研究报告

基于高通量测序和可培养方法的勐海发酵普洱茶细菌多样性分析

  • 张欣 ,
  • 姚粟 ,
  • 白飞荣 ,
  • 田海霞 ,
  • 赵婷 ,
  • 马跃 ,
  • 刘海新 ,
  • 李颂 ,
  • 郝彬秀 ,
  • 王春玲
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  • 1 (中国食品发酵工业研究院有限公司,中国工业微生物菌种保藏管理中心,北京,100015)
    2 (中国茶叶有限公司,北京,102209)
硕士,工程师(姚粟教授级高级工程师为通讯作者,E-mail:milly@china-cicc.org)。

收稿日期: 2018-02-04

  网络出版日期: 2018-10-30

基金资助

北京市科技计划课题(Z161100000616012)

Analysis on bacterial diversity in Menhai fermented Pu-erh tea by high throughput sequencing and culture method

  • ZHANG Xin ,
  • YAO Su ,
  • BAI Fei-Rong ,
  • TIAN Hai-Xia ,
  • ZHAO Ting ,
  • MA Yue ,
  • LIU Hai-xin ,
  • LI Song ,
  • HAO Bin-Xiu ,
  • WANG Chun-Ling
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  • 1(China Center of Industrial Culture Collection, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing 100015, China)
    2(China Tea Co., Ltd., Beijing 102209, China)

Received date: 2018-02-04

  Online published: 2018-10-30

摘要

以勐海地区百中堂茶厂发酵普洱茶为研究对象,采用高通量测序技术,并结合传统可培养方法,对普洱茶从晒青毛茶原料到发酵完成过程中9个时间节点的细菌多样性进行了研究。结果表明,普洱茶发酵过程中细菌多样性丰富。高通量测序数据分析到61个属,212个OTU,发酵前期以欧文氏菌属(Erwinia sp.)、泛菌属(Pantoea sp.)和假单胞菌属(Pseudomonas sp.)为主,而发酵中、后期芽胞杆菌属(Bacillus sp.)、葡萄球菌属(Staphylococcus sp.)、短杆菌属(Brevibacterium sp.)、考克氏菌属(Kocuria sp.)及小短杆菌属(Brachybacterium sp.)为优势菌群稳定存在。通过可培养方法,从发酵过程中分离到细菌18个属,30个种,优势菌属包括微杆菌属(Microbacterium sp.)、葡萄球菌属(Staphylococcus sp.)、小短杆菌属(Brachybacterium sp.)、考克氏菌属(Kocuria sp.)、泛菌属(Pantoea sp.)和芽胞杆菌属(Bacillus sp.)等。

本文引用格式

张欣 , 姚粟 , 白飞荣 , 田海霞 , 赵婷 , 马跃 , 刘海新 , 李颂 , 郝彬秀 , 王春玲 . 基于高通量测序和可培养方法的勐海发酵普洱茶细菌多样性分析[J]. 食品与发酵工业, 2018 , 44(9) : 15 -21 . DOI: 10.13995/j.cnki.11-1802/ts.017024

Abstract

The fermented Pu-erh tea collected from Baizhongtang tea factory in the area of Menhai was chosen as the research object, bacterial diversity of 9 stages during the post-fermentation process from sundried green tea to Pu-erh ripe tea was studied by high throughput sequencing combined with the traditional culture method. The results showed that the bacterial diversity was rich during the fermentation. 212 OTU belonging to 61 genera were detected by high-throughput sequencing technology from the whole process of Pu-erh tea fermentation. Erwinia sp., Pantoea sp. and Pseudomonas sp. were the main genera in the early stage. However, in the middle and late stage Bacillus sp., Staphylococcus sp., Brevibacterium sp., Brevibacterium sp. Kocuria sp. and Brachybacterium sp. were the dominant microflora. 30 species belonging to 18 genera were isolated by the cultured method. The dominant genera included Microbacterium sp., Staphylococcus sp., Brachybacterium sp., Kocuria sp., Pantoea sp. and Bacillus sp..

