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Food Science and Technology International

http://fst.sagepub.com/ Progress in antimicrobial activities of chitin, chitosan and its oligosaccharides: a systematic study needs for food applications
J. Dutta, S. Tripathi and P.K. Dutta Food Science and Technology International 2012 18: 3 originally published online 27 September 2011 DOI: 10.1177/1082013211399195 The online version of this article can be found at: http://fst.sagepub.com/content/18/1/3

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Article

Progress in antimicrobial activities of chitin, chitosan and its oligosaccharides: a systematic study needs for food applications
J. Dutta1, S. Tripathi2 and P.K. Dutta2

Abstract In recent years, active biomolecules such as chitosan and its derivatives are undergoing a significant and very fast development in food application area. Due to recent outbreaks of contaminations associated with food products, there have been growing concerns regarding the negative environmental impact of packaging materials of antimicrobial biofilms, which have been studied. Chitosan has a great potential for a wide range of applications due to its biodegradability, biocompatibility, antimicrobial activity, nontoxicity and versatile chemical and physical properties. It can be formed into fibers, films, gels, sponges, beads or nanoparticles. Chitosan films have been used as a packaging material for the quality preservation of a variety of foods. Chitosan has high antimicrobial activities against a wide variety of pathogenic and spoilage microorganisms, including fungi, and Gram-positive and Gram-negative bacteria. A tremendous effort has been made over the past decade to develop and test films with antimicrobial properties to improve food safety and shelf-life. This review highlights the preparation, mechanism, antimicrobial activity, optimization of biocide properties of chitosan films and applications including biocatalysts for the improvement of quality and shelf-life of foods.

Keywords Chitin, chitosan, oligosaccharides, antimicrobial activity, pathogenic microorganisms, mechanism, Gram-positive and Gram-negative bacteria, food application
Date received: 7 September 2010; revised: 30 November 2010

INTRODUCTION
Once we talk about antimicrobial activity, it is always directed to its applicability. Very recently, the research is focused into development of materials with lm-forming capacities and those having antimicrobial properties which help to improve food safety and shelf-life. Antimicrobial packaging is one of the most promising active packaging systems that have been found highly eective in killing or inhibiting spoilage and pathogenic microorganisms that contaminate foods (Salleh et al., 2007). In this context, chitosan lms have shown great promise for their application in food preservation. It is well known that microbial alternation is responsible for
Food Science and Technology International 18(1) 334 ! The Author(s) 2011 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1082013211399195 fst.sagepub.com

the enormous losses of food and hence, over the years, various chemical and physical processes have been developed to extend the shelf-life of foods. The antimicrobial activity limits or prevents microbial growth by extending the lag period and reducing the growth rate or decreases live counts of microorganisms (Han, 2000). Currently, food application of an antimicrobial packaging system is limited due to the availability of suitable antimicrobials, new polymer materials, regulatory concerns and appropriate testing methods (Jin and Zhang, 2008). In particular, polymeric bioactive lms laced with an assortment of antimicrobial agents have been found
Department of Chemistry, Disha Institute of Management and Technology, Raipur 400701, India 2 Department of Chemistry, Motilal Nehru National Institute of Technology, Allahabad 211004, India Corresponding author: P.K. Dutta, Department of Chemistry, Motilal Nehru National Institute of Technology, Allahabad 211004, India Email: pkd_437@rediffmail.com
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Food Science and Technology International 18(1)

CH2OH H OH O O Conc. NaOH Deacetylation H

CH2OH O OH O

H Chitin

NHCOCH3

NH2 Chitosan

Figure 1. Structure of chitin and chitosan.

Table 1. Active properties of edible films and coatings Different additives for improvement of edible films and coatings Pigments, light absorbers, shininess salts and other food additives like citric acid, oleic acid, etc., flavors, spices, antimicrobial and antioxidant agents Improvement of property Color, transparency, roughness, sticking, microbe resistance, etc.

ingredient for the production of active bio-based lms and to summarize the dierent methods used for the preparation of chitosan-based lms and their perspectives in the modern food packaging technology. The active properties of edible lms and coatings are described in Table 1.

to be very eective and practical in applications. Till date, a number of articles have been published describing the nature of dierent materials used in making lms and their eectiveness in food preservation (Aider, 2010; Alvarez, 2000; Appendini and Hotchkiss, 2002; Cha and Chinnan, 2004; Cooksey, 2005; Coma, 2008a,b; Cutter, 2002a,b; Cutter, 2006; Dutta and Dutta, 2010; Dutta et al., 2009; Ozdemir and Floros, 2004; Quintavalla and Vicini, 2002; Suppakul et al., 2003). Present review aims to summarize all the known methods of formation of chitosan-based lms with antimicrobial properties and discuss their mode of action and applicability, particularly in the area of food preservation in one place. The structure of chitin and chitosan is shown in Figure 1. To control the great economic losses from the spoilage of foods each year, the attention of the people all over the world has been focused on preservation of the food as a protection from various microorganisms. In this direction, bioactive polymeric lms have been found very eective as antimicrobial agents and thus their applications and research work on them are getting more importance today. This review is an attempt to create awareness about the progress in the antimicrobial activities of chitin and chitosan in the case of food applications and the literature survey made from the year 1957 to present. More specically, the aim of this review was to highlight the potential of chitosan as an 4

CHITIN, CHITOSAN AND THEIR OLIGOSACCHARIDES VERSUS OTHER POLYMERS IN ANTIMICROBIAL ACTIVITY
Polysaccharide lms are derived from various natural materials which impart gel-forming ability to a variety of lms. The polysaccharide-derived lms, due to excellent gas permeability properties, exhibit desirable modied atmospheres and thus enhance the shelf-life of the product without creating anaerobic conditions (Baldwin et al., 1995; Ben and Kurth, 1995; Cutter and Sumner, 2002; Glicksman, 1983; Nisperos-Carriedo, 1994; Whistler and Daniel, 1990). Furthermore, polysaccharide lms and coatings can be used to extend the shelf-life of muscle foods by preventing dehydration, oxidative rancidity and surface browning (NisperosCarriedo, 1994). Particularly, the use of biopolymer and bio-based polymer lms as antimicrobial delivery systems to reduce undesirable bacteria in foodstus is not a novel concept. Various approaches have been proposed and demonstrated for the use of these lms to deliver compounds to a variety of food surfaces, including muscle foods. For example, antimicrobial compounds such as organic acids (acetic propionic, benzoic, sorbic, lactic and lauric), potassium sorbate, bacteriocins (nisin and lacticin), grape seed extracts, spice extracts (thymol, p-cymene and cinnamaldehyde), thiosulnates (allicin), enzymes (peroxidase and lysozyme), proteins (conalbumin), isothiocyanates (allylisothiocyanate), antibiotics (imazalil), fungicides (benomyl), chelating agents

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Dutta et al. (EDTA), metals (silver) or parabens (heptylparaben) could be added to edible lms to reduce bacteria in solution, on culture media, or on a variety of muscle foods (Cha and Chinnan, 2004; Cutter, 2002a,b; Whistler and Daniel, 1990; Han, 2000). Further studies on edible lm made from starch, carrageenan or alginate and addition of antifungal compounds, organic acids, potassium sorbate or the bacteriocin nisin, were more eective for reducing levels of foodborne organisms when immobilized via solution method (Baron and Sumner, 1994; Cutter and Siragusa, 1996, 1997; Dawson et al., 1996; Meyer et al., 1959; Padgett et al., 1998; Siragusa and Dickson, 1992, 1993). The use of biopolymers and biobased polymer lms on a variety of food surfaces and the result-oriented observation of dierent workers are given in Table 2. The above studies indicate that the application of biopolymers, bio-based polymers, edible gels, lms or coatings incorporated with food preservatives and/or natural antimicrobial compounds has a signicant role in nding out the practical applications in the food industry. This specic information demonstrates the feasibility and applicability for incorporating various antimicrobial compounds with a range of inhibitory activity directly into bio-based or edible packaging materials for use in controlling food spoilage as well as enhancing microbial safety of muscle foods. Table 3 focuses on the study of chitosan lms for packaging lms for processed meats and seafood, as well as those combined with various ingredients. Due to their inherent properties, coupled with the ability to form lms, alone or in combination with other polymers, chitin, chitosan and oligosaccharides are desirable food packaging materials. Chitooligomers, a class of chitosans with degree of polymerization <20, are known to have some special biological activities such as antibacterial activities (Jeon et al., 2001), and antitumor and immune-enhancing eects (Jeon and Kim, 2002; Tian et al., 2010; Tokoro et al., 1988). enriched with galangal extract; sweet potato starch lms incorporated with potassium sorbate on chitosan; and chitosan/methyl cellulose lms have also been reported for antibacterial study. Most recently, Tripathi et al. (2009, 2010) have synthesized chitosanbased antimicrobial lms for food applications employing the supercritical carbon dioxide and microwave technique. The novelty of this method lies in achieving the lm formation without the addition of any crosslinker or plasticizer. Some typical preparative techniques are enumerated in the following sections. Preparation of chitosan-coating solution enriched with cinnamon oil Chitosan solution was prepared with 2% (w/v) chitosan in 1% (v/v) acetic acid. To achieve complete dispersion of chitosan, the solution was stirred at room temperature for 3 h. The solution in beakers was placed on a hotplate/magnetic stirrer and glycerol added to chitosan at 0.75 mL/g concentration as a plasticizer and stirred for 10 min. The resultant chitosan-coating solution was ltrated through a Whatman No. 3 lter paper to remove any undissolved particles. Then, the cinnamon oil, mixed with Tween 80, to help in distributing and completely incorporating the cinnamon oil, was added to the chitosan solution. The nal coating forming solution consisted of 2% chitosan, 1% acetic acid, 0.75% glycerol, 0.2% Tween 80 and 1.5% cinnamon oil. The nal coating forming solution was homogenized under aseptic conditions at 21 600 rpm for 1 min. The control solution was prepared without the addition of cinnamon oil (Ojagh et al., 2010). Preparation of environment-friendly films based on chitosan and tetrahydrocurcuminoid derivatives Homogeneous chitosan films. A 2% (w/v) lm-forming solution was obtained by dispersing chitosan in a 1% (w/w) acetic acid aqueous solution (pH 3.5). The solution was ltered through a Bu chner funnel and then degassed under reduced pressure for 1 h. Films were obtained by casting the solutions onto polystyrene plates which were dried for 12 h under a laminar-ow hood at room temperature. Thanks to degasication and drying under laminar-ow, even at room temperature, most of the volatile acetic acid could be removed from the thin lms (Portes et al., 2009). Chitosantetrahydrocurcuminoid films. Film-forming solutions (2%) were prepared by adding chitosan (2 g) and tetrahydrocurcuminoid (THC; 20 mg) to 100 mL of a 1% acetic acid aqueous solution (pH 3.5) under 5
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PREPARATION OF CHITOSAN-BASED ANTIMICROBIAL FILMS/COATINGS


Various methods are employed to prepare chitosan lms and coatings for food packaging applications. Solution casting method is one of the popular methods. As a general practice, chitosan lms are prepared with dierent kinds of oils like cinnamon oil, tea tree essential oil (TTEO); tetrahydrocurcuminoid derivatives; blends with ethylenevinyl alcohol copolymer, PVP or PEO, Gliadins. The methods of preparation of novel hydroxypropyl methylcellulose (HPMC) edible lms with chitosan/tripolyphosphate nanoparticles; nanoparticles/ plasticized-starch composites; edible chitosan lms

Table 2. Use of biopolymer and bio-based polymer films in a variety of food surfaces and the result-oriented observations of different researchers Basic matrix Carrageenan film Edible coatings from waxes and cellulose ethers Beef carcass tissue Ingredients Antibiotics and antifungal compounds Antimycotic agents Activity and observation To reduce bacteria by 2 log 10 (99%) on poultry References Meyer et al. (1959) Hotchkiss (1995)

Organic acids, calcium alginate Potassium sorbate and lactic acid Bacteriocin nisin compared to nisinonly controls Calcium alginate containing nisin Nisin and lysozyme

Edible cornstarch film Calcium alginate gels on lean and adipose beef surfaces Pork Edible heat-set and cast films made from corn zein or soy protein Corn zein films

More efficacious for reducing levels of L. monocytogenes, S. typhimurium, and E. coli O157:H7 when immobilized To inhibit S. typhimurium and E. coli O157:H7 on poultry Resulted in greater and sustained bacteriocin activity when the tissues were ground and stored under refrigerated conditions for up to 7 days Reductions in pathogen populations Exhibit activity against E. coli and L. plantarum

Siragusa and Dickson (1992, 1993) Baron and Sumner (1994) Cutter and Siragusa (1996, 1997)

Fang and Lin (1994) Dawson et al. (1996) and Padgett et al. (1998) Hoffman et al. (2001)

Plastic-based packaging films treated with methylcellulose/ HPMC-based solutions Gelatin-based coatings

EDTA, lauric acid, nisin and combinations of the three compounds Nisin surface of hot dogs

Significant reductions of Listeria monocytogenes in solution

Listeria monocytogenes could be inhibited >2 log 10 (99%)

Franklin et al. (2004)

