Extraction of chitin and chitosan from shell and operculum of ...

International Journal of Medicine and Medical Sciences Vol 1(5) pp.198-205, May, 2009 Available online http://www.academicjournals.org/ijmms 2009 Academic Journals

Full Length Research paper

Extraction of chitin and chitosan from shell and operculum of mangrove gastropod Nerita (Dostia) crepidularia Lamarck Chendur Palpandi*, Vairamani Shanmugam and Annaian Shanmugam Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai – 608 502, India. Accepted 21 April, 2009

Today, several companies are producing chitin and chitosan products on a commercial scale; the majority are located in Japan, where more than 100 billion tons of chitosan are manufactured each year from the shells of crabs and shrimps, an amount that account for ~90% of the global chitosan market (approximately four trillion yen). Keeping the importance of chitin and chitosan in mind, an attempt has been made on the extraction of chitin from the shell and operculum of Nerita crepidularia and conversion of chitin into chitosan through deactylation. The chitin was prepared from shell and operculum of N. crepidularia (by demineralization and deprotenization) and chitosan by the deacetylation of chitin. The yield of chitin and chitosan was found to be 23.91 and 35.43% and 31.14 and 44.29%. The FT – IR spectrum of chitin and chitosan was also confirming the presence of chitin and chitosan in the shell and operculum of N. crepidularia. The results of the present study pave the way and provide the baseline information for the utilization of chitin and chitosan in the development of drugs, “artificial skin” as in Japan and in food industries apart from opening in avenue of research to the future researchers. Key words: Shell, operculum, chitin, chitosan, FT – IR. INTRODUCTION Chitin is one of the most abundant natural polysaccharides produced by many living organisms; it is usually found as a component of crustacean shell. This polymer consists of a linear chain of poly (β1→ 4) – N – acetyl D – glucosamine), first identified in 1884 (Rinaudo, 2006). It is a nitrogen containing polysaccharide, related chemically to the cellulose that forms a semitransparent horny substance. But this polymer is not soluble in usual solvents and for its use, chemical modification are performed (Patil and Satam, 2002). Three different polymers of chitin are found in nature. – Chitin, the most abundant in nature, has a structure of antiparallel chains and is found in the crabs, shrimp and lobsters; whereas – chitin found in squid has intrasheet hydrogen bonding by parallel chains. However, – chitin has a mixture of parallel and antiparallel chains, which is a combination of – and – chitin (Jang et al., 2004).

*Corresponding author. E-mail: [email protected]. Tel.: 04144 – 243223, 09944224044.

Chitin and chitosan are nontoxic and biodegradable. Diabetic ulcers were also showed immediate response to chitosan. It is potentially useful for applications in the medicine, pharmacy and agriculture and also as biosorbent materials forth uptake of metal ions from polluted water as well as for analytical application (Sahiwa and Aiba, 2004; Rashidova et al., 2004; Batista - Banos et al., 2006). The chitosan scavenging action for chloride ions also prevents blood pressure elevation via control of the angiotension converting enzymes (Okuda, 1995). Hence, present study is to clarify and compare the physico – chemical properties of chitin and chitosan prepared from shell and operculum of Nerita crepidularia. MATERIALS AND METHODS N. crepidularia specimens were collected from Parangipettai (Lat. N 11º 29.45’ Long. 79º 46.028’) mangrove areas and the shell and operculum were removed from the animal. They were washed, dried and pulverized with pestle and mortar into fine powder and used for further studies. Chitin was extracted from the shell and operculum following the method of Takiguchi (1991a) by demineralising and deproteinising the powder. Chitosan was extracted from

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Table1. Wave length of the main bands obtained for the standard chitin and -chitin extracted from N. crepidularia.

Vibration modes OH out – of – plane bending NH out – of – plane bending Ring stretching CH3 wagging along chain CO stretching CO stretching Asymmetric in – phase ring stretching mode CH2 bending and CH3 deformation Amide II band Amide I band CH stretching Symmetric CH3 stretching and asymmetric CH2 stretching NH stretching OH stretching

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– chitin (cm ) 690 752 896 952 1026 1073 1116 1418 1563 1661 2878 2930 3268 3439

Shell 699 713 854 1083 1478 1788 2853 2923 3395

Operculum 699 712 861 908 1082 1483 1788 2853 2921 3401

Figure 1. Showing FT – IR spectrum of standard chitin.

the chitin through deacetylation process following the method of Takiguchi (1991b). FT- IR spectroscopy of solid samples of N. crepidularia relied on a Bio-Rad FTIS - 40 model USA. Sample (10 g) was mixed with 100 g of dried Potassium Bromide (KBr) and compressed to prepare a salt disc (10 mm diameter) for reading the spectrum further.

