LWT - Food Science and Technology 130 (2020) 109573

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Antioxidant and antibacterial activities of cassava starch and whey protein T

blend films containing rambutan peel extract and cinnamon oil for active
packaging
Rungsima Chollakupb, Siraprapa Pongburoosa, Watthana Boonsonga, Nattaporn Khanoonkonb,
Kunat Kongsinb,c, Rungsinee Sothornvitd, Prakit Sukyaic, Udomlak Sukattab,
Nathdanai Harnkarnsujarita,∗
a
Department of Packaging and Materials Technology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand
b
Kasetsart Agricultural and Agro-Industrial Product Improvement Institute, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand
c
Biotechnology of Biopolymers and Bioactive Compounds Special Research Unit, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak,
Bangkok, 10900, Thailand
d
Department of Food Engineering, Faculty of Engineering at Kamphaengsaen, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom, 73140, Thailand

ARTICLE INFO ABSTRACT

Keywords: Blending of cassava starch and whey protein isolate (WPI) films containing plant extracts showed modified
Food packaging properties and release behaviors. Effects of native starch (NS) and acetylated starch (AS) on film properties,
Edible film antioxidant and antibacterial activities were investigated. WPI and WPI-starch films contained ethanolic frac-
Antioxidant tions of rambutan peel extract (RPE) and cinnamon oil (CO). Mechanical, physical and water barrier properties
Antimicrobial
of blend films depended on starch type and contents. Corilagin and (E)-cinnamaldehyde were the highest active
Starch
Whey protein
components in RPE and CO, respectively. FTIR revealed hydrophobic interaction and hydrogen bonding between
starch-protein-polyphenols. Protein conformation was altered giving modified amide I and amide II bands.
Active compounds reduced water vapor permeability depending on starch types that governed CO dispersion.
Starch, particularly AS, enhanced release of polyphenols and DPPH· scavenging activity in water and 50%
ethanol. Antibacterial activity of the blend films differed between in vitro (disc diffusion method) and real food
(salami) and was governed by the phenolic release and food components, respectively. WPI films showed the
lowest in vitro antibacterial activity but the highest efficacy to delay microbial growth in salami. Release me-
chanism controlled the antioxidant capacity of active protein-starch films, whereas antibacterial capacity in
foods strongly depended on food matrix components.

1. Introduction animal feed. Rambutan plants are a rich source of polyphenolic com-
pounds that have health benefits and prevent chronic disease due to
Whey protein and starch are potent biomaterials to produce bio- their antioxidant power (Perera, Appleton, Ying, Elendran, &
degradable and edible food packaging. The positive environmental Palanisamy, 2012; Thitilertdecha, Teerawutgulrag, Kilburn, &
impacts of edible food packaging drive investigations of alternative Rakariyatham, 2010). Essential oils are natural compounds used to
environmentally friendly bio-based materials. Moreover, development produce active packaging that exhibit antimicrobial activity against a
of active bioplastic and edible packaging effectively extends shelf-life as variety of microorganisms including gram-positive and gram-negative
an efficient solution to reduce food waste. Antioxidant and anti- bacteria, yeast and molds (Balaguer, Lopez-Carballo, Catala, Gavara, &
microbial functions are major developments for controlled-release ap- Hernandez-Munoz, 2013; Sanla-Ead, Jangchud, Chonhenchob &
plications in active bioplastic packaging (Jariyasakoolroj, Leelaphiwat, Suppakul, 2012). Natural plant extracts are rich sources of active
& Harnkarnsujarit, 2018). compounds and exhibit antioxidant and antimicrobial activities.
Rambutan (Nephelium lappaceum) peels generated as waste from the Starch is abundant at low cost and many physical and chemical
canned fruit industry are fractionated, purified and used as low-cost modifications are available. However, high hydrophilicity of hydroxyl

∗
Corresponding author.
E-mail address: [email protected] (N. Harnkarnsujarit).

https://doi.org/10.1016/j.lwt.2020.109573
Received 6 February 2020; Received in revised form 6 May 2020; Accepted 8 May 2020
Available online 05 June 2020
0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
R. Chollakup, et al. LWT - Food Science and Technology 130 (2020) 109573

Fig. 1. Chromatogram of (A) rambutan peel extract (RPE) and (B) cinnamon oil determined by HPLC and GC-MS, respectively.