Lysozyme, nisin and EDTA ham and sausage Bacteriocins, nisin and bovine

Ibrinogen/thrombinbased gel (Fibrimex)

Collagen (Coffi) films (NICF) on hot dog surfaces Milk proteins for edible films and coatings for foods, whey protein films Pullulan films as produced by fungi during fermentation. Pullulan films made from the exopolysaccharides of Aureobasidium pulluans

Nisin

Essential oils of oregano, rosemary and garlic Lysozyme and disodium EDTA

To control spoilage and pathogenic organisms such as L. sakei, Leuconostoc mesenteroides, L. monocytogenes and S. typhimurium Provide an added antimicrobial and advantage to restructured raw meat products that incorporate surface tissues into the product interior or as a delivery system for antimicrobials to meat surfaces Brochothrix thermosphacta and L. monocytogenes were inhibited following treatments with subjected to temperature abuse as well as long-term refrigerated storage Antimicrobials were treated and evaluated against E. coli O157:H7, S. aureus, Salmonella enteritidis, L. monocytogenes and L. plantarum Evaluated for antimicrobial effectiveness against E. coli and L. plantarum. The resulting films with a neutral pH were composed of glucans and polysaccharides, and were water soluble, transparent, and exhibited low oxygen permeability. These antimicrobial films were stable for up to 21 days during cold storage and could inhibit the E. coli under laboratory conditions

Gill (2000)

Cutter and Siragusa (1997)

Cutter and Miller (2004)

Seydim (2006)

Kandemir et al. (2005)

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Dutta et al.
Table 3. Chitosan for packaging films: activities and observations of various researchers Chitosan films combined with various ingredients Processed meats and seafood, as well as nisin and coated onto the surfaces of paper Edible film made from 3% or 5% chitosan and yam starch Activities and observations For inhibiting microorganisms References Vartiainen et al. (2004)

Chitosan-treated films made with 3% or 5% chitosan

Nisin into chitosan

Chitosan coatings

Evaluated an against S. enteritidis in suspensions. When applied directly to cell suspensions, 1% chitosan reduced the pathogen > 4 log10 CFU/mL (or 99.99%). Reduced populations of S. enteritidis >1 log10 CFU/mL (or 90%). Chitosan-treated films made with 5% chitosan were the most efficient means of treatment for inhibiting S. enteritidis in solution To inhibit L. monocytogenes. In solution and in agar diffusion assays, the antimicrobial film inhibited the pathogen, but no further studies were conducted in meat systems Reduced microbial contamination on shrimp and oysters, respectively

Durango et al. (2006)

Cooksey (2005)

Simpson et al. (1997) Chen et al. (1998)

vigorous agitation. The solutions were ltered and cast as in the procedure used to make homogeneous chitosan lms. Ten thickness values were taken randomly at different positions on each lm. Film thicknesses were found to be 30 4 l mm. A good repeatability of the absorbances directly measured on dierent portions of the lms showed that the THCs were distributed homogeneously. Antibacterial chitosan-based blends with ethylenevinyl alcohol (EVOH) copolymer Chitosan polysaccharide dispersions were prepared in 2% (v/v) acetic acid to a nal concentration of 3% (w/v) and stirred at 37  C for approximately 3 h. The chitosoniumacetate solutions were ltered through polyester cloth to remove residues of insoluble particles and then autoclaved before their further use for blending. Pure chitosoniumacetate lms were obtained by casting at 37  C, 80  C and 120  C. In addition, pure EVOH lms were obtained by the same method at 80  C. Composite lms of EVOH29/LMWchitosan with high ratios of polysaccharide (i.e., 50 and 80 wt%) were obtained under various temperature conditions (37  C, 80  C and 120  C). Blends of EVOH with lower contents of chitosan did not form good lms but 80/20 (wt%) EVOH/ chitosoniumacetate blends could be nally obtained by adding an excess of glacial acetic acid to the EVOH solution to a nal concentration of 2% (w/v). Nevertheless, the higher chitosan concentration blends, i.e., 50 and 80 wt%, were also obtained using the same

solution of EVOH and acetic acid for comparison purposes. Typical pure and composite lms with a thickness of ca. 50 lm were obtained (Fernandez-Saiz et al., 2010). Preparation of chitosan/PVP and chitosan/PEO blend films Chitosan solution was prepared with 1% (w/w) chitosan in 1% (v/v) acetic acid, stirred overnight at room temperature and ltered through Miracloth to remove impurities. PVP and PEO were separately dissolved in d.i. water to form 1% (w/w) solutions. Aqueous solutions of the individual polymers were mixed to prepare a series of chitosan/PVP and chitosan/PEO blend solutions with weight ratios 100/0, 75/25, 50/50, 25/75 and 0/100. Aliquots of 10 g of lm-forming solutions were poured into 50 mm-diameter polystyrene Petri dishes and the solvent was evaporated in a vacuum oven at 38  C under 17 kPa pressure for 24 h. The dried lms were peeled from the Petri dishes and conditioned in desiccators at 25  C and 20% relative humidity (RH) prior to testing (Li et al., 2010). Preparation of novel HPMC edible films with chitosan/tripolyphosphate nanoparticles Preparation of chitosantripolyphosphate nanoparticles. The chitosantripolyphosphate (CSTPP) nanoparticles were prepared on the ionic gelation of CS with TPP anions. Chitosan was dissolved in acetic acid solution at concentrations of 3.00 and 4.41 mg/mL. 7

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Food Science and Technology International 18(1) The concentration of acetic acid in aqueous solution was, in all cases, 1.5 times that of chitosan. Under magnetic stirring at room temperature, 28 mL sodium TPP aqueous solution with concentrations 1.2 and 2.1 mg/ mL were added into 70 mL chitosan solution. The preparations were mixed with a homogenizer at 6000 rpm with continuous addition of TPP solution at the rate of 1 mL/min. The zone of opalescent suspension was further examined as nanoparticles (de Moura et al., 2009). Preparation of solutions for film casting. The HPMC solution (control lm) was obtained by dissolving 3.0 g of HPMC in 100 mL of distilled water under magnetic stirring for 12 h. The HPMC: water ratio in all lmforming solutions was 3:97 to study the eect of particle size and CSTPP concentration in the HPMC lm matrix. The HPMC lms with CSTPP nanoparticles were obtained by addition of 3.0 g of HPMC in 100 mL of nanoparticle solution (recently synthesized) under magnetic stirring for 12 h. After the solutions were prepared, the asks were kept closed for 6 h to prevent microbubble formation in the lms. The solutions were then poured in a glass plate (30 30 cm2) covered with Mylar (Polyester lm) for lm casting preparation. The solutions were cast at a wet thickness of 0.5 mm onto plates using casting bars and the plates placed on a leveled surface at room temperature and let to dry for 24 h. After drying, the lms were removed and conditioned in sealed plastic bags stored at room temperature. Preparation of chitosan nanoparticles/ plasticized-starch composites Preparation of chitosan nanoparticles. Chitosan nanoparticles (CN) were fabricated on the basis of ionotropic gelation between chitosan and sodium tripolyphosphate with some modications (Shu and Zhu, 2000; Tsai et al., 2008). Chitosan was dissolved in 2% (v/v) acetic acid to obtain a 1% (w/v) chitosan solution. Under vigorous stirring at room temperature, 8 mL of 1% (w/v) TPP aqueous solutions was added dropwise, at the rate of 1 mL/min, into 200 mL of chitosan solution. The mixture was stirred for 30 min, and treated with sonication for 30 min. The suspension was subsequently centrifuged at 12,000 rpm for 20 min. The precipitate was washed with water and centrifuged again twice. The precipitate was washed in ethanol and then dried (Chang et al., 2010). Processing of glycerol/potato starch/chitosan composites. Chitosans were dispersed in a solution of distilled water (100 mL) and glycerol (1.5 g) and ultrasonicated for 0.5 h before adding 5 g of potato starch. The chitosan ller loading level (0, 1, 2, 4, 6 and 8 wt%) 8
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was based on the amount of potato starch. The mixture was heated at 90  C for 0.5 h with constant stirring in order to plasticize the starch. To obtain the glycerol/ potato starch (GPS)/chitosan composite lms, the mixture was cast into a dish and placed in an air-circulating oven at 50  C until dry (about 6 h). The composite lms were preconditioned in a climate chamber at 25  C and 50% RH for at least 48 h prior to the testing. Water content of the lms was about 10 wt%. Preparation of chitosanTTEO composite films Preparation of the film-forming dispersions. Chitosan (1% w/w) was dispersed in an aqueous solution of glacial acetic acid (0.5% w/w) at 25  C. After an overnight agitation, TTEO was added to the chitosan (CH) solution to reach a nal concentration of 0%, 0.5%, 1% and 2% (w/w). CHTTEO mixtures were emulsied at room temperature using a rotorstator homogenizer at 13,500 rpm for 4 min. These emulsions were vacuum degasied at room temperature with a vacuum pump. The experimental design was made taking into account the maximum levels of TTEO which could be incorporated into the matrix without oil phase separation during nchez-Gonza lez et al., 2010). the lm drying (Sa Preparation of films. Films were obtained by the casting procedure given as follows: lm-forming dispersions (FFDs) were poured onto a framed and leveled polytetrauoroethylene (PTFE) plate (j 15 cm) and dried under atmospheric conditions for 48 h. Film thickness was controlled by pouring the amount of FFD that will provide a surface density of solids in the dry lms of 56 g/ m2 in all formulations. Dry lms were peeled o from the casting surface and preconditioned in desiccators at 20  C. Preparation of blend films of gliadins and chitosan A total of 100 g of wheat gluten powder was suspended in 350 mL pure ethanol and 150 mL distilled water was transferred into the suspension under stirring. The mixture was magnetic stirred for 1 h and centrifuged at 3000 rpm for 15 min at 25  C. The supernatant containing the gliadin-rich fraction was collected as the lmforming solution. This method was dierent from the general way that crude wheat gluten was dispersed in 70% (v/v) aqueous ethanol and stirred for about 12 h to extract glianidins (Hernandez-Munoz et al., 2004a,b; Song et al., 2009). Glycerol, as plasticizer, was added into the supernatant in a content of 20 g/ 100 g dry protein. About 2 g of CS were dispersed in 97 g water before 1 g acetic acid was added to the mixture under stirring to give a CS content of 2.0 wt%. By doing

Dutta et al. this, clotting of CS in acetic acid aqueous solution could be avoided. Glycerol plasticizer was added into the solution in a content of 20 g/100 g CS. The gliadin and CS solutions were mixed and stirred for 20 min. The weight content of CS, wCS, dened as the weight ratio of CS to total weight of CS plus gliadins, was varied from 0 to 100 wt%. The nal solution was poured onto horizontal plastic dishes and dried at 25  C. The dried lms were carefully peeled o from the dishes and preconditioned at 25  C at 57.5% RH for 3 days at least before testing. Preparation of edible chitosan films enriched with galangal extract Preparation of chitosan solution. In this method, 1.5% (w/v) chitosan solution was prepared by dissolving chitosan in 1% (v/v) acetic acid under constant stirring at 300 rpm using a magnetic stirrer at room temperature for 24 h. Then, 25% glycerol (w/w chitosan) was added into the chitosan solution; stirring was continued at room temperature for 1 h. After mixing, the solution was centrifuged for 15 min at 12 400 rpm by a refrigerated centrifuge to remove undissolved impurities and bubbles in the solution (Mayachiew et al., 2010). Preparation of galangal extract. Galangal powder (10 g dry basis), dried by a tray dryer at 40  C with particle size in the range 125425 mm, was extracted with 100 mL of 95% (v/v) ethanol (Oonmetta-aree et al., 2006). The extract was ltered through a lter paper; the ltrate was collected and concentrated by a rotary evaporator at 40  C for 10 min and kept at 4  C in a dark bottle until its use (Mayachiew and Devahastin, 2008a). Preparation of antimicrobial chitosan films. Galangal extract was added to the chitosan solution at concentrations 0.3, 0.6 and 0.9 g/100 g. These concentrations were selected based on a minimum inhibitory concentration (MIC) of the extract against Staphylococcus aureus (Mayachiew and Devahastin, 2008a). The nal concentrations of galangal extract in the lms were 126, 252 and 378 mg/g lm, respectively. The mixture was homogenized by a bench top homogenizer at 9500 rpm for 2 min. The lm solution (21 g) was poured on an acrylic plate with dimensions 13 10 cm2 to cast an antimicrobial lm. Drying of the lm was performed by four methods, which are ambient air drying (30  C), hot air drying at 40  C, vacuum drying and low-pressure superheated steam drying at 70  C, 80  C and 90  C at 10 kPa, following the methods of Mayachiew and Devahastin (2008b). After drying, the lms were conditioned for at least 48 h in desiccators at a RH of 53% containing saturated salt solution of magnesium nitrate.

