INTRODUCTION:

Chitosan is a fiber-like substance derived from chitin, a homopolymer of β-(1, 4)-linked N-acetyl-D-glucosamine. Chitin is the second most profuse organic compound in nature after Cellulose. Chitosan is the deacetylated form of chitin, i.e. poly-(1, 4)-2-amido-2- deoxy-β-D-glucose. The actual difference between chitin and chitosan is the acetyl content of the polymer. Chitosan having a free amino group is the most useful derivative of chitin¹.

Chitosan is used as a flocculants, clarifier, thickener, fibre, film, affnity chromatography column matrix, gas-selective membrane, plant disease resistance promoter, anti-cancer agent, wound healing promoting agent and antimicrobial agent. It can be used in pet food and GRAS (generally regarded as safe) status has been applied for. It is used as a processing aid and is being trialed for applications in fruit preservation, wound dressings, cosmetics, artificial organs and pharmaceuticals². Chitosan is usually prepared from chitin and chitin has been found in a wide range of natural resources (crustaceans, fungi, insects, annelids, mollscs, coelenterate etc.)³

Figure 1: Structure of Cellulose, Chitin, and Chitosan

As seen in Figure 1, the only difference between chitosan and cellulose is the amine (-NH₂) group in the position C-2 of chitosan instead of the hydroxyl (-OH) group found in cellulose. However, distinct plant fiber, chitosan possesses positive ionic charges, which give it the aptitude to chemically combine with negatively charged fats, lipids, cholesterol, metal ions, proteins, and macromolecules. As the natural renewable resource with a number of unique properties, chitosan is now attracting more and more scientific and industrial significance from diversified fields such as chemistry, medicine, pharmacology, biotechnology, biochemistry, food and textile sciences. Properties such as biodegradability, biocompatibility, non-toxicity, wound healing, antimicrobial activity, adsorption and ability to form films, and metal ion chelation⁴ have generated much research work.

Prawns shell and shrimp shell that is an abundant byproduct of fish market are also a good source of chitin and hence chitosan (deacetylated chitin). Prawns and shrimp shells are removed from the prawns and shrimp during processing and are currently regarded as waste so the raw material is cheap.

Generally, four main steps are involved in the manufacturing of chitosan Viz: i) Decalcification, ii) Deproteinization, iii) Decolouration and iv) Deacetylation⁵. But, chtin extracted from prawns and shrimp shell is almost white in colour, so this change in chitosan extraction procedure should reduce the cost of processing. Therefore in the present study the attempt were made to manufacture the Chitosan from voluntarily available waste material by applying modified method. It was compared with commercially available Chitosan which helps to obtain valuable Chitosan with equivalent characteristics as Commercial. This is obtained product from cheaper source can be utilize for various purposes as mentioned above.

EXPERIMENTAL:

Materials:

Chemicals and reagents :

Sodium hydroxide (NaOH), Hydrochloric acid (HCl), Acetic acid (CH₃COOH), Sulphuric acid, sodium acetate, Ethanol, Methanol, Benzene, Chloroform, Petroleum ether, Acetone and Distilled water. All chemicals are supplied by S.D. Fine chemicals and are of AR grade.

Source:

The raw material (RM) as Shrimp, Prawn and Crab shells were amassed from the local fish market as per the requirements of experiments. (Wadala and Kalwa fish market- Maharashtra India). The commercial Chitosan was obtained from Mhatani Chitosan Pvt. Ltd (Gujarat-India).

Methods:

Extraction of chitosan from Shrimp, Prawn and Crab shells by modified method

Chitosan was manufactured from different RM used as Shrimp, Prawn and Crab shells in the laboratory. Raw material was brought from the local fish market which is the waste material. The wastes were thoroughly washed with water and then sun dried to get completely dried shells. Now, these shells were crushed into the powder.

During extraction of chitosan from raw material; four main steps were followed namely i) Decalcification, ii) Deproteinization, iii) Decolouration and iv) Deacetylation. From RM, chitin were obtained after Decolouration process, however, the isolation of chitin specifically consists of only two steps: demineralization (DM) and deproteinization (DP), which involves dissolution of calcium carbonate with HCl and the removal of proteins with alkali.

