RESUMO

Soybean is one of the most important commodities in the world, being applied in feed crops and food, pharmaceutical industries in different ways. Soy is rich in isoflavones that in aglycone forms have exhibited significant anti-obesity and anti-lipogenic effects. Obesity is a global problem as several diseases have been related to this worldwide epidemic. The aim of this work was to verify the effect of free and immobilized ß-glucosidase, testing Lentikats, and sol-gel as carriers. Moreover, we wanted to examine if the different types of hydrolysis would generate extracts with distinct biological activity concerning lipid accumulation, PPAR-α regulation, and TNF-α, IL-6, and IL-10 concentrations using in vitro assays. Our results show that all formulations of ß-glucosidase could hydrolyze soy isoflavones. Thus, after 24 h of incubation, daidzein content increased 2.6-, 10.8-, and 12.2-fold; and genistein content increased 11.7, 11.4, and 11.4 times with the use of free enzyme, Lentikats®, and sol-gel immobilized enzyme, respectively. Moreover, both methodologies for enzyme immobilization led to promising forms of biocatalysts for application in the production of soy extracts rich in isoflavones aglycones, which are expected to bring about health benefits. A mild lipogenic effect was observed for some concentrations of extracts, as well as a slight inhibition in PPAR-α expression, although no significant differences were noticeable in the cytokines TNF-α, IL-10, and IL-6 as compared with the control.

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BACKGROUND: Propolis has been used as a natural health product mainly due to the presence of polyphenols, flavonoids, phenolic aldehydes, amino acids, vitamins and others bioactive constituents. To this natural substance are attributed different biological and pharmacological properties which are influenced by its chemical composition and organoleptic properties. The aim of this work was to evaluate the physicochemical properties and parameters of green propolis collected during a period of six years (2008-2013) in the state of Minas Gerais, located at the southeastern region of Brazil. METHODS: The methodology were in accordance with Brazilian legislation on the identity and quality standards of propolis. The evaluated parameters of hydroalcoholic from green propolis were total flavonoids, antioxidant activity - DPPH method, oxidation index, wax content, humidity and insoluble impurities. RESULTS: Propolis samples collected in different seasons during the years 2008 to 2013 presented mean values of total flavonoids (3.4 ± 0.11 mg/g), antioxidant activity DPPH (4.76 ± 0.16 µg/mL), oxidation index (3, 4 ± 0.33 seconds) and wax (15.14 ± 0.78% m/m), which are in accordance with Brazilian legislation. CONCLUSION: Green propolis did not show abrupt seasonal changes during the six years of investigation, and may be considered as an adequate functional ingredient.

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ß-Glucosidases represent an important group of enzymes due to their pivotal role in various biotechnological processes. One of the most prominent is biomass degradation for the production of fuel ethanol from cellulosic agricultural residues and wastes, where the use of immobilized biocatalysts may prove advantageous. Within such scope, the present work aimed to evaluate the feasibility of entrapping ß-glucosidase in either sol-gel or in Lentikats supports for application in cellobiose hydrolysis, and to perform the characterization of the resulting bioconversion systems. The activity and stability of the immobilized biocatalyst over given ranges of temperature and pH values were assessed, as well as kinetic data, and compared to the free form, and the operational stability was evaluated. Immobilization increased the thermal stability of the enzyme, with a 10 °C shift to an optimal temperature in the case of sol-gel support. Mass transfer hindrances as a result of immobilization were not significant, for sol-gel support. Lentikats-entrapped glucosidase was used in 19 consecutive batch runs for cellobiose hydrolysis, without noticeable decrease in product yield. Moreover, encouraging results were obtained for continuous operation. In the overall, the feasibility of using immobilized biocatalysts for cellobiose hydrolysis was established.

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This study concerned the production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans strain 191. In shaken flasks the maximum yield of chitinase was 6.9 U/mL after 72 h of cultivation at 25ºC and 200 rpm. In a 5 L fermenter with 1.5 vvm aeration, the highest yield obtained was 4.19 U/mL after 168 h of fermentation at 25ºC and 200 rpm, and using 3 vvm, it was 4.38 U/mL after 144 h of fermentation. The chitinase (61 KDa) was purified about 6.65 times by Sepharose CL 4B 200 gel filtration with a yield of 46.61 percent. The purified enzyme was able to lyse the cell walls of some fungi and to form protoplasts.

