In previous studies carried out in our laboratory, the fungus Thermomucor indicae-seudaticae N31, isolated by our group,
produced a protease that specifically hydrolysed κ-casein during milk clotting ( Merheb-Dini, Gomes, Boscolo, & da Silva, selleck compound 2010). This led us to develop studies with this enzyme using it as a coagulant for Prato cheese making. The properties of the resultant cheeses during ripening were compared with cheeses manufactured with a traditional commercial coagulant, since after a cheese is made, some of the coagulant remains in the cheese block and its activity contributes to the proteolysis that takes place during ripening ( Guinee & Wilkinson, 1992). The fungus, T. indicae-seudaticae N31, obtained from the Laboratory of Applied Biochemistry and Microbiology – IBILCE – UNESP, was maintained in Sabouraud dextrose agar medium (Oxoid) and prior to use it was inoculated in 250 ml Erlenmeyer flasks containing Sabouraud with 0.2% casein and incubated at 45 °C for 2 days for complete growth. Enzyme production was carried out according to Merheb-Dini et al.
(2010) using wheat bran as substrate and a fermentation period of 24 h. After extraction, 1.116 ml of enzymatic extract was concentrated to 112 ml through ultrafiltration for use in cheese making. Cheeses Protein Tyrosine Kinase inhibitor were made from 15 l of pasteurised cow’s milk (Laticínio Saboroso, São José do Rio Preto-SP): the milk was warmed to 32 °C before adding 7.5 ml of 50% calcium chloride, 12 ml of starter (LL50 A, composed of strains Lactococcus lactis ssp lactis and Lactococcus lactis ssp cremoris), 1.05 ml urucum colourant, sorbic acid (1.8 g in 90 ml of distilled water), and finally coagulant Ha-la (Chr. Hansen)
– process H or coagulant from T. indicae-seudaticae N31 – process T (the amount of coagulant added was standardised to equal milk-clotting activity of approximately 45 min). After coagulation (45 min for both treatments), the curd was cut into 0.3–0.5 cm3 cubes which 5-Fluoracil supplier were then submitted to slow continuous mixing for 15 min (1st mixing), followed by removal of part of the whey (30%) and further heating of the curd to 38 °C with the addition of 80 °C water (17%). The curd was mixed again for another 15 min (2nd mixing) followed by complete whey removal then placed in plastic moulds and pressed. The cheeses were turned upside down after the first 30 min and then pressed for 24 h in a vertical press, with stainless steel weights. Cheeses were then removed from the press and from the moulds and were placed in 18% (NaCl) brine solution for 5 h at 4 °C. Finally, they were dried at 9 °C/24 h, weighed, sealed under vacuum in heat-shrinkable plastic bags and stored at 9 °C/80% relative humidity for 60 days. Two processes were carried out, one using the commercial coagulant (control) and the other substituting the commercial coagulant for the protease from T. indicae-seudaticae N31.