CHAPTER ONE

INTRODUCTION
1.1 Background of the Study
Pectinase is a group of enzymes that catalyze a degradation of pectin through depolymeration
(hydrolases and lyases) and de-esterification (esterases) reactions (Pedrolli et al., 2009). Based
on their mode of action, the pectinase is classified into three types: pectin esterase, hydrolases
and lyases. Pectin esterase catalyze the de-esterification of the methoxyl group of pectins,
forming pectic acid. Hydrolases (polygalacturonases and polymethylgalacturonases) catalyze the
hydrolytic cleavage of α-1, 4glycosidic linkage in pectic acid and pectin. Lyases
(polygalacturonate lyase and polymethylgalacturonate lyase) catalyze the cleavage of α -1, 4-
glycosidic linkage in pectic acid and pectin, forming unsaturated galacturonates and methyl
galacturonates (Garg et al., 2016).
Pectinases share about 25% in global sales of food enzymes (Tapre and Jain, 2014). Pectinases
are widely used in food industries, such as fruit processing industry for extraction and
clarification of fruit juice, preparation of fruit cordials (Merín et al., 2015), enhance process
efficiency of wine making and wine quality (Merín et al., 2015), oil extraction and fermentation
of coffee, cocoa and tea (Tapre and Jain, 2014). Since pectinases are widely used enzyme for
different industrial application, it is necessary to improve pectinase production, including
selection for potential source of pectinase. Many experiments have attempted to increase the
performance of enzyme production (Molina et al., 2011). The new microbes with high
extracellular pectinase activity, stability over wide range of temperature and pH for a longer
period of time, with their cost-effective production have been the focus of recent research (Garg
et al., 2016). Mango peel Mango is an important tropical fruit, cultivated in many tropical
regions and distributed worldwide. India is largest producer of mango amongst 90 countries in
the world, with its production of 18, 643, 000 metric tons on 2, 209, 000 hectare area which is
35.8% of total fruit area (DACFW 2015; Reddy and Saritha 2015). Mango peel, one of the major
by-products from the mango pulp industry, constitutes about 20–25% of the mango fruit
processing waste and is discarded during the process. It is a rich source of pectin, which
comprises 12.2–21.2% pectin with degrees of esterification from 56.3 to 65.6% (Cheok et
al. 2016). Mango peel has been used in SSF for pectinase production by A. foetidus (Kumar et
al. 2012). The substrate mango peel was supplemented with salt solution consisting of (g/l)

1
(NH4)2SO4, 25; MgSO4, 0.6; FeSO4, 0.4; urea, 3; peptone, 5 and KH2PO4, 6 at pH 7.0 for use in
SSF. Reddy and Saritha (2016) also used mango fruit processing waste (0.5%) for production of
pectinase by Enterobacter sp. PSTB-1in SmF.
Pectinolytic enzymes are a group of related enzymes that hydrolyse pectic substances. Pectin is a
complex polysaccharide present in the middle lamella of plant cell walls. It is composed of
multiple units of D-Galacturonic acid linked by α (1, 4) glycosidic linkage. Pectinolytic enzymes
have been reported in higher plants (Nighojkar et al. 1994; Jolie et al. 2010) and microorganisms
including bacteria and fungi (Uzuner and Cekmecelioglu 2015; Patidar et al. 2016; Rebello et
al. 2017). Pectin is completely digested by three major enzymes: pectin methylesterase
(pectinesterase; EC: 3.1.1.11), pectinase (polygalacturonase; EC: 3.1.1.15) and pectin lyase (EC:
4.2.2.10) to release galacturonic acids and its oligomers (Combo et al. 2012). In nature,
microorganisms have been endowed with vast potential. They produce a range of enzymes,
which have been exploited commercially over the years. It has been reported that microbial
enzymes account for 25% of total global enzyme sales (Jayani et al. 2005). The pectic substances
can be converted by means of microorganisms or their enzymes into constituent
monosaccharides or specific oligosaccharides without the production of undesirable by-products
(Zykwinska et al. 2008; Martínez et al. 2009).
Pectinases are known for their tremendous potential in various industries. Pectin methylesterase
and endo-polygalacturonase have important role in softening of fruits, extraction and
clarification of juices, preparing gel, food manufacturing, retting of textile fibers, extraction of
olive oil, protoplast isolation, etc. (Kashyap et al. 2001; Jayani et al. 2005; Kohli and
Gupta 2015). Galacturonic acid, produced by action of pectinolytic enzymes, has various
applications in industries mainly in pharmaceutical industries. It is used for the production of
vitamin C as acidic agent in food industries and as washing powder agent in chemical industries
(Molnar et al. 2009; Burana-Osot et al. 2010). Almost all the commercial preparations of
pectinases are produced from fungal sources (Kertesz 1951). Filamentous fungi
especially Aspergillus niger is the major producer of acidic pectinase used mainly in fruit juice
and wine industries (Kashyap et al. 2001).
The utilization of SSF processes is interesting for pectinase production by fungi and has the
advantages of low water requirement, high productivity, lesser chance of contamination, cost
effectiveness and simpler fermentation technology (Pandey et al. 2000; Viniegra-González et

