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Progress in antimicrobial activities of chitin, chitosan and its oligosaccharides: a systematic study
needs for food applications
J. Dutta, S. Tripathi and P.K. Dutta
Food Science and Technology International 2012 18: 3 originally published online 27 September 2011
DOI: 10.1177/1082013211399195

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Article

Progress in antimicrobial activities of chitin,

chitosan and its oligosaccharides: a systematic
study needs for food applications

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

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

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

the enormous losses of food and hence, over the years,

INTRODUCTION
Once we talk about antimicrobial activity, it is always
directed to its applicability. Very recently, the research is
focused into development of materials with film-forming
capacities and those having antimicrobial properties
which help to improve food safety and shelf-life.
Antimicrobial packaging is one of the most promising
active packaging systems that have been found highly
effective in killing or inhibiting spoilage and pathogenic
microorganisms that contaminate foods (Salleh et al.,
2007). In this context, chitosan films have shown great
promise for their application in food preservation. It is
well known that microbial alternation is responsible for
Food Science and Technology International 18(1) 3–34
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DOI: 10.1177/1082013211399195
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various chemical and physical processes have been
developed to extend the shelf-life of foods. The antimi-
crobial activity limits or prevents microbial growth by
extending the lag period and reducing the growth rate or
decreases live counts of microorganisms (Han, 2000).
Currently, food application of an antimicrobial packag-
ing system is limited due to the availability of suitable
antimicrobials, new polymer materials, regulatory con-
cerns and appropriate testing methods (Jin and Zhang,
2008). In particular, polymeric bioactive films laced with
an assortment of antimicrobial agents have been found
1
Department of Chemistry, Disha Institute of Management and
Technology, Raipur 400701, India
2
Department of Chemistry, Motilal Nehru National Institute of
Technology, Allahabad 211004, India
Corresponding author:
P.K. Dutta, Department of Chemistry, Motilal Nehru National
Institute of Technology, Allahabad 211004, India
Email: pkd_437@rediffmail.com

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Food Science and Technology International 18(1)
CH2OH
O O
H H
O Conc. NaOH
OH
Deacetylation
H NHCOCH3
n n
Chitin
Figure 1. Structure of chitin and chitosan.
Table 1. Active properties of edible films and coatings
Different additives for
improvement of edible films Improvement of
and coatings property
Pigments, light absorbers, Color, transparency,
shininess salts and other food roughness, sticking,
additives like citric acid, oleic microbe resistance, etc.
acid, etc., flavors, spices,
antimicrobial and antioxidant
agents
to be very effective and practical in applications. Till
date, a number of articles have been published describ-
ing the nature of different materials used in making
films 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 and
Dutta, 2010; Dutta et al., 2009; Ozdemir and Floros,
2004; Quintavalla and Vicini, 2002; Suppakul et al.,
2003). Present review aims to summarize all the known
methods of formation of chitosan-based films with anti-
microbial properties and discuss their mode of action
and 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 all
over the world has been focused on preservation of the
food as a protection from various microorganisms. In
this direction, bioactive polymeric films have been found
very effective as antimicrobial agents and thus their
applications and research work on them are getting
more importance today. This review is an attempt to
create awareness about the progress in the antimicrobial
activities of chitin and chitosan in the case of food appli-
cations and the literature survey made from the year
1957 to present. More specifically, the aim of this
review was to highlight the potential of chitosan as an
4
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CH2OH
O
OH
H NH2
Chitosan
ingredient for the production of active bio-based films
and to summarize the different methods used for the
preparation of chitosan-based films and their perspec-
tives in the modern food packaging technology.
The active properties of edible films and coatings are
described in Table 1.
CHITIN, CHITOSAN AND THEIR
OLIGOSACCHARIDES VERSUS OTHER
POLYMERS IN ANTIMICROBIAL
ACTIVITY
Polysaccharide films are derived from various natural
materials which impart gel-forming ability to a variety
of films. The polysaccharide-derived films, due to excel-
lent gas permeability properties, exhibit desirable
modified atmospheres and thus enhance the shelf-life
of the product without creating anaerobic conditions
(Baldwin et al., 1995; Ben and Kurth, 1995; Cutter and
Sumner, 2002; Glicksman, 1983; Nisperos-Carriedo,
1994; Whistler and Daniel, 1990). Furthermore, poly-
saccharide films and coatings can be used to extend the
shelf-life of muscle foods by preventing dehydration,
oxidative rancidity and surface browning (Nisperos-
Carriedo, 1994).
Particularly, the use of biopolymer and bio-based
polymer films as antimicrobial delivery systems to
reduce undesirable bacteria in foodstuffs is not a novel
concept. Various approaches have been proposed and
demonstrated for the use of these films to deliver com-
pounds to a variety of food surfaces, including muscle
foods. For example, antimicrobial compounds such as
organic acids (acetic propionic, benzoic, sorbic, lactic
and lauric), potassium sorbate, bacteriocins (nisin and
lacticin), grape seed extracts, spice extracts (thymol,
p-cymene and cinnamaldehyde), thiosulfinates (allicin),
enzymes (peroxidase and lysozyme), proteins (conalbu-
min), isothiocyanates (allylisothiocyanate), antibiotics
(imazalil), fungicides (benomyl), chelating agents

(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 and
Daniel, 1990; Han, 2000). Further studies on edible film
made from starch, carrageenan or alginate and addition
of antifungal compounds, organic acids, potassium sor-
bate or the bacteriocin nisin, were more effective for
reducing 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 and
Dickson, 1992, 1993). The use of biopolymers and bio-
based polymer films on a variety of food surfaces and the
result-oriented observation of different workers are
given 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/or
natural antimicrobial compounds has a significant role
in finding out the practical applications in the food
industry. This specific information demonstrates the fea-
sibility and applicability for incorporating various anti-
microbial compounds with a range of inhibitory activity
directly into bio-based or edible packaging materials for
use in controlling food spoilage as well as enhancing
microbial safety of muscle foods. Table 3 focuses on
the study of chitosan films for packaging films for pro-
cessed meats and seafood, as well as those combined
with various ingredients.
Due to their inherent properties, coupled with
the ability to form films, alone or in combination
with 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-BASED
ANTIMICROBIAL FILMS/COATINGS
Various methods are employed to prepare chitosan films
and coatings for food packaging applications. Solution
casting method is one of the popular methods. As a gen-
eral practice, chitosan films are prepared with different
kinds of oils like cinnamon oil, tea tree essential oil
(TTEO); tetrahydrocurcuminoid derivatives; blends
with ethylene–vinyl alcohol copolymer, PVP or PEO,
Gliadins. The methods of preparation of novel hydro-
xypropyl methylcellulose (HPMC) edible films with
chitosan/tripolyphosphate nanoparticles; nanoparticles/
plasticized-starch composites; edible chitosan films
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Dutta et al.
enriched with galangal extract; sweet potato starch
films incorporated with potassium sorbate on
chitosan; and chitosan/methyl cellulose films have also
been 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 microwave
technique. The novelty of this method lies in achieving
the film formation without the addition of any cross-
linker or plasticizer.
Some typical preparative techniques are enumerated
in the following sections.
Preparation of chitosan-coating solution
enriched with cinnamon oil
Chitosan solution was prepared with 2% (w/v) chitosan
in 1% (v/v) acetic acid. To achieve complete dispersion
of chitosan, the solution was stirred at room tempera-
ture for 3 h. The solution in beakers was placed on a
hotplate/magnetic stirrer and glycerol added to chitosan
at 0.75 mL/g concentration as a plasticizer and stirred
for 10 min. The resultant chitosan-coating solution was
filtrated through a Whatman No. 3 filter paper to
remove any undissolved particles. Then, the cinnamon
oil, mixed with Tween 80, to help in distributing and
completely incorporating the cinnamon oil, was added
to the chitosan solution. The final coating forming solu-
tion consisted of 2% chitosan, 1% acetic acid, 0.75%
glycerol, 0.2% Tween 80 and 1.5% cinnamon oil. The
final coating forming solution was homogenized under
aseptic conditions at 21 600 rpm for 1 min. The control
solution was prepared without the addition of cinnamon
oil (Ojagh et al., 2010).
Preparation of environment-friendly films based
on chitosan and tetrahydrocurcuminoid
derivatives
Homogeneous chitosan films. A 2% (w/v) 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 Büchner funnel and then
degassed under reduced pressure for 1 h. Films were
obtained by casting the solutions onto polystyrene
plates which were dried for 12 h under a laminar-flow
hood at room temperature. Thanks to degasification and
drying under laminar-flow, even at room temperature,
most of the volatile acetic acid could be removed from
the thin films (Portes et al., 2009).
Chitosan–tetrahydrocurcuminoid films. Film-forming
solutions (2%) were prepared by adding chitosan (2 g)
and tetrahydrocurcuminoid (THC; 20 mg) to 100 mL of
a 1% acetic acid aqueous solution (pH 3.5) under
5
Table 2. Use of biopolymer and bio-based polymer films in a variety of food surfaces and the result-oriented observations
of different researchers

Basic matrix Ingredients Activity and observation References

Carrageenan film Antibiotics and anti- To reduce bacteria by 2 log 10 (99%) on Meyer et al. (1959)
fungal compounds poultry
Edible coatings from Antimycotic agents – Hotchkiss (1995)
waxes and cellulose
ethers
Beef carcass tissue Organic acids, More efficacious for reducing levels of L. Siragusa and
calcium alginate monocytogenes, S. typhimurium, and E. coli Dickson (1992,
O157:H7 when immobilized 1993)
Edible cornstarch Potassium sorbate To inhibit S. typhimurium and E. coli O157:H7 Baron and Sumner
film and lactic acid on poultry (1994)
Calcium alginate Bacteriocin nisin Resulted in greater and sustained bacteriocin Cutter and Siragusa
gels on lean and compared to nisin- activity when the tissues were ground and (1996, 1997)
adipose beef only controls stored under refrigerated conditions for up to
surfaces 7 days
Pork Calcium alginate Reductions in pathogen populations Fang and Lin (1994)
containing nisin
Edible heat-set and Nisin and lysozyme Exhibit activity against E. coli and L. Dawson et al. (1996)
cast films made from plantarum and Padgett et al.
corn zein or soy (1998)
protein
Corn zein films EDTA, lauric acid, Significant reductions of Listeria Hoffman et al. (2001)
nisin and combina- monocytogenes in solution
tions of the three
compounds
Plastic-based pack- Nisin surface of hot Listeria monocytogenes could be inhibited Franklin et al. (2004)
aging films treated dogs >2 log 10 (99%)
with methylcellulose/
HPMC-based
solutions
Gelatin-based Lysozyme, nisin and To control spoilage and pathogenic organ- Gill (2000)
coatings EDTA ham and isms such as L. sakei, Leuconostoc mesen-
sausage teroides, L. monocytogenes and S.
typhimurium
Ibrinogen/thrombin- Bacteriocins, nisin Provide an added antimicrobial and advan- Cutter and Siragusa
based gel (Fibrimex) and bovine tage to restructured raw meat products that (1997)
incorporate surface tissues into the product
interior or as a delivery system for antimicro-
bials to meat surfaces
Collagen (Coffi) films Nisin Brochothrix thermosphacta and L. monocyto- Cutter and Miller
(NICF) on hot dog genes were inhibited following treatments (2004)
surfaces with subjected to temperature abuse as well
as long-term refrigerated storage
Milk proteins for Essential oils of Antimicrobials were treated and evaluated Seydim (2006)
edible films and oregano, rosemary against E. coli O157:H7, S. aureus,
coatings for foods, and garlic Salmonella enteritidis, L. monocytogenes and
whey protein films L. plantarum
Pullulan films as pro- Lysozyme and diso- Evaluated for antimicrobial effectiveness Kandemir et al.
duced by fungi dium EDTA against E. coli and L. plantarum. (2005)
during fermentation. The resulting films with a neutral pH were
Pullulan films made composed of glucans and polysaccharides,
from the exopoly- and were water soluble, transparent, and
saccharides of exhibited low oxygen permeability. These
Aureobasidium antimicrobial films were stable for up to
pulluans 21 days during cold storage and could inhibit
the E. coli under laboratory conditions

6
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 Dutta et al.

