0961-9534/93 $6.00 + 0.00 C 1993 Pergamon Press Ltd &moss and BioenergyVol 5, No 2, pp 145-153,1993 Printed in Great Britain All rights reserved EXTRACTION AND CHARACTERIZATION FROM CRUSTACEANS N ACOSTA*, C JItiNEZt, BoRAutand V OF CHITIN A HERAS*$ ?? 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, /.Iand 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 environmental 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 relationship 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 rigidbody least-squares smoothing methods The 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 chitosan 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, potentiometry, 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 ideal supporting materials Traditionally, should be inert and have no effect on the kinetic behaviour of the biocatalyst they were intended to host However, comparative studieP have shown dramatic differences in performance between enzymes supported on various materials In this respect, chitin possesses excellent properties for immobilizing enzymes In this work, chitin was isolated from various natural sources Samples were characterized by IR spectroscopy and scanning electron microscopy, as well as from the degree of acetylation, in order to test them as supports on immobilization of enzymes MATERIALS AND METHODS 2.1 Materials Chitin was obtained from crustacean shells of different sources including Cuban lobsters 145 N ACOSTAet al 146 (Polinurus vulgaris), Sanhicar prawns (Penaeus caramote), Norway lobsters (Nephrops norvegicus), 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 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 by Scharlau (Barcelona, Spain) and sodium Grinding hypochlorite was obtained from Panreac (Barcelona, Spain) All of the above chemicals were of analytical reagent grade 2.2 Methods 2.2.1 Isolation of chitin The isolation procedure 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 were ground to 24mm pieces and stored at 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): and sieving 1N HCL at room temperature for 2h 15% NaOH Deproteinization Extraction at 65°C for 3h with acetone Dilute NaOCL for 15min at room temperature Washing Scheme and drying Isolation of chitin Extraction and characterization (a) Demineralization Shell particles were demineralized with N HCl at room temperature and a solid-to-solvent ratio of 1: 15 (w/v) under continuous stirring for h, and then washed repeatedly with distilled water to neutralize excess acid After filtering, particles of OS-2 mm diameter were obtained (b) Deproteination Demineralized shell particles were brought into contact with a 15% NaOH solution at 65°C and a solid-to-solvent ratio of 1: 10 (w/v) for 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-tosolvent ratio of 1: 10 (w/v) and room temperature 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 following procedures were used to characterize the previously obtained chitin: (a) IR spectroscopy Infrared spectra of the samples on KBr were recorded between 400 and 4000 cm-’ on a Bomen MB100 IR spectrophotometer For this purpose, mg of dry sample was mixed with 10 g of also dry KBr in order to make a 100mg pellet (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 scanning electron microscope (c) Estimation of the degree of acetylation The degree of acetylation of chitin was measured by using a previously reported method.’ RESULTS AND DISCUSSION 3.1 Chitin yields Table lists the yields with which chitin was obtained from the various sources As can be seen, they ranged between 14% and 23.8% (on dry weight basis) The best results in this respect (23.2-23.8%) were provided by common lobsters, prawns, Norway lobsters and squills, followed by Fusarium oxysporum and, finally, Spanish and American crayfish These results are consistent with the fact that prawns, Norway lobsters and squills belong to the same species, whereas crayfish not-the last two of chitin 141 Table I Chitin yield of various sources Sourcef Yield (%) Lobster Prawn Norway lobster Squills Spanish crayfish American crayfish 14.2 23.2 23.6 23.8 14.5 14.1 Fusarium oxysporum 15.0* *Dry weight of chitin/wet weight of mycelia order: Polinurus vulgaris, Penaeus Vn caramote, Nephrops norvegicus, Squilla mantis Astecus jluviabilis, Astecus cambarus probably contain different amounts of carbonates 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 reported by other authors’ and to that of chitin from Fusarium oxysporum and other fungi.” 3.2 Characterization of the chitin samples The physico-chemical properties of the chitin samples were studied by IR spectroscopy, scanning electron microscopy and the degree of acetylation in order to characterize them as potential biotechnological supports 3.2.1 IR spectroscopy Figure (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 previous 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) The spectrum of chitin from Fusarium 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 between methyl groups Also, chitin from Spanish crayfish differed from the rest in the intensity and width of the band at 1420 cm -‘ Beran et al.,13 purified chitin from fungi, showing that, in this case, the relative intensity of the bands at 1630 and 955 cm-’ increases As can be seen, the intensity of the bands at 1630 and 955 cm-’ was smaller for chitin from Fusarium oxysporum than for the others It thus seems that the procedure used to isolate chitin from this fungus yielded less pure chitin than did crustacean shells The presence of additional impurities may be the root of the interferences encountered in the spectrophotometric determi- 148 N ACOSTAet al Wavenumbers (cm-l) “0 j P 41 3000 Wavenumbrre 2000 (cm-l) Fig Infrared spectra of chitin samples from various sources (a) I Squills (Sqda mantis); Lobster (Polinurus vulgaris); Norway lobster (Nephrops norvegicus); Prawn (Penaeus caramore) (b) Fusarium oxysporum; Commercial product; Spanish crayfish (Asrecus~uviabilis) nation of the number of free -NH, groups, as shown below 3.2.2 Scanning electron microscopy Figure shows the scanning electron micrographs obtained 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, according 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 However, this difference from prawn and Spanish crayfish chitin in the photographed surface was not reflected in the IR spectra where Extraction and characterization Fig 2(a) Fig 2(b) of chitin 149 50 Fig 2(c) Fig 2(d) Extraction and characterization of chitin Fig 2(e) Fig 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) (d) Commercial product (e) Fusariu~ oxysporu& 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, (particularly those of Fusarium oxysporum chitin) The bands corresponding to the p-structure, 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 preferentially a-structured since its surface shows sharper fibres, whereas that commercially available 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 14NNMR or 13C-NMR spectroscopy, or even UV spectrophotometry at 199 nm.4 However, the determination is hindered by the fact that the polymer is insoluble in most common organic solvents 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 heterogeneous 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 N ACOSTAer al 152 Table Amount of phenol adsorbed in monolayer form by the various chitin samples Sourcet Lobster Prawn Norway lobster Squills Spanish crayfish American crayfish XIII (mol g-’ chitin) x 10e6 5.4 5.8 5.6 3.2 3.3 3.4 Fusarium oxysporum Commercial product 3.1 ?In order: Polinurus vulgaris, Penaeus caramote, Nephrops norvegicus, Squilla mantis, Astecus fluviabilis, Astecus cam barus 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 behaviour 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 REFERENCES The amount of titrant adsorbed by our samples at each point along the isotherm was determined spectrophotometrically using the method of Marinas et a1.9-22over the concentration 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 dissolved 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 concentration of dissolved titrant in equilibrium with the amount of adsorbed titrant, X) Table 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 amounts of phenol in monolayer form (ca x 10P6mol gg’ chitin) On the other hand, the remaining samples adsorbed an average x 10m6mol g-’ chitin Taking into account that the interaction between amino groups and phenol conforms to a 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 Chitin from Fusarium oxysporum did not allow the amount of phenol adsorbed to be determined because the final absorbance of the phenol solutions brought into contact with the solid 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