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16ПРЕДСТАВЛЕНИЕ НАУЧНОЙ РАБОТЫ
CHITIN PURIFICATION FROM SHRIMP SHELL WASTE BY CHEMICAL DEACETYLATION
AllaM Ayman Yunes, Dolgonova Natalya Vadimovna, The Astrakhan state Technical university, g. Astrakhan
E-mail: [email protected]
Abstract. Due to the multiplications of chitosan particularly in food products. This paper was performed to achieve the optimum parameters involved in shrimp shell processing. The major procedure for obtaining chitosan is based on the alkaline deacetylation of chitin with a strong alkaline solution. The obtained chitin was converted into the more useful soluble chitosan by stepping into solutions of NaOH of various concentrations and for extended periods of time, then the alkali chitin was heated which dramatically reduced the time of deacetylation. Isolation of chitin itself from different sources is affected by the source. To produce chitosan deacetylation of chitin is required and the optimum parameters were 40% (10 M NaOH) at 90°C for 2hr. The produced shrimp shells chitosan under these conditions had 83.53%, 521.65% and 405.65% degree of deacetylation, water and fat binding capacities respectively.
Keywords: Chitosan, degree of deacetylation, isolation, concentrations.
Introudaction
Approximately 70% of the landed value of shellfish is rejected as offal. This abundant waste material has either to be discarded or converted to value added products, and this has led to the production of several useful biochemical and nutrients, such as chitin, pigments and seafood peptones from these by products. It is frequently present as a cell wall material in plants, and in the cuticle of animals. In addition, chitins in animal tissues are frequently calcified, such as in the case of shellfish. Some fungi contain chitosan; however, it is commercially produced by the deacetylation of chitin. Chitosan is a natural Bio-polymer derived by deacetylation of chitin, a major component of the shells of crustacean such as crab, shrimp, and
ВЕСТНИК НАУКИ И ТВОРЧЕСТВА
crayfish. During the past several decades, chitosan has been receiving increased attention for its commercial applications in biomedical, food, and chemical industries. Chitosan is now widely produced commercially from crab and shrimp shells wastes. Several techniques to extract chitin from different sources have been reported. The most common method is referred to as the chemical procedure [1].
Therefore, this study aimed to evaluate define the optimum parameters (acid / alkaline concentration, temperature and time) to extract chitosan from shrimp shells. The chemical compositions, functional properties of shrimp shells chitosan were studied.
Material and methods
Shells of green shrimp Penaeus semisulcatus were purchased from the Abou Ghalli Company for trading and exporting Alabour market, Egypt. The shells were manually scraped (free of loose tissue), collected and brought to the laboratory in the same day. Whenever, the shells were brought to the laboratory it freeze immediately (at -18°C) and stored for further analysis. The shells were first washed several times with tap water and rinsed several times with distilled water. The rinsed shells were • dehydrated in an electric draft oven at 45 °C tell drying. The dried shells were grounded in a grinder (Braun Biotech International GMBH. D.34212 Melsungen, Germany) to pass through a 1.6 mm sieves and stored at 4°C in tight dark glasses till it was subjected to demineralization and deproteinization process. Demineralization was carried out using 1M HCl at 45 °C for 2 hr with a solution/solid ratio of 1/15 v/w. The HCl, which showed the highest ash reduction rate (2M) was applied during determination of the optimum temperature (45°C).The temperature (45°C) resulted in the highest ash reduction rate was applied during determination of the optimum treatment time (2hr). Deproteinization was carried out using NaOH concentrations (1M NaOH) at a temperature of 75 °C for 4 hr with a solution/solid ratio of 1/15 w/v. The NaOH which showed the highest protein reduction rate (1M) was applied during determination of the optimum temperature (75°C). A preliminary experiment to define the optimum deacetylation condition of shrimp shell chitin was carried out using 50g dried shrimp shell chitin (demineralized and deprotienized). Deacetylation was carried out using different NaOH concentrations (40% NaOH) at a temperature of 90 °C for 4hr with a solution/solid ratio of 1/15 v/w. The procedure [2] was followed in the determination of moisture (method No. 32.1.03), crude fat (method No. 32.1.13), Crude fiber (method No. 32.1.15), Crude protein (method No. 32.1.22), and total ash (method No. 32.1.05). Total carbohydrate content was calculated by difference.
Measurement of Degree of Deacetylation (% DD). The acid-base titration method was used to determine the DD from the amino group content in chitosan. Chitosan (0.3 g) was dissolved in 30 ml of HCl standard solution (0.1 mol/L). Methyl
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orange and aniline blue mixing indicators were added [3].
