Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water

Food Chemistry 122 (2010) 161–166

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Development and evaluation of a novel biodegradable film made from chitosanand cinnamon essential oil with low affinity toward water

Seyed Mahdi Ojagh a, Masoud Rezaei a,*, Seyed Hadi Razavi b, Seyed Mohamad Hashem Hosseini b

a Dept. of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iranb Dept. of Food Science and Engineering, Faculty of Engineering and Agricultural Technology, University of Tehran, Iran

a r t i c l e i n f o

Article history:Received 9 August 2009Received in revised form 18 January 2010Accepted 11 February 2010

Keywords:Chitosan filmCinnamonAntimicrobialPhysicalMechanicalScanning electron microscopy

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.02.033

* Corresponding author. Address: Dept. of FisheriesTarbiat Modares University, P.O. Box 46414-356, Noor3; fax: +98 122 6253499.

E-mail address: [email protected] (M. Rezae

a b s t r a c t

Combining antimicrobial agents such as plant essential oils directly into a food packaging is a form ofactive packaging. In this work chitosan-based films containing cinnamon essential oil (CEO) at level of0.4%, .0.8%, and 1.5% and 2% (v/v) were prepared to examine their antibacterial, physical and mechanicalproperties. Scanning electron microscopy was carried out to explain structure–property relationships.Incorporating CEO into chitosan-based films increased antimicrobial activity. CEO decreased moisturecontent, solubility in water, water vapour permeability and elongation at break of chitosan films. It is pos-tulated that the unique properties of the CEO added films could suggest the cross-linking effect of CEOcomponents within the chitosan matrix. Electron microscopy images confirmed the results obtained inthis study.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Microbial growth on food surfaces is a major cause of foodspoilage. There have been remarkable developments in recentyears in the polymeric packaging films incorporated with antimi-crobial agents for improving the preservation of packaged foods.These films possess the potential for improving microbial stabilityof foods by acting on the food surface, upon contact (Cha, Choi,Chinnan, & Park, 2002).

Chitosan is the deacetylated (to varying degrees) form of chitinwhich is the second most abundant natural biopolymer after cellu-lose, largely widespread in living organisms such as crustacean,insects, and fungi. It is a linear binary heteropolysaccharide com-posed of b-1,4-linked glucosamine and N-acetylglucosamine, andis determined as a non-toxic, a biodegradable and a biocompatiblepolymer (Beverlya, Janes, Prinyawiwatkula, & No, 2008; Shahidi,Arachchi, & Jeon, 1999). Interestingly some antibacterial and anti-fungal activities have been described for chitosan and modifiedchitosan such as more soluble and biodegradable polymers withthe knowledge that this aminopolysaccharide was able to reducemicrobial growth (Tsai, Wu, & Su, 2000). One of the reasons for

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, Faculty of Marine Sciences,, Iran. Tel.: + 98 122 6253101/

i).

the antimicrobial efficacy of chitosan is its positively charged ami-no group which interacts with negatively charged microbial cellmembranes, leading to the leakage of proteinaceous and otherintracellular constituents of the microorganisms (Jeon, Kamil, &Shahidi, 2002; Shahidi et al., 1999). Chitosan-based films are excel-lent oxygen barriers; however, due to their hydrophilic nature,they have poor moisture barrier properties (Caner, Vergano, &Wiles, 1998).

Many spices and herbs and their extracts possess antimicrobialactivity, which minimise questions regarding their safe use in foodproducts. Essential oils and their constituents have wide spectra ofantimicrobial action. The composition, structure as well as func-tional groups of the oils play an important role in determining theirantimicrobial activity (Holley & Patel, 2005). Usually compoundswith phenolic groups are most effective (Dorman & Deans, 2000).Among these, the oils of clove, thyme, cinnamon, rosemary, sageand vanillin have been found to be most consistently effectiveagainst microorganisms. Because of the effect of direct additionof essential oils to food on sensory characteristics of added food,incorporation of essential oils to films may have supplementaryapplications in food packaging (Ojagh, Rezaei, Razavi, & Hosseini2010; Seydim & Sarikus, 2006).

