New Aspects of the Extraction of Chitin from Squid Pens - American

In this work, we studied the extraction of β chitin from squid pens. The characterization by ICP of the raw material confirmed the significant absenc...
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Biomacromolecules 2004, 5, 559-564

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New Aspects of the Extraction of Chitin from Squid Pens Ge´ raldine Chaussard and Alain Domard* Laboratoire des Mate´ riaux Polyme` res et des Biomate´ riaux - UMR-CNRS 5627, Domaine scientifique de la Doua, Baˆ timent ISTIL, 15, Bd. A. Latarjet, 69622 Villeurbanne Cedex, France Received October 9, 2003; Revised Manuscript Received January 7, 2004

In this work, we studied the extraction of β chitin from squid pens. The characterization by ICP of the raw material confirmed the significant absence of calcium carbonate and then the possibility to definitely avoid a step of demineralization responsible for chain degradation. The kinetics studies associated with the extraction of lipid parts showed the important role played by the latter structures on the limitation of the protein extraction by a simple treatment in an aqueous solution of 1 M NaOH. The role of various parameters such as the size of the particles, time and number of steps, and the concentration in sodium hydroxide was studied. The determination of the molecular characteristics (molecular weight and degree of acetylation) allowed us to define the optimal conditions of deproteinization in relation with the best preservation of these characteristics. Introduction Chitin is a word referring to the series of the linear copolymers of linked β, (1 f 4) glucosamine and Nacetylglucosamine. Depending on the origin, the content in the latter residue, within 70 and 95%, is responsible for its water insolubility. This polymer is largely widespread in biomass and is present in numerous living species, especially in the cuticles of arthropods or the endoskeletons of cephalopods.1 These sources have the particularity to contain chitin belonging to two crystallographic forms named R and β. In the first case, the chains are organized according to a succession of antiparallel planes containing parallel chain segments. All of these chains are linked together thanks to a dense network of hydrogen bonding. In the second case, although, all of the chains are parallel, whatever the considered axis, hydrogen bonds between the chains of the successive planes are absent.2 Then, β chitin exhibits a higher accessibility to water and thus to water-soluble reagents. It is also characterized by a low mineralization, especially by the absence of calcium carbonate, which, in contrast, is largely present in the R chitin constituting the cuticles of arthropods. As a consequence, in the former case, we could imagine to avoid the demineralization step, which, in most cases, is responsible for a decrease of the molecular weight. In relation with its location in endoskeletons, β chitin is not really pigmented and should not be contaminated by heavy metals. Thus, for particular applications requiring high molecular weights and/or a high purity, the sources containing β chitin can be considered as particularly appropriate. In previous works,3,4 we studied the demineralization and deproteinization of the cuticles of shrimp shells by means of various methods and we defined the conditions to produce chitin with the molecular characteristics and purity optimal either for a direct use or to be deacetylated in view of the * To whom correspondence should be addressed. E-mail: alain.domard@ univ-lyon1.fr.

chitosan production. In this paper, we did the same kind of study on the deproteinization step of a source of squid pens. It particularly consisted of investigating the role of various parameters such as the time and length of the particles on the final characteristics (degree of acetylation, molecular weight, protein and ash contents). Thus, it was interesting to compare the fundamental parameters studied for the extraction of chitin from the two types of sources. Experimental Section Raw Material. Squid pens (loligo) were a gift from France Chitine. They were collected in India in the Arabian Sea. Just after fishing, the pens were extracted carefully then washed thoroughly with tap water, dried in air and sent immediately to our laboratory. Once received, they were frozen and preserved at -30 °C in a freezer until their use. Just before use, they were grounded in liquid nitrogen to avoid a possible thermal degradation, simultaneously sieved at different sizes, and then dried under reduced pressure. The water content was deduced from thermogravimetric analysis on a Du Pont thermo-balance. The weight loss was measured under argon atmosphere between 20 and 150 °C. The ramp of temperature was of 2 °C/min. Ash and Mineral Contents. The ash content was deduced from the difference of weights before and after a thermal treatment of the product in an oven. A succession of temperature raises and of intervals at constant temperature were operated up to 900 °C. The minerals were analyzed in detail by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and ICP-mass spectrometry (ICP-MS). Determination of the Degree of N-Acetylation (DA) of Chitin. Chitin samples were dissolved in DCl/D2O (20% w/w) with vigorous stirring for 8 h at 50 °C. These conditions were necessary to sufficiently depolymerize chitin, thus allowing the full solubilization of the polymer. The spectra were recorded on a Bruker AC 200 spectrometer (200 MHz for 1H) at 298 K. The DA was calculated from the ratio of

