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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 filmsfabricated by incorporation of phenolic compounds and dicarboxylicacids

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

Article history:Received 28 November 2014Received in revised form4 March 2015Accepted 10 March 2015Available online 18 March 2015

Keywords:ZeinChitosanComposite filmsAntioxidant activityAntimicrobial activity

* Corresponding author. Tel.: þ886 5 2717599; fax:E-mail address: ymweng@mail.ncyu.edu.tw (Y.-M.

http://dx.doi.org/10.1016/j.lwt.2015.03.0300023-6438/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

Edible films and coatings can not only function as barriers of water vapor, gases and volatile compoundsbut also serve as carriers of functional ingredients. Composite edible films fabricated with zein andchitosan as base materials and supplemented with phenolic compounds (ferulic acid or gallic acid) anddicarboxylic acids (adipic acid or succinic acid) were prepared. The composite films exerted better watervapor barrier and mechanical properties. While the recovery percentage of phenolic compounds fromcomposite films was 71e84%, the recovery of dicarboxylic acids was 48e65%. The composite films alsopossessed antioxidant activities as shown by DPPH and ABTS free radical scavenging tests. The antimi-crobial activity of composite filmwas demonstrated by using Staphylococcus aureus and Escherichia coli astest microorganisms.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Edible films and coatings offer the opportunity to effectivelycontrol mass transfer among different component within a foodsystem or between the food and its environment (Hernandez-Izquierdo & Krochta, 2008). Moreover, active packaging materialscan be obtained when functional ingredients are incorporated intoedible films and coatings (Rojas-Graü, Soliva-Fortuny, & Martín-Belloso, 2009). The functionalities of active packaging materialsinclude nutrient supplementation, antimicrobial activity, antioxi-dant activity, etc. The commonly used base materials for ediblefilms and coatings are polysaccharides (such as starch, cellulosederivatives, pectin and chitosan), proteins (such as gelatin, cerealproteins, milk casein and soy protein) and lipophilic materials (suchas glycerides, and bee wax); the above materials can be used eitherindividually or in combination (Nesterenkoa, Alric, Silvestre, &Durrieu, 2013). Zein and chitosan with good film forming charac-teristics and relatively good barrier properties are commonly usedfor edible films and coatings (Bourtoom, 2008).

Zein is the major storage protein in corn and accounts about44e79% endosperm protein (Lawton, 2002; Paramawati, Yoshino,

þ886 5 2717596.Weng).

& Isobe, 2001). Because of the high composition of hydrophobicamino acids (such as leucine and alanine) and acidic amino acid(glutamic acid, 21e26%), zein does not dissolve in water and dilutesalt solution but dissolve in 60e95% ethanol (Chen, Ye,& Liu, 2014).Zein film exerts better barrier against transmission of water vaporand volatile components when compared with other types ofprotein-based edible films (Ozcalik & Tihminlioglu, 2013). How-ever, the brittleness is the major disadvantage of zein film. Theresearches have been conducted to incorporate plasticizers in filmforming formula to reduce the brittleness (Lawton, 2002;Paramawati et al., 2001; Tillekeratne & Easteal, 2000). Further-more, functional additives have also been included in zein to conferedible films with multiple activities. Phenolic compounds wereadded into zein to improve the mechanical properties and to in-crease the antioxidant activities (Arcan & Yemenicio�glu, 2011).Polyvalent organic acids had been added into zein forming mixtureto improve mechanical properties because of the interaction be-tween the terminal amino acids (serine or threonine) of zeinmolecules and the polyvalent organic acids (Selling & Sessa, 2007).

Chitosan, a copolymer of N-acetyl glucosamine and glucosaminelinked by b-1-4 linkage, is derived from chitin by partial deacety-lation. Chitin presents mainly in the fungal cell wall and theexoskeleton of crustaceans and insects (Bourtoom, 2008). Chitosandissolves in dilute organic acids such as formic acid, acetic acid,

S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121116

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 carboxylicgroup (eCOO�) can further form ionic bond with ammoniumgroups (eNH3

þ). The combination effects are presented as enhanceof solubility of chitosan in acid solution and increase of film me-chanical properties (Chen et al., 2007; Kim, Son, & Kim, 2006).Dicarboxylic organic acid used in chitosan film forming solutionswill further improve the mechanical properties since ionic-crosslinking among chitosan molecules can be established (Hamdineet al., 2005).

