Otimização, purificação e caracterização da peptidase coagulante obtida por fermentação submersa pelo Thermomucor indicae-seudaticae N31 e produção de queijo mussarela
Carregando...
Data
2017-03-27
Autores
Silva, Bruna Lima da [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Peptidases coagulantes microbianas estão sendo cada vez mais utilizadas na indústria láctica para produção de queijos. Produzir uma enzima coagulante com elevada Razão (R) entre a atividade coagulante (AC) ou específica e atividade proteolítica (AP) inespecífica tem se tornado o objetivo de muitos estudos, já que a atividade inespecífica pode prejudicar a qualidade do produto final. O presente estudo objetivou otimizar a produção submersa, aumentar a escala de fermentação, purificar e caracterizar e a aplicar a enzima coagulante do Thermomucor indicae-seudaticae N31 na produção do queijo mussarela. A aplicação do Plackett-Burman e Doehlert design para a otimização da AC resultou no aumento de 11,66 vezes (10,46 para 122,0 U/mL). A melhor condição de produção da enzima em Erlenmeyer foi entre 4,1 - 4,7 % de farelo de trigo, 40,5 a 41,75 ºC °C, 0,4 % (NH4)2SO4, 0,4 % MgSO4.7H2O e 0,1 % NH4NO3, 150 rpm, 96 horas de incubação e pH 5,5. O melhor período para produção da enzima em biorreator com agitação mecânica ocorreu em 96 horas e pH 6,48, apresentando uma Razão AC/AP de 647. A enzima apresentou resistência aos repetidos processos de congelamento e descongelamento. O processo de purificação consistiu da concentração em dialisador tangencial e cromatografia de troca iônica em resina Q-Sepharose com tampão acetato 0,02 mol/L e pH 4,6, apresentando uma banda homogênea em SDS-PAGE. A enzima purificada apresentou pH ótimo 5,3 (R=786), temperatura ótima 65 °C (R=3417), ativação da atividade coagulante durante 24 horas de exposição em pH 4,0 (R=5023) e 4,5 (R=3518) e estabilidade térmica até 55 °C durante 5 horas de exposição (R=480). O CaCl2 influenciou positivamente até a concentração de 0,06 mol/L (AC=101 U/mL). A produção do queijo mussarela foi realizada com o coagulante do Thermomucor indicae-seudaticae N31 (QT) e com um coagulante comercial (QC). Os queijos foram armazenados por um período de 120 dias. As análises de Umidade, Acidez, Gordura, GES, Cinzas, Nitrogênio, Proteína total, NS pH 4.6, NS TCA, Sal, Eletroforese e capacidade de derretimento foram realizadas em períodos específicos ao longo do período de armazenamento. A acidez foi maior no QT enquanto a proteína foi maior no QC em todos os períodos analisados. O NS pH 4.6 e o NS TCA também foram maiores no QT em todos os períodos analisados, exceto no primeiro dia, no qual não foi verificado diferença significativa. Os dois tratamentos não apresentaram diferença em relação ao teor de Umidade, Gordura, GES, Cinzas e Sal. O perfil eletroforético demonstrou que houve maior proteólise no QT. A capacidade de derretimento não 8 diferiu no período do primeiro dia, porém foi maior no QT até o fim da estocagem. Os resultados demonstram que a produção da renina microbiana do Thermomucor indicae-seudaticae N31 pôde ser otimizada, produzida em maior escala no biorreator de bancada e purificada por cromatografia de troca iônica. Sua aplicação na produção do queijo mussarela, resultou em produto de boa qualidade. Estes resultados confirmam que a peptidase coagulante do Thermomucor indicae-seudaticae N31 apresenta potencial para aplicação tecnológica.
