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Chitin, poly ( b- ( 1-4 ) -N-acetyl-D-glucosamine ) , is a natural polyose of major importance. It was foremost discovered by Braconnot ( 1811 ) , a professor of natural history. He isolated chitin from mushrooms by handling it with warm base. Subsequently Odier ( 1823 ) A found chitin while analyzing beetling cuticles and named “ chitin ” after Grecian word “ chiton ” ( tunic, envelope ) . The silk worm was besides discovered as a beginning 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 20th century, research on chitin was largely directed toward the survey of its happening in life beings. Finally in 1981 Austin and his coworkers came up with a completed information on the beginnings of chitin which is widely distributed in Marine invertebrates ( Figure 1 ) , insects, Fungis, and barm ( 1981 ) . However, chitin is non present in higher workss and higher animate beings. By and large, the shell of selected crustacean was reported by Knorr ( 1984 ) to dwell of 30-40 % protein, 30-50 % Ca carbonate and Ca phosphate, and 20-30 % chitin. Chitin is widely available from a assortment of beginning among which, the chief beginning is shellfish waste such as runts, pediculosis pubis, and crawfish ( Allan et al. , 1979 ) . It besides exists of course in a few species of Fungi.

Chitin occurs in nature as ordered crystalline microfibrils organizing structural constituents in the exoskeleton of arthropods or in the cell walls of Fungis and barm. It is besides produced by a figure of other populating beings in the lower works and animate being lands, functioning in many maps where support and strength are required. ( Rinaudo, 2006 ) . The construction of chitin has been described ( Fig. 1 ) . In footings of its construction, chitin is associated with proteins and, hence, high in protein contents. Chitin filaments are embedded in a matrix of Ca carbonate and protein. The matrix is proteinaceous, where the protein is hardened by a tanning procedure ( Muzzarrelli, 1977 ) . Surveies of Ashford et al. , ( 1977 ) demonstrated that chitin represents 14-27 % and 13-15 % of the dry weight of runt and crab processing wastes, severally.

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Fig. 1. Seafood Produces Considerable Sums of Waste which are the Principal Source

of Chitin

2.2. Features and Structure of Chitin

Chitin is made up of extremely extended H bonded concatenation and is semi-crystalline in construction of chitin . Chitin is a structural biopolymer, which has a function correspondent to that of collagen in the higher animate beings and cellulose in tellurian workss . Plants produce cellulose in their cell walls and insects and crustaceans produce chitin in their shells ( Muzzarelli, 1986 ) . Cellulose and chitin are, therefore, two of import and structurally related polyoses that provide structural unity and protection to workss and animate beings, severally . Chitin occurs in nature as ordered crystalline microfibrils organizing structural constituents in the exoskeleton of arthropods or in the cell walls of Fungis and barm ( Raabe 2007 ) . In crustaceans, Chitin polymers tend to organize rod like filaments or crystallites that are balanced by H bonds formed between the aminoalkane and carbonyl groups.

X-ray diffraction analysis suggests that chitin is a polymorphous substance that occurs in three different crystalline alterations, termed I± , ? and I? chitin. They chiefly differ in the grade of hydration, in the size of the unit cell and in the figure of chitin ironss per unit cell . In the I± signifier, all ironss exhibit an anti-parallel orientation ; in the ? signifier the ironss are arranged in a parallel mode ; in the I? signifier sets of two parallel strands alternate with individual anti-parallel strands. Chitin is found to happen as hempen stuff embedded in a six stranded protein spiral . The polymorphous signifiers of chitin differ in the wadding and mutual oppositions of next ironss in consecutive sheets ; in the termed I± signifier, all ironss are aligned in a parallel mode, which is non the instance in ? signifier and I? chitin. The molecular order of chitin depends on the physiological function and tissue features. In both constructions, the chitin ironss are organized in sheets where they are tightly held by a figure of intra-sheet H bonds with the ?- and I? ironss packed in antiparallel agreements Rinaudo. ( 2008 ) .

