Research On Optimizing Technological Parameter Of Enzymatic Preparation Of Antihypertensive Peptides Derived From Swine Hemoglobin And Hypertensive Activities In Vitro

Several antihypertensive peptides are generated from swine hemoglobin with protease. Reports shows that the peptides with high antihypertensive effect and safty, it is going to be widely used as a kind of natural antihypertensive agents.In the study, the inhibitory percentages of AngiotensinⅠ-Converting Enzyme (ACE) of enzymatic hydrolysates with trypsin, pepsin and papain were used to select the most suitable enzyme. Swine hemoglobin was used as the substrate. Determining ACE inhibotory activities and degree of hydrolysis of enzymatic hydrolysate produced with 3 proteinases.The results at 6h showed:the degree of hydrolysis of hydrolysates ranged in a desend order was papain, pepsin, trypsin while ACE inhibotory activities of hydrolysates ranged in a desend order was pepsin, trypsin, papain. Papain was excluded firstly. Secondly, comparing the hydrolyzing degree, pepsin was higher than trypsin. As a result, trypsin was choosen to hydrolyse swine hemoglobin.Single factor level test was adopted to optimize the hydrolysis conditions (substrate concentration, temperature, pH, enzyme dosage and hydrolysis time) of swine hemoglonbin hydrolysates by trypsin. The optimum levels of factors were used to design orthogonal experiment. The results showed that the high ACE inhibitory activity of trypsin hydrolysates would be obtained at substrate concentration 10%,40℃,pH9.0, enzyme dosage 2000U/g and hydrolyzing 6h. The ACE inhibition was 62.54% in this condition. Swine hemoglobin was hydrolysed in the optimum condition by trypsin.3 components prepared by ultrafiltration from hydrolysates.Testing the activities of components of different molecular weight. The results showed that:ACE inhibition of different components ranged in a desend order was Lc (MWCO<5KDa), Oc (original hydrolysates), Mc (5KDa10KDa), and the molecular weight of the highest ACE inhibitory activitiy component was mostly less than 5KDa.The components of less than 5KDa molecular weight were prepared for 7 different solutions with concentrations (4,8,12,16,20,24,28μg/ml).Captopril was prepared for 7 different solutions with concentrations (0.4,0.8,1.2,1.6,2.0,2.4,2.8ng/ml).Testing the ACE inhibitory activites of each solution respectively. The results show that the ACE inhibitiory IC50 of components of molecular weight less than 5KDa was 13.69μg/mL,to illustrate that there were high ACE inhibitory activity peptides in the component. The ACE inhibitory IC50 of Captopril was corresponded with some published papers. The result validated the method of ACE inhibitory activity determination was feasible, and showed the scientificity of the determining method of ACE inhibitiory activity.In the digestion simulating experiment. Pepsin (P), chymotrypsin (C), trypsin (T) were used to prepare 3 simulating conditions:(P), (P+C), (P+C+T).2 components (Oc, Lc)were treated in the 3 conditions. Testing the ACE inhibitory activites of each solution respectively. The results of the Lc before and after processing showed no evident differences while the results of the original hydrolysates before and after processing had significant difference.The component of Lc (MWCO<5KDa) was stable in the digestion. The study made experimental basis for the further study of antihypertensive peptides in the food industry, and provided reliable experimental data for developing swine hemoglobin antihypertensive peptides in the future.Meanwhile, a new way of deep processing of swine blood was explored.