International Journal of Biological Macromolecules 108 (2018) 444–454

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Preparation and characterization of biocomposite film based on

chitosan and kombucha tea as active food packaging
Azam Ashrafi a , Maryam Jokar b,∗ , Abdorreza Mohammadi Nafchi a
a
Food Biopolymer Research Group, Food Science and Technology Department, Damghan Branch, Islamic Azad University, Damghan, Iran
b
Research group for Nano-Bio Science, National Food Institute, Technical University of Denmark, Denmark

a r t i c l e i n f o a b s t r a c t

Article history: An active film composed of chitosan and kombucha tea (KT) was successfully prepared using the solvent
Received 14 June 2017 casting technique. The effect of incorporation of KT at the levels 1%–3% w/w on the physical and func-
Received in revised form tional properties of chitosan film was investigated. The antimicrobial activity of chitosan/KT film against
23 November 2017
Escherichia coli and Staphylococcus aureus was evaluated using agar diffusion test, and its antioxidant
Accepted 5 December 2017
activity was determined using DPpH assay. The results revealed that incorporation of KT into chitosan
Available online 6 December 2017
films improved the water vapor permeability (from 256.7 to 132.1 g cm−2 h−1 KPa−1 mm) and enhanced
the antioxidant activity of the latter up to 59% DPpH scavenging activity. Moreover, the incorporation
Keywords:
Kombucha tea
of KT into the chitosan film increased the protective effect of the film against ultra violet (UV). Fourier
Chitosan transform infrared spectroscopic analysis revealed the chemical interactions between chitosan and the
Minced beef polyphenol groups of KT. In a minced beef model, chitosan/KT film effectively served as an active pack-
aging and extended the shelf life of the minced beef as manifested in the retardation of lipid oxidation
and microbial growth from 5.36 to 2.11 log cfu gr−1 in 4 days storage. The present work demonstrates
that the chitosan/KT film not only maintains the quality of the minced beef but also, retards microbial
growth significantly, extending the shelf life of the minced beef meat up to 3 days; thus, chitosan/KT film
is a potential material for active food packaging.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction Chitosan, poly-␤-(1/4) N-acetyl-d-glucosamine, yielded from

Conventional plastics are derived from petroleum, which entails
serious environmental concerns. Biodegradable films and coatings
represent an interesting alternative to conventional plastic mate-
rials, which is why several biopolymers have been exploited to
develop materials for eco-friendly food packaging [1]. In response
to the consumer demand and market trends, the area of active pack-
aging is becoming increasingly significant. Active packaging is an
innovative concept that can be defined as a mode of packaging in
which the package, the product, and the environment interact to
prolong shelf life or enhance safety or sensory properties, while
maintaining the quality of the product [2]. Active packaging has
been applied for fresh and processed foods. One of the common
methods to develop active food packaging is to incorporate active
compounds, such as antioxidants and antimicrobial agents, into
packaging materials.
∗ Corresponding author.
E-mail address: marjok@food.dtu.dk (M. Jokar).
deacetylation of chitin, a biopolymer that is abundant in a vari-
ety of crustacean shells, such as crab, crawfish and shrimp shells
[3]. Chitosan as one of the most abundant renewable polymers has
been studied in various fields due to its unique properties, and
one of them is the area of edible coatings and films, which have
been emerged as effective and eco-friendly methods to extend the
shelf life of food products [4]. Chitosan has been found to be non-
toxic, biodegradable, bio functional, biocompatible in addition to
antimicrobial characteristics [5–8]. Chitosan has the advantage of
being able to incorporate substances such as minerals or vitamins
and possesses functional properties as compared with other bio-
based food packaging materials [9–11]. Due to chitosan intrinsic
antimicrobial properties, it has a great potential for applications
as antimicrobial films and coatings [12,13] .The exact antimicro-
bial action mechanism of chitosan is still unknown, but different
mechanisms have been proposed; interaction between positively
charged chitosan molecules and negatively charged microbial cell
membranes causing the leakage of proteins and other intracellu-
lar constituents [14]. Although chitosan is a promising biopolymer,
there are limiting factors in food packaging application because
of high molecular weight of chitosan which resulted in low sol-

