Cogumelos da Amazônia como fonte de protease para aplicação na indústria de queijo

Abstract

The increase in world cheese production, the scarcity of animal rennet, associated with the rejection of genetically modified proteases, encourages the search for new sources of milkclotting enzymes. Among fungi, edible mushrooms have stood out as alternative sources of peptidases that can be used as milk-clotting. This research aimed to evaluate the synthesis of proteases by six edible mushrooms from DPUA Culture Collection and select a milk-clotting producer species with physicochemical characteristics for cheese production. The submerged fermentation was carried out in GYP medium (glucose, yeast extract and peptone) with 0.5% (w/v) of gelatine. The edible mushroom that produced proteases with higher coagulant ratio in the liquid medium was cultivated in Amazon agroindustrial wastes as substrate: angelim sawdust, cupuaçu, exocarp of tucumã, banana leaf and açai seeds, supplemented with rice bran. The proteolytic activity was determined using 1% (w/v) of azocasein as substrate and the determinination of milk-clotting activity was used 10% (w/v) of skimmed milk solution as substrate. Some parameters were used to evaluate the influence of milk-clotting enzymes production (pH of the culture medium, fermentation time, size and age of the inoculum). The enzymes were characterized according to optimums pH and temperature, pH and temperature stabilities, and effect of metallic ions and inhibitors. The cytotoxic effect of the selected mushroom crude extract was evaluated in human MRC-5 fibroblast. The analysis of centesimal and microbiological composition was determined in the elaborated cheese. In submerged fermentation conditions, Pleurotus albidus was the best protease producer (34.00 U/mL) and the synthesis of theses enzymes were stimulated by culture age (10 days) e inoculum volume [5% (p/v)]. The enzymes showed optimum activities at pH 5 and 60°C, with stabilies at pH 5 to 8 and 30°C to 60°C, being classified as cysteine and serine proteases. The milk-clotting enzymes from Pleurotus albidus expressed the highest coagulant ratio (21.60) with maximum activity at 60 °C and pH 6. Iodoacetic acid was the inhibitor of higher action in the proteolytic activity suggesting the presence of cysteine proteases. Pleurotus albidus milk-clotting enzymes showed no toxicity against human fibroblasts (MRC5). In the solid fermentation, Pleurotus albidus presented high enzyme production in the mixture composed by açai seeds and rice bran (90:10, w/w). The milk-clotting enzymes demonstrated coagulant ratio of 14.58, abundant whey formation and strong coagulation in milk solution. The best conditions to milk-clotting enzymes production were: inoculum with 10 days of age, culture medium at pH 6 and inoculum size of 2.5% (p/v). The enzymes exhibited maximum catalytic activity at pH 5 and 55 ° C with stability until 45°C. They were completely inhibited by iodoacetic acid. The cheese produced with Pleurotus albidus milk-clotting enzymes as coagulant agent demonstrated contents of moisture, lipid and protein of 55%, 20% and 18%, respectively. The microbiological analysis showed that the cheese was under the standards required by Brazilian legislation. Pleurotus albidus is a potential source of coagulant that can be applied in dairy industry, especially in the manufacture of cheese.