Chitosan 8
Surinder P Chawla Sweetie R Kanatt and A K Sharma
Contents
1 Introduction 220
2 Chitin 220
3 Chitosan 222
4 Sources 223
5 Production of Chitosan 224
6 Novel Methods for Preparation of Chitosan 226
7 Characterization of Prepared Chitosan and Its Properties 227
8 Modifications of Chitosan 230
9 Chitosan Depolymerization 232
10 Applications of Chitosan 233
11 Antimicrobial Activity 233
12 Antioxidant Activity 235
13 Chitosan as Edible Coating for Fruits and Vegetables 235
14 Chitosan as Functional Additive in Muscle Foods 236
15 Chitosan as Clearing Agent in Wines and Vinegars 237
16 Chitin as Feed Additive 237
17 Chitosan as LipidLowering Agent 237
18 Biomedical Applications 238
19 Chitosan in Water Treatment 239
20 Chitosan Application in Agriculture 240
21 Regulations and Commercial Applications 241
22 Conclusion and Future Research Needs 242
References 243
SP Chawla (*) • SR Kanatt • AK Sharma
Food Technology Division Bhabha Atomic Research Centre Mumbai India
email spchawla@barcgovin
# Springer International Publishing Switzerland 2015
KG Ramawat JM Me´rillon (eds) Polysaccharides
DOI 1010079783319162980_13
219Abstract
Chitin is the most abundant natural amino polysaccharide and is next to cellulose
in abundance on the planet Chitosan is obtained by deacetylation of chitin
Chitosan is being researched by academic and industrial scientists as an
underutilized resource and as a new functional material of high potential in
various fields The purpose of this chapter is to give an overview of chitosan
production characterization modification and applications
Keywords
Chitin • Chitosan • Polysaccharide • Deacetylation • Antimicrobial • Gel forma
tion • Biopolymer • Active packaging • Metal chelation • Food additive •
Biomedical • Biosorbent • Biodegradability
1 Introduction
Polysaccharides are polymeric carbohydrate molecules composed of long chains of
monosaccharide units bound together by glycosidic linkages and on hydrolysis give
the constituent monosaccharides or oligosaccharides They have linear to highly
branched structure Their major role in organism is to store energy or to give
structural support Starch and glycogen are examples of storage polysaccharide
and cellulose and chitin are examples of structural polysaccharides
2 Chitin
Henri Braconnot a French professor discovered chitin in 1811 and named it
fungine In 1823 Odier found the same material in insects and plants and named
it chitin After cellulose chitin is the most abundant natural polysaccharide avail
able on the planet Chitin is similar to cellulose in chemical structure (Fig 1) and in
biological function Both polymers mainly serve as structural components
supporting cell and body surfaces cellulose strengthens the cell wall of plant
cells whereas chitin contributes to the mechanical strength of fungal cell walls
and exoskeletons of arthropods (Rudall and Kenchington 1973)
It has been estimated that at least 11 Â 1013 kg of chitin is present in the
biosphere However its use has been limited because it is insoluble in most
solvents and relatively difficult to isolate from natural sources in pure form
under economically viable conditions Chitin is a white hard inelastic nitroge
nous polysaccharide found in the exoskeletonaswellasintheinternalstructures
of invertebrates It is a linear cationic polymer of Nacetylglucosamine residues
with β14linkage Chitin occurs in three polymorphic forms α βandγbut
αchitin is the most abundant (Khoushab and Yamabhai 2010) The arrangement
of the chains is found to depend on the origin of the chitin αChitin is present in
fungal and yeast cell walls insect cuticles egg shells of nematodes and rotifers
220 SP Chawla et althe radulae of mollusks and cuticles of arthropods This form of chitin is also
present in krill lobster crab tendons and shells and shrimp shells as well as in
other marine organisms such as the harpoons of cone snails and the filaments
ejected by Phacocystis seaweed βChitin is found in the pen and cuticle of squid
and the diatom Thalassiosira fluviatilisInαchitin sheets are formed by
intermolecular hydrogen bonding in parallel chains Interchain hydrogen bonding
occurs between sheets in different directions There is also intermolecular hydro
gen bonding between CH2OH groups which is believed to be the cause for the
lack of swelling of αchitin in water βChitin has a monoclinic unit cell with
polysaccharide chains attached in a parallel manner (Gardner and Blackwell
1975) In βchitin hydrogen bonding occurs only within sheets not between
sheets as in αchitin This is thought to be responsible for the swelling of
βchitin as water can be included between the sheets γChitin is said to be a
combination of α and β structure rather than a third polymorph (Robert 1992)
Chitin has <10 degree of acetylation 7 nitrogen content nitrogencarbon
ratio of 0146 and molecular weight of 1–25 Â 106 Da corresponding to a degree
of polymerization of ca 5000–10000 which differ in the arrangement of their
molecular chains
During biosynthesis of chitin monomers of Nacetylglucosamine are joined in a
reaction catalyzed by the membraneintegral enzyme chitin synthase a member of
the family of glycosyltransferases The polymerization requires UDP–Nacetylglu
cosamine as a substrate and divalent cations as cofactors Chitin formation can be
divided into three distinct steps In the first step the catalytic domain of chitin
synthase facing the cytoplasmic site forms the polymer The second step involves
the translocation of the nascent polymer across the membrane and its release into
the extracellular space The third step completes the process as single polymers
spontaneously assemble to form crystalline microfibrils of varying diameter and
length (Merzendorfer 2006)
Fig 1 Chemical structure of cellulose and chitin
8 Chitosan 221In the chitin crystal structure the chains form hydrogenbonded sheets linked by
CO and HNgroups In addition each chain has intramolecular hydrogen bonds
between the neighboring sugar rings the carbonyl group bonds to the hydroxyl group
on C6 There is also a second hydrogen bond between the OHgroup on C3 and the
ring oxygen similar to that in cellulose (Minke and Blackwel 1978) This extensive
hydrogen bonding shown in Fig 2 enhances the stiffness of the chitin chain
With only one known exception the chitin of diatoms chitin is found in nature
crosslinked to other structural components The chitin microfibrils combine with
other sugars proteins glycoproteins and proteoglycans to form fungal septa and
cell walls as well as arthropod cuticles and peritrophic matrices notably in crusta
ceans and insects (Kozloff 1990) In animals chitin is associated with proteins
while in fungal cell wall it is associated with glucans mannans or other poly
saccharides In fungal walls it is found covalently bound to glucans either directly
or via peptide bridges (Roberts 1992) In insects and other invertebrates the chitin
is always associated with specific proteins with both covalent and noncovalent
bonding to produce the observed ordered structures
3 Chitosan
Chitosan is obtained by deacetylation of chitin It consists of Dglucosamine linked
to Nacetyl Dglucosamine by β14glycosidic bond (Fig 3) The distribution of
these subunits depends on the method of preparation of chitosan In chitosan
degree of deacetylation ranges from 40 to 98 and the molecular weight ranges
between 5 Â 104 Da and 2 Â 106 Da
H
O
O
O
O
O
O
O
O
O
HO
O
O
OH
O
O
H
NH NH
NHHO
O
HO
HO
NH NH
H
O
O
O
O
O HO
H
H2C
H3C
H3C
H2C
H3C
H2C
H3C
H2C
H2C
H3C
Fig 2 Chemical structure of chitin shown with its intramolecular hydrogen bonds (dotted lines)
O O
O
O
OO
OH OH
HO HO NH
CH3
NH2
Fig 3 Chemical structure of chitosan
222 SP Chawla et alIntense research and development work is being carried out on chitosan as it is
