Study On Bacterial Diversity And Functional Genes In Traditional Fermented Dairy Products

Traditional fermented dairy products have a long history of production and consumption;they are valuable carriers for microorganisms such as lactic acid bacteria(LAB).Koumiss has high nutritional value and unique medicinal properties;and thus it is also of important research and application values.Laboratory cultivation and routine metagenomics approaches are limited to profile microbes that have growth advantage in specific ecological environment,but can not accurately reflect the microbial diversity in traditional fermented dairy products.Recently,single cell-based techniques have received much attention because of their potential in microbial diversity research.They are advantageous in characterizing low-abundant and non-culturable microorganisms in natural environments.In this study,metagenomics analysis based on the single cell amplification technique was used to identify the bacterial diversity and functional genome in koumiss.This study randomly selected thirty koumiss(collected from Mongolia and Inner Mongolia of China)bacterial dilution samples and applied the single cell amplification technique on them.Illumina sequencing technology was used for the metagenomics analysis of koumiss samples,and a total of 24 bacterial species were identified.In addition to the common species,such as Lactobacillus helveticus,Lactococcus lactis,Lactobacillus kefiranofaciens,Streptococcus parauberis,and Enterococcus faecium,we successfully detected three rare species with relatively lower abundance in the koumiss samples.namely Lactobacillus otakiensis,Streptococcus macedonicus,and Ruminococcus torques.To improve the detection sensitivity,the samples were further analyzed by the PacBio single-molecule real-time sequencing technology(SMRT).The results showed that the use of this technique could further improve the precision in profiling the koumiss bacterial diversity.A total of 62 species were identified.The species identified by the PacBio SMRT sequencing was 2.58 times higher than that of the Illumina sequencing technology.In addition to common taxa,six other low-abundant species were found,namely Lactobacillus gallinarum,Lactobacillus parakefiri,Lactobacillus parabuchneri,Leuconostoc citreum,Lactobacillus sunkii,and Lactobacillus hamsteri.In fact,apart from the common koumiss bacteria such as LAB and acetic acid bacteria,we detected trace amounts of pathogenic bacteria,e.g.Klebsiella pneumonia,Mycobacterium orygis,Salmonella enterica,Streptococcus gallolyticus,Streptococcus alactolyticus,Ktuyvera cryocrescens,and Acinetobacter lwoffii.Meanwhile,some other valuable species with biotechnology potential were uncovered,such as Macrococcus caseolyticus and Lysobacter xinjiangensis.These results have not only expanded our knowledge in the koumiss bacterial composition,but also implicated new ways in detecting contaminatingbacteria in the industrial production process.To fully explore the function of the microbial coded-genes in koumiss,the Kyoto Encyclopedia of Genes and Genome(KEGG),Clusters of Orthologous Groups of proteins(COG),and the Carbohydrate Activity Enzyme(CAZy)databases were used for gene annotation.The results showed that many of these genes were involved in amino acid transport and metabolism,as well as carbohydrate and metabolism.Especially,koumiss bacteria coded a large number of genes relating to lactose metabolism,which are important for the bacterial proliferation and the formation of unique flavor of koumiss.In addition,koumiss bacteria also coded a large number of genes that belong to the glycoside hydrolase family and the glycosyltransferase family.The carbohydrate-active enzymes have important biotechnology potential.In conclusion,the integration of the single cell amplification technology and metagenomics analysis does not only reveal the bacterial composition in traditional fermented dairy products quickly and accurately,it also provides novel insights into microbial diversity and functional genome of traditional fermented dairy products.