In our process (modified method), we have by-pass the decolouration step. Now the important step is manufacturing of chitosan from chitin, i.e. deacetylation step. After deacetylation chitosan was obtained and its chemical and physical characterization was carried out and it was compared with standard Chitosan.

Shrimp, Prawn and Crab shells (RM)

Decalcification (Aqueous HCl)

Deproteination (Aqueous NaOH)

Chitin

Deacetylation (Hot concentrated NaOH)

CHITOSAN

Figure 2: Preparation of Chitin and Chitosan from developed method

Characterization of manufactured chitosan from developed method

The developed Chitosan was undertaken for its chemical as well as physical characterization as follows:

Chemical Characteristics of developed Chitosan

1. Degree of deacetylation⁶

The powder was tested for degree of deacetylation by titration. Chitosan (0.5 g) was dissolved in 20 ml of 0.3N Hydrochloric Acid. After adding 400 ml of distilled water, this solution was titrated with 1N NaOH solution (Sigma Aldrich St.Lousis, MO). A titration curve of pH against NaOH titration volume was generated. The curve’s inflection points were found for each indicated transition. The volume of NaOH at each inflection points was applied to the equation:

% NH₂=16.1 (y-x)/M

Where, M is the weight of chitosan used (0.5 grams in this study), x is the first inflection point on the graph of measured pH vs. transition volume, y is the second inflection point.

Three samples were run for each chitosan. The average of three readings was taken. The DDAs obtained by titration were similar to those of commercial chitosan.

2. Molecular weight determination

The molecular weight of Chitosan was determined by measuring the viscosity of 0.05-0.15% chitosan dissolved in 0.5 mol/1acetic acid and 0.2 mol/1 sodium acetate solutions using an Oswald viscometer. The molecular weight was calculated by the Mark-Houwink equation:

η =KmM^α,

Where Km =3.5 *10^-4and α =0.76.

For pH-metric titration, dried chitosan was crushed to powder. The aqueous suspension of this powder was titrated with HCL⁷

3. Nitrogen Content:

Nitrogen content is estimated by Kjeldahl’s Method⁸

Physical characteristics of Chitosan

1. Determination of % yield of extract

The manufactured Chitosan was oven dried at 110 °C. The extracted sample was weighed for calculating the % yield by the following equation:

Yield (%) = (W₁ x 100) / W₂

Where,

W₁ - Weight of extracted product after oven drying and

W₂ - Weight of raw material.

2. Determination of Viscosity;

The viscosity was studied using Brookfield viscometer with a helipath stand and the spindle. The flow behavior of 1% chitosan solutions in 1% acetic acid was tabulated.

3. Ash Content:

Chitosan ash content was determined using a constant weight crucible. The temperature of the Muffle furnace was kept at 450^oC. The ash percentage was calculated by the equation:

Ash % = (W₂-W₀/W₁-W₀)*100

Where W₀ is the constant weight of crucible, W₁ is the weight of sample and crucible, W₂ is the weight of ash and crucible.

4. Solubility:

Chitosan was checked for its solubility in water; dilute mineral acids such as hydrochloric acid, sulphuric acid, nitric acid and acetic acid, commonly available organic solvents (alcohol, acetone, ether, benzene, and chloroform), dilute sodium hydroxide and other alkaline solutions.

Test method: 200 mg polymer was placed in contact with 15ml of the solvent and stirred gently with the help of a magnetic stirrer. The end point was taken as the complete dissolution of the polymer in the solvent.

5. Determination of % purity and % insolubles:

An accurately weighed (2 gm) sample of chitosan was transferred to a beaker. 100 ml of 2% acetic acid was added with constant stirring to it. It was stirred till solution was affected. The resulting viscous solution was decanted and the insoluble matter was transferred carefully to a previously weighed gooch crucible. It was dried till constant weight at 110⁰C. The crucible, after cooling, was reweighed on a sensitive balance. The percentage insolubles were calculated as followed:

% insolubles = (C- B/10)* 100

Where, C = weight of crucible + insoluble matter,

B = weight of empty crucible

% purity = 100 - % insolubles

6. Determination of Ph:

The pH of 1% solution of chitosan in 1% acetic acid was determined on a Digital pH meter.