The overall objective of the current study was to improve anti-microbial efficacy of chitosan-based films by incorporating cinna-mon essential oils (CEO). Mechanical and physical propertieswere characterised, and antimicrobial efficacy was assessedagainst five pathogenic and spoilage bacteria. The effects of CEO

162 S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166

on the antimicrobial, physical and mechanical properties of filmswere evaluated and explained in terms of their microstructures.

2. Materials and methods

2.1. Bacterial strains and maintenance

The bacterial strains used in this study were Listeria monocytog-enes PTCC1163, Escherichia coli PTCC1399, Lactobacillus plantarumPTCC1058, Lactobacillus sakei PTCC1272 and Pseudomonas fluores-cens ATCC 17482. These bacteria were obtained from Persian TypeCulture Collection (Tehran, Iran) on nutrient agar slants and kept at4 �C. Subculturing was carried out each 14 days to maintain bacte-rial viability. Overnight cultures of L. plantarum and L. sakei weregrown in MRS (Man-Rogosa-Sharpe) broth at 37 and 30 �C at CO2

chamber (New Brunswick Scientific Co, Edison, NJ), respectively,and L. monocytogenes, E. coli, and Ps. fluorescens were grown andagitated at 140–150 rpm in an incubator shaker for 24 h in BHI(brain heart infusion) broth at 37 �C. All the media were purchasedfrom Merck Co (Darmstadt, Germany). The bacterial population inall the inoculated media was more than 1 � 109 CFU/ml after 24 hincubation.

2.2. GC–MS analysis of cinnamon essential oil

Cinnamon (Cinnamomum zeylanicum) essential oil was obtainedfrom Zardband Co. (Tehran, Iran). GC/MS analysis was carried outon a Hewlett Packard 6890 (II) coupled to an HP 5973 mass spec-trometer detector with electron impact ionisation (70 eV) (Agilent,Palo Alto, CA, USA). The analysis was carried out using DB-5 fusedsilica capillary column (60 m, 0.25 mm I.D.; 0.25 lm film thickness,J&W Scientific Inc., Rancho Cordova, CA, USA). The temperatureprogram was 10 min at 60 �C, then to 250 �C at 5 �C/min, held for10 min. Other operating conditions were as follows: carrier gas, he-lium (99.999%), with a flow rate of 1.1 ml/min; injection volume of1 ll and split ratio, 1:50. Essential oil components were identifiedby comparison of their retention indices (RI) relative to (C6–C24)n-alkanes with those of authentic compounds under the same con-ditions (Davies, 1990). Further identification was made by match-ing their mass spectra fragmentation patterns with those stored inthe Wiley/NBS mass spectral library and other published massspectra (Adams, 2001). Moreover, relative percentage of the com-ponent cinnamon oil was calculated by normalisation of the baseGC–mass peak.

2.3. Determination of MIC and MBC of CEO against bacterial strains

Minimum inhibitory concentration (MIC) and minimum bacte-ricidal concentration (MBC) of CEO were determined according tothe method of Kim, Marshall, and Wei (1995).

2.4. Preparation of antimicrobial films

Chitosan-based film was prepared by dissolving crab shellchitosan (medium molecular weight (190–310 kDa), 75–85%deacetylated, Sigma Chemical Co., St. Louis, MO., USA) in an aque-ous solution (1% v/v) of glacial acetic acid to a concentration of2% (w/v) while stirring on a magnetic stirrer/hot plate. The solutionwas stirred with low heat (at 40 �C) which typically required 6 hstirring. The resultant chitosan solution was filtered through aWhatman No. 3 filter paper to remove any undissolved particles.After filtration the solution was returned to the magnetic stirrer/hot plate and glycerol (Sigma Chemical Co., St. Louis, MO, USA)was added to a level of 0.75 ml/g chitosan as a plasticiser. The plas-ticiser was mixed into the solution for 30 min. Then Tween 80 at