The antimicrobial activities of chitosan have been reported inthe literature (Chen & Zhao, 2012; Katas, Mohamad, & Zin, 2011).Under acidic conditions, the positively charged eNH3

þ groups ofchitosan will interact with the negatively charged bacterial cellcoverings and result in bactericidal effects through the interferenceof cell permeability and interrupting the integrity of cell structure(Chang, Chen, & Tan, 2011). Active food packaging materials basedon chitosan have been applied to foods to preserve qualities(Kanatt, Rao, Chawla,& Sharma, 2013). However, films and coatingsconstructed solely with polysaccharide, such as chitosan, are notgood water vapor and gas barriers because of the presence of hy-drophilic groups (Bourtoom, 2008).

A modern trend for developing active edible films is to combinedifferent base materials and to incorporate multiple functional in-gredients (Rojas-Graü et al., 2009). For example, chitosan has beenused to form composite films with polysaccharides (tapioca starch,hydroxypropyl methylcellulose, or pectin) and proteins (round scadprotein, whey protein, or fish gelatin) (Aider, 2010). Moreover, zeinand chitosan were used to form microspheres (Müller et al., 2011)or composite films (Escamilla-García et al., 2013; Li, Ren, Wang, Xu,& Wang, 2011). However, no research in the literature reported thecombination of zein/phenolic compounds and chitosan/dicarbox-ylic acids to fabricate composite films with antioxidant and anti-microbial activities. In the present study, composite films withacceptable physicochemical properties and possessing antioxidantand antimicrobial activities were successfully fabricated by usingzein/phenolic compounds and chitosan/dicarboxylic acids.

2. Materials and methods

2.1. Preparation of zein/chitosan composite films

Defatted zein was prepared according to the method describedby Padgetti, Han, and Dawson (2000). Briefly, zein (SigmaeAldrich,St. Louis, MO, USA) was mixed with 10 times of hexane (w/v) andstirred at room temperature for 2 h. After filtration, the residuehexane 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 deacetyla-tion 90%) was purchased from SigmaeAldrich (St. Louis, MO, USA)and used directly without pretreatments.

In previously published researches, phenolic compounds wereadded into zein to improve the mechanical properties and to in-crease the antioxidant activities (Arcan & Yemenicio�glu, 2011) anddicarboxylic organic acid was used in chitosan film forming solu-tions to improve the film mechanical properties (Hamdine et al.,2005). Based on the preliminary trials conducted in our labora-tory, the zein/chitosan composite film was prepared as follows. Aportion of 0.9 g zeinwas thoroughly dissolved in 10 ml 95% ethanol(Taiwan Tobacco andWine Co., Taipei, Taiwan). After the addition of257 ml glycerol (Choneye Pure Chemical, Taipei, Taiwan), themixture was heated (80 �C, 3 min) and cooled down to roomtemperature. Gallic acid or ferulic acid (SigmaeAldrich, St. Louis,

MO, USA) at the level of 246 mg/g zein was added into the zeinsolution. Acetic, succinic and adipic acids were purchased fromChem-Service (Chester, PA, USA). A portion of 0.3 g chitosan wasthoroughly dissolved in 30 ml 1% acetic acid containing either 0.5%adipic acid or 0.5% succinic acid. The zein and chitosan solutionswere degassed separately in the vacuum oven at room temperaturefor 1 h. And then, the chitosan solution was slowly added into zeinsolution with gentle mixing. The zein/chitosan composite film wasobtained by transferring the mixture into a plastic petri dish (id.14.5 cm) and drying in a gel drier (SE1200 Easy Breeze Air Gel-dryerSystem, Hoefer, San Francisco, CA, USA) for 24 h. All edible filmswere stored in a dehumidifier (SL-450CA, Moisture Buster, Tai-chung, Taiwan) at RH 40% and 25 �C.

2.2. Measurement of film thickness

The film thickness was measured with a thickness micrometer(SM-114, Teclock, Okaya, Japan). The thickness of each piece of filmwas the average of thickness of ten randomly selected spots. Thethickness of each type of film was represented by mean ± SD ofthree films.