7. Determination of FTIR spectroscopy:

For determination of a material in FTIR machine, diffuse reflectance measuring instrument which is one of the accessories of the FTIR machine was used. This instrument is used to measure reflectance spectrum of solid sample. In the diffuse reflectance method, the emitted infrared light repeats transmission and irregular reflection together with specular reflection in sample, and it then goes out in various directions. The scattering light is measured based on the principle that a spectrum similar to the absorption spectrum can be obtained since scattering light comes out after being absorbed by the sample.

In this case, the structure of chitosan was confirmed by comparing the infrared spectrum of the product with the standard and also with that of chitin.

RESULTS AND DISCUSSION:

Table 1: % yield obtained during processing of RM

Process	Product	% Yield
Process	Product	Shrimp	Prawn	Crab
Decalcification	-	63.3	55.4	17.3
Deproteinization	Chitin	24.5	19.4	8.1
Deacetylation	Chitosan (Low DD)	18.2	16.0	-
Deacetylation	Chitosan (High DD)	12.4	10.4	-

Table 2: Chemical characteristics of developed and commercial chitosan

Properties	Chitosan
Properties	Shrimp	Prawn	Commercial
Degree of Deacetylation (DD)	85.86	88.32	90.16
Molecular weight	43019.57	57845.97	124999.78
Nitrogen content (%)	6.88	7.10	7.22

Table: 3 Physical properties of developed and commercial chitosan

Properties	Chitosan
Properties	Shrimp	Prawn	Commercial
Ash content	1.1	1.48	1.65
% Insolubles	2.36	1.78	1.12
% Purity	97.64	98.22	98.88
pH	4.6	4.13	4.8

Figure 3: viscosity studies in manufactured and commercial chitosan

Table 4: Solubility of developed and commercial chitosan in different solvents

Solvent	Solubility of Chitosan
Solvent	Shrimp	Prawn	Commercial
Water	Insoluble	Insoluble	Insoluble
Dil. HCl	Soluble	Soluble	Soluble
Dil. HNO₃	Soluble	Soluble	Soluble
Dil. H₂SO₄	Soluble	Soluble	Soluble
Dil. HCOOH	Soluble	Soluble	Soluble
Dil. CH₃COOH	Soluble	Soluble	Soluble
Dil. Sodium hydroxide	Insoluble	Insoluble	Insoluble
Methanol	Insoluble	Insoluble	Insoluble
Ethanol	Insoluble	Insoluble	Insoluble
Acetone	Insoluble	Insoluble	Insoluble
Ether	Insoluble	Insoluble	Insoluble
Chloroform	Insoluble	Insoluble	Insoluble
Benzene	Insoluble	Insoluble	Insoluble
Petroleum ether	Insoluble	Insoluble	Insoluble
Dil. Acetic acid +ethanol	Soluble	Soluble	Soluble
Dil. Acetic acid +methanol	Soluble	Soluble	Soluble
Dil. Acetic acid + IPA	Soluble	Soluble	Soluble

Table 5: viscosity study of developed and commercial chitosan

% of solution in 1% acetic acid	viscosity of Chitosan in cps
% of solution in 1% acetic acid	Shrimp	Prawn	Commercial
0.1%	31	35	43
0.2%	42	47	56
0.4%	50	54	63
0.6%	62	68	74
0.8%	72	78	82
1%	79	85	91

Figure 4: FTIR of Commercial Chitosan

Figure 5: FTIR of Chitosan prepared from Shrimp shell using developed method

Fig: 6 FTIR of Chitosan prepared from Prawns shell using developed method

Table 1 shows the % yield of at various stage of processing of prawns, shrimp and crab shells. It is observed that in case of crab shell the percent yield after decalcification was 17.3 % and after deproteinization was 8.1%. This is due to the fact that the crab shells contain large amount of proteins and minerals in it which get dissolved in Demineralization and deproteinization step. Also, it is very difficult to get ample amount of crabs shell from the market in terms of wastes. So, it is not considered for further processing of chitosan.