level of 0.2% (v/v) of essential oil was added as an emulsifier to as-sist essential oil dissolution in film forming solutions. After 1 h ofstirring, CEO was added to chitosan solution to reach a final con-centration of 0.4%, 0.8%, 1.5% and 2% (v/v) as essential oil concen-tration per film in emulsifying equipment (IKA T25-Digital UltraTurrax, Staufen, Germany) at 7000 rpm for 2 min. After cooling toroom temperature, the film forming solutions were degassed undervacuum for 5 min. The film forming solutions (160 ml) were castedon the centre of 27 � 27 cm2 glass plates, and then dried for 30 h atambient conditions (25 �C). Dried films were peeled and stored in adesiccator at 25 �C and 51% relative humidity until evaluation. Sat-urated magnesium nitrate solution was used to meet required rel-ative humidity.

2.5. Determination of antimicrobial effects of films

The agar diffusion method was used for determining the anti-bacterial effects of films on bacterial strains. The films were cutinto 15 mm diameter discs with a circular knife. Film cuts wereplaced on BHI agar for L. monocytogenes, E. coli and Ps. fluorescensand on MRS agar for L. plantarum and L. sakei. Agar plates had pre-viously been seeded with 0.1 ml of an overnight broth culture ofindicator strains. The plates were incubated at appropriate temper-ature for 24 h in the appropriate incubation chamber. The diameterof the zone of inhibition was measured with a caliper to the nearest0.02 mm. The whole zone area was calculated then subtractedfrom the film disc area and this difference in area was reportedas the ‘‘zone of inhibition” (Seydim & Sarikus, 2006).

2.6. Determination of physical properties of films

2.6.1. ThicknessThickness of the films was determined using a digital coating

thickness gauge (Elcometer A300 FNP 23, Elcometer InstrumentLtd., Manchester, England) to the nearest 0.001 mm. Informed val-ues are an average of at least ten random locations of the filmsheets. The means were calculated and used in the determinationof mechanical and physical properties.

2.6.2. Moisture contentMoisture content of films was determined measuring weight

loss of films, upon drying in an oven at 110 �C until a constantweight was reached (dry sample weight).

2.6.3. Water vapour permeability coefficient (WVPC)Standard method E 96 was used to determine WVPC with a 75%

relative humidity (RH) gradient at 25 �C (ASTM, 1995). Diffusioncells containing anhydrous calcium chloride desiccant (0% RH, as-say cup) or nothing (control cup) were sealed by the test film(0.00287 m2 film area). To maintain a 75% RH gradient across thefilm, a sodium chloride-saturated solution (75% RH) was used inthe desiccator. The RH inside the cell was always lower than out-side. This difference in RH corresponds to a driving force of1753.55 Pa, expressed as water vapour partial pressure. Water va-pour transport was determined from the weight gain of the diffu-sion cell at a steady state of transfer. Changes in the weight of thecell were recorded to the nearest 0.0001 g and plotted as a functionof time. The slope of the weight vs time plot was divided by theeffective film area to obtain the water vapour transmission rate(WVTR). This was multiplied by the thickness of the film and di-vided by the pressure difference to obtain the water vapour perme-ability coefficient (WVPC). All WVPC values were corrected for airgap distance between calcium chloride and film surface by correct-ing the values of WVTR, according to the equations of Gennadios,Weller, and Gooding (1994).

S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166 163

2.6.4. Surface properties measurementsContact angle measurements were performed with water using

a goniometer (Kruss G23, Germany). A small drop of distilled waterwas deposited on the film surface. The contact angle is defined asthe angle between the film surface and the tangent line at the pointof contact of the water droplet with the surface. For each filmtype, at least five measurements were made and the average wastaken.

2.6.5. Film solubility in waterPieces of film of 1 � 3 cm2 were cut from each film and weighed

to the nearest 0.0001 g. The solubility in water of the differentchitosan films was measured from immersion assays under con-stant agitation in 50 ml of distilled water for 6 h at 25 �C. Theremaining pieces of film after immersion were dried at 110 �C toconstant weight (Final dry weight). The initial dry weight wasdetermined by thermal processing at 110 �C to constant weight.Solubility in water (%) was calculated by using the following equa-tion (Eq. (1)):

Solubility in water ð%Þ

¼ Initial dry weight� Final dry weightInitial dry weight

� �� 100 ð1Þ

Table 1Essential oil composition of Cinnamomum zeylanicum.