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, as For inhibiting microorganisms Vartiainen et al. (2004)
well as nisin and coated onto the
surfaces of paper
Edible film made from 3% or 5% Evaluated an against S. enteritidis in suspen- Durango et al. (2006)
chitosan and yam starch sions. When applied directly to cell suspensions,
1% chitosan reduced the pathogen > 4 log10
CFU/mL (or 99.99%).
Chitosan-treated films made with 3% Reduced populations of S. enteritidis
or 5% chitosan >1 log10 CFU/mL (or 90%).
Chitosan-treated films made with 5% chitosan
were the most efficient means of treatment for
inhibiting S. enteritidis in solution
Nisin into chitosan To inhibit L. monocytogenes. In solution and in Cooksey (2005)
agar diffusion assays, the antimicrobial film
inhibited the pathogen, but no further studies
were conducted in meat systems
Chitosan coatings Reduced microbial contamination on shrimp and Simpson et al. (1997)
oysters, respectively Chen et al. (1998)

vigorous agitation. The solutions were filtered and cast
as in the procedure used to make homogeneous chitosan
films. Ten thickness values were taken randomly at dif-
ferent positions on each film. Film thicknesses were
found to be 30  4 l mm. A good repeatability of the
absorbances directly measured on different portions of
the films showed that the THCs were distributed
homogeneously.
Antibacterial chitosan-based blends with
ethylene–vinyl alcohol (EVOH) copolymer
Chitosan polysaccharide dispersions were prepared
in 2% (v/v) acetic acid to a final concentration of 3%
(w/v) and stirred at 37  C for approximately 3 h. The
chitosonium–acetate solutions were filtered through
polyester cloth to remove residues of insoluble particles
and then autoclaved before their further use for blending.
Pure chitosonium–acetate films were obtained by casting
at 37  C, 80  C and 120  C. In addition, pure EVOH films
were obtained by the same method at 80  C. Composite
films of EVOH29/LMW–chitosan with high ratios of
polysaccharide (i.e., 50 and 80 wt%) were obtained
under various temperature conditions (37  C, 80  C and
120  C). Blends of EVOH with lower contents of chito-
san did not form good films but 80/20 (wt%) EVOH/
chitosonium–acetate blends could be finally obtained
by adding an excess of glacial acetic acid to the EVOH
solution to a final concentration of 2% (w/v).
Nevertheless, the higher chitosan concentration blends,
i.e., 50 and 80 wt%, were also obtained using the same
solution of EVOH and acetic acid for comparison pur-
poses. Typical pure and composite films with a thickness
of ca. 50 lm were obtained (Fernandez-Saiz et al., 2010).
Preparation of chitosan/PVP
and chitosan/PEO blend films
Chitosan solution was prepared with 1% (w/w) chitosan
in 1% (v/v) acetic acid, stirred overnight at room tem-
perature and filtered through MiraclothÕ to remove
impurities. PVP and PEO were separately dissolved in
d.i. water to form 1% (w/w) solutions. Aqueous solu-
tions of the individual polymers were mixed to prepare a
series of chitosan/PVP and chitosan/PEO blend solu-
tions with weight ratios 100/0, 75/25, 50/50, 25/75 and
0/100. Aliquots of 10 g of film-forming solutions were
poured into 50 mm-diameter polystyrene Petri dishes
and the solvent was evaporated in a vacuum oven at
38  C under 17 kPa pressure for 24 h. The dried films
were peeled from the Petri dishes and conditioned in
desiccators at 25  C and 20% relative humidity (RH)
prior to testing (Li et al., 2010).
Preparation of novel HPMC edible films with
chitosan/tripolyphosphate nanoparticles
Preparation of chitosan–tripolyphosphate nano-
particles. The chitosan–tripolyphosphate (CS–TPP)
nanoparticles were prepared on the ionic gelation of
CS with TPP anions. Chitosan was dissolved in acetic
acid solution at concentrations of 3.00 and 4.41 mg/mL.

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Food Science and Technology International 18(1)
The concentration of acetic acid in aqueous solution
was, in all cases, 1.5 times that of chitosan. Under mag-
netic stirring at room temperature, 28 mL sodium TPP
aqueous solution with concentrations 1.2 and 2.1 mg/
mL were added into 70 mL chitosan solution. The prep-
arations were mixed with a homogenizer at 6000 rpm
with continuous addition of TPP solution at the
rate of 1 mL/min. The zone of opalescent suspension
was further examined as nanoparticles (de Moura
et al., 2009).
Preparation of solutions for film casting. The HPMC
solution (control film) was obtained by dissolving 3.0 g
of HPMC in 100 mL of distilled water under magnetic
stirring for 12 h. The HPMC: water ratio in all film-
forming solutions was 3:97 to study the effect of particle
size and CS–TPP concentration in the HPMC film
matrix. The HPMC films with CS–TPP nanoparticles
were obtained by addition of 3.0 g of HPMC in
100 mL of nanoparticle solution (recently synthesized)
under magnetic stirring for 12 h. After the solutions were
prepared, the flasks were kept closed for 6 h to prevent
microbubble formation in the films. The solutions were
then poured in a glass plate (30  30 cm2) covered with
Mylar (Polyester film) for film casting preparation. The
solutions were cast at a wet thickness of 0.5 mm onto
plates using casting bars and the plates placed on a
leveled surface at room temperature and let to dry for
24 h. After drying, the 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 ionotropic
gelation between chitosan and sodium tripolyphosphate
with some modifications (Shu and Zhu, 2000; Tsai et al.,
2008). Chitosan was dissolved in 2% (v/v) acetic acid to
obtain a 1% (w/v) chitosan solution. Under vigorous
stirring at room temperature, 8 mL of 1% (w/v) TPP
aqueous solutions was added dropwise, at the rate of
1 mL/min, into 200 mL of chitosan solution. The mix-
ture was stirred for 30 min, and treated with sonication
for 30 min. The suspension was subsequently centri-
fuged at 12,000 rpm for 20 min. The precipitate was
washed with water and centrifuged again twice. The pre-
cipitate was washed in ethanol and then dried (Chang
et al., 2010).
Processing of glycerol/potato starch/chitosan
composites. Chitosans were dispersed in a solution of
distilled water (100 mL) and glycerol (1.5 g) and ultra-
sonicated for 0.5 h before adding 5 g of potato starch.
The chitosan filler loading level (0, 1, 2, 4, 6 and 8 wt%)
8
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was based on the amount of potato starch. The mixture
was heated at 90  C for 0.5 h with constant stirring in
order to plasticize the starch. To obtain the glycerol/
potato starch (GPS)/chitosan composite films, the mix-
ture was cast into a dish and placed in an air-circulating
oven at 50  C until dry (about 6 h). The composite films
were preconditioned in a climate chamber at 25  C and
50% RH for at least 48 h prior to the testing. Water
content of the films was about 10 wt%.
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 overnight
agitation, TTEO was added to the chitosan (CH) solu-
tion to reach a final concentration of 0%, 0.5%, 1% and
2% (w/w). CH–TTEO mixtures were emulsified at
room temperature using a rotor–stator homogenizer at
13,500 rpm for 4 min. These emulsions were vacuum
degasified at room temperature with a vacuum pump.
The experimental design was made taking into account
the maximum levels of TTEO which could be incorpo-
rated into the matrix without oil phase separation during
the film drying (Sánchez-González et al., 2010).
Preparation of films. Films were obtained by the casting
procedure given as follows: film-forming dispersions
(FFDs) were poured onto a framed and leveled polyte-
trafluoroethylene (PTFE) plate (j ¼ 15 cm) and dried
under atmospheric conditions for 48 h. Film thickness
was controlled by pouring the amount of FFD that will
provide a surface density of solids in the dry films of 56 g/
m2 in all formulations. Dry films were peeled off from
the casting surface and preconditioned in desiccators
at 20  C.
Preparation of blend films of gliadins and
chitosan
A total of 100 g of wheat gluten powder was suspended
in 350 mL pure ethanol and 150 mL distilled water was
transferred into the suspension under stirring. The mix-
ture was magnetic stirred for 1 h and centrifuged at
3000 rpm for 15 min at 25  C. The supernatant contain-
ing the gliadin-rich fraction was collected as the film-
forming solution. This method was different from the
general way that crude wheat gluten was dispersed in
70% (v/v) aqueous ethanol and stirred for about 12 h
to extract glianidins (Hernandez-Munoz et al.,
2004a,b; Song et al., 2009). Glycerol, as plasticizer,
was added into the supernatant in a content of 20 g/
100 g dry protein. About 2 g of CS were dispersed in
97 g water before 1 g acetic acid was added to the mixture
under stirring to give a CS content of 2.0 wt%. By doing

this, clotting of CS in acetic acid aqueous solution could
be avoided. Glycerol plasticizer was added into the
solution in a content of 20 g/100 g CS. The gliadin
and CS solutions were mixed and stirred for 20 min.
The weight content of CS, wCS, defined as the weight
ratio of CS to total weight of CS plus gliadins, was
varied from 0 to 100 wt%. The final solution was
poured onto horizontal plastic dishes and dried at
25  C. The dried films were carefully peeled off from
the dishes and preconditioned at 25  C at 57.5% RH
for 3 days at least before testing.
Preparation of edible chitosan films enriched
with galangal extract
Preparation of chitosan solution. In this method, 1.5%
(w/v) chitosan solution was prepared by dissolving chit-
osan in 1% (v/v) acetic acid under constant stirring at
300 rpm using a magnetic stirrer at room temperature
for 24 h. Then, 25% glycerol (w/w chitosan) was
added into the chitosan solution; stirring was contin-
ued at room temperature for 1 h. After mixing, the
solution was centrifuged for 15 min at 12 400 rpm by
a refrigerated centrifuge to remove undissolved
impurities and bubbles in the solution (Mayachiew
et al., 2010).
Preparation of galangal extract. Galangal powder (10 g
dry basis), dried by a tray dryer at 40  C with particle size
in the range 125–425 mm, was extracted with 100 mL of
95% (v/v) ethanol (Oonmetta-aree et al., 2006). The
extract was filtered through a filter paper; the filtrate
was collected and concentrated by a rotary evaporator
at 40  C for 10 min and kept at 4  C in a dark bottle until
its use (Mayachiew and Devahastin, 2008a).
Preparation of antimicrobial chitosan films. Galangal
extract was added to the chitosan solution at concentra-
tions 0.3, 0.6 and 0.9 g/100 g. These concentrations were
selected based on a minimum inhibitory concentration
(MIC) of the extract against Staphylococcus aureus
(Mayachiew and Devahastin, 2008a). The final concen-
trations of galangal extract in the films were 126, 252 and
378 mg/g film, respectively. The mixture was homoge-
nized by a bench top homogenizer at 9500 rpm for
2 min. The film solution (21 g) was poured on an acrylic
plate with dimensions 13  10 cm2 to cast an antimicro-
bial film. Drying of the film was performed by four meth-
ods, which are ambient air drying (30  C), hot air drying
at 40  C, vacuum drying and low-pressure superheated
steam drying at 70  C, 80  C and 90  C at 10 kPa, follow-
ing the methods of Mayachiew and Devahastin (2008b).
After drying, the films were conditioned for at least 48 h
in desiccators at a RH of 53% containing saturated salt
solution of magnesium nitrate.
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Dutta et al.
Preparation of sweet potato starch films
incorporated with potassium sorbate or
chitosan
In this method, 4 g of sweet potato starch was dispersed
in 100 mL H2O, moderately stirred for 20 min at room
temperature and then heated to 100  C for over 30 min.
After gelatinization, glycerol was added as a plasticizer
at a concentration of 3% (w/w, on dry basis of the
weight of starch) and the resulting dispersion subjected
to further mixing for 5 min. To prepare the antimicrobial
film, potassium sorbate or chitosan was added at differ-
ent concentrations (0, 5, 10, 15 g/100 g starch) at one
time during the mixing period. Before antimicrobial
agents were added into starch paste, potassium sorbate
was dissolved in 20 mL water and chitosan was dissolved
in 20 mL 1% acetic acid aqueous solution. For potas-
sium sorbate-incorporated film, the pH of the gel-like
mixture was adjusted to 4.5 by the addition of 2 M
HCl. When chitosan was incorporated into the film,
the pH of the gel-like mixture was 4.0 because of the
acetic acid used in preparing chitosan solution. After
being degassed under vacuum, the warm mixture was
cast on framed glass plates, and then dried at 50  C for
4 h. Starch film, without antimicrobial agents, was also
prepared in the same way and used as a control (Shen
et al., 2010).
Preparation of chitosan/methyl
cellulose films
Chitosan solution was prepared by dissolving 1.5 g of
chitosan in 100 mL of 1% acetic acid solution. In this
method, 1.5 g of methyl cellulose was dissolved in 50%
ethanol. Also, 1 g of polyethylene glycol 400 was used as
a plasticizer. Solutions of chitosan and methyl cellulose
were mixed in a beaker with a stir bar and heated to
72  C. Stearic acid, 0.075 g, was added to improve the
water barrier properties of the film. Vanillin, 0.9 g, was
incorporated after the temperature of the solution
reached its melting point (83  C). The film-forming solu-
tion was filtered through a cheese cloth to remove the
undissolved parts, homogenized with a homogenizer,
degassed, cast onto glass plates and dried at 40  C for
42 h. Dried 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 of
components and film composition and structure. The
choice of film-forming substance and/or active additive
is made based on the objective, the nature of the food
product and/or the application method.
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Food Science and Technology International 18(1)

