Food Chemistry 114 (2009) 1173–1182

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Review

Perspectives for chitosan based antimicrobial films in food applications

P.K. Dutta a,*, Shipra Tripathi a, G.K. Mehrotra a, Joydeep Dutta b
a
Department of Chemistry, Motilal Nehru National Institute of Technology, Allahabad 211004, India
b
Regenerative Medicine, Reliance Life Sciences Pvt. Ltd., R-282, TTC Area of MIDC, Thane Belapur Road, Rabale, Navi Mumbai-400701, India

a r t i c l e i n f o a b s t r a c t

Article history: Recently, increasing attention has been paid to develop and test films with antimicrobial properties in
Received 17 March 2008 order to improve food safety and shelf life. Active biomolecules such as chitosan and its derivatives have
Received in revised form 20 September a significant role in food application area in view of recent outbreaks of contaminations associated with
2008
food products as well as growing concerns regarding the negative environmental impact of packaging
Accepted 7 November 2008
materials currently in use. Chitosan has a great potential for a wide range of applications due to its bio-
degradability, biocompatibility, antimicrobial activity, non-toxicity and versatile chemical and physical
properties. Thus, chitosan based films have proven to be very effective in food preservation. The presence
Keywords:
Chitosan
of amino group in C2 position of chitosan provides major functionality towards biotechnological needs,
Bioactive films particularly, in food applications. Chitosan based polymeric materials can be formed into fibers, films,
Food packaging material gels, sponges, beads or even nanoparticles. Chitosan films have shown potential to be used as a packaging
Antimicrobial activity material for the quality preservation of a variety of food. Besides, chitosan has widely been used in anti-
Review microbial films to provide edible protective coating, in dipping and spraying for the food products due to
its antimicrobial properties. Chitosan has exhibited high antimicrobial activity against a wide variety of
pathogenic and spoilage microorganisms, including fungi, and Gram-positive and Gram-negative bacte-
ria. The present review aims to highlight various preparative methods and antimicrobial activity includ-
ing the mechanism of the antimicrobial action of chitosan based films. The optimisation of the biocidic
properties of these so called biocomposites films and role of biocatalysts in improvement of quality
and shelf life of foods has been discussed.
Ó 2008 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1174
2. Scope and objective of the present review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1174
3. Functionality and composition of edible films and coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1174
4. Preparation of chitosan based antimicrobial films/coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175
4.1. Preparation of chitosan/starch film by using supercritical carbon dioxide treatment (Tripathi, Mehrotra, & Dutta, unpublished results) 1175
4.1.1. Preparation of chitin whiskers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175
4.1.2. Supercritical fluid (SCF) drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175
4.1.3. Preparation of chitosan/starch film . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175
4.2. Preparation of antimicrobial chitosan-potato starch film by using microwave treatment (Tripathi et al., 2008). . . . . . . . . . . . . . . . . . . 1175
4.3. Preparation of starch/chitosan blend film under the action of irradiation (Zhai, Zhao, Yoshii, & Kume, 2004) . . . . . . . . . . . . . . . . . . . . 1175
4.4. Preparation of chitosan film enriched with oregano essential oil (Chi, Zivanovic, & Penfield, 2006) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1176
4.5. Preparation of-chitosan-oleic acid edible coatings (Vargas, Albors, Chiralt, & Gonzalez-M, 2006) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1176
4.6. Preparation of water soluble chitosan and amylose film (Suzuki et al., 2005) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1176
5. Mechanism of antimicrobial action of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1176
6. Antimicrobial activity of chitosan films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1177
7. Factors affecting the antimicrobial activity of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1179
8. Optimization of the biocide properties of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1179
9. Applications as bio-packaging for food preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1179

* Corresponding author. Tel.:+915322271829; fax: +915322271272.

E-mail address: pkd_437@yahoo.com (P.K. Dutta).

0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.11.047
1174
9.1. General concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2. Homogeneous coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3. Biocomposites films and multilayer systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4. Biocatalysts in food packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
There has been a growing interest in recent times to develop
materials with film-forming capacity and having antimicrobial
properties which help improve food safety and shelf life. Antimi-
crobial packaging is one of the most promising active packaging
systems that have been found highly effective in killing or inhibit-
ing spoilage and pathogenic microorganisms that contaminate
foods (Salleh, Muhamad, & Khairuddin, 2007). In this context,
chitosan films have shown great promise for their application in
food preservation. It is well-known that microbial alternations
are responsible for the enormous losses in food and hence, over
the years, various chemical and physical processes have been
developed to extend shelf-life of foods. Among such processes ade-
quate packaging of food products is a fundamental factor in their
conservation and marketing phases. Thus, packaging is not only
crucial but actually preponderant for food quality preservation.