% yield of prawns shell after decalcification and demineralization step is 55.4% and 19.4% respectively and that of shrimp shell is 63.3% and 24.5% respectively. The chitin percent yield of prawns shell is less because the flesh is firmly attached to the shell which is difficult to remove while washing. These proteins get dissolved in alkali step and hence the yield is poor as compared to shrimp

Table 2 shows the Degree of Deacetylation of manufactured chitosan and was compared against the Commercial one. Degree of deacetylation was carried out by volumetric analysis in which we get the percentage of amino groups formed in the deacetylation process. With the referred deacetylation process the Degree of Deacetylation (DDA) obtained for prawn shell was 88.32% and for shrimp shell was 85.86 % and that of commercial chitosan was 90%. It was found that after deacetylation of manufactured chitosan from prawns and shrimp shell is around 90%, which is comparable to that of commercial chitosan.

Value of molecular weight of manufactured chitosan was compared against the commercial chitosan which is shown in Table 2. This Molecular Weight was determined by Oswald’s Viscometer. The molecular weight of chitosan from prawns shell is somewhat higher than that of chitosan from shrimp shell as shown in the table. The molecular weight obtained is distinctly lower for both prawns shell and shrimp shell than that obtained by the commercial chitosan. This may be due to the process and raw material variables for different samples under comparison. However it is worth mentioning that for the desired work the lower degree of polymerization of chitosan polymer as obtained can be used without any difficulty.

Also Table 2 shows the values of nitrogen content of manufactured chitosan and commercial chitosan. The nitrogen content of the chitosan derived from prawns shell obtained is slightly more than that of chitosan from shrimp shell prawn shell, this may due to its varying nature the values also do vary. Nitrogen content of manufactured chitosan (both Prawn and Shrimp) is almost equivalent to that of commercial chitosan showing satisfactory formation of chitosan.

Table 3 shows the different chemical properties of manufactured chitosan. From the table it can be noted that, purity of the compound is 99.17%, ash content is 1.48% and pH of 1% solution in 1% acetic acid is found to be 4.13. The % insolubles in 1% acetic acid solution are 0.83%. % yield of the final product is 10.4%. It can be concluded from the table that there are no batch to batch variations in the ash value, % purity and % insolubles of the manufactured Chitosan. Also it can be concluded that chitosan can be synthesized in the laboratory from shrimp and prawns shell with high reproducibility and yield.

The results of the solubility studies with various solvents are shown in Table no 4. The solubility of prawns, shrimp and commercial grade chitosan is similar for all the solvents. As seen from the table chitosan is insoluble in water, dil. NaOH, ether, chloroform, benzene and acetone. It was found to be soluble in dilute mineral acids such as HCl and HNO₃. It is also soluble in dil. acetic acid. Solvents along with dil. acetic acid show complete solubility of chitosan. Solubility of chitosan derived from shrimp takes little longer time than chitosan derived from prawn which takes more time than commercial chitosan. This is due the impurities present in developed chitosan is more compared to commercial chitosan.

Table no. 5 shows the viscosities of manufactured chitosan and commercial chitosan at different concentrations.

Viscosity of Chitosan from prawn shell is more at different concentration (0.1-1%) than that of chitosan from shrimp shell. This is because viscosity is proportional to the molecular weight of the sample and molecular weight of chitosan from prawns shell is higher than that of chitosan from shrimp shell. Figure 1 shows the graphical representation of prawns, shrimp and commercial chitosan at different concentrations. From graph it is clear that viscosity of chitosan increases as the concentration increases at it is quite linear. Also, commercial chitosan has higher viscosity than developed chitosan.

IR investigations are widely used for studies of chitosan. Figure 2-5 shows the FTIR spectra’s of chitosan manufactured from prawns and shrimp shells and that of commercial chitosan are recorded. FTIR investigations show that chitosan has the characteristic absorption at 1550.66, 1674.10c m^-1 and in the vicinity of 3085.89 and 3265.26cm^-1 and corresponds to the stretching vibration of C=O and NH in (NHCOCH₃), respectively. The FTIR of manufactured chitosan shows the band emerging at 1654.81 cm^-1become weaker, and the new absorption feature observed around 1620cm^-1 (bending vibration of NH of R-NH2) indicates an increase in degree of deacetylation. The peaks due to C-H stretching vibrations are observed at 2916.10 and 2858.31cm^-1 and bending vibrations at 1415.65 and 1375.15cm^-1. The FTIR spectra’s of commercial chitosan and chitosan from prawns and shrimp shells are quite similar.

