Biomedical potential of chitosan-silver nanoparticles with special reference to antioxidant, antibacterial, hemolytic and in vivo cutaneous wound healing effects.

The aim of the present study was to prepare chitosan-PVA-silver nanoparticles (CS-AgNPs) through green method. Chitosan and PVA polymers acted as stabilizing agents. DLS and TEM analyses showed that CS-AgNPs were homogeneously dispersed in matrix with an average size of 190-200 nm. The CS-AgNPs were tested for their antioxidant and antibacterial properties and the results revealed that they exhibited higher antioxidant activity than CS powder. Moreover, CS-AgNPs were characterized by a low cytotoxicity effect at 5-200 µg/ml against Chinese Hamster Ovary (CHO-K1) cells. In addition, the prepared CS-Ag NPs were found to promote significantly the wound healing, as determined by the wound contraction ratio and histological examination. A significant improvement in wound healing progression and in oxidative stress damage were observed for CS, CS-PVA and CS-AgNPs-treated wound tissues, when compared to control and CICAFLORA®-treated groups. The wound healing effect could be attributed to the antibacterial and antioxidant synergy of AgNPs and CS. Results strongly support the possibility of using CS-AgNPs for wound care applications.

Chitin extraction from blue crab (Portunus segnis) and shrimp (Penaeus kerathurus) shells using digestive alkaline proteases from P. segnis viscera.

Since chitin is closely associated with proteins, deproteinization is a crucial step in the process of extracting chitin. Thus, this research aimed to extract chitin from Portunus segnis and Penaeus kerathurus shells by means of crude digestive alkaline proteases from the viscera of P. segnis, regarding deproteinization step, as an alternative to chemical treatment. Casein zymography revealed that five caseinolytic proteases bands exist, suggesting the presence of at least five different major proteases. The optimum pH and temperature for protease activity were pH 8.0 and 60°C, respectively, using casein as a substrate. The crude enzymes extract was highly stable at low temperatures and over a wide range of pH from 6.0 to 12.0. The crude alkaline protease extract was found to be effective in the deproteinization of blue crab and shrimp shells, to produce chitin. The best efficiency in deproteinization (84.69±0.65% for blue crab shells and 91.06±1.40% for shrimp shells) was achieved with an E/S ratio of 5U/mg of proteins after 3h incubation at 50°C. These results suggest that enzymatic deproteinization of crab and shrimp wastes by fish endogenous alkaline proteases could be a potential alternative in the chitin production process.

Alkaline proteases from a newly isolated Micromonospora chaiyaphumensis S103: Characterization and application as a detergent additive and for chitin extraction from shrimp shell waste.

The present study was undertaken to characterize the extracellular thermostable serine alkaline proteases from newly actinomycete strain Micromonospora chaiyaphumensis S103 and to describe their evaluation in commercial detergents and shrimp waste deproteinization. This proteolytic crude extract was active and stable in alkaline solution. It was extremely stable in the pH range of 5.0-12.0. The optimum pH and temperature were 8.0 and 70°C, respectively, using casein as a substrate. The thermoactivity and thermostability of proteases were enhanced by the addition of 5mM Ca2+. Proteases from S103 were also used for shrimp wastes deproteinization in the process of chitin preparation. The percent of protein removal after 3h hydrolysis at 45°C with an enzyme/substrate ratio of 20U/mg had reached 93%. Furthermore, S103 crude enzyme was stable towards several organic solvents and retained 100% of its original activity after 90days of incubation in the presence of methanol, hexane, acetone, and DMSO. These properties make S103 proteases an ideal choice for application in detergent formulations, chitin production, and enzymatic peptide synthesis.

Structural analysis, and antioxidant and antibacterial properties of chitosan-poly (vinyl alcohol) biodegradable films.

The development and characterization of biodegradable blend films based on chitosan and poly (vinyl alcohol) for possible use in a variety of biological activities are reported. Fourier transform infrared spectroscopy (FTIR) spectra of chitosan-poly (vinyl alcohol) (Ch/PVA) films showed characteristics peaks shifting to a lower frequency range due to hydrogen bonding between -OH of PVA and -NH2 of chitosan. The chitosan and PVA polymers presented good compatibility. The morphology study of chitosan and composite films showed a compact and homogenous structure. The tensile strength and elongation at break increased with PVA content. In fact, the highest tensile strength and elongation at break (53.58 MPa and 454 %) occurs with pure PVA film. The results showed that PVA incorporation in the blends contributes to increase the intermolecular interactions, thus improving the mechanical properties. In addition, the prepared films demonstrated high antioxidant activities monitored by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging, reducing power, and ß-carotene bleaching activity. Nevertheless, PVA addition reduced antioxidant and antibacterial activities against Gram-positive and Gram-negative bacteria tested.

Optimization of proteins and minerals removal from shrimp shells to produce highly acetylated chitin.

