Optimization Of The Fermentation Process Of High-Yielding ACE Inhibitory Peptides Fermented Milk And Analysis Of Their Peptide Profiles

Hypertension is an important risk factor for cardiovascular and cerebrovascular diseases.With changes in modern lifestyle habits,the incidence of hypertension has been increasing year by year,and it has become a common chronic disease.Angiotensin-I converting enzyme（ACE）is a dipeptidyl carboxypeptidase with a dual-domain structure that plays a crucial physiological role in the regulation of blood pressure in the body.Inhibiting the biological activity of ACE can effectively reduce blood pressure.Therefore,the development of fermented milk with ACE inhibitory activity has broad application value and prospects in the food industry.In this study,casein fortification and the use of advantageous strains that produce ACE inhibitory peptides as adjunct cultures were used to improve the ACE inhibitory activity of fermented milk.Single-factor experiments and response surface experiments were used to optimize the production process of high-yield ACE inhibitory peptide fermented milk.The effects of sodium caseinate and Lactiplantibacillus plantarun M11（Lb.plantarun M11）on the physicochemical and quality characteristics of the fermented milk,as well as the peptide production capacity,were explored,and the peptide sequences in the fermented milk were identified and analyzed.The main research results are as follows:（1）Analysis and calculation of 29 amino acid sequences of milk proteins showed thatβ-casein variant B（BIOPEP ID:1100）had the highest frequency of bioactive peptide occurrence（ΣA=1.6651）and ACE inhibitory peptide occurrence(A_ACE=0.6268).In addition,β-casein(A_ACE=0.5844),α-casein(A_ACE=0.4677),andκ-casein(A_ACE=0.4421)all exhibited good potential as sources of ACE inhibitory peptides and are excellent sources for the production of ACE inhibitory peptides.Therefore,it is speculated that casein reinforcement of fermented milk can enhance its ACE inhibitory activity.（2）Four strains with good acid and bile tolerance and acid production capacity selected by the research group were studied for their protease activity,protein hydrolysis ability,peptide content,and ACE inhibition activity.The ACE inhibition rate of Lb.plantarum M11 fermented milk reached 76.19±0.52%,significantly higher than the other three groups（P<0.05）,and it had the highest protease activity（89.37 U/m L）,protein hydrolysis ability(OD_340nm=0.141±0.007),and peptide content（0.61±0.02 mg/m L）.It is a strain that produces ACE-inhibiting peptides.（3）Based on the single-factor experiments on the inoculation ratio,inoculation amount,fermentation temperature,and addition amount of sodium caseinate,the response surface methodology was used to optimize the production process of high ACE inhibitory peptide fermented milk.The optimal process conditions were determined to be as follows:an inoculation ratio of Lb.plantarum M11 and commercial starter YO-MIX 883 at 2:1,an inoculation amount of1.5×10⁷CFU/m L,a fermentation temperature of 40℃,and an addition amount of sodium caseinate of 2%.Under these conditions,the ACE inhibition rate of the fermented milk can reach83.15%.（4）The effect of adding sodium caseinate,Lb.plantarum M11,and their combination on the properties of fermented milk was studied.The results showed that the lactose utilization of fermented milk significantly increased（P<0.05）with the addition of sodium caseinate and Lb.plantarum M11.The high viable cell counts were maintained throughout the storage period（8.14lg CFU/m L after 28 days）.The water-holding capacity was enhanced（82.05±0.06%after 28 days）,and the hardness,viscosity,cohesion,and viscosity index were significantly increased（P<0.05）.Rheological properties were improved,and ACE inhibition rate was 83.15±0.52%.These findings suggest that the ACE inhibitory activity of fermented milk can be significantly increased without significant adverse effects on its flavor.（5）Nano LC-MS/MS was used to identify peptide sequences in fermented milk.The results showed that there were 6,8,10,and 13 peptides with confirmed ACE inhibitory activity in the 883,883+M11,883-CS,and 883+M11-CS groups,respectively.Among the peptides not included in the ACE inhibitory peptide database,YPFPGPIH had a smaller molecular weight（927.18 Da）,hydrophobicity（hydrophobic value of 0.11）,was non-toxic,non-potentially allergenic,and could resist digestion by gastrointestinal fluids.NILRFF had the highest Peptide Ranker score（0.93）,a smaller molecular weight（802.14 Da）,hydrophobicity（hydrophobic value of 0.01）,and was non-toxic.FVAPFPEVFGK,APFPEVFGK,ARHPHPHLSFM,HLPLPLL,and RHPHPHLSFM all produced ACE inhibitory peptides after simulated digestion by gastrointestinal fluids and were non-toxic.Therefore,YPFPGPIH,NILRFF,FVAPFPEVFGK,APFPEVFGK,ARHPHPHLSFM,HLPLPLL,and RHPHPHLSFM were considered to be biologically active peptides with ACE inhibitory potential.Molecular docking results showed that NILRFF and ARHPHPHLSFM had binding energies of-10.7 Kcal/mol and-9.8 Kcal/mol,respectively,and could strongly interact with the active site of ACE.It is suggested that it may have good ACE inhibitory activity.