-
an
ho, Unreet,
Antimicrobial activity
d w, phs wsans ag
incorporating 0.5 g/100 g gallic acid. Inclusion of 0.5 g/100 g
gallic acid also signicantly decreased water
ediblesteadital pod con
Chitosan is a natural polysaccharide produced by deacetylationof
chitin, which is the structural element of the crustaceans
shell,insects cuticle and cell walls of fungi. Chitosan lms have
beensuccessfully developed and used for packaging foods such as
fruits,vegetables, and meats (Chien, Sheu, & Yang, 2007;
Darmadji &Izumimoto, 1994; Moreira et al., 2011). The elastic
and transparent
oo, & Chen, 2008).lm against food-sodium benzoate,oextend
the shelf-corporation of anantimicrobial ac-
rganic acids, inor-have been incor-
porated into plastic packaging materials (Suppakul et al.,
2003).However, because of environmental problems associated
withchemicals and plastics and the health concerns of the
consumers,extensive studies have been conducted to use natural
bioactiveagents including antimicrobial enzymes, essential oils,
bacteriocins,and phenolic compounds in biodegradable or edible
packagingmaterials (Coma, 2008; Ramos-Garcia et al., 2012; Vodnar,
2012).For instance, edible chitosan lms containing lactoferrin as a
nat-ural antimicrobial agent were developed and shown to
exhibit
* Corresponding author. Tel.: 1 313 577 3444; fax: 1 313 577
8616.
Contents lists availab
ce
w.e
LWT - Food Science and Technology 57 (2014) 83e89E-mail address:
[email protected] (K. Zhou).properties, such as antioxidant and
antimicrobial properties, beyondtheir essential mechanical
properties (Bajpai, Chand, & Chaurasia,2010; Suppakul, Miltz,
Sonneveld, & Bigger, 2003). Antimicrobialpackaging is
showingagreatpotential in the futureof
activepackagingsystemsthrough itspromisingproposed
impactonshelf-lifeextensionand food safety, via controlling
spoilage and the growth of pathogenicmicroorganisms (Moreira,
Pereda, Marcovich, & Roura, 2011). There-fore, research on new
functional edible and biodegradable packagingmaterials should yield
numerous potential applications.
the antiseptic level desired by packers (Ye, NeetFor example, to
enhance the efcacy of chitosanborne pathogens, nisin, potassium
sorbate, andhavebeen incorporated into the chitosancoating tlife of
frankfurters (Samelis et al., 2002). The inadditional antimicrobial
agent could enhance itstivity and expand the scope of its
application.
Different antimicrobial chemicals such as oganic gases, metals
or ammonium compoundsquality food products (Bravin, Peressini,
& Sensidoni, 2006). Newlydeveloped packaging materials often
have additional functional
(Durango et al., 2006). However, for certain food products,
thelimited antimicrobial activity of pure chitosan lms does not
reachMechanical propertiesEdible lm
1. Introduction
The interest in the development offor food packaging has
recently beennicant concerns about environmenbiodegradable
packaging materials an0023-6438/$ e see front matter 2013 Elsevier
Ltd.http://dx.doi.org/10.1016/j.lwt.2013.11.037vapor permeability
(WVP) and oxygen permeability (OP). Microstructure of the lms was
investigated byFourier transform infrared spectroscopy (FT-IR) and
scanning electron microscopy (SEM) and it wasfound that gallic acid
was dispersed homogenously into the chitosan matrix.