Solubility, Water Binding Capacity (WBC) and Fat Binding Capacity (FBC) of chitosan. Solubility. Chitosan (0.1 g ) was placed into a centrifuge tube (known weight) then dissolved in 10 ml of 1% acetic acid for 30 min using an incubator shaker operating at 240 rpm and 25°C. The solution was then immersed in a boiling water bath for 10 minutes, cooled to room temperature (25oC) and centrifuged at 10,000 rpm for 10 min. The supernatant was decanted. The residue particles were washed with distilled water (25ml) then centrifuged at 10,000 rpm [4]. The supernatant was removed and the residue dried at 60oC for 24hr. Finally, weighed the dried residue and the percentage of solubility was calculated as followed:
(Initial weight of tube + chitosan) - (Final weight of tube + chitosan)
Solubility of Chitosan (%) = X 100
(Initial weight of tube + chitosan) - (Initial weight of tube)
Water and fat binding capacity. Water binding capacity (WBC) and fat binding capacity (FBC) of chitosans were measured using the method of [3]. Briefly, • the procedure was carried out by weighing a centrifuge tube containing 0.5 g sample, adding 10 mL of water or corn oil, and mixing on a vortex mixer for 1 min to disperse the sample. The contents were left at ambient temperature for 30 min with shaking for 5s every 10 min and centrifuged at 3200 rpm for 25 min. The supernatant was decanted and the tube was weighed again. WBC and FBC were calculated using the following formula:
WBC (%) = [water bound (g)/sample weight (g)] x 100.
FBC (%) = [fat bound (g)/sample weight (g)] x 100.
Statistical analysis. Statistical analysis was done using analysis of variance (ANOVA), and least significant difference (LSD) were obtained to compare the means of treatments, using Costat version 6.311 (Copyright 1998 - 2005, CoHort Software). Duncan's multiple range test was used to compare between the treatments means [5].
Results and discussion
Proximate composition of crude shrimp shells (moisture, protein, fat, ash, carbohydrates, and fiber content), was presented in Table 1. The protein content was 36.63%, while ash content was 44.96%. Our results showed a low content of lipids (4.85%), while shrimp shells total fiber and total carbohydrates content were 6.18 % and 7.38 %, respectively.
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Table 1
Chemical composition of crude shrimp shells (on dry weight basis)
Samples Moisture % Protein % Fat % Carbohydrates % Ash % Fiber %
Crud shrimp 13.05 36.63 4.85 7.38 44.96 6.18
Deacetylation treatment (chitosan production). Effect of NaOH concentration. Increasing the concentration of NaOH up to 10M resulted in a significant (p<0.05) increase in the degree of deacetylation Table 2. However, the degree of deacetylation was significantly (p<0.05) decreased by NaOH concentration increasing than 10 M. Water binding capacity was significantly (p<0.05) increased with increasing of NaOH concentration up to 10M. While, it was significantly (p<0.05) decrease when the concentration of NaOH increased up to 12.5M. The same trend was observed in fat binding capacity. Solubility was significantly (p<0.05) affected by concentration of NaOH. Increasing the concentration of NaOH resulted in • a significant (p<0.05) decreased in solubility.
Table 2
Effect of different concentrations of NaOH on the degree of deacetylation and functional properties of shrimp shells chitosan (Means in the same column with different letters are significantly different at (p<0.05))
NaOH concentration (M) Degree of Deacetylation ( % DD) Water Binding Capacity % Fat Binding Capacity % Solubility %
2.5 49.87 e 501.20 bc 422.89 c 67.03 a
5 53.20 d 489.94 c 412.32 d 62.51 b
7.5 60.53 c 511.01 b 464.73 b 55.37 c
10 75.83 a 526.49 a 487.27 a 51.40 d
12.5 72.83 b 506.22 b 415.63 d 47.39 e
LSD 2.55 11.64 4.17 2.60
Effect of temperature. Effect of temperature on the degree of deacetylation and functional properties of shrimp shells chitosan were presented in Table 3. The degree of deacetylation was not significantly (p>0.05) affected by the temperature of extraction up to 60°C. However, more than 60°C resulted in a significant (P<0.05)
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increase in the degree of deacetylation. The highest (p<0.05) water binding capacity was noticed when the chitin extracted at 90°C (528.53%) while the lowest (p>0.05) value was observed when the chitin treated at 45°C (450.60%). The fat binding capacity had the similar trend of water binding capacity. Solubility was significantly (p<0.05) affected by temperature. Solubility was significantly (p<0.05) increased with temperature up to 60 °C.
