Food Science and Technology International 2012 Dutta 3 34

http://fst.sagepub.com/International

Food Science and Technology

http://fst.sagepub.com/content/18/1/3The online version of this article can be found at:

 DOI: 10.1177/1082013211399195

2012 18: 3 originally published online 27 September 2011Food Science and Technology InternationalJ. Dutta, S. Tripathi and P.K. Duttaneeds for food applications

Progress in antimicrobial activities of chitin, chitosan and its oligosaccharides: a systematic study  

Published by:

http://www.sagepublications.com

On behalf of: 

  Consejo Superior de Investigaciones Científicas (Spanish Council for Scientific Research)

can be found at:Food Science and Technology InternationalAdditional services and information for    

  http://fst.sagepub.com/cgi/alertsEmail Alerts:

http://fst.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

What is This? 

- Sep 27, 2011 OnlineFirst Version of Record 

- Feb 10, 2012Version of Record >>

by guest on December 26, 2013fst.sagepub.comDownloaded from by guest on December 26, 2013fst.sagepub.comDownloaded from

Article

Progress in antimicrobial activities of chitin,chitosan and its oligosaccharides: a systematicstudy needs for food applications

J. Dutta1, S. Tripathi2 and P.K. Dutta2

AbstractIn recent years, active biomolecules such as chitosan and its derivatives are undergoing a significant and veryfast development in food application area. Due to recent outbreaks of contaminations associated with foodproducts, there have been growing concerns regarding the negative environmental impact of packaging mate-rials of antimicrobial biofilms, which have been studied. Chitosan has a great potential for a wide range ofapplications due to its biodegradability, biocompatibility, antimicrobial activity, nontoxicity and versatile chem-ical and physical properties. It can be formed into fibers, films, gels, sponges, beads or nanoparticles. Chitosanfilms have been used as a packaging material for the quality preservation of a variety of foods. Chitosan hashigh 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 todevelop and test films with antimicrobial properties to improve food safety and shelf-life. This review highlightsthe preparation, mechanism, antimicrobial activity, optimization of biocide properties of chitosan films andapplications including biocatalysts for the improvement of quality and shelf-life of foods.

KeywordsChitin, chitosan, oligosaccharides, antimicrobial activity, pathogenic microorganisms, mechanism, Gram-posi-tive and Gram-negative bacteria, food application

Date received: 7 September 2010; revised: 30 November 2010

INTRODUCTION

Once we talk about antimicrobial activity, it is alwaysdirected to its applicability. Very recently, the research isfocused into development of materials with film-formingcapacities and those having antimicrobial propertieswhich help to improve food safety and shelf-life.Antimicrobial packaging is one of the most promisingactive packaging systems that have been found highlyeffective in killing or inhibiting spoilage and pathogenicmicroorganisms that contaminate foods (Salleh et al.,2007). In this context, chitosan films have shown greatpromise for their application in food preservation. It iswell known that microbial alternation is responsible for

the enormous losses of food and hence, over the years,various chemical and physical processes have beendeveloped to extend the shelf-life of foods. The antimi-crobial activity limits or prevents microbial growth byextending the lag period and reducing the growth rate ordecreases live counts of microorganisms (Han, 2000).Currently, food application of an antimicrobial packag-ing system is limited due to the availability of suitableantimicrobials, new polymer materials, regulatory con-cerns and appropriate testing methods (Jin and Zhang,2008). In particular, polymeric bioactive films laced withan assortment of antimicrobial agents have been found

1Department of Chemistry, Disha Institute of Management andTechnology, Raipur 400701, India2Department of Chemistry, Motilal Nehru National Institute ofTechnology, Allahabad 211004, India

Corresponding author:P.K. Dutta, Department of Chemistry, Motilal Nehru NationalInstitute of Technology, Allahabad 211004, IndiaEmail: [email protected]

Food Science and Technology International 18(1) 3–34! The Author(s) 2011 Reprints and permissions:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/1082013211399195fst.sagepub.com

by guest on December 26, 2013fst.sagepub.comDownloaded from

to be very effective and practical in applications. Tilldate, a number of articles have been published describ-ing the nature of different materials used in makingfilms and their effectiveness 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 andDutta, 2010; Dutta et al., 2009; Ozdemir and Floros,2004; Quintavalla and Vicini, 2002; Suppakul et al.,2003). Present review aims to summarize all the knownmethods of formation of chitosan-based films with anti-microbial properties and discuss their mode of actionand applicability, particularly in the area of food pres-ervation in one place. The structure of chitin and chit-osan is shown in Figure 1.

To control the great economic losses from the spoil-age of foods each year, the attention of the people allover the world has been focused on preservation of thefood as a protection from various microorganisms. Inthis direction, bioactive polymeric films have been foundvery effective as antimicrobial agents and thus theirapplications and research work on them are gettingmore importance today. This review is an attempt tocreate awareness about the progress in the antimicrobialactivities of chitin and chitosan in the case of food appli-cations and the literature survey made from the year1957 to present. More specifically, the aim of thisreview was to highlight the potential of chitosan as an

ingredient for the production of active bio-based filmsand to summarize the different methods used for thepreparation of chitosan-based films and their perspec-tives in the modern food packaging technology.The active properties of edible films and coatings aredescribed in Table 1.

CHITIN, CHITOSAN AND THEIROLIGOSACCHARIDES VERSUS OTHERPOLYMERS IN ANTIMICROBIALACTIVITY

Polysaccharide films are derived from various naturalmaterials which impart gel-forming ability to a varietyof films. The polysaccharide-derived films, due to excel-lent gas permeability properties, exhibit desirablemodified atmospheres and thus enhance the shelf-lifeof the product without creating anaerobic conditions(Baldwin et al., 1995; Ben and Kurth, 1995; Cutter andSumner, 2002; Glicksman, 1983; Nisperos-Carriedo,1994; Whistler and Daniel, 1990). Furthermore, poly-saccharide films and coatings can be used to extend theshelf-life of muscle foods by preventing dehydration,oxidative rancidity and surface browning (Nisperos-Carriedo, 1994).

Particularly, the use of biopolymer and bio-basedpolymer films as antimicrobial delivery systems toreduce undesirable bacteria in foodstuffs is not a novelconcept. Various approaches have been proposed anddemonstrated for the use of these films to deliver com-pounds to a variety of food surfaces, including musclefoods. For example, antimicrobial compounds such asorganic acids (acetic propionic, benzoic, sorbic, lacticand lauric), potassium sorbate, bacteriocins (nisin andlacticin), grape seed extracts, spice extracts (thymol,p-cymene and cinnamaldehyde), thiosulfinates (allicin),enzymes (peroxidase and lysozyme), proteins (conalbu-min), isothiocyanates (allylisothiocyanate), antibiotics(imazalil), fungicides (benomyl), chelating agents

OH

CH2OH

OH

H

O

NHCOCH3 n

Conc. NaOH

Deacetylation

OH

CH2OH

OH

H

O

NH2 nChitin Chitosan

Figure 1. Structure of chitin and chitosan.

Table 1. Active properties of edible films and coatings

Different additives forimprovement of edible filmsand coatings

Improvement ofproperty

Pigments, light absorbers,shininess salts and other foodadditives like citric acid, oleicacid, etc., flavors, spices,antimicrobial and antioxidantagents

Color, transparency,roughness, sticking,microbe resistance, etc.

Food Science and Technology International 18(1)

4

by guest on December 26, 2013fst.sagepub.comDownloaded from

(EDTA), metals (silver) or parabens (heptylparaben)could be added to edible films to reduce bacteria in solu-tion, on culture media, or on a variety of muscle foods(Cha and Chinnan, 2004; Cutter, 2002a,b; Whistler andDaniel, 1990; Han, 2000). Further studies on edible filmmade from starch, carrageenan or alginate and additionof antifungal compounds, organic acids, potassium sor-bate or the bacteriocin nisin, were more effective forreducing levels of foodborne organisms when immobi-lized 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 andDickson, 1992, 1993). The use of biopolymers and bio-based polymer films on a variety of food surfaces and theresult-oriented observation of different workers aregiven in Table 2.

The above studies indicate that the application of bio-polymers, bio-based polymers, edible gels, films or coat-ings incorporated with food preservatives and/ornatural antimicrobial compounds has a significant rolein finding out the practical applications in the foodindustry. This specific information demonstrates the fea-sibility and applicability for incorporating various anti-microbial compounds with a range of inhibitory activitydirectly into bio-based or edible packaging materials foruse in controlling food spoilage as well as enhancingmicrobial safety of muscle foods. Table 3 focuses onthe study of chitosan films for packaging films for pro-cessed meats and seafood, as well as those combinedwith various ingredients.

Due to their inherent properties, coupled withthe ability to form films, alone or in combinationwith other polymers, chitin, chitosan and oligosac-charides are desirable food packaging materials.Chitooligomers, a class of chitosans with degree of poly-merization<20, are known to have some special biolog-ical activities such as antibacterial activities (Jeon et al.,2001), and antitumor and immune-enhancing effects(Jeon and Kim, 2002; Tian et al., 2010; Tokoro et al.,1988).

PREPARATION OF CHITOSAN-BASEDANTIMICROBIAL FILMS/COATINGS

Various methods are employed to prepare chitosan filmsand coatings for food packaging applications. Solutioncasting method is one of the popular methods. As a gen-eral practice, chitosan films are prepared with differentkinds of oils like cinnamon oil, tea tree essential oil(TTEO); tetrahydrocurcuminoid derivatives; blendswith ethylene–vinyl alcohol copolymer, PVP or PEO,Gliadins. The methods of preparation of novel hydro-xypropyl methylcellulose (HPMC) edible films withchitosan/tripolyphosphate nanoparticles; nanoparticles/plasticized-starch composites; edible chitosan films

enriched with galangal extract; sweet potato starchfilms incorporated with potassium sorbate onchitosan; and chitosan/methyl cellulose films have alsobeen reported for antibacterial study. Most recently,Tripathi et al. (2009, 2010) have synthesized chitosan-based antimicrobial films for food applications employ-ing the supercritical carbon dioxide and microwavetechnique. The novelty of this method lies in achievingthe film formation without the addition of any cross-linker or plasticizer.

Some typical preparative techniques are enumeratedin the following sections.

Preparation of chitosan-coating solutionenriched with cinnamon oil

Chitosan solution was prepared with 2% (w/v) chitosanin 1% (v/v) acetic acid. To achieve complete dispersionof chitosan, the solution was stirred at room tempera-ture for 3 h. The solution in beakers was placed on ahotplate/magnetic stirrer and glycerol added to chitosanat 0.75mL/g concentration as a plasticizer and stirredfor 10min. The resultant chitosan-coating solution wasfiltrated through a Whatman No. 3 filter paper toremove any undissolved particles. Then, the cinnamonoil, mixed with Tween 80, to help in distributing andcompletely incorporating the cinnamon oil, was addedto the chitosan solution. The final coating forming solu-tion consisted of 2% chitosan, 1% acetic acid, 0.75%glycerol, 0.2% Tween 80 and 1.5% cinnamon oil. Thefinal coating forming solution was homogenized underaseptic conditions at 21 600 rpm for 1min. The controlsolution was prepared without the addition of cinnamonoil (Ojagh et al., 2010).

Preparation of environment-friendly films basedon chitosan and tetrahydrocurcuminoidderivatives

Homogeneous chitosan films. A 2% (w/v) film-form-ing solution was obtained by dispersing chitosan in a 1%(w/w) acetic acid aqueous solution (pH 3.5). The solu-tion was filtered through a Buchner funnel and thendegassed under reduced pressure for 1 h. Films wereobtained by casting the solutions onto polystyreneplates which were dried for 12 h under a laminar-flowhood at room temperature. Thanks to degasification anddrying under laminar-flow, even at room temperature,most of the volatile acetic acid could be removed fromthe thin films (Portes et al., 2009).

Chitosan–tetrahydrocurcuminoid films. Film-formingsolutions (2%) were prepared by adding chitosan (2 g)and tetrahydrocurcuminoid (THC; 20mg) to 100mL ofa 1% acetic acid aqueous solution (pH 3.5) under

Dutta et al.

5

by guest on December 26, 2013fst.sagepub.comDownloaded from

Table 2. Use of biopolymer and bio-based polymer films in a variety of food surfaces and the result-oriented observationsof different researchers

Basic matrix Ingredients Activity and observation References

Carrageenan film Antibiotics and anti-fungal compounds

To reduce bacteria by 2 log 10 (99%) onpoultry

Meyer et al. (1959)

Edible coatings fromwaxes and celluloseethers

Antimycotic agents – Hotchkiss (1995)

Beef carcass tissue Organic acids,calcium alginate

More efficacious for reducing levels of L.monocytogenes, S. typhimurium, and E. coliO157:H7 when immobilized

Siragusa andDickson (1992,1993)

Edible cornstarchfilm

Potassium sorbateand lactic acid

To inhibit S. typhimurium and E. coli O157:H7on poultry

Baron and Sumner(1994)

Calcium alginategels on lean andadipose beefsurfaces

Bacteriocin nisincompared to nisin-only controls

Resulted in greater and sustained bacteriocinactivity when the tissues were ground andstored under refrigerated conditions for up to7 days

Cutter and Siragusa(1996, 1997)

Pork Calcium alginatecontaining nisin

Reductions in pathogen populations Fang and Lin (1994)

Edible heat-set andcast films made fromcorn zein or soyprotein

Nisin and lysozyme Exhibit activity against E. coli and L.plantarum

Dawson et al. (1996)and Padgett et al.(1998)

Corn zein films EDTA, lauric acid,nisin and combina-tions of the threecompounds

Significant reductions of Listeriamonocytogenes in solution

Hoffman et al. (2001)

Plastic-based pack-aging films treatedwith methylcellulose/HPMC-basedsolutions

Nisin surface of hotdogs

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

Franklin et al. (2004)

Gelatin-basedcoatings

Lysozyme, nisin andEDTA ham andsausage

To control spoilage and pathogenic organ-isms such as L. sakei, Leuconostoc mesen-teroides, L. monocytogenes and S.typhimurium

Gill (2000)

Ibrinogen/thrombin-based gel (Fibrimex)

Bacteriocins, nisinand bovine

Provide an added antimicrobial and advan-tage to restructured raw meat products thatincorporate surface tissues into the productinterior or as a delivery system for antimicro-bials to meat surfaces

Cutter and Siragusa(1997)

Collagen (Coffi) films(NICF) on hot dogsurfaces

Nisin Brochothrix thermosphacta and L. monocyto-genes were inhibited following treatmentswith subjected to temperature abuse as wellas long-term refrigerated storage

Cutter and Miller(2004)

Milk proteins foredible films andcoatings for foods,whey protein films

Essential oils oforegano, rosemaryand garlic

Antimicrobials were treated and evaluatedagainst E. coli O157:H7, S. aureus,Salmonella enteritidis, L. monocytogenes andL. plantarum

Seydim (2006)

Pullulan films as pro-duced by fungiduring fermentation.Pullulan films madefrom the exopoly-saccharides ofAureobasidiumpulluans

Lysozyme and diso-dium EDTA

Evaluated for antimicrobial effectivenessagainst E. coli and L. plantarum.The resulting films with a neutral pH werecomposed of glucans and polysaccharides,and were water soluble, transparent, andexhibited low oxygen permeability. Theseantimicrobial films were stable for up to21 days during cold storage and could inhibitthe E. coli under laboratory conditions

Kandemir et al.(2005)

6

by guest on December 26, 2013fst.sagepub.comDownloaded from

vigorous agitation. The solutions were filtered and castas in the procedure used to make homogeneous chitosanfilms. Ten thickness values were taken randomly at dif-ferent positions on each film. Film thicknesses werefound to be 30 � 4 l mm. A good repeatability of theabsorbances directly measured on different portions ofthe films showed that the THCs were distributedhomogeneously.

Antibacterial chitosan-based blends withethylene–vinyl alcohol (EVOH) copolymer

Chitosan polysaccharide dispersions were preparedin 2% (v/v) acetic acid to a final concentration of 3%(w/v) and stirred at 37 �C for approximately 3 h. Thechitosonium–acetate solutions were filtered throughpolyester cloth to remove residues of insoluble particlesand then autoclaved before their further use for blending.Pure chitosonium–acetate films were obtained by castingat 37 �C, 80 �C and 120 �C. In addition, pure EVOHfilmswere obtained by the same method at 80 �C. Compositefilms of EVOH29/LMW–chitosan with high ratios ofpolysaccharide (i.e., 50 and 80wt%) were obtainedunder various temperature conditions (37 �C, 80 �C and120 �C). Blends of EVOH with lower contents of chito-san did not form good films but 80/20 (wt%) EVOH/chitosonium–acetate blends could be finally obtainedby adding an excess of glacial acetic acid to the EVOHsolution to a final concentration of 2% (w/v).Nevertheless, the higher chitosan concentration blends,i.e., 50 and 80wt%, were also obtained using the same

solution of EVOH and acetic acid for comparison pur-poses. Typical pure and composite films with a thicknessof ca. 50 lm were obtained (Fernandez-Saiz et al., 2010).

Preparation of chitosan/PVPand chitosan/PEO blend films

Chitosan solution was prepared with 1% (w/w) chitosanin 1% (v/v) acetic acid, stirred overnight at room tem-perature and filtered through Miracloth� to removeimpurities. PVP and PEO were separately dissolved ind.i. water to form 1% (w/w) solutions. Aqueous solu-tions of the individual polymers were mixed to prepare aseries of chitosan/PVP and chitosan/PEO blend solu-tions with weight ratios 100/0, 75/25, 50/50, 25/75 and0/100. Aliquots of 10 g of film-forming solutions werepoured into 50mm-diameter polystyrene Petri dishesand the solvent was evaporated in a vacuum oven at38 �C under 17 kPa pressure for 24 h. The dried filmswere peeled from the Petri dishes and conditioned indesiccators at 25 �C and 20% relative humidity (RH)prior to testing (Li et al., 2010).

Preparation of novel HPMC edible films withchitosan/tripolyphosphate nanoparticles

Preparation of chitosan–tripolyphosphate nano-particles. The chitosan–tripolyphosphate (CS–TPP)nanoparticles were prepared on the ionic gelation ofCS with TPP anions. Chitosan was dissolved in aceticacid solution at concentrations of 3.00 and 4.41mg/mL.

