Journal of Food Engineering 111 (2012) 343350

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Journal of Food Engineering

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A novel active bionanocomposite lm incorporating rosemary essential oil and nanoclay into chitosan
Mehdi Abdollahi a, Masoud Rezaei a,, Gholamali Farzi b
a b

Department of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, P.O. Box 46414-356, Noor, Iran Department of Material and Polymer Engineering, Faculty of Engineering, Sabzevar Tarbiat Moallem University, Sabzevar, Iran

a r t i c l e

i n f o

a b s t r a c t
Montmorillonite (MMT) nanoclay and rosemary essential oil (REO) were incorporated into chitosan lm to improve its physical and mechanical properties as well as antimicrobial and antioxidant behavior. The MMT weight percent relative to chitosan was varied from 1 to 5 and was activated by three REO levels (0.5%, 1%, and 1.5% v/v), and their impact on physical, mechanical, and barrier properties of the chitosan lms was investigated. Total phenolic and antimicrobial activity were also evaluated. Microstructure of chitosan/MMTREO nanocomposites was characterized through X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The results showed that incorporating MMT and REO into chitosan improves water gain, water vapor permeability, and solubility of the chitosan lm by more than 50%. It was also shown that the combined effect of clay and REO improves signicantly the tensile strength and elongation of chitosan (p < 0.05). The XRD and FTIR results conrmed that the improvements are related to the MMT exfoliation and good interaction between chitosan and MMT in the presence of REO. Antimicrobial properties of the lms also improved by REO incorporation in 1.5% v/v. 2012 Elsevier Ltd. All rights reserved.

Article history: Received 9 August 2011 Received in revised form 4 February 2012 Accepted 7 February 2012 Available online 16 February 2012 Keywords: Bionanocomposite Chitosan Montmorillonite Rosemary essential oil Food packaging

1. Introduction Biopolymers have been widely investigated over the last two decades because they can be a viable solution to the waste disposal of foods plastic packaging materials. Furthermore, biopolymer lms are excellent vehicles for incorporating a wide variety of additives, such as antioxidants, antifungal agents, antimicrobials, colours, and other nutrients (Rhim and Ng, 2007; Wong et al., 1994), and so these biodegradable materials can also improve food quality and extend shelf life by minimizing microbial growth in the product. Chitosan, a deacetylated derivative of chitin, is the second most abundant polysaccharide found in the nature after cellulose. It has been found to be non-toxic, biodegradable, biofunctional, and biocompatible (Rinaudo, 2006; Shahidi et al., 1999; Srinivasa and Tharanathan, 2007). Chitosan brings some advantages over other biomolecule-based active polymers used as packaging materials because of its antibacterial behavior and bivalent minerals chelating ability (Aider, 2010). However, poor mechanical and gas barrier properties, and weak water resistance limit its application particularly in the presence of water and humidity (Wang et al., 2005; Xu et al., 2006). As for other biopolymers, many strategies have been explored to improve the barrier and mechanical properties of chitosan Corresponding author. Tel.: +98 122 6254986; fax: +98 122 6253499.
E-mail address: rezai_ma@modares.ac.ir (M. Rezaei). 0260-8774/$ - see front matter 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2012.02.012

based biodegradable lms. These include the addition of plasticizers and salt, chemical modication of hydroxyl groups, cross-linking of polysaccharides, the use of suitable solvent, the change of pH, the addition of different polysaccharides, and blending with other polymers (Bourtoom and Chinnan, 2008; Ghosh et al., 2010; Hoagland and Parris, 1996; Park and Zhao, 2004; Srinivasa et al., 2007; Li et al., 2010; Gmez-Estaca et al., 2011). Recently, polymerclay nanocomposites have received signicant attention as an alternative to conventional lled polymers, because of their ability for nano-scale dispersion, which brings signicant improvement in mechanical and physical properties compared to micro-scale polymer composites. Several studies have reported amelioration of mechanical properties (Lavorgna et al., 2010; Xu et al., 2006), thermal stability (Darder et al., 2003; Wang et al., 2005), functional properties (Rhim et al., 2006), barrier properties (Casariego et al., 2009; Rhim et al., 2006), and water solubility (Casariego et al., 2009) of chitosan lms via incorporation of nanoclay into chitosan in the range of 15 wt.%. Montmorillonite is the most widely studied type of clay which is a hydrated alumina-silicate layered clay consisting of an edge-shared octahedral sheet of aluminum hydroxide between two silica tetrahedral layers. The imbalance of the surface negative charges is compensated by exchangeable cations (typically Na+ and Ca2+) (Azeredo, 2009). On the other hand, antibacterial and antioxidant activities of essential oils (EOs) have long been acknowledged, but the food industry has recently been paying more attention to their