RESULTS Chitin The yield of chitin from the shell and operculum of N. crepidularia was found to be 23.91 and 35.43% (Figures 1 to 3 and Table 1).

Chitosan The yield of chitosan from the shell and operculum of N. crepidularia was found to 31.14 and 44.29% (Figures 4 to 6 and Table 2). FT – IR spectral analysis Chitin The FT – IR spectrum of the standard chitin contains 14 major peaks (Figure); whereas the FT – IR spectrum of the chitin from the shell and operculum of N. crepidularia were obtained and the effective peaks were compared

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Table 2. Wave length of the main bands obtained for the standard chitosan and chitosan extracted from N. crepidularia.

Vibration mode 2HPO4 (NH) Amide III -3 PO4 4 PO3 OH group (monomer) (-NH2) Amide II Structural unit

Chitosan 891.41 897.41 1026.63 1259.54 1422.73 1587.94 3377.95

Shell 856 896 1032 1264 1408 3377

Operculum 893 893 1021 1255 1420 1581 3404

Figure 2. Showing FT – IR spectrum of chitin (shell) from N. crepidularia.

with that of the standard chitin. The wave lengths of the main bands observed in the infrared of the alpha – chitin, in the present study, is depicted in Figures 1 to 3 and Table 1. Chitosan The FT – IR spectrum of the standard chitosan showed seven major peaks at the ranges of 893, 893, 1021, -1 1255, 1420, 1581 and 3404 cm ; where as the chitosan

sample (shell and operculum) from N. crepidularia also showed seven major peaks (Figures 4 to 6 and Table 2). DISCUSSION Poulicek (2003) has been studied the operculi of 114 species of gastropods representing 38 families have been examined for the presence of chitin using a quantitative enzymatic method along with qualitative tests. All the species having calcified operculi so far analysed (28

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Figure 3. Showing FT – IR spectrum of chitin (operculum) from N. crepidularia.

Figure 4. Showing the FT – IR spectrum of standard chitosan.

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Figure 5. Showing FT – IR spectrum of chitosan (shell) from N. crepidularia.

species) were relatively rich in chitin and neighbouring species had a similar composition. No chitin was found in the non-calcified, corneous operculi (86 species). The level of calcification was the unique character found to be correlated to the presence of chitin in the operculum; no other morphological parameters showed any reciprocity. Within the calcified operculi, chitin is restricted to the mineralized matrix. The tanned corneous sheet seems completely devoid of chitin. The possibility of a direct relation between calcification processes and the presence of chitin in the mineralizing matrix of some molluscan hard structures is discussed. Peters (1971) has studied the chitin in the radulae of ten species of the following groups: Placophora (three), Gastropoda (five), Scaphopoda (one) and Cephalopoda (one). Tolaimate et al. (2003) reported that the more concentrated acid solution and larger reaction times would not show any improvement of the efficiency of the demineralization reaction. The persistence of minerals during demineralization reaction would be related not only to concentration and duration effects (in relation with an optimal swelling of chitin) but also to the process, either continuous or performed by stages. In the present study, the colour of the chitin from N. crepidularia shell was pure white and operculum was pink It was supported in the earlier study of Tolaimate et al.

(2003) that crustaceans colour was slightly pink. Cuttlebone of Salvia officinalis was found to contain 24% of chitin (Tolaimate et al., 2000, 2003), whereas in general the squid/octopus reported 3 – 20% of chitin (Patil and Satam, 2002). Comparatively in the present investtigation, the yield of chitin from shell and operculum of N. crepidularia was higher (23.91 and 35.43%) to the above. But in certain other species of squids, the chitin yield was comparatively higher than that of the present study. 36 to 37% of – chitin content was about 40% of the original weight of the dried pens of L. vulgaris (Tolaimate et al., 2000, 2003) and 40 to 42% of chitin was extracted from the pens of L. sanpaulensis and L. pei (Lavall et al., 2004). Nair and Madavan (1989) and Das et al. (1996) demonstrated that the crab, S. serrata and P. pelagicus had 16.07 and 20.19% of chitin from the body shell waste. Whereas Faster and Hachman (1987) and Broussignae (1968) isolated very low percentage (0.5 and 20%) of chitin from the cuticle and body shell waste, respectively. But in the present study, the yield of chitin from the shell and operculum of N. crepidularia was higher (23.91%) shell and 35.43% (Operculum). Subasinghe (1999) noticed more chitin yield in snow crab legs (32%) than claws (24%). Further Das et al. (1996) also reported the same trend in the other portu-