2
al. 2003). Research on the selection of suitable solid substrates for SSF has mainly been centered
on agricultural and industrial residues due to their potential advantages for filamentous fungi,
which are capable of penetrating into the hardest of these solid substrates. In addition to the
utilization of this agro-industrial waste, it provides alternative substrates and helps in solving the
pollution problems (Pandey 2003).
Due to the wide applications of polygalacturonase and pectin methylesterase, there is a need to
highlight recent developments on several aspects related to their production in SSF. Microbial
sources, production, characterization and application of pectinases have been reviewed (Kashyap
et al. 2001; Jayani et al. 2005; Favela-Torres et al. 2006; Sharma et al. 2013). However, the most
common sources of pectinolytic microorganisms, recent development in SSF process for
polygalacturonase and pectin methylesterase production, and their assay methods have not been
considered until now.
1.2 Statement of the Problem
Mango (Mangifera indica) contain substantial amount of pectin having a high gelling grade
(Bhardwaj and Garg, 2010). In the processing of mango products, mango peels is a major by-
product which ends up as a waste product. However, mango peels can be used as a valuable,
economic and abundant media source for the commercial production of natural enzymes such as
pectinases. Pectin acts as an inducer for the production of pectinolytic enzymes by microbial
systems. Mango peel is one of the major by-products from the mango pulp processing industries.
During the processing of mango fruit, peel and stone are generated as waste (40–50% of total
fruit mass). Waste generated from mango processing constitutes 20–25% peel, which was found
to be a good source for the extraction of pectin of good quality, with a high degree of
esterification and phenolic compounds (Berardini et al., 2005). Pectin acts as the inducer for the
production of pectinase enzymes by microbial systems, and pectin-rich mango Peels are
considered to be a good source for pectinase production and ideal substrate for the
decomposition of mango peels by microorganisms (Kumar et al., 2012). The current commercial
sources of pectinase comprise a wide variety of bacteria, yeast and filamentous fungi (Yazid et
al., 2011). Amande and Adebayo-Tayo (2012) noted that among the filamentous fungi
Aspergillus spp. are the strains of choice for polygalacturonase production.
1.3 Justification

3
Pectinases are widely used in food industries for juice extraction and clarification, coffee and tea
fermentation, oil extraction, improvement of chromacity and stability of red wines. This research
project will help to determine pectinolytic filamentous fungi from decomposed mango peels.
These produce a huge amount of waste throughout the year. These waste materials can be
constructively used to produce important products including pectin and pectinases as a
replacement for dumping them in the environment where they might at times generate pollution.
This is a promising way of converting wastes to wealth.
1.4 Aim and Objectives of the Study
1.4.1 Aims
The aim of this study is to isolate and characterize of pectinolytic filamentous fungi from
decomposed mango peels..
1.4.2 Objectives:
i. To isolate yeasts from mango peels samples decomposed mango peels
ii. To screen for the ability of yeast isolates to ferment xylose or arabinose decomposed
mango peels
iii. To study the physiological characteristics of selected isolates from decomposed mango
peels

4
 CHAPTER TWO
LITERATURE REVIEW
2.1 Introduction
Mangifera indica (MI), also known as mango, aam, it has been an important herb in the
Ayurvedic and indigenous medical systems for over 4000 years. Mangoes belong to genus
Mangifera which consists of about 30 species of tropical fruiting trees in the flowering plant
family Anacardiaceae. According to ayurveda, varied medicinal properties are attributed to
different parts of mango tree (Shah et al., 2010). Mango is one of the most popular of all tropical
fruits. Mangiferin, being a polyphenolic antioxidant and a glucosyl xanthone, it has strong
antioxidant, anti-lipid peroxidation, immunomodulation, cardiotonic, hypotensive, wound
healing, antidegenerative and antidiabetic activities. Various parts of plant are used as a
dentrifrice, antiseptic, astringent, diaphoretic, stomachic, vermifuge, tonic, laxative and diuretic
and to treat diarrhea, dysentery, anaemia, asthma, bronchitis, cough, hypertension, insomnia,
rheumatism, toothache, leucorrhoea, haemorrhage and piles (Guha et al., 2016). All parts are
used to treat abscesses, broken horn, rabid dog or jackal bite, tumour, snakebite, stings, datura
poisoning, heat stroke, miscarriage, anthrax, blisters, wounds in the mouth, tympanitis, colic,
diarrhea, glossitis, indigestion, bacillosis, bloody dysentery, liver disorders, excessive urination,
tetanus and asthma (Muanza et al., 2015).
Ripe mango fruit is considered to be invigorating and freshening. The juice is restorative tonic
and used in heat stroke. The seeds are used in asthma and as an astringent. Fumes from the
burning leaves are inhaled for relief from hiccups and affections of the throat. The bark is
astringent, it is used in diphtheria and rheumatism, and it is believed to possess a tonic action on
mucus membrane. The gum is used in dressings for cracked feet and for scabies. It is also
considered anti-syphilitic. The kernels are converted into flour after soaking in water and
eliminating the astringent principles. Most parts of the tree are used medicinally and the bark
also contains tannins, which are used for the purpose of dyeing.