Table 3
Effect of different temperatures on the degree of deacetylation and functional
properties of shrimp shells chitosan
Temp (° C) Degree of Deacetylation (% DD) WaterBinding Capacity % Fat Binding Capacity % Solubility %
30 °C 74.97 c 498.43 d 414.17 b 54.31 c
45 °C 75.16 c 450.60 e 391.36 d 60.02 b
60 °C 76.67 c 502.56 c 385.52 e 64.55 a
75 °C 79.28 b 510.51 b 407.33 c 57.70 b
90 °C 82.76 a 528.53 a 512.91 a 50.41 d
L.S.D 2.55 2.40 5.07 2.70
Effect of time. No significant (p>0.05) differences were detected in the DD (%) between the chitin treated for 4hr (76.83%) and that treated for 5hr Table 4. The highest value of WBC was obtained when the shrimp shells chitin treated for 2 hr (537.71%), while the lower water binding capacity was detected when shrimp chitin treated for 5 hr (491.27%). Fat binding capacity (%) showed the highest value when shrimp shells chitin treated for 4 hr (424.48%), however it showed low values by using 1 hr and 2 hr (311.17% and 345.89%) respectively. The effect of heating time on the degree of deacetylation and % solubility of shrimp shells chitosan showed clear decrease in both parameters due to the increase of time. The results (Tables 2, 3, and 4) indicated that the optimum condition for producing shrimp shells chitosan were extraction with 10M NaOH at 90°C for 2hr. Shrimp shells chitosan produced under the optimum condition had 83.53% degree of deacetylation, 521.65% water binding capacity and 405.65% fat binding capacity. It seems that the purity for the product to be considered as chitosan was 80.5%. Accordingly, all crab chitosans were nearly pure chitosans. For the purity of crab chitosan products, the reaction time of 60 min was sufficient.Water binding (WBC) and fat binding capacities (FBC) of commercial chitosan are lower than the extracted chitosan. Water and fat binding capacities of
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ВЕСТНИК НАУКИ И ТВОРЧЕСТВА
different commercial chitosan were reported as 458-805% and 314-535%, respectively.Water Binding capacity and FBC of six commercial chitosan products observed by [6] were in the range of355-611% and 217-477%, respectively. The WBC (492.67%) and FBC (383.04%) of commercial chitosan in the present study were compatible to those reported by [7].
Table 4
Effect of different times on the degree of deacetylation and functional properties of shrimp shells chitosan (Means in the same column with different letters are
significantly different at (p<0.05))
Time (hr) Degree of Deacetylation (% DD) Water Binding Capacity % Fat Binding Capacity % Solubility %
1 hr 85.35 ab 519.75 c 311.17 e 53.79 b
2 hr 86.67 a 537.71 a 345.89 d 59.79 a
3 hr 81.94 b 517.36 c 418.46 b 48.55 c
4 hr 76.83 c 531.69 b 424.48 a 43.71 d
5 hr 78.01 c 491.27 d 383.76 c 40.99 e
L.S.D 3.67 2.40 2.08 1.34
The effect of optimum conditions for producing shrimp shells chitosan (10 M NaOH at 90 °C for 2hr), on the degree of deacetylation and some function properties. Shrimp shells chitosan produced under the optimum condition had 83.53% degree of deacetylation, 521.65% water binding capacity and 405.65% fat binding capacity. The solubility of the produced chitosan reached to 55.65%. on the source and preparation procedure, DD may range from 30% to 90%. Water binding (WBC) and fat binding capacities (FBC) of commercial chitosan are lower than the extracted chitosan. Water and fat binding capacities of different commercial chitosan were reported as 458805% and 314-535%, respectively, by [6, 7]. WBC and FBC of six commercial chitosan products observed by [7] were in the range of355-611% and 217-477%, respectively. The WBC (492.67%) and FBC (383.04%) of commercial chitosan.
Conclusion
The best condition to production of shrimp shell chitosan was 10M NaOH at 90 ° C for 2hr. The optimum conditions for producing shrimp shells chitosan (10 M NaOH at 90 °C for 2hr), on the degree of deacetylation and some functional properties. Shrimp shells chitosan produced under the optimum condition had 83.53% degree of deacetylation, 521.65% water binding capacity and 405.65% fat binding capacity. The solubility of the produced chitosan reached to 55.65%.
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