Preparation of sweet potato starch films incorporated with potassium sorbate or chitosan In this method, 4 g of sweet potato starch was dispersed in 100 mL H2O, moderately stirred for 20 min at room temperature and then heated to 100  C for over 30 min. After gelatinization, glycerol was added as a plasticizer at a concentration of 3% (w/w, on dry basis of the weight of starch) and the resulting dispersion subjected to further mixing for 5 min. To prepare the antimicrobial lm, potassium sorbate or chitosan was added at dierent concentrations (0, 5, 10, 15 g/100 g starch) at one time during the mixing period. Before antimicrobial agents were added into starch paste, potassium sorbate was dissolved in 20 mL water and chitosan was dissolved in 20 mL 1% acetic acid aqueous solution. For potassium sorbate-incorporated lm, the pH of the gel-like mixture was adjusted to 4.5 by the addition of 2 M HCl. When chitosan was incorporated into the lm, the pH of the gel-like mixture was 4.0 because of the acetic acid used in preparing chitosan solution. After being degassed under vacuum, the warm mixture was cast on framed glass plates, and then dried at 50  C for 4 h. Starch lm, without antimicrobial agents, was also prepared in the same way and used as a control (Shen et al., 2010). Preparation of chitosan/methyl cellulose films Chitosan solution was prepared by dissolving 1.5 g of chitosan in 100 mL of 1% acetic acid solution. In this method, 1.5 g of methyl cellulose was dissolved in 50% ethanol. Also, 1 g of polyethylene glycol 400 was used as a plasticizer. Solutions of chitosan and methyl cellulose were mixed in a beaker with a stir bar and heated to 72  C. Stearic acid, 0.075 g, was added to improve the water barrier properties of the lm. Vanillin, 0.9 g, was incorporated after the temperature of the solution reached its melting point (83  C). The lm-forming solution was ltered through a cheese cloth to remove the undissolved parts, homogenized with a homogenizer, degassed, cast onto glass plates and dried at 40  C for 42 h. Dried lms were peeled o and conditioned at 25 2  C, 50 5% RH for at least 48 h prior to use (Sangsuwan et al., 2008).

ANTIMICROBIAL ACTIVITY
Functional eciency strongly depends on the nature of components and lm composition and structure. The choice of lm-forming substance and/or active additive is made based on the objective, the nature of the food product and/or the application method. 9

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Food Science and Technology International 18(1)


Table 4. Inhibitory activities of chitosan Types of study Surface spoilage bacteria Activities and observations References Ouattara et al. (2000a); Savard et al. (2002); No et al. (2002); Coma et al. (2003); Gerasimenko et al. (2004) Siragusa and Dickson (1992); Helander et al. (2001); Tsai and Su (1999); Tsai et al. (2000); No et al. (2002); Coma et al. (2003); Darmadji and Izumimoto (1994) Papineau et al. (1991) Li et al. (2006)

Various pathogen food strains

Potential bactericide in liquid medium, against some plant pathogenic bacteria

Studied the bioactivity of chitosan in liquid medium and reported that 0.01% chitosan is sufficient to inhibit the growth of some spoilage bacteria such as B. subtilis, E. coli, Pseudomonas fragi and S. aureus in liquid medium Significant inactivation of the Gram-negative bacteria E. coli and the Gram-positive enterotoxigenic S. aureus by two commercially available water-soluble chitosan salts chitosan lactate and chitosan hydroglutamate By markedly inhibiting the growth of Xanthomonas sp. A decrease from 9.3 to 3.6 log CFU/mL of X. axonopodis pv. Poinsettiicola was obtained using chitosan, compared to the control. These authors observed an increase in the bacterial activity by the addition of 0.5% NaCl

Chitosan has been studied in terms of bacteriostatic/ bactericidal activity to control the growth of a wide variety of bacteria. Chitosan has several advantages over other types of disinfectants because, according to Kim and Choi (1998), it possesses a higher antibacterial activity and a broader spectrum of activity. The inhibitory activity of chitosan is given in Table 4 (Inhibitory activity of chitosan). Chitosan hydroglutamate showed more eective antimicrobial properties with a decrease in S. aureus population of approximately 6 log cycles within 60 min of exposure for a chitosan concentration of 0.2 mg/mL. It also reported that the application of high hydrostatic pressure (2380 odm) to chitosan-treated cultures of both bacterial strains resulted in additional inactivation. Chung and Chen (2008) also investigated the antibacterial activity of low-molecular weight chitosan (Mw 30 kDa) by assessing the mortality rates of Escherichia coli and S. aureus and demonstrated that chitosan can destroy the cell structure of both bacterial cells, resulting in the leakage of enzymes and nucleotides from dierent cell locations. The MIC values of chitosan for dierent food bacteria are listed: Bacillus cereus (1000 ppm), Erwinia sp. (500 ppm), Pseudomonas uorescens (500 ppm), E. coli (20 ppm), Micrococcus luteus (20 ppm) and S. aureus (20 ppm). As reported in the literature, chitosan seemed to be more active against Gram-positive than against Gramnegative bacteria. Coma et al. (2003) assessed the potential of chitosan coating especially active against the growth of two food pathogens, S. aureus, Listeria monocytogenes and one strain involved in food alteration, Pseudomonas aeruginosa. The study was conducted on 10

a model of agar medium and on a real cheese food product. Numeration on model agar medium showed a total inhibition of the development of selected Gram-positive bacteria and 77% inhibition on Pseudomonas growth. This result poses the problem of a possible microbial selection related to the dierent sensitivities of microorganisms. Moreover, other parameters could have an impact on the bactericidal eect of chitosan. The age of a bacterial culture aected its susceptibility to chitosan (Tsai and Su, 1999). As a result, E. coli cells in the exponential phase are most sensitive to chitosan. These authors also showed that higher temperature (37  C) increased the impact against the target strains. Another parameter identied as signicantly inuential is the molecular weight. Jeon et al. (2001) showed that chitooligosaccharide eectively blocked the growth of the tested bacteria although their eects were lower than that of chitosan. These authors mentioned that for an eective inhibition, the molecular weight should be 10 kDa. The pH and the quaternization degree of the chitosan were the inuential parameters. The majority of studies showed that an increase of deacetylation (DA) degree and a decrease in pH improved the bioactive properties of chitosan. The occulation and adsorption behavior of chitosans to E. coli cells were not observed; however, such behaviors were noticed by applying uorescence labeled to chitosans and monitoring changes in zeta potential values of the bacterial with chitosan coating. Without chitosan coating cells, it was increased approximately by 40% if pH increased from 5 to 6.5 (Strand et al., 2003). Despite their low charge density, the chitosans with higher deacetylation degree adsorbed

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Dutta et al. in higher amounts and reversed the cell surface charge most eectively. Finally, the chitosans with low-molecular weight adsorbed the most. The same authors reported that ionic strength did not aect the adsorption of highly acetylated chitosan (DA 0.49), whereas for chitosan with a DA of 0.01, adsorption increased with ionic strength. The combination of occulation and adsorption data clearly showed that charge neutralization was not the main occulation mechanism. Several results pointed to bridging as one dominating mechanism for occulation. Study demonstrated that in vitro antimicrobial eciency is not always proven in food, due to the highly reactive nature of the polycationic chitosan, which could interact with proteins, fats and other anionic substances in foods. Darmadji and Izumimoto (1994), however, observed that the growth of spoilage bacteria in meat was inhibited by 0.51.0% chitosan during incubation at 30  C for 48 h or storage at 4  C for 10 days. Studies based on UV absorption indicated that chitosan causes considerable losses of proteinic material to the Pythium oaroecandrum at pH 5.8 (Helander et al., 2001; Liu et al., 2004). Chitosan also acts as a chelating agent that selectively binds trace metals and thereby inhibits the production of toxins and microbial growth (Cuero et al., 1991). It also activates several defense processes in the host tissue (El Ghaouth et al., 1992), acts as a water binding agent and inhibits various enzymes. Binding of chitosan with DNA and inhibition of mRNA synthesis occurs through chitosan penetration toward the nuclei of the microorganisms and interference with the synthesis of mRNA and proteins (Sudarshan et al., 1992). It has been proposed that when chitosan is liberated from the cell wall of fungal pathogens by plant host hydrolytic enzymes, it then penetrates to the nuclei of fungi and interferes with RNA and protein synthesis (Hadwiger et al., 1985). A microscopic examination of Saccharomyces unisporus after treatment with chitosan-salt with a polymerization degree of 25 showed agglutination of a refractive substance on the entire cell wall (Savard et al., 2002). When chitosanase was added to the culture media containing chitosan-salt, they could not observe refractive substances. In this study, an interaction between chitosan and the cell wall was observed. In vitro evaluation of antimicrobial activities of carboxymethyl chitosan (CM), quarternized chitosan (Q) and quarternized carboxymethyl chitosan A series of quaternized carboxymethyl chitosan (CMQ), the sample number and the characterization were listed out in Table 5 (Sun et al., 2005). A Gram-positive
Table 5. Sample numbers and characterization of different CMQ Sample no. DS of CM DS of Q Sample no. Mw (105) CM3Q1 CM3Q2 CM3Q3 CM1Q2 CM2Q2 CM4Q2 0.73 0.73 0.73 0.45 0.56 0.86 0.78 0.59 0.32 0.59 0.59 0.59

CM3Q2-1 CM3Q2-2 CM3Q2-3 CM3Q2-4

4.72 2.28 0.45 0.11

bacterium S. aureus and a Gram-negative bacterium E. coli were used and inoculated on a gel containing 1% peptone, 2% agar, 3% meat extract, and 0.5% NaCl for this experiment. The agar plate method was used to determine the MICs of CM, Q and CMQ as follows: the samples were prepared at a concentration of 1% (w/v) and then autoclaved at 121  C for 25 min. Duplicate twofold serial dilutions of each sample were added to nutrient broth (beef extract 5 g, peptone10 g to 1000 mL distilled water, pH 7.0) for nal concentrations 0.1%, 0.05%, 0.025%, 0.0125%, 0.00625%, and 0.00313%. Some samples were prepared and diluted by the same way except for nal concentrations 0.00065% and 0.00033%. The culture of each bacterium was diluted by sterile distilled water to 105106 CFU/mL. A loop of each suspension was inoculated on the nutrient medium with sample or control added. After inoculation, the plates were incubated at 37  C for 72 h, the colonies counted and the MIC values obtained. The MIC was considered to be the lowest concentration that completely inhibited against agar plates on comparison, disregarding a single colony or a faint haze caused by the inoculum (Speciale et al., 2002).

Antimicrobial activity of CM and CMQ The antimicrobial activities of CM and CMQ are shown in Tables 6 and 7. It was found that these samples showed eective antimicrobial activities against not only E. coli but also S. aureus which were used in the test, although dierences existed among them. Generally, the samples had more eective inhibition on S. aureus than E. coli. The fact may be attributed to their dierent cell walls. In S. aureus, a typical Grampositive bacterium, the cell wall is fully composed of peptide polyglycogen. The peptidoglycan layer is composed of networks with plenty of pores, which allow foreign molecules to come into the cell without diculty. But in E. coli, a typical Gram-negative bacterium, the cell wall is made up of a thin membrane of peptide polyglycogen and an outer membrane is constituted of lipopolysaccharide, lipoprotein and phospholipids. Because 11

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Food Science and Technology International 18(1)


Table 6. The antimicrobial activities of chitosan (CS), carboxymethyl chitosan (CM), quarternized chitosan (Q) and quarternized carboxymethyl chitosan (CMQ) Samples CM Q CMQ DS of CM 0.46 0.45 DS of Q 0.60 0.59 Mw(105) 4.30 3.89 4.51 Escherichia coli 0.05 0.0125 0.00625 Staphylococcus aureus 0.1 0.025 0.0125

