A Multi-Omics Study On Microbail Communities And Proteome In Fermention Of Pu-Erh Tea

The key factors for the formation of the special properties of Pu-erh tea are microorganisms, microbial proteins and changes of the chemical compositions in the fermentation process. To investigate these key factors, 36 fermented samples of two fermentations of Pu-erh tea were collected from Defeng tea factory, Yunnan. And five studies were developed as follow: (1) Structure and dynamic of microbial communities in the fermented tea leaves were studied through a metagenomics approach based on Illumina MiSeq sequencing; (2) The microbial proteins were investigated through metaproteomic approach by liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS); (3) The chemical constituents in the fermented tea leaves were detected by Ultraviolet spectrophotometric method and High Performance Liquid Chromatography (HPLC); (4) The microbial proteins in fermentation of Pu-erh tea were verified by pure culture fermentation and protein identification; (5) The enzymes be responsible for formation of the special properties of Pu-erh tea were screened by search of functions of identified proteins in KEGG database. The main results were as follow:(1) The study of structure and dynamic of microbial communities in the fermented sample of Pu-erh teaThe structure and dynamic of microbial communities in the fermented tea leaves were revealed through extraction of microbial DNA, PCR amplification of V4-V5 region of bacterial 16S rRNA gene and ITS region of fungal 18S rRNA gene, Illumina MiSeq high throughput sequencing and bioinformatics analysis. The dominant bacteria were Enterobacteriaceae in the raw material and in the initial stages of fermentation. The dominant bacteria were changed to Bacillaceae at the last stages of fermentation. The dominant fungi in raw material and initial stage of fermentations were Aspergillus. Fungi in Rasamsonia and Thermomyces were multiplied at the middle stage of fermentation, and became as dominant fungi with Aspergillus. Fungi belonged in Aspergillus were dominant in last stage of fermentation.(2) Study of microbial proteins in fermented samples of Pu-erh tea27-379 bacterial proteins and 189-1893 fungal proteins were identified through a metaproteomic approach composed of Tris-phenol extraction, trypsin hydrolysis, LC-MS/MS analysis, and search in the SwissProt database of microbial proteins by Proteome Discoverer 1.4. The main identified bacterial proteins were produced by Proteobacteria, and main identified fungal proteins were produced by Aspergillus. The major of Molecular Function, Cellular Component and Biological Process of identified proteins were catalytic activity, cytoplasm and metabolic process, respectively.(3) Study of the chemical compositions of fermented sample of Pu-erh teaDetection of Ultraviolet spectrophotometric method and HPLC showed that the contents of tea polyphenols, catechins, and amino acids were decreased significantly (P<0.05). Sensory evaluation showed that the taste of tea infusion became mellow; the color of tea infusion and leaves became reddish-brownish, the aroma became stale flavor.(4) Study on proteome produced by dominate fungi in the tea mediumA. niger, A. tamari, A. fumigatus and Blastobotrys adansenioivor were inoculated into liquor and solid tea medium, respectively.16-1916 proteins were identified in liquor and 493-1632 in solid fermentations through LC-MS/MS based proteomic analysis. These results confirmed that the dominate fungi can produce a variety of proteins in fermentation of Pu-erh tea. The Molecular Function, Cellular Component and Biological Process of identified proteins were catalytic activity, cytoplasm and metabolic process, respectively.(5) The microbial enzymes involved in formation of the special properties of Pu-erh tea98 enzymes involved in formation of the special properties of Pu-erh tea were identified by search on KEGG database. Such as, Alpha-xylosidase, Beta-glucosidase, Pectin lyase, etc., which involved in degradation of polysaccharide; Catechol 1,2-dioxygenase, Phenol 2-monooxygenase, etc. which involved in polyphenol metabolism; Adenylosuccinate synthetase, Adenylosuccinate lyase, etc., which involved in caffeine metabolism. The chemical reaction catalyzed by these enzymes, and the role in the formation of the special properties of Pu-erh tea are one of focus in future study.In conclusion, the microbial community and proteins in two fermentation of Pu-erh tea were demonstrated by metagenomics and metaproteomics approaches. Enzymes involved in the formation of the special properties of Pu-erh tea, including enzymes are responsible for degradation of polysaccharide, polyphenol and caffeine metabolism were identified. It gave novel theoretical knowledge and important clue for study the role of microorganisms in the formation of the special properties of Pu-erh tea.