2013 Elsevier Ltd. All rights reserved.
and biodegradable lmsly increasing due to sig-llution caused by
non-sumer demand for high
chitosan lms are known for their solid mechanical properties
andselective permeability for gases (Pereda, Amica, &Marcovich,
2012).Moreover, they are less sensitive to water in comparison
withhydroxylpropyl methylcellulose lms (Sebti, Chollet,
Degraeve,Noel, & Peyrol, 2007). These non-toxic, biodegradable,
andbiocompatible lms also have unique antimicrobial
propertiesChitosanGallic acidKeywords:subtilis. Chitosan lms
incorporated with 1.5 g/100 g gallic acid showed the strongest
antimicrobialactivity. It was also found that tensile strength (TS)
of chitosan lm was signicantly increased whenThe antimicrobial,
mechanical, physicalof chitosanegallic acid lms
Xiuxiu Sun a, Zhe Wang b, Hoda Kadouh a, Kequan ZaDepartment of
Nutrition and Food Science, Wayne State University, Detroit, MI
48202b School of Biological and Agricultural Engineering, Jilin
University, No. 5988 Renmin St
a r t i c l e i n f o
Article history:Received 5 August 2013Received in revised form18
November 2013Accepted 27 November 2013
a b s t r a c t
Chitosan lms incorporateantimicrobial, mechanicalcontaminate
food productprepared gallic acidechitocrobial activities of the
lm
LWT - Food Scien
journal homepage: wwAll rights reserved.d structural
properties
u a,*
ited StatesChangchun, Jilin 130025, China
ith various concentrations of gallic acid were prepared and
investigated forysical and structural properties. Four bacterial
strains that commonlyere chosen as target bacteria to evaluate the
antimicrobial activity of thelms. The incorporation of gallic acid
signicantly increased the antimi-ainst Escherichia coli, Salmonella
typhimurium, Listeria innocua and Bacillus
le at ScienceDirect
and Technology
lsevier .com/locate/ lwt
-
e ansignicant antimicrobial activity against both Listeria
mono-cytogenes and Escherichia coli O157:H7 (Brown, Wang, & Oh,
2008).Chitosan-based formulations with lime or thyme essential
oil,beeswax, and oleic acid were found effective in inhibiting E.
coliDH5a (Ramos-Garcia et al., 2012). Others have incorporated
oleo-resins and tea extracts into chitosan lms to improve their
anti-microbial activity against L. monocytogenes (Vodnar,
2012).
The use of phenolic compounds and extracts in active
packagingattracts a particular interest since these compounds show
potentantimicrobial activity in food systems and their intake can
make acontribution to human health (Komes, Horzic, Belscak, Ganic,
&Vulic, 2010). Gallic acid is a widely available phenolic acid
thathas been shown to possess strong antimicrobial
activity(Chanwitheesuk, Teerawutgulrag, Kilburn, &
Rakariyatham, 2007).Gallic acid extracted from Caesalpinia
mimosoides Lamk (Legumi-nosae) exhibited the activity against the
bacteria Salmonella typhiand Staphylococcus aureus with MIC values
of 2.50 and 1.250 g/L,respectively (Chanwitheesuk et al., 2007).
Gallic acid puried fromthe owers of Rosa chinensis Jacq. has also
been shown to possessignicant antibacterial activity against
pathogenic Vibrios species(Li et al., 2007). All of these reports
in the literature have indicatedpromising potential in using gallic
acid to develop antimicrobialpackaging materials against pathogens
and spoilage bacteria.
In addition, gallic acid appears to enhance elasticity, thus
actingas a plasticizer and eliminates classical brittleness and
exibilityproblems (Alkan et al., 2011; Hager, Vallons, &
Arendt, 2012). Gallicacid incorporation during the formation of
chitosanegallic acidpolymers yielded a conjugate with a superior
hydroxyl radicalscavenging capacity (Pasanphan, Buettner, &
Chirachanchai, 2010).This is an encouraging aspect of gallic acid
used in manufacturingfood packaging chitosan lms. Thus, our purpose
is to evaluate thepotential to develop a new cost-effective edible
chitosan lm withimproved antimicrobial and mechanical properties by
incorpo-rating a widely accessible natural antimicrobial
compound.
2. Materials and methods
2.1. Film-making materials
Chitosan (95e98% deacetylated, MV 8.0 105 Da) (Moreiraet al.,
2011) and glacial acetic acid (99%, analytical reagent grade)were
obtained from SigmaeAldrich Co. (St. Louis, MO, USA); Glyc-erol, as
a plasticizing agent, and gallic acid, as an antimicrobial
agent,were purchased from Fisher Scientic Inc. (Pittsburgh, PA,
USA).