2.3. Water vapor transmission

The measurement of water vapor transmission (WVT) wasconducted according to Tillekeratne and Easteal (2000) withmodifications. The film was fixed to a cup filled with approximate25 g dried silica gel beads. After weighted (W1), the cup was placedin a temperature and humidity control chamber (HRMB-80, TaichyTechnology Ltd., Nantou, Taiwan) at 25 �C and 75% RH for 24 h. Thecup was removed from the humidity chamber and weighted (W2)again. WVT was calculated with following equation.

WVT�g mm

.m2 24 h

�¼ ðW2 �W1Þ �mm

area

2.4. Mechanical properties

The film mechanical properties including maximum force,maximum elongation, tensile strength, break value and breakstrength were measured according to the Official Method of theChinese National Standards CNS 10591 (CNS, 2008) with a universaltesting machine (AI-2500, Gotech, Taichung, Taiwan). The teststrips with dimension of 100 � 10 mm were cut from compositefilm and conditioned in an environment of 23 ± 2 �C and RH65 ± 5% for two days. The test conditions were initial grip gaugelength 80 mm and grip separation rate 50 mm/min. Triplicatesamples were used for each type of film.

2.5. The measurement of color parameters

The color parameters (L*, a* and b*) of composite film weremeasured with a color meter (NE4000, Nippon Denshoku In-dustries, Tokyo, Japan). Before measurement, white tile was usedfor calibration. The color parameters of each piece of film were theaverage of 5 randomly selected spots. Triplicate samples were usedfor each type of composite film.

2.6. Determination of phenolic compounds and dicarboxylic acidsin composite films

The determination of phenolic compounds was conducted ac-cording to the method described by Yao et al. (2002) with modifica-tions. A portion of 50 mg composite film and 10 ml methanol were

S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121 117

placed in a plastic centrifuge tube and thoroughly mixed for 30 min.After centrifugation (10,400 � g, CT15RT centrifuge, Pantech In-struments, Taipei, Taiwan) for 10 min at 4 �C, the supernatant wasfiltered through a 0.45 mmmembrane filter and analyzed with HPLC.TheHPLCapparatus consisted of an L-7100pumpand an L-7455diodearray detector (Hitachi, Tokyo, Japan). A LichroXART RP-1e column(4.6 � 150 mm, 5 mm; Merck, Darmstadt, Germany) was used. Themobilephaseconsistedof0.3%formicacid in2%aqueousmethanol (A)and methanol (B). With flow rate of 1 ml/min, the mobile phasegradient was as follow: 85% (A) and 15% (B) initially, (B) increasingfrom 15% to 32% in 0e10 min, (B) increasing from 32% to 35% in10e12min, (B)holdingat35%in12e15min, (B) increasing from35%to50% in 15e25min, (B) decreasing from 50% to 15% in 25e30min, and(B) holding at 15% in 30e35 min. The injection volumewas 20 ml anddetectionwavelengthwas260nm. The contents phenolic compoundswerecalculatedagainstcalibrationcurvesestablishedwith ferulic acidand gallic acid. The recovery percentage of each individual phenoliccompound was calculated based on the theoretical amount used forthe preparation of composite film.

The analysis of dicarboxylic acid was conducted according to themethod reportedbyShirai et al. (2001)withmodifications. Aportionof 200 mg composite film and 10ml distilled water weremixed in acentrifuge tube for 30min. After centrifugation (10,400� g, 4 �C and30min), the supernatant was filtered through a 0.45 mmmembranefilter and analyzed with HPLC. A Sugar Shodex SH1011 column(8.0 � 300 mm, Showa Denko, Kanagawa, Japan) was used. Sulfuricacid solution (20 mM) was used as the mobile phase and the flowrate was 0.6 ml/min. The injection volume was 20 ml and detectionwavelength was 210 nm. The contents of dicarboxylic acids werecalculated against calibration curves established with adipic acid orsuccinic acid. The recovery percentage of each individual dicarbox-ylic acidwas calculatedbasedon the theoretical amountused for thepreparation of composite film.

