In-vitro And In-vivo Evaluation Of ACE Inhibitory Bioactive Peptides Of Fermented Milk To Reduce Hypertension

Food is considered as a fuel for human body.Food originated from animal or plant source,contains certain level of bioactive compounds that affect human body,positively.In case of vascular diseases,human body responds to certain bioactive peptides of Plants as well as animal origin.These bioactive peptides can be derived either from source of food by applying different techniques e.g.by the exploitation of hydrolyzing enzymes,microbial enzymes or proteinases.Recently,Scientists are trying to exploit bioactive peptides of foods to eradicate or minimize the load of cardiovascular diseases like hypertension.Usefulness of bioactive peptides in reducing hypertension with the inhibition of ACE is an established fact because ACE is considered as main causative agent in growing hypertension and other cardiovascular disorders.The main goal of this study was,in-vitro and in-vivo evaluation of fermented buffalo milk for the production and evaluation of novel ACE inhibitory peptides on the basis of serum protein level,and to evaluate fermented goat milk for the production and evaluation of novel ACE inhibitory peptides in-vitro and in-vivo by optimizing fermentation time.Due to better profile of protein and availability worldwide,goat and buffalo milk was used to design this study.After defatting and lyophilization,milk protein was targeted for further analysis.After evaluating the health status of buffaloes by running hemto-chemical tests,four young dairy animals(D-22,D-26,D-30 and D-34)were selected on the basis of serum protein level for further analysis and fermented with LC Lacobacillus Casei).In goats,milk protein was,then,fermented with LH(Lactobacillus Helveticus)by optimizing the incubation time to 8,16,24 and 36 h.Following are the main results of our study.1-D-22 was used as control in buffaloes to compare the ACE inhibition trend,and S-8(incubated for 8 hours)was used as control in goats.After co-culturing of buffalo skim milk with LC for 24 h,peptide samples were purified via ultrafiltration by using Molecular weight cut-off;10000 Da(PM-10)membrane.2-Protein hydrolysis was confirmed by applying nin-hydrin reaction and SDS-PAGE.Milk of 34-months old animal(D-34)showed greater ACE inhibitory activity in-vitro via ACE inhibitory assay.On the other hand,ACE inhibitory peptides were purified from fermented samples via ultrafiltration.Molecular weight cut-off;10000 Da(PM-10)and ultracel 3K membranes were used to perform ultrafiltration.3-Sample with 24 hours incubation time was considered as best hydrolyzed as compared to others after applying nin-hydrin reaction and SDS-PAGE.4-ACE inhibitory assay further validated the authenticity of S-24(incubated for 24 hours)in inhibiting ACE,in-vitro.Furthermore,Q exactive Hybrid Quadrapole-Orbitrap Mass Spectrometry was used to determine molecular structure and amino acid sequence of ACE inhibitory peptides in both groups of animals.Peptides were identified with the help of MaxQuant search database.5-Finally,Peptides with higher concentration of Proline,Lysine and aromatic amino acid were identified in D-34.Higher concentration of hydrophobic aliphatic amino acids in PIN-94 and PIN-332 indicated the usefulness of D-34 as ACE-inhibitor in fermented buffalo milk protein sample.While,Three peptides in S-24,VLPVPQKAVPQ,VLPVPQKVVPQ and TQTPVVVPPFLQPEIMGVPKVKE containing functional amino acid structure,had been identified with highest ACE inhibitory activity on the basis of intensity,size and higher concentration of hydrophobic amino acids.6-In-vivo analysis was performed in SHRs to check the effects of those abundant peptides with the application of telemetric method.Reduction in systolic blood pressure after 12-14 hours and diastolic blood pressure was recorded after 14-16 hours in D-34 while decrease in systolic and diastolic blood pressure was observed in S-24 after 10-12 and 8-10 h,respectively.It was established in the light of observations that small peptides with functional amino acids can be used as part of functional food to inhibit ACE and can serve as a remedy to reduce blood pressure.In this way,fermented milk protein can be used as nutraceutical for hypertensive patients with health promoting effect.Industrial production on affordable price of this functional food can bring a better change in the lives of hypertensive and cardiovascular patients.