The Symbiotic Relationship Between Lactic Acid Bacteria And Yeast In Traditional Horse Milk And Its Application In Horse Milk Fermentation

Lactic acid bacteria and yeast are the microbial group of traditional fermented horse milk.In this study,the functional strains were enriched by subculture methode and high-throughput sequencing method was used to analyze the microbial structure and the succession trend of dominant strains in the process of subculture.After the dominant strains are obtained,lactic acid bacteria and yeasts with symbiotic relationship are selected and the mechanism of flavor formation of symbiotic strains in fermented horse milk was studied by metabolomics methods.This research provides an important theoretical basis for the development of industrialized production technology for fermented horse milk.At the same time,the study of the interaction of symbiotic bacteria has an important role for revealing the adaptive evolution mechanism of microorganisms in the milk.1.Research on microbial diversity and subculture in traditional fermented horse milk(1)The diversity of bacteria in traditional fermented horse milk:Three different origin of traditional fermented horse milks mainly included:Lactobacillus,Enterobacter,Lactococcus,Acetobacter,Citrobacter,Escherichia coli Shigella,and the absolute dominant bacterial genus was Lactobacillus Genus.After subculture,the dominant bacterial phyla was Firmicutes,and the dominant genus included Lactobacillus,and its abundance value was in the range of 93.7%-99.7%.(2)The diversity of fungi in traditionalfermented horse milk:Three different origin of traditional fermentd horse milk mainly included Dirk Saccharomyces,Kazakhstan Saccharomyces,Kluyveromyces,Dipodia,Pichia,and Mucor at the genus classification level.The absolute predominant fungus genus was Dekkeromyces.After subculture,the dominant phyla was Ascomycota,and Dekkeromyces was the absolute dominant fungus in the these samples.(3)The subculture from T1 to T4 is a process of cultivating dominant strains.Acetobacter gradually disappeared and Lactobacillus gradually enriched,and its abundance value was close to 1.Kluyveromyces gradually disappeared,and Dekkeromyces was enriched.Through the isolation and identification of lactic acid bacteria and yeasts in T4 samples,a total of 96 lactic acid bacteria were identified,of which 80 strains of horse milk-like bacillus(Lactobacillus kefiranofaciens),accounting for 83.3%;62 yeast strains,of which 32 were Dekkera anomala Strains,accounting for 51.6%.Lactobacillus and Dekkeromyces were the dominant genera,which was consistent with the results of high-throughput sequencing.2.Screening of symbiotic lactic acid bacteria and yeasts and optimization of fermentation conditionsLactic acid bacteria(L157)and yeast(Y5),which have symbolic effect,have beed obtained by Screening from identified 96 strains of lactic acid bacteria and 62 strains of yeasts.Through single factor and orthogonal experiments,the fermentation conditions of symbiotic strains were optimized.Taking the number of viable yeast,the viable number of lactic acid bacteria and the sensory evaluation score as indicators,the optimal fermentation condition was 8%(v/v)of noculation amount,inoculation ratio(lactic acid bacteria: yeasts = 1:1),first at 30? 150 rpm,a total of 48 h of fermentation.3.The symbiosis mechanism of lactic acid bacteria and yeast(1)The symbiosis relationship between Y5 and L157 only existed in mare's milk,and there was no similar symbiosis relationship between them in cow milk.The aseptic fermentation broth of L157 promotes the growth of Y5,and consistent with this,the aseptic fermentation broth of Y5 also promotes the growth of L157.The lactose in mare's milk was hydrolyzed by lactase and cofermented by Y5 and L157.The results showed that the number of viable bacteria of Y5 and L157 did not increase.The activity of ?-galactosidase was measured,and the results showed that the utilization rate of lactose was increased during the cofermentation process,and the activity of ?-galactosidase was increased to 0.427 U/mg.(2)The metabolites in 12 horse milk samples were analyzed,and a total of 1150metabolites were obtained.Differential metabolite screening,principal component analysis,and KEGG function annotation and enrichment were carried out.Compared with group Y and group YL,there are 202 metabolites that are significantly upregulated,such as carboxylic acid and its derivatives.The main enrichment pathways include biosynthesis of valine,leucine and isoleucine,and arginine Compared with proline metabolism,pyruvate metabolism,and propionate metabolism;compared with the L group and the YL group,there are 135 metabolites significantly up-regulated,such as phenyllactic acid,asparagine and proline,which are mainly enriched metabolic pathways Including amino acid biosynthesis,pyruvate metabolism.