enhancing antimicrobial activity of chitosan films

8/13/2019 Enhancing Antimicrobial Activity of Chitosan Films

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LWT 38 (2005) 859865

Enhancing antimicrobial activity of chitosan films 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 film 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 againstS. aureus,L. monocytogenes, andB. cereus. At these levels,

the films were physically acceptable in term of appearance, mechanical and physical properties. GO components did not affect the

physical and mechanical properties of chitosan films 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 film; 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 contamina-

tion 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 limita-tions because the active substances could be neutralized,

evaporated or diffused inadequately into the bulk of

food (Torres, Motoki, & Karel, 1985; Siragusa &

Dickson, 1992).

Edible films or coatings have been investigated for

their abilities to retard moisture, oxygen, aromas, and

solute transports (Gennadios& Weller, 1990). It is one

of the most effective methods to maintain food quality.

This is further improved by film carrying food additives

such as antioxidants, antimicrobial, colorants, flavors,

fortified 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 (Ouat-

tara 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, biodegrad-

able, biofunctional, biocompatible and have anti-

microbial characteristics (Wang, 1992; Darmadji &

Izumimoto, 1994; Jongrittiporn, Kungsuwan, & Rak-

shit, 2001). One of the reasons for the antimicrobial

character of chitosan is its positively charged amino

group which interacts with negatively charged microbial

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0023-6438/$30.00 r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

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Corresponding author. Tel.: +66 25246110; fax: +66 25246200.

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cell membranes, leading to the leakage of proteinaceous

and other intracellular constituents of the microorgan-

isms (Shahidi, Arachchi, & Jeon, 1999). In the Gram-

positive 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 films are easily prepared by evaporating from

dilute acid solutions (Park, Marsh, & Rhim, 2002). A

number of studies on the antimicrobial characteristics of

films 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, bacter-

iocins 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-contain-

ing compound such as allicin, diallyl disulfide and diallyl

trisulfide that possess better antimicrobial activity than

the corresponding ground form (Nychas, 1995). Potas-

sium 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 beincorporated into cellulose, whey protein isolate, soy

protein isolate, egg albumen, wheat gluten, hydroxy-

prophyl methylcellulose and zein film (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-asso-

ciated antimicrobial agents is an active area of research.

This study was done to improve antimicrobial efficacy

of edible film based on chitosan by incorporating garlic

oil, potassium sorbate and nisin. Mechanical and

physical properties were characterized, and antimicro-bial efficacy was assessed against five food pathogenic

bacteria. The functional groups interactions of these

three antimicrobials with the chitosan-based films were

also studied.

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-

ichia coliTISTR 73, Staphylococcus aureus TISTR 29,

Salmonella typhimurium TISTR 292, Listeria monocyto-

genes S 0273, and Bacillus cereus TISTR 747 were

obtained from the culture collection at Thailand

Institute of Scientific 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 films, a loopful of bacteria

from agar slant was taken and inoculated into 50 ml of

nutrient broth in 125 ml flask. The flask 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 film

Chitosan edible film was prepared by dissolving 1 g of

shrimp chitosan (molecular weight of 900,000 to

1,000,000 Dalton, degree of deacetylation approxi-

mately 95%) in 100 ml of 1% acetic acid solution. The

solution was then filtered 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 film forming solution at

various levels to obtain antimicrobial efficacy and

acceptable physical nature of the films. The solutions

were then casted in a 12cm 16 cm polyacrylic plates

and dried at 40 1C for 2024 h. The dry films obtained

were peeled off and stored in a chamber at 50% RH and

25 1C until evaluation.

2.3. Physical characteristics of edible film

The average thickness of edible film (mm) was

determined by measuring it at several points with ahand micrometer (Mitutoyo Corp., Japan). Tensile

strength (TS) and elongation at break (E) of films 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, films were cut into 1.5 cm 10 cm strips. The

films 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 film. Percent E was calculated based on the

length extended and original length of the films.

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Water vapor permeability (WVP) was determined

gravimetrically using a modified ASTM procedure as

used by Gontard, Duchez, Cuq, and Guilbert (1994).

