Development And Identification Of Angiotensin Converting Enzyme Inhibitory Peptide From Casein

Now the ACE inhibitory peptide, one kind of the most popular biologically active peptides, is attracting more and more the scientists'attention. Milk and the diary product contain all kinds of essential amino acid which make them one of the most nutrient food, meanwhile some ACE inhibitory peptides have been developed from it. Here, the new ACE inhibitory peptides were obtained from casein by simulating the gastrointestinal digestion. At first, the macroporus adsorption resin DA201-C and Sephadex G-10 were applied to separated the enzyme hydrolysate. Then, the fraction named 7-3 possessed the highest ACE inhibitory rate was obtained after two-step RP-HPLC. The method to develop ACE inhibitory peptide from casein was proved to be effective, and new thought and approach were provided for the utilizing of milk and development of ACE inhibitory peptides. Meanwhile, some basis was provided for the structure-effect relationship. The mainly content of this research is displayed as follows:Casein was hydrolyzed by pepsin at first, then by trypsin andα-chymotrpsin as the second step, simulating the mammal gastrointernal digestion process. The optimum condition of pepsin hydrolysis was: [E]/[S]=6%, pH=1.8, T=37℃, t=2 h. The ACE inhibitory activity assay revealed an value of 84.5% when the supernatant was diluted 10 times, the molecular weight(MW) distributed between 200 D~3000 D, the next enzyme hydrolysis process with trypsin andα-chymotrpsin accomplished under the following conditions: trypsin(6%):α-chymotrpsin(3%)=2:1, pH=7.8, T=48℃, t=5h, The ACE inhibitory activity assay revealed an value of 85.9% when the supernatant was diluted 10 times, the molecular weight distributed between 100 D~1000 D. The result proved that ACE inhibitory peptides with high activity of low MW could be yielded by simulating gastrointernal digestion.Then the enzyme hydrolysate was separated with macroporus adsorption resin DA201-C.The peptide was absorbed on the resin and eluted in turns by ethanol of different concentration: 25%, 50%, 75% and 100%. The optimum condition of absorbance was described as below: when the peptide concentration was 20 mg/mL, resin weight was 30 g per 100 mL peptide solution, t=4 h; elution condition parameter: the volume of ethanol was the same as the peptide solution, t=2 h. Elution fraction of ethanol of 75% shows the highest ACE inhibitory activity. It was 89.16% when the concentration was 0.5 mg/mL.Next, the elution fraction of ethanol of 75% was separated further by sephadex G10. Two peaks was obtained and F2 showed higher ACE inhibitory activity. The ACE inhibitory rate of F2 was 42.7% when the concentration was 0.2 mg/mL.Next, the component 7-3 with the highest ACE inhibitory activity was purified through two step semi-RP-HPLC and IC50 was 26.5μg/mL.The component 7-3 may contain TFLW,NGGDECDTPT and FPYGTAPDSW after analysed with LC-ESI/MS/MS.