Stability And Preparation Of ACE-inhibitory Peptides From Cyclina Sinensis

Cyclina sinensis as the materials,In order to select the suitable detection method,this paper try to use the high performance liquid chromatography（HPLC）method and ultravio let spectrophotometry（UV）method to determine ACE inhibitory activity in vitro.Through the method validation,select linear relationship,detection limit,limit of quantification,precision and recovery rate to become the test indexes.Preparation of angiotensin-convert ing enzyme（ACE）inhibitory peptides from Cyclina sinensis by enzymatic hydrolysis was explored.The suitable protease was chosen and the effects of the temperature,time,p H,enzyme dosage and solid-liquid ratio were examined using single factor method coupled with L₁₆（4⁵）orthogonal array design based on ACE inhibition rate.And purification with ultrafiltration technology,fast protein purification system and high performance liquid chromatography.The amino acid sequence of ACE inhibitory peptides were identified by the N-terminal degradation and the inhibition kinetics of ACE inhibitory peptides were studied.Finally,the stability of ACE inhibitory peptides under different temperatures,p H and gastrointestinal simulation were explored.The experimental results showed that HPLC and UV on ACE inhibitory activity had no significant difference（P>0.05）.Method of validation indicated that,An excellent linearity over the range of 0.001-1 mmol/L（R²=0.9997）was observed.The HPLC method has good accuracy and reproducibility,the recoveries of hippuric acid ware96.85%-100.43%.The optimum enzyme is trypsin,the optimum preparation conditions were temperature 45℃,reaction time 9 h,p H 8.0,enzyme dosage 1200 u/g and solid-liquid ratio 1:2,under which the ACE inhibition rate of the hydrolysates was 36.8%.After the separation and purification,a single component which ACE inhibitory rate of87.58%,the amino acid sequence was identified as Trp-Pro-Met-Gly-Phe（TPMGP）,and the molecular weight was 636.83 Da.TPMGP had the high ACE inhibitory activity(IC₅₀=0.5025 mg/m L).The IC₅₀ of captopril was 27.3 n M,and the literature reported that IC₅₀ of captopril was 23-35 n M.It showed that the determination method has better accuracy.The Lineweaver-Burk about ACE inhibition experiment showed that ACE inhibitory peptides and captopril on the ACE inhibitory pattern were competitive inhibition.The stability about temperature,p H and gastrointestinal simulation experiments show that ACE inhibitory peptides were stable under 0～100℃and p H between 2 to 8,it have poor tolerance to pepsin but trypsin has better.