groups in the starch matrix leads to poor water adsorption and in- Nakhon Pathom, Thailand) were washed, dried and ground into powder
stability (Mali, Sakanaka, Yamashita, & Grossmann, 2005; Rhim, before Soxhlet extraction using ethanol for 1 h, as described by
Gennadios, Weller, Cezeirat, & Hanna, 1998). Mixing starch with pro- Nanthakumar, Udhayasankar, Ashadevi, Arumugasamy, and Shalimol
tein containing several hydrophobic side chains can improve stability. (2014). The extracted solvents were filtered using Whatman no.1 filter
Previous research formulated and characterized the properties of var- paper and dried using a vacuum rotary evaporator (Buchi, Switzerland).
ious proteins including casein, gelatin, albumin, soy and whey in Rambutan peel extract (RPE) was obtained from ethanol crude extract
combination with starch films (Galus, Mathieu, Lenart, & Debeaufort, and determined for phenolic compounds using high-performance liquid
2012; Rhim et al., 1998; (Jagannath et al., 2003); Sun, Sun, & Xiong, chromatography (HPLC, Shimadzu, Japan) as described by
2013). However, scant investigations have considered the effects of Thitilertdech and Rakariyatham (2011) (Supplementary data).
protein-starch blends on antioxidant and antimicrobial activities of
active films. Cinnamon is a potent essential oil and produces active 2.2. Cinnamon oil extraction and analysis
edible packaging when incorporated in whey protein concentrate
(Bahram et al., 2014), hagfish skin (Kim, Beak, & Song, 2018), chitosan- Cinnamon (Cinnamomum zeylanicum) bark (Nguan-Sun, Bangkok)
carboxymethyl cellulose (Noshirvani et al., 2017) and cassava starch was ground to a powder and extracted for cinnamon oil (CO) using
(Souza, Goto, Mainardi, Coelho, & Tadini, 2013). However, no in- hydrodistillation. The cinnamon bark powder was boiled with distilled
vestigations have previously been conducted on cinnamon oil in- water in a flask and the system was connected with a condenser and
corporation in protein-starch blends or the use of rambutan peel extract cooler. The CO was derived after distillation at 100 °C for 6 h. Water
in active packaging. was removed from the oil mixture by addition of anhydrous sodium
Here, the effects of native and acetylated cassava starch and blend sulfate for 20 min and compositions of the obtained CO were de-
ratios on the antioxidant and antibacterial activities of starch and whey termined using a gas chromatography-mass spectrometer (GC-MS;
protein blend films were investigated. Moreover, properties of films Shimadzu QP 5050 A). One microliter of CO was injected into a DB-5
containing rambutan peel extract and cinnamon essential oil as active column (internal diameter 0.25 μm and length 6 m) using a split in-
compounds were compared to those films without active compounds jection mode. Temperature was held at 60 °C for 3 min followed by a
(non-active). Physical, mechanical and water barrier properties of the stepwise increase to 150 °C at a rate of 4 °C∙min−1. Helium gas was used
films were also determined for application as active food packaging of as the carrier with a flow rate of 1.2 ml min−1. The spectrum was re-
salami as a representative of high protein and lipid food. Findings corded and compared with the standard spectrum of Wiley 7, NIST 12
supported the controlled-release applications of active edible films from and NIST 62 library in the mass range of 40–400.
starch and protein-based materials.
2.3. Film preparation
2. Materials and methods
Edible films were prepared from whey protein isolate (WPI)
2.1. Rambutan peel extraction and analysis (Provon® 292: Glanbia Nutritionals Inc., USA), native cassava starch
(NS, Tong Chan Registered Ordinary Partnership, Thailand) and
Rambutan peels (cultivar Rongrean, from Malee Sampran Co., Ltd., acetylated cassava starch (AS, DS of 0.01–0.03, Siam Modified Starch

2
R. Chollakup, et al. LWT - Food Science and Technology 130 (2020) 109573

2.4. Mechanical properties

Tensile properties of the films were determined according to ASTM

D882 using an Instron Universal Testing Machine (Shimadzu AGS5kN,
Japan). The films were cut into 1.5 × 10 cm strips and placed between
grips at a distance of 8 cm. Mechanical properties of the five different
samples were determined with a load cell of 50 kg⋅m⋅s−2 at a rate of
50 mm min−1.

2.5. Barrier properties

2.5.1. Contact angle

Water contact angle was measured (Data Physics Instruments
OCA20, Germany). Films (1 cm × 10 cm) were placed on the stage and
contacted with 0.5 mm3 of distilled water using a microsyringe. The
images were immediately captured and determined for contact angle
from 10 replicates.

2.5.2. Water vapor permeability (WVP)

Water vapor transmission rate (WVTR) was determined for 5 re-
plicates by the cup method (Huntrakul & Harnkarnsujarit, 2020) fol-
lowing ASTM E96 (supplementary data).

2.6. FTIR

Infrared spectra of films were collected using attenuated total re-

flectance Fourier transform infrared (ATR-FTIR) spectroscopy (Tensor
27 FTIR, Bruker Corporation, Germany) as described by Huntrakul and
Harnkarnsujarit (2020) (supplementary data).

2.7. Release of phenolic and DPPH radical scavenging activity

Films were determined for release of phenolic compounds and an-

tioxidant capacity at different times. Triplicate films (2 cm × 2 cm)
were immersed in 30 cm3 of distilled water and 0.5 g g−1 ethanol at
room temperature. Water and ethanol (0.1 cm3) were collected at dif-
ferent times and determined for total phenolic contents and DPPH ra-
dical scavenging activity using a spectrophotometer as described by
Panrong, Karbowiak, and Harnkarnsujarit (2019) (Supplementary
data).

2.8. Antibacterial capacity

Fig. 2. Mechanical properties as (A) tensile strength, (B) elongation at break,
(C) water contact angle, and (D) water vapor permeability (WVP) of WPI, WPI- Antibacterial properties of Bacillus cereus, Staphylococcus aureus and
NS and WPI-AS at different ratios between WPI:starch namely 5:0 ( WPI5), 4:1 Escherichia coli were determined using the disc diffusion method. The
( WPI4-NS1 and WPI4-AS1) and 3:2 ( WPI3-NS2 and WPI3:AS2) in non- microorganisms were transferred to sterile peptone solution at different
active and active (containing RPE and CO mixture at a ratio of 1:1) films. dilutions of 105, 106 and 107 cfu cm−3 and nutrient broth before
Different letters indicate significant difference among non-active or active films. swabbing on nutrient agar. Films (6 mm diameter) were placed on
nutrient agar and incubated at 37 °C for 24 h. Inhibition zones were
Co., Ltd., Thailand) blends. The suspensions contained 0.05 g g−1 of determined in triplicate using a micrometer.
different WPI: starch ratios namely 5:0 (WPI5), 4:1 (WPI4-NS1 and
WPI4-AS1) and 3:2 (WPI3-NS2 and WPI3-AS2). Glycerol (0.5 g g so- 2.9. Active packaging of food products
lids−1) was added as a plasticizer. The ‘active’ films were prepared by
adding 0.04 g g−1 of mixtures of RPE and CO at a ratio of 1:1. This ratio Slices of salami (~40 mm diameter and 3 mm thickness) were
was selected based on trial experiments indicating synergistic anti- placed between two film layers and determined for appearance and
microbial inactivation. RPE (0.02 g g−1) was added to the mixtures total viable count (TVC) during storage for 10 d at room temperature.
before adjusting the pH to 7 with 2 m−3⋅mol sodium hydroxide Salami (25 g) was filled in stomacher bags containing peptone solution
(NaOH). The mixtures were heated to 90 °C for 30 min in a water bath (0.1 g cm−3, 225 cm3) and subjected to a stomacher for 1 min. The
while stirring. The CO was added to solutions and further stirred for mixtures were diluted with peptone and poured onto Petrifilm™
15 min at 1500 rpm. The solutions were poured onto Teflon covered Aerobic Count Plates (3 M™, USA). The Petrifilms were incubated at
plates (12 cm × 12 cm) and dried in a hot air oven at 50 °C for 13 h. 37 °C for 48 h. Duplicate samples were counted for viable colonies on
The films were peeled and stored in a desiccator containing potassium day 5 and day 10 of storage.
carbonate (K2CO3) corresponding to 50%RH. Films without RPE and Color (L*, a* and b*) of triplicate salami samples was determined
CO mixtures were also prepared following the same procedure. using a colorimeter (MiniScan XE, Hunter Associates Laboratory Inc.,
USA) during storage for 10 d under standard illuminant D65 and 10°
observer. Images were taken in a box equipped with two LED light

