Preparation of a crude chitosanase from blue crab viscera as well as its application in the production of biologically active chito-oligosaccharides from shrimp shells chitosan.

In this study, a digestive chitosanase from blue crab (Portunus segnis) viscera was extracted, characterized and applied. The crude chitosanase showed optimum activity at pH 4.0 and 60 °C and retained >80% of its activity over a pH range from 3.0 to 10.0. Subsequently, the crude chitosanase was applied to produce bioactive varying molecular weight (Mw) and acetylation degree chitosan-depolymerization products (CDP) with specially sequences composition determined by MALDI-TOF MS owing to an endo-cleavage mode. This hydrolysis process allowed to the preparation, after 24 h of incubation at 40 °C, of a low Mw water soluble CDP (H 24h, <4.4 kDa) with DP up to 6 and a high Mw CDP (C 24h, 142.19 kDa). Following their physicochemical characterization, the functional properties, antioxidant and antimicrobial activities of CDP were investigated. Interestingly, as compared to the native chitosan, CDP, especially low Mw derivatives (H 24h) exhibited potent antioxidant activities, while high Mw derivatives, especially C 24h, markedly inhibited the growth of all tested bacteria and fungi. These results may provide novel insights into the efficiency of chitosan depolymerisation using the Portunus segnis digestive crude chitosanase as a simple, inexpensive and easily method to produce bioactive chitosan-derivatives and that this bioactivity depends highly on their attractive characteristics.

Chitin extraction from crab shells by Bacillus bacteria. Biological activities of fermented crab supernatants.

Crab shells waste were fermented using six protease-producing Bacillus species (Bacillus subtilis A26, Bacillus mojavensis A21, Bacillus pumilus A1, Bacillus amyloliquefaciens An6, Bacillus licheniformis NH1 and Bacillus cereus BG1) for the production of chitin and fermented-crab supernatants (FCSs). In medium containing only crab shells, the highest demineralization DM was obtained with B. licheniformis NH1 (83±0.5%) and B. pumilus A1 (80±0.6%), while the highest deproteinization (DP) was achieved with A1 (94±1%) followed by NH1 (90±1.5%) strains. Cultures conducted in medium containing crab shells waste supplemented with 5% (w/v) glucose, were found to remarkably promote demineralization efficiency, and enhance slightly deproteinization rates. FTIR spectra of chitins showed the characteristics bands of α-chitin. FCSs showed varying degrees of antioxidant activities which were in a dose-dependent manner (p<0.01). In fact, FCS produced by B. amyloliquefaciens An6 exhibited the highest DPPH free radical-scavenging activity (92% at 4 mg/ml), while the lowest hydroxyl radical-scavenging activity (60% at 4 mg/ml) was obtained with B. subtilis A26 hydrolysates. However, the highest reducing power (OD700nm=2 at 0.5 mg/ml) was obtained by B.amyloliquefaciens An6 hydrolysates. These results suggest that crab hydrolysates are good sources of natural antioxidants. Further, FCSs were found to exhibit antibacterial activity against Gram-positive and Gram-negative bacteria.

Use of a fractional factorial design to study the effects of experimental factors on the chitin deacetylation.

Chitosan is obtained by deacetylation of chitin. Chitosan versatility is directly related to the polymer's characteristics depending on the deacetylation process. The aim of this research was to study the parameters influencing deacetylation and to elucidate their effect on acetylation degree (DA) and molecular weight (MW). The effect on chitosan DA was investigated using a fractional factorial design 2(7-3) with seven factors and two variation levels. The tested factors were: X1=number of successive baths, X2=reaction time, X3=temperature, X4=alkali reagent, X5=sodium borohydride, X6=the atmospheric conditions and X7=alkali concentration. A mathematical model was investigated corresponding to the following relation y=7.469-1.344X1-1.094X2-3.094X3+1.906X4+0.656X5+0.906X6-1.031X7+0.469X1X2-0.781X3X4+0.906X1X3X4 with R2=0.99. This model allows fixing experimental conditions for each desired DA. To study the effect on chitosan MW, only atmospheric conditions and use of sodium borohydride as an oxygen scavenger were investigated. The use of sodium borohydride and nitrogen atmosphere was found to have a protective effect against chitosan degradation during deacetylation.

