Antimicrobial activity against foodborne pathogens of chitosan biopolymer films of different molecular weights

LWT - Food Science and Technology 44 (2011) 565e569

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Antimicrobial activity against foodborne pathogens of chitosan biopolymer films of different molecular weights Kyung W. Kim a, B.J. Min b,1, Young-Teck Kim a, *, Robert M. Kimmel a, Kay Cooksey a, S.I. Park c a

Department of Packaging Science, B-212 Poole Agricultural Center, Clemson University, Clemson, SC 29634-0320, United States Department of Food and Nutrition Science, Tuskegee University, Tuskegee, AL, United States c Department of Packaging, Yonsei University, Wonju, Kangwon-do, 220-710, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 September 2009 Received in revised form 30 July 2010 Accepted 2 August 2010

Antimicrobial activity against Listeria monocytogenes, Escherichia coli 0157:H7 and Samonella typhimurium of chitosan biopolymer films (CBFs) prepared with four different viscosities of chitosans (10, 40, 100 and 200 mPa s) were investigated by agar diffusion assay. The films were also characterized with measurements of color, tensile strength (TS), % elongation (EL), water vapor permeability and oxygen permeability. CBFs prepared with 100 mPa s chitosan showed an antimicrobial effect only on 104 cfu/mL inoculation of L. monocytogenes while other viscosities showed an antilisterial effect on all concentrations (104e106 cfu/mL) of L. monocytogenes. CBFs prepared with 10 mPa s (CBF-10) and 40 mPa s (CBF-40) chitosans showed an inhibitory effect against E. coli 0157:H7 and S. typhimurium only at the 104 cfu/mL concentration. CBFs prepared with the two higher viscosity chitosans did not show any effect regardless of bacterial level. TS and EL of the CBFs increased with increasing viscosity up to 100 mPa s. Molecular weight distribution was found to be positively correlated with viscosity. The oxygen permeability of the CBFs increased with increasing viscosity of chitosans, but water vapor transmission rate was not similarly affected. In conclusion, CBFs were more effective at inhibition of L. monocytogenes than S. typhimurium and E. Coli O157:H7. Molecular weight of chitosan must be chosen selectively to control the target foodborne pathogens. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Chitosan Molecular weight L. monocytogenes S. typhimurium

1. Introduction Chitosan, poly-b-(1 / 4) N-acetyl-D-glucosamine, is obtained by deacetylation of chitin, a biopolymer that is abundant in a variety of crustacean shells, such as crab shells, crawfish shells and shrimp shells. Since chitosan is decomposable and biocompatible (Coma et al., 2002) as well as exhibiting inhibitory effects against fungi, yeast and various bacteria (Muzzarelli et al., 1990; Roades & Roller, 2000; Roller & Covill, 1999, Sebti, Martial-Gros,Carnet-pantiez, Grelier, & Coma, 2005), it has been used as an antimicrobial agent to improve food safety and quality. Antimicrobial effects of chitosan in various environments against gram-positive and gram-negative bacteria have been studied by changing media, emulsion and food systems (Chhabra, Huang, Frank, Chmielewski, & Gates, 2006; Roades & Roller, 2000; Zivanovic, Shuang, & Draughon, 2004). For example, the growth of Zygosacchromyces bailii was studied * Corresponding author. Tel.: þ1 864 656 5689; fax: þ1 864 656 4395. E-mail address: [email protected] (Y.-T. Kim). 1 Author Byung Jin Min has equivalently contributed to this paper as a first author. 0023-6438/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.08.001

