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LWT 38 (2005) 859865 www.elsevier.com/locate/lwt

Enhancing antimicrobial activity of chitosan lms by incorporating garlic oil, potassium sorbate and nisin
Y. Pranoto, S.K. Rakshit, V.M. Salokhe
School of Environment, Resources and Development, Food Engineering and Bioprocess Technology, Asian Institute of Technology, P.O. Box 4, Klong Luang, Pathumthani 12120, Thailand Received 20 August 2004; received in revised form 28 September 2004; accepted 30 September 2004

Abstract Antimicrobial effect of chitosan edible lm incorporating garlic oil (GO) was compared with conventional food preservative potassium sorbate (PS) and bacteriocin nisin (N) at various concentrations. This activity was tested against food pathogenic bacteria namely Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Listeria monocytogenes and Bacillus cereus. Mechanical and physical properties were characterized and Fourier Transform Infrared (FTIR) was also performed to determine functional groups interactions between the matrix and added agent. Incorporation of GO up to levels at least 100 ml/g, PS at 100 mg/g or N at 51,000 IU/g of chitosan were found to have antimicrobial activity against S. aureus, L. monocytogenes, and B. cereus. At these levels, the lms were physically acceptable in term of appearance, mechanical and physical properties. GO components did not affect the physical and mechanical properties of chitosan lms as it did not have any interaction with the functional groups of chitosan as measured by FTIR. r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial chitosan lm; Garlic oil; Potassium sorbate; Nisin; Food pathogenic bacteria

1. Introduction Food quality and safety are major concerns in the food industry as consumers prefer fresher and minimally processed products. In particular, bacterial contamination of ready to eat products is of concern to human health. Antibacterial sprays or dips have been done to overcome those contaminations (Ouattara, Simard, Piette, Begin, & Holley, 2000). However, direct surface application of antibacterial substances has some limitations because the active substances could be neutralized, evaporated or diffused inadequately into the bulk of food (Torres, Motoki, & Karel, 1985; Siragusa & Dickson, 1992). Edible lms or coatings have been investigated for their abilities to retard moisture, oxygen, aromas, and
Corresponding author. Tel.: +66 25246110; fax: +66 25246200.

E-mail address: rakshit@ait.ac.th (S.K. Rakshit).

solute transports (Gennadios & Weller, 1990). It is one of the most effective methods to maintain food quality. This is further improved by lm carrying food additives such as antioxidants, antimicrobial, colorants, avors, fortied nutrient and spices (Pena & Torres, 1991; Han, 2000). In many cases, the agents being carried are slowly released into the food surface and therefore remain at high concentration for extended period of time (Ouattara et al., 2000; Coma, Sebti, Pardon, Deschamps, & Pichavant, 2001). Chitosan, b-1,4 linked glucosamine and N-acetyl glucosamine, is prepared by deacetylation of chitin. Chitosan has been proved to be nontoxic, biodegradable, biofunctional, biocompatible and have antimicrobial characteristics (Wang, 1992; Darmadji & Izumimoto, 1994; Jongrittiporn, Kungsuwan, & Rakshit, 2001). One of the reasons for the antimicrobial character of chitosan is its positively charged amino group which interacts with negatively charged microbial

0023-6438/$30.00 r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2004.09.014