Table 3. Chitosan for packaging films: activities and observations of various researchers

Chitosan films combined with vari-ous ingredients Activities and observations References

Processed meats and seafood, aswell as nisin and coated onto thesurfaces of paper

For inhibiting microorganisms Vartiainen et al. (2004)

Edible film made from 3% or 5%chitosan and yam starch

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

Evaluated an against S. enteritidis in suspen-sions. When applied directly to cell suspensions,1% chitosan reduced the pathogen> 4 log10CFU/mL (or 99.99%).Reduced populations of S. enteritidis>1 log10 CFU/mL (or 90%).Chitosan-treated films made with 5% chitosanwere the most efficient means of treatment forinhibiting S. enteritidis in solution

Durango et al. (2006)

Nisin into chitosan To inhibit L. monocytogenes. In solution and inagar diffusion assays, the antimicrobial filminhibited the pathogen, but no further studieswere conducted in meat systems

Cooksey (2005)

Chitosan coatings Reduced microbial contamination on shrimp andoysters, respectively

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

Dutta et al.

7

by guest on December 26, 2013fst.sagepub.comDownloaded from

The concentration of acetic acid in aqueous solutionwas, in all cases, 1.5 times that of chitosan. Under mag-netic stirring at room temperature, 28mL sodium TPPaqueous solution with concentrations 1.2 and 2.1mg/mL were added into 70mL chitosan solution. The prep-arations were mixed with a homogenizer at 6000 rpmwith continuous addition of TPP solution at therate of 1mL/min. The zone of opalescent suspensionwas further examined as nanoparticles (de Mouraet al., 2009).

Preparation of solutions for film casting. The HPMCsolution (control film) was obtained by dissolving 3.0 gof HPMC in 100mL of distilled water under magneticstirring for 12 h. The HPMC: water ratio in all film-forming solutions was 3:97 to study the effect of particlesize and CS–TPP concentration in the HPMC filmmatrix. The HPMC films with CS–TPP nanoparticleswere obtained by addition of 3.0 g of HPMC in100mL of nanoparticle solution (recently synthesized)under magnetic stirring for 12 h. After the solutions wereprepared, the flasks were kept closed for 6 h to preventmicrobubble formation in the films. The solutions werethen poured in a glass plate (30 � 30 cm2) covered withMylar (Polyester film) for film casting preparation. Thesolutions were cast at a wet thickness of 0.5mm ontoplates using casting bars and the plates placed on aleveled surface at room temperature and let to dry for24 h. After drying, the films were removed and condi-tioned in sealed plastic bags stored at room temperature.

Preparation of chitosan nanoparticles/plasticized-starch composites

Preparation of chitosan nanoparticles. Chitosan nano-particles (CN) were fabricated on the basis of ionotropicgelation between chitosan and sodium tripolyphosphatewith some modifications (Shu and Zhu, 2000; Tsai et al.,2008). Chitosan was dissolved in 2% (v/v) acetic acid toobtain a 1% (w/v) chitosan solution. Under vigorousstirring at room temperature, 8mL of 1% (w/v) TPPaqueous solutions was added dropwise, at the rate of1mL/min, into 200mL of chitosan solution. The mix-ture was stirred for 30min, and treated with sonicationfor 30min. The suspension was subsequently centri-fuged at 12,000 rpm for 20min. The precipitate waswashed with water and centrifuged again twice. The pre-cipitate was washed in ethanol and then dried (Changet al., 2010).

Processing of glycerol/potato starch/chitosancomposites. Chitosans were dispersed in a solution ofdistilled water (100mL) and glycerol (1.5 g) and ultra-sonicated for 0.5 h before adding 5 g of potato starch.The chitosan filler loading level (0, 1, 2, 4, 6 and 8wt%)

was based on the amount of potato starch. The mixturewas heated at 90 �C for 0.5 h with constant stirring inorder to plasticize the starch. To obtain the glycerol/potato starch (GPS)/chitosan composite films, the mix-ture was cast into a dish and placed in an air-circulatingoven at 50 �C until dry (about 6 h). The composite filmswere preconditioned in a climate chamber at 25 �C and50% RH for at least 48 h prior to the testing. Watercontent of the films was about 10wt%.

Preparation of chitosan–TTEO composite films

Preparation of the film-forming dispersions. Chitosan(1% w/w) was dispersed in an aqueous solution of gla-cial acetic acid (0.5% w/w) at 25 �C. After an overnightagitation, TTEO was added to the chitosan (CH) solu-tion to reach a final concentration of 0%, 0.5%, 1% and2% (w/w). CH–TTEO mixtures were emulsified atroom temperature using a rotor–stator homogenizer at13,500 rpm for 4min. These emulsions were vacuumdegasified at room temperature with a vacuum pump.The experimental design was made taking into accountthe maximum levels of TTEO which could be incorpo-rated into thematrix without oil phase separation duringthe film drying (Sanchez-Gonzalez et al., 2010).

Preparation of films. Films were obtained by the castingprocedure given as follows: film-forming dispersions(FFDs) were poured onto a framed and leveled polyte-trafluoroethylene (PTFE) plate (j¼ 15 cm) and driedunder atmospheric conditions for 48 h. Film thicknesswas controlled by pouring the amount of FFD that willprovide a surface density of solids in the dry films of 56 g/m2 in all formulations. Dry films were peeled off fromthe casting surface and preconditioned in desiccatorsat 20 �C.

Preparation of blend films of gliadins andchitosan

A total of 100 g of wheat gluten powder was suspendedin 350mL pure ethanol and 150mL distilled water wastransferred into the suspension under stirring. The mix-ture was magnetic stirred for 1 h and centrifuged at3000 rpm for 15min at 25 �C. The supernatant contain-ing the gliadin-rich fraction was collected as the film-forming solution. This method was different from thegeneral way that crude wheat gluten was dispersed in70% (v/v) aqueous ethanol and stirred for about 12 hto 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 in97 g water before 1 g acetic acid was added to themixtureunder stirring to give a CS content of 2.0wt%. By doing

Food Science and Technology International 18(1)

8

by guest on December 26, 2013fst.sagepub.comDownloaded from

this, clotting of CS in acetic acid aqueous solution couldbe avoided. Glycerol plasticizer was added into thesolution in a content of 20 g/100 g CS. The gliadinand CS solutions were mixed and stirred for 20min.The weight content of CS, wCS, defined as the weightratio of CS to total weight of CS plus gliadins, wasvaried from 0 to 100wt%. The final solution waspoured onto horizontal plastic dishes and dried at25 �C. The dried films were carefully peeled off fromthe dishes and preconditioned at 25 �C at 57.5% RHfor 3 days at least before testing.

Preparation of edible chitosan films enrichedwith galangal extract

Preparation of chitosan solution. In this method, 1.5%(w/v) chitosan solution was prepared by dissolving chit-osan in 1% (v/v) acetic acid under constant stirring at300 rpm using a magnetic stirrer at room temperaturefor 24 h. Then, 25% glycerol (w/w chitosan) wasadded into the chitosan solution; stirring was contin-ued at room temperature for 1 h. After mixing, thesolution was centrifuged for 15min at 12 400 rpm bya refrigerated centrifuge to remove undissolvedimpurities and bubbles in the solution (Mayachiewet al., 2010).

Preparation of galangal extract. Galangal powder (10 gdry basis), dried by a tray dryer at 40 �Cwith particle sizein the range 125–425mm, was extracted with 100mL of95% (v/v) ethanol (Oonmetta-aree et al., 2006). Theextract was filtered through a filter paper; the filtratewas collected and concentrated by a rotary evaporatorat 40 �C for 10min and kept at 4 �C in a dark bottle untilits use (Mayachiew and Devahastin, 2008a).

Preparation of antimicrobial chitosan films. Galangalextract was added to the chitosan solution at concentra-tions 0.3, 0.6 and 0.9 g/100 g. These concentrations wereselected based on a minimum inhibitory concentration(MIC) of the extract against Staphylococcus aureus(Mayachiew and Devahastin, 2008a). The final concen-trations of galangal extract in the films were 126, 252 and378mg/g film, respectively. The mixture was homoge-nized by a bench top homogenizer at 9500 rpm for2min. The film solution (21 g) was poured on an acrylicplate with dimensions 13 � 10 cm2 to cast an antimicro-bial film.Drying of the filmwas performed by fourmeth-ods, which are ambient air drying (30 �C), hot air dryingat 40 �C, vacuum drying and low-pressure superheatedsteam drying at 70 �C, 80 �C and 90 �C at 10 kPa, follow-ing the methods of Mayachiew and Devahastin (2008b).After drying, the films were conditioned for at least 48 hin desiccators at a RH of 53% containing saturated saltsolution of magnesium nitrate.

Preparation of sweet potato starch filmsincorporated with potassium sorbate orchitosan

In this method, 4 g of sweet potato starch was dispersedin 100mL H2O, moderately stirred for 20min at roomtemperature and then heated to 100 �C for over 30min.After gelatinization, glycerol was added as a plasticizerat a concentration of 3% (w/w, on dry basis of theweight of starch) and the resulting dispersion subjectedto further mixing for 5min. To prepare the antimicrobialfilm, potassium sorbate or chitosan was added at differ-ent concentrations (0, 5, 10, 15 g/100 g starch) at onetime during the mixing period. Before antimicrobialagents were added into starch paste, potassium sorbatewas dissolved in 20mLwater and chitosan was dissolvedin 20mL 1% acetic acid aqueous solution. For potas-sium sorbate-incorporated film, the pH of the gel-likemixture was adjusted to 4.5 by the addition of 2MHCl. When chitosan was incorporated into the film,the pH of the gel-like mixture was 4.0 because of theacetic acid used in preparing chitosan solution. Afterbeing degassed under vacuum, the warm mixture wascast on framed glass plates, and then dried at 50 �C for4 h. Starch film, without antimicrobial agents, was alsoprepared in the same way and used as a control (Shenet al., 2010).

Preparation of chitosan/methylcellulose films

Chitosan solution was prepared by dissolving 1.5 g ofchitosan in 100mL of 1% acetic acid solution. In thismethod, 1.5 g of methyl cellulose was dissolved in 50%ethanol. Also, 1 g of polyethylene glycol 400 was used asa plasticizer. Solutions of chitosan and methyl cellulosewere mixed in a beaker with a stir bar and heated to72 �C. Stearic acid, 0.075 g, was added to improve thewater barrier properties of the film. Vanillin, 0.9 g, wasincorporated after the temperature of the solutionreached its melting point (83 �C). The film-forming solu-tion was filtered through a cheese cloth to remove theundissolved parts, homogenized with a homogenizer,degassed, cast onto glass plates and dried at 40 �C for42 h. Dried films were peeled off and conditioned at 25�2 �C, 50 � 5% RH for at least 48 h prior to use(Sangsuwan et al., 2008).

ANTIMICROBIAL ACTIVITY

Functional efficiency strongly depends on the nature ofcomponents and film composition and structure. Thechoice of film-forming substance and/or active additiveis made based on the objective, the nature of the foodproduct and/or the application method.

Dutta et al.

9

by guest on December 26, 2013fst.sagepub.comDownloaded from

Chitosan has been studied in terms of bacteriostatic/bactericidal activity to control the growth of a wide vari-ety of bacteria. Chitosan has several advantages overother types of disinfectants because, according to Kimand Choi (1998), it possesses a higher antibacterial activ-ity and a broader spectrum of activity. The inhibitoryactivity of chitosan is given in Table 4 (Inhibitory activ-ity of chitosan). Chitosan hydroglutamate showed moreeffective antimicrobial properties with a decrease inS. aureus population of approximately 6 log cycleswithin 60 min of exposure for a chitosan concentrationof 0.2 mg/mL. It also reported that the application ofhigh hydrostatic pressure (2380 odm) to chitosan-trea-ted cultures of both bacterial strains resulted in addi-tional inactivation. Chung and Chen (2008) alsoinvestigated the antibacterial activity of low-molecularweight chitosan (Mw ¼ 30 kDa) by assessing the mor-tality rates of Escherichia coli and S. aureus and demon-strated that chitosan can destroy the cell structure ofboth bacterial cells, resulting in the leakage of enzymesand nucleotides from different cell locations. The MICvalues of chitosan for different food bacteria are listed:Bacillus cereus (1000 ppm), Erwinia sp. (500 ppm),Pseudomonas fluorescens (500 ppm), E. coli (20 ppm),Micrococcus luteus (20 ppm) and S. aureus (20 ppm).

As reported in the literature, chitosan seemed to bemore active against Gram-positive than against Gram-negative bacteria. Coma et al. (2003) assessed the poten-tial of chitosan coating especially active against thegrowth of two food pathogens, S. aureus, Listeria mono-cytogenes and one strain involved in food alteration,Pseudomonas aeruginosa. The study was conducted on

a model of agar medium and on a real cheese food prod-uct. Numeration on model agar medium showed a totalinhibition of the development of selected Gram-positivebacteria and 77% inhibition on Pseudomonas growth.This result poses the problem of a possible microbialselection related to the different sensitivities of microor-ganisms. Moreover, other parameters could have animpact on the bactericidal effect of chitosan. The ageof a bacterial culture affected its susceptibility to chito-san (Tsai and Su, 1999). As a result, E. coli cells in theexponential phase are most sensitive to chitosan. Theseauthors also showed that higher temperature (37 �C)increased the impact against the target strains.Another parameter identified as significantly influentialis the molecular weight. Jeon et al. (2001) showed thatchitooligosaccharide effectively blocked the growth ofthe tested bacteria although their effects were lowerthan that of chitosan. These authors mentioned thatfor an effective inhibition, the molecular weight shouldbe 10 kDa. The pH and the quaternization degree of thechitosan were the influential parameters. The majorityof studies showed that an increase of deacetylation (DA)degree and a decrease in pH improved the bioactiveproperties of chitosan. The flocculation and adsorptionbehavior of chitosans to E. coli cells were not observed;however, such behaviors were noticed by applying fluo-rescence labeled to chitosans and monitoring changes inzeta potential values of the bacterial with chitosan coat-ing. Without chitosan coating cells, it was increasedapproximately 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

Table 4. Inhibitory activities of chitosan

Types of study Activities and observations References

Surface spoilagebacteria

Ouattara et al. (2000a); Savardet al. (2002); No et al. (2002);Coma et al. (2003);Gerasimenko et al. (2004)

Various pathogenfood strains

Studied the bioactivity of chitosan in liquid medium andreported that 0.01% chitosan is sufficient to inhibit the growthof some spoilage bacteria such as B. subtilis, E. coli,Pseudomonas fragi and S. aureus in liquid mediumSignificant inactivation of the Gram-negative bacteriaE. coli and the Gram-positive enterotoxigenic S. aureus by twocommercially available water-soluble chitosan salts – chitosanlactate and chitosan hydroglutamate

Siragusa and Dickson (1992);Helander et al. (2001); Tsaiand Su (1999); Tsai et al.(2000); No et al. (2002); Comaet al. (2003); Darmadji andIzumimoto (1994)Papineau et al. (1991)

Potential bactericidein liquid medium,against some plantpathogenic bacteria

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 thecontrol. These authors observed an increase in the bacterialactivity by the addition of 0.5% NaCl

Li et al. (2006)

Food Science and Technology International 18(1)

10

by guest on December 26, 2013fst.sagepub.comDownloaded from

in higher amounts and reversed the cell surfacecharge most effectively. Finally, the chitosans withlow-molecular weight adsorbed the most. The sameauthors reported that ionic strength did not affect theadsorption of highly acetylated chitosan (DA 0.49),whereas for chitosan with a DA of 0.01, adsorptionincreased with ionic strength. The combination of floc-culation and adsorption data clearly showed that chargeneutralization was not themain flocculationmechanism.Several results pointed to bridging as one dominatingmechanism for flocculation. Study demonstrated thatin vitro antimicrobial efficiency is not always proven infood, due to the highly reactive nature of the polycatio-nic chitosan, which could interact with proteins, fats andother anionic substances in foods. Darmadji andIzumimoto (1994), however, observed that the growthof spoilage bacteria in meat was inhibited by 0.5–1.0%chitosan during incubation at 30 �C for 48 h or storage at4 �C for 10 days.

Studies based on UV absorption indicated that chit-osan causes considerable losses of proteinic material tothe Pythium oaroecandrum at pH 5.8 (Helander et al.,2001; Liu et al., 2004). Chitosan also acts as a chelatingagent that selectively binds trace metals and therebyinhibits the production of toxins and microbial growth(Cuero et al., 1991). It also activates several defense pro-cesses in the host tissue (El Ghaouth et al., 1992), acts asa water binding agent and inhibits various enzymes.Binding of chitosan with DNA and inhibition ofmRNA synthesis occurs through chitosan penetrationtoward the nuclei of the microorganisms and interfer-ence with the synthesis of mRNA and proteins(Sudarshan et al., 1992).

It has been proposed that when chitosan is liberatedfrom the cell wall of fungal pathogens by plant hosthydrolytic enzymes, it then penetrates to the nuclei offungi and interferes with RNA and protein synthesis(Hadwiger et al., 1985).

A microscopic examination of Saccharomyces uni-sporus after treatment with chitosan-salt with a poly-merization degree of 25 showed agglutination of arefractive substance on the entire cell wall (Savardet al., 2002). When chitosanase was added to the culturemedia containing chitosan-salt, they could not observerefractive substances. In this study, an interactionbetween chitosan and the cell wall was observed.

In vitro evaluation of antimicrobial activities ofcarboxymethyl chitosan (CM), quarternizedchitosan (Q) and quarternized carboxymethylchitosan

A series of quaternized carboxymethyl chitosan (CMQ),the sample number and the characterization were listedout in Table 5 (Sun et al., 2005). A Gram-positive

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 forthis experiment.

The agar plate method was used to determine theMICs of CM, Q and CMQ as follows: the sampleswere prepared at a concentration of 1% (w/v) and thenautoclaved at 121 �C for 25min. Duplicate twofold serialdilutions of each sample were added to nutrient broth(beef extract 5 g, peptone10 g to 1000mL distilled water,pH 7.0) for final concentrations 0.1%, 0.05%, 0.025%,0.0125%, 0.00625%, and 0.00313%. Some samples wereprepared and diluted by the same way except for finalconcentrations 0.00065% and 0.00033%. The culture ofeach bacterium was diluted by sterile distilled water to105–106CFU/mL. A loop of each suspension was inoc-ulated on the nutrient medium with sample or controladded. After inoculation, the plates were incubated at37 �C for 72 h, the colonies counted and the MIC valuesobtained. TheMIC was considered to be the lowest con-centration that completely inhibited against agar plateson comparison, disregarding a single colony or a fainthaze caused by the inoculum (Speciale et al., 2002).

