The Diversity Of Eurotiums Sp.in Dark Tea And Its Effect On The Quality Improvement

Dark tea is a unique fermented tea in China,which is mainly divided into Shaanxi Fuzhuan brick tea,Anhua Dark tea,Yunnan Pu'er tea,Guangxi Liubao tea,Sichuan Kang brick tea,Guizhou side selling dark tea.Dark tea has become an essential drink in the daily life of ethnic minorities in the northwest frontier because of its rich aroma,full taste and unique effect of eliminating fat and reducing greasy.The formation of the unique taste flavor and health care effect of dark tea is related to the unique fermentation process of dark tea pile fermentation and flooding.Its essence is a series of complex biotransformation reactions,especially jinhua,which is actually a large class of Eurotiums SP.,which plays a key role in the formation of dark tea quality.The unique taste of dark tea can not be separated from the formation of pile fermentation and Eurotium sp.in the fermentation stage,its metabolites have good antibacterial activity,antioxidant activity and anti-tumor activity,Consequently,Eurotium SP.can be used to develop health products as well as medicinec.The classification of Eurotiums SP.in dark tea is vague and classified into Jinhua fungus in different shapes,so as to be identified in dark tea.At present,there are many microbes involved in the fermentation of dark tea,the interaction between strains and its mechanism need to be analyzed.dark tea pile fermentation is an open natural fermentation,affected by environment and workshop microclimate,there,the quality and safety of dark tea have been debated.Synthetic probiotics can not only improve the taste and flavor of tea,but also improve the quality of tea.artificial synthesis of probiotics and accurate control of fermentation by inoculation are important ways to solve safety problems and ensure the quality of dark tea.The main findings are as follows:1.The morphological classification,multi-gene system classification and fingerprint superimposition analysis were carried out in Jingyang Fuzhuan Dark tea,Hunan Anhua dark tea,Guangxi Liubao tea and Yunnan Pu'er tea.From Anhua dark tea,15 strains of Eurotiums SP.were isolated,5 strains of Eurotiums SP.were isolated from Jingyang Fuzhuan dark tea,3 strains of Eurotiums SP.were isolated from Yunnan Shengpu tea and 1 strain was isolated from Liubao tea.The DNA sequences obtained from these 24strains were compared BLAST the NCBI Gene Bank database,Ah9,Ah7,Ah6,LB1and Ah5.Combined with the HPLC fingerprint,the identification and classification of Eurotiums SP.at the composition level,with the aid of external induce(blue light irradiation LG?hydrogen peroxide induce SYS).Superposition analysis of Ah5-ZC,Ah6-ZC,Ah7-ZC,Ah9-ZC,LB1-ZC fingerprints,Ah5-ZC found a unique absorption peak at RT=4.339 min;Ah9-ZC showed one unique absorption peak at RT=35.536 min,and two unique absorption peaks at RT=2.327 min,8.148min.By Superposition analysis fingerprints of the Ah5-LG,Ah6-LG,Ah7-LG,Ah9-LG,LB1-LG cultured by blue light irradiation,One unique absorption peak was found at RT=22.132 min of Ah5-LG;The results showed that the Ah6-LG had four unique absorption peaks min,7.120 min,7.819 min,16.526 min the four time points of 5.853;Ah9-LG showed 2 unique absorption peaks at RT=8.071 min,38.141 min two time points.Analysis of the fingerprint of the Ah5-SYS,Ah6-SYS,Ah7-SYS,Ah9-SYS,LB1-SYS of H₂O₂-induced culture,Ah9-SYS peak area of the four peaks,at RT=6.874min,7.567 min,8.672 min,9.396 min is significantly different from that of the corresponding RT.For Ah5-LGSYS,Ah6-LGSYS,Ah7-LGSYS,Ah9-LGSYS,LB1-LGSYS fingerprints,The Ah9-LGSYS was found to have a special absorption peak min at RT=4.452 min.2.By co-culture and screening of Eurotium amstelodami and Bacillus amyloliquefaciens,Hanson yeast,Saccharomyces cerevisiae and Wickerhamomyces anomalus,In this paper,the most obvious effect of co-culture was found in the group of Eurotium amstelodami and Bacillus amyloliquefaciens.High performance liquid phase analysis was carried out to obtain the co-cultured new peaks of Eurotium amstelodami and Bacillus amyloliquefaciens,Five new absorption peaks were detected by co-culture of Eurotium amstelodami and Bacillus amyloliquefaciens,the corresponding RT time was 10.097 min,21.690min,22.315 min,25.679 min,32.472min.These peaks were not found in single culture of Eurotium