The antimicrobial packagings have been used to control microbial
growth in a food ingredient using packaging materials and edible
films and coatings that contain antimicrobial agents and sometime
by using techniques that modify the atmosphere within the pack-
age. Because of the increase in consumer demand for minimally
processed, preservative-free products, the preservative agents
must be applied to packaging in such a way that only low levels
of preservatives come into contact with the food. In order to meet
this demand, the film or coating technique is considered to be
more effective though little cumbersome to apply. A greater
emphasis on safety features associated with the addition of antimi-
crobial agents is gaining ground as one of the emerging areas for
development in packaging technology and it has likely to play a
major role in the next generation of ‘active’ packaging systems
(Brody, 2001). Active packaging is the packaging system possessing
attributes beyond basic barrier properties that are achieved by
adding active ingredients in the packaging system and /or using
functionally active polymers. When the packaging system acquires
antimicrobial activity, the packaging system (or material) limits or
prevents microbial growth by extending the lag period and reduc-
ing the growth rate or decreases live counts of microorganisms
(Han, 2000). The primary goals of a conventional packaging system
such as safety assurance, quality maintenance, and shelf-life exten-
sion, exactly take a reversed order for an antimicrobial packaging
system. Food security has always been a big issue worldwide and
antimicrobial packaging could play a major role in food security
assurance. Currently, food application of an antimicrobial packag-
ing system is limited due to the availability of suitable antimicro-
bials, new polymer materials, regulatory concerns, and appropriate
testing methods (Jin & Zhang, 2008).
Edible films or coatings have been investigated for their abilities
to retard moisture, oxygen, aromas and solute transports
(Gennadios & Weller, 1990). Edible and biodegradable films are
always not meant to totally replace the synthetic packaging films
(Krochta & Johnston, 1997) though it is one of the most effective
methods of maintaining food quality. Usually film-forming sub-
stances are based on proteins, polysaccharides lipids and resins
or a combination of these (Greener-Donhowe & Fennema, 1994).
P.K. Dutta et al. / Food Chemistry 114 (2009) 1173–1182
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Chitosan, a linear polysaccharide consisting of (1,4)-linked 2-
amino-deoxy-b-D-glucan, is a deacetylated derivative of chitin,
which is the second most abundant polysaccharide found in nature
after cellulose. Chitosan has been found to be nontoxic, biodegrad-
able, biofunctional, biocompatible in addition to having antimicro-
bial characteristics (Darmadji & Izumimoto, 1994; Jayakumar,
Nwe, Tokura, & Tamura, 2007; Jayakumar, Prabaharan, Reis, &
Mano, 2005; Jayakumar, Reis, & J. F., 2006; Jongrittiporn,
Kungsuwan, & Rakshit, 2001; Wang, 1992). As compared with
other bio-based food packaging materials, chitosan has the advan-
tage of being able to incorporate functional substances such as
minerals or vitamins and possesses antibacterial activity (Chen,
Zheng, Wang, Lee, & Park, 2002; Jeon, Kamil, & Shahidi, 2002;
Möller, Grelier, Pardon, & Coma, 2004). In view of these qualities,
chitosan films have been used as a packaging material for the
quality preservation of a variety of food (Park & Zhao, 2004; Suyatma,
Tighzert, & Copinet, 2005; Tsai & Su, 1999; Wu, Zivanovic,
Draughon, Conway, & Sams, 2005). Recently, a chitosan-starch film
has been prepared using microwave treatment which may find
potential application in food packaging (Tripathi, Mehrotra, &
Dutta, 2008). A wide variety of chitosan based antimicrobial films
have recently been well documented (Tripathi et al., 2008).
2. Scope and objective of the present review
The spoilage of foods each year all over the world incurs great
economic losses. In order to preserve the food from microorgan-
isms, various antimicrobial based products and processes have
been developed. In particular, polymeric bioactive films laced with
assortment of antimicrobial agents have been found very effective
and practical in applications. Till date, a number of review articles
have been published describing the nature of different materials
used in making such films and their effectiveness in food preserva-
tion (Alvarez, 2000; Appendini & Hotchkiss, 2002; Cha & Chinnan,
2004; Cooksey, 2005; Cutter, 2002; Cutter, 2006; Ozdemir & Floros,
2004; Quintavalla & Vicini, 2002; Suppakul, Miltz, Sonneveld, &
Bigger, 2003). Present review aims to summarise all the known
methods of formation of chitosan based films with antimicrobial
properties and discuss their subsequent applicability in the area
of food preservation.
3. Functionality and composition of edible films and coatings
The polymer films are already in use for food packaging are
polyethylene (PE), polyvinyl chloride (PVC), polyvinyl alcohol
(PVA), polylactic acid (PLA), nylon, and others. Binding of antimi-
crobials to polymeric surfaces has been achieved by different
means, ranging from simply spreading antimicrobial solutions
onto the polymer surface or by more sophisticated means such
as combining the antimicrobials with binders. These binders can
be of a cellulosic, or an acrylic co-polymer nature. Sometimes the
antimicrobials were covalently attached, with natural and syn-
thetic cross-linkers like genipine, glutaraldehyde, formaldehyde
etc. For some films plasticizers were considered necessary, and
 P.K. Dutta et al. / Food Chemistry 114 (2009) 1173–1182
Table 1
Active properties of edible films and coatings.
Encapsulation or Improvement of Individual protection of small
carriage mechanical resistance pieces of food
Flavors, spices Improvement of Separation of food by individual
appearance portion
Antimicrobial, color soluble package for pre-dosed food
antioxidant ingredients or additives
agents
Pigments, light Shininess
absorbers
Salts Transparency
Other food Roughness
additives
Sticking
for this purpose glycerol, propylene glycol (PG) or polyethylene
glycol (PEG) is added.
Edible packagings must have some functional and specific prop-
erties. Indeed, first of all they have to be selective toward mass
transfers, but in some cases they have active properties, or they
can be both selective and active. Selective properties of edible films
and coatings are responsible for retarding the organic vapours
(aromas, solvents), water vapour, solute (lipids, salts, additives,
pigments), and gases (oxygen, carbon dioxide, nitrogen).