3
R. Chollakup, et al. LWT - Food Science and Technology 130 (2020) 109573

Fig. 3. FTIR spectra of WPI, WPI-NS and WPI-AS at different ratios between WPI:starch namely 5:0 (WPI5), 4:1 (WPI4-NS1 and WPI4-AS1) and 3:2 (WPI3-NS2 and
WPI3:AS2) in non-active and active (containing RPE and CO mixture at a ratio of 1:1) films.

bulbs to control light exposure. 3. Results and discussion

3.1. RPE and CO compositions

2.10. Statistical analysis
Rambutan peel contained high amounts of phenolic compound
A completely randomized experimental design was used and a one- mixtures that contributed to antioxidant and antimicrobial activities.
way analysis of variance (ANOVA) was performed using SPSS version HPLC analysis indicated major phenolic components in ethanol ex-
20 (SPSS Inc., USA). Significant differences among samples were de- tracted RPE namely geraniin, corilagin, ellagic acid and gallic acid at
termined using Duncan's multiple range test at a confidence level of 146.3, 69.1, 25.91 and 3.2 mg g−1, respectively (Fig. 1A).
95% (p ≤ 0.05).

4
R. Chollakup, et al. LWT - Food Science and Technology 130 (2020) 109573

Table 1 Pseudomonas aeruginosa, Salmonella Enteritidis, Candida albicans, Sac-

Wavenumbers of FTIR peak for O–H stretching, amide I and amide II bands of charomyces cerevisiae and Zygosaccharomyces rouxii in methylcellulose
WPI, WPI-NS and WPI-AS at different ratios between WPI:starch namely 5:0 films (Sanla-Ead et al., 2012). Trial experiments showed that a mixture
(WPI5), 4:1 (WPI4-NS1 and WPI4-AS1) and 3:2 (WPI3-NS2 and WPI3-AS2) in of RPE and CO (1:1) gave synergistic antimicrobial functions. Minimum
non-active and active (containing RPE and CO mixture at a ratio of 1:1) films.
inhibitory concentrations (MICs) for Bacillus sp., Staphylococcus aureus
Films Wavenumbers (cm−1) and Escherichia coli were 1,600, 1600 and 6400 μg cm3 for RPE, re-
spectively, while RPE-CO mixtures gave identical MIC values of
O–H band amine I band amine II band
400 μg cm3. Therefore, this mixture was added to WPI, WPI-NS and
WPI5 3274 1630 1543 WPI-AS films to produce active packaging, giving a brownish film color
WPI4-NS1 3277 1638 1543 as shown in Supplementary Fig. 2 and Supplementary Table 1.
WPI4-AS1 3280 1638 1543
WPI3-NS2 3281 1638 1543
WPI3-AS2 3277 1638 1544 3.2. Mechanical properties
WPI5-ACTIVE 3279 1632 1543
WPI4-NS1- ACTIVE 3281 1638 1548
WPI4-AS1- ACTIVE 3281 1637 1547 Fig. 2 shows the tensile properties of WPI, WPI-NS and WPI-AS films
WPI3-NS2- ACTIVE 3284 1638 1556 (non-active) and those containing RPE and CO mixtures (active).
WPI3-AS2- ACTIVE 3284 1638 1547 Thickness of WPI, WPI-NS and WPI-AS films (0.130 ± 0.015 mm to
0.140 ± 0.015 mm) increased with dispersion of RPE and CO, ranging
from 0.177 ± 0.021 mm to 0.192 ± 0.020 mm (Supplementary
Thitilertdecha et al. (2010) and Perera et al. (2012) indicated that
Table 1). Different starch types and contents showed insignificant ef-
geraniin was the major component in ethanolic extracts of rambutan
fects on film thickness in both active and non-active films. Addition of
peels, contributing to 21–57% of the peel extract. RPE showed strong
NS and AS greatly reduced tensile strength (TS) due to the non-homo-
antioxidant capacity as lipid peroxidation inhibition and DPPH•
geneous network of protein-starch blends and increased the hydro-
scavenging (8.7 IC50 μgcm−3), ABTS (0.4 IC50 μgcm−3) and FRAP (5.8
philicity of starch components. Starch contained high amounts of hy-
Fe (II) g−1) capacity due to high levels of polyphenolic compounds
droxyl groups which contributed to higher water adsorption and
(Khonkarn, Okonogi, Ampasavate, & Anuchapreeda, 2010;
plasticization than protein films (Mali et al., 2005). Higher water
Thitilertdecha et al., 2010).
plasticization led to decreased TS due to reduced bonding between
The GC-MS analysis revealed CO components as shown in Fig. 1B.
hydrophilic polymer networks. Moreover, WPI3-AS2 had the lowest TS,
(E)-Cinnamaldehyde was a major component (69.7%) followed by α-
indicating that increasing AS led to decreased TS. Acetylation in-
pinene, borneol acetate, 1,8-cineole and benzaldehyde, respectively.
troduced acetyl groups which replaced the hydroxyl groups on starch
Cinnamaldehyde is a major volatile essential oil giving antimicrobial
molecules. Bulky acetyl groups possibly reduced bonding between
activity against several microorganisms including Penicillium expansum
polymer chains due to steric hindrance and, therefore, contributed to
and Aspergillus niger in gliadin films (Balaguer et al., 2013), and Bacillus
reduced TS.
cereus, Enterococcus faecalis, Listeria monocytogenes, Micrococcus luteus,
Increased starch content led to increased elongation (EB) as higher
Staphylococcus aureus, Aeromonas hydrophila, Escherichia coli,
water plasticization led to increased flexibility. Moreover, the side

Fig. 4. Release of phenolic compounds into (A) water, (B) 50% ethanol and DPPH radical scavenging activity, (C) water and (D) 50% ethanol of WPI, WPI-NS and
WPI-AS at different ratios between WPI:starch namely 5:0 (× WPI5), 4:1 (♦ WPI4-NS1 and ■ WPI4-AS1) and 3:2 (● WPI3-NS2 and ▲WPI3-AS2) in active
(containing RPE and CO mixture at a ratio of 1:1) films at different ratios. Error bars embedded in the symbols indicate standard deviation.