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cell membranes, leading to the leakage of proteinaceous and other intracellular constituents of the microorganisms (Shahidi, Arachchi, & Jeon, 1999). In the Grampositive bacteria, the major constituent of its cell wall is peptidoglycan and very little protein. The cell wall of Gram-negative bacteria on the other hand is thinner but more complex and contains various polysaccharides, proteins and lipids beside peptidoglycan. The cell wall of Gram-negative bacteria also has outer membrane which constitutes the outer surface of the wall (Black, 1996). Chitosan lms are easily prepared by evaporating from dilute acid solutions (Park, Marsh, & Rhim, 2002). A number of studies on the antimicrobial characteristics of lms made from chitosan have been carried out earlier (Chen, Yeh, & Chiang, 1996; Ouattara et al., 2000; Coma, Martial-Gros, Garreau, Copinet, & Deschamps, 2002). Antimicrobial agents such as organic acids, bacteriocins and spice extracts have been tested for their ability to control meat spoilage (Abugroun, Cousin, & Judge, 1993; Hotchkiss, 1995; Miller, Call, & Whiting, 1993). Garlic oil is mainly composed of sulfur-containing compound such as allicin, diallyl disulde and diallyl trisulde that possess better antimicrobial activity than the corresponding ground form (Nychas, 1995). Potassium sorbate is active against yeast, mould and many bacteria (Meyer, Suhr, Nielsen, & Holm, 2002). Nisin is a bacteriocin produced by Lactococcus lactis subsp. lactis. It has antimicrobial activity against a broad spectrum of Gram-positive bacteria. Nisin has widely been used in the food industry as a safe and natural preservative and has been studied of its suitability to be incorporated into cellulose, whey protein isolate, soy protein isolate, egg albumen, wheat gluten, hydroxyprophyl methylcellulose and zein lm (Coma et al., 2001; Ko, Janes, Hettiarachchy, & Johnson, 2001; Janes, Kooshesh, & Johnson, 2002). The development of complementary methods to inhibit the growth of pathogenic bacteria such as packaging material-associated antimicrobial agents is an active area of research. This study was done to improve antimicrobial efcacy of edible lm based on chitosan by incorporating garlic oil, potassium sorbate and nisin. Mechanical and physical properties were characterized, and antimicrobial efcacy was assessed against ve food pathogenic bacteria. The functional groups interactions of these three antimicrobials with the chitosan-based lms were also studied.

ichia coli TISTR 73, Staphylococcus aureus TISTR 29, Salmonella typhimurium TISTR 292, Listeria monocytogenes S 0273, and Bacillus cereus TISTR 747 were obtained from the culture collection at Thailand Institute of Scientic and Technological Research (TISTR) whereas L. monocytogenes was obtained from the culture collection of Department of Fisheries, Bangkok, Thailand. The bacterial cultures were grown on the nutrient agar slant and kept at 4 1C. Monthly subculture was carried out to maintain bacterial viability. In the preparation of seeding culture for antimicrobial test of edible lms, a loopful of bacteria from agar slant was taken and inoculated into 50 ml of nutrient broth in 125 ml ask. The ask was then incubated at 125 rpm in an incubator shaker (Edmund Bu hler TH 25) at 37 1C for 24 h. A dilution series was taken to meet required bacterial population for seeding by using sterile distilled water. 2.2. Preparation of antimicrobial edible lm Chitosan edible lm was prepared by dissolving 1 g of shrimp chitosan (molecular weight of 900,000 to 1,000,000 Dalton, degree of deacetylation approximately 95%) in 100 ml of 1% acetic acid solution. The solution was then ltered through a silk screen to remove undissolved material. The three antimicrobial agents garlic oil (obtained from ABBRA Co. Ltd., Bangkok, Thailand), potassium sorbate (Fluka Chemie GmbH, Buchs) and nisin having activity 1020 IU/mg (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were incorporated into chitosan lm forming solution at various levels to obtain antimicrobial efcacy and acceptable physical nature of the lms. The solutions were then casted in a 12 cm 16 cm polyacrylic plates and dried at 40 1C for 2024 h. The dry lms obtained were peeled off and stored in a chamber at 50% RH and 25 1C until evaluation. 2.3. Physical characteristics of edible lm The average thickness of edible lm (mm) was determined by measuring it at several points with a hand micrometer (Mitutoyo Corp., Japan). Tensile strength (TS) and elongation at break (E) of lms were tested according to the ASTM standard method by using Lloyd Instrument Testing Machine type LRX 5K (Lloyd Instrument, Ltd., Fareham, UK). In preparing samples, lms were cut into 1.5 cm 10 cm strips. The lms were held parallel with an initial grip separation of 5 cm and then pulled apart at a head speed of 25 mm/ min. TS was calculated by dividing the maximum force at break (read from machine or chart) by cross-sectional area of lm. Percent E was calculated based on the length extended and original length of the lms.

2. Materials and methods 2.1. Organisms and cultures Five food pathogenic bacteria, which are typical meat product contaminants were used in this study. Escher-

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Water vapor permeability (WVP) was determined gravimetrically using a modied ASTM procedure as used by Gontard, Duchez, Cuq, and Guilbert (1994). The WVP was calculated as follows: WVP Dwx=ADtp2 p1 ; (1)