In most cases, the water barrier efficiency of films is desirable to
retard the surface dehydration of fresh (meat, fruits and vegeta-
bles) or frozen products. The water absorption inducing the caking
in food powder or the loss of crispness in dried cakes, for example,
could be delayed by coatings. The control of gas exchanges, partic-
ularly of oxygen, allows better control of the ripening of fruits or to
significantly reduce the oxidation of oxygen-sensitive foods and
the rancidity of polyunsaturated fats. Organic vapour transfers
have to be minimised in order to retain aroma compounds in the
product during storage or to prevent solvent penetration in foods,
which induces toxicity or off- flavoring. The penetration of oil dur-
ing frying and of sucrose or sodium chloride during osmotic dehy-
dration can be limited by an edible film. One of the more
interesting applications of edible films and coatings is their use in-
side a composite food to control mass transfers between different
compartments of the product, such as to reduce water migration in
a pie. Further, the effect of UV light that involves radical air reac-
tions in foods could also be significantly reduced. In the latter case,
the efficiency of the film to prevent light effect can be improved by
the addition of pigments or light absorbers. Thus, when films are
used as carriers or used for encapsulation of food additives or
ingredients, they become active (Table 1). Edible packagings can
improve mechanical properties of food to facilitate handling and
carriage. Furthermore, sensorial characteristics such as color, shin-
iness, transparency, roughness or sticking can be improved.
Functional efficiency strongly depends on the nature of compo-
nents and film composition and structure. The choice of film-form-
ing substance and/or active additive is made based on the
objective, the nature of the food product and /or the application
method.
4. 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 general practice, chitosan films
are prepared with cross-linked by agylcone geniposidic acid (Mi
et al., 2006); ternary chitosan-glucomann-nisin (Li, Kennedy, Peng,
Yie, & Xie, 2006); blending of ferulic acid incorporated starch-
chitosan (Mathew & Abraham, 2008); incorporation of garlic oil,
potassium sorbate and nisin (Pranoto et al., 2005). The derivatised
1175
chitosan such as O-carboxymethylated chitosan blended with cel-
lulose from LiCl/N,N-dimethylacetamide solution (Li, Zhuang, Liu,
Guan, & Yao, 2002) has also been reported for antibacterial study.
Most recently, the authors (Tripathi et al., 2008) have synthesised
chitosan based antimicrobial films for food applications employing
supercritical carbon dioxide and microwave technique. The novelty
of this method lies in achieving the film formation without addi-
tion of any cross-linker or plasticizer.
Some typical preparative techniques are enumerated below:
4.1. Preparation of chitosan/starch film by using supercritical carbon
dioxide treatment (Tripathi, Mehrotra, & Dutta, unpublished results)
4.1.1. Preparation of chitin whiskers
Chitin (1 g) was treated in 3N hydrochloric acid (100 mL) and
stirred at 105 °C for 3 h to obtain the colloidal solution. The resi-
dues were collected after centrifugation and treated with hydro-
chloric acid for two times. Finally, the residues were dialysed in
distiled water until they were neutral. The prepared chitin was
then subjected to solvent-exchange into acetone and ethyl alcohol
prior to sc.CO2 treatment.
4.1.2. Supercritical fluid (SCF) drying
The solvent-exchange products were placed inside a sealed
chamber of the SCF reactor (Thorr Co., USA). The temperature
and pressure were raised to 40 °C and 100 bar respectively. Further
pressure was raised to 200 bar. The reaction was left for 2 h and a
flow of CO2 was then applied through the sample in order to re-
place all the organic solvents with CO2. The pressure was then re-
leased slowly to the atmosphere.
4.1.3. Preparation of chitosan/starch film
The supercritical carbon dioxide treated chitin was stirred in
NaOH and Na2CO3 at 150 °C respectively and left at room temper-
ature overnight. The residues were collected and the treatment
was repeated for two times to result in the total alkaline treating
time for 21 h. The crude product was dialysed in distiled water un-
til it was neutral. The prepared material was then subjected to sol-
vent-exchange into acetone and ethyl alcohol prior to sc.CO2
treatment. The solvent-exchange products were then treated with
sc. CO2. The sc. CO2 treated chitosan was mixed with starch in 3:1
ratio in aqueous AcOH solution. After obtaining the clear homoge-
neous solution, it was casted on glass substrate to obtain free hand
films.
4.2. Preparation of antimicrobial chitosan-potato starch film by using
microwave treatment (Tripathi et al., 2008)
Chitosan solution was prepared in 1% (v/v) glacial acetic acid
solution. The prepared solution was stirred overnight at room tem-
perature and filtered through synthetic cloth. Starch solution was
prepared by dissolving starch powder in hot water. Chitosan solu-
tion was mixed with starch solution and then stirred at room tem-
perature for few hours. The final film-forming solution was poured
into a petri dish. The films from chitosan and potato starch were
successfully prepared by microwave treatment. Plasticizers were
not used for making chitosan-potato starch film.
Some other preparative methods for chitosan films are as
follows:
4.3. Preparation of starch/chitosan blend film under the action of
irradiation (Zhai, Zhao, Yoshii, & Kume, 2004)
Chitosan solution was prepared in acetic acid solution (chito-
san: acetic acid = 5:4). Starch powder was mixed with glycerol
homogenously with the above prepared chitosan solution to form
1176 P.K. Dutta et al. / Food Chemistry 114 (2009) 1173–1182
15% starch and chitosan semisolid gel-like mixtures by heating at
100 °C for 2 h. The gel-like mixtures in hot state were cold pressed
to prepare wet starch/chitosan films.