Antimicrobial activity of CM and CMQ

The antimicrobial activities of CM and CMQ are shownin Tables 6 and 7. It was found that these samplesshowed effective antimicrobial activities against notonly E. coli but also S. aureus which were used in thetest, although differences existed among them.Generally, the samples had more effective inhibitionon S. aureus than E. coli. The fact may be attributed totheir different cell walls. In S. aureus, a typical Gram-positive bacterium, the cell wall is fully composed ofpeptide polyglycogen. The peptidoglycan layer is com-posed of networks with plenty of pores, which allowforeignmolecules to come into the cell without difficulty.But in E. coli, a typical Gram-negative bacterium, thecell wall is made up of a thin membrane of peptide poly-glycogen and an outer membrane is constituted of lipo-polysaccharide, lipoprotein and phospholipids. Because

Table 5. Sample numbers and characterization of differentCMQ

Sample no. DS of CM DS of Q Sample no. Mw (�105)

CM3Q1 0.73 0.78

CM3Q2 0.73 0.59 CM3Q2-1 4.72

CM3Q3 0.73 0.32 CM3Q2-2 2.28

CM1Q2 0.45 0.59 CM3Q2-3 0.45

CM2Q2 0.56 0.59 CM3Q2-4 0.11

CM4Q2 0.86 0.59

Dutta et al.

11

by guest on December 26, 2013fst.sagepub.comDownloaded from

of the bilayer structure, the outer membrane is a poten-tial barrier against foreign molecules.

Compared with CM, Q and CMQ in Table 7, CMQhad much better antimicrobial activities, whose MICvalues were 4–8 times lower than those of CM and 2–4 times lower than those of Q. It was noticed that theintroduction of carboxymethyl and quarternized groupsto the chitosan chain greatly enhanced the antimicrobialactivity of the CMQ.We can deduce that carboxymethyland quaternary ammonium groups are in synergisticeffect.

As given in Table 6, compared with CM1Q2, CM2Q2,CM3Q2 and CM4Q2, which have the same degree of sub-stitution of quaternized group, the authors found noclear effect of DS value of carboxymethyl group on anti-microbial activity. Compared with CM3Q1, CM3Q2 andCM3Q3, with similar degree of carboxymethyl groupsubstitution, it can be observed that their antimicrobialactivities were enhanced with increase of the DS.Compared with CM3Q2-1, CM3Q2-2, CM3Q2-3 andCM3Q2-4, which have same degrees of substitution forboth carboxymethyl and quaternized groups, the resultsdemonstrated that their antimicrobial activities wereaffected by their molecular weights remarkably. Lowermolecular weight resulted in better antimicrobial ability,and when the molecular weight was below 1 � 104, theantimicrobial activity of CMQ was strong and the MICvalues reached 0.00313%.

The development of complementary methods to inhi-bit the growth of pathogenic bacteria such as packaging

material-associated antimicrobial agents is an activearea of research. A number of studies on the antimicro-bial characteristics of films made from chitosan havebeen carried out earlier (Chen et al., 1996; Coma et al.,2002; Ouattara et al., 2000a). Among other polymers,chitosan has received a significant attention as an anti-microbial film-forming agent for food preservation tothe researchers due to its biodegradability, biocompati-bility, cytotoxicity and antimicrobial activity. Chitosanfilms are easily prepared by the evaporation of its diluteacid solutions (Park et al., 2002). Chitosan has beenstudied in terms of bacteriostatic/bactericidal activityto control the growth of a wide variety of bacteria.In the Gram-positive bacteria, the major constituent oftheir cell wall is peptidoglycan and a little amount ofprotein. The cell wall of Gram-negative bacteria on theother hand is thinner but more complex and containsvarious polysaccharides, proteins and lipids beside thepeptidoglycan. Also, the cell wall of Gram-negative bac-teria has an outer membrane which constitutes the outersurface of the wall (Black, 1996).

The antimicrobial effect of konjac glucomannanedible film was improved by incorporating chitosanand nisin (Li et al., 2006). In this study, antimicrobialefficacy was assessed against four food pathogenic bac-teria namely E. coli, S. aureus, L. monocytogenes and B.cereus. Antimicrobial activity tests of edible films werecarried out using the agar diffusion method.

In this method, the film cuts are placed on MuellerHinton agar plates, which were previously seeded with

Table 7. The antimicrobial activities of CMQ with different molecular structure factors

Samples DS of CMC DS of QC Mw (�105) Escherichia coli Staphylococcus aureus

CM1 Q2 0.45 0.59 4.51 0.00625 0.0125

CM2 Q2 0.56 0.59 4.64 0.00625 0.0125

CM3 Q2 0.73 0.59 4.72 0.00625 0.00625

CM4 Q2 0.86 0.59 4.66 0.00625 0.0125

CM3 Q1 0.73 0.78 4.21 0.0125 0.0125

CM3 Q3 0.73 0.32 4.83 <0.00625 0.00625

CM3 Q2-1 0.73 0.59 4.72 0.00625 0.0125

CM3 Q2-2 0.73 0.59 2.28 0.00625 0.00625

CM3 Q2-3 0.73 0.59 0.45 0.00313 0.00313

CM3 Q2-4 0.73 0.59 0.11 <0.00313 0.00313

Table 6. The antimicrobial activities of chitosan (CS), carboxymethyl chitosan (CM), quarternized chitosan (Q) and quar-ternized carboxymethyl chitosan (CMQ)

Samples DS of CM DS of Q Mw(�105) Escherichia coli Staphylococcus aureus

CM 0.46 – 4.30 0.05 0.1

Q – 0.60 3.89 0.0125 0.025

CMQ 0.45 0.59 4.51 0.00625 0.0125

Food Science and Technology International 18(1)

12

by guest on December 26, 2013fst.sagepub.comDownloaded from

0.1mL of inoculums containing indicator microorgan-isms in the range 105–106CFU/mL. The antimicrobialeffect of chitosan or incorporating nisin was found to bemuch better than that of konjac glucomannan incorpo-rating nisin at each corresponding concentration andthere existed significant difference (p< 0.05). However,there was no significant difference in the antimicrobialeffect between chitosan and chitosan incorporatingnisin. Incorporating chitosan into konjac glucomannanfilm therefore improved not only physical properties butalso antimicrobial activity.

The characteristics of chitosan film was evaluated bycross-linking with naturally occurring aglycone genipo-sidic acid (Mi et al., 2006). In this study, a comparativestudy was performed between chitosan film withoutcross-linking (fresh), the glutaraldehyde-cross-linkedchitosan film and aglycone geniposidic acid-cross-linked chitosan film. A 50 mL bacterial broth (E. coli orS. aureus) was seeded onto film and cultured. The freshand glutaraldehyde-cross-linked chitosan films wereused as control. It has been proposed that the interactionbetween the polycationic chitosan and the negativelycharged surface of bacteria may alter the permeabilityof the bacterial wall and lead to the leakage of intracel-lular electrolytes and proteins. The results suggested thatcross-linking of chitosan films did not alter their anti-bacterial capability. This may be due to the fact that thecross-linking degrees of glutaraldehyde and aglyconegeniposidic acid (aGSA) cross-linked chitosan filmsused in this part of the study were relatively low(<18%, with a concentration of cross-linking agent0.8mM). The aGSA-cross-linked chitosan film dis-played a relatively lower water vapor permeability, alower cytotoxicity, and a slower degradation rate thanthe glutaraldehyde-cross-linked film. It was finally con-cluded that the aGSA-cross-linked chitosan film may bea promising material as an edible film for foodpackaging.

The shelf-life of food was extended by ferulic acid-incorporated starch-chitosan blend films (Mathew andAbraham, 2008). Incorporation of ferulic acid has beenfound to improve the barrier properties and tensilestrength of starch-chitosan blend films and signifi-cantly enhance the lipid peroxide inhibition capacity.This study has helped to improve the performance ofpolysaccharide-based films for the storage of highlipid-containing ingredients.

The antimicrobial activity of chitosan films enhancedby incorporation of garlic oil, potassium sorbate andnisin (Pranoto et al., 2005). The antimicrobial activitywas tested against food pathogenic bacteria namelyE. coli, S. aureus, Salmonella typhimurium, L. monocyto-genes and B. cereus. Antimicrobial tests have beencarried out using agar diffusion method. The agardiffusion test is a method commonly used to examine

antimicrobial activity regarding the diffusion of thecompound tested through water-containing agarplate. Incorporating antimicrobial agents into chitosanedible films thus improves the antimicrobial efficacy ofchitosan, as diffused antimicrobial activity would add tothe nonmigrated antimicrobial potency of chitosan. Itwas concluded that garlic oil incorporated into chitosanfilm led to an increase in its antimicrobial efficacy, andhas little effect on the mechanical and physical proper-ties of chitosan films. Overall, the incorporation of garlicoil into chitosan film had the desirable characteristics ofacting as a physical and antimicrobial barrier to foodcontamination.

Two types of O-carboxymethylated chitosan (O-CMCh)/cellulose polyblends were prepared using LiCl/N, N-dimethylacetamide solution (Li et al., 2002).Antimicrobial activity of the blend films against E. coliwas evaluated by using the optical density (OD)method.The smaller the OD of the medium, the higher was theantimicrobial activity of the film. It was observed thatthe antimicrobial activity of the blend films enhances ifthe O-CMCh contamination was raised. Both blendfilms exhibited satisfactory antibacterial activitiesagainst E. coli, even with O-CMCh concentration2wt%.

Antibacterial assessment of chitosan-starch

Inhibitory effects of starch solution and chitosan-starchfilm against E. coli, S. aureus and Bacillus subtilis areshown in Figures 2(a)–(c), and 3(a)–(c). The inhibitoryeffect was measured based on clear zone surroundingcircular film strips/solution. Measurement of clearzone diameter included diameter of film strips/solutions.Therefore, the values were always higher than the diam-eter of film strips/solutions whenever clearing zone waspresent. If there is no clear zone surrounding, weassumed that there is no inhibitory zone, and further-more, 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 film did not show inhibitory effects againstall the tested microorganisms. The chitosan-starch film-forming 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 athigher concentration did not reveal a significantincreased inhibitory effect. It was generally caused bythe maximum capability of chitosan polymer to carryactive agents beside the occurrence of interaction phe-nomenon between the functional groups. The antimicro-bial effect of chitosan occurred without migration ofactive agents. As chitosan is in solid form, only organ-isms in direct contact with the active sites of chitosan are

Dutta et al.

13

by guest on December 26, 2013fst.sagepub.comDownloaded from

inhibited. Chitosan is incapable of diffusing through theadjacent agar media. The agar diffusion test is a methodcommonly used to examine the antimicrobial activityregarding the diffusion of the compound tested throughwater-containing agar plate. The diffusion itself isdependent on the size, shape and polarity of the diffusionmaterial. The chemical structure and the cross-linkinglevel of the films also affect this phenomenon. The

chitosan–starch solution shows stronger inhibitoryeffect against E. coli and B. subtilis than S. aureus.Furthermore, it was found that the bioactive chitosan–potato starch film can be used to extend the food shelf-life.

Incorporating chitosan and lauric acid into starch-based film showed more effective antimicrobial abilityagainst B. subtilis and E. coli (Salleh et al., 2007). In thisstudy, the authors studied the incorporation of chitosanand lauric acid into starch-based films; obvious effectstoward inhibition of B. subtilis and E. coli have beenobserved while the film had synergistic antimicrobialeffect when chitosan and lauric acid were combined.Antimicrobial starch-based film incorporated withlauric acid and chitosan showed good flexibility thanwhen purely starch-based film was formulated andformed (Figure 4). Inhibition of bacterial growth wasexamined using two methods, i.e., zone of inhibitiontest on solid media and liquid culture test (OD

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).

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).

Table 8. Diameters of inhibitory zone of the solution andfilm against E. coli, S. aureus and B. subtilis

Test culture

Diameter (cm) of inhibitory zone

In the solution In the film

Escherichia coli 0 1.5

Staphylococcus aureus 0 1.2

Bacillus subtilis 0 1.4

Food Science and Technology International 18(1)

14

by guest on December 26, 2013fst.sagepub.comDownloaded from

measurements). The inhibitory activity was measuredbased on the diameter of the clear inhibition zone. Inthe solutions of starch and chitosan with differentmixing ratios (w/w), 8:2 and 9:1 were the most effectivemixing ratios which had greater inhibition on both B.subtilis and E. coli than other solutions in agar plate andliquid culture test. The control (pure wheat starch) andantimicrobial (AM) film (incorporated with chitosanand lauric acid) were produced by casting method.

The antimicrobial effectiveness of control (purewheat starch) and AM film incorporated with chitosanand lauric acid are shown in Figure 5(a) and (b). A wideclear zone on solid media was observed for B. substilisgrowth inhibition whereas inhibition for E. coli was notas effective as B. substilis. From the liquid culture test,

the AM films clearly demonstrated a better inhibitionagainst B. substilis than E. coli.

The tensile properties of the antimicrobial starch-based film has been improved by the addition of chito-san. These antimicrobial starch-based films can be usedto extend food shelf-life.

Antimicrobial Activity of chitosan–polyvinylalcohol film

Inhibitory effects of chitosan–polyvinyl alcohol (PVA)solution and chitosan–PVA film against E. coli, S.aureus and B. subtilis were studied by Tripathi et al.(2009). The inhibitory effect was measured based onclear zone surrounding circular film strips/solution.Measurement of clear zone diameter includedmeasuringthe diameter of film strips/solutions; therefore, thevalues were always higher than the diameters of filmstrips/solutions whenever clearing zone was present. Ifthere is no clear zone surrounding, the authors assumedthat there is no inhibitory zone, and furthermore, thediameter was valued as zero. In terms of surroundingclearing zone, chitosan–PVA film did not show inhibi-tory effects against all tested microorganisms. The chit-osan–PVA film-forming solution showed a clearinhibitory zone against E. coli, S. aureus and B. subtilis,respectively. The chitosan–PVA solution shows strongerinhibitory effects against E. coli and B. subtilis than S.aureus. Furthermore, it was found that the bioactivechitosan–PVA film can be used to extend food shelf-life. Chitosan-based antimicrobial films consisting ofchitosan and PVA were prepared by solution castingmethod. These results pointed out that there is amolecular miscibility between PVA and chitosan.

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).

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

Dutta et al.

15

by guest on December 26, 2013fst.sagepub.comDownloaded from

Chitosan-based antimicrobial film may be a promisingmaterial as a packaging film.

Preservation of vacuum-packaged processedmeats

The feasibility of improving the preservation of vacuum-packaged processed meats during refrigerated storageachieved by the use of an antimicrobial film designedto gradually release antimicrobial agents at the productsurface (Ouattara et al., 2000b). The antimicrobial filmswere applied onto bologna, regular cooked ham or pas-trami. The activity of the various films for inhibitingbacterial growth was tested against indigenous lacticacid bacteria and Enterobacteriaceae and againstLactobacillus sakei or Serratia liquefaciens, surface-inoculated onto the meat products. The growth ofEnterobacteriaceae and S. liquefaciens was delayed bythe application of the antimicrobial film. It was foundthat the inhibition of indigenous Enterobacteriaceae wasmore extensive at the surface of bologna than at thesurface of pastrami, irrespective of film type. It is dueto the fact that bologna contains efficient water-bindingagents, and so exudes little water during storage.

The moisture and high lipid contents of bolognahelped the diffusion of the oregano essential oil (EO)from the chitosan film matrix into the product (Chiet al., 2006). Sensory evaluation suggested that additionof 45 ppm or less of oregano oil to bologna would beacceptable to consumers. In conclusion, the gas chroma-tography mass spectroscopy analysis showed that757.7 � 99.7 ppm carvacrol was extracted from thefilm-forming solution prepared without Tween� 20and only 364.7 � 39.9 ppm from the film-forming solu-tion with the emulsifier. Different levels of carvacrolwere detected in the presence of Tween� 20 due to theinteraction of the amphiphilic emulsifier’s molecule withboth chitosan and oregano EO compounds. It is con-cluded that incorporation of an emulsifier in chitosan–oregano EO films may slow the losses of volatilecompounds of the oil and help to control the release ofactive compounds into the product.

The antimicrobial properties of crawfishchitosan

Antimicrobial activities of crawfish chitosan film formu-lations against seven pathogenic bacteria, L. monocyto-genes, B. cereus, Shigella sonnei, E. coli (O157:H7), S.aureus, S. typhimurium and Vibrio vulnificus, wereexpressed in terms of zone inhibition. The zone inhibi-tion assay revealed primarily three types of observa-tions, namely, (1) defaced films without any clear orinhibition zones which could be attributed to theabsence of any inhibitory activity, (2) clear zones

without inhibitory zones which could be attributed tobacteriostatic activity and (3) clear inhibition zone rep-resenting bactericidal inhibition by films. Some of thetests are enumerated as follows (Nadarajah, 2005).

Minimum inhibitory activity. All chitosan acetate filmswere defaced with L. monocytogenes and this was wellin agreement with the report of Coma et al. (2002).However, it was reported that 8% film-forming solu-tion (chitosan in acetic acid) incorporated in agarmedium (v/v) completely inhibited L. monocytogenes.Chitosan acetate films were also defaced with B.cereus, and V. vulnificus lawns, indicating that theywere ineffective in controlling these bacteria. However,all chitosan acetate films showed bacteriostatic effectsagainst S. aureus, S. sonnei, S. typhimurium and E. coliO157:H7, as indicated by their clear zones i lawns.

The chitosan formate films were also ineffective incontrolling B. cereus and V. vulnificus, as indicated bydefaced films by these bacterial lawns. Nevertheless, thechitosan formate films showed inhibitory effects againstS. aureus, S. sonnei, S. typhimurium and E. coliO157:H7and bacteriostatic activity against L. monocytogenes.

However, compared to the chitosan citrate films, theinhibitory effects of chitosan formate films were lowerand the thickness of the inhibitory zone was in therange 0.49–1.64mm compared to 0.78–6.0mm of chit-osan citrate films. All chitosan citrate films exhibitedprominent inhibitory effects on all seven pathogenicbacteria. All chitosan citrate films showed distinctiveinhibition zones against all tested pathogenic bacteriaand the inhibition zones were considerably thicker thanthose produced by chitosan formate films. Also, theinhibitory effects of chitosan citrate films were remark-ably higher for S. aureus and V. vulnificus, as indicatedby thicker inhibition zones accounting for more than4 mm. The chitosan citrate films were the only filmswith antimicrobial effects against B. cereus and V. vul-nificus. The higher inhibitory activity shown by all chit-osan citrate films can be attributable to completesolubility of chitosan which could make them morereactive against bacterial cells.

Antimicrobial activity. The quantitative analysis of anti-microbial activity for selected chitosan films were carriedout as follows.

Acetate films with demineralized þ decolorized þdeacetylated (DMCA) chitosan, formate films withdeproteinized þ demineralized þ decolorized þ deace-tylated (DPMCA) chitosan and citrate films with demi-neralized þ deacetylated (DMA) chitosan.