5
R. Chollakup, et al. LWT - Food Science and Technology 130 (2020) 109573

Inhibition zone (mm) for Bacillus cereus, Escherichia coli and Staphylococcus aureus of WPI, WPI-NS and WPI-AS at different ratios between WPI:starch namely 5:0 (WPI5), 4:1 (WPI4-NS1 and WPI4-AS1) and 3:2 (WPI3-NS2
chains of proteins contained several functional groups that contributed

0.21 ab
0.13bc
to inter- and intra-molecular interactions and bonding including dis-

0.23a
0.14a
0.08c
ulfide bonds, hydrogen bonding and hydrophobic interaction giving a

±
±
±
±
±
more rigid structure than starch. Therefore, reduced WPI content con-

1.72
1.84
2.03
2.16
2.19
current with increased starch led to higher flexibility and EB.
105

0 Active films gave diverse results, suggesting interaction of RPE and

CO on tensile properties. WPI4-NS1-active and WPI4-AS1-active
showed increased TS, coincident with reduced EB values and suggested
0.17 ab
0.32 ab
0.21 b

0.22a
0.18a
that addition of starch in the presence of RPE and CO formed more rigid
chain networks. Amylose formed helical structures with an inner hy-
±
±
±
±
±
drophobic core in the presence of hydrophobic compounds. Therefore,
1.91
2.18
2.09
2.40
2.45
106

inclusion of CO into the starch helix formed amylose-lipid complexes

0

which reduced hydrophilicity and enhanced rigidity of the starch net-

work (Gelders, Goesaert, & Delcour, 2006). Consequently, reduced
water plasticization in starch contributed to less flexibility. Moreover,
0.41a
0.39a
0.15a
0.16a
0.23a

hydrophobicity of the oil phase reduced water plasticization and led to

S. aureus

±
±
±
±
±

a stiffer film network, as previously found in soy protein films (Otoni,

1.97
2.16
2.23
2.41
2.42

Avena-Bustillos, Olsen, Bilbao-Sáinz, & McHugh, 2016). However, in-

107

creased NS and AS in WPI3-NS2-active and WPI3-AS2-active reduced

TS, suggesting that a further increase in starch led to reduced network
strength. The presence of non-complex amylose structures with in-
2.38 ± 0.04 b

2.94 ± 0.14a
3.14 ± 0.27a
3.02 ± 0.20a
2.6 ± 0.25 b

creased NS and AS reduced TS values, as found in non-active film

systems. Moreover, active films had lower EB than non-active films.
Incorporation of RPE and CO gave non-homogeneity and decreased
and WPI3-AS2) in non-active and active (containing RPE and CO mixture at a ratio of 1:1) films at different dilutions of 107, 106 and 105.

5
10

continuity of film networks led to reduced EB. Active compounds re-

0

duced the TS of WPI films, indicating plasticization effects in protein

matrices. Similarly, Bahram et al. (2014) reported decreased TS and EB
0.31a
0.25a
0.11a
0.47a
0.23a

with incorporation of cinnamon essential oil that induced a hetero-

geneous matrix. Results indicated diverse interaction between starch
±
±
±
±
±

and protein phases with RPE and CO compounds.

2.32
2.21
2.42
2.56
2.64
6
10

3.3. Contact angle and water vapor permeability

WPI5 non-active films showed the lowest contact angle (Fig. 2C),
0.13 b
0.31a
0.20a
0.23a
0.11c

indicating the lowest surface hydrophobicity. Contact angle for WPI5

film was 58°, and within the values previously reported namely 30°
±
±
±
±
±
E. coli

(Galus & Kadzińska, 2016) and 94° (Basiak, Lenart, & Debeaufort,
1.50
1.85
2.45
2.63
2.64
7
10

2017). Different values probably attributed to diverse surface struc-

tures. Addition of starch increased contact angle in WPI non-active
films. Contact angle values in blend films concurred with Basiak et al.
0.34 ab
0.07bc
0.14bc

(2017) namely 86° and 89° for 20% and 40% starch in WPI films, re-
0.10a
0.11c

Different letters indicate significant difference within the same column (p ≤ 0.05).

spectively. Results suggested that starch-protein interaction possibly

modified the surface energy of the films which enhanced hydro-
±
±
±
±
±
1.42
1.70
1.71
2.16
1.96

phobicity of the surface. Moreover, wettability of hydrophilic surfaces

5
10

increased with increased roughness (Quéré, 2008). Incorporation of

starch possibly increased smoothness due to strong hydrogen bonding
of polymers, hence reducing surface roughness giving increased contact
0.48 ab

0.19 ab
0.23 b

0.23a

angle. Moreover, WPI3-AS2 film gave the highest contact angle because
0.11c

of the hydrophobically modified groups which enhanced surface hy-

±
±
±
±
±

drophobicity.
1.49
2.32
2.20
2.56
2.68

Addition of RPE and CO was expected to improve hydrophobicity of

6
10

the films (Bahram et al., 2014; Galus & Kadzińska, 2016). However, the
contact angle greatly decreased possibly due to the heterogeneous
surface and hence increased roughness due to dispersed RPE solids. RPE
0.38 ab
0.21 b
0.25 b

0.32a
0.09c

and CO reduced contact angle in active films depending on starch

components. NS gave a higher contact angle than WPI5 films, while AS
B. cereus

±
±
±
±
±

gave the lowest values. NS gave a smoother film surface, while bulky
1.49
2.25
2.15
2.58
2.74
7

acetyl groups formed a rougher surface in the presence of RPE and CO.
10

Results suggested that the organization of RPE and CO with starch

molecules modified surface roughness and hence surface hydro-
phobicity.
WPI4-NS1- ACTIVE

WPI3-NS2- ACTIVE
WPI4-AS1- ACTIVE

WPI3-AS2- ACTIVE

WPI5, WPI4-NS1 and WPI4-AS1 films had identical WVP values.