Fig.2. Structure of Cellulose and Chitin

2.3. Biodegradation of tiger shrimp shell by Lactic acid agitation for extraction of Chitin

Every twelvemonth tones of sea nutrient waste is dumped onto the shores of the sea and lagunas or in the interior mangrove country environing the sea for these are the parts where maximal sea nutrient cultivation is done. These countries are the hub of figure of little and big graduated table seafood industries which deal with culturing and processing of seafood. This immense sum of sea nutrient waste is fouling the environing land and H2O and is consuming the fresh H2O supply. Dumping of Seafood waste leads to accretion of deposits doing organic pollution which causes physical perturbation of hydrological governments ensuing in a figure of ecological jobs which include transition and debasement of costal ecosystem. ( Mathew and Nair, 2006 )

The demineralisation of crustaceous shells have been chemically performed utilizing 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 assorted research workers. However, the chemical methods are expensive and damaging to the environment taking to effluent jobs . The Traditional method of chitin readying from crustaceous waste affecting the usage of bases and acids for demineralisation, make the method ecologically rough and a cause of pollution ( Rao et al. , 2000 )

It besides reduces the chitin quality to certain extent ( Simpson et al. 1994 ; Healy et al. , 1994 ) largely such procedures depolymerising chitin to a higher extent taking to the formation of a deacetylated signifier of chitin called chitosan.

Wet shell

a†“

Washing and drying

a†“

Crunching and screening

3.5 % NaOH ( w/v ) for 2 H at 65oC, solid: dissolver ( 1:10, w/v ) a†“

Deproteinization

a†“

Washing

1 N HCl for 30 min at room temp. , solid: dissolver ( 1:15, w/v ) a†“

Demineralization

a†“

Washing

a†“

Decoloration

a†“

Washing and Drying

a†“

Chitin

Fig. 3. Traditional chitin Production Flow Scheme ( No and Meyers, 1995 )

Biotechnological procedure of lactic acerb agitation of crustacean shell waste is a powerful tool to get the better of the environmental jobs. Agitation of crustaceous shells utilizing lactic acid bacterium is besides an attractive method which lowers the pH of the medium and facilitates the demineralisation of minerals and the hydrolysis of proteins while go forthing the associated chitin integral. Thus this procedure besides helps in a safe recovery of chitin as the fermented residue. Besides, agitation of crustaceous bio waste to retrieve chitin well replaces the expensive and non environmentally friendly chemical procedure .

Lactic acid bacterial agitation of shrimp waste for chitin recovery was studied with lactose or cassava infusion as extra beginnings of saccharide for natural energy ( Hall and Silva 1992 ) . Raw caputs of Africa river shrimp were fermented with Lactobacillus plantarum utilizing cane molasses ( Fagbenro 1996 ) . Treatment of minced waste of scampi in the presence of glucose by a civilization of Lactobacillus paracasei strain A3 was investigated ( Zakaria et al. 1998 ) . The primary object of all these surveies was demineralisation of the natural stuffs along with which deproteinisation took topographic point ( Shirai et al. 2001 ) . The effectivity of demineralisation was exaggerated by the increasing inoculant sums supplied. Besides, the proportion of glucose was important for the lactic acid agitation by the bacterial strain to demineralise the shell wastes ( Shirai et al. 2001 and Rao et Al. 2002 ) .

The demineralized and deproteinized chitin has a light pink colour due to the presence of astaxanthin pigment. When faded merchandise is desired, this pigment can be eliminated by the decolorization utilizing decoloring agents. The ensuing chitin is indissoluble in most organic dissolvers ; nevertheless, its deacetylated derivative chitosan is soluble in some acids. The subsequent transition of chitin to chitosan is by and large achieved by intervention with concentrated Na hydrated oxide solution ( 40-50 % ) at 100°C or higher for 30 proceedingss to take some or all of the ethanoyl group groups from the polymer ( No and Meyers, 1995 ) .

Lactic acid bacterial agitation for demineralisation has besides been on occasion reported for runt waste ( Shirai et al. 2001 ) crayfish exoskeleton ( Bautista et al. 2001 ) and scampi waste ( Zakaria et al. 1998 ) . However, demineralisation by lactic acerb agitation of tiger shrimp shell waste along with the word picture of the ensuing chitin has been less studied in relation to glucose concentration and inoculant sum. In the present work, we evaluated the demineralisation of tiger shrimp shell waste by lactic acid bacterial agitation with assorted concentrations of inoculant and glucose and qualify the fermented residue the chitin by powerful techniques such as X-Ray diffraction, FTIR, SEM and TGA.