No. Compound RI % of the total peak area

1 a-Pinene 936 2.18

2.6.6. Surface colour measurementsFilm colour was determined by a colourimeter (Minolta CR 300

Series, Minolta Camera Co., Ltd., Osaka, Japan). The CIELab scalewas used, lightness (L) and chromaticity parameters a (red–green)and b (yellow–blue) were measured. Measurements were per-formed by placing the film sample over the standard white plate(L* = 93.49, a* = �0.25 and b* = �0.09). Colour differences (DE)were calculated by the following equation (Eq. (2)):

DE ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðL� � LÞ2ða� � aÞ2ðb� � bÞ2

qð2Þ

where L*, a* and b* are the colour parameter values of the standardand L, a and b are the colour parameter values of the sample.

2 Camphene 950 0.143 Myrcene 982 1.034 a-Phellandrene 1001 0.545 d-3-Carene 1010 0.946 P-cymene 1016 2.857 1,8-Cineole 1026 3.328 Linalool 1086 6.469 Z-Cinnamaldehyde 1188 0.2910 E-Cinnamaldehyde 1247 60.4111 Hexagerman 1312 0.4812 Eugenol 1334 3.1913 a-Copaene 1386 0.3414 Coumarine 1398 0.4315 E-Cynnamyl acetate 1413 2.0116 b-Caryophyllene 1431 3.5017 a-Humulene 1463 0.2718 O-methoxycinnamaldehyde 1492 3.6319 Caryophyllene oxide 1585 0.34

2.7. Determination of mechanical properties of films

The mechanical properties of films including tensile strength(MPa) and elongation at break (%) were performed at 25 �C and51% RH. Tests were performed using a Testometric MachineM350-10CT (Testometric Co. Ltd., Rochdale, Lancs., England)according to the ASTM method D882-91 (ASTM, 1996). In prepar-ing samples, films were cut into 1 � 10 cm2 strips. The films wereheld parallel with an initial grip separation of 5 cm and then pulledapart at a head speed of 25 mm/min. Elongation at break (deforma-tion divided by initial grip separation and multiplying by 100) andmaximum force were obtained from force versus deformationcurves. Tensile strength was calculated by dividing maximum forceby film cross section (thickness �width).

20 Benzyl benzoate 1736 0.6521 Equilin 2049 0.91

Table 2MIC and MBC values of CEO against five tested bacterial strains.

Bacteria L.monocytogenes

E. coli Ps.fluorescens

L.plantarum

L.sakei

MIC (ppm) 500 500 500 500 250MBC

(ppm)>1500 >1500 1000 >1500 1000

2.8. Scanning electron microscopy

The chitosan-based films were mounted on the specimenholder with aluminium tape and then sputtered with gold inBAL-TEC SCD 005 sputter coater (BAL-TEC AG, Balzers, Liechten-stein). All the specimens were examined with a Philips XL 30 scan-ning electron microscope (Philips, Eindhoven, Netherlands) underhigh vacuum condition and at an accelerating voltage of 20.0 kV.Samples were photographed at tilt angles of 60–90� to the electronbeam for the views in the cross section.

2.9. Statistical analysis

The triplicate data were performed to an analysis of variance forthe significance of added essential oils using MSTATC programs(version 2.10, East Lansing, MI, USA). Duncan’s multiple range testswas used to compare the difference among means at the level of0.05.

3. Results and discussion

3.1. Identification of volatile components from CEO

GC–MS analytical data of major compounds in CEO are shownin Table 1. E-Cinnamaldehyde was a predominant componentand accounted for 60.41% of the total peak area. The other compo-nents were linalool (6.46%), ortho-methoxycinnamaldehyde(3.63%), b-caryophyllene (3.5%), 1,8-cineole (3.32%), and eugenol(3.19%).

