CHITIN AND CHITOSAN
Introduction
Chitin is a structural polysaccharide widely found in nature. Chitin occurs as
highly ordered microfibrils in many species, in a variety of arrangements, from diatom spines to cell wall components of many fungi and yeast. It is also a principal
component in the exoskeleton of insects and marine invertebrates such as Arthropoda and Mollusca. Chitin is a homopolymer of 1-4 linked 2-acetamido-2-deoxyβ-D-glucopyranose, although some of the glucopyranose residues are deacetylated
and occur as 2-amino-2-deoxy-β-D-glucopyranose. When chitin is deacetylated to
about 50% of the free amine form, it is referred to as chitosan. Figure 1 indicates
the copolymeric nature of this material. Chitosan is rarely found in nature but does
occur in dimorphic fungi such as Mucor rouxii wherein it is formed by the action
of a deacetylase enzyme on chitin. Most chitosan is obtained by the chemical or
enzymatic treatment of chitin obtained from the shell of commercially harvested
Arthropoda, such as shrimp and crab.
Chitin was first isolated from mushroom tissue and named “fungine” in 1811
by Braconnot, a French botanist. A similar material was isolated by Odier from
insect exoskeleton, which he termed “chitine” (1). Chitin is considered the second
most plentiful biomaterial, following cellulose. The annual production of chitin
biomass has been estimated at 1 × 1013 kg worldwide (2). This has led to considerable scientific and technological interest in chitin and chitosan. Chitosan has
become the preferred commercial form of this material as it is more tractable
than chitin. Chitin is insoluble in most common solvents, whereas chitosan dissolves in many common aqueous acidic solutions. Chitosan has found applications
in many primary industries such as agriculture, paper, textiles, and wastewater
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Fig. 1. The chemical structure of chitin and chitosan, a copolymer of the N-acetyl sugar
and the amino sugar. Chitin occurs as mostly the “x” or N-acetyl form. Chitosan occurs as
the “y” or amino form.
treatment. Chitosan is also under study for medical and pharmaceutical uses. It
has also become a popular nutritional dietary additive (3).
Biosynthesis
The synthesis of any polysaccharide involves the addition of an activated
monomeric sugar to the end of a growing polysaccharide chain by an enzyme
mediated mechanism (4). In higher plants, algae, and photosynthetic bacteria
(autotrophic organisms), polysaccharides are produced by a reductive metabolic
pathway beginning with water, carbon dioxide, and light. In nonphotosynthetic
bacteria, lower plants, fungi, and animals (heterotrophic organisms), they are
synthesized from ingested foods. Chitin synthase catalyzes the polymerization of
activated N-acetyl-glucosamine monomer (5,6).
Most studies of chitin synthesis and microfibril assembly have involved easily cultured unicellular fungi, protists, and algae, which have simple biochemical
pathways. Insect and fungi chitin biosyntheses have been perceived as a route to
the development of new pesticides as well as for monitoring the environmental impact of these biocides in crustacea (4). Post-polymerization deacetylation by chitin
deacetylase converts chitin to chitosan in nature (7). The completion of the biological cycle is accomplished with chitinases and chitosanases, which ultimately
degrade chitin and chitosan to their corresponding sugars (8–10).
Chitin and chitosan occur in a wide range of disparate organisms
(Table 1). In crustaceans and insects most of the biosynthesis of chitin occurs
in the layer of epidermal cells lying just under the cuticle (exoskeleton). Chitin
usually occurs in the presence of other cellular materials such as glucans, proteins, and calcium carbonate. One of the purest forms of chitin is the spike of
some centric diatoms, such as Thalassiosira fluviatilis (5). Some algae also produce a 100% N-acetylated chitin (17). Chitosan is always obtained by the action of
a deacetylase on chitin. Figure 2 illustrates the high degree of organization found
in native chitin microfibrils.
