RESUMEN
The aim of this study was to optimize the mechanical and texture properties of edible film improving its antibacterial property after adding rosemary essential oil (REO) using a Doehlert matrix. Films with the highest mechanical properties were acquired using a polymer composition of 65.2% glycerol, 24.3% gelatin, 10.0% chitosan and 0.5% pectin. This composition provided the highest elongation at break, tensile strength and texture values, which were respectively 51.60 ± 6.04%, 8.53 ± 2.36 MPa and 13.67 ± 1.43. The antibacterial activity of REO enriched films against Bacillus subtilis, Staphylococcus aureus, Enterococcus aerogenes, Enterococcus faecalis and Escherichia coli was enhanced when applying a mixture of 1.995 and 1.250 mg/g of two REO extracted from two rosemary different varieties. The structural, optical and barrier properties of the films were evaluated. To conclude, the enriched film showed potential coatings for controlling most common food borne bacteria growth during the food storage.
Asunto(s)
Quitosano/química , Gelatina/química , Aceites Volátiles/química , Pectinas/química , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Bovinos , Composición de Medicamentos , Fenómenos Mecánicos , Aceites Volátiles/farmacologíaRESUMEN
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.
Asunto(s)
Quitosano/química , Quitosano/farmacología , Acetilación , Anexinas/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Resistencia a Antineoplásicos , Humanos , Concentración de Iones de Hidrógeno , Peso Molecular , Neoplasias de la Vejiga UrinariaRESUMEN
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%).
Asunto(s)
Exoesqueleto/química , Quitina/química , Crustáceos/química , Minerales/química , Proteínas/química , Acetilación , Animales , Bacillus/enzimología , Biodegradación Ambiental , Péptido Hidrolasas/químicaRESUMEN
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.
Asunto(s)
Antiinfecciosos/farmacología , Antioxidantes/farmacología , Organismos Acuáticos/química , Quitosano/aislamiento & purificación , Quitosano/farmacología , Animales , Antifúngicos/farmacología , Antineoplásicos/farmacología , Compuestos de Bifenilo/química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Quitina/farmacología , Hongos/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Oxidación-Reducción/efectos de los fármacos , Picratos/química , Espectroscopía Infrarroja por Transformada de Fourier , beta Caroteno/químicaRESUMEN
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.
Asunto(s)
Exoesqueleto/química , Antibacterianos/farmacología , Antioxidantes/farmacología , Braquiuros/química , Quitina/aislamiento & purificación , Mezclas Complejas/farmacología , Animales , Antibacterianos/química , Antioxidantes/química , Bacillus/efectos de los fármacos , Bacillus/enzimología , Proteínas Bacterianas/metabolismo , Compuestos de Bifenilo/antagonistas & inhibidores , Mezclas Complejas/química , Pruebas Antimicrobianas de Difusión por Disco , Fermentación , Glucosa/metabolismo , Glucosa/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/crecimiento & desarrollo , Hidrólisis , Radical Hidroxilo/antagonistas & inhibidores , Picratos/antagonistas & inhibidoresRESUMEN
This review describes the most common methods for recovery of chitin from marine organisms. In depth, both enzymatic and chemical treatments for the step of deproteinization are compared, as well as different conditions for demineralization. The conditions of chitosan preparation are also discussed, since they significantly impact the synthesis of chitosan with varying degree of acetylation (DA) and molecular weight (MW). In addition, the main characterization techniques applied for chitin and chitosan are recalled, pointing out the role of their solubility in relation with the chemical structure (mainly the acetyl group distribution along the backbone). Biological activities are also presented, such as: antibacterial, antifungal, antitumor and antioxidant. Interestingly, the relationship between chemical structure and biological activity is demonstrated for chitosan molecules with different DA and MW and homogeneous distribution of acetyl groups for the first time. In the end, several selected pharmaceutical and biomedical applications are presented, in which chitin and chitosan are recognized as new biomaterials taking advantage of their biocompatibility and biodegradability.
Asunto(s)
Organismos Acuáticos/química , Quitina/química , Quitosano/química , Acetilación , Animales , Quitina/aislamiento & purificación , Quitina/farmacología , Quitosano/aislamiento & purificación , Quitosano/farmacología , Humanos , Peso Molecular , SolubilidadRESUMEN
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-ß-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.
Asunto(s)
Alginatos/química , Alginatos/aislamiento & purificación , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Fenómenos Químicos , Phaeophyceae/química , Algas Marinas/química , Alginatos/farmacología , Antioxidantes/farmacología , Compuestos de Bifenilo/química , Dicroismo Circular , Roturas del ADN de Cadena Simple/efectos de los fármacos , Emulsiones/química , Depuradores de Radicales Libres/química , Ácido Glucurónico/química , Ácido Glucurónico/aislamiento & purificación , Ácido Glucurónico/farmacología , Ácidos Hexurónicos/química , Ácidos Hexurónicos/aislamiento & purificación , Ácidos Hexurónicos/farmacología , Concentración de Iones de Hidrógeno , Radical Hidroxilo/química , Peso Molecular , Oxidación-Reducción , Picratos/química , Reología , Espectroscopía Infrarroja por Transformada de Fourier , Túnez , Viscosidad , beta Caroteno/químicaRESUMEN
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.
