Proteases fúngicas: produção utilizando farelo de soja e farinhas de banana, caracterização enzimática e aplicação em farinha de grilo
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Data
2020-03-24
Autores
Koike, Meliane Akemi
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Universidade Estadual Paulista (Unesp)
Resumo
Resíduos e subprodutos agroindustriais são extensivamente estudados a fim de se reaproveitar seu potencial biológico e agregar valor. A fermentação em estado sólido (FES) é uma das formas de utilizar estes resíduos e subprodutos para obtenção de enzimas, como as proteases. Este trabalho objetivou estudar a produção de proteases fúngicas por FES utilizando farelo de soja e farinhas de banana, caracterizar bioquimicamente as proteases mais promissoras e, por fim, aplicar na hidrólise da farinha de grilo (Gryllus assimilis), avaliando os produtos de hidrólise. O estudo da composição do meio de cultivo foi conduzido através de um delineamento experimental, o planejamento de misturas, para definir a proporção ótima de parte sólida dos substratos de fermentação. Posteriormente, realizou-se a cinética de produção de protease por FES, a caracterização bioquímica das proteases mais promissoras quanto ao pH e temperatura ótimos de atuação, bem como a influência de íons metálicos e L-cisteína em diferentes concentrações. Por meio de relargagem (salting-out), fez-se a purificação parcial das enzimas, seguida de diálise e liofilização, para então serem aplicadas na hidrólise da farinha de grilo. O fator de hidrólise, a atividade antioxidante e o perfil eletroforético dos produtos de hidrólise foram avaliados. O planejamento de misturas resultou em duas proporções com altas atividades proteolíticas, 50 % de farelo de soja (FS) e 50 % de farinha de casca de banana madura (FCBM) e um terço de cada componente estudado (farelo de soja, farinha de casca de banana madura e farinha de banana verde) para os dois micro-organismos estudados: Trichoderma koningii (INCQS 40331) e Aspergillus oryzae (ATCC 1003 / INCQS 40068). Foi possível obter um modelo quadrático apenas para o A. oryzae, com p<0,15. A cinética de produção enzimática em 168 h revelou que em 48 h a atividade proteolítica atingiu o seu máximo, na mistura de substratos contendo 50 % de FS e 50 % de FCBM. Por meio da caracterização bioquímica foram definidos temperatura ótima das proteases de 40oC produzidas por ambos micro-organismos e pH ótimo de 6,0 para protease de T. koningii e pH 7,0 para protease de A. oryzae. Os íons metálicos e L-cisteína não resultaram em efeito positivo na atividade proteolítica. A análise dos sobrenadantes dos hidrolisados obtidos por ação das proteases de ambos fungos revelaram potencial antioxidante, com destaque para os hidrolisados da protease de A. oryzae, correspondente a 101,09 µM de TE. A eletroforese em gel dos hidrolisados indicou proteínas menores que 14,4 kDa, corroborando a ação hidrolítica das proteases do A. oryzae na farinha de grilo. Palavras-chaves: banana, soja, proteases fúngicas, hidrólise, farinha de grilo, antioxidante
Agro-industrial waste and by-products are extensively studied aiming to add value and explore their full biological potential. Solid state fermentation (SSF) is a technique that use agro-industrial waste and by-products and may be used to obtain enzymes, such as proteases. This study aimed to produce fungal proteases by SSF using soybean bran and banana flours as substrate, characterize biochemically and apply most promising proteases to hydrolysis of cricket flour (Gryllus assimilis), and evaluate the hydrolysis products. The composition of the culture medium was evaluated through an experimental design to define the optimal proportion of solid part of the fermentation substrates. Subsequently, the protease production kinetics were performed by SSF, the most promising proteases were characterized biochemically according to the optimal pH and temperature, as well as according to the influence of metal ions and L-cysteine in different concentrations. Proteases were partially purified by salting-out, followed by dialysis and lyophilization and applied in hydrolysis of the cricket flour. The hydrolysis factor, the antioxidant activity and the electrophoretic profile of the hydrolysis products were evaluated. The mixture design resulted in two combinations with high proteolytic activities, 50% soybean bran (SB) and 50% ripe banana peel flour (RBPF) and one third of each component (soybean bran, ripe banana peel flour and green banana flour) for the microorganisms Trichoderma koningii (INCQS 40331) and Aspergillus oryzae (ATCC 1003 / INCQS 40068). It was possible to obtain a quadratic model only for A. oryzae (p <0.15). The kinetics of proteases showed that after 48 h the proteolytic activity reached its maximum in the mixture of substrates containing 50% of SB and 50% of RBPF. Biochemical characterization showed that the optimum temperature was 40°C for both fungi proteases, as well as pH 6.0 optimum for T. koningii protease and pH 7.0 optimum for A. oryzae protease. Metal ions and L-cysteine did not result in a positive effect on proteolytic activity. The analysis of hydrolysate supernatants obtained by both fungi proteases showed antioxidant potential, with emphasis on the hydrolysates of A. oryzae protease, corresponding to 101.09 µM of TE. Gel electrophoresis of hydrolysates indicated proteins smaller than 14.4 kDa, corroborating the hydrolytic action of A. oryzae proteases in cricket flour.
Agro-industrial waste and by-products are extensively studied aiming to add value and explore their full biological potential. Solid state fermentation (SSF) is a technique that use agro-industrial waste and by-products and may be used to obtain enzymes, such as proteases. This study aimed to produce fungal proteases by SSF using soybean bran and banana flours as substrate, characterize biochemically and apply most promising proteases to hydrolysis of cricket flour (Gryllus assimilis), and evaluate the hydrolysis products. The composition of the culture medium was evaluated through an experimental design to define the optimal proportion of solid part of the fermentation substrates. Subsequently, the protease production kinetics were performed by SSF, the most promising proteases were characterized biochemically according to the optimal pH and temperature, as well as according to the influence of metal ions and L-cysteine in different concentrations. Proteases were partially purified by salting-out, followed by dialysis and lyophilization and applied in hydrolysis of the cricket flour. The hydrolysis factor, the antioxidant activity and the electrophoretic profile of the hydrolysis products were evaluated. The mixture design resulted in two combinations with high proteolytic activities, 50% soybean bran (SB) and 50% ripe banana peel flour (RBPF) and one third of each component (soybean bran, ripe banana peel flour and green banana flour) for the microorganisms Trichoderma koningii (INCQS 40331) and Aspergillus oryzae (ATCC 1003 / INCQS 40068). It was possible to obtain a quadratic model only for A. oryzae (p <0.15). The kinetics of proteases showed that after 48 h the proteolytic activity reached its maximum in the mixture of substrates containing 50% of SB and 50% of RBPF. Biochemical characterization showed that the optimum temperature was 40°C for both fungi proteases, as well as pH 6.0 optimum for T. koningii protease and pH 7.0 optimum for A. oryzae protease. Metal ions and L-cysteine did not result in a positive effect on proteolytic activity. The analysis of hydrolysate supernatants obtained by both fungi proteases showed antioxidant potential, with emphasis on the hydrolysates of A. oryzae protease, corresponding to 101.09 µM of TE. Gel electrophoresis of hydrolysates indicated proteins smaller than 14.4 kDa, corroborating the hydrolytic action of A. oryzae proteases in cricket flour.
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Palavras-chave
Banana, Soja, Proteases fúngicas, Hidrólise, Farinha de grilo, Antioxidante, Soy, Fungal proteases, Hydrolysis, Cricket flour, Antioxidant