In animals, chitin biosynthesis begins with glucose. Glucose is phosphorylated, aminated, N-acetylated, and then converted to N-acetyl-uridine diphosphate (UDP)-glucosamine. Chitin is polymerized by the activated monomer Nacetyl-UDP-glucosamine. The polymerization is driven by the scission of the
phosphoester bond. A similar pathway is involved in fungi. The gene fragments
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Table 1. Sources of Chitin and Chitosan
Source structure
Chitin
Insects
Cuticle
Ovipositors
Beetle cocoon
Crustaceans
Crab shell
Shrimp shell
Squid
Ommastrephes pen
Loligo stomach wall
Centric Diatoms
Thalassiosira fluviatilis
Algae
Fungi
Mucor rouxi
Aspergillis nidulans
Fungi
Mucor rouxi
Deacetylated chitin
Shrimp shell
Type
Reference
α-Chitin
α-Chitin
γ -Chitin
11
12
13
α-Chitin
α-Chitin
6
14
β-Chitin
γ -Chitin
15
16
β-Chitin (100% N-acetylated)
α-Chitin
5
17
α-Chitin
α-Chitin
18
19
60–92% deacetylated
20
M w up to 1.6 × 106 Da.
21
coding for chitin synthase have been sequenced for many fungi and classified (23). Chitin synthase is an integral membrane protein. It is oriented such
that its carboxyl terminus is exposed to the cytoplasmic side of the membrane
(24).
Chitin deacetylase catalyzes the hydrolysis of the N-acetyl group in chitin. A
chitin deacetylase has been purified from M. rouxii and identified as a high mannose glycoprotein requiring (at minimum) a tetramer of chitin sugars for catalysis
(7). Chitin deacetylase is found outside the plasma membrane and is associated
with the nascent chitin chains during fungal chitin biosynthesis. Chitin deacetylation may also be a fungal defense mechanism, as chitosan is more resistant to
plant chitinase defense mechanisms.
Isolation
Chitin and chitosan rarely occur in a pure, easily isolated form. A substantial effort
has been made to develop chemical, mechanical, and enzymatic methods to obtain
purified materials (25). The usual method of obtaining chitin involves the chemical
treatment of shell fish wastes from the crab and shrimp industries. The first step is
to demineralize the shell with dilute hydrochloric acid at room temperature. This
is followed with a deproteinization step with warm dilute caustic. This yields a
partially deacetylated chitin, which may then be further deacetylated to chitosan.
Figure 3 shows the underlying chitin matrix in the crab shell and its microfibrillar
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Fig. 2. Chitin usually occurs in the form of microfibrils in many organisms. Shown here
are β-chitin microfibrils from Tevnia jerichonana, a deep sea hydrothermal vent worm.
The insert is a typical electron diffractogram oriented with respect to the bundle. The
image was obtained by transmission electron microscopy (TEM) after protein removal (22).
Reproduced with permission.
structure. This chitin is termed α-chitin because of its crystal structure (see next
section). Treatment of this chitin with 50% NaOH for 1–3 h at 120◦ C gives a 70%
deacetylated chitin (chitosan), which is soluble in many dilute acids. Repeating
this step can give deacetylation values up to 98%.
A more reactive form of chitin is obtained from squid pens (15). This β-chitin
(see next section) is easily isolated and has a looser chain packing in the crystal,
accounting for its higher reactivity and solubility in formic acid. The isolation of
β-chitin is accomplished by first washing the squid pens in 1 M HCl for 12 h,
followed by a 12-h treatment with 2 M NaOH. The final step is to heat the pens
at 100◦ C for 4 h in fresh 2 M NaOH. This procedure yields 35% chitin from the
mass of the pens. The degree of acetylation may be up to 92%.
Properties
Physical. Chitin occurs naturally as one of three crystalline forms, known
as α-, β-, and γ -chitin. The unit cell parameters, determined by x-ray diffraction,
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Fig. 3. Chitin and chitosan are commonly isolated from the shells of crab and shrimp.