amstelodami or Bacillus amyloliquefaciens.3.UHPLC-Triple TOF/MS metabonomics was used to analyze the production of new compounds in co-culture of Eurotium amstelodami and Bacillus amyloliquefaciens.The original data obtained after UHPLC-Triple TOF/MS analysis were annotated with 825 metabolites,including 277 metabolites annotated to KEGG database and 662 metabolites annotated to HMDB and Lipidmaps database.A statistical annotation of compounds into the HMDB database of 617 metabolites,And matched to 13 HMDB super classes,Of these,there are 188 metabolites,112metabolites belonging to organic acids and their derivatives,98 metabolites belong to organic heterocyclic compounds,All three accounted for about 64.50%of all HMDB annotated metabolites.861,864,867 and 868 metabolites were detected in Control,CE,CM and CL groups,of which 2,1,1 and 1 metabolites were unique.There were 263different metabolites in CE and Control,of which 185 were upregulated and 78 were downregulated;There were 238 different metabolites in CM and Control,Of which 39were upregulated and 199 were downregulated;There were 203 differential metabolites in CL and Control,of which170 were upregulated and 33 were downregulated.The cluster analysis and KEGG pathway enrichment analysis of differential metabolites before and after co-culture revealed significant differences in the expression of differential metabolites before and after co-culture.the related pathways of Control and CE groups of differential metabolites included purine metabolism,cyanoamino acid metabolism,arginine and proline metabolism,phenylalanine metabolism,galactose metabolism,?-alanine metabolism,tyrosine metabolism,pyrimidine metabolism,histidine metabolism and aminoacyl biosynthesis,lysine biosynthesis,phenylalanine,tyrosine and tryptophan biosynthesis.At the same time,through the study of the changes of metabolites in different co-culture periods,the metabolites were significantly different in different co-culture stages,and the related metabolic pathways were galactose metabolism,caffeine metabolism and so on.4.In this part,Eurotium amstelodami and Bacillus amyloliquefaciens co-culture ear-(CE),co-culture mid-(CM),co-culture late(CL)as experimental materials,combined with Eurotium amstelodami rice fermentation products(Control)as control.Through RNA-seq technology and Illumina sequencing platforms,of which 23709 Unigene sequences annotated,12402 Unigenes sequences annotated into the metabolic term of the GO classification,and The 13573 unigene sequences are annotated in the kegg metabolic classification of the 40892 Unigene.Three experimental and control samples were analyzed for differential expression of two genes.It turns out,CE vs Control there were 3307 differential genes between the two samples,of which2037 were significantly upregulated and 1270 were significantly downregulated;CM vs Control there were 2623 differential genes between the two samples,of which 2393 were significantly upregulated,230 significantly downregulated genes;Between CL vs Control two samples,there were 1041 differentially expressed genes,Of which 832were significantly upregulated and 210 significantly downregulated genes.The CL difference genes were the smallest and the CE difference genes were the most relative to Control sample.Therefore,CE vs Control two samples will be annotated and enriched GO differentially expressed genes.For GO enrichment analysis,There are significant enrichment processes such as lipid metabolism,cellular lipid metabolism,lipid metabolism,and lipid biosynthesis.For KEGG enrichment,Significant enrichment is strong:tyrosine metabolism,glyceride metabolism,phenylalanine metabolism.Therefore,the co-culture of Eurotium amstelodami and Bacillus amyloliquefaciens had a significant effect on the secondary metabolism of Eurotium amstelodami,which mainly focused on the process of middle lipid metabolism,amino acid metabolism and enzyme activity.5.We have created a new tea drink by combining the synthetic microflora of Eurotium amstelodami,Bacillus amyloliquefaciens,Wickerhamomyces anomalus,Saccharomyces cerevisiae and Hanson yeast with black tea and dark tea processes to give full play to the biotransformation of tea components by microorganisms.through sensory evaluation,the green gas disappeared,the amber color was bright,the sweet back was good,and the quality was improved very well.