2.7. Antioxidant activities

DPPH free radical scavenging activity of composite film wasmeasured according to the method described byWangcharoen andMorasuk (2007) with modifications. A portion of 10 mg film and10 ml methanol were mixed in a plastic centrifuge tube. Themixture was kept at room temperature for 30 min with occasionalvortexing. The mixture was centrifuged (10,400 � g, CT15RTcentrifuge, Pantech Instruments, Taipei, Taiwan) for 10 min at 4 �C.An aliquot of 0.3 ml supernatant, 0.6 ml 0.8 mM DPPH (Sigma-eAldrich, St. Louis, MO, USA) solution and 5.1 ml methanol weremixed and kept at room temperature for 1 min. Then, the absor-bance at 515 nmwas measured. The DPPH scavenging activity wascalculated against a calibration curve established with Trolox andexpressed as mg Trolox equivalent/g film.

ABTS free radical scavenging activity of composite film wasmeasured according to the method described by Carrasco-Castillaet al. (2012) with modifications. ABTSþ� (final concentration7 mM) was prepared by dissolving ABTS (SigmaeAldrich, St. Louis,MO, USA) in 2.45 mM potassium persulfate and kept at dark for12e16 h. Prior to use, the absorbance of solution at 734 nm wasadjusted to 0.70 ± 0.02 with methanol. An aliquot of 0.05 ml su-pernatant and 5 ml diluted ABTSþ� solution were mixed and incu-bated for 6 min before the absorbance at 734 nm was measured.The activity was expressed as mg Trolox equivalent/g film bycalculating against a calibration curve established with Trolox.

2.8. Antimicrobial activity

Staphylococcus aureus BCTC13962 and Escherichia coliBCRC10675 were obtained from the Bioresource Collection and

Research Center (Shin-Chu, Taiwan). The working cultures weremaintained on the nutrient agar (NA; Merck, Darmstadt, Germany)plates and subcultured every 2 weeks. The single colony was pickedfrom NA plate and inoculated into 50 ml sterilized nutrient broth(NB; Merck, Darmstadt, Germany). After incubation overnight at37 �C, the cell density was adjusted to 104 CFU/ml with freshsterilized 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 mgfilm were mixed and incubated at 37 �C for 180 min. A portion of0.1 ml mixture was sampled and plated with NA. The plates wereincubated at 37 �C for 2 days and the colonies were counted. Bac-terial suspension (0.5 ml) and sterilized NB (4.5 ml) without com-posite film was used as the control. The percentage of inhibitionwas calculated according the following equation:

Inhibitionð%Þ ¼CFU

.mlcontrol � CFU

.mlsample

CFU=mlcontrol� 100%

2.9. Statistical analysis

The data were presented as mean ± SD. One-way ANOVA andDuncan's multiple range test were used to analyze the differenceamong groups (SPSS software, 12.0, SPSS Inc., Chicago, IL, USA).Differences were considered to be statistically significant when Pvalues were less than 0.05.

3. Results and discussion

With series of preliminary trials, the best combinations of chi-tosan/dicarboxylic acid and zein/phenolic compound for compositefilm formation were determined (Table 1). However, the combi-nation of chitosan without dicarboxylic acids and zein withphenolic compounds failed to form composite films. Since dicar-boxylic organic acid used in film forming formulation couldimprove the film mechanical properties by forming ionic-crosslinking among chitosan (Hamdine et al., 2005), the lack of crosslinking might explain the failure. Thus, there were six types of zein/chitosan composite films used for further studies (Table 1).

3.1. The thickness of zein/chitosan composite films

The thickness of the six types of zein/chitosan composite filmswas between 0.103 ± 0.003 mm to 0.113 ± 0.009 mm (Table 2). Nosignificant difference in film thickness was detected among alltypes of zein/chitosan composite films containing differentphenolic compounds and dicarboxylic acids. Our results were inconsistent with a previous report by Arcan and Yemenicio�glu(2011); gallic acid at various concentrations did not significantlyaffect the thickness of zein films. The interaction between chitosanand carboxylic acids is determined by ionic interaction betweenfunctional groups of chitosan and carboxylic acids and hydrophobicinteractions between internal domains of chitosan helices and thehydrocarbon chain of carboxylic acids (Shamov et al., 2002). Theresults suggest that phenolic compounds could be distributed inthe film matrix without affecting the film thickness. Moreover,dicarboxylic acids might interact with amino groups in chitosanmolecular structure through the formation of ionic bonds and heldthe chitosan molecules close to each other.

