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LWT - Food Science and Technology 63 (2015) 115e121

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LWT - Food Science and Technology


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Antioxidant and antimicrobial edible zein/chitosan composite lms


fabricated by incorporation of phenolic compounds and dicarboxylic
acids
Siang-Ying Cheng, Be-Jen Wang, Yih-Ming Weng*
Department of Food Science, National Chiayi University, 300 University Road, 60004 Chiayi, Taiwan

a r t i c l e i n f o

a b s t r a c t

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,

* Corresponding author. Tel.: 886 5 2717599; fax: 886 5 2717596.


E-mail address: ymweng@mail.ncyu.edu.tw (Y.-M. Weng).
http://dx.doi.org/10.1016/j.lwt.2015.03.030
0023-6438/ 2015 Elsevier Ltd. All rights reserved.

& Isobe, 2001). Because of the high composition of hydrophobic


amino acids (such as leucine and alanine) and acidic amino acid
(glutamic acid, 21e26%), zein does not dissolve in water and dilute
salt solution but dissolve in 60e95% ethanol (Chen, Ye, & Liu, 2014).
Zein lm exerts better barrier against transmission of water vapor
and volatile components when compared with other types of
protein-based edible lms (Ozcalik & Tihminlioglu, 2013). However, the brittleness is the major disadvantage of zein lm. The
researches have been conducted to incorporate plasticizers in lm
forming formula to reduce the brittleness (Lawton, 2002;
Paramawati et al., 2001; Tillekeratne & Easteal, 2000). Furthermore, functional additives have also been included in zein to confer
edible lms with multiple activities. Phenolic compounds were
added into zein to improve the mechanical properties and to inlu, 2011).
crease the antioxidant activities (Arcan & Yemeniciog
Polyvalent organic acids had been added into zein forming mixture
to improve mechanical properties because of the interaction between the terminal amino acids (serine or threonine) of zein
molecules and the polyvalent organic acids (Selling & Sessa, 2007).
Chitosan, a copolymer of N-acetyl glucosamine and glucosamine
linked by b-1-4 linkage, is derived from chitin by partial deacetylation. Chitin presents mainly in the fungal cell wall and the
exoskeleton of crustaceans and insects (Bourtoom, 2008). Chitosan
dissolves in dilute organic acids such as formic acid, acetic acid,

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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

placed in a plastic centrifuge tube and thoroughly mixed for 30 min.


After centrifugation (10,400  g, CT15RT centrifuge, Pantech Instruments, Taipei, Taiwan) for 10 min at 4  C, the supernatant was
ltered through a 0.45 mm membrane lter and analyzed with HPLC.
The HPLC apparatus consisted of an L-7100 pump and an L-7455 diode
array detector (Hitachi, Tokyo, Japan). A LichroXART RP-1e column
(4.6  150 mm, 5 mm; Merck, Darmstadt, Germany) was used. The
mobile phase consisted of 0.3% formic acid in 2% aqueous methanol (A)
and methanol (B). With ow rate of 1 ml/min, the mobile phase
gradient was as follow: 85% (A) and 15% (B) initially, (B) increasing
from 15% to 32% in 0e10 min, (B) increasing from 32% to 35% in
10e12 min, (B) holding at 35% in 12e15 min, (B) increasing from 35% to
50% in 15e25 min, (B) decreasing from 50% to 15% in 25e30 min, and
(B) holding at 15% in 30e35 min. The injection volume was 20 ml and
detection wavelength was 260 nm. The contents phenolic compounds
were calculated against calibration curves established with ferulic acid
and gallic acid. The recovery percentage of each individual phenolic
compound was calculated based on the theoretical amount used for
the preparation of composite lm.
The analysis of dicarboxylic acid was conducted according to the
method reported by Shirai et al. (2001) with modications. A portion
of 200 mg composite lm and 10 ml distilled water were mixed in a
centrifuge tube for 30 min. After centrifugation (10,400  g, 4  C and
30 min), the supernatant was ltered through a 0.45 mm membrane
lter and analyzed with HPLC. A Sugar Shodex SH1011 column
(8.0  300 mm, Showa Denko, Kanagawa, Japan) was used. Sulfuric
acid solution (20 mM) was used as the mobile phase and the ow
rate was 0.6 ml/min. The injection volume was 20 ml and detection
wavelength was 210 nm. The contents of dicarboxylic acids were
calculated against calibration curves established with adipic acid or
succinic acid. The recovery percentage of each individual dicarboxylic acid was calculated based on the theoretical amount used for the
preparation of composite lm.
2.7. Antioxidant activities
DPPH free radical scavenging activity of composite lm was
measured according to the method described by Wangcharoen and
Morasuk (2007) with modications. A portion of 10 mg lm and
10 ml methanol were mixed in a plastic centrifuge tube. The
mixture was kept at room temperature for 30 min with occasional
vortexing. The mixture was centrifuged (10,400  g, CT15RT
centrifuge, Pantech Instruments, Taipei, Taiwan) for 10 min at 4  C.
An aliquot of 0.3 ml supernatant, 0.6 ml 0.8 mM DPPH (SigmaeAldrich, St. Louis, MO, USA) solution and 5.1 ml methanol were
mixed and kept at room temperature for 1 min. Then, the absorbance at 515 nm was measured. The DPPH scavenging activity was
calculated against a calibration curve established with Trolox and
expressed as mg Trolox equivalent/g lm.
ABTS free radical scavenging activity of composite lm was
measured according to the method described by Carrasco-Castilla
et al. (2012) with modications. ABTS (nal concentration
7 mM) was prepared by dissolving ABTS (SigmaeAldrich, St. Louis,
MO, USA) in 2.45 mM potassium persulfate and kept at dark for
12e16 h. Prior to use, the absorbance of solution at 734 nm was
adjusted to 0.70 0.02 with methanol. An aliquot of 0.05 ml supernatant and 5 ml diluted ABTS solution were mixed and incubated for 6 min before the absorbance at 734 nm was measured.
The activity was expressed as mg Trolox equivalent/g lm by
calculating against a calibration curve established with Trolox.

