Chitin deproteinization

Carbamate derivatives (Table 1) of cellulose, chitin, amylose, amylopectin, and dextran were prepared using the isocyanates described in Part A of the Experimental Section. Amylose, amylopectin, dextran, and cellulose were obtained from Polysciences, Inc. and used without further purification. Chitin, obtained from Eastman Kodak, was decalcified and deproteinated by the method reported by Haye r prior to use. 

FIGURE 2.21 NMR solid-state spectra of (a) a-chitin from deproteinized lobster tendon, (b) p-chitin from dried deproteinized tube of Tevnia jerichonana. 

One of the major uses of chitin fibers is as sutures for surgery. Another important use is the production of paper by applying deproteinized ground chitin particles from a homogenized suspension to a continuous paper making machine. 

Chitin is a homopolymer of AT-acetyl-D-glucosamine residues and is a major structural component in the exoskeletons of crustaceans, mollusks, arthropods, and the cell walls of numerous fungi and algae. Owing to its widespread presence in both terrestrial and aquatic organisms, chitin is second only to cellulose as the most abundant biopolymer on the Earth (Shahidi and Abuzaytoun, 2005). On a dry weight basis, shrimp, crab, lobster, prawn, and crayfish have been reported to contain between 14% and 35% chitin, while deproteinized dry shell waste of Antarctic krill contains approximately 40% crude chitin (Haard et al, 1994). Crustaceans are the primary sources of chitin used in industry. Chitin can be extracted from shellfish and crustacean waste by mixing with a dilute add to induce demineralization, followed by a deproteini-zation step in a hot alkaline solution (Synowiecki and Al-Khateeb, 2003). 

Despite the various potential applications of chitosan, there are several drawbacks. For example, the costs of production often outweigh the economic benefits of its application. Extraction yields for chitosan from waste are generally very low ( 3-5% of raw material) and production is limited by seasonal variations in crustacean harvesting. Also, additional waste streams are created during the alkali deproteinization of chitin during isolation (Ludlow, 2001 Synowiecki and Al-Khateeb, 2003). One interesting... 

Conversion of chitin into chitosan involves the deacetylation process, which is a harsh treatment usually performed with concentrated sodium hydroxide solution. Chitin flakes are treated in suspension with aqueous 40-50% caustic solution at 80-120°C with constant stirring for 4-6 h and this treatment is repeated once or more than once for obtaining high-amino-content product. To avoid depolymerization due to oxidation, sodium borohydrate is added. Excess alkali is drained off and the mixture is washed with water several times until it is free from alkali. Most of the alkali is then used either in deproteinization or in deacetylation. Excess water is removed in screw press and the wet chitosan cake is either sun dries or in drier at 60°C. Chitosan thus obtained is in the form of flakes and can be pulverized to powder. The flowchart for the manufacture of chitosan from the starting material (crustacean shells) is shown in Fig. 19.5. 

One of the most studied biosorbent is chitin, which is an abundant biopolymer found in crustaceans, insects and fungus. This biopolymer is commercially purified by alkaline deproteinization, acid demineralization and decoloration by organic solvents of crustaceans wastes (Pastor, 2004). An additional stronger alkaline treatment of chitin produces deacetylated chitin. If the acetylation degree (DA) decreases at 39% or less, the biopolymer is named chitosan. Hence, the DA of chitin is variable and depends on the process conditions (alkali concentration, contact time, temperature, etc.), which produces DA values from 100 to 0%. Because of this, chitin is known as the biopolymer which has a DA from 100 to 40% likewise, when the chitinous biopolymer has DA lower than 40%, the biopolymer is named chitosan. Chitosan is, therefore, a biopolymer with structure very similar to that of chitin (see Figure 2) however, chitosan solubility is much greater, especially in acid mediums. 

Chitin, Chitosan, Demineralization, Deproteinization, Deacetylation, Degree of acetylation. Metal ions complexation. Polyelectrolyte complex. Biomaterial, Rocculation, Antimicrobial activity... 

Chitin isolation processes are generally performed through the following consecutive steps raw material conditioning, protein extraction (deproteinization), removal of inorganic components (demineralization) and decolouration. This sequence is preferred if the isolated protein is to be used as food additive for livestock feeding. Otherwise, demineralization can be carried out first [10]. A brief account of these processes will be given below. A more detailed description of chitin isolation (and chitosan preparation) can be found elsewhere [5, 8, 11]. 

The processes and conditions for the extraction of chitin and chitosan from mushroom were nearly same in the methods of Crestini et al. (1996) and Pochanavanich and Suntomsuk (2002), and were different in Mario et al. (2008) and Yen and Mau (2007a). Crestini et al. (1996) and Pochanavanich and Snntornsuk (2002) used IM NaOH at 121°C for 0.25 h for deproteination and the chitosan was extracted from the collected alkaline insoluble material using 1% acetic acid at 95°C for 8-14h. Mario et al. (2008) used 1M NaOH at 40°C for 15-17h for deproteination and the chitosan was extracted from the collected alkaline insoluble material using 5% acetic acid at 90°C for 3 h. The total yield of chitin, i.e., 85-196 mg/g of dried mushroom and only a low yield of chitosan, 10-40 mg/g of dried mushroom were obtained from different species of mushrooms by both chitosan extraction procedures (Table 1.3). 