Table 4. Inhibitory activities of chitosan

Types of study Activities and observations References

Surface spoilage Ouattara et al. (2000a); Savard

bacteria et al. (2002); No et al. (2002);
Coma et al. (2003);
Gerasimenko et al. (2004)
Various pathogen Studied the bioactivity of chitosan in liquid medium and Siragusa and Dickson (1992);
food strains reported that 0.01% chitosan is sufficient to inhibit the growth Helander et al. (2001); Tsai
of some spoilage bacteria such as B. subtilis, E. coli, and Su (1999); Tsai et al.
Pseudomonas fragi and S. aureus in liquid medium (2000); No et al. (2002); Coma
Significant inactivation of the Gram-negative bacteria et al. (2003); Darmadji and
E. coli and the Gram-positive enterotoxigenic S. aureus by two Izumimoto (1994)
commercially available water-soluble chitosan salts – chitosan Papineau et al. (1991)
lactate and chitosan hydroglutamate
Potential bactericide By markedly inhibiting the growth of Xanthomonas sp. Li et al. (2006)
in liquid medium, A decrease from 9.3 to 3.6 log CFU/mL of X. axonopodis pv.
against some plant Poinsettiicola was obtained using chitosan, compared to the
pathogenic bacteria control. These authors observed an increase in the bacterial
activity by the addition of 0.5% NaCl

Chitosan has been studied in terms of bacteriostatic/
bactericidal activity to control the growth of a wide vari-
ety of bacteria. Chitosan has several advantages over
other types of disinfectants because, according to Kim
and Choi (1998), it possesses a higher antibacterial activ-
ity and a broader spectrum of activity. The inhibitory
activity of chitosan is given in Table 4 (Inhibitory activ-
ity of chitosan). Chitosan hydroglutamate showed more
effective antimicrobial properties with a decrease in
S. aureus population of approximately 6 log cycles
within 60 min of exposure for a chitosan concentration
of 0.2 mg/mL. It also reported that the application of
high hydrostatic pressure (2380 odm) to chitosan-trea-
ted cultures of both bacterial strains resulted in addi-
tional inactivation. Chung and Chen (2008) also
investigated the antibacterial activity of low-molecular
weight 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 of
both bacterial cells, resulting in the leakage of enzymes
and nucleotides from different cell locations. The MIC
values 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 be
more active against Gram-positive than against Gram-
negative bacteria. Coma et al. (2003) assessed the poten-
tial of chitosan coating especially active against the
growth 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 total
inhibition of the development of selected Gram-positive
bacteria and 77% inhibition on Pseudomonas growth.
This result poses the problem of a possible microbial
selection related to the different sensitivities of microor-
ganisms. Moreover, other parameters could have an
impact on the bactericidal effect of chitosan. The age
of a bacterial culture affected its susceptibility to chito-
san (Tsai and Su, 1999). As a result, E. coli cells in the
exponential phase are most sensitive to chitosan. These
authors also showed that higher temperature (37  C)
increased the impact against the target strains.
Another parameter identified as significantly influential
is the molecular weight. Jeon et al. (2001) showed that
chitooligosaccharide effectively blocked the growth of
the tested bacteria although their effects were lower
than that of chitosan. These authors mentioned that
for an effective inhibition, the molecular weight should
be 10 kDa. The pH and the quaternization degree of the
chitosan were the influential parameters. The majority
of studies showed that an increase of deacetylation (DA)
degree and a decrease in pH improved the bioactive
properties of chitosan. The flocculation and adsorption
behavior of chitosans to E. coli cells were not observed;
however, such behaviors were noticed by applying fluo-
rescence labeled to chitosans and monitoring changes in
zeta potential values of the bacterial with chitosan coat-
ing. Without chitosan coating cells, it was increased
approximately by 40% if pH increased from 5 to 6.5
(Strand et al., 2003). Despite their low charge density,
the chitosans with higher deacetylation degree adsorbed

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in higher amounts and reversed the cell surface
charge most effectively. Finally, the chitosans with
low-molecular weight adsorbed the most. The same
authors reported that ionic strength did not affect the
adsorption of highly acetylated chitosan (DA 0.49),
whereas for chitosan with a DA of 0.01, adsorption
increased with ionic strength. The combination of floc-
culation and adsorption data clearly showed that charge
neutralization was not the main flocculation mechanism.
Several results pointed to bridging as one dominating
mechanism for flocculation. Study demonstrated that
in vitro antimicrobial efficiency is not always proven in
food, due to the highly reactive nature of the polycatio-
nic chitosan, which could interact with proteins, fats and
other anionic substances in foods. Darmadji and
Izumimoto (1994), however, observed that the growth
of spoilage bacteria in meat was inhibited by 0.5–1.0%
chitosan during incubation at 30  C for 48 h or storage at
4  C for 10 days.
Studies based on UV absorption indicated that chit-
osan causes considerable losses of proteinic material to
the Pythium oaroecandrum at pH 5.8 (Helander et al.,
2001; Liu et al., 2004). Chitosan also acts as a chelating
agent that selectively binds trace metals and thereby
inhibits the production of toxins and microbial growth
(Cuero et al., 1991). It also activates several defense pro-
cesses in the host tissue (El Ghaouth et al., 1992), acts as
a water binding agent and inhibits various enzymes.
Binding of chitosan with DNA and inhibition of
mRNA synthesis occurs through chitosan penetration
toward the nuclei of the microorganisms and interfer-
ence with the synthesis of mRNA and proteins
(Sudarshan et al., 1992).
It has been proposed that when chitosan is liberated
from the cell wall of fungal pathogens by plant host
hydrolytic enzymes, it then penetrates to the nuclei of
fungi and interferes with RNA and protein synthesis
(Hadwiger et al., 1985).
A microscopic examination of Saccharomyces uni-
sporus after treatment with chitosan-salt with a poly-
merization degree of 25 showed agglutination of a
refractive substance on the entire cell wall (Savard
et al., 2002). When chitosanase was added to the culture
media containing chitosan-salt, they could not observe
refractive substances. In this study, an interaction
between chitosan and the cell wall was observed.
In vitro evaluation of antimicrobial activities of
carboxymethyl chitosan (CM), quarternized
chitosan (Q) and quarternized carboxymethyl
chitosan
A series of quaternized carboxymethyl chitosan (CMQ),
the sample number and the characterization were listed
out in Table 5 (Sun et al., 2005). A Gram-positive
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Dutta et al.
Table 5. Sample numbers and characterization of different
CMQ
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
bacterium S. aureus and a Gram-negative bacterium E.
coli were used and inoculated on a gel containing 1%
peptone, 2% agar, 3% meat extract, and 0.5% NaCl for
this experiment.
The agar plate method was used to determine the
MICs of CM, Q and CMQ as follows: the samples
were prepared at a concentration of 1% (w/v) and then
autoclaved at 121  C for 25 min. Duplicate twofold serial
dilutions of each sample were added to nutrient broth
(beef extract 5 g, peptone10 g to 1000 mL distilled water,
pH 7.0) for final concentrations 0.1%, 0.05%, 0.025%,
0.0125%, 0.00625%, and 0.00313%. Some samples were
prepared and diluted by the same way except for final
concentrations 0.00065% and 0.00033%. The culture of
each bacterium was diluted by sterile distilled water to
105–106 CFU/mL. A loop of each suspension was inoc-
ulated on the nutrient medium with sample or control
added. After inoculation, the plates were incubated at
37  C for 72 h, the colonies counted and the MIC values
obtained. The MIC was considered to be the lowest con-
centration that completely inhibited against agar plates
on comparison, disregarding a single colony or a faint
haze caused by the inoculum (Speciale et al., 2002).
Antimicrobial activity of CM and CMQ
The antimicrobial activities of CM and CMQ are shown
in Tables 6 and 7. It was found that these samples
showed effective antimicrobial activities against not
only E. coli but also S. aureus which were used in the
test, although differences existed among them.
Generally, the samples had more effective inhibition
on S. aureus than E. coli. The fact may be attributed to
their different cell walls. In S. aureus, a typical Gram-
positive bacterium, the cell wall is fully composed of
peptide polyglycogen. The peptidoglycan layer is com-
posed of networks with plenty of pores, which allow
foreign molecules to come into the cell without difficulty.
But in E. coli, a typical Gram-negative bacterium, the
cell 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
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Food Science and Technology International 18(1)

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

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

of the bilayer structure, the outer membrane is a poten-
tial barrier against foreign molecules.
Compared with CM, Q and CMQ in Table 7, CMQ
had much better antimicrobial activities, whose MIC
values were 4–8 times lower than those of CM and 2–
4 times lower than those of Q. It was noticed that the
introduction of carboxymethyl and quarternized groups
to the chitosan chain greatly enhanced the antimicrobial
activity of the CMQ. We can deduce that carboxymethyl
and quaternary ammonium groups are in synergistic
effect.
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 no
clear effect of DS value of carboxymethyl group on anti-
microbial activity. Compared with CM3Q1, CM3Q2 and
CM3Q3, with similar degree of carboxymethyl group
substitution, it can be observed that their antimicrobial
activities were enhanced with increase of the DS.
Compared with CM3Q2-1, CM3Q2-2, CM3Q2-3 and
CM3Q2-4, which have same degrees of substitution for
both carboxymethyl and quaternized groups, the results
demonstrated that their antimicrobial activities were
affected by their molecular weights remarkably. Lower
molecular weight resulted in better antimicrobial ability,
and when the molecular weight was below 1  104, the
antimicrobial activity of CMQ was strong and the MIC
values reached 0.00313%.
The development of complementary methods to inhi-
bit the growth of pathogenic bacteria such as packaging
material-associated antimicrobial agents is an active
area of research. A number of studies on the antimicro-
bial characteristics of films made from chitosan have
been carried out earlier (Chen et al., 1996; Coma et al.,
2002; Ouattara et al., 2000a). Among other polymers,
chitosan has received a significant attention as an anti-
microbial film-forming agent for food preservation to
the researchers due to its biodegradability, biocompati-
bility, cytotoxicity and antimicrobial activity. Chitosan
films are easily prepared by the evaporation of its dilute
acid solutions (Park et al., 2002). Chitosan has been
studied in terms of bacteriostatic/bactericidal activity
to control the growth of a wide variety of bacteria.
In the Gram-positive bacteria, the major constituent of
their cell wall is peptidoglycan and a little amount of
protein. The cell wall of Gram-negative bacteria on the
other hand is thinner but more complex and contains
various polysaccharides, proteins and lipids beside the
peptidoglycan. Also, the cell wall of Gram-negative bac-
teria has an outer membrane which constitutes the outer
surface of the wall (Black, 1996).
The antimicrobial effect of konjac glucomannan
edible film was improved by incorporating chitosan
and nisin (Li et al., 2006). In this study, antimicrobial
efficacy was assessed against four food pathogenic bac-
teria namely E. coli, S. aureus, L. monocytogenes and B.
cereus. Antimicrobial activity tests of edible films were
carried out using the agar diffusion method.
In this method, the film cuts are placed on Mueller
Hinton agar plates, which were previously seeded with

