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2751
deacetylation degree, the higher the solubility. Above
pH 7.0, chitosan solubility is low and precipitation or
gelation tends to occur.
b-(1!3)(1!4)-glucanases, proteases, lysozyme and lip-
ases.9–13 These findings have opened the possibility of
developing novel efficient and economically feasible
industrial processes for hydrolyzing chitosan.
Although chitosan shows a number of interesting
functional properties in different areas, the above men-
tioned features regarding its high molecular weight
and viscosity of aqueous solutions, and the special con-
ditions (acidic media) required to achieve solubilization
in water, limit the best part of its potential uses.2 In this
respect, chitosan oligosaccharides, because of their
shorter chain length, display a reduced viscosity and
are soluble in aqueous media at pH values close to neu-
trality, which increases their bioavailability and opens a
wide range of new potential applications.
Chitosan oligosaccharides are bioactive compounds
with many uses in the fields of food, health and agricul-
ture. They have been claimed to have a great number of
effects and activities, including among others: prebiotic,
antimicrobial, antitumoural, tissue recovery stimulation,
The use of chitosanases and some of the above
enzymes in the production of chitosan oligosaccharides
has been assessed at laboratory scale, using enzyme
reactors both in batch and in continuous configura-
tions.8,14–17 In most cases, the resulting products show
high molecular weight (>10 kDa) and, when low poly-
merization degree oligosaccharides have been obtained,
their yield has been poor and with predominance of the
smallest sized species (2–4 residues) and monomers.
In this paper, an efficient procedure for the produc-
tion of low- and medium-size chitosan oligosaccharides,
in high yields and almost free of monomers, starting
from a high deacetylation degree chitosan as substrate
and using pepsin, a low cost commercial enzyme, as
catalyst is described. The products resulting from
chitosan hydrolysis are expected to be suitable for most
of the reported chitosan applications, where the large
molecular weight of the polymer limits its use.
antidiabetic,
immunostimulant,
antiinflammatory,
calcium absorption acceleration, antimutagenic, antioxi-
dant and activator of plant resistance towards insect and
pathogen attack.2,3
Chitosan oligosaccharides can be obtained by chemi-
cal or enzymatic hydrolysis of the chitosan chains.
Chemical hydrolysis is carried out by two alternative
methods: acid hydrolysis with concentrated acids4 or
oxidative degradation with hydrogen peroxide.5 Both
methods have been applied successfully to chitosan
degradation, which occurs almost quantitatively, but
show some drawbacks,2 including the difficulty to obtain
low polymerization degree oligosaccharides because
high polydispersity mixtures predominate, and to con-
trol the extent of hydrolysis, which frequently results
in hydrolysates containing a high ratio of monosaccha-
rides. In addition, the harsh reaction conditions
required, such as elevated temperatures and high reagent
concentrations, may cause environmental problems and
often result in the formation of chemically modified
oligosaccharides.
Alternatively to the aggressive chemical hydrolysis,
chitosan may also be hydrolyzed in a milder way using
enzymes. Enzyme catalyzed chitosan hydrolysis is more
specific and allows a greater control of the extent of
reaction and, therefore, of the product size. The specific
enzymes intended to catalyze chitosan hydrolysis would
be chitosanases.6–8 These enzymes, however, show a
reduced commercial availability and, as a consequence,
are rather expensive, which limit their industrial use.
In the search for alternative enzymes to chitosanases,
showing original specificities different from chitosan
hydrolysis, but able to catalyze the hydrolysis of this
polysaccharide and, most important, commercially
available in great quantities and at reduced cost,
several enzymes have been found to fulfil these require-
ments, including cellulases, hemicellulases, pectinases,
2. Results and discussion
2.1. Hydrolysis of chitosan by commercial enzymes
In an attempt to develop an efficient process for the pro-
duction of chitosan oligosaccharides on a large scale,
that is, with a good yield of low- and medium-chain
length species and reduced levels of monomers, we
studied the hydrolysis of chitosan catalyzed by several
commercial enzymes previously reported to do it. The
enzymes assayed included cellulase, hemicellulase,
papain, bromelain, pepsin, protease type XIV from
Streptomyces griseus, lysozyme and lipase A, with
chitosanase as control. Unlike most of the previous stud-
ies of this kind, which used chitosan with deacetylation
degrees ranging from 70% to 85% and resulted in high
levels of dimers and monomers, our rationale was to
use a very high deacetylation degree (93%) chitosan in
order to avoid a too extensive hydrolysis of the polymer.
Except hemicellulase, all the enzymes assayed showed
an appreciable activity under the standard reaction con-
ditions, as determined by viscosimetry and reducing end
assay. Results are shown in Table 1.
According to the viscosimetry assays, all enzymes,
apart from lysozyme and papain, showed a chitosan
hydrolyzing activity comparable, and even greater, to
that of chitosanase, reaching a viscosity decrease higher
than 80–85% in 20 h. The effects of enzyme action could
be observed early, occurring the greatest viscosity
decreases in the first hour of hydrolysis.
Reducing end formation was slower than viscosity
decrease and, in general, paralleled to it, although both