3A, lanes 1 and 2), indicating the occurrence of hydrolysis with the generation of a remarkably intense 17-kDa polypeptide band (Fig. 3A, lane 3). The peak in the densitogram for αs-casein

band after incubation with positive control chymosin was higher than that obtained after incubation with PP (Fig. 3A, lanes 1 and 2), indicating that αs-casein was more hydrolysed by PP than by chymosin. Low reduction of β-casein band intensity was observed only after 24-h incubation with Crenolanib clinical trial PP and chymosin (Fig. 3B, lane 1). Hydrolysis by PP generated several polypeptides with molecular mass between 7 and 19 kDa (Fig. 3B, lane 2), while cleavage by chymosin resulted mainly in polypeptides with very low molecular masses (Fig. 3B, lane 3). Reduction in intensity of κ-casein band due to hydrolysis by PP after 10, 30, 60 and 120 min (Fig. 3C, lane 1) was accompanied by an increase in the intensity Crizotinib chemical structure of a 16-kDa polypeptide band, which probably corresponds

to para-κ-casein (Fig. 3C, lane 2). No other peak of intensity in the region of κ-casein band was detected after incubation with PP and chymosin for 24 h, revealing total degradation of protein (Fig. 3C, lane 1). In addition, the para-κ-casein band intensity was strongly reduced after 24-h incubation with PP and chymosin (Fig. 3C, lane 2). Chymosin cleaves a single peptide bond in κ-casein, producing insoluble para-κ-casein and a C-terminal glycopeptides (Fox and Stepaniak, 1993 and Rao et al., 1998). Extracts from sunflower (Helianthus annuus), as well as from albizia (Albizia lebbeck) and S. dubium seeds, have been proved to hydrolyse κ-casein to para-κ-casein Y-27632 2HCl ( Ahmed et al., 2010 and Egito et al., 2007). Curd constituents include αs-, β- and para-κ-caseins (Abreu, 2005). The detection of para-κ-casein on SDS–PAGE after casein hydrolysis by PP and

the fact that milk-clotting activity of PP was detected only in the presence of calcium suggests that milk coagulation was probably due to the degradation of κ-casein, leading to the collapse of the micellar structure and aggregation of αs- and β-caseins under the influence of calcium, resulting in gel formation (Merin, Talpaz, & Fishman, 1989). PP from M. oleifera flowers is a potentially useful tool in cheese production processes, since it did not promote extensive hydrolysis of αs- and β-caseins. The speed of hydrolysis of caseins influences the yield, consistency as well as flavour of cheese, and slow degradation of αs- and β-caseins is guarantee of production of a firm curd, which is what occurs when chymosin is used, as mentioned above ( Bruno et al., 2010 and Fox, 1989). Plant rennets which promote extensive proteolysis of caseins are inappropriate for cheese production, because the generated peptides confer a bitter taste ( Lo Piero et al., 2002 and Macedo et al., 1996). Caseinolytic activity on azocasein significantly increased after heating of PP at 50 °C, while loss of this activity was detected after heating of PP at 60 °C (Table 1).