On the right-hand side is the original, cooked crab shell. On the left-hand side is the
underlying matrix of chitin, after the proteins and minerals have been removed. Chitin is
biosynthesized as an ordered assembly of chains yielding microfibrils, which are seen in
the scanning electron micrograph insert. The scale bar in the insert is 2000 nm.
are given in Table 2. The α- and γ -chitin forms are defined as antiparallel stack
unit cell structures. The β-form is similar to Cellulose I and assigned a parallel stack structure (16). Chitin is mostly found as either the α- or β-form. The
α-form has a strong, three-dimensional hydrogen bond network, which makes the
swelling and dissolution of α-chitin difficult. The β-form lacks hydrogen bonding
between the stacked planes of the parallel chitin chains. This allows for the easy
formation of hydrates and accounts for the higher reactivity of the β-chitin.
Chitosan is also found in different crystalline or polymorphic forms. The unit
cell dimensions for these different forms are shown in Table 2. They are obtained by
different processing conditions. Chitosan is not usually found completely deacetylated; hence, it has some of the characteristics of a random copolymer. When the
deacetylation of the chitin is less than 90%, the crystallization is hindered, and
the chitosan has a lower degree of crystallinity than the original chitin.
Table 2. Unit Cell Parameters for Chitin and Chitosan
Unit cell parameters
Form
α-Chitin
β-Chitin
γ -Chitin
Chitosan I
Chitosan II
3
Source
Density, g/cm
a, nm
b, nm
c, nm
Reference
Lobster tendon
Diatom spines
Loligo stomach
Shrimp shell
Shrimp shell
1.46
1.46
1.47
1.23
1.18
0.474
0.485
0.47
0.776
0.44
1.886
0.926
2.84
1.091
1.00
1.032
1.038
1.03
1.03
1.03
26
27
16
28
29
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The two polymeric characteristics that are the most important for
chitin/chitosan solubility are the degree of deacetylation and the molecular weight.
Chitin is insoluble in most solvent systems. Chitosan, having at least 50% of its
repeat groups deacetylated, is soluble in aqueous acidic solution existing as a
randomly coiled cationic polyelectrolyte. Chitin and chitosan have an extensive
hydrogen bonded network in the solid state, requiring solvents which either induce
interchain repulsions or disturb intermolecular hydrogen bonding for dissolution.
Protonation of the amine group found in the deacetylated repeat units of
chitosan provides a means of introducing interchain coulombic repulsions promoting dissolution. Highly deacetylated chitosan, having a pK a of approximately
6.5, requires a pH lower than 6 for complete dissolution. However, with increasing acetyl content the pK a of residual amine groups increases and is believed
to approach a value slightly greater than 7.5 (29). As a result of this pK a shift,
chitosan with a degree of deacetylation of 50% is soluble in neutral water. Upon
further increases in acetyl content, the interpolymeric attractions dominate, and
the material (chitin) is insoluble in water with the addition of acid despite the protonation of accessible amine groups. Chitosan is not soluble in diprotic acids such
as sulfuric acid. The divalent sulfate anion facilitates interchain ionic attractions
preventing dissolution and forming ionic cross-links.
Chitin is soluble in N,N-dimethylacetamide–5% LiCl, N-methyl-2pyrrolidone–5% LiCl, and mixtures of trichloroacetic acid with chlorinated hydrocarbons (ie, chlorinated methanes and ethanes) (30). Generally, the solubilities
of chitin and chitosan decrease with increases in molecular weight. Oligomers of
chitin and chitosan with a degree of polymerization of 8 or less are water soluble regardless of pH. Many derivatives of chitin and chitosan, which have wide
solubility characteristics, have been synthesized.
