EXTRACTION AND CHARACTERIZATION OF CHITIN FROM CRUSTACEANS

The physico-chemical properties of chitin from the different sources were studied by IR spectroscopy and scanning electron microscopy, and its degree of acetylation was determined.. Key

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EXTRACTION AND CHARACTERIZATION

FROM CRUSTACEANS

OF CHITIN

N ACOSTA*, C JItiNEZt, V BoRAut and A HERAS*$

??Departamento de Quimica Fisica Farmackutica, Facultad de Farmacia, Instituto Pluridisciplinar,

Universidad Complutense, E-28040 Madrid, Spain TDepartamento de Quimica Organica, Facultad de Ciencias, Universidad de Cordoba, Avda San Albert0

Magno s/n, E-14004 Cbrdoba, Spain

(Received I March 1993; revised received 28 June 1993; accepted 14 July 1993)

Ahstraet-Chitin was isolated from various natural sources including Cuban lobsters, Sanlircar prawns,

Norway lobsters, Squills, Spanish crayfish, American crayfish and Fusarium oxysporum with a yield of 14-25% on a dry basis

The physico-chemical properties of chitin from the different sources were studied by IR spectroscopy

and scanning electron microscopy, and its degree of acetylation was determined The chitin thus obtained

is suitable for biotechnological applications (e.g as supporting material for immobilizing enzymes)

Keywords-Chitin, chitin isolation, IR spectroscopy, scanning electron microscopy, degree of acetylation

INTRODUCTION

The term “chitin” is used to designate fibrillar

1,4-linked 2-acetamido-2-deoxy-/3-D-glucan

This substance can be acetylated to a variable

extent and occurs in three polymorphic forms

(a, /.I and y) and various degrees of crystallinity

The term “chitosan” encompasses a wide range

of partially deacetylated derivatives of chitin

The composition of chitin and its chitosan

content varies with its source, as well as with the

particular season, habitat and other environ-

mental conditions.’

Chitin and chitosan are the only naturally

abundant polysaccharides with markedly basic

properties In fact, chitin is a constituent of the

outer structure of insects, fungi and crustaceans

Chitin is also significant because of its relation-

ship to some components of foods of animal,

and fungal origin, and its potential medical and

pharmaceutical uses Fungal chitin is readily

available for a variety of current and potential

uses in diverse fields.*

The structure of a- and /I-chitin has been

elucidated by the X-ray diffraction using rigid-

polarity of neighbouring chains (anti-parallel in

a-chitin and parallel in /I-chitin) has also been

determined, as has the hydrogen bond network.’

The degree of acetylation of chitin and chi-

tosan is a major parameter for their chemical

IAuthor to whom correspondence should be addressed

characterization which can be determined by NMR of the solid and IR spectroscopy, poten- tiometry, mass spectrometry, and chemical or enzymatic titration.4

Chitosan is highly reactive at its primary amino group and its primary and secondary hydroxyl functions Both chitosan and chitin are very hard solids that are insoluble in most organic solvents and possess good mechanical properties In addition, they are biodegradable and biocompatible, and very scarcely toxic, so they make excellent supports for acid and basic reagents and enzymes

should be inert and have no effect on the kinetic behaviour of the biocatalyst they were intended

shown dramatic differences in performance be- tween enzymes supported on various materials

In this respect, chitin possesses excellent proper- ties for immobilizing enzymes

In this work, chitin was isolated from vari- ous natural sources Samples were character- ized by IR spectroscopy and scanning electron microscopy, as well as from the degree of acety- lation, in order to test them as supports on immobilization of enzymes

2 MATERIALS AND METHODS

2.1 Materials

Chitin was obtained from crustacean shells

of different sources including Cuban lobsters

145

(Polinurus vulgaris), Sanhicar prawns (Penaeus

caramote), Norway lobsters (Nephrops norvegi-

cus), squills (Squilla mantis), Spanish crayfish

(Astecus Juviabilis), and American crayfish

(Astecus cambarus), specimens of which were

collected from the waste of a local seafood

restaurant Fusarium oxysporum was cultivated

on potato dextrose broth medium at 25°C for

4 days The procedure used for this purpose was

based on one described in detail elsewhere.7 The

end products were freeze-dried

Commercially available chitin was purchased

from Sigma, while hydrochloric acid, sodium

hydroxide, acetone, potassium bromide, sodium

chloride and glutaraldehyde were supplied by

Merck Phenol and cyclohexane were provided

Grinding and sieving

(Barcelona, Spain) All of the above chemicals were of analytical reagent grade

2.2 Methods

2.2.1 Isolation of chitin The isolation pro- cedure was applied three times to each type of sample Prior to use, shells from the various sources were boiled for ca 12 h in order to remove soluble organics and binding protein, and then dried at 80°C for 24 h The dried shells

room temperature

The procedure used to isolate chitin was a modified version of a previously reported one.7

It involved the following steps (see Scheme 1):

