Chitin: Applications, Composition and Properties

Chitin Applications, Composition and Properties1. IntroductionChitin, poly (b-(1-4)-N-acetyl-D-glucosamine), is a natural polysaccharide of major importance. It was first ascertained by Braconnot (1811), a professor of natural history. He isolated chitin from mushrooms by treating it with warm alkali. Later Odier (1823)found chitin while subject expanseing beetle cuticles and named chitin after classical word chiton (tunic, envelope). The silk worm was in addition discovered as a source of chitin when Lassaigne (1843) isolated it from the Bombyx mori. The monomeric unit of chitin (N-acetyl glucosamine) became known because of the work of Ledderhose in 1878. In the first half of the twentieth century, research on chitin was mostly directed toward the study of its occurrence in living organisms. Finally in 1981 Austin and his coworkers came up with a completed data on the sources of chitin which is widely distributed in marine invertebpaces (Figure 1), insects, fungi, and yeast (19 81). However, chitin is not present in higher plants and higher animals. Generally, the shell of selected crustaceous was reported by Knorr (1984) to consist of 30-40% protein, 30-50% calcium carbonate and calcium phosphate, and 20-30% chitin. Chitin is widely available from a variety of source among which, the principal source is shellfish emaciate such as shrimps, crabs, and crawfish (Allan et al., 1979). It to a fault exists naturally in a few species of fungi.Chitin occurs in nature as ordered crystalline microfibrils orchestrateing structural components in the exoskeleton of arthropods or in the carrell walls of fungi and yeast. It is also scramd by a number of other living organisms in the lower plant and animal kingdoms, overhaul in many functions where reinforcement and strength be required. (Rinaudo, 2006). The structure of chitin has been described (Fig. 1). In terms of its structure, chitin is associated with proteins and, therefore, high in protein contents. Chitin fibrils are embedded in a matrix of calcium carbonate and protein. The matrix is proteinaceous, where the protein is hardened by a tanning process (Muzzarrelli, 1977). Studies of Ashford et al., (1977) demonstrated that chitin represents 14-27% and 13-15% of the dry weight of shrimp and crab process devours, respectively.2.2. Characteristics and Structure of ChitinChitin is made up of extremely extended hydrogen bonded chain and is semi-crystalline in structure of chitin Rinaudo (2006) Kurita (2001). Chitin is a structural biopolymer, which has a role analogous to that of collagen in the higher animals and cellulose in terrestrial plants Muzzarelli, (1977) Mayer, (1996). Plants produce cellulose in their cell walls and insects and crustaceans produce chitin in their shells (Muzzarelli, 1986). Cellulose and chitin are, thereof, two important and structurally related polysaccharides that provide structural honor and protection to plants and animals, respectively Muzzarelli (198 6) and Roberts (1992). Chitin occurs in nature as ordered crystalline microfibrils forming structural components in the exoskeleton of arthropods or in the cell walls of fungi and yeast (Raabe 2007). In crustaceans, Chitin polymers tend to form rod like fibrils or crystallites that are balanced by hydrogen bonds formed between the amine and carbonyl pigeonholings.X-ray diffraction synopsis suggests that chitin is a polymorphic substance that occurs in three different crystalline modifications, termed , and chitin. They mainly differ in the degree of hydration, in the size of it of the unit cell and in the number of chitin handcuffs per unit cell Rudall and Kenchington, (1973) Kramer and Koga, (1986). In the form, all chains exhibit an anti-parallel orientation in the form the chains are ordered in a parallel manner in the form sets of two parallel strands alternate with single anti-parallel strands. Chitin is found to occur as fibrous natural embedded in a six stranded pro tein helix http//meyersgroup.ucsd.edu, 2006. The polymorphic forms of chitin differ in the packing and polarities of adjacent chains in successive sheets in the termed form, all chains are aligned in a parallel manner, which is not the case in form and chitin. The molecular order of chitin depends on the physiological role and tissue characteristics. In both structures, the chitin chains are organized in sheets where they are tightly held by a number of intra-sheet hydrogen bonds with the - and chains packed in antiparallel ar personaments Rinaudo. (2008).2.3. Bio humiliation of tiger prawn shell by Lactic acid hullabaloo for extraction of ChitinEvery year tones of sea food for thought