https://doi.org/10.1016/j.ijbiomac.2017.12.028
0141-8130/© 2017 Elsevier B.V. All rights reserved.
 A. Ashrafi et al. / International Journal of Biological Macromolecules 108 (2018) 444–454
ubility [15]. Chitosan has been also considered as poor water
barrier properties due to hydrophilic characteristics. Improvement
of antioxidant, antimicrobial, and solubility characteristics of chi-
tosan may lead to further advantages in active food packaging
applications [16].
kombucha tea is a refreshing beverage obtained by the fermen-
tation of sugared tea with a symbiosis of Acetobacters, including
Acetobacter xylinum, Acetobacter xylinoides, Bacterium Glu- con-
icum, Acetobacter aceti, Acetobacter pasteurianus and various yeasts,
such as the Schizosaccharomyces pombe, Saccharomycodes ludwigii,
Kloeckera apiculata, Saccharomyces cerevisiae, Zygosaccharomyces
bailii, Brettanomyces bruxellensis, Brettanomyces lambicus, Bret-
tanomyces custersii, Candida and Pichia species [17,18].
The tea fungus broth is composed of two portions; a floating cel-
lulosic pellicle layer and the sour liquid broth. Acetic acid, ethanol,
and gluconic acid are the major components of the liquid broth
[19], other minor constituents such as lactic acid, glucuronic acid,
phenolic components which includes catechin, epicatechin, epicat-
echin gallate, gallocatechin, epigallocatechin and epigallocatechin
gallate [20]. Groups of vitamin B and enzymes are also present [21];
Some of its advantageous effects have already been demonstrated
such as: anti-microbial, anti-oxidant, anti-carcinogenic [22], anti-
diabetic treatment for gastric ulcers [23]. It has also shown to
have impact on immune response [24] and liver detoxification [25].
Steinkraus et al [26]. showed that the antimicrobial activity of KT
against Helicobacter pylori, Escherichia coli, Staphylococcus aurous,
and Agrobacterium umefaciens made with a low tea usage level
(4.4 g l−1 ) could be attributed to the acetic-acid content.
Previous studies showed that antioxidant activity of chitosan
could be improved through incorporating of natural substances,
such as plant extracts [27], green tea extract [28], rosemary essen-
tial oil [29], Rosemary and oxygen scavenger [30], grape seed
extract [31], and the addition of vitamin E and ␣- tocopherol
[32,33], thymus moodier or thymus piper Ella essential oils [34] in
to chitosan-based film. KT could be used as a promising source of
antimicrobial and antioxidant ingredients to incorporate chitosan
film. Therefore, The main objective of this study was to i) pro-
duce chitosan film containing KT, ii) assess the antibacterial and
antioxidant activities of chitosan films containing KT, iii) investi-
gate the effect of KT on physical properties of chitosan film and
iv)application of chitosan film containing KT as potential active
packaging to extend shelf life of minced beef.
2. Material and methods
2.1. Materials
Black tea (Camellia sinensis L.) and tea fungus samples were
purchased from local markets in Iran. Chitosan (with molecular
weight of 310000–375000 Da and 75–85% deacetylation degree)
was purchased from Sigma Aldrich (USA). DPpH (2,2-diphenyl-1
picryhydrazyl), TBA (thiobarbituric acid), acetic acid, and methanol
were also obtained from Sigma Aldrich (USA). Peptone water, Plate
Count Agar, Baird Parker agar, and egg yolk tellurite were obtained
from HiMedia Laboratories (Bombay, India). All other chemicals
were analytical grade and from Merck (Darmstadt, Germany).
2.2. Preparation
2.2.1. Preparation of KT
Four gram of black tea, were added to 1l boiling water and
allowed to infuse for about 5 min after which the infusions were
filtered through sterile sieve. Sucrose (100 gr) was dissolved in hot
tea and then was left to cool to room temperature. A certain amount
of tea (200 ml) was poured into 500 ml glass jars that had been pre-
445
viously sterilized at 121 ◦ C for 20 min. The cool tea was inoculated
aseptically with 3% (w/v) of freshly grown tea fungus that had been
cultured in the same medium for 35days and 10% (v/v) of previ-
ously fermented liquid tea broth. The jar was carefully covered with
a clean cloth and fastened properly. The fermentation was carried
out in a dark incubator at 24 ± 3 ◦ C for about 18 days [35].
2.2.2. Preparation of films
Chitosan film was prepared by dissolving chitosan (high molec-
ular weight, 310000–375000 Da, 75–85% deacetylated, Sigma-
Aldrich, Steinheim, Germany) in an acetic acid aqueous solution (1%
v/v, Sigma-Aldrich,Steinheim, Germany) while stirring on a mag-
netic stirrer/hot plate, following the indications of Ojagh et al. [16]
with some modifications. The chitosan solution was stirred at room
temperature until it was completely dissolved for 24 h. The resul-
tant chitosan solution was filtered through a Whatman No. 3 filter
paper to remove any undisclosed particles. Then, KT at the concen-
trations of 1%, 2%, and 3% (w/w) was added to chitosan aqueous
solution. The solution stirred for 60 min at 300 rpm and 30 ◦ C and
then the appropriate amount of solution was distributed into petri
dishes for casting and dried at 50 ◦ C and 30% relative humidity for
24 h. The peeled films were kept in a chamber at room temperature
and 75% relative humidity for 48 h prior to experimental use.
2.3. Characterization
2.3.1. Chemical analysis of KT
Chemical analysis of KT compounds was determined by the
method described by Malbaša et al. procedure [36]. Dry matter con-
tent was measured after drying at 150 ◦ C for 24 h. Ash content was
measured after mineralization at 550 ◦ C. The pH of the samples was
measured with an electronic pH meter (Orion 290A, ThermoFisher
Scientific, USA). Acidity of KT was determined by titration with
a standard solution of sodium hydroxide and phenolphthalein as
indicator [37].
2.3.2. DPpH radical scavenging assay
DPpH assay performed according to Yen & Chen [38] procedure.
A certain amount of KT (0.025 ml) was diluted to 4 ml of methanol
and then 0.6 ml of DPpH solution (a-diphenyl-b-picrylhydrazyl,
1 mM in methanol) was added. The mixture was then incubated at
room temperature for 30 min and then the absorbance at 517 nm
was determined [38].
The antioxidant activity of the chitosan/KT films was evaluated
by assaying the scavenging of free radical of DPpH following the
method of Blois [39], Siripatrawan & Harte [28],Wang et al. [40]
with some modification. 3 ml of film extract solution were mixed
with 1 ml of 1 mM methanol solution of DPpH . After shaken in an
oscillator for 1 min, the mixture was incubated in dark at room tem-
perature for 30 min. The UV absorbance of the DPpH assay solution
at 517 nm was measured.
The scavenging capacity of KT and films was calculated by the
following equation:
ADPPH − As
DPPH scavengi ng activity (%) = × 100 (1)
ADPPH
Where ADPpH is the absorbance value at 517 nm of the methanol
solution of DPpH and As is the absorbance value at 517 nm of the
DPpH assay solution. Tests were performed three times for each
specimen and the average values were taken.
2.3.3. Determination of the amount of total phenolic
The amount of total phenolic compounds was determined by
the method described by Jayabalan et al [41]. 0.1 ml of KT was
transferred to a 100 ml Erlenmeyer flask and the final volume was
adjusted to 46 ml by addition of distilled water. Afterward, 1 ml
446 A. Ashrafi et al. / International Journal of Biological Macromolecules 108 (2018) 444–454
of Folin–Ciocalteau reactive solution was added and incubated at
room temperature for 3 min 3 ml of 2% sodium carbonate solution
was added and the mixture was shaken on a shaker for 2 h at room
temperature. The absorbance was measured at 760 nm. Gallic acid
was used as the standard for the calibration curve. The phenolic
compound content was expressed as Gallic acid equivalent[42].
2.3.4. Ultraviolet visible spectroscopy (UV)
UV-vis spectra of films were performed using UV–vis spec-
trophotometer (Model UV-1601, Shimadzu Co., Kyoto, Japan). The
analysis was performed by continuous scanning from 300 to
800 nm. Each film specimen was cut into a rectangular piece and
placed directly in a test cell and measurements were performed
using air as the reference. A spectrum of each film was obtained at
wavelengths between 300 and 800 nm.
2.3.5. FTIR analysis
Fourier Trans form Infrared (FTIR) spectrometry (ATR mode) was
carried out to observe the structural interactions of chitosan films
containing KT. The FTIR spectra of chitosan films were recorded
from 4000 to 500 cm −1 at resolution of 4 cm−1 using an FTIR spec-
trometer (PerkinElmer 1760X, Waltham, Massachusetts USA).
2.3.6. Color properties
The color of the films was evaluated by using a Hunter Lab
Color Flex (Xinlian Creation Electronic Co. Ltd., Shanghai, China).
The color values of L* (luminosity), a* (negative-green; positive red)
and b* (negative-blue; positive-yellow) were measured. A standard
plate CX 2064 was used as standard. The total color difference (E)
and Chroma (C) was calculated as follows:
 ples in the medium was recorded as an indication of inhibition of
E = a2 + b2 + l2