The above-selected films were also tested with furtherinhibition zone assays with more controls. The nisinspots used as the positive control produced more prom-inent clear zones with L. monocytogenes, and S. aureus

Food Science and Technology International 18(1)

16

by guest on December 26, 2013fst.sagepub.comDownloaded from

lawns representing the Gram-positive bacteria andvague spots with S. sonnei and S. typhimurium lawnsrepresenting the Gram-negative bacteria. Regardless ofthe type of bacteria, controls such as chitosan solutions,acid solutions and the polyvinyl chloride plastic failed toproduce any clear or inhibition zones indicating thatthey were ineffective in inhibiting the above-mentionedfood pathogenic bacteria. This substantiates the claimthat the direct application of antimicrobial agents, suchas chitosan and acid solutions used in our studies, ontofood surfaces is less effective due to loss of antimicrobialactivity caused by leaching onto the food, enzymaticactivity and reaction with other food components(Jung et al., 1992; Ouattara et al., 2000a; Ray, 1992).Hence, the use of packaging films or coating as amatrix to deliver antimicrobial agents becomes impor-tant. Such packaging or coating can maintain a highconcentration of antimicrobial agents on a food surfaceand 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 agree-ment with the inhibition zone assays (the survivor lognumber CFU/mL of L. monocytogenes inoculated ontothe surface of the selected chitosan films).

Chitosan citrate film: L. monocytogenes was moresusceptible to chitosan formate or chitosan acetatefilms. 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 overthe entire 24 h period incubation.

Further, the chitosan formate films caused about1 log CFU/mL reduction of inoculum at 2 h of incuba-tion and maintained a 1 log CFU/mL reduction over24 h of incubation. The rate of reduction of microbialcount was poor with both chitosan acetate and formatefilms as there was no significant difference in microbialcount between 2 and 4 h of incubation and between 4and 8 h of incubation. Organic acids with smaller molec-ular weights have higher antimicrobial activities andundissociated smaller molecules of formic (46.03Da)and acetic (60.05Da) acids may enter the bacterial cellseasily to change the internal pH of the organisms(Eswaranandam et al., 2004). Undissociated larger mol-ecules of citric acid (192.13Da) may not enter into thecells effectively. Such a trend was not observed in thestudy (Nadarajah, 2005) and the result was in contrary.

It indicates that chitosan films made of organic acidsmay behave as one entity rather than separate entities,i.e., as a career matrix containing an antimicrobialagent. Several studies have demonstrated that antimi-crobial edible films can reduce bacterial levels on meatproducts (Table 9).

In most of these studies, antibacterial activity againstL. monocytogenes was attempted with added antimicro-bials. Some of the major findings of the work for craw-fish chitosan are as follows.

The chitosan citrate film producing more than a4.4 log reduction in L. monocytogenes was a commend-able achievement. As with the case L. monocytogenes,the chitosan citrate films showed higher antibacterialactivity against S. aureus.The chitosan citrate films pro-duced more than a 5 log reduction in S. aureuswithin 4 hof incubation and maintained its inhibitory effectthrough out the incubation period. The chitosan acetatefilms produced a poor inhibition with less than 1 logreduction at 24 h. The chitosan formate filmsmaintainedabout 1 log reduction for up to 4 h. At 24 h incubation,chitosan formate films produced more than a 5 logreduction similarly observed for chitosan citrate films.

Relatively very little research work has been dedi-cated to formulate edible films active against S. aureus(Table 10).

All these studies indicate the importance ofhaving added antimicrobials in the films to controlS. aureus.However, the crawfish chitosan citrate andformate films which contained no added antimicrobialscould produce more than a 5 log reduction. Further, theinhibitory effects of chitosan citrate and formatefilms against S. aureus were higher than that against L.monocytogenes. Along with L. monocytogenes, S. typhi-murium has been considered as a microbiological hurdle

Table 9. Studies on antimicrobial edible films and bacteriallevels on meat products

Types of study, activityand observation References

Organic acids more effective againstL. monocytogenes on beef carcasstissue when immobilized in calciumalginate than when used as a sprayor dip

Siragusa andDickson (1992,1993)

Zein films, impregnated with nisin,lauric acid and EDTA and tested withbroth cultures of L. monocytogenes,reduced the bacterial counts over5 logs after 48 h

Hoffman et al. (2001)

Zein films containing nisin produceda 4.5–5 log reduction on L. monocy-togenes inoculated onto chickenbreast tenders CFU/mL withoutrefrigeration for 16 days

Janes et al. (2002)

Impregnation the surface of meatcasing with pediocin powder toproduce a 1–3 log reduction ofL. monocytogenes on ham, turkeybreast and beef compared to inoc-ulated controls

Ming et al. (1997)

Dutta et al.

17

by guest on December 26, 2013fst.sagepub.comDownloaded from

for a long time. As with L. monocytogenes and S. aureus,a similar trend of inhibition was observed with S. typhi-murium. The chitosan citrate films produced more than3.4 log reduction in S. typhimurium within 2 h of incuba-tion, and reduction in counts reached 3.85 log at 4 h and4.83 log at 8 h incubation. The chitosan acetate filmswere less effective with about 1 log reduction at 24 h.

There was no significant (p> 0.05) change in the S.typhimurium count from 2h to 24 h for chitosan acetatefilms. The chitosan formate films maintained about2.7 log reduction up to 8 h and then produced a signifi-cant (p< 0.05) increased inhibition (3.7 log) between 8and 24 h of incubation.

The effects of edible films on S. typhimurium havebeen given in Table 11.

Compared to these published data, reduction of S.typhimurium in this study by more than 4.7 log by chit-osan citrate film and 3.7 log by chitosan formate film isoutstanding.

As with L. monocytogenes, and S. aureus, chitosanacetate films produced minimal inhibition against S.sonnei. The chitosan formate films accounted for about1 log reduction at 4 h of incubation and 2.6 log reductionat 24 h. The citrate films showed the highest antibacterialactivity against S. sonnei with more than 5 log reductionat 8 h of incubation.

Major finding of the overall work

This study confirms that crawfish chitosan can be usedto develop antimicrobial edible films effective againstboth Gram-positive and Gram-negative food patho-genic bacteria. Chitosan acetate films showed poorinhibitory effects against L. monocytogenes, S. aureus,S. typhimurium and S. sonnei.Although chitosan acetatefilms outweighed other films in terms of their mechanicalproperties, they demonstrated minimal antibacterialeffects similar to bacteriostatic effects with negligiblebacterial reduction over a period of 24 h. Chitosan for-mate films were effective against S. aureus, S. typhimur-ium and S. sonnei, causing more than 5, 3.7 and 2.5 logreductions at 24 h incubation, respectively. Chitosanformate films produced poor inhibitory effect againstL. monocytogenes with less than 1 log reduction at 24 hincubation. Based on antibacterial and packaging prop-erties, chitosan formate films can be used as antibacterialpackaging to control S. aureus, S. typhimurium and S.sonnei, except L. monocytogenes. Chitosan citrate filmswere highly effective against L. monocytogenes, S.aureus, S. typhimurium, and S. sonnei. The effect of chit-osan citrate films against L. monocytogenes and S.aureus was prominent with more than 5 log reductionwithin 4 h of incubation. Furthermore, chitosan citratefilms indicated their potential antibacterial effectsagainst B. cereus and V. vulnificus as indicated by thezone inhibition tests. This study indicates the possibilityof formulating an antibacterial edible film, especiallycrawfish chitosan citrate film, active against a broadspectrum of bacteria (Nadarajah, 2005).

Bacterial growth susceptibility

Bacterial growth susceptibility was determined by theMIC method. Drops of chitin derivatives of differentconcentrations were applied to the surface of agaroseplates containing cultures of bacteria in nutrientdextrose medium or LB medium for phytopathogenicbacteria and E. coli, respectively. MIC was defined asthe lowest concentration of chitin derivatives thatinhibited bacterial growth after overnight

Table 10. Report to formulate edible films that are activeagainst S. aureus

Types of study, activityand observation References

Polyethelene film containinggrapefruit seed extract showed aninhibitory effect against S. aureus asindicated by a 2.5–7.0 mm inhibitionzone by the agar diffusion method

Ha et al. (2001)

A 1.5 log and 2.8 log reduction ofS. aureus in cheese and ham bynisin-absorbed bioactive inserts

Scanell et al.(2000)

An edible packaging made ofcellulosic esters, fatty acids andnisin produced up to 88 mmdiameter inhibition zone onS. aureus.Further, they reported thataddition of fatty acid reduced theinhibitory activity

Coma et al.(2001)

Table 11. Report to formulate edible films on S.typhimurium

Types of study, activityand observation References

A 4.3 log reduction ofS. typhimurium on inoculated broilerskin exposed to nisin-coatedpolyvinyl chloride film

Natrajan andSheldon (2000)

A 4.23 log reduction ofS. typhimurium in pads treatedwith nisin formulations

Sheldon et al.(1996)

Further, applied nisin formulationsto S. typhimurium inoculated on traypads and demonstrated 3.1 logreduction

Sheldon (2001)

Food Science and Technology International 18(1)

18

by guest on December 26, 2013fst.sagepub.comDownloaded from

incubation of the agarose plates at 37 �C (Struszczyk andPospieszny, 1997).

In another experiment, the effect of chitin derivativeson Pseudomonas syringae pv. phaseolicola was testedusing the hypersensitive reaction (HR) of tobacco.Mixtures of bacterium and chitin derivatives at finalconcentration 5 � 107 CFU/mL and 0.05wt%, respec-tively, were injected into leaves of tobacco Xanthi nc.Suspensions of the bacterium in distilled water or solu-tions of chitin derivatives in distilled water were used ascontrols.

Water-soluble chitin oligomers, chitosan, chitosansulfates and carboxymethyl chitosan were used in thisresearch. Chitosan was dissolved in the acetic acid andother chitin derivatives in distilled water. The reactionsof all the solutions were adjusted to pH ¼ 5.5–6.0 withpotassium hydroxide. Cationic chitin derivatives, i.e.,chitin oligomers and chitosan, inhibited the growth ofthe Gram-positive bacteria: Corynebacterium michiga-nense subsp. michiganense and C. michiganense subsp.insidiosum, and Gram-negative bacteria: Xanthomonascampestris pv. Phaseoli, P. syringae pv. Phaseolicola,P. syringae pv. tomato, Erwinia amylovora, Erwinia car-otovora subsp. carotovora and Agrobacterium tumefa-ciens at concentrations of the range 0.01–0.3wt%.However, both derivatives were less effective against E.coli. Anionic chitin derivatives, i.e., chitosan sulfate andcarboxymethyl chitosan at a concentration of 1.5wt%were not effective against any of the bacterial tested.When cationic derivatives were added to the bacteriasuspension, flocculation was observed. The HR ofplants is widely used for quick demonstration of bacte-rial pathogenicity (Klement, 1963). When the tobaccoleaves were injected by a mixture of P. syringae pv.Phaseolicola and chitin derivatives, HR was prevented.

Chemical depolymerization

Chitosan oligosccahrides have received attentionbecause of their versatile biological properties. Thosehave lower viscosities, and low molecular weights andare soluble in aqueous solution. They seem to be readilyadsorbed in vivo (Chatterjee et al., 2005).

Chemical treatment of chitosan using strong acids,e.g. HNO2 and HCl is a very common and fast methodto produce a series of chitooligomers. However, thismethod has some disadvantages such as high cost andthe low yield of chitosan oligosaccharides with degree ofdepolymerization (DP) from DP2 to DP5 because ofrandom cleavage resulting in mostly monosaccharides.The irradiation effects on chitosan in acetic acid solutionwith various dose rates and the yield of chitosan oligo-mers were investigated (Choi et al., 2002). Low molecu-lar weight chitosans were prepared at different reactiontemperatures and times using 85% phosphoric acid that

resulted in the decrease of viscosity average molecularweight from 21.4� 104 to 7.1� 104. Depolymerizationof chitosan by the use of HNO2 is a homogeneous reac-tion where the number of glycosidic bonds broken isstochiomeric to the amount of HNO2 used (Jia andShen, 2002). The hydrolysis of chitosan with strongHCl was studied over a range of acid concentrationsand temperatures. There have been very few reports onthe degradation of chitosan by free redicals. Nordveidtet al. (1994) demonstrated that the viscosity of chitosansolution decreased rapidly in the presence of H2O2 andFeCl3 probably due to random depolymerization ofchitosan (Chen et al., 1997).

Several biological activities of chitosan depend on thedegree of polymerization. According to Liu et al.(2006),the main factors affecting the antibacterial activity ofchitosan are molecular weight and concentration.Recent studies on chitosan have attracted interest forconverting it into oligosaccharides because they arenot only water-soluble but also they are believed tohave greater antimicrobial activity. Chitosan has amean molecular mass of up to 1MDa, which corre-sponds to a chain length of approximately 5000 units,but there is considerable variation between commercialbatches. The molecular mass of native chitin is usuallyhigher than 106 Da, whereas the molecular mass of thecommercial chitosan is often observed between 105 and12� 105 Da (Muzzarelli, 1973). During the process ofdeacetylation, the hard conditions tend to degrade anddepolymerize chitosan (No et al., 2002). Medium- andlow-Mw chitosan can be obtained by chemical or enzy-matic hydrolysis of the high-Mw polymer. The chemicalhydrolysis is usually achieved using strong acids, whichis an unexpensive and rapid method. Its drawback is thenecessity to purify extensively the low-Mw chitosanproducts for biological applications due to the toxicityof the reagents used for the reaction. Hydrogen peroxidetreatment (No et al., 2002) and ultrasonication(Czechowska-Biskup et al., 2005) could be also used.The extent of hydrolysis is, however, rather difficult tocontrol (Plouffe et al, 1997).

Enzymatic depolymerization

Enzymatic depolymerization seems to be a bettermethod to prepare chito oligosaccharides.Microorganisms have been found to possess chitosanaseactivity. Among bacteria, Bacillus and Streptomycesstrains are most often studied. Studies on fungal chito-sanase are less reported (Cheng and Li, 2000).

The growing consumer demand for foods withoutchemical preservatives has led people to indulge ineffortstoward the discovery of new natural antimicro-bials (No et al., 2002). In this context, the antimicrobialactivities of chitosan and their derivatives against

Dutta et al.

19

by guest on December 26, 2013fst.sagepub.comDownloaded from

different groups of microorganisms, such as bacteria,yeast and fungi have received considerable attention inrecent years. Antibacterial activities of six chitosans andsix chitosan oligomers with different molecular weightswere examined against four Gram-negative (E. coli, P.fluorescens, S. typhimurium and Vibrio parahaemolyti-cus) and seven Gram-positive bacteria (L. monocyto-genes, Bacillus mageterium, B. cereus, S. aureus,Lactobacillus plantarum, Lactobacillus brevis andLactobacillus bulgaricus). Chitosans showed higher anti-bacterial activities than chitosan oligomers and mark-edly inhibited growth of most bacteria tested althoughinhibitory effects differed with Mws of chitosan and theparticular bacterium. Chitosan generally showed stron-ger bactericidal effects with Gram-positive bacteria thanGram-negative one in the presence of 0.1% chitosan(Wang, 1992). The MIC of chitosans ranged from0.05% to >0.1% depending on the bacteria and Mwsof chitosan.

A solution can be obtained by the use of enzymes toproduce bioconversions. The chitooligosaccharides pro-duced by the enzymatic hydrolysis of chitosan are widelyused in the food, agricultural and pharmaceuticalfields because of their various physiological activities.Chitinase (EC 3.2.1.14) is an important chitin-degradingenzyme which is involved in bioconversion processes ofwastes from crustaceans and in plant protection by pre-serving them from chitin-containing pathogens such asfungi (Decleire et al., 1997). Chitosanase (EC 3.2.1.132)is defined as an enzyme that catalyses random hydro-lysis of ß-1, 4 linkages between GlchitosanAc andGlchitosan residues in a partially N-acetylated chitosan.Chitosanase was distinguished from chitinase on thebasis of its ability to hydrolyze Glchitosan–Glchitosan.As specified by Seki et al. (1997), chitosanaseswere subdivided into three subclasses, characterizedby the ability to split Glchitosan–Glchitosan andGlchitosanAc–Glchitosan linkages (subclass I), onlyGlchitosan–Glchitosan linkages (subclass II) andGlchitosan–Glchitosan and Glchitosanc–GlchitosanAclinkages (subclass III). Chitosanase can be definedas the enzyme which requires at least one Glchitosanresidue at either side of hydrolyzing linkages inpartially N-acetylated chitosan but not GlchitosanAc–GlchitosanAc bonds. To date, many chitosanases havebeen found in a variety of microorganisms, includingparticularly bacteria (Kurakake et al., 2000; Pelletierand 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 enzy-matic cleavage of glycosidic linkage in chitosan(Muzzarelli et al., 1995; Pantaleone et al., 1992;Yalpani and Pantaleone, 1994).

At first, various investigators reported molecularweight relationships of antibacterial activity by chitosanand there are some reports that chitosan is more effectivein inhibiting the growth of bacteria than chitosan oligo-mers. Hirano and Nagao (1989) studied the relationshipbetween the degree of polymerization of chitosan andthe inhibition grade against 18 phytopathogens. Thetest materials were the lactate salt of high-molecularweight chitosan (Mw 400 kDa, with 95% of deacetyla-tion) and chitosan oligosaccharides, with a degree ofpolymerization (DP) in the range 2–8. The growth of13 fungi was inhibited more than 10% by the high-molecular weight chitosan and 6% by chitosan oligosac-charides. These authors observed that a decrease in thedegree of polymerization of chitosan resulted indecreases in the number of inhibited fungal species.Previously, Kendra and Hadwiger (1984) demonstratedthat the maximal antifungal activity of chitosan wasexhibited by chitosan oligomers of seven or more resi-dues. In contrast to these results, Avadi et al.(2004) men-tioned that chitosan oligomers with a DP 30 possessantimicrobial activity against a number of bacteria,whereas low-DP oligomers are ineffective. Jeon et al.(2001) studied the bioactivities of three chitosans, high,medium and low-molecular weight chitosans with a Mwvalues in the range 24–7 and 6–1 and 5 kDa, respectively.The profile of low molecular weight consisted of oligo-saccharides with DP in the range from pentamer to hep-tamer. They observed that the efficacy on the growth ofE. coli increased with molecular weight.

Chemical modifications

Even though chitosan is quite attractive as a biopoly-mer with distinctive physicochemical properties andbiological activities, it is currently utilized to only thelimited extent. The delay in application study is partlyascribable to the difficulty in controlled modificationsbecause of the insoluble nature in organic solvent andmultifunctionality of chitosan. However, many kindsof modification reactions have been exploited toincrease the antimicrobial properties of chitosan. Thegrowing demand for a more accurate control of poly-saccharide properties has prompted the developmentof numerous techniques for selective modifications.The amino and two hydroxyl groups present in therepeating unit of chitosan are the targets of differentchemical modifications (Hirano et al., 1987). As aresult, the functionality of linear polysaccharides is sig-nificantly affected by the presence, level and distribu-tion of substituents along the main chain. As specifiedby Rabea et al. (2003), chitosan and chitin are com-mercially interesting compounds because of their highnitrogen contents (6.89%) compared to syntheticallysubstituted cellulose (1.25%). This makes chitosan a

Food Science and Technology International 18(1)

20

by guest on December 26, 2013fst.sagepub.comDownloaded from

useful chelating agent. However, these naturally abun-dant materials are also limited in their reactivity andprocessability.