Chitin and derivatives used for biomedical or pharmaceutical applications require a high level of purity and quality that are difficult to achieve. In this study, we propose to optimize the extraction of chitin in order to obtain pure product keeping a structure as close as possible to the native form. Thus, demineralization step was firstly optimized using response surface methodology. In the optimized conditions predicted by the model, the obtained chitin has an acetylation degree (DA) and a demineralization degree (DM) equal to 99% and 100%, respectively. Then, different microbial and fish crude alkaline proteases were tested for their efficiency in deproteinization. Crude alkaline proteases giving the highest deproteinization degrees (DP), Bacillus mojavensis A21 and Scorpaena scrofa, were selected for chitin extraction. The obtained DP was 88±2% and 83±1%, respectively. At the end, effect of the use of mixed enzymatic treatment with the two selected crude enzymes and the order of demineralization/deproteinization steps were tested. The results demonstrated that two separated steps in enzymatic treatments realized on demineralized sample give the best DP (96%) preserving the DA (99%).

Digestive alkaline proteases from thornback ray (Raja clavata): Characteristics and applications.

This study describes the characterization of a crude protease extract from thornback ray (Raja clavata) and its evaluation in liquid detergent and in deproteinizattion of shrimp waste. At least five clear caseinolytic proteases bands were observed in a zymogram. The crude protease showed optimum activity at pH 8.0 and 50 °C, and it was highly stable over pH range from 8.0 to 11.0. Proteolytic enzymes were very stable in non-ionic surfactants and in the presence of oxidizing agents, maintaining 70% of their activity after incubation for 1 h at 30 °C in the presence of 1% sodium perborate. In addition, they showed high stability and compatibility with various liquid laundry-detergents available in the Tunisian market. The crude extract retained 100% of its activity after preincubation for 60 min at 30 °C in the presence of Nadhif Perfect, Textil and Carrefour laundry detergents. Further, proteases from R. clavata viscera were used for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3 h hydrolysis at 45 °C with an enzyme/substrate ratio of 30 U/mg of proteins was 74%. These results suggest that enzymatic deproteinization of shrimp wastes by fish endogenous alkaline proteases could be applicable to the chitin production process.

Characterization and In Vitro Evaluation of Cytotoxicity, Antimicrobial and Antioxidant Activities of Chitosans Extracted from Three Different Marine Sources.

Chitins in the α and ß isomorphs were extracted from three Tunisian marine sources shrimp (Penaeus kerathurus) waste, crab (Carcinus mediterraneus) shells and cuttlefish (Sepia officinalis) bones. The obtained chitins were transformed into chitosans, the acid-soluble form of chitin. Chitosans were characterized and their biological activities were compared. Chitosan samples were then characterized by Fourier transform infrared spectroscopy (FTIR). The results showed that all chitosans presented identical spectra. Antimicrobial, antioxidant, and antitumor activities of the extracted chitosans were investigated. In fact, cuttlefish chitosan showed the highest DPPH radical-scavenging activity (83 %, 5 mg/ml), whereas it was 79 % and 76 % for shrimp and crab chitosans, respectively. However, in linoleate-ß-carotene system, cuttlefish and crab chitosans exerted higher antioxidant activity (82 % and 70 %, respectively), than shrimp chitosan (49 %). Chitosans were tested for their antimicrobial activities against three Gram-negative and four Gram-positive bacteria and five fungi. Chitosans markedly inhibited growth of most bacteria and fungi tested, although the antimicrobial activity depends on the type of microorganism and on the source of chitin. In addition, chitosans showed high antitumor activity which seemed to be dependent on the chitosan characteristics such as acetylation degree and especially the molecular weight.

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.

Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan.

Chitin was recovered through enzymatic deproteinization of the shrimp processing by-products. Different microbial and fish viscera proteases were tested for their deproteinization efficiency. High levels of protein removal of about 77±3% and 78±2% were recorded using Bacillus mojavensis A21 and Balistes capriscus proteases, respectively, after 3h of hydrolysis at 45°C using an enzyme/substrate ratio of 20U/mg. Therefore, these two crude proteases were used separately for chitin extraction and then chitosan preparation by N-deacetylation. Chitin and chitosan samples were then characterized by 13 Cross polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy and compared to samples prepared through chemical deproteinization. All chitins and chitosans showed identical spectra. Chitosans prepared through enzymatic deproteinization have practically the same acetylation degree but higher molecular weights compared to that obtained through chemical process. Antimicobial, antioxidant and antitumoral activitities of chitosan-M obtained by treatment with A21 proteases and chitosan-C obtained by alkaline treatment were investigated. Results showed that both chitosans inhibited the growth of most Gram-negative, Gram-positive bacteria and fungi tested. Furthermore, both chitosans exhibited antioxidant and antitumor activities which was dependent on the molecular weight.

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.