Aqueous Two Phase Systems (ATPS) composed of 2-propanol + inorganic salt (Na2SO4/MgSO4) + water, at different temperatures T = (20, 30, 40 and 50) °C were applied in the partition studies of the textile dyes Yellow, Blue Royal and Red Dianix CC. The thermodynamic parameters of the free transfer energy (∆Gtr), enthalpy (∆Htr) and entropy (∆Str), respectively, were determined for dye partitioning, using the Van’t Hoff equation. The values of the partition coefficients of textile dyes Yellow, Royal Blue and Red Dianix CC, ranged from 14.21 to 1610.00, 15.15 to 994.00 and 17.32 to 311.75, respectively; and with dye extraction yields ranging from 93.17 to 99.93; 94.53 to 99.89 and 97.19 to 99.64, respectively. Based on the ∆Gtr results, it was verified that the dyes partition process was thermodynamically spontaneous, in all evaluated systems. For the 2-propanol + sodium sulfate ATPS, there was a prevalence of the enthalpic contribution over the entropic contribution (∆Htr > ∆Str), while for the 2-propanol + magnesium sulfate systems, a greater predominance of the entropic contribution was observed (∆Htr < ∆Str). The recovery yields (Y) values obtained were above 90% for all systems.

RESUMO A busca por proteases de origem vegetal aumenta gradualmente devido à sua diversidade e estabilidade em ampla faixa de pH e temperatura. As flores da alcachofra são uma fonte vegetal proteolítica já estudada, porém suas folhas são escassas nesse aspecto. Assim, o objetivo dessa pesquisa foi a obtenção de extratos das folhas da alcachofra com diferentes tampões e métodos de extração como fonte alternativa proteolítica e coagulante vegetal, bem como a separação e comparação do perfil proteico desses extratos. A metodologia utilizada foi baseada na extração com tampão de citrato de sódio (pH 3), acetato de sódio (pH 5) e tris-HCl (pH 7), por agitação mecânica (AM) e ultrassom (US); determinação de proteínas; atividade proteolítica (AP) e atividade específica (AE); atividade de coagulação do leite (ACL) e potencial de substituição do coalho (PSC); análise de cromatografia liquida de alta eficiência (HPLC) com detector UV-Vis e análise de componentes principais (ACP). Os extratos das folhas Cynara scolymus apresentaram resultados elevados no extrato com Citrato-US para os parâmetros AP (14,38), AE (19,71), ACL (440) e PSC (30,60) comparados aos demais tratamentos. Os extratos com citrato e acetato tiveram tempo de coagulação rápido (máx 3 min). A análise de HPLC possibilitou a separação dos diferentes compostos proteicos presentes nos extratos e picos mais expressivos nas amostras com Citrato-AM e Acetato-AM; e picos isolados para o Citrato-US. Conclui-se que os extratos das folhas da alcachofra com o tampão citrato e acetato atribuíram resultados satisfatórios para atuarem como coagulante vegetal, bem como para realização de novos estudos para purificação de enzimas proteolíticas e aplicação em queijos.

ABSTRACT The search for origin plant-based proteases increases gradually due to their diversity and stability over a wide range of pH and temperature. Artichoke (Cynara scolymus) flowers are a proteolytic vegetable source already studied, but their leaves are scarce in this respect. Thus, the objective of this research was to obtain extracts of artichoke leaves with different buffers and extraction methods as an alternative proteolytic source and plant coagulant, as well as the separation and comparison of the protein profile of these extracts. The methodology used was based on extraction with sodium citrate buffer (pH 3), sodium acetate (pH 5) and Tris-HCl (pH 7) by mechanical stirrer (MS) and ultrasound (US); protein determination; proteolytic activity (PA) and specific activity (SA); milk clotting activity (MCA) and rennet substitution potential (RSP); high- performance liquid chromatography analysis (HPLC) with UV-Vis detector and principal component analysis (PCA). Extracts of Cynara scolymus leaves showed high results with Citrate-US for the parameters PA (14.38), SA (19.71), MCA (440) and RSP (30.60) compared to other treatments. The extracts with citrate and acetate presented a quick coagulation time (max 3 min). The HPLC analysis enabled the separation of the different protein compounds present in the extracts and most expressive peaks in the samples with Citrate-MS and Acetate-MS; and isolated peaks for Citrate-US. It was concluded that extracts of artichoke leaves with citrate and acetate buffer attributed satisfactory results to act as plant coagulant, as well as to carry out further studies for the purification of proteolytic enzymes and application in cheeses.

Abstract The α-amylase enzyme was produced by solid state fermentation (SSF) and its partition behavior in Aqueous Two-Phase Systems (ATPS) was determined using systems consisting of polyethylene glycol (PEG) and potassium phosphate buffer. To determine the best conditions for α-amylase production, a central composite design was constructed for two independent variables: fermentation time and moisture content, and for the partition study, a face-centered central composite design (CCF) with four independent variables: PEG molecular weight, pH, temperature and partition time. It was indicated that the best enzyme production conditions were a fermentation time of 35.0 hours and 45.7% moisture content. From the results obtained, it was found that increasing the polymer molecular weight promotes a reduction of the enzyme concentration in the upper phase, increasing its concentration in the lower phase. It was also observed that the partition coefficients increased with increasing pH values and a reduction in temperature.