Chemical. A large number of chitin and chitosan derivatives have been
synthesized through modification of the primary (C-6) and secondary (C-3) hydroxyl groups present on each repeat unit, including amine (C-2) functionality existing on deacetylated units (31). Reactions typical of hydroxyl and amine groups
(such as acylations with acid chlorides and anhydrides) including urethane and
urea formation respectively, are feasible with isocyanates. The primary amine can
be quaternized by alkyl iodides or converted to an imine with a variety of aldehydes and ketones that can subsequently be reduced to an N-alkylated derivative.
Chitin and chitosan are reactive with a variety of alkyl chlorides after treatment
with concentrated NaOH. Important derivatives such as carboxymethylated chitin
and chitosan are commonly produced in this manner with the addition of sodium
chloroacetate.
Chitin and chitosan have been chemically modified by graft copolymerization using a variety of monomers (“grafting from”) and telechelic polymers
(“grafting onto”). “Grafting from” techniques have predominantly utilized either a
free-radical mechanism with vinyl monomers such as styrene, methyl methacrylate, methyl acrylate, acrylic acid, and acrylamide, or a ring-opening mechanism
(initiated by free amine groups) with D,L-alanine and γ -methyl L-glutamate Ncarboxyanhydrides (32). Vinyl grafting has been initiated with techniques involving the ceric ion (Ce4+ ), Fenton’s reagent (Fe2+ /H2 O2 ), and γ -irradiation.
“Grafting onto” methods have bonded poly(ethylene glycol) (33) or poly(2-methyl-2oxazoline) (34) chain ends to the trunk polymer. For example, poly(ethylene glycol)
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was synthesized with an activated carboxylic acid chain end capable of forming
an amide linkage with free amine groups along the chitin/chitosan backbone. The
range of possible chitin and chitosan modifications, using either lower molecular weight reagents or graft copolymerizations, allows modification of chitin and
chitosan solubility, miscibility with other materials, conductivity, and moisture
absorbency.
Biological. A wide range of hydrolytic enzymes lead to the biodegradability of chitin and chitosan. Within an organism, directed degradation by chitinases
are involved in fungal autolysis and the molting of arthropod cuticle. Other chitinases are abundantly secreted into the environments of plants and microbes as
part of their defense and feeding mechanisms. In bacteria, chitinases play a role in
nutrition and parasitism. The major groups of enzymes degrading chitin and chitosan have been classified and described (4,8–10). Many studies have also utilized
lysozyme, an easily obtainable hydrolytic enzyme, found in the lymphoid system
of vertebrates. Lysozyme shows hydrolytic activity over a range of deacetylation
values for chitin and chitosan.
Generally, the complete hydrolysis of chitin to N-acetyl-glucosamine requires
the consecutive action of two chitinase enzymes. These are frequently found together in plant and animal species as well as bacteria and fungi. Usually, the
chitin is degraded to oligosaccharides and N,N -diacetylchitobiose by endochitinase enzymes. Chitobiases then hydrolyze these products to monomeric N-acetylglucosamine. Exochitinases, which directly degrade chitin to chitobiose, are also
known. The same is also found for chitosanases.
In arthropods, synthesis and degradation of chitin occurs at different stages
of the molting cycle and is hormonally controlled. During the molt, the rate of
chitin synthesis is slowed by molting hormones (ecdysteroids) that also stimulate
the production of chitinases. Many animals and marine microorganisms have a
high digestive capacity for chitin. In seawater, the presence of secreted chitinases
from microorganisms leads to the near total degradation of crustacean chitin in
shallow water sediments within 4 months in temperate regions.
Processing
Chitin is usually obtained as a powder or a flake upon isolation from crab shell,
fungi, or algae. Further processing is difficult because of the lack of simple solvents for chitin. However, chitosan is easily solubilized and formed into shaped
articles such as films (membranes), fibers, and gel microspheres (30,35). Chitosan
is conveniently dissolved in an aqueous 5 vol% acetic acid solution, in the form of
a cationic polyelectrolyte. This ability to develop basic properties in solution gives
chitosan its uniqueness among common polysaccharides. Chitin and chitosan are
also easily treated with acids and hydrolytic enzymes to yield useful sugars and
oligomers.
