Chitin and chitosan preparation from shrimp shells using optimized enzymatic deproteinization

Different crude microbial proteases were applied for chitin extraction from shrimp shells. A Box–Behnken design with three variables and three levels was applied in order to approach the prediction of optimal enzyme/substrate ratio, temperature and incubation time on the deproteinization degree with Bacillus mojavensis A21 crude protease. These optimal conditions were: an enzyme/substrate ratio of 7.75 U/mg, a temperature of 60 °C and an incubation time of 6 h allowing to predict 94 ± 4% deproteinization. Experimentally, in these optimized conditions, a deproteinization degree of 88 ± 5% was obtained in good agreement with the prediction and larger than values generally given in literature. The deproteinized shells were then demineralized to obtain chitin which was converted to chitosan by deacetylation and its antibacterial activity against different bacteria was investigated. Results showed that chitosan dissolved at 50 mg/ml markedly inhibited the growth of most Gram-negative and Gram-positive bacteria tested.     

Diversity of terrestrial isopods in the northern Tunisian wetlands

To evaluate the influence of wetland types on the distribution of terrestrial isopods, species richness, relative abundance and diversity indices were studied in the supralittoral zone of 95 wetlands in the north‐western of Tunisian dorsal, belonging to six types: lagoon, hill reservoir, river, dam, lake and sebkha. We tested the following hypothesis: (i) is isopod diversity influenced by wetland types? (ii) is isopod diversity influenced by bioclimatic zones? and (iii) what are the environmental factors influencing isopod distribution? A total of 3255 individuals belonging to twenty species of terrestrial isopods were captured. Species richness differs significantly between wetland types. A highly significant positive relationship between species richness and both humidity and altitudinal gradient was described. The dendrogram of similarities showed a divergence of the lagoons compared to the remaining wetland types.     

Identification and natural functions of cyclic lipopeptides from Bacillus amyloliquefaciens An6

Lipopeptides constitute a structurally diverse group of metabolites produced by various bacterial and fungal genera. In the past decades, research on lipopeptides has been fueled by their surfactant activities. However, natural functions of lipopeptides compounds have received considerably less attention. The aim of this study was to isolate and identify the lipopeptides from Bacillus amyloliquefaciens An6, and further evaluate their biological activities. An6 lipopeptides were detected by PCR using degenerated primers and MALDI‐TOF‐MS. An6 strain was found to produce surfactin, fengycin, and bacillomycin. Following their purification, the in vitro antioxidant activity of An6 lipopeptides was studied through different assays. The scavenging effect on 1,1‐diphenyl‐2‐picrylhydrazyl radicals at a dosage of 0.75 mg/mL was 81%. Its reducing power was concentration‐dependant and reached a maximum of 1.07 at 2.5 mg/mL. Moreover, they showed a strong inhibition of β‐carotene bleaching. An6 lipopeptides mixture was also found to display significant antimicrobial activity against several Gram‐positive, Gram‐negative bacteria, and fungal strains. An6 lipopeptides were insensitive to proteolytic enzymes, stable between pH 4.0 and 12.0, and resistant to high temperature. Our results provided enough evidence proving that An6 lipopeptides could be used as functional‐food components.     

Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes

A protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa MN7. The strain was found to produce proteases when it was grown in media containing only shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. The use of 60 g/l SWP resulted in a high protease production. Elastase, the major protease produced by P. aeruginosa MN7, was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration, and ultrafiltration using a 10-kDa cut-off membrane, with a 5.2-fold increase in specific activity and 38.4% recovery. The molecular weight of the purified elastase was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for protease activity were 60 degrees C and 8.0, respectively. The activity of the enzyme was totally lost in the presence of ethylene glycol tetraacetic acid, suggesting that the purified enzyme is a metalloprotease. The purified enzyme was highly stable in the presence of organic solvents, retaining 100% of its initial activity after 60 days of incubation at 30 degrees C in the presence of dimethyl sulfoxide and methanol. The lasB gene, encoding the MN7 elastase, was isolated and its DNA sequence was determined.     

Optimization of alkaline protease production by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis> ES1 in <Emphasis Type="Italic">Mirabilis jalapa</Emphasis> tuber powder using statistical experimental design

Medium composition and culture conditions for the bleaching stable alkaline protease production by Aspergillus clavatus ES1 were optimized. Two statistical methods were used. Plackett-Burman design was applied to find the key ingredients and conditions for the best yield. Response surface methodology (RSM) including full factorial design was used to determine the optimal concentrations and conditions. Results indicated that Mirabilis jalapa tubers powder (MJTP), culture temperature, and initial medium pH had significant effects on the production. Under the proposed optimized conditions, the protease experimental yield (770.66 U/ml) closely matched the yield predicted by the statistical model (749.94 U/ml) with R (2)=0.98. The optimum operating conditions obtained from the RSM were MJTP concentration of 10 g/l, pH 8.0, and temperature of 30 degrees C, Sardinella heads and viscera flour (SHVF) and other salts were used at low level. The medium optimization contributed an about 14.0-fold higher yield than that of the unoptimized medium (starch 5 g/l, yeast extract 2 g/l, temperature 30 degrees C, and pH 6.0; 56 U/ml). More interestingly, the optimization was carried out with the by-product sources, which may result in cost-effective production of alkaline protease by the strain.     

Preparation and testing of Sardinella peptones: application to lipase production by Staphylococcus sp

Production of lipase by Staphylococcus sp. in media containing fish peptones from sardinelle (Sardinella aurita) prepared in the laboratory was studied. Lipase production is strongly affected by lipids present in fish flours. Fish peptones prepared from dIgresed whole flesh was an excellent substrate for lipase production. A comparison of lipase production in media containing fish peptones or high quality commercial peptones indicated that fish peptones enhanced enzyme formation.