considered to be a material of great futuristic potential with immense possibilities for
structural modifications to impart desired properties and functions The presence of
reactive amino groups at C2 atom and the hydroxyl group at atom C3 and C6 on
chitosan is useful in a wide application in various industries The positive attributes of
excellent biocompatibility and admirable biodegradability with ecological safety and
low toxicity with versatile biological activities such as antimicrobial activity and low
immunogenicity have provided ample opportunities for further development
4 Sources
Chitosan can be extracted from insects yeast mushroom cell wall of fungi and marine
shellfish such as crab lobster krill cuttlefish shrimp and squid pens (Table 1) In
shellfish chitin forms the outer protective coating as a covalently bound network with
proteins and some metals and carotenoids Shrimps are in general sold headless and
Table 1 Contents of chitin in different commercially important organism
Organism W (chitin)
Cancer (crab) 721a
Carcinus (crab) 642b
Paralithodes (king crab) 350b
Callinectes (blue crab) 140c
Crangon and Pandalus (shrimp) 17–40
Alaska shrimp 280d
Nephro (lobster) 698a
Homarus (lobster) 60–75a
Lepas (goose barnacle) 583a
Bombyx (silk worm) 442a
Mollusks
Clam 61
Shell oysters 36
Squid pen 410
Krill deproteinized shells 402
Fungi
Penicillium notatum 185e
Penicillium chrysogenum 201e
Mucor rouxii 445
Lactarius vellereus 190
Adapted from Jo et al (2011) and Kurita (2006)
aBased on the mass of the organic cuticle
bWith respect to the body dry mass
cCompared to the body fresh mass
dCompared to the total mass of the cuticle
eRelative to the dry mass of the cell wall
8 Chitosan 223often peeled of the outer shells and tail Crustacean shells consist of 30–40 proteins
30–50 calcium carbonate and 20–30 chitin and also contain pigments
(astaxanthin canthaxanthin lutein and βcarotene) These proportions vary with
species and with seasons Shrimp prawn and crab wastes are the principal source of
commercial chitin and chitosan production The increase in consumption of shellfish
and the expansion of aquaculture have led to a tremendous increase in the quantity of
shrimp and prawn being processed and hence in the amount of waste available for
chitinchitosan production Using mycelium waste from fermentation processes as a
source of chitin and chitosan still remains a vast and as yet untapped potential source
5 Production of Chitosan
Majority of chitosan available globally is produced by the USA Japan Norway
Thailand India Australia and Poland The production of chitosan involves various
steps such as preparation of the chitin from the biological material followed by the
deacetylation that would result in chitosan Thus typical production of chitosan
from crustacean shell generally consists of four basic steps demineralization
deproteinization decoloration and deacetylation Demineralization and deprotei
nization steps are interchangeable in terms of order The exoskeleton of crustacean
is a major starting material used for commercial production of chitosan Typical
flow chart for manufacture of chitosan is given below (Fig 4)
51 Demineralization
The mineral content in the exoskeleton of crustacean is not the same for all species
of crustaceans Demineralization is generally carried out using acids such as
hydrochloric acid nitric acid acetic acid or formic acid (up to 10 ) at room
temperature with agitation to dissolve calcium carbonate as calcium chloride
However hydrochloric acid is the preferred acid and is used at a concentration of
02–2 M for 1–48 h at temperatures varying from 0 C to 100 C Demineralization
for 1–3 h using dilute (1–8 ) hydrochloric acid at room temperature produces
appreciable amounts of calcium chloride A solidtosolvent ratio of 115 (wv) is
usually used The ash content of the demineralized shell is an indicator of the
effectiveness of the demineralization process
52 Deproteinization
Chitin occurs naturally in association with protein The protein is bound by covalent
bonds to chitin through aspartyl or histidyl residues or both thus forming stable
complexes (Attwood and Zola 1967) Deproteinization of chitin is usually carried
out by alkaline treatment The shells are treated with sodium or potassium hydrox
ide at 65–100 C at a minimum shelltoalkali ratio of 14 for periods ranging from
1 to 12 h Under these conditions the protein becomes detached from the solid
224 SP Chawla et alcomponent of the shrimp waste Relatively high ratios of solidtoalkali solution
of 110 or 120 with proper agitation are used to increase the deproteinization
efficiency To prevent oxidation of the products the process is usually carried
out in a nitrogen atmosphere and in the presence of sodium borohydride
(NaBH4) After completion of deproteinization step the protein hydrolysate is
removed easily by separation of the solids from the protein slurry by filtration
Prolonged alkaline treatment under severe conditions causes depolymerization and
deacetylation
53 Decoloration
Chitin obtained after the demineralization and deproteinization of shell waste is a
colored product For commercial acceptability the chitin needs to be decolorized or
bleached to yield cream white chitin powder (No et al 1989) The pigment in the
Deproteinisation
Washing
Demineralisation
Deacetylation
Washing
Decolouration
Drying
Milling
Chitosan
Fig 4 Flow chart for
chitosan production
8 Chitosan 225crustacean shells forms complexes with chitin Fox (1973) found one
4ketoßcarotene and three 4 40diketoßcarotene derivatives firmly bound to
the exoskeletal chitin of red kelp crab The level of association of chitin and
pigments varies from species to species among crustacean The residues are decol
orized using solvents andor oxidants (Acosta et al 1993) During the process of
decoloration the chemical used should not affect the physicochemical or functional
properties of chitin and chitosan No et al (1989) were able to prepare a nearwhite
colored crawfish chitin by extraction with acetone which was dried for 2 h at
ambient temperature followed by bleaching with 0315 (vv) sodium
hypochloride solution (containing 525 available chlorine) for 5 min with a
solidtosolvent ratio of 110 (wv) based on dry shell
54 Deacetylation
Deacetylation is the process to convert chitin to chitosan by removal of acetyl
group There are several critical factors that affect the extent of deacetylation
including temperature and time of deacetylation alkali concentration prior treat
ments applied to chitin isolation atmosphere (air or nitrogen) ratio of chitin to
alkali solution density of chitin and the particle size Considering all these as
necessary conditions the ideal process condition of deacetylation should yield a
chitosan that is not degraded and is soluble in dilute acetic acid in minimal time
(Muzzarelli et al 1980) The Nacetyl groups cannot be removed by acidic reagents
without hydrolysis of the polysaccharide thus alkaline methods must be employed
for Ndeacetylation (Muzzarelli 1977) Severe alkaline hydrolysis treatments are
required due to the resistance of groups imposed by the trans arrangement of the
C2C3 substituents in the sugar ring It is generally achieved by treatment with
concentrated sodium or potassium hydroxide solution (40–60 ) usually at 80–140 C
for 30 min or longer using a solidtosolvent ratio of 110 (wv) to remove some
or all of the acetyl groups from the polymer (No and Meyers 1989) Sodium
hydroxide is the preferred alkali After deacetylation the chitosan is washed to
completely remove alkali and is dried to give flakes The material should be low in
protein and ash Production of chitosan by chemical processes has several disadvan