Economic Aspects
A worldwide industry has developed around the production and uses of chitin
and chitosan. Shrimp and crab shell wastes pose local waste disposal problems
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in many areas. The seasonal harvesting of these crustacea has created problems for an economic, year-round chemical production of chitin and chitosan,
in some regions though. In the United States, there are several producers who
manufacture and import chitosan. These include Vanson, Inc., DCV, Inc., Biopolymer Engineering, Inc., and Marine Polymer Technologies. There are also industries manufacturing chitin and chitosan in many countries in almost every continent. In particular, Japan, China, and Norway have well-established companies.
Chitin and chitosan are still in the stage of a speciality chemical and is sold mostly
in the form of chitosan powder, at $25–50/kg, depending on purity. The primary
uses are in the water treatment, paper, and agricultural industries. It is also
sold as a dietary additive worldwide. Speciality producers offer very pure chitin
and chitosan derived from algae and squid pen. These are primarily employed in
biomedical research and biomedical products.
Specifications and Analytical Methods
Chitin and chitosan are natural products, commercially available from many
sources as mentioned previously. It is usually found in the presence of proteins,
calcium carbonate, and perhaps other polysaccharides. Thus, its quality ranges
from crude extracts to high purity material. Many of the properties of chitin and
chitosan are dependent on the degree of deacetylation. The distribution of N-acetyl
groups in these polymers is also expected to have an influence on the properties.
Most spectroscopic and titration methods have been employed to determine
these values and were listed by Rathke (36). The structural differences between
chitin polymorphs have been studied by nmr and ir (37,38). Conductometric titrations have been confirmed by solid state nmr to be an accurate but easy method
to determine the degree of deacetylation of chitosan (39).
The molecular weight and its distribution are also important. Commercial
chitosan samples may have molecular weights up to 4 × 106 Da. The Mark
Houwink equation [η] = kM v a is used to obtain a viscosity average molecular
weight. However, the values of k and a are most likely functions of the degree of
deacetylation (21). Gel permeation chromatography is also suited for the determination of chitosan molecular weight and polydispersity (21).
Health and Safety Factors
The benefits of chitosan as a dietary fiber have been recognized (3,40,41). Chitin
and chitosan have very low toxicity. Studies on the oral administration of chitosan indicates that it may have beneficial actions on osteoarthrosis, body weight
control, and the lowering of blood plasma cholesterol levels. Chitosan’s complexation with bile acids helps block the absorption of lipids. Both materials have been
granted approval for different food additive uses for humans and animals in several countries. The corresponding sugars of these polysaccharides, glucosamine,
and N-acetyl-glucosamine are also of interest. In many circumstances it is these
sugars which have the observed biological activity. Tissue and animal studies
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indicate that chitin and chitosan promote wound healing and increase immune
response, but these claims need to be validated by human clinical trials. Certain
medical precautions should be observed, however, with long-term ingestion of high
doses of chitosan to avoid potential adverse metabolic effects (3).
Uses
Chitin is mainly used as a powder and as a precursor to chitosan. However, the
unusual aminopolysaccharide structure of chitosan has led to many potential applications. The ease of processing chitosan into shaped articles, coupled with its
ease of chemical derivatization, makes it a versatile material. The specificity of
chitin and chitosan’s structure is also important for many biological applications
involving the binding and purification of many proteins.
Chitin and chitosan have potential in agricultural applications because of
their ability to increase crop yields. The presence of chitosan appears to enhance
plant reproduction (42), and coating seeds with chitin/chitosan activates the secretion of plant chitinases into the environment providing extra protection from
harmful pests.