waste is dumped onto the shores of the sea and lagoons or in the inner mangrove area surrounding the sea for these are the regions where maximum sea food cultivation is done. These areas are the hub of number of small and large scale seafood industries which deal with culturing and processing o f seafood. This huge amount of sea food waste is polluting the surrounding land and water and is depleting the new-fashioned water supply. Dumping of Seafood waste leads to accumulation of sediments ca utilise organic pollution which causes physical disturbance of hydrological regimes resulting in a number of ecological problems which include change and degradation of costal ecosystem. ( Mathew and Nair, 2006)The demineralization of crustacean shells have been chemically performed using concentrated acids such as HCl (Whistler et al., 1962), H2SO4 (Peniston and Johnson, 1978), CH3COOH (Bautisa et al., 2000) and HCOOH (Horowitz et al., 1957) by various researchers. However, the chemical manners are high-priced and detrimental to the environment leading to effluent problems Shirai (2001) and Fagberno (1996). The Traditional method of chitin preparation from crustacean waste involving the use of alkalis and acids for demineralization, make the method ecologically harsh and a cause of pollution (Rao et al., 2000)It also reduces the chitin quality to certain extent (Simpson et al. 1994 Healy et al., 1994) mostly such processes depolymerising chitin to a higher extent leading to the formation of a deacetylated form of chitin called chitosan.Biotechnological process of lactic acid fermentation of crustacean shell waste is a powerful tool to overcome the environmental problems. Fermentation of crustacean shells using lactic acid bacteria is also an attractive method which lowers the pH of the medium and facilitates the demineralization of minerals and the hydrolysis of proteins while leaving the associated chitin intact. Thus this process also helps in a safe recovery of chitin as the fermented residue. Also, fermentation of crustacean bio waste to recover chitin considerably replaces the expensive and non environmentally friendly chemical process Rao et al., (2000), Shirai et al., (2001) and anteroom et al., (1992) .Lactic acid bacterial fermentation of shrimp waste for chitin recovery was study with lactose or cassava extract as additional sources of clams for natural energy (Hall and Silva 1992). Raw heads of Africa river prawn were fermented with Lactobacillus plantarum using cane molasses (Fagbenro 1996). Treatment of minced waste of scampi in the presence of glucose by a culture of Lactobacillus paracasei striving A3 was investigated (Zakaria et al. 1998). The primary object of all these studies was demineralization of the raw materials on with which deproteinisation took place (Shirai et al. 2001). The effectiveness of demineralization was exaggerated by the increasing inoculum amounts supplied. Also, the proportion of glucose was fundamental for the lactic acid fermentation by the bacterial strain to demineralize the shell wastes (Shirai et al. 2001 and Rao et al. 2002).The demineralized and deproteinized chitin has a light pink color due to the presence of astaxanthin pigment. When decolorise product is desired, this pigmen t can be eliminated by the decolorization using bleaching agents. The resulting chitin is insoluble in most organic solvents however, its deacetylated derivative chitosan is soluble in some acids. The subsequent conversion of chitin to chitosan is generally achieved by treatment with concentrated sodium hydroxide solution (40-50%) at 100C or higher for 30 legal proceeding to remove some or all of the acetyl groups from the polymer (No and Meyers, 1995).Lactic acid bacterial fermentation for demineralization has also been occasionally reported for shrimp waste (Shirai et al. 2001) crayfish exoskeleton (Bautista et al. 2001) and scampi waste (Zakaria et al. 1998). However, demineralization by lactic acid fermentation of tiger prawn shell waste along with the characterization of the resulting chitin has been less studied in relation to glucose concentration and inoculum amount. In the present work, we evaluated the demineralization of tiger prawn shell waste by lactic acid bacterial f ermentation with various concentrations of inoculum and glucose and characterize the fermented residue the chitin by powerful techniques such as X-Ray diffraction, FTIR, SEM and TGA.From the literature it is evident that the limitations of the chemical method for the degradation of sea food can be largely overcome by the biologic method of demineralization and hence research interest has been shown in new-fangled years in this direction. Lactic