Files
Abstract
A microplate kinetic assay was conducted to examine the degradtion of substrate N-[3-(2-furyl) acryloyl]-L-phenylalanylglycylglycine (FAPGG) by angiotensin I converting enzyme (ACE) to furylacryloylphenylalanine (FAP) and glycylglycine (GG). The slopes of resulting kinetic curves for the first 15 min were used to calculate ACE inhibition. For food protein hydrolysates, ACE inhibition began to plateau after peanut was hydrolyzed for 2.5 h and cowpea for 1 h. A Box-Behnken statistical screening experiment with three levels was performed to evaluate the effects of hydrolysis factors pH, temperature and enzyme to substrate (E/S) ratio on ACE inhibitory activities of Alcalase hydrolysates of peanut and cowpea flours using response surface methodology (RSM). The factor pH did not show significant influence, whereas hydrolysis temperature and E/S ratio had significant effects on ACE inhibitory activities over their ranges studied. Response surface modelings and optimial hydrolysis conditions were determined. Peanut and cowpea hydrolysates obtained after 6 h of digestion under central point conditions by Alcalase were used to isolate ACE inhibitory peptides. After a series of purifications by membrane separation, primary and secondary reverse-phase high performance liquid chromatographies, the active peptides were found to be KAFR for peanut and FF for cowpea sequenced by a matrix-assisted laser desorption and ionization (MALDI) tandem TOF-TOF (time-of-flight) mass spectrometer. Quantitative structure-activity relationship (QSAR) of ACE inhibitory dipeptides was studied by partial least square (PLS) regression based on four sets of amino acid descriptor scales. The activies of collected dipeptides including the isolated dipeptide FF from Alcalase hydrolysate of cowpea were predicted. A potent ACE inhibitory dipeptide was proposed to have a large and hydrophobic amino acid such as Trp and Phe at the C-terminus and a nonpolar amino acid such as Val, Leu and Ile or possibly a positively charged amino acid such as Arg and Lys at the N-terminus.