3.2. Water vapor transmission

The water vapor transmission of zein/chitosan composite filmsis shown in Table 2. As compared to the composite films withoutphenolic compounds (A film and S film), significant decrease of

Table 1Fabrication of zein/chitosan composite films.

Film formation solution containing chitosana Film formation solution containing zeinb Notation of zein/chitosan composite film

Chitosan dissolved in 1% acetic acid without dicarboxylic acid Zein dissolved in ethanol with phenolic compound Failed to form filmChitosan dissolved in 1% acetic acid containing 0.5% adipic acid Zein dissolved in ethanol without phenolic compound A filmChitosan dissolved in 1% acetic acid containing 0.5% adipic acid Zein dissolved in ethanol containing ferulic acidc AF filmChitosan dissolved in 1% acetic acid containing 0.5% adipic acid Zein dissolved in ethanol containing gallic acidc AG filmChitosan dissolved in 1% acetic acid containing 0.5% succinic acid Zein dissolved in ethanol without phenolic compound S filmChitosan dissolved in 1% acetic acid containing 0.5% succinic acid Zein dissolved in ethanol containing ferulic acidc SF filmChitosan dissolved in 1% acetic acid containing 0.5% succinic acid Zein dissolved in ethanol containing gallic acidc SG film

a 0.3 g Chitosan dissolved in 30 ml mixed acid solution.b 0.9 g Zein and 257 ml glycerol dissolved in 10 ml 95% ethanol.c Phenolic compound used at the level of 246 mg/g zein.

S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121118

water vapor transmission was detected for the composite filmscontaining phenolic compounds (AF film, AG film, SF film and SGfilm). Since dicarboxylic acid could hold chitosan moleculestogether, the less hydrophilic phenolic compounds could furtherprevent the diffusion of water vapor through the composite filmmatrix. With regard to the effect of dicarboxylic acids, better watervapor barrier capacity was detected for composite films preparedwith adipic acid (AF film and AG film) than prepared with succinicacid (SF film and SG film). It might be explained by that adipic acidwith larger molecular weight and longer carbon chain is morehydrophobic than succinic acid. On the other hand, considering theeffect of phenolic compounds on water vapor transmission, addi-tion of gallic acid or ferulic acid made the films less permeable,which might be accounted by less hydrophilic characteristic ofphenolic compounds. However, the effect of phenolic compoundsseemed to be affected by the dicarboxylic acid used in the filmforming solutions.

Water vapor barrier property of edible film is affected by variousfactors including the nature of film forming material, type ofplasticizer/additive and film preparation process. Ghanbarzadehet al. (2006) reported that the water vapor transmission wasinfluenced by hydrophilic-hydrophobic characteristic of the filmforming materials and the steric hindrance of film molecularstructure. It is reported the water vapor transfer process dependedon the simultaneous actions of water diffusivity and solubility in apolymeric matrix (Zhong, Song,& Li, 2011). The types of plasticizersused in the biopolymer films also influenced the water vapor bar-rier properties (Tillekeratne & Easteal, 2000). Water vapor barrierproperty of chitosan filmwas influenced by the organic acid used todissolve chitosan (Park, Marsh, & Rhim, 2002). Kim et al. (2006)reported the similar results that acid types (formic, acetic of pro-pionic acids) significantly affected the water vapor barrier propertyof chitosan films.