WPI5-ACTIVE

This indicated that protein networks played key roles in WVP when
starch was increased up to 20% of the film content. WVP values sig-
Table 2

WPI5
Films

nificantly increased in WPI3-NS2 and WPI3-AS2 non-active films due to

the high amount of starch components (Fig. 2D). WPI composed of

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R. Chollakup, et al. LWT - Food Science and Technology 130 (2020) 109573

Fig. 5. Qualities of salami namely (A) total viable count (TVC), (B) appearance and (C) color values, packaged in WPI, WPI-NS and WPI-AS films at different ratios
between WPI:starch namely 5:0 (WPI5), 4:1 (WPI4-NS1 and WPI4-AS1) and 3:2 (WPI3-NS2 and WPI3-AS2) in non-active and active (containing RPE and CO mixture
at a ratio of 1:1) films stored at 25 °C for 5 and 10 d. Different letters indicate significant difference within the same day of storage. Color can be seen in the online
version. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

several amino acids with both hydrophilic and hydrophobic side chains. polyphenols. The presence of many hydroxyl groups and hydrogen
Conversely, starch contained large numbers of hydroxyl groups that bonding in starch and polyphenols caused a broader O–H peak than in
contributed to increased hydrophilicity and water gain, thereby accel- WPI5 films (Koupantsis, Pavlidou, & Paraskevopoulou, 2016).
erating water vapor diffusion through the film matrices (Galus et al., Fig. 3 shows that WPI5 had intense band peaks at 1045 cm−1 and
2012; Jagannath, Nanjappa, Das Gupta, & Bawa, 2003). Moreover, re- 1117 cm−1. These were identical to glycerol, indicating no reaction
sults showed no correlation between surface hydrophobicity and WVP, between glycerol and whey protein through covalent linkages
suggesting that the WVP of WPI and starch blend films was mainly (Guerrero & De la Caba, 2010). Conversely, addition of NS and AS
governed by water vapor diffusion. Accordingly, affinity between water slightly shifted these bands to lower wavenumbers, confirming inter-
vapor and surface gave minor effects on WVP of blend films. action between glycerol and starch. Plasticized starch with glycerol
All active films showed decreased WVP due to enhanced hydro- typically showed a peak at 1150 cm−1 attributed to C–O stretching of
phobicity of dispersed CO droplets and RPE and retarded water vapor the C–O–H group, whereas two distinct peaks at 1080 cm−1 and
diffusion through the matrices. Moreover, cinnamaldehyde droplets 1020 cm−1 were attributed to C–O bond stretching of the C–O–C group
reduced total pore volume in the films, leading to lower WVP (Otoni of the anhydro glucose ring. These band intensities increased with
et al., 2016). Increased starch content slightly reduced WVP possibly starch content regardless of starch types. RPE and CO showed no effects
due to enhanced CO dispersion by formation of amylose-lipid com- on the IR spectra in wavenumbers ranging from 900 cm−1 to
plexes that also reduced exposure of the hydrophilic hydroxyl groups. 1200 cm−1, typical of the fingerprint region for carbohydrate compo-
nents.
Typical major absorption bands of protein were peptide linkages of
3.4. FTIR amide I and amide II located at approximately 1650 cm−1 and
1540 cm−1, respectively. Amide I related to C]O stretching and cou-
The FTIR spectra revealed characteristic peaks of starch-protein- pling of the bending of the N–H bond, while stretching of the C–N bond
glycerol blends (Fig. 3). Bands at 3100 to 3400 cm−1 and 2700 to contributed to amide II bands (Guerrero & De la Caba, 2010; Zhang
3000 cm−1 were attributed to O–H and C–H stretching vibrations, re- et al., 2013). Intensity of the band at 1630 cm−1 (amide I) was higher
spectively (Zhang et al., 2013). The O–H band shifted to a higher wa- than the band at 1530 cm−1 (amide II) in all films. Addition of RPE and
venumber (Table 1) concurrent with a broader peak with addition of CO increased the intensity of amide II and led to a smaller difference
starch. Active components suggested hydrogen bonding of the hydroxyl between amide I and amide II intensities in active films.
groups between starch-protein and active compounds, particularly

