Preparation Of Angiotensin-Converting Enzyme Inhibitory Peptides By Hydrolysis Of Cattle Bone

In this paper, angiotensin-converting enzyme inhibitory peptides were prepared by hydrolysis cattle bone. Therefore, the work was carried out from the following aspects:The changes of bone protein by softening process and nutritional value of cattle bone meal by preparating in the laboratory were studied. The hydrolysis effect of three kinds of enzymes which were alkaline protease, neutral protease and trypsin were better than others. The bone protease solution conditions impacted on the kinetic parameters. According to the dynamic test, the optimum conditions of alkaline protease acted on the bone protein at 55℃and pH 12 and the kinetic parameters was 0.10824 g/L·min for Vmax and 11.71892g/L for Km. The optimum conditions of Neutral protease was at 45℃and pH 7 and the Vmax was 0.04724g/L·min with Km for 14.35711g/L at the same time. The optimum conditions of Trypsin was at 55℃,pH 9 and this time Vmax for 0.05073g/L·min and Km for 4.31516g/L,The crude enzyme solution of angiotensin-converting enzyme (ACE) was extracted from fresh lung. After purification, the specific activity of ACE enzyme solution was improved to 5.21u. The relationship between the degree of hydrolysis and the rate of ACE inhibition was researched. The results were that the regulation was inconsistent in different enzymes. On this basis, the alkaline protease was selected as a tool to preparate ACE inhibitory peptide from cattle bone meal. Optimum hydrolysis conditions were obtained with an enzyme to substrate ratio (E:S) of 0.04 for 3 h at 50℃and pH 10.The molecular weight distribution of hydrolysates was determined by Sephadex G-25 chromatography. There are three peaks in which molecular weight of the peakⅢis less than 1000 Da, the half maximal inhibitory concentration (IC50) was determined as 0.561mg/ml. Ultrafiltrate (5000Da) and hydrolysates were qualitative analyzed by amino acid analyzers. Digestive stability test shows that ACE inhibitory activity is stability because the mixture can not be hydrolysis easily by digestive enzyme.ACE inhibition kinetics study found that the ultra-filtrate was a competitive inhibitor.Scatter and non-linear regression model demonstrates that the proportion of hydrophobic amino acids and aromatic amino acids in protein have great impacted on ACE inhibitory activity of hydrolysis.Based on the above conclusions, macroporous resin and activated carbon were used for enrichment of hydrophobic amino acids and aromatic amino acid to improve ACE inhibitory activity of bone peptide. The optimum sample concentration of macroporous resin purification was 50mg/ml.100% ethanol eluted fraction obtained the highest ACE inhibitory activity and its half-inhibitory concentration (IC50) as 0.45mg/ml, the yield of component as 24.59%.The optimum sample concentration of activated carbon purification was 30mg/ml.100% ethanol and 5% acetic acid elution fraction obtained IC50 as 0.59mg/ml.