Metatranscriptomics And Bacteria Community Structure Analysis Of Koumiss’s Fermentation Process

The development of china’s Horse Industry is in transforming condition from the traditional horse industry to the modern form. Product horse industry is one kind of modern horse industry, included to utilizing meat,mare,blood and hippurate. Horse milk products, especially the Koumiss have broad prospects for development. Koumiss in China has2,000years of history.It have not only essential fatty acids, highly unsaturated fatty acid content, it is appropriate to humen digested, also have could lowering blood pressure, treatment of tuberculosis, anemia, gastrointestinal diseases,et al. It’s medical care value is consistent with the needs of the modern consumer. However, a large number of studies Koumiss lack of attention to their traditional fermentation process.Understanding the structure and function of the dynamic changes of gene expression of Koumiss’s microbial community could help us optimizing the community structure, community adjustment function, but also provide a theoretical basis for researching Koumiss’s food safety, clinical applications, improving controllability of traditional fermentation. In this study, fermented mare’s milk acid3.5L, puddled300times every2.5h and puddled1500times a day, fermentation temperature is maintained within the range of22±2℃.Under these conditions, continuous fermentation time96h, samples taken once every12h to prepare for future study. First, a sample of each period Koumiss’s physiological and biochemical parameters had been measured. Secondly, the six samples selected during the fermentation period, extracted the metagenomics DNA, PCR amplification of16S rDNA for454pyrosequencing, to analyze bacterial community structure succession in the Koumiss fermentation process.Finally, selecting the samples of12h,48h and96h in fermentation process for RNA-Seq high-throughput sequencing, building metatranscriptomic library of Koumiss to understanding the situation of functional gene expression in the fermentation process. The main findings were as follows:1. pH value of the fermentation process were decline, the rapid acidification of the initial fermentation, post-fermentation slow decline and reaching the lowest3.54; The contents of total protein、fats、amino acids、fatty acids during fermentation were increased, to achieve the highest value at72h; Lactose content decreased, while the lactic acid content increases in the fermentation process. Glucose, galactose,acetic acid, propionic acid at a low level during fermentation. Butyric acid was not detected at all sampl.2. The highest diversity of bacterial fermentation at the begining. Then droping. Highest value of bacteria’s abundance were72h.3. Firmicutes and Proteobacteria was the dominant phylum in Koumiss fermentation.4. Lactobacillus and Lactococcus were dominant bacterial genus. With the extension of the fermentation time,12h.24h Lactobacillus content showing a rising trend, while at72h and96h of its content showing a downward trend; Lactococcus presented with rising trends at fermentation; Acetobacter levels showed an increasing trend.5. By high-throughput sequencing,obtaining12.2G high-quality short sequences and assembling64,210merge-Unigenes,59,339Unigenes could determining the direction and their CDS region. The remaining sequences were predicted Coding regions by useing ESTscan software,there may be2,230new protein-coding sequences.6. GO functional classification analysis showed that the fermentation process is closely related to the metabolic process, catalytic activity and cellular process, etc.; COG classification results displayed by the function, in the fermentation process of Koumiss involved amino acid transport and metabolism-related genes,replication, recombination and repair genes play a key role in the regulation;KEGG metabolic pathway analysis results, metabolic pathways, biosynthesis of secondary metabolites and microbial metabolism in diverse environments pathway were major signaling pathways.7. Based on GO functional enrichment analysis of genes differentially expressed results, initial fermentation to medium fermentation, metabolic process,cellular process, organic substance metabolic process is the main biological processes, meanwhile organic cyclic compound binding,heterocyclic compound binding,small molecule binding related genes expression active; Since the medium to the end of fermentation, cellular process,cellular macromolecule metabolic process,single-organism cellular process genes activly expressed, and molecules functional related genes,such as structural molecule activity,structural constituent of ribosome,protein binding genes activly expressed.8. Differentially expressed genes pathway enrichment analysis indicating,during from the initial fermentation to the medium fermentation, Ribosome pathways is the dominant metabolic pathways of this period; Since the medium to the end of fermentation, ether lipid metabolism,aminobenzoate degradation,teroid biosynthesis,mRNA surveillance pathway occuring major roles.