Table 7. The antimicrobial activities of CMQ with different molecular structure factors Samples CM1 Q2 CM2 Q2 CM3 Q2 CM4 Q2 CM3 Q1 CM3 Q3 CM3 Q2-1 CM3 Q2-2 CM3 Q2-3 CM3 Q2-4 DS of CMC 0.45 0.56 0.73 0.86 0.73 0.73 0.73 0.73 0.73 0.73 DS of QC 0.59 0.59 0.59 0.59 0.78 0.32 0.59 0.59 0.59 0.59 Mw (105) 4.51 4.64 4.72 4.66 4.21 4.83 4.72 2.28 0.45 0.11 Escherichia coli 0.00625 0.00625 0.00625 0.00625 0.0125 <0.00625 0.00625 0.00625 0.00313 <0.00313 Staphylococcus aureus 0.0125 0.0125 0.00625 0.0125 0.0125 0.00625 0.0125 0.00625 0.00313 0.00313

of the bilayer structure, the outer membrane is a potential barrier against foreign molecules. Compared with CM, Q and CMQ in Table 7, CMQ had much better antimicrobial activities, whose MIC values were 48 times lower than those of CM and 2 4 times lower than those of Q. It was noticed that the introduction of carboxymethyl and quarternized groups to the chitosan chain greatly enhanced the antimicrobial activity of the CMQ. We can deduce that carboxymethyl and quaternary ammonium groups are in synergistic eect. As given in Table 6, compared with CM1Q2, CM2Q2, CM3Q2 and CM4Q2, which have the same degree of substitution of quaternized group, the authors found no clear eect of DS value of carboxymethyl group on antimicrobial activity. Compared with CM3Q1, CM3Q2 and CM3Q3, with similar degree of carboxymethyl group substitution, it can be observed that their antimicrobial activities were enhanced with increase of the DS. Compared with CM3Q2-1, CM3Q2-2, CM3Q2-3 and CM3Q2-4, which have same degrees of substitution for both carboxymethyl and quaternized groups, the results demonstrated that their antimicrobial activities were aected by their molecular weights remarkably. Lower molecular weight resulted in better antimicrobial ability, and when the molecular weight was below 1 104, the antimicrobial activity of CMQ was strong and the MIC values reached 0.00313%. The development of complementary methods to inhibit the growth of pathogenic bacteria such as packaging 12

material-associated antimicrobial agents is an active area of research. A number of studies on the antimicrobial characteristics of lms made from chitosan have been carried out earlier (Chen et al., 1996; Coma et al., 2002; Ouattara et al., 2000a). Among other polymers, chitosan has received a signicant attention as an antimicrobial lm-forming agent for food preservation to the researchers due to its biodegradability, biocompatibility, cytotoxicity and antimicrobial activity. Chitosan lms are easily prepared by the evaporation of its dilute acid solutions (Park et al., 2002). Chitosan has been studied in terms of bacteriostatic/bactericidal activity to control the growth of a wide variety of bacteria. In the Gram-positive bacteria, the major constituent of their cell wall is peptidoglycan and a little amount of 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 the peptidoglycan. Also, the cell wall of Gram-negative bacteria has an outer membrane which constitutes the outer surface of the wall (Black, 1996). The antimicrobial eect of konjac glucomannan edible lm was improved by incorporating chitosan and nisin (Li et al., 2006). In this study, antimicrobial ecacy was assessed against four food pathogenic bacteria namely E. coli, S. aureus, L. monocytogenes and B. cereus. Antimicrobial activity tests of edible lms were carried out using the agar diusion method. In this method, the lm cuts are placed on Mueller Hinton agar plates, which were previously seeded with

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Dutta et al. 0.1 mL of inoculums containing indicator microorganisms in the range 105106 CFU/mL. The antimicrobial eect of chitosan or incorporating nisin was found to be much better than that of konjac glucomannan incorporating nisin at each corresponding concentration and there existed signicant dierence (p < 0.05). However, there was no signicant dierence in the antimicrobial eect between chitosan and chitosan incorporating nisin. Incorporating chitosan into konjac glucomannan lm therefore improved not only physical properties but also antimicrobial activity. The characteristics of chitosan lm was evaluated by cross-linking with naturally occurring aglycone geniposidic acid (Mi et al., 2006). In this study, a comparative study was performed between chitosan lm without cross-linking (fresh), the glutaraldehyde-cross-linked chitosan lm and aglycone geniposidic acid-crosslinked chitosan lm. A 50 mL bacterial broth (E. coli or S. aureus) was seeded onto lm and cultured. The fresh and glutaraldehyde-cross-linked chitosan lms were used as control. It has been proposed that the interaction between the polycationic chitosan and the negatively charged surface of bacteria may alter the permeability of the bacterial wall and lead to the leakage of intracellular electrolytes and proteins. The results suggested that cross-linking of chitosan lms did not alter their antibacterial capability. This may be due to the fact that the cross-linking degrees of glutaraldehyde and aglycone geniposidic acid (aGSA) cross-linked chitosan lms used in this part of the study were relatively low (<18%, with a concentration of cross-linking agent 0.8 mM). The aGSA-cross-linked chitosan lm displayed a relatively lower water vapor permeability, a lower cytotoxicity, and a slower degradation rate than the glutaraldehyde-cross-linked lm. It was nally concluded that the aGSA-cross-linked chitosan lm may be a promising material as an edible lm for food packaging. The shelf-life of food was extended by ferulic acidincorporated starch-chitosan blend lms (Mathew and Abraham, 2008). Incorporation of ferulic acid has been found to improve the barrier properties and tensile strength of starch-chitosan blend lms and signicantly enhance the lipid peroxide inhibition capacity. This study has helped to improve the performance of polysaccharide-based lms for the storage of high lipid-containing ingredients. The antimicrobial activity of chitosan lms enhanced by incorporation of garlic oil, potassium sorbate and nisin (Pranoto et al., 2005). The antimicrobial activity was tested against food pathogenic bacteria namely E. coli, S. aureus, Salmonella typhimurium, L. monocytogenes and B. cereus. Antimicrobial tests have been carried out using agar diusion method. The agar diusion test is a method commonly used to examine antimicrobial activity regarding the diusion of the compound tested through water-containing agar plate. Incorporating antimicrobial agents into chitosan edible lms thus improves the antimicrobial ecacy of chitosan, as diused antimicrobial activity would add to the nonmigrated antimicrobial potency of chitosan. It was concluded that garlic oil incorporated into chitosan lm led to an increase in its antimicrobial ecacy, and has little eect on the mechanical and physical properties of chitosan lms. Overall, the incorporation of garlic oil into chitosan lm had the desirable characteristics of acting as a physical and antimicrobial barrier to food contamination. Two types of O-carboxymethylated chitosan (OCMCh)/cellulose polyblends were prepared using LiCl/ N, N-dimethylacetamide solution (Li et al., 2002). Antimicrobial activity of the blend lms against E. coli was evaluated by using the optical density (OD) method. The smaller the OD of the medium, the higher was the antimicrobial activity of the lm. It was observed that the antimicrobial activity of the blend lms enhances if the O-CMCh contamination was raised. Both blend lms exhibited satisfactory antibacterial activities against E. coli, even with O-CMCh concentration 2 wt%. Antibacterial assessment of chitosan-starch Inhibitory eects of starch solution and chitosan-starch lm against E. coli, S. aureus and Bacillus subtilis are shown in Figures 2(a)(c), and 3(a)(c). The inhibitory eect was measured based on clear zone surrounding circular lm strips/solution. Measurement of clear zone diameter included diameter of lm strips/solutions. Therefore, the values were always higher than the diameter of lm strips/solutions 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 (Tripathi et al., 2010). The results were observed and noted as follows (Table 8). In terms of surrounding clearing zone, chitosan potato starch lm did not show inhibitory eects against all the tested microorganisms. The chitosan-starch lmforming solution showed a clear inhibitory zones of 1.5, 1.2 and 1.4 cm against E. coli, S. aureus and B. subtilis, respectively. However, increasing level of starch at higher concentration did not reveal a signicant increased inhibitory eect. It was generally caused by the maximum capability of chitosan polymer to carry active agents beside the occurrence of interaction phenomenon between the functional groups. The antimicrobial eect of chitosan occurred without migration of active agents. As chitosan is in solid form, only organisms in direct contact with the active sites of chitosan are 13
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Food Science and Technology International 18(1)

Figure 2. Inhibitory effects of chitosan-starch solution against: (a) E. coli, (b) S. aureus and (c) B. subtilis. Source: Tripathi et al., (2008); reprinted with permission from Asian Chitin Journal).

Figure 3. Inhibitory effects of chitosan-starch film against: (a) E. coli, (b) S. aureus and (c) B. subtilis. Source: Tripathi et al., (2008); reprinted with permission from Asian Chitin Journal). Table 8. Diameters of inhibitory zone of the solution and film against E. coli, S. aureus and B. subtilis Diameter (cm) of inhibitory zone Test culture In the solution In the film 1.5 1.2 1.4

Escherichia coli 0 Staphylococcus aureus 0 Bacillus subtilis 0

inhibited. Chitosan is incapable of diusing through the adjacent agar media. The agar diusion test is a method commonly used to examine the antimicrobial activity regarding the diusion of the compound tested through water-containing agar plate. The diusion itself is dependent on the size, shape and polarity of the diusion material. The chemical structure and the cross-linking level of the lms also aect this phenomenon. The 14

chitosanstarch solution shows stronger inhibitory eect against E. coli and B. subtilis than S. aureus. Furthermore, it was found that the bioactive chitosan potato starch lm can be used to extend the food shelflife. Incorporating chitosan and lauric acid into starchbased lm showed more eective antimicrobial ability against B. subtilis and E. coli (Salleh et al., 2007). In this study, the authors studied the incorporation of chitosan and lauric acid into starch-based lms; obvious eects toward inhibition of B. subtilis and E. coli have been observed while the lm had synergistic antimicrobial eect when chitosan and lauric acid were combined. Antimicrobial starch-based lm incorporated with lauric acid and chitosan showed good exibility than when purely starch-based lm was formulated and formed (Figure 4). Inhibition of bacterial growth was examined using two methods, i.e., zone of inhibition test on solid media and liquid culture test (OD

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Dutta et al. measurements). The inhibitory activity was measured based on the diameter of the clear inhibition zone. In the solutions of starch and chitosan with dierent mixing ratios (w/w), 8:2 and 9:1 were the most eective mixing ratios which had greater inhibition on both B. subtilis and E. coli than other solutions in agar plate and liquid culture test. The control (pure wheat starch) and antimicrobial (AM) lm (incorporated with chitosan and lauric acid) were produced by casting method. The antimicrobial eectiveness of control (pure wheat starch) and AM lm incorporated with chitosan and lauric acid are shown in Figure 5(a) and (b). A wide clear zone on solid media was observed for B. substilis growth inhibition whereas inhibition for E. coli was not as eective as B. substilis. From the liquid culture test, the AM lms clearly demonstrated a better inhibition against B. substilis than E. coli. The tensile properties of the antimicrobial starchbased lm has been improved by the addition of chitosan. These antimicrobial starch-based lms can be used to extend food shelf-life. Antimicrobial Activity of chitosanpolyvinyl alcohol film Inhibitory eects of chitosanpolyvinyl alcohol (PVA) solution and chitosanPVA lm against E. coli, S. aureus and B. subtilis were studied by Tripathi et al. (2009). The inhibitory eect was measured based on clear zone surrounding circular lm strips/solution. Measurement of clear zone diameter included measuring the diameter of lm strips/solutions; therefore, the values were always higher than the diameters of lm strips/solutions whenever clearing zone was present. If there is no clear zone surrounding, the authors assumed that there is no inhibitory zone, and furthermore, the diameter was valued as zero. In terms of surrounding clearing zone, chitosanPVA lm did not show inhibitory eects against all tested microorganisms. The chitosanPVA lm-forming solution showed a clear inhibitory zone against E. coli, S. aureus and B. subtilis, respectively. The chitosanPVA solution shows stronger inhibitory eects against E. coli and B. subtilis than S. aureus. Furthermore, it was found that the bioactive chitosanPVA lm can be used to extend food shelflife. Chitosan-based antimicrobial lms consisting of chitosan and PVA were prepared by solution casting method. These results pointed out that there is a molecular miscibility between PVA and chitosan.

Figure 4. A translucent starch-based film incorporated with lauric acid and chitosan. Source: (Salleh et al., (Muhamad and Khairuddin, 2007); reprinted with permission from Asian Chitin Journal).

Figure 5. Inhibition areas of: (a) control film and (b) AM incorporated film. Source: Salleh et al., Muhamad and Khairuddin, (2007); reprinted with permission from Asian Chitin Journal).