using degraded chitosan (0.1 g/L) for four days in laboratory media, indicating the antifungal effects of chitosan (Roller & Covill, 1999). Chitosan has been tested in food applications such as strawberries, bell peppers and tomatoes (El Ghaouth, Arul, & Ponnampalam, 1991a; El Ghaouth, Arul, & Ponnampalam, 1991b; El Ghaouth, Ponnampalam, Castaigne, & Arul, 1992; Jian & Li, 2001). It was reported that N-Carboxybutyl chitosan showed inhibition activity against many different gram-positive and gram-negative bacteria (Muzzarelli et el. 1990). The growth of L. monocytogenes Scott A and L. monocytogenes 310 were effectively suppressed by a chitosan polysaccharide oil/water emulsion (; Zivanovic, Shuang, et al., 2004). The growth of Staphylococcus aureus was strongly inhibited in raw oysters coated with 2 g/100 mL chitosan dissolved in 0.5 g/100 mL hydrochloric acid (Chhabra et al., 2006). Chitosan has mucoadhesive properties which are revealed by the spreading ability of chitosan over a mucus layer and also through its positive ionic interactions with the negative charges of the mucus or of the cell surface membranes (Lehr, Bouwstra, Schacht, & Junginger, 1992; Lehr, Bouwstra, Schacht, & Junginger, 1993). The antimicrobial action of chitosan involves destruction of the inner and outer membranes of bacteria by the releasing of

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intracellular components (Liu, Du, Wang, & Sun, 2004). Also, chitosan changes the outer membrane state by some linking effects and also that it modifies the cell surface. This modification may cause the weakening of the cell function of gram-negative bacteria (Helander, Nurmiaho-Lassila, Ahvenainen, Rhoades, & Roller, 2001). Antimicrobial efficacy of chitosan is not only affected by the degree of deacetylation (DA) and by solubility but also by its molecular weight (MW). In common with other polymers, viscosity is an important factor in the conventional determination of the molecular weight of chitosan. In general, higher MW results in the higher viscosity. Viscosity is affected by DA, MW, concentrations of the solution, ionic strength, pH and temperature. Viscosity is specifically used for determining commercial and industrial applications of chitosans in complex biological environments such as food systems. Many chitosan manufacturers deliver their products in a variety of viscosities. The effects of different MW of the chitosan on bacteria have been examined by many researchers. Most studies have been carried out in solution or in oil/water emulsions, rather than in a dried format such as a biopolymer film. For examples, it has been reported that six chitosans (28, 224, 470, 746, 1106 and 1671 kDa) and six chitosan oligomers solutions (1, 2, 4, 7, 10 and 22 kDa) showed antibacterial effects against gram-positive and gramnegative bacteria, with a tendency to exhibit better antimicrobial effect by the chitosans than by the chitosan oligomers. The extent of the antimicrobial activity against each tested bacteria was dependent upon the molecular weight of the chitosan (No, Park, Lee, & Meyers, 2002). Kim, Thomas, Lee, and Park (2003) showed that the antimicrobial effect against bacteria (E. coli , S. aures, Bacillus. subtilis and S. typhymurium) in media was dependent on the MW of chitosans prepared as native chitosan (120 kDa), degraded chitosan (30 kDa), O-carboxymethyl chitosan (435.5 kDa) from native chitosan and O-carboxymethyl chitosan (78.6 kDa MW) from degraded chitosan. O-carboxymethyl chitosan produced from degraded chitosan had the greatest antimicrobial effect. Chitosan of approximately 150 kDa in a polysaccharide oil-in-water emulsion system showed more inhibition against S. typhimurium than an oligosaccharide emulsion system (Zivanovic, Basurto, Chi, Davidson, & Weiss, 2004). The antimicrobial effect of chitosan (450 kDa) film enriched with essential oils on the growth of bacteria has been examined (Zivanovic, Shuang, et al., 2004), with the finding that chitosan films showed up to 3 log reductions of Listeria monocytogenes and E. coli after 5 days storage in a bolgnawrap package, while oregano-enhanced chitosan films (1 g/100 mL and 2 g/100 mL) showed 4 log reductions against the same pathogens (Zivanovic, Shuang, et al., 2004). In most studies, the antimicrobial activity of chitosan has been tested using direct addition of chitosan into the solution and/or emulsion type model system. Up to now, the antimicrobial activity of chitosan biopolymer films with different MWs using a diffusion assay has rarely been discussed. The objective of the present study is to characterize the properties of chitosan biopolymer films (CBFs) prepared at various viscosities with different MWs and to investigate their antimicrobial activity on E. coli, L. monocytogenes and S. typhimurium in the culture medium using relatively low to medium chitosan MW and viscosity in a diffusion assay. 2. Materials and methods 2.1. Materials Four different chitosans with low to medium viscosity (10, 40, 100 and 200 mPa s) were purchased from Kimitsu Co. (Tokyo, Japan). Their degrees of deacetylation (DA, %) were 82.32%, 80.77%,