C= a2 + b2
∗
Where L = lstandard ∗
−lsampled , a = a∗standard a∗sampled and b =
∗ ∗
bstandard − bsampled . The color parameter values of the standard plate
are L * = 9223 and a * = −1.28, b * =1.22. Five measurements were
taken on each film, one at the center and four around the perimeter
[40].
2.3.7. Water solubility (WS)
The water solubility of the films was defined as the percentage
of solved specimen dry matter after 24 h of immersion in distilled
water and measured according to the method used by [40].The
chitosan/KT films, previously equilibrated at 75% RH, were cut to
1 × 4 cm strips, and immersed in 50 ml of distilled water and stirred
at100 rpm. After 24 h, the strips were taken out and dried at 105 ◦ C
for 24 h and the final dry weight (wf ) was measured. The initial
dry weight (wi ) was determined by drying the strips in an oven at
105 ◦ C to constant weight. All the tests were conducted in triplicate
and the means were reported. The water solubility was calculated
according to the following equation:
Wi Wf
WS(%) = × 100
Wi
Where wi is the initial dry matter and wf is the final dry matter
2.3.8. Water vapor permeability (WVP)
The WVP of the films was determined gravimetrically according
to the standard test method ASTM E96 [43] with some modifica-
tions. Glass cup, with 45 mm external diameter, 30 mm of internal
diameter, 35 mm of height, was used. The Glass cup was filled with
8 mm distilled water and sealed by the films, then placed into a
desiccator at room. The Glass cup was weighed periodically (every
2 h for 18 h) using an analytical balance with a precision of 0.1 mg
(Mettler-Toledo Instrument) .WVP was determined by using the
following equation:
m × x
WVP (g cm−2 h−1 KPa−1 mm) = (4)
A × t × p
Where m was the weight of water permeated through the film
(g), X was the thickness of the film (mm), A was the permeation
area (7/065), was the time of permeation (h), and was water vapor
pressure difference across the film (kPa).
2.3.9. Antimicrobial activity
Escherichia coli PTCC 1330, Staphylococcus aurous PTCC1112 and
strains were obtained from Iran Industrial and scientific research
center. For recovery of lyophilized culture, the desired culture
contained in plastic bead was aseptically transferred into a tube
containing 5 ml of Nutrient Broth (Oxide Ltd., England) and main-
tained in incubator at 37 ◦ C for 24 h for bacteria. The working stock
culture was maintained on tryptone soy agar (Oxoid Ltd., England)
slants at 4 ◦ C in a refrigerator [44]. Antimicrobial efficiency of chi-
tosan/KT films was evaluated using an agar diffusion method as
described by Appendini and Hotchkiss. [45]
In the agar diffusion test, a single bacterial colony for each
microorganism was used to inoculate a 100-ml sterile nutrient
broth, which was grown aerobically overnight at 37 ◦ C for bacte-
ria in incubator (New Brunswick C24, USA) at 90 rpm to give a
final concentration of 18.76×108 for E. coli, 23.36 × 107 for S. aureus.
Each film (about 0.5 × 0.5 cm) sample was placed on a tryptone soy
agar plate surface seeded with 1 ml of peptone water-diluted test
culture. The tryptone soy agar plates were incubated for 1 day at
37 ◦ C for bacteria cultures.The clear zone formed around the sam-
the microbial species [46].
2.4. Application
(2)
2.4.1. Treatment of minced meat samples
According to the antibacterial activity of KT, the chitosan/KT film
was used to packaging of mince beef samples. The fresh minced
beef samples were collected from a local supermarket (Tehran, Iran)
and were kept at 0 ◦ C in an ice box for 1 h before packaging with
chitosan film and chitosan/KT films at KT concentration of 1% and 2%
and 3% (w/w). The minced beef contained 56.90 ± 0.60% moisture,
24.91 ± 0.91 fat, 16.84 ± 0.17% protein, and 1.15 ± 0.04% ash. The
chemical composition of minced meat was determined using AOAC
standard methods [47].
The minced beef mixture was divided into five treatment groups
consisted of 1) control samples (C), 2)samples packaged with chi-
tosan films (CH), 3) samples packaged with chitosan containing
1% (w/w) KT (CH+1%KT), 4) samples packaged with chitosan con-
taining 2% (w/w) KT (CH+2%KT), and 5) samples packaged with
chitosan containing 3% (w/w) KT (CH+3%KT). Minced beef from
each treatment was weighed into 100 ± 0.5 g portions and formed
into individual patties using sterile plastic petri plates (9 cm diam-
eter). The film materials were applied to the upper and bottom
surfaces of the patties except for control (C). Each sample after pack-
(3)
aging was stored in polyethylene bag at refrigerated temperature
(4 ◦ C) until analysis.
2.4.2. Microbial analysis
To determine total counts, each sample (10 g) was aseptically
homogenized with 90 ml of 0.1% sterile peptone water for 2 min
with a Seward stomacher lab blender (West Sussex, England). Total
counts were determined using plate count agar (total mesophilic
and psychrotrophic bacteria). 1 ml of homogenized sample in pep-
tone water inoculated using pour plate method. The incubation
time was 37 ◦ C for 48 h. For total Staphylococcus counts, Baird Parker
 A. Ashrafi et al. / International Journal of Biological Macromolecules 108 (2018) 444–454
agar supplement with egg yolk tellurite was used. Colonies were
counted after 48 h at 37 ◦ C.
2.4.3. pH analysis
10 g Samples were homogenized with 90 ml of distilled water in
a blender (pH ILIPS, HR2870) for 1 min. Then pH of the mixture was
measured directly using a pH-meter (FE20, Mettler-Toledo Instru-
ments Co., Ltd., China). The pH measurements were performed at
days 1, 2, 3, 4, 5 and 6 of refrigerated storage.
2.4.4. Determination of 2-thiobarbituric acid (TBA)
Lipid oxidation was measured by thiobarbituric acid reactive
substances (TBARS) as previously reported [48] .In brief, the sam-
ple (1 g) was homogenized with a mixed solution (5 ml) of 0.25 M
hydrochloric solution containing 0.375% (w/v) thiobarbituric acid
and 15% (w/v) trichloroacetic acid. The mixture was heated in a
water bath at 100 ◦ C for 10 min, and cooled in iced water. The sam-
ple mixture was centrifuged (5785 rpm for 20 min) and analyzed
with an UV- spectrophotometer at 532 nm using a standard curve
of malonaldehyde (0–2 ppm). TBARS was expressed in unit of mg
malondialdehyde/kg sample (mg MDA/kg meat).
2.4.5. Sensory evaluation
The samples were evaluated by an experienced thirty one
−member panel. Panelists were chosen on the basis of previous
experience in consuming minced beef. Furthermore, a preparatory
session was held prior to testing so that each panel member could
thoroughly discuss and clarify each attribute to be evaluated. Test-
ing was initiated after the panelists agreed on the specifications.
Samples (100 g) were cooked in a microwave oven at high power
(700 W) for 4 min. Quantitative descriptive analysis was used to
obtain four attributes to describe the samples. Odor attributes were
assessed first, followed by appearance (color and slime) flavor and
overall acceptability.
