| 16SrDNA alignment show that Brevundimonas aurantiaca, Millisia brevis, Novosphingobium sp, Clostridium ultunense, Pseudomonas veronii, Lactobacillus plantarum, Klebsiella pneumonia and Uncultured epsilon proteobacteriutn, Uncultured Saprospiraceae bacterium, Uncultured Myxococcales bacterium, Uncultured Rhizobiales bacterium and six kinds of unknown taxonomic status of unculturable bacteria were detected. This study for the first time shows a detailed spectrum of bacteria at different time periods during the Fahua-fermentation process.6. To explore fungal community structure and species composition in the pile-fermentation process of Fuzhuan Brick Tea(FBT), the fungal variable region of 18S rDNA was amplified in different periods of pile-fermentation process. Amplified products were separated by denaturing gradient gel electrophoresis(DGGE) and the bands were cloned, sequenced and aligned. The results show that fungal species are rich in pile-fermentation process of FBT, Wallemia sebi, Candida sp, Candida tropicalis, Lodderomyces sp, Debaryomyces hansenii, Pichia kudriavzevii, Saccharomyces cerevisiae, Cryptococcus sp, Rhodotorula graminis, Eurotium amstelodami, Aspergillus glaucus,Rhizomucor miehei, Rhizomucorpusillus, Galactomyces geotrichum, Candida ontarioensis, Trichosporon dermatis, Penicillium decumbens and Aspergillus penicillioides were detected. Two distinctive kinds of fungal communities, correlating to the pile-fermentation times:0-24 h and 24-48 h, were found. The fungi Shannon-Wiener Index gradual tended to increase during 0-16 h in pile-fermentation process of FBT,16-24 h slightly reduced, after 40 h increased to the maximum 2.348,40-48 h still maintained a higher level. There are dominant fungi Lodderomyces sp and Debaryomyces hansenii, sub-dominant fungi Eurotium amstelodami throughout the pile-fermentation process of FBT, and sub-dominant fungi Wallemia muriae, Debaryomyces hansenii and Saccharomyces cerevisiae were appeared in the later of pile-fermentation. The 18S rDNA alignment results also proved that there are 3 strains Wallemia sebi and 7 strains Eurotium amstelodami, meaning a wide variety of subspecies and ecotype within the dominant fungi. This study shows a detailed spectrum of fungi at different time periods during the pile-fermentation process.7. To explore fungi community structure and species in Fahua-fermentation process of Fuzhuan Brick Tea, the fungi variable region of 18S rDNA were amplified in different periods of Fahua-fermentation process. Amplified products were separated by denaturing gradient gel electrophoresis(DGGE) and the bands were cloned, sequenced and aligned. The results show that two distinctive kinds of fungi community structure, correlating to the fermentation times:0-8 d and 10-14d, were found. The dominant fungi of the former are Wallemia muriae, Pichia kudriavzevii, Candida sp., and the later are Eurotium amstelodami and Wallemia muriae. The results of variable region of 18S rDNA alignment show that Wallemia sebi, Wallemia muriae, Debaryomyces hansenii, Saccharomyces cerevisiae, Candida sp., Candida tropicalis, Lodderomyces sp., Pichia kudriavzevii, Rhodotorula graminis, Rhizomucor pusillus, Cryptococcus sp., Eurotium amstelodami, Aspergillus penicillioides, Rhizomucor miehei, Candida ontarioensis, Trichosporon dermatis, Penicillium decumbens, Galactomyces geotrichum, Aspergillus glaucus, Eurotium herbariorum were detected. The 18S rDNA alignment results also prove that there are 3 strains Wallemia sebi and 5 strains Eurotium amstelodami, meaning a wide variety of subspecies and ecotype within the dominant fungi.This study for the first time shows a detailed spectrum of fungi at different time periods during the Fahua-fermentation process.To study of Fuzhuan Brick Tea(FBT) microbial community structure and diversity in the process of FBT fermentation, dominant fungi Eurotium cristatum were identificatied; morphology and microstructure of dominant fungi Eurotium cristatum were compared; 5 strains of a dominant fungi Eurotium cristatum were selected based on the morphological characteristics for dominant fungi diversity of random amplified polymorphic DNA(RAPD) analysis; to explore the bacteria and fungi communities