84.71% and 84.32%, respectively. DA and viscosity values were provided by the manufacturer. All microbial cultures were generously given from the laboratory of Dr P. L. Dawson (Department of food science and human nutrition, Clemson University, Clemson, SC USA). All chemicals used were of analytical grade or the highest grade available and were obtained either from SigmaeAldrich or Merck (Darmstadt, Germany). 2.2. Determination of MWs of chitosan The MWs of chitosans except for 200 mPa s chitosan was determined using High-performance size-exclusion chromatography together with Multiangle laser light scatteringerefractive index system. The system consisted of a pump (P2000, Spectra System, San Jose, CA), an injection valve (model 7021, Rheodyne, Cotati, CA), a guard column (TSK PWH, Tosoh Corp., Tokyo, Japan), and SEC column (TSK Gel 3000PW, 7.8  600 mm, Tosoh Corp., Tokyo, Japan) detectors, Multiangle laser light-scattering (LS; Dawn DSP-F, Wyatt Technology, Santa Barbara, CA, USA), and refractive index (RI; Shodex SE71, Tokyo, Japan) detectors. Output volatage of RI and LS at the 18 angles were collected and used to calculate MW using Astra 4.50 software (Kim et al., 2003). 2.3. Film preparation Four different CBFs (10, 40, 100 and 200 mPa s) were fabricated by a casting technique. Chitosan (3 g) and glycerol (0.6 g) were combined with 150 mL distilled water containing 1 (mL/100 mL) of acetic acid. The pH value of the chitosan solution was adjusted to 5.4. The chitosan solution was stirred at room temperature until it was completely dissolved, and then poured on a teflon sheet (25  25 cm) attached to a glass plate. The solutions were poured through cheese cloth to remove contaminants and bubbles. The films used in the subsequent experiments were allowed to dry and then peeled from the glass plates. 2.4. Physical properties of CBFs Film color was measured using a Minolta Chroma Meter (CR-400, Minolta Corporation, Ramsey, NJ) after calibration using a calibration tile. The CIE L*, a* and b* values of the chitosan films were measured. A SATEC Material Testing System (Model No. T1000, Instron Co., USA) was used to measure tensile strength (TS), percent elongation (%E) at break and energy at break (J) according to ASTM Standard Method D 882 (2001). Samples were 1.53 cm  10 cm and film thickness was measured using a digital micrometer (Precision micrometer, Testing Machines Inc., USA). Testing speed was 100 mm/min. TS and percent elongation at break were calculated by the Bluehill2 software system (Version 2.52, Instron, Co., USA) 2.5. Oxygen permeability Oxygen permeability (OP) was measured by using a MOCON OXTRAN 2/20 Permeability Tester (Modern Controls, Inc., USA) according to the American Society of Testing and Materials Standard (ASTM) Method D3985 (ASTM, 2005a). Testing was performed at 25  C and 50% RH; gas flow rate was fixed at 10 mL/min. The equipment was calibrated before use using a reference film provided by the manufacturer. Oxygen permeability (OP) is reported as m$cc O2/m2$day at 101.325 kPa. The mean value of three separate measurements is reported. Oxygen permeability was calculated by multiplying these values by film thickness. Film thickness was measured immediately upon removal of the film

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Table 1 Color properties of chitosan biopolymer films prepared with various viscosities of chitosan.