2.2. Film preparation
The edible lms were prepared by dissolving 1 g of chitosan in100
g of 1% acetic acid solution and stirred, at room temperature,until
chitosanwas completely dissolved. Glycerol at 0.3 g/100 g wasadded
as a plasticizer. Film without gallic acid was designated aslm 0
(F0) which was used as a control. Gallic acid was added atvarying
concentrations: 0.5 g/100 g in lm 1 (F1), 1.0 g/100 g in lm2 (F2)
and 1.5 g/100 g in lm 3 (F3), respectively. Equal volumes(150 mL)
of the lm solutions were spread on glass plates(200 200 mm) and
dried for 12 h at 35 2 C in an incubator(New Brunswick Scientic
Excella* E24Fisher Scientic Inc. PA,USA). The lms were removed from
the glass plate with a thinspatula and conditioned at 23 2 C and 50
2% relative humidity(RH) before running further tests.
2.3. Bacterial strains and cultures
Two gram-negative bacteria: E. coli 0157:H7 (ATCC 43895) and
X. Sun et al. / LWT - Food Scienc84Salmonella typhimurium (ATCC
19585) and 2 g-positive bacteria:Bacillus subtilis (ATCC 1254) and
Listeria innocua (F4078) were used.E. coli was incubated in
LuriaeBertani (LB) broth media, B. subtilisand L. innocua were
incubated in Nutrient broth media, andS. typhimurium was incubated
in Brain-heart infusion (BHI) brothmedia at 37 C for 24 h.
2.4. Antimicrobial activity
Antimicrobial properties of the crafted lms were determinedby
the log reduction method with a slight modication(Ravishankar, Zhu,
Olsen, McHugh, & Friedman, 2009). Briey,culture medium broth
was inoculated with certain amount ofsuspension of bacteria. The
bacterial concentration in the seedingculture was approximately 6
108 CFU/mL. Serial dilutions of thesuspension were performed and
the optical density values weretested to achieve a standard curve.
Square lm pieces (20 20mm)were sterilized and introduced into a
test tube containing 5 mLfresh suspension of bacteria and incubated
at 37 C for 24 h. Opticaldensity of culture media was measured at
620 nm using a PerkineElmer HTS 7000 Bio Assay reader, and cell
concentrations weredetermined. All samples/standards were run in
triplicates.
2.5. Film thickness (FT)
FT was measured with a 0e25 mm dial thickness gage with
anaccuracy of 0.01 mm in ve random locations for each lm. Av-erages
were calculated for mechanical properties, water vaporpermeability
and oxygen permeability.
2.6. Mechanical properties
Tensile strength (TS) and elongation at break (EB) tests
wereperformed at room temperature (23 2 C) using a universaltesting
machine (PARAM XLW (B) Auto Tensile Tester, Jinan, China)with a 200
N load cell according to the standard testing methodASTMD882-01
(ASTM, 2001). Sample lms, previously equilibratedat 23 2 C and 50
2% RH, were cut into strips 15 mmwide and130mm long. Five specimens
from each lmwere tested. The initialgrip separation andmechanical
crosshead speed were set at 80mmand 50 mm/min, respectively.
TS (MPa) was calculated using the following equation:TS Fmax/A;
where Fmax is the maximum load (N) needed to pull
the sample apart; A is cross-sectional area (m2) of the
samples.EB (%) was calculated using the following equation:EB L=80
100; where L is the lm elongation (mm) at the
moment of rupture; 80 is the initial grip length (mm) of
samples.
2.7. Physical properties
2.7.1. Water vapor permeability (WVP)The WVP of the lms was
determined by a Water Vapor
Permeability Tester (PERME TSY-TIL, Labthink Instruments Co.,
Ltd,Jinan, China) according to the standard testing method
ASTME96-95 (ASTM, 1995). Test cups were 2/3 lled with
distilledwater. The test cups were tightly covered with circular lm
sam-ples. Difference in water vapor pressure between the inside
andoutside of the cup causes water vapor diffusion through the
sample.For each sample, ve replicates were tested. The weight of
the cupswas measured at 1 h intervals for 24 h. Simple linear
regressionwasused to estimate the slope of weight loss versus time
plot.
WVP (g m1 s1 Pa1) was calculated using the followingequation
(Sztuka & Kolodziejska, 2009): WVP (WVTR L)/Dp;whereWVTR (water
vapor transmission rate) is slope/lm test area(g/m2 s); L is lm
thickness (m); Dp is partial water vapor pressure
d Technology 57 (2014) 83e89difference (Pa) between the two
sides of the lm.