Deproteinization

Extraction with acetone

Washing and drying

1N HCL at room tempera-

ture for 2h

15% NaOH at 65°C for 3h

Dilute NaOCL for 15min at

room temperature

(a) Demineralization Shell particles were

demineralized with 1 N HCl at room tempera-

ture and a solid-to-solvent ratio of 1: 15 (w/v)

under continuous stirring for 2 h, and then

washed repeatedly with distilled water to neu-

tralize excess acid After filtering, particles of

OS-2 mm diameter were obtained

(b) Deproteination Demineralized shell par-

ticles were brought into contact with a 15%

NaOH solution at 65°C and a solid-to-solvent

ratio of 1: 10 (w/v) for 3 h, after which they were

washed with distilled water and filtered

(c) Bleaching The product thus obtained was

extracted into acetone in order to remove the

pigment astaxanthin’ and then allowed to dry at

room temperature Deproteinated samples were

bleached in 15% v/v NaClO/HCl at a solid-to-

solvent ratio of 1: 10 (w/v) and room tempera-

ture for 15 min, and subsequently washed and

dried at 80°C for 12 h The chitin thus obtained

was stored at 25°C prior to characterization

2.2.2 Characterization procedures The fol-

lowing procedures were used to characterize the

previously obtained chitin:

Table I Chitin yield of various sources

American crayfish 14.1

Fusarium oxysporum 15.0*

*Dry weight of chitin/wet weight of mycelia

Vn order: Polinurus vulgaris, Penaeus caramote, Nephrops norvegicus, Squilla mantis Astecus jluviabilis, Astecus cambarus

probably contain different amounts of carbon- ates and other salts and in addition to proteins,”

so their weight yields were lower

The chitin yields obtained from crustacean shells are comparable to those previously re- ported by other authors’ and to that of chitin

3.2 Characterization of the chitin samples

(a) IR spectroscopy Infrared spectra of the

samples on KBr were recorded between 400 and

4000 cm-’ on a Bomen MB100 IR spectropho-

tometer For this purpose, 8 mg of dry sample

was mixed with 10 g of also dry KBr in order to

make a 100mg pellet

The physico-chemical properties of the chitin samples were studied by IR spectroscopy, scan- ning electron microscopy and the degree of acetylation in order to characterize them as potential biotechnological supports

(b) Scanning electron microscopy (SE&i) The

SEM technique was used to characterize the

surface of chitin particles Thus, dried particles

were coated with Au-Pd on a SEM Coating

Unit PS3 under a nitrogen atmosphere for 70 s

and then examined under an ISI-SX-25 scan-

ning electron microscope

3.2.1 IR spectroscopy Figure 1 (a, b) shows the IR spectra of the chitin samples All of them are very similar, particularly as regards the characteristic bands at 3450, 3265, 3102, 1666,

1622, 1574, 1435, 1430, 1361, 1315, 1250, 1113,

1020, 951 and 887cm-‘, consistent with pre- vious observations of Gow et al.” on a-chitin; however, no bands were observed at 972 or

632 cm-’ (these two are typical of fi-chitin) (c) Estimation of the degree of acetylation The

degree of acetylation of chitin was measured by

using a previously reported method.’

3 RESULTS AND DISCUSSION

3.1 Chitin yields

oxysporum was different from the rest Thus, the relative intensity of the bands between 2968 and

2850 cm-’ was different from those of the other chitins, which suggests a different interaction

Spanish crayfish differed from the rest in the intensity and width of the band at 1420 cm -‘ Table 1 lists the yields with which chitin was Beran et al.,13 purified chitin from fungi, obtained from the various sources As can be showing that, in this case, the relative intensity seen, they ranged between 14% and 23.8% (on of the bands at 1630 and 955 cm-’ increases As dry weight basis) The best results in this respect can be seen, the intensity of the bands at 1630

lobsters, prawns, Norway lobsters and squills, ium oxysporum than for the others It thus seems followed by Fusarium oxysporum and, finally, that the procedure used to isolate chitin from Spanish and American crayfish These results this fungus yielded less pure chitin than did are consistent with the fact that prawns, crustacean shells The presence of additional Norway lobsters and squills belong to the same impurities may be the root of the interferences species, whereas crayfish do not-the last two encountered in the spectrophotometric determi-

“0

.j

P

3

41

Wavenumbers (cm-l)

Wavenumbrre (cm-l)

Fig 1 Infrared spectra of chitin samples from various sources (a) I Squills (Sqda mantis); 2 Lobster

(Polinurus vulgaris); 3 Norway lobster (Nephrops norvegicus); 4 Prawn (Penaeus caramore) (b)

