Study On The Production Of ACE And DPP-Ⅳ Inhibitory Peptides From Mare’s Milk Fermented By Lactic Acid Bacteria And Yeast

Traditional fermented milk contains a variety of beneficial microorganisms such as lactic acid bacteria and yeasts.It is important to study the microbial community structure of the traditional fermented milk,to clarify the interaction of different microorganisms during its the fermentation process,to reveal the flavor function and physiological activity of microbial strains,which have important theoretical significance and application value in exploring microbial resources and establishing industrial technology of its production.In this study,samples of five different type of fermented milk products,Eedem(ED),Tarag(TR),Airag(AR),and Chige(CG),were collected from Xilin Gol,Inner Mongolia.Firs,the microbial community structure and the diversity of all products were determined by high-throughput sequencing.Second,the inhibiting activity of ACE and DPP-Ⅳ of the milk either fermented by lactic acid bacteria and yeast alone or the milk co-fermented by them have been evaluated.Optimization of peptide producing conditions for co-culture fermentation was performed based on the response surface method,and ACE and DPP-Ⅳ inhibitory peptides were separated and purified.Finally,the interaction of lactic acid bacteria and yeast in the co-culture fermentation system is then discussed.The results of the study are as follows:1.study on the microbial diversity in Inner Mongolian traditional fermented milk(1)Bacterial diversity in Inner Mongolian traditional fermented milk : the order of bacterial microbial community diversity is: ZH>HR>CG>AR>ED,and the order of fungal community diversity is ED>HR>CG>AR>ZH.As a whole,the diversity and richness of bacterial microbial communities are higher than fungi.The dominant bacterial phylum in these five traditional fermented milk samples are Firmicutes and Proteobacteria.At the genus level,the dominant genus in the CG,HR,and AR groups is Lactobacillus and Acetobacter,the dominant genus in the ED and ZH group is Lactococcus,the bacterial communities structure between CG,HR,AR and ED,ZH are quite different.As a whole,the bacterial communities of the 15 traditional fermented milks basically has no geographical differencesat the genus classification level.Among the 5 groups of samples,the bacterial communities in CG,HR and AR were very close,while that of ED and ZH was different.1.Study on Microbial Diversity of Inner Mongolian Traditional Fermented Milk(2)The fungal diversity in Inner Mongolian traditional fermented milk: the fungal microbial community diversity ranks as: ED>HR>CG>AR>ZH.The fungal phylum includes Ascomycota and Basidiomycota.Basidiomycota mainly exist in the ED group.A total of 20 different genera have been identified at the genus level,of which Dipodascus,Dekkera,Kazachstania,and Pichia are the dominant genera.Overall,the fungal communities in the five traditional fermented milks has certain regional differences at the genus level.The fungal communities in AQ and XS are similar,but it is significantly different from DW.The fungal communities included in the five different groups of samples is quite different.2.Isolation of lactic acid bacteria and yeast from traditional fermented milk from Inner Mongolia and screening of active peptides from its fermenting products.In this study,889 strains of lactic acid bacteria and 400 strains of yeast were isolated from five kinds of fermented milk samples.10 strains of lactic acid bacteria and 8 strains of yeast were screened out.The isolated strains were preliminarily identified by 16 S r RNA and 26 S r RNA gene sequence homology analysis.10 strains of lactic acid bacteria reached the maximum degree of proteolysis after 60 hours of fermentation.Among them,the ACE and DPP-Ⅳ inhibitory activity of the polypeptide from the horse milk fermented by L8 strain was significantly higher than that of fermented by other strains.α-glucosidase inhibitory activity of the polypeptide from the horse milk fermented by L7 strain was significantly higher than that of polypeptided from milk fermented by other strains.Y7 strain has a strong ability to hydrolyze horse milk protein,and the ACE and DPP-Ⅳ inhibitory activity polypeptide from fermented horse milk fermented by Y7 strain is significantly higher than other strains.Polypeptides form horse milk fermented by each of 8 strains of yeast had no inhibitory activity onα-glucosidase.3.Optimization of L8 and Y7 co-fermentation for horse milk producing peptide,interaction study and peptide separation and purification(1)Through the optimization of single factor and response surface test,the best cofermenting horse milk conditions for Y7 and L8 were obtained where the highest inhibitory activity of ACE and DPPⅣ can get at 29℃,52 h,6% of inoculation amount,and 115 rpm of rotation speed.(2)In the co-fermentation system of Y7 and L8,Y7 significantly inhibited the growth of L8.Compared with L8 single-bacteria fermentation,the number of L8 viable bacteria in mixed fermentation is significantly reduced,the p H is increased,the abilities to produce oxalic acid,malic acid,lactic acid,acetic acid,and galactose are significantly reduced,and the lactose metabolism is significantly reduced.L8 played a important role in promoting Y7.Compared with Y7 single-bacteria fermentation,the number of viable Y7 bacteria and the degree of proteolysis in mixed fermentation increased significantly.The ACE and DPP-Ⅳ inhibition rates of the peptides obtained in the co-fermentation system were significantly higher than those of single-bacteria fermentation.(3)Separation and purification of ACE and DPP-Ⅳ inhibitory peptides.Firstly,peptides were separated by AKTA dextran G-25 gel chromatography,part of the separated components were used for gastrointestinal simulated digestion,and part of them were used by semi-preparation RP-HPLC for further separation and purification.Simulated digestion of gastrointestinal peak of AKTA No.1 and further separation and purification performed by RP-HPLC.The ACE half-inhibition concentrations before digestion and after gastric digestion and gastrointestinal digestion were 4.77 mg/m L,3.11 mg/m L and 2.02 mg/m L,respectively.The DPP-Ⅳ were 11.15 mg/m L,7.52 mg/m L and 5.25 mg/m L,respectively.The half-inhibitory concentrations of ACE and DPP-Ⅳ of the fractions that further separated and purified by RP-HPLC were 0.243 mg/m L and 0.946 mg/m L,respectively.