Caseinolytic

activity was also determined according to Sousa and Malcata (1998) using bovine αs-, β-, and κ-caseins purchased from Sigma–Aldrich, USA. PP (50 μl, 1.7 mg of protein) was added to αs-, β- or κ-casein solutions (1 ml, 10 mg of protein) in 0.1 M sodium phosphate buffer, pH 6.5 and reaction was allowed to proceed at 37 °C. Aliquots of 10 and 900 μl from the reaction mixtures were retrieved within 10, 30, 60, 120 min and 24 h of incubation. The aliquots of 10 μl were heated at 100 °C for 5 min and submitted to SDS–PAGE as described in Section 2.5. The aliquots of 900 μl LGK-974 cell line were evaluated for absorbance at 366 nm after addition of 10% (w/v) trichloroacetic acid (200 μl) and centrifugation (9,000 g, 10 min, 4 °C). One unit of caseinolytic activity was defined as the amount of enzyme that promoted a 0.01 increase in absorbance. Chymosin (50 μl, 10 μg; Chy-Max® Liquid, buy INK 128 Chr. Hansen, Denmark) and 0.15 M NaCl were used as positive and negative controls, respectively. Hydrolysis of αs-, β- or κ-caseins by

PP and chymosin (positive control) were evaluated by SDS–PAGE using 15% (w/v) polyacrylamide gels (Laemmli, 1970). Aliquots (10 μl) from reaction mixtures described in the Section 2.4, and molecular mass markers (SigmaMarker™ kit from Sigma–Aldrich, USA, containing the standard proteins: bovine serum albumin, 66,000 Da; glutamic Niclosamide dehydrogenase from bovine liver, 55,000 Da; ovalbumin from chicken egg, 45,000 Da,; glyceraldehyde 3-phosphate dehydrogenase from rabbit muscle, 36,000 Da; carbonic anhydrase from bovine erythrocytes, 29,000 Da; trypsinogen from bovine pancreas, 24,000 Da; trypsin inhibitor from soybean, 20,000 Da; α-lactalbumin from bovine milk, 14,200 Da; and aprotinin from bovine lung, 6,500 Da) were applied on gel. After running and staining with 0.02% (v/v) Coomassie Brilliant Blue in 10% acetic acid, the gels were dehydrated and scanned. The densitograms were obtained using the software Scion Image Beta 4.02.2 (Scion Corporation,

Frederick, MD, USA) and indicated the intensity of polypeptide bands. The substrate (10% skim milk, Molico®, Nestlé, Brazil) was prepared in distilled water or in 10 mM CaCl2 in water, and pH was adjusted at 6.5. The milk (2.0 ml) was incubated with flower extract (0.3 ml, 9.0 mg of protein), PP (0.3 ml, 9.8 mg of protein) or 60% supernatant fraction (0.3 ml, 9.0 mg of protein) at 37 °C, and curd formation was observed. The end point was recorded when the full separation between whey and curd was observed. One milk-clotting unit was defined as the amount of enzyme that clots 2 ml of the substrate within 180 min. Chymosin and 0.15 M NaCl were used as positive and negative controls, respectively. Milk-clotting activity was also determined using skim milk (10% w/v) heated at 30, 50 and 70 °C.