Microbial Community Structure During Pu-erh Tea Fermentation And Clone Of Functional Enzyme Genes

The pile fermentation is the critical process during Pu-erh tea manufacture.Microorganism and their extracellular enzymes play a decisive role in the quality formation ofPu-erh tea. Most researches were studied on the identifications, growth characteristic and thequantity of mold and yeast that obtained by separation and selection from culture medium.The bacterial and fungal community structures and their dynamic changes, including theirrelationship are rarely reported. Besides, the dominant enzymes activity was highly relatedwith the microbial community structure. However, most of the reports tell about the generalidea of the enzyme source without further studies. In this paper, PCR-DGGE technology wasapplied to analyze the bacterial and fungal community structures and changes during thePu-erh tea fermentation. Meanwhile，several strains were isolated from Pu-erh tea andidentified by16S rRNA and18S rRNA sequencing, respectively. The laccase and tannasegenes in the isolates were identified by PCR reaction. This paper provide theory and datasupport for the further researches on the microbial function mechanism during Pu-erh teafermentation.The main results are as follow:1. Repeated freezing in liquid nitrogen and thawing was determined to be used to extractthe total DNA from Pu-erh tea microorganisms, which reflect the microbial diversity wellaccording to the DGGE profiles.2. PCR-DGGE results show that,3groups of bacteria, including γ-proteobacteria,β-proteobacteria and Bacilli presented during the Pu-erh tea fermentation, while2groups offungi including Eurotiales and Saccharomycetales presented.3. Microbial community structures between tea pile surface and inner have no significantdifference. Bacteria and fungi population changed obviously after the second fermentationstage. Bacillus increased to be the predominant bacteria at the later stage, and Bacillusthermoamylovorans existed in the whole fermentation, while partial bacteria disappearedduring the process. Aspergillus niger was the dominant fungus at the initial stage while Arxulaadeninivorans was at the later stage. Aspergillus fumigatus existed in the whole fermentationand changed little.4. Bacteria and fungi were dependent on and competitive to each other. Aspergillus nigergrowed rapidly and promoted the propagating of Arxula adeninivorans, and bacteria wereinhibited so that some kinds of them decreased or disappeared. 5. One strain of bacteria was isolated from culture and identified to be an unknownspecies of Bacillus by16S rRNA sequencing, which was also98%similar to Bacillusshackletonii. Two strains of aspergillus were isolated and indentified to be Aspergillus nigerand Aspergillus fumigatus by18S rRNA sequencing. Similarly, one strain of yeast wasseparated and identified to be Arxula adeninivorans.6. Primers were designed and successfully amplified the laccase genes from Aspergillusniger, the laccase genes and tannase genes from Aspergillus fumigatus. This Aspergillus nigerand this Aspergillus fumigatus may secrete laccase during the fermentation and dedicate to thered, yellow and bright feature of Pu-erh tea. The Aspergillus fumigatus also may secretetannase and dedicate to the formation of simple catechins and gallic acid.