3. Results and discussion 3.1. Mechanical and physical properties of chitosanbased lms Mechanical and physical properties of chitosan edible lms comprising of TS, elongation at break (E), WVP and total color different (DE) are shown in Table 1. It shows the effect of different concentrations of antimicrobial agents incorporated into edible lm and the resultant change in the properties. In the range of antimicrobial agents concentration studied, a greater reduction of TS was shown by incorporating potassium sorbate (PS) and nisin (N) compared to incorporating garlic oil (GO). Incorporating PS at 150 mg/g of chitosan reduced TS from 37.03 into 13.94 MPa, while with N at 102,000 IU/g chitosan reduced from 37.03 into 16.57 MPa. Incorporation of GO reduced slightly TS of chitosan lm. A signicant (po0.05) reduction of TS was revealed by addition of GO at 400 ml/g of chitosan which reduced TS value from 37.03 to 28.97 MPa. This result conrms the outcome of the report by Cagri, Ustunol, and Ryser (2001), who had concluded earlier that incorporation of additives other than crosslinking agents generally lowers TS value. Similarly, incorporation of GO up to 400 ml/g into chitosan lm did not signicantly affect (po0.05) E value. On the other hand, in the range of concentrations studied, incorporating PS and N affected E value. Addition of PS at 100150 mg/g of chitosan increased E from 3.45% to approximately 9.90%, and it decreased when concentration of potassium sorbate was increased into 200 mg/g of chitosan. Similar pattern occurred on chitosan lm incorporated with N. In the range of N concentration investigated, E value was increased more than four times. Highest nisin incorporated at 204,000 IU/g of chitosan increased E from 3.45% to 30.72%. WVP is a measure of ease of the moisture to penetrate and pass through a material. GO did not signicantly (po0.05) affect water WVP on chitosan lm. Addition of PS at 150 mg/g of chitosan signicantly (po0.05) increased WVP from 0.02309 to 0.03363 g m/m2 day kPa. N at level of 153,000 IU/g of chitosan signicantly increased WVP value of chitosan from 0.02309 to 0.02762 g m/m2 day kPa. In general, the WVP value increased as antimicrobial agents added were higher. The antimicrobial agents contributed to extend intermolecular interaction and furthermore, loosening the compactness of the structure. This enhanced moisture passing through the edible lms and thereby increases WVP values of the lms. However, this did not occur prominently when chitosan lm incorporated with GO which is a hydrophobic material (Ross, Ogara, Hill, Sleightholme, & Maslin, 2001). Hydrophilic groups in the lm material tend to cause poor moisture barrier

where Dw is the weight of water absorbed in the cup (g), Dt the time for weight change (day), A the area of the exposed lm (m2), x the lm thickness (m), p2 p1 the vapor pressure differential across the lm (kPa), and calculated based on relative humidity and temperature inside and outside the cup. The WVP value was expressed in g m/m2 day kPa. Samples were monitored for their surface color by using a Color and Color Differential Meter model TCPIIIA (Tokyo Denshoku Co. Ltd., Japan). Measurements were taken as average of at least three points of each sample. Total color difference (DE) was calculated as follows: q DE L L2 a a2 b b2 ; (2) where L*, a* and b* are the standard values of white plate, L, a, and b are values of samples measured.

2.4. Antimicrobial assay Antimicrobial activity test of edible lms was carried out using agar diffusion method. Edible lms were cut into a disc form of 17 mm diameter using a circular knife. Film cuts were placed on Mueller Hinton agar (Merck, Darmstadt, Germany) plates which had been previously seeded with 0.1 ml of inoculum containing indicator microorganisms in the range of 105106 CFU/ ml. The plates were then incubated at 37 1C for 24 h. The diameter of inhibitory zone surrounding lm discs as well as contact area of edible lms with agar surface were then measured.

2.5. FTIR analysis The spectra of chitosan lms (control and those incorporating the three antimicrobial substances) were recorded by a Fourier Transform Infrared (FTIR) spectrometry (Perkin Elmer System 2000R) at room temperature at National Science and Technology Development Agency (NSTDA), Thailand. Light source of transmittance was in the middle range infrared 5004000 cm1. Detector used was TGS (Tri-GlycineSulfate) with resolution 4 cm1. The spectra obtained were used to determine possible interactions of functional groups between chitosan with garlic oil, potassium sorbate or nisin.