15
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Food Science and Technology International 18(1) Chitosan-based antimicrobial lm may be a promising material as a packaging lm. Preservation of vacuum-packaged processed meats The feasibility of improving the preservation of vacuumpackaged processed meats during refrigerated storage achieved by the use of an antimicrobial lm designed to gradually release antimicrobial agents at the product surface (Ouattara et al., 2000b). The antimicrobial lms were applied onto bologna, regular cooked ham or pastrami. The activity of the various lms for inhibiting bacterial growth was tested against indigenous lactic acid bacteria and Enterobacteriaceae and against Lactobacillus sakei or Serratia liquefaciens, surfaceinoculated onto the meat products. The growth of Enterobacteriaceae and S. liquefaciens was delayed by the application of the antimicrobial lm. It was found that the inhibition of indigenous Enterobacteriaceae was more extensive at the surface of bologna than at the surface of pastrami, irrespective of lm type. It is due to the fact that bologna contains ecient water-binding agents, and so exudes little water during storage. The moisture and high lipid contents of bologna helped the diusion of the oregano essential oil (EO) from the chitosan lm matrix into the product (Chi et al., 2006). Sensory evaluation suggested that addition of 45 ppm or less of oregano oil to bologna would be acceptable to consumers. In conclusion, the gas chromatography mass spectroscopy analysis showed that 757.7 99.7 ppm carvacrol was extracted from the lm-forming solution prepared without Tween 20 and only 364.7 39.9 ppm from the lm-forming solution with the emulsier. Dierent levels of carvacrol were detected in the presence of Tween 20 due to the interaction of the amphiphilic emulsiers molecule with both chitosan and oregano EO compounds. It is concluded that incorporation of an emulsier in chitosan oregano EO lms may slow the losses of volatile compounds of the oil and help to control the release of active compounds into the product. The antimicrobial properties of crawfish chitosan Antimicrobial activities of crawsh chitosan lm formulations against seven pathogenic bacteria, L. monocytogenes, B. cereus, Shigella sonnei, E. coli (O157:H7), S. aureus, S. typhimurium and Vibrio vulnicus, were expressed in terms of zone inhibition. The zone inhibition assay revealed primarily three types of observations, namely, (1) defaced lms without any clear or inhibition zones which could be attributed to the absence of any inhibitory activity, (2) clear zones 16
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without inhibitory zones which could be attributed to bacteriostatic activity and (3) clear inhibition zone representing bactericidal inhibition by lms. Some of the tests are enumerated as follows (Nadarajah, 2005). Minimum inhibitory activity. All chitosan acetate lms were defaced with L. monocytogenes and this was well in agreement with the report of Coma et al. (2002). However, it was reported that 8% lm-forming solution (chitosan in acetic acid) incorporated in agar medium (v/v) completely inhibited L. monocytogenes. Chitosan acetate lms were also defaced with B. cereus, and V. vulnicus lawns, indicating that they were ineective in controlling these bacteria. However, all chitosan acetate lms showed bacteriostatic eects against S. aureus, S. sonnei, S. typhimurium and E. coli O157:H7, as indicated by their clear zones i lawns. The chitosan formate lms were also ineective in controlling B. cereus and V. vulnicus, as indicated by defaced lms by these bacterial lawns. Nevertheless, the chitosan formate lms showed inhibitory eects against S. aureus, S. sonnei, S. typhimurium and E. coliO157:H7 and bacteriostatic activity against L. monocytogenes. However, compared to the chitosan citrate lms, the inhibitory eects of chitosan formate lms were lower and the thickness of the inhibitory zone was in the range 0.491.64 mm compared to 0.786.0 mm of chitosan citrate lms. All chitosan citrate lms exhibited prominent inhibitory eects on all seven pathogenic bacteria. All chitosan citrate lms showed distinctive inhibition zones against all tested pathogenic bacteria and the inhibition zones were considerably thicker than those produced by chitosan formate lms. Also, the inhibitory eects of chitosan citrate lms were remarkably higher for S. aureus and V. vulnicus, as indicated by thicker inhibition zones accounting for more than 4 mm. The chitosan citrate lms were the only lms with antimicrobial eects against B. cereus and V. vulnicus. The higher inhibitory activity shown by all chitosan citrate lms can be attributable to complete solubility of chitosan which could make them more reactive against bacterial cells. Antimicrobial activity. The quantitative analysis of antimicrobial activity for selected chitosan lms were carried out as follows. Acetate lms with demineralized decolorized deacetylated (DMCA) chitosan, formate lms with deproteinized demineralized decolorized deacetylated (DPMCA) chitosan and citrate lms with demineralized deacetylated (DMA) chitosan. The above-selected lms were also tested with further inhibition zone assays with more controls. The nisin spots used as the positive control produced more prominent clear zones with L. monocytogenes, and S. aureus

Dutta et al.
Table 9. Studies on antimicrobial edible films and bacterial levels on meat products Types of study, activity and observation Organic acids more effective against L. monocytogenes on beef carcass tissue when immobilized in calcium alginate than when used as a spray or dip Zein films, impregnated with nisin, lauric acid and EDTA and tested with broth cultures of L. monocytogenes, reduced the bacterial counts over 5 logs after 48 h Zein films containing nisin produced a 4.55 log reduction on L. monocytogenes inoculated onto chicken breast tenders CFU/mL without refrigeration for 16 days Impregnation the surface of meat casing with pediocin powder to produce a 13 log reduction of L. monocytogenes on ham, turkey breast and beef compared to inoculated controls References Siragusa and Dickson (1992, 1993)

Hoffman et al. (2001)

Janes et al. (2002)

Ming et al. (1997)

lawns representing the Gram-positive bacteria and vague spots with S. sonnei and S. typhimurium lawns representing the Gram-negative bacteria. Regardless of the type of bacteria, controls such as chitosan solutions, acid solutions and the polyvinyl chloride plastic failed to produce any clear or inhibition zones indicating that they were ineective in inhibiting the above-mentioned food pathogenic bacteria. This substantiates the claim that the direct application of antimicrobial agents, such as chitosan and acid solutions used in our studies, onto food surfaces is less eective due to loss of antimicrobial activity caused by leaching onto the food, enzymatic activity and reaction with other food components (Jung et al., 1992; Ouattara et al., 2000a; Ray, 1992). Hence, the use of packaging lms or coating as a matrix to deliver antimicrobial agents becomes important. Such packaging or coating can maintain a high concentration of antimicrobial agents on a food surface and it allows low migration into food (Ouattara et al., 2000b; Siragusa and Dickson, 1992; Torres et al., 1985). Results of the direct inoculation study were in agreement with the inhibition zone assays (the survivor log number CFU/mL of L. monocytogenes inoculated onto the surface of the selected chitosan lms). Chitosan citrate lm: L. monocytogenes was more susceptible to chitosan formate or chitosan acetate lms. It reduced the bacterial count by 5.34 log CFU/ mL within 4 h of incubation and accounted for more

than 4.47 log CFU/mL reduction of inoculum in 24 h. It caused only marginal reduction of the inoculum, accounting for less than 1 log CFU/mL reduction over the entire 24 h period incubation. Further, the chitosan formate lms caused about 1 log CFU/mL reduction of inoculum at 2 h of incubation and maintained a 1 log CFU/mL reduction over 24 h of incubation. The rate of reduction of microbial count was poor with both chitosan acetate and formate lms as there was no signicant dierence in microbial count between 2 and 4 h of incubation and between 4 and 8 h of incubation. Organic acids with smaller molecular weights have higher antimicrobial activities and undissociated smaller molecules of formic (46.03 Da) and acetic (60.05 Da) acids may enter the bacterial cells easily to change the internal pH of the organisms (Eswaranandam et al., 2004). Undissociated larger molecules of citric acid (192.13 Da) may not enter into the cells eectively. Such a trend was not observed in the study (Nadarajah, 2005) and the result was in contrary. It indicates that chitosan lms made of organic acids may behave as one entity rather than separate entities, i.e., as a career matrix containing an antimicrobial agent. Several studies have demonstrated that antimicrobial edible lms can reduce bacterial levels on meat products (Table 9). In most of these studies, antibacterial activity against L. monocytogenes was attempted with added antimicrobials. Some of the major ndings of the work for crawsh chitosan are as follows. The chitosan citrate lm producing more than a 4.4 log reduction in L. monocytogenes was a commendable achievement. As with the case L. monocytogenes, the chitosan citrate lms showed higher antibacterial activity against S. aureus.The chitosan citrate lms produced more than a 5 log reduction in S. aureus within 4 h of incubation and maintained its inhibitory eect through out the incubation period. The chitosan acetate lms produced a poor inhibition with less than 1 log reduction at 24 h. The chitosan formate lms maintained about 1 log reduction for up to 4 h. At 24 h incubation, chitosan formate lms produced more than a 5 log reduction similarly observed for chitosan citrate lms. Relatively very little research work has been dedicated to formulate edible lms active against S. aureus (Table 10). All these studies indicate the importance of having added antimicrobials in the lms to control S. aureus.However, the crawsh chitosan citrate and formate lms which contained no added antimicrobials could produce more than a 5 log reduction. Further, the inhibitory eects of chitosan citrate and formate lms against S. aureus were higher than that against L. monocytogenes. Along with L. monocytogenes, S. typhimurium has been considered as a microbiological hurdle 17

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Food Science and Technology International 18(1)


Table 10. Report to formulate edible films that are active against S. aureus Types of study, activity and observation Polyethelene film containing grapefruit seed extract showed an inhibitory effect against S. aureus as indicated by a 2.57.0 mm inhibition zone by the agar diffusion method A 1.5 log and 2.8 log reduction of S. aureus in cheese and ham by nisin-absorbed bioactive inserts An edible packaging made of cellulosic esters, fatty acids and nisin produced up to 88 mm diameter inhibition zone on S. aureus.Further, they reported that addition of fatty acid reduced the inhibitory activity References Ha et al. (2001)

Scanell et al. (2000) Coma et al. (2001)

Compared to these published data, reduction of S. typhimurium in this study by more than 4.7 log by chitosan citrate lm and 3.7 log by chitosan formate lm is outstanding. As with L. monocytogenes, and S. aureus, chitosan acetate lms produced minimal inhibition against S. sonnei. The chitosan formate lms accounted for about 1 log reduction at 4 h of incubation and 2.6 log reduction at 24 h. The citrate lms showed the highest antibacterial activity against S. sonnei with more than 5 log reduction at 8 h of incubation. Major finding of the overall work This study conrms that crawsh chitosan can be used to develop antimicrobial edible lms eective against both Gram-positive and Gram-negative food pathogenic bacteria. Chitosan acetate lms showed poor inhibitory eects against L. monocytogenes, S. aureus, S. typhimurium and S. sonnei. Although chitosan acetate lms outweighed other lms in terms of their mechanical properties, they demonstrated minimal antibacterial eects similar to bacteriostatic eects with negligible bacterial reduction over a period of 24 h. Chitosan formate lms were eective against S. aureus, S. typhimurium and S. sonnei, causing more than 5, 3.7 and 2.5 log reductions at 24 h incubation, respectively. Chitosan formate lms produced poor inhibitory eect against L. monocytogenes with less than 1 log reduction at 24 h incubation. Based on antibacterial and packaging properties, chitosan formate lms can be used as antibacterial packaging to control S. aureus, S. typhimurium and S. sonnei, except L. monocytogenes. Chitosan citrate lms were highly eective against L. monocytogenes, S. aureus, S. typhimurium, and S. sonnei. The eect of chitosan citrate lms against L. monocytogenes and S. aureus was prominent with more than 5 log reduction within 4 h of incubation. Furthermore, chitosan citrate lms indicated their potential antibacterial eects against B. cereus and V. vulnicus as indicated by the zone inhibition tests. This study indicates the possibility of formulating an antibacterial edible lm, especially crawsh chitosan citrate lm, active against a broad spectrum of bacteria (Nadarajah, 2005). Bacterial growth susceptibility Bacterial growth susceptibility was determined by the MIC method. Drops of chitin derivatives of dierent concentrations were applied to the surface of agarose plates containing cultures of bacteria in nutrient dextrose medium or LB medium for phytopathogenic bacteria and E. coli, respectively. MIC was dened as the lowest concentration of chitin derivatives that bacterial growth after overnight inhibited

Table 11. Report to formulate edible films on S. typhimurium Types of study, activity and observation A 4.3 log reduction of S. typhimurium on inoculated broiler skin exposed to nisin-coated polyvinyl chloride film A 4.23 log reduction of S. typhimurium in pads treated with nisin formulations Further, applied nisin formulations to S. typhimurium inoculated on tray pads and demonstrated 3.1 log reduction References Natrajan and Sheldon (2000)

Sheldon et al. (1996) Sheldon (2001)

for a long time. As with L. monocytogenes and S. aureus, a similar trend of inhibition was observed with S. typhimurium. The chitosan citrate lms produced more than 3.4 log reduction in S. typhimurium within 2 h of incubation, and reduction in counts reached 3.85 log at 4 h and 4.83 log at 8 h incubation. The chitosan acetate lms were less eective with about 1 log reduction at 24 h. There was no signicant (p > 0.05) change in the S. typhimurium count from 2 h to 24 h for chitosan acetate lms. The chitosan formate lms maintained about 2.7 log reduction up to 8 h and then produced a signicant (p < 0.05) increased inhibition (3.7 log) between 8 and 24 h of incubation. The eects of edible lms on S. typhimurium have been given in Table 11. 18

