Biotransformation Of EGCG By Culturable Microorganisms In Fuzhuan Brick Tea

Fuzhuan brick tea(FBT)is a unique kind of microbial fermented tea.The main stage involved in microbial fermentation is the processes of piling and “flowering”,among the“flowering” is a key process for the quality of FBT.Therefore,it is particularly important to research the microbial species and the transformation of tea components by these microbial for explaining the quality forming mechanism of FBT.In this study,we selected the samples of FBT and those at the piling and “flowering”stages.As many bacterial and fungal strains as possible were isolated by streak plate method,then identified by morphological features and the BLAST of DNA sequencing techniques.These microorganisms were inoculated in the solid-state fermentation system containing epigallocatechin gallate(EGCG),theanine and caffeine.After a certain time of incubation,we used organic reagents to extract metabolites of various microbial respectively,and acquired the MS data on a UPLC-Q-Orbitrap mass spectrometer.The data were comprehensively and deeply analyzed using the Feature-Based Molecular Networking(FBMN)to illustrate the differences of metabolites and the rules of biotransformation of EGCG between bacteria and fungi in the fermentation system.The results of this paper are as follows.Firstly,we isolated 29 bacterial strains and23 fungal strains from FBT,and identified that the bacteria can be classified into 3 phyla,3classes,3 orders,5 families and 5 genera.These genera of bacteria were mainly from Bacillius,Paenibacillus,Pseudomonas,Acinetobacter and Microbacterium.Most bacteria strains belonged to Firmicutes in the three phyla which accounted for about 90%(26strains)of the total bacterial species.Among the five genera,Bacillus was the most common that accounted for 86 %(25 strains)of the isolated bacteria.The fungal strains isolated and identified in this study were annotated as 3 phyla,5 classes,5 orders,7families and 8 genera.It mainly includes the genera of Eurotium,Aspergillus,Penicillium,Cladosporium,Bjerkandera,Candida,Cyberlindnera,Trametes and Rhizomucor.Most of the fungi isolated in the experiment(18 strains)belong to Ascomycetes,including the dominant fungus Eurotium cristatum.Secondly,the LC-MS data indicated that there are some similarities and significant differences between bacteria and fungi in the biotransformation of EGCG.These microorganisms both have the ability to hydrolyze gallate and to metabolize or bioconvert EGCG.The differences were shown in metabolic ability and structural modification.Thefungi had stronger ability to convert EGCG than bacteria.The products of EGCG modification by fungi were mainly fuzhuanin A,fuzhuanin D and teasperin,while the main bioconverted flavan-3-ols of bacteria were xanthocerin and fuzhuanin B.In addition,although the MS intensities of flavoalkaloids were strong,whereas we also can clearly found this intensities in control without microbial.The conclusion is the reaction from EGCG and theanine to flavoalkaloids is able to initiated without microbial enzymes.Microbial fermentation is the driving force for the characteristic flavour and functions of all brick teas,including FBT.It is possible that the microbial conversion on the original compounds in tea,especially the key flavour compound EGCG,is related to the unique flavour and functions in these dark teas.Therefore,the research on the separation and identification of microorganisms in FBT,and the biotransformation of EGCG by these microorganisms are determines EGCG is the substrates of flavan-3-ol A-ring derivatives that replace by N-ethyl pyrrolidone or B-ring fission derivatives.The transformation power of flavan-3-ol A-ring derivatives is spontaneous organic chemical reactions,some of B-ring fission derivatives Mainly from the transformation of microorganisms.