Several alkylated chitosans are reported to be synthe-sized. Kim et al. (1997) preparedN-alkyl chitosan deriv-atives by introducing alkyl groups into the amine groupsof chitosan via Schiff’s base intermediates. Indeed, thefree amine groups of the chitosan react with aldehydes togive the Schiff’s base (Figure 6) in homogeneous med-iums such as acetic acid and methanol (Hirano, 1997).

Long alkyl chains (until C12) can be introduced onthe chitosan. Quaternization of the N-alkyl chitosanderivatives could be carried out using a halogenoalcanein the presence of sodium hydroxide (Belalia et al., 2008;Jia et al., 2001). As shown in Figure 7,MeI could be usedto produce water-soluble cationic polyelectrolytes andnovel chitosan derivatives, with quaternary ammoniumsalt (Belalia et al., 2008; Kim et al., 1997).

Their antibacterial activities against S. aureus wereexplored by the viable cell count method in acetatebuffer at pH 6. The antibacterial activities of the quater-nary ammonium salt increased with increase in the chainlength of the alkyl substituent, and this increased activitycould be ascribed to the contribution of the increasedhydrophobic properties of the derivatives. In addition,Avadi et al. (2004) prepared a quaternized chitosan (i.e.,N-diethylmethyl chitosan, DEMC) based on a modified

two-step process. With a degree of quaternization of79%, the DEMC exhibited a higher antibacterial activ-ity than chitosan against E. coli. However, the antimi-crobial effects of both compounds were pH dependentand an increase in concentration of acetic acid resultedin a significant decrease in MIC, determined by turbidi-metric method. As a result, the antibacterial activities ofchitosan and DEMC are higher in 1% acetic acid incomparison with the lower levels of acetic acid concen-tration, 0.25%, for example. The MIC of DEMC isdecreased from 500 to 62.5 mg/mL when the medium ischanged from water to 1% acetic acid solution. Theauthors mentioned that this is due to the target site ofthe polycation, i.e., the negatively charged surface of thebacteria cell. Therefore, the polycation DEMC with ahigh charge density interacts with the bacteria more thanwhat chitosan itself does. Other chitosan derivativessuch as N,N,N-trimethyl chitosan, N-propyl-N,N-dimethyl chitosan and N-furfuryl-N,N-dimethyl chito-san were prepared and tested for their activities againstE. coli (Jia et al., 2001). It was shown that the antibac-terial activity of quaternary ammonium chitosan inacetic acid medium stronger than that in wateragainst E. coli and is stronger than that of chitosanitself. More recently, Chi et al. (2007) preparedChitosan-N-2-hydroxypropyl trimethyl ammoniumchloride by the reaction of chitosan with glycidyl

O

OH

NHCH3

HO

OO

CH3I, NaOH, NaI

N-methyl-2-pyrrolidinone

O

OH

N

HO

OO

N-methylchitosan

CH3CH3 H3C

I

N,N,N-trimethylchitosan

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

O

OH

NH2

RCHO O

OH

HO

N

O

OHO

CHR

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

Dutta et al.

21

by guest on December 26, 2013fst.sagepub.comDownloaded from

trimethylammonium chloride. The chitosan derivatives,with different molecular weights, near 1.7, 35.7, 90.2 and415.5 kDa, showed biocidal activity on S. aureus andStaphylococcus epidermidis, B. subtilis and Candida albi-cans. These authors observed that the chitosan with amolecular weight of 415.5 kDa exhibits a slightly lowerbiocidal activity on C. albicans than others. However, itseems that high-molecular weight chitosan derivativeshad high biocidal activities on the Gram-positive bacte-ria, with a decreasing biocidal effect with decreasingmolecular weight from 90 to 1.7 kDa. Nevertheless,derivatives with molecular weights from 90 to 1.7 kDadid not exhibit any biocidal effect against E. coli and P.aeruginosa even at concentrations up to 10mg/mL. Inanother study, Kim et al. (1997) synthesized diethylami-noethyl–chitosan (DEAE–chitosan) from deacetylationof a diethylaminoethyl–chitin (DEAE18 chitin) by intro-ducing DEAE groups onto the C(6)–OH in chitin. Thedeacetylation was conducted by heating in aqueous 10%sodium hydroxide containing sodium borohydride. Inaddition, DEAE–chitin was quaternized to producetriethylaminoethyl–chitin (TEAE–chitin). Their antibac-terial activities against S. aureus and E. coli were evalu-ated using colony count by means of the shake flaskmethod. The antibacterial activities were found toincrease in the order DEAE–chitin, DEAE–chitosanand TEAE–chitin. To obtain copolymers with zwitter-ionic property, Jung et al. (1999) prepared water-solubleanionic chitosan moieties and investigated their

antimicrobial activity. Mono(2-methacryloyl oxyethyl)acid phosphate (chitosan–MAP) and vinylsulfonic acidsodium salt (chitosan–VSS) were grafted onto chitosan(Figure 8). Concerning their antimicrobial activities, chit-osan–MAP and chitosan–VSS exhibited inhibition valuesof the growth of C. albicans of about 95% and 75%,respectively. Both chitosan derivatives showed the samehigh pH-dependence. As a result, if the pH changed from5.7 to 6.2, their antimicrobial properties dropped to 10–15%, which was less than the activity of the parent chit-osan.AgainstTrichophyton rubrum, theMAPgrafting ledto a low negative impact on antimicrobial activity,whereas the anionic chitosan showedmuch enhanced bio-active action against Trichophyton violaceum, comparedto unmodified chitosan. The authors suggested than thisselectivity could result from the structural affinitybetween the wall of microbial strains and the chitosanor its derivatives. A possible reason might be that thewall of microorganisms consisted of chitin, chitosan orß-glycan.

Other chitosan derivatives are sulfonated and sulfo-benzoyl chitosans. The antibacterial effects of the chem-ical modifications were evaluated and compared withthose of 69% deacetylated chitosan by Chen et al.(1998). MIC values of sulfonated chitosan (0.63%sulfur content) against Shigella dysenteriae, Aeromonashydrophila, S. typhimurium, and B. cereus were found tobe lower than those of the deacetylated chitosan. A highsulfur content in sulfonated chitosan adversely

O

NH2

OH

CH2OH

O

NH

OH

CH2OH

O

O

Chitosan O

HN

OH

CH2OH

O

(CH2)13CH3H3C

COOCH2CH2OP(OH)2

OO

CH2

CH2

SO3Na

Chitosan-g-VSS

Chitosan-g-MAP

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

Food Science and Technology International 18(1)

22

by guest on December 26, 2013fst.sagepub.comDownloaded from

influenced its antibacterial effect. Sulfobenzoyl chitosanexhibited excellent water solubility and an antibacterialeffect comparable to those of sulfonated chitosan.Concerning the antifungal properties, Cuero et al.(1991) showed that aqueous solutions of N-carboxy-methylchitosan suppressed both growth and aflatoxinproduction by Aspergillus flavus and Aspergillus parasi-ticus in submerged culture. Five chemically modifiedchitosans were tested for their antifungal activitiesagainst Saprolegnia parasitica by Muzzarelli et al.(2001) using the radial growth assay in chitosan-bearingagar and the fungal growth assay in chitosan-bearingbroth. Members of the genus Saprolegnia are responsi-ble for the infections of fish and eggs in aquaculturefacilities. Results indicated that methylpyrrolidinonechitosan,N-carboxymethyl chitosan andN-phosphono-methyl chitosan exerted effective fungistatic actionagainst the target strain. Electron microscopy observa-tions provided evidence of ultrastructural alterations,damaged fungal structures, uptake of modified chito-sans, and hyphal distortion and retraction. As a result,chemical modifications of chitosan with respect to theamine site are numerous and relatively easy taking intoaccount the reactivity of the primary amine. However, topreserve the amine groups in order to maintain the bio-active properties, various modification procedures haverecently been described, which yield C-6 modified chit-osan derivatives.

MECHANISM OF ANTIMICROBIALACTION

The different mechanisms have been proposed whereasthe exact mechanism of the antimicrobial action ofchitin, chitosan and their derivatives is still unknown(Rabea et al., 2003). The mechanisms of the antimicro-bial activity of chitosan were different for Gram-positiveand Gram-negative bacteria (Zheng and Zhu, 2003). Inthis study, they differentiated the effect of chitosan on S.aureus (Gram-positive) and on E. coli (Gram-negative).For Gram-positive S. aureus, the antimicrobial activityincreased with increase themolecular weight of chitosan.Besides, for Gram-negative E. coli, the antimicrobialactivity increased with decrease in molecular weight.The authors suggested the following two different mech-anisms for the antimicrobial activity: (1) in the case of S.aureus, the chitosan on the surface of the cell can form apolymer membrane, which inhibits nutrients from enter-ing the cell and, (2) for E. coli, where chitosan of lowermolecular weight entered the cell through pervasion.

The antimicrobial mechanisms of CM, Q and CMQare suggested as: on one hand, the positive charge of thegroup at C-2 resulted in a polycationic structure whichcan be expected to interact with the predominantly anio-nic components (lipopolysaccharides and proteins) of

the microorganisms’ surface (Helander et al., 2001).The interaction resulted in great alteration of the struc-ture of outer membrane which caused release of a majorproportion of proteinaceous material from the cells(Helander et al., 1998); when the quarternized groupwas introduced onto the molecular chain, the positivecharge was strengthened. On the other hand, when car-boxymethyl group was introduced along the molecularchain, the presence of a molecular structure with hydro-philic ends and weak interaction forming betweenhydrophilic ends and chitosan enhances the antimicro-bial activity.

Because of the positive charge on the C-2 of the glu-cosamine monomer below pH 6, chitosan is more solu-ble and has a better antimicrobial activity than chitin(Chen et al., 1998). The exact mechanism of the antimi-crobial action of chitin, chitosan and their derivatives isstill imperfectly known, but different mechanisms havebeen proposed (Rabea et al., 2003). One of the reasonsfor the antimicrobial character of chitosan is its posi-tively charged amino group which interacts with nega-tively charged microbial cell membranes, leading to theleakage of proteinaceous and other intracellular constit-uents of the microorganisms (Shahidi et al., 1999).Chitosan acted mainly on the outer surface of bacteria.At a lower concentration (0.2mg/mL), the polycationicchitosan does probably bind to the negatively chargedbacterial surface to cause agglutination, while at higherconcentrations, the larger number of positive chargesmay have imparted a net positive charge to the bacterialsurfaces to keep them in suspension (Papineau et al.,1991; Sudarshan et al., 1992).

A strong attachment of heterologous bacteria to thewalls in tobacco leaves is essential to elicit the HR.Therefore, frommechanistic point of views, it is possiblethat chitin derivatives prevent the attachment of bacte-rial cells into the plant cell walls or affect their survival inthe intercellular spaces.

Chitosan acted mainly on the outer surface of thebacteria. The obvious antibacterial effects can be attrib-uted to the formation of polyelectrolyte complexesbetween the polycationic agent and the bacterial cell sur-face (Muzzarelli et al., 1990). Indeed, interactionbetween positively charged chitosan molecules and neg-atively charged microbial cell membranes leads to theleakage of proteinaceous and other intracellular constit-uents. Studies based on UV absorption indicated thatthe chitosan causes considerable losses of proteinicmaterial to Pythium oaroecandrum, at a pH 5.8(Helander et al, 2001; Liu et al., 2004). Different behav-iors were reported, dependent on the chitosan concen-tration (Rabea et al., 2003). At a lower concentration(<0.2mg/mL), the polycationic chitosan does probablybind to the negatively charged bacterial surface to causeagglutination, while at higher concentrations, the larger

Dutta et al.

23

by guest on December 26, 2013fst.sagepub.comDownloaded from

number of positive charges may have imparted a netpositive charge to the bacterial surfaces to keep themin suspension. Savard et al. (2002), by a microscopicexamination of Saccharomyces unisporus after treatmentwith chitosan-salt with a polymerization degree of25, showed agglutination of a refractive substance onthe entire cell wall. When chitosanase was added to theculture media containing chitosan-salt, they could notobserve refractive substances. This result suggested aninteraction between chitosan and the cell wall. Inanother study, chitosan caused leakage of glucose andlactate deshydrogenase from E. coli cells (Tsai and Su,1999). These results support the hypothesis that themechanism of chitosan antibacterial action involvescross-linkage between the polycations of chitosan andthe anions on the bacterial surface that change the mem-brane permeability. As already mentioned, chitosancoatings adjusted to pH 5.0 totally inhibited Gram-positive bacterial surface growth such as L. monocyto-genes and S. aureus. However, Gram-negative microbialstrains such as P. aeruginosa overcame the active chito-san protection, and the development was not completelyexcluded (Coma et al., 2003). Therefore, the microbio-logical target of protonated chitosan’s action would bethe cytoplasmic membrane of sensitive cells. Cellulardamage can lead to the disruption of the cellularintegrity of the membrane. The outer membrane ofGram-negative bacteria could act as a barrier and beresponsible for preventing chitosan from reaching thecytoplasmic membrane. Although the cytoplasmicmembrane should be sensitive to chitosan, the outermembrane protects the cells. Zheng and Zhu (2003)also indicated that the mechanisms of the antimicrobialactivity of chitosan were different between Gram-positive and Gram-negative bacteria. They showed, ina comparative study, that the effect of chitosan wasdifferent on S. aureus (Gram-positive) and on E. coli(Gram-negative). To E. coli, the antimicrobial activitywas enhanced as the molecular weight decreased. It wasobvious 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 increas-ing molecular weight of chitosan. The inhibiting effectwas fairly obvious for higher ones, such as 305 kDa, evenif the concentration was quite small. The authorssuggested two possible mechanisms for antimicrobialactivity: (1) the chitosan on the surface of the cell canform a polymer membrane, which prevents nutrientsfrom entering the cell and (2) chitosan of lower molecu-lar weight entered the cell through pervasion. For S.aureus, the dominant mechanism is the former, whilefor E. coli, the latter mechanism seems more likely. Inaddition, the chitosan has also the faculty to bind speci-fically with some macromolecules. It can thus inhibitvarious enzymes, bind to the DNA and inhibit the

synthesis of the mRNA after penetration of the chitosanin the core of the microorganisms (Hadwiger et al.,1985). Epifluorescence microscopy results showed a pos-sible action of chitosan during a short duration of timeon the synthesis of nucleic acids and especially on therelative proportion of RNA compared with DNA(Coma et al., 2003). This impact was followed by anadaptative mechanism of the cells. Binding of chitosanwith DNA and inhibition of mRNA synthesis occurthrough chitosan penetration toward the nuclei of themicroorganisms and interference with the synthesis ofmRNA and proteins (Rabea et al., 2003).

It has been postulated that the antimicrobial action ofchitosan occurs as a result of several mechanisms.Chung andChen (2008) studied the antibacterial activityof chitosan with respect to the extent of damaged ormissing cell walls and the degree of leakage of enzymesand nucleotides from different cellular locations. First,the addition of chitosan to the bacterial suspensionseemed to have a stronger impact on the Gram-negativeE. coli than on theGram-positiveS. aureus in terms of theleakage of enzymes. In addition, the experimental resultrevealed that the antimicrobial action of chitosan notonly involves a reaction with the cell wall of the bacteriabut also may affect the structure of the phospholipidbilayer in the cell membrane, thereby changing the per-meability of the cell membrane, resulting in the release ofsome of the cellular components. This action was furtherenhanced when chitosan with a high degree of deacetyla-tion was used. To gain a better understanding of themechanism by which chitosan functions as a bactericide,the cells were also subjected to a known antibiotic whichreacts with the anionic phosphate group of phospholipidsin the cell membrane, destroying the cell membrane struc-ture and affecting its permeability. In parallel, the cellswere subjected to EDTA, a chemical chelating agentthat destroys the structure of the cell wall by chelatingwith the Ca2þ orMg2þ present in the cell wall. As a result,chitosanwas found to react with both the cell wall and thecell membrane of E. coli, but not simultaneously, indicat-ing that the inactivation by chitosan occurs via a two-stepsequential mechanism: an initial separation of the cellwall from its cell membrane, followed by cell membranedestruction. Concerning the bioactivity of the chitosanagainst phytopathogenic fungi, it could be related withits potential elicitor of many plant defense responses,including for example the accumulation of chitinases orby producing phytoalexins, defined as ‘substances withantibiotic activity that function as growth inhibitors ofphytopathogenic organisms, chiefly fungi’ (Bade andWick, 1987). In a study on Botrytis cinerea andRhizopus stolonifer, chitosan-induced morphologicalchanges were characterized by excessive hyphal branch-ing and abnormal aerial surface hyphal growth comparedto the control (El Ghaouth et al., 1992). As a result,

Food Science and Technology International 18(1)

24

by guest on December 26, 2013fst.sagepub.comDownloaded from

chitosan appears to play a dual function, by interferingdirectly with fungal growth and also by activating severalbiological processes in plant tissues. Benhamou et al.(1994) applied chitosan to decrease the infection withFusarium oxysporum. Biopolymer coating was appliedon seed prior to fungal inoculation. Chitosan at concen-trations ranging from 0.5 to 1mg/mL was used for theultrastructural and cytochemical investigations. Theauthors observed that after a pretreatment with chitosan,the root tissues at sites of fungal penetration were alwaysassociated with the expression of plant defense reactions.In the epidermis, cells showed typical signs of necrosischaracterized by marked disorganization of the cyto-plasm. The pathogen was detected in the outer cortexwhere its development was halted. Fungal cells sufferedfrom serious damage and were frequently encircled by anelectron-densematerial. In the noncolonized inner cortex,strong host reactionswere detected thatweremainly asso-ciated with the deposition of two types of materials thatdiffered in their electron densities. Gold cytochemistrywith a ß-1,3-glucanase and a laccase showed that themore electron-dense material was phenolic in nature,whereas the other material, occurring either as depositsinserted between the phenolic aggregates or as globularstructures lining the host cell walls, was made of ß-1,3-glucans. These observations bring further evidence thatchitosan is an active inducer of plant defense reactionsand, thus, has the potential to become a powerful alter-native means of disease control.

FACTORS AFFECTING THEANTIMICROBIAL ACTIVITY

There are various factors, such as the intrinsic andextrinsic properties of chitosan: molecular weight,degree of polymerization, deacetylation, solubility andhigher charge density that affect the antimicrobial activ-ity of chitosan (Ralston et al., 1964; Sekiguchi, 1994;Uchida, 1989).

