Optimization of Chitin Extraction from Shrimp Shells - American

Aline Percot, Christophe Viton, and Alain Domard*. Laboratoire des Matériaux Polyme`res et des Biomatériaux, UMR-CNRS 5627, Bâtiment ISTIL,. Domain...
0 downloads 0 Views 88KB Size
Biomacromolecules 2003, 4, 12-18

12

Articles Optimization of Chitin Extraction from Shrimp Shells Aline Percot, Christophe Viton, and Alain Domard* Laboratoire des Mate´ riaux Polyme` res et des Biomate´ riaux, UMR-CNRS 5627, Baˆ timent ISTIL, Domaine Scientifique de la Doua, 15 Bd. Andre´ Latarjet, 69622 Villeurbanne Cedex, France Received June 25, 2002; Revised Manuscript Received October 4, 2002

The aim of this paper is to define optimal conditions for the extraction of chitin from shrimp shells. The kinetics of both demineralization and deproteinization with, in the latter case, the role of temperature are studied. The characterization of the residual calcium and protein contents, the molecular weights, and degrees of acetylation (DA) allows us to propose the optimal conditions as follows. The demineralization is completely achieved within 15 min at ambient temperature in an excess of HCl 0.25 M (with a solid-to-liquid ratio of about 1/40 (w/v)). The deproteinization is conveniently obtained in NaOH 1 M within 24 h at a temperature close to 70 °C with no incidence on the molecular weight or the DA. In these conditions, the residual content of calcium in chitin is below 0.01%, and the DA is almost 95%. Introduction Sources of chitin are estimated to be as abundant as those for cellulose with a yearly production of approximately 10101012 T.1 Chitin is a polysaccharide corresponding to linear copolymers of β(1f4)-linked 2-amino-2-deoxy-D-glucan and 2-acetamido-2-deoxy-D-glucan. Chitin, especially its main derivative chitosan, has numerous applications, for example, in agriculture, biomedicine, paper making, and food industries.2 Some applications require specific architectures, and the effectiveness of the polymers for these applications was shown to depend on the molecular weight distribution and the degree of acetylation (DA).3-5 A cost-effective, fast, and easily controlled industrial process for producing chitins of high molecular weight and DA still remains to be developed. The main sources of raw material for the production of chitin are cuticles of various crustaceans, principally crabs and shrimps. However, chitin in biomass is closely associated with proteins, minerals, lipids, and pigments. They all have to be quantitatively removed to achieve the high purity necessary for biological applications. Although many methods can be found in the literature for the removal of proteins and minerals, detrimental effects on the molecular weight and DA cannot be avoided with any of these extraction processes.6 Therefore, a great interest still exists for the optimization of the extraction to minimize the degradation of chitin, while, at the same time, bringing the impurity levels down to a satisfactory level for specific applications. Demineralization is generally performed by acids including HCl, HNO3, H2SO3, CH3COOH, and HCOOH but HCl seems to be the preferred reagent and is used with a concentration between 0.275 and 2 M for 1-48 h at * To whom correspondence should be addressed.

temperatures varying from 0 to 100 °C.1 Madhavan and Ramachandran Nair7 have shown that the viscosity of the chitosan obtained decreases with the treatment time in HCl, suggesting a decrease of the molecular weight with time. Deproteinization of chitin is usually performed by alkaline treatments, although other effective reagents have been reported. Typically, raw chitin is treated with approximately 1 M aqueous solutions of NaOH for 1-72 h at temperatures ranging from 65 to 100 °C.1 An interesting alternative method involves the enzymatic degradation of proteins. However, the residual protein satisfactory level, ranging approximately from 1% to 7%, remains higher and the reaction time is longer compared to that of the chemical way. These drawbacks make the enzymatic method8 unlikely to be applied industrially before progress is made in making the process more efficient. In this first paper on the study of the production of chitin, we propose an optimized method of extraction for the production of biological-grade chitin with both high molecular weights and DA. The kinetics of demineralization and deproteinization using two classical methods have been studied to achieve these objectives. The purity level of chitin was followed by the evaluation of the calcium and protein contents as a function of the reaction time. In parallel, the influence of both treatments on the preservation of the chitin structure was studied from the control of the molecular weight and DA after each step. Materials and Methods Raw Materials and Preparation. Shells of marine shrimp Parapenaeopsis stylifera were provided by France-Chitine. The tiny brown shrimp, which is common all over the Indian Ocean, originates in our case from the port of Jakham (India)

