1. INTRODUCTION:

Chitin (b-1, 4-poly-N-acetyl-D-glucosamine) is the second most common polymer after cellulose in nature, existing in the shells of crustaceans like crab, shrimp and lobster as well as in the exoskeleton of marine zooplankton, the cuticle of insects and the cell walls of fungicide. Chitosan (poly-b-1,4-2-amino-2-deoksi-b-D-glu-kopiranoz)is derived by deacetylation of chitin^1-3. Due to its biodegradability, biocompatibility, nontoxic and wound healing properties and haemostatic activity, chitosan has received increased attention as one of the promising renewable polymeric materials for various applications^4-5. Chitosan is a natural nontoxic biopolymer derived by deacetylation of chitin, a chief component of the shells of crustacea such as crab, shrimp, and craw ﬁsh. In recent years, applications of chitosan to the ﬁelds of chemical engineering, medicine, food, nutrition, pharmaceuticals, environmental protection and agriculture have received considerable attention^6-7. In aquaculture, chitosan is utilized as an immunostimulants to protect salmonids against bacterial disease (Brook trout (Salvelinus fontinalis) against Aeromonas salmonicida and Yersinia ruckeri ⁸, Rainbow trout (Oncorhynchus mykiss) against A. salmonicida and Vibrio anguillarum⁹.

The waste amount of the shell not used in the factories presents a big potential. This waste is normally likely to affect human health adversely as it could lead to environmental pollution. For this reason, the utilization of this source would not only be beneﬁcial to the industry, but to the community health as well.

The aim of the present study was to perform a characterization of the chitosan extracted from Paratelphusa hydrodromous shells with chemical methods. In the characterization of the chitosan, the yield, moisture and ash contents, degree of deacetylation, water and fat binding capacities were measured. In addition, evaluate the antibacterial activity of chitosan against Yersinia ruckeri fish pathogenic bacteria was investigated in this study.

2. MATERIALS AND METHOD:

2.1. Collection of Crab raw materials:

The fresh water crabs (Paratelphusa hydrodromous) were collected from Bhavani River in Erode district were washed and dried under sun for a day. The specimens were washed repeatedly in Fresh water to remove all the dirt and sand. They were then taken to the laboratory the viscera and tissues were removed. The exoskeleton of the crustaceans were thoroughly washed with running tap water to remove sand adhered to it, and placed in hot air oven at 60⁰C for 24 hours. The exoskeleton were subjected to shade dry for 2 days and then placed in hot air oven at 60⁰C for 24 hours. The samples were weighed separately from the raw carapace samples, which was packed in polythene covers and kept in airtight containers.

2.2. Extraction of chitosan:

The shells contain approximately 30-40% protein, 30-50% calcium carbonate, and 20-30% chitin on dry basis¹⁰. These portions vary with crustacean species and seasons^11-12. Isolation of chitosan from crab shell wastes involves three steps demineralization (DM), deproteinization (DP), and deacetylation (DA).

2.2.1 Deproteinization:

The shells were deproteinized with 5% NaOH at the ratio of shell to solution of 1:10 (w/v) at 120-130^oC for 3hrs. The deproteinized shells were filtered and washed with tap water until NaOH was washed off completely, then dried overnight in a hot air oven at 55-60^oC.

2.2.2 Demineralization:

The deproteinized shells were demineralized by continuously agitating with 5% HCl at the ratio of 1:10 (w/v, shell to solution) overnight at room temperature.

2.2.3 Deacetylation:

The shells were filtered and washed with tap water until neutral. Then deacetylation of chitosan was carried out by hydrolyzing with 47% NaOH at the ratio of 1:20 (w/v, chitin to solvent) at 120-130^oC for an hour. This product was washed and dried overnight at 55- 60^oC¹².

2.3. Characterization of chitosan:

2.3.1 Yield

The chitosan yield was calculated by comparing the weight measurements of the raw material to the chitosan obtained after treatment.