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application as natural antimicrobials and antioxidants (Du Plooy et al., 2009; Snchez-Gonzlez et al., 2010a). The main advantage of EOs application is their greater activity as compared with the effects of the individual active compounds, probably due to the synergistic effects (Bakkali et al., 2008; Snchez-Gonzlez et al., 2010b; Burt, 2004). Among essential oils the largest number of reports on the effective antioxidant properties pertains to extracts from plants belonging to family Labiatae, particularly rosemary (Rosmarinus ofcinalis L.) (Waszkowiak, 2008). Phenolic compounds such as carnosol, carnosoic acid, rosmanol, rosmadial, epirosmanol, rosmadiphenol, rosmarinic acid, etc. are considered to have antioxidant ability (Bozin et al., 2007). Major components of REO which have showed antimicrobial properties are a-pinene (225% of composition), bornyl acetate (017%), camphor (2 14%), and 1,8-cineole (389%) (Burt, 2004). Although several reports on chitosan/clay nanocomposites have appeared in the literature, to the best of our knowledge there are no reported data on the combined effect of nanoclay and antioxidant/antimicrobial compounds like EOs. It seems that the combination of nanoclay and an antimicrobial/antioxidant compound in chitosan lms can be suitable due to its acceptable structural integrity and barrier properties imparted by the nanocomposite matrix, and the antimicrobial/antioxidant properties contributed by the natural antimicrobial/antioxidant agents impregnated within. In addition, such nanocomposite lms prepared based upon natural biopolymers are environmentally friendly with all the benets one may expect from biopolymer and nanocomposite packaging materials (Rhim and Ng, 2007). In the present study, the combined effects of montmorillonite nanoclay and rosemary essential oil on the physico-chemical and antimicrobial properties of chitosan composite lms have been investigated. 2. Materials and methods 2.1. Materials Crab shell chitosan (medium molecular weight, 190310 kDa, 7585% deacetylated, SigmaAldrich Chemical Co., USA) and Na+montmorillonite (MMT) from Southern Clay Products, USA were used as received. Glacial acetic acid and Tween 80 were purchased from Merck, Germany. Rosemary essential oil was obtained from Barij Essence Pharmaceutical Co., Kashan, Iran, and stored in dark container at 4 C until used. 2.2. Sample preparation 2.2.1. Preparation of chitosan/clay-rosemary solutions Aqueous solution of chitosan was prepared by dissolving 20 g of chitosan powder in 1000 mL of aqueous acetic acid solution (1%, v/ v) to obtain a 2% w/v chitosan solution, using a magnetic stirring plate at 90 C and 1250 rpm for 20 min and then cooled to room temperature. Nanocomposite samples were prepared according to methods reported by Xu et al. (2006). Selected amounts of clay (1, 3, and 5 w% on solid chitosan) were dispersed in 100 mL of 1% (v/v) aqueous acetic acid solution and vigorously stirred for 24 h. Afterward 150 mL of chitosan solution was added to the clay solutions and the mixture was stirred for 4 h. Then Tween 80 at a level of 0.2% w/v of essential oil, as an emulsier, was added to the mixture and stirred at 40 C for 30 min. Finally appropriate amounts of rosemary essential oil (REO) were added to the solution, to reach a nal concentration of 0.5%, 1%, 1.5% v/v, on the basis of neat chitosan solution, and homogenized with Ultra-Turrax (IKA T25-Digital Ultra-Turrax, Staufen, Germany) at 7000 rpm for 2 min. After cooling the resulting mixture at room temperature, it was degassed under vacuum for 5 min in order to remove all bubbles.

2.2.2. Preparation of nanocomposite lms The chitosan/clay-rosemary solutions (160 mL) were cast in simple cubic molds made from Teon-coated steel with dimension of 25.5 28.5 cm, then dried for 72 h at ambient conditions (T = 25 C and RH = 55% 2%) to prepare nanocomposite lms. Dried lms were then peeled and stored in a desiccator containing saturated magnesium nitrate solution at 25 C and 52.89% relative humidity until evaluation. 2.3. Sample characterization 2.3.1. X-ray diffraction (XRD) characterization The structure of nanoparticle and its nanocomposites was evaluated by XRD measurement. XRD patterns were taken with a Philips XPert MPD Diffractometer (Eindhoven, Netherlands), with Co Ka radiation at a wavelength of 1.544 nm, at 40 kV and 30 mA. Chitosan/clay-rosemary lms were scanned over the range of diffraction angle 2h = 112, with a scan speed of 1/min at room temperature. 2.3.2. Fourier transform infrared (FTIR) spectra FTIR spectra were collected in transmission mode by using a Bruker Equinox 55 FTIR spectrometer (Ettlingen, Germany) in the range of 4004000 cm1 at a resolution of 4 cm1. 2.3.3. Measurement of lm thickness The thickness of the samples was determined with a manual digital micrometer (0.001 mm, Mitutoyo, Mizonokuchi, Japan). Measurements were repeated in ten different regions of each sample. Average values were calculated and used in water vapor permeability and tensile properties calculations. 2.3.4. Water resistance The solubility of the lm was determined as the percentage of soluble matter to initial dry matter of lm sample (Tunc et al., 2007). The initial dry matter of samples (4 4 cm) was determined by drying lms at 105 C for 24 h. The lms were then immersed in 50 mL distilled water and then placed in a shaker incubator at 25 C and stirred for 24 h at 250 rpm. Then, the samples were ltered through Whatman No. 1 lter paper. The papers containing any insolubilized lm were dried at 105 C for 24 h. The lm solubility (%) was calculated using the following equation:

Solubility in water% Initial dry weight Final dry weight 100=Initial dry weight 1

2.3.5. Calculation of water vapor permeability The water vapor permeability (WVP) of the lms was measured gravimetrically according to the ASTM E9692 method as described by Casariego et al. (2009). The tested lm was sealed on the top of a glass permeation cell (internal diameter = 3 cm). The cell contained distilled water (100% RH; 2.337 103 Pa vapor pressure at 20 C) placed in a desiccator. It was maintained at 20 C and 1.5% RH (28.044 Pa water vapor pressure) with silica gel, and air was stirred in the desiccators. Weight loss of the permeation cell was determined at intervals of 2 h for 10 h. The weight loss of the cell was considered equal with the transferred water through the lm and adsorbed by the desiccant. The slope of weight loss vs. time was obtained by linear regression. The WVP was then calculated as follows:

WVP WVTR L=DP

Where WVTR (water vapor transmission rate, kg/m2 s1) is the measured slope, L is the mean lm thickness (m), and DP is the partial water vapor pressure difference (Pa) between the two sides of the lm. This test was repeated three times for each specimen to conrm its repeatability.

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2.3.6. Mechanical properties Tensile strength (TS) and elongation at break (E%) of the lm samples were determined according to ASTM standard method D 88202 with an Instron Universal Testing Machine (Model 200, Hiwa Engineering Co., Iran). The lm samples were cut in rectangular specimens (2.54 10 cm). Initial grip separation was set at 50 mm, and cross-head speed was set at 50 mm/min. This test was repeated ve times for each specimen to conrm its repeatability. 2.3.7. Surface colour measurement Colour properties of lms were measured using a colour meter from BYK Gardner, USA. Measurements are expressed as L (lightness), a (red/green), and b (yellow/blue). The parameters were determined by placing lm samples on a standard plate (L = 94.63, a = 0.88, and b = 0.65). Colour difference (DE) and whiteness index (WI) were calculated with respect to standard plate parameters by using following Eqs. (3) and (4), respectively.

q DE Da 2 Db 2 DL 2 q 2 100 L 2 a 2 b

WI 100

2.3.10. Total phenolic assay Total phenolic content of the lms was studied using the Folin Ciocalteu method as described by Siripatrawan and Harte (2010) with some modication. FolinCiocalteu phenol reagent was used to obtain a crude estimate of the amount of phenolic groups present in the lms. Phenolic compounds undergo a complex redox reaction with phosphotungstic and phosphomolybdic acids present in the FolinCiocalteu reaction. The color development is due to the transfer of electrons at basic pH to reduce the phosphomolybdic/phosphotungstic acid complexes (Curcio et al., 2009). 50 mg of each lm sample were dissolved in 3 ml of methanol, and 0.1 ml of lm extract solution was mixed with 7 ml distilled water and 0.5 ml of FolinCiocalteu reagent (Merck Company, Darmstadt, Germany). After preserving the mixture for 8 min at room temperature, 1.5 ml of saturated sodium carbonate solution and 0.9 ml of distilled water were added to it. The mixture was stored in darkness and at room temperature for 2 h. The absorbance values were then measured at 765 nm using a spectrophotometer (Lambda 25, PerkinElmer, Fremont, CA, USA). A calibration curve was drawn using gallic acid in specic concentrations and the total phenolic content of the lms was expressed as Gallic acid equivalents. This test was repeated ve times for each specimen and the Gallic acid equivalent value was reported as mean SD of triplicate. 2.4. Statistical Analysis

