高通量测序技术分析浓香型白酒中温曲和高温曲的细菌群落结构
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摘要
本文通过Illumin公司Miseq 2×300 bp测序平台对浓香型白酒中温曲和高温曲进行高通量测序分析,发现以丰度>0.01%为阈值,中温曲中细菌共得到7个门,高温曲15个门,中温曲共得到19个属,高温曲26个属。首次从中温曲中检测出的类群有Pediococcus(小球菌属),Pantoea(泛菌属),Melghirimyces。高温曲中检测出的类群有Streptophyta,Brevibacterium(短杆菌属),Thermus(栖热菌属),Saccharopolyspora,Citrobacter(柠檬酸杆菌属),Streptococcus(链球菌),Brevundimonas(短波单胞菌属),Allobacillus。此外研究发现高温曲中的细菌较中温曲的细菌相比Bacillus(芽孢杆菌属)和Thermoactinomyces(高温放线菌属)显著上升,而Kroppenstedtia和Lactobacillus(乳杆菌属)显著下降,可能是与张弓酒独特的制曲工艺有关。本研究建立了一套高通量测序技术分析浓香型白酒大曲细菌群落结构的方法,为建立浓香型白酒大曲微生物信息数据库,优化大曲发酵工艺和提升品质提供理论依据。
The medium temperature Daqu and high temperature Daqu of Luzhou-flavor Liqu were analyzed by High-throughput sequencing through Illumin Miseq 2×300 bp sequencing platform, which revealed that 7 phyla and 19 genera were obtained from medium temperature Daqu and 15 phyla and 26 genera were obtained from high temperature Daqu at the threshold of abundance > 0.01%. The first group detected from the medium temperature Daqu was Pediococcus, Pantoea and Melghirimyces. The strains detected in the high temperature Daqu were Streptophyta, Brevibacterium, Thermus, Saccharopolyspora, Citrobacter, Streptococcus, Brevundimonas, and Allobacillus. In addition, the study also found that Bacillus and Thermoactinomyces in the high temperature Daqu were significantly increased compared to the bacteria in medium temperature Daqu, while Kroppenstedtia and Lactobacillus were significantly decreased, probably due to the unique production Daqu technology of Zhanggong. In this study, we established a high-throughput sequencing technique to analyze the bacterial community structure of Luzhou-flavor liquor, which provided theoretical basis for establishing microorganism information database of Luzhou-flavor liquor, optimizing Daqu fermentation technology and improving quality.
The medium temperature Daqu and high temperature Daqu of Luzhou-flavor Liqu were analyzed by High-throughput sequencing through Illumin Miseq 2×300 bp sequencing platform, which revealed that 7 phyla and 19 genera were obtained from medium temperature Daqu and 15 phyla and 26 genera were obtained from high temperature Daqu at the threshold of abundance > 0.01%. The first group detected from the medium temperature Daqu was Pediococcus, Pantoea and Melghirimyces. The strains detected in the high temperature Daqu were Streptophyta, Brevibacterium, Thermus, Saccharopolyspora, Citrobacter, Streptococcus, Brevundimonas, and Allobacillus. In addition, the study also found that Bacillus and Thermoactinomyces in the high temperature Daqu were significantly increased compared to the bacteria in medium temperature Daqu, while Kroppenstedtia and Lactobacillus were significantly decreased, probably due to the unique production Daqu technology of Zhanggong. In this study, we established a high-throughput sequencing technique to analyze the bacterial community structure of Luzhou-flavor liquor, which provided theoretical basis for establishing microorganism information database of Luzhou-flavor liquor, optimizing Daqu fermentation technology and improving quality.