2.3.2 Moisture Content:

Moisture content of the chitosan was determined by the gravimetric method¹³. The water mass was determined by drying the sample to constant weight and measuring the sample after and before drying. The water mass (or weight) was the difference between the weights of the wet and oven dry samples. Procedures were as follows: weighed and recorded weight of aluminium dish, placed 1.0g of chitosan sample in duplicates in the metal aluminium dish, recorded weight of dish with sample, then placed the sample with the lid (filter paper to prevent or minimize contamination) in the oven. The temperature adjusted in oven to 60^oC, and dried the samples for 24 hrs was taken the sample from the oven and placed it in a desiccator until it cools to room temperature. The sample was weighted.

Calculated moisture content as:

(wet weight, g - dry weight, g) x 100

% Of moisture ________________________________

content = (Wet weight, g)

2.3.3 Water Binding Capacity (WBC):

WBC of chitosan was measured using a modified method of Wang and Kinsella¹⁴. WBC was initially carried out by weighing a centrifuge tube containing 0.5 g of sample and adding 10 ml of water, mixing on a vortex mixer for 1 min to disperse the sample. The sample contents were left at ambient temperature for 30 min with intermittent shaking for 5 s every 10 min and centrifuged (at 3,500 rpm (6,000 x g) for 25 min. The supernatant was decanted and the tube was weighed again.

WBC was calculated as follows:

WBC (%) = [water bound (g)/ initial sample weight (g)] x 100.

2.3.4 Fat Binding Capacity (FBC):

FBC of chitosan was measured using a modified method of Wang and Kinsella¹⁴. FBC was initially carried out by weighing a centrifuge tube containing 0.5 g of sample, adding 10 ml of olive oil was added mixed with a vortex mixer for 1 min. The contents were left at ambient temperature for 30 min with shaking for 5 seconds every 10 min and centrifuged for 25 min. After the supernatant was decanted, the tube was weighed again. FBC was calculated as follows:

FBC (%) = [fat bound (g)/ initial sample weight (g)] x 100.

2.3.5 Fourier Transform Infrared spectroscopy (FTIR):

In order to detect the structural changes incurred in the chitosan samples after chemical treatment with sodium nitrite or acetic anhydride, the FTIR spectra of the chitosan sample were acquired before and after treatment using a Fourier transform infrared spectrophotometer. The lyophilized powders were analyzed using the KBr method. Two mg of chitosan powder was mixed with 198 mg of KBr, and pressed into a pellet under a pressure of 13 tons for 10 min.

The FTIR spectra were measured in KBr pellets in the transmission mode in the range 400–4000 cm^-1. The DA was calculated ratio of A1655 and A3450 are the absorbance of bands at 1655 and 3450 cm-1 respectively.

2.3.6 Solubility:

Chitosan powder (0.1 g in triplicate) were placed into a centrifuge tube (known weight) then dissolved with 10 ml of 1% acetic acid for 30 min using an incubator shaker operating at 240 rpm and 25^oC. The solution was then immersed in a boiling water bath for 10 minutes, cooled to room temperature (25^oC) and centrifuged at 10,000 rpm for 10 min. The supernatant was discarded. The undissolved particles were washed in distilled water (25ml) then centrifuged at10, 000 rpm. The supernatant was removed and undissolved pellets were dried at 60^oC for 24hr. Finally, weighed the particles and determined the percentage solubility. Solubility (%) calculated as:

(Initial weight of

tube + chitosan)

(Final weight of tube + chitosan)

X 100

(Initial weight of

tube + chitosan)

(Initial weight of tube)

2.4. Antibacterial assay:

Antibacterial activity was measured following the method of Zheng & Zhu¹⁵with slight modification. Nutrient agar plates were prepared for culture of Yersinia ruckeri bacteria. 100 μl bacterial suspensions were spread on the plates followed by 100 μl of chitosan preparation in 5% acetic acid (pH 5.5 to 6.0). Controls were identical except that 100 μl of acetic acid solution (pH 5.5 to6.0) replaced the chitosan solution. All plates were incubated at 28 ± 1°C for 24 h before the total number of colonies was enumerated. Inhibition rate (η) was calculated using the equation

η (%) = N₁- N₂×100

N₁

Where N1 and N2 are the amount of colonies developed on the control and experimental plates, respectively.