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Dutta et al. incubation of the agarose plates at 37  C (Struszczyk and Pospieszny, 1997). In another experiment, the eect of chitin derivatives on Pseudomonas syringae pv. phaseolicola was tested using the hypersensitive reaction (HR) of tobacco. Mixtures of bacterium and chitin derivatives at nal concentration 5 107 CFU/mL and 0.05 wt%, respectively, were injected into leaves of tobacco Xanthi nc. Suspensions of the bacterium in distilled water or solutions of chitin derivatives in distilled water were used as controls. Water-soluble chitin oligomers, chitosan, chitosan sulfates and carboxymethyl chitosan were used in this research. Chitosan was dissolved in the acetic acid and other chitin derivatives in distilled water. The reactions of all the solutions were adjusted to pH 5.56.0 with potassium hydroxide. Cationic chitin derivatives, i.e., chitin oligomers and chitosan, inhibited the growth of the Gram-positive bacteria: Corynebacterium michiganense subsp. michiganense and C. michiganense subsp. insidiosum, and Gram-negative bacteria: Xanthomonas campestris pv. Phaseoli, P. syringae pv. Phaseolicola, P. syringae pv. tomato, Erwinia amylovora, Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens at concentrations of the range 0.010.3 wt%. However, both derivatives were less eective against E. coli. Anionic chitin derivatives, i.e., chitosan sulfate and carboxymethyl chitosan at a concentration of 1.5 wt% were not eective against any of the bacterial tested. When cationic derivatives were added to the bacteria suspension, occulation was observed. The HR of plants is widely used for quick demonstration of bacterial pathogenicity (Klement, 1963). When the tobacco leaves were injected by a mixture of P. syringae pv. Phaseolicola and chitin derivatives, HR was prevented. Chemical depolymerization Chitosan oligosccahrides have received attention because of their versatile biological properties. Those have lower viscosities, and low molecular weights and are soluble in aqueous solution. They seem to be readily adsorbed in vivo (Chatterjee et al., 2005). Chemical treatment of chitosan using strong acids, e.g. HNO2 and HCl is a very common and fast method to produce a series of chitooligomers. However, this method has some disadvantages such as high cost and the low yield of chitosan oligosaccharides with degree of depolymerization (DP) from DP2 to DP5 because of random cleavage resulting in mostly monosaccharides. The irradiation eects on chitosan in acetic acid solution with various dose rates and the yield of chitosan oligomers were investigated (Choi et al., 2002). Low molecular weight chitosans were prepared at dierent reaction temperatures and times using 85% phosphoric acid that resulted in the decrease of viscosity average molecular weight from 21.4 104 to 7.1 104. Depolymerization of chitosan by the use of HNO2 is a homogeneous reaction where the number of glycosidic bonds broken is stochiomeric to the amount of HNO2 used (Jia and Shen, 2002). The hydrolysis of chitosan with strong HCl was studied over a range of acid concentrations and temperatures. There have been very few reports on the degradation of chitosan by free redicals. Nordveidt et al. (1994) demonstrated that the viscosity of chitosan solution decreased rapidly in the presence of H2O2 and FeCl3 probably due to random depolymerization of chitosan (Chen et al., 1997). Several biological activities of chitosan depend on the degree of polymerization. According to Liu et al.(2006), the main factors aecting the antibacterial activity of chitosan are molecular weight and concentration. Recent studies on chitosan have attracted interest for converting it into oligosaccharides because they are not only water-soluble but also they are believed to have greater antimicrobial activity. Chitosan has a mean molecular mass of up to 1 MDa, which corresponds to a chain length of approximately 5000 units, but there is considerable variation between commercial batches. The molecular mass of native chitin is usually higher than 106 Da, whereas the molecular mass of the commercial chitosan is often observed between 105 and 12 105 Da (Muzzarelli, 1973). During the process of deacetylation, the hard conditions tend to degrade and depolymerize chitosan (No et al., 2002). Medium- and low-Mw chitosan can be obtained by chemical or enzymatic hydrolysis of the high-Mw polymer. The chemical hydrolysis is usually achieved using strong acids, which is an unexpensive and rapid method. Its drawback is the necessity to purify extensively the low-Mw chitosan products for biological applications due to the toxicity of the reagents used for the reaction. Hydrogen peroxide treatment (No et al., 2002) and ultrasonication (Czechowska-Biskup et al., 2005) could be also used. The extent of hydrolysis is, however, rather dicult to control (Ploue et al, 1997). Enzymatic depolymerization Enzymatic depolymerization seems to be a better method to prepare chito oligosaccharides. Microorganisms have been found to possess chitosanase activity. Among bacteria, Bacillus and Streptomyces strains are most often studied. Studies on fungal chitosanase are less reported (Cheng and Li, 2000). The growing consumer demand for foods without chemical preservatives has led people to indulge in eortstoward the discovery of new natural antimicrobials (No et al., 2002). In this context, the antimicrobial activities of chitosan and their derivatives against 19
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Food Science and Technology International 18(1) dierent groups of microorganisms, such as bacteria, yeast and fungi have received considerable attention in recent years. Antibacterial activities of six chitosans and six chitosan oligomers with dierent molecular weights were examined against four Gram-negative (E. coli, P. uorescens, S. typhimurium and Vibrio parahaemolyticus) and seven Gram-positive bacteria (L. monocytogenes, Bacillus mageterium, B. cereus, S. aureus, Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus bulgaricus). Chitosans showed higher antibacterial activities than chitosan oligomers and markedly inhibited growth of most bacteria tested although inhibitory eects diered with Mws of chitosan and the particular bacterium. Chitosan generally showed stronger bactericidal eects with Gram-positive bacteria than Gram-negative one in the presence of 0.1% chitosan (Wang, 1992). The MIC of chitosans ranged from 0.05% to >0.1% depending on the bacteria and Mws of chitosan. A solution can be obtained by the use of enzymes to produce bioconversions. The chitooligosaccharides produced by the enzymatic hydrolysis of chitosan are widely used in the food, agricultural and pharmaceutical elds because of their various physiological activities. Chitinase (EC 3.2.1.14) is an important chitin-degrading enzyme which is involved in bioconversion processes of wastes from crustaceans and in plant protection by preserving them from chitin-containing pathogens such as fungi (Decleire et al., 1997). Chitosanase (EC 3.2.1.132) is dened as an enzyme that catalyses random hydrolysis of -1, 4 linkages between GlchitosanAc and Glchitosan residues in a partially N-acetylated chitosan. Chitosanase was distinguished from chitinase on the basis of its ability to hydrolyze GlchitosanGlchitosan. As specied by Seki et al. (1997), chitosanases were subdivided into three subclasses, characterized by the ability to split GlchitosanGlchitosan and GlchitosanAcGlchitosan linkages (subclass I), only GlchitosanGlchitosan linkages (subclass II) and GlchitosanGlchitosan and GlchitosancGlchitosanAc linkages (subclass III). Chitosanase can be dened as the enzyme which requires at least one Glchitosan residue at either side of hydrolyzing linkages in partially N-acetylated chitosan but not GlchitosanAc GlchitosanAc bonds. To date, many chitosanases have been found in a variety of microorganisms, including particularly bacteria (Kurakake et al., 2000; Pelletier and Sygusch, 1990; Rivas et al., 2000; Sikorski et al., 2006; You et al., 2003) and fungi (Cheng and Li, 2000; Eom and Lee, 2003; Zheng and Zhu, 2003). Moreover, several other hydrolytic enzymes, such as lysozyme, cellulases and papain, were found to catalyze the enzymatic cleavage of glycosidic linkage in chitosan (Muzzarelli et al., 1995; Pantaleone et al., 1992; Yalpani and Pantaleone, 1994). 20
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At rst, various investigators reported molecular weight relationships of antibacterial activity by chitosan and there are some reports that chitosan is more eective in inhibiting the growth of bacteria than chitosan oligomers. Hirano and Nagao (1989) studied the relationship between the degree of polymerization of chitosan and the inhibition grade against 18 phytopathogens. The test materials were the lactate salt of high-molecular weight chitosan (Mw 400 kDa, with 95% of deacetylation) and chitosan oligosaccharides, with a degree of polymerization (DP) in the range 28. The growth of 13 fungi was inhibited more than 10% by the highmolecular weight chitosan and 6% by chitosan oligosaccharides. These authors observed that a decrease in the degree of polymerization of chitosan resulted in decreases in the number of inhibited fungal species. Previously, Kendra and Hadwiger (1984) demonstrated that the maximal antifungal activity of chitosan was exhibited by chitosan oligomers of seven or more residues. In contrast to these results, Avadi et al.(2004) mentioned that chitosan oligomers with a DP 30 possess antimicrobial activity against a number of bacteria, whereas low-DP oligomers are ineective. Jeon et al. (2001) studied the bioactivities of three chitosans, high, medium and low-molecular weight chitosans with a Mw values in the range 247 and 61 and 5 kDa, respectively. The prole of low molecular weight consisted of oligosaccharides with DP in the range from pentamer to heptamer. They observed that the ecacy on the growth of E. coli increased with molecular weight. Chemical modifications Even though chitosan is quite attractive as a biopolymer with distinctive physicochemical properties and biological activities, it is currently utilized to only the limited extent. The delay in application study is partly ascribable to the diculty in controlled modications because of the insoluble nature in organic solvent and multifunctionality of chitosan. However, many kinds of modication reactions have been exploited to increase the antimicrobial properties of chitosan. The growing demand for a more accurate control of polysaccharide properties has prompted the development of numerous techniques for selective modications. The amino and two hydroxyl groups present in the repeating unit of chitosan are the targets of dierent chemical modications (Hirano et al., 1987). As a result, the functionality of linear polysaccharides is signicantly aected by the presence, level and distribution of substituents along the main chain. As specied by Rabea et al. (2003), chitosan and chitin are commercially interesting compounds because of their high nitrogen contents (6.89%) compared to synthetically substituted cellulose (1.25%). This makes chitosan a

Dutta et al.

OH RCHO O HO NH2 O HO

OH O O CHR

Figure 6. Schiffs base obtained from the reaction between free amino groups of chitosan and aldehyde.

OH O O HO NHCH3

OH O O HO N CH3 CH3 H 3C I

CH3I, NaOH, NaI N-methyl-2-pyrrolidinone

N-methylchitosan

N,N,N-trimethylchitosan

Figure 7. Synthesis of N,N,N-trimethylchitosan. Source: Belalia et al. (2008).

useful chelating agent. However, these naturally abundant materials are also limited in their reactivity and processability. Several alkylated chitosans are reported to be synthesized. Kim et al. (1997) prepared N-alkyl chitosan derivatives by introducing alkyl groups into the amine groups of chitosan via Schis base intermediates. Indeed, the free amine groups of the chitosan react with aldehydes to give the Schis base (Figure 6) in homogeneous mediums such as acetic acid and methanol (Hirano, 1997). Long alkyl chains (until C12) can be introduced on the chitosan. Quaternization of the N-alkyl chitosan derivatives could be carried out using a halogenoalcane in the presence of sodium hydroxide (Belalia et al., 2008; Jia et al., 2001). As shown in Figure 7, MeI could be used to produce water-soluble cationic polyelectrolytes and novel chitosan derivatives, with quaternary ammonium salt (Belalia et al., 2008; Kim et al., 1997). Their antibacterial activities against S. aureus were explored by the viable cell count method in acetate buer at pH 6. The antibacterial activities of the quaternary ammonium salt increased with increase in the chain length of the alkyl substituent, and this increased activity could be ascribed to the contribution of the increased hydrophobic properties of the derivatives. In addition, Avadi et al. (2004) prepared a quaternized chitosan (i.e., N-diethylmethyl chitosan, DEMC) based on a modied

two-step process. With a degree of quaternization of 79%, the DEMC exhibited a higher antibacterial activity than chitosan against E. coli. However, the antimicrobial eects of both compounds were pH dependent and an increase in concentration of acetic acid resulted in a signicant decrease in MIC, determined by turbidimetric method. As a result, the antibacterial activities of chitosan and DEMC are higher in 1% acetic acid in comparison with the lower levels of acetic acid concentration, 0.25%, for example. The MIC of DEMC is decreased from 500 to 62.5 mg/mL when the medium is changed from water to 1% acetic acid solution. The authors mentioned that this is due to the target site of the polycation, i.e., the negatively charged surface of the bacteria cell. Therefore, the polycation DEMC with a high charge density interacts with the bacteria more than what chitosan itself does. Other chitosan derivatives such as N,N,N-trimethyl chitosan, N-propyl-N,Ndimethyl chitosan and N-furfuryl-N,N-dimethyl chitosan were prepared and tested for their activities against E. coli (Jia et al., 2001). It was shown that the antibacterial activity of quaternary ammonium chitosan in acetic acid medium stronger than that in water against E. coli and is stronger than that of chitosan itself. More recently, Chi et al. (2007) prepared Chitosan-N-2-hydroxypropyl trimethyl ammonium chloride by the reaction of chitosan with glycidyl 21

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Food Science and Technology International 18(1)

CH2OH O OH O

NH CH2OH O OH NH2
Chitosan

H3C

(CH2)13CH3 COOCH2CH2OP(OH)2

O Chitosan-g-MAP CH2OH O OH O

HN CH2 CH2 SO3Na Chitosan-g-VSS

Figure 8. Mono (2-methacryloyl oxyethyl) acid phosphate (chitosan-MAP) and vinylsulfonic acid sodium salt (chitosanVSS) grafted onto chitosan synthesized by Jung et al. (1999).

trimethylammonium chloride. The chitosan derivatives, with dierent molecular weights, near 1.7, 35.7, 90.2 and 415.5 kDa, showed biocidal activity on S. aureus and Staphylococcus epidermidis, B. subtilis and Candida albicans. These authors observed that the chitosan with a molecular weight of 415.5 kDa exhibits a slightly lower biocidal activity on C. albicans than others. However, it seems that high-molecular weight chitosan derivatives had high biocidal activities on the Gram-positive bacteria, with a decreasing biocidal eect with decreasing molecular weight from 90 to 1.7 kDa. Nevertheless, derivatives with molecular weights from 90 to 1.7 kDa did not exhibit any biocidal eect against E. coli and P. aeruginosa even at concentrations up to 10 mg/mL. In another study, Kim et al. (1997) synthesized diethylaminoethylchitosan (DEAEchitosan) from deacetylation of a diethylaminoethylchitin (DEAE18 chitin) by introducing DEAE groups onto the C(6)OH in chitin. The deacetylation was conducted by heating in aqueous 10% sodium hydroxide containing sodium borohydride. In addition, DEAEchitin was quaternized to produce triethylaminoethylchitin (TEAEchitin). Their antibacterial activities against S. aureus and E. coli were evaluated using colony count by means of the shake ask method. The antibacterial activities were found to increase in the order DEAEchitin, DEAEchitosan and TEAEchitin. To obtain copolymers with zwitterionic property, Jung et al. (1999) prepared water-soluble anionic chitosan moieties and investigated their 22

antimicrobial activity. Mono(2-methacryloyl oxyethyl) acid phosphate (chitosanMAP) and vinylsulfonic acid sodium salt (chitosanVSS) were grafted onto chitosan (Figure 8). Concerning their antimicrobial activities, chitosanMAP and chitosanVSS exhibited inhibition values of the growth of C. albicans of about 95% and 75%, respectively. Both chitosan derivatives showed the same high pH-dependence. As a result, if the pH changed from 5.7 to 6.2, their antimicrobial properties dropped to 10 15%, which was less than the activity of the parent chitosan. Against Trichophyton rubrum, the MAP grafting led to a low negative impact on antimicrobial activity, whereas the anionic chitosan showed much enhanced bioactive action against Trichophyton violaceum, compared to unmodied chitosan. The authors suggested than this selectivity could result from the structural anity between the wall of microbial strains and the chitosan or its derivatives. A possible reason might be that the wall of microorganisms consisted of chitin, chitosan or -glycan. Other chitosan derivatives are sulfonated and sulfobenzoyl chitosans. The antibacterial eects of the chemical modications were evaluated and compared with those of 69% deacetylated chitosan by Chen et al. (1998). MIC values of sulfonated chitosan (0.63% sulfur content) against Shigella dysenteriae, Aeromonas hydrophila, S. typhimurium, and B. cereus were found to be lower than those of the deacetylated chitosan. A high sulfur content in sulfonated chitosan adversely