Production and biochemical characterization of a high maltotetraose (G4) producing amylase from Pseudomonas stutzeri AS22.

Amylase production and biochemical characterization of the crude enzyme preparation from Pseudomonas stutzeri AS22 were evaluated. The highest α-amylase production was achieved after 24 hours of incubation in a culture medium containing 10 g/L potato starch and 5 g/L yeast extract, with initial pH 8.0 at 30°C under continuous agitation at 200 rpm. The optimum temperature and pH for the crude α -amylase activity were 60°C and 8.0, respectively. The effect of different salts was evaluated and it was found that both α -amylase production and activity were Ca(2+)-dependent. The amylolytic preparation was found to catalyze exceptionally the formation of very high levels of maltotetraose from starch (98%, w/w) in the complete absence of glucose since the initial stages of starch hydrolysis (15 min) and hence would have a potential application in the manufacturing of maltotetraose syrups.

Composition, functional properties and in vitro antioxidant activity of protein hydrolysates prepared from sardinelle (Sardinella aurita) muscle.

Composition, functional properties and in vitro antioxidative activities of protein hydrolysates prepared from muscle of sardinelle (Sardinella aurita) were investigated. Sardinelle protein hydrolysates (SPH) were obtained by treatment with crude enzyme preparations from Bacillus pumilus A1 (SPHA1), Bacillus mojavensis A21 (SPHA21) and crude enzyme extract from sardinelle (Sardinella aurita) viscera (SPHEE). The protein hydrolysates SPHA1, SPHA21 and SPHEE contained high protein content 79.1%, 78.25% and 74.37%, respectively. The protein hydrolysates had an excellent solubility and possessed interfacial properties, which were governed by their concentrations. The antioxidant activities of protein hydrolysates at different concentrations were evaluated using various in vitro antioxidant assays, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power assay, chelating activity, ß-carotene bleaching and DNA nicking assay. All protein hydrolysates showed varying degrees of antioxidant activity. SPHA21 had the highest DPPH radical scavenging activity (89% at 6 mg/ml) and higher ability to prevent bleaching of ß-carotene than SPHA1 and SPHEE (p < 0.05). However, SPHEE exhibited the highest metal chelating activity (89% at 1 mg/ml) and the strongest protection against hydroxyl radical induced DNA breakage (p < 0.05).

Structural differences between chitin and chitosan extracted from three different marine sources.

Three marine sources of chitin from Tunisia were investigated. Structural differences between α-chitin from shrimp (Penaeus kerathurus) waste, crab (Carcinus mediterraneus) shells, and ß-chitin from cuttlefish (Sepia officinalis) bones were studied by the (13)C NMR, FTIR, and XRD diffractograms. The (13)C NMR analysis showed a splitting of the C3 and C5 carbon signals for α-chitin, while that of ß-chitin was merged into a single resonance. The bands contour of deconvoluted and curve-fit FTIR spectra showed a more detailed structure of α-chitin in the region of O-H, N-H and CO stretching regions. IR and (13)C NMR were used to determine the chitin degree of acetylation (DA). XRD analysis indicated that α-chitins were more crystalline polymorph than ß-chitin. Shrimp chitin was obtained with a good yield (20% on raw material dry weight) and no residual protein and salts. Chitosans, with a DA lower than 20% and relatively low molecular masses were prepared from the wet chitins in the same experimental conditions. They were perfectly soluble in acidic medium. Nevertheless, chitin and chitosan characteristics were depending upon the chitin source.

Optimization of chitin extraction from shrimp waste with Bacillus pumilus A1 using response surface methodology.

