Abstract: The aim of this study was to prepare ACE inhibitory peptides with low molecular mass from casein by stepwise hydrolysis with a single enzyme followed by double enzymes as a mimic for the gastrointestinal tract digestive system.  Peptides with high ACE inhibitory activity were first produced by hydrolyzing casein under optimized operating conditions and then further hydrolyzed by trypsin and chymotrypsin together into small peptides still having high ACE inhibitory activity. The optimal conditions for pepsin hydrolysis were enzyme/substrate ratio of 6%, substrate concentration of 0.015 g/mL, initial hydrolysis pH of 1.8, hydrolysis temperature of 37 ℃ and hydrolysis time of 2 h, and the ACE inhibitory rate of the 10-fold diluted hydrolysate obtained was 84.5%. The optimal conditions for further hydrolysis by trypsin and chymotrypsin together were trypsin/chymotrypsin ratio of 2:1 (m/m), initial hydrolysis pH of 7.8, hydrolysis temperature of 48℃ and hydrolysis time of 5 h, and the resulting 10-fold diluted hydrolysate showed an ACE inhibitory rate of 85.9% and a molecular mass of less than 500 D. Thus, the stepwise enzymatic hydrolysis process proposed in this study can enable the preparation of casein-derived small peptides with high ACE inhibitory activity.

Key words: casein, angiotensin-converting enzyme (ACE), active peptide, stepwise hydrolysis