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Addition of starch caused a peak shift of the amide I band from The increased starch of WPI3-NS2 and WPI3-AS2 showed a similar re-
1630 cm−1 to 1638 cm−1, regardless of starch type and concentration lease concentration, indicating reduced impact of acetate-modified
(Table 1). Payne and Veis (1988) reported deconvolution of amide I groups at a higher starch ratio (40%). Increased hydrophilicity of starch
bands into different peaks depending on conformation of the protein molecules played a major role in phenolic release at higher starch
structure. Shift of the amide I band suggested changes in the relative concentration. Results indicated that starch types and concentration
intensity of these protein components due to structural changes of the modified the rate and contents of phenolic release.
protein backbone (Byler & Susi, 1986). The band at 1638 cm−1 in- Polyphenols gave strong antioxidant functions to the films as con-
tensified with increased NS or AS, suggesting the role of starch on firmed by DPPH assay. DPPH radical scavenging activity of the films
changes in protein conformation. Bands centered at 1654 cm−1, was monitored in water and 50% ethanol media. Ethanol gave a fast
1675 cm−1 and 1690 cm−1 were assigned to α-helix, β-turn and β- release of DPPH radical scavenging activity, reaching a constant value
sheet structure, respectively ((Fu, Griebenow, Hsieh, Klibanov, & after 25 min. Release rate depended on starch type and concentration.
Langera, 1999); Chu, Liu, & Lin, 2001). Accordingly, the shift to a Release of antioxidant activity was lower in water, with a slightly in-
higher wavenumber of the amide I band suggested changes of α-helix creased radical scavenging activity after 25 min. NS and AS affected the
into β-structure of the protein component. Moreover, active films rate and content of antioxidant activity, similar to the release of phe-
showed an intensified shoulder at higher wavenumbers and a wider nolic compounds. CO and RPE contained high amounts of phenolic
peak of the amide I band. Widening of the amide I band was attributed compounds, especially geraniin that contributed to DPPH radical
to formation of hydrogen bonds and exposure of functional groups from scavenging activity.
the interior of the protein, caused by unfolding of protein owing to the
strengthening of the β-sheet protein structure (Koupantsis et al., 2016). 3.6. Antibacterial activity
Results suggested that RPE and CO, which contained high polyphenols
and lipid phase, interacted with protein molecules causing conforma- WPI non-active films showed no antibacterial activity, while addi-
tion changes. tion of RPE and CO enhanced antibacterial activity against B. cereus, E.
NS and AS showed no effects on amide II maximum in all films, coli and S. aureus (Table 2). Films containing CO inhibited micro-
suggesting no interaction between O–H groups of starch and N–H organisms that caused degradation of the cell wall, leakage of cyto-
groups in WPI (Table 1). However, amide II showed a slight shift of plasmic cell contents and disrupted dehydrogenase activity of mi-
band maximum toward higher wavenumbers with addition of RPE and tochondria and the plasma membrane leading to cell death (Noshirvani
CO. Moreover, the shoulder of amide II bands greatly decreased. Pro- et al., 2017). Increased starch content improved the antibacterial ac-
tein-polyphenol associations are formed by several weak interactions tivity of active films. Protein-polyphenol interaction limited the release
(mainly hydrophobic) between side chains of amino acids and poly- of polyphenolic compounds and led to a lower inhibition zone in WPI5-
phenol aromatic rings (surface phenomenon) which are complemented active films. Moreover, starch was more hydrophilic than protein and
by hydrogen bonding (Von Staszewski, Pilosof, & Jagus, 2011). RPE this possibly improved water affinity and solubility and led to higher
contained several phenolic compounds, particularly corilagin and ger- release. Nevertheless, AS and NS gave similar effects on antibacterial
aniin (Fig. 1). Results suggested the presence of protein-polyphenol activity, except for E. coli at a WPI starch ratio of 4:1. WPI4-AS1 showed
interaction in active films. Moreover, hydrophobic interaction between a larger inhibition zone than WPI4-NS1, coincident with the higher
protein and lipid components in CO modified the structure of the pro- release of phenolic compounds in water media. The release study
tein backbone, resulting in modification of the amide band. clarified the effects of water exposure on release behavior of the films.
Agar media is a high-moisture semi-solid matrix; therefore, water mi-
3.5. Release of phenolic compounds and DPPH radical scavenging activity gration from agar caused polymer swelling, and interaction with phe-
nolic compounds led to antibacterial release. Increased total phenolic
Phenolic compounds are major components that promote anti- content in water indicated higher release due to starch, corresponding
microbial and antioxidant capacity of films. Total phenolic contents in to an increased inhibition zone. Similar results were observed in all
film solutions were monitored and results indicated the release of types of microorganisms. RPE polyphenols and CO gave antibacterial
polyphenol from RPE and CO (Fig. 4). Rate of phenolic release in water activity depending on the release of polyphenols into water-based
differed in the first 25 min. Phenolic contents in water only slightly media (Balaguer et al., 2013; Sanla-Ead et al., 2012). Results also
increased after 25 min of the test, indicating controlled-release of starch suggested that release properties were a major controlling factor of
components in the early stage. Release behavior was also investigated antibacterial function for in vitro measurement.
in 50% ethanol, representing food components with less polarity than
water. Ethanol media showed higher release of polyphenols because 3.7. Packaging of salami
RPE as the ethanol-extracted fraction dissolved easily in ethanol.
Moreover, concentration of phenolic compounds increased at higher NS TVC in salami was determined during storage for 10 d (Fig. 5A).
and AS contents in both water and 50% ethanol. Starch contained WPI5 non-active films showed the highest TVC of up to 5.1 log cfu∙g−1
higher hydroxyl groups that partly dissolved in water and led to higher salami at 10 d, while WPI5 active films gave the lowest microbial
release of phenolic compounds. Exposure of hydroxyl groups in starch growth during storage. Active films reduced microbial growth due to
with water also caused swelling of polymer matrices that accelerated RPE and CO that contained several polyphenols and essential oils which
further water diffusion. Consequently, interaction between solvent and contributed to antibacterial activity.
polyphenols enhanced migration. Increased starch led to higher microbial growth in WPI blend films.
Von Staszewski et al. (2011) showed that hydrophobic interactions Higher starch content enhanced the release of polyphenols in aqueous
and hydrogen bonding between polyphenols and whey protein de- media. This was in agreement with increased inhibition zone, con-
creased antioxidant and antimicrobial effects of green tea polyphenols. tributing to reduced microbial growth, as shown by the disc diffusion
Protein-polyphenol interaction reduced free polyphenol and led to re- method. However, application in food (salami) revealed diverse results.
duced release of phenolic compounds into water (Giménez, De Lacey, Rezaeigolestani et al. (2017) recorded a difference in antimicrobial
Pérez-Santín, López-Caballero, & Montero, 2013). Interaction between activity of films between the disc diffusion method and real foods.
WPI and starch reduced bonding between protein and polyphenols led Several factors including the mixture of microorganisms, microbial
to higher release. Moreover, AS showed higher phenolic release in both load, package permeability and oxygen and water vapor transfer af-
media. The bulky acetate group inhibited closer packaging of the fected microbial growth, leading to different antimicrobial activity
polymer chain and increased medium access through the film matrices. (Jariyasakoolroj et al., 2018; Rezaeigolestani et al., 2017). Moreover,

