No abstract

The objective of this research was to compare the effect of 2 fungal proteases, one that is already commercially established as a milk-clotting agent and another produced at the laboratory scale, on Prato cheese composition, protein and fat recovery, yield, and sensory characteristics. Cheeses were produced according to the traditional protocol, using protease from the fungus Thermomucor indicae-seudaticae N31 and commercial coagulant from Rhizomucor spp. as clotting agents. A 2×6 factorial design with 3 replications was performed: 2 levels of coagulants and 6 levels of storage time. After 5, 12, 19, 33, 43, and 53d of refrigerated storage (12°C), cheeses were monitored for proteolysis, firmness, and casein degradation by capillary electrophoresis. Sensory acceptance was evaluated after 29d of manufacturing. The different coagulants did not statistically affect Prato cheese composition, protein and fat recovery, and yield. Both cheeses presented good sensory acceptance. Proteolysis increased and firmness decreased for both cheeses during the storage time, as expected for Prato cheese. Caseins were well separated by capillary electrophoresis and the results showed, with good resolution, that the cheeses exhibited similar protein hydrolysis profile. Both cheeses presented good sensory acceptance. The gathered data showed that the protease from T. indicae-seudaticae N31 presented similar action compared with the commercial enzyme, indicating its efficiency as clotting agent for Prato cheese manufacture.

Prato cheeses were manufactured using coagulant from Thermomucor indicae-seudaticae N31 or a commercial coagulant. Cheeses were characterised using the following analysis: yield; fat; acidity; moisture; ash; salt; pH; total nitrogen; total protein; NS-pH 4.6/NTÃ100; NS-TCA 12%/NTÃ100; casein electrophoresis; and RP-HPLC. The results were statistically analysed and revealed that the proteolytic indices were not significantly different throughout the 60 days of ripening of cheeses made with either coagulant. Even though there were some quantitative differences in the peptide profile of cheeses, the enzyme from T. indicae-seudaticae N31 was used in the production of good quality Prato cheese without having to change the established technological parameters of the process.

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)).

The aim of this study was to explore the use of a new coagulant from Thermomucor indicae-seudaticae N31 for the manufacture of a high-cooked starter-free cheese variety, by evaluating its physicochemical and functional characteristics in comparison to cheeses made with a traditional commercial coagulant. Coalho cheese was successfully produced with the new protease as it exhibited comparable characteristics to the one produced using the commercial enzyme: pH behavior during manufacture; cheese composition; protein and fat recovery; and cheese yield. In addition, during storage, melting was low and not affected by storage time; the increase of TCA 12% soluble nitrogen (% of total nitrogen) was lower than half of that of pH 4.6 soluble nitrogen (% of total nitrogen); concentration of β-CN significantly decreased, whereas αs1 -CN concentration was not affected by storage time.