Characterization of a novel protease from Bacillus cereus and evaluation of an eco-friendly hydrolysis of a brewery byproduct

Proteases and proteolytic enzymes constitute one of the most important groups of enzymes and are attracting worldwide attentionin attempts to exploit their physiological and biotechnological applications. In this study, partial purifications and biochemicaland antimicrobial characterizations of a protease from Bacillus cereus spp., originally isolated from fermented cabbage, werecarried out. The crude extract obtained after purification, involving ammonium sulphate precipitation and dialysis, was designatedas a partially purified protease (PPP). The obtained PPP had a specific activity of 0.395?2.539 U/g at 32 °C, with maximumactivities for the fractions precipitated at 60 and 80% ammonium sulphate. The PPP activity ranged between 20 and 55 °C, withan optimum temperature at 40 °C. At 60 °C, the PPP retained more than 30% of its activity. The optimum pH for the PPP wasachieved at pH 9, indicating the alkaline source of the enzyme. Protease production was specifically dependent on the calciumconcentration in the culture medium. Also the robustness of the protease on brewer?s spent grain hydrolysis was demonstrated.This suggests a potential eco-friendly application of the enzyme. Finally, it was found that the PPP inhibited the growth ofEscherichia coli O157:H7. This novel property of the PPP liberated by the B. cereus spp. could provide important future benefitsto industry.Fil: Kotlar, Catalina Elena. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Ponce, Alejandra Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química; ArgentinaFil: Roura, Sara Ines. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentin

Improvement of functional and antimicrobial properties of brewery byproduct hydrolysed enzymatically

The aim of this study is to investigate the characteristics of brewer's spent grain (BSG) hydrolysates. Hydrolysis was performed using a Bacillus cereus sp. extracellular peptidase. The proteins were hydrolysed for 2, 8 and 24 h, achieving degree of hydrolysis ranging from 2.65 to 21.80%. During enzymatic hydrolysis, average peptide chain length decreased rapidly and the soluble forms increased. Solubility of the hydrolysates showed a good correlation with turbidity. The water/oil holding capacity, the emulsifying properties and the foaming expansion were analysed, and improved functional properties were found respect to the control. For the studied hydrolysates concentrations no gel formation were obtained. However, BSG hydrolysates exhibited also desirable rheological properties making their good candidate for many food formulations. Thermal characterization revealed the energy recovery by the enzymatic hydrolysis process. The understanding of hydrolysates antimicrobial properties may lead to utilize their as potent natural antimicrobial against Escherichia coli O157:H7. In the light of the results, hydrolysates made from BSG can be converted into a high value protein food ingredient.Fil: Kotlar, Catalina Elena. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; ArgentinaFil: Ponce, Alejandra Graciela. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Roura, Sara Ines. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

A Peptidase Enzyme from Bacillus cereus with Antimicrobial Properties: Optimizing the Immobilization in Chitosan Beads Using Box-Behnken Design

Enzymes are exploited as catalysts in many industrial, biomedical, and analytical processes. There has been considerable interest in the development of carrier systems for enzyme immobilization because immobilized enzymes have enhanced stability compared to soluble enzymes, and can easily be separated from the reaction. In the current study, microbial peptidases liberated by B. cereus were immobilized in cross-linked chitosan beads and characterized using azocasein as a substrate. The Box-Behnken design was applied to determine the optimal conditions to maximize proteolytic activity. An empirical second-order model was determined by multiple regression analysis of the experimental data to describe the relationship between tested variables and the response. The determination coefficients (R2) were above 90%. Under optimal conditions (2.2 mm bead diameter, 1.06 enzyme/ bead ratio, 5.82% v/v glutaraldehyde and 18°C) the proteolytic activity was 0.938 U/ml. The retained immobilized enzyme can be reused up to five times. The storage stability of immobilized peptidases at 4°C was up to 10 days, while at 32°C the enzyme lost its activity within three days. Finally, novel antimicrobial properties of the immobilized peptidases were found. These results could have important benefit for the food industry.Fil: Kotlar, Catalina Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química; ArgentinaFil: Roura, Sara Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química; ArgentinaFil: Ponce, Alejandra Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química; Argentin

Isolation and characterization of bacterial strains with a hydrolytic profile with potential use in bioconversion of agroindustial by-products and waste

There is a trend towards the use of novel technologies nowadays, mainly focused on biological processes, for recycling and the efficient utilization of organic residues that can be metabolized by different microorganisms as a source of energy. In the present study the isolation of bacterial strains from six different agro-industrial by-products and waste was performed with the objective of evaluating their hydrolytic capacities and suitability for use in bioconversion of specific substrates. The 34 isolated strains were screened in specific culture media for the production of various hydrolytic enzymes (lipase, protease, cellulase, and amylase). It was found that 28 strains exhibited proteolytic activity, 18 had lipolytic activity, 13 had caseinolytic activity, 15 had amylolytic activity, and 11 strains exhibited cellulolytic activity. The strains that showed the highest hydrolytic capacities with biotechnological potential were selected, characterized genotipically, and identified as Bacillus, Serratia, Enterococcus, Klebsiella, Stenotrophomonas, Lactococcus, and Escherichia genera. It was concluded that the strain isolates have a high potential for use in the bioconversion of agro-industrial waste, both as a pure culture and as a microbial consortium

Development of a microbial consortium for dairy wastewater treatment

The wastewater from the dairy industries usually contains high concentrations of contaminants and, since the volume generated is also high, the total contaminant load is very significant. Among the available options for treatment, biological degradation looks like the most promising one. Furthermore, the supplementation of the native microbial populations with external microorganisms with high specific degradation rates (bio-augmentation) has demonstrated to improve the performance of treatment. The main objective of this research was to select a combination of bacteria to improve the aerobic treatment of dairy processing wastewater. For this purpose, eleven fat/protein-degrading microorganisms belonging to the genera Bacillus, Serratia, Lactococcus, Enterococcus, Stenotrophomonas, Klebsiella and Escherichia, were evaluated as potential degrading bacteria using a Plackett-Burman design. Assays were carried out to select the strains that most significantly influenced the degradation of wastewater and biomass yield, in terms of COD removal. A simulated dairy industry effluent was used as culture medium. Four strains were selected as potential members of the microbial consortium: Lactococcus garvieae, Bacillus thuringiensis, Escherichia coli and Stenotrophomonas sp. The optimal operation temperature and pH range of the selected consortium were 32°C and 6 ∼ 8, respectively. The degradation percentages reached with the selected consortium were 80.67 and 83.44% at 24 and 48 h, respectively. The selected consortium significantly improved the degradation of the dairy wastewater, and the degradation degree achieved by this consortium was higher than by using the strains individually.Fil: Mazzucotelli, Cintia Anabela. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química. Grupo de Investigación en Ingeniería en Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Durruty, Ignacio. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kotlar, Catalina Elena. Universidad Nacional de La Plata. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moreira, Maria del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química; ArgentinaFil: Ponce, Alejandra Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química; ArgentinaFil: Roura, Sara Ines. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin