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Article history:
Received 28 November 2014
Received in revised form
4 March 2015
Accepted 10 March 2015
Available online 18 March 2015
Edible lms and coatings can not only function as barriers of water vapor, gases and volatile compounds
but also serve as carriers of functional ingredients. Composite edible lms fabricated with zein and
chitosan as base materials and supplemented with phenolic compounds (ferulic acid or gallic acid) and
dicarboxylic acids (adipic acid or succinic acid) were prepared. The composite lms exerted better water
vapor barrier and mechanical properties. While the recovery percentage of phenolic compounds from
composite lms was 71e84%, the recovery of dicarboxylic acids was 48e65%. The composite lms also
possessed antioxidant activities as shown by DPPH and ABTS free radical scavenging tests. The antimicrobial activity of composite lm was demonstrated by using Staphylococcus aureus and Escherichia coli as
test microorganisms.
2015 Elsevier Ltd. All rights reserved.
Keywords:
Zein
Chitosan
Composite lms
Antioxidant activity
Antimicrobial activity
1. Introduction
Edible lms and coatings offer the opportunity to effectively
control mass transfer among different component within a food
system or between the food and its environment (HernandezIzquierdo & Krochta, 2008). Moreover, active packaging materials
can be obtained when functional ingredients are incorporated into
edible lms and coatings (Rojas-Gra, Soliva-Fortuny, & MartnBelloso, 2009). The functionalities of active packaging materials
include nutrient supplementation, antimicrobial activity, antioxidant activity, etc. The commonly used base materials for edible
lms and coatings are polysaccharides (such as starch, cellulose
derivatives, pectin and chitosan), proteins (such as gelatin, cereal
proteins, milk casein and soy protein) and lipophilic materials (such
as glycerides, and bee wax); the above materials can be used either
individually or in combination (Nesterenkoa, Alric, Silvestre, &
Durrieu, 2013). Zein and chitosan with good lm forming characteristics and relatively good barrier properties are commonly used
for edible lms and coatings (Bourtoom, 2008).
Zein is the major storage protein in corn and accounts about
44e79% endosperm protein (Lawton, 2002; Paramawati, Yoshino,
116
S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121
propionic acid and lactic acid (Hamdine, Heuzey, & Begin, 2005;
Shamov, Bratskaya, & Avramenko, 2002). The carboxylic groups
(eCOOH) can serve as proton donor to neutralize the amine groups
(eNH2) in chitosan. Furthermore, the ionized form of carboxylic
group (eCOO) can further form ionic bond with ammonium
groups (eNH
3 ). The combination effects are presented as enhance
of solubility of chitosan in acid solution and increase of lm mechanical properties (Chen et al., 2007; Kim, Son, & Kim, 2006).
Dicarboxylic organic acid used in chitosan lm forming solutions
will further improve the mechanical properties since ionic-cross
linking among chitosan molecules can be established (Hamdine
et al., 2005).
The antimicrobial activities of chitosan have been reported in
the literature (Chen & Zhao, 2012; Katas, Mohamad, & Zin, 2011).
Under acidic conditions, the positively charged eNH
3 groups of
chitosan will interact with the negatively charged bacterial cell
coverings and result in bactericidal effects through the interference
of cell permeability and interrupting the integrity of cell structure
(Chang, Chen, & Tan, 2011). Active food packaging materials based
on chitosan have been applied to foods to preserve qualities
(Kanatt, Rao, Chawla, & Sharma, 2013). However, lms and coatings
constructed solely with polysaccharide, such as chitosan, are not
good water vapor and gas barriers because of the presence of hydrophilic groups (Bourtoom, 2008).
A modern trend for developing active edible lms is to combine
different base materials and to incorporate multiple functional ingredients (Rojas-Gra et al., 2009). For example, chitosan has been
used to form composite lms with polysaccharides (tapioca starch,
hydroxypropyl methylcellulose, or pectin) and proteins (round scad
protein, whey protein, or sh gelatin) (Aider, 2010). Moreover, zein
and chitosan were used to form microspheres (Mller et al., 2011)
or composite lms (Escamilla-Garca et al., 2013; Li, Ren, Wang, Xu,
& Wang, 2011). However, no research in the literature reported the
combination of zein/phenolic compounds and chitosan/dicarboxylic acids to fabricate composite lms with antioxidant and antimicrobial activities. In the present study, composite lms with
acceptable physicochemical properties and possessing antioxidant
and antimicrobial activities were successfully fabricated by using
zein/phenolic compounds and chitosan/dicarboxylic acids.