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0.1 mL of inoculums containing indicator microorgan-
isms in the range 105–106 CFU/mL. The antimicrobial
effect of chitosan or incorporating nisin was found to be
much better than that of konjac glucomannan incorpo-
rating nisin at each corresponding concentration and
there existed significant difference (p < 0.05). However,
there was no significant difference in the antimicrobial
effect between chitosan and chitosan incorporating
nisin. Incorporating chitosan into konjac glucomannan
film therefore improved not only physical properties but
also antimicrobial activity.
The characteristics of chitosan film was evaluated by
cross-linking with naturally occurring aglycone genipo-
sidic acid (Mi et al., 2006). In this study, a comparative
study was performed between chitosan film without
cross-linking (fresh), the glutaraldehyde-cross-linked
chitosan film and aglycone geniposidic acid-cross-
linked chitosan film. A 50 mL bacterial broth (E. coli or
S. aureus) was seeded onto film and cultured. The fresh
and glutaraldehyde-cross-linked chitosan films were
used as control. It has been proposed that the interaction
between the polycationic chitosan and the negatively
charged surface of bacteria may alter the permeability
of the bacterial wall and lead to the leakage of intracel-
lular electrolytes and proteins. The results suggested that
cross-linking of chitosan films did not alter their anti-
bacterial capability. This may be due to the fact that the
cross-linking degrees of glutaraldehyde and aglycone
geniposidic acid (aGSA) cross-linked chitosan films
used in this part of the study were relatively low
(<18%, with a concentration of cross-linking agent
0.8 mM). The aGSA-cross-linked chitosan film dis-
played a relatively lower water vapor permeability, a
lower cytotoxicity, and a slower degradation rate than
the glutaraldehyde-cross-linked film. It was finally con-
cluded that the aGSA-cross-linked chitosan film may be
a promising material as an edible film for food
packaging.
The shelf-life of food was extended by ferulic acid-
incorporated starch-chitosan blend films (Mathew and
Abraham, 2008). Incorporation of ferulic acid has been
found to improve the barrier properties and tensile
strength of starch-chitosan blend films and signifi-
cantly enhance the lipid peroxide inhibition capacity.
This study has helped to improve the performance of
polysaccharide-based films for the storage of high
lipid-containing ingredients.
The antimicrobial activity of chitosan films enhanced
by incorporation of garlic oil, potassium sorbate and
nisin (Pranoto et al., 2005). The antimicrobial activity
was tested against food pathogenic bacteria namely
E. coli, S. aureus, Salmonella typhimurium, L. monocyto-
genes and B. cereus. Antimicrobial tests have been
carried out using agar diffusion method. The agar
diffusion test is a method commonly used to examine
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Dutta et al.
antimicrobial activity regarding the diffusion of the
compound tested through water-containing agar
plate. Incorporating antimicrobial agents into chitosan
edible films thus improves the antimicrobial efficacy of
chitosan, as diffused antimicrobial activity would add to
the nonmigrated antimicrobial potency of chitosan. It
was concluded that garlic oil incorporated into chitosan
film led to an increase in its antimicrobial efficacy, and
has little effect on the mechanical and physical proper-
ties of chitosan films. Overall, the incorporation of garlic
oil into chitosan film had the desirable characteristics of
acting as a physical and antimicrobial barrier to food
contamination.
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. coli
was evaluated by using the optical density (OD) method.
The smaller the OD of the medium, the higher was the
antimicrobial activity of the film. It was observed that
the antimicrobial activity of the blend films enhances if
the O-CMCh contamination was raised. Both blend
films exhibited satisfactory antibacterial activities
against E. coli, even with O-CMCh concentration
2 wt%.
Antibacterial assessment of chitosan-starch
Inhibitory effects of starch solution and chitosan-starch
film against E. coli, S. aureus and Bacillus subtilis are
shown in Figures 2(a)–(c), and 3(a)–(c). The inhibitory
effect was measured based on clear zone surrounding
circular film strips/solution. Measurement of clear
zone diameter included diameter of film strips/solutions.
Therefore, the values were always higher than the diam-
eter of film strips/solutions whenever clearing zone was
present. If there is no clear zone surrounding, we
assumed that there is no inhibitory zone, and further-
more, the diameter was valued as zero (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 against
all 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 at
higher concentration did not reveal a significant
increased inhibitory effect. It was generally caused by
the maximum capability of chitosan polymer to carry
active agents beside the occurrence of interaction phe-
nomenon between the functional groups. The antimicro-
bial effect of chitosan occurred without migration of
active agents. As chitosan is in solid form, only organ-
isms in direct contact with the active sites of chitosan are
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Food Science and Technology International 18(1)

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

Figure 3. Inhibitory effects of chitosan-starch film against: (a) E. coli, (b) S. aureus and (c) B. subtilis.
Source: Tripathi et al., (2008); reprinted with permission from Asian Chitin Journal).

Table 8. Diameters of inhibitory zone of the solution and chitosan–starch solution shows stronger inhibitory
film against E. coli, S. aureus and B. subtilis effect against E. coli and B. subtilis than S. aureus.
Furthermore, it was found that the bioactive chitosan–
Diameter (cm) of inhibitory zone
potato starch film can be used to extend the food shelf-
Test culture In the solution In the film life.
Incorporating chitosan and lauric acid into starch-
Escherichia coli 0 1.5
based film showed more effective antimicrobial ability
Staphylococcus aureus 0 1.2 against B. subtilis and E. coli (Salleh et al., 2007). In this
Bacillus subtilis 0 1.4 study, the authors studied the incorporation of chitosan
and lauric acid into starch-based films; obvious effects
toward inhibition of B. subtilis and E. coli have been
inhibited. Chitosan is incapable of diffusing through the observed while the film had synergistic antimicrobial
adjacent agar media. The agar diffusion test is a method effect when chitosan and lauric acid were combined.
commonly used to examine the antimicrobial activity Antimicrobial starch-based film incorporated with
regarding the diffusion of the compound tested through lauric acid and chitosan showed good flexibility than
water-containing agar plate. The diffusion itself is when purely starch-based film was formulated and
dependent on the size, shape and polarity of the diffusion formed (Figure 4). Inhibition of bacterial growth was
material. The chemical structure and the cross-linking examined using two methods, i.e., zone of inhibition
level of the films also affect this phenomenon. The test on solid media and liquid culture test (OD

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measurements). The inhibitory activity was measured
based on the diameter of the clear inhibition zone. In
the solutions of starch and chitosan with different
mixing ratios (w/w), 8:2 and 9:1 were the most effective
mixing ratios which had greater inhibition on both B.
subtilis and E. coli than other solutions in agar plate and
liquid culture test. The control (pure wheat starch) and
antimicrobial (AM) film (incorporated with chitosan
and lauric acid) were produced by casting method.
The antimicrobial effectiveness of control (pure
wheat starch) and AM film incorporated with chitosan
and lauric acid are shown in Figure 5(a) and (b). A wide
clear zone on solid media was observed for B. substilis
growth inhibition whereas inhibition for E. coli was not
as effective as B. substilis. From the liquid culture test,
Figure 4. A translucent starch-based film incorporated
with lauric acid and chitosan.
Source: (Salleh et al., (Muhamad and Khairuddin, 2007);
reprinted with permission from Asian Chitin Journal).
Figure 5. Inhibition areas of: (a) control film and (b) AM incorporated film.
Source: Salleh et al., Muhamad and Khairuddin, (2007); reprinted with permission from Asian Chitin Journal).
Downloaded from fst.sagepub.com at Gebze Yuksek Teknoloji Enstitu on April 25, 2014
Dutta et al.
the AM films clearly demonstrated a better inhibition
against 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 used
to extend food shelf-life.
Antimicrobial Activity of chitosan–polyvinyl
alcohol 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 on
clear zone surrounding circular film strips/solution.
Measurement of clear zone diameter included measuring
the diameter of film strips/solutions; therefore, the
values were always higher than the diameters of film
strips/solutions whenever clearing zone was present. If
there is no clear zone surrounding, the authors assumed
that there is no inhibitory zone, and furthermore, the
diameter was valued as zero. In terms of surrounding
clearing zone, chitosan–PVA film did not show inhibi-
tory effects against all tested microorganisms. The chit-
osan–PVA film-forming solution showed a clear
inhibitory zone against E. coli, S. aureus and B. subtilis,
respectively. The chitosan–PVA solution shows stronger
inhibitory effects against E. coli and B. subtilis than S.
aureus. Furthermore, it was found that the bioactive
chitosan–PVA film can be used to extend food shelf-
life. Chitosan-based antimicrobial films consisting of
chitosan and PVA were prepared by solution casting
method. These results pointed out that there is a
molecular miscibility between PVA and chitosan.
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Food Science and Technology International 18(1)
Chitosan-based antimicrobial film may be a promising
material as a packaging film.
Preservation of vacuum-packaged processed
meats
The feasibility of improving the preservation of vacuum-
packaged processed meats during refrigerated storage
achieved by the use of an antimicrobial film designed
to gradually release antimicrobial agents at the product
surface (Ouattara et al., 2000b). The antimicrobial films
were applied onto bologna, regular cooked ham or pas-
trami. The activity of the various films for inhibiting
bacterial growth was tested against indigenous lactic
acid bacteria and Enterobacteriaceae and against
Lactobacillus sakei or Serratia liquefaciens, surface-
inoculated onto the meat products. The growth of
Enterobacteriaceae and S. liquefaciens was delayed by
the application of the antimicrobial film. It was found
that the inhibition of indigenous Enterobacteriaceae was
more extensive at the surface of bologna than at the
surface of pastrami, irrespective of film type. It is due
to the fact that bologna contains efficient water-binding
agents, and so exudes little water during storage.
The moisture and high lipid contents of bologna
helped the diffusion of the oregano essential oil (EO)
from the chitosan film matrix into the product (Chi
et al., 2006). Sensory evaluation suggested that addition
of 45 ppm or less of oregano oil to bologna would be
acceptable to consumers. In conclusion, the gas chroma-
tography mass spectroscopy analysis showed that
757.7  99.7 ppm carvacrol was extracted from the
film-forming solution prepared without TweenÕ 20
and only 364.7  39.9 ppm from the film-forming solu-
tion with the emulsifier. Different levels of carvacrol
were detected in the presence of TweenÕ 20 due to the
interaction of the amphiphilic emulsifier’s molecule with
both chitosan and oregano EO compounds. It is con-
cluded that incorporation of an emulsifier in chitosan–
oregano EO films may slow the losses of volatile
compounds of the oil and help to control the release of
active compounds into the product.
The antimicrobial properties of crawfish
chitosan
Antimicrobial activities of 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, were
expressed in terms of zone inhibition. The zone inhibi-
tion assay revealed primarily three types of observa-
tions, namely, (1) defaced films without any clear or
inhibition zones which could be attributed to the
absence of any inhibitory activity, (2) clear zones
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without inhibitory zones which could be attributed to
bacteriostatic activity and (3) clear inhibition zone rep-
resenting bactericidal inhibition by films. Some of the
tests are enumerated as follows (Nadarajah, 2005).
Minimum inhibitory activity. All chitosan acetate films
were defaced with L. monocytogenes and this was well
in agreement with the report of Coma et al. (2002).
However, it was reported that 8% film-forming solu-
tion (chitosan in acetic acid) incorporated in agar
medium (v/v) completely inhibited L. monocytogenes.
Chitosan acetate films were also defaced with B.
cereus, and V. vulnificus lawns, indicating that they
were ineffective in controlling these bacteria. However,
all chitosan acetate films showed bacteriostatic effects
against S. aureus, S. sonnei, S. typhimurium and E. coli
O157:H7, as indicated by their clear zones i lawns.
The chitosan formate films were also ineffective in
controlling B. cereus and V. vulnificus, as indicated by
defaced films by these bacterial lawns. Nevertheless, the
chitosan formate films showed inhibitory effects against
S. aureus, S. sonnei, S. typhimurium and E. coliO157:H7
and bacteriostatic activity against L. monocytogenes.
However, compared to the chitosan citrate films, the
inhibitory effects of chitosan formate films were lower
and the thickness of the inhibitory zone was in the
range 0.49–1.64 mm compared to 0.78–6.0 mm of chit-
osan citrate films. All chitosan citrate films exhibited
prominent inhibitory effects on all seven pathogenic
bacteria. All chitosan citrate films showed distinctive
inhibition zones against all tested pathogenic bacteria
and the inhibition zones were considerably thicker than
those produced by chitosan formate films. Also, the
inhibitory effects of chitosan citrate films were remark-
ably higher for S. aureus and V. vulnificus, as indicated
by thicker inhibition zones accounting for more than
4 mm. The chitosan citrate films were the only films
with antimicrobial effects against B. cereus and V. vul-
nificus. The higher inhibitory activity shown by all chit-
osan citrate films can be attributable to complete
solubility of chitosan which could make them more
reactive against bacterial cells.
Antimicrobial activity. The quantitative analysis of anti-
microbial activity for selected chitosan films were carried
out as follows.
Acetate films with demineralized þ decolorized þ
deacetylated (DMCA) chitosan, formate films with
deproteinized 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 further
inhibition zone assays with more controls. The nisin
spots used as the positive control produced more prom-
inent clear zones with L. monocytogenes, and S. aureus
 Dutta et al.