tages such as environmental pollution inconsistent molecular weights and degree of
acetylation
6 Novel Methods for Preparation of Chitosan
The conventional harsh conditions used for extraction could adversely affect the
quality of the chitin Novel methods are being developed to replace conventional
demineralization and deproteinization to extract chitin from crustacean waste The
use of enzymes in the deproteinization step has been extensively studied Shrimp
waste deproteinized using Aspergillus niger washed dried and then demineralized
using acetic or lactic acid produced by fermentation from low cost biomass such as
226 SP Chawla et alcheese whey has been reported (Rinaudo 2006) A number of microorganisms such
as Bacillus subtilis Lactobacillus helveticus Pseudomonas aeruginosa Lactoba
cillus paracasei Lecanicillium fungicola and Penicillium chrysogenum have been
utilized for demineralization (Choorit et al 2008 Oh et al 2008) These microor
ganisms are responsible for the precipitation of organic salts such as calcium
lactate which is easily removed from media by wash out Deproteinization is
also carried out with the aid of proteolytic activities of some microorganisms
The calcium magnesium and potassium acetates obtained as byproducts are
suggested as possible deiceing agents while the calcium and potassium lactates
could find applications as food preservatives Enzymatic deacetylation by using
fungal chitin deacetylase also has commercial potential
7 Characterization of Prepared Chitosan and Its Properties
71 Molecular Weight
One of the most fundamental parameters characterizing a macromolecule is its
molecular weight Knowledge of the molecular weight of polysaccharides is of
fundamental importance for the understanding of their applications and their role in
living systems The molecular weight of chitosan depends largely on the conditions
of deacetylation and can be determined by methods such as chromatography
(Bough et al 1978) light scattering (Muzzarelli 1977) and viscometry (Maghami
and Roberts 1988) Viscometry is the simplest and most popular method to deter
mine molecular weight of chitosan The method however has the disadvantage of
not being absolute because it relies on the correlation between the values of intrinsic
viscosity with those of molecular weight Chitosan is available commercially with
molecular weight ranging from 10000 to 1000000 Da
72 Viscosity
Viscosity of chitosan increases with increase in its molecular weight and concen
tration Increasing the degree of deacetylation also increases the viscosity
(Skaugrud 1991) This can be explained by the fact that high and low deacetylated
chitosan have different conformations in aqueous solution Chitosan has an
extended conformation with a more flexible chain when it is highly deacetylated
because of the charge repulsion in the molecule However the chitosan molecule
has a rodlike shape or coiled shape at low degree of deacetylation due to the low
charge density in polymer chain The viscosity of chitosan solution is also affected
by factors such as concentration and temperature As the chitosan concentration
increases and the temperature decreases the viscosity increases Chitosan viscosity
decreases with an increased time of demineralization due to depolymerization
(Moorjani et al 1975) Similarly No et al (1999) demonstrated that chitosan
viscosity is considerably affected by physical (grinding heating autoclaving
8 Chitosan 227ultrasonication) and chemical (ozone) treatments Viscosity of chitosan solution
stored at 4 C is found to be relatively stable
73 Solubility
Solubility characteristics of chitosan are based on its degree of deacetylation High
degree of deacetylation shows higher solubility and low degree of deacetylation
shows poor solubility (Heux et al 2000) It has swelling characteristics due to much
weaker intermolecular hydrogen bonding ascribable to the parallel arrangement of
the main chains Chitosan solubility depends on the amount of protonated amino
groups in the polymeric chain and therefore on the proportion of acetylated and
nonacetylated Dglucosamine units Its cationic nature is unique relative to other
neutral or negatively charged polysaccharides Chitosan is a strong base possessing
primary amino group with a pKa value of 63 The pH of solution substantially
alters the charged state and properties of chitosan (Yi et al 2005) At low pH the
amines get protonated and become positively charged and that makes chitosan a
watersoluble cationic polyelectrolyte On the other hand as the pH increases above
6 chitosan amines become deprotonated and the polymer loses its charge and
becomes insoluble At higher pH precipitation or gelation tends to occur and the
chitosan solution forms polyion complex with anionic hydrocolloid resulting in gel
formation (Kurita 1998) The soluble–insoluble transition occurs at its pKa value
around pH between 6 and 65 Chitosan can easily form quaternary nitrogen salts at
low pH values So organic acids such as acetic formic and lactic acids can
dissolve chitosan The most commonly used solvent for chitosan is 1 acetic
acid at about pH 40 (Rinaudo et al 1999) Chitosan is also soluble in 1
hydrochloric acid and dilute nitric acid but insoluble in sulfuric and phosphoric
acids Thus solubility of chitosan is related to the degree of deacetylation the ionic
concentration pH the nature of the acid used for protonation and the distribution
of acetyl groups along the chain as well as the conditions of isolation and drying of
the polysaccharide The high molecular weight of chitosan which results in poor
solubility at neutral pH and its high solution viscosity limits its use in the food
cosmetics agriculture and health industry (Xia et al 2011)
74 Degree of Deacetylation
Degree of deacetylation (DD) has often been cited as an important parameter that
determines many physiochemical and biological properties of chitosans such as
crystallinity hydrophilicity degradation and cell response Degree of deacetylation
of chitosan is generally controlled by processing of the native polymer with alkali and
with increasing time and temperature to obtain the highest degree of deacetylation
(>90) materials During the deacetylation reaction the acetyl group of the chitin
reacts with NaOH and produces an amine group This is a reversible reaction and
when NaOH concentration is increased the reaction is biased toward the forward
228 SP Chawla et aldirection by producing more chitosan As a result deacetylation will increase In the
deacetylation process acetyl groups are removed from the polymers randomly
resulting in a final polymer that has a random distribution of acetyl glucosamine
and glucosamine units The biopolymer is characterized as either chitin or chitosan
according to the deacetylation which is determined by the proportion of Dglucos
amine and Nacetyl Dglucosamine Various methods have been reported for the
determination of the degree of deacetylation of chitosan such as (1) spectroscopy
(infrared ultraviolet or 1H 13C 15N nuclear magnetic resonance) (2) conventional
methods (various types of titration conductometry potentiometry ninhydrin assay
adsorption of free amino groups of chitosan by picric acid) and (3) destructive
methods (elemental analysis or acid or enzymatic hydrolysis of chitin or chitosan)
followed by colorimetric methods or highperformance liquid chromatography
pyrolysis gas chromatography and thermal analysis using differential scanning
calorimetry Of these 1H NMR has been found to be simple rapid and more precise
than many of the other methods (Rinaudo 2006)
75 Crystallinity
One of the major physical characteristics that determine the functional properties of
chitosan is the crystallinity (Trang et al 2006) Crystallinity has been found to have
