| In this study,the whole genome of Lactobacillus delbrueckii subsp.lactis QS 306was determined,the bacterial protease identified and the correlation between the two analysed.At the same time,the peptides in fermented milk before and after ultrahigh pressure treatment were identified by proteomics and the distribution of the potentially inhibitory peptides of ACE was investigated.Then the ACE inhibitory peptides with high activity and good stability were screened and their in vitro mechanism of action was analysed by molecular docking and molecular dynamics simulations.Finally,the animal model SHR was selected to investigate the antihypertensive effect of the inhibitory peptides of ACE in vivo and their potential antihypertensive mechanism.The specific research content is as follows:(1)The chromosome length of Lactobacillus delbrueckii QS306 is 2,007,516 bp,and there are 2,024 protein-coding sequences(CDSs)with an average G+C content of49.7%.By functional gene analysis,the genes Lac Z and Lac A were found to be related to lactose metabolism,as well as the phosphotransferase system(PTS),which is beneficial for sugar metabolism of Lactobacillus delbrueckii in fermented milk.Related genes of the proteolytic system were found,including Pep O,Pep Q,Pep N,Pep C,Pep T,Pep F,Pep DA and Pep P,as well as a complete Opp operon Opp ABCDF,17 separate oligopeptide binding protein genes Opp A,Dpp A1,which is beneficial for the transport of hydrophobic dipeptides/tripeptides,and the protease gene Clp P;with strong proteolytic capabilities.Identification of QS306 protease chromatography revealed 20 types of aminopeptidase,endopeptidase,tripeptidase,dipeptidase and Clp protease,and 9 types of peptide transporters.The identification of whole genome and protease provided help for the study of ACE inhibitory peptides in fermented milk.(2)Fermented milk of Lactobacillus delbrueckii QS306 was treated with ultra-high pressure and it was found that the ACE inhibitory activity of each component after treatment(UHP-FM)was significantly higher than that of the untreated group(FM)(P<0.05).There are 1464 and 1431 polypeptide sequences in UHP-FM and FM,belonging to53 and 49 types of precursor proteins,respectively,and the isoelectric points of the polypeptides are mainly at p H 5-7 and p H 8-10.There are 455 short peptides in UHP-FM and 425 short peptides in FM.The isoelectric points of short peptides are mostly in the range of p H 5-6,and the net charge is mainly positively charged or uncharged.The numbers of N-terminal amino acid peptides and C-terminal peptides are 115 and 116,respectively.214 short peptides with high activity values were screened out,including130 new peptides with potential ACE inhibitory activity.(3)Peptide Cutter was used to predict 43 new peptides with good stability,of which22 short peptides had ACE semi-inhibitory concentration of less than 10 mmol/L,and the ACE inhibitory activity of VAPFP was the best 10.56μmol/L.The short peptides were flexibly docked to ACE,and based on the binding energy,scoring,molecular structure and semi-inhibitory concentration of ACE inhibitory peptides,VAPFP,HLPLP,AARP,YRP,WRP,PYPQR,PAAP and WSR were screened out.Eight ACE inhibitory peptides were analysed for their inhibitory types,of which AARP,HLPLP,PAAP,WRP and WSR were competitive.PYPQR,VAPFP and YRP inhibit non-competitively,and the affinity of the peptide with ACE is YRP>WRP>WSR,AARP>PAAP,HLPLP>VAPFP>PYPQR,respectively.Simulated gastrointestinal digestion,WRP(after gastrointestinal protease treatment there was no significant change(P>0.05),but the ACE inhibitory activity of other short peptides remained above 85%.By combining RMSD,RMSF,β-factor,free energy landscape and hydrogen bonding in molecular dynamics simulation,it is found that the binding of AARP,PAAP,VAPFP and WRP with ACE is more stable,and the free energy of binding of the eight systems calculated by the method MM-PBSA is only HLPLP,which is not effective.Finally,ADMET and TOPKAT were used to predict toxicity.Eight short peptides had high metabolic activity in vivo,and none of them were carcinogenic,potentially developmentally toxic and carcinogenic to rodents,with VPFP being the mildest.In combination with the semi-inhibitory concentration,molecular docking,molecular dynamics simulation,stability and toxicity of VAPFP,VAPFP was selected for further in vivo verification.(4)After a five-week intervention with VAPFP,the blood pressure of rats with essential hypertension(SHR)was significantly reduced(P<0.05).When the cardiac section of SHR was examined,the pathological changes in the negative control group were obvious,and the pathological changes in the VAPFP group were the least.However,the kidney section showed that the tissues of the VAPFP group and the empty control group were in good condition and had no obvious pathological changes,while all other groups had varying degrees of pathological changes.The key enzymes and metabolites of RAAS metabolism in the blood of SHR were determined by enzyme immunoassay.It was found that VAPFP inhibited the activities of renin and angiotensin and decreased the production of angiotensinⅠ,angiotensinⅡand aldosterone,while the production of NO increased and water and Na~+were effectively excreted,which lowered blood pressure.Furthermore,angiotensin converting enzyme 2(ACE2)in the renin-angiotensin system(RAS)was found in proteomics analysis of rat heart tissue.Blood from SHR was measured using metabonomics.It was found that metabolism and synthesis of amino acids and biosynthesis of primary bile acids were the major metabolic pathways affecting the intervention of VAPFP.Arginine,tyrosine,lysine,L-ornithine,bile acid,glycocholic acid,glycochenodeoxycholic acid and betaine lowered blood pressure to varying degrees.In this study,the mechanism of ACE inhibitory peptides in fermented milk of Lactobacillus delbrueckii QS06 was systematically evaluated in vitro and in vivo,which provided a reference method for the future study of ACE inhibitory peptides from milk. |
PDF Full Text Request