Study Of Angiotensin I-converting Enzyme Inhibitory Peptides From Acetes Chinensis Protein

Trypsin, S5317 acid protease, low-temperature alkaline protease from F.lavobacterium, 3942 neutral protease and 2709 alkaline protease were used to hydrolyze Acetes Chinensis protein, respectively. According to analyze soluble N determination, total N determination and inhibitory activity against Angiotensin I-Converting Enzyme (ACE) of hydrolysates, low-temperature alkaline protease from Flavobacterium and 3942 neutral protease were selected for the proper enzymes to prepare ACE inhibitory peptides. In order to look for the optimal hydrolysis conditions of low-tempreture alkaline protease from Flavobacterium and 3942 neutral protease, orthogonal experiment were processed. Low-temperature alkaline protease from Flavobacterium hydrolysate F4 and 3942 neutral protease hydrolysate F7 have the most potent ACE inhibitory activities respectively. IC₅₀ value of F4 and F7 are 5mg/mL, 8.6mg/mL respectively. F4 and F7 with good flavor are resistant to degradation by digestant protease and ACE to maintain ACE inhibitory activity. So F4 and F7 can apply to functional foods for preventing hypertension caused by ACE and for therapeutic proposes.The acute antihypertensive effects of hydrolysate F4 and F7 derived from Acetes Chinensis, which have ACE inhibitory activity in vitro, on renovascular hypertensive rats were investigated. Renovascular hypertensive rats models were made with two-kidney one clip assay. Changes of arterial blood pressure and plasma angiotensin â…¡ (Ang â…¡) were measured respectively. The results revealed F4 and F7 markedly reduced the arterial blood pressure of renovascular hypertensive rats, as well as plasma Ang â…¡ . This indicate that primary mechanism of the acute antihypertensive effects caused by Acetes Chinesis hydrolysate might beassociated with its actions on decreasing the production of Angll.After F4 was dialysed with dialyzer molecular-weight cut-off 2000 and 1000 Da, it was found that active fraction with ACE inhibitory activity was the peptides which have a molecular-weight below 1000 Da. Then the active fraction was separated and purified by Sephadex G-15 and SP-Sephadex C-50. The most potent fraction was then purified by reverse-phase high-performance liquid chromatography (HPLC) C18 preparative column to get pure sample FIA. FIA was affirmed by amino acid composition analysis and amino acid sequence analysis to be an new ACE inhibitory peptide Pro-Arg-Tyr. F7 was separated and purified by Sephadex G-25 and SP-Sephadex C-50 successively. The most potent fraction was concentrated and further purified by preparative HPLC to get pure sample FI. FI was identified as an new ACE inhibitory peptide Ser-Pro after the application of amino acid composition analysis and amino acid sequence analysis. ACE inhibition pattern of F7 and F4 was investigated by Lineweaver-Burk plot respectively. It was found that F7 and F4 bind competitively with the substrate at the active site of ACE.The ACE inhibitory peptides from shrimp sauce as a kind of fermented Acetes Chinensis were investigated. HWE (water extract), HWEE80 (80% ethanol extract after water extraction) and E80 (ethanol extract) were prepared from shrimp sauce. HWE, HWEE80 and E80 were determined to have potent ACE inhibitory activity. HWE, HWEE80 and E80 are resistant to degradation by digestant protease and have higher activities after preincubation with ACE.