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862 Y. Pranoto et al. / LWT 38 (2005) 859865 Table 1 Tensile strength (TS), elongation at break (E), water vapor permeability (WVP) and total color difference (DE) of chitosan lms incorporated with garlic oil, potassium sorbate and nisin Antimicrobial agents TS (MPa) 37.0371.29a 35.3674.99ab 33.82+1.47ab 31.2574.64abc 28.9771.92bcd 26.4079.72cd 26.3273.17cd 13.9471.37e 13.5471.11e 23.7076.29df 16.5772.21e 17.5372.09ef 13.5871.33e E2 (%) 3.4570.34a 2.9971.15a 2.30770.62a 3.0271.52a 2.4671.30a 3.1470.77a 9.9772.62bc 9.8574.28bc 4.8971.66ab 14.1372.88cd 16.0078.54d 28.7873.06e 30.7271.81e WVP (g m/ m2 day kPa) 0.0230972.18 103a 0.0229671.99 103a 0.0258974.26 103a 0.0257174.97 103a 0.0280374.64 103ab 0.0236173.97 103a 0.0258171.98 103a 0.0336372.30 103b 0.0438474.13 103c 0.0239777.79 103a 0.0252574.84 103a 0.0276271.97 103ab 0.0342072.45 103b (DE) 9.2870.76a 8.5671.75a 8.4271.33a 9.8571.72a 8.4571.73a 11.5971.21ab 13.7272.38b 25.1972.91c 24.7474.56c 9.2971.24a 10.6970.60ab 9.1071.32a 13.4071.67b

Control Garlic oil (ml/g of chitosan) 100 200 300 400 Potassium sorbate (mg/g of chitosan) 50 100 150 200 Nisin (103 IU/g chitosan) 51 102 153 204

af Mean7standard deviation (n=3). Means in same column with different superscript letters are signicantly different (po0.05). TS is tensile strength; E is elongation at break; WVP is water vapor permeability; DE is total color different.

(Cagri et al., 2001). Incorporation of GO into chitosan lm material thus did not increase WVP value. Total color difference was observed by reading DE value which is formulated by L, a and b values, which represent black to white, green to red and blue to yellow, respectively. White plate was used as a color reference. Chitosan lm produced was slightly yellow and transparent. Its transparency was reduced as the antimicrobial agents were incorporated. Chitosan lm or control had DE value 9.28. Incorporation of GO in the range studied did not signicantly (po0.05) change DE value. PS and N affected DE of chitosan lm produced. PS at 100 mg/g of chitosan started to signicantly (po0.05) increase DE by giving value 13.72. Higher addition of PS to 150 mg/g of chitosan led to a great change on DE to become 25.19. Incorporating N at 204,000 IU/g of chitosan revealed DE value of 13.40, which was signicantly higher than that of the control. Overall it seems that the incorporation of antimicrobials into the lm leads to moderate changes in lms physical properties. 3.2. Antimicrobial activity The details of antimicrobial activity of chitosan edible lms incorporated with GO, PS and N against E. coli, S. aureus, S. typhimurium, L. monocytogenes and B. cereus are shown in Table 2. The indicator bacteria used for examination are common meat product contaminants. The inhibitory activity was measured based on clear zone surrounding circular lm strips. Measurement of clear zone diameter included diameter of lm strips, therefore, the values were always higher than the

diameter of lm strips (17 mm) whenever clearing zone was present. If there is no clear zone surrounding, we assumed that there is no inhibitory zone, and furthermore, the diameter was valued as zero. Contact area was used to evaluate growth inhibition underneath lm discs in direct contact with target microorganisms in agar. In terms of surrounding clearing zone, the control chitosan lm did not show inhibitory effect against all tested microorganisms. Incorporating GO into chitosan lm revealed antimicrobial effect. The inhibitory zones were markedly high for S. aureus, L. monocytogenes and B. cereus. It also reduced bacterial growth underneath lm of E. coli and S. typhimurium. Inhibitory zone increased by the increase of GO incorporated. L. monocytogenes was the most sensitive against GOincorporated lm followed by S. aureus and B. cereus. Chitosan lm incorporated with PS showed antimicrobial activity against S. aureus, L. monocytogenes and B. cereus. There was no effect on E. coli and S. typhimurium whether in its inhibitory zone or underneath lm. Increasing PS level higher than 100 mg/g of chitosan did not signicantly improve antimicrobial effect. The lack of increment on antimicrobial effect by increasing PS level was due to chemical interaction between amino group of chitosan and carboxyl group of preservative (Chen et al., 1996). Therefore, it hindered the release of PS to inhibit microorganism surrounding lm strips during agar diffusion assay. Functional groups interaction between PS and chitosan would be discussed latter (section FTIR analysis). Similar to GO and PS, incorporating N did not show inhibitory zone on E. coli and Salmonella typhimurium. However,