5
2.1.1 Taxonomical Classification

Kingdom : Plantae
Class : Mangoliopsida
Phylum : Mangoliophyta
Order : Sapindales
Family : Anacardiaceae
Genus : Mangifera
Species : Indica
Species of mango:
Mangifera altissima Mangifera persiciformis
Mangifera caesia Mangifera camptosperma
Mangifera casturi Mangifera decandra
Mangifera foetida Mangifera indica
Mangifera griffi thii Mangifera laurina
Mangifera kemanga Mangifera macrocarpa
Mangifera longipes Mangifera odorata
Mangifera mekongensis Mangifera quadrifi da
Mangifera pajang Mangifera similis
Mangifera siamensis Mangifera sylvactia
Mangifera torquenda Mangifera zeylanica
Mangifera applanata Mangifera swintonioides

6
2.1.2 Botanical description
MI is a large evergreen tree in the anacardiaceae family that grows to a height of 10-45 m, dome
shaped with dense foliage, typically heavy branched from a stout trunk. The leaves are spirally
arranged on branches, linear-oblong, lanceolate – elliptical, pointed at both ends, the leaf blades
mostly about 25-cm long and 8-cm wide, sometimes much larger, reddish and thinly fl accid
when fi rst formed and release an aromatic odour when crushed. The infl orescence occurs in
panicles consisting of about 3000 tiny whitish-red or yellowish – green fl owers. The fruit is a
well known large drupe, but shows a great variation in shape and size. It contains a thick yellow
pulp, single seed and thick yellowish – red skin when ripe. The seed is solitary, ovoid or oblong,
encased in a hard, compressed fi brous endocarp.
2.1.3 Habitat
It is native tropical Asia and has been cultivated in the Indian subcontinent for over 4000 years
and is now found naturalized in most tropical countries. Parts used: Roots, bark, leaves, fruits,
seeds, flowers and kernels are used.
2.1.4 Synonyms
Sanskrit: Ambrah; Madhuulii; Madhuula; Madhuulaka; English: Mango; Hindi: Aam; French:
mangot; mangue; manguier; Portuguese: manga; mangueira; Dutch: manja; Tamil: Ambiram;
Mambazham; Mambalam; Mangai;
Punjabi: Amb; Wawashi; Gujarati: Ambo, Keri; Marvo (unripe); Kashmiri: Amb; Malayalam:
Amram; Choothaphalam; Manga;
Manpalam; Mavu; Marathi: Amchur; Amba
2.2 Phytochemistry
Chemical constituents of MI are always of an interest. The different chemical constituents of the
plant, especially the polyphenolics, flavonoids, triterpenoids. Mangiferin a xanthone glycoside
major bio-active constituent, isomangiferin, tannins and gallic acid derivatives. The bark is
reported to contain protocatechic acid, catechin, mangiferin, alanine, glycine, γ-aminobutyric
acid, kinic acid, shikimic acid and the tetracyclic triterpenoids cycloart-24-en-3β, 26diol, 3-
ketodammar-24 (E)-en20S, 26-diol, C-24 epimers of cycloart-25 en 3β, 24, 27-triol and
cycloartan-3β, 24, 27-triol (Scartezzini and Speroni, 2010).

7
Figure 1: Structure of Mangiferin
Indicoside A and B, manghopanal, mangoleanone, friedelin, cycloartan-3β-30-diol and
derivatives, mangsterol, manglupenone, mangocoumarin, n-tetacosane, n-heneicosane, n-
triacontane and mangiferolic acid methyl ester and others isolated from stem bark of MI (Khan
et al., 2013). Mangostin, 29-hydroxy mangiferonic acid and mangiferin have been isolated from
the stem bark together with common flavonoids (Shankarnarayanan et al., 2019). The flower
yielded alkyl gallates such as gallic acid, ethyl gallate, methyl gallate, n-propyl gallate, n-pentyl
gallate, n-octyl gallate, 4-phenyl gallate, 6-phenyl-n-hexyl gallate and dihydrogallic acid (Khan
and Khan, 2018). Root of mango contains the chromones, 3-hydroxy-2-(4’-methylbenzoyl)-
chromone and 3-methoxy-2(4’-methyl benzoyl)-chromone. The leaf and fl ower yield an
essential oil containing humulene, elemene, ocimene, linalool, nerol and many others. The fruit
pulp contains vitamins A and C, β-carotene and xanthophylls. An unusual fatty acid, cis-9, cis-
15-octadecadienoic acid was isolated from the pulp lipids of mango. Phenolic Antioxidants, Free
Sugars and Polyols isolated and analyzed from Mango (MI) Stem Bark. All structures were
elucidated by ES-MS and NMR spectroscopic methods. Quantitative analysis of the compounds
has been performed by HPLC, and mangiferin was found to be the predominant component
(Nunez et al., 2012).
Polyphenols have been characterized in mango puree concentrate by HPLC with diode array and
mass spectrometric detection (Andreas et al., 2010). A rapid method was developed for
quantitative determination of beta-carotene, including cis-isomers, in dried mango (Pott et al.,
2013). HPLC method was developed to determine carotenoids in Taiwanese mango (Chen et al.,
2014). 5-Alkyl- and 5-alkenylresorcinols, as well as their hydroxylated derivatives, extracted
from mango (MI) peels, purified on polyamide and characterized by highperformance liquid
chromatography/atmospheric pressure chemical ionization mass spectrometry (HPLC/APcI-MS)