The antimicrobial activities of chitosan and chitosan-based films increases by decreasing pH. This effect canbe considered as synergetic for the reasons of the hurdleeffect of the acid stress on the bacterial cells (Rhoadesand Roller, 2000).

The mechanism of the antimicrobial activity of chit-osan and its derivatives is well studied. As theory, it hasbeen suggested that the positive charge of the aminegroup (NH3

þ) at pH values lower than the pKa (pH<6.3) at which this functional group carries 50% of itstotal electric charge allows the interactions with nega-tively charged microbial cell membranes, a phenomenonwhich is susceptible to cause a leakage of intracellularconstituents (Helander et al., 2001). However, even ifthis is a well-recognized explanation of chitosan

antimicrobial effect, there is no direct evidence demon-strating this behavior against bacteria.

Lower pH increases the antimicrobial activity ofchitosan for much the same reasons, in addition to the‘hurdle effect’ of inflicting acid stress on the target organ-isms (Rhoades andRastall, 2000). Surroundingmatrix isthe greatest single influence on antimicrobial activity.Being cationic, chitosan has the potential to bind tomany food components such as alginates, pectins, pro-teins and inorganic polyelectrolytes such as polypho-sphate (Kubota and Kikuchi, 1998). Solubility can bedecreased using high concentrations of low-molecularweight electrolytes such as sodium halides, sodium phos-phate and organic anions (Roberts, 1992).

Sorption capacity of chitosan films was significantlyaffected by the moisture content of the chitosan-basedfilms. Authors reported that a decrease of water contentdecreased the total amount of the active sites that canparticipate in the sorption phenomenon. Antibacterialactivities of the produced chitosan-based films have beenevaluated against E. coli and S aureus and it has beenshown that diameters of the inhibition zones were 5 and3 times higher than those of control for E. coli and Saureus, respectively. Authors showed that moisture con-tent of chitosan films had significant effect on their bac-tericide effect which decreased on decreasing filmmoisture content. Decrease of film moisture contentfrom 22% (w/w) down to 12% (w/w) decreased the bac-tericide activity by 2.5 times. This was demonstrated bythe decreased diameter of the inhibition zone. This find-ing can be exploited in the cheese-making industry forcheese coating with chitosan films in the maturationchambers in order to avoid mold and pathogenicbacteria growth on cheese surface (Buzinova andShipovskaya, 2008).

The effect of the molecular weight on some antibac-terial and antifungal activities has been explored (Chen,1998). Chitosans with molecular weights ranging from10,000 to 100,000 have been found to be helpful inrestraining the growth of bacteria. In addition, chitosanwith an average molecular weight of 9300 was effectivein restraining E. coli, whereas that with a molecularweight of 2200 helped in accelerating the growth(Tokura et al., 1994). Moreover, the antibacterial activ-ity of chitosan is influenced by its degree of deacetyla-tion, its concentration in solution and the pH of themedium. Antibacterial activities were also found to beincreasing in the order N,O-carboxymethylated chito-san, chitosan and O-carboxymethylated chitosan (Liuet al., 2001).

In addition to the formation of gas-permeable films,chitosan has a dual function: (1) to direct the interfer-ence of fungal growth and (2) to activate several defenseprocesses (Bai et al., 1988). These defense mechanismsinclude accumulation of chitinases, synthesis of

Dutta et al.

25

by guest on December 26, 2013fst.sagepub.comDownloaded from

proteinase inhibitors and lignification and induction ofcallous synthesis (El Ghaouth et al., 2000). Whenapplied on wounded wheat leaves, chitosan-induced lig-nifications and consequently restricted the growth ofnonpathogenic fungi in wheat. Chitosan inhibited thegrowth of A. flavus and aflatoxin production in liquidculture, preharvest maize and groundnut, and it alsoenhanced phytoalexin production in germinatingpeanut (Cuero et al., 1991a,b). Chitosan has also beenfound to inhibit growth and toxin production byAlternaria alternata fungal species lycopersici in culture(Bhaskara et al., 1998; Dornenburg and Knorr, 1997).

Chitosan solution at 0.10mg/mL markedly inhibitedthe growth of Xanthomonas pathogenic bacteria (iso-lated from Euphorbia pulcherrima) from different geo-graphical origins (Li et al., 2008). The antibacterialactivity of chitosan solution against Xanthomonas axo-nopodis pv. poinsettiicola (strain R22580) increased withthe increase of chitosan concentration up to 0.10mg/mL. The antibacterial activity of chitosan solution at0.05mg/mL was enhanced by NaCl.

The antibacterial activity of chitosan was investigatedby assessing the mortality rates of E. coli and S. aureusbased on the extent of damaged or missing cell walls andthe degree of leakage of enzymes and nucleotides fromdifferent cellular locations (Chung and Chen, 2008). Theinactivation of E. coli by chitosan occurred via a two-step sequential mechanism: an initial separation of thecell wall from its cell membrane, followed by destructionof the cell membrane.

The antibacterial activities of chitosan nanoparticlesand copper-loaded nanoparticles against E. coli,Salmonella choleraesuis, S. typhimurium and S. aureuswere evaluated by the calculation of MIC and minimumbactericidal concentration (MBC; Qi et al., 2004). Theobtained results showed that chitosan nanoparticles andcopper-loaded nanoparticles could inhibit the growth ofvarious bacteria tested. Their MIC values were less than0.25mg/mL and the MBC values of nanoparticlesreached 1mg/mL. They reported that the exposure ofS. choleraesuis to the chitosan nanoparticles led to thedisruption of cell membranes and the leakage ofcytoplasm.

OPTIMIZATION OF THE BIOCIDEPROPERTIES

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 forP. aeruginosa, 68%) andmorethan 98% against Gram-positive bacteria such asStreptococcus mutans, M. luteus, S. aureus and B. sub-tilis. Chitooligosaccharides showed a less bactericidaleffect, with 71%, 56% and 60% against E. coli

O-157.for the high-, medium- and low-molecularweight samples, respectively. Another test was doneagainst the P. aeruginosa strain, with bactericidal effectsof about 47%, 35% and 22% for the high-, medium- andlow-molecular weight chitosans, respectively. Tokuraet al. (1997) prepared chitosan oligomers of averagemolecular weights 9.3 and 2.2 kDa by nitrous acid deg-radation followed by the reduction of 2,5-anhydroman-nose terminal by sodium borohydrate. Although the9.3 kDa provided the growth inhibition of E. coli, thechitosan 2.2 kDa was not a growth inhibitor but agrowth accelerator. It has been suggested that the smal-ler oligomers serve as nutrients for bacteria, whereas thehigher oligomers are toxic by virtue of their charge-mediated adhesion to the cell membrane, which in turnprevents the uptake of nutrients through the cell wall.Liu et al. (2006) specified that the molecular weight ofchitooligosaccharides is critical for microorganism inhi-bition and must be higher than 10 kDa. Several studiesshowed that the impact of the Mw on the bioactivity isdependent on the concentration of the biocide. Theyinvestigated the bioactivities of chitosans of differentmolecular weights, from 55 to 155 kDa, against E. coliwith the same degree of deacetylation (80%). The mech-anism of antibacterial activity was the flocculation of thestrains. These authors observed that at high concentra-tion (over 200 ppm), the antibacterial activities of eachchitosan sample were almost the same and all the bacte-ria could be killed. At low concentration (20 ppm), therewas no antibacterial activity and chitosan could pro-mote the growth of E. coli. But at the middle range con-centrations (50–100 ppm), there were some differencesbetween chitosans of different molecular weights in theantibacterial activation. Indeed, the authors concludedthat at high concentrations (200, 500 and 1000 ppm) anda low concentration (20 ppm), the antibacterial activityof chitosan had no relationship to the molecular weight.But at concentrations ranging from 50 to 100 ppm, theantibacterial activity of low-molecular weight chitosan ishigher than that of the high-molecular weight samples.Qin et al. (2006) also showed that the molecular weightdependence of the antimicrobial activity of chitosan wasmore pronounced at a low concentration. The action ofchitosans with molecular weights Mw from 1.4 to400 kDa on the growth of S. aureus, E. coli and C. albi-cans was explored by microcalorimetry. The chitosanswith middle range values of Mw, 78 and 48 kDa, hadhigher inhibitory effect than others. Chitooligomer1.4 kDa promoted the growth ofC. albicans, but slightlyinhibited growth of the bacteria. The 400 kDa, with thehighestMw, exhibited amuchweaker inhibitory effect incomparison with the chitosan at 78 kDa. It seems thatthe water-insoluble chitosans with Mw around 50 kDawere the optimum ones for antimicrobial action in theirtested samples. In addition, Zheng and Zhu (2003)

Food Science and Technology International 18(1)

26

by guest on December 26, 2013fst.sagepub.comDownloaded from

observed that for chitosan with molecular weight below300 kDa, the antimicrobial effect on S. aureus wasstrengthened as the molecular weight increased. In con-trast, the effect on E. coli was weakened. In parallel todepolymerization, chemical modifications of chitosanhave been attempted to improve its antimicrobialproperties.

The influence on biocide performance of someunprecedented physicochemical features of chitosancast films such as film thickness, pH of the nutrientbroth, film neutralization, film autoclave sterilizationand temperature exposure was analyzed against S.aureus and in some experiments also againstSalmonella spp. The work demonstrates, for the firsttime, the influence of the release or positive migrationof protonated glucosamine fractions from the biopoly-mer into the microbial culture as the responsible eventfor the antimicrobial performance of the biopolymerunder the studied conditions. From the results, a reli-able and reproducible method for the determination ofthe bactericidal activity of chitosan-based films wasdeveloped in an attempt to standardize the testingconditions for the optimum design of active antimicro-bial food packaging films and coating applications.The optimization of biocide properties of chitosanwill be useful for its application in the design ofactive films of interest in the food area (Fernandez-Saiz et al., 2008).

CHITOSAN FOR IMMOBILIZATION

Enzymatic catalysis in nonaqueous solvents has gainedconsiderable interest for the preparation of natural prod-ucts, pharmaceuticals, fine chemicals and food ingredients(Carrea and Riva, 2000; Faber and Franssen, 1993;Margolin, 1993; Ru et al., 2000). Lipases (glycerol estershydrolase, E.C.3.1.1.3) have been widely used to produceorganic chemicals, biosurfactants, oleochemicals, agro-chemicals, paper, cosmetics, fine chemicals and pharma-ceuticals (Sharma et al., 2001). Chitosan has been used asamatrix for immobilization of lipases (Alsarra et al., 2002)and many other enzymes (Krajewska, 2004). Enzymesbound to sugars, or sugar-based polymers like chitosanare stabilized during lyophilization and in nonaqueousenvironments. This may be due to a reduction of auto-lysis, that is a multipoint attachment limiting enzymedistortions or microenvironmental effects (Wang et al.,1992).

Elemental analysis and Raman spectra measure-ments of the lipase, supports and immobilized lipasesystems gave evidence of the presence of enzymes onsupports. Chitosan supports with internal surface area(m2/g) among 3.31 and 1.26 were obtained. Regardlessof these low values, acceptable protein load(0.61–3.21%) and esterification initial rates were

achieved (0.88–2.75mmol/min/g of protein; Orregoet al., 2010).

CHITOSAN NANOCOMPOSITE

Nowadays, the nanocomposites are receiving moreattention from the researchers, because of their moreeffective action in penetrating and disrupting bacterialcell membranes to conquer the battle of pathogenic bac-teria (Yacoby and Benhar, 2008). Many works on theuse of nanochitosans to prevent food spoilage have beenreported in a recent review (Friedman and Juneja, 2010).

Chitosan/vermiculite (VMT) nanocomposites canenhance the thermal stability of chitosan nanocompo-sites dramatically due to the well dispersion of acid-modified VMT (HCl-modified VMT, HVMT) andbetter interaction between HVMT and chitosan in thefabricated nanocomposites (Zhang et al., 2009).

Chitosan-based nanocomposite films, especiallysilver-containing ones, showed a promising range ofantimicrobial activities (Rhim et al., 2006). The chit-osan/OREC nanocomposites films provide promisingapplications as antimicrobial agents, water-barriercompounds, anti-ultraviolet compounds and drug-controlled release carriers in antimicrobial food pack-aging (Wang et al., 2007). From the antimicrobialactivity test, it was found that the chitosan–clay nano-composites showed a synergistic effect in the antimi-crobial activity against to E. coli and S. aureus (Hanet al., 2010).

Antimicrobial studies showed that the nanocompo-sites could strongly inhibit the growth of a wide varietyof microorganisms, including Gram-positive bacteria,Gram-negative bacteria and fungi; more importantly,they exhibited good antimicrobial capacity in whichevermedium, in weak acid, water or weak base. As theamount of Montmorillonite increased, the nanocompo-sites had better inhibitory effect on microorganisms,especially Gram-positive bacteria. The lowest MICvalues of the nanocomposites against S. aureus and B.subtilis were less than 0.00313% (w/v) under all the con-ditions (Wang et al., 2007).

CONCLUSIONS

Chitin, chitosan and its oligosaccharides played veryimportant roles in the application of antimicrobial mate-rials and food packaging. The systematic study towardantimicrobial activity of chitin, chitosan and its oligo-saccharides would be a promising tool for the futureimprovement of food quality and preservation duringprocessing and storage because the antimicrobial pack-aging can be helpful in extending the food shelf-life.The combination of other film-forming and coatingmaterials may provide the functional properties for a

Dutta et al.

27

by guest on December 26, 2013fst.sagepub.comDownloaded from

better food shelf-life. The understanding of the factorsaffecting the antimicrobial activity, mechanism of anti-microbial action, and optimization of the biocide prop-erties of chitin, chitosan and their oligosaccharideswould be an added advantage to use these materials ina better way.

ACKNOWLEDGMENTS

We thank UGC, New Delhi and Royal Society of Chemisty(RSC), UK for giving the Research Fund Grant Award-2009to PKD. We also sincerely thank the different researchers whopublished their works in different journals, magazine, disser-

tation, doctoral degree work and elsewhere which have been agreat source for resource materials toward compiling thereview 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 constructivesuggestions.

REFERENCES

Aider M (2010). Chitosan application for active bio-based

films production and potential in the food industry:

Review. LWT-Food Science & Technology 43(6): 837–842.

Alsarra IA, Betigeri SS, Zhang H, Erans BA and Neau SH.

(2002). Molecular weight and degree of deacetylation

effects on lipase-loaded chitosan bead characteristics.

Biomaterials 23(17): 3637–3644.Alvarez MF. (2000). Review: Active food packaging. Food

Science and Technology International 6(2): 97–108.Appendini P and Hotchkiss JH. (2002). Review of antimicro-

bial food packaging. Innovative Food Science and Emerging

Technologies 3(2): 113–126.

Avadi MR, Sadeghi AMM, Tahzibi A, Bayati K, Pouladzadeh

M,Zohuriaan-MehrMJ, et al. (2004).Diethylmethyl chitosan

asanantimicrobial agent: Synthesis, characterizationandanti-

bacterial effects. European Polymer Journal 40(7): 1355–1361.

Bade ML and Wick RL. (1987). Protecting crops and wildlife

with chitin and chitosan. In: Yalpani M (ed.) Industrials

Polysaccharides: Genetic, Engineering, Structure/Property

Relations and Applications. Amsterdam: Elsevier, 450.Bai RK, Huang MY and Jiang YY. (1988). Selective perme-

abilities of chitosan-acetic acid complex membrane and

chitosan-polymer complex membranes for oxygen and

carbon dioxide. Polymer Bulletin 20(1): 83–88.Baldwin EA, Nisperos NO and Baker RA. (1995). Use of

edible coatings to preserve quality of lightly (and slightly)

processed products. Critical Reviews in Food Science and

Nutrition 35(6): 509–524.

Baron JK and Sumner SS. (1994). Inhibition of Salmonella typhi-

murium and Escherichia coli O157:H7 by an antimicrobial

containing edible film.Journal ofFoodProtection56: 916–920.Belalia R, Grelier S, Benaissa M and Coma V. (2008). New

bioactive biomaterials based on quaternized chitosan.

Journal of Agricultural and Food Chemistry 56(5): 1582–1588.Ben A and Kurth LB. (1995). Edible film coatings for meat

cuts and primal. In: Meat 95. Proceedings of the Australian

Meat Industry Research Conference. CSIRO Division of

Food Sci & Technol, Cannon Hill, Qld.

Benhamou N, Lafontaine PJ and Nicole M. (1994). Induction

of systemic resistance to fusarium crown and root rot in

tomato plants by seed treatment with chitosan.

Phytopathology 84(12): 1432–1444.Bhaskara MV, Arul J, Essaid AB, Anger P, Richard C and

Castaigne F. (1998). Effect of chitosan on growth and toxin

production by Alternaria alternata f. sp. lycopersici.

Biocontrol Science and Technology 8(1): 33–43.Black JG. (1996). Microbiology: Principles and Application.

New Jersey: Prentice-Hall, Inc, 80–82.Buzinova DA and Shipovskaya AB. (2008). Sorption and sac-

tericide properties of chitosan films. Izvestya of Saratov

University 8(2). doi:547.458:544.723.Carrea G and Riva S. (2000). Properties and synthetic appli-

cations of enzymes in organic solvents. Angewede Chemie

International Edition 39(13): 2226–2254.Cha DS and Chinnan MS. (2004). Biopolymer-based antimi-

crobial packaging: A review. Critical Reviews in Food

Science and Nutrition 44(4): 223–237.

Chang Peter R, Jian R, Yu J and Ma X. (2010). Fabrication

and characterisation of chitosan nanoparticles/plasticised-

starch composites. Food Chemistry 120(3): 736–740.Chatterjee B, Guha AK, Chatterjee S, Chatterjee S and

Mukherjee R. (2005). Chitosan: A journey from chemistry

to biology. In: Dutta PK (ed.) Chitin and Chitosan:

Opportunities and Challenges. India: SSM International

Pub., 35–58.Chen T. (1998). Specific properties and use of chitosan.

Symposium on Nature Marine Product and Nature

Biological Medicine. 282–284, Beijing, China.Chen RH, Chang JR and Shyru JS. (1997). Effects of ultra-

sonic condition and storage in acidic solutions on changes

in molecular weight and polydispersity of treated chitosan.

Carbohydrate Research 299(4): 287–294.Chen CS, Liau WY and Tsai GJ. (1998). Antibacterial effects

of N-sulfonated and N-sulfobenzoyl chitosan and applica-

tion to oyster preservation. Journal of Food Protection

61(9): 1124–1128.Chen MC, Yeh GH and Chiang BH. (1996). Antimicrobial

and physicochemical properties ofmethylcellulose and chit-

osan films containing a preservative. Journal of Food

Processing and Preservation 20(5): 279–390.Cheng CY and Li YK. (2000). AnAspergillus chitosanase with

potential for large-scale preparation of chitosan oligosac-

charides. Biotechnology and Applied Biochemistry 32(3):

197–203.