acid fermentation of crustaceans shell waste has been reported to be studied as a potential biological method of degradation (P Mathew and KGR. Nair, 2006)2.4. Factors Affecting Production of Chitin by Lactic Acid Fermentation2.4.1. Effect of Initial Glucose Concentration and Inoculation Level of Lactic Acid bacterium on Tiger Prawn Shell Waste FermentationAmount of starter culture and initial glucose concentration are critical parts in the fermentation of tiger prawn shell waste fermentation. A amend proportion of initial glucose and st arter culture concentrations increase the amount of lactic acid produced and thus increased the % demineralization. Glucose is a readily fermentable sugar and hence chosen as the source of carbon for the microbes in most of the studies. Glucose concentration is a highly important parameter of fermentation and hence chitin production. harmonise to Jung et al. (2004) Microbial growth and hence acidification of the broth during fermentation is highly dependent on glucose concentration.Lactobacillus sp. has the potential to produce lactic acid and other organic acids. Using organic acids such as lactic and/or acetic acids for the demineralization process is a bright idea since organic acids in order to produce low cost biomass, purified chitin and reduce the harmful to the environment (Jung et al., 2005,Rao et al., 2000, Sunita et al.,2009). According to Hong et al. (1999) the production of organic acids by the lactic acid bacterium L. plantarum decreased the pH and made the environme nt selective against spoilage microorganisms. Zakaria et al. (1998) had also reported that the decaying of the raw crustacean waste materials can be controlled through the selection of microorganisms having a high capacity to produce organic acids. Further Shirai et al. (2001) reported that the selection of the correct micro organism is an important factor for the acidification of crab shell waste and for suppressing the growth of spoilage organisms.Cira et al., (2002) reported that lactic acid bacteria fermentation with the 10% inoculums was helpful in attaining a pH of around pH 5 after day 3. On the other hand it was reported by Shirai et al. (2001) that lactic acid fermentation of shrimp wastes which contained 10% glucose and a 5% inoculum of Latobacillus sp. B2 lowered from to pH 4.5. Therefore medium pH likely depends on the content of the energy source such as glucose and sucrose and the other factor least considered but of great importance is the solid to liquid ratio. Lower the solid to liquid ratio higher is the rate of demineralization. As the solid concentration increases the concentration of slurry increases resulting in reduced mass transfer and hence poor demineralization occurs. (Kyung. et al., 2008). The selection of the potential microbe along with the correct proportion of the additional starter is very important for the lactic acid bacterial fermentation to demineralize the raw shell wastes (Shirai et al. 2001 Rao et al. 2002) along with the correct propotion of solid to liquid ratio (Kyung.et.al. 2008).2.4.2. Temperature of FermentationApplication of microorganisms or enzymes to extract chitin from marine crustacean wastes is a current research trend for bio-conversion of wastes into useful biomass (Bhaskar et al., 2006). From his study he analyzed that a temperature of 35C resulted in lowest pH conditions of pH 3.7 and highest % demineralization of about 92%. Kyung et al., (2008) reported that a temperature of 30C gave the highest % demin eralizatuion.2.4.3. Particle SizeParticle size in chitin productions has sparked controversial reports on its effect on chitin quality. Some agree that small particle size is better than large particle size. According to Bough et al. (1978), smaller particle size (1mm) results in higher demineralization % with a chitin product of both higher viscousness and molecular weight than that of larger particle size (above 2 to 6.4 mm). The larger particle sizes require longer swelling time resulting in a sluggish deacetylation rate.2.5. Process Optimization by TaguchiTaguchi method of production optimization is a purely statistical approach to analyze scientific data establish on statistical factorials. Taguchi experimental design offers remarkable advantages by examining a group of factors simultaneously and extracting as much quantitative information as can be extracted with a few experimental trials Stone and Veevers, (1994) and Houng et al., 2006. But yet only a few reports are availa ble on the application of Taguchis method in the field of biotechnology (Cobb and Clarkson, 1994 and Han et al., 1998).2.6. Characterization and Physiochemical study of