Preparation,Inhibition Mechanism And Stability Of ACE Inhibitory Peptides Derived From Maize Germ

Hypertension is the most common non-communicable chronic disease in the world and a major cause of heart disease,stroke,kidney failure,early death and disability.Angiotensin Converting Enzyme(ACE)inhibitors are widely used in the prevention and control of hypertension as first-line drugs in clinical practice.Food-derived ACE inhibitory peptides has been widely concerned by researchers because their natural origin,safety and effectiveness.High value utilization of food raw materials and their by-products to prepare new bioactive peptides has broad prospects for the development of new antihypertensive drugs or functional food ingredients.In this paper,maize germ was used as raw material.Firstly,the extraction conditions of corn germ protein were optimized.Then,the effects of different enzymatic hydrolysis conditions on the ACE inhibitory activity of proteolytic products were investigated to determine the optimal process conditions for the preparation of maize germ ACE inhibitory peptides.Secondly,the hydrolysate of maize germ ACE inhibitory peptide was isolated,purified and its structure was identified to explore the activity mechanism of maize germ ACE inhibitory peptides.The protective effect of ACE inhibitory peptide on hypertension-impaired HUVEC and its mechanism were further explored.Finally,the stability of ACE inhibitory peptide under different conditions was studied.The main research contents and results are as follows:(1)maize germ was extracted as protein by alkaline method.The effects of extraction conditions such as solid-liquid ratio,temperature,alkali solubility time and alkali solubility p H value on the extraction rate of maize germ protein were investigated.The optimal extraction conditions were determined by response surface methodology(RSM)as follows:solid-liquid ratio of 1:18,p H 9.5,temperature 53?and time 122 min.The ACE inhibitory peptides of maize germ were prepared by enzymatic method.The effects of protease types and enzymatic hydrolysis process on the ACE inhibitory activity of the hydrolysates were investigated.The results showed that the enzymatic product of flavor protease had the highest ACE inhibition activity.The optimum enzymatic hydrolysis conditions were determined as follows:substrate concentration of 3%,enzymatic hydrolysis time 120 min,p H 6,temperature of 40?,and enzymatic dosage of 13300 U/g.(2)Maize germ protein hydrolysates were divided into three components(F1,F2,and F3)by Sephadex G-25 chromatography,the F3 showed the most potent ACE inhibitory rate.F3was separated into three fractions(F3-1,F3-2,and F3-3)by ion exchange chromatography,of which F3-1 with most potent ACE inhibitory rate.Four novel ACE inhibitory peptides were identified by capillary high performance liquid chromatography-electrospray combined with ion trap Orbitrap(LC-ESI-Orbitrap-MS),and their amino acid sequences were Pro-Trp(PW,1.48 m M),Val-Thr-Leu(VTLL,2.24 m M),Leu-Pro-Gly-Pro(LPGP,2.07 m M),Ser-Pro-Gly-Thr-Ala-Phe((SPGTAF,2.31 m M)and their activity was verified by synthesis.Kinetics studies showed that PW was found to be a non-competitive inhibitor,and VTLL,LPGP,SPGTAF competitive were competitive inhibitor.Furthermore,molecular docking results revealed that the high inhibitory activities of ACE inhibitory peptides were mainly attributable to coordination with Zn(II)and the formation of hydrogen bonds interactions with the ACE.(3)CCK-8(Cell Counting Kit-8)assay was used to detect cell viability,and it was found that PW,VTLL and LPGP had no significant cytotoxicity to HUVEC.The HUVEC injury model induced by Ang?was constructed.It was found that PW,VTLL and LPGP could improve the cell viability and balance the contents of ET-1 and NO,which had a protective effect on HUVEC induced by Ang?.To investigate the mechanism of LPGP on damaged HUVEC,it was found that LPGP could decrease the contents of ET-1 and ROS in a concentration-dependent manner,reduce the rate of apoptosis,regulate the contents of MDA and SOD,and significantly increase the contents of NO and NOS(P<0.05).LPGP can increase the relative protein levels of p-e NOS and p-AKT when the total protein of e NOS and Akt does not change.By further adjusting the Akt signaling pathway,it was found that LPGP could realize cell proliferation through the AKT pathway,improve the expression of p-e NOS,reduce the contents of ET-1 and ROS,reduce the content of oxidative stress factor MDA,increase the content of SOD,and increase the production of NO and NOS.By increasing the relative expression level of p-AKT,Modulate the mechanism of apoptosis,thereby ameliorating Ang II-mediated HUVEC cell injury.(4)The activity changes of ACE inhibitory peptides PW,VTLL and LPGP in food environment(thermal treatment,p H,food additives,metal ions,food preservatives)and in gastrointestinal environment were studied.The results show that PW,VTLL and LPGP can maintain high activity under heat treatment.VTLL were significantly affected by alkaline conditions(P<0.05).When food additives are added to the food system,the ACE inhibitory activities of PW,VTLL and LPGP are easily affected by high glucose concentration,but remain stable under citric acid treatment.The activity of PW decreased slightly when Na Cl concentration was higher than 4%,while VTLL and LPGP were able to tolerate the effects of high salt concentration.PW,VTLL and LPGP can retain high activity when exposed to K⁺,Ca²⁺and Mg²⁺.Potassium sorbate,a food preservative,can be used as one of the additives of these functional foods,while sodium benzoate has a certain effect on the ACE inhibition activity.In simulated digestion in vitro,the ACE inhibitory activities of PW,VTLL and LPGP had certain resistance to gastrointestinal digestive enzymes.