2.3.8. Swelling Swelling of the clay and REO containing chitosan lms was evaluated by determining their water sorption according to the method explained by Lavorgna et al. (2010). The sample was cut into small pieces (2 2 cm), desiccated overnight (containing silica gel), and weighed to determine their dry mass. The weighed samples were placed in closed beakers containing 30 mL of water (pH = 7) and stored at 25 C. The kinetic of swelling was evaluated by periodically measuring the weight increment of the samples. The lms wet surface was gently blotted with a tissue before weighing with a balance accurate to 0.0001 g. The weighing was continued until equilibrium state. The procedure was repeated three times for each sample to conrm its repeatability. The water gain of each sample was calculated as follows:

The difference between factors and levels was evaluated by the analysis of variance (ANOVA). Duncans multiple range tests were used to compare the means to identify which groups were signicantly different from other groups (p < 0.05). All data are presented as mean SD. 3. Results and discussion 3.1. XRD analysis of chitosan nanocomposites Wide-angle X-ray diffraction is a classical method for determining the gallery height (d-spacing distance) in clay particles. During intercalation, the insertion of polymer into the organoclay galleries forces the platelets apart and increases the d-spacing, resulting in a shift of the diffraction peak to lower angles (Xu et al., 2006). Results of XRD characterization of chitosan and chitosan/MMT nanocomposite are shown in Fig. 1. in the text. The crystalline structure of chitosan is strongly dependent on its processing treatment, as well as its origin and molecular constitution, such as its degree of deacetylation and molecular weight (Rhim et al., 2006). However, the data reported in Fig. 1 show lower intensity for the characteristic crystallinity peak of chitosan for composite lms at 2h = 10.18, which means the crystallinity of chitosan is slightly reduced by the addition of the MMT. This agrees with previously reported results of Lavorgna et al. (2010). MMT had a characteristic peak at 2h = 8.56 (d0 0 1 = 11.93 ). With the addition of 1 and 3 wt.% MMT to chitosan, the reection peak disappeared, indicating formation of an exfoliated structure, which was disordered and not detectable by XRD. Further increasing the amount of MMT to 5 wt.% gave a broad peak at 2h = 5.82 (d0 0 1 = 17.59 ). This was much lower than that of pristine MMT, suggesting that intercalation occurs together with some exfoliation (Xu et al., 2006). Fig. 1 shows that the structure of nanocomposites is not affected by the presence of the REO. These results are in good agreement with those obtained by other investigators (Lavorgna et al., 2010; Rhim et al., 2006; Xu et al., 2006). It was shown that inorganic and hydrophilic MMT in low concentration (15%) could easily be dispersed in the chitosan solution to form a favorable interaction with hydrophilic chitosan. Strong polar interactions,

Water Gain% Weight of wet film Weight of dry film 100=Weight of dry film 5

2.3.9. Antibacterial activity of the essential oil, MMT, lm solutions, and lm disks Antibacterial properties of REO, lm-forming solution and disks were studied using the agar diffusion method (Gmez-Estaca et al., 2010). Five different pathogenic and spoilage bacteria including Listeria monocytogenes (Persian Type Culture Collection (PTCC) 1163), Pseudomonas putida (PTTC 1694), Streptococcus agalactiae (PTCC 1768), Escherichia coli (PTCC 1533), and Lactococcus lactis (PTCC 1336) were used for testing. Bacterial strains were cultured overnight in Brain Heart Infusion Broth (Scharlua, Spain) at 37 C. 30 lL of REO, MMT solution, and different lm-forming solutions were poured into Mueller Hinton (Scharlua, Spain) agar wells (5 mm diameter), after their plates had been seeded with 0.1 ml of inoculums by swab containing approximately 106107 CFU/ml of the indicated bacteria. In the same way, lms were punched into discs of 6 mm diameter, and then placed on the plates. Next, the plates were incubated in chamber at 37 C for 24 h. After incubation the microbial growth was observed and the degree of inhibition was expressed as follows; +++: totally inhibited; ++: partially inhibited; +: slightly inhibited; : no inhibition as explained by Gmez-Estaca et al. (2010).

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Fig. 2. FTIR spectra of MMT (a), chitosan (b), chitosan-MMT3% (c), and chitosanMMT3%REO1% (d) lms.

Fig. 1. XRD patterns of MMT powder and chitosan-based nanocomposite lms.

especially hydrogen bonding, critically affect the formation of intercalated and exfoliated hybrids (Vaia and Giannelis, 1997).