3.2. MIC and MBC of CEO

MIC and MBC values (antibacterial properties) of CEO are pre-sented in Table 2. The lower concentration of the essential oil couldfully inhibit the growth or could almost kill L. sakei (MIC 250 lg/mL and MBC 1000 lg/mL) and Ps. fluorescens (MIC 500 lg/mLand MBC 1000 lg/mL). However, MIC and MBC of the essentialoil for the other three bacteria were 500 and more than 1500 lg/mL, respectively.

This is not supported by many other reports on the greater sus-ceptibility of gram-positive bacteria to inhibitory effect of essentialoils and their components (Oussalah, Caillet, Saucier, & Lacroix,2007; Shan, Cai, Brooks, & Corke, 2007). Similar results have been

164 S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166

reported by Kim et al. (1995) who found that L. monocytogenes wasmore resistant to the inhibitory effects of eleven essential oil con-stitutes than the gram-negative bacteria tested under the samecondition, including E. coli, E. coli O157:H7, Salmonella typhimuriumand Vibrio vulnificus. It seems that the variability of the inhibitoryeffect of essential oil may be due to differences between strainsof the same bacterial species. This hypothesis was confirmed bySivropoulou et al. (1996) with two strains of Staphylococcus aureusin the presence of carvacrol and thymol.

Table 4Physical properties of chitosan films incorporated with CEO.

3.3. Antimicrobial properties of films

Effects of CEO addition on antimicrobial properties of chitosan-based films are shown in Table 3. When antimicrobial agents areincorporated into films, these materials diffuse through agar geland result in clear zone around the film cuts.

Incorporation of CEO into chitosan-based films at higher than0.4% (v/v) exhibited a clear inhibitory zone by the absence of bac-terial growth around the film cuts. At CEO concentration of 0.4% (v/v) the clear zone of inhibition was not observed with L. plantarum.As the concentration increased, the zone of inhibition also in-creased significantly (p < 0.05). However, increasing level of CEOat its high concentration did not reveal significant an increasedinhibitory. It was generally caused by the compactness of chitosanfilm network containing CEO because of the occurrence of func-tional groups interaction phenomenon. The active component ofCEO is cinnamaldehyde (Matan et al., 2006). As shown in Table 1,cinnamaldehyde constitutes more than 60% of CEO. Sublethal con-centrations of cinnamaldehyde have been found to inhibit produc-tion of amylase and proteases by Bacillus cereus (Thoroski, Blank, &Biliaderis, 1989).

Chitosan control films did not show inhibitory zone in bacterialstrains tested. Despite antimicrobial activity of chitosan because ofits innate characteristic, this effect of chitosan occurred without

Table 3Antibacterial activity (inhibitory zone) of chitosan films incorporated with CEOagainst gram-positive and gram-negative bacteria.

Bacteria CEO conc. (v/v) in filmsolution (%)

Inhibitory zone*

(mm2)

L. monocytogenes (Gram +) Control 0c

0.4 38.17 ± 1.00b

0.8 39.80 ± 0.94b

1.5 53.05 ± 1.84a

2 52.31 ± 2.65a

L. plantarum (Gram +) Control 0c

0.4 0c

0.8 43.6 ± 0.94b

1.5 49.80 ± 2.77a

2 53.64 ± 3.07a

L. sakei (Gram +) Control 0c

0.4 39.60 ± 2.36b

0.8 41.08 ± 1.44b

1.5 56.69 ± 2.82a

2 57.23 ± 1.50a

Ps. fluorescens (Gram �) Control 0c

0.4 34.68 ± 1.71b

0.8 36.91 ± 1.32b

1.5 41.60 ± 2.09a

2 43.60 ± 1.21a

E. coli (Gram �) Control 0d

0.4 36.31 ± 0.77c

0.8 38.90 ± 0.94b

1.5 51.72 ± 1.15a

2 51.04 ± 2.12a

* Means in each column with different superscript letters are significantly different(p < 0.05). Control is a film disc containing no essential oil.

migration of active agents. Chitosan does not diffuse through theadjacent agar media in agar diffusion test method; so that onlyorganisms in direct contact with the active sites of chitosan areinhibited (Coma et al., 2002).