3.3. Color analysis

In practical applications, color quality might influence theappearance of edible films which in turn affect the acceptance offoods to consumers. The L*, a* and b* values of composite films are

Table 2Thickness and water vapor transmission of zein/chitosan composite films prepared with

Film typea Thickness (mm) Water vapor transmission(g mm/cm2 24 h)

A film 0.104 ± 0.004A 0.071 ± 0.007BAF film 0.108 ± 0.006A 0.022 ± 0.003EAG film 0.113 ± 0.009A 0.022 ± 0.004ES film 0.103 ± 0.003A 0.085 ± 0.003ASF film 0.103 ± 0.003A 0.054 ± 0.007CSG film 0.107 ± 0.005A 0.036 ± 0.008D

Data were presented as mean ± S.D. of triplicate samples.Data in the same column with different letter were significantly different (p < 0.05).

a The formulation of film as described in Table 1.

shown in Table 2. The composite films without phenolic acidpossessed the highest brightness (expressed by relative high L*

value); for example the L* values for A film and S film were65.41 ± 1.04 and 55.08 ± 3.06, respectively. With the addition ofphenolic compounds, the L* values of composite films (AF film, AGfilm, SF film and SG film) significantly decreased. The results reflectthe fact that composite films became darker when phenolic com-pounds were included in the film formulation. Du, Olsen, Avena-Bustillos, Friedman, and McHugh (2011) reported that the inclu-sion of phenolic compounds darkened the edible films. Ascompared between adipic acid and succinic acid, the reduction ofbrightness caused by the former was greater than that caused bythe latter. As a general trend, the addition of phenolic compoundscaused a decrease of blueness for composite films as indicated bydecreased b* values.

3.4. Mechanical properties

The mechanical properties of zein/chitosan composite films areshown in Table 3. As compared to composite films without phenoliccompounds (A film and S film), all tested parameters of the com-posite films were significantly increased when phenolic com-pounds were included in the film forming formulation (AF film, AGfilm, SF film and AG film).

The tensile strength of composite films containing succinic acid(SF film and SG film) was higher than composite films containingadipic acid (AF film and AG film). With regard to tensile strength,the results indicated that succinic acid is the better dicarboxylicacid than adipic acid for fabrication of stronger composite films.The break value and break strength were also increased whenphenolic compounds were incorporated into the composite films.For break value and break strength, SF films possessed the highestvalues of 0.206 ± 0.029 N and 0.216 ± 0.029 MPa, respectively. It isassumed that phenolic compounds serving as plasticizerscontribute to increase the break value and break strength. In thecase of maximum elongation, combination of gallic acid anddicarboxylic acid conferred the highest values for the compositefilms (22.31 ± 7.23% and 22.71 ± 3.20% for AG film and SG film,respectively). Less increase on the maximum elongation was

different dicarboxylic acid solvent and phenolic compounds.

Color parameters

L* a* b*

65.41 ± 1.04A �1.44 ± 0.11A 13.05 ± 0.05A27.97 ± 1.85D �3.87 ± 0.28B 8.97 ± 0.50B27.83 ± 1.46D �1.00 ± 0.66AB 4.66 ± 0.49C55.08 ± 3.06B �0.94 ± 2.28AB 10.98 ± 1.28AB35.96 ± 4.27C �3.02 ± 2.52B 10.44 ± 4.10AB31.92 ± 4.27CD �2.22 ± 1.96B 8.40 ± 1.62B

Table 3Mechanical properties of zein/chitosan composite films prepared with different dicarboxylic acid solvent and phenolic compounds.

Film typea Maximum force (N) Tensile strength (MPa) Break value (N) Break strength (MPa) Maximum elongation (%)

A film 1.324 ± 0.226C 1.353 ± 0.226C 0.088 ± 0.029B 0.088 ± 0.029CD 3.185 ± 2.083DAF film 1.981 ± 0.579B 1.873 ± 0.490B 0.167 ± 0.069A 0.157 ± 0.069B 13.14 ± 6.323BCAG film 1.883 ± 0.167B 1.608 ± 0.147BC 0.157 ± 0.020A 0.137 ± 0.010BC 22.31 ± 7.231AS film 0.824 ± 0.098D 0.726 ± 0.078D 0.059 ± 0.020B 0.049 ± 0.020D 8.471 ± 2.966CDSF film 2.481 ± 0.118A 2.589 ± 0.127A 0.206 ± 0.029A 0.216 ± 0.029A 17.30 ± 8.351ABSG film 1.883 ± 0.137B 1.667 ± 0.127BC 0.167 ± 0.000A 0.147 ± 0.000B 22.71 ± 3.202A

Data were presented as mean ± S.D. of 5 samples.Data in the same column with different letter were significantly different (p < 0.05).

a The formulation of film as described in Table 1.

