Dynamics Of Bacterial Diversity And Functional Genes During Koumiss Fermentation

Koumiss,as a fermented mare milk beverage,has been popular in Central Asia and Eastern Europe due toits nutritional richness and important economical value.Although it is known that the structure of koumiss microbiota is unique;however,until now,there have only been few in-depth studies focusing on the koumiss core bacterial microbiota,dynamics of the diversity and functional changes of these microbial communities during the fermentation process.Firstly,this study applied the PacBio SMRT sequencing technology to profile the koumiss core microbiota at the species level.Secondly,samples have been taken from 5 selected local families of a pasturing area of the Chinese minority group at different time points in order to reveal the dynamics and the regulation of the microbial communities during the traditional fermentation process using a metagenomics approach.This study provides a comprehensive and in-depth mechanistic overview of the dynamics of koumiss microbiota in relation to its quality.The presentworkhas provided novel data in this field and will act as theoretical guidance for improving both the quality of traditionally fermented products and the process of their industrial production.The main results are as follow:(1)The koumiss bacterial microbiota mainly consisted of four species,i.e.Lactobacillus helveticus,Lactobacillus kefiranofaciens,Lactobacillus gallinarum,and Acetobacter pasteurianus,belonging to the phyla Proteobacteria and Firmicutes.Among the 11 koumiss samples,25 core OTUs could be identified,which was equivalent to 43.64%of all high-quality sequences.Twenty-four of these core OTUs were Lactobacillus helveticus.Thus,we can conclude that a core microbiota is shared across the koumiss samples,although the microbiota composition of individual samples might vary.(2)The bacteria community playsa crucial role during koumissproduction and fermentation processes.To reveal the dynamics of the koumiss bacterial structure and biodiversity during fermentation,the full-length 16s rRNA genes of 22 samples collected from 5 sampling sites and fermentation times were sequenced bySMRT sequencing in the present study.The results identified a total of 148 species that belonged to82 genera and 8 phyla of bacteria.The results suggest that the structural difference in the bacterial community was linkto the difference insampling location.The microbial composition was most significantly different between the group D samples compared with those of the other groups.Moreover,between 0-9 hours of koumiss fermentation,the relative abundance of Lactobacillus helveticus gradually increased and reached a peak at 9 h,followed by a decline.The relative abundances of the Enterococcus faecalis,Enterococcus durans,and Enterococcus casseliflavus increased gradually throughout the fermentation process,and reached a maximum level at 24 h.(3)At the gene level,the sample functional genes of different time points were mainly made up of the COG categories of L(Replication,recombination and repair),E(Amino acid transport and metabolism),J(Translation,ribosomal structure and biogenesis),and G(Carbohydrate transport and metabolism).The COG categories of F(Nucleotide transport and metabolism),M(Cell wall/membrane/envelope biogenesis),V(Defense mechanisms),Q(Secondary metabolites biosynthesis,transport and catabolism),L(Replication,recombination and repair),and U(Intracellular trafficking,secretion,and vesicular transport)were significantly and positively correlated between each other.In addition,the COG categories and koumiss nutrient components were closely associated.The results of the KEGG database matching showed that 92.7%of all KOs were found across the first 9 hours of fermentation;and there was no significant increase in the number of KOs during this period.As the fermentation went on,the gene richness and metabolic pathway distribution of the koumiss samples would change to a certain extent.Overall speaking,there was a greater difference in the relative microbial gene abundance between the fresh milk(B1)and the milk that had just been inoculated with the starter(B2).With the prolongation of fermentation time,the change in the functional gene set slowed down.