Teflon sheet Chitosan biopolymer film

mPa s

MWb (kDa)

Lc*

Experimental value*

M.Va e

10 40 100 200

e 29.1  0.0 91.2  0.1 119.1  0.4 N/A

30 90 120 z300a

91.7  89.8  89.7  91.2  89.6 

ad*

0.0 0.2 0.1 0.1 0.1

0.9 0.4 0.1 0.7 0.5

be*

    

0.0 0.1 0.0 0.0 0.0

1.2 5.7 4.2 0.0 2.7

    

0.0 0.2 0.0 0.0 0.0

*

P < 0.05. Values were the means  standard deviation of 3 replications (n ¼ 3). a M.V: Manufacturer’s value. b MW: Molecular Weight. c Brightness. d Redness. e Yellowness.

sample from the testing cell using a digital micrometer (Precision micrometer, Testing Machines Inc., USA). 2.6. Water vapor permeability A Mocon Permatran-W3/31 was used to measure the water vapor transmission rate at 25  C and 50% RH according to ASTM F1249 (ASTM, 2005b). The test film was masked with aluminum foil giving a 5 cm2 test area. The nitrogen gas flow was set at 100 sccm (standard cubic centimeters per minute). The equipment was calibrated using a reference film provided by the manufacturer. The water vapor permeability coefficient (WVP) was reported as thickness$gram of water vapor/m2$day at 101.325 kPa according to WVP (mg/m2$day) ¼ WVTR  (film thickness/difference in partial pressure of vapor (DP)). P is 101.325 kPa in this study. 2.7. Antimicrobial activity The three foodborne pathogens L. monocytogenes, E. coli 0157:H7 and S. typhimurium were used as test organisms to determine antimicrobial activity via the agar diffusion technique. L. monocytogenes was stored in a brain heart infusion (BHI) broth with 20 g/100 mL glycerol at 70  C until used. The thawed culture (0.1 mL) was transferred to 10 mL of BHI broth and grown in a shaker at 37  C for 18 h. A second transfer of 0.1 mL of culture into 10 mL of BHI broth was grown in a shaker at 37  C for 18 h and used for the experiment. The same procedure using a tryptic soy agar (TSA) was used for E. coli 0157:H7 and S. typhimurium. To prevent CBFs from curling up when placed on agar, CBFs were preconditioned in sterilized water for 1 s. The preconditioned circular samples (6 mm diameter) were placed on the surface of solid soft agar with the test organism at three different levels (104, 105, and 106 cfu/mL). Brain Heart Infusion Soft Agar (BHA) was used for L. monocytogenes and Tryptic Soy Soft Agar (TSA) was used for E. coli 0157:H7 and S. typhimurium. After incubation at 37  C for 48 h, the antimicrobial effect of CBFs prepared with various viscosities in different MWs was observed. After peeling off the circular CBFs on the surface of soft agars used, the inhibition zone was optically determined by observing whether bacterial cells grew in the contact area.

2.8. Statistical analysis Each experiment was performed three times in duplicates. Data were analyzed using the general linear models procedure in the SAS software program (Statistical Analysis System Institute Inc., Cary, N.C., U.S.A.). Multiple mean comparisons were carried out by Duncan’s multiple range tests (P < 0.05). 3. Results and discussion 3.1. Physical properties The MW of chitosan is highly correlated to viscosity as expected (Table 1) based on previous literature (Whistler & Bemiller, 1997 pp. 91e115). Our experimental values for MW are identical with the manufacturer’s reported values. However, we were unable to determine the MW of 200 mPa s chitosan because the viscosity of this sample was too high for our system. In addition, it was impossible to purify the 200 mPa s chitosan before fabricating the biopolymer film due to too high and too broad molecular weight distribution. Therefore, the manufacturer’s approximate MW value (z300 kDa) for 200 mPa s chitosan was used for further tests. All CBFs tested in our study showed very similar color characteristics as shown in Table 1. There is no difference in brightness (L*). Color of all CBFs except for CBF-100 agrees well with previous characterization of chitosan films (e.g. slightly acidic smell, yellow tinted color and transparent characteristics) (Nadarajah, Prinyawiwatkul, No, Sathivel, & Xu, 2006). CBF-100 was clear and transparent rather than yellowish. Compared to the other CBFs, the thickness of CBF-200 (with highest viscosity and MW) was significantly (P < 0.05) higher. As viscosity increased from 10 to 100, the tensile strength (TS) and energy at break (EB) of chitosan increased from 44.47 to 106.51 MPa and 0.40e1.23 J, respectively. But, TS and EB of CBF-200 were significantly lower than that of CBF-100. It is believed that this is an undesirable effect of the broad molecular weight distribution and lower purity of 200 mPa s chitosan according to the manufacturer’s data. A similar phenomenon was observed in the mechanical properties. The percent elongation of CBFs increased

Table 2 Mechanical properties of chitosan biopolymer films prepared with various viscosities of chitosan.