引文
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[19]乔晓梅,赵景龙,杜小威,等.高通量测序法对清香大曲真菌群落结构的分析[J].酿酒科技,2015,4:28-31QIAO Xiao-mei,ZHAO Jing-long,DU Xiao-wei,et al.Analysis of fungal communities of Qingxiang daqu by barcoded pyrosequencing[J].Liquor-Making Science and Technology,2015,4:28-31
[20]Fadrosh D W,Ma B,Gajer P,et al.An improved dual-indexing approach for multiplexed 16S r RNA gene sequencing on the Illumina Mi Seq platform[J].Microbiome,2014,2(1):6
[21]Xu C L,Sun R,Qiao X J,et al.Protective effect of glutamine on intestinal injury and bacterial community in rats exposed to hypobaric hypoxia environment[J].World Journal of Gastroenterology:WJG,2014,20(16):4662
[22]Zhou H W,Li D F,Tam N F Y,et al.BIPES,a cost-effective high-throughput method for assessing microbial diversity[J].The ISME Journal,2011,5(4):741-749
[23]Schloss P D,Westcott S L,Ryabin T,et al.Introducing mothur:open-source,platform-independent,communitysupported software for describing and comparing microbial communities[J].Applied and Environmental Microbiology,2009,75(23):7537-7541
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[25]王明跃,张文学.浓香型白酒两个产区窖泥微生物群落结构分析[J].微生物学通报,2014,41(8):1498-1506WANG Ming-yue,ZHANG Wen-xue.Analysis of microbial community structure in pit mud from two Chinese Luzhou-flavor liquor producing areas[J].Microbiology,2014,41(8):1498-1506
[26]孟镇,熊正河,钟其顶,等.应用PCR-DGGE技术解析白酒大曲细菌群落结构[J].食品与发酵工业,2010,10:159-162MENG Zhen,XIONG Zheng-he,ZHONG Qi-ding,et al.Application of PCR-DGGE to analysis of bacteria community structure of Daqu[J].Food and Fermentation Industries,2010,10:159-162
[27]张明春,曹敬华,向苇,等.白云边酿酒大曲微生物分析研究[J].酿酒科技,2010,2:65-67ZHANG Ming-chun,CAO Jing-hua,XIANG Wei,et al.Analysis of microbes in Baiyunbian Daqu[J].Liquor-Making Science and Technology,2010,2:65-67
[28]高亦豹,王海燕,徐岩.利用PCR-DGGE未培养技术对中国白酒高温和中温大曲细菌群落结构的分析[J].微生物学通报,2010,37(7):999-1004GAO Yi-bao,WANG Hai-yan,XU Yan.PCR-DGGE analysis of the bacterial community of Chinese liquor high and medium temperature Daqu[J].Microbiology,2010,37(7):999-1004
[29]汤斌,刘金英,周庆武,等.免培养技术对浓香型白酒大曲中细菌多样性的影响[J].食品与发酵工业,2011,37(9):36-40TANG Bin,LIU Jin-ying,ZHOU Qing-wu,et al.Phylogenetic diversity analyse of bacteria in Gujing-flavor liquor Daqu using culture independent method[J].Food and Fermentation Industries,2011,37(9):36-40
[30]明红梅,董瑞丽,许德富,等.浓香型大曲中优势菌的分离及初步鉴定[J].酿酒科技,2013,12:63-66MING Hong-mei,DONG Rui-li,XU De-fu,et al.Separation and preliminary identification of dominant microbes in Nong-flavor Daqu[J].Liquor-Making Science and Technology,2013,12:63-66
[31]熊小毛,严楠峰,黄莹娜,等.兼香型白云边酒不同工艺高温大曲差异性分析[J].酿酒科技,2014,1:006XIONG Xiao-mao,YAN Nan-feng,HUANG Ying-na,et al.Analysis of the difference in physiochemical indexes and microbial quantity of Baiyunbian high-temperature Daqu produced by different techniques[J].Liquor-Making Science and Technology,2014,1:006
[32]李德林,张宿义,毛振宇,等.PCR-DGGE对浓香型白酒糟醅微生物群落结构解析[J].酿酒科技,2014,3:25-27LI De-lin,ZHANG Su-yi,MAO Zhen-yu,et al.Analysis of microbial communities in fermented grains of Nong-xiang type Baijiu(liquor)by PCR-DGGE[J].Liquor-Making Science and Technology,2014,3:25-27
[33]黄永光,黄平,涂华彬.窖泥微生物总DNA的提取纯化研究[J].酿酒科技,2004,3:41-42HUANG Yong-guang,HUANG Ping,TU Hua-bin.Research on extraction and purification of total DNA of microbes in pit mud[J].Liquor-Making Science and Technology,2004,3:41-42
[34]Lonvaud-Funel A.Lactic acid bacteria in the quality improvement and depreciation of wine[M].Lactic Acid Bacteria:Genetics,Metabolism and Applications.Springer Netherlands,1999
[35]Yoon J H,Park Y H.Phylogenetic analysis of the genus Thermoactinomyces based on 16S r DNA sequences[J].International Journal of Systematic and Evolutionary Microbiology,2000,50(3):1081-1086
[36]姚粟,葛媛媛,李辉,等.利用非培养技术研究芝麻香型白酒高温大曲的细菌群落多样性[J].食品与发酵工业,2012,38(6):1-6YAO Su,GE Yuan-yuan,LI Hui,et al.Analysis on bacterial communities in high temperature Daqu of sesame flavor liquor through culture-tree Approach[J].Food and Fermentation Industries,2012,38(6):1-6
[37]孙西玉,潘春梅.张弓酒典型风格及其成因研究[J].酿酒科技,2006,9:94-95SUN Xi-yu,PAN Chun-mei.Studies on the typical style of Zhanggong liquor and its origin[J].Liquor-Making Science and Technology,2006,9:94-95