Table 9. Studies on antimicrobial edible films and bacterial than 4.47 log CFU/mL reduction of inoculum in 24 h.
levels on meat products It caused only marginal reduction of the inoculum,
accounting for less than 1 log CFU/mL reduction over
Types of study, activity
and observation References the entire 24 h period incubation.
Further, the chitosan formate films caused about
Organic acids more effective against Siragusa and 1 log CFU/mL reduction of inoculum at 2 h of incuba-
L. monocytogenes on beef carcass Dickson (1992, tion and maintained a 1 log CFU/mL reduction over
tissue when immobilized in calcium 1993) 24 h of incubation. The rate of reduction of microbial
alginate than when used as a spray count was poor with both chitosan acetate and formate
or dip
films as there was no significant difference in microbial
Zein films, impregnated with nisin, Hoffman et al. (2001) count between 2 and 4 h of incubation and between 4
lauric acid and EDTA and tested with
and 8 h of incubation. Organic acids with smaller molec-
broth cultures of L. monocytogenes,
reduced the bacterial counts over
ular weights have higher antimicrobial activities and
5 logs after 48 h undissociated smaller molecules of formic (46.03 Da)
Zein films containing nisin produced Janes et al. (2002)
and acetic (60.05 Da) acids may enter the bacterial cells
a 4.5–5 log reduction on L. monocy- easily to change the internal pH of the organisms
togenes inoculated onto chicken (Eswaranandam et al., 2004). Undissociated larger mol-
breast tenders CFU/mL without ecules of citric acid (192.13 Da) may not enter into the
refrigeration for 16 days cells effectively. Such a trend was not observed in the
Impregnation the surface of meat Ming et al. (1997) study (Nadarajah, 2005) and the result was in contrary.
casing with pediocin powder to It indicates that chitosan films made of organic acids
produce a 1–3 log reduction of may behave as one entity rather than separate entities,
L. monocytogenes on ham, turkey i.e., as a career matrix containing an antimicrobial
breast and beef compared to inoc- agent. Several studies have demonstrated that antimi-
ulated controls crobial edible films can reduce bacterial levels on meat
products (Table 9).
In most of these studies, antibacterial activity against
lawns representing the Gram-positive bacteria and L. monocytogenes was attempted with added antimicro-
vague spots with S. sonnei and S. typhimurium lawns bials. Some of the major findings of the work for craw-
representing the Gram-negative bacteria. Regardless of fish chitosan are as follows.
the type of bacteria, controls such as chitosan solutions, The chitosan citrate film producing more than a
acid solutions and the polyvinyl chloride plastic failed to 4.4 log reduction in L. monocytogenes was a commend-
produce any clear or inhibition zones indicating that able achievement. As with the case L. monocytogenes,
they were ineffective in inhibiting the above-mentioned the chitosan citrate films showed higher antibacterial
food pathogenic bacteria. This substantiates the claim activity against S. aureus.The chitosan citrate films pro-
that the direct application of antimicrobial agents, such duced more than a 5 log reduction in S. aureus within 4 h
as chitosan and acid solutions used in our studies, onto of incubation and maintained its inhibitory effect
food surfaces is less effective due to loss of antimicrobial through out the incubation period. The chitosan acetate
activity caused by leaching onto the food, enzymatic films produced a poor inhibition with less than 1 log
activity and reaction with other food components reduction at 24 h. The chitosan formate films maintained
(Jung et al., 1992; Ouattara et al., 2000a; Ray, 1992). about 1 log reduction for up to 4 h. At 24 h incubation,
Hence, the use of packaging films or coating as a chitosan formate films produced more than a 5 log
matrix to deliver antimicrobial agents becomes impor- reduction similarly observed for chitosan citrate films.
tant. Such packaging or coating can maintain a high Relatively very little research work has been dedi-
concentration of antimicrobial agents on a food surface cated to formulate edible films active against S. aureus
and it allows low migration into food (Ouattara et al., (Table 10).
2000b; Siragusa and Dickson, 1992; Torres et al., 1985). All these studies indicate the importance of
Results of the direct inoculation study were in agree- having added antimicrobials in the films to control
ment with the inhibition zone assays (the survivor log S. aureus.However, the crawfish chitosan citrate and
number CFU/mL of L. monocytogenes inoculated onto formate films which contained no added antimicrobials
the surface of the selected chitosan films). could produce more than a 5 log reduction. Further, the
Chitosan citrate film: L. monocytogenes was more inhibitory effects of chitosan citrate and formate
susceptible to chitosan formate or chitosan acetate films against S. aureus were higher than that against L.
films. It reduced the bacterial count by 5.34 log CFU/ monocytogenes. Along with L. monocytogenes, S. typhi-
mL within 4 h of incubation and accounted for more murium has been considered as a microbiological hurdle

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Food Science and Technology International 18(1)

Table 10. Report to formulate edible films that are active Compared to these published data, reduction of S.
against S. aureus typhimurium in this study by more than 4.7 log by chit-
osan citrate film and 3.7 log by chitosan formate film is
Types of study, activity
and observation References outstanding.
As with L. monocytogenes, and S. aureus, chitosan
Polyethelene film containing Ha et al. (2001) acetate films produced minimal inhibition against S.
grapefruit seed extract showed an sonnei. The chitosan formate films accounted for about
inhibitory effect against S. aureus as 1 log reduction at 4 h of incubation and 2.6 log reduction
indicated by a 2.5–7.0 mm inhibition at 24 h. The citrate films showed the highest antibacterial
zone by the agar diffusion method
activity against S. sonnei with more than 5 log reduction
A 1.5 log and 2.8 log reduction of Scanell et al. at 8 h of incubation.
S. aureus in cheese and ham by (2000)
nisin-absorbed bioactive inserts
An edible packaging made of Coma et al. Major finding of the overall work
cellulosic esters, fatty acids and (2001)
nisin produced up to 88 mm
This study confirms that crawfish chitosan can be used
diameter inhibition zone on to develop antimicrobial edible films effective against
S. aureus.Further, they reported that both Gram-positive and Gram-negative food patho-
addition of fatty acid reduced the genic bacteria. Chitosan acetate films showed poor
inhibitory activity inhibitory effects against L. monocytogenes, S. aureus,
S. typhimurium and S. sonnei. Although chitosan acetate
films outweighed other films in terms of their mechanical
properties, they demonstrated minimal antibacterial
Table 11. Report to formulate edible films on S. effects similar to bacteriostatic effects with negligible
typhimurium bacterial reduction over a period of 24 h. Chitosan for-
mate films were effective against S. aureus, S. typhimur-
Types of study, activity
and observation References ium and S. sonnei, causing more than 5, 3.7 and 2.5 log
reductions at 24 h incubation, respectively. Chitosan
A 4.3 log reduction of Natrajan and formate films produced poor inhibitory effect against
S. typhimurium on inoculated broiler Sheldon (2000) L. monocytogenes with less than 1 log reduction at 24 h
skin exposed to nisin-coated incubation. Based on antibacterial and packaging prop-
polyvinyl chloride film erties, chitosan formate films can be used as antibacterial
A 4.23 log reduction of Sheldon et al. packaging to control S. aureus, S. typhimurium and S.
S. typhimurium in pads treated (1996) sonnei, except L. monocytogenes. Chitosan citrate films
with nisin formulations
were highly effective against L. monocytogenes, S.
Further, applied nisin formulations Sheldon (2001) aureus, S. typhimurium, and S. sonnei. The effect of chit-
to S. typhimurium inoculated on tray
osan citrate films against L. monocytogenes and S.
pads and demonstrated 3.1 log
reduction
aureus was prominent with more than 5 log reduction
within 4 h of incubation. Furthermore, chitosan citrate
films indicated their potential antibacterial effects
against B. cereus and V. vulnificus as indicated by the
for a long time. As with L. monocytogenes and S. aureus, zone inhibition tests. This study indicates the possibility
a similar trend of inhibition was observed with S. typhi- of formulating an antibacterial edible film, especially
murium. The chitosan citrate films produced more than crawfish chitosan citrate film, active against a broad
3.4 log reduction in S. typhimurium within 2 h of incuba- spectrum of bacteria (Nadarajah, 2005).
tion, and reduction in counts reached 3.85 log at 4 h and
4.83 log at 8 h incubation. The chitosan acetate films
Bacterial growth susceptibility
were less effective with about 1 log reduction at 24 h.
There was no significant (p > 0.05) change in the S. Bacterial growth susceptibility was determined by the
typhimurium count from 2 h to 24 h for chitosan acetate MIC method. Drops of chitin derivatives of different
films. The chitosan formate films maintained about concentrations were applied to the surface of agarose
2.7 log reduction up to 8 h and then produced a signifi- plates containing cultures of bacteria in nutrient
cant (p < 0.05) increased inhibition (3.7 log) between 8 dextrose medium or LB medium for phytopathogenic
and 24 h of incubation. bacteria and E. coli, respectively. MIC was defined as
The effects of edible films on S. typhimurium have the lowest concentration of chitin derivatives that
been given in Table 11. inhibited bacterial growth after overnight