2.5. Statistical analysis
The difference between factors and levels was evaluated by the
analysis of variance (ANOVA). Tukey’s multiple range tests were
used to compare the means to identify which groups were signifi-
cantly different from other groups (p < 0.05) using Minitab 14.0. All
data are presented as mean ± standard deviation.
3. Results and discussion
3.1. Characterization of KT
3.1.1. Chemical properties
Table 1 shows the chemical characteristics of KT, wherein KT
contained 5% dry matter and 1.23% ash and showed an acidity of
0.39% (acetic acid). The pH of KT decreased during fermentation.
It decreased rapidly from 5.0 to 3.0 within 3 days of fermentation
and continued to gradually decrease for 18 days. The pH decreased
from 5.0 to 3.0 due to increased concentration of organic acids
produced by bacteria and fungi during fermentation. The fermen-
tation broth apparently demonstrates a buffering capacity. In the
Table 2
DPPH scavenging ability and phenolic compounds during kombucha fermentation.
Fermentation time(days)
1 3
DPPH scavenging ability (%) 13.21 ± 0.04a 20.31 ± 0.04a
Phenolic compounds(mg ml−1 ) 3.55 ± 0.24a 4.50 ± 0.72ab
Values are recorded as mean ± standard deviation.
Means followed by different lowercase letters in each row are significantly different (p<0.05) in fermentation days.
447
Table 1
Chemical properties of KT.
properties Values (%)
Dry matter content 5.12 ± 0.66
Ash 1.23 ± 0.22
Acidity 0.39 ± 0.07
pH 3.13 ± 0.21
Values are recorded as mean ± standard deviation.
fermentation process, carbon dioxide was released slowly at first
and then at a much faster rate after 2–3 days. The aqueous solution
of carbon dioxide induces production of amphiprotic hydrocarbon-
ate anion HCO3 −1 , which easily reacts with hydrogen ions (H+̂from
organic acids), preventing further changes in H+ concentration and
contributing to a buffering characteristics of the system. This phe-
nomenon is attributed to the slight decrease in pH after 3 days
[49,50].
3.1.2. DPpH radical scavenging assay
In the present study, the scavenging ability of KT during fer-
mentation was determined using hydroxyl radical scavenging assay
(DPpH). The DPpH scavenging abilities of KT increased with fer-
mentation time (Table 2). The scavenging properties of KT directly
depend on the components produced during fermentation, as well
as on tea components. The compound responsible for the maxi-
mum scavenging ability is possibly transformed into compounds
with less potential scavenging abilities, and such transformation
results from structural modifications induced by enzymes liber-
ated by bacteria and yeast in the tea fungus consortium [41].
The antioxidant activities of individual phenolic compounds pos-
sibly depend on structural factors, such as number of phenolic
hydroxyl or methoxyl groups, flavone hydroxyl, keto groups, and
free carboxylic groups, as well as on other structural features.
Dihydroxylation in both rings in catechin, myricetin, quercein, and
epicatechin is necessary in antioxidant activity as reported in var-
ious lipid systems [13]. Dziedzic and Hudson found that steric
hindrance in phenolic hydroxyl groups by addition of methoxyl
groups enhances antioxidant activity [51].
Glycosylation reduced the antioxidant activity of quercetin,
cyanidin, pelargonidin, and peonidin. Addition of a sugar moi-
ety decreased the activity of aglycon, and addition of a second
moiety further decreased the activity probably due to steric hin-
drance resulting from addition of sugar moieties [41]. Tsuda et al.
[52] found that the antioxidant activity of cyanidin is greather
than that of cyanidin 3-glycoside in linoleic acid system. Partt and
Hudson noted that 3-glycosides of flavonoids demonstrates the
same or sometimes less activity than their corresponding aglycons
[53]. These findings clearly show that structural modification of
tea components possibly occur during KT fermentation, resulting
in enhanced scavenging of nitrogen and superoxide radicals [54].
Dueñas et. al [55] demonstrated that the bioactive polyphenolic
compounds of lentils were modified due to exogenous applica-
tion of enzymes, such as phytase, agalactosidase, and tannase.
Moreover, they demonstrated the increased antioxidant activity of
enzyme-treated lentils. Thus, the enzymes liberated by bacteria and
6 9 12 15 16
27.70 ± 0.08c 36.21 ± 0.04d 40.32 ± 0.02e 47.33 ± 0.02e 55.33 ± 0.01f
5.25 ± 0.24bc 5.81 ± 0.02cd 6.55 ± 0.40d 7.42 ± 0.00d 8.46 ± 0.16e
448 A. Ashrafi et al. / International Journal of Biological Macromolecules 108 (2018) 444–454
Fig. 1. FTIR spectra of chitosan (CH)/KT films.
yeast during KT fermentation are more likely to degrade the com-
plex polyphenols into small molecules, which in turn increase the
amount of total phenolic compounds [55]. The antioxidant prop-
erties of most KT samples showed time-dependent profiles, and
prolonged fermentation is not recommended because of accumu-
lation of organic acids, which possibly reach harmful levels as a
result of direct consumption [22].
3.1.3. Total phenolic compounds
Table 2 shows the changes in concentration of total phenolic
compounds in KT during fermentation. Total phenolic compounds
progressively increased with fermentation time. Phenolic com-
pounds are called high-level antioxidants because of their ability
to scavenge free radical and active oxygen species, such as sin-
glet oxygen, superoxide free-radicals, and hydroxyl radicals [56].
Complex phenolic compounds in black tea are possibly degraded
in the acidic environment of KT and by the enzymes liberated by
bacteria and yeast in tea fungus consortium. Jayabalan et al. [57]
found that epicatechin isomers are degraded during KT fermen-
tation. Friedman [58] reported the resistance pattern of catechin
and epigallocatechin at a pH range of 3–11. Zhu et al. [59] found
that flavonoids and proanthocyanidins are more stable under acidic
conditions. Aflavin is unstable in alkaline solution but stable in
acidic solutions [54]. Duenas et al. [60] demonstrated that bioactive
polyphenolic compounds of lentils were modified due to exoge-
nous application of enzymes, such as phytase, agalactosidase, and
tannase. Moreover, they demonstrated the increased antioxidant
activity of enzyme-treated lentils. Thus, the enzymes liberated by
bacteria and yeast during kombucha fermentation possibly degrade
complex polyphenols into small molecules, which in turn results in
the increase in total phenolic compounds.
3.2. Characterization of the films
3.2.1. Fourier transform infrared spectroscopy (FTIR)
Fig. 1 shows the FTIR results for chitosan film and different
KT concentrations. The chitosan film spectrum was very similar
to that in [61]. The broad band at 3268.23 cm−1 corresponds to
OH stretching, whereas the band at 1541.15 cm −1 corresponds
to N H bending (amide II). A small peak at 1631.77 cm −1 was
due to C O stretching (amide I), and a peak at 993.33 cm−1 sug-
gested the presence of an ether group in the film. Changes in the