117

Research Center (Shin-Chu, Taiwan). The working cultures were


maintained on the nutrient agar (NA; Merck, Darmstadt, Germany)
plates and subcultured every 2 weeks. The single colony was picked
from NA plate and inoculated into 50 ml sterilized nutrient broth
(NB; Merck, Darmstadt, Germany). After incubation overnight at
37  C, the cell density was adjusted to 104 CFU/ml with fresh
sterilized NB based on a pre-established turbidity calibration curve.
An aliquot of 0.5 ml cell suspension, 4.5 ml sterile NB and 2.5 mg
lm were mixed and incubated at 37  C for 180 min. A portion of
0.1 ml mixture was sampled and plated with NA. The plates were
incubated at 37  C for 2 days and the colonies were counted. Bacterial suspension (0.5 ml) and sterilized NB (4.5 ml) without composite lm was used as the control. The percentage of inhibition
was calculated according the following equation:

Inhibition%

.
.
CFU mlcontrol  CFU mlsample
CFU=mlcontrol

 100%

2.9. Statistical analysis


The data were presented as mean SD. One-way ANOVA and
Duncan's multiple range test were used to analyze the difference
among groups (SPSS software, 12.0, SPSS Inc., Chicago, IL, USA).
Differences were considered to be statistically signicant when P
values were less than 0.05.
3. Results and discussion
With series of preliminary trials, the best combinations of chitosan/dicarboxylic acid and zein/phenolic compound for composite
lm formation were determined (Table 1). However, the combination of chitosan without dicarboxylic acids and zein with
phenolic compounds failed to form composite lms. Since dicarboxylic organic acid used in lm forming formulation could
improve the lm mechanical properties by forming ionic-cross
linking among chitosan (Hamdine et al., 2005), the lack of cross
linking might explain the failure. Thus, there were six types of zein/
chitosan composite lms used for further studies (Table 1).
3.1. The thickness of zein/chitosan composite lms
The thickness of the six types of zein/chitosan composite lms
was between 0.103 0.003 mm to 0.113 0.009 mm (Table 2). No
signicant difference in lm thickness was detected among all
types of zein/chitosan composite lms containing different
phenolic compounds and dicarboxylic acids. Our results were in
lu
consistent with a previous report by Arcan and Yemeniciog
(2011); gallic acid at various concentrations did not signicantly
affect the thickness of zein lms. The interaction between chitosan
and carboxylic acids is determined by ionic interaction between
functional groups of chitosan and carboxylic acids and hydrophobic
interactions between internal domains of chitosan helices and the
hydrocarbon chain of carboxylic acids (Shamov et al., 2002). The
results suggest that phenolic compounds could be distributed in
the lm matrix without affecting the lm thickness. Moreover,
dicarboxylic acids might interact with amino groups in chitosan
molecular structure through the formation of ionic bonds and held
the chitosan molecules close to each other.
3.2. Water vapor transmission

