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Polysaccharides are widely distributed in nature. They are important in different fields since they possess unique structures and characteristics that are different from typical synthetic polymers. Among many kinds of polysaccharides, cellulose and chitin are the most important biomass resources. Chitin was first isolated by Frenchman Braconnot in 1811 from mushrooms and was named ‘‘fungine’’. A material identical to fungine was found in insects in 1821 by Odier, he named it as ‘‘chitine’’ (Madhavan, 1992). Chitin, poly(β-(1→4)-N-acetyl-D-glucosamine), is a hard and colourless biopolymer. It has a role analogous to that of collagen in the higher animals and cellulose in terrestrial plants (Mayer and Sarikaya, 2002).
The scope of this study was to optimize physical and nutritional parameters influencing deproteination (DP) and demineralization (DM) process of shrimp shell wastes employing the statistical approach of Response Surface Methodology followed by a scale-up of the fermentation process under the optimized conditions. The prepared chitin and chitosan was purified and its characteristic properties were determined.
Results of the study can be summarized in as follows:
1. Sampling, Screening, isolation of proteolytic isolates. 70 bacterial cultures were isolated from different sources (soil rhizoshere samples of different plants and shrimp shell waste) on shrimp shell agar medium (med. 3).Nine isolates were selected based on the diameter of their clear zone on the skim milk agar in addition to Bacillus subtilis 262 as a reference strain. The selected bacterial isolates have code number Sh1, Sh2, Sh4, Sa3, B1, G2, Sb4, T4 and C3 were tested for deproteinization (DP) and demineralization (DM) of shrimp shell waste.One isolate (Sb4) forming the largest clearing zone was selected for further experiments as a promising candidate for DP andDM.
2. Identification of proteolytic bacteria sp. Based on morphological characteristics, gram staining, endospore formation and further confirmation by sequencing the 16S-rRNA gene, the isolate Sb4 was identified as Bacillus subtilis NA12 (99 % identity).
3. Screening of factors affecting the production of proteases by “one-variable at-a-time” approach
a. Effect of carbon sources. In fermentation medium (med. 4), where brown sugar was replaced by the three tested carbon sources (glucose, fructose and sucrose) in order to evaluate their efficiency on the rates of DP and DM of shrimp shell waste fermented by B. subtilis NA12. Also, the carbon source was removed from med. 4.According to the results of statistical analysis, sucrose was selected as the best carbon source to be applied for the following experiments and the medium will be designated as M.m.4-1.
b. Effect of sucrose concentration. Different sucrose concentrations ranged from 5 to 20% (w/v) were conductedto study their impact on the rates of DP and DM.10 % (w/v) sucrose was selected as the best concentration to be applied in the following experiments and the medium will be designated as M.m.4-2.
c. Effect of shrimp shell waste concentration. Shrimp shell waste (SSW) concentration ranged from 5 to 20 % (w/v) was investigated to figure out its effect on DP and DM rates of shrimp shell waste fermented by B. subtilis NA12. The highest rate of DP and DM wasobtained at 5 % (w/v) SSW, therefore it was selected to be applied in the following experiments and the medium will be designated as M.m.4.
d. Effect of inoculum size. The impact of different inoculum size (5 % control, 10 % and 15 % with CFU/ml = 35 x 108) on the rates of DP and DM were investigated. Inoculum size of 15 % (v/v) was the optimum for proteases production, achieving the highest DP and DM.
e. Effect of incubation period. Shrimp shell medium were inoculated with 24h inoculum of B. subtilis NA12, incubated at 35oC in a rotary shaking incubator at 100 rpm during 6 days of incubation time, samples were taken ever 48h. Results revealed that the highest level of DP % and DM % were 93.38 % and DM 82.48 %, respectively was achieved after 6 days of incubation. Therefore, this cultivated medium (modified medium No. 4) will be recommended to use for chitin extraction from fermented shrimp shell waste by B. subtilis NA12 at 35°C for 6 days at 100 rpm.
4. Impact of gamma irradiation on the viability of selected strain.
B. subtilis NA12 was exposed to different γ-irradiation doses ranging from 2.0 to 10.0 kGy in order to evaluate their efficiency on the cell viability which express as CFU (colony forming unit). B. subtilis NA12 was exposed to the sub-lethal dose (determined from the resulting dose response curve) being 16 kGy, which produced variant named B. subtilis NA12-V. The DP % and DM % was increased by 1.02 fold by B. subtilis NA12-V than the parent strain (B. subtilis NA12).
5. Statistical screening of physical factors using SPSS software.
a. All tested physical factors were significant in which they affected deproteinization (DP) and demineralization (DM) process, either positively or negatively.
b. Sucrose concentrations, shrimp concentrations, inoculum size and incubation period were tested for their interactive effects on DP and DM process using response surface methodology (RSM).
6. Statistical screening of factors affecting deproteinization and demineralization of SSW using Box-Behnken Design (BBD).
from the previous experiments the significant fermentation conditions that mostly affected the rates of deproteinization (DP) and demineralization (DM) were sucrose concentration, shrimp shell concentration, inoculum size and fermentation time. Box-Behnken Design (BBD) was used to optimize the above mentioned significant parameters for deproteinization and demineralization of shrimp shell by B. subtilis NA12. It was found that a sucrose concentration of 5 %, SSW of 12.5 %, inoculum size of 10 % (35 x 108 CFU/ml) and fermentation time of 7 days; had a predicted value of DP of 97.65 % whereas the actual experiment gave 96.37 %. The predicted value of DM of 82.94 % whereas the actual experiment gave 82.19 %.
7. Convert extracted chitin from shrimp shell waste to chitosan.
Chitin was extracted from shrimp shell waste by fermentation using B. subtilis NA12 and converted to chitosan by chemical method (deaceytlation). The influence of using γ-irradiation on the time needed for deaceytlation process, degree of deacetylation and average molecular weight (MW) was investigated. The obtained results shows that
a. The irradiation dose at 35 kGy, causes the reduction time of deacetylation process by a factor of four and half fold comparing with non-irradiated samples.
b. Degree of deacetylation (DDA) for chitosan was determined by potentiometric titration. The best DDA for the converted chitosan was 87.9 % at 35kGy.
c. The molecular weight of chitosan decreases constantly with increasing radiation dose from 1.9x106 (non-irradiated) to 3.7x104 (at 35 kGy).
8. characterization of chitin and chitosan.
The characteristics of chitin and chitosan were studied using some characterizing tools. Results could be summarized as follows:
a. The structure of prepared chitin and chitosan was confirmed by FT-IR.
b. Thermogravemetric analysis (TGY) data showed that chitin exists as a stable structure toward thermal decomposition than chitosan.
c. The crystallinity of the prepared chitin and chitosan was determined by XRD analysis. The result shows that, chitin more crystalline than chitosan.
9. Applicationof chitosan as antibacterial agent.
The prepared chitosan samples at different γ-irradiation doses with different MW (from 4.8 x 105 to 3.7 x 104 g/mol) and different DDA values (from 84.4 % to 87.9 %) were tested as an antimicrobial agent against some pathogenic bacteria isolates such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp. and Staphylococcus aureus.Chitosan obtained at 35 kGy with MW 3.7 x 104 g/mol and DDA 87.9 % has the highest antimicrobial activity against E. coli (5.4 ± 0.2), K. sp. (5.4 ± 0.12), Staph. aureus (3.5 ± 0.21) and Ps. aeruginosa (1.4 ± 0.06).