8
for the first time (Knödler et al., 2017). Xanthophyll esters, carotenes, and tocopherols has been
identified and quantified in the fruit of seven mexican mango cultivars by liquid
chromatography-atmospheric pressure chemical ionization-time-of-flight mass spectrometry
(LC-(APcI (+))-MS) (Ornelas-Paz Jde et al., 2017). A simple, precise, and rapid HPTLC method
was established for quantitative determination of the bioactive marker compound mangiferin in
the stem bark and leaves of MI. The method was validated for selectivity, linearity, precision,
accuracy, and robustness (Subha et al., 2017). The natural C-glucoside xanthone mangiferin (2-
C-β-Dgluco-pyranosyl-1, 3, 6, 7-tetrahydroxyxanthone; C19H18O11; Mw, 422.35; melting point,
anhydrous 271°C) has been reported in various parts of MI leaves, fruits, stem bark, heartwood
and roots. The presence of a phenolic compound from leaves of MI which was named as
homomangifirin (Subbarayan et al., 2016).
2.3 Pharmacology
Although a lot of pharmacological investigations have been carried out based on the ingredients
present but a lot more can still be explored, exploited and utilized. A summary of the fi ndings of
these studies is presented below.
2.4 Anti-oxidant
Reactive oxygen species (ROS) possess a strong oxidizing effect and induce damage to
biological molecules, including proteins, lipids and DNA, with concomitant changes in their
structure and function (Seifried et al., 2017).(17) The major nutritional antioxidants, vitamin E,
vitamin C and β-carotene, may be benefi cial to prevent several chronic disorders (Diplock et al.,
2018) considerable interest has arisen in the possible reinforcement of antioxidant defenses, both
for chemoprevention and treatment purposes. The extract showed a powerful scavenging activity
of hydroxy radicals and acted as a chelator of iron. It also showed a signifi cant inhibitory effect
on the peroxidation of rat brain phospholipid and prevented DNA damage caused by bleomycin
or copper-phenenthroline systems (Martinez et al., 2010)) The interaction of Vimang (MI
extract) with Fe (III) was studied and the results justify the high effi ciency of Vimang as an
agent protecting from iron-induced oxidative damage (Pardo-Andreu et al., 2016). The work has
been carried out to investigate the pulp composition of four mango cultivars (Haden, Tommy
Atkins and Ubá) at the ripening stage in relation to three components with antioxidant potential
(total phenolics, carotenoids and ascorbic acid). These results corroborated previous information
that mangoes are a good source of antioxidants in human diet (Rocha Ribeiro et al., 2009). In

9
vitro antioxidant and free radical scavenging properties of a stem bark aqueous extract of mango
tree (MI), whose formulations are used in Cuba as food supplements under the brand name of
Vimang, Luminolenhanced chemiluminescence was used to elucidate the effect of this extract on
the generation of reactive oxygen species in PMA- or zymosan-stimulated human
polymorphonuclear leukocytes and on superoxide radicals generated in the hypoxanthine–
xanthine oxidase reaction. Part of this MI extract antioxidant activity could be ascribed to the
presence of mangiferin as its main component. The iron-complexing ability of Vimang as a
primary mechanism for protection of rat liver mitochondria against Fe 2+ -citrate-induced
lipoperoxidation was reported. The results are of pharmacological relevance since Vimang could
be a potential candidate for antioxidant therapy in diseases related to abnormal intracellular iron
distribution or iron overload (Pardo Andreu et al., 2015). The protective abilities of MI stem
bark extract (Vimang) 50250 mgkg(-1), mangiferin 50 mgkg(-1) and selected antioxidants
(vitamin C 100 mgkg(-1), vitamin E 100 mgkg(-1)and beta-carotene 50 mgkg(-1)) against the
12-O-tetradecanoylphorbol13-acetate (TPA)-induced oxidative damage in serum, liver, brain as
well as in the hyper-production of reactive oxygen species (ROS) by peritoneal macrophages
was compared (Sanchez et al., 2015).
2.4.1 Anti-diabetic
A 50% ethanolic extract of the leaves of MI produced a signifi cant hypoglycemic effect at a
dose of 250 mg/kg, both in normal and streptozotocin-induced diabetic animals. The stimulation
of β-cells to release insulin was thought to be part of the mechanism of action (Sharma et al.,
2017).) The effect of the aqueous extract of the leaves of MI on blood glucose level in
normoglycaemic, glucose - induced hyperglycaemic and streptozotocin (STZ)induced diabetic
rats has been assessed. The results indicate that the aqueous extract of the leaves of MI possess
hypoglycaemic activity. This action may be due to an intestinal reduction of the absorption of
glucose (Aderibigbe et al., 2019). The leaves of MI used for antidiabetic properties using
normoglycaemic, glucose-induced hyperglycaemia and streptozotocin (STZ) induced diabetic
mice. The aqueous extract of the leaves of MI possess hypoglycaemic activity (Aderibigbe et al.,
2011). The effect of mango (MI) ingestion on blood glucose levels of normal and diabetic rats
has been studied. The results from this research suggest that mango fl our can possibly help in
the treatment of diabetes. The stem-bark of aqueous extract of MI was used to examine the
antiinfl ammatory, analgesic and antidiabetic properties. The different chemical constituents of