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R. Chollakup, et al. LWT - Food Science and Technology 130 (2020) 109573

salami contained a lipid phase which highly diffused to the hydro- Theeraphorn Panrong are gratefully acknowledged for technical sup-
phobic region of whey protein networks, resulting in higher release or port.
exposure of RPE and CO. The release of CO and RPE in disc diffusion
methods was also highly controlled by the hydrophilic phase of the Appendix A. Supplementary data
water-based agar media. Accordingly, WPI5 gave a lower microbial
count of 0.3–0.4 log cfu∙g−1 than WPI4-NS1 and WPI4-AS1. Supplementary data to this article can be found online at https://
The appearance of salami (Fig. 5B) darkened during storage. Salami doi.org/10.1016/j.lwt.2020.109573.
also showed a decrease in a*, indicating reduced redness during storage
(Fig. 5C). L* and b* decreased and increased during storage, respec- References
tively due to browning which caused dark pigments. Conversely, salami
packaged in active films effectively retained color of the products, Bahram, S., Rezaei, M., Soltani, M., Kamali, A., Ojagh, S. M., & Abdollahi, M. (2014).
concurrent with significantly higher a* values than non-active films on Whey protein concentrate edible film activated with cinnamon essential oil. Journal
of Food Processing and Preservation, 38(3), 1251–1258.
day 5. WPI5-active films gave the least TVC value; however, the color of Balaguer, M. P., Lopez-Carballo, G., Catala, R., Gavara, R., & Hernandez-Munoz, P.
salami changed after 10 d of storage. Higher color retention was found (2013). Antifungal properties of gliadin films incorporating cinnamaldehyde and
in higher starch contents. AS gave slightly higher color retention than application in active food packaging of bread and cheese spread foodstuffs.
International Journal of Food Microbiology, 166(3), 369–377.
NS. Results were in agreement with the release of polyphenols. RPE and Basiak, E., Lenart, A., & Debeaufort, F. (2017). Effects of carbohydrate/protein ratio on
CO contained several polyphenols that effectively inhibited oxidation of the microstructure and the barrier and sorption properties of wheat starch–whey
lipid and myoglobin which led to browning. Exposure of the meat protein blend edible films. Journal of the Science of Food and Agriculture, 97(3),
858–867.
surface to green tea polyphenols also enhanced the redness of bacon Byler, D. M., & Susi, H. (1986). Examination of the secondary structure of proteins by
(Panrong et al., 2019). Results suggested the significance of meat sur- deconvolved FTIR spectra. Biopolymers: Original Research on Biomolecules, 25(3),
face exposure to RPE and CO on the film surface. Diffusion and release 469–487.
Chu, H. L., Liu, T. Y., & Lin, S. Y. (2001). Effect of cyanide concentrations on the sec-
of active compounds from the film matrices were significant for color
ondary structures of protein in the crude homogenates of the fish gill tissue. Aquatic
retention. Toxicology, 55(3), 171–176.
Fu, K., Griebenow, K., Hsieh, L., Klibanov, A. M., & Langera, R. (1999). FTIR character-
4. Conclusions ization of the secondary structure of proteins encapsulated within PLGA micro-
spheres. ournal of Controlled Release, 58(3), 357–366.
Galus, S., & Kadzińska, J. (2016). Whey protein edible films modified with almond and
Antioxidant and antibacterial activities of WPI and starch blend walnut oils. Food Hydrocolloids, 52, 78–86.
films containing RPE and CO depended on the release and polarity of Galus, S., Mathieu, H., Lenart, A., & Debeaufort, F. (2012). Effect of modified starch or
maltodextrin incorporation on the barrier and mechanical properties, moisture sen-
the food matrices, respectively. NS and AS strongly affected WPI film sitivity and appearance of soy protein isolate-based edible films. Innovative Food
properties and release behavior. RPE contained large amounts of ger- Science & Emerging Technologies, 16, 148–154.
aniin with mixed polyphenolic compounds, whereas CO was mainly Gelders, G. G., Goesaert, H., & Delcour, J. A. (2006). Amylose− lipid complexes as
controlled lipid release agents during starch gelatinization and pasting. Journal of
composed of (E)-cinnamaldehyde which exhibited antioxidant and an- Agricultural and Food Chemistry, 54(4), 1493–1499.
tibacterial activities. These active compounds strongly affected TS and Giménez, B., De Lacey, A. L., Pérez-Santín, E., López-Caballero, M. E., & Montero, P.
EB of films due to matrix heterogeneity and increased stiffness. Starch- (2013). Release of active compounds from agar and agar–gelatin films with green tea
extract. Food Hydrocolloids, 30(1), 264–271.
WPI-polyphenol complexes governed the release behavior of phenolic Guerrero, P., & De la Caba, K. (2010). Thermal and mechanical properties of soy protein
compounds in water and ethanol media due to hydrophobic interaction films processed at different pH by compression. Journal of Food Engineering, 100(2),
and H-bonding. The active compounds greatly influenced film proper- 261–269.
Huntrakul, K., & Harnkarnsujarit, N. (2020). Effects of plasticizers on water sorption and
ties, while high starch contents (40%) gave significant impact on
aging stability of whey protein/carboxy methyl cellulose films. Journal of Food
properties of non-active films. Acetylated starch enhanced release be- Engineering, 272, 109809.
haviors in aqueous media. Antibacterial activity, as determined by the Jagannath, J. H., Nanjappa, C., Das Gupta, D. K., & Bawa, A. S. (2003). Mechanical and
disc diffusion method, increased in agreement with increased poly- barrier properties of edible starch–protein‐based films. Journal of Applied Polymer
Science, 88(1), 64–71.
phenolic release in water media. However, antibacterial activity of the Jariyasakoolroj, P., Leelaphiwat, P., & Harnkarnsujarit, N. (2018). Advances in research
films on salami as a real food model was strongly dependent on food and development of bioplastic for food packaging. Journal of the Science of Food and
components rather than release behavior in aqueous medium. Agriculture. https://doi.org/10.1002/jsfa.9497.
Khonkarn, R., Okonogi, S., Ampasavate, C., & Anuchapreeda, S. (2010). Investigation of
Moreover, all active films showed redness stabilization of salami during fruit peel extracts as sources for compounds with antioxidant and antiproliferative
storage. Findings indicated that antioxidant capacity of active protein- activities against human cell lines. Food and Chemical Toxicology, 48(8–9),
starch films was controlled by release mechanisms, whereas anti- 2122–2129.
Kim, H., Beak, S.-E., & Song, K. B. (2018). Development of a hagfish skin gelatin film
bacterial capacity in foods was strongly dependent on food matrix containing cinnamon bark essential oil. LWT- Food Science and Technology, 96,
components. 583–588.
Koupantsis, T., Pavlidou, E., & Paraskevopoulou, A. (2016). Glycerol and tannic acid as
applied in the preparation of milk proteins–CMC complex coacervates for flavour
CRediT authorship contribution statement
encapsulation. Food Hydrocolloids, 57, 62–71.
Mali, S., Sakanaka, L. S., Yamashita, F., & Grossmann, M. V. E. (2005). Water sorption and
Rungsima Chollakup: Conceptualization, Writing - review & mechanical properties of cassava starch films and their relation to plasticizing effect.
Carbohydrate Polymers, 60(3), 283–289.
editing. Siraprapa Pongburoos: Investigation. Watthana Boonsong:
Nanthakumar, R., Udhayasankar, M. R., Ashadevi, V., Arumugasamy, K., & Shalimol, A.
Investigation. Nattaporn Khanoonkon: Supervision. Kunat Kongsin: (2014). In vitro antimicrobial activity of aqueous and ethanol extracts of
Methodology. Rungsinee Sothornvit: Conceptualization, Writing - Rhinacanthus nasutus-a medicinal plant. International Journal of Pharmaceutical,
review & editing. Prakit Sukyai: Writing - review & editing. Udomlak Chemical and Biological Sciences, 4, 164–166.
Noshirvani, N., Ghanbarzadeh, B., Gardrat, C., Rezaei, M. R., Hashemi, M., Le Coz, C.,
Sukatta: Methodology, Investigation. Nathdanai Harnkarnsujarit: et al. (2017). Cinnamon and ginger essential oils to improve antifungal, physical and
Conceptualization, Methodology, Investigation, Supervision, Writing - mechanical properties of chitosan-carboxymethyl cellulose films. Food Hydrocolloids,
original draft, Writing - review & editing. 70, 36–45.
Otoni, C. G., Avena-Bustillos, R. J., Olsen, C. W., Bilbao-Sáinz, C., & McHugh, T. H.
(2016). Mechanical and water barrier properties of isolated soy protein composite
Acknowledgements edible films as affected by carvacrol and cinnamaldehyde micro and nanoemulsions.
Food Hydrocolloids, 57, 72–79.
Panrong, T., Karbowiak, T., & Harnkarnsujarit, N. (2019). Thermoplastic starch and green
This research was financially supported by Kasetsart University tea blends with LLDPE films for active packaging of meat and oil-based products.
Research and Development Institute (KURDI), Thailand. Miss Food Packaging and Shelf Life, 21, 100331.
Netchanok Kimbuatong, Miss Thitarat Pinainitisartra and Miss Payne, K. J., & Veis, A. (1988). Fourier transform IR spectroscopy of collagen and gelatin