参考文献

[1] 边纪平.勐海:普洱茶名重天下[J].中国品牌,2017(2):78-79.
[2] 罗勇,徐平.西双版纳勐海县普洱茶产业发展现状初探[J].云南科技管理,2008,21(1):38-39.
[3] 云南省人民政府茶叶产业办公室.GB/T 22111—2008地理标志产品 普洱茶[S].北京:中国标准出版社, 2008.
[4] 朱广鑫,周红杰,赵明.普洱茶发酵技术研究进展[J].江西农业学报,2011,23(5):80-85.
[5] 张冬英,施兆鹏,刘亚林.普洱茶药理作用研究进展[J].福建茶叶,2005(1):43-44.
[6] 李敏.普洱茶品质形成的研究[D].广州:华南农业大学, 2006:50.
[7] 周红杰,李家华,赵龙飞,等.渥堆过程中主要微生物对云南普洱茶品质形成的研究[J].茶叶科学,2004,24(3):212-218.
[8] XU X,YAN M,ZHU Y.Influence of fungal fermentation on the development of volatile compounds in the Puer tea manufacturing process[J].Engineering in Life Sciences,2005,5(4):382-386.
[9] 张偎,肖海军,梁俊涛,等.普洱茶发酵过程中一种优势菌种的分离鉴定及其变化规律[J].食品工业科技,2012,33(19):88-91.
[10] 董文明,谭超,付晓萍,等.5种成品普洱茶中微生物的分离及其产酶特性研究[J].食品科技,2013(6):22-25.
[11] 姚静,陈迪,郑晓燕,等.普洱茶渥堆发酵过程中细菌种群的分离与分子鉴定[J].安徽农业科学,2013,41(6):2 667-2 668.
[12] 王辉,任丽,李亚莉,等.普洱茶发酵过程中不同层间细菌群落结构研究[J].食品安全质量检测学报,2015(5):1 567-1 574.
[13] 杨晓苹,罗剑飞,刘昕,等.普洱茶固态发酵过程中微生物群落结构及变化[J].食品科学, 2013,34(19):142-147.
[14] ABE M,TAKAOKA N,IDEMOTO Y,et al.Characteristic fungi observed in the fermentation process for Puer tea[J].International Journal of Food Microbiology,2008,124(2):199-203.
[15] 赵明,马燕,张冬莲,等.普洱熟茶后发酵过程细菌多样性的16S rRNA基因文库研究[C].中国科协年会第20分会场:科技创新与茶产业发展论坛.北京:中国科学技术协会学会学术部,2013.
[16] JI Y J,LEE S H,KIM J M,et al.Metagenomic analysis of kimchi,a traditional korean fermented food[J].Applied & Environmental Microbiology,2011,77(7):2 264-2 274.
[17] CAUCHIE E,GAND M,KERGOURLAY G,et al.The use of 16S rRNA gene metagenetic monitoring of refrigerated food products for understanding the kinetics of microbial subpopulations at different storage temperatures:the example of white pudding[J].International Journal of Food Microbiology,2017,247:70-78.
[18] NI K,WANG Y, CAI Y,et al.Natural lactic acid bacteria population and silage fermentation of whole-crop wheat[J]. Asian-Australasian Journal of Animal Sciences,2015,28(8):1 123-1 132.
[19] YOON S H,HA S M,KWON S,et al.Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies[J].International Journal of Systematic & Evolutionary Microbiology,2017,67(5):1 613-1 617.
[20] ZHANG Xin,LIU Yang,LIN Yafang,et al.Novosphingobium clariflavum sp. nov. isolated from a household product plant[J]. International Journal of Systematic & Evolutionary Microbiology,2017,67(9):3 150-3 155.
[21] WALTERS W,HYDE E R,BERGLYONS D,et al.Improved bacterial 16S rRNA gene(V4 and V4-5)and fungal internal transcribed spacer marker gene primers for microbial community surveys[J].Msystems,2016,1(1):e00009-15.
[22] FADROSH D W,BING M,GAJER P,et al.An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform[J].Microbiome,2014,2(1):6.
[23] MAGO T,SALZBERG S L.FLASH:fast length adjustment of short reads to improve genome assemblies[J].Bioinformatics, 2011,27(21):2 957-2 963.
[24] EDGAR R C.UPARSE:Highly accurate OTU sequences from microbial amplicon reads[J].Nature Methods,2013,10(10):996.
[25] ABARENKOV K,NILSSON R H,LARSSONK H,et al.The UNITE database for molecular identification of fungi-recent updates and future perspectives[J].New Phytologist,2010,186(2):281-285.