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Y. Pranoto et al. / LWT 38 (2005) 859865 863 Table 2 Antimicrobial activity of chitosan lms containing garlic oil, potassium sorbate and nisin against food pathogenic bacteria of E. coli, S. aureus, S. typhimurium, L. monocytogenes, and B. cereus Antimicrobial agents E. coli Gram () Inhibitory Control 0a Contact 7 7 + + S. aureus Gram (+) Inhibitory 0a 20.3973.77b 31.1773.77f 34.4673.28f 32.5475.21f 21.1570.59bc 22.0470.32bcd 19.6770.07b 20.0270.75b 22.6770.29bcd 25.3370.58de 23.8370.29cde 26.5070.50e Contact + + + + + + + + + + + + S. typhimurium Gram () Inhibitory 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a Contact 7 + + + 7 7 7 + L. monocytogenes Gram (+) Inhibitory 0a 26.4771.72d 34.7370.42g 34.6771.29g 40.8370.52h 21.0671.25b 24.3372.08c 22.9470.66c 22.9071.61bc 28.6771.15e 29.8370.29e 32.1770.29f 31.8370.29f Contact 7 + + + + + + + + + + + + B. cereus Gram (+) Inhibitory 0a 21.5670.56bc 30.7873.15e 34.8373.41f 28.4170.36e 21.4471.57b 24.1570.82cd 21.4271.26b 21.8871.69bcd 22.1771.04bcd 22.8370.29bcd 24.5071.00d 22.8370.29bcd Contact + + + + + + + + + + + + +

Garlic oil (ml/g of chitosan) 100 0a 200 0a 300 0a 400 0a

Potassium sorbate (mg/g of chitosan) 50 0a 100 0a 150 0a 200 0a Nisin ( 103 IU/g of chitosan) 51 0a 102 0a 153 0a 204 0a
af

7 7 7 +

Mean+standard deviation (n=3). Means in same column with different superscript letters are signicantly different (p o0.05). Inhibitory is inhibitory zone surrounding lm discs, measured diameter in mm; Contact is contact area under lm discs on agar surface. indicates growth in the area, +indicates no growth; Control is a plain lm disc without antimicrobial agent incorporation.

N-incorporated chitosan lm revealed growth inhibition underneath lm discs on these organisms. Among inhibited microorganisms, L. monocytogenes was the most sensitive and susceptible to N, same results as reported by investigators (Ming, Weber, Ayres, & Sandine, 1997; Gill & Holley, 2000; Cha, Cooksey, Chinnan, & Park, 2003). Incorporation of N at the lowest level of 51,000 IU/g of chitosan already showed a clear inhibitory zone of 28.67 mm dia. However, increasing level of nisin at higher concentration did not reveal signicant an increased inhibitory, as also observed when incorporated with GO and N at their high certain levels. It was generally caused by the maximum capability of chitosan polymer to carry active agents beside the occurrence of functional groups interaction phenomenon. Observation on the contact area, it revealed that incorporating nisin into chitosan lm revealed inhibitory effect shown by limited growth underneath lm for all bacteria. Also, the antimicrobial agents were obviously more effective against Grampositive bacteria than the Gram-negative bacteria studied. This is to be expected as the cell wall structures of these categories of bacteria are different and Grampositive bacteria are more sensitive to such agents (Nychas, 1995; Black, 1996). In general, chitosan lm itself showed some antimicrobial effect even though it did not reveal inhibitory zone in any microorganisms tested. It was obviously revealed by the limited growth of L. monocytogenes and

B. cereus underneath chitosan lm discs. This is reasonable as chitosan has the innate characteristic of antimicrobial activity itself (Wang, 1992; Darmadji & Izumimoto, 1994; Jongrittiporn et al., 2001). According to Brody, Strupinsky, and Kline (2001), the antimicrobial effect of chitosan occurred without migration of active agents. As chitosan is in a solid form, therefore, only organisms in direct contact with the active sites of chitosan is inhibited. Chitosan is incapable to diffuse through the adjacent agar media (Coma et al., 2002). The agar diffusion test is a method commonly used to examine antimicrobial activity regarding the diffusion of the compound tested through water-containing agar plate. The diffusion itself is dependent on the size, shape and polarity of the diffusing material. The chemical structure and the crosslinking level of the lms also affect this phenomenon (Cagri et al., 2001). When antimicrobial agents are incorporated, there will be diffusing materials through agar gel, and furthermore, resulting clearing zone on the bacterial growth. Incorporating antimicrobial agents into chitosan edible lm thus improves antimicrobial efcacy of chitosan, as diffused antimicrobial actively would add to nonmigrated antimicrobial potency of chitosan. 3.3. FTIR analysis The FTIR has been used to study the interaction between lm and antimicrobial agents incorporated.