Chi W, Qin C, Zeng L, Li W and Wang W. (2007).

Microbiocidal activity of chitosan-N-2-hydroxypropyl tri-

methyl ammonium chloride. Journal of Applied Polymer

Science 103(6): 3851–3856.Chi S, Zivanovic S and Penfield MP. (2006). Application

of Chitosan films enriched with oregano essential oil on

bologna- active compounds and sensory attributes. Food

Science and Technology International 12(2): 111–117.

Choi WS, Ahn KJ, Lee DW, Byan MW and Park HJ. (2002).

Preparation of chitosan oligomers by irradiation. Polymer

Degradation and Stability 78(3): 533–538.

Food Science and Technology International 18(1)

28

by guest on December 26, 2013fst.sagepub.comDownloaded from

Chung Y-C and Chen C-Y. (2008). Antibacterial characteris-tics and activity of acid-soluble chitosan. BioresourceTechnology 99(8): 2806–2814.

Coma V. (2008a). A review: Bioactive packaging technologiesfor extended shelf life of meat-based products. MeatScience 78(1): 90–103.

Coma V. (2008b). Bioactive chitosan-based substances and

films. Current Research and Developments on Chitin andChitosan in Biomaterials Science. Vol. 1, Trivandrum,Kerala, India: Research Signpost, 21–52.

Coma V, Deschamps A and Martial-Gros A. (2003).Bioactive packaging materials from edible chitosanpolymer-antimicrobial activity assessment on dairy related

contaminants. Journal of Food Science 68(9): 2788–2792.Coma V, Martial-Gros A, Garreau S, Copinet A and

Deschamps A. (2002). Edible antimicrobial films based

on chitosan matrix. Journal of Food Science 67(3):1162–1169.

Coma V, Sebti I, Pardon P, Deschamps A and Pichavant FH.(2001). Antimicrobial edible packaging based on cellulosic

ethers, fatty acids and nisin incorporation to inhibit Listeriainnocua and Staphylococcus aureus. Journal of FoodProtection 64(4): 470–475.

Cooksey K. (2005). Effectiveness of antimicrobial food pack-aging materials. Food Additives and Contaminants 22(10):980–987.

Cuero R, Duffus E, Osuji G and Pettit R. (1991a). Aflatoxincontrol in preharvest maize: Effects of chitosan and twomicrobial agents. Journal of Agricultural Science 117(2):165–169.

Cuero RG, Osuji G and Washington A. (1991b). N-carboxy-methyl chitosan inhibition of aflatoxin production: Role ofzinc. Biotechnology Letters 13(6): 441–444.

Cutter CN. (2002a). Microbial control by packaging: Areview. Critical Reviews in Food Science and Nutrition42(2): 151–161.

Cutter CN. (2002b). Incorporation of antimicrobials intopackaging materials. In: Proceedings of the 55th AnnualReciprocal Meat Conference. Columbia, MO: University

of Missouri, 83–87.Cutter CN. (2006). Opportunities for bio-based packaging

technologies to improve the quality and safety of freshand further processed muscle foods. Meat Science 74(1):

131–142.Cutter CN andMiller BJ. (2004). Incorporation of nisin into a

collagen film retains antimicrobial activity against Listeria

monocytogenes and Brochothrix thermosphacta associatedwith a ready-to-eat meat product. Journal of the Associationof Food and Drug Officials 68(4): 64–77.

Cutter CN and Siragusa GR. (1996). Reduction ofBrochothrix thermosphacta on beef surfaces followingimmobilization of nisin in calcium alginate gels. Letters inApplied Microbiology 23(1): 9–12.

Cutter CN and Siragusa GR. (1997). Growth of Brochothrixthermosphacta in ground beef following treatments withnisin in calcium alginate gels. Food Microbiology 14(5):

425–430.Cutter CN and Sumner SS. (2002). Application of edible coat-

ings on muscle foods. In: Gennadios A (ed.) Protein-based

Films and Coatings. Boca Raton, FL: CRC Press, 467–484.

Czechowska-Biskup R, Rokita B, Lotfy S, Ulanski P andRosiak JM. (2005). Degradation of chitosan and starchby 360-kHz ultrasound Carbohydrate. Polymers 60(2):

175–184.Darmadji P and Izumimoto M. (1994). Effect of chitosan in

meat preservation. Meat Science 38(2): 243–254.Dawson PL, Acton JC, Han IY, Padgett T, Orr R and Larsen

T. (1996). Incorporation of antibacterial compounds intoedible and biodegradable packaging films.Activities Reportof the Research and Development Associates 48: 203–210.

de Moura Marcia R, Aouada Fauze A, Avena-BustillosRoberto J, McHugh Tara H, Krochta John M andMattoso Luiz HC. (2009). Improved barrier and mechani-

cal properties of novel hydroxypropyl methylcelluloseedible films with chitosan/tripolyphosphate nanoparticles.Journal of Food Engineering 92(4): 448–453.

DecleireM, Callebaut A,DeCatW, VanHuynhN,Khoury C,Le Goffic F, et al. (1997). Comparison of endochitinaseactivities and antifungal properties from nine Serratia spe-cies. In: Domard A, Roberts AF and Varum KM (eds)

Advances in Chitin Science. Vol. 7(II), Jacques, Andre,Lyon (France).

Devlieghere F, Vermeiren L, Bockstal A and Debevere J.

(2000). Study on antimicrobial activity of food packagingmaterial containing potassium sorbate. Acta Alimentaria29(2): 137–146.

Dornenburg H and Knorr D. (1997). Evaluation of elicitor-and high-pressure-induced enzymatic browning utilizingpotato (solanum tuberosum) suspension cultures as amodel system for plant tissues. Journal of Agricultural and

Food Chemistry 45(10): 4173–4177.DurangoAM, Soarres NFF, Benevides S, Teixeria J, Carvalho

M,Wobeto C, et al. (2006). Development and evaluation of

an edible antimicrobial films based on yam starch and chit-osan. Packaging Technology and Science 19(1): 55–59.

Dutta J andDutta PK. (2010). Antimicrobial activity of chitin,

chitosan and their oligosaccharides. In: Kim SK (ed.)Chitin, Chitosan, Oligosaccharides and Their Derivatives.New York: CRC Press, 195–214.

Dutta PK, Tripathi S, Mehrotra GK and Dutta J. (2009).Perspectives for chitosan based antimicrobial films infood applications. Food Chemistry 114(4): 1173–1182.

El Ghaouth A, Arul J, Asselin A and Benhamou N. (1992).

Antifungal activity of chitosan on post-harvest pathogens:Induction of morphological and cytological alternations inRhizopus stolonifer. Mycological Research 96(9): 769–779.

El Ghaouth A, Smilanick JL, Brown GE, Ippolito A,Wisniewski M and Wilson CL. (2000). Application ofCandida saitoana and glycolchitosan for the control of

postharvest diseases of apple and citrus fruit under semi-commercial conditions. Plant Disease 84(3): 243–248.

Eom TK and Lee KM. (2003). Characteristics of chitosanasesfrom Aspergillus fumigatus KB-1. Archives of Pharmacal

Research 26(12): 1036–1041.Eswaranandam S, Hettiarachchy NS and Johnson MG.

(2004). Antimicrobial activity of citric, lactic, malic, or tar-

taric acids and nisin-incorporated soy protein film againstListeria monocytogenes, Escherichia coli 0157:H7 andSalmonbella gaminara. Journal of Food Science 69(3):

M79–M84.

Dutta et al.

29

by guest on December 26, 2013fst.sagepub.comDownloaded from

Faber K and Franssen MCR. (1993). Prospects for theincreased application of biocatalysts in organic transforma-tions. Trends in Biotechnology 11(11): 461–469.

Fang TJ and Lin LW. (1994). Growth of Listeria monocyto-genes and Pseudomonas fragi on cooked pork in amodifiedatmosphere packaging/nisin combination system. Journalof Food Protection 57(6): 479–485.

Fernandez-Saiz P, Lagaron JM and Ocio MJ. (2008).Optimization of the biocide properties of chitosan for itsapplication in the design of active films of interest in the

food area. Food Hydrocolloids 23(3): 913–921.Fernandez-Saiz P, Ocio MJ and Lagaron JM. (2010).

Antibacterial chitosan-based blends with ethylene vinyl

alcohol copolymer. Carbohydrate Polymers 80(3): 874–884.Franklin NJ, Cooksey KD and Getty KJK. (2004). Inhibition

of Listeria monocytogenes on the surface of individually

packaged hot dogs with a packaging film coating contain-ing nisin. Journal of Food Protection 67(3): 480–485.

Friedman M and Juneja V. (2010). Review of antimicrobialand antioxidative activities of chitosan in food. Journal of

Food Protection 73(9): 1737–1761.Gerasimenko DV, Avdienko ID, Bannikova GE, Yu Zueva O

and Varlamov VP. (2004). Antibacterial effect of water-

soluble low-molecularweight chitosan on different micro-organisms. Applied Biochemistry and microbiology 40(3):253–257.

Gill AO (2000) Application of lysozyme and nisin to controlbacterial growth on cured meat products. MS thesis, TheUniversity of Manitoba, Winnipeg, MB, Canada.

GlicksmanM. (1983). FoodHydrocolloids. Vol. 3, Boca Raton,

FL: CRC Press.Ha JU, Kim YM and Lee DS. (2001). Multilayered antimicro-

bial polyethylene films applied to the packaging of ground

beef. Packaging Technology and Science 14(2): 55–62.Hadwiger LA, Kendra DF, Fristensky BW and Wagoner W.

(1985). Chitosan both activated genes in plants and inhibits

RNA synthesis in fungi. In: Muzzarelli RAA, Jeuniaux Cand Gooday GW (eds) Chitin in Nature and Technology.New York: Plenum Press, 209–214.

Han JH. (2000). Antimicrobial food packaging. FoodTechnology 54(3): 56–65.

Han YS, Lee SH, Choi KH and Park I. (2010). Preparationand characterization of chitosan–clay nanocomposites with

antimicrobial activity. Journal of Physics and Chemistry ofSolids 71(4): 464–467.

Helander IM, Latva KK and Lounatmaa K. (1998).

Permeabilizing action of polyethyleneimine on Salmonellatyphimurium involves disruption of the outer membraneand interactions with lipopolysaccharide. Microbiology

144(2): 385–390.Helander IM, Nurmiaho-Lassila EL, Ahvenainen R, Rhoades

J and Roller S. (2001). Chitosan disrupts the barrier prop-erties of the outer membrane of Gram-negative bacteria.

International Journal of Food Microbiology 71(2–3):235–244.

Hernandez-Munoz P, Villalobos R and Chiralt A. (2004a).

Effect of cross-linking using aldehydes on properties ofglutenin-rich films. Food Hydrocolloids 18(3): 403–411.

Hernandez-Munoz P, Villalobos R and Chiralt A. (2004b).

Effect of thermal treatments on functional properties of

edible films made from wheat gluten fractions. Food

Hydrocolloids 18(4): 647–654.

Hirano S. (1997). Chitin and chitosan: Molecular and biolog-

ical functions newly generated by chemical modifications.

In: Domard A, Roberts AF and VarumKM (eds)Advances

in Chitin Science. Vol. 7(II): 10, Jacques Andre, Lyon

(France).

Hirano S andNagaoN. (1989). Effects of chitosan, pectic acid,

lysozyme and chitinase on the growth of several phytopath-

ogens. Agricultural and Biological Chemistry 53(11):

3065–3066.Hirano S, Sato N, Yoshida S and Kitagawa S. (1987).

Chemical modification of chitin and chitosan and their

novel applications. In: Yalpani M (ed.) Industrials

Polysaccharides: Genetic, Engineering, Structure/Property

Relations and Applications. Amsterdam, The Netherlands:

Elsevier, 163.Hoffman KL, Han IY and Dawson PL. (2001). Antimicrobial

effects of corn zein films impregnated with nisin, lauric acid,

and EDTA. Journal of Food Protection 64(6): 885–889.

Hotchkiss JH. (1995). Safety considerations in active packag-

ing. In: Rooney ML (ed.) Active Food Packaging. New

York: Blackie Academic and Professional, 238–255.Janes ME, Kooshesh S and Johnson MG. (2002). Control of

Listeria monocytogenes on the surface of refrigerated,

ready-to-eat chicken coated with edible zein film coatings

containing nisin and/or calcium propionate. Journal of

Food Science 67(7): 2754–2757.Jeon YJ and Kim SK. (2002). Antitumor activity of chitosan

oligosaccharides produced in ultrafiltration membrane

reactor system. Journal of Microbiology Biotechnology 12:

503–507.

Jeon YJ, Park PJ and Kim SK. (2001). Antimicrobial effect of

chitooligosaccharides produced by bioreactor. Carbo-

hydrate Polymers 44(1): 71–76.Jia Z and Shen D. (2002). Effect of reaction temperature and

reaction time on the preparation of low-molecular-weight

chitosan using phosphoric acid. Carbohydrate Polymers

49(4): 393–396.Jia Z, Shen D and Xu W. (2001). Synthesis and antibacterial

activities of quaternary ammonium salt of chitosan.

Carbohydrate Research 333(1): 1–6.

Jin T and ZhangH. (2008). Biodegradable polylactic acid poly-

mer with nisin for use in antimicrobial food packaging.

Journal of Food Science 73(3): M127–M134.Jung DS, Bodyfelt FW and Daechel MA. (1992). Influence of

fat and emulsifiers on the efficacy of nisin in inhibiting

Listeria monocytogens in fluid milk. Journal of Dairy

Science 75(2): 387–393.

Jung BO, Kim CH, Choi KS, Lee YM and Kim JJ. (1999).

Preparation of amphiphilic chitosan and their antimicro-

bial activities. Journal of Applied Polymer Science 72(13):

1713–1719.Kandemir N, Yemenicioglu A, Mecitoglu C, Elmaci ZS,

Arslanoglu A and Goksungur Y. (2005). Production of

antimicrobial films by incorporation of partially purified

lysozyme into biodegradable films of crude exopolysac-

charides obtained from Aureobasidium pullulans fermen-

tation. Food Technology and Biotechnology 43(4): 343–350.

Food Science and Technology International 18(1)

30

by guest on December 26, 2013fst.sagepub.comDownloaded from

Kendra DF and Hadwiger LA. (1984). Characterisation of thesmallest chitosan oligomer that is maximally antifungal toFusarium solani and elicits pisatin formation in isum sati-

vum. Experimental Mycology 8(3): 276–281.Kim CH, Cho JW and Chun HJ. (1997). Synthesis of chitosan

derivatives with quaternary ammonium salt and their anti-bacterial activity. Polymer Bulletin 38(4): 387–393.

Kim C and Choi KS. (1998). Synthesis and properties of car-boxyalkyl chitosan derivatives. Journal of Industrial andEngineering Chemistry 4(1): 19–25.

Kim C, Kim SY and Choi KS. (1997). Synthesis and antibac-terial activity of water-soluble chitin derivatives. Polymersfor Advanced Technologies 8(5): 319–325.

Klement Z. (1963). Rapid detection of the pathogenicity of phy-topathogenic pseudomonas. Nature 199(4890): 299–300.

Krajewska B. (2004). Application of chitin- and chitosan-

based materials for enzyme immobilizations: A review.Enzyme and Microbial Technology 35(2–3): 126–139.

Kubota N and Kikuchi Y. (1998). Macromolecular complexesof chitosan. In: Dumitriu S (ed.) Polysaccharides:

Structural Diversity and Functional Versatility. New York,NY, USA: Marcel Dekker Inc, 595–628.

Kurakake M, Nakagawa SYK, Sugihara M and Komaki T.

(2000). Properties of chitosanase from Bacillus cereus S1.Current Microbiology 40(1): 6–9.

Li B, Kennedy JF, Peng JL, Yie X and Xie BJ. (2006).

Preparation and performance evaluation of glucomannan-chitosan-nisin ternary antimicrobial blend film. Carbo-hydrate Polymers 65(4): 488–494.

Li B, Wang X, Chen R, Huangfu W and Xie G. (2008).

Antibacterial activity of chitosan solution againstXanthomonas pathogenic bacteria isolated fromEuphorbia pulcherrima. Carbohydrate Polymers 72(2):

287–292.Li Z, Zhuang XP, Liu XF, Guan YL and Yao KD. (2002).

Study on antibacterial O-carboxymethylated chitosan/

cellulose blend film from LiCl/N, N-dimethylacetamidesolution. Polymer 43(4): 1541–1547.

Li J, Zivanovic S, Davidson PM and Kit K. (2010).

Characterization and comparison of chitosan/PVP andchitosan/PEO blend films. Carbohydrate Polymers 79(3):786–791.

Liu N, Chen XG, Park HJ, Liu CG, Liu CS, Meng XH, et al.

(2006). Effect of MW and concentration of chitosan onantibacterial activity of Escherchia coli. CarbohydratePolymers 64(1): 60–65.

Liu H, Du Y, Wang X and Sun L. (2004). Chitosan kills bac-teria through cell membrane damage. International Journalof Food Microbiology 95(2): 147–155.

Liu XF, Guan YL, Yang DZ, Li Z and Yao KD. (2001).Antibacterial action of chitosan and carboxymethylatedchitosan. Journal of Applied Polymer Science 79(7):1324–1335.

Margolin AL. (1993). Enzymes in the synthesis of chiral drugs.Enzyme and Microbial Technology 15(4): 266–280.

Mathew S and Abraham TE. (2008). Characterisation of feru-

lic acid incorporated starch-chitosan blend films. FoodHydrocolloids 22(5): 826–835.

Mayachiew P and Devahastin S. (2008a). Antimicrobial and

antioxidant activities of Indian gooseberry and galangal

extracts. LWT – Food Science and Technology 41(7):1153–1159.

Mayachiew P and Devahastin S. (2008b). Comparative evalu-

ation of physical properties of edible chitosan films pre-pared by different drying methods. Drying Technology26(2): 176–185.

Mayachiew P, Devahastin S, Mackey BM and Niranjan K.

(2010). Effects of drying methods and conditions on anti-microbial activity of edible chitosan films enriched withgalangal extract. Food Research International 43(1):

125–132.Meyer RC, Winter AR and Weister HH. (1959). Edible pro-

tective coatings for extending the shelf life of poultry. Food

Technology 13(2): 146–148.Mi F-L, Huang C-T, Liang H-F, Chen M-C, Chiu Y-L and

Chen CH. (2006). Physicochemical, antimicrobial, and

cytotoxic characteristics of a chitosan film cross-linked bya naturally occurring cross-linking agent, aglycone genipo-sidic acid. Journal of Agricultural and Food Chemistry 54(9):3290–3296.