structure, dynamic change diversity, denaturing gradient gel electrophoresis(DGGE) was used in pile-fermentation, Fahua-fermentation of Xiang yi brand FBT process, which were producrd at FBT countries designated production enterprise, Hunan tea factory co., LTD. production lines. Main results are as follows:1. After the separation, purification and counting of the fungi in XIANG-YI brand FBT through gradient dilution coating, five of the strain, namely,2301,2401,3302,4304 and 5402 were obtained. Results showed that the dominant bacteria was 5402, which reached 4.97><106 per gram the dried tea sample. Others are non-predominant bacterium, relatively less in the district of brick tea. Based on the morphological characteristics and the result of sequences alignment of 18S rDNA, strain 2301,2401,3302,4304 and 5402 was identified as Penicillium purpurogenum, Penicillium chrysogenum, Penicillium sp., Aspergillus sojae and Eurotium herbariorum respectively.2. To study morphological diversity of different brand Fuzhuan brick tea(FBT), Morphological and microstructure characteristics of the 23 strains dominant fungi {Eurotium cristatum) were identificatied, which were isolatied and purificatied from FBT samples around the Hunan, Hubei, Guangxi, Guizhou, Zhejiang, Jiangsu, Shanxi, Xinjiang eight provinces FBT enterprises. The results of experiment indicated that the dominant fungi Eurotium cristatum can be divided into five types:type Ⅰ including G3, G8, G9, G11, G12, G14, G17, G19, G21 and G23; Type Ⅱincluding G7 and G16; Type Ⅲincluding G13, G15, G20, G22 and G24; Type Ⅳincluding G2 and G18; Type V including G4, G5, G6 and G10.3. Eurotium cristatum, the dominant Fungi in the fermentation process of Fuzhuan brick-tea, is the key of Fuzhuan brick-tea special quality formation. To explore the diversity of dominant fungi Eurotium cristatum all in the Fuzhuan brick-tea over the country, random amplified polymorphic DNA(RAPD) was adopted for G3, G9, G13, G15 and G19, which were separated from Shanxi, Hubei, Guangxi, Hunan province. The results show that the maximum genetic distance is G3 and G13(14), the genetic similarity coefficient is minimum(48.15%); while the minimum genetic distance is G15 and G19(2), the genetic similarity coefficient is maximum (92.59%); the difference of between of those dominant fungi Eurotium cristatum were obvious.4. In order to study bacterial community structure and species in Pile-fermentation process of Fuzhuan Brick Tea, the bacterial V3 variable region of 16SrDNA were amplified in different periods of Pile-fermentation Process, the denaturing gradient gel electrophoresis(DGGE) was used, the DGGE bands were cloned, sequenced and alignmented. The results show that,24 hours of pile-fermentation is the critical point. Before and after that the point their bacterial community structure is similar, but bacterial community structure was significantly different before 24 hours and after 24 hours; from the V3 variable region of 16SrDNA alignment results show that Millisia brevis, Novosphingobium sp., Brevundimonas aurantiaca, Pseudomonas veronii, Clostridium ultunense, Klebsiella pneumonia, Lactobacillus plantarum, uncultured Epsilon proteobacterium, Uncultured Saprospiraceae bacterium, Uncultured Myxococcales bacterium, Uncultured Rhizobiales bacterium and six kind of unknown taxonomic status of unculturable bacteria were detected. That black tea pile-fermentation has a large number of bacteria, and the bacterial community dynamic change process more stable. Experimental results show that a large number of bacteria were involved in Fuzhuan Brick Tea pile-fermentation, and the bacterial community dynamic change process was relatively stable.5. In order to explore bacterial community structure and species in Fahua-fermentation process of Fuzhuan Brick Tea, the bacterial V3 variable region of 16SrDNA were amplified in different periods of Fahua-fermentation process. Amplified products were separated by DGGE and the bands were cloned, sequenced and aligned. Three distinctive kinds of bacterial community structure, correlating to the fermentation times:0-4 d,6-8 d and 10-14d, were found, The results of V3 variable region of... |
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