2.8. Antimicrobial activity


Staphylococcus aureus BCTC13962 and Escherichia coli
BCRC10675 were obtained from the Bioresource Collection and

The water vapor transmission of zein/chitosan composite lms


is shown in Table 2. As compared to the composite lms without
phenolic compounds (A lm and S lm), signicant decrease of

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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

Film formation solution containing zeinb

Notation of zein/chitosan composite lm

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

without dicarboxylic acid


containing 0.5% adipic acid
containing 0.5% adipic acid
containing 0.5% adipic acid
containing 0.5% succinic acid
containing 0.5% succinic acid
containing 0.5% succinic acid

dissolved
dissolved
dissolved
dissolved
dissolved
dissolved
dissolved

in
in
in
in
in
in
in

ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol

with phenolic compound


without phenolic compound
containing ferulic acidc
containing gallic acidc
without phenolic compound
containing ferulic acidc
containing gallic acidc

0.3 g Chitosan dissolved in 30 ml mixed acid solution.


0.9 g Zein and 257 ml glycerol dissolved in 10 ml 95% ethanol.
Phenolic compound used at the level of 246 mg/g zein.

water vapor transmission was detected for the composite lms


containing phenolic compounds (AF lm, AG lm, SF lm and SG
lm). Since dicarboxylic acid could hold chitosan molecules
together, the less hydrophilic phenolic compounds could further
prevent the diffusion of water vapor through the composite lm
matrix. With regard to the effect of dicarboxylic acids, better water
vapor barrier capacity was detected for composite lms prepared
with adipic acid (AF lm and AG lm) than prepared with succinic
acid (SF lm and SG lm). It might be explained by that adipic acid
with larger molecular weight and longer carbon chain is more
hydrophobic than succinic acid. On the other hand, considering the
effect of phenolic compounds on water vapor transmission, addition of gallic acid or ferulic acid made the lms less permeable,
which might be accounted by less hydrophilic characteristic of
phenolic compounds. However, the effect of phenolic compounds
seemed to be affected by the dicarboxylic acid used in the lm
forming solutions.
Water vapor barrier property of edible lm is affected by various
factors including the nature of lm forming material, type of
plasticizer/additive and lm preparation process. Ghanbarzadeh
et al. (2006) reported that the water vapor transmission was
inuenced by hydrophilic-hydrophobic characteristic of the lm
forming materials and the steric hindrance of lm molecular
structure. It is reported the water vapor transfer process depended
on the simultaneous actions of water diffusivity and solubility in a
polymeric matrix (Zhong, Song, & Li, 2011). The types of plasticizers
used in the biopolymer lms also inuenced the water vapor barrier properties (Tillekeratne & Easteal, 2000). Water vapor barrier
property of chitosan lm was inuenced by the organic acid used to
dissolve chitosan (Park, Marsh, & Rhim, 2002). Kim et al. (2006)
reported the similar results that acid types (formic, acetic of propionic acids) signicantly affected the water vapor barrier property
of chitosan lms.
3.3. Color analysis
In practical applications, color quality might inuence the
appearance of edible lms which in turn affect the acceptance of
foods to consumers. The L*, a* and b* values of composite lms are

shown in Table 2. The composite lms without phenolic acid


possessed the highest brightness (expressed by relative high L*
value); for example the L* values for A lm and S lm were
65.41 1.04 and 55.08 3.06, respectively. With the addition of
phenolic compounds, the L* values of composite lms (AF lm, AG
lm, SF lm and SG lm) signicantly decreased. The results reect
the fact that composite lms became darker when phenolic compounds were included in the lm formulation. Du, Olsen, AvenaBustillos, Friedman, and McHugh (2011) reported that the inclusion of phenolic compounds darkened the edible lms. As
compared between adipic acid and succinic acid, the reduction of
brightness caused by the former was greater than that caused by
the latter. As a general trend, the addition of phenolic compounds
caused a decrease of blueness for composite lms as indicated by
decreased b* values.
3.4. Mechanical properties
The mechanical properties of zein/chitosan composite lms are
shown in Table 3. As compared to composite lms without phenolic
compounds (A lm and S lm), all tested parameters of the composite lms were signicantly increased when phenolic compounds were included in the lm forming formulation (AF lm, AG
lm, SF lm and AG lm).
The tensile strength of composite lms containing succinic acid
(SF lm and SG lm) was higher than composite lms containing
adipic acid (AF lm and AG lm). With regard to tensile strength,
the results indicated that succinic acid is the better dicarboxylic
acid than adipic acid for fabrication of stronger composite lms.
The break value and break strength were also increased when
phenolic compounds were incorporated into the composite lms.
For break value and break strength, SF lms possessed the highest
values of 0.206 0.029 N and 0.216 0.029 MPa, respectively. It is
assumed that phenolic compounds serving as plasticizers
contribute to increase the break value and break strength. In the
case of maximum elongation, combination of gallic acid and
dicarboxylic acid conferred the highest values for the composite
lms (22.31 7.23% and 22.71 3.20% for AG lm and SG lm,
respectively). Less increase on the maximum elongation was