an effect on metal sorption Piron et al (1997) found that the crystallinity of
chitosan controlled the sorption rate and total uptake of uranyl concluding that
sorption was only possible in the amorphous domains and not in the crystalline
domains The crystallinity of the polymer can also control the accessibility of the
amine groups (Guibal 2004) The crystallinity of chitosan is determined by Xray
diffraction (XRD) in which the pattern produced by the diffraction of Xrays
through the closely spaced lattice of atoms in a crystal is recorded and then
analyzed to reveal the nature of the lattice
76 Complex Formation with Metals
Chitosan exhibits superior metal ion sequestering ability than chitin It has reactive
amino group and hydroxyl group and chelates many transition metal ions Chelation
is related to the amino content as well as to the distribution of the amino group The
nature of the cation is very important in the mechanism of interaction (Rhazi
et al 2002) Various processes such as adsorption ion exchange and chelation
have been considered as the mechanisms responsible for complex formation
between metal ions and chitosan The type of interaction prevailing depends on
the metal its chemistry and the pH Under heterogenous conditions at pH less than
6 chitosan acts as a poly(monodentate) ligand while at a higher pH it behaves as a
poly(bidentate) ligand forming chelates However in solution the formation of
complexes in which two amino groups belonging to the same chain or different
chains coordinated to the same metal ion can also take place
8 Chitosan 2298 Modifications of Chitosan
Chitosan can be modified to improve its physicochemical properties to suit various
applications Modification of chitosan is possible due to the presence of several
functional groups in the polymer (Fig 5) It has both reactive amino and hydroxyl
groups that can be used to chemically alter its properties under mild reaction
conditions The main goals of modifying chitosan are to provide derivatives that
are soluble at neutral and basic pH values to control hydrophobic cationic and
anionic properties as well as to attach various functional groups and ligands
(Mourya and Inamdar 2008) Strong intramolecular and intermolecular hydrogen
bonds exist in chitosan to form random orientations The dissociation and reorga
nization of these hydrogen bonds by chemical modification facilitate the production
of novel molecular conformations in the forms of solutions hydrogels fibers films
and sponges (Tokura et al 1996)
81 Acylation
A variety of acylation reactions are possible with chitosan Acylation with long
chain aliphatic carboxylic acid chlorides such as hexanoyl dodecanoyl and
tetradecanoyl chlorides give derivatives with a high degree of acylation Nacyla
tion of chitosan with fatty acid (C6–C16) chlorides increased its hydrophobic
character Such acylated products are soluble in chloroform (Fujii et al 1980)
Chitosan with a higher degree of deacetylation is more susceptible for acylation
owing to a decrease in hydrogen bonding Nacyl chitosan has the ability for longer
retention in body and resistance to digestible enzymes like lysozyme and chitinase
and is more biocompatible than native chitosan (Hirano and Yagi 1980)
82 Graft Copolymerization
Graft copolymerization reaction introduces side chains and makes various molec
ular designs possible thus affording novel types of tailored hybrid materials
composed of chitosan and synthetic polymers The properties of the graft copoly
mers can be controlled by molecular structure length and number of side chains
attached Grafting of chitosan allows the formation of functional derivatives by
OH
OH
HO
HO O
O
O
O
Primary amino function
Primary hydroxy function
Secondary hydroxy function
O
NH2
NH2
Fig 5 Functional groups in chitosan that can be modified
230 SP Chawla et alcovalent binding of a molecule the graft onto the chitosan backbone The swelling
behavior of chitosan at different pH has been improved by graft polymerization of
vinylic monomers such as acrylic acid acrylamide and acrylonitrile onto chitosan
(Borzacchiello et al 2001 Mahdavinia et al 2004) Super absorbents (absorb
aqueous solutions up to hundreds of times their own dry weight) have been prepared
by grafting these resins with chitosan (Nge et al 2004) and have possible applica
tions in infant diapers feminine hygiene products agriculture and other special
ized areas (Dutkiewicz 2002) Different types of chitosan graft copolymers have
been prepared for use as flocculants paperbinder strengtheners and slowrelease
drug carrier Polyethylene glycol (PEG) has been grafted onto chitosan to prepare
watersoluble chitosan derivatives that have been used as carrier of anticancer
drugs Phosphorylated chitosan synthesized by grafting mono(2methacryloyl
oxyethyl) acid phosphate onto chitosan improved antimicrobial activities (Jung
et al 1999)
83 Carboxymethyl Chitosans
It is an amphoteric polymer is a derivative of chitosan and is prepared under
controlled reaction conditions It can be synthesized by reductive alkylation
wherein the amino group of chitosan is reacted with the carbonyl group of aldehyde
glyoxylic acid and then hydrogenated by reaction with NaBH4 or NaCNBH3 to give
carboxymethyl chitosans It can also be prepared by direct alkylation using
monohalocarboxylic acids such as monochloroacetic acid in alkaline medium
Carboxymethyl chitosans have enhanced biological and physicochemical proper
ties compared to chitosan and hence have promising biomedical applications
(Mohan et al 2012)
84 Nmethylene Phosphonic Chitosans
These are anionic derivatives with amphoteric character and are synthesized under
various conditions and proved to have good complexing efficiency for cations such
as Ca2+ and those of transition metals (Cu (II) Cd (II) Zn (II) etc) (Heras
et al 2001) The complexation provides corrosion protection for metal surfaces
These derivatives are also modified and grafted with alkyl chains to obtain amphi
philic properties that have potential applications in cosmetics
85 CarbohydrateBranched Chitosan
Carbohydrates can be grafted on the chitosan backbone at the C2 position by
reductive alkylation disaccharides such as cellobiose and lactose (having a reduc
ing end group) are introduced in the presence of a reductant on chitosan in the
open chain form These derivatives are water soluble Carbohydrates can also be
8 Chitosan 231introduced without ring opening on the C6 position These derivatives are important
as they are recognized by the corresponding specific lectins and thus could be used
for drug targeting (Morimoto et al 2001)
86 Alkylated Chitosans
Alkylated chitosans are very important as amphiphilic polymers based on poly
saccharides They exhibit surface activity and increase considerably the viscosity of
aqueous solution due to hydrophobic interchain interactions Alkyl chitosans are
compatible with neutral and cationic surfactants (Yang et al 2002)
9 Chitosan Depolymerization
The main limitations in the use of chitosan in several applications are its high
viscosity and low solubility at neutral pH Low molecular weight chitosans and
oligomers can be prepared by hydrolysis of the polymer chains For some specific
applications these smaller molecules have been found to be much more useful
(Rege and Block 1999) Chitosan depolymerization can be carried out chemically
enzymatically or physically
91 Chemical Depolymerization
It is mainly carried out by acid hydrolysis using HCl or by oxidative reaction using
HNO2 and H2O2 (Prashanth and Tharanathan 2007) It has been found to be specific
in the sense that HNO2 attacks the amino group of Dunits with subsequent
cleavage of the adjacent glycosidic linkage
92 Enzymatic Depolymerization
In the case of enzymatic depolymerization low molecular weight chitosan with