O presente estudo visou a produção, purificação e aplicação da quitinase extracelular da linhagem Cellulosimicrobium cellulans 191. A maior produção de quitinase em frascos agitados foi 6,9 U/mL após 72 h de fermentação a 25ºC e 200 rpm. Em fermentador de 5 L utilizando aeração de 1,5 vvm, a maior atividade da enzima foi 4,19 U/mL após 168 h de fermentação a 25ºC e 200 rpm; e com 3 vvm, foi obtido 4,38 U/mL após 144 h de fermentação. A quitinase (61 KDa) foi purificada cerca de 6,65 vezes em coluna de filtração em gel Sepharose CL 4B 200 com um rendimento de 46,61 por cento. A enzima purificada foi capaz de lisar a parede celular de alguns fungos e formar protoplastos.

RESUMO

Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell walldegradingenzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showedthat the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of(NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC.

Cellulosimicrobium cellulans é um microrganismo que produz uma variedade de enzimas que hidrolisam a parede celular de leveduras: β-1,3-glucanase, protease e quitinase. Célulasdesidratadas de Saccharomyces cerevisiae foram usadas como fonte de carbono e nitrogênio para o crescimento celular e produção de protease. Os componentes do meio de cultura: KH2PO4, KOH e células de levedura desidratadas mostraram efeitos significativos (p<0,05) no planejamento experimental fracionário. Um segundo planejamento foi preparado usandodois fatores: pH e porcentagem de células de levedura desidratadas. Os resultados mostraram que o meio de cultura para a produção máxima de protease foi 0,2 g/L de MgSO4.7H2O;2,0 g/L de (NH4)2SO4 e 8% de células de levedura desidratadas em tampão fosfato 0,15M e pH 8,0. A produção máxima de protease alcalina foi 7,0 ± 0,27 U/mL no ponto central. A proteasebruta apresentou atividade ótima a 50ºC e pH 7,0-8,0; e foi estável a 50ºC.

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The strain Klebsiella sp. K18 produces the enzyme glucosyltransferase and catalyses the conversion of sucrose to palatinose, an alternative sugar that presents low cariogenicity. Response Surface Methodology was successfully employed to determine the optimal concentration of culture medium components. Maximum glucosyltransferase production (21.78 U mL-1) was achieved using the optimized medium composed by sugar cane molasses (80 g L-1), bacteriological peptone (7 g L-1) and yeast extract (20 g L-1), after 8 hours of fermentation at 28ºC. The conversion of sucrose to palatinose was studied utilizing immobilized cells in calcium alginate. The effects of the alginate concentration (2-4%), cell mass concentration (20-40%) and substrate concentration (25-45%) were evaluated and the yield of palatinose was approximately 62.5%.

A linhagem Klebsiella sp. K18 produz a enzima glicosiltransferase que catalisa a conversão de sacarose em palatinose, um açúcar alternativo que apresenta baixa cariogenicidade. Metodologia de Superfície de Resposta foi empregada com sucesso para determinar a concentração ótima dos componentes do meio de cultivo. A máxima produção deglicosiltransferase (21,78 U mL-1) foi obtida utilizando o meio de cultivo otimizado composto por melaço de cana de açúcar (80 g L-1), peptona bacteriológica (7 g L-1) e extrato de levedura (20 g L-1), após 8 horas de fermentação a 28ºC. A conversão desacarose em palatinose foi estudada utilizando células imobilizadas em alginato de cálcio. Os efeitos da concentração de alginato (2-4%), concentração de massa celular (20-40%) e concentração de substrato (25-45%) foram avaliados e a porcentagem de palatinose foi de aproximadamente 62,5%.

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Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, ß-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showed that the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of (NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC.

RESUMO

The strain Klebsiella sp. K18 produces the enzyme glucosyltransferase and catalyses the conversion of sucrose to palatinose, an alternative sugar that presents low cariogenicity. Response Surface Methodology was successfully employed to determine the optimal concentration of culture medium components. Maximum glucosyltransferase production (21.78 U mL(-1)) was achieved using the optimized medium composed by sugar cane molasses (80 g L(-1)), bacteriological peptone (7 g L(-1)) and yeast extract (20 g L(-1)), after 8 hours of fermentation at 28°C. The conversion of sucrose to palatinose was studied utilizing immobilized cells in calcium alginate. The effects of the alginate concentration (2-4%), cell mass concentration (20-40%) and substrate concentration (25-45%) were evaluated and the yield of palatinose was approximately 62.5%.

RESUMO

This study concerned the production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans strain 191. In shaken flasks the maximum yield of chitinase was 6.9 U/mL after 72 h of cultivation at 25°C and 200 rpm. In a 5 L fermenter with 1.5 vvm aeration, the highest yield obtained was 4.19 U/mL after 168 h of fermentation at 25°C and 200 rpm, and using 3 vvm, it was 4.38 U/mL after 144 h of fermentation. The chitinase (61 KDa) was purified about 6.65 times by Sepharose CL 4B 200 gel filtration with a yield of 46.61%. The purified enzyme was able to lyse the cell walls of some fungi and to form protoplasts.

RESUMO

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.

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