In vitro antibacterial assay was carried out by disc diffusion technique sterile discs with 4mm diameter were impregnated with known amount test samples of the chitosan and positive control contained a standard antibiotic (Ampcillin) disc. Negative controls not comprised sterile disc only. The impregnated discs along with control (incorporated with solvent alone) were placed at the center of Agar Plates, seeded with test bacterial cultures. After incubation at room temperature (37°C) for 24 hrs for bacterial plates, antimicrobial activity was showed in terms of diameter of Zone of inhibition which was measured in mm using caliper or a scale and recorded.

3. RESULTS AND DISCUSSION:

The present study to investigate various physiochemical properties of chitosan extracted from Paratelphusa hydrodromous. The results of yield, moisture, water and fat binding capacities of chitosan extracted from Paratelphusa hydrodromous shells were analysed (Table 1).

Table 1: Characteristics of Chitosan (%)

Yield	39.23
Moisture	0.48±0.18
Water binding capacity (WBC)	619±50
Fat Binding Capacity (FBC)	525±20
Solubility	80.8±10.7

The yield of chitosan was being about 39.23%. A chitosan yield of 14.6% was reported from the carapax of Penaeus monodon¹⁶. The moisture content of the chitosan was found to be 0.48±0.18%. The result shows similarities with the moisture content of chitosan obtained from different sources (Artemia urmiana, snow crab processing) ^17-18.

According to Rout¹⁹ WBC for CS ranges between 581 to 1150% with an average of 702%. In the present study the water binding capacity of chitosan was 619%. Similar result has been reported by Cho et al.²⁰ but No et al.²¹ reported lower results of 355 - 611%. Rout (2001) also reported that the process of decolouration causes a decrease in WBC of Chitosan than those of unbleached crawfish chitosan. Table-1 shows that the WBC of samples is 619±50%. No et al.²² reported that the physicochemical characteristics of chitin and Chitosan influence their functional properties, which vary with species and preparation procedures. Knorr⁴ noticed that differences in WBC between chitinous polymers possibly were due to dissimilarities in crystallinity, amount of salt forming groups and the residual protein content of the products. In the present study HPTLC result shows the protein level of chitosan.

The fat binding capacity of and fresh water crab shell (Paratelphusa hydrodromous) was measured using olive oil. The fat binding capacity of crab chitosan measured 525±20%. The range of FBC found in the present study (525%) was slightly similar to that reported ¹⁹ and slightly higher than that (217 - 403%) explained²⁰. Several studies reported a correlation between physicochemical and functional properties of Chitosan.

Various absorption bands within the 4000-400 cm range were recorded in the FTIR spectra of chitosan, prepared from crab shell. Different stretching vibration bands were observed in the range 3444.87-2227.94 cm^-1related to (N-H) in (NH) associated to primary amines. The presence of methyl group in NHCOCH₃range at1654.92 to 1627 cm^-1. CH₂ in CH2OH group was observed in 1427.32cm^-1 stretch. Glycosidic (C-O-C) linkage was observed in 1153.48cm^-1. The presence of CH₂ and CO groups were observed in the 1072.42cm^-1and 605cm^-1. Parasakthi²³ observed the FTIR peaks at 534.61, 1024.16, 1321.88, 1380.81 and 1640.40 cm^-1 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, 2853, 2921 and 3401 cm^-1 were coincided with that of the shell and operculum samples confirming the presence of chitin.

In the present study the anti-bacterial activity of Yersinia ruckeri was investigated (Table 2). Results showed that there was an increase in antimicrobial activity with increasing chitosan concentration. The highest concentration (0.75%) of chitosan used inhibited Yersinia ruckeri by 85.61±12.85%. 0.50% concentration of chitosan inhibited Yersinia ruckeri to 72.31±10.59% than 0.25% concentration of chitosan which inhibited 44.20±10.8%.