-
2.7.2. Oxygen permeability (OP)OP of the lms was determined by a
Gas Permeability Tester
(GDP-C) (Brugger Feinmechanik GmbH, Germany) according to
thestandard testing method ASTM D3985-05 (ASTM, 2005). An ediblelm
was mounted in a gas transmission cell to form a sealed
semi-barrier between chambers. Oxygen enters the cell on one side
ofthe lm from a chamber which is at a specic high pressure
andleaves from the other which is at a specic lower pressure with
acontrolled ow rate (100 mL/min). The lower pressure chamberwas
initially evacuated and the transmission of oxygen through thetest
specimen was indicated by an increase of pressure. For eachsample,
at least ve replicates were tested. OP (mol m1 s1 Pa1)was
calculated using the following equation (Ayranci &
Tunc,2003):
OP (M L)/(A T Dp); where M is the volume of gaspermeated through
the lm (mol); L is lm thickness (m); A is thearea of the exposed lm
surface (m2); T is the measured time in-terval (s); Dp is
difference (Pa) between the two sides of the lm.
2.8. Microstructure properties
2.8.1. Fourier transform infrared spectroscopy (FT-IR)FT-IR was
recorded on a Spectrum 400 FT-IR spectrometer
(PerkinElmer Inc., USA). Films were placed on the steel plate
andmeasured directly in a spectral range of 650e4000 cm1 at
theresolution of 4 cm1, and the average of 128 scans was taken
foreach sample.
examined at an accelerating voltage of 15 KV and
magnied10,000.
2.9. Statistical analysis
Analysis of variance (ANOVA) was carried out using SPSS
soft-ware (version 17). When the p-value was less than or equal to
0.05,the results were considered signicant.
3. Results and discussion
3.1. Antimicrobial properties
To examine the antimicrobial properties of the studied
ediblelms, E. coli, S. typhimurium, B. subtilis, and L. innocua,
which arevery signicant pathogens in the food industry, were
tested. Theresults are shown in Fig. 1. The edible lms incorporated
withdifferent concentrations of gallic acid signicantly improved
theantimicrobial activities of the chitosan lm against all the
testedbacteria (p < 0.05). The log reduction increases with the
increase ofgallic acid concentration, which illustrates the
antimicrobial ac-tivity of gallic acid.
The results show that the log reductions of B. subtilis,
rangedfrom 1.24 to 5.75, are demonstrated to be higher than other
bac-teria. The minimum inhibitory concentration (MIC) of
chitosanagainst B. subtilis is 0.10 g/L (Yadav & Bhise, 2004).
The log re-ductions of E. coli ranges from 0.57 to 2.31. The MIC of
chitosan
ly lacid;
X. Sun et al. / LWT - Food Science and Technology 57 (2014)
83e89 852.8.2. Scanning electron microscopy (SEM)The lms were cut
into small pieces (10 10 mm), dried and
mounted on aluminum stubs using a double-sided adhesive
carbontape and sputtered with a thin layer of gold. Microstructures
of thesurface and cross-section of the dried lms were observed by
aScanning ElectronMicroscope (SEM, JSM-6510LV-LGS, JEOL Co.,
Ltd.USA) and Field Emission Scanning Electron Microscope
(FESEM,JSM-7600F, JEOL Co., Ltd. USA), respectively. All samples
were
Fig. 1. Antimicrobial properties of the edible gallic
acidechitosan versus chitosan-onS. typhimurium (d)). F0 represents
the edible lm casted from chitosan without gallic
represents edible lm casted from chitosan with 1.0 g/100 g
gallic acid (w/v); F3 representsletters indicate signicant
difference (p < 0.05).against E. coli is 0.75 mg/mL (Tao, Qian,
& Xie, 2011) and gallic aciddemonstrated signicant
antimicrobial activity against E. coli(MIC 1 g/L) (Binutu &
Cordell, 2000). Combining gallic acid withchitosan shows a potent
antimicrobial effect according to our re-sults. The log reductions
of S. typhimurium ranged from 1.07 to 1.75.Furthermore, the
combination of gallic acid in chitosan lmsexhibited obvious
reduction in the growth of L. innocua, resulting inan approximate
2.5-log reduction. Listeria growth inhibition wasrecorded for
gallic acid at 0.45 g/L (Aissani, Coroneo, Fattouch, &
ms (The log reduction of cell number of B. subtilis (a), L.
