In Cheddar cheese, the peptide αs1-CN f1–23 is further

hydrolysed by proteinases from Lactococcus lactis ssp. cremoris into smaller peptides, which present bitter taste ( Singh et al., 2003). Since Prato cheese is also made with this starter culture, it is probable that this hydrolysis also occurs, affecting TCA 12%-SN. Thus, pH 4.6-SN and TCA 12%-SN in Prato cheese were essentially affected by residual chymosin, plasmin and by proteolytic enzymes of lactic acid bacteria. According to Sousa et al. (2001), since proteolysis is one of the TGF-beta inhibitor main biochemical events during the ripening of cheese, it is desirable to include a general assay for proteolysis, such as the determination of soluble N as % of total N, as has been done. If the objectives of the study cover investigating the effect of one of the agents of proteolysis in cheese, such as different types of coagulants, the methodology should be chosen so as to emphasise the level of proteolysis caused by that agent. In this case, for example, proteolysis evolution could be followed by urea–polyacrylamide gel electrophoresis (urea–PAGE) and the peptide profiles of the pH 4.6-soluble fraction should be determined by reverse phase-high performance liquid chromatography (RP-HPLC). Therefore, INK128 proteolysis was assayed by the frequently used method of monitoring casein proteolytic processes:

polyacrylamide RAS p21 protein activator 1 gel electrophoresis using urea, which makes possible to visualise the integrity of casein fractions during ripening (Fig. 2). In Fig. 2A, two main casein groups were identified in the urea–PAGE: αs1-casein, with higher electrophoretic mobility and β-casein, with lower mobility (Silva & Malcata, 2004). The region of family αs2-casein can also be seen, whose electrophoretic

mobilities is between caseins αs1 and β (Sgarbieri, 2005). Fig. 2B shows casein degradation in cheeses made with commercial coagulant (H1–H60) and with coagulant from T. indicae-seudaticae N31 (T1–T60) during 60 days of ripening. Degradation of αs1-casein is seen, more pronounced in cheeses made with commercial coagulant, showing that the hydrolysis of casein molecules is specific for the type of coagulant used ( Lawrence et al., 1987). Degradation of β-casein is also seen, more intense in cheeses made with coagulant from T. indicae-seudaticae N31, with formation of its hydrolysis products, the γ-caseins, which accumulate in cheese ( Singh et al., 2003). Plasmin is one of the agents responsible for the proteolysis during cheese ripening acting especially in the initial stages along with residual coagulant, liberating peptides which will serve as substrate for proteinases from starter and non starter bacteria ( Fox, 1989 and Visser, 1993). Besides plasmin, chymosin also acts on β-casein, on the bond between Leu192 and Tyr193 ( Visser, 1993). It can also be seen in Fig.