S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121 119

detected for composite films containing ferulic acid (13.14 ± 6.32%and 17.30 ± 8.35% for AF film and SF film, respectively).

Acid types used to dissolve chitosan could influence the me-chanical properties of chitosan composite films (Park et al., 2002;Zhong et al., 2011). In polymeric films, flexibility and extensibilityare enhanced by using plasticizers because of reducing intermo-lecular forces and increasing the mobility of the molecular chains(Tillekeratne & Easteal, 2000). The hydrophilic groups of phenoliccompounds decreased the hydrophobic interaction among zeinmolecule and increased the molecular mobility that lowered thebrittleness and improved the flexibility of zein films (Arcan &Yemenicio�glu, 2011). Similar results were reported when galac-tose was used as the plasticizer of zein films. This could be attrib-uted to the increase of polymer chains association in film matrix byplasticization at low level of plasticizer (Ghanbarzadeh et al., 2006).Furthermore, the tensile strength of chitosan films prepared withlactic acid was lower than that of chitosan film prepared with aceticacid (Kim et al., 2006).

Although dicarboxylic acids were designated to dissolve chito-san in this study, they might also interact with zein and affect thegeneral mechanical properties of the composite films. Selling andSessa (2007) reported that polycarboxylic acids were used asplasticizers for zein films and affected the tensile strength withoutforming cross link. However, multivalent acids were assumed toserve as cross-linking agents and improved the wet strength ofcellulosic materials (Kim et al., 2006). Adipic acid and succinic acidused in our study might exert the cross-linking activity for chitosanbut not for zein.

Tensile strength and elongation of chitosan films were signifi-cantly affected by the organic acid used to dissolve chitosan (Parket al., 2002). Kim et al. (2006) reported the similar results thatacid types (formic, acetic of propionic acids) significantly affectedthe tensile strength and elongation at break of chitosan films. Theaddition of plasticizer reduced the tensile strength and increasedthe elongation of zein films (Paramawati et al., 2001). The elonga-tion at break was increased when ferulic acid was added intoprotein-based edible film; and it was assumed that cross-link was

Table 4Recovery of phenolic compounds and dicarboxylic acids and the antioxidant activities of

Film typea Phenolic compoundsb Dicarboxy

Recovery (%) Recovery

A film 0 51.46 ± 6AF film 71.6 ± 1.9 48.65 ± 1AG film 77.6 ± 2.7 49.97 ± 1S film 0 65.41 ± 3SF film 71.5 ± 1.1 59.81 ± 1SG film 84.6 ± 1.3 50.05 ± 6

All data are presented as mean ± S.D. of triplicate samples.a The formulation of film as described in Table 1.b Ferulic acid for AF film and SF film; gallic acid for AG film and SG film. The formulatc Adipic acid for A film, AF film and AG film; succinic acid for S film, SF film and SG fid For antioxidant activity, the results are expressed as mg Trolox equivalent/g film. Da

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

3.5. The recovery of phenolic acids and dicarboxylic acids fromcomposite films

The recovery of phenolic acids and dicarboxylic acids fromcomposite films is shown in Table 4. While the recovery of ferulicacid was greater than 71%, the recovery of gallic acid was greaterthan 77%. The high recovery indicated that the high retention ofphenolic compounds during the film fabrication process. Arcan andYemenicio�glu (2011) reported that about 88% gallic acid wasreleased from zein film.

In general, the recovery of dicarboxylic acids was lower thanthat of phenolic compounds. It is assumed that dicarboxylic acidsmight have higher affinity to chitosan and protein. As for thecomparison of two dicarboxylic acids, succinic acid showed slightlyhigher the recovery than that of adipic acid. Relatively low watersolubility might be the reason for low recovery rate.