Table 2: Antibacterial activity

Samples	Diameter (mm) of inhibitory zone against Yersinia ruckeri
Control	No inhibition
Chitosan	0.57±0.1**
Ampicillin (positive control)	0.73±0.3*

* Significant and ** Highly Significant

The antimicrobial activity of chitosan has been studied extensively; it has been shown that chitosan acts by disrupting the barrier properties of the outer membrane of gram negative bacteria²⁴. The minimum inhibitory concentration (MIC) of chitosan ranged from 0.05% to more than 0.1% depending on the examined gram negative bacteria which were Escherichia coli, Pseudomonas fluorescens, Salmonella typhimurium and Vibrio parahaemolyticus²⁵. The present investigation confirms and supports the earlier findings regarding usefulness of chitosan as an antimicrobial agent. This is proved that the natural chitosan and its derivatives were having antibacterial and/or antifungal characteristics^6,26-29resulted in their use in commercial disinfectants. According to literature^30-31, chitosan possesses antimicrobial activity against a number of Gram-negative and Gram-positive bacteria.

4. CONCLUSIONS:

This study investigated the physicochemical characteristics of chitosan extracted from the shells of Paratelphusa hydrodromous waste, which is discarded without being used and causes environmental pollution. Constitute a significant amount of waste in nature, the production of chitosan from crab shells, natural antibiotics could be developed and used to preventing from the pathogenic bacteria. And their non toxic antibacterial property also could be used as preservatives in the food industries to avoid food spoilages and food borne diseases.

5. REFERENCES:

1. Knorr D. Use of chitinous polymers in food- A challenge for food research and development. Food Technology. 38; 1984: 85-97.

2. No HK and Meyers SP. Crawﬁsh chitosan as a coagulant in recovery of organic compounds from seafood processing streams. Journal of Agricultural and Food Chemistry. 37(3); 1989: 580–583.

3. Ruiz-Herrera J. The distribution and quantitative importance of chitin in fungi. In: Edited by R. A. A. Muzzarelli, E. R. Pariser. Proceedings of the ﬁrst international conference on chitin/chitosan. MIT Sea Grant Report MITSG78-7, Index No. 78-307-Dmb Cambridge: Massachusetts Institute of Technology. 1978; pp. 11–21.

4. Knorr D. Functional properties of chitin and chitosan. Journal of Food Science. 47; 1982: 593-595.

5. Yen MT. Yang JH and Mau JL. Physicochemical characterization of chitin and chitosan from crab shells. Carbohydrate Polymers. 75; 2009: 15–21.

6. Chung YC. Wang HL. Chen YM. and Li SL. Eﬀect of abiotic factors on the antibacterial activity of chitosan against waterborne pathogens. Bioresource Technology. 88; 2003: 179–184.

7. Devlieghere F. Vermeulen A and Debevere J. Chitosan: Antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables. Food Microbiology. 21; 2004: 703–714.

8. Anderson DP and Siwicki AK. Duration of protection against Aeromonas salmonicida in brook trout immunostimulated with glucan or chitosan by injection or immersion. The Progressive Fish-Culturist. 56; 1994: 258-261.

9. Siwicki AK. Anderson DP and Rumsey GL Dietary in- take of immunostimulants by rainbow trout aﬀects non-speciﬁc immunity and protection against furunculosis. Vetinary Immunology Immunopathology. 41; 1994: 125–139.

10. Johnson EL. and Peniston. Q.P. Utilization of shellfish waste for chitin and chitosan production. In Chemistry and Biochemistry of Marine Food Products edited by Martin RE. Flick GJ. Hebard CE. Ward DR. AVI Publishing:Westport, CT. Chapter 19; 1982.

11. Green JH. and Mattick JF. Fishery waste management. In Food Processing Waste Management, Edited by Green, J.H. and Kramer, A. AVI Publishing Co. Westport, CT. 1979; pp 202-227.

12. Madhavan, P., and Nair, K.G.R. (1974). Utilization of prawn waste isolation of chitin and its conversion to chitosan. Fish Technology 11: 50-53.