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Dutta et al. inuenced its antibacterial eect. Sulfobenzoyl chitosan exhibited excellent water solubility and an antibacterial eect comparable to those of sulfonated chitosan. Concerning the antifungal properties, Cuero et al. (1991) showed that aqueous solutions of N-carboxymethylchitosan suppressed both growth and aatoxin production by Aspergillus avus and Aspergillus parasiticus in submerged culture. Five chemically modied chitosans were tested for their antifungal activities against Saprolegnia parasitica by Muzzarelli et al. (2001) using the radial growth assay in chitosan-bearing agar and the fungal growth assay in chitosan-bearing broth. Members of the genus Saprolegnia are responsible for the infections of sh and eggs in aquaculture facilities. Results indicated that methylpyrrolidinone chitosan, N-carboxymethyl chitosan and N-phosphonomethyl chitosan exerted eective fungistatic action against the target strain. Electron microscopy observations provided evidence of ultrastructural alterations, damaged fungal structures, uptake of modied chitosans, and hyphal distortion and retraction. As a result, chemical modications of chitosan with respect to the amine site are numerous and relatively easy taking into account the reactivity of the primary amine. However, to preserve the amine groups in order to maintain the bioactive properties, various modication procedures have recently been described, which yield C-6 modied chitosan derivatives. the microorganisms surface (Helander et al., 2001). The interaction resulted in great alteration of the structure of outer membrane which caused release of a major proportion of proteinaceous material from the cells (Helander et al., 1998); when the quarternized group was introduced onto the molecular chain, the positive charge was strengthened. On the other hand, when carboxymethyl group was introduced along the molecular chain, the presence of a molecular structure with hydrophilic ends and weak interaction forming between hydrophilic ends and chitosan enhances the antimicrobial activity. Because of the positive charge on the C-2 of the glucosamine monomer below pH 6, chitosan is more soluble and has a better antimicrobial activity than chitin (Chen et al., 1998). The exact mechanism of the antimicrobial action of chitin, chitosan and their derivatives is still imperfectly known, but dierent mechanisms have been proposed (Rabea et al., 2003). One of the reasons for the antimicrobial character of chitosan is its positively charged amino group which interacts with negatively charged microbial cell membranes, leading to the leakage of proteinaceous and other intracellular constituents of the microorganisms (Shahidi et al., 1999). Chitosan acted mainly on the outer surface of bacteria. At a lower concentration (0.2 mg/mL), the polycationic chitosan does probably bind to the negatively charged bacterial surface to cause agglutination, while at higher concentrations, the larger number of positive charges may have imparted a net positive charge to the bacterial surfaces to keep them in suspension (Papineau et al., 1991; Sudarshan et al., 1992). A strong attachment of heterologous bacteria to the walls in tobacco leaves is essential to elicit the HR. Therefore, from mechanistic point of views, it is possible that chitin derivatives prevent the attachment of bacterial cells into the plant cell walls or aect their survival in the intercellular spaces. Chitosan acted mainly on the outer surface of the bacteria. The obvious antibacterial eects can be attributed to the formation of polyelectrolyte complexes between the polycationic agent and the bacterial cell surface (Muzzarelli et al., 1990). Indeed, interaction between positively charged chitosan molecules and negatively charged microbial cell membranes leads to the leakage of proteinaceous and other intracellular constituents. Studies based on UV absorption indicated that the chitosan causes considerable losses of proteinic material to Pythium oaroecandrum, at a pH 5.8 (Helander et al, 2001; Liu et al., 2004). Dierent behaviors were reported, dependent on the chitosan concentration (Rabea et al., 2003). At a lower concentration (<0.2 mg/mL), the polycationic chitosan does probably bind to the negatively charged bacterial surface to cause agglutination, while at higher concentrations, the larger 23
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MECHANISM OF ANTIMICROBIAL ACTION


The dierent mechanisms have been proposed whereas the exact mechanism of the antimicrobial action of chitin, chitosan and their derivatives is still unknown (Rabea et al., 2003). The mechanisms of the antimicrobial activity of chitosan were dierent for Gram-positive and Gram-negative bacteria (Zheng and Zhu, 2003). In this study, they dierentiated the eect of chitosan on S. aureus (Gram-positive) and on E. coli (Gram-negative). For Gram-positive S. aureus, the antimicrobial activity increased with increase the molecular weight of chitosan. Besides, for Gram-negative E. coli, the antimicrobial activity increased with decrease in molecular weight. The authors suggested the following two dierent mechanisms for the antimicrobial activity: (1) in the case of S. aureus, the chitosan on the surface of the cell can form a polymer membrane, which inhibits nutrients from entering the cell and, (2) for E. coli, where chitosan of lower molecular weight entered the cell through pervasion. The antimicrobial mechanisms of CM, Q and CMQ are suggested as: on one hand, the positive charge of the group at C-2 resulted in a polycationic structure which can be expected to interact with the predominantly anionic components (lipopolysaccharides and proteins) of

Food Science and Technology International 18(1) number of positive charges may have imparted a net positive charge to the bacterial surfaces to keep them in suspension. Savard et al. (2002), by a microscopic examination of Saccharomyces unisporus after treatment with chitosan-salt with a polymerization degree of 25, showed agglutination of a refractive substance on the entire cell wall. When chitosanase was added to the culture media containing chitosan-salt, they could not observe refractive substances. This result suggested an interaction between chitosan and the cell wall. In another study, chitosan caused leakage of glucose and lactate deshydrogenase from E. coli cells (Tsai and Su, 1999). These results support the hypothesis that the mechanism of chitosan antibacterial action involves cross-linkage between the polycations of chitosan and the anions on the bacterial surface that change the membrane permeability. As already mentioned, chitosan coatings adjusted to pH 5.0 totally inhibited Grampositive bacterial surface growth such as L. monocytogenes and S. aureus. However, Gram-negative microbial strains such as P. aeruginosa overcame the active chitosan protection, and the development was not completely excluded (Coma et al., 2003). Therefore, the microbiological target of protonated chitosans action would be the cytoplasmic membrane of sensitive cells. Cellular damage can lead to the disruption of the cellular integrity of the membrane. The outer membrane of Gram-negative bacteria could act as a barrier and be responsible for preventing chitosan from reaching the cytoplasmic membrane. Although the cytoplasmic membrane should be sensitive to chitosan, the outer membrane protects the cells. Zheng and Zhu (2003) also indicated that the mechanisms of the antimicrobial activity of chitosan were dierent between Grampositive and Gram-negative bacteria. They showed, in a comparative study, that the eect of chitosan was dierent on S. aureus (Gram-positive) and on E. coli (Gram-negative). To E. coli, the antimicrobial activity was enhanced as the molecular weight decreased. It was obvious that 0.25% chitosan solution (Mw < 5 kDa) could inhibit the growth of E. coli. In contrast, for S. aureus, the antimicrobial activity increased with increasing molecular weight of chitosan. The inhibiting eect was fairly obvious for higher ones, such as 305 kDa, even if the concentration was quite small. The authors suggested two possible mechanisms for antimicrobial activity: (1) the chitosan on the surface of the cell can form a polymer membrane, which prevents nutrients from entering the cell and (2) chitosan of lower molecular weight entered the cell through pervasion. For S. aureus, the dominant mechanism is the former, while for E. coli, the latter mechanism seems more likely. In addition, the chitosan has also the faculty to bind specically with some macromolecules. It can thus inhibit various enzymes, bind to the DNA and inhibit the 24
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synthesis of the mRNA after penetration of the chitosan in the core of the microorganisms (Hadwiger et al., 1985). Epiuorescence microscopy results showed a possible action of chitosan during a short duration of time on the synthesis of nucleic acids and especially on the relative proportion of RNA compared with DNA (Coma et al., 2003). This impact was followed by an adaptative mechanism of the cells. Binding of chitosan with DNA and inhibition of mRNA synthesis occur through chitosan penetration toward the nuclei of the microorganisms and interference with the synthesis of mRNA and proteins (Rabea et al., 2003). It has been postulated that the antimicrobial action of chitosan occurs as a result of several mechanisms. Chung and Chen (2008) studied the antibacterial activity of chitosan with respect to the extent of damaged or missing cell walls and the degree of leakage of enzymes and nucleotides from dierent cellular locations. First, the addition of chitosan to the bacterial suspension seemed to have a stronger impact on the Gram-negative E. coli than on the Gram-positive S. aureus in terms of the leakage of enzymes. In addition, the experimental result revealed that the antimicrobial action of chitosan not only involves a reaction with the cell wall of the bacteria but also may aect the structure of the phospholipid bilayer in the cell membrane, thereby changing the permeability of the cell membrane, resulting in the release of some of the cellular components. This action was further enhanced when chitosan with a high degree of deacetylation was used. To gain a better understanding of the mechanism by which chitosan functions as a bactericide, the cells were also subjected to a known antibiotic which reacts with the anionic phosphate group of phospholipids in the cell membrane, destroying the cell membrane structure and aecting its permeability. In parallel, the cells were subjected to EDTA, a chemical chelating agent that destroys the structure of the cell wall by chelating with the Ca2 or Mg2 present in the cell wall. As a result, chitosan was found to react with both the cell wall and the cell membrane of E. coli, but not simultaneously, indicating that the inactivation by chitosan occurs via a two-step sequential mechanism: an initial separation of the cell wall from its cell membrane, followed by cell membrane destruction. Concerning the bioactivity of the chitosan against phytopathogenic fungi, it could be related with its potential elicitor of many plant defense responses, including for example the accumulation of chitinases or by producing phytoalexins, dened as substances with antibiotic activity that function as growth inhibitors of phytopathogenic organisms, chiey fungi (Bade and Wick, 1987). In a study on Botrytis cinerea and Rhizopus stolonifer, chitosan-induced morphological changes were characterized by excessive hyphal branching and abnormal aerial surface hyphal growth compared to the control (El Ghaouth et al., 1992). As a result,

Dutta et al. chitosan appears to play a dual function, by interfering directly with fungal growth and also by activating several biological processes in plant tissues. Benhamou et al. (1994) applied chitosan to decrease the infection with Fusarium oxysporum. Biopolymer coating was applied on seed prior to fungal inoculation. Chitosan at concentrations ranging from 0.5 to 1 mg/mL was used for the ultrastructural and cytochemical investigations. The authors observed that after a pretreatment with chitosan, the root tissues at sites of fungal penetration were always associated with the expression of plant defense reactions. In the epidermis, cells showed typical signs of necrosis characterized by marked disorganization of the cytoplasm. The pathogen was detected in the outer cortex where its development was halted. Fungal cells suered from serious damage and were frequently encircled by an electron-dense material. In the noncolonized inner cortex, strong host reactions were detected that were mainly associated with the deposition of two types of materials that diered in their electron densities. Gold cytochemistry with a -1,3-glucanase and a laccase showed that the more electron-dense material was phenolic in nature, whereas the other material, occurring either as deposits inserted between the phenolic aggregates or as globular structures lining the host cell walls, was made of -1,3glucans. These observations bring further evidence that chitosan is an active inducer of plant defense reactions and, thus, has the potential to become a powerful alternative means of disease control. antimicrobial eect, there is no direct evidence demonstrating this behavior against bacteria. Lower pH increases the antimicrobial activity of chitosan for much the same reasons, in addition to the hurdle eect of inicting acid stress on the target organisms (Rhoades and Rastall, 2000). Surrounding matrix is the greatest single inuence on antimicrobial activity. Being cationic, chitosan has the potential to bind to many food components such as alginates, pectins, proteins and inorganic polyelectrolytes such as polyphosphate (Kubota and Kikuchi, 1998). Solubility can be decreased using high concentrations of low-molecular weight electrolytes such as sodium halides, sodium phosphate and organic anions (Roberts, 1992). Sorption capacity of chitosan lms was signicantly aected by the moisture content of the chitosan-based lms. Authors reported that a decrease of water content decreased the total amount of the active sites that can participate in the sorption phenomenon. Antibacterial activities of the produced chitosan-based lms have been evaluated against E. coli and S aureus and it has been shown that diameters of the inhibition zones were 5 and 3 times higher than those of control for E. coli and S aureus, respectively. Authors showed that moisture content of chitosan lms had signicant eect on their bactericide eect which decreased on decreasing lm moisture content. Decrease of lm moisture content from 22% (w/w) down to 12% (w/w) decreased the bactericide activity by 2.5 times. This was demonstrated by the decreased diameter of the inhibition zone. This nding can be exploited in the cheese-making industry for cheese coating with chitosan lms in the maturation chambers in order to avoid mold and pathogenic bacteria growth on cheese surface (Buzinova and Shipovskaya, 2008). The eect of the molecular weight on some antibacterial and antifungal activities has been explored (Chen, 1998). Chitosans with molecular weights ranging from 10,000 to 100,000 have been found to be helpful in restraining the growth of bacteria. In addition, chitosan with an average molecular weight of 9300 was eective in restraining E. coli, whereas that with a molecular weight of 2200 helped in accelerating the growth (Tokura et al., 1994). Moreover, the antibacterial activity of chitosan is inuenced by its degree of deacetylation, its concentration in solution and the pH of the medium. Antibacterial activities were also found to be increasing in the order N,O-carboxymethylated chitosan, chitosan and O-carboxymethylated chitosan (Liu et al., 2001). In addition to the formation of gas-permeable lms, chitosan has a dual function: (1) to direct the interference of fungal growth and (2) to activate several defense processes (Bai et al., 1988). These defense mechanisms include accumulation of chitinases, synthesis of 25
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FACTORS AFFECTING THE ANTIMICROBIAL ACTIVITY


