Study On Preparation Of Angiotensin-I-converting Enzyme Inhibitory Pepetides From Enzymatic Hydrolysis Of Pinctada Martensii Protein

Pinctada martensii was one of the most important economic shellfish in our country’ssaltwater culturing. Pinctada martensii meat was rich in amino acids, for exampleTyrosine(Tyr), Valine(Val), Alanine(Ala), Isoleucine(Ile), Leucine(Leu), Proline(Pro),Tryptophan(Trp), Phenylalanine(Phe) and so on.Thirty kinds of protein sequence in Pinctada martensii meat were selected, which werealmost expressed in muscle tissue, and then these sequence were compared with the knownangiotensin-converting enzyme(ACE) inhibitory peptides, The results showed that someprotein sequences of Pinctada martensii meat contain a large number of ACE inhibitorypeptides, most of which were short chain peptides, such as the dipeptide, tripeptide and so on.The condition of mobile phase, the dosage and concentration of sample, the dosage ofthe termination reaction acid, adding sequence of N-Hippuryl-His-Leu hydrate(HHL) andACE were explored. The optimized condition was determined: the mobile phase wasacetonitrile:water=25:75(v/v), water contained0.05%TFA, the dosage and concentration ofsample: the total reaction volume was130μL, made up of30μL of5mmol/L HHL,10μLhydrolysate,30μL of100mU/mL ACE, and10μL of100mmol/L HCl. The method thatHHL added firstly was adopted. The method for determining the inhibitory activity of ACEwas modified by eliminated the effect of interfering peaks of some samples.Four commercial proteases (Papain, Pepsin, Alkali protease, Protemax) and self-madeenzyme were employed to hydrolyse Pinctada martensii meat. The ACE inhibitory, proteinrecovery, degree of hydrolysis of these hydrolyses were determined to compare the efficiencyof hydrolysis. And then3kinds of proteases was selected for further study. In conclusion,pepsin was selected to be the optimized enzyme. By orthogonal experiment, the optimizedconditions was determined: the hydrolysis temperature was37℃,the pH value were3, thehydrolysis time were35h.Effect of simulated gastrointestinal digestion on the ACE inhibitory of hydrolysate wasstudied. The hydrolysate was made under the optimized conditions. The ACE inhibitoryincreased to65.64%after hydrolyzing by pepsin for2h, and the ACE inhibitory decreased to52.57%after hydrolyzing by tyrisin for2h, as the ACE inhibitory changed little after thesimulated gastrointestinal digestion, so it indicated that ACE inhibitory of hydrolysateremained comparatively stable after simulated gastrointestinal digestion.