Characterization Of Angiotensin I Converting Enzyme Inhibition Peptides From Lactobacillus Delbrueckii QS306 Fermented Milk

In this paper,Lactobacillus delbrueckii QS306 was used as the research object,and the inhibitory rate of angiotensin converting enzyme（ACE）was used as an index to isolate,purify,identify and analyze amino acid sequence of ACE inhibitory peptide in fermented skim milk.The ACE inhibitory peptides were synthesized by Fmoc solid-phase synthesis and the spatial structure was determined.The effect of digestive enzymes on ACE inhibitory activity was analyzed.At the same time,the effect of ultra-high pressure treatment on the ACE inhibitory activity and flavor of Lactobacillus delbrueckii QS306 fermented milk was studied.The results of the study are as follows:（1）Isoelectric precipitation method was used to extract the whey of Lactobacillus delbrueckii QS306 fermented milk and the whey ACE IC50 was 0.57 mg/mL.The whey was separated by ultrafiltration centrifuge tubes（10 kDa,3 kDa）and Sephadex chromatography.The ACE IC₅₀ was 0.426 mg/mL,0.324 mg/mL,and 0.051 mg/mL.After glucan gel chromatography,the sample with good separation and high activity was purified.（2）RP-HPLC purified the sample with high ACE inhibitory activity and the result showed that the chromatographic sample was a mixture of hydrophobic peptides of different molecular weights.The ACE inhibitory activity of component C5 was found to be highes and the ACE IC₅₀ is 12.87μg/mL.（3）The amino acid sequence of the purified high-ACE inhibitory active fraction C5was determined by LC-MS/MS,a pentapeptide sequence LPYPY（Leu-Pro-Tyr-Pro-Tyr）with a molecular weight of 651.326 Da was identified.This sequence was derived fromκ-f（77-81）in bovine milk protein and was first found in fermented milk;prediction of the stability of the polypeptide with class 28 enzymes in the protein database found only pepsin,trypsin,and K-Protease has an effect on it.（4）LPYPY was synthesized using Fmoc solid-phase synthesis method.The ACE IC₅₀0 was 83.94μg/mL,LPYPY was scanned by circular dichroism and analysis by JWSSE32 software.The secondary structure of the pentapeptide LPYPY was:beta-pleated sheet 74.6%,turn 12.5%,Random coil 7.2%,α-helix 5.7%.（5）Using trypsin,chymotrypsin,pepsin of pH 1.2 and pH 2,to simulated gastrointestinal digestion tests on LPYPY.The ACE inhibition rate were 70.85%,75.68%,94.4%,63.2%and 70.85%.Gastrointestinal enzymes had enzymatic hydrolysis of post-synthetic ACE inhibitory peptides.Except for pepsin with a pH of 2,there were significant differences among other treatments.（6）The treatment of Lactobacillus delbrueckii QS306 fermented milk with ultra-high pressure showed that the inhibition rate of ACE in fermented milk after ultra-high pressure treatment was significantly improved,and its taste had no significant effect.The effect of skim milk was better than that of whole milk.