There are various factors, such as the intrinsic and extrinsic properties of chitosan: molecular weight, degree of polymerization, deacetylation, solubility and higher charge density that aect the antimicrobial activity of chitosan (Ralston et al., 1964; Sekiguchi, 1994; Uchida, 1989). The antimicrobial activities of chitosan and chitosanbased lms increases by decreasing pH. This eect can be considered as synergetic for the reasons of the hurdle eect of the acid stress on the bacterial cells (Rhoades and Roller, 2000). The mechanism of the antimicrobial activity of chitosan and its derivatives is well studied. As theory, it has been suggested that the positive charge of the amine group (NH3) at pH values lower than the pKa (pH <6.3) at which this functional group carries 50% of its total electric charge allows the interactions with negatively charged microbial cell membranes, a phenomenon which is susceptible to cause a leakage of intracellular constituents (Helander et al., 2001). However, even if this is a well-recognized explanation of chitosan

Food Science and Technology International 18(1) proteinase inhibitors and lignication and induction of callous synthesis (El Ghaouth et al., 2000). When applied on wounded wheat leaves, chitosan-induced lignications and consequently restricted the growth of nonpathogenic fungi in wheat. Chitosan inhibited the growth of A. avus and aatoxin production in liquid culture, preharvest maize and groundnut, and it also enhanced phytoalexin production in germinating peanut (Cuero et al., 1991a,b). Chitosan has also been found to inhibit growth and toxin production by Alternaria alternata fungal species lycopersici in culture (Bhaskara et al., 1998; Dornenburg and Knorr, 1997). Chitosan solution at 0.10 mg/mL markedly inhibited the growth of Xanthomonas pathogenic bacteria (isolated from Euphorbia pulcherrima) from dierent geographical origins (Li et al., 2008). The antibacterial activity of chitosan solution against Xanthomonas axonopodis pv. poinsettiicola (strain R22580) increased with the increase of chitosan concentration up to 0.10 mg/ mL. The antibacterial activity of chitosan solution at 0.05 mg/mL was enhanced by NaCl. The antibacterial activity of chitosan was investigated by assessing the mortality rates of E. coli and S. aureus based on the extent of damaged or missing cell walls and the degree of leakage of enzymes and nucleotides from dierent cellular locations (Chung and Chen, 2008). The inactivation of E. coli by chitosan occurred via a twostep sequential mechanism: an initial separation of the cell wall from its cell membrane, followed by destruction of the cell membrane. The antibacterial activities of chitosan nanoparticles and copper-loaded nanoparticles against E. coli, Salmonella choleraesuis, S. typhimurium and S. aureus were evaluated by the calculation of MIC and minimum bactericidal concentration (MBC; Qi et al., 2004). The obtained results showed that chitosan nanoparticles and copper-loaded nanoparticles could inhibit the growth of various bacteria tested. Their MIC values were less than 0.25 mg/mL and the MBC values of nanoparticles reached 1 mg/mL. They reported that the exposure of S. choleraesuis to the chitosan nanoparticles led to the disruption of cell membranes and the leakage of cytoplasm. O-157.for the high-, medium- and low-molecular weight samples, respectively. Another test was done against the P. aeruginosa strain, with bactericidal eects of about 47%, 35% and 22% for the high-, medium- and low-molecular weight chitosans, respectively. Tokura et al. (1997) prepared chitosan oligomers of average molecular weights 9.3 and 2.2 kDa by nitrous acid degradation followed by the reduction of 2,5-anhydromannose terminal by sodium borohydrate. Although the 9.3 kDa provided the growth inhibition of E. coli, the chitosan 2.2 kDa was not a growth inhibitor but a growth accelerator. It has been suggested that the smaller oligomers serve as nutrients for bacteria, whereas the higher oligomers are toxic by virtue of their chargemediated adhesion to the cell membrane, which in turn prevents the uptake of nutrients through the cell wall. Liu et al. (2006) specied that the molecular weight of chitooligosaccharides is critical for microorganism inhibition and must be higher than 10 kDa. Several studies showed that the impact of the Mw on the bioactivity is dependent on the concentration of the biocide. They investigated the bioactivities of chitosans of dierent molecular weights, from 55 to 155 kDa, against E. coli with the same degree of deacetylation (80%). The mechanism of antibacterial activity was the occulation of the strains. These authors observed that at high concentration (over 200 ppm), the antibacterial activities of each chitosan sample were almost the same and all the bacteria could be killed. At low concentration (20 ppm), there was no antibacterial activity and chitosan could promote the growth of E. coli. But at the middle range concentrations (50100 ppm), there were some dierences between chitosans of dierent molecular weights in the antibacterial activation. Indeed, the authors concluded that at high concentrations (200, 500 and 1000 ppm) and a low concentration (20 ppm), the antibacterial activity of chitosan had no relationship to the molecular weight. But at concentrations ranging from 50 to 100 ppm, the antibacterial activity of low-molecular weight chitosan is higher than that of the high-molecular weight samples. Qin et al. (2006) also showed that the molecular weight dependence of the antimicrobial activity of chitosan was more pronounced at a low concentration. The action of chitosans with molecular weights Mw from 1.4 to 400 kDa on the growth of S. aureus, E. coli and C. albicans was explored by microcalorimetry. The chitosans with middle range values of Mw, 78 and 48 kDa, had higher inhibitory eect than others. Chitooligomer 1.4 kDa promoted the growth of C. albicans, but slightly inhibited growth of the bacteria. The 400 kDa, with the highest Mw, exhibited a much weaker inhibitory eect in comparison with the chitosan at 78 kDa. It seems that the water-insoluble chitosans with Mw around 50 kDa were the optimum ones for antimicrobial action in their tested samples. In addition, Zheng and Zhu (2003)

OPTIMIZATION OF THE BIOCIDE PROPERTIES


The bactericidal activities of chitosan (Mw 685 kDa) against various bacteria were more than 99% against Gram-negative bacteria such as E. coli O-157, S. typhimurium(except for P. aeruginosa, 68%) and more than 98% against Gram-positive bacteria such as Streptococcus mutans, M. luteus, S. aureus and B. subtilis. Chitooligosaccharides showed a less bactericidal eect, with 71%, 56% and 60% against E. coli 26

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Dutta et al. observed that for chitosan with molecular weight below 300 kDa, the antimicrobial eect on S. aureus was strengthened as the molecular weight increased. In contrast, the eect on E. coli was weakened. In parallel to depolymerization, chemical modications of chitosan have been attempted to improve its antimicrobial properties. The inuence on biocide performance of some unprecedented physicochemical features of chitosan cast lms such as lm thickness, pH of the nutrient broth, lm neutralization, lm autoclave sterilization and temperature exposure was analyzed against S. aureus and in some experiments also against Salmonella spp. The work demonstrates, for the rst time, the inuence of the release or positive migration of protonated glucosamine fractions from the biopolymer into the microbial culture as the responsible event for the antimicrobial performance of the biopolymer under the studied conditions. From the results, a reliable and reproducible method for the determination of the bactericidal activity of chitosan-based lms was developed in an attempt to standardize the testing conditions for the optimum design of active antimicrobial food packaging lms and coating applications. The optimization of biocide properties of chitosan will be useful for its application in the design of active lms of interest in the food area (FernandezSaiz et al., 2008). achieved (0.882.75 mmol/min/g of protein; Orrego et al., 2010).

CHITOSAN NANOCOMPOSITE
Nowadays, the nanocomposites are receiving more attention from the researchers, because of their more eective action in penetrating and disrupting bacterial cell membranes to conquer the battle of pathogenic bacteria (Yacoby and Benhar, 2008). Many works on the use of nanochitosans to prevent food spoilage have been reported in a recent review (Friedman and Juneja, 2010). Chitosan/vermiculite (VMT) nanocomposites can enhance the thermal stability of chitosan nanocomposites dramatically due to the well dispersion of acidmodied VMT (HCl-modied VMT, HVMT) and better interaction between HVMT and chitosan in the fabricated nanocomposites (Zhang et al., 2009). Chitosan-based nanocomposite lms, especially silver-containing ones, showed a promising range of antimicrobial activities (Rhim et al., 2006). The chitosan/OREC nanocomposites lms provide promising applications as antimicrobial agents, water-barrier compounds, anti-ultraviolet compounds and drugcontrolled release carriers in antimicrobial food packaging (Wang et al., 2007). From the antimicrobial activity test, it was found that the chitosanclay nanocomposites showed a synergistic eect in the antimicrobial activity against to E. coli and S. aureus (Han et al., 2010). Antimicrobial studies showed that the nanocomposites could strongly inhibit the growth of a wide variety of microorganisms, including Gram-positive bacteria, Gram-negative bacteria and fungi; more importantly, they exhibited good antimicrobial capacity in whichever medium, in weak acid, water or weak base. As the amount of Montmorillonite increased, the nanocomposites had better inhibitory eect on microorganisms, especially Gram-positive bacteria. The lowest MIC values of the nanocomposites against S. aureus and B. subtilis were less than 0.00313% (w/v) under all the conditions (Wang et al., 2007).

CHITOSAN FOR IMMOBILIZATION


Enzymatic catalysis in nonaqueous solvents has gained considerable interest for the preparation of natural products, pharmaceuticals, ne chemicals and food ingredients (Carrea and Riva, 2000; Faber and Franssen, 1993; Margolin, 1993; Ru et al., 2000). Lipases (glycerol esters hydrolase, E.C.3.1.1.3) have been widely used to produce organic chemicals, biosurfactants, oleochemicals, agrochemicals, paper, cosmetics, ne chemicals and pharmaceuticals (Sharma et al., 2001). Chitosan has been used as a matrix for immobilization of lipases (Alsarra et al., 2002) and many other enzymes (Krajewska, 2004). Enzymes bound to sugars, or sugar-based polymers like chitosan are stabilized during lyophilization and in nonaqueous environments. This may be due to a reduction of autolysis, that is a multipoint attachment limiting enzyme distortions or microenvironmental eects (Wang et al., 1992). Elemental analysis and Raman spectra measurements of the lipase, supports and immobilized lipase systems gave evidence of the presence of enzymes on supports. Chitosan supports with internal surface area (m2/g) among 3.31 and 1.26 were obtained. Regardless of these low values, acceptable protein load (0.613.21%) and esterication initial rates were

CONCLUSIONS
Chitin, chitosan and its oligosaccharides played very important roles in the application of antimicrobial materials and food packaging. The systematic study toward antimicrobial activity of chitin, chitosan and its oligosaccharides would be a promising tool for the future improvement of food quality and preservation during processing and storage because the antimicrobial packaging can be helpful in extending the food shelf-life. The combination of other lm-forming and coating materials may provide the functional properties for a 27

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Food Science and Technology International 18(1) better food shelf-life. The understanding of the factors aecting the antimicrobial activity, mechanism of antimicrobial action, and optimization of the biocide properties of chitin, chitosan and their oligosaccharides would be an added advantage to use these materials in a better way.
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ACKNOWLEDGMENTS
We thank UGC, New Delhi and Royal Society of Chemisty (RSC), UK for giving the Research Fund Grant Award-2009 to PKD. We also sincerely thank the dierent researchers who published their works in dierent journals, magazine, dissertation, doctoral degree work and elsewhere which have been a great source for resource materials toward compiling the review in the present form. We apologize if some of the content from the above resource is/are similar during presentation. We thank the journal reviewers for their constructive suggestions.

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