Asunto(s)
Quitina/química , Acetilación , Quitosano/metabolismo , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , ViscosidadRESUMEN
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.
Asunto(s)
Exoesqueleto/química , Quitosano/aislamiento & purificación , Quitosano/farmacología , Crustáceos/química , Péptido Hidrolasas/metabolismo , Animales , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antioxidantes/aislamiento & purificación , Antioxidantes/metabolismo , Antioxidantes/farmacología , Bacterias/efectos de los fármacos , Línea Celular Tumoral , Quitosano/metabolismo , Hongos/efectos de los fármacos , Ratones , Minerales/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Antibacterianos , Bacterias/efectos de los fármacos , Quitosano/química , Quitosano/farmacología , Hongos/efectos de los fármacos , Acetilación , Antibacterianos/química , Antibacterianos/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Recuento de Colonia Microbiana , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad Microbiana , Peso MolecularRESUMEN
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.
Asunto(s)
Braquiuros/química , Quitina/química , Quitina/aislamiento & purificación , Quitosano/química , Quitosano/aislamiento & purificación , Decapodiformes/química , Penaeidae/química , Acetilación , Exoesqueleto/química , Exoesqueleto/metabolismo , Animales , Minerales/aislamiento & purificación , Peso Molecular , Péptido Hidrolasas/metabolismo , ViscosidadRESUMEN
The present study describes the characterization of crude protease extract from zebra blenny (Salaria basilisca) and its evaluation in liquid detergent and shrimp waste deproteinization. At least five caseinolytic proteases clear bands were observed in zymogram. 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 6.0 to 11.0. Proteolytic enzymes showed extreme stability towards non-ionic surfactants (5 % Tween 80 and 5 % Triton X-100) and oxidizing agents (1 % sodium perborate), and relative stability towards anionic surfactant (1 % Sodium dodecyl sulfate (SDS)). They also showed high stability and compatibility with various laundry liquid detergents from Tunisian market. Furthermore, the crude enzyme 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 50 % (v/v) dimethylsulfoxide (DMSO). Further, proteases from zebra blenny viscera were found to be effective in the deproteinization of shrimp wastes. The protein removal after 3 h at 40 °C with an enzyme/substrate ratio (E/S) of 5 U/mg protein was about 77 %.
RESUMEN
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.
Asunto(s)
Exoesqueleto/química , Bacillus/metabolismo , Quitina/química , Decápodos/química , Animales , Biodegradación Ambiental , Modelos TeóricosRESUMEN
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.
Asunto(s)
Exoesqueleto/química , Bacillus/metabolismo , Quitina/aislamiento & purificación , Decápodos/química , Depuradores de Radicales Libres/aislamiento & purificación , Residuos Industriales , Quelantes del Hierro/aislamiento & purificación , Animales , Bacillus/crecimiento & desarrollo , Compuestos de Bifenilo/química , Quitina/biosíntesis , Quitina/química , Medios de Cultivo/química , Fermentación , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/metabolismo , Glucosa/química , Quelantes del Hierro/metabolismo , Quelantes del Hierro/farmacología , Oxidación-Reducción , Péptido Hidrolasas/biosíntesis , Picratos/químicaRESUMEN
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.
Asunto(s)
Biotecnología/métodos , Elastasa Pancreática/aislamiento & purificación , Elastasa Pancreática/metabolismo , Pseudomonas aeruginosa/enzimología , Secuencia de Aminoácidos , Exoesqueleto/química , Animales , Bovinos , Quitina/química , Estabilidad de Enzimas , Remoción del Cabello , Concentración de Iones de Hidrógeno , Metales/farmacología , Datos de Secuencia Molecular , Compuestos Orgánicos/farmacología , Elastasa Pancreática/química , Elastasa Pancreática/genética , Pseudomonas aeruginosa/genética , Análisis de Secuencia , Solventes/farmacología , TemperaturaRESUMEN
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.
Asunto(s)
Estructuras Animales/química , Quitina/aislamiento & purificación , Modelos Químicos , Penaeidae/anatomía & histología , Pseudomonas aeruginosa/metabolismo , Residuos , Estructuras Animales/anatomía & histología , Animales , Quitina/análisis , Fermentación , Explotaciones Pesqueras , Espectroscopía de Resonancia MagnéticaRESUMEN
A solvent-stable protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa A2. The strain was found to produce high level of protease activity when grown in media containing only fresh shrimp waste (FSW) or shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. Maximum protease activities 17,000 and 12,000 U/mL were obtained with 80 g/L SWP and 135 g/L FSW, respectively. The optimum temperature and pH for protease activity were 60 °C and 8.0, respectively. The crude protease, at different enzyme/substrate (E/S) ratio, was tested for the deproteinization of shrimp waste to produce chitin. The crude enzyme of P. aeruginosa A2 was found to be effective in the deproteinization of shrimp waste. The protein removals after 3 h hydrolysis at 40 °C with an E/S ratio of 0.5 and 5 U/mg protein were about 56% and 85%, respectively. (13)C CP/MAS-NMR spectral analysis of the chitin prepared by treatment with the crude protease was carried out and was found to be similar to that of the commercial α-chitin. These results suggest that enzymatic deproteinization of shrimp waste by A2 protease could be applicable to the chitin production process.