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

Water vapor transmission


(g mm/cm2 24 h)
0.071
0.022
0.022
0.085
0.054
0.036

0.007B
0.003E
0.004E
0.003A
0.007C
0.008D

Data were presented as mean S.D. of triplicate samples.


Data in the same column with different letter were signicantly different (p < 0.05).
a
The formulation of lm as described in Table 1.

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

Maximum force (N)

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

Tensile strength (MPa)


1.353
1.873
1.608
0.726
2.589
1.667

Break value (N)

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

Break strength (MPa)


0.088
0.157
0.137
0.049
0.216
0.147

0.029CD
0.069B
0.010BC
0.020D
0.029A
0.000B

Maximum elongation (%)


3.185
13.14
22.31
8.471
17.30
22.71

2.083D
6.323BC
7.231A
2.966CD
8.351AB
3.202A

Data were presented as mean S.D. of 5 samples.


Data in the same column with different letter were signicantly different (p < 0.05).
a
The formulation of lm as described in Table 1.

formed between protein and ferulic acid (Ou, Wang, Tang, Huang, &
Jackson, 2005).

detected for composite lms containing ferulic acid (13.14 6.32%


and 17.30 8.35% for AF lm and SF lm, respectively).
Acid types used to dissolve chitosan could inuence the mechanical properties of chitosan composite lms (Park et al., 2002;
Zhong et al., 2011). In polymeric lms, exibility and extensibility
are enhanced by using plasticizers because of reducing intermolecular forces and increasing the mobility of the molecular chains
(Tillekeratne & Easteal, 2000). The hydrophilic groups of phenolic
compounds decreased the hydrophobic interaction among zein
molecule and increased the molecular mobility that lowered the
brittleness and improved the exibility of zein lms (Arcan &
lu, 2011). Similar results were reported when galacYemeniciog
tose was used as the plasticizer of zein lms. This could be attributed to the increase of polymer chains association in lm matrix by
plasticization at low level of plasticizer (Ghanbarzadeh et al., 2006).
Furthermore, the tensile strength of chitosan lms prepared with
lactic acid was lower than that of chitosan lm prepared with acetic
acid (Kim et al., 2006).
Although dicarboxylic acids were designated to dissolve chitosan in this study, they might also interact with zein and affect the
general mechanical properties of the composite lms. Selling and
Sessa (2007) reported that polycarboxylic acids were used as
plasticizers for zein lms and affected the tensile strength without
forming cross link. However, multivalent acids were assumed to
serve as cross-linking agents and improved the wet strength of
cellulosic materials (Kim et al., 2006). Adipic acid and succinic acid
used in our study might exert the cross-linking activity for chitosan
but not for zein.
Tensile strength and elongation of chitosan lms were signicantly affected by the organic acid used to dissolve chitosan (Park
et al., 2002). Kim et al. (2006) reported the similar results that
acid types (formic, acetic of propionic acids) signicantly affected
the tensile strength and elongation at break of chitosan lms. The
addition of plasticizer reduced the tensile strength and increased
the elongation of zein lms (Paramawati et al., 2001). The elongation at break was increased when ferulic acid was added into
protein-based edible lm; and it was assumed that cross-link was

3.5. The recovery of phenolic acids and dicarboxylic acids from


composite lms
The recovery of phenolic acids and dicarboxylic acids from
composite lms is shown in Table 4. While the recovery of ferulic
acid was greater than 71%, the recovery of gallic acid was greater
than 77%. The high recovery indicated that the high retention of
phenolic compounds during the lm fabrication process. Arcan and
lu (2011) reported that about 88% gallic acid was
Yemeniciog
released from zein lm.
In general, the recovery of dicarboxylic acids was lower than
that of phenolic compounds. It is assumed that dicarboxylic acids
might have higher afnity to chitosan and protein. As for the
comparison of two dicarboxylic acids, succinic acid showed slightly
higher the recovery than that of adipic acid. Relatively low water
solubility might be the reason for low recovery rate.