characteristic spectral peaks affect physical blending and chemi-
cal interactions when two components are mixed [62]. An obvious
change was observed, that is, a new peak emerged at 1550 cm−1
(carbonyl peaks), which was attributed to the presence of amine
acids in KT. These results are consistent with the results of the
study on physico-chemical interaction between chitosan and cat-
echin [63], who found that the amount of amine functional groups
in chitosan decreases when chitosan is incorporated with catechin.
 A. Ashrafi et al. / International Journal of Biological Macromolecules 108 (2018) 444–454
Fig. 2. UV visible spectra of chitosan/KT films.
Table 3
Water solubility (WS) and Water vapor permeability of chitosan/KT films .
Film samples Water solubility Water vapor permeability × 10−7 g
(WS) (%) cm−2 h−1 kPa−1 mm
CH 31.66 ± 0.08a 256.67 ± 0.88a
CH+1%KT 59.67 ± 1.20b 137.33 ± 0.88b
CH+2%KT 68.66 ± 0.88c 134.21 ± 0.55bc
CH+3%KT 77.33 ± 2.40d 132.30 ± 0.57c
Values are recorded as mean ± standard deviation.
Means followed by different lowercase letters in each column are significantly dif-
ferent (p<0.05) among chitosan (CH)/KT films.
Similar findings were observed [64] in the formation of covalent
bonds between gallic acid and chitosan and between catechin and
chitosan.
3.2.2. UV visible spectroscopy
One of the desired characteristics of a packaging material is
that it should protect food from the effects of light, especially UV
radiation. The films were scanned at wavelengths ranging from
300 nm to 800 nm to determine their transmission properties, and
the percentage light transmittance was recorded (Fig. 2). The film
samples demonstrated low transmission of UV light, especially at
280 nm. Chitosan films without KT demonstrated a low percent
transmittance, suggesting that chitosan films with KT are good
barrier for UV light, a powerful lipid-oxidizing agent in food sys-
tems. At 400 nm, the control films showed transmittance values of
approximately 10%–20%, whereas KT-supplemented films showed
a further reduced transmittance values, suggesting that films added
with KT display improved light barrier properties. These results are
consistent with the findings of [65], who reported the light barrier
properties of gelatin-based edible film were added with antioxidant
extract from murta.
3.2.3. Water vapor permeability
Film permeability depends on the chemical structure and
morphology of the film, the nature of the permeant, and the tem-
perature of the environment. Factors affecting WVP of chitosan
films include the molecular weight and degree of deacetylation
of chitosan, the type and amount of plasticizers added, and the
preparation conditions, which influence the network structure of
chitosan films. Table 3 shows the WVP of the chitosan/KT films.
After addition of KT, WVP of the chitosan/KT films decreased sig-
nificantly (p < 0.05). The permeability coefficient decreased from
257.6 g cm−2 h−1 KPa−1 mm to 132.1 × 107 g cm−2 h−1 KPa−1 mm.
Reduction of WVP of the chitosan/KT film is due to the interac-
tion between chitosan and KT via covalent bonding. Despite the
hydrophilic character of chitosan which led to increasing of water
449
solubility (WS), WVP decreased by adding KT into chitosan films.
The possible reason is interaction between chitosan and KT which
showed by FTIR spectra. This interaction reduces the availability of
hydrogen groups to form hydrophilic groups and diminishes water
interaction. Spencer et al. reported similar results for polyphe-
nol protein complexes, wherein polyphenols formed hydrogen and
covalent bonds with polar groups (e.g., aminoandhydroxyl groups)
of the polypeptide surface, resulting in a layer of polyphenol pro-
tein complexes with reduced hydrophilicity [66]. Curcio et al. [64]
also observed the formation of covalent bonds between gallic acid
antioxidant and chitosan as verified by FTIR. A similar finding was
observed by (Park and Zhao 2004) in a study on incorporation
of mineral and vitamin into a chitosan-based film. They found
that by adding mineral or vitamin into the film matrix, interac-
tion among adjacent molecules structures increased, resulting in
reduced diffusivity of water vapor transmission rate through a film
matrix and in reduced hydrophilic tendency of the films. More-
over, Gomez et al. reported the availability of hydrogen groups
due to cross-linking between tuna-fish gelatin and antioxidant
extracts of murta leaves [65]. Similar results were found in [28],
wherein incorporation of green tea and tea polyphenols into chi-
tosan films improved the water vapor barrier properties of films.
The FTIR spectra of the chitosan films changed when green tea was
incorporated, suggesting that some interactions occurred between
chitosan and the polyphenols from green tea polyphenols. Despite
the hydrophobic character of ␣-tocopherol, WVP values increased
when ␣-tocopherol was added into chitosan films [33].
3.2.4. Water solubility (WS)
As shown in Table 3, WS of the films was significantly affected
by KT incorporation (p < 0.05); thus, an increase in KT-to-chitosan
ratio from 0% to 3% considerably improved the solubility of the film
from 31.67 for CH (plain chitosan film) to 77.33 for CH+3%KT. WS
of chitosan films incorporated with Aloe vera gel increased with
increased amount of Aloe vera gel [68]. Moreover, Wang et al. [40]
reported that addition of polyphenols into chitosan films generally
increases the WS of chitosan-based films.
3.2.5. Color properties
Color properties are important in the appearance of films.
Table 4 shows the values of L*, a*, and b* for the chitosan/KT films.
Incorporation of KT notably reduced the L* values; the effect of KT
concentration on L* values, which ranged from 1% to 2%, was not
significant (p > 0.05). Given that L = 0 represents black and L = 100
represents white, a low L* value indicates film darkening. In addi-
tion, with the increase in KT concentration, the values of a* and
b* increased significantly (p < 0.05), indicating the tendency of the
film to show red and yellow coloration. Moreover, the total color
difference increased when KT concentration increased, and higher
E* values indicated increased coloration of the films. The con-
trol chitosan film (without KT) showed the highest C* value, which
increased significantly with increased KT concentration (p < 0.05),
consistent with the results reported by Wang et al [40]. By contrast,
incorporation of KT into the chitosan films led to higher degree of
darkening and enhanced red and yellow attributes.
3.2.6. Antimicrobial activity
Table 5 shows the antimicrobial activities of chitosan/KT films
against Escherichia coli and Staphylococcus aureus. The inhibitory
effect of the film toward these bacteria was dependent on
KT concentration. The chitosan +3%KT films demonstrated high
antimicrobial properties. The results showed that the antimicro-
bial effect of the films was significantly higher toward E. coli (Gram