Chitosan biopolymer film

mPa s

Thickness (mm)

Tensile Strength (MPa)

Elongation (%)

Energy at break (J)

10 40 100 200

0.044b 0.042b 0.045b 0.051a

44.47d 65.80b 106.51a 52.66c

20.64b 30.93a 34.06a 24.77b

0.40bc 0.68b 1.23a 0.57b

   

0.005 0.002 0.002 0.003

   

3.15 3.25 7.87 4.31

   

1.82 1.27 2.37 1.81

   

0.03 0.17 0.14 0.06

Values were the means  standard deviation of 3 replications (n ¼ 3). Means followed by the same lowercase letters in the same column were not significantly different (P > 0.05).

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from 20.64 to 34.06% when the viscosity increased from 10 to 100 mPa s. Therefore, the mechanical properties of CBFs correlated well with viscosity (10e100 mPa s).

Table 4 Antimicrobial effect of chitosan biopolymer films against L. monocytogenes, E. coli 0157:H7 and S. typhimurium, by an agar diffusion technique. Chitosan (mPa s)

3.2. Gas barrier properties Oxygen permeability of CBFs was proportional to viscosity (p < 0.05), while water vapor permeability was not statistically affected by the viscosity (Table 3). This is in good agreement with previous values (Muzzarelli, Isolati, & Ferrero, 1974; Park, Marsh, & Rhim, 2002). It was reported that oxygen permeabilities of films prepared with relatively narrow viscosity range chitosans (10, 30, and 50 mPa s) were not systematic, giving the highest value at 50 mPa s (Park et al., 2002). In our study, oxygen permeability of CBFs showed a positive relationship over a relatively wide range of viscosity (Table 2). 3.3. Antimicrobial activity of chitosan films The antimicrobial effects of CBFs prepared with different viscosity of chitosans against three different bacteria are shown in Table 4. L. monocytogenes was used as a test organism for grampositive bacteria while E. coli 0157:H7 and S. typhimurium were used for gram-negative bacteria tests. Regardless of inoculum levels from 104 to 106 cfu/mL and the viscosity of chitosans, L. monocytogenes was strongly inhibited by all CBFs prepared with 10, 40 and 200 mPa s chitosan. CBF-100 showed antilisterial effect at only 104 cfu/mL Coma et al. (2002) tested antimicrobial effects of chitosan film forming solutions, coatings and films against L. monocytogenes and Listeria innocua during different time periods at 37  C. They found that two bacteria were completely inhibited based on film forming solution and coating method instead of film type. According to the results shown in Table 4, CBF-10 and CBF-40 inhibited the bacterial growth of both E. coli 0157:H7 and Samonella typhimurium at only 104 cfu/mL, while two other higher viscosity CBFs did not show any antimicrobial effects at all of the inoculum levels (105 and 106 cfu/mL) used in our study. Park, Daeschel, and Zhao (2004) found that shrimp-derived chitosan films prepared with 11 mPa s viscosity chitosan had an antimicrobial effect against E. coli during the storage time, when they determined the antimicrobial activity of lysozyme-based chitosan film (Park et al., 2004). These results indicated that low viscosity chitosan will be relatively effective against gram-negative bacteria. Contrary to our observation, No et al. (2002) reported that the growth of E.Coli was effectively inhibited by relatively high MW (224 kDa) chitosan instead of low MW. In their reports, S. typhimurium was not inhibited at both 224 kDa and various low molecular weights of chitosan (10, 22 and 28 kDa) and L. monocytogenes was successfully inhibited over a very broad MW range from 1 kDa to 1671 kDa, as we observed in our tests. These variations could be related to other factors such as the

Table 3 Oxygen and water vapor permeability of chitosan biopolymer films prepared with various viscosities of chitosan.

