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: kzhou@wayne.edu (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