Simultaneous monitoring of twelve angiotensin I converting enzyme inhibitory peptides during enzymatic β-casein hydrolysis using Lactobacillus peptidases

An enzyme extract from Lactobacillus helveticus ATCC 15009 was used for β-casein hydrolysis. The enzymatic release of twelve X-Pro and X-Pro-Pro peptides was monitored over time by reversed phase LC–MS analysis after dabsylation of the samples. This allowed the separation and quantification of the structural isomers isoleucine-proline and leucine-proline. All peptides demonstrated inhibitory effects on the angiotensin converting enzyme (ACE) reaction in vitro. Most of the X-Pro dipeptides reached their maximum concentrations after 24 h of hydrolysis, whereas the maximum concentration of the isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) was observed after 72 h. The theoretical yield of each peptide was calculated and compared with the maximum amount obtained during the course of hydrolysis. Here, the highest yield (10.44%, 1.65 mg L−1) was observed for the X-Pro dipeptide alanine-proline; IPP and VPP had much lower maximum yields. Furthermore, three dipeptides, threonine-proline, methionine-proline and leucine-proline, were identified as novel ACE-inhibitors.