Incorporating chitin and chitosan into different aspects of the food industry
has received considerable attention (43). The antibacterial and antifungal properties of chitin and chitosan have potential in reducing the amount of synthetic food
preservatives. N,O-carboxymethylated chitosan, a water soluble derivative, has
also been found to possess antifungal activity. The most probable means of application are either through packaging wraps or directly as food coatings. Chitin and
chitosan films are improvements to synthetic wraps as a result of the reduction
in oxygen permeability and increased moisture transfer. Chitosan films have also
decreased the level of browning in physically damaged fruits.
In regard to nutritional value, chitin and chitosan oligomers have been observed to lower cholesterol and exhibit antitumor activity. “Fat-blockers” containing chitosan are currently on the market (40). The amine groups along the polymer backbone form carboxylate-protonated amine complexes with fats which are
subsequently excreted from the body because of chitosan’s lack of digestibility in
humans.
Chitosan has potential applications in various wastewater treatments (44).
Chitosan is a natural chelating polymer as a result of the amine group (C-2) and
the adjacent hydroxyl group (C-3), and thus could be used to remove transition
metal ions from wastewater streams. Some important factors that affect the level
of ion removal are the degree of deacetylation, pH, and surface area of the chitosan
substrate. Since protons compete for amine sites, the metal ions can be removed
from the chitosan backbone by lowering the pH. Chitosan beads cross-linked with
gluteraldehyde appear to be a more practical physical form because of its lack of
solubility at low pH and its higher surface area.
Chitosan can be used as a flocculating agent for food processing streams.
Having a partial positive charge in water, chitosan can break down food particles
comprised of a protein-based colloidal suspension possessing a partial negative
charge. Suspended solids can be coagulated by chitosan, collected, and used as a
protein source for animal feed.
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Anionic dyes can be removed from textile effluent streams at acidic pH with
chitosan through protonated amine complexation with anionic dye sites. Phenols
are common waste products in paper processing. Application of mushroom enzyme
tyrosinase to the stream specifically converts phenols into quinones, which can
subsequently be absorbed by chitosan. Toxic polychlorinated biphenols (PCBs),
commonly used in plastic processing and lubricants, are a significant source of
water contamination. Although the nature of the interaction is not currently
known, chitosan treatment shows potential in lowering PCB concentrations.
Research on the use of chitosan for drug delivery indicates promise in oral,
parenteral, transdermal, ocular, and nasal applications (45). Generally, chitosan
has low toxicity with excellent mucoadhesion properties. Cross-linked chitosan
has potential in tablet form because of its low solubility in the stomach. The same
effect can be obtained by incorporating hydrophobic coatings on chitosan microspheres. Chitosan gels and films exhibit sustained release of various drugs useful
for wound dressings and colon treatments. Many other examples of drug delivery
applications of chitosan have been reviewed (46), and chitosan peroral peptide
delivery systems have been described (47). Other pharmaceutical applications of
chitosan and the mechanisms of action for various in vitro and in vivo models have
been outlined and discussed (48).
The direct application of chitin and chitosan to wounds stimulates several
different physiological activities (49). The presence of chitin and chitosan on the
wound surface stimulates macrophage activity for the secretion of lysozyme and
human chitinase, enzymes which breakdown chitin-based pathogens to prevent
infection. The chitin/chitosan present in the wound dressing is thus simultaneously hydrolyzed to chitooligomers, which further stimulate collagen deposition
and other macrophage functions, namely nitric oxide and tumor necrosis factor
production.
Hemodialysis utilizes film and hollow fiber membranes to remove solutes and
water from patients suffering from kidney failures. The excellent fiber and film
forming properties of chitosan have led to many studies on its use for this purpose
(30,50). Chitosan may be blended with other polymers to give permeability control
and to improve blood compatibility.
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SAMUEL M. HUDSON
DAVID W. JENKINS
North Carolina State University
CHLOROSULFONATED POLYETHYLENE.
See ETHYLENE POLYMERS, CHLOROSULFONATED.
CHLOROTRIFLUOROETHYLENE POLYMERS.
See FLUOROCARBON ELASTOMERS.