The WVP was calculated as follows:

WVP Dwx=ADtp2p1; (1)

whereDwis the weight of water absorbed in the cup (g),

Dt the time for weight change (day), A the area of the

exposed film (m2), x the film thickness (m), p2p1 the

vapor pressure differential across the film (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 TC-

PIIIA (Tokyo Denshoku Co. Ltd., Japan). Measure-

ments were taken as average of at least three points of

each sample. Total color difference (DE) was calculated

as follows:

DE

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiL L2 a a2 b b2

q ; (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 films was carried

out using agar diffusion method. Edible films were cut

into a disc form of 17 mm diameter using a circularknife. Film cuts were placed on Mueller Hinton agar

(Merck, Darmstadt, Germany) plates which had been

previously seeded with 0.1ml 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 film discs as

well as contact area of edible films with agar surface

were then measured.

2.5. FTIR analysis

The spectra of chitosan films (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-Glycine-

Sulfate) with resolution 4 cm1. The spectra obtained

were used to determine possible interactions of func-

tional groups between chitosan with garlic oil, potas-

sium sorbate or nisin.

3. Results and discussion

3.1. Mechanical and physical properties of chitosan-

based films

Mechanical and physical properties of chitosan edible

films comprising of TS, elongation at break (E), WVPand total color different (DE) are shown inTable 1. It

shows the effect of different concentrations of anti-

microbial agents incorporated into edible film 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 film. A significant (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 confirms 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 400ml/g into

chitosan film did not significantly 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 increasedinto 200 mg/g of chitosan. Similar pattern occurred on

chitosan film incorporated with N. In the range of N

concentration investigated, Evalue 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 significantly

(po0.05) affect water WVP on chitosan film. Addition

of PS at 150 mg/g of chitosan significantly (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 significantlyincreased 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 inter-

molecular interaction and furthermore, loosening the

compactness of the structure. This enhanced moisture

passing through the edible films and thereby increases

WVP values of the films. However, this did not occur

prominently when chitosan film incorporated with GO

which is a hydrophobic material (Ross, Ogara, Hill,

Sleightholme, & Maslin, 2001). Hydrophilic groups in

the film material tend to cause poor moisture barrier

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(Cagri et al., 2001). Incorporation of GO into chitosan

film 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 film produced was slightly yellow and trans-

parent. Its transparency was reduced as the antimicrobialagents were incorporated. Chitosan film or control had

DEvalue 9.28. Incorporation of GO in the range studied

did not significantly (po0.05) change DEvalue. PS and N

affected DEof chitosan film produced. PS at 100 mg/g of

chitosan started to significantly (po0.05) increaseDEby

giving value 13.72. Higher addition of PS to 150 mg/g of

chitosan led to a great change on DEto become 25.19.

Incorporating N at 204,000 IU/g of chitosan revealedDE

value of 13.40, which was significantly higher than that of

the control. Overall it seems that the incorporation of

antimicrobials into the film leads to moderate changes in

films physical properties.

3.2. Antimicrobial activity

The details of antimicrobial activity of chitosan edible

films 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 film strips. Measurement of

clear zone diameter included diameter of film strips,

therefore, the values were always higher than the

diameter of film strips (17 mm) whenever clearing zone

was present. If there is no clear zone surrounding, we

assumed that there is no inhibitory zone, and further-

more, the diameter was valued as zero. Contact area was

used to evaluate growth inhibition underneath film discs

in direct contact with target microorganisms in agar.

In terms of surrounding clearing zone, the control

chitosan film did not show inhibitory effect against alltested microorganisms. Incorporating GO into chitosan

film revealed antimicrobial effect. The inhibitory zones

were markedly high forS. aureus,L. monocytogenesand

B. cereus. It also reduced bacterial growth underneath

film of E. coli and S. typhimurium. Inhibitory zone

increased by the increase of GO incorporated. L.

monocytogenes was the most sensitive against GO-

incorporated film followed by S. aureus and B. cereus.