3.4. Physical properties of films

The effects of incorporating CEO on the physical properties ofchitosan films are shown in Table 4. Thin films were easily re-moved from the cast plate. Thickness of films varied between0.095 and 0.107 mm, as shown in Table 4.

In general, the moisture content value decreased as CEO wasincorporated into chitosan-based film, which is attributed to com-pactness of film network. CEO caused to formation covalent bondsbetween the functional groups of chitosan chains, leading to a de-crease in the availability of hydroxyl and amino groups and limit-ing polysaccharide–water interactions by hydrogen bonding andresulting in a decrease of moisture content value of edible films(Park & Zhao, 2004). As CEO concentration increased, the moisturecontent of films decreased significantly (p < 0.05).

Chitosan control films were compact, and the film surface had asmooth surface without pores or cracks (Fig. 1A and B). Controlfilm had water vapour permeability coefficient (WVPC) value2.250 � 10�10 g s�1 m�1 Pa s�1. Same results were obtained byWong, Gastineau, Gregorski, Tillin, and Pavlath (1992), Caneret al. (1998) and Seydim and Sarikus (2006). Incorporation ofCEO into chitosan-based films decreased WVPC of films. Besidethe hydrophobic nature of CEO which could affect the hydro-philic/hydrophobic property of the films, the physical factors hadan influence on the WVPC of the films enriched with CEO. WhenCEO was incorporated into the chitosan film formulation, the sur-

Physical properties* CEO conc. (v/v) infilm solution (%)

Thickness (mm) Control 0.095 ± 0.0025b

0.4 0.098 ± 0.0024b

0.8 0.104 ± 0.0029a

1.5 0.105 ± 0.0032a2 0.107 ± 0.0039a

Moisture content (%) Control 20.82 ± 1.82a

0.4 18.72 ± 2.11a

0.8 14.04 ± 0.93b

1.5 10.82 ± 1.47c

2 8.47 ± 1.53c

Water vapor permeability coefficient(g s�1 m�1 Pa s�1 � 10�10)

Control 2.250 ± 0.074a

0.4 1.352 ± 0.152b

0.8 1.234 ± 0.040b

1.5 1.014 ± 0.040c

2 1.003 ± 0.067c

Water contact angle (�) Control 37.3 ± 2.25d

0.4 39.6 ± 1.89d

0.8 45.3 ± 1.38c

1.5 59.2 ± 2.77b

2 70.3 ± 1.42a

Solubility in water (%) Control 23.2 ± 1.09a

0.4 21.6 ± 0.65a

0.8 16.8 ± 0.85b

1.5 13.6 ± 1.55c

2 10.4 ± 0.94d

Total colour difference (DE) Control 17.35 ± 0.98d

0.4 17.83 ± 0.47d

0.8 21.29 ± 0.55c

1.5 23.35 ± 0.87b

2 25.62 ± 0.58a

* Means in each column with different superscript letters are significantly different(p < 0.05).

Fig. 1. Scanning electronic microscopic images of chitosan control film (A) and (B), and film containing CEO at level of 1.5% (C) and (D) surfaces and cross sections,respectively.

Table 5Mechanical properties of chitosan films incorporated with CEO.

Mechanical properties* CEO conc. (v/v) in film solution (%)

Tensile strength (MPa) Control 10.97 ± 0.54e

0.4 13.35 ± 1.23d

0.8 17.43 ± 1.08c

1.5 24.10 ± 1.47b

2 29.23 ± 2.25a

Elongation at break (%) Control 24.73 ± 1.86a

0.4 16.57 ± 0.77b

0.8 11.26 ± 1.39c

1.5 6.42 ± 0.63d

2 3.58 ± 0.35e

* Means in each column with different superscript letters are significantly different(p < 0.05).

S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166 165

face view suggested a sheet like and dense structure, while thecross section revealed the sheets stacked in compact layers(Fig. 1C and D), which showed CEO incorporated uniformly in thematrix.

