Screening Of Novel Antihypertensive Peptides By Directed Hydrolysis In Qula Casein Based On The Structural Informatics

Hypertension is a considerable public health problem worldwide and poses a major risk factor that contributes to the pathogenesis of cardiocascular diseases.Angiotension I-converting enzyme(ACE)plays an important role in the blood pressure regulation system.In recent years,a variety of ACEI(ACE inhibitory)peptides derived from food proteins were reported as natural alternative bioactive peptides through the enzyme hydrolysis.Multi-steps isolation and purification are common strategies for the generation of novel ACEI peptides.However,this strategy is labor-intensive and costly.With more and more ACEI peptides reported,the relationship between their structural characteristics and inhibitory activity has been elucidated.Therefore,screening of ACEI peptides based on the structural informatics has promoted the development and utilization of novel ACEI peptides.Qula casein was selected as a precursor to produce bioactive peptides.The analysis of the amino acid species of Qula casein revealed that the proportions of hydrophobic amino acids,basic amino acids and aromatic amino acids were 41.85%,12.05%and 8.35%,respectively.The existence of these amino acids most likely strengthens the inhibitory activity of peptides to ACE.Then,the distribution of bioactive peptides in Qula casein was analyzed and found that many ACEI peptides were contained in Qula casein,and the total frequency of ACEI peptides(?AacE inhibition)was 2.1632,which indicated that Qula casein can be used as a precursor for producing ACEI peptides.In addition,an in silico proteolysis of Qula casein using single(thermolysin-Th,alcalase-Al,papain-Pa,proteinase K-Pr,trypsin-Tr and chymotrypsin-Ch)or combined enzymes to obtained ACE inhibitory peptides was investigated.Compared with the ? AACE inhibition values of single or combined enzyme to hydrolyse Qula casein,three single enzyme groups and five two-enzyme combination groups yielded high ? AACE inhibition values:Pr(0.1750),Pa(0.1608),Th(0.1537),Th+Pa(0.2856),Tr+Pr(0.2399),Ch+Pa(0.2387),Ty+Th(0.2338)and Pa+Al(0.2199).Then,Qula casein was hydrolyzed using various single enzymes and two-enzyme combinations.The hydrolysates were separated by ultrafiltration and found that the ACEI activities was mainly attributed to peptide components with lower than 3 kDa.Among the single enzyme groups,the hydrolysates derived from thermolysin showed the highest ACEI activity(IC50 8.8±1.6 ?g/mL),and for the two-enzyme combination groups,hydrolysates derived from Th+Al(IC50 2.6±0.6?g/mL)and Th+Pr(IC50 4.5±0.2 ?g/mL)had relatively low IC50 values.The relationship between ACEI activity and the ? AACE inhibition was also analysed.The results found that thermolysin showed a positive correlation,and Th+Pr and Th+Al showed a negative correlation.These results indicated that the high ACEI peptides may be contained in these two-enzyme combinations.Lineweaver-Burk plots were used to elucidate ACE inhibition kinetics of 3 kDa permeates of the hydrolysates and found that the hydrolysates exhibited mixed competitive inhibition.The peptide sequences in the hydrolysates from single enzyme and two-enzyme combinations(Th+Pr and Th+Al)were identified by LC-MS/MS.Then,the quantitative structure-activity relationship(QSAR)models were established based on the known ACEI peptides for predicting potential ACEI peptides in hydrolysates and totally 12 potential ACEI peptides were obtained.The molecular docking analysis was performed to further screen the peptides which can bind to the active site of ACE.Finally,7 potential ACEI peptides were obtained.Among these peptides,13 hydrogen bonds were formed between KYIPIQ and ACE.These ACEI peptides were synthesized by solid-phase synthesis,and the ACEI activities were determined.Among these peptides,KYIPIQ existed in thermolysin,Th+Pr and Th+Al hydrolysates showed the highest ACE inhibitory activity(IC50:7.3 ?mol/L).Peptide KYIPIQ exhibited a competitive inhibition pattern,which indicated KYIPIQ can bind to the active site of ACE.Finally,the effect of the peptide KYIPIQ on the production of nitric oxide(NO)in HUVECs and its transport pathway across monolayers of Caco-2 cells was explored.Treatment with KYIPIQ increased NO synthesis and induced endothelial nitric oxide synthase(eNOS)phosphorylation by the Akt activation in HUVECs.Furthermore,the results indicated that paracellular transport was the main transcellular parthway of KYIPIQ across Caco-2 cell monolayers.The peptide KYIPIQ was also performed with spontaneously hypertensive rats(SHRs)with oral administration and it could significantly reduce the systolic blood pressure of SHRs to 162.7±6.2 mmHg after 4 h administration.The novel ACEI peptides screened by structural informatics exhibited high ACEI activities,and the results in this study indicated that Qula casein can be used as precursor to produce ACEI peptides.The antihypertensive efficacy and transcellular pathway of novel ACEI peptide KYIPIQ derived from Qula casein hydrolysates was clarified in this study.