negative) than toward S. aureus (Gram positive). These results are
consistent with several findings, which showed that chitosan exerts
antimicrobial activity against a wide range of target microorgan-
450 A. Ashrafi et al. / International Journal of Biological Macromolecules 108 (2018) 444–454
Table 4
Color values of the chitosan/KT films .
Film samples a b
CH −4.63 ± 0.14a 12.11 ± 0.5a
CH+1%KT 26.79 ± 0.85b 31.54 ± 1.67b
CH+2%KT 29.52 ± 0.32b 37.89 ± 1.06c
CH+3%KT 34.66 ± 0.88c 39.38 ± 1.45d
Values are recorded as mean ± standard deviation.
Means followed by different lowercase letters in each column are significantly different (p < 0.05) among chitosan (CH)/KT films.
Table 5
Antioxidant activity and antimicrobial activity of chitosan/KT films .
Film samples DPPH radical scavenging activity(%)
CH 6.19 ± 0.57a
CH+1%KT 29.15 ± 1.21b
CH+2%KT 44.56 ± 0.37c
CH+3%KT 59.20 ± 1.21d
Values are recorded as mean ± standard deviation.
Means followed by different lowercase letters in each column are significantly different (p < 0.05) among chitosan (CH)/KT films.
isms. Antimicrobial activity of chitosan varies considerably with the
type of chitosan, particularly on the degree of deacetylation, molec-
ular weight, target organism, and the conditions of the medium
in which chitosan is applied, as well as on pH and ionic strength.
Moreover, the solutes present are susceptible to react with chitosan
through electrostatic interaction and/or covalent binding, possibly
screening or completely blocking the reactivity of the active amine,
thereby affecting antimicrobial efficiency [12]. Low pH values (up to
5.5) increase the antimicrobial activity of chitosan because of solu-
bility and protonation of chitosan is increased under acidic pH[12].
For example [69]. reported that chitosan solution at 0.10 mg ml−1
markedly inhibited the growth of the pathogenic Xanthomonas bac-
teria of different geographical origins, whereas [70] reported that
chitosan demonstrated good inhibitory activities against Bacillus
subtilis, E. coli, and S. aureus. By contrast, Ojagh et al. [16] and Wang
et al. [40] reported that pure chitosan film did not form signifi-
cant inhibition zone when used against E. coli and S. aureus. KT
exerts antimicrobial activity against B. subtilis, E. coli, S. aureus,
Helicobacter pylori, Salmonella typhimurium, S. aureus, and Agrobac-
terium tumefaciens as reported by [71,26] against Bacillus cereus as
reported by [71], and against Shigella sonnei, Salmonella enteritidis,
and E. coli as reported by [71,50]. Black and green teas are the usual
and best substrates for the preparation of KT drinks (Jayabalan,
Marimuthu, and Swaminathan 2007) [49]. The antimicrobial activ-
ity of KT is imparted by organic acids, primarily acetic acid, and
such activity was eliminated when the samples were neutralized
[71]. Despite the potential of KT as antimicrobial agent, its usage is
often restricted because of their intense aroma and that it possibly
changes the organoleptic properties of food. Direct incorporation
of KT into chitosan is a convenient methodology to achieve antimi-
crobial activity. This work is the first to analyze the antibacterial
activity of edible films added with KT. [72] showed that the shelf life
of food has been extended by nanoparticles loaded with cinnamon
essential oil incorporated chitosan films. Similarly, incorporation
of garlic oil and nisin enhances the antimicrobial activity of chi-
tosan film [73]. Another study [74] reported on the antibacterial
properties of chitosan edible films incorporated with maqui berry,
and such antibacterial properties are possibly related to bioactive
compounds in maqui extracts, such as phenolic acids, flavonoids,
or anthocyanin.
3.2.7. Antioxidant activity
Table 5 shows the DPpH free radical scavenging activity of the
chitosan/KT films. The chitosan film without KT showed a low DPpH
free radical scavenging activity, similar to that reported by Gen-
C L E
14.6 ± 0.5a 87.29 ± 0.47a 15.43 ± 0.56a
41.60 ± 1.34b 36.56 ± 1.34b 75.32 ± 1.32b
48.17 ± 0.96c 35.26 ± 2.31b 78.09 ± 2.33c
50.91 ± 1.10d 21.65 ± 2.56c 80.04 ± 1.89d
Inhibitory zone(mm) Staphylococcus aureus Inhibitory zone(mm) Escherichia coli
8.66 ± 0.33a 6.21 ± 0.51a
13.20 ± 0.50b 15.33 ± 0.33b
15.21 ± 0.00b 20.11 ± 0.05c
17.66 ± 0.88c 20.11 ± 0.05c
skowsky et al. [74] and Jridi et al. [75]. The scavenging mechanism of
chitosan is related to the fact that a free radical reacts with residual
free amino (NH 2) groups to form stable macromolecule radicals,
and the NH 2 groups can form ammonium groups by absorb-
ing hydrogen ion from the solution [76]. Moreover, KT presents
promising antioxidant properties, which are useful in improving
the antioxidant activity of chitosan films. The phenolic compounds
present in KT impart KT its antioxidant activity because polyphe-
nols improves scavenging and antioxidant activities [41]. However,
incorporating KT significantly enhanced (p < 0.05) the free radi-
cal scavenging activity of the chitosan films, and the free radical
scavenging activity increased with KT concentration. The highest
scavenging activity was demonstrated by the chitosan/KT film with
3% KT.
3.3. Effects of KT/chitosan film on minced beef samples
3.3.1. Microbiological analysis
Table 6 shows the microbial counts in the control and in other
samples packaged in active chitosan/KT films. The initial total
bacterial count of the control sample was 3.3 log cfu g−1 , which
increased to 4.93 log cfu g−1 after 3 days. In the samples treated
with KT coatings, the total bacterial counts decreased to 4.76, 4.33,
and 4.1 log cfu g −1 on day 6 (p < 0.05). This result indicated that
the shelf life of stored minced beef packaged in chitosan/KT can
be extended up to 6 days. The Staphylococcus growth counts were
also inhibited by packaging in chitosan containing KT (Table 7). S.
aureus counts were significantly reduced when the meat sample
was wrapped in chitosan/KT films (p < 0.05).
3.3.2. pH analysis
Fig. 3 shows the pH values of the samples packaged in chi-
tosan/KT films during storage. The initial pH value was 5.6, and
the pH values of the samples slightly decreased, except for those of
the control, during storage. No significant difference (p > 0.05) was
observed between the pH values of the samples coated with chi-
tosan/KT films on day 6. The pH values of the samples coated with
chitosan/KT films decreased on the first 4 days and then increased
until the end of the storage period. Growth of lactic acid bacte-
ria in packed mince samples resulted in pH reduction [77,78]. This
phenomenon is possibly caused by the increase in volatile bases
(e.g., ammonia and trimethylamine) produced by either microbial
or endogenous enzymes [79]. The kombucha component of the chi-
tosan/KT films effectively prohibited enzyme activity, so low pH
of the samples was maintained. Moreover, the pH of the samples
 A. Ashrafi et al. / International Journal of Biological Macromolecules 108 (2018) 444–454 451