In order to produce a kind of antibacterial films, the wet films
prepared through above methods were irradiated further at room
temperature by electron beam (EB). The wet starch/chitosan films
were dried at room temperature to gain starch/chitosan films.
4.4. Preparation of chitosan film enriched with oregano essential oil
(Chi, Zivanovic, & Penfield, 2006)
Chitosan stock solution was prepared with 1.5% w/w chitosan in
1.5% v/v acetic acid. The solution was stirred overnight at room
temperature, filtered through MiraclothÒ and sterilized at 121 °C
for 15 min. At first the essential oil was mixed with TweenÒ 20
and then added to the chitosan stock solution. The final film-form-
ing solutions were homogenised under aseptic conditions at
21,600 rpm for 1 min and poured into sterile petri dishes. The films
were dried under 5 psi vacuum at 30 °C.
4.5. Preparation of-chitosan-oleic acid edible coatings (Vargas, Albors,
Chiralt, & Gonzalez-M, 2006)
Chitosan (1%, w/v) was mixed in acetic acid solution (1% v/v) at
40 °C. Tween 80 at 0.1% (v/v) was added to improve wettability.
The solution was stirred for 8 h and then oleic acid added to chito-
san solution to reach a final concentration of 0, 1, 2 and 4% (v/v).
These mixtures were emulsified at 13500 rpm for 4 min. In order
to guarantee the stability of the emultions, pH was adjusted to 5
with 1N NaOH (Jumaa, Furkert, & Muller, 2002).
4.6. Preparation of water soluble chitosan and amylose film (Suzuki
et al., 2005)
Aqueous amylose solution (1%) was prepared by dissolving
amylose powder in hot water (80 °C). A 1% salt free aqueous water
soluble chitosan (WSC) was obtained by dialysis of an WSC solu-
tion that included NaCl, produced by the neutralisation of a dilute
hydrochloric acid solution of WSC with NaOH. Each film having a
thickness of 40–50 lm was prepared by casting the amylose,
WSC, or their mixed solutions at 60 °C. A film of fully deacetylated
chitosan was obtained by casting a 0.1 M aqueous AcOH solution of
fully deacetylated chitosan (1%) on a Kapton (polyimide) film. The
resulting acidic chitosan film was neutralised with 1 M aqueous
NaOH followed by washing with water and then dried.
5. Mechanism of antimicrobial action of chitosan
Because of the positive charge on the C2 of the glucosamine
monomer below pH 6, chitosan is more soluble and has a better
antimicrobial activity than chitin (Chen, Liau, & Tsai, 1998). The ex-
act mechanism of the antimicrobial action of chitin, chitosan, and
their derivatives is still imperfectly known, but different mecha-
nisms have been proposed (Rabea, Badawy, Stevens, Smagghe, &
Steurbaut, 2003). One of the reasons for the antimicrobial charac-
ter of chitosan is its positively charged amino group which inter-
acts with negatively charged microbial cell membranes, leading
to the leakage of proteinaceous and other intracellular constituents
of the microorganisms (Shahidi, Arachchi, & Jeon, 1999). Chitosan
acted mainly on the outer surface of bacteria. At a lower concentra-
tion (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, Hoover, Knorr, &
Farkas, 1991; Sudarshan, Hoover, & Knorr, 1992).
Studies based on UV absorption indicated that the chitosan
causes considerable losses of proteinic material to the Pythium
oaroecandrum at pH 5.8 (Helander, Nurmiaho-Lassila, Ahvenainen,
Rhoades, & Roller, 2001; Liu, Du, Wang, & Sun, 2004). Chitosan also
acts as a chelating agent that selectively binds trace metals and
thereby inhibits the production of toxins and microbial growth
(Cuero, Osuji, & Washington, 1991b). It also activates several de-
fence processes in the host tissue (El Ghaouth, Arul, Asselin, &
Benhamou, 1992), acts as a water binding agent, and inhibits var-
ious enzymes. Binding of chitosan with DNA and inhibition of
mRNA synthesis occurs through chitosan penetration toward the
nuclei of the microorganisms and interference with the synthesis
of mRNA and proteins (Sudarshan et al., 1992).
It has been proposed that when chitosan is liberated from the
cell wall of fungal pathogens by plant host hydrolytic enzymes, it
then penetrates to the nuclei of fungi and interferes with RNA
and protein synthesis (Hadwiger, Kendra, Fristensky, & Wagoner,
1985).
A microscopic examination of Saccharomyces unisporus after
treatment with chitosan-salt with a polymerisation degree of 25,
showed agglutination of a refractive substance on the entire cell
wall (Savard, Beauliu, Boucher, & Champagne, 2002). When chito-
sanase was added to the culture media containing chitosan-salt,
they could not observe refractive substances. In this study, there
was an interaction between chitosan and the cell wall.
The mechanisms of the antimicrobial activity of chitosan were
different for Gram-positive and Gram-negative bacteria (Zheng &
Zhu, 2003). In this study they differentiated the effect of chitosan
on S. aureus (Gram-positive) and on Escherichia coli (Gram-nega-
tive). For Gram-positive S. aureus, the antimicrobial activity in-
creased on increasing the molecular weight of chitosan. Besides,
for Gram-negative E. coli, the antimicrobial activity increased on
decreasing molecular weight. The authors suggested the following
two different mechanisms for the antimicrobial activity: (1) In case
of S. aureus, the chitosan on the surface of the cell can form a poly-
mer membrane, which inhibits nutrients from entering the cell
and, (2) For, E. coli, where chitosan of lower molecular weight en-
tered the cell through pervasion.