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incubation of the agarose plates at 37  C (Struszczyk and
Pospieszny, 1997).
In another experiment, the effect of chitin derivatives
on Pseudomonas syringae pv. phaseolicola was tested
using the hypersensitive reaction (HR) of tobacco.
Mixtures of bacterium and chitin derivatives at final
concentration 5  107 CFU/mL and 0.05 wt%, 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 as
controls.
Water-soluble chitin oligomers, chitosan, chitosan
sulfates and carboxymethyl chitosan were used in this
research. Chitosan was dissolved in the acetic acid and
other chitin derivatives in distilled water. The reactions
of all the solutions were adjusted to pH ¼ 5.5–6.0 with
potassium hydroxide. Cationic chitin derivatives, i.e.,
chitin oligomers and chitosan, inhibited the growth of
the Gram-positive bacteria: Corynebacterium michiga-
nense subsp. michiganense and C. michiganense subsp.
insidiosum, and Gram-negative bacteria: Xanthomonas
campestris pv. Phaseoli, P. syringae pv. Phaseolicola,
P. syringae pv. tomato, Erwinia amylovora, Erwinia car-
otovora subsp. carotovora and Agrobacterium tumefa-
ciens at concentrations of the range 0.01–0.3 wt%.
However, both derivatives were less effective against E.
coli. Anionic chitin derivatives, i.e., chitosan sulfate and
carboxymethyl chitosan at a concentration of 1.5 wt%
were not effective against any of the bacterial tested.
When cationic derivatives were added to the bacteria
suspension, flocculation was observed. The HR of
plants is widely used for quick demonstration of bacte-
rial pathogenicity (Klement, 1963). When the tobacco
leaves were injected by a mixture of P. syringae pv.
Phaseolicola and chitin derivatives, HR was prevented.
Chemical depolymerization
Chitosan oligosccahrides have received attention
because of their versatile biological properties. Those
have lower viscosities, and low molecular weights and
are soluble in aqueous solution. They seem to be readily
adsorbed in vivo (Chatterjee et al., 2005).
Chemical treatment of chitosan using strong acids,
e.g. HNO2 and HCl is a very common and fast method
to produce a series of chitooligomers. However, this
method has some disadvantages such as high cost and
the low yield of chitosan oligosaccharides with degree of
depolymerization (DP) from DP2 to DP5 because of
random cleavage resulting in mostly monosaccharides.
The irradiation effects on chitosan in acetic acid solution
with 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 reaction
temperatures and times using 85% phosphoric acid that
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Dutta et al.
resulted in the decrease of viscosity average molecular
weight from 21.4  104 to 7.1  104. Depolymerization
of chitosan by the use of HNO2 is a homogeneous reac-
tion where the number of glycosidic bonds broken is
stochiomeric to the amount of HNO2 used (Jia and
Shen, 2002). The hydrolysis of chitosan with strong
HCl was studied over a range of acid concentrations
and temperatures. There have been very few reports on
the degradation of chitosan by free redicals. Nordveidt
et al. (1994) demonstrated that the viscosity of chitosan
solution decreased rapidly in the presence of H2O2 and
FeCl3 probably due to random depolymerization of
chitosan (Chen et al., 1997).
Several biological activities of chitosan depend on the
degree of polymerization. According to Liu et al.(2006),
the main factors affecting the antibacterial activity of
chitosan are molecular weight and concentration.
Recent studies on chitosan have attracted interest for
converting it into oligosaccharides because they are
not only water-soluble but also they are believed to
have greater antimicrobial activity. Chitosan has a
mean molecular mass of up to 1 MDa, which corre-
sponds to a chain length of approximately 5000 units,
but there is considerable variation between commercial
batches. The molecular mass of native chitin is usually
higher than 106 Da, whereas the molecular mass of the
commercial chitosan is often observed between 105 and
12  105 Da (Muzzarelli, 1973). During the process of
deacetylation, the hard conditions tend to degrade and
depolymerize chitosan (No et al., 2002). Medium- and
low-Mw chitosan can be obtained by chemical or enzy-
matic hydrolysis of the high-Mw polymer. The chemical
hydrolysis is usually achieved using strong acids, which
is an unexpensive and rapid method. Its drawback is the
necessity to purify extensively the low-Mw chitosan
products for biological applications due to the toxicity
of the reagents used for the reaction. Hydrogen peroxide
treatment (No et al., 2002) and ultrasonication
(Czechowska-Biskup et al., 2005) could be also used.
The extent of hydrolysis is, however, rather difficult to
control (Plouffe et al, 1997).
Enzymatic depolymerization
Enzymatic depolymerization seems to be a better
method to prepare chito oligosaccharides.
Microorganisms have been found to possess chitosanase
activity. Among bacteria, Bacillus and Streptomyces
strains are most often studied. Studies on fungal chito-
sanase are less reported (Cheng and Li, 2000).
The growing consumer demand for foods without
chemical preservatives has led people to indulge in
effortstoward the discovery of new natural antimicro-
bials (No et al., 2002). In this context, the antimicrobial
activities of chitosan and their derivatives against
19
Food Science and Technology International 18(1)
different groups of microorganisms, such as bacteria,
yeast and fungi have received considerable attention in
recent years. Antibacterial activities of six chitosans and
six chitosan oligomers with different molecular weights
were 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 and
Lactobacillus bulgaricus). Chitosans showed higher anti-
bacterial activities than chitosan oligomers and mark-
edly inhibited growth of most bacteria tested although
inhibitory effects differed with Mws of chitosan and the
particular bacterium. Chitosan generally showed stron-
ger bactericidal effects with Gram-positive bacteria than
Gram-negative one in the presence of 0.1% chitosan
(Wang, 1992). The MIC of chitosans ranged from
0.05% to >0.1% depending on the bacteria and Mws
of chitosan.
A solution can be obtained by the use of enzymes to
produce bioconversions. The chitooligosaccharides pro-
duced by the enzymatic hydrolysis of chitosan are widely
used in the food, agricultural and pharmaceutical
fields because of their various physiological activities.
Chitinase (EC 3.2.1.14) is an important chitin-degrading
enzyme which is involved in bioconversion processes of
wastes from crustaceans and in plant protection by pre-
serving them from chitin-containing pathogens such as
fungi (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 and
Glchitosan residues in a partially N-acetylated chitosan.
Chitosanase was distinguished from chitinase on the
basis of its ability to hydrolyze Glchitosan–Glchitosan.
As specified by Seki et al. (1997), chitosanases
were subdivided into three subclasses, characterized
by the ability to split Glchitosan–Glchitosan and
GlchitosanAc–Glchitosan linkages (subclass I), only
Glchitosan–Glchitosan linkages (subclass II) and
Glchitosan–Glchitosan and Glchitosanc–GlchitosanAc
linkages (subclass III). Chitosanase can be defined
as the enzyme which requires at least one Glchitosan
residue at either side of hydrolyzing linkages in
partially N-acetylated chitosan but not GlchitosanAc–
GlchitosanAc bonds. To date, many chitosanases have
been found in a variety of microorganisms, including
particularly bacteria (Kurakake et al., 2000; Pelletier
and Sygusch, 1990; Rivas et al., 2000; Sikorski et al.,
2006; You et al., 2003) and fungi (Cheng and Li, 2000;
Eom and Lee, 2003; Zheng and Zhu, 2003). Moreover,
several other hydrolytic enzymes, such as lysozyme,
cellulases and papain, were found to catalyze the enzy-
matic cleavage of glycosidic linkage in chitosan
(Muzzarelli et al., 1995; Pantaleone et al., 1992;
Yalpani and Pantaleone, 1994).
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At first, various investigators reported molecular
weight relationships of antibacterial activity by chitosan
and there are some reports that chitosan is more effective
in inhibiting the growth of bacteria than chitosan oligo-
mers. Hirano and Nagao (1989) studied the relationship
between the degree of polymerization of chitosan and
the inhibition grade against 18 phytopathogens. The
test materials were the lactate salt of high-molecular
weight chitosan (Mw 400 kDa, with 95% of deacetyla-
tion) and chitosan oligosaccharides, with a degree of
polymerization (DP) in the range 2–8. The growth of
13 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 the
degree of polymerization of chitosan resulted in
decreases in the number of inhibited fungal species.
Previously, Kendra and Hadwiger (1984) demonstrated
that the maximal antifungal activity of chitosan was
exhibited by chitosan oligomers of seven or more resi-
dues. In contrast to these results, Avadi et al.(2004) men-
tioned that chitosan oligomers with a DP 30 possess
antimicrobial 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 Mw
values 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 of
E. coli increased with molecular weight.
Chemical modifications
Even though chitosan is quite attractive as a biopoly-
mer with distinctive physicochemical properties and
biological activities, it is currently utilized to only the
limited extent. The delay in application study is partly
ascribable to the difficulty in controlled modifications
because of the insoluble nature in organic solvent and
multifunctionality of chitosan. However, many kinds
of modification reactions have been exploited to
increase the antimicrobial properties of chitosan. The
growing demand for a more accurate control of poly-
saccharide properties has prompted the development
of numerous techniques for selective modifications.
The amino and two hydroxyl groups present in the
repeating unit of chitosan are the targets of different
chemical modifications (Hirano et al., 1987). As a
result, the functionality of linear polysaccharides is sig-
nificantly affected by the presence, level and distribu-
tion of substituents along the main chain. As specified
by Rabea et al. (2003), chitosan and chitin are com-
mercially interesting compounds because of their high
nitrogen contents (6.89%) compared to synthetically
substituted cellulose (1.25%). This makes chitosan a
 Dutta et al.

OH OH

RCHO
O
O O
O HO
HO
N CHR

NH2

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

OH OH

O O
O CH3I, NaOH, NaI O
O O
HO HO
N-methyl-2-pyrrolidinone
NHCH3 N I
CH3 CH3
H 3C

N-methylchitosan N,N,N-trimethylchitosan

Figure 7. Synthesis of N,N,N-trimethylchitosan.

Source: Belalia et al. (2008).

useful chelating agent. However, these naturally abun-
dant materials are also limited in their reactivity and
processability.
Several alkylated chitosans are reported to be synthe-
sized. Kim et al. (1997) prepared N-alkyl chitosan deriv-
atives by introducing alkyl groups into the amine groups
of chitosan via Schiff’s base intermediates. Indeed, the
free amine groups of the chitosan react with aldehydes to
give 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 on
the chitosan. Quaternization of the N-alkyl chitosan
derivatives could be carried out using a halogenoalcane
in the presence of sodium hydroxide (Belalia et al., 2008;
Jia et al., 2001). As shown in Figure 7, MeI could be used
to produce water-soluble cationic polyelectrolytes and
novel chitosan derivatives, with quaternary ammonium
salt (Belalia et al., 2008; Kim et al., 1997).
Their antibacterial activities against S. aureus were
explored by the viable cell count method in acetate
buffer at pH 6. The antibacterial activities of the quater-
nary ammonium salt increased with increase in the chain
length of the alkyl substituent, and this increased activity
could be ascribed to the contribution of the increased
hydrophobic properties of the derivatives. In addition,
Avadi et al. (2004) prepared a quaternized chitosan (i.e.,
N-diethylmethyl chitosan, DEMC) based on a modified
two-step process. With a degree of quaternization of
79%, the DEMC exhibited a higher antibacterial activ-
ity than chitosan against E. coli. However, the antimi-
crobial effects of both compounds were pH dependent
and an increase in concentration of acetic acid resulted
in a significant decrease in MIC, determined by turbidi-
metric method. As a result, the antibacterial activities of
chitosan and DEMC are higher in 1% acetic acid in
comparison with the lower levels of acetic acid concen-
tration, 0.25%, for example. The MIC of DEMC is
decreased from 500 to 62.5 mg/mL when the medium is
changed from water to 1% acetic acid solution. The
authors mentioned that this is due to the target site of
the polycation, i.e., the negatively charged surface of the
bacteria cell. Therefore, the polycation DEMC with a
high charge density interacts with the bacteria more than
what chitosan itself does. Other chitosan derivatives
such as N,N,N-trimethyl chitosan, N-propyl-N,N-
dimethyl chitosan and N-furfuryl-N,N-dimethyl chito-
san were prepared and tested for their activities against
E. coli (Jia et al., 2001). It was shown that the antibac-
terial activity of quaternary ammonium chitosan in
acetic acid medium stronger than that in water
against E. coli and is stronger than that of chitosan
itself. More recently, Chi et al. (2007) prepared
Chitosan-N-2-hydroxypropyl trimethyl ammonium
chloride by the reaction of chitosan with glycidyl

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Food Science and Technology International 18(1)
CH2OH
O O
OH
NH2
Chitosan
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).
trimethylammonium chloride. The chitosan derivatives,
with different molecular weights, near 1.7, 35.7, 90.2 and
415.5 kDa, showed biocidal activity on S. aureus and
Staphylococcus epidermidis, B. subtilis and Candida albi-
cans. These authors observed that the chitosan with a
molecular weight of 415.5 kDa exhibits a slightly lower
biocidal activity on C. albicans than others. However, it
seems that high-molecular weight chitosan derivatives
had high biocidal activities on the Gram-positive bacte-
ria, with a decreasing biocidal effect with decreasing
molecular weight from 90 to 1.7 kDa. Nevertheless,
derivatives with molecular weights from 90 to 1.7 kDa
did not exhibit any biocidal effect against E. coli and P.
aeruginosa even at concentrations up to 10 mg/mL. In
another study, Kim et al. (1997) synthesized diethylami-
noethyl–chitosan (DEAE–chitosan) from deacetylation
of a diethylaminoethyl–chitin (DEAE18 chitin) by intro-
ducing DEAE groups onto the C(6)–OH in chitin. The
deacetylation was conducted by heating in aqueous 10%
sodium hydroxide containing sodium borohydride. In
addition, DEAE–chitin was quaternized to produce
triethylaminoethyl–chitin (TEAE–chitin). Their antibac-
terial activities against S. aureus and E. coli were evalu-
ated using colony count by means of the shake flask
method. The antibacterial activities were found to
increase in the order DEAE–chitin, DEAE–chitosan
and TEAE–chitin. To obtain copolymers with zwitter-
ionic property, Jung et al. (1999) prepared water-soluble
anionic chitosan moieties and investigated their
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CH2OH
O O
OH
NH
H3C (CH2)13CH3
COOCH2CH2OP(OH)2
O
O
Chitosan-g-MAP
CH2OH
O O
OH
HN CH2
CH2
SO3Na
Chitosan-g-VSS
antimicrobial activity. Mono(2-methacryloyl oxyethyl)
acid phosphate (chitosan–MAP) and vinylsulfonic acid
sodium salt (chitosan–VSS) were grafted onto chitosan
(Figure 8). Concerning their antimicrobial activities, chit-
osan–MAP and chitosan–VSS exhibited inhibition values
of the growth of C. albicans of about 95% and 75%,
respectively. Both chitosan derivatives showed the same
high pH-dependence. As a result, if the pH changed from
5.7 to 6.2, their antimicrobial properties dropped to 10–
15%, which was less than the activity of the parent chit-
osan. Against Trichophyton rubrum, the MAP grafting led
to a low negative impact on antimicrobial activity,
whereas the anionic chitosan showed much enhanced bio-
active action against Trichophyton violaceum, compared
to unmodified chitosan. The authors suggested than this
selectivity could result from the structural affinity
between the wall of microbial strains and the chitosan
or its derivatives. A possible reason might be that the
wall of microorganisms consisted of chitin, chitosan or
ß-glycan.
Other chitosan derivatives are sulfonated and sulfo-
benzoyl chitosans. The antibacterial effects of the chem-
ical modifications were evaluated and compared with
those of 69% deacetylated chitosan by Chen et al.
(1998). MIC values of sulfonated chitosan (0.63%
sulfur content) against Shigella dysenteriae, Aeromonas
hydrophila, S. typhimurium, and B. cereus were found to
be lower than those of the deacetylated chitosan. A high
sulfur content in sulfonated chitosan adversely

