The Preparation Of ACE Inhibitory Peptide From Mare's Milk Casein By Fermentation And Enzymatic Hydrolysis

Hypertension is a public cardiovascular disease,which is a key risk factor of leading to cerebral infarction,myocardial infarction and renal failure.Although the blood pressure lowering drugs used clinically can maintain the stability of blood pressure very well,long-term use of these drugs will bring varying degrees of side effects to the kidneys and other organs.Angiotensin Converting Enzyme(ACE)is related to two blood pressure regulating systems in the human body,namely Renin-Angiotensin System(RAS)and Kinin-Nitric Oxide System(Kinin-Nitric Oxide).System,KNOS).The RAS system is the main system for regulating blood pressure,and ACE is the core enzyme of the RAS system.Therefore,inhibiting ACE is an effective way to lower blood pressure.In recent years,there has been increasing attention to food-derived ACE inhibitory peptides due to their safety,effectiveness,and non-toxic side effects.There have been studies on the isolation and purification of ACE inhibitory peptides from milk proteins,but most of them have focused on the study of cow milk.In this study,mare's milk casein was used as the raw material,and the yeast with high ACE inhibitory peptide production was combined with optimized protease hydrolysis conditions to prepare ACE inhibitory peptides.The studies on separation,purification and identification of peptides were carried out.The results of the studies are as follows:(1)The casein and whey protein of mare's milk were separated,and the ACE inhibition rate after the digestion of the simulated gastrointestinal digestion was measured.The results showed that the ACE inhibition rate of casein digesta was significantly higher than that of whey protein.A yeast strain was selected from Koumiss,and the ACE inhibitory activity of the fermented mare's milk casein product was 47.56%.The strain was identified as Ka.unispora from Kazakhstan by ITS phylogenetic analysis,and named as Ka.unispora KU530 strain,or KU530 for short.(2)Five commercial proteases,namely alkaline protease,neutral protease,trypsin,papain and flavour protease,have been utilized to hydrolyze mare's milk casein.Using ACE inhibition rate as an indicator,the trypsin hydrolysate showed the highest ACE inhibition rate of 58.15%.Single factor and response surface design methods were used to optimize the conditions of trypsin hydrolysis of mare's milk casein.The results showed that the optimal condition was:the casein concentration was 20 mg/m L,p H 7.6,enzyme addition E/S was 6.8%,at 36°C for hydrolyzing 1.5 h.Among them,temperature has the greatest influence on the ACE inhibition rate,followed by p H,and the enzyme addition had the less effect on ACE inhibiting activity.The optimized ACE inhibition rate reached 70.17%,which is an increase by12.02%compared to the unoptimized one.(3)Combination of fermentation with protease hydrolysis:the product of mare's milk casein fermenting by KU530 were subsequently hydrolyzed by trypsin under the optimized conditions.The ACE inhibitory activity was significantly higher than that of fermenting by KU530 alone(35.77%higher)and hydrolyzing by trypsin alone(13.16%higher).The final product for combination of fermentation with protease hydrolysis of mare's milk casein was separated and purified,and the R₂-2-2component was obtained as a single peak.The R₂-2-2 component was identified by MALDI/TOF-TOF-MS/MS and four new ACE inhibitory peptides were identified,namelyTVDMESTEVVTEK,VNNQALPQPIER,andPQPIERT TPKGEKFPSMSEAR.These four peptides used to carry out molecular docking study and the results showed that the peptides can bind to the ACE active site to form a stable complex and interact with them to form hydrogen bonds.It is speculated that these four peptides can well inhibit ACE activity,and It will be subjected to solid phase synthesis.