5 Fusarium oxysporum; 6 Commercial product; 7 Spanish crayfish (Asrecus~uviabilis)

nation of the number of free -NH, groups, as

shown below

3.2.2 Scanning electron microscopy Figure 2

shows the scanning electron micrographs ob-

tained for the dry samples As can be seen, the

surface appearance depends on the type (family,

species) of crustacean concerned

Thus, the surface of chitin from lobster and

Spanish crayfish consists of fibres that form

parallel thread networks This is consistent with

our IR results as regards the bands at 3265, 1630

and 955cm-’ for the a-structure, which, ac- cording to Blackwell,’ forms thread groups that

in turn make up images such as those observed

in our micrographs The surface of chitin from prawn shows scarcely fibrillar material and a somewhat granular structure which is described

in the literature as a chitin-protein complex.‘4

Spanish crayfish chitin in the photographed surface was not reflected in the IR spectra where

Fig 2(a)

Fig 2(b)

Fig 2(c)

Fig 2(d)

(d) Commercial product

the bands for this sample had an a-structure

identical with that of chitin from lobster

The surface of commercially available chitin

and that obtained from Fusarium oxysporum is

somewhat different, they have a granular rather

than fibrillar appearance This can be ascribed

to the polymorphic character of chitin, which is

also consistent with their IR bands: those of the

%-structure are weaker and more ill-defined,

chitin) The bands corresponding to the p-struc-

ture, which forms no fibres as no hydrogen

bonds are established between threads-so they

can swell and form hydrates-are also observed

From the above results and those obtained by

IR spectroscopy, one might conclude that chitin

from lobster and Spanish crayfish is preferen-

tially a-structured since its surface shows

sharper fibres, whereas that commercially avail-

able and fungal chitin, is more granular, which

is consistent with a p-structure or a less marked

a-structure

3.2.3 Degree of acetylution The degree of

acetylation of chitin can be determined by 14N-

NMR or 13C-NMR spectroscopy, or even UV

determination is hindered by the fact that the

polymer is insoluble in most common organic

solvents

Fig 2(e) Fig 2 Scanning electron micrographs of dry surfaces of chitin from various sources (b) Lobster

(Polinurus ru~pris), (b) Prawn (Penaeus carumofe), (c) Spanish crayfish (Astecus fluuiuhilis)

(e) Fusariu~ oxysporu&

Some authors use IR spectroscopy’5-~‘s or a benzylation procedure” to determine the degree

of 0-acetylation and N-acetylation of chitin These methods, however, may be subject to major experimental errors

There are a number of available hetero- geneous catalysis methods for the determination

of acid and basic surface sites.20.2’ Essentially, all entail measuring the amount of titrant (and acid

or base) retained in the solid monolayers On the assumption that each titrant molecule is adsorbed at one active site, the number of acid

or basic surface sites can readily be calculated

In dilute enough solutions, the titrant can act

as a gas and its adsorption on a solid be fitted

to a Langmuir isotherm of the form:

c/s =&++

m where X is the amount of titrant adsorbed per gram of solid at a given temperature, b the Langmuir constant, X,,, the amount of titrant adsorbed in monolayer form per gram of solid, and C the dissolved titrant concentration in equilibrium with the adsorbed concentration, X

By plotting C/x against c (an amount) one obtains a straight line whose slope provides X,,, ,

a measure of the solid acidity or basicity at a given temperature

Table 2 Amount of phenol adsorbed in monolayer form by

the various chitin samples

XIII Sourcet (mol g-’ chitin) x 10e6

Fusarium oxysporum

?In order: Polinurus vulgaris, Penaeus caramote, Nephrops

norvegicus, Squilla mantis, Astecus fluviabilis, Astecus cam -

barus

samples at each point along the isotherm was

method of Marinas et a1.9-22 over the concen-

tration range where Beer’s law was obeyed

Active sites in chitin were titrated with pyridine

dissolved in cyclohexane, while basic sites

(-NH, groups) were titrated with phenol dis-

solved in cyclohexane

Identical results were obtained if x (the

amount of titrant adsorbed per gram of solid)

was determined by plotting X vs C (the concen-

tration of dissolved titrant in equilibrium with

the amount of adsorbed titrant, X)

Table 2 lists the X,,, values obtained in the

titrations with phenol dissolved in cyclohexane

As can be seen, chitin from common lobster,

prawn and Norway lobster adsorbed the largest

5 x 10P6 mol gg’ chitin) On the other hand, the

3 x 10m6 mol g-’ chitin Taking into account

that the interaction between amino groups and

phenol conforms to a 1: 1 stoichiometry, chitin

from the former group of samples (the first

three in Table 2) and that from prawn in

particular, contains the most deacetylated units

in its structure

allow the amount of phenol adsorbed to be

determined because the final absorbance of the

phenol solutions brought into contact with the

solid exceeded the initial absorbance, so the

amine was not retained at the surface, but

adsorbed dissolved species that were responsible

As noted earlier, the IR spectrum of Fwarium oxysporum chitin was different from the rest Consequently, the above-mentioned impurities, which are not removed in the purification of chitin, are responsible for the peculiar be- haviour of this sample

Acknowledgements-The authors wish to thank Dr M I G Roncero for kindly supplying the Fusarium oxysporum used Financial support from the Spanish CICYT (Project FAR 88-0276/2) and the Programa Iberoamericano 1990 is also gratefully acknowledged

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