The isolation of chitin from shellfish waste consists of three steps deproteinization (DP), demineralization (DM), and decolorization (DC) whereby the order of the first two steps is generally considered irrelevant if protein or pigment recovery is not an objective (Shahidi and Synowiecki 1991). Chitin is further deacetylated (DA) to make chitosan or other products for a wide array of applications. Both chemical and enzymatic non-continuous batch methods are widely used on an industrial scale for the production of chitin, chitosan, and COS. 

Teng et al. (2001) reported that proteolytic fungal fermentation of shrimp shells is a simple, effective, and inexpensive approach of chitin production from shrimp shells and fnngal mycelia. The results suggest that deproteinization and demineralization occnrs nnder those conditions. Sini et al. (2007) reported that Bacillus subtilis is an efficient starter cnltme from fermentation of shrimp shells. About 84% of the protein and 72% of the minerals were ranoved from the fermented residne at the end of fermentation. 

In bacterial fermentation for chitin and chitosan production, the most often applied strains are Lactobacillus sp., Bacillus sp., Pseudomonas sp and S. marcescens. The microbial DP process is little efficient, ranging between 50% and 85% DP rate depending on materials, used microorganism, fermentation type, and time. Rao et al. (2000) cultured shrimp biowaste with L. plantarum and achieved 75% DP. Bautista et al. (2001) achieved 81.5% DP from crayfish using Lactobacillus pentosus 4023. Fermentation of crab shell wastes with 10% S. marcescens FS-3 inoculum resulted in DP of 84% and DM of 47% at 7 days culture (Jo et al. 2008). Squid pen for the preparation of P-chitin were deproteinized by 73% for 3 days with Bacillus sp. TKU004 (Wang et al. 2006). Also, the shrimp shells were deproteinized by 75% and 87% at 30°C for 6 days with Candida parapsilosis and Pseudomonas maltophilia, respectively (Chen 2001). 

Chen, H. C. 2001. Microbial deproteinization for chitin isolation. In T. Uragami, K. Kurita, and T. Fukamizo (eds.), Chitin and Chitosan, pp. 165-169. Kodansha Scientific Ltd. Tokyo, Japan. 

Jo, G. H., Jung, W. J., Kuk, J. H. et al. 2008. Screening of protease-producing Serratia marcescens FS-3 and its application to deproteinization of crab shell waste for chitin extraction. Carbohydr. Polym. 74 504—508. 

Waldeck, J., Daum, G., Bisping, B., and F. Meinhardt. 2006. Isolation and molecular characterization of chi-tinase-deficient Bacillus licheniformis strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin. Appl. Environ. Microbiol. 72 7879-7885. 

Wang, S. L., Kao, T. Y., Wang, C. L. et al. 2006. A solvent stable metalloprotease produced by Bacillus sp. TKU004 and its application in the deproteinization of squid pen for P-chitin preparation. Enzyme Microb. Technol. 39 724-731. 

Xu, Y, Gallert, C., and J. Winter. 2008. Chitin purification from shrimp wastes by microbial deproteination and decalcification. Appl. Microbiol. Biot. 19 69,1-691. 

The yield of chitin was 15-20%. In this case, the protein layer was unprotected because the deproteinization precedes the decalcification. That is why during demineralization more hydrolysis and loss of material in the solid chitin fraction occurred... 

Decalcification first with 4% HCl for either 2 or 12 h, gave colored matter and the protein still remained bound to the solid matrix The yield of chitin was 20-27%, which is attributed to the strong adherence of chitin to protein, leading to less hydrolysis of the backbone because the decalcification precedes the deproteinization... 

Source of chitin Demineralization process Deproteinization process References... 

Mahmoud NS, Ghaly AE, Arab F (2007) Unconventional approach for demineralization of deproteinized crustacean shells for chitin production. Am J Biochem Biotechnol 3 1-9... 

Chitin and chitosan rarely occur in a pure, easily isolated form. A substantial effort has been made to develop chemical, mechanical, and enzymatic methods to obtain purified materials (25). The usual method of obtaining chitin involves the chemical treatment of shell fish wastes from the crab and shrimp industries. The first step is to demineralize the shell with dilute hydrochloric acid at room temperature. This is followed with a deproteinization step with warm dilute caustic. This yields a partially deacetylated chitin, which may then be further deacetylated to chitosan. Figure 3 shows the underlying chitin matrix in the crab shell and its microfibrillar... 

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