10.1021/bm034401t CCC: $27.50 © 2004 American Chemical Society Published on Web 02/14/2004

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Biomacromolecules, Vol. 5, No. 2, 2004

Chaussard and Domard

Table 1. Weight % of the Main Constituents of Shrimp Shells Compared to Those of Squid Pens Studied in This Worka species

water (%)

chitin (%)

proteins (%)

extractibles (%)

ash (%)

squid pens shrimp shells3

13 ( 0.4 11 ( 0.4

32.5 ( 2 17 ( 2

42.5 ( 2 7(2

2 ( 0.5 4.5 ( 0.5

2.4 ( 0.4 35.4 ( 0.4

a The water content was determined from a thermo-gravimetric analysis; the chitin content, after a prolonged treatment in sodium hydroxide; the protein content, from the nitrogen content (see below); the extractible parts, from the sum of the extracts obtained in chloroform and in a mixture chloroform/ methanol (2:1 v/v); and the ash content, after mineralization in an oven up to 900 °C. Each value is the result of two independent experiments including at least 2 measurements.

the methyl proton signal of (1 f 4)-2-acetamido-2-deoxyβ-D-glucan residues with reference to the H-2 to H-6 proton signals of the whole structure. For the solid-state 13C NMR spectroscopy, the spectra were obtained on lyophilized samples with CP-MAS techniques (cross polarization, magic angle spinning) using a Bruker DSX400 instrument working at 100.6 MHz. Typical conditions were 90 RF pulse, 4.5 µs; contact time, 2.5 ms; pulse repetition, 2s; MAS rate, 5 kHz; 4096 scans were acquired. The measurements were performed at room temperature. The DA was calculated by comparison between the integrated areas of the methyl group carbon (δ 24 ppm) and the C2-C6 signals (δ 56-105 ppm). Determination of the Intrinsic Viscosity of Chitin. Chitin samples were dissolved at about 0.25 mg/mL in N,Ndimethylacetamide (DMAc) containing 5% lithium chloride (LiCl). The viscosity was measured using an automatic capillary viscometer, Viscologic TI 1 SEMATech (diameter 0.8 mm) at 25 °C. Studies of Crystallinity. The crystallinity of chitin was studied with a Siemens D500 diffractometer and evaluated thanks to the computation software DIFFRAC PLUS-EVA (Bruker AXS). Results and Discussions 1. Composition of the Raw Materials. It is first interesting to compare the contents in major components present in the raw materials coming from shrimp shells and squid pens (Table 1). Although these contents can vary from one fishing place to another, it remains that the main differences come from the considered animal species.1 The differences observed between the two sources essentially concern the mineral and protein contents, which necessarily influence the water content. These contents are also possibly influenced by the crystalline structure. Indeed, β chitin is well-known to be much more sensitive to water, in relation to a weaker network of hydrogen bonds compared to R chitin.5 A panoramic analysis of the content of anions or cations present in the squid pens was obtained thanks to ICP measurements. The results are reported in Table 2. Concerning the squid pens, we may notice several interesting things. (i) The content in transition metals is very low and, whatever the case, largely below the upper limit of toxicity. This confirms that, contrarily to what can occur with a cuticle of arthropod, a squid pen is not really polluted. Moreover, some metals such as Ti, Fe, Cu, and Zn, in low amount, are necessary to the animal life. (ii) The content in carbonate ions is very weak. Since it can be attributed either to the water used to wash the pens and/or to each of the present cations, the part attributable to

Table 2. Content in Anions and Cations in Squid Pens and in β Chitin after Deproteinizationa detected species anion or cation

in squid pens (ppm)

in chitin (ppm)

HCO3FClNO2 BrNO3PO43SO42Al Ti Mn Cr Hg Ni Cu Sr Cd Zr Ba Na K Mg Fe Ca Zn Sn

135 55 19700