Ming X, Webber GH, Ayres JW and Sandine WE. (1997).Bacteriocins applied to food packaging materials to inhibitListeria monocytogenes on meats. Journal of Food Science

62(2): 413–415.Muzzarelli RAA. (1973). Natural chelating polymers.

In: Muzzarelli RAA (ed.) Natural Chelating Polymers:

Alginic Acid, Chitin, and Chitosan. Oxford, UK:Pergamon Press, 83–95.

Muzzarelli RAA, Muzzarelli C, Tarsi R, Miliani M,Gabbanelli F and Cartolari M. (2001). Fungistatic activity

of modified chitosans against Saprolegnia parasitica.Biomacromolecules 2(1): 165–169.

Muzzarelli RAA, Tarsi R, Filippini O, Giovanetti E, Biagini G

and Varaldo P. (1990). Antimicrobial properties of N-car-boxybutyl chitosan. Antimicrobial Agents Chemthery34(19): 2019–2023.

Muzzarelli RAA, Xia W, Tomasetti M and Ilari P. (1995).Depolymerization of chitosan and substtuted chitosanswith the aid of a wheat germ lipase preparation. Enzyme

and Microbial Technology 17(6): 541–545.Nadarajah K. (2005). Development and characterization of

antimicrobial edible films from crawfish chitosan.Louisiana, USA: PhD dissertation, Louisiana State

University.Natrajan N and Sheldon BW. (2000). Efficacy of nisin-coated

polymer films to inactive Salmonella typhimurium on fresh

broiler skin. Journal of Food Protection 63(9): 1189–1196.Nisperos-Carriedo MO. (1994). Edible coatings and films

based on polysaccharides. In: Krochta JM, Baldwin EA

and Nisperos-Carriedo MO (eds) Edible Coatings andFilms to Improve Food Quality. Lancaster, PA: TechnomicPublishing Company, 305–335.

No HK, Park NY, Lee SH and Meyers SP. (2002).

Antibacterial activity of chitosans and chitosan oligomerswith different molecular weights. International Journal ofFood Microbiology 74(1–2): 65–72.

Nordtveit RJ, Varum KM and Smidsrod O. (1994).Degradation of fully water soluble partially N-acetylatedchitosan with Lysozyme. Carbohydrate Polymers 23(4):

253–260.

Dutta et al.

31

by guest on December 26, 2013fst.sagepub.comDownloaded from

Ojagh SM, Rezaei M, Razavi SH and Hosseini SMH. (2010).

Effect of chitosan coatings enriched with cinnamon oil on

the quality of refrigerated rainbow trout. Food Chemistry

120(1): 193–198.Oonmetta-aree J, Suzuki T, Gasaluck P and Eumkeb G.

(2006). Antimicrobial properties and action of galangal

Alpinia galanga Linn. on Staphylococcus aureus. LWT –

Food Science and Technology 39(10): 1214–1220.Orrego CE, Salgado N, Valencia JS, Giraldo GI, Giraldo OH

and Cardona CA. (2010). Novel chitosan membranes as

support for lipases immobilization: Characterization

aspects. Carbohydrate Polymers 79(1): 9–16.Ouattara B, Simard RE, Piette G, Begin A and Holley RA.

(2000a). Diffusion of acetic and propionic acids from chit-

osan-based antimicrobial packaging films. Journal of Food

Science 65(5): 768–773.Ouattara B, Simard RE, Piette G, Begin A and Holley RA.

(2000b). Inhibition of surface spoilage bacteria in pro-

cessed meats by application of antimicrobial films prepared

with chitosan. International Journal of Food Microbiology

62(1–2): 139–148.Ozdemir M and Floros JD. (2004). Active food packaging

technologies. Critical Reviews in Food Science and

Nutrition 44(3): 185–193.Padgett T, Han IY and Dawson PL. (1998). Incorporation of

foodgrade antimicrobial compounds into biodegradable

packaging films. Journal of Food Protection 61(10):

1330–1335.Pantaleone D, Yalpani M and Scollar M. (1992). Unusual sus-

ceptibility of chitosan to enzymic hydrolysis. Carbohydrate

Research 237(12): 325–332.Papineau AM, Hoover DG, Knorr D and Farkas DF. (1991).

Antimicrobial effect of water-soluble chitosans with high

hydrostatic pressure. Food Biotechnology 5(1): 45–57.

Park SY, Marsh KS and Rhim JW. (2002). Characteristics of

different molecular weight chitosan films affected by the

type of organic solvents. Journal of Food Science 67(1):

194–197.Pelletier A and Sygusch J. (1990). Purification and character-

ization of three chitosanase activities from Bacillus mega-

terium P1. Applied and Environmental Microbiology 56(4):

844–848.

Plouffe B, Sane AT and Brzezinski R. (1997). Engineering of

the chitosanase from Steptomyces sp N174 for an easy

immobilization on cellulose. In: Domard A, Roberts AF

and Varum KM (eds) Advances in Chitin Science.

Vol. 7(II): 236, Jacques Andre, Lyon (France).

Portes E, Gardrat C, Castellan A and Coma V. (2009).

Environmentally friendly films based on chitosan and tet-

rahydrocurcuminoid derivatives exhibiting antibacterial

and antioxidative properties. Carbohydrate Polymers

76(4): 578–584.

Pranoto Y, Rakshit SK and Salokhe VM. (2005). Enhancing

antimicrobial activity of chitosan films by incorporating

garlic oil, potassium sorbate and nisin. Lebensmittel-

Wissenschaft und-Technologie 38(8): 859–865.Qi L, Xu Z, Jiang X, HuC and Zou X. (2004). Preparation and

antibacterial activity of chitosan nanoparticles. Carbo-

hydrate Research 339(16): 2693–2700.

Qin C, Li H, Xiao Q, Liu Y, Zhu J and Du Y. (2006). Water-solubility of chitosan and its antimicrobial activity.Carbohydrate polymers 63(3): 367–374.

Quintavalla S and Vicini L. (2002). Antimicrobial food pack-aging in meat industry. Meat Science 62(3): 373–380.

Rabea EI, Badawy MET, Stevens CV, Smagghe G andSteurbaut W. (2003). Chitosan as antimicrobial agent:

Applications and mode of action. Biomacromolecules 4(6):1457–1465.

Ralston GB, Tracey MV and Wrench PM. (1964). The inhibi-

tion of fermentation in baker’s yeast by chitosan.Biochimica and Biophysica Acta 93: 652–655.

Ray B. (1992). Nisin of Lactococcus lactis subsp. Lactis as a

food biopreservative. In: Ray B and Daeschel M (eds) FoodBiopreservatives of Microbial Origins. Boca Raton, FL:CRC Press Inc, 207–264.

Rhim JW, Hong SI, Park HM andNg PK. (2006). Preparationand characterization of chitosan-based nanocompositefilms with antimicrobial activity. Journal of Agriculturaland Food Chemistry 54(16): 5814–5822.

Rhoades J and Rastall B(2000). Chitosan as an AntimicrobialAgent. Available at: www.fpi-international.com/articles/ingredients_additives/032_FTI007.pdf.

Rhoades J and Roller S. (2000). Antimicrobial actions ofdegraded and native chitosan against spoilage organismsin laboratory media and foods. Applied and

Environmental Microbiology 66(1): 80–84.Rivas LA, Parro V, Moreno-Paz M and Mellado RP. (2000).

The Bacillus subtilis 168 csn gene encodes a chitosanasewith similar properties to a Streptomyces enzyme.

Microbiology 146(Pt. 11): 2929–2936.Roberts GAF. (1992). Chitin Chemistry. Indianapolis, IN,

USA: Macmillan, 274–301.

Ru MT, Hirokane SY, Lo AS, Dordick H, Reimer JA andClark DS. (2000). On the salt-induced activation of lyoph-ilized enzymes in organic solvents: Effect of salt cosmotro-

picity on enzyme activity. Journal of American ChemicalSociety 122(8): 1565–1571.

Salleh E, Muhamad I and Khairuddin N. (2007). Preparation,

characterization and antimicrobial analysis of antimicro-bial starch-based film incorporated with chitosan andlauric acid. Asian Chitin Journal 3: 55–68.

Sanchez-Gonzalez L, Gonzalez-Martınez C, Chiralt A and

Chafer M. (2010). Physical and antimicrobial propertiesof chitosan–tea tree essential oil composite films. Journalof Food Engineering 98(4): 443–452.

Sangsuwan J, Rattanapanone N and Rachtanapun P. (2008).Effect of chitosan/methyl cellulose films on microbialand quality characteristics of fresh-cut cantaloupe and

pineapple. Postharvest Biology and Technology 49(3):403–410.

Savard T, Beauliu C, Boucher I and Champagne CP. (2002).Antimicrobial action of hydrolyzed chitosan against spoil-

age yeasts and lactic acid bacteria of fermented vegetables.Journal of Food Protection 65(5): 828–833.

Scanell AGM, Hill C, Ross RP, Marx S, Hartmeier W and

Arendt EK. (2000). Development of bioactive food pack-aging materials using immobilized bacteriocins Lacticin3147 and Nisaplin�. International Journal of Food

Microbiology 60(2–3): 241–249.

Food Science and Technology International 18(1)

32

by guest on December 26, 2013fst.sagepub.comDownloaded from

Seki K, Kuriyama H, Okuda T and Uchida Y. (1997).Molecular cloning of the gene encoding chitosanase fromBacillus amyloliquefaciens UTK. In: Domard A, Roberts

AF and Varum KM (eds) Advances in Chitin Science.Vol. 7(II): 284, Jacques Andre, Lyon (France).

Sekiguchi S. (1994). Molecular weight dependency of antimi-crobial activity by chitosan oligomers. In: Nishinari K and

Doi E (eds) Food Hydrocolloids: Structures, Properties andFunctions. New York, NY, USA: Plenum Press, 71–76.

Seydim AC. (2006). Antimicrobial activity of whey protein

based edible films incorporated with oregano, rosemary,and garlic essential oils. Food Research International39(5): 639–644.

Shahidi F, Arachchi JKV and Jeon YJ. (1999). Food applica-tion of chitin and chitosans. Trends in Food Science andTechnology 10(2): 37–51.

Sharma R, Chisti Y and Banerjee UC. (2001). Production,purification, characterization, and applications of lipases.Biotechnology Advances 19(8): 627–662.

Sheldon BW. (2001). Development of an inhibitory absorbent

cellulose gum tray pads for reducing spoilage microorgan-isms and the risk of cross contamination. Poultry Science80(1): 17.

Sheldon BW, Hale SA and Beard BM. (1996). Efficacy ofincorporating nisin-based formulations into absorbentmeat tray pads to control pathogens and spoilage organ-

isms. Poultry Science 75(Suppl. 1): 7.Shen XL, Jia MW, Yonghong C and Guohua Z. (2010).

Antimicrobial and physical properties of sweet potatostarch films incorporated with potassium sorbate or chito-

san. Food Hydrocolloids 24(4): 285–290.Shu XZ and Zhu KJ. (2000). A novel approach to prepare

tripolyphosphate/ chitosan complex beads for con-

trolled release drug delivery. International Journal ofPharmaceutics 201(1): 51–58.

Sikorski P, SorbottenA,Horn SJ, Eijsink VG andVarumKM.

(2006). Serratia marcessens chitinases with tunnel-shapedsubstrate-binding grooves show endo activity and differentdegrees of processivity during enzymatic hydrolysis of chit-

osan. Biochemistry 45(31): 9566–9574.Simpson BK, Gagne N, Ashie INA and Noroozi E. (1997).

Utilization of chitosan for preservation of raw shrimp.Food Biotechnology 11(1): 25–44.

Siragusa GR and Dickson JS. (1992). Inhibition of Listeriamonocytogenes on beef tissue by application of organicacids immobilized in a calcium alginate gel. Journal of

Food Science 57(2): 293–296.Siragusa GR and Dickson JS. (1993). Inhibition of Listeria

monocytogenes, Salmonella typhimurium and Escherichia

coli O157:H7 on beef muscle tissue by lactic or acetic acidcontained in calcium alginate gels. Journal of Food Safety13(2): 147–158.

Song YH, Li LF and Zheng Q. (2009). Influence of methylcel-

lulose on properties of wheat gliadin film cast from aqueousethanol. Food Science and Biotechnology 18(4): 910–916.

Speciale A, Musumeci R, Blanddino G,Milazzo I, Caccamo F

and Nicoletti G. (2002). Minimal inhibitory concentrationsand time-kill determination of moxifloxacin againstaerobic and anaerobic isolates. International Journal of

Antimicrobial Agents 19(2): 111–118.

Strand SP, Varum KM and Ostgaard K. (2003). Interactionsbetween chitosans and bacterial suspensions: Adsorptionand flocculation. Colloids and Surfaces B: Biointerfaces

27(1): 71–81.Struszczyk H and Pospieszny H. (1997). New applications of

chitin and its derivatives in plant protection. In: GoosenMFA (ed.) Applications of Chitin and Chitosan Chapter

11. Lancaster, UK: Technomic publishing Co, 171–184.SudarshanNR,HooverDGandKnorrD. (1992).Antibacterial

action of chitosan. Food Biotechnology 6(3): 257–272.

Suppakul P, Miltz J, Sonneveld K and Bigger SW. (2003).Active packaging technologies with an emphasis on antimi-crobial packaging and its applications. Journal of Food

Science 68(2): 408–420.Sun L, Du Y, Chen X, Li J and Shi X. (2005). Quarternized

carboxymethyl chitosan: A new approach to chemically

modified chitosan III: Antimicrobial activity and applica-tion as pulp-cup. Asian Chitin Journal 1: 39–48.

Tian M, Chen F, Ren D, Yu X, Zhang X, Zhong R andWanaC. (2010). Preparation of a series of chitooligomers and

their effect on hepatocytes. Carbohydrate Polymers 79(1):137–144.

Tian F, Liu Y, Hu K and Zhao B. (2004). Study of the depo-

lymerization behaviour of chitosan by hydrogen peroxide.Carbohydrate Polymers 57(1): 31–37.

Tokoro A, Suzuki K, Matsumoto T, Mikami T, Suzuki S and

Suzuki M. (1988). Chemotactic response of human neutro-phils of N-acetylchitohexaose in vitro. Microbiology andImmunology 32(4): 387–395.

Tokura S, Miuray Y, Johmen M, Nishi N and Nishimura SI.

(1994). Induction of drug specific antibody and the con-trolled release of drug by 6-O-carboxymethyl-chitin.Journal of Controlled Release 28(1–3): 235–241.

Tokura S, UeonK,Miyazaki S andNishi N. (1997).Molecularweight dependent antimicrobial activity by chitosan.Macromolecular Symposia 120: 1–9.

Torres JA, Motoki M and Karel M. (1985). Microbial stabili-zation of intermediate moisture food surfaces I control ofsurface preservative concentration. Journal of Food

Processing and Preservation 9(2): 75–92.Tripathi S, Mehrotra GK, Tripathi CKM, Banerjee B, Joshi

AK and Dutta PK. (2008). Chitosan based bioactive film:Functional properties towards biotechnological needs.

Asign Chitin Journal 4: 29–36.Tripathi S, Mehrotra GK and Dutta PK. (2009). New

approach for using chitosan based ntimicrobial film in

food packaging. Proceedings of the Advances in ChemicalEngineering. India: Tata Macmillan, 69–73.

Tripathi S, Mehrotra GK and Dutta PK. (2010). Preparation

and physicochemical study of antimicrobial chitosan starchfilm via supercritical CO2 method. National Seminaron Contemporary Research in Material Science andChemical Biology. University of Allahabad, Allahabad.

Tsai ML, Bai SW and Chen RH. (2008). Cavitation effectsversus stretch effects resulted in different size and polydis-persity of ionotropic gelation chitosan–sodium tripolyphos-

phate nanoparticle.Carbohydrate Polymers 71(3): 448–457.Tsai GJ and Su WH. (1999). Antibacterial activity of shrimp

chitosan against Escherichia coli. Journal of Food

Protection 62(3): 239–243.

Dutta et al.

33

by guest on December 26, 2013fst.sagepub.comDownloaded from

Tsai GJ,WuZY and SuWH. (2000). Antibacterial activity of achitooligosaccharide mixture prepared by cellulase diges-tion of shrimp chitosan and its application to milk preser-

vation. Journal of Food protection 63(6): 747–752.Uchida Y. (1989). Preparation of chitosan oligomers with

purified chitosanase and its application. In: Skjak-BraekG, Anthonsen T and Sandford P (eds) Chitin and

Chitosan: Sources, Chemistry, Biochemistry, PhysicalProperties and Applications. London, UK: ElsevierApplied Science, 373–382.

Vartiainen J, Motion R, Kulonen K, Ratto M, Skytta E andAdvenainen R. (2004). Chitosan-coated paper: Effects ofnisin and different acids on the antimicrobial activity.

Journal of Applied Polymer Science 94(3): 986–993.WangGH. (1992). Inhibition and inactivation of five species of

foodborne pathogens by chitosan. Journal of Food

Protection 55(11): 916–919.Wang X, Du Y, Luo J, Lin B and Kennedy JF. (2007).

Chitosan/organic rectorite nanocomposite films:Structure, characteristic and drug delivery behaviour.

Carbohydrate Polymers 69(1): 41–49.Wang P, Hill TG,WartchowCA,HustonME, Oehler LM and

Smith MB. (1992). New carbohydrate-based materials for

the stabilization of proteins. Journal of the American

Chemical Society 114(1): 378–380.

Whistler RL and Daniel JR. (1990). Functions of polysaccha-

rides in foods. In: Branen AL, Davidson PM and Salminen

S (eds) Food Additives. New York: Marcel Dekker,

395–424.Yacoby I and Benhar I. (2008). Antibacterial nanomedicine.

Nanomedicine 3(3): 329–341.Yalpani M and Pantaleone D. (1994). An examination of the

unusual susceptibilities of aminoglycans to enzymatic

hydrolysis. Carbohydrate Research 256(1): 159–175.

You YJ, Jo KJ, Jin YL, Kim KY, Shim JH, Kim YW, et al.

(2003). Characterization and kinetics of 45-kDa chitosa-

nase from Bacillus sp. P16. Bioscience Biotechnology and

Biochemistry 67(9): 1875–1882.Zhang K, Xu J, Wang KY, Cheng L, Wang J and Liu B.

(2009). Preparation and characterization of chitosan nano-

composites with vermiculite of different modification.

Polymer Degradation and Stability 94(12): 2121–2127.

Zheng LY and Zhu JF. (2003). Study of antimicrobial activity

of chitosan with different molecular weight. Carbohydrate

Polymers 54(4): 527–530.

Food Science and Technology International 18(1)

34

by guest on December 26, 2013fst.sagepub.comDownloaded from