Study On ACE And DPP-? Inhibitory Peptides In Camel Milk Co-fermented By Lactic Acid Bacteria And Yeast

Food-derived active peptides have become a research highlights in the field of chronic disease prevention and treatment due to their wide sources,non-toxic side effects and low cost.Studies have proved that fermented camel milk has various life properties such as regulating blood pressure and lowering blood sugar.In recent years,there have been more and more studies on the use of fresh and fermented camel milk in the adjuvant therapy of diabetes and hypertension,but there are fewer reports on the relationship between the biological activity of fermented milk and the bacteria for fermenting.In this study,lactic acid bacteria and yeasts from traditional fermented milk were used to ferment camel milk to screen out strains with high production of angiotensinconverting enzyme(ACE)and dipeptidyl peptidase ?(DPP-?)inhibitory peptides,Active small molecule peptides were isolated,identified and synthesized,and studied their inhibitory mechanisms,which provide important data for the research of fermented camel milk lowering blood sugar and blood pressure mechanism.The research results obtained are as follows:1.Screening and identification of active lactic acid bacteria and yeastsAccording to the growth of strains in camel milk,173 strains of lactic acid bacteria and 70 strains of yeasts from traditionally fermented milk in Inner Mongolia were screened,and 141 strains of lactic acid bacteria and 62 strains of yeasts that could grow in and ferment camel milk were obtained.Milk antioxidant activity,?-glucosidase,ACE and DPP-? inhibitory activity were tested.Five lactic acid bacteria and four strains of yeasts with high ACE inhibitory activity were initially screened,and 5 lactic acid bacteria and 3 yeasts with high DPP-? inhibitory activity were screened.Taking the inhibition rate of ACE and DPP-? as an indicator,the screened strains were subjected to combined fermentation to obtain a combination with a higher inhibition rate of ACE and DPP-?—Lactococcus lactis strain L108 and Kluyveromyces lipolytica marxianus strain Y15.2.Optimization of conditions for co-fermentation of camel milk by lactic acid bacteria and yeast to produce ACE and DPP-? inhibitory peptidesAccording to single factor and response surface tests,the optimal conditions for co-fermentation of L108 and Y15 were deter mined as temperature of 30?,fermentation time for 87 h,and rotation speed as 153 rpm.Under the optimal conditions,the ACE inhibition rate of fermented camel milk was 35.44% higher than that of using single L108,and 20.12% higher than that of fermenting by single Y15;DPP-? inhibition rate was 30.55% higher than L108,and 17.24% higher than fermenting by single Y15.In the co-fermentation system,the number of viable bacteria of Y15 increased by 1.8% compared with single-bacteria fermentation,and the growth and metabolism of L108 played a role in promoting Y15.The activity of co-fermented camel milk was significantly improved after in vitro simulated gastrointestinal digestion.The half-inhibitory concentration of ACE decreased from 15.349 mg/m L to2.825 mg/m L;the half-inhibitory concentration of DPP-? decreased from 30.12mg/m L to 6.82 mg/m L.3.Separation,identification and synthesis of ACE and DPP-? inhibitory peptides(1)The ACE and DPP-? inhibitory peptides produced by fermented camel milk were separated and purified by ultrafiltration,dextran gel chromatography and RPHPLC.The fraction <3 KDa separated by the cut-off ultrafiltration membrane has the highest inhibition rate of ACE and DPP-?;the fraction <3 KDa is selected and separated by AKTA Dextran G-10 gel chromatography,and the peak No.3 with the highest ACE inhibition rate was obtained.Peak No.1 with the highest inhibition rate of DPP-?;two components were selected for further separation and purification by semi-preparative RP-HPLC,and finally the single peak 5-2 with the highest ACE inhibition rate and the single peak D3-2 with the highest inhibition rate of DPP-? were separated.(2)The isolated single-peak sequence was identified,and the ACE and DPP-? inhibitory activities of the peptide sequence were predicted through BIOPEP.The ACE inhibitory peptides VFGK,VYPYYG and DPP-? inhibitory peptides PHPALLAP,FGGY with higher BIOPEP scores were selected for synthesizing.The half-inhibitory concentrations of the ACE inhibitory peptides VFGK and VYPYYG were deter mined in vitro to be 1067±0.45 ?mol/L and 484±0.98 ?mol/L,both of which were competitive inhibition;the half-inhibitory concentrations of the DPP-? inhibitory peptides PHPALLAP and FGGY were 920±0.49 ?mol/L and 1946±0.58 ?mol/L,respectively,are also competitive inhibition.(3)The interaction sites and forces between synthetic peptides VFGK,VYPYYG and ACE were analyzed by molecular docking technology;the interaction sites and forces between synthetic peptides PHPALLAP,FGGY and DPP-? were analyzed.The results showed that hydrogen bond is the main driving factor that inhibits the binding of the peptide to the two enzymes.Among them,VFGK combined with His344,Glu372 and His348 of ACE enzyme to form three hydrogen bonds,and it also forms a hydrogen bond with the active site Zn2+ of the ACE enzyme.VYPYYG combined with His314 and Met184 of ACE enzyme to form two hydrogen bonds Hydrogen bond,and combined with Pro368 and Arg479 of the ACE enzyme to form a hydrogen bond.FGGY combines with Ser1318,Glu893,Glu894 and Tyr1350 of DPP-? enzyme to form 5 hydrogen bonds;PHPALLAP combines with Arg1248 of DPP-? enzyme to form hydrogen bonds.And it is close to the active site of DPP-? enzyme,so there may be exists van der Waals forces.