3.2. FTIR analysis of chitosan/clay nanocomposites Fig. 2 shows the FTIR spectra of MMT, chitosan, and chitosan/ MMT nanocomposite lms. The peak of AlAlOH stretching vibration at 3626 cm1 found in the spectrum of MMT is typical for smectites with high amount of Al in the octahedral layer. The peaks at 3426 and 1639 cm1 were the HOH stretching and bending vibrations of the adsorbed water, respectively. The sharp peak at 1034 cm1 was attributed to SiO stretching frequency (Tun and Duman, 2008) which could act as hydrogen-bonding sites for functional groups of chitosan. These characteristic peaks have shifted to lower frequencies in nanocomposites which recommend some interaction between functional groups of chitosan and MMT (Katti et al., 2008). It is also important to note that the peaks between 3500 and 3000 cm1 correspond to the stretching vibration of free hydroxyl and to asymmetric and symmetric stretching of the NH bonds in the amino group (Siripatrawan and Harte, 2010), respectively. These are stronger in neat chitosan lm (Fig. 2b) compared to those incorporated with clay (Fig. 2c) and REO (Fig. 2d). The bands appearing between 2750 and 3000 in the spectrum of chitosan lm are due to stretching vibrations of CH bond in CH2 (m = 2930 cm1) and CH3 (m = 2870 cm1) groups (Paluszkiewicz et al., 2010) respectively. In addition, two strong bands at 1541

and 1403 cm1, associated with OH in-plane bending, are less discernible in the lms incorporated with clay and REO. These peaks attened more with incorporating clay and REO. This may be due to hydrogen bonding between the OH group in MMT and functional groups in REO ingredients and the NH and OH groups in chitosan (Wang et al., 2008). In the spectra of the nanocomposites, the NH bonded to the O H vibration band at 3209 cm1 in chitosan, and shifted towards lower frequency (m = 3202 cm1) in nanocomposite with 3% MMT. This fact indicates that NH2 and OH groups of chitosan formed hydrogen bonds with the OH group of MMT (Wang et al., 2006). It is further shifted to lower frequency (m = 3130 cm1) for chitosan/clay 1% nanocomposite containing 1% REO, which shows that hydrogen-bonding interaction between chitosan and clay is enhanced in the presence of REO in accordance with other results (Tang et al., 2009). A shift towards lower frequency in the absorption bands at 3209 in the FTIR spectra was coincidental with the decrease in peaks at 35003000, 1541, and 1403 cm1. This observation presents an assumption that there could be a particular arrangement in the lms due to the interactions of MMT and REO ingredients functional groups with hydroxyl and amino groups in chitosan matrix (Siripatrawan and Harte, 2010). 3.3. Film appearance characterization The colour of the packaging is an important factor in terms of general appearance and consumer acceptance (Bourtoom and Chinnan, 2008). The result of colour measurements for chitosan lms are shown in Table 1. All of the lms were transparent, with a slight yellowish tint according to the amounts observed for b of the lms. The results were in good agreement with the results of Rhim et al. (2006). Chitosan lm lightness increased signicantly by incorporating MMT but REO did not affect the nanocomposites lightness. Casariego et al. (2009) reported a similar result about incorporating MMT in chitosan lm. Red/green (a), yellow/blue (b) coordinates, and total colour differences (DE) signicantly increased by incorporating REO and increased as REO increased. Whiteness (WI) increased in the presence of MMT and decreased when REO was incorporated. The results are in agreement with the results of Ojagh et al. (2010) and Siripatrawan and Harte (2010).

M. Abdollahi et al. / Journal of Food Engineering 111 (2012) 343350 Table 1 Surface colour parameters of chitosan-based nanocomposite lms. Concentration MMT (%w/w chitosan) Control 1 1 1 1 3 3 3 3 5 5 5 5 REO (%v/v) 0 0.5 1 1.5 0 0.5 1 1.5 0 0.5 1 1.5 82.51b 0.28 82.63b 1.95 81.95b 1.64 82.12b 1.74 82.70b 0.96 85.21b 1.02 85.42a 1.61 87.50a 1.14 85.67a 1.68 85.94a 1.04 84.28a 1.02 86.09a 2.24 86.19a 2.04 0.17d 0.04 0.67c 0.11 1.09b 0.21 1.22b 0.15 1.35b 0.24 0.70c 0. 07 1.11b 0.15 2.10a 0.49 1.93ab 0.36 0.87bc 0.20 1.96ab 0.36 1.57ab 0.14 2.51a 0.51 7.54f 0.90 6.44f 0.54 9.42de 1.70 9.17de 1.37 11.22c 1.43 7.86def 0.77 10.71ce 2.60 11.22bce 0.20 12.12abce 1.18 8.54abc 0.30 13.37a 0.90 11.17bce 1.01 13.00a 1.03 13.96abc 0.66 10.38d 1.31 16.26a 2.23 14.60abc 2.20 16.15a 1.65 12.01bcd 0.77 15.43a 1.33 15.56a 0.68 16.24a 0.99 11.76cd 0.75 16.50a 1.45 13.60abc 2.19 15.20ab 1.65 L a b

347

DE

WI

80.94abcd 0.59 84.62a 1.52 80.21bcd 1.96 80.33bcd 2.16 79.16cd 1.57 82.81abc 0.57 79.56 cd 1.43 81.90abcd 1.25 82.61abcd 0.99 83.51ab 0.85 80.01cd 1.15 82.10abcd 2.4 80.77bc 2.10

a-e Different superscripts within the same column indicate signicant differences among formulations (p < 0.05).Montmorillonte (MMT), Rosemary Essential oil (REO), Colour difference (DE) and Whiteness index (WI).

