Purification, Activity and Sequence of Angiotensin I Converting Enzyme Inhibitory Peptide from Alcalase Hydrolysate of Peanut Flour

Peanut hydrolysate obtained after 6 h of digestion by Alcalase was used to isolate angiotensin I converting enzyme (ACE) inhibitory peptides. After centrifugation and ultrafiltration through a 0.2 mu m nylon filter, the hydrolysate was filtered through the polyethersulfone membrane with a molecular weight cutoff (MWCO) of 10 kDa. The resulting permeate was then separated by primary reverse-phase high performance liquid chromatography (RP-HPLC). Eluate was divided into six major fractions according to eluation time, The fraction with eluting time 50-60 min showed the most potent ACE inhibition and was subjected to further purification by the secondary RP-HPLC. Four peaks were found to have strong ACE inhibitory activities, and their IC50 values were determined. Peptide mass for the most potent peak was obtained by matrix-assisted laser desorption and ionization (MALDI), and sequence was determined by MALDI tandem TOF-TOF (time-of-flight) mass spectrometer (MS/MS) to be Lys-Ala-Phe-Arg.