Research On Production And Purification Of Angiotensin Converting Enzyme Inhibitory Peptides From Casein

A higher angiotensin I converting enzyme(ACE)'s activity is the "key factor of hypertension in human bodies.The ACE's activity centre can be combined with ACE inhibitory peptides(ACEIP) competitively so as to lower blood pressure.The existing research show that these peptides' molecular weigh are between 200～2000 Da and 2～10 amino acid recidues composed peptides are more common.Recent years, research on preparation of ACE inhibitory peptides from food proteins have been widely developing at home and abord.In Japan,the relative peptides products have listed on the market.At present,study of this field is gradually on the rise,but industrial production has not been developed.ACE inhibitory peptides with high inhibiting activity were prepared in this study by screening of the best protease for casein hydrolysis and optimization of the enzymatic hydrolysis condition.Besides,isolation and purification of ACE inhibitory peptides were performed,and the molecular weigh as well as structure of the peptide with the highest activity were researched.The mainly results were as follows:1.According to the method of Cushman etc.,the detection of ACE inhibitory peptides' activity in vitro was improved.The amount of ACE was adjusted,and the influence of HHL and hydrolysate on the testing results was counteracted.The reliability,repeatability and accuracy of this method were identified to demonstrate it's convenient and feasible.2.Casein was hydrolyzed by pepsin,compound protease,flavored protease, trypsinase,Alcalase respectively under their optimal condition.The best protease Alcalase was screened out for ACEIP's preparation by the comparation of hydrolysis process and hydrolysate's inhibiting activity.5 hours later,the hydrolysate's inhibiting activity reached the highest level with the inhibition rate of 56.1±0.7%.3.Alcalase was chosed for the preparation of ACE inhibitor(ACEI).Response surface methodology was used to optimize the enzymatic hydrolysis condition,which based on 4 factors,namely temperature,enzyme/substrate ratio,concentration of substrate and pH value at their different levels respectively with the hydrolysate's ACE inhibition rate as index.A multivariant quadratic regession model was established and the optimum conditions were as follows:temperature 60℃,concentration of casein 7.11%,Alcalase/casein ratio 7.83%,pH7.0.The hydrolysate's ACE inhibition rate reached 70.5%under this conditions,which increased 25.7%contrasted to the intial. The IC₅₀ was 0.54mg/mL.4.ACEIP were prepared by casein hydrolysis under the optimum conditions.The enzymatic hydrolysate was concentrated by ultra-filtration membrane with molecular weight cut-off 3kDa,and further purified by Sephadex G-15 gel chromatography.One isolated component was analysed by RP-HPLC and ESI-MS.The results showed that the ACE inhibiting activity of enzymatic hydrolysate increased 8.8%,and IC₅₀ decreased 0.15mg/mL by ultra-filtration,seven active components were obtained by Sephadex G-15,where the molecular weigh of the fourth was mostly between 182-752Da,and the IC₅₀ was 0.057mg/mL.