3.4. Water resistance Water resistance is an important property of biodegradable or edible lms for applications in food protection where water activity is high, or when the lm must be in contact with water permanently as long as it acts as food protective lm, e.g., to avoid exudation of fresh or frozen products (Bourtoom and Chinnan, 2008). As the results presented in Table 2. indicates, chitosan lm showed a low solubility value of (17.97 0.6) similar to that reported by Ojagh et al. (2010). When MMT was added to the lm, a signicant (p < 0.05) decrease in water solubility was observed (Table 2). This can be justied by the fact that MMT nanoclay brings signicant barrier properties due to its disk-like morphology and there are of course some specic interactions between chitosan and MMT that stabilize the lm structure. In the other word, water could not break hydrogen bonds between MMT layers and chitosan sufciently which caused the lower solubility in the nanocomposites (Tun and Duman, 2010). Similar results were reported by Casariego et al. (2009). When REO was added to the chitosan clay nanocomposite matrix the solubility of the lm further decreased. The lowest water solubility was observed for nanocomposite lms containing 3% clay and 1% REO (9.55 0.78). This fact is highly attributable to cross-linking effects of REO components to esters and/or amide groups (Ojagh et al., 2010). It was supported by FTIR spectra that showed the interaction of chitosan-functional groups with MMT and further interactions in the presence of REO. Higher cross-linking density in chitosan leads to a matrix with lower water solubility results, which is benecial

when product integrity and water resistance are intended (Hosseini et al., 2009). 3.5. Swelling Knowledge of the swelling is also important for predicting stability and quality changes during packaging and storage of food product (Srinivasa et al., 2007b). Swelling of chitosan and its nanocomposites are displayed in Fig. 3. As can be seen in these gures, chitosan lm absorbed large amounts of water (about 1400%) in the rst few minutes, and swelled and crumpled before reaching equilibrium (Fig. 3 A). But all nanocomposites reached equilibrium and the amount of sorbed water was less than chitosan lm. The lowest amount of water sorption relates to chitosan nanocomposite containing 3% MMT. The result was in contrast with result of Lavorgna et al. (2010) that showed chitosan/clay nanocomposite had more water sorption compared to neat chitosan lm. Fig. 3 B, C, and D show swelling of REO-incorporated chitosan nanocomposites. As can be seen from this gure, water sorption of REO containing nanocomposites is 85% less than lms not containing REO. In nanocomposites containing 1 and 5% MMT, the lowest amount of water sorption was observed in 1.5% REO (Fig. 3B and D) but in lms with 3% MMT it was detected in 1% REO (Fig. 3 B). As explained previously, the hydrophobic nature of REO may increase lm hydrophobicity. Moreover, this low water sorption is also most likely to be due both to the formation of the cross-link network induced by the hydrogen bonds between the chitosan and REO and good intercalation of chitosan molecules into the silicate galleries (Lavorgna et al., 2010), as conrmed by FTIR analysis.

Table 2 Summary of water vapor permeability, and mechanical and physical properties for chitosan-based nanocomposite lms. Concentration MMT (%w/w chitosan) Control 1 1 1 1 3 3 3 3 5 5 5 5 REO (%v/v) 0 0 0.5 1 1.5 0 0.5 1 1.5 0 0.5 1 1.5 0.049 0.005 0.049 0.004 0.050 0.006 0.051 0.003 0.051 0.005 0.050 0.004 0.050 0.003 0.051 0.004 0.052 0.005 0.051 0.004 0.051 0.006 0.052 0.005 0.052 0.005 17.97a 0.60 11.70defg 1.66 12.37def 1.02 11.11efg 1.23 10.75efg 1.18 15.03bc 0.96 12.53de 1.38 9.55g 0.78 12.31def 0.75 15.50b 1.00 10.40fg 1.47 12.83cde 0.54 13.90bcd 0.29 0.71a 0.01 0.43c 0.01 0.36f 0.01 0.42c 0.00 0.41cd 0.02 0.39de 0.01 0.47b 0.01 0.30g 0.01 0.31g 0.02 0.45b 0.00 0.36ef 0.01 0.39d 0.02 0.35f 0.01 60.80d 8.60 63.74cd 3.38 72.02bc 6.98 74.66ab 2.90 64.86bcd 1.47 72.54bc 1.46 73.48abc 4.39 71.45bcd 3.78 71.33bcd 5.34 69.33bcd 5.22 83.00a 3.47 65.71bcd 7.48 74.71ab 4.84 3.79 cd 0.54 3.56cd 0.54 4.28bc 0.53 3.91bc 0.74 5.47a 0.71 3.44bcd 0.22 4.11bc 0.11 4.31bc 0.27 4.83ab 0.49 3.01d 0.52 4.10bc 0.61 3.42cd 0.09 4.31bc 0.47 Thickness (mm) Solubility (%) WVP (g/msPa) 1010 TS (MPa) E (%)

