, 481
Self infrared, 67, 85–87
assembly, 98, 100–101, 110, 126 mass, 5, 76
healing, 111, 117, 119–122, 125, 471 NMR, 87, 294
Serratia marcescens, 191, 195, 199, 207, UV, 67
209–211, 237 UV/VIS, 87
Serum glucose levels, 160 Spinning, 399, 402
Shape pseudo‐dry, 403
memory Splenocytes, 153
effects, 125–127, 131 Spoilage, 174–175, 338, 355, 362–363
polymers, 125, 127, 131 Sponges, 35–37, 40–43, 54
triple, 127, 129, 131 Spray, 179–180, 306
Shape recovery, 127 coating, 175
pH‐triggered, 126 drying, 275, 333, 357
ratio, 126 Squid pen, 5
ultrasound triggered, 126 Standard, 71, 294, 296, 307
water‐/solvent‐ triggered, 126 method, 43, 49, 54
Shear rate, 408, 410 Staphylococcus aureus, 76, 155, 170–173,
Shelf life, 174–175, 295, 316, 321, 324, 356–357, 175–176, 303, 304, 306, 355, 360,
361–363, 376, 378–380, 385 382, 385
Shrimp, 9, 14, 22–23, 25, 38, 155, 297, 316, Starch, 136, 230, 250, 322, 397, 407–408, 412,
351, 363 414, 450
Signalling molecules, 146 Steam
Silica, 36, 41, 44, 50, 324, 331 explosion, 23, 237
chitin, 40, 43, 50 saturated, 295
Single displacement, 191 sterilisation, 295–296
Size exclusion chromatography, 63, 83, 294, 481 Sterilisation, 293, 295–296, 301, 304, 308
Sizing agent, 398, 406–408, 410–412, 420 Stimuli‐responsive materials, 125
Skin, 74, 121, 157, 235, 250, 266–267, 296, 305, 353 Stomach, 248, 268–269, 316
wound, 301, 306 Stratum corneum, 266–267
Small intestine, 158, 260, 265 Stratum granulosum, 267
Soil, 10, 133, 180, 248, 435 Streptococcus mutans, 173
activation, 181 Structural characterisation, 82, 164, 198, 234,
pathogens, 177, 181 293, 318
Index 509
Structure TEMPO‐laccase oxidation, 248, 464