innocua (b), E. coli (c), andF1 represents edible lm casted from
chitosan with 0.5 g/100 g gallic acid (w/v); F2
edible lm casted from chitosan with 1.5 g/100 g gallic acid
(w/v). Bars with different
-
Caboni, 2012). The diameters of the zone of inhibition (mm)
ofchitosan against E. coli and B. subtilis were 18 mm and 40
mmrespectively (Yadav & Bhise, 2004), which veried that B.
subtilis ismore sensitive than E. coli to chitosan.
Furthermore, the lm showed a higher effectiveness againstB.
subtilis and L. innocua compared to E. coli and S. typhimuriumwhich
may be rationalized by the characteristic difference of theouter
membrane between Gram-positive bacteria and Gram-negative bacteria
(Ramos et al., 2012).
3.2. Mechanical properties
20% to 6% of chitosan lms indicated that the incorporation
ofcellulose whiskers into the chitosan matrix resulted in strong
in-teractions betweenmatrix and ller, which restricted the motion
ofthe matrix (Li, Zhou, & Zhang, 2009).
3.3. Physical properties
3.3.1. Water vapor permeability (WVP)Table 2 shows there was a
signicant difference between the
WVP values of F0eF3 lms incorporated with different gallic
acidconcentrations (p < 0.05). When the added gallic acid was
below1.0 g/100 g, the WVP values of the lms decreased
signicantly
X. Sun et al. / LWT - Food Science and Technology 57 (2014)
83e8986Mechanical properties are important to edible lms,
becauseadequate mechanical strength ensures the integrity of the lm
andits freedom from minor defects (Murillo-Martinez,
Pedroza-Islas,Lobato-Calleros, Martinez-Ferez, & Vernon-Carter,
2011). Table 1shows mechanical property values of four edible lms
after con-ditioning at 23 2 C and 50 2% RH. Differences in the TS
and EBof F0, F1, F2 and F3 were observed and could be attributed to
theaddition of gallic acid interacting with chitosan and forming
newlinkages that affect lm structure.
Our chitosan control lm (F0) had TS and EB values of13.876 MPa
and 32.36%, respectively (Table 1). These values arecomparable to
the previous reports with TS and EB in the range of12e20 MPa and
17e42%, respectively (Vargas, Albors, Chiralt,
&Gonzalez-Martinez, 2009). The TS and EB of chitosan lms
areaffected by the type of chitosan used, the presence of glycerol,
andthe temperature during lm drying (Pereda et al., 2012).
Interest-ingly, the incorporation of 0.5 g/100 g and 1.0 g/100 g
gallic acid intochitosan lms signicantly increased its TS (P <
0.05). The additionof a relatively lower dose of gallic acid (F1)
exhibited the highest TSamong the lms, which could be attributed to
the formation ofintermolecular hydrogen bonding between the NH3 of
the chitosanbackbone and the OH- of gallic acid (Sun et al., 2011).
The inter-molecular hydrogen bonding between chitosan and gallic
acidcould enhance the cross-linkage, which decreases the
molecularmobility and the free volume of chitosan (Pasanphan
&Chirachanchai, 2008). This phenomenon was reported by
otherresearchers in similar systems. For example, the cross-linking
ofchitosaneolive oil emulsion as well as chitosaneoleic acid
lmsresulted in an increased TS due to the enhancement of the
struc-tural bonds in the polymer network (Pereda et al., 2012;
Vargaset al., 2009). However, when the added concentration of
gallicacid is higher than 0.5 g/100 g, the TS of the resulting
lmsdecreased with increasing gallic acid concentration. As we can
see,the TS of F3 (9.207 MPa) was lower than that of F0 (13.876
MPa). Itis possible that the excessive gallic acid scattered in the
lm crackthe inner structure of the lm (Figs. 3d and 4d).