Microbial peptidases coagulants are being increasingly used in the dairy industry for cheese production. Producing a clotting enzyme with high ratio (R) between milk-clotting activity (MCA) or specific activity and proteolytic activity (PA) or non-specific has become the object of many studies, since non-specific activity may damage the quality of the final product. The present study aimed to optimize submerged production, increase fermentation scale, purify and characterize and to apply the milk-clotting enzyme produced by Thermomucor indicae-seudaticae N31 in the production of mozzarella cheese. The application of Plackett-Burman and Doehlert design to the optimization of MCA resulted in an increase of 11.66 times (10.46 to 122.0 U/mL). The best enzyme production condition in Erlenmeyer was between 4.1 - 4.7 % wheat bran, 40.5 to 41.75 °C, 0.4% (NH4)2SO4, 0.4% MgSO4.7H2O and 0.1% NH 4NO3, 150 rpm, 96 hours of incubation and pH 5.5. The best period for the production of the enzyme in a mechanical agitation bioreactor occurred in 96 hours and pH 6.48, presenting a Ratio MCA/PA of 647. The enzyme showed resistance to repeated freezing and thawing processes. The purification process consisted of the concentration in a tangential dialyzer and ion exchange chromatography on Q-Sepharose resin with 0.02 mol/L acetate buffer and pH 4.6, presenting a homogeneous band on SDS-PAGE. The purified enzyme had an optimum pH of 5.3 (R = 786), optimum temperature 65 ° C (R = 3417), activation of the milk-clotting activity during 24 hours of exposure at pH 4.0 (R = 5023) and 4.5 (R = 3518) and thermal stability at 55 °C for 5 hours of exposure (R = 480). CaCl2 positively influenced until the concentration of 0.06 mol/L (MCA = 101 U/mL). The production of mozzarella cheese was performed with the coagulant Thermomucor indicae-seudaticae N31 (TC) and with a commercial coagulant (CC). The cheeses were stored for a period of 120 days. The analyzes of moisture, acidity, fat, GES, ash, total nitrogen, total protein, NS-pH 4.6, NS-TCA, salt, casein electrophoresis and melting capacity of cheese were performed at specific periods throughout the storage period. The acidity was higher in the TC while the protein was higher in the CC in all periods analyzed. NS-pH 4.6 and NS-TCA were also higher on TC in all the periods analyzed, except on the first day, in which there wasn't significant difference. The two treatments showed no difference in relation to moisture, fat, GES, ash and salt content. The electrophoretic profile showed that there was more proteolysis in the TC. The melting capacity did not differ on the first day, but was higher in the TC until the end of storage. 10 The results demonstrated that the production of the microbial renin of Thermomucor indicae-seudaticae and N31 could be optimized, produced in larger scale in the bench bioreactor and purified by ion exchange chromatography. Its application in the production of mozzarella cheese resulted in good quality product. These results confirm that the milk-clotting peptidase of Thermomucor indicae-seudaticae and N31 presents potential for technological application.
Microbial peptidases coagulants are being increasingly used in the dairy industry for cheese production. Producing a clotting enzyme with high ratio (R) between milk-clotting activity (MCA) or specific activity and proteolytic activity (PA) or non-specific has become the object of many studies, since non-specific activity may damage the quality of the final product. The present study aimed to optimize submerged production, increase fermentation scale, purify and characterize and to apply the milk-clotting enzyme produced by Thermomucor indicae-seudaticae N31 in the production of mozzarella cheese. The application of Plackett-Burman and Doehlert design to the optimization of MCA resulted in an increase of 11.66 times (10.46 to 122.0 U/mL). The best enzyme production condition in Erlenmeyer was between 4.1 - 4.7 % wheat bran, 40.5 to 41.75 °C, 0.4% (NH4)2SO4, 0.4% MgSO4.7H2O and 0.1% NH 4NO3, 150 rpm, 96 hours of incubation and pH 5.5. The best period for the production of the enzyme in a mechanical agitation bioreactor occurred in 96 hours and pH 6.48, presenting a Ratio MCA/PA of 647. The enzyme showed resistance to repeated freezing and thawing processes. The purification process consisted of the concentration in a tangential dialyzer and ion exchange chromatography on Q-Sepharose resin with 0.02 mol/L acetate buffer and pH 4.6, presenting a homogeneous band on SDS-PAGE. The purified enzyme had an optimum pH of 5.3 (R = 786), optimum temperature 65 ° C (R = 3417), activation of the milk-clotting activity during 24 hours of exposure at pH 4.0 (R = 5023) and 4.5 (R = 3518) and thermal stability at 55 °C for 5 hours of exposure (R = 480). CaCl2 positively influenced until the concentration of 0.06 mol/L (MCA = 101 U/mL). The production of mozzarella cheese was performed with the coagulant Thermomucor indicae-seudaticae N31 (TC) and with a commercial coagulant (CC). The cheeses were stored for a period of 120 days. The analyzes of moisture, acidity, fat, GES, ash, total nitrogen, total protein, NS-pH 4.6, NS-TCA, salt, casein electrophoresis and melting capacity of cheese were performed at specific periods throughout the storage period. The acidity was higher in the TC while the protein was higher in the CC in all periods analyzed. NS-pH 4.6 and NS-TCA were also higher on TC in all the periods analyzed, except on the first day, in which there wasn't significant difference. The two treatments showed no difference in relation to moisture, fat, GES, ash and salt content. The electrophoretic profile showed that there was more proteolysis in the TC. The melting capacity did not differ on the first day, but was higher in the TC until the end of storage. 10 The results demonstrated that the production of the microbial renin of Thermomucor indicae-seudaticae and N31 could be optimized, produced in larger scale in the bench bioreactor and purified by ion exchange chromatography. Its application in the production of mozzarella cheese resulted in good quality product. These results confirm that the milk-clotting peptidase of Thermomucor indicae-seudaticae and N31 presents potential for technological application.
Descrição
Palavras-chave
Coagulante microbiano, Farelo de trigo, Biorreator, Troca iônica, Queijo mozzarella, Microbial coagulant, Wheat bran, Bioreactor, Ion exchange, Mozzarella cheese