RESUMO: Palma forrageira foi usada como fonte de nutrientes para a produção de enzimas celulolíticas durante a fermentação em estado sólido com auxílio dos fungos filamentosos Aspergillus niger e Rhizopus sp. Os efeitos da ativiade de água, do tempo de fermentação e da temperatura sobre a produção enzimática foram determinados segundo o protocolo padrão de Delineamento Box-Behnken. A condição ideal para produção das enzimas endoglucanase pelo fungo Aspergillus niger foi 70,35 h de fermentação, atividade de água de 0,875 e temperatura de 29,56 ° C enquanto que para o fungo Rhizopus sp. foi 68,12 h e 30,41 ° C. Entretanto, a celulase total produzida pelo fungo Rhizopus sp. foi otimizada em 74,27 h de fermentação e temperatura de 31,22 ° C e para o fungo Aspergillus niger foi 72,48 h e 27,86 ° C. A atividade de água só tinha uma influência significativa sobre a endoglucanase para Aspergillus niger. A alta estabilidade dos extratos enzimáticos em diferentes tepenraturas e pH's foram confirmadas após caracterização enzimática, indicando viabilidade em aplicações industriais. Os ensaios de desativação cinética indicaram que essas enzimas permaneceram ativas após o congelamento do extrato bruto. Com base nos resultados, utilizar a palma forrageira com matéria prima em bioprocessos é uma alternativa excelente para a produção de enzimas termoestaveis.

ABSTRACT: Prickly palm cactus husk was used as a solid-state fermentation support substrate for the production of cellulolytic enzymes using Aspergillus niger and Rhizopus sp. A Box-Behnken design was used to evaluate the effects of water activity, fermentation time and temperature on endoglucanase and total cellulase production. Response Surface Methodology showed that optimum conditions for endoglucanase production were achieved at after 70.35 h of fermentation at 29.56°C and a water activity of 0.875 for Aspergillus niger and after 68.12 h at 30.41°C for Rhizopus sp. Optimum conditions for total cellulase production were achieved after 74.27 h of fermentation at 31.22°C for Aspergillus niger and after 72.48 h and 27.86°C for Rhizopus sp. Water activity had a significant effect on Aspergillus niger endoglucanase production only. In industrial applications, enzymatic characterization is important for optimizing variables such as temperature and pH. In this study we showed that endoglucanase and total cellulase had a high level of thermostability and pH stability in all the enzymatic extracts. Enzymatic deactivation kinetic experiments indicated that the enzymes remained active after the freezing of the crude extract. Based on the results, bioconversion of cactus is an excellent alternative for the production of thermostable enzymes.

Neste trabalho, foram analisados o efeito da atividade de água (0,88, 0,94 e 0,97) e do tempo de fermentação (24, 48, 72, 96 e 120 horas) sobre a atividade cinética das enzimas celulolíticas, produzidas durante a fermentação em estado sólido do resíduo do beneficiamento de manga com a utilização da espécie fúngica Aspergillus niger. A fermentação em estado sólido foi realizada a 35oC em estufa bacteriológica. Os resultados estatísticos indicaram que a melhor atividade para a enzima CMCase foi de 7,26U g-1 após 74,51 horas de fermentação, enquanto que para a enzima FPase esse valor foi de 2,55U g-1. Após 98,52 horas, através dos resultados obtidos pela aplicação da metodologia de superfície de resposta, ambas as enzimas apresentaram melhores resultados em aproximadamente 0,928 de atividade de água. Nos gráficos de Pareto, observamos que o tempo de fermentação tem maior efeito sobre a atividade da enzima CMCase, enquanto que a variável atividade de água exerce maior efeito sobre a atividade da enzima FPase. Durante a fermentação, o fungo secretou as enzimas sem a necessidade de qualquer indutor além do resíduo de manga e água.

The present paper analyses the effects of water activity (0.88, 0.94 and 0.97) and of fermentation time (24, 48, 72, 96 and 120 hours) on the kinetic activity of enzymes cellulolytic, produced during the solid state fermentation of waste from the improvement of mango, with the aid of fungus species Aspergillus niger. Solid state fermentation was carried out at 35°C inside a bacteriological incubator. The statistical results indicated that the best activity for enzyme CMCase was 7.26U g-1 after 74.51 hours of fermentation, whereas for enzyme FPase was 2.55U g-1 after 98.52 hours, both presenting best results in approximately 0.928 of water activity. Pareto charts have showed that fermentation time has greater effect over the activity of enzyme CMCase, while the water activity variable has greater effect over enzyme FPase activity. During fermentation the fungus synthesized the enzymes without the need of inductors other than mango residue and water.