From the literature it is apparent that the restrictions of the chemical method for the debasement of sea nutrient can be mostly overcome by the biological method of demineralisation and therefore research involvement has been shown in recent old ages in this way. Lactic acerb agitation of crustaceans shell waste has been reported to be studied as a possible biological method of debasement ( P Mathew and KGR. Nair, 2006 )

2.4. Factors Affecting Production of Chitin by Lactic Acid Fermentation

2.4.1. Consequence of Initial Glucose Concentration and Inoculation Level of Lactic Acid Bacteria on Tiger Prawn Shell Waste Fermentation

Sum of starter civilization and initial glucose concentration are critical factors in the agitation of tiger shrimp shell waste agitation. A right proportion of initial glucose and starter civilization concentrations increase the sum of lactic acid produced and therefore increased the % demineralisation. Glucose is a readily fermentable sugar and therefore chosen as the beginning of C for the bugs in most of the surveies. Glucose concentration is a extremely of import parametric quantity of agitation and hence chitin production. Harmonizing to Jung et Al. ( 2004 ) Microbial growing and hence acidification of the stock during agitation is extremely dependent on glucose concentration.

Lactobacillus sp. has the possible to bring forth lactic acid and other organic acids. Using organic acids such as lactic and/or acetic acids for the demineralisation procedure is a promising thought since organic acids in order to bring forth low cost biomass, purified chitin and cut down the harmful to the environment ( Jung et al. , 2005, Rao et al. , 2000, Sunita et al.,2009 ) . Harmonizing to Hong et Al. ( 1999 ) the production of organic acids by the lactic acid bacteria L. plantarum decreased the pH and made the environment selective against spoilage micro-organisms. Zakaria et Al. ( 1998 ) had besides reported that the decaying of the natural crustacean waste stuffs can be controlled through the choice of micro-organisms holding a high capacity to bring forth organic acids. Further Shirai et Al. ( 2001 ) reported that the choice of the right micro being is an of import factor for the acidification of crab shell waste and for stamp downing the growing of spoilage beings.

Cira et al. , ( 2002 ) reported that lactic acid bacteriums agitation with the 10 % inoculant was helpful in achieving a pH of around pH 5 after twenty-four hours 3. On the other manus it was reported by Shirai et Al. ( 2001 ) that lactic acerb agitation of runt wastes which contained 10 % glucose and a 5 % inoculant of Latobacillus sp. B2 lowered from to pH 4.5. Therefore medium pH likely depends on the content of the energy beginning such as glucose and saccharose 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 demineralisation. As the solid concentration increases the concentration of slurry additions ensuing in decreased mass transportation and therefore hapless demineralisation occurs. ( Kyung. et al. , 2008 ) . The choice of the possible bug along with the right proportion of the extra starting motor is really of import for the lactic acid bacterial agitation to demineralise the natural shell wastes ( Shirai et al. 2001 ; Rao et Al. 2002 ) along with the right propotion of solid to liquid ratio ( Kyung.et.al. 2008 ) .

2.4.2. Temperature of Agitation

Application of micro-organisms or enzymes to pull out chitin from marine crustaceous wastes is a current research tendency for bio-conversion of wastes into utile biomass ( Bhaskar et al. , 2006 ) . From his survey he analyzed that a temperature of 35A°C resulted in lowest pH conditions of pH 3.7 and highest % demineralisation of approximately 92 % . Kyung et al. , ( 2008 ) reported that a temperature of 30A°C gave the highest % demineralizatuion.

2.4.3. Atom Size

Particle size in chitin productions has sparked controversial studies on its consequence on chitin quality. Some agree that little atom size is better than big atom size. Harmonizing to Bough et Al. ( 1978 ) , smaller atom size ( 1mm ) consequences in higher demineralisation % with a chitin merchandise of both higher viscousness and molecular weight than that of larger atom size ( above 2 to 6.4 millimeter ) . The larger atom sizes require longer swelling clip ensuing in a slower deacetylation rate.

2.5. Procedure Optimization by Taguchi

Taguchi method of production optimisation is a strictly statistical attack to analyse scientific informations based on statistical factorials. Taguchi experimental design offers singular advantages by analyzing a group of factors at the same time and pull outing every bit much quantitative information as can be extracted with a few experimental tests . But yet merely a few studies are available on the application of Taguchi ‘s method in the field of biotechnology ( Cobb and Clarkson, 1994 and Han et al. , 1998 ) .