The decrease of EB values in F1eF3 lms indicated that
theincorporation of gallic acid into the chitosan lm resulted in
astrong reaction between ller and matrix, which decreased EB bythe
motion restriction of the matrix. The decreased EB values from
Table 1Mechanical properties of the edible gallic acidechitosan
and chitosan-only lms.
Film code FT (mm) TS (MPa) EB (%)
F0 0.107 0.006b 13.876 0.604c 32.36 1.18aF1 0.108 0.009b 23.773
0.453a 33.15 2.53aF2 0.111 0.001b 18.394 1.405b 25.56 0.58bF3 0.141
0.001a 9.207 0.616d 10.97 0.95c
F0 represents edible lm casted from chitosan without gallic
acid; F1 representsedible lm casted from chitosan with 0.5 g/100 g
gallic acid (w/v); F2 representsedible lm casted from chitosan with
1.0 g/100 g gallic acid (w/v); F3 represents
edible lm casted from chitosan with 1.5 g/100 g gallic acid
(w/v). Superscripts insame column with different letters indicate
signicant differences (p < 0.05).(p < 0.05) with increasing
gallic acid concentrations, which couldbe because the bulky benzene
ring group of gallic acid obstructs theinter- and intra-molecular
hydrogen bond network of chitosan(Pasanphan & Chirachanchai,
2008). However, when the concen-tration of gallic acid was higher
than 1.0 g/100 g, the WVP of thelm increased (p < 0.05), which
may be related to the excessivegallic acid scattered in the lm
(Figs. 3d and 4d) which subse-quently decreased the intermolecular
forces between polymerchains and increased the free volume and
segmental motions(Sothornvit & Krochta, 2001). In addition,
carboxyl groups andhydroxyl groups of gallic acid are hydrophilic
groups, which mightpromote water transfer in the matrix
(Sanchez-Gonzalez, Chafer,Chiralt, & Gonzalez-Martinez,
2010).
The WVP values of our crafted lms were in the similar range
ofthe previous reports (Pereda et al., 2012; Sanchez-Gonzalez et
al.,2010). In general, the WVP of chitosan lms is lower than that
ofcorn-zein lm and wheat gluten lm, but higher than that
ofhydroxypropylmethyl cellulose lm (Park & Chinnan,
1995).Nonetheless, the WVP values of the lms are all in the order
of1010 g m s1 m2 Pa1, which are qualied for preventingmigration of
moisture from fruits or vegetables.
3.3.2. Oxygen permeability (OP)Oxygen is an essential component
of lipid oxidation, which
decreases food quality and shortens shelf life (Sothornvit &
Krochta,2000). The OP values of the chitosan edible lms are shown
inTable 2. The incorporation of gallic acid into the lms plays
animportant role in the improvement of OP. From the results, the
OPvalue of F1 is the lowest, which is signicantly different from
otherlms (p< 0.05). The OP value of F3 is 1.39 1018 mol m1 s1
Pa1,being the highest, indicates that F3 is not qualied for good
oxygenprevention properties compared with the other lms. The high
OPvalue of F3 might be due to the non-cross-linking gallic acid
par-ticles scattered in the lm which may have decreased the
inter-molecular forces between polymer chains, thus increasing the
freevolume and segmental motions (Sothornvit & Krochta, 2001),
andresulting in the formation of pores. This result can also be
veriedby Figs. 3d and 4d, where obvious pores are shown. The OP
valuesof these lms ranging from 0.50 to 1.46 1018 mol m1 s1 Pa1
Table 2WVP and OP of the edible gallic acidechitosan and
chitosan-only lms.
Film code FT (mm) WVP (g m1 s1
Pa1) 1010OP (mol m1 s1
Pa1) 1018
F0 0.107 0.006b 2.52 0.03b 1.35 0.03aF1 0.108 0.009b 2.24 0.05c
0.56 0.06cF2 0.111 0.001b 2.23 0.04c 0.90 0.03bF3 0.141 0.001a 3.71
0.07a 1.39 0.07a
F0 represents edible lm casted from chitosan without gallic
acid; F1 representsedible lm casted from chitosan with 0.5 g/100 g
gallic acid (w/v); F2 representsedible lm casted from chitosan with
1.0 g/100 g gallic acid (w/v); F3 represents
edible lm casted from chitosan with 1.5 g/100 g gallic acid
(w/v). Superscripts insame column with different letters indicate
signicant differences (p < 0.05).