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Figs. 13 depict the spectra of the chitosan lms incorporated with different antimicrobial agents at varying levels used in the study. All spectra show similar patterns with the peaks at 3400 cm1 and 10301155 cm1 broadened. Absorption in this area indicates stretching of the OH and NH bonds at 3400 cm1, and CO bonds at 10301155 cm1. In addition, absorption peaks at 2880 cm1 region, around 15501590 cm1 and 1400 cm1 correspond to CH stretching, amine groups (NH2) and carboxyl groups (COO), respectively. These indicated that there was no major structural change in the chitosan polymer. The spectra of chitosan lms incorporated with different levels of GO (Fig. 1) shows the same pattern on their informative peaks as the control chitosan lms. This

Fig. 3. Spectra of Fourier Transform Infrared (FTIR) of chitosan edible lms. (A) Chitosan lm, (B) chitosan lm incorporated with nisin 51,000 IU/g, (C) chitosan lm incorporated with 102,000 IU/g, (D) chitosan lm incorporated with 153,000 IU/g, and (E) chitosan lm incorporated with 204,000 IU/g of chitosan.

Fig. 1. Spectra of Fourier Transform Infrared (FTIR) of chitosan edible lms. (A) chitosan lm, (B) chitosan lm incorporated with garlic oil 100 ml/g, (C) chitosan lm incorporated with garlic oil 200 ml/ g, (D) chitosan lm incorporated with garlic oil 300 ml/g, and (E) chitosan incorporated with garlic oil 400 ml/g of chitosan.

Fig. 2. Spectra of Fourier Transform Infrared (FTIR) of chitosan edible lms. (A) chitosan lm, (B) chitosan lm incorporated with potassium sorbate 50 mg/g, (C) chitosan lm incorporated with 100 mg/g, (D) chitosan lm incorporated with 150 mg/g, and (E) chitosan incorporated with potassium sorbate 200 mg/g of chitosan.

indicates that there is no interaction between active groups of GO with functional groups of chitosan. However, in the spectrum of chitosan lms incorporated with PS (Fig. 2), a change in the amide I band at 1638 cm1 appears obviously. It is sharper with the increase of PS incorporated indicating some interaction between amine group of chitosan and carboxyl group of sorbate as reported earlier by Chen et al. (1996). Besides the peak around 1386 cm1 was stronger and sharper, and can be attributed to accumulation of carboxylate groups (COO). The spectra of chitosan lms incorporated with various levels of nisin are shown in Fig. 3. This is similar to the chitosan lm incorporated with PS on the amine peak region. The amide I band at 1638 cm1 increased with the increase in the amount of nisin incorporated. This is probably due to interaction between amine group of chitosan and functional groups contained in nisin leading to covalent bonds and hence an increase in peak size. However there was no observable change in carboxyl group region (1400 cm1). The infrared spectral data support mechanical and physical as well as antimicrobial properties data of chitosan lms incorporated with the three antimicrobial agents. When chitosan lms are incorporated with GO, there is no modication on the functional groups of chitosan. There is thus no signicant change on the mechanical and physical properties. The active compounds are free to inhibit microorganisms in the antimicrobial test. On the other hand, PS modied functional groups of chitosan, therefore, they signicantly changed mechanical and physical properties of chitosan lms produced. The presence of interaction between chitosan and PS led to a lower inhibitory effect as observed in antimicrobial assay. Incorporation of nisin into chitosan lm had a little effect on the

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functional groups change of the chitosan lm. Therefore, it did not change much on physical properties of chitosan lm produced and it also showed inhibitory effect due to the availability of free active nisin.

4. Conclusions Chitosan has great potential to improve its antimicrobial property by incorporating antimicrobial agents. Garlic oil incorporated into chitosan lm led to an increase in its antimicrobial efcacy, and had little effect on mechanical and physical properties of chitosan lms. However, the applications of garlic oil into lms will depend on the type of food where its avor is not a problem. Overall, the incorporation of garlic oil into chitosan lm has the desirable characteristic of acting as a physical and antimicrobial barrier to food contamination. FTIR studies data support the results obtained from other tests. References
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