9
R. Chollakup, et al. LWT - Food Science and Technology 130 (2020) 109573

solutions: Deconvolution of the amide I band for conformational studies. Biopolymers: Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 54(2),
Original Research on Biomolecules, 27(11), 1749–1760. 346–352.
Perera, A., Appleton, D., Ying, L. H., Elendran, S., & Palanisamy, U. D. (2012). Large scale Sun, Q., Sun, C., & Xiong, L. (2013). Mechanical, barrier and morphological properties of
purification of geraniin from Nephelium lappaceum rind waste using reverse-phase pea starch and peanut protein isolate blend films. Carbohydrate Polymers, 98(1),
chromatography. Separation and Purification Technology, 98, 145–149. 630–637.
Quéré, D. (2008). Wetting and roughness. Annual Review of Materials Research, 38, 71–99. Thitilertdecha, N., Teerawutgulrag, A., Kilburn, J. D., & Rakariyatham, N. (2010).
Rezaeigolestani, M., Misaghi, A., Khanjari, A., Basti, A. A., Abdulkhani, A., & Fayazfar, S. Identification of major phenolic compounds from Nephelium lappaceum L. and their
(2017). Antimicrobial evaluation of novel poly-lactic acid based nanocomposites antioxidant activities. Molecules, 15(3), 1453–1465.
incorporated with bioactive compounds in-vitro and in refrigerated vacuum-packed Thitilertdech, N., & Rakariyatham, N. (2011). Phenolic content and free radical scaven-
cooked sausages. International Journal of Food Microbiology, 260, 1–10. ging activities in rambutan during fruit maturation. Scientia Horticulturae, 129,
Rhim, J. W., Gennadios, A., Weller, C. L., Cezeirat, C., & Hanna, M. A. (1998). Soy protein 247–252.
isolate–dialdehyde starch films. Industrial Crops and Products, 8(3), 195–203. Von Staszewski, M., Pilosof, A. M., & Jagus, R. J. (2011). Antioxidant and antimicrobial
Sanla‐Ead, N., Jangchud, A., Chonhenchob, V., & Suppakul, P. (2012). Antimicrobial performance of different Argentinean green tea varieties as affected by whey pro-
Activity of cinnamaldehyde and eugenol and their activity after incorporation into teins. Food Chemistry, 125(1), 186–192.
cellulose‐based packaging films. Packaging Technology and Science, 25(1), 7–17. Zhang, N., Liu, X., Yu, L., Shanks, R., Petinaks, E., & Liu, H. (2013). Phase composition
Souza, A. C., Goto, G. E. O., Mainardi, J. A., Coelho, A. C. V., & Tadini, C. C. (2013). and interface of starch–gelatin blends studied by synchrotron FTIR micro-spectro-
Cassava starch composite films incorporated with cinnamon essential oil: scopy. Carbohydrate Polymers, 95(2), 649–653.
Antimicrobial activity, microstructure, mechanical and barrier properties.

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