Chitin2.6.1. X-Ray Diffraction AnalysisThe crystalline structures of chitin are differently presented according to the raw materials. XRD is low cost and user friendly method to accurately characterize the kind of chitin extracted from a particular species. Chitin has three different crystalline polymorphic forms according to the derived material chitin, chitin, and chitin. The structures of the and forms differ only in that the heaps of chains are arranged alternately antiparallel in chitin, whereas they are all parallel in chitin. The structures of chitin, chitin, Sugiyama et al., (1999) and Syed et al., 1999 have been determined by X-ray diffraction (XRD). According to the crystalline structure of chitin suggested by Rudall (1963) and (1967.) chitin has strong intersheet and intrasheet hydrogen bonding, and chitin chitin has weak hydrogen bonding by intrasheets. Therefore, in contrast to chitin, chitin is characterized by a weak intermolecular force, Lee et al., 1996. Not much information is available regarding the crystalline study of chitin by X ray diffraction technique. The XRD profiles of chitin samples easily help to distinguish the different forms of chitin based on the peaks and crystallinity. It has been found that chitin has three to four sharp crystalline reflections at 9.6, 19.6, 21.1, and 23.7 whereas chitin , has two broad crystalline reflections at 9.1 and 20.3 within the 2 range of 5-35. These results also support that the crystallinity of chitin is less than that of chitin because of the parallel structure. chitin has a more rigid crystalline structure because of its intersheets and intrasheets, and its structure exists as a stable structure with neither a crystalline phase transition nor thermal decomposition ( Jang et al., 2004).2.6.2. FTIR Spectrophotom eter MeasurementsDifferent methods have been used for the purpose of measuring the degree of deacetylation of chitin for eg. the linear potentiometric titration, ninhydrin test, hydrogen bromide titrimetry, near-infrared spectroscopy, thermonuclear magnetic resonance spectroscopy, infrared spectroscopy, and first derivative UV-spectrophotometry. Among all the tests stated above FTIR is one of the potential methods to determine the degree of deacetylation of the sample. It is far easier yet highly sensitive compared to the other processes. The process of removal of acetyl groups from the molecular chain of chitin is called deacetylation, it leaves behind a high degree chemical reactive amino group (-NH2). Thus the physicochemical properties of chitin highly depend on the degree of deacetylation (DD) hence it determines its appropriate applications. (Khan et al., 2002) Degree of deeacetylation also affects the biodegradability and immunological activity (Tolaimate et al., 2003). The degree of deacetylation can also be used to differentiate between chitin and chitosan because it helps to know the amount of free amino groups in the polysaccharides. A degree of deacetylation of 75% or above in Chitin is generally known as chitosan (Knaul et al., 1999).2.6.3. TGAThe thermal degradation of chitin or chitosan with a broad range of DD has received little attention (GuinesiCavalheiro, 2006 Kittur, Prashanth, Sankar, Tharanathan, 2002). There are less reports on the thermal degradation process of chitin/chitosan and its derivatives than on chemical and enzymatic degradation (De Britto Campana-Filho, 2004 Holme, Foros, Pettersen, Dornish, Smidsrod, 2001 Hong et al., 2007 Neto et al., 2005 Qu, Wirsen, Albertsson, 2000 Wanjin, Cunxin, Donghua,2005). Thus to examine the thermal degradation of chitin with a broad range of DD, thermogravimetric analysis (TGA) is a highly useful technique. It has also been reported that with an increase in the rate of deacetylation the t emperature of degradation decreases (Young et al., 2009).2.7. Application of ChitinChitin and chitosan has several distinctive biological properties, including biocompatibility and biodegradability, cellularbinding capability, acceleration of wound healing, hemostatic properties, and anti-bacterial properties (Cho, Cho, Chung, Yoo, Ko, 1999 Muzzarelli, 1993 Tomihata Ikada, 1997).Some of the important industrial applications of chitin have been listed below in Table 1.Different industrial applications of chitinWaste Water Treatment removal of metal ions, flocculant/coagulant, protein, dyeFood IndustryThickener and gelling agent, animal feed additive.MedicalWound and bone healing, blood cholesterol control, skin burn gardeningSeed Coat, Fertiliser, Controlled agrochemical release.CosmeticsMoisturizer, face, hand, and body creams, bath lotion, etcBiotechnologyEnzyme immobilization, protein separation, cell recovery.