Chitin extraction from shrimp shells by biological treatment, using the Bacilli Bacillus pumilus A1, is a non-polluting method and offers the opportunity to preserve the exceptional qualities of chitin and its derivatives. However, the major disadvantage of the fermentative way is the low efficiency of demineralization and deproteinization. The aim of this study is to improve the yield of extraction which depends on many factors, such as the medium composition and the physical parameters. In order to look for the optimal conditions, a Plackett and Burman design was carried out to screen eight factors influencing the deproteinization and demineralization efficiencies. The four most influencing variables were then examined to achieve the optimization using a central composite design. The results obtained showed that the optimal conditions were: shrimp shell concentration of 70 g/l, glucose concentration of 50 g/l, pH of 5.0 incubated with 0.225 OD of B. pumilus A1 inoculum, at 35 °C and 150 rpm for 6 days in 500 ml flask containing 100 ml of working volume. These conditions led to 88% of demineralization and 94% of deproteinization. (13)C CP/MAS NMR spectral analysis of the chitin prepared was carried out and was found to be similar to that of the commercial α-chitin.

Chitin extraction from shrimp shell waste using Bacillus bacteria.

The ability of six protease-producing Bacillus species (Bacillus pumilus A1, Bacillus mojavencis A21, Bacillus licheniformis RP1, Bacillus cereus SV1, Bacillus amyloliquefaciens An6 and Bacillus subtilis A26) to ferment media containing only shrimp shell waste, for chitin extraction, was investigated. More than 80% deproteinization was attained by all the strains tested. However, demineralization rates not exceeding 67% were registered. Cultures conducted in media containing shrimp shell waste supplemented with 5% (w/v) glucose were found to remarkably promote demineralization efficiency, without affecting deproteinization rates. The antioxidant activities of hydrolysates, at different concentrations, produced during fermentation in medium supplemented with glucose, were determined using different tests: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method, reducing power assay and chelating activity. All hydrolysates showed varying degrees of antioxidant activity. Hydrolysate produced by B. pumilus A1 exhibited the highest DPPH radical scavenging activity, with an IC(50) value of 0.3 mg/ml. Highest reducing power (DO 700 nm=1.55 at 1.5 mg/ml) and metal chelating activity (98% at 5mg/ml) were obtained with B. pumilus A1 and B. licheniformis RP1 hydrolysates, respectively.

Pseudomonas aeruginosa A2 elastase: purification, characterization and biotechnological applications.

An extracellular protease from Pseudomonas aeruginosa A2 grown in media containing shrimp shell powder as a unique source of nutriments was purified and characterized. The enzyme was purified to homogeneity from culture supernatant by ultrafiltration, Sephadex G-100 gel filtration and Sepharose Mono Q anion exchange chromatography, with a 2.23-fold increase in specific activity and 64.3% recovery. The molecular mass of the enzyme was estimated to be 34 kDa. Temperature and pH with highest activity were 60 °C and 8.0, respectively. The protease activity was inhibited by EDTA suggesting that the purified enzyme is a metalloprotease. The enzyme is stable in the presence of organic solvents mainly diethyl ether and DMSO. The lasB gene, encoding the A2 elastase, was isolated and its DNA sequence was determined. The A2 protease was tested for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3 h hydrolysis at 40 °C with an enzyme/substrate (E/S) ratio of 5 U/mg protein was about 75%. Additionally, A2 proteolytic preparation demonstrated powerful depilating capabilities of hair removal from bovine skin. Considering its promising properties, P. aeruginosa A2 protease may be considered a potential candidate for future use in several biotechnological processes.

Solvent-stable digestive alkaline proteinases from striped seabream (Lithognathus mormyrus) viscera: characteristics, application in the deproteinization of shrimp waste, and evaluation in laundry commercial detergents.

Alkaline proteases from the viscera of the striped seabream (Lithognathus mormyrus) were extracted and characterized. Interestingly, the crude enzyme was active over a wide range of pH from 6.0 to 11.0, with an optimum pH at the range of 8.0-10.0. In addition, the crude protease was stable over a broad pH range (5.0-12.0). The optimum temperature for enzyme activity was 50 °C. The crude alkaline proteases showed stability towards various surfactants and bleach agents and compatibility with some commercial detergents. It was stable towards several organic solvents and retained more than 50% of its original activity after 30 days of incubation at 30 °C in the presence of 25% (v/v) dimethyl sulfoxide, N,N-dimethylformamide, diethyl ether, and hexane. The crude enzyme extract was also tested for shrimp waste deproteinization in the preparation of chitin. The protein removal with a ratio enzyme/substrate of 10 was about 79%.