Table 6
Effect of Chitosan/KT films on changes in microbial counts (log cfu gr−1 ) of minced beef during storage at 4 ◦ C.

Attribute Treatment

1 2 3 4 5 6

Total Control 3.31 ± 0.18a 4.16 ± 0.31a 4.93 ± 0.08a ND ND ND

counts CH 2.53 ± 0.17a 3.13 ± 0.03b 4.52 ± 0.2a 4.93 ± 0.08a ND ND
CH+1%KT 1.76 ± 0.08b 2.43 ± 0.12bc 2.93 ± 0.08b 3.93 ± 0.15b 4.13 ± 0.07a 4.76 ± 0.08a
CH+2%KT 1.74 ± 0.11b 2.26 ± 0.09c 2.80 ± 0.05b 3.53 ± 0.09c 3.86 ± 0.04a 4.33 ± 0.12a
CH+3%KT 1.40 ± 0.05b 1.76 ± 0.08c 2.10 ± 0.05c 2.46 ± 0.15d 3.36 ± 0.08b 4.14 ± 0.32a

Staphylococcus Control 3.23 ± 0.08a 4.46 ± 0.14a 5.26 ± 0.027a ND ND

CH 2.76 ± 0.24a 3.63 ± 0.145b 4.60 ± 0.43a 5.36 ± 0.17a ND ND
CH+1%KT 1.76 ± 0.08b 2.26 ± 0.08bc 2.93 ± 0.08b 3.43 ± 0.2b 4.46 ± 0.17a 4.92 ± 0.23a
CH+2%KT 1.53 ± 0.05b 2.06 ± 0.08c 2.23 ± 0.08bc 3.12 ± 0.11b 3.93 ± 0.08a 4.13 ± 0.03b
CH+3%KT 1.40 ± 0.11b 1.70 ± 0.15c 1.80 ± 0.05c 2.11 ± 0.11c 2.73 ± 0.12b 3.26 ± 0.08c

Values are recorded as mean ± standard deviation.

Means followed by different lowercase letters in each column are significantly different (p<0.05) among chitosan (CH)/KT films.
ND: not-detected.

Table 7
Effect of Chitosan/KT films on changes in sensory evaluation of minced beef during storage at 4 ◦ C.