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Figure 6. Showing the FT –IR spectrum of chitosan (operculum) from N. crepidularia.

nids crab (S. serrata and P. pelagicus). They reported that the yield of chitin was more in the shell of legs (16.07 and 20.19%) than in body (11.6 and 13.51%) and claw (10.42 and 11.66%). Thus the different raw materials vary in biochemical composition in terms of the quantity of chitin and non – chitinous fraction. Besides this, the non – chitinous materials affect the properties of chitin by interacting with it (Skaugrud and Sargent, 1990). The proportion of chitin and the non – chitinous fractions varies with species and with season (Green and Martrick, 1979) apart from different body parts (Thirunavukkarasu, 2005). The usefulness of chitosan as a wound healing accelerator and its effectiveness in protecting wound from bacterial invasion by suppressing bacterial proliferation and its effectiveness against typhoid producing microorganisms was well studied by Yadav and Bhise (2004). Different raw materials vary in biochemical composition in terms of the quantity of chitin and non – chitinous fractions. Besides this, the non – chitinous materials affect the properties of chitin by interacting with it (Skaugrud and Sargent, 1990). The proportion of chitin and the non – chitinous fractions varies with species and with season (Green and Martrick, 1979) apart from different body parts (Thirunavukkarasu, 2005). In the different body parts of the exoskeleton of portunid crab, Scylla tranquebarica such as carapace, claw

and legs, the chitosan yield was varying, that is, 6.59, 4.12 and 8.42%, respectively (Thirunavukkarasu, 2005). Varghese (2002) reported a chitosan yield of 4.4 to 8.3% in different size groups of mantis shrimp H. nelanoura. Sosa et al. (1991) reported that the N – carboxymethylchitosan N, O – sulfate, a heparin-like polysaccharide derived from N-carboxymethyl chitosan by a random sulfation reaction, was also shown to inhibit HIV – 1 replication and viral binding with CD4. Gentili et al. (2006) observed that inoculants produced with chitin and chitosan flake as a carrier material allows the development of biofilm, providing a protective niche to the bacterial strain and resulting in a long shelf life and in a high crude oil degrading activity in natural seawater. The properties of chitosan depend on various intrinsic parameters such as percentage of degree of deacetylation (DDA) and molecular weight and thereby on the uses of chitosan (Domand, 1988; Rinaudo and Domard, 1988). The quality parameters of chitosan vary from procedure to consumer. At present a standard set of quality parameters for chitin and chitosan is lacking (Hirano, 1988; Cho et al., 1998) and No et al. (2000) analysed these qualities for different commercially available chitins and chitosans and found that they differed with the products. Besides this, numerous methods are being used for the estimation of the same parameters leading to discrepancies (Rinau-


