| The Oyster ( Crassostrea gigas ) meet was degisted by different proteases and the angiotensin coverting enzyme inhibitory peptides were separated and partially purified from oyster in this study. The whole work included three parts described below.1. A rapid HPLC method was developed for the determination of Angiotensin I-Converting Enzyme (ACE) inhibitory activity. The hippuric acid formed from the ACE catalyzed hydrolysis of HGG was separated and identified by HPLC method (ODS C18 column (5μm, 150 mm×4.6 mm), eluting solution: acetonitrile and water (20:80, V/V, containing 0.5‰methanoic acid), flow rate: 1 mL/min, detection at 228 nm). The developed calibration curve of hippuric acid displayed good linearity over the concentration range from 0.1μg/mL to 500μg/mL with a correlation coefficient equaling 0.9999. The average spike recoveries (3 replicates) at 3 levels ranged from 89.77% to 99.57%, with a relative standard deviation (RSD) from 0.01% to 1.52%. By testing the inhibitory activity of different ACE inhibitors, the HPLC method was proved to be rapid, accurate and suitable for analysis of ACE inhibitory activity in vitro.2. Using HPLC method to determine ACE activity, the ACE reaction conditions catalyzed by the ACE isolated from pig lung were optimized. The results indicated that the optimal reaction time of ACE with its substrate HGG was 30 minutes by measuring the amount of hippuric acid liberated from HGG in the ACE reaction system. The optimal concentration of the substrate HGG and ACE were 5 mmol/L and 12.5 mg/mL, respectively. In order to study the preparation of antihypertensive peptide derived from oyster, seven enzymes including flavourzyme, papain, pepsin, alcalase, acidic protease, trypsin and neutrase were applied to hydrolyze oyster. The effect of time on the products hydrolyzed by each of proteases was also investigated on the basis of the degree of hydrolysis (DH). The results showed that the degree of hydrolysis approximately reached the peak at 4 hours after the hydrolysis started. Among the proteases, neutral protease had the strongest hydrolysis activity with a degree of hydrolysis approaching to 89.88%. Contrarily, the degree of hydrolysis of papain protease was the lowest at 34.51%. The relationship between the inhibitory rate of ACE and the degree of hydrolysis of oyster degraded by the different proteases was examined. The results showed that the ACE inhibitory rates of the different hydrolysates could rise to a high level at the first 15 minutes, while they revealed a slow increase or even decrease with time prolonging. The hydrolysates produced by pepsin were stable in 12 hours at least and possessed the highest ACE inhibitory ratio (96.99%) under the conditions of 1200 U enzymatic concentration per gram substrate for 4 hours.3. Pepsin hydrolysis products were purified by gel filtration through G-75. Two peaks, A and B, were obtained. SDS-PAGE analysis showed that the molecular weight of peak B, which had the high ACE inhibitory activity, was far below 6 kDa and the protein band was dispersed and smeared. Then peak B was separated using G-25 and got peak C. The peak C was further separated by RP-HPLC method under the following separating system: ODS C18 column (5μm, 150 mm×4.6 mm), elution with acetonitrile and water (15:85, V/V, containing 0.5‰phosphoric acid) at a flow rate of 0.8 mL/min, detection at 265 nm. Three individual peaks were isolated and the molecular weights of the main component of each peak as assessed by HPLC-MS were 973.583 Da, 1086.715 Da and 1353.821 Da, respectively. Finally, DSC thermal denaturation temperature analysis was conducted and the results indicated that each component separated from RP-HPLC was a mixture of different peptides. Thus, to obtain a single peptide, further purification work need to be done in the future. |
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