The effect of the molecular weight on some antibacterial and
antifungal activities has been explored (Chen, 1998). Chitosan with
a molecular weight ranging from 10 000 to 100 000 have been
found to be helpful in restraining the growth of bacteria. In addi-
tion, chitosan with an average molecular weight of 9300 was effec-
tive in restraining E. coli, whereas that with a molecular weight of
2200 helped in accelerating the growth (Tokura, Miuray, Johmen,
Nishi, & Nishimura, 1994). Moreover, the antibacterial activity of
chitosan is influenced by its degree of deacetylation, its concentra-
tion in solution, and the pH of the medium. Antibacterial activities
were also found to be increasing in the order N,O-carboxymethy-
lated chitosan, chitosan, and O-carboxymethylated chitosan (Liu,
Guan, Yang, Li, & Yao, 2001).
In addition to the formation of gas-permeable films, chitosan
has a dual function: (a) to direct the interference of fungal growth
and (b) to activate several defence processes (Bai, Huang, & Jiang,
1988). These defence mechanisms include accumulation of chitin-
ases, synthesis of proteinase inhibitors, and lignification and induc-
tion of callous synthesis (El Ghaouth et al., 2000). When applied on
wounded wheat leaves, chitosan induced lignifications and conse-
quently restricted the growth of nonpathogenic fungi in wheat.
Chitosan inhibited the growth of A. flavus and aflatoxin production
in liquid culture, pre-harvest maize, and groundnut, and it also
enhanced phytoalexin production in germinating peanut (Cuero,
Duffus, Osuji, & Pettit, 1991a; Cuero, Osuji, et al., 1991b). Chitosan
has also been found to inhibit growth and toxin production by
A. alternata fungal species lycopersici in culture (Bhaskara et al.,
1998; Dornenburg & Knorr, 1997).
 P.K. Dutta et al. / Food Chemistry 114 (2009) 1173–1182
Chitosan solution at 0.10 mg/mL markedly inhibited the growth
of Xanthomonas pathogenic bacteria (isolated from Euphorbia pul-
cherrima) from different geographical origins (Li, Wang, Chen, Hua-
ngfu, & Xie, 2008). The antibacterial activity of chitosan solution
against Xanthomonas axonopodis pv. poinsettiicola (strain R22580)
increased with the increase of chitosan concentration up to 0.10
mg/mL. The antibacterial activity of chitosan solution at 0.05 mg/
mL was enhanced by NaCl.
The antibacterial activity of chitosan was investigated by
assessing the mortality rates of E. coli and S. aureus based on the
extent of damaged or missing cell walls and the degree of leakage
of enzymes and nucleotides from different cellular locations
(Chung & Chen, 2008). The inactivation of E. coli by chitosan oc-
curred via a two-step sequential mechanism: an initial separation
of the cell wall from its cell membrane, followed by destruction of
the cell membrane.
6. Antimicrobial activity of chitosan films
The antimicrobial activity of chitosan was observed against a
wide variety of microorganisms including fungi, and some bacteria.
The antimicrobial action is influenced by intrinsic factors such as
the type of chitosan, the degree of chitosan polymerization, the
host, the natural nutrient constituency, the chemical or nutrient
composition of the substrates or both, and the environmental con-
ditions (e.g., substrate water activity or moisture or both). The
development of complementary methods to inhibit the growth of
pathogenic bacteria such as packaging material-associated antimi-
crobial agents is an active area of research. There has been increas-
ing interest in antimicrobial edible packaging materials. A number
of studies on the antimicrobial characteristics of films made from
chitosan have been carried out earlier (Chen, Yeh, & Chiang,
1996; Coma, Martial-Gros, Garreau, Copinet, & Deschamps, 2002;
Ouattara, Simard, Piette, Begin, & Holley, 2000a, 2000b). Among
other polymers, chitosan has received a significant attention as
antimicrobial film-forming agent for food preservation to the
researchers due to its biodegradability, biocompatibility, cytotoxic-
ity, and antimicrobial activity. Chitosan films are easily prepared
by evaporation of its dilute acid solutions (Park, Marsh, & Rhim,
2002). Chitosan has been studied in terms of bacteriostatic/bacte-
ricidal 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 peptidoglycan. Also, the cell wall of Gram-negative bacteria
has an outer membrane which constitutes the outer surface of the
wall (Black, 1996).
The antimicrobial effect of konjac glucomannan edible film has
been improved by incorporating chitosan and nisin (Li et al., 2006).
In this study, antimicrobial efficacy has been assessed against four
food pathogenic bacteria namely E. coli, Staphylococcus aureus, Lis-
teria monocytogenes, and Bacillus cereus. Antimicrobial activity tests
of edible films have been carried out using the agar diffusion
method.
In the above method, the film cuts are placed on Mueller Hinton
agar plates, which have been previously seeded with 0.1 ml of
inoculums containing indicator microorganisms in the range of
105–106 CFU/ml. (CFU is colony-forming unit) . The antimicrobial
effect of chitosan or KC2 incorporating nisin have been found much
better than that of konjac glucomannan incorporating nisin at each
corresponding concentration and there existed significant differ-
ence (p < 0.05). However, there was no significant difference on
the antimicrobial effect between chitosan and KC2 incorporating
nisin. Incorporating chitosan into konjac glucomannan film (KC2)
1177
therefore improved not only physical properties but also antimi-
crobial activity.
