, 2010, Anema et al., 2005 and Ishak et al., 2006). Milk-clotting activity exerted by PP did not change when milk was heated up to 30 and 50 °C. However, the activity using milk heated up to 70 °C VX809 as substrate was higher (3.6 U) than when non-heated

milk (1.8 U) was used. Similarly, the milk-clotting activities from goat (Capra hircus) chymosin and C. scolymus flower extracts have been reported to reach the highest value when the milk was heated up to temperatures above 50 °C ( Chazarra et al., 2007 and Kumar et al., 2006). Protein aggregation by heating of milk has been related to the increasing of milk clotting activity ( Nájera, Renobales, & Barron, 2003). Bovine αs-, β-, and κ-caseins were used as substrates to determine the specificity of caseinolytic activity from PP. The enzyme reactions were monitored by absorbance at 366 nm. Fig. 2A shows that hydrolysis of κ-casein by PP started after 30 min of incubation, while degradation

of αs- and β-casein could only be detected after 60 min. Incubation for longer periods (120 min and 24 h) did not lead to any considerable improvement in degradation of αs- and β-caseins by PP, though hydrolysis of κ-casein increased over 4 times (Fig. 2A). Oppositely, milk-clotting enzymes from C. cardunculus flowers have been reported to hydrolyse αs-casein better than β-casein, and was less effective in cleaving κ-casein ( Ordiales et al., 2012). Chymosin is the major enzyme of calf rennet, and it has been extensively used in the dairy industry to produce a stable curd with good flavour due to its GSK1120212 purchase high specificity for the κ-casein (Rao, Tanksale, Ghatge, & Deshpande, 1998). Thus, this enzyme was used as a benchmark positive control. Specificity of PP for bovine caseins was similar to that

of chymosin, which extensively cleaved κ-casein and promoted very slight hydrolysis of αs- and β-caseins (Fig. 2B). On the other hand, the time course of κ-casein hydrolysis by PP was slower than that by chymosin (Fig. 2). However, unlike chymosin, PP is a partially purified protease preparation PLEK2 and thus the protein concentration reflects the amount of flower extract proteins that were precipitated with ammonium sulphate. The molecular masses of bovine αs-, β-, and κ-caseins on SDS–PAGE were between 20 and 25 kDa (Fig. 3), values that were similar to those reported by Dalgleish (1990). The degrees of casein hydrolysis by PP and chymosin were also evaluated by the reduction of αs-, β-, and κ-caseins bands on SDS–PAGE, since peptides from casein proteolysis can be quantified by gel scanning, followed by densitometry (Cavalli et al., 2008 and Franco et al., 2001). The densitogram revealed that the intensities of αs-casein bands (Fig. 3A, lanes 1 and 2) did not fall after incubation with PP for 10 to 120 min.