10.1021/bm025602k CCC: $25.00 © 2003 American Chemical Society Published on Web 11/05/2002

Biomacromolecules, Vol. 4, No. 1, 2003 13

Optimization of Chitin Extraction

located on the Arabian Sea. The shrimps were kept on ice for 2 or 3 days before being peeled; the shells were scraped free of loose tissue and washed individually in lightly salted water. The following procedure was chosen by the producer as an optimal treatment for a long time preservation of the raw material. The shells were then separated from cephalothoraxes, salted (10 kg of NaCl per 500 g of shell), and dried in the sun (25-30 °C) for 3 days. Prior to use, the shrimp shells were washed thoroughly in distilled water until the conductivity reached that of water. The shells were then freeze-dried and cryo-ground under liquid nitrogen. The powder thus obtained was sieved, and the fraction below 80 µm was used hereafter. Characterization of Shrimp Shells and Intermediary Products. The water content in the obtained powders was estimated by thermogravimetric analysis using a Du Pont Instrument TGA 2000 with 10-20 mg samples and a temperature ramp of 2 °C/min. The percentage of proteins remaining in chitin was determined by two different methods. First of all, the nitrogen content was measured by elemental analysis and the percentage of proteins calculated from the following equation P% ) (N% - 6.9) × 6.25

(1)

where P% represents the percentage of proteins remaining in the obtained powder and N% represents the percentage of nitrogen measured by elemental analysis with 6.9 corresponding to the theoretical percentage of nitrogen in fully acetylated chitin and 6.25 corresponding to the theoretical percentage of nitrogen in proteins. In the case of the crude shrimp shells, no accurate values could be obtained because of the great amount of calcium carbonate present. For this special case, the amount of proteins was measured using the amino acid analysis. Two milligrams of sample was hydrolyzed with HCl 6 M in the presence of trifluoroacetic acid for 45 min at 150 °C under vacuum. The samples were then solubilized in a buffer, and an aliquot was used for analysis on a Beckman system 6300 amino acid analyzer. The percentage of proteins was calculated from the total amino acid weight. The lipid content was estimated after a Soxlhet extraction with chloroform/methanol (2/1, v/v) and subsequent gravimetric analysis of the shrimp shells. The ash content was determined by slowly heating a sample to 900 °C with stages at 120 and 340 °C and weighing the remaining product after cooling in a desiccator. For the minerals, in addition to ashes analysis, levels of calcium and magnesium were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and ICP mass spectrometry (ICP-MS). Prior to analysis, the solid samples were digested in concentrated sulfuric acid in a microwave reactor until complete dissolution had occurred. 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, as