amorphous, 98 Tenacity, 405–406, 420
Bouligand, 479–480 Tensile strength, 125, 295, 322, 354, 359, 363,
chemical, 2, 62, 176, 263, 276, 296, 352, 405–406, 411–413, 420, 464
430, 444 Tetanus toxoid antigen, 268
crystal, 43, 47, 199, 202, 443 Tetrahedral oxyanion intermediate, 201
3D, 98, 472 Textile
lattice, 41 antimicrobial, 176, 417
molecular, 61–63, 74–76, 176 auxiliaries, 400, 405, 407–408, 415
multilayered, 48, 98–101, 357 coating, 414, 417, 419
oriented, 98–99 fabrics, 395–396, 398, 414
sheath‐like, 104, 304 industry, 176, 395–396
skeletal, 39, 41 material, 301, 407
supramolecular, 263–264 medical, 176, 401
Structure‐function relationship, 81–82, 87, processing, 395, 411
91–92 production, 397–398, 400, 419
Subcritical water treatment, 237 Th2
Subsite, 91, 194–195, 202–204 dominated allergic response, 149, 153, 155
specificity, 88, 91 skewing, 146, 153
Substitution degree, 133, 248, 251 Therapeutic
Substrate agents, 260, 269, 271
accessibility, 213, 215 effects, 259–260, 265
assisted mechanism, 191, 195 range, 260
binding, 190, 193–195, 198–199, 204–205, Thermophiles, 216
208–209 Thiol‐ene/yne conjugation reactions, 114
cleavage‐site specificities, 198 Titanium, 304, 436, 470
distortion, 199 Tissue
Succinic anhydride, 269, 417, 449 engineering, 51–54, 114, 116, 306
Superabsorbents, 131, 133–136 regeneration, 108, 111, 291, 301
Supercritical water treatment, 237 TNFα, 147–148, 153, 156, 158, 163
Surface Tooth, 173
charge, 177, 268, 334, 411, 440, 462 Topical
tension, 410–411 administration, 270
volume ratio, 269, 275 delivery, 270
Sustained release, 260, 263, 275–276 haemostatic agent, 304, 308
Swelling behaviour, 107, 441 instillation, 270
Synergistic effects, 133, 199, 236, 254, 306, 376 Toxic, 133, 176, 315–316, 334, 338, 360, 397,
Synthetic polymers, 131, 246, 254, 322, 399, 419, 402, 417, 430, 434–435, 437, 439
430, 462 Toxicity, 116, 122, 131–132, 246, 260–261, 265,
296, 307, 316, 360, 434
Tablets, 71, 160, 269, 271, 275, 470 Toxin, 173, 177, 268, 338
Taxonomical classification, 198 Traceability, 292
T‐cells, 147, 156–158 Transdermal delivery, 260, 266–267
Teichoic acid, 170, 319, 355 Transfection, 259, 263–264, 276
Temperature, 170, 236, 295, 410, 434 Transglutaminase, 252, 333, 414
deacetylation, 21–23 Transpiration, 179
deproteination, 15, 25 Triglycerides, 160, 162, 321
demineralization, 14, 39 Tripolyphosphate, 99, 305, 333, 416, 450
dissolving chitosan, 25 Tumour growth, 149, 157–158
thermosensitive sol‐gel transition, 104 Tyrosinase, 116, 250, 252
510 Index
Ultrafiltration, 75, 429, 447, 450–452 solubility, 98, 103, 234–235, 250, 359, 450
membrane, 432, 446, 448–450 soluble polymers, 251, 450
polymer‐enhanced, 450–451 treatment, 232, 234, 237, 430, 450,
process, 432, 446–447, 452 483, 485
Ultrasonic uptake, 132–133, 135–136, 412, 466
treatment, 45 vapour, 307
waves, 45, 417 permeability, 174, 377–378
Ultrasonication, 237 resistance, 179, 380
Ultrasound, 45–46, 54, 126–127, 271, 359 Wear resistance, 102, 415
UV‐irradiation, 275, 376 Weaving, 400, 419, 421
Weight
Vacuum packaging, 175, 363 average, 86
Validation, 149, 292–293, 297, 307–308 electrophoretic mobility, 89
Vapour permeability, 304 molar mass, 63
Verification, 292–294, 297, 307–308 loss, 158, 163–164, 234, 361, 376–382, 384
Verongula gigantea, 36, 43 Well‐being, 276
Vinyl monomers, 132, 136 Wetting, 407, 410
Virulence, 156–157 Wide‐angle X‐ray scattering, 76
factor, 207–208 Wine, 175, 235, 316, 338
Viruses, 76–77, 177–178, 198–199 Wound
Viscometer, 66, 70 dressing, 72, 171, 241, 292–297, 301–308, 398,
Viscose, 399 417, 471
fibres, 404 gel, 297
Viscosimetry, 86 nonwoven, 297
Viscosity, 66–70, 194–195, 408, 410, 447 powder, 297
average, 86 sponge/foam, 297
molecular weight, 86 healing, 145, 171–172, 174, 275, 291,
number, 66–67 297, 301–302, 305–306, 398,
471, 484
Wastewater, 28, 214, 232, 364, 400, 413–414, treatment, 174, 261, 306
418, 429–430, 434–435, 437, 440, 445,
450–452 X‐ray diffraction, 87, 481
synthetic, 439–440 Xylanase, 75
treatment, 136, 408, 430, 432, 444, 452
adsorption, 430, 432–433 Yarn, 399, 401, 404
coagulation/flocculation, 430, 440, blend, 400, 406
443–444 Yeast, 10, 35, 307, 361–362, 376
membrane separation, 430, 446, 452
Water Zero‐order release kinetics, 260
consumption, 179 Zero‐waste circular economy, 230
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Chitin and

Titles in the Series:

Renewables‐Based Technology: Sustainability Assessment

Handbook of Natural Colorants

Surfactants from Renewable Resources

Industrial Applications of Natural Fibres: Structure, Properties and Technical Applications

Thermochemical Processing of Biomass: Conversion into Fuels, Chemicals and Power

Biorefinery Co‐Products: Phytochemicals, Primary Metabolites and Value‐Added Biomass