Asunto(s)
Quitina/aislamiento & purificación , Decápodos , Metaloproteasas/química , Pseudomonas aeruginosa/enzimología , Animales , Quitina/química , Cinética , Metaloproteasas/metabolismo , Pseudomonas aeruginosa/metabolismo , Solventes/metabolismo , TemperaturaRESUMEN
The aim of this work was to study some biochemical characteristics of crude alkaline protease extracts from the viscera of goby (Zosterisessor ophiocephalus), thornback ray (Raja clavata), and scorpionfish (Scorpaena scrofa), and to investigate their applications in the deproteinization of shrimp wastes. At least four caseinolytic proteases bands were observed in zymogram of each enzyme preparation. The optimum pH for enzymatic extracts activities of Z. ophiocephalus, R. clavata, and S. scrofa were 8.0-9.0, 8.0, and 10.0, respectively. Interestingly, all the enzyme preparations were highly stable over a wide range of pH from 6.0 to 11.0. The optimum temperatures for enzyme activity were 50°C for Z. ophiocephalus and R. clavata and 55°C for S. scrofa crude alkaline proteases. Proteolytic enzymes showed high stability towards non-ionic surfactants (5% Tween 20, Tween 80, and Triton X-100). In addition, crude proteases of S. scrofa, R. clavata, and Z. ophiocephalus were found to be highly stable towards oxidizing agents, retaining 100%, 70%, and 66%, respectively, of their initial activity after incubation for 1 h in the presence of 1% sodium perborate. They were, however, highly affected by the anionic surfactant SDS. The crude alkaline proteases were tested for the deproteinization of shrimp waste in the preparation of chitin. All proteases were found to be effective in the deproteinization of shrimp waste. The protein removals after 3 h of hydrolysis at 45°C with an enzyme/substrate ratio (E/S) of 10 were about 76%, 76%, and 80%, for Z. ophiocephalus, R. clavata, and S. scrofa crude proteases, respectively. These results suggest that enzymatic deproteinization of shrimp wastes by fish endogenous alkaline proteases could be applicable to the chitin production process.
RESUMEN
Chitin is a polysaccharide found in abundance in the shell of crustaceans. In this study, the protease from Bacillus cereus SV1 was applied for chitin extraction from shrimp waste material of Metapenaeus monoceros. A high level of deproteinization 88.8% +/- 0.4 was recorded with an E/S ratio of 20. The demineralization was completely achieved within 6 h at room temperature in HCl 1.25 M, and the residual content of calcium in chitin was below 0.01%. (13)C CP/MAS-NMR spectral analysis of chitin prepared by the enzymatic deproteinization of shrimp wastes was found to be similar to that obtained by alkaline treatment and to the commercial alpha-chitin. The degree of N-acetylation, calculated from the spectrum, was 89.5%. Chitin obtained by treatment with crude protease from B. cereus was converted to chitosan by N-deacetylation, and the antibacterial activity of chitosan solution against different bacteria was investigated. Results showed that chitosan solution at 50 mg/mL markedly inhibited the growth of most Gram-negative and Gram-positive bacteria tested. Furthermore, the antioxidant potential of the protein hydrolysates obtained during enzymatic isolation of chitin was evaluated using various in vitro assays. All the samples exerted remarkable antioxidant activities. These results suggest that enzymatic deproteinization of the shrimp shell wastes, using B. cereus SV1 protease, could be applicable to the chitin production process.
Asunto(s)
Bacillus cereus/enzimología , Quitina/metabolismo , Quitosano/metabolismo , Crustáceos/metabolismo , Péptido Hidrolasas/metabolismo , Hidrolisados de Proteína/metabolismo , Animales , Antibacterianos/aislamiento & purificación , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antioxidantes/farmacología , Quitina/aislamiento & purificación , Quitosano/aislamiento & purificación , Quitosano/farmacología , Industria de Alimentos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Residuos Industriales , Hidrolisados de Proteína/aislamiento & purificación , Hidrolisados de Proteína/farmacologíaRESUMEN
A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30 degrees C in media containing casein as carbon source (14,000 U ml(-1)). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60 degrees C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K (m) and K (cat) of the purified enzyme using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide were 0.158 mM and 1.14 x 10(5) min(-1), respectively. The catalytic efficiency (K (cat) /K (m)) was 7.23 x 10(8) min(-1) M(-1). The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.