10
the plant, especially the polyphenolics, flavonoids, triterpenoids, mangiferin, and other chemical
compounds present in the plant may be involved in the observed antiinfl ammatory, analgesic,
and hypoglycemic effects of the plant's extract. The results of this experimental animal study
lend pharmacological credence to the suggested folkloric uses of the plant in the management
and control of painful, arthritic and other infl ammatory conditions, as well as in the
management of adult-onset type 2 diabetes mellitus in some rural African communities (Ojewole
et al., 2015). Investigations were carried out to evaluate the effect of MI on glucose absorption
using a rat intestinal preparation in situ. The ethanol extracts of stem-barks reduced glucose
absorption gradually during the whole perfusion period in type 2 rats (Amrita et al., 2009). In
glucose-loaded normal rats, mangiferin induces a significant improvement in oral glucose
tolerance but without alteration of basal plasma glucose levels (Muruganandan et al., 2015)
these studies show that mangiferin (10 and 20 mg/kg, i.p.) exhibits potent antidiabetic,
antihyperlipidemic, antiatherogenic and antioxidant properties without causing hypoglycaemia;
mangiferin would then offer a greater therapeutic benefit for the management of diabetes
mellitus and diabetic complications associated with abnormalities in lipid profiles. It has been
reported that long standing hyperglycaemia with diabetes mellitus leads to the formation of
advanced glycosylated end-products which are involved in the generation of ROS, leading to
oxidative damage, particularly to heart and kidney (Rolo et al., 2016).
2.4.2 Antiviral activity
In vitro the effect of mangiferin was studied against Herpes simplex virus type 2; mangiferin
does not directly inactivate HSV-2 but inhibits the late event in HSV-2 replication (Zhu et al.,
2013). In vitro mangiferin was also able to inhibit HSV-1 virus replication within cells (Zheng
et al., 2010) and to antagonize the cytopathic effects of HIV (Guha et al., 2016).
2.4.3 Anthelmintic and Anti-Allergenic Activity
Anthelminthic and antiallergic activities of MI stem bark components Vimang and mangiferin
was investigated in mice experimentally infected with nematodes, Trichinella spiralis. The study
was carried out to fi nd out anti-allergic properties of vimang and mangiferin, a C-
glucosylxanthone isolated from extract of MI. The results constitute the anti-allergic properties
of Vimang on allergic models, as well as suggesting that this natural extract could be
successfully used in the treatment of allergic disorders. Mangiferin, the major compound of
Vimang, contributes to the anti-allergic effects of the extract (Rivera et al., 2016).

11
2.4.4 Antiparasitic Activity
In a neonatal mouse model, mangiferin at 100 mg/kg has a similar inhibitory activity on
Cryptosporidium parvum than the same dose (100 mg/kg) of an active drug, paromomycin
(Perrucci et al., 2016).
2.4.5 Antibone Resorption
Four water extracts of Kampo formulae were screened for their inhibitory effect on bone
resorption induced by parathyroid hormone in organ culture of neonatal mouse parietal bones.
Mangiferin isolated and tested in vitro showed a signifi cant inhibitory effect on this model (Li
et al., 2018).
2.4.6 Anti-Tumor-Anti-HIV
The signifi cant cytotoxic activities has been demonstrated by the stem bark extract of mango
against the breast cancer cell lines MCF 7, MDA-MB-435 and MDA-N, as well as against a
colon cancer cell line (SW-620) and a renal cancer cell line (786-0) (Muanza et al., 2015). The
ethanol/water (1:1) extract of dried aerial parts of mango administered intraperitoneally to mice
at a dose of 250.0 mg/ kg was inactive on Leuk-P388 (Aswal et al., 2014). In vitro, mangiferin
dose- and time-dependently inhibited the proliferation of K562 leukemia cells and induced
apoptosis in K563 cells line, probably through down-regulation of bcr/abl gene expression.
These results suggest that mangiferin has a potential as a naturally-occurring chemopreventive
agent (Yoshimi et al., 2011).
2.4.7 Antispasmodic and Antipyretic Activity
The stem bark extract of MI was evaluated for antiplasmodial activity against Plasmodium yoelii
nigeriensis. The extract was also screened for antipyretic activity in mice. The extract exhibited a
schizontocidal effect during early infection, and also demonstrated repository activity. A
reduction in yeast-induced hyperpyrexia was also produced by the extract (Awe et al., 2018).
The in vitro antimalarial activity of chloroform: methanol (1:1) extract of MI was evaluated. The
extract showed a good activity on P. falciparum in vitro with a growth inhibition of 50.4% at 20
μg/mL (Bidla et al., 2014).
2.4.8 Immunomodulatory
Immunomodulatory activity of alcoholic extract of stem bark of MI was investigated in mice. It
is concluded that test extract is a promising drug with immunostimulant properties. Mangiferin
mediates the down-regulation of NF-κB, suppresses NF-κB activation induced by inflammatory