The characteristics of chitosan film have been evaluated by
cross-linking with naturally occurring aglycone geniposidic acid
(Mi et al., 2006). In this study, a comparative study was performed
between chitosan film without cross-linking (fresh), the glutaral-
dehyde-cross-linked chitosan film and aglycone geniposidic acid-
cross-linked chitosan film. A 50 lL bacterial broth (E. coli or S. aur-
eus) has been seeded onto film and cultured. The fresh chitosan
film and glutaraldehyde-cross-linked chitosan films have been
used as control. It has been proposed that the interaction between
the polycationic chitosan and the negatively charged surface of
bacteria may alter the permeability of the bacterial wall and lead
to the leakage of intracellular electrolytes and proteins. The results
suggested that cross-linking of chitosan films did not alter their
antibacterial capability. This may be due to the fact that the
cross-linking degrees of glutaraldehyde and aglycone geniposidic
acid (aGSA) cross-linked chitosan films used in this part of the
study were relatively low (<18%, with a concentration of cross-
linking agent of 0.8 mM). The aGSA-cross-linked chitosan film
has displayed a relatively lower water vapour permeability, a low-
er cytotoxicity, and a slower degradation rate than the glutaralde-
hyde-cross-linked film. It has been finally concluded that the
aGSA-cross-linked chitosan film may be a promising material as
an edible film for food packaging.
The shelf-life of food has been extended by ferulic acid incorpo-
rated starch-chitosan blend films (Mathew & Abraham, 2008).
Incorporation of ferulic acid has been found to improve the barrier
properties and tensile strength of starch-chitosan blend films and
significantly enhanced the lipid peroxide inhibition capacity.
The surface pictures obtained from scanning electron micro-
scope has revealed smooth structured films for both control and
ferulic acid incorporated films indicating good compatibility of
the components and the plasticizer. On comparing the cross-sec-
tion of the control blend films it could be seen that the blend film
had certain discontinuous zones and small pores while the latter
films were more compact due to the networking introduced by
ferulic acid. 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 film has been enhanced
by incorporation of garlic oil, potassium sorbate and nisin (Pranoto,
Rakshit, & Salokhe, 2005). The antimicrobial activity has been
tested against food pathogenic bacteria namely E. coli, Staphylococ-
cus aureus, Salmonella typhimurium, L. monocytogenes and B. cereus.
Antimicrobial tests have been carried out using agar diffusion
method. The agar diffusion test is a method commonly used to
examine antimicrobial activity regarding the diffusion of the com-
pound tested through water-containing agar plate. Incorporating
antimicrobial agents into chitosan edible films thus improves anti-
microbial efficacy of chitosan, as diffused antimicrobial activity
would add to non-migrated antimicrobial potency of chitosan. It
has been concluded that garlic oil incorporated into chitosan film
led to an increase in its antimicrobial efficacy, and has little effect
on mechanical and physical properties of chitosan films.
Overall, the incorporation of garlic oil into chitosan film has the
desirable characteristic of acting as a physical and antimicrobial
barrier to food contamination.
Two types of O-carboxymethylated chitosan/cellulose polybl-
ends have been prepared by using LiCl/N,N-dimethylacetamide
solution (Li et al., 2002). Antimicrobial activity of the blend films
against E. coli has been 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 has been observed that the antimicrobial activity of the blend
films enhances if the O-CMCh contamination is raised. Both blend
1178 P.K. Dutta et al. / Food Chemistry 114 (2009) 1173–1182

films exhibit satisfactory antibacterial activity against E. coli, even
with the O-CMCh concentration of 2 wt.% (see Fig. 1).
The antibacterial activity of chitosan-starch film using micro-
wave treatment has been carried out using agar plate diffusion
method (Tripathi et al., 2008). The antibacterial activity of the film
and their same solution has been evaluated against three different
test cultures viz. gram negative bacteria E. coli, gram positive bac-
teria S. aureus and gram positive bacteria Bacillus subtilis. It was
found that the solution of chitosan-starch showed inhibitory effect
against above said test cultures but film proved to be negative
(Fig. 2a–c and Fig. 3a–c).
Incorporating chitosan and lauric acid into starch based film
showed more effective antimicrobial ability against B. subtilis and
E. coli (Salleh et al., 2007). In this study, incorporating chitosan
and lauric acid into starch based film, obvious effects towards inhi-
bition of B. subtilis and E. coli have been observed while the film
had synergistic antimicrobial effect when chitosan and lauric acid
were combined. Antimicrobial starch-based film incorporated with
lauric acid and chitosan showed good flexibility than when purely
starch-based film was formulated and formed (Fig. 4). Inhibition of
CH2OH CH2OH
H
O H
O pastrami. The activity of the various films for inhibiting bacterial
O Conc. NaOH
OH OH
Deacetylation
H NHCOCH3 H NH2
n n
Chitin Chitosan
Fig. 1. Structure of chitin and chitosan.
bacterial growth was examined using two methods, i.e. zone of
inhibition test on solid media and liquid culture test (optical den-
sity measurements). The inhibitory activity was measured based
on the diameter of the clear inhibition zone. The solution of starch
and chitosan with different mixing ratio (w/w) 8:2 and 9:1 were
the most effective mixing ratio which had greater inhibition on
both B. subtilis and E. coli than other solution in agar plate and li-
quid culture test. The control (pure wheat starch) and 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 Fig. 5a and Fig. 5b. A wide clear zone on solid media was ob-
served for B. substilis growth inhibition whereas inhibition for
E. coli was not as effective as B. substilis. From the liquid culture
test, the AM films clearly demonstrated a better inhibition against
B. substilis than E. coli.
The tensile properties of the antimicrobial starch-based film
had been improved by the addition of chitosan. These antimicro-
bial starch-based films can be used to extend food shelf-life.