2.6. Word picture and Physiochemical survey of Chitin

2.6.1. X-Ray Diffraction Analysis

The crystalline constructions of chitin are otherwise presented harmonizing to the natural stuffs. XRD is low cost and user friendly method to accurately qualify the sort of chitin extracted from a peculiar species. Chitin has three different crystalline polymorphic signifiers harmonizing to the derived stuff I± chitin, I? chitin, and I? chitin. The constructions of the I± and I? signifiers differ merely in that the hemorrhoids of ironss are arranged alternately antiparallel in I± chitin, whereas they are all analogues in I? chitin. The constructions of I± chitin, I? chitin, Sugiyama et al. , ( 1999 ) and Syed et al. , 1999 ; have been determined by X-ray diffraction ( XRD ) . Harmonizing to the crystalline construction of chitin suggested by Rudall ( 1963 ) and ( 1967. ) I± chitin has strong intersheet and intrasheet H bonding, and I? chitin chitin has weak H bonding by intrasheets. Therefore, in contrast to I± chitin, I? chitin is characterized by a weak intermolecular force, Lee et al. , 1996. Not much information is available sing the crystalline survey of I? chitin by X beam diffraction technique. The XRD profiles of chitin samples easy help to separate the different signifiers of chitin based on the extremums and crystallinity. It has been found that I± chitin has three to four crisp crystalline contemplations at 9.6, 19.6, 21.1, and 23.7A° whereas I? chitin, has two wide crystalline contemplations at 9.1 and 20.3A° within the 2I? scope of 5-35A° . These consequences besides support that the crystallinity of I? chitin is less than that of I± chitin because of the parallel construction. I± chitin has a more stiff crystalline construction because of its intersheets and intrasheets, and its construction exists as a stable construction with neither a crystalline stage passage nor thermic decomposition ( Jang et al. , 2004 ) .

2.6.2. FTIR Spectrophotometer Measurements

Different methods have been used for the intent of mensurating the grade of deacetylation of chitin for eg. the additive potentiometric titration, ninhydrin trial, H bromide titrimetry, near-infrared spectrometry, atomic magnetic resonance spectrometry, infrared spectrometry, and first derivative UV-spectrophotometry. Among all the trials stated above FTIR is one of the possible methods to find the grade of deacetylation of the sample. It is far easier yet extremely sensitive compared to the other procedures. The procedure of remotion of acetyl groups from the molecular concatenation of chitin is called deacetylation, it leaves behind a high grade chemical reactive amino group ( -NH2 ) . Thus the physicochemical belongingss of chitin extremely depend on the grade of deacetylation ( DD ) hence it determines its appropriate applications. ( Khan et al. , 2002 ) Degree of deeacetylation besides affects the biodegradability and immunological activity ( Tolaimate et al. , 2003 ) . The grade of deacetylation can besides be used to distinguish between chitin and chitosan because it helps to cognize the sum of free amino groups in the polyoses. A grade of deacetylation of 75 % or above in Chitin is by and large known as chitosan ( Knaul et al. , 1999 ) .

Fig.4- Structure of Chitin and Chitosan

The undermentioned expression by Domszy and Roberts ( 1985 ) is extremely used for ciphering the grade of deacetylation.

DD = 100 –

A= % Absorption

2.6.3. TGA

The thermic debasement of chitin or chitosan with a wide scope of DD has received small attending ( Guinesi & A ; Cavalheiro, 2006 ; Kittur, Prashanth, Sankar, & A ; Tharanathan, 2002 ) . There are fewer studies on the thermic debasement procedure of chitin/chitosan and its derived functions than on chemical and enzymatic debasement ( De Britto & A ; Campana-Filho, 2004 ; Holme, Foros, Pettersen, Dornish, & A ; Smidsrod, 2001 ; Hong et al. , 2007 ; Neto et al. , 2005 ; Qu, Wirsen, & A ; Albertsson, 2000 ; Wanjin, Cunxin, & A ; Donghua,2005 ) . Therefore to analyze the thermic debasement of chitin with a wide scope of DD, thermohydrometric analysis ( TGA ) is a extremely utile technique. It has besides been reported that with an addition in the rate of deacetylation the temperature of debasement lessenings ( Young et al. , 2009 ) .

2.7. Application of Chitin

Chitin and chitosan has several typical biological belongingss, including biocompatibility and biodegradability, cellularbinding capableness, acceleration of lesion healing, styptic belongingss, and anti-bacterial belongingss ( Cho, Cho, Chung, Yoo, & A ; Ko, 1999 ; Muzzarelli, 1993 ; Tomihata & A ; Ikada, 1997 ) .Some of the of import industrial applications of chitin have been listed below in Table 1.

Table1. Different industrial applications of chitin

Waste Water Treatment

Removal of metal ions, flocculant/coagulant, protein, dye

Food Industry

Thickener and gelling agent, carnal provender additive.

Medical

Wound and bone healing, blood cholesterin control, skin burn

Agribusiness

Seed Coat, Fertiliser, Controlled agrochemical release.

Cosmetics

Moisturizer, face, manus, and organic structure picks, bath lotion, etc

Biotechnology

Enzyme immobilisation, protein separation, cell recovery.

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