2. Materials and methods
2.1. Preparation of zein/chitosan composite lms
Defatted zein was prepared according to the method described
by Padgetti, Han, and Dawson (2000). Briey, zein (SigmaeAldrich,
St. Louis, MO, USA) was mixed with 10 times of hexane (w/v) and
stirred at room temperature for 2 h. After ltration, the residue
hexane was further removed by placing zein in a vacuum oven
(CVO-30, Hsin-Chien-Hsing Co., Tainan, Taiwan) at 30 C overnight.
Chitosan (molecular weight 50,000e190,000, degree of deacetylation 90%) was purchased from SigmaeAldrich (St. Louis, MO, USA)
and used directly without pretreatments.
In previously published researches, phenolic compounds were
added into zein to improve the mechanical properties and to inlu, 2011) and
crease the antioxidant activities (Arcan & Yemeniciog
dicarboxylic organic acid was used in chitosan lm forming solutions to improve the lm mechanical properties (Hamdine et al.,
2005). Based on the preliminary trials conducted in our laboratory, the zein/chitosan composite lm was prepared as follows. A
portion of 0.9 g zein was thoroughly dissolved in 10 ml 95% ethanol
(Taiwan Tobacco and Wine Co., Taipei, Taiwan). After the addition of
257 ml glycerol (Choneye Pure Chemical, Taipei, Taiwan), the
mixture was heated (80 C, 3 min) and cooled down to room
temperature. Gallic acid or ferulic acid (SigmaeAldrich, St. Louis,
MO, USA) at the level of 246 mg/g zein was added into the zein
solution. Acetic, succinic and adipic acids were purchased from
Chem-Service (Chester, PA, USA). A portion of 0.3 g chitosan was
thoroughly dissolved in 30 ml 1% acetic acid containing either 0.5%
adipic acid or 0.5% succinic acid. The zein and chitosan solutions
were degassed separately in the vacuum oven at room temperature
for 1 h. And then, the chitosan solution was slowly added into zein
solution with gentle mixing. The zein/chitosan composite lm was
obtained by transferring the mixture into a plastic petri dish (id.
14.5 cm) and drying in a gel drier (SE1200 Easy Breeze Air Gel-dryer
System, Hoefer, San Francisco, CA, USA) for 24 h. All edible lms
were stored in a dehumidier (SL-450CA, Moisture Buster, Taichung, Taiwan) at RH 40% and 25 C.
2.2. Measurement of lm thickness
The lm thickness was measured with a thickness micrometer
(SM-114, Teclock, Okaya, Japan). The thickness of each piece of lm
was the average of thickness of ten randomly selected spots. The
thickness of each type of lm was represented by mean SD of
three lms.
2.3. Water vapor transmission
The measurement of water vapor transmission (WVT) was
conducted according to Tillekeratne and Easteal (2000) with
modications. The lm was xed to a cup lled with approximate
25 g dried silica gel beads. After weighted (W1), the cup was placed
in a temperature and humidity control chamber (HRMB-80, Taichy
Technology Ltd., Nantou, Taiwan) at 25 C and 75% RH for 24 h. The
cup was removed from the humidity chamber and weighted (W2)
again. WVT was calculated with following equation.
.
W W mm
2
1
WVT g mm m2 24 h
area
2.4. Mechanical properties
The lm mechanical properties including maximum force,
maximum elongation, tensile strength, break value and break
strength were measured according to the Ofcial Method of the
Chinese National Standards CNS 10591 (CNS, 2008) with a universal
testing machine (AI-2500, Gotech, Taichung, Taiwan). The test
strips with dimension of 100 10 mm were cut from composite
lm and conditioned in an environment of 23 2 C and RH
65 5% for two days. The test conditions were initial grip gauge
length 80 mm and grip separation rate 50 mm/min. Triplicate
samples were used for each type of lm.
2.5. The measurement of color parameters
The color parameters (L*, a* and b*) of composite lm were
measured with a color meter (NE4000, Nippon Denshoku Industries, Tokyo, Japan). Before measurement, white tile was used
for calibration. The color parameters of each piece of lm were the
average of 5 randomly selected spots. Triplicate samples were used
for each type of composite lm.