The feasibility of improving the preservation of vaccum-pack-
aged processed meats during refrigerated storage by use of an anti-
microbial film designed to gradually release antimicrobial agents
at the product surface (Ouattara et al., 2000a, 2000b). The antimi-
crobial films were applied onto bologna, regular cooked ham or
O growth were tested against indigenous lactic acid bacteria and
Enterobacteriaceae, and against Lactobacillus sakei or Serratia lique-
faciens, surface-inoculated onto the meat products. The growth of
Enterobacteriaceae and S. liquefaciens was delayed by application
of the antimicrobial film. It was found that the inhibition of indig-
enous 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.

Fig. 2. Inhibitory effect of chitosan-starch solution against (a) E. coli, (b) S. aureus, and (c) B. Subtilis (Tripathi et al., 2008).

Fig. 3. Inhibitory effect of chitosan-starch film against (a) E. coli, (b) S. aureus, and (c) B. Subtilis (Tripathi et al., 2008).
 P.K. Dutta et al. / Food Chemistry 114 (2009) 1173–1182
Fig. 4. A translucent starch-based film incorporated with lauric acid and chitosan
(Salleh et al., 2007; reprinted with permission from Asian Chitin Journal).
Fig. 5. Inhibition area of (a) control film and (b) AM incorporated film (Salleh et al.,
2007; reprinted with permission from Asian Chitin Journal).
The moisture and high lipid content of bologna helped the dif-
fusion of the oregano essential oil 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 chromatogra-
phy mass spectroscopy (GCMS) analysis showed that 757.7 ±
99.7 ppm carvacrol was extracted from the film-forming solution
prepared without TweenÒ 20 and only 364.7 ± 39.9 ppm from
the film-forming solution with the emulsifier. Different levels of
carvacrol were detected in the presence of TweenÒ 20 due to the
interaction of the amphiphilic emulsifier’s molecule with both
chitosan and oregano EO compounds. It is concluded that incorpo-
ration of an emulsifier in chitosan-oregano EO (essential oil) films,
may slow losses of volatile compounds of the oil and help control
the release of active compounds into the product.
7. Factors affecting the antimicrobial activity of chitosan
There are various factors, such as intrinsic and extrinsic, that af-
fects the antimicrobial activity of chitosan. It has been demon-
strated that lower molecular weight chitosan (of less than 10
kDa) have greater antimicrobial activity than native chitosans
(Uchida & et al., 1989). Furthermore, a degree of polymerisation
of at least seven is required; lower molecular weight fractions have
little or no activity (Ralston, Tracey, & Wrench, 1964; Uchida et al.,
1989). Highly deacetylated chitosans are more antimicrobial than
those with a higher proportion of acetylated amino groups, due
to increased solubility and higher charge density (Sekiguchi &
et al., 1994).
Lower pH increases the antimicrobial activity of chitosan for
much the same reasons, in addition to the ‘hurdle effect’ of inflict-
ing acid stress on the target organisms (Rhoades & Rastall, 2000).
Surrounding matrix is the greatest single influence on antimicro-
1179
bial activity. Being cationic, chitosan has the potential to bind to
many food components such as alginates, pectins, proteins and
inorganic polyelectrolytes such as polyphosphate (Kubota & Kiku-
chi, 1998). Solubility can be decreased by using high concentra-
tions of low molecular weight electrolytes such as sodium
halides, sodium phosphate and organic anions (Roberts, 1992).
8. Optimization of the biocide properties of chitosan
The influence on biocide performance of some unprecedented
physicochemical features of chitosan cast films such as film thick-
ness, pH of the nutrient broth, film neutralization, film autoclave
sterilization and temperature exposure were analysed against
Staphylococcus aureus and in some experiments also against Salmo-
nella spp. The work demonstrates for the first time the influence of
the release or positive migration of protonated glucosamine frac-
tions from the biopolymer into the microbial culture as the respon-
sible event for the antimicrobial performance of the biopolymer
under the studied conditions. From the results, a reliable and
reproducible method for the determination of the bactericidal
activity of chitosan based films was developed in an attempt to
standardise the testing conditions for the optimum design of active
antimicrobial 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, Lagaron, & Ocio, 2008).
9. Applications as bio-packaging for food preservation
9.1. General concepts
One of the best attributes of a good packaging film is the pre-
vention of microbial spoilage due to pathogens during extended
storage. Chitosans have been found useful as bio-packaging mate-
rials in the form of bioactive films for food preservation. Further-
more, the bioactive chitosan films potentially serve as a vehicle
to incorporate and enhance the food value along with other addi-
tives such as flavoring, coloring, antioxidant and antimicrobial
agents (Tharanathan, 2003). In addition to serving as packaging
material, bioactive Chitosan films have also been used as an edible
coatings which help preserve the food for a longer duration.