Chitosan film incorporated with PS showed antimicro-

bial 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 under-neath film. Increasing PS level higher than 100 mg/g of

chitosan did not significantly 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

film 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. coliand Salmonella typhimurium. However,

ARTICLE IN PRESS

Table 1

Tensile strength (TS), elongation at break (E), water vapor permeability (WVP) and total color difference (DE) of chitosan films incorporated with

garlic oil, potassium sorbate and nisin

Antimicrobial agents TS (MPa) E2 (%) WVP (g m/

m2 daykPa)

(DE)

Control 37.0371.29a 3.4570.34a 0.0230972.18 103a 9.2870.76a

Garlic oil (ml/g of chitosan)

100 35.3674.99ab 2.9971.15a 0.0229671.99 103a 8.5671.75a

200 33.82+1.47ab 2.30770.62a 0.0258974.26 103a 8.4271.33a

300 31.2574.64abc 3.0271.52a 0.0257174.97 103a 9.8571.72a

400 28.9771.92bcd 2.4671.30a 0.0280374.64 103ab 8.4571.73a

Potassium sorbate (mg/g of chitosan)

50 26.4079.72cd 3.1470.77a 0.0236173.97 103a 11.5971.21ab

100 26.3273.17cd 9.9772.62bc 0.0258171.98 103a 13.7272.38b

150 13.9471.37e 9.8574.28bc 0.0336372.30 103b 25.1972.91c

200 13.5471.11e 4.8971.66ab 0.0438474.13 103c 24.7474.56c

Nisin (103 IU/g chitosan)

51 23.7076.29df 14.1372.88cd 0.0239777.79 103a 9.2971.24a

102 16.5772.21e 16.0078.54d 0.0252574.84 103a 10.6970.60ab

153 17.5372.09ef 28.7873.06e 0.0276271.97 10

3ab 9.1071.32a

204 13.5871.33e 30.7271.81e 0.0342072.45 103b 13.4071.67b

afMean7standard deviation (n=3). Means in same column with different superscript letters are significantly different (po0.05).

TS is tensile strength; Eis elongation at break; WVP is water vapor permeability; DE is total color different.



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N-incorporated chitosan film revealed growth inhibition

underneath film 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 significant 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 chitosanfilm revealed inhibitory effect shown by limited growth

underneath film for all bacteria. Also, the antimicrobial

agents were obviously more effective against Gram-

positive 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 Gram-

positive bacteria are more sensitive to such agents

(Nychas, 1995;Black, 1996).

In general, chitosan film itself showed some anti-

microbial effect even though it did not reveal inhibitory

zone in any microorganisms tested. It was obviously

revealed by the limited growth ofL. monocytogenesand

B. cereus underneath chitosan film discs. This is

reasonable as chitosan has the innate characteristic of

antimicrobial activity itself (Wang, 1992; Darmadji &

Izumimoto, 1994;Jongrittiporn et al., 2001). According

toBrody, Strupinsky, and Kline (2001), the antimicro-bial 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 films also

affect this phenomenon (Cagri et al., 2001). Whenantimicrobial agents are incorporated, there will be

diffusing materials through agar gel, and furthermore,

resulting clearing zone on the bacterial growth. Incor-

porating antimicrobial agents into chitosan edible film

thus improves antimicrobial efficacy of chitosan, as

diffused antimicrobial actively would add to non-

migrated antimicrobial potency of chitosan.

3.3. FTIR analysis

The FTIR has been used to study the interaction

between film and antimicrobial agents incorporated.

ARTICLE IN PRESS

Table 2

Antimicrobial activity of chitosan films 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 S. aureus S. typhimurium L. monocytogenes B. cereus

Gram () Gram (+) Gram () Gram (+) Gram (+)

Inhibitory Contact Inhibitory Contact Inhibitory Contact Inhibitory Contact Inhibitory Contact

Control 0a 0a 0a 0a 7 0a +

Garlic oil (ml/g of chitosan)

100 0a 7 20.3973.77b + 0a 7 26.4771.72d + 21.5670.56bc +

200 0a 7 31.1773.77f + 0a + 34.7370.42g + 30.7873.15e +

300 0a + 34.4673.28f + 0a + 34.6771.29g + 34.8373.41f +

400 0a + 32.5475.21f + 0a + 40.8370.52h + 28.4170.36e +

Potassium sorbate (mg/g of chitosan)