a-g Different superscripts within the same column indicate signicant differences among formulations (p < 0.05). Montmorillonte (MMT), Rosemary Essential oil (REO), Water Vapor Permeability (WVP),Tensile Strength (TS), Elongation .

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Fig. 3. Water sorption proles of (A) chitosan lm without REO: (j) CS, () CS/MMT 1%, (N) CS/MMT 3%, and (d) CS/MMT 5%, (B) lm containing 1% MMT: (j) CS/MMT 1%, () CS/MMT 1/REO 0.5%, (N) CS/MMT 1/REO 1%, and (d) CS/MMT 1/REO 1.5%, (C) lm containing 3% MMT: (j) CS/MMT 3%, () CS/MMT 3/REO 0.5%, (N) CS/MMT3/ REO 1%, and (d) CS/MMT3/REO 1.5%, (D) lm containing 5% MMT: (j) CS/MMT 1%, () CS/MMT5/REO 0.5%, (N) CS/MMT 5/REO 1%, and (d) CS/MMT 5/REO 1.5%.

3.6. Water vapor permeability WVP is one of the most important parameters for biodegradable lms. This parameter was studied to evaluate the combined effect of clay and REO on the barrier properties of the chitosan lm. The WVP of chitosan-based lms is shown in Table 2. As can be seen from this table, when 1 or 3 w% MMT was incorporated into chitosan, WVP decreased about 40 or 45%, respectively. The presence of REO resulted in further decreasing of WVP. It was demonstrated that for chitosan nanocomposite containing 3 w% of clay, and 1% or 1.5% REO, WVP decreases by 58%. As mentioned previously, it is believed that clay improves the barrier properties of polymers due to its disk-like morphology and its creation of a tortuous path for the molecule diffusion (Rhim et al., 2006). This is the case for decreasing the WVP of chitosan/clay nanocomposites compared to pure chitosan. A signicant decrease of WVP relates to the presence of ordered dispersed nanoparticle layers with relatively large aspect ratios in chitosan (Cussler et al.,1988; Yano et al., 1997). This can be seen from the XRD pattern of the chitosan/MMT. Further decreasing WVP in the presence of REO relates to the hydrophobic nature of REO, which could affect the hydrophilic/ hydrophobic property of the lms. It may be that the hydrogen and covalent interactions between the chitosan network and REO ingredients limit the availability of hydrogen groups to form hydrophilic bonding with water, subsequently leading to a decrease in the afnity of chitosan lm to water and decreasing WVP (Siripatrawan and Harte, 2010). This hypothesis was supported by FTIR spectra in which the interaction of chitosan functional groups with MMT in the presence of REO is shown.

Despite the low percentages (1 and 3 w%) of MMT could decrease WVP of chitosan/MMT nanocomposite, when the amount of MMT increased to 5 w%, WVP of chitosan/MMT, nanocomposite did not improve. This can be attributed to the quality of clay dispersion in chitosan. It seems that orientation of nanoclay changes once its concentration reaches a certain level and/or some aggregation of nanoclay probably occurs (Casariego et al., 2009). As the XRD pattern showed under this condition, exfoliation of chitosan MMT occurs together with some intercalation (Fig. 1). 3.7. Mechanical properties Mechanical properties of chitosan-based lms are presented in Table 2. The result showed that the tensile strength (TS) of chitosan lm increased signicantly by incorporating MMT into chitosan up to 3 w% but adding more MMT could not improve it. As can be seen from Table 2, TS increased about 20% and 15% when the concentration of MMT was 3 and 5 w%, respectively. However, for similar samples Elongation (E%) decreased slightly. Xu et al. (2006) and Lavorgna et al. (2010) also found that the TS of the chitosan lm containing various concentrations of MMT was signicantly higher than that of chitosan. This improvement in the TS of nanocomposite in a low amount of MMT may be attributed to the uniform dispersion of MMT in chitosan matrix and a strong interaction between chitosan and MMT. The decrease in TS at high concentration of MMT may be caused by some aggregation of MMT particles with high surface energy (Xu et al., 2006). The TS of nanocomposite improved signicantly by incorporating REO, even at a low amount of REO. The highest TS was observed for nanocomposite with 5% of MMT and 0.5% of REO. TS value for