high water solubility is produced by several enzymes such as chitinase chitosanase
gluconase and some proteases (Cabrera and Cutsem 2005) Nonspecific enzymes
including lysozyme cellulase lipase amylase and pectinase that are capable of
depolymerizing chitosan are also used Enzymatic methods for the hydrolysis of
chitosan are performed in gentle conditions and the molecular weight distribution
of the product can be controlled (Jeon et al 2001)
93 Physical Depolymerization
Physical depolymerization yielding dimers trimers and tetramers has been carried
out by radiation (Co60 gamma rays) but low yields have been achieved High
pressure homogenization is a novel method employed for the depolymerization of
232 SP Chawla et alchitosan (Mistry et al 2012) Chitosan has been physically modified in a variety of
ways resulting in conditioned forms such as powders nanoparticles gel beads
gels fibers and sponge (Denkbas 2006)
10 Applications of Chitosan
A lot of research is being carried out by both academic and industrial scientists on
applications of chitosan This can be seen by a number of relevant research papers
and patents on the subject Chitosan and its derivatives have varied applications in
agriculture food processing biotechnology chemistry cosmetics dentistry med
icine textiles veterinary medicine and environmental sciences The polyelectro
lyte nature and the presence of reactive functional groups are responsible for the
gelforming ability high adsorption capacity biodegradability and antimicrobial
properties of chitosan which in turn are essential for its commercial applications
11 Antimicrobial Activity
Chitosan displays a broadspectrum antimicrobial activity against bacteria
molds and yeasts It is effective against both Grampositive and Gram
negative foodborne microorganisms including Aeromonas hydrophila Bacillus
cereus B licheniformis B subtilis Clostridium perfringens Brochothrix spp
Enterobacter sakazakii Lactobacillus spp Listeria monocytogenes Pseudomonas
spp Salmonella typhimurium S enteritidis Serratia liquefaciens Staphylococcus
aureus and Escherichia coli O157H7 the yeasts Candida Saccharomyces and
Rhodotorula and the molds Aspergillus Penicillium and Rhizopus The chitosan
and its derivatives are effective against plant pathogenic bacteria such as
A tumefaciens C fascians E amylovora E carotovora P solanacearum and
S lutea and fungi A alternata B fabae F oxysporum P digitatum
P debaryanum and R solani (Vishnukumar et al 2005 Venugopal 2011)
The exact mechanism of antibacterial activity of chitosan is not fully understood
and several factors contribute toward this Three models have been proposed to
explain the antimicrobial action of chitosan The most satisfactory model suggests
that the antimicrobial effect of chitosan is due to its polycationic nature In an acid
environment the NH2 groups in the C2 position of chitosan protonates to yield NH3+
which binds to negatively charged carboxylate (–COO–) groups located on the
surface of the bacterial and fungal cell surfaces causing disruption of the
barrier properties of the outer membranes of the microorganisms followed by
leakage of cell components (Tsai and Su 1999) This hypothesis is supported
by electron microscopy studies that show binding of chitosan to outer membrane
of bacteria (Raafat et al 2008) The pH of the microenvironment in which
chitosan functions determines the relative concentrations (ratios) of unprotonated
and protonated amino groups At a pH ~ pKa 50 of amino group are protonated
At pH 55 the positively charged amino group contributes 90 and at pH 45 99
8 Chitosan 233The antimicrobial effectiveness of chitosan appears to be highest below pH 60
where the protonated form predominates and where chitosan is most soluble
Second proposed mechanism is based on ability of chitosan to bind with
microbial DNA leading to inhibition of the mRNA and protein synthesis (Sebti
et al 2005) In this hypothesis chitosan molecules are assumed to be able to pass
through the bacterial cell wall composed of multilayers of crosslinked murein and
reach the plasma membrane This theory is supported by confocal laser scanning
microscopy where the presence of chitosan oligomers (a chain with few number of
monomer units) inside E coli exposed to chitosan under different conditions has
been demonstrated (Lui et al 2001)
The third mechanism is based on ability of chitosan to chelate metals It is well
known that chitosan has excellent metalbinding capacities where the amine groups
in the chitosan molecules are responsible for the uptake of metal cations by
chelation this results in reduced microbial growth and toxin synthesis (Goy
et al 2009) This mechanism is likely to be more efficient at high pH values
where positive ions are bounded to chitosan since the amine groups are
unprotonated and the electron pair on the amine nitrogen is available for donation
to metal ions
The ability of chitosan to form gasimpermeable coating interferes with fungal
growth It inhibits different developmental stages such as mycelial growth sporu
lation spore viability and germination and the production of fungal virulence
factors (El Ghaouth et al 1992)
The derivatives of chitosan such as Ntrimethyl sulfonated chitosan and
chitose oligomers have been reported to demonstrate antibacterial activities against
Bacillus subtilis Pseudomonas aeruginosa Staphylococcus aureus S epidermidis
Klebsiella pneumoniae and Proteus vulgaris to different extents (Venugopal
2011)
111 Factors Affecting Antimicrobial Activity
The antimicrobial activity of chitosan depends on its molecular weight degree of
deacylation pH of solution and of course the target organism
Molecular weight The antimicrobial activity of chitosan increases as the molec
ular weight increases However it is difficult to find a clear correlation between
molecular weight and antimicrobial activity of chitosan when comparisons are
between different studies This is mainly attributed to the fact that many investiga
tors have used an uncertain term for low MW (LMW) and high MW (HMW)
chitosan without indicating exactly its MW There are reports that conclude posi
tive negative and neutral effects of MW on antimicrobial activity of chitosan
(Badawy and Rabea 2011)
Degree of deacetylation The antimicrobial activity of chitosan is directly
proportional to the degree of deacetylation of chitosan The increase in degree of
deacetylation means the increased number of amino groups on chitosan As a result
chitosan has an increased number of protonated amino groups in an acidic condition
234 SP Chawla et aland dissolves in water completely which leads to an increased chance of interaction
between chitosan and negatively charged cell walls of microorganisms (Sekiguchi
et al 1994)
The pH The antimicrobial activity of chitosan is strongly affected by the pH At
lower pH there is an increase in the number of protonated amino groups on
chitosan in addition to the hurdle effect of inflicting acid stress on the target
organisms (Badawy and Rabea 2011)
Temperature The incubation temperature also has an effect on the antimicrobial
activity of chitosan Higher temperature (37 C) has been shown to enhance its
antimicrobial activity compared to refrigeration temperatures (Kong et al 2010)
Cations Antimicrobial action of chitosan is inhibited by divalent cations in the
order of Ba+2 >Ca+2 >Mg+2 It is proposed that the cations form complexes with
chitosan and consequently the reduced available amino groups of chitosan lead to
the reduced bactericidal effect (Badawy and Rabea 2011)
Chitosan posses a number of characteristics that make it a suitable antimicrobial
polymer for various industrial applications These include the following (1) easy