3.6. Antioxidant activities


Ferulic acid has been used in protein-based edible lm to exert
antioxidant properties (Ou et al., 2005). Gallic acid incorporated
into zeinecarnauba wax composites lm showed antioxidant aclu, 2013). In this study,
tivity (nalan, Arcan, Kore, & Yemeniciog
DPPH and ABTS free radical scavenging activities of zein/chitosan
composite lms are shown in Table 4. Trolox, an antioxidant with
wide diversity of antioxidant activities, was used as the common
reference standard for these two antioxidant tests (Thaipong,
Boonprakob, Crosby, Cisneros-Zevallos, & Byrne, 2006). Composite lms containing no phenolic compounds (A lm and S lm)
basically showed no antioxidant activity. The addition of phenolic
compounds in lm forming formulation signicantly increased the
antioxidant activity of composite lms. As compared the effects
between ferulic acid and gallic acid on the antioxidant activity of

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

All data are presented as mean S.D. of triplicate samples.


a
The formulation of lm as described in Table 1.
b
Ferulic acid for AF lm and SF lm; gallic acid for AG lm and SG lm. The formulation for A lm and S lm did not contain phenolic compound.
c
Adipic acid for A lm, AF lm and AG lm; succinic acid for S lm, SF lm and SG lm.
d
For antioxidant activity, the results are expressed as mg Trolox equivalent/g lm. Data in the same column with different letter were signicantly different (p < 0.05).

120

S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121

chitosan has been generally regarded as better Gram () bacteria


inhibitor because of higher negative charge generally found on the
outer membrane of this type of bacteria, the antimicrobial activity
of phenolic compounds in edible lms might be contributed to the
effects (nalan et al., 2013). Moreover, the types of acid used to
prepare chitosan lm also inuenced the antimicrobial activity
(Chen & Zhao, 2012).
4. Conclusions
Edible lms or coatings are potential alternatives for food
packaging. Active packaging materials can be obtained when
functional ingredients are incorporated into edible lms and
coatings. A modern trend for developing active edible lms is to
combine different base materials and to incorporate multiple
functional ingredients. In this study, successful preparation of zein/
chitosan composite lms with improved mechanical properties and
water vapor barrier properties is demonstrated. Since the composite lms contained phenolic compounds and chitosan, the
composite lms exert multiple functionality including antioxidant
and antimicrobial activities. In the practical applications, the lm
forming formulation can be used either as edible lms or as edible
coatings to enhance food quality and safety.
Acknowledgments
The nancial support (Grant No. NSC102-2313-B-415-007) from
National Science Council, Taiwan is gratefully acknowledged.

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).

composite lms (AF lm vs. AG lm and SF lm vs. SG lm),


stronger activities were found when gallic acid was used.

3.7. Antimicrobial activities


S. aureus (a Gram-positive bacterium which is the causative
microorganism of food borne intoxication) and E. coli (a common
food sanitation indicator microorganism) are used as the representative microorganisms for detection of antimicrobial activity of
edible lms (Kanatt et al., 2013; Zhong et al., 2011). The inhibitory
effects of zein/chitosan composite lms on the growth of S. aureus
and E. coli are shown in Fig. 1. While the inhibition percentages for A
lm and S lm against E. coli and S. aureus were around 10e15% and
25e30%, the inclusion of phenolic compounds in composite lms
signicantly increased the antimicrobial activity as indicated by the
results found for AF lm, AG lm, SF lm and SG lm.
Chitosan was reported to inhibit the growth of both Grampositive and Gram-negative bacteria (Chen & Zhao, 2012; Katas
et al., 2011; Qi, Xu, Jiang, Hu, & Zou, 2004). On the other hand,
phenolic compounds are biocidal agents by interacting with the
surface of the cell and causing cell death through disintegration of
the cell membrane and release of the intracellular constituents
~ oz-Bonilla &
leading to cell death or inhibition of cell growth (Mun
ndez-Garca, 2012). It is interesting to note that the inhibitory
Ferna
activity of zein/chitosan lms against S. aureus was higher than that
against E. coli. The possible antimicrobial mechanism of chitosan is
that the interaction between anionic groups on bacterial cell surface and cationic chitosan molecules (Qi et al., 2004). Although

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