do and Domard, 1988). Hence there exists a need for standard analytical parameters and methodologies that are recognized by all. In the study of IR spectra of N – acetyl – D – glucoseamine and D – glucosamine Brugnerotto et al. (2001) reported the repeating units in these polymers; comparing both spectra, it could be appreciated that a specific band -1 appears at 1320 cm for N – acetylglucosamine. The -1 band located at 2900 cm often used in the literature as reference band to analyze chitin and chitosan, must be excluded as, for glucosamine, it may not be distinguished from the background. As reference peak Brugnerotto et al. (2001) evaluated two possibilities, either the large -1 band centered at 3350 cm (very near to that at 3450 cm 1 -1 chosen for polymers) or the 1420 cm band, which also seems to be suitable from the comparison of the two monomers. The amide I band shows a well defined peak at 1650 -1 -1 cm with a minor shoulder at 1625 and 3450 cm (cor-1 responding to that located at 3350 cm for the structural -1 units) as the reference one and the band at 1320 cm characteristics of –OH, -NH2, - CO groups were chosen to measure the extent of N – acetylation (Brugnerotto et al., 2001). The stretching band of the OH groups involved in hy-1 drogen bonds 0.3 – H…0-5 occurs at 3440 cm (Pearson et al., 1960; Focher et al., 1992). The C=O stretching re-1 gion of the amide moiety between 1600 and 1500 cm for -1 – chitin, the amide I band is split at 1656 and 1621 cm for – chitin. In contrast, the amide band is unique in -1 both chitin allomorphs at 1556 cm for – chitin and -1 -1 1560 cm – chitin. The band at 1656 cm , which occurs at similar wavelength in polyamides and proteins, is commonly assigned to stretching of the C=O group hydrogen bonded to N – H of the neighboring intra sheet chain (Focher et al., 1992). Parasakthi (2004) observed the FT – IR peaks at -1 534.61, 1024.16, 1321.88, 1380.81 and 1640.40 cm in chitin from the shell of S. aculeata which resembles the peaks of crab carapace, legs and the claw. Whereas in the chitin sample of N. crepidularia (shell and operculum) the peaks at 699, 713, 854, 1083, 1478, 1788, 2853, 2923, 3395 and 699, 712, 854, 908, 1082, 1483, 1788, -1 2853, 2921 and 3401 cm were also coincided with that of the shell and operculum samples confirming the presence of chitin. In the study of chitosan and chitin from the carapace, shell of claw and legs in S. tranquebarica. Saraswathy et al. (2001) observed the major absorption band between -1 1220 and 1020 cm which represents the free amino group (-NH2) at C2 position of glucosamine, a major group present in chitosan. Further the sample showed the absorption bands for the free amino group between -1 1023.15 and 1259.99 cm (carapace), 1022.58 and -1 -1 1260.30 cm (claw) and 1022.41 and 1260.20 cm (leg) -1 when the peak at 1384 cm represents the –C-O stretching of primary alcoholic group (-CH2 - OH). The primary

alcoholic group was represented by a band in 1378.54 -1 (carapace), 1380.14 (claw) and 1379.92 cm (legs), respectively. The absorbance bands of 3450, 2878, 1420, 1655 and -1 1320 cm indicated the hydroxyl stretching, C–H stretching, C–H deformations, C=O stretching in secondary amide (amide I) and C–N– stretching in secondary amide (amide III), respectively (de Velde and Kiekens, 2004). In the present study also the same absorbance bands were observed at 3377, 1581, 1420, 1255, 1032, 896 and 893 cm-1. A more detected structure in the 3500 – 3000 cm-1 region was observed for – chitin compared to – chitin (Focher et al., 1992). In the present investigation also the structural stretching band was observed at the range of 3470 cm-1. Chitosan is the only pseudo natural cationic polymer and thus, it finds many applications that follow from its unique character (flocculants for protein recovery, depollution, etc.). Being soluble in aqueous solutions, it is largely used in different applications as solutions, gels or films and fibers. Byun et al. (2005) found that chitosan and their oligomers are capable of inhibiting – secratase activity and this activity depends on their molecular weight and degree of deacetylation. – secratase enzyme plays a major role in the occurrence of Alzheimer’s disease and chitosan oligomers are the first reported carbohydrate – secratase inhibitors. The characterization of a chitosan sample requires the determination of its average DA. When the degree of deacetylation of chitin is about 50% (depending on the origin of the polymer), it becomes soluble in aqueous acidic media and is called chitosan. The solubilization occurs by protonation of the –NH2 function on the C-2 position of the D-glucosamine repeat unit, whereby the polysaccharide is converted to a polyelectrolyte in acidic media (Rinaudo et al., 2006). Conclusion In the present study, the – chitin has been extracted from shell and operculum of N. crepidularia. Then chitosan was isolated from chitin with good yield. The results of the present study pave the way and provide the base-line information for the utilization of chitin and chitosan in the development of drugs, “artificial skin” as in Japan and in food industries apart from opening in avenue of research to the future researchers. In India further collaboration with the industries and other pharmaceutical companies may be sought to develop many products by using the chitin and chitosan from the shell and operculum of N. crepidularia which is being thrown as waste in the processing industries. So the shell and operculum could be wisely utilized to obtain useful products. ACKNOWLEDGEMENTS The authors are thankful to the Director, CAS in Marine Biology and authorities of Annamalai University for provi-

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