12
agents, including tumor nuclear factor (TNF), increases the intracellular glutathione (GSH)
levels and potentiates chemotherapeutic agent-mediated cell death; this suggests a possible role
in combination therapy for cancer. It is likely that these effects are mediated through mangiferin
ROS quenching and GSH rising; increased intracellular (GSH) levels are indeed known to
inhibit the TNFinduced activation of NF-κB (Manna et al., 2019).
2.4.9 Anti-Diarrhoeal
The potential anti-diarrhoeal activity of methanolic (MMI) and aqueous (AMI) extracts of seeds
of MI has been evaluated in experimental diarrhoea, induced by castor oil and magnesium
sulphate in mice. The results illustrate that the extracts of MI have significant anti-diarrhoeal
activity and part of the activity of MMI may be attributed to its effect on intestinal transit
(Sairam et al., 2013).
2.4.10 Anti-Inflammatory
An ethanolic (95%) extract of the seed kernel of MI exhibited signifi cant anti-inflammatory
activity in acute, subacute and chronic cases of inflammation. The MI leaf extract exhibited
antibacterial activity against Bacillus subtilis, staphylococcus albus and Vibrio cholera (Das et
al., 2019). Analgesic and anti-inflammatory effects of MI extract (Vimang) has studied. The
polyphenols found in the extract were found to account for the activity reported (Garrido et al.,
2011) In vivo and in vitro anti-infl ammatory activity of MI extracts (VIMANG) was
investigated. MI extract, administered topically (0.5-2 mg per ear), reduced ear edema induced
by arachidonic acid (AA) and phorbol myristate acetate (PMA, ED50 = 1.1 mg per ear) in mice.
The results represent an important contribution to the elucidation of the mechanism involved in
the anti-inflammatory and anti-nociceptive effects reported by the standard MI extract VIMANG
(Garrido et al., 2014).)
2.4.11 Anti-Bacterial and Antifungal Activity
In an in vitro agar diffusion technique, mangiferin showed activity against 7 bacterial species,
Bacillus pumilus, B. cereus, Staphylococcus aureus, S. citreus, Escherichia coli, Salmonella
agona, Klebsiella pneumoniae, 1 yeast (Saccharomyces cerevisiae) and 4 fungi (Thermoascus
aurantiacus, Trichoderma reesei, Aspergillus fl avus and A. fumigatus) (Stoilova et al., 2014).)
2.4.12 Anti-Microbial
The antimicrobial activities of methanolic extracts of P. guajava and MI have been investigated.
The results show that P. guajava and MI extracts exhibited antimicrobial activities at a

13
concentration of 20 mg/ml. Overall, P. guajava extract show more antimicrobial activity than
MI extract against tested organisms (Akinpelu and Onakoya, 2016).
2.4.13 Hepatoprotective
Chemopreventive properties of lupeol and mango pulp extract (MPE) was evaluated against 7,
12-dimethylbenz (a) anthracene (DMBA) induced alteration in liver of Swiss albino mice.
Lupeol/ MPE was found to be effective in combating oxidative stress induced cellular injury of
mouse liver by modulating cell-growth regulators (Prasad et al., 2017).
2.5 Application of enzyme in fruit juice clarification
Pectinolytic enzymes are one of the upcoming enzymes of fruit industries. Fruit juices are
naturally cloudy mainly due to the presence of pectin polysaccharides (Sharma et al. 2017). The
high concentration of pectin leads to colloid formation in the juice, which leads to create problem
in the processing of clear fruit juices. The role of acidic pectinases in bringing down the
cloudiness and bitterness of fruit juices is well established (Kashyap et al. 2001; Jayani et
al. 2005). In an unripe fruit, pectin is bound to cellulose micro fibrils in the cell wall. Such pectin
is insoluble and hence confers rigidity to cell walls. However, during ripening the structure of
pectin is altered by naturally occurring enzymes in the fruits. As a result of this, the pectin
becomes more soluble and softens the plant tissues (Caffall and Mohnen 2009). Therefore, pectin
degrading enzymes have been used to clarify following important fruit juices:
Apple juice Pectinolytic enzymes produced by Aspergillus sp. have been widely used for
purification of apple juice. Amin et al. (2016) reported 73.78% reduction in turbidity of apple
juice by exo-polygalacturonase produced by P. notatum. Yuan et al. (2011) also used
polygalacturonase of Penicillium sp. and reported 4.5% reduction in viscosity and 71.8%
increased the light transmission. Apple juice clarification by polygalacturonase produced
by Aspergillus sp. has been reported in recent literature (Mahmoodi et al. 2017; Dey et al. 2014;
Kant et al. 2013; Sandri et al. 2015). Mahmoodi et al. (2017) reported 7.2% reduction in apple
juice viscosity by A. niger pectinase. Dey et al. (2014) treated apple juice with polygalacturonase
produced by A. awamori Nakazawa and reported 38% reduction in viscosity and 93% increase in
transmission at 660 nm when it was incubated at 50 °C for 2 h. Apple juice treated with
polygalacturonase produced by A. niger showed the maximum clarification by increase in %
transmission from 2.5 to 20.4 upon overnight incubation (Kant et al. 2013). A 90% decrease in
apple juice turbidity has been reported by Sandri et al. (2015) using A. niger pectinase enzyme