-
show a better oxygen prevention property compared to wheatgluten
lm (34.6 1018 mol m1 s1 Pa1) and soy protein lm(31.5 1018 mol m1 s1
Pa1) (Choi & Han, 2002; Mehyar & Han,2004).
3.4. Microstructure properties
3.4.1. Fourier transform infrared spectroscopy (FT-IR)FT-IR
spectroscopy was employed to analyze the hydrogen
bonds in the lms. The FT-IR spectra of control lms and
lmscontaining gallic acid were shown in Fig. 2. Fig. 2a shows the
F0 lmspectrum, which is similar to the chitosan lms developed
byothers (Li et al., 2009).
To facilitate the coupling reaction with primary amine groups
inchitosan, the carboxylic group of gallic acid is activated by
con-verting the carboxylic acid group into ester, as reported
previously(Lee et al., 2005). Gallic acid could be conjugated at
C-2 to obtain anamide linkage, or at C-3 and C-6 to obtain an ester
linkage(Pasanphan & Chirachanchai, 2008). The spectra of F1, F2
and F3lms showed signicant peaks around 1700 cm1 and 1640 cm1,while
F0 did not. These peaks correspond to ester and amidelinkages
between chitosan and gallic acid, respectively (Pasanphan&
Chirachanchai, 2008). Detected ester and amide linkages areunlikely
due to either gallic acid or chitosan individually (Yu et
al.,2011). These results suggest the conjugation of the gallate
groupwith chitosan in the lms. A sharp peak at 3267 cm1, detected
onlyin F3 but not in the other lms, corresponds to eOH group.
The
Fig. 2. FT-IR spectra of the edible gallic acid-chitosan and
chitosan-only lms (a.represents the edible lm casted from
chitosanwithout gallic acid; b. represents ediblelm casted from
chitosan with 0.5 g/100 g gallic acid (w/v); c. represents edible
lmcasted from chitosan with 1.0 g/100 g gallic acid (w/v); d.
represents edible lm castedfrom chitosan with 1.5 g/100 g gallic
acid (w/v)).
Fig. 3. SEM of surface of the edible gallic acidechitosan and
chitosan-only lms (a. represecasted from chitosanwith 0.5 g/100 g
gallic acid (w/v); c. represents edible lm casted from cwith 1.5
g/100 g gallic acid (w/v)).
X. Sun et al. / LWT - Food Science and Technology 57 (2014)
83e89 87nts the edible lm casted from chitosan without gallic acid;
b. represents edible lm
hitosanwith 1.0 g/100 g gallic acid (w/v); d. represents edible
lm casted from chitosan
-
peaks at 1610 cm1, 1201 cm1 and 1021 cm1 referred to the
C]O,CeO, and OeH respectively. These peaks demonstrated the
pres-ence of eCOOH in F3, which indicates the existence of
excessivegallic acid in F3. From these results, it can be concluded
that thegallate group of gallic acid was successfully cross-linked
with chi-tosan via amide and ester linkages for F1 and F2, though
there wasmore than enough unreacted gallic acid in F3 (Figs. 3d and
4d).
3.4.2. Scanning electron microscopy (SEM)SEMwas employed to
observe thelms surfacemorphologyand
cross-section as well as the homogeneity of the composite,
thepresenceof voids, and thehomogeneous structureof thelms (Khanet
al., 2012). The surface and cross-sectionmorphologies of thelmsare
shown in Figs. 3 and 4, respectively. Fig. 3a and b shows aat
andsmooth appearance and a good compact structure of the F0 and
F1lms, respectively,which indicates that themixtures of chitosan
andglycerol, aswell as chitosan, glycerol and gallic acid are
homogenousin these lms. This is further supported by Fig. 4a and b,
where thecross-sectionmorphologies of both F0 and F1 lms are also
smooth.In Fig. 3c, the appearance of a white spot suggests some
heteroge-neity in the chitosan matrix when gallic acid was
incorporated intochitosan. This phenomenon is further veriedbyFig.