and abundant availability (2) longterm storage stability at the temperature of its
intended application (3) it does not decompose to andor emit toxic products (4) it
is not toxic or irritating to handlers and (5) it is biocidal to a broadspectrum of
pathogenic microorganisms
12 Antioxidant Activity
Chitosan and its derivatives have been reported to have strong antioxidant activity
They control lipid oxidation by scavenging free radicals which can be attributed to
their ability to chelate metals The antioxidant effects of chitin and chitosan are
dependent on their molecular weight viscosity and degree of deacetylation
(Venugopal 2011)
13 Chitosan as Edible Coating for Fruits and Vegetables
The edible films and coatings are used to extend shelf life and improve quality of
food products At present edible films based on cellulose and proteins are being
used for the purpose They provide good reduction of O2 and CO2 partial pressure
but are not so good for moisture transfer between food and the surrounding
environment Chitosan forms tough longlasting flexible semipermeable films
that can be used as food wraps for extending their shelf life
Fruits and vegetables undergo a number of physiological changes during
postharvest storage These include tissue softening increase in sugar levels deg
radation of chlorophyll and synthesis and degradation of volatile flavor com
pounds Controlling respiration rate significantly improves the storability and
shelf life of fresh produce as a certain level of respiration activity is required to
prevent plant tissues from senescing and dying In minimally processed agricultural
8 Chitosan 235products the most important quality attributes contributing to marketability are
appearance color texture flavor nutritional content and microbial quality The
marketability of these products therefore demands efficient control of these quality
changes Due to its barrier properties chitosan film can prevent moisture loss and
drip formation retain color and flavor attributes and improve microbial quality
thereby extending the shelf life of a variety of fruits and vegetables Rather than
packaging produce within a chitosan film dipping the produce in a dilute solution
of chitosan and dilute acetic acid can be performed The technique also allows the
incorporation of additives such as vitamin E rosemary oleoresin calcium and
potassium to enhance the efficiency of treatment (Aider 2010) The efficacy of
treatment is demonstrated in strawberries bell peppers cucumbers peaches pears
and kiwifruit (BautistaBanosa et al 2006)
14 Chitosan as Functional Additive in Muscle Foods
Chitosan is used as an additive in flesh foods to control flavor loss microbial
growth and oxidation resulting in extended shelf life When cooked flesh foods
are stored a warmedover flavor develops which is perceived as loss of freshness
Chitosan is capable of preventing this flavor deterioration due to its antioxidant
activity (No et al 2007 Venugopal 2011) Ncarboxymethyl chitosan (NCMC) and
its lactate and acetate derivatives are effective in controlling the oxidation and
offflavor development in cooked meat at refrigerated temperatures Research by
the US Department of Agriculture has revealed that NCMC is useful as preservative
in flesh foods It can be sprinkled on gravies or meat products NCMC is very useful
in preserving microwavable or quickly prepared foods as well as in preventing
development of the warmedover flavor of institutional foods It is advantageous
to use as it is itself tasteless blends well with foods as a colorless ingredient and is
nontoxic and nonallergenic It is used as a glazing compound prior to flashfreezing
of many flesh foods to inhibit surface oxidation and enhance shelf life Meat and
poultry processors use NCMC as a postslaughter perfusion and as a longterm
flavor and storage preservative (Flick and Martin 2000) Textural properties of
surimi products can also be improved by addition of chitosan in combination with
other additives (Benjakul et al 2001 GomezGuillien et al 2005)
Chitosan as coating for eggs Chitosan coating of eggs can provide a protective
barrier against moisture and CO2 transfer from the albumen through the egg shell
thus extending the shelf life of eggs It prevents weight loss and enhances Haugh
unit and yolk index values indicating improved albumen and yolk quality of eggs
respectively The coated eggs can be preserved for up to 5 weeks at 25 C which is
at least 3 weeks longer than that observed for control uncoated eggs Overall
consumer acceptability of coated eggs did not differ from that for control and
commercial eggs (Bhale et al 2003)
Chitosan as additive in bakery and dairy products Chitosan and chitin can be
used as food additives in cookies noodles and bread to improve their texture
These effects are due to the ability of chitosan to control starch retrogradation
236 SP Chawla et alMicrocrystalline chitin has a positive effect on emulsion stability in addition
to increasing the specific loaf volume of white bread and proteinfortified
breads (No et al 2007) Maillard reaction products (MRPs) prepared from
chitosan and xylose extend the shelf life of fresh noodles (Huang et al 2007)
Chitosan–lysozyme (CL) film is reported to prevent growth of Listeria
monocytogenes Escherichia coliorPseudomonas fluorescens in preinoculated
mozzarella cheese (Duan et al 2007)
15 Chitosan as Clearing Agent in Wines and Vinegars
Browning due to oxidation is one of the most common defects affecting white
wines It can be minimized by using adsorbents to reduce phenolic compounds
Chitosan is useful for the clarification of wine and vinegars It exhibits high affinity
to a number of phenolic compounds particularly cinnamic acid and prevents
browning in a variety of white wines (Spagna et al 1996)
16 Chitin as Feed Additive
Chitin has a growthpromoting effect on broiler chickens It increases average live
weight and dressed weight and decreases wastage during dressing in broiler
chickens The use of chitin as a source of dietary fiber in chicken feed promotes
the growth of bifidobacteria in the guts (Hirano et al 1990) Similarly feeds
containing chitin and glucosamine could also be used in aquaculture for improved
growth of cultured fish (Kono et al 1987) Chitin hydrolysates produced through
the digestion of crustacean waste by chitinases are used as a carbon source for the
cultivation of yeast that can convert chitin oligosaccharides into singlecell proteins
(Carroad and Tom 1978) The yeast could be utilized as feed component
17 Chitosan as LipidLowering Agent
Chitosan is used as a dietary ingredient due to its ability to reduce serum choles
terol It reduces lipid absorption by binding neutral lipids such as cholesterol and
other sterols by means of hydrophobic interactions Because of this inhibitory
activity on fat absorption chitosan acts as fat scavenger in the digestive tract and
eliminates fat and cholesterol via excretion (Luo and Wang 2013) Chitosan
satisfies the requirements of dietary fiber including nondigestibility in the upper
GI tract high viscosity and high waterbinding ability in the lower GI tract From a
physiological standpoint the prime function of a dietary fiber is to lower cholesterol
levels and to promote the loss of body weight through a reduction of intestinal lipid
absorption It differs from other dietary fibers in that it possesses a positive ionic
charge which has the ability to bond chemically with the negatively charged lipids
fats and bile acids It is desirable that its prolonged use as fiber in diets should be
8 Chitosan 237monitored to ensure that it does not disturb the intestinal flora or interfere in the
absorption of micronutrients particularly lipidsoluble vitamins and minerals and
