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Valorização do soro do leite pela obtenção de péptidos bioativos
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Abstract(s)
As proteínas do soro do leite são uma fonte de péptidos bioativos capazes de modular respostas fisiológicas no organismo. A melhor forma de obter estes péptidos bioativos é através da hidrólise enzimática. Neste estudo selecionou-se 3 bactérias produtoras de serina proteases (2 Bacillus cereus, isolados s3B e s10B; 1 B. mycoides, isolado s102A), pertencentes ao banco de bactérias da Universidade dos Açores. Os extratos enzimáticos dos isolados foram produzidos em meio mínimo salino e meio mínimo suplementado com proteína do soro do leite, utilizando bactérias em suspensão e bactérias imobilizadas. Os extratos enzimáticos produzidos foram usados para hidrolisar as proteínas do soro do leite comercial e proteínas diretamente separadas do soro do leite de uma industria local produtora de queijo (UNILEITE). Esses hidrolisados foram fracionados por exclusão molecular (Superdex 75) num sistema de FPLC e testados para atividade antibacteriana, antioxidante e antitrombótica. Foi identificada a atividade antibacteriana contra Micrococcus luteus numa fração de 4 a 10.5 kDa do isolado s10B, obtendo-se um halo de 2 mm com 69.4 μg de proteína. Usou-se os testes DPPH e ABTS para testar a atividade antioxidante. Os hidrolisados obtidos com os 3 isolados produziram uma fração peptídica de 1.2 kDa com atividade antioxidante superior a 70% a uma concentração de 25 μg/ml de proteína. O IC50 da fração obtida com as enzimas dos isolados s3B, s10B e s102A foi calculado em 18.85, 18.35 e 15.44 μg/ml, respetivamente. A atividade inibidora da lise do coagulo foi obtida pela medição do retardamento do tempo de lise da euglobulina. Todos os hidrolisados das proteínas do soro do leite testados produziram atividade inibidora da trombólise. A maior atividade foi obtida na fração peptídica de 3.5 e 10.7 kDa do hidrolisado obtido com o isolado s102A, com retardamento superior a 3 horas na lise do coágulo, em comparação com controlo. A atividade inibidora da coagulação foi medida também pelo ensaio da euglobulina. Neste teste, a mesma fração do isolado s102A (3.5 e 10.7 kDa) apresentou uma atividade inibitória da formação de coágulo com um IC50 de 27.75 e depois de parcialmente purificada por troca iónica, apresentou um IC50 de 7.91 μg/ml.
As atividades identificadas no presente trabalho têm grande potencial de aplicação Biotecnológica. Os péptidos antimicrobianos e antioxidantes poderão ser incorporados em alimentos, para aumentar o seu tempo de prateleira, e os péptidos com atividade anticoagulante poderão ser utilizados como suplemento alimentar ou no fabrico de alimentos funcionais.
ABSTRACT: Whey proteins are a source of bioactive peptides able to modulate physiological responses in the body. The best way to get these bioactive peptides by enzymatic hydrolysis. In this study, we selected 3 serine protease-producing bacteria (2 Bacillus cereus, isolated s3B and s10B; 1 b. mycoides, isolated s102A), belonging to the bacteria at the University of the Azores. The enzymatic extracts of the isolates were produced in minimal saline medium and minimal medium supplemented with whey protein, using suspended bacteria and immobilized bacteria. The enzymatic extracts produced were used to hydrolyze commercial whey proteins and proteins directly separated from whey from a local cheesemaking industry (UNILEITE). These hydrolysates were fractionated by molecular exclusion (Superdex 75) a FPLC system and tested for antimicrobial activity, antioxidant and Antithrombotic. It was identified the antibacterial activity against Micrococcus luteus at a fraction of the 4 to 10.5 isolate s10B kDa, with a halo of 2 mm with 69.4 μg of protein. It used the DPPH test and ABTS to test the antioxidant activity. The hydrolysates obtained with the 3 isolates produced a peptide fraction of 1.2 kDa with antioxidant activity greater than 70% at a concentration of 25 μg/ml of protein. The IC50 of the fraction obtained with the enzymes of the s3B, s10B and s102A isolates was calculated at 18.85, 18.35 and 15.44 μg/ml, respectively. The inhibitory activity of clot Lysis was obtained by measuring the time delay of lysis of euglobulin. All hydrolyzed whey proteins tested produced inhibitory activity of thrombolysis. The highest activity was obtained in the peptide fraction of 3.5 and 10.7 kDa of the hydrolysate obtained with isolated s102A, exceeding 3 hours delay in lysis of the clot, compared with control. Coagulation inhibitor activity was measured by the euglobulin assay. In this test, the same fraction of isolated s102A (3.5 and 10.7 kDa) presented an inhibitory activity of clot formation with an IC50 of 27.75 and after partially purified by ion exchange, presented an IC50 of 7.91 μg/ml. The activities identified in this study have great potential to Biotechnology application. Antimicrobial peptides and antioxidants could be incorporated in food, to increase the time of your shelf, and peptides with Anticoagulant activity can be used as a food supplement or in the manufacture of functional foods.