3.6. Antioxidant activities

Ferulic acid has been used in protein-based edible film to exertantioxidant properties (Ou et al., 2005). Gallic acid incorporatedinto zeinecarnauba wax composites film showed antioxidant ac-tivity (Ünalan, Arcan, Kore, & Yemenicio�glu, 2013). In this study,DPPH and ABTS free radical scavenging activities of zein/chitosancomposite films are shown in Table 4. Trolox, an antioxidant withwide diversity of antioxidant activities, was used as the commonreference standard for these two antioxidant tests (Thaipong,Boonprakob, Crosby, Cisneros-Zevallos, & Byrne, 2006). Compos-ite films containing no phenolic compounds (A film and S film)basically showed no antioxidant activity. The addition of phenoliccompounds in film forming formulation significantly increased theantioxidant activity of composite films. As compared the effectsbetween ferulic acid and gallic acid on the antioxidant activity of

zein/chitosan composite films.

lic acidsc Antioxidant activityd

(%) DPPH ABTS

.31 4.08 ± 0.92C 11.15 ± 10.8C

.89 98.61 ± 6.42B 276.2 ± 43.4B

.49 315.4 ± 2.50A 455.5 ± 35.9A

.19 8.542 ± 2.10C 0.82 ± 0.83C

.26 96.85 ± 3.18B 246.8 ± 29.4B

.23 321.4 ± 3.88A 449.7 ± 33.6A

ion for A film and S film did not contain phenolic compound.lm.ta in the same column with different letter were significantly different (p < 0.05).

Fig. 1. The growth inhibition (%) of Escherichia coli (A) and Staphylococcus aureus (B) byzein/chitosan composite films prepared with different dicarboxylic acids and phenoliccompounds. Film types as described in Table 1. Data are presented as means of trip-licate samples, error bar: SD. Data with different letter are significantly different(p < 0.05).

S.-Y. Cheng et al. / LWT - Food Science and Technology 63 (2015) 115e121120

composite films (AF film vs. AG film and SF film vs. SG film),stronger activities were found when gallic acid was used.

3.7. Antimicrobial activities

S. aureus (a Gram-positive bacterium which is the causativemicroorganism of food borne intoxication) and E. coli (a commonfood sanitation indicator microorganism) are used as the repre-sentative microorganisms for detection of antimicrobial activity ofedible films (Kanatt et al., 2013; Zhong et al., 2011). The inhibitoryeffects of zein/chitosan composite films on the growth of S. aureusand E. coli are shown in Fig.1.While the inhibition percentages for Afilm and S film against E. coli and S. aureuswere around 10e15% and25e30%, the inclusion of phenolic compounds in composite filmssignificantly increased the antimicrobial activity as indicated by theresults found for AF film, AG film, SF film and SG film.

Chitosan was reported to inhibit the growth of both Gram-positive and Gram-negative bacteria (Chen & Zhao, 2012; Kataset al., 2011; Qi, Xu, Jiang, Hu, & Zou, 2004). On the other hand,phenolic compounds are biocidal agents by interacting with thesurface of the cell and causing cell death through disintegration ofthe cell membrane and release of the intracellular constituentsleading to cell death or inhibition of cell growth (Mu~noz-Bonilla &Fern�andez-García, 2012). It is interesting to note that the inhibitoryactivity of zein/chitosan films against S. aureuswas higher than thatagainst E. coli. The possible antimicrobial mechanism of chitosan isthat the interaction between anionic groups on bacterial cell sur-face and cationic chitosan molecules (Qi et al., 2004). Although

chitosan has been generally regarded as better Gram (�) bacteriainhibitor because of higher negative charge generally found on theouter membrane of this type of bacteria, the antimicrobial activityof phenolic compounds in edible films might be contributed to theeffects (Ünalan et al., 2013). Moreover, the types of acid used toprepare chitosan film also influenced the antimicrobial activity(Chen & Zhao, 2012).

4. Conclusions

Edible films or coatings are potential alternatives for foodpackaging. Active packaging materials can be obtained whenfunctional ingredients are incorporated into edible films andcoatings. A modern trend for developing active edible films is tocombine different base materials and to incorporate multiplefunctional ingredients. In this study, successful preparation of zein/chitosan composite filmswith improvedmechanical properties andwater vapor barrier properties is demonstrated. Since the com-posite films contained phenolic compounds and chitosan, thecomposite films exert multiple functionality including antioxidantand antimicrobial activities. In the practical applications, the filmforming formulation can be used either as edible films or as ediblecoatings to enhance food quality and safety.

Acknowledgments

The financial support (Grant No. NSC102-2313-B-415-007) fromNational Science Council, Taiwan is gratefully acknowledged.

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