2.6. Determination of phenolic compounds and dicarboxylic acids
in composite lms
The determination of phenolic compounds was conducted according to the method described by Yao et al. (2002) with modications. A portion of 50 mg composite lm and 10 ml methanol were
S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121
117
Inhibition%
.
.
CFU mlcontrol CFU mlsample
CFU=mlcontrol
100%
118
S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121
Table 1
Fabrication of zein/chitosan composite lms.
Film formation solution containing chitosana
Chitosan
Chitosan
Chitosan
Chitosan
Chitosan
Chitosan
Chitosan
Zein
Zein
Zein
Zein
Zein
Zein
Zein
Failed to form lm
A lm
AF lm
AG lm
S lm
SF lm
SG lm
a
b
c
dissolved
dissolved
dissolved
dissolved
dissolved
dissolved
dissolved
in
in
in
in
in
in
in
1%
1%
1%
1%
1%
1%
1%
acetic
acetic
acetic
acetic
acetic
acetic
acetic
acid
acid
acid
acid
acid
acid
acid
dissolved
dissolved
dissolved
dissolved
dissolved
dissolved
dissolved
in
in
in
in
in
in
in
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
Table 2
Thickness and water vapor transmission of zein/chitosan composite lms prepared with different dicarboxylic acid solvent and phenolic compounds.
Film typea
A lm
AF lm
AG lm
S lm
SF lm
SG lm
Thickness (mm)
0.104
0.108
0.113
0.103
0.103
0.107
0.004A
0.006A
0.009A
0.003A
0.003A
0.005A
0.007B
0.003E
0.004E
0.003A
0.007C
0.008D
Color parameters
L*
65.41
27.97
27.83
55.08
35.96
31.92
a*
1.04A
1.85D
1.46D
3.06B
4.27C
4.27CD
1.44
3.87
1.00
0.94
3.02
2.22
b*
0.11A
0.28B
0.66AB
2.28AB
2.52B
1.96B
13.05
8.97
4.66
10.98
10.44
8.40
0.05A
0.50B
0.49C
1.28AB
4.10AB
1.62B
S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121
119
Table 3
Mechanical properties of zein/chitosan composite lms prepared with different dicarboxylic acid solvent and phenolic compounds.
Film typea
A lm
AF lm
AG lm
S lm
SF lm
SG lm
1.324
1.981
1.883
0.824
2.481
1.883
0.226C
0.579B
0.167B
0.098D
0.118A
0.137B
0.226C
0.490B
0.147BC
0.078D
0.127A
0.127BC
0.088
0.167
0.157
0.059
0.206
0.167
0.029B
0.069A
0.020A
0.020B
0.029A
0.000A
0.029CD
0.069B
0.010BC
0.020D
0.029A
0.000B
2.083D
6.323BC
7.231A
2.966CD
8.351AB
3.202A
formed between protein and ferulic acid (Ou, Wang, Tang, Huang, &
Jackson, 2005).
Table 4
Recovery of phenolic compounds and dicarboxylic acids and the antioxidant activities of zein/chitosan composite lms.
Film typea
A lm
AF lm
AG lm
S lm
SF lm
SG lm
Phenolic compoundsb
Dicarboxylic acidsc
Antioxidant activityd
Recovery (%)
Recovery (%)
DPPH
0
71.6
77.6
0
71.5
84.6
51.46
48.65
49.97
65.41
59.81
50.05
4.08
98.61
315.4
8.542
96.85
321.4
1.9
2.7
1.1
1.3
6.31
1.89
1.49
3.19
1.26
6.23
ABTS
0.92C
6.42B
2.50A
2.10C
3.18B
3.88A
11.15
276.2
455.5
0.82
246.8
449.7
10.8C
43.4B
35.9A
0.83C
29.4B
33.6A
120
S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121
Fig. 1. The growth inhibition (%) of Escherichia coli (A) and Staphylococcus aureus (B) by
zein/chitosan composite lms prepared with different dicarboxylic acids and phenolic
compounds. Film types as described in Table 1. Data are presented as means of triplicate samples, error bar: SD. Data with different letter are signicantly different
(p < 0.05).
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