Islem Younes

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 β-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 β-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 β-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.

Influence of acetylation degree and molecular weight of homogeneous chitosans on antibacterial and antifungal activities

The results given in the literature are conflicting when considering the relationship between antimicrobial activity and chitosan characteristics. To be able to clarify, we prepared fifteen homogeneous chitosans with different acetylation degrees (DA) and molecular weights (MW) by reacetylation of a fully deacetylated chitin under homogeneous conditions. They were tested at different pH values for their antimicrobial activities against four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi), four Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis and Micrococcus luteus) and three fungi (Aspergillus niger, Fusarium oxysporum and Alternaria solani). Chitosans markedly inhibited growth of most bacteria and fungi tested, although the inhibitory effect depends on the type of microorganism and on the chitosan characteristics (DA and MW) with minimum inhibitory concentrations in the range of 0.001 to 0.1 w%. Considering chitosan efficiency on bacteria, our series of data clearly show that the lower DA and the lower pH give the larger efficiency. Antibacterial activity was further enhanced for Gram-negative bacteria with decreasing MW, whereas, opposite effect was observed with the Gram-positive. Concerning the antifungal activity, the influence of chitosan characteristics was dependent on the particular type of fungus. Fungal growth decreased with increasing MW for F. oxysporum and decreasing DA for A. solani, but no MW or DA dependences were observed with A. niger.

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.

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.

Shrimp waste fermentation with Pseudomonas aeruginosa A2: Optimization of chitin extraction conditions through Plackett–Burman and response surface methodology approaches

A Box-Bhenken design with four variables (shrimp shell concentration (SSC), glucose concentration, incubation time and inoculum size) and three levels was used for the determination of the deproteinization and demineralization efficiencies in fermented shrimp shells by Pseudomonas aeruginosa A2. The fermentation variables were selected in accordance with Plackett-Burman design. Maximum demineralization of 96%, with about 89% of protein removal occurs under the following conditions: SSC 50 g/l, glucose 50 g/l, 5 days and inoculum of 0.05 OD. This environment friendly method (biological treatment) can be considered as an effective pretreatment to produce a high-quality chitin.

Structural, physicochemical and antioxidant properties of sodium alginate isolated from a Tunisian brown seaweed

An original sodium alginate from Tunisian seaweed (Cystoseira barbata) was purified and characterized by circular dichroism (CD) and ATR-FTIR spectroscopies. ATR-FTIR spectrum of C. barbata sodium alginate (CBSA) showed the characteristic bands of mannuronic (M) and guluronic acids (G). The M/G ratio was estimated by CD (M/G = 0.59) indicating that CBSA was composed of 37% mannuronic acid and 63% guluronic acid. The analysis of viscosity of CBSA showed evidence of pseudoplastic fluid behaviour. The emulsifying capacity of CBSA was evaluated at different concentrations (0.25-3%), temperatures (25-100 °C) and pH (3.0-11.0). Compared to most commercial emulsifiers, the emulsion formulated by CBSA was found to be less sensitive to temperature changes and more stable at acidic pH. CBSA was examined for antioxidant properties using various antioxidant assays. CBSA exhibited important DPPH radical-scavenging activity (74% inhibition at a concentration of 0.5 mg/ml) and considerable ferric reducing potential. Effective hydroxyl-radical scavenging activity (82% at a concentration of 5 mg/ml) and potent protection activity against DNA breakage were also recorded for CBSA. However, in the linoleate-β-carotene system, CBSA exerted moderate antioxidant activity (60% at a concentration of 1.5 mg/ml). Therefore, CBSA can be used as a natural ingredient in food industry or in the pharmaceutical field.

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.

Cytotoxicity of chitosans with different acetylation degrees and molecular weights on bladder carcinoma cells

The purpose of this research was to evaluate the cytotoxicity of chitosans with different degrees of acetylation (DA) and molecular weights (MW), as well as the effect of their positive ionic charges controlled by pH on bladder carcinoma cells (RT112 and RT112cp) using the tetrazolium salt colorimetric (MTT) assay. Our data showed that all chitosan samples were cytotoxic on RT112 and RT112cp cells with a higher cytotoxicity obtained at lower pH. Further, it was found that the toxicity increased with increasing DA. However, no significant difference in cytotoxicity between chitosans with different molecular weights was observed. Annexin V-FITC staining test was then used to study and quantify the induction of apoptosis. Data shows that chitosans induce apoptosis of RT112 and RT112cp cells with the same dependence with DA.

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.

Golden Grey Mullet (Liza aurata) Alkaline Proteases: Biochemical Characterization, Application in the Shrimp Wastes Deproteinization, Laundry Commercial Detergents, and Preparation of Antioxidant Protein Hydrolysate

Digestive alkaline proteinases from golden grey mullet (Liza aurata) were extracted and characterized. The crude alkaline protease showed optimum activity at pH 8.0 and 60°C, and it was highly stable over a wide range of pH from 4.0 to 10.0, retaining more than 80% activity after incubation for 1 h at 4°C. The alkaline proteases showed extreme stability toward nonionic and anionic surfactants after preincubation for 1 h at 25°C and relative stability toward oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various solid and liquid detergents. Further, proteases from golden grey mullet viscera were found to be effective in the deproteinization of shrimp wastes. The protein removal after 3 h at 45°C with an enzyme/substrate (E/S) ratio of 10 U/mg protein was about 76%. The golden grey mullet proteases were also shown to be efficient in the production of antioxidant protein hydrolysate.