Chitosan Biopolymer film

mPa s

Water Vapor Permeability (m$g/m2$day)

Oxygen Permeability (108m cc/m2$day)

10 40 100 200

6,477ab 6,404ab 6,644a 6,303b

1.77d 2.44c 2.98b 3.48a

   

236 202 95 93

   

0.04 0.03 0.02 0.01

Values were the means  standard deviation of 3 replications (n ¼ 3) and measured at 101.325 kPa. Means followed by the same lowercase letters in the same column were not significantly different (P > 0.05).

Bacteria

cfu/mL

10

40

100

200

L. monocytogenes

104 105 106 104 105 106 104 105 106

þ þ þ þ   þ  

þ þ þ þ   þ  

þ        

þ þ þ      

E. coli 0157:H7

S. typhimurium

þ; No bacterial growth in contact area, ; Bacterial growth in contact area. Each observation was performed three times in duplicates, after incubation at 37  C for 48 h.

degree of deacetylation of chitosan and type of test (e.g. film diffusion test or solution test). CBFs can be useful for inhibition of gram-positive and gramnegative bacteria and relatively low viscosity-based CBFs (CBF-10 and -40) will be effective in improving food safety (Table 4). Based on our observations, it was presumed that the antimicrobial effects of CBFs prepared with relatively low MW and viscosity chitosan are caused by direct diffusion and interaction of small chitosan molecules from the films with the test medium at the contact area, since CBFs prepared in our system did not include any antimicrobial agents. It implies that the diffusion of small MW and low viscosity chitosan molecules could be a major mechanism of antimicrobial activity of chitosan polymer film on the agar gel system. 4. Conclusions Based on this study, it was confirmed that viscosity is highly correlated to MW. Chitosan with a narrow molecular weight distribution is more useful to control film quality and antimicrobial activity. The effect of CBFs on growth of foodborne pathogens can be successfully determined by the agar diffusion test. According to our results, the growth of L. monocytogenes was affected by a broad range of viscosity and MW chitosans. To successfully control the growth of gram-negative bacteria at higher concentrations, the characteristics of chitosan should be carefully selected in advance. References ASTM. (2001). Standard test method for tensile properties of thin plastic sheeting (D882). In Annual book of ASTM. Philadelphia, PA: American Society for Testing and Materials. ASTM. (2005a). Standard test method for oxygen gas transmission rate through plastic film and sheeting using a colorimetric sensor (D3985). In Annual book of ASTM. Philadelphia, PA: American Society for Testing and Materials. ASTM. (2005b). Standard test method for water vapor transmission rate through plastic film and sheeting using a modulated infrared sensor (F1249-06). In Annual book of ASTM. Philadelphia, PA: American Society for Testing and Materials. Chhabra, P., Huang, Y. W., Frank, J. F., Chmielewski, R., & Gates, K. (2006). Fate of staphylococcus aureus, salmonella enterica serovar typhimurium, and vibiro vulnificus in raw oysters treated with chitosan. Journal of Food Protection, 69, 952e959. Coma, V., Martial-Gros, A., Garreau, S., Copinet, F., Salin, F., & Deschamps, A. (2002). Edible antimicrobial films based on chitosan matrix. Journal of Food Science, 67, 1162e1169. El Ghaouth, A., Arul, J., & Ponnampalam, R. (1991a). Chitosan coating effect on storability and quality of fresh strawberries. Journal of Food Science, 56, 1618e1620. El Ghaouth, A., Arul, J., & Ponnampalam, R. (1991b). Use of chitosan coating to reduce water loss and maintain quality of cucumber and bell pepper fruits. Journal of Food Processing and Preservation, 15, 359e368. El Ghaouth, A., Ponnampalam, R., Castaigne, F., & Arul, J. (1992). Chitosan coating to extend the storage life of tomatoes. HortScience, 27, 1016e1018.

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