influenced its antibacterial effect. Sulfobenzoyl chitosan
exhibited excellent water solubility and an antibacterial
effect 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 aflatoxin
production by Aspergillus flavus and Aspergillus parasi-
ticus in submerged culture. Five chemically modified
chitosans were tested for their antifungal activities
against Saprolegnia parasitica by Muzzarelli et al.
(2001) using the radial growth assay in chitosan-bearing
agar and the fungal growth assay in chitosan-bearing
broth. Members of the genus Saprolegnia are responsi-
ble for the infections of fish and eggs in aquaculture
facilities. Results indicated that methylpyrrolidinone
chitosan, N-carboxymethyl chitosan and N-phosphono-
methyl chitosan exerted effective fungistatic action
against 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 the
amine site are numerous and relatively easy taking into
account the reactivity of the primary amine. However, to
preserve the amine groups in order to maintain the bio-
active properties, various modification procedures have
recently been described, which yield C-6 modified chit-
osan derivatives.
MECHANISM OF ANTIMICROBIAL
ACTION
The different mechanisms have been proposed whereas
the exact mechanism of the antimicrobial action of
chitin, chitosan and their derivatives is still unknown
(Rabea et al., 2003). The mechanisms of the antimicro-
bial activity of chitosan were different for Gram-positive
and Gram-negative bacteria (Zheng and Zhu, 2003). In
this 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 activity
increased with increase the molecular weight of chitosan.
Besides, for Gram-negative E. coli, the antimicrobial
activity increased with decrease in molecular weight.
The authors suggested the following two different mech-
anisms for the antimicrobial activity: (1) in the case of S.
aureus, the chitosan on the surface of the cell can form a
polymer membrane, which inhibits nutrients from enter-
ing the cell and, (2) for E. coli, where chitosan of lower
molecular weight entered the cell through pervasion.
The antimicrobial mechanisms of CM, Q and CMQ
are suggested as: on one hand, the positive charge of the
group at C-2 resulted in a polycationic structure which
can be expected to interact with the predominantly anio-
nic components (lipopolysaccharides and proteins) of
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Dutta et al.
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 major
proportion of proteinaceous material from the cells
(Helander et al., 1998); when the quarternized group
was introduced onto the molecular chain, the positive
charge was strengthened. On the other hand, when car-
boxymethyl group was introduced along the molecular
chain, the presence of a molecular structure with hydro-
philic ends and weak interaction forming between
hydrophilic 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 is
still imperfectly known, but different mechanisms have
been proposed (Rabea et al., 2003). One of the reasons
for the antimicrobial character of chitosan is its posi-
tively charged amino group which interacts with nega-
tively charged microbial cell membranes, leading to the
leakage 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 (0.2 mg/mL), the polycationic
chitosan does probably bind to the negatively charged
bacterial surface to cause agglutination, while at higher
concentrations, the larger number of positive charges
may have imparted a net positive charge to the bacterial
surfaces to keep them in suspension (Papineau et al.,
1991; Sudarshan et al., 1992).
A strong attachment of heterologous bacteria to the
walls in tobacco leaves is essential to elicit the HR.
Therefore, from mechanistic point of views, it is possible
that chitin derivatives prevent the attachment of bacte-
rial cells into the plant cell walls or affect their survival in
the intercellular spaces.
Chitosan acted mainly on the outer surface of the
bacteria. The obvious antibacterial effects can be attrib-
uted to the formation of polyelectrolyte complexes
between the polycationic agent and the bacterial cell sur-
face (Muzzarelli et al., 1990). Indeed, interaction
between positively charged chitosan molecules and neg-
atively charged microbial cell membranes leads to the
leakage of proteinaceous and other intracellular constit-
uents. Studies based on UV absorption indicated that
the chitosan causes considerable losses of proteinic
material to Pythium oaroecandrum, at a pH 5.8
(Helander et al, 2001; Liu et al., 2004). Different behav-
iors were reported, dependent on the chitosan concen-
tration (Rabea et al., 2003). At a lower concentration
(<0.2 mg/mL), the polycationic chitosan does probably
bind to the negatively charged bacterial surface to cause
agglutination, while at higher concentrations, the larger
23
Food Science and Technology International 18(1)
number of positive charges may have imparted a net
positive charge to the bacterial surfaces to keep them
in suspension. Savard et al. (2002), by a microscopic
examination of Saccharomyces unisporus after treatment
with chitosan-salt with a polymerization degree of
25, showed agglutination of a refractive substance on
the entire cell wall. When chitosanase was added to the
culture media containing chitosan-salt, they could not
observe refractive substances. This result suggested an
interaction between chitosan and the cell wall. In
another study, chitosan caused leakage of glucose and
lactate deshydrogenase from E. coli cells (Tsai and Su,
1999). These results support the hypothesis that the
mechanism of chitosan antibacterial action involves
cross-linkage between the polycations of chitosan and
the anions on the bacterial surface that change the mem-
brane permeability. As already mentioned, chitosan
coatings adjusted to pH 5.0 totally inhibited Gram-
positive bacterial surface growth such as L. monocyto-
genes and S. aureus. However, Gram-negative microbial
strains such as P. aeruginosa overcame the active chito-
san protection, and the development was not completely
excluded (Coma et al., 2003). Therefore, the microbio-
logical target of protonated chitosan’s action would be
the cytoplasmic membrane of sensitive cells. Cellular
damage can lead to the disruption of the cellular
integrity of the membrane. The outer membrane of
Gram-negative bacteria could act as a barrier and be
responsible for preventing chitosan from reaching the
cytoplasmic membrane. Although the cytoplasmic
membrane should be sensitive to chitosan, the outer
membrane protects the cells. Zheng and Zhu (2003)
also indicated that the mechanisms of the antimicrobial
activity of chitosan were different between Gram-
positive and Gram-negative bacteria. They showed, in
a comparative study, that the effect of chitosan was
different on S. aureus (Gram-positive) and on E. coli
(Gram-negative). To E. coli, the antimicrobial activity
was enhanced as the molecular weight decreased. It was
obvious that 0.25% chitosan solution (Mw < 5 kDa)
could inhibit the growth of E. coli. In contrast, for S.
aureus, the antimicrobial activity increased with increas-
ing molecular weight of chitosan. The inhibiting effect
was fairly obvious for higher ones, such as 305 kDa, even
if the concentration was quite small. The authors
suggested two possible mechanisms for antimicrobial
activity: (1) the chitosan on the surface of the cell can
form a polymer membrane, which prevents nutrients
from entering the cell and (2) chitosan of lower molecu-
lar weight entered the cell through pervasion. For S.
aureus, the dominant mechanism is the former, while
for E. coli, the latter mechanism seems more likely. In
addition, the chitosan has also the faculty to bind speci-
fically with some macromolecules. It can thus inhibit
various enzymes, bind to the DNA and inhibit the
24
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synthesis of the mRNA after penetration of the chitosan
in the core of the microorganisms (Hadwiger et al.,
1985). Epifluorescence microscopy results showed a pos-
sible action of chitosan during a short duration of time
on the synthesis of nucleic acids and especially on the
relative proportion of RNA compared with DNA
(Coma et al., 2003). This impact was followed by an
adaptative mechanism of the cells. Binding of chitosan
with DNA and inhibition of mRNA synthesis occur
through chitosan penetration toward the nuclei of the
microorganisms and interference with the synthesis of
mRNA and proteins (Rabea et al., 2003).
It has been postulated that the antimicrobial action of
chitosan occurs as a result of several mechanisms.
Chung and Chen (2008) studied the antibacterial activity
of chitosan with respect to the extent of damaged or
missing cell walls and the degree of leakage of enzymes
and nucleotides from different cellular locations. First,
the addition of chitosan to the bacterial suspension
seemed to have a stronger impact on the Gram-negative
E. coli than on the Gram-positive S. aureus in terms of the
leakage of enzymes. In addition, the experimental result
revealed that the antimicrobial action of chitosan not
only involves a reaction with the cell wall of the bacteria
but also may affect the structure of the phospholipid
bilayer in the cell membrane, thereby changing the per-
meability of the cell membrane, resulting in the release of
some of the cellular components. This action was further
enhanced when chitosan with a high degree of deacetyla-
tion was used. To gain a better understanding of the
mechanism by which chitosan functions as a bactericide,
the cells were also subjected to a known antibiotic which
reacts with the anionic phosphate group of phospholipids
in the cell membrane, destroying the cell membrane struc-
ture and affecting its permeability. In parallel, the cells
were subjected to EDTA, a chemical chelating agent
that destroys the structure of the cell wall by chelating
with the Ca2þ or Mg2þ present in the cell wall. As a result,
chitosan was found to react with both the cell wall and the
cell membrane of E. coli, but not simultaneously, indicat-
ing that the inactivation by chitosan occurs via a two-step
sequential mechanism: an initial separation of the cell
wall from its cell membrane, followed by cell membrane
destruction. Concerning the bioactivity of the chitosan
against phytopathogenic fungi, it could be related with
its potential elicitor of many plant defense responses,
including for example the accumulation of chitinases or
by producing phytoalexins, defined as ‘substances with
antibiotic activity that function as growth inhibitors of
phytopathogenic organisms, chiefly fungi’ (Bade and
Wick, 1987). In a study on Botrytis cinerea and
Rhizopus stolonifer, chitosan-induced morphological
changes were characterized by excessive hyphal branch-
ing and abnormal aerial surface hyphal growth compared
to the control (El Ghaouth et al., 1992). As a result,