[26] J GREGORY C,JUSTIN K,JESSE S.QIIME allows analysis of high-throughput community sequencing data[J].Nature Methods,2010,7(5):335-336.
[27] 葛慈斌,刘波,陈梅春,等.不同年份陈年普洱茶中芽胞杆菌的多样性[J].农业生物技术学报, 2017, 25(8):1 336-1 348.
[28] FAEZI GM, TAYERI A. Isolation and characterization of polyphenol oxidase and peroxidase-producing Bacillus strainer from fully fermented tea (Camellia sinensis)[J].World J Microbial Biotechnol,2007,24(3):1 327-1 332.
[29] WEI C L,CHAO S H,TSAI W B,et al.Analysis of bacterial diversity during the fermentation of inyu, a high-temperature fermented soy sauce, using nested PCR-denaturing gradient gel electrophoresis and the plate count method[J].Food Microbiology,2013,33(2):252-261.
[30] PRAJAPATI K A,SHILPKAR P,DUNGRECHIYA A.Alkaline protease production from:Sorghum vulgare by Staphylococcus sciuri[J].Journal of Pure & Applied Microbiology,2015,9(1):441-447.
[31] 赵亚娟.富硝芹菜粉在发酵肉制品中使用功效的研究[D].无锡:江南大学,2012:21.
[32] SONDERGAAD A K,STAHNKE L H.Growth and aroma production by Staphylococcus xylosus, S. carnosus and S. equorum-a comparative study in model systems[J].International Journal of Food Microbiology,2002,75(1-2):99-109.
[33] LATORREMORATALLA M L,BOSCHFUSTE J,Bovercid S,et al.Contribution of enterococci to the volatile profile of slightly-fermented sausages[J]. LWT-Food Science and Technology,2011,44(1):145-152.
[34] O’HALLORAN R,BOCKELMANN W,O'CUINN G.Purification of an extracellular proteinase from Staphylococcus sciuri found on the surface of Tilsit cheese[J].Biochemical Society Transactions,1998,26(1):S29-S29.
[35] CORBIERE M S,LEROY S,TALON R.Staphylococcal community of a small unit manufacture traditional dry fermented sausages[J]. International Journal of Food Microbiology,2006,108(2):210-217.
[36] SCHLEIFER K H,KILPPER-BALZ R,DEVERIESE L A.Staphylococcus arlettae sp. nov., S. equorum sp. nov. and S. k1oosii sp. nov.:Three new coagulase-negative, novobiocin-resistant species from animals[J]. Systematic & Applied Microbiology,1984,5(4):501-509.
[37] 陈廷涛.发酵乳和豆豉中微生物菌群DGGE分析及益生作用研究[D].南昌:南昌大学,2013:34.
[38] TANAKA Y,WATANABE J,MOGI Y.Monitoring of the microbial communities involved in the soy sauce manufacturing process by PCR-denaturing gradient gel electrophoresis[J].Food Microbiology,2012,31(1):100-106.
[39] ONDA T,YANAGIDA F,TSUJI M,et al.Time series analysis of aerobic bacterial flora during Miso fermentation.[J].Letters in Applied Microbiology,2003,37(2):162.
[40] EUNJIN P,SEONGWOON R,MINSOO K,et al.Kocuria koreensis sp. nov., isolated from fermented seafood[J].International Journal of Systematic & Evolutionary Microbiology, 2010,60(1):140-143.
[41] BRENNAN N M,BROWN R, GOODFELLOW M,et al.Microbacterium gubbeenense sp. nov., from the surface of a smear-ripened cheese[J]. International Journal of Systematic & Evolutionary Microbiology,2001,51(6):1 969-1 976.
[42] ZHANG Guoxia,ZENG Guoqu,CAI Xiaowei,et al.Brachybacterium zhongshanense sp. nov., a cellulose-decomposing bacterium from sediment along the Qijiang River, Zhongshan City, China[J].International Journal of Systematic & Evolutionary Microbiology,2007,57(11):2 519-2 524.
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