The value of the contact angle with water indicates how hydro-phobic the surface is. It is well-known that the water contact anglewill increase with increasing surface hydrophobicity. The higherhydrophilicity of the control film is attributed to the water bindingcapacity of plasticiser (glycerol) and functional groups of chitosan.According to Table 4, addition of CEO increased water contact angleof films and resulted in decreasing hydrophilicity of the chitosanfilms which might be due to the loss of free functional groups (ami-no and hydroxyl groups).

Chitosan control film had low solubility in water below 24% at25 �C after 6 h dipping. Same result was reported by García, Pinotti,Martino, and Zaritzky (2004). Control film in general was veryhydrophilic thus absorbed water quickly and resulted in swelling.Incorporation of CEO into the chitosan film formulation at levelof 1.5% and 2% (v/v) led to 41% and 55% reduction in solubility inwater respectively. This phenomenon is due to the cross-linking ef-fects of CEO components leading to esters and/or amide groups.Cross-linking in the chitosan film matrix resulted in a decrease insolubility in water and produced films with low affinity towardwater which is beneficial when product integrity and water resis-tance are intended. Cross-linking of chitosan-hydroxy propylmethyl cellulose based films using citric acid (polycarboxylic acid)as the cross-linking agent, was reported by Möller, Grelier, Pardon,and Coma (2004).

The colour of the films may influence the consumer acceptabil-ity of a product (Sivarooban, Hettiarachchy, & Johnson, 2008). Totalcolour differences (DE) of chitosan films are shown in Table 4.Visually, chitosan films had a slightly yellow appearance. Controlfilm had DE value 17.35. Its transparency was reduced as theCEO was incorporated. Incorporating CEO revealed DE values,which were significantly higher than that of the control. The valuesof chromaticity parameter b of chitosan films incorporated withCEO were higher than that of control film (data not shown). These

results agreed with visual observations. Nevertheless, the sensorystudies should be conducted for evaluating consumer acceptabilityof this colour change.

3.5. Mechanical properties of films

The effect of incorporating CEO on mechanical properties ofchitosan films is presented in Table 5. Chitosan control film hadtensile strength value 10.97 MPa. Incorporation of CEO into chito-san films increased tensile strength values significantly (p < 0.05).As previously mentioned, a strong interaction between the poly-mer and the CEO produced a cross-linker effect, which decreasesthe free volume and the molecular mobility of the polymer. Thisphenomenon led to a sheet like structure (Fig. 1C). Arrangementof stacking layers of CEO added chitosan sheets (Fig. 1D) meansthat in these films a compact structure was present thus increasedcontinuities within the polysaccharide network leading to decreasein elongation at break. There are also possibilities such as decreasein moisture content of films incorporated with CEO during the filmproduction, thus leading to the decrease in strain and the increase

166 S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166

in tensile strength. The results of moisture content measurementsare shown in the Table 4. According to Gontard, Guilbert, and Cuq(1993) and Pouplin, Redl, and Gontard (1999) water is the mostubiquitous and uncontrollable plasticiser for most hydrocolloid-based films because of its ability to modify the structure of naturalpolymers. Thus, the plasticisation effect of water could not be neg-ligible for these films, and their plasticisation efficiency does notonly come from intrinsic plasticiser action of glycerol. Differencesbetween data of chitosan films obtained in the current study andthose of reported in literature (Caner et al., 1998; García et al.,2004) may be attributed to chitosan composition and suppliers,plasticiser presence and film preparation.

4. Conclusions

Chitosan is a promising biopolymer for active food packaging.Its sensitivity to moisture can be offset by blending it with CEO.The results of this study showed that a unique compatibility canbe achieved between chitosan and CEO. The incorporation of CEOimproved the antibacterial properties of chitosan. Films containingCEO are useful for coating of highly perishable foods such as fishand poultry. The other edible films may be ruptured upon contactwith wet surfaces. Functional groups interaction phenomenon inedible films has critical effect on their antibacterial, physical andmechanical properties which are important in food packagingapplications. Moreover, the antimicrobial effect of CEO enrichedfilms should be determined on an entire model food.

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