Bio‐Based Plastics: Materials and Applications

Introduction to Wood and Natural Fiber Composites

Cellulosic Energy Cropping Systems

Introduction to Chemicals from Biomass, 2nd Edition

Cellulose Nanocrystals: Properties, Production and Applications

Nanoporous Catalysts for Biomass Conversion

Thermochemical Processing of Biomass: Conversion into Fuels, Chemicals and Power,

Waste Valorization: Waste Streams in a Circular Economy

Process Systems Engineering for Biofuels Development

Biobased Packaging: Material, Environmental and Economic Aspects

LAMBERTUS A.M. VAN DEN BROEK

Limit of Liability/Disclaimer of Warranty

Library of Congress Cataloging‐in‐Publication data applied for

Cover Design: Wiley

Set in 10/12pt Times by SPi Global, Pondicherry, India

List of Contributors xvii

1 Sources of Chitin and Chitosan and their Isolation 1

2 Methods of Isolating Chitin from Sponges (Porifera) 35

2.3 The Modern Approach to Chitin Isolation from Sponges 40

3 Physicochemical Properties of Chitosan and its Degradation Products 61

4 New Developments in the Analysis of Partially Acetylated Chitosan

6 Beneficial Health Effects of Chitin and Chitosan 145

7 Antimicrobial Properties of Chitin and Chitosan 169

8 Enzymes for Modification of Chitin and Chitosan 189

9 Chitin and Chitosan as Sources of Bio‐Based Building Blocks

9.2.1 Chitosan Production 232

10 Chemical and Enzymatic Modification of Chitosan to Produce New

11 Chitosan‐Based Drug Delivery Systems 259

12 The Application of Chitin and its Derivatives for the Design of Advanced

13 Food Applications of Chitosan and its Derivatives 315

14 Potential of Chitosans in the Development of Edible Food Packaging 349

14.2.1 Definitions and Interests 353

15 The Use of Chitosan‐Based Nanoformulations for Controlling Fungi

16 Chitosan Application in Textile Processing and Fabric Coating 395

16.4.2 Chitosan Spinning Processes 402

17 Chitin and Chitosan for Water Purification 429

18 Chitosan for Sensors and Electrochemical Applications 461

19 Marketing and Regulations of Chitin and Chitosan from Insects 477

Artur Bartkowiak Center of Bioimmobilisation and Innovative Packaging Materials,

Bogdan Ionel Tamba A&B Pharm Corporation, Roman, Neamt ̦, Romania

Lambertus A.M. van den Broek and Carmen G. Boeriu

Chitin and Chitosan: Properties and Applications, First Edition.

1.1 Chitin and Chitosan

1.1.2 Different Crystalline Forms of Chitin

1.2 Sources of Chitin and Chitosan

% (w/w) chitin in biomass

0004461239.INDD 6 10/26/2019 1:16:41 PM

0004461239.INDD 7 10/26/2019 1:16:41 PM

% (w/w) chitin in biomass

Cuttlefish (Cephalopoda) Sepia spp. Shells 91.25 1.35 7.4 [26]

0004461239.INDD 8 10/26/2019 1:16:41 PM

List of Contributors xvii

1 Sources of Chitin and Chitosan and their Isolation 1

2 Methods of Isolating Chitin from Sponges (Porifera) 35

2.3 The Modern Approach to Chitin Isolation from Sponges 40

3 Physicochemical Properties of Chitosan and its Degradation Products 61

6 Beneficial Health Effects of Chitin and Chitosan 145

7 Antimicrobial Properties of Chitin and Chitosan 169

8 Enzymes for Modification of Chitin and Chitosan 189

9.2.1 Chitosan Production 232

11 Chitosan‐Based Drug Delivery Systems 259

13 Food Applications of Chitosan and its Derivatives 315

14 Potential of Chitosans in the Development of Edible Food Packaging 349

14.2.1 Definitions and Interests 353

16 Chitosan Application in Textile Processing and Fabric Coating 395

16.4.2 Chitosan Spinning Processes 402

17 Chitin and Chitosan for Water Purification 429

18 Chitosan for Sensors and Electrochemical Applications 461

19 Marketing and Regulations of Chitin and Chitosan from Insects 477