ABSTRACT: Whey proteins are a source of bioactive peptides able to modulate physiological responses in the body. The best way to get these bioactive peptides by enzymatic hydrolysis. In this study, we selected 3 serine protease-producing bacteria (2 Bacillus cereus, isolated s3B and s10B; 1 b. mycoides, isolated s102A), belonging to the bacteria at the University of the Azores. The enzymatic extracts of the isolates were produced in minimal saline medium and minimal medium supplemented with whey protein, using suspended bacteria and immobilized bacteria. The enzymatic extracts produced were used to hydrolyze commercial whey proteins and proteins directly separated from whey from a local cheesemaking industry (UNILEITE). These hydrolysates were fractionated by molecular exclusion (Superdex 75) a FPLC system and tested for antimicrobial activity, antioxidant and Antithrombotic. It was identified the antibacterial activity against Micrococcus luteus at a fraction of the 4 to 10.5 isolate s10B kDa, with a halo of 2 mm with 69.4 μg of protein. It used the DPPH test and ABTS to test the antioxidant activity. The hydrolysates obtained with the 3 isolates produced a peptide fraction of 1.2 kDa with antioxidant activity greater than 70% at a concentration of 25 μg/ml of protein. The IC50 of the fraction obtained with the enzymes of the s3B, s10B and s102A isolates was calculated at 18.85, 18.35 and 15.44 μg/ml, respectively. The inhibitory activity of clot Lysis was obtained by measuring the time delay of lysis of euglobulin. All hydrolyzed whey proteins tested produced inhibitory activity of thrombolysis. The highest activity was obtained in the peptide fraction of 3.5 and 10.7 kDa of the hydrolysate obtained with isolated s102A, exceeding 3 hours delay in lysis of the clot, compared with control. Coagulation inhibitor activity was measured by the euglobulin assay. In this test, the same fraction of isolated s102A (3.5 and 10.7 kDa) presented an inhibitory activity of clot formation with an IC50 of 27.75 and after partially purified by ion exchange, presented an IC50 of 7.91 μg/ml. The activities identified in this study have great potential to Biotechnology application. Antimicrobial peptides and antioxidants could be incorporated in food, to increase the time of your shelf, and peptides with Anticoagulant activity can be used as a food supplement or in the manufacture of functional foods.
Description
Dissertação de Mestrado, Ciências Biomédicas, 06 de março de 2018, Universidade dos Açores.
Keywords
Hidrólise Enzimática Péptido Bioativo Proteína Soro de Leite Bioactive Peptides Enzymatic Hydrolysis
Citation
Paiva, Tiago Machado. "Valorização do soro do leite pela obtenção de péptidos bioativos". 2018. 82 p.. (Dissertação de Mestrado em Ciências Biomédicas). Ponta Delgada: Universidade dos Açores, 2017. [Consult. Dia Mês Ano]. Disponível em www:<http://hdl.handle.net/10400.3/4687>.