4c,where somebands are presented. Fig. 3d and 4d show abundant
plaques andobvious pores which interrupt the inner structure of the
lm (F3),therefore reducing the tensile strength and elongation at
break by33.6% and 66.1% compared to the pure chitosan lm (F0),
respec-tively. The interrupted inner structure also affects the
permeabilityof the lm (F3): the water vapor permeability and oxygen
perme-ability were increased by 47.2% and 3.0%, respectively.
Overall, these
gures suggest that thelmswith lower concentrationsof gallic
acid(F1 and F2) have bettermechanical and barrier properties
comparedto the lm added with 1.5 g/100 g gallic acid (F3).
Meanwhile, ourresults agreewith the concept that surface properties
are importantto the barrier properties oflms,where a homogeneous
and smoothsurface is usually preferred (Wang et al., 2013). Water
permeabilityand moisture sensitivity of edible lm were directly
affected by itssurface properties and hydrophobicity (Wu, Sakabe,
& Isobe, 2003).For instance, lms casted from unmodied zein
showed higherwater permeability and moisture sensitivity than
modied zeinlms partially because the former lms had larger water
surfacecontact angles, while the modied zein lms had stronger
surfacehydrophobicity through the acylation reaction (Shi, Huang,
Yu, Lee,& Huang, 2011).
4. Conclusions
The results of this study suggest that chitosan lms
incorpo-rated with gallic acid improved the antimicrobial
properties ofthe lm signicantly, and the lms reduced microbial
growth by2.5-log reduction. Furthermore, incorporation of lower
concen-trations of gallic acid (0.5 g/100 g) increased the TS of
the chi-tosan lm by 71.3%. It also improved the barrier properties
ofchitosan lm by reducing WVP and OP by 11.1% and
58.5%,respectively. Surface morphology of the lm with lower
gallicacid concentration revealed a homogeneous structure.
Overall,chitosan lms with gallic acid could be used as novel
foodpackaging material due to their excellent antimicrobial
andmechanical properties.
s (a.
X. Sun et al. / LWT - Food Science and Technology 57 (2014)
83e8988Fig. 4. SEM of the cross-section of the edible gallic
acidechitosan and chitosan-only lm
lm casted from chitosan with 0.5 g/100 g gallic acid (w/v); c.
represents edible lm castedchitosan with 1.5 g/100 g gallic acid
(w/v)).represents the edible lm casted from chitosan without gallic
acid; b. represents edible
from chitosan with 1.0 g/100 g gallic acid (w/v); d. represents
edible lm casted from
-
Acknowledgments
Authors recognize and appreciate the nancial support from
by protein-polysaccharide complexes for producing edible lms:
rheological,mechanical and water vapour properties. Food
Hydrocolloids, 25(4), 577e585.
Park, H. J., & Chinnan, M. S. (1995). Gas and water-vapor
barrier properties of ediblelms from protein and cellulosic
materials. Journal of Food Engineering, 25(4),497e507.
X. Sun et al. / LWT - Food Science and Technology 57 (2014)
83e89 89Wayne State University Graduate Research Fellow (UGRF).
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The antimicrobial, mechanical, physical and structural
properties of chitosangallic acid films1 Introduction2 Materials
and methods2.1 Film-making materials2.2 Film preparation2.3
Bacterial strains and cultures2.4 Antimicrobial activity2.5 Film
thickness (FT)2.6 Mechanical properties2.7 Physical properties2.7.1
Water vapor permeability (WVP)2.7.2 Oxygen permeability (OP)
2.8 Microstructure properties2.8.1 Fourier transform infrared
spectroscopy (FT-IR)2.8.2 Scanning electron microscopy (SEM)
2.9 Statistical analysis
3 Results and discussion3.1 Antimicrobial properties3.2
Mechanical properties3.3 Physical properties3.3.1 Water vapor
permeability (WVP)3.3.2 Oxygen permeability (OP)
3.4 Microstructure properties3.4.1 Fourier transform infrared
spectroscopy (FT-IR)3.4.2 Scanning electron microscopy (SEM)
4 ConclusionsAcknowledgmentsReferences