14
extract. Yang et al. (2011) also treated apple juice with Bispora sp. pectinase and reported 84%
increase in transmittance and reduction in viscosity by 7.7%. Baron et al. (2006) studied effect of
high pressure on apple juice clarification by pectin methylesterase. Rajdeo et al. (2016) reported
apple juice clarification with immobilized pectinase. The properties of apple juice treated with
immobilized enzyme were similar to those of that treated with free pectinase.
Orange and mosambi juice Citrus fruits viz. oranges, lemon, and grapefruit contain the highest
reported concentrations of pectin in their tissues (Galant et al. 2014). In commercially prepared
citrus juices, pectin accounts for approximately one-third of the insoluble material that is found
within the juice cloud (Baker and Bruemmer 1969). Pectin from orange is only partially
methylated because of the large amounts of pectin methylesterase which remove methoxyl group
from pectin (Kashyap et al. 2001; Maran et al. 2013). In the presence of calcium ions, insoluble
calcium pectate is formed in orange juice leading to the undesirable precipitation of haze
particles. Tounsi et al. (2016) reported citrus fruit juice clarification using polygalacturonase
of Penicillium occitanis. Anand et al. (2017) reported orange juice clarification using exo-
polygalacturonase produced by A. niger. Diaz et al. (2013) reported 95% clarification in orange
juice on addition of mixture of pectinase, xylanase and CMCase produced in tray reactor by A.
awamori. Rai et al. (2004) have obtained 89% clarified mosambi juice by a pectinase
of Aspergillus niger using enzyme protein concentration 0.004 g/l for 99 min, at temperature of
42 °C.
Passion Fruit Juice This juice has commercial importance due to its pleasant unique aroma and
flavor and high nutritional values (Cheok et al. 2016). Brazil is the largest producer of passion
fruit in the world and about 50% of the production is used in the juice processing industry
(Canteri et al. 2012). Jiraratananon and Chanachai (1996) observed a viscosity reduction of 18%
with enzymatic treatment of passion fruit juice with pectinase. Domingues et al. (2012) reported
passion fruit juice clarification using chitosan treatment. The juice is centrifuged at 4000 rpm
followed by coagulation/flocculation process with chitosan at 300 ppm and at pH 6.
Banana Juice The turbidity of banana juice is caused mainly by the polysaccharides in the juice
such as pectin and starch. It is a major challenge to process banana after its harvesting
(Mohapatra et al. (2011). Pectin makes the clarification process difficult because of its fibre such
as molecular structure. Cheng et al. (2017) used acidic endo-polygalacturonase produced
by Penicillium oxalicum CZ1028 for banana juice clarification at 45 °C for 1 h. Cheng et al.

15
(2017) reported 31.76% reduction in viscosity and 6% increase in transmission at 660 nm.
Barman et al. (2014) reported banana juice clarification by pectinase produced by A. niger. The
optimum results (0.10 OD at 660 nm) were obtained when raw banana juice was incubated for
60 min with 2% concentration of partially purified pectinase. Sagu et al. (2013) optimized
conditions for banana juice clarification using commercial pectinase and reported 33 °C and
108 min for best results. Lee et al. (2006) employed response surface methodology to clarify
banana juice in which optimum conditions for clarification are found to be 0.084% enzyme
concentration, incubation temperature of 43.2 °C and incubation time of 80 min.
Lemon juice Maktouf et al. (2014) have reported lemon juice clarification using Penicillium
occitanis pectinase. The optimum treatment conditions reported were 600 U/l enzyme
concentration, 45 min and 30 °C. Under optimized conditions, 77% reduction of viscosity and
47% reduction in turbidity is reported.
Mango juice Pectinase from A. foetidus has been reported for mango juice clarification (Kumar
et al. 2012). Mango juice was treated with 20 ml of crude enzyme preparation (specific activity
228 IU/ml). The maximum mango juice clarification (92.5%) was obtained at temperature of
40 °C and 150 min incubation time. Cheng et al. (2017) used acidic endo-polygalacturonase
produced by Penicillium oxalicum CZ1028 for mango juice clarification at 45 °C for 1 h and
reported 5% increase in transmission at 660 nm.
Pineapple juice Pectin methylesterase produced by A. tubingensis in SSF has been reported for
pineapple juice clarification (Patidar et al. 2016). It was found that increase in amount of enzyme
from 10 to 100 U increased the juice clarification from 3.1 to 19.5% and decreased the pH from
4.3 to 3.0 at 30 °C. Tochi et al. (2009) reported pineapple juice clarification by commercially
available A. niger pectinase (Sigma- Aldrich) which showed reduction in turbidity from 1.5 to
0.8 at 35 °C. de Carvalho et al. (2008) reported 5% change in pineapple juice sugar content due
to the addition of pectinase and cellulase followed by cross flow micro- and ultra-filtration to
maintain the nutritional quality of pineapple juice.
Blue berry juice Taking into consideration the high nutritional potential, blueberry juice has been
clarified by Sandri et al. (2015). Pectinase of A. niger produced in SSF has been used for the
clarification of blueberry juices.
Guava juice South Africa, India and Hawaii are major producers of Guava (Kashyap et al. 2001).
Kant et al. (2013) reported guava juice clarification using A. niger polygalacturonase. In this

