Preparation Of Angiotensin I-Converting Enzyme Inhibitory Peptides From Pinctada Martensii Meat By Protease Hydrolysis

Hypertension is a major risk factor of cardiovascular diseases, such as heart failure, apoplexy, coronary disease and myocardial infarction. Angiotensin I-converting enzyme inhibitory (ACEI) is one kind of important drugs for prevention and treatment of hypertension, but chemical synthesis of ACEI is extremely easy to cause queasiness, vomit, diarrhea and other side effects. Therefore, angiotensin I-converting enzyme inhibitor peptides from food protein has to be especially noticeable because of its advantages which includes safty, little side effects, easily absorbed, and so on, and become one of the focuses in the bioactive peptides research fields.In this article, six different proteolytic enzymes were employed to hydrolyze Pinctada martensii meat to produce the hydrolysates under their suitable condition, and the high performance liquid chromatography method was developed for determination of the inhibitory activity of angiotensin I-converting enzyme (ACE) in hydrolysates. The result indicated that hydrolysis of Pinctada martensii meat with alkaline proteinase produced the highest ACE inhibitory activity. Therefore, alkaline proteinase was established for further study on optimization of hydrolysis conditions. And the degree of hydrolysis was determined on 21% when hydrolysate has the highest ACE inhibitory activity. On this basis, the factors, including the enzymatic quantity, concentration of substrate, temperature, pH and reaction time, were inspected respectively on DH. The result showed that the optimum conditions of alkaline proteinase to hydrolyze Pinctada martensii meat was: enzymatic concentration at0.175 g·L^-1, concentration of substrate at 35 g·L^-1, temperature at 45℃, pH on 9.5 and hydrolysis time at 240 min. Detected by SDS-polyacrylamide gel electrophoresis, the result about weight of molecular indicated that lots of peptides from hydrolysate was less than 14.4 kD.Based on the experimental data and empirical formula, the dynamics model about hydrolyzing of Pinctada martensii meat by alkaline proteinase was deduced respectively. And the results of general kinetic equation for this hydrolysis process were suggested: DH=5.757ln[1+(44.023E₀/S₀-0.0448)t] and r=(253.44E₀-0.2576S₀)exp[-0.1738(DH)]·Meanwhile, some kinetic parameters were obtained respectively: Enzymic inactivation constant k_d is 51.737 min^-1, the hydrolysis activation energyâ–³E_A is 57.05 kJ·mol^-1 and theinactivation energyâ–³E_D is 63.30 kJ·mol^-1. Compared theoretical value with actual value, thedynamic model was proved very good agreement with experimental result, and showed its good practical value.The 10 KD ultrafiltration membrane was used to separate ACEI initially. At this time, the optimum process parameters were determined: The pressureâ–³P at 0.4 MPa, the initialconcentration of substrate is 25 mg·mL^-1, pH on 7.0, operating temperature for 35℃.Meanwhile, compared inhibitory activity of solutions with before and after ultrafiltration, the result showed that ultrafiltration was able to enrich higher ACE inhibitory activity components of hydrolysate effectively.