proposed by Hirai et al.,9 from the ratio of the methyl proton signal of (1f4)-2-acetamido-2-deoxy-β-D-glucan residues with reference to the H-2 to H-6 proton signals of the whole structure. For samples containing proteins, solid-state 13C NMR spectroscopy was used. Indeed, in this case, the too high amount of proteins makes too difficult the interpretation of the proton NMR spectra. 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 as follows: 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 C2C6 signals (δ 56-105 ppm).9 Determination of the Intrinsic Viscosity of Chitin. Chitin samples were solubilized at about 0.25 mg/mL in N,Ndimethylacetamide (DMAc) containing 5% lithium chloride (LiCl).10,11 The viscosity was measured using an automatic capillary viscometer, Viscologic TI 1 SEMATech (diameter 0.8 mm), at 25 °C. Kinetics of Demineralization. Demineralization was carried out in dilute HCl solutions. Typically, the shrimp shells were soaked in HCl 0.25 M at ambient temperature with various solution-to-solid ratios under constant stirring. The demineralization kinetics were followed by monitoring the pH as a function of time in the supernatant. The characteristics of the obtained chitin as a function of the demineralization time were studied by retrieving a representative sample of known volume from the dispersion of shrimp shell particles at 2, 6, 13, 30, 60, 180, 360, and 1440 min using a syringe with a large needle. The heterogeneous samples were then filtered under vacuum on paper, and the supernatant was analyzed for the pH and the calcium content (by ICP-AES). The recovered demineralized shrimp shell powder was washed to neutrality and freeze-dried. Following the demineralization step, the partly demineralized shrimp shells were deproteinized with a solution of NaOH 1 M under vigorous stirring using 30 mL of solution per gram of demineralized shells. After 24 h of reaction, the solid samples were washed to neutrality and freeze-dried. The calcium content in the purified chitin was measured by ICP-AES. Kinetics of Deproteinization. The kinetic studies of deproteinization were performed on demineralized shrimp shell powder (Figure 1). Quantitative demineralization was carried out in HCl 1 M at a room temperature corresponding to 22 ( 1 °C with a solution-to-solid ratio of 10 mL/g. After 24 h, the demineralized shrimp shell powder was removed by filtration, washed to neutrality, and freeze-dried. Deproteinization kinetics studies were then performed by addition of NaOH 1 M to decalcified powder at a solution-to-solid ratio of 15 mL/g, and a representative sample was taken at 5, 20, 60, 180, 300, and 1440 min. The partly deproteinized shrimp shell powder was removed in each sample by filtration, washed to neutrality, and freeze-dried, while the supernatant was analyzed for the protein content.

14

Biomacromolecules, Vol. 4, No. 1, 2003

Percot et al.

Figure 1. Overall process for the preparation of chitin from salted shrimp shells.

The release of proteins in the supernatant was followed by the measurement of the absorbance at 280 nm, characteristic of the tryptophan residues present in the protein composition. The absorbance was measured on an Uvikon (UV-vis.) spectrophotometer 941 (Kontron Instruments). Shrimp shell proteins were recovered by a classical process of deproteinization on demineralized shrimp shells in NaOH 1 M for 24 h with a solution-to-solid ratio of 15 mL/g (Figure 1). After filtration, the supernatant containing the proteins was dialyzed against pure water for 1 week and lyophilized. The protein content in the recovered powder was determined by amino acid analysis and found to be close to 60% (w/w). These proteins were used to plot a calibration curve giving a straight line with the following equation abs ) Cp × 1.7

r2 ) 0.999 73

(2)

where abs corresponds to the UV absorbance measured at 280 nm, Cp corresponds to the protein concentration in mg/ mL, and r corresponds to the correlation coefficient. The weight of proteins released could be then expressed as a weight percentage of the initial weight of decalcified shrimp shells. The deproteinization was also studied as a function of temperature. In this case, NaOH 1 M was added to demineralized shrimp shells with a solution-to-solid ratio of 15 mL/g. The deproteinization was carried out for 24 h at various temperatures. The protein content was measured in the supernatant and in the obtained chitin. Results and Discussion The overall process for the preparation of shrimp chitin is given in Figure 1. The raw shrimp shells contain about 20% of chitin and other components reported in Table 1. While CaCO3 is the major inorganic component, some magnesium is also present in a low proportion. Demineralization (usually performed in concentrated acid) and deproteinization (in aqueous NaOH) are therefore the critical steps

Table 1. Composition of Crude Shrimp Shells, Demineralized Shrimp Shells (24 h in HCl 1 M), and Chitin (24 h in NaOH 1 M) composition

shrimp shells (wt %)

water crude protein crude fat ash (as oxide) calcium magnesium

11.3 ( 0.4 7c 6(2 35.49 ( 0.04 19a 1a

demineralized shrimp shells (wt %) 6.3 20d

chitin (wt %) 6.9 ( 0.1