50 0a 21.1570.59bc + 0a 21.0671.25b + 21.4471.57b +

100 0a 22.0470.32bcd + 0a 24.3372.08c + 24.1570.82cd +

150 0a 19.6770.07b + 0a 22.9470.66c + 21.4271.26b +

200 0a 20.0270.75b + 0a 22.9071.61bc + 21.8871.69bcd +

Nisin ( 103 IU/g of chitosan)

51 0a

7 22.6770.29bcd

+ 0a

7 28.6771.15e

+ 22.1771.04bcd

+102 0a 7 25.3370.58de + 0a 7 29.8370.29e + 22.8370.29bcd +

153 0a 7 23.8370.29cde + 0a 7 32.1770.29f + 24.5071.00d +

204 0a + 26.5070.50e + 0a + 31.8370.29f + 22.8370.29bcd +

afMean+standard deviation (n=3). Means in same column with different superscript letters are significantly different (p o0.05).

Inhibitory is inhibitory zone surrounding film discs, measured diameter in mm; Contact is contact area under film discs on agar surface. indicates

growth in the area, +indicates no growth; Control is a plain film disc without antimicrobial agent incorporation.



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Figs. 13 depict the spectra of the chitosan films

incorporated with different antimicrobial agents at

varying levels used in the study. All spectra show similar

patterns with the peaks at 3400 c m1 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

. Inaddition, absorption peaks at 2880 cm1 region, around

15501590 cm1 and 1400cm1 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 films incorporated with different

levels of GO (Fig. 1) shows the same pattern on their

informative peaks as the control chitosan films. This

indicates that there is no interaction between active

groups of GO with functional groups of chitosan.

However, in the spectrum of chitosan films 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 byChen 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 films incor-

porated with various levels of nisin are shown inFig. 3.This is similar to the chitosan film 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 inter-

action between amine group of chitosan and fun-

ctional 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 films incorporated with the three antimicrobialagents. When chitosan films are incorporated with GO,

there is no modification on the functional groups of

chitosan. There is thus no significant change on the

mechanical and physical properties. The active com-

pounds are free to inhibit microorganisms in the

antimicrobial test. On the other hand, PS modified

functional groups of chitosan, therefore, they signifi-

cantly changed mechanical and physical properties of

chitosan films 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 film had a little effect on the

ARTICLE IN PRESS

Fig. 1. Spectra of Fourier Transform Infrared (FTIR) of chitosanedible films. (A) chitosan film, (B) chitosan film incorporated with

garlic oil 100 ml/g, (C) chitosan film incorporated with garlic oil 200ml/

g, (D) chitosan film incorporated with garlic oil 300ml/g, and (E)

chitosan incorporated with garlic oil 400 ml/g of chitosan.

Fig. 2. Spectra of Fourier Transform Infrared (FTIR) of chitosan

edible films. (A) chitosan film, (B) chitosan film incorporated with

potassium sorbate 50mg/g, (C) chitosan film incorporated with

100 mg/g, (D) chitosan film incorporated with 150 mg/g, and (E)

chitosan incorporated with potassium sorbate 200 mg/g of chitosan.

Fig. 3. Spectra of Fourier Transform Infrared (FTIR) of chitosan

edible films. (A) Chitosan film, (B) chitosan film incorporated with

nisin 51,000 IU/g, (C) chitosan film incorporated with 102,000IU/g,

(D) chitosan film incorporated with 153,000IU/g, and (E) chitosan

film incorporated with 204,000IU/g of chitosan.



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functional groups change of the chitosan film. There-

fore, it did not change much on physical properties of

chitosan film produced and it also showed inhibitory

effect due to the availability of free active nisin.

4. Conclusions

Chitosan has great potential to improve its antimicro-

bial property by incorporating antimicrobial agents. Garlic

oil incorporated into chitosan film led to an increase in its

antimicrobial efficacy, and had little effect on mechanical

and physical properties of chitosan films. However, the

applications of garlic oil into films will depend on the type

of food where its flavor is not a problem. Overall, the

incorporation of garlic oil into chitosan film 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.

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enhancing antimicrobial activity of chitosan films

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