chitosan appears to play a dual function, by interfering
directly with fungal growth and also by activating several
biological processes in plant tissues. Benhamou et al.
(1994) applied chitosan to decrease the infection with
Fusarium oxysporum. Biopolymer coating was applied
on seed prior to fungal inoculation. Chitosan at concen-
trations ranging from 0.5 to 1 mg/mL was used for the
ultrastructural and cytochemical investigations. The
authors observed that after a pretreatment with chitosan,
the root tissues at sites of fungal penetration were always
associated with the expression of plant defense reactions.
In the epidermis, cells showed typical signs of necrosis
characterized by marked disorganization of the cyto-
plasm. The pathogen was detected in the outer cortex
where its development was halted. Fungal cells suffered
from serious damage and were frequently encircled by an
electron-dense material. In the noncolonized inner cortex,
strong host reactions were detected that were mainly asso-
ciated with the deposition of two types of materials that
differed in their electron densities. Gold cytochemistry
with a ß-1,3-glucanase and a laccase showed that the
more electron-dense material was phenolic in nature,
whereas the other material, occurring either as deposits
inserted between the phenolic aggregates or as globular
structures lining the host cell walls, was made of ß-1,3-
glucans. These observations bring further evidence that
chitosan is an active inducer of plant defense reactions
and, thus, has the potential to become a powerful alter-
native means of disease control.
FACTORS AFFECTING THE
ANTIMICROBIAL ACTIVITY
There are various factors, such as the intrinsic and
extrinsic properties of chitosan: molecular weight,
degree of polymerization, deacetylation, solubility and
higher charge density that 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 can
be considered as synergetic for the reasons of the hurdle
effect of the acid stress on the bacterial cells (Rhoades
and Roller, 2000).
The mechanism of the antimicrobial activity of chit-
osan and its derivatives is well studied. As theory, it has
been suggested that the positive charge of the amine
group (NH3þ) at pH values lower than the pKa (pH
<6.3) at which this functional group carries 50% of its
total electric charge allows the interactions with nega-
tively charged microbial cell membranes, a phenomenon
which is susceptible to cause a leakage of intracellular
constituents (Helander et al., 2001). However, even if
this is a well-recognized explanation of chitosan
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Dutta et al.
antimicrobial effect, there is no direct evidence demon-
strating this behavior against bacteria.
Lower pH increases the antimicrobial activity of
chitosan for much the same reasons, in addition to the
‘hurdle effect’ of inflicting acid stress on the target organ-
isms (Rhoades and Rastall, 2000). Surrounding matrix is
the greatest single influence on antimicrobial activity.
Being cationic, chitosan has the potential to bind to
many food components such as alginates, pectins, pro-
teins and inorganic polyelectrolytes such as polypho-
sphate (Kubota and Kikuchi, 1998). Solubility can be
decreased using high concentrations of low-molecular
weight electrolytes such as sodium halides, sodium phos-
phate and organic anions (Roberts, 1992).
Sorption capacity of chitosan films was significantly
affected by the moisture content of the chitosan-based
films. Authors reported that a decrease of water content
decreased the total amount of the active sites that can
participate in the sorption phenomenon. Antibacterial
activities of the produced chitosan-based films have been
evaluated against E. coli and S aureus and it has been
shown that diameters of the inhibition zones were 5 and
3 times higher than those of control for E. coli and S
aureus, respectively. Authors showed that moisture con-
tent of chitosan films had significant effect on their bac-
tericide effect which decreased on decreasing film
moisture content. Decrease of film moisture content
from 22% (w/w) down to 12% (w/w) decreased the bac-
tericide activity by 2.5 times. This was demonstrated by
the decreased diameter of the inhibition zone. This find-
ing can be exploited in the cheese-making industry for
cheese coating with chitosan films in the maturation
chambers in order to avoid mold and pathogenic
bacteria growth on cheese surface (Buzinova and
Shipovskaya, 2008).
The effect of the molecular weight on some antibac-
terial and antifungal activities has been explored (Chen,
1998). Chitosans with molecular weights ranging from
10,000 to 100,000 have been found to be helpful in
restraining the growth of bacteria. In addition, chitosan
with an average molecular weight of 9300 was effective
in restraining E. coli, whereas that with a molecular
weight 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 the
medium. Antibacterial activities were also found to be
increasing in the order N,O-carboxymethylated chito-
san, chitosan and O-carboxymethylated chitosan (Liu
et 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 defense
processes (Bai et al., 1988). These defense mechanisms
include accumulation of chitinases, synthesis of
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Food Science and Technology International 18(1)
proteinase inhibitors and lignification and induction of
callous synthesis (El Ghaouth et al., 2000). When
applied on wounded wheat leaves, chitosan-induced lig-
nifications and consequently restricted the growth of
nonpathogenic fungi in wheat. Chitosan inhibited the
growth of A. flavus and aflatoxin production in liquid
culture, preharvest maize and groundnut, and it also
enhanced phytoalexin production in germinating
peanut (Cuero et al., 1991a,b). Chitosan has also been
found to inhibit growth and toxin production by
Alternaria alternata fungal species lycopersici in culture
(Bhaskara et al., 1998; Dornenburg and Knorr, 1997).
Chitosan solution at 0.10 mg/mL markedly inhibited
the growth of Xanthomonas pathogenic bacteria (iso-
lated from Euphorbia pulcherrima) from different geo-
graphical origins (Li et al., 2008). The antibacterial
activity of chitosan solution against Xanthomonas axo-
nopodis pv. poinsettiicola (strain R22580) increased with
the increase of chitosan concentration up to 0.10 mg/
mL. The antibacterial activity of chitosan solution at
0.05 mg/mL was enhanced by NaCl.
The antibacterial activity of chitosan was investigated
by assessing the mortality rates of E. coli and S. aureus
based on the extent of damaged or missing cell walls and
the degree of leakage of enzymes and nucleotides from
different cellular locations (Chung and Chen, 2008). The
inactivation of E. coli by chitosan occurred via a two-
step sequential mechanism: an initial separation of the
cell wall from its cell membrane, followed by destruction
of the cell membrane.
The antibacterial activities of chitosan nanoparticles
and copper-loaded nanoparticles against E. coli,
Salmonella choleraesuis, S. typhimurium and S. aureus
were evaluated by the calculation of MIC and minimum
bactericidal concentration (MBC; Qi et al., 2004). The
obtained results showed that chitosan nanoparticles and
copper-loaded nanoparticles could inhibit the growth of
various bacteria tested. Their MIC values were less than
0.25 mg/mL and the MBC values of nanoparticles
reached 1 mg/mL. They reported that the exposure of
S. choleraesuis to the chitosan nanoparticles led to the
disruption of cell membranes and the leakage of
cytoplasm.
OPTIMIZATION OF THE BIOCIDE
PROPERTIES
The bactericidal activities of chitosan (Mw 685 kDa)
against various bacteria were more than 99%
against Gram-negative bacteria such as E. coli O-157,
S. typhimurium(except for P. aeruginosa, 68%) and more
than 98% against Gram-positive bacteria such as
Streptococcus mutans, M. luteus, S. aureus and B. sub-
tilis. Chitooligosaccharides showed a less bactericidal
effect, with 71%, 56% and 60% against E. coli
26
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O-157.for the high-, medium- and low-molecular
weight samples, respectively. Another test was done
against the P. aeruginosa strain, with bactericidal effects
of about 47%, 35% and 22% for the high-, medium- and
low-molecular weight chitosans, respectively. Tokura
et al. (1997) prepared chitosan oligomers of average
molecular weights 9.3 and 2.2 kDa by nitrous acid deg-
radation followed by the reduction of 2,5-anhydroman-
nose terminal by sodium borohydrate. Although the
9.3 kDa provided the growth inhibition of E. coli, the
chitosan 2.2 kDa was not a growth inhibitor but a
growth accelerator. It has been suggested that the smal-
ler oligomers serve as nutrients for bacteria, whereas the
higher oligomers are toxic by virtue of their charge-
mediated adhesion to the cell membrane, which in turn
prevents the uptake of nutrients through the cell wall.
Liu et al. (2006) specified that the molecular weight of
chitooligosaccharides is critical for microorganism inhi-
bition and must be higher than 10 kDa. Several studies
showed that the impact of the Mw on the bioactivity is
dependent on the concentration of the biocide. They
investigated the bioactivities of chitosans of different
molecular weights, from 55 to 155 kDa, against E. coli
with the same degree of deacetylation (80%). The mech-
anism of antibacterial activity was the flocculation of the
strains. These authors observed that at high concentra-
tion (over 200 ppm), the antibacterial activities of each
chitosan sample were almost the same and all the bacte-
ria could be killed. At low concentration (20 ppm), there
was 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 differences
between chitosans of different molecular weights in the
antibacterial activation. Indeed, the authors concluded
that at high concentrations (200, 500 and 1000 ppm) and
a low concentration (20 ppm), the antibacterial activity
of chitosan had no relationship to the molecular weight.
But at concentrations ranging from 50 to 100 ppm, the
antibacterial activity of low-molecular weight chitosan is
higher than that of the high-molecular weight samples.
Qin et al. (2006) also showed that the molecular weight
dependence of the antimicrobial activity of chitosan was
more pronounced at a low concentration. The action of
chitosans with molecular weights Mw from 1.4 to
400 kDa on the growth of S. aureus, E. coli and C. albi-
cans was explored by microcalorimetry. The chitosans
with middle range values of Mw, 78 and 48 kDa, had
higher inhibitory effect than others. Chitooligomer
1.4 kDa promoted the growth of C. albicans, but slightly
inhibited growth of the bacteria. The 400 kDa, with the
highest Mw, exhibited a much weaker inhibitory effect in
comparison with the chitosan at 78 kDa. It seems that
the water-insoluble chitosans with Mw around 50 kDa
were the optimum ones for antimicrobial action in their
tested samples. In addition, Zheng and Zhu (2003)

observed that for chitosan with molecular weight below
300 kDa, the antimicrobial effect on S. aureus was
strengthened as the molecular weight increased. In con-
trast, the effect on E. coli was weakened. In parallel to
depolymerization, chemical modifications of chitosan
have been attempted to improve its antimicrobial
properties.
The influence on biocide performance of some
unprecedented physicochemical features of chitosan
cast films such as film thickness, pH of the nutrient
broth, film neutralization, film autoclave sterilization
and temperature exposure was analyzed against S.
aureus and in some experiments also against
Salmonella spp. The work demonstrates, for the first
time, the influence of the release or positive migration
of protonated glucosamine fractions from the biopoly-
mer into the microbial culture as the responsible event
for the antimicrobial performance of the biopolymer
under the studied conditions. From the results, a reli-
able and reproducible method for the determination of
the bactericidal activity of chitosan-based films was
developed in an attempt to standardize the testing
conditions for the optimum design of active antimicro-
bial food packaging films and coating applications.
The optimization of biocide properties of chitosan
will be useful for its application in the design of
active films of interest in the food area (Fernandez-
Saiz et al., 2008).
CHITOSAN FOR IMMOBILIZATION
Enzymatic catalysis in nonaqueous solvents has gained
considerable 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 esters
hydrolase, E.C.3.1.1.3) have been widely used to produce
organic chemicals, biosurfactants, oleochemicals, agro-
chemicals, paper, cosmetics, fine chemicals and pharma-
ceuticals (Sharma et al., 2001). Chitosan has been used as
a matrix for immobilization of lipases (Alsarra et al., 2002)
and many other enzymes (Krajewska, 2004). Enzymes
bound to sugars, or sugar-based polymers like chitosan
are stabilized during lyophilization and in nonaqueous
environments. This may be due to a reduction of auto-
lysis, that is a multipoint attachment limiting enzyme
distortions or microenvironmental effects (Wang et al.,
1992).
Elemental analysis and Raman spectra measure-
ments of the lipase, supports and immobilized lipase
systems gave evidence of the presence of enzymes on
supports. Chitosan supports with internal surface area
(m2/g) among 3.31 and 1.26 were obtained. Regardless
of these low values, acceptable protein load
(0.61–3.21%) and esterification initial rates were
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Dutta et al.
achieved (0.88–2.75 mmol/min/g of protein; Orrego
et al., 2010).
CHITOSAN NANOCOMPOSITE
Nowadays, the nanocomposites are receiving more
attention from the researchers, because of their more
effective action in penetrating and disrupting bacterial
cell membranes to conquer the battle of pathogenic bac-
teria (Yacoby and Benhar, 2008). Many works on the
use of nanochitosans to prevent food spoilage have been
reported in a recent review (Friedman and Juneja, 2010).
Chitosan/vermiculite (VMT) nanocomposites can
enhance the thermal stability of chitosan nanocompo-
sites dramatically due to the well dispersion of acid-
modified VMT (HCl-modified VMT, HVMT) and
better interaction between HVMT and chitosan in the
fabricated nanocomposites (Zhang et al., 2009).
Chitosan-based nanocomposite films, especially
silver-containing ones, showed a promising range of
antimicrobial activities (Rhim et al., 2006). The chit-
osan/OREC nanocomposites films provide promising
applications as antimicrobial agents, water-barrier
compounds, anti-ultraviolet compounds and drug-
controlled release carriers in antimicrobial food pack-
aging (Wang et al., 2007). From the antimicrobial
activity 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 (Han
et al., 2010).
Antimicrobial studies showed that the nanocompo-
sites could strongly inhibit the growth of a wide variety
of microorganisms, including Gram-positive bacteria,
Gram-negative bacteria and fungi; more importantly,
they exhibited good antimicrobial capacity in whichever
medium, in weak acid, water or weak base. As the
amount of Montmorillonite increased, the nanocompo-
sites had better inhibitory effect on microorganisms,
especially Gram-positive bacteria. The lowest MIC
values of the nanocomposites against S. aureus and B.
subtilis were less than 0.00313% (w/v) under all the con-
ditions (Wang et al., 2007).
CONCLUSIONS
Chitin, chitosan and its oligosaccharides played very
important roles in the application of antimicrobial mate-
rials and food packaging. The systematic study toward
antimicrobial activity of chitin, chitosan and its oligo-
saccharides would be a promising tool for the future
improvement of food quality and preservation during
processing and storage because the antimicrobial pack-
aging can be helpful in extending the food shelf-life.
The combination of other film-forming and coating
materials may provide the functional properties for a
27
Food Science and Technology International 18(1)
better food shelf-life. The understanding of the factors
affecting the antimicrobial activity, mechanism of anti-
microbial action, and optimization of the biocide prop-
erties of chitin, chitosan and their oligosaccharides
would be an added advantage to use these materials in
a better way.
ACKNOWLEDGMENTS
We thank UGC, New Delhi and Royal Society of Chemisty
(RSC), UK for giving the Research Fund Grant Award-2009
to PKD. We also sincerely thank the different researchers who
published their works in different journals, magazine, disser-
tation, doctoral degree work and elsewhere which have been a
great source for resource materials toward compiling the
review in the present form. We apologize if some of the content
from the above resource is/are similar during presentation.
We thank the journal reviewers for their constructive
suggestions.
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