16
study, addition of enzyme increased the sugar content and % transmission at 650 nm, whereas
the pH of the juice decreased. The % transmission at 650 nm and mg % sugar content increased
from 1.7 to 20.4 and 1.9–4.8, respectively.
Date syrup Dates are important products in the hot desert regions of the world and are marketed
globally as a high-value fruit. It always plays an important part in the economic and social lives
of the people of these regions (Abbès et al. 2011). The commercial quality of date syrup
increases on addition of pectinase. The use of pectinase and cellulase enzyme (50U/5U) gave the
highest recovery of total soluble solids and the lowest turbidity compared with control sample.
Papaya juice Cheng et al. (2017) used acidic endo-polygalacturonase produced by Penicillium
oxalicum CZ1028 for banana juice clarification at 45 °C for 1 h. Cheng et al. (2017) reported
78.36% reduction in viscosity and 43.3% increase in transmission at 660 nm. Tu et al. (2013)
reported reduction in viscosity of papaya juice by 17.6%, and increased its transmittance by
59.1% when papaya juice was treated with Achaetomium sp. polygalacturonase. Tu et al. (2013)
also reported higher efficiency with a synergy degree of more than 1.25, when juice was treated
with commercial pectin methylesterase and polygalacturonase produced by Achaetomium sp.
Grape juice Li et al. (2017) used combination of two polygalacturonase (exo- and endo-)
produced by Talaromyces leycettanus in ratio of 1:4 for grape juice clarification and reported
140% pectin-degrading efficiency with light transmittance improvement from 14 to 82%.
Sreenath and Santhanam (1992) reported 25% reduction in viscosity when white grape juice was
treated with commercial A. niger pectinase.
Bitter gourd juice (Karela Juice) Bitter gourd exhibits significant anti-diabetic activity. Bitter
gourd is tropical medicinal plant which belongs to the Cucurbitaceae family. Its fruit bitter
melon or karela has a distinct bitter taste (Deshaware et al. 2017). The optimum conditions, i.e.,
pectinase concentration (10.2 ml/kg), incubation time (140 min) and temperature (48.8 °C)
resulted in juice yield of 82%, α–amylase and α-glucosidase inhibition activity of 23 and 59%,
respectively.

17
 CHAPTER THREE
MATERIALS AND METHODS
3.1 Reagents
This research used modified pectic agar medium to isolate and screen a pectinolytic fungi (Jayani
et al., 2013). The composition of modified pectin agar medium (g/l, wt/vol.) will be: Pectin 5.00,
K2HPO4 0.50, MgSO4.7H2O 0.10, NaCl 0.20, CaCl2.2H2O 0.20, FeCl3.6H2O 0.01, Yeast extract
1.00, Agar 20.00.

3.3 Collection of Samples

The mango peels will be collected from sold mangos sold in Sangere Futy Girei Local
Government Area. Samples will be collected in a sterile clean container when freshly prepared
and taken immediately to the SLT lab under aseptic condition. Each sample collected will be
properly labeled and transported to the Microbiology Laboratory in aseptic condition, where they
will be processed for Pectinolytic Fungi examination.

3.4 Isolation and Screening of Pectinolytic Fungi

The fungi will be isolated from mango peels collected in Sangere-Futy Girei Local Government
Area. A slice of the peels will be put on pectic agar medium and had been incubated for 2-3
days. Whereas, the different fungal colonies will be inoculated on potato dextrose agar (PDA) for
purification. The spores of pure culture will be maintained in PDA medium in test tubes sealed
with parafilm and stored at 4 °C for further use. The screening of the pectinolytic activity will be
conducted based on a clear zone formation on pectic agar medium after it will be stained by
Cetyl trimethyl ammonium bromide. Fungal isolates will be inoculated on pectic agar medium
and had been incubated at a room temperature for 48 hours. .

3.5 Identification of Pectinolytic Fungi

Pectinolytic fungi will be identified based on macroscopic and microscopic morphological
characters. These characters will be compared to Pictorial Atlas of Soil and Seed Fungi, Second
Edition (Watanabe , 2002) until genus level.
3.6 Data Analysis

18
The data were entered into the computer and statistical analysis was done using ANOVA
provided by the SAS version 9.1 and significant difference examined for different parameters
and between pectinolytic fungal isolates at 95% confidence level of significance and at p <0.05.

19
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