that it does not have any other negative effects Chitosan shows an LD50 (median
lethal dose) of around 16 gkg comparable to the salt and glucose values ensuring
safety for longterm use (Singla and Chawla 2001)
18 Biomedical Applications
Chitosan due its polyelectrolyte nature gelforming capability biodegradability
biocompatibility nontoxicity to living tissues and antimicrobial and antitumor
properties has extensive applications in medicine It is used in hemodialysis mem
branes artificial skin hemostatic agents and drug delivery systems The property
of chitosan to form gels at a slightly acid pH gives chitosan its antacid and antiulcer
activities Chitin and chitosan oligosaccharides when intravenously injected
enhance antitumor activity by activating macrophages
Chitosan as control release system Chitosan has an advantage of forming
covalent or ionic bonds with the crosslinking agents building a sort of network
where the active substance is retained Consequently these bonds carry advantages
in terms of controlled release (Estevinho et al 2013) Depending on the cross
linker the major interactions involved in the formation of the network are covalent
or ionic bonds Covalent crosslinking leads to the formation of hydrogels or
microparticles with a permanent network structure because irreversible chemical
bonds are formed This type of linkage allows absorption of water andor bioactive
compounds without dissolution and allows its release by diffusion The addition of
a second polymer as encapsulating agent makes possible the pHcontrolled delivery
(Berger et al 2004) Crosslinking compounds used to create covalent bonds are
molecules that have at least two reactive functional groups that allow the formation
of linkage between polymeric chains The most common crosslinkers used with
chitosan are dialdehydes such as glyoxal and in particular glutaraldehyde But they
are known to be toxic For example glutaraldehyde is known to be neurotoxic and
glyoxal is known to be mutagenic Hence even if microparticles are purified before
usage the presence of free unreacted dialdehydes cannot be completely excluded
and will induce toxic effects (Estevinho et al 2013) Other covalent crosslinkers
for chitosan such as diethyl squarate oxalic acid or genipin have been investigated
to overcome this problem Ionically crosslinked microparticles or hydrogels are
more biocompatible and well tolerated Ionically crosslinked chitosan hydrogels or
microparticles exhibit a greater swelling sensitivity to pH changes compared to
covalently crosslinked ones This fact broadens their potential application since
dissolution can be regulated by pH conditions (Berger et al 2004)
Chitosan with its positive charges reacts with polyanionic compounds forming
polyelectrolytic complexes that can easily incorporate active substances
Tripolyphosphate citrate sulfate and phosphate are used to prepare this kind of
complexes They are normally well tolerated and biocompatible with the human
238 SP Chawla et alorganism showing advantages in terms of applications for food and pharmaceutical
industry (Berger et al 2004 Gupta and Jabrail 2006)
Biotechnological application of chitosan Chitin and chitosan have been
found to be useful as a matrix for immobilization of various enzymes for the
processing of such products as wine and sugar the synthesis of organic compounds
(Ravikumar 2000) and the construction of sophisticated biosensors for in situ
measurements of environmental pollutants and metabolite control in artificial
organs (Krajewska 2004)
Chitosan as drug delivery matrix Chitosan is considered to be the drug carrier
for the twentyfirst century For effective drug delivery it is being used in the form
of microspheres microparticles nanoparticles granules gels or films Chitosan
microspheres are useful for the controlled release of antibodies antihypertensive
agents anticancer agents protein and peptide drugs vaccines and nutraceutical
compounds (Dash et al 2011)
Chitosan as wound healing agent Due to bacteriostatic and fungistatic proper
ties of chitosan it is used as a wound healing agent in skin ointments Chitosan
implanted in animal tissues encourages wound healing and hemostatic activities
Biocompatible wound dressings derived from chitin are available in the form of
hydrogels xerogels powders composites and films (Gavhane et al 2013)
19 Chitosan in Water Treatment
Water gets polluted due to metal ions inorganic anions phenolic compounds dyes
and radioactive isotopes Many of these water pollutants are toxic and can enter
the human food chain The toxic heavy metal ions are discharged into the environ
ment through different industrial activities The high adsorption potential of
chitosan is attributed to (1) high hydrophilicity due to a large number of hydroxyl
groups of glucose units (2) the presence of a large number of functional groups
(3) the high chemical reactivity of these groups and (4) flexible structure of the
polymer chain
Chitosan and its derivatives are being successfully used in water treatment to
remove lead copper and cadmium from drinking water due to complex formation
between the amino group and heavy metal ions (Bhatnagar and Sillanp€a€a 2009)
Radionuclides are an important category of metals in terms of environmental
impact and interest from nuclear industry Chitosan is an excellent biosorbent to adsorb
radionuclide from aqueous solution in an acid environment (Wang and Chen 2014)
Dyes are usually present in the effluents of textile leather paper and dye
manufacturing industries These effluents are not only toxic to the aquatic biota
but also disturb the natural equilibrium by reducing photosynthetic activity of water
in streams Some dyes are reported to cause allergy dermatitis skin irritation and
cancer in humans The removal of dyes from effluents before they are released into
natural water bodies is important Chitosanbased biosorbents have an extremely
high affinity for many classes of dyes (Crini and Badot 2008)
8 Chitosan 239Phenol and substituted phenols cause unpleasant taste and odor in drinking water
and can exert negative effects on different biological processes The ubiquitous
nature of phenols their toxicity even in trace amounts and the stricter environ
mental regulations make it necessary to develop processes for the removal of
phenols from wastewaters Chitin and chitosan derivatives can remove phenol
and substituted phenols from water (Bhatnagar and Sillanp€a€a 2009) The pH
primarily affected the degree of ionization of phenol and the surface properties of
chitin The functional groups of chitosan are protonated at low pH values and
resulted in a stronger attraction for negatively charged ions in the adsorption
medium Phenol being weakly acidic is partially ionized in solution These ions
are negatively charged and are attracted due to electrostatic forces by the protonated
amino groups of chitosan As the pH increases the overall surface charge of
chitosan becomes negative and adsorption decreases The equilibrium uptake
of phenol is also affected by temperature due to the enlargement of pore size or
creation of some new active sites on the adsorbent surface due to bond rupture In
comparison with activated charcoal chitosan is more efficient in the removal of
polychlorinated biphenyls from contaminated water (Venugopal 2011)
Inorganic anions are also an important class of aquatic pollutants and various
inorganic anions are found in potentially harmful concentrations in drinking water
sources The removal of these pollutants from drinking water supplies is an
emerging issue In recent years chitin and chitosan derivatives have been success
fully utilized for some anion removal from water (Bhatnagar and Sillanp€a€a 2009)
Chitosan is currently employed in domestic sewage treatment systems in con
