Produção de proteases coagulantes por espécies de Pleurotus em resíduos vegetais da Amazônia

Abstract

Fungal species are already available in the market and the mushrooms have been reported as producers of milk coagulating enzymes for cheese production. The objective of this study was to investigate the growth of Pleurotus florida DPUA 1534 and Pleurotus ostreatus DPUA 1533 in vegetable substrate to select a species with potential proteolytic coagulant for the application in the food industry. The results of vertical mycelial growth, although P. florida DPUA 1534 and P. ostreatus DPUA 1533 have colonized natural residues tested, P. florida DPUA 1534 was the species that showed a significant increase (p> 0.05) after 15 days given that determined the choice of this species for the production of the mushroom. In producing the substrate formulated based on cupuaçu bark supplemented with rice bran 20%, 20 mycelial inoculum discs (Ø = 10 mm) resulted in better biological efficiency (14.2%) and the remaining analyzes were performed with P. florida DPUA 1534 produced under those conditions. The analysis showed the mushroom highlighted in the following nutritional characteristics P. florida DPUA 1534 proteins (25.94%), carbohydrate (49.72%), iron (89.82%) and zinc (85.08%) and the presence of all essential amino acids. In the crude extract of the mushroom P. florida DPUA 1534 to coagulant activity and the AUC ratio was 67.68 and 2.15, respectively. Optimum activity was shown at pH 7.0 at 40 ° C. The enzyme extract was stable between 25 ° C and 40 ° C and at pH between 4 and 10 showed about 80 to 90% of activity in the first 90 minutes of incubation. The enzymatic extract showed susceptibility to inhibitors of the serine protease class (90%), metalloprotease (93%), cysteine proteases (92%) and aspartic protease (94%). The most effective compound was Pepstatin A, suggesting the presence of aspartic protease enzymes showing aspartic residues in the active site, indicating the possibility of its application in the dairy industry. Regarding toxicity results showed that P. florida DPUA 1534 produces proteases with biological activity on Artemia salina (LC50 = 25,5μg) and showed hemolytic activity in blood of sheep trading in ascending order with respect to extract concentrations (0, 2 to 1.0 mL), LC50 = 363,88μg and LC50 = 241,51μg after 1h and 24h of incubation, respectively. The in vivo test indicated that the extract when administered at a dose of 1000 mg.Kg-1 in rats, no deaths or clinical signs of toxicity in any of the treated animals did not cause significant changes in haematological and biochemical parameters when compared to the group control, and histological analyzes revealed no acute toxicity under the experimental conditions. Therefore, the extract of P. florida DPUA 1534 showed potential for use as coagulating milk.