M. Abdollahi et al. / Journal of Food Engineering 111 (2012) 343350 Table 3 Antibacterial activity of REO, MMT, lm-forming solutions (FFS), and lm discs. Clay con. (%) Pure REO MMT MMT MMT FFS well 1 3 5 3 3 3 Film disk 3 3 3 REO con. (%) 0 0 0 Control 0.5 1 1.5 Control 0.5 1 1.5 L. lactis + + P. putida + + + S. agalactiae ++ + + ++ + + E. coli ++ + + +

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L. monocytogenes ++ + ++ + +

: No inhibited; +: slightly inhibited; ++: partially inhibited.

this nanocomposite is about 36.5% higher than pure chitosan lm. The E% increased signicantly by incorporating REO up to 44%, and it seems that REO has shown a plasticizing ability. As described for swelling, perhaps the presence of REO in low amounts changes the hydrogen-bonding network within the material and allows better interaction between nanoller and matrix. 3.8. Antibacterial activity and total phenolic content Antibacterial activity of REO, MMT, lm forming solutions, and lm discs is shown in Table 3. Regarding their extra protective outer membrane, gram-negative bacteria are usually considerably more resistant to antibacterial agents than their gram-positive counterparts. In this regard, the essential oil showed its best antibacterial activity in the disc diffusion test on gram-positive bacteria (i.e., L. monocytogenes, S. agalactiae). The results are in agreement with the results of other published data (Bozin et al., 2007; Gmez-Estaca et al., 2010). However, its inhibitory activity on gram-negative bacteria (especially on E. coli) was also notable and interesting. Bozin et al. (2007) also reported good inhibitory activity for REO against E. coli strain. As explained before, the high antimicrobial properties of REO are mainly related to phenol diterpenes, such as carnosic acid, carnosol, rosmanol, isorosmanol, and rosmarinic acid (Bozin et al., 2007; Tre et al., 2008). MMT did not show antibacterial activity in the well test but we could not nd reported results about this test for comparison. Neat chitosan did not show antimicrobial properties in neither solution form nor in lm form. These results coincide with the results of Ojagh et al. (2010) and Zivanovic et al. (2005) about chitosan lm. This effect of chitosan may be related to the fact that chitosan does not diffuse through the adjacent agar media in the agar diffusion test method, so that only organisms in direct contact

with the active sites of chitosan are inhibited (Coma et al., 2002). Chitosan/clay nanocomposite without REO did not show antibacterial properties, which was in agreement with the results of Rhim and Ng (2007). Films solution and discs containing 1% and 1.5% REO could show antibacterial activity in comparison to neat chitosan. Total phenolic content of chitosan-based lms was shown in Fig. 4. As expected, total phenol content of chitosan lm increased signicantly by incorporating REO and the highest value (2.23 mg galic acid/g sample) was for the lm formulated with clay 3% and REO1.5% which was in agreement with other reported results (Gmez-Estaca et al., 2009; Norajit et al., 2010; Siripatrawan and Harte, 2010). In general, it has been showed in many recent studies that the antioxidant activity of plants is caused mainly by phenolic compounds. The effect of phenolic compounds on lipid molecules may depend on structural factors, like the number of phenolic hydroxyl or methoxyl groups, avone hydroxyl, keto groups, free carboxylic groups, and other structural features (Jayabalan et al., 2008). 4. Conclusions It was shown that incorporation of very low amounts of nanoclay into chitosan signicantly improves its physical and mechanical properties. XRD pattern showed MMT exfoliation and FTIR spectra demonstrated good interaction between chitosan and MMT. The compatibility of REO with chitosan/MMT nanocomposite was conrmed in order to produce an active bionanocomposite for food packaging. Good interaction between chitosan and MMT could improve WVP, water sensitiveness, and mechanical properties of the chitosan lms. In addition, chitosan nanocomposites containing REO exhibit antimicrobial properties and more phenol content, which make them very interesting for food preservation. In the near future, our group will report the results of the antimicrobial and antioxidant aspects of chitosan nanocomposites. Acknowledgments We are grateful to Dr. Badiei for preparing some experimental facilities for this research. We also thank to Dr. Zarei for graciously providing some raw materials. References
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Fig. 4. Total phenol content of chitosan-based lms.

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