9.2. Homogeneous coatings
Edible coatings could reduce moisture transfer, restrict oxygen
uptake, lower respiration, retard ethylene production, seal in fla-
vour volatiles and carry additional functional ingredients (such as
antioxidants and antimicrobial agents) that retard microbial
growth and potential discoloration. Many authors have investi-
gated chitosan coatings for their potential to enhance the quality
and extend the storage life of food products (Coma, 2008; Coma,
Deschamps, & Martial-Grosc, 2003; Darmadji & Izumimoto, 1994;
Fisk, Silver, Strik, & Zhao, 2008; Rabea et al., 2003; Rhoades &
Roller, 2000; Ribeiro, Vicente, Teixeira, & Miranda, 2007; Sathivel,
Liu, Huang, & Prinyawiwatkul, 2007).
Different types of edible packagings can be obtained as a func-
tion of the composition and the manufacturing technique. Indeed,
homogeneous films with a smooth surface are obtained from
homogeneous solutions of polysaccharides or proteins, or from
molten lipids. Some mixtures of proteins and polysaccharides
make homogeneous edible packagings if all components are com-
pletely soluble in water or in a hydroalcoholic solution.
The linear structure of polysaccharides such as cellulose, amy-
lose or chitosan, renders their films tough, flexible and transparent
(Tharanathan, 2003). Pure chitosan films are generally cohesive,
1180 P.K. Dutta et al. / Food Chemistry 114 (2009) 1173–1182
compact and the film surface has a smooth contour without pores
or cracks (Coma et al., 2002; Wong, Gastineau, Gregorski, Tillin, &
Pavlath, 1992). Chitosan films such as many polysaccharide based
films, tend to exhibit fat and oil resistance and selective permeabil-
ity to gases but lack resistance to water transmission (Bordenave,
Grelier, & Coma, 2007; Sebti, Martial-Gros, Carnet-Pantiez, Grelier,
& Coma, 2006). This is due to strongly hydrophilic character of
these biopolymers, leading to an interaction with water molecules.
9.3. Biocomposites films and multilayer systems
Polymer blending is one of the useful ways to obtain new mate-
rials with required properties and there has been great scientific
and commercial progress in the area of food applications. Compos-
ite packagings are defined as films or coatings whose structure is
heterogeneous, that is, composed of a continuous matrix with
some inclusions, such as lipidic globules in case of an emulsion,
or solid particles in case of non-soluble substances (fibers, hydro-
phobic proteins), or composed of several layers. Usually, multilay-
ered films have better mechanical and barrier efficiencies than
emulsion-based films and coatings but their manufacturing re-
quires additional step of spreading or lamination and drying for
each layer. Their use, therefore, in an industrial plan does not seem
very practical because of too many steps in their making. On the
contrary, emulsion-based edible films provide nearly the same
properties, while only requiring one operation in their preparation.
In preparation of the bioactive konjac glucomannan edible films
by the incorporation of chitosan or nisin, it has been demonstrated
that there exists a strong intermolecular hydrogen bonding be-
tween chitosan and konjac glucomannan.
In order to improve moisture barrier properties, chitosan has of-
ten been combined with fatty acids. The chitosan-lauric acid films
exhibited higher moisture barrier properties than chitosan-pal-
mitic or stearic acid (Wong et al., 1992). Fatty acids from butyric
to octanoic acids were tested at a ratio 2:1 (w/w chitosan/fatty
acid). The authors concluded that the degree of water permeability
does not follow the increasing chain length of the fatty acid. The
incorporation of lauric acid decreased the permeability by 49% as
compared to chitosan film while palmitic acid increased the per-
meability by 67%.
9.4. Biocatalysts in food packaging
Since the market for minimally processed foods is broadening,
packaging is being employed as part of the strategies that actively
contribute to the food preservation and transformation concepts.
One opportunity is given by the incorporation of biocatalysts, usu-
ally in prefabricated carriers, where they can fully retain their
activity. These biocatalysts can either be repeatedly used within
the packaging or, under controlled conditions, migrate to the prod-
uct. The potential use of packaging composed of classic polymers
with functional surfaces or recently introduced biopolymers have
shown good perspectives for their controlled release when directly
used in contact with the product or as part of the combined strat-
egies. Moreover, new nanostructured materials (modified clays,
LBL assemblies, electrospun fibers) are being implemented as part
of more innovative solutions. The review (Fernández, Cava, Ocio, &
Lagarón, 2008) summarises most recent developments and upcom-
ing possibilities of incorporating biocatalysts in biopolymers tradi-
tionally used in food packaging applications.
10. Conclusions
Antimicrobial packaging is very promising system for the future
improvement of food quality and preservation during processing
and storage. Antimicrobial packaging can also be helpful in extend-
ing the food shelf-life. Chitosan has offered itself as a versatile and
promising biodegradable polymer for food packaging. In addition,
chitosan possesses immense potential as a antimicrobial packaging
material owing to its antimicrobial activity and non-toxicity. The
functional properties of chitosan films can be improved when
chitosan films are combined with other film- forming materials.
Inherent antibacterial properties and film-forming ability of chito-
san make it an ideal choice for use as a biodegradable antimicrobial
packaging material that can be used to improve the storability of
perishable foods. It has convincingly been proved that chitosan
films exhibit good antimicrobial activity which can help extend
the food shelf life. Hence, it is going to be no surprise if we witness
a widespread use of chitosan films in tomorrow’s food packagings.
Acknowledgements
One of us (Shipra Tripathi) is grateful to Prof.Arun B.Samaddar,
Director, M. N. National Institute of Technology, Allahabad, India,
for financial assistance in the form of Institute Research
Fellowship.
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