CONCLUSION:

Chitosan from prawn shell and shrimp shell can easily be processed in the laboratory as the raw material is abundantly available in the fish market at very low or negligible cost. The results of chitosan manufactured using prawn and shrimp shell is comparable with the commercial chitosan and has considerable potential. It is inherently purer than another chitosan source; it does not contain large amounts of calcium carbonate and carotenoids. It does contain large amounts of protein but these can be removed easily and should not possess any problems with allergenicity. The purity of chitosan from prawns shell makes it particularly suitable for the high quality sector of the Chitosan functional properties⁹. The FTIR, physical and chemical properties of the prepared chitosan confirmed that it can be used commercially in the different fields such as food supplement, additive, drug preparation as well as water treatment. The preparation of chitosan from crustacean processing waste (shells) would successfully minimize the environmental pollutants.

ACKNOWLEDGEMENTS:

The authors are thankful to the Department of Oils, Oleochemicals and Surfactants Technology,Institute of Chemical Technology, Matunga, for supporting this research work and for providing us infrastructural facilities and University Grant Commission (UGC) for funding the project.

REFERENCES:

1. Joydeep Dutta and P.K.Dutta, Chitin and Chitosan: Functional Biopolymers of the 21^st century, Chitin and Chitosan: Opportunities and Challenges. Edited by P.K.Dutta, SSM International Publication, 2005; pp. 13.

2. Brine CJ, Sandford PA, Zikakis JP, Advances in Chitin and Chitosan, London: Elsevier Applied Science. 1991:1- 491

3. Muzzarelli RAA, Chitin, Encyclopedia of Polymer Science and Engineering. 3; 1990: 430-441

4. Bishnu Pada Chatterjee et al; Chitosan: A Journey from Chemistry to Biology, Chitin and Chitosan: Opportunities and Challenges. Edited by P.K.Dutta SSM International Publication, 2005, pp 36-41.

5 Joydeep Dutta and P.K.Dutta, Chitin and Chitosan: Functional Biopolymers of the 21^st century, Chitin and Chitosan: Opportunities and Challenges. Edited by P.K.Dutta, SSM International Publication, 2005, pp 4-5

6. Virginia Hamilton et al; Two-Three dimensional Chitosan Substrates for Chondrocyte Culture, Chitin and Chitosan: Opportunities and Challenges. Edited by P.K.Dutta SSM International Publication, 2005, pp 200.

7. K.V. Harish Prashanth, F.S. Kittur and R.N. Tharanathan, Solid state structure of chitosan prepared under different N-deacetylating conditions, Carbohydrate Polymer. 50 (1); 2002: 27-33.

8. Kjeldahl, J, Journal of Analytical chemistry. 22; 1883: 366-382

9. Shepherd R, Reader S, Falshaw A, Chitosan functional properties, Glycoconjugate Journal.14; 1997: 535-542.

Received on 25.07.2012 Modified on 18.08.2012

Asian J. Research Chem. 5(8): August, 2012; Page 1078-1083

Chitosan	Commercial	Prawn shell	Shrimp shell
FTIR Spectra cm^-1	3429.20 (N-H stretch) and (O-H stretch), 3259.47 (NH₂ band), 2858.31(C-H stretch), 1654.81 (C=O stretch), 1554.52(N-H bend), 1415.65(C-H bend), 1068.49(C-N stretch)	3350.33 cm^-1(N-H Stretch),3298.05 cm^-1(NH₂ band), 2869.88 cm^-1(C-H stretch), 1645.17 cm^-1(C=O stretch),1562.23 cm^-1(N-H bend), 1415.65 cm^-1(C-H bend), 1062.70 cm^-1(C-N stretch), 1026.06 cm^-1(C-N stretch), 877.55 cm^-1 (N-H wag)	3359.77 (N-H stretch) and(O-H stretch), 3298.05 (NH₂ band), 2869.88 (C-H stretch), 1645.17 (C=O stretch), 1562.23 (N-H bend), 1415.65 (C-H bend), 1062.70 (C-N stretch), 877.55 (N-H wag)