junction with other settling aids such as alum or bentonite clay to promote coagu
lation and settling of colloidal and other suspended solids The polyelectrolyte is
added at the rate of 1–2 ppm but can also be employed alone without alum when the
concentration is raised to around 10 ppm Being positively charged it is very
effective at agglomerating the negatively charged sludge particles (Venugopal
2011) Chitosan is also employed as a coagulant in the treatment of wastewater
from food industries The production of surimi generates a large amount of wash
water that contains sizeable amounts of proteins showing high turbidity Chitosan
treatment of surimi wash water results in the recovery of soluble proteins The
protein recovery is further increased by adding a complex of chitosan and alginate
It is also used as coagulant to treat wastewater from milk processing plants
Recovered proteins have application in food and feed industry (Wibowo
et al 2005)
20 Chitosan Application in Agriculture
Chitin and chitosan also have potential in agriculture with regard to controlling
plant diseases They are active against soil fungi viruses bacteria and other pests
Addition of chitin and chitosan alters the environmental conditions in the rhizo
sphere and phyllosphere to shift the microbial balance in favor of beneficial
240 SP Chawla et alorganisms and to the detriment of plant pathogens Fragments from chitin and
chitosan are known to have eliciting activities for a variety of defense responses
in host plants including the accumulation of phytoalexins pathogenrelated
(PR) proteins proteinase inhibitors lignin synthesis and callose formation
(El Hadrami 2010)
21 Regulations and Commercial Applications
Chitosan is used as a food quality enhancer in a number of countries Chitosan
preparations in tablet capsule and powder form are being used in healthcare
industry In the European market chitosan is sold in the form of dietary capsules
to assist weight loss and in some countries such as Japan it is added to various
foods (eg noodles potato crisps biscuits) Chitosanfortified fruit juices and
chocolates are marketed in the USA The role of chitosan as fiber is challenged
by popular fiber products such as oats soy and bran In spite of some limitations
chitosan promises to offer innovative applications in diverse areas of food
processing and other fields (Venugopal 2011)
In the USA the 1994 Dietary Supplement Health and Education Act permits use
of chitosan as a food supplement without premarket approval as long as no health
claims are made The use of chitin and chitosan as ingredients in foods or pharma
ceutical products however requires standardization of identity purity and stabil
ity Manufacturers should consider filing petitions with agencies such as Food
Chemical Codex US Pharmacopoeia European Pharmacopoeia and Japan Phar
macopoeia These organizations establish methods to identify specific products and
standards of purity for pharmaceutical and drug use Such standards will be
necessary for future expansion of the use of chitin and chitosan (Heinze
et al 2005) Chitin and chitosan have been approved for pesticide and seed
treatments as fertilizer and as animal feed additives The US Environmental
Protection Agency has approved the use of commercially available chitosan for
wastewater treatment up to a maximum level of 10 mgL
Chitin and chitosan products fall within the lowest level of concern for toxico
logical testing Being naturally present in living organisms chitin and its
deacetylated derivative chitosan are considered safe The available literature on
chitin and chitosan suggests a low order of toxicity based on chemical structure and
animal studies Like several highmolecularweight food polymers of natural origin
such as cellulose and carrageenan chitin and chitosan are not expected to be
digested or absorbed from the human gastrointestinal tract To date chitosan
appears to be clinically well tolerated The safety of chitooligomers prepared by
the enzymatic depolymerization of chitosan has been reported in a shortterm mice
feeding study No mutagenicity has been reported as judged by the Ames test
mouse bone marrow cell micronucleus test and mouse sperm abnormality test
A 30day feeding studies did not show any abnormal symptoms and clinical signs or
deaths in rats No significant differences are reported in body weight food
8 Chitosan 241consumption food availability hematology values clinical chemistry values or
organbody weight ratios No abnormality of any organ was found during histo
pathological examination (Qin et al 2006)
22 Conclusion and Future Research Needs
Chitosan is a versatile biopolymer that has a variety of commercial applications
However individual research reports have used chitosans from various sources
with varying physicochemical properties Hence the question arises as to how to
globally produce chitosans with consistent properties Each batch of chitosan
produced from the same manufacturer may differ in its quality For proper quality
control in the chitosan production there is a critical need to establish less expensive
and reliable analytical methods especially for the evaluation of molecular weight
and degree of deacetylation Functional properties of chitosan vary with molecular
weight and degree of deacetylation With proper modification of chitosan its
functional properties and biological activities can be further enhanced and more
applications are being developed
Chitosan with different structures shows different biological activities and not
all the biological activities are found in one kind of chitosan Each special type of
bioactive chitosan should be developed for its potential application Moreover
many studies carried out on chitosan and chitooligosaccharide bioactivity have
not provided detailed molecular mechanisms Hence it is difficult to explain
exactly how these molecules exert their activities Therefore future research should
be directed toward understanding their molecularlevel details which may provide
insights into the unknown biochemical functions of chitosan and chitooligo
saccharide as well as help accelerate their future applications The traditional
chitosan production process is costly thus limiting wider applications of chitosan
Simplification of chitosan production for example by elimination of deprotei
nization andor demineralization or by reduction of reaction time required for
deproteinization and demineralization would considerably reduce production cost
due to reduction in chemical usage process time and voluminous wastewater
discharge The typical astringentbitter taste of chitosan limits its use as a food
additive or preservative Incorporation of Larginine and adenosine monophosphate
both considered as GRAS can be used to mask or minimize this effect and should
be further investigated Inherent antibacterialantifungal properties and film
forming ability of chitosan make it ideal for use as biodegradable antimicrobial
packaging material One major drawback of chitosan film is its high sensitivity to
humidity and thus it may not be appropriate for use when it is in direct contact with
moist foods More research is needed to develop antimicrobial chitosan films that
are less sensitive to humidity Numerous researches conducted on food applications
of chitosans have been done at a small or laboratory scale Further research on
quality and shelf life of foods containing or coated with chitosan should be
conducted on scaleup with large volumes typical of commercial conditions
242 SP Chawla et alThis would provide a more realistic and practical information required for actual
commercialization of food products containing or coated with chitosans
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