13. Black CA Methods of Soil Analysis: Part I physical and mineralogical properties. American Society of Agronomy, Madison, Wisconsin. 1965.

14. Wang JC and Kinsella JE. Functional properties of novel proteins: Alfalfa leaf protein. Journal of Food Science. 41;1976: 286-292.

15. Zheng LY. Zhu JF. Study on antimicrobial activity of chitosan with different molecular weights. Carbohydrate Polymer. 54; 2003: 527- 530.

16. Hongpattarakere T and Riyaphan O. Effect of deacetylation conditions on antimicrobial activity of chitosans prepared from carapace of black tiger shrimp (Penaeus monodon). Songklanakarin. Journal of Science and Technology. 30(1); 2008: 1–9.

17. Kamil JYVA. Jeon YJ. and Shahidi F. Antioxidative activity of chitosan of different viscosity in cooked comminuted ﬂesh of herring (Clupea harengus), Food Chemistry. 79; 2002: 69–77.

18. Tajik H. Moradi M. Rohani SMR. Efrani AM and Jalali FSS. Preparation of chitosan from brine shrimp (Artemia urmiana) cyst shells and effects of different chemical processing sequences on the physicochemical and functional properties of the product. Molecules. 13; 2008: 1263–1274.

19. Rout SK. Physicochemical, functional, and spectroscopic analysis of crawfish chitin and chitosan as affected by process modification. Dissertation Louisiana State University: Baton Rouge, LA, USA. 2001.

20. Cho YI. No HK. Meyers SP. Physicochemical characteristics and functional properties of various commercial chitin and chitosan products. Journal of Agricultural Food Chemistry. 46; 1998:3839-3843.

21. No HK. Kim S.H. Lee SH. Park NP and Prinyawiwatkul W. Stability and antibacterial activity of chitosan solutions affected by storage temperature and time. Carbohydrate Polymers. 65; 2006: 174–178.

22. No HK. Lee SH. Park NY. Meyers SP. Comparison of physicochemical, binding, and antibacterial properties of chitosans prepared without and with deproteinization process. Journal of Agriculture and Food Chemistry. 51; 2003: 7659-7663.

23. Parasakthi MS. Extraction of chitin from two cephalopods. M.Sc., Dissertation, Annamalai University. India. 2004; p.18.

24. Ohtakara AM. Izume M. and Mitsutomi. Action of microbial chitosanases on chitosan with different degrees of deacetylation. Agricultural and Biological Chemistry. 52; 1988: 3181-3182.

25. No HK. Park NY. Lee SH and Meyers SP. Antibacterial activity of chitosan and chitosan oligomers with different molecular weights. International Journal of Food Microbiology. 74; 2002: 65-72.

26. El-Ghaouth A. Ponnampalam R. Castaigne F. Arul J. Chitosan coating to extend the storage life of tomatoes. Horticulture Science. 27; 1992: 1016-1018.

27. Kim CH. Kim SY. Choi KS. Synthesis and antibacterial activity of water-soluble chitin derivatives. Polymer Advanced Technology. 8; 1997: 319-325.

28. Papineau AM. Hoover DG. Knorr D. Farkas DF. Antimicrobial effect of water-soluble chitosans with high hydrostatic pressure. Food Biotechnology. 5; 1991: 45-47

29. Sudarshan NR. Hoover DG and Knorr D. Antibacterial action of chitosan. Food Biotechnology. 6;1992: 257-272.

30. Jeon YJ. Park PJ and Kim SK. Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohydrate Polymer. 44; 2001: 71-76.

31. Ueno K. Yamaguchi T. Sakairi N. Nishi N. Tokura S. Antimicrobial activity by fractionated chitosan oligomers. In: Domard, A., Roberts, G.A.F. and Varum, K.M., Editors. Jacques Andre, Lyon, Advances in chitin Science. 2; 1997: 156-161.

Received on 06.09.2014 Accepted on 22.09.2014

Asian J. Res. Pharm. Sci. 4(3): July-Sept. 2014; Page 125-128