Attribute Treatment Days

1 2 3 4 5 6

Red Control 3.81 ± 0.31a 3.62 ± 0.21a 2.13 ± 0.12c – – –

color CH 3.70 ± 0.14a 3.63 ± 0.18a 3.21 ± 0.133b 3.30 ± 0.25b – –
CH1%KT 3.72 ± 0.16a 3.72 ± 0.16a 4.00 ± 0.00a 4.10 ± 0.00a 3.21 ± 0.11a 3.35 ± 1.60a
CH2%KT 3.5 ± 0.31a 3.80 ± 0.3a 4.01 ± 0.02a 4.01 ± 0.00a 4.00 ± 0.00a 4.00 ± 0.00a
CH3%KT 3.90 ± 0.21a 3.80 ± 0.28a 4.10 ± 0.01a 4.20 ± 0.00a 4.02 ± 0.00a 4.00 ± 0.010a

Odour Control 4.11 ± 0.0a 3.80 ± 0.13a 2.20 ± 0.13b – – –

CH 3.80 ± 0.13ab 3.72 ± 0.13a 3.61 ± 0.23a 2.40 ± 0.16b – –
CH1%KT 4.11 ± 0.01a 3.71 ± 0.16a 3.42 ± 0.16a 3.30 ± 0.15a 2.80 ± 0.31a 2.90 ± 0.21a
CH2%KT 4.12 ± 0.01a 3.90 ± 0.21a 3.81 ± 0.30a 3.40 ± 0.26a 3.11 ± 0.1a 3.10 ± 0.18a
CH3%KT 4.01 ± 0.01a 3.50 ± 0.26a 3.33 ± 0.20a 2.71 ± 0.21a 1.81 ± 0.21b 1.60 ± 0.26a

Flavor Control 4.13 ± 0.03a 3.80 ± 0.13a 3.60 ± 0.11a – – –

CH 4.11 ± 0.00a 3.90 ± 0.11a 3.60 ± 0.12a 2.42 ± 0.12a – –
CH1%KT 4.12 ± 0.00a 3.80 ± 0.11a 3.42 ± 0.11a 3.31 ± 0.15a 3.11 ± 0.21a 3.10 ± 0.18a
CH2%KT 4.11 ± 0.01a 3.80 ± 0.15a 3.41 ± 0.26a 3.13 ± 0.31 3.12 ± 0.22a 2.90 ± 0.24a
CH3%KT 4.00 ± 0.01a 3.70 ± 0.13a 3.55 ± 0.16a 3.40 ± 0.31b 2.60 ± 0.16b 2.80 ± 0.24a

Overall Control 4.03 ± 0.01a 3.81 ± 0.13a 2.23 ± 0.13a – – –

accept- CH 4.02 ± 0.01a 3.71 ± 0.13a 3.61 ± 0.20a 2.40 ± 0.16b – –
abil- CH1%KT 4.10 ± 0.01a 3.70 ± 0.16a 3.40 ± 0.16a 3.30 ± 0.15a – –
ity CH2%KT 4.12 ± 0.01a 3.90 ± 0.21a 3.81 ± 0.32a 3.40 ± 0.26a 3.11 ± 0.11a 3.12 ± 0.18a
CH3%KT 4.00 ± 0.01a 3.50 ± 0.26a 3.30 ± 0.20a 3.40 ± 0.22a 2.60 ± 0.16b 2.80 ± 0.24a

Values are recorded as mean ± standard deviation.

Means followed by different lowercase letters in each column are significantly different (p < 0.05)) among chitosan (CH)/KT films.

Fig. 3. pH of samples coated with chitosan (CH)/KT films during refrigerated storage at 4 ◦ C.
452 A. Ashrafi et al. / International Journal of Biological Macromolecules 108 (2018) 444–454
Fig. 4. TBA value of samples coated with chitosan (CH)/KT films during refrigerated storage at 4 ◦ C.
decreased with increased KT concentration in the films during stor-
age. This phenomenon is due to the synergistic antibacterial effect
of KT and chitosan.
3.3.3. Lipid oxidation
Lipid oxidation is one of the important factors related to the
deterioration of food quality, such as undesirable rancid off-flavors
and poisoning. In addition, lipids are easily oxidized in the pres-
ence of light, heat, and enzymes. Lipid oxidation is normally found
in stored meat products [80]. Free radical products in lipid oxi-
dation create by the attack of oxygen at the double bond in fatty
acids. Primary lipid oxidation products, hydroperoxides, continue
the polymeric secondary oxidation to aldehyde, ketone and alco-
hol compounds. Thiobarbituric acid reactive substances are a good
index for secondary lipid oxidation products [81]. Fig. 4 illustrates
the influence of different KT concentrations on TBA values of the
samples during refrigerated storage for 6 days. TBA value on day 1
was 0.46 mg malonaldehyde Kg−1 . TBA in all of the samples signif-
icantly increased throughout the storage period (p < 0.05). Connell
[82] and Wenjiao et al. [83] reported that a TBA value of 2 mg Kg−1
is the limit. The control reached a TBA of 0.17 mg Kg−1 at day 3,
corresponding to the sensor results for off-odor. The TBA values of
the samples coated with chitosan/KT films were lower than those
of the control (p < 0.05). Moreover, Ojagh et al. [16] reported that
rainbow trout fillets treated with chitosan-cinnamon oil reached a
TBA of 0.21 mg Kg−1 , which is significantly lower than those of the
control or chitosan-coated samples. These findings demonstrated
the synergistic antioxidant activity of KT and chitosan. Studies have
shown that the interactivity of amino groups with fat derivatives
and the chelation effect of chitosan with metal ions inhibited lipid
oxidation [84].
The samples coated with chitosan+3%KT showed the lowest
TBA values (0.31 malondialdehyde mg kg−1 ) after 6 days of stor-
age, demonstrating that chitosan/KT at 3% concentration inhibited
lipid oxidation.
3.3.4. Sensory analysis
Table 7 shows the sensory scores of the minced beef samples
coated with chitosan/KT films. The acceptable scores for color, odor,
and overall acceptability were consistent with the microbial growth
and TBA values (Section 3.3.3). The off-odor of all the samples signif-
icantly increased during refrigerated storage (p < 0.05). Other study
showed that the TBA value of 1.5 was closely related to percepti-
ble and unacceptable off-odor of meat [85]. Thus, chitosan+ 3%KT
coating effectively delayed the off-odor formation in minced beef
samples.
4. Conclusions
Chitosan/KT film was prepared in water without any chemi-
cal modification or the use of organic solvents. This study showed
that active packaging of chitosan containing KT may be applied to
extend shelf life of foods. Chitosan is a versatile and promising
biodegradable polymer material for food packaging. In addition,
chitosan possesses immense potential as an antimicrobial packag-
ing material due to its antimicrobial activity and non-toxicity. The
results of this study suggested that functional properties of chi-
tosan films improve when combined with KT. The incorporation of
KT caused interactions between chitosan and KT, making the chi-
tosan films darker. The WVP of the chitosan film decreased and the
antioxidant activity of the chitosan film significantly increased after
addition of KT, making the chitosan/KT films a potential material
for active food packaging. This innovative chitosan/KT film devel-
oped without the use of organic solvents or chemical reagents may
be used an active food packaging material.
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