[2]胡承,邬捷锋,沈才洪,等.浓香型(泸型)大曲的研究及其应用[J].酿酒科技,2004,1:33-36HU Cheng,WU Jie-feng,SHEN Cai-hong,et al.Research on Luzhou-flavor Daqu starter and its application[J].Liquor-Making Science and Technology,2004,1:33-36
[3]程光胜.中国酒曲微生物利用的发展现状[J].酿酒科技,2014,3:122-124CHENG Guang-sheng.Present situations of the utilization of microbes in Chinese Jiuqu[J].Liquor-Making Science and Technology,2014 3:122-124
[4]邢钢,敖宗华,邓波.大曲中微生物研究和检测进展[J].酿酒科技,2012,12:86-89XING Gang,AO Zong-hua,DENG Bo.Research and detection progress of microbes in Daqu[J].Liquor-Making Science and Technology,2012,12:86-89
[5]胡佳,邓斌,张文学,等.浓香型白酒曲药中细菌组成及系统学分析[J].酿酒科技,2007,5:17-19HU Jia,DENG Bin,ZHANG Wen-xue,et al.Phylogenetic analysis of bacterial composition in Luzhou-flavor liquor Quyao[J].Liquor-Making Science and Technology,2007,5:17-19
[6]Amann R I,Ludwig W,Schleifer K H.Phylogenetic identification and in situ detection of individual microbial cells without cultivation[J].Microbiological Reviews,1995,59(1):143-169
[7]Nogales B,Moore E R B,Llobet-Brossa E,et al.Combined use of 16S ribosomal DNA and 16S r RNA to study the bacterial community of polychlorinated biphenyl-polluted soil[J].Applied and Environmental Microbiology,2001,67(4):1874-1884
[8]Curtis T P,Sloan W T,Scannell J W.Estimating prokaryotic diversity and its limits[J].Proceedings of the National Academy of Sciences,2002,99(16):10494-10499
[9]Li R,Zhu H,Ruan J,et al.De novo assembly of human genomes with massively parallel short read sequencing[J].Genome Research,2010,20(2):265-272
[10]Yang F,Zeng X,Ning K,et al.Saliva microbiomes distinguish caries-active from healthy human populations[J].The ISME Journal,2012,6(1):1-10
[11]秦楠,栗东芳,杨瑞馥.高通量测序技术及其在微生物学研究中的应用[J].微生物学报,2011,51(4):445-457QIN Nan,LI Dong-fang,YANG Rui-fu.Next generation sequencing technologies and the application in microbiologyA review[J].Acta Microbiologica Sinica,2011,51(4):445-457
[12]Soon W W,Hariharan M,Snyder M P.High-throughput sequencing for biology and medicine[J].Molecular Systems Biology,2013,9(1):640
[13]Maccallum I,Przybylski D,Gnerre S,et al.ALLPATHS 2:small genomes assembled accurately and with high continuity from short paired reads[J].Genome Biology,2009,10(10):1
[14]Andersson A F,Lindberg M,Jakobsson H,et al.Comparative analysis of human gut microbiota by barcoded pyrosequencing[J].Plo S One,2008,3(7):e2836
[15]Chu H,Fierer N,Lauber C L,et al.Soil bacterial diversity in the Arctic is not fundamentally different from that found in other biomes[J].Environmental Microbiology,2010,12(11):2998-3006
[16]陈玲,袁玉菊,曾丽云,等.16S r DNA克隆文库法与高通量测序法在浓香型大曲微生物群落结构分析中的对比研究[J].酿酒科技,2015,12:33-36CHEN Ling,YUAN Yu-ju,ZENG Li-yun,et al.16S r DNA clone library vs.high-throughput sequencing method in the analysis of the microbial communities in Nongxiang Daqu[J].Liquor-Making Science and Technology,2015,12:33-36
[17]Wang C,Shi D,Gong G.Microorganisms in Daqu:a starter culture of Chinese Maotai-flavor liquor[J].World Journal of Microbiology and Biotechnology,2008,24(10):2183-2190
[18]杨代永,范光先,汪地强,等.高温大曲中的微生物研究[J].酿酒科技,2007,5:37-38YANG Dai-yong,FAN Guang-xian,WANG Di-qiang,et al.Microbes in high temperature starter[J].Liquor-Making Science and Technology,2007,5:37-38
[19]乔晓梅,赵景龙,杜小威,等.高通量测序法对清香大曲真菌群落结构的分析[J].酿酒科技,2015,4:28-31QIAO Xiao-mei,ZHAO Jing-long,DU Xiao-wei,et al.Analysis of fungal communities of Qingxiang daqu by barcoded pyrosequencing[J].Liquor-Making Science and Technology,2015,4:28-31
[20]Fadrosh D W,Ma B,Gajer P,et al.An improved dual-indexing approach for multiplexed 16S r RNA gene sequencing on the Illumina Mi Seq platform[J].Microbiome,2014,2(1):6
[21]Xu C L,Sun R,Qiao X J,et al.Protective effect of glutamine on intestinal injury and bacterial community in rats exposed to hypobaric hypoxia environment[J].World Journal of Gastroenterology:WJG,2014,20(16):4662
[22]Zhou H W,Li D F,Tam N F Y,et al.BIPES,a cost-effective high-throughput method for assessing microbial diversity[J].The ISME Journal,2011,5(4):741-749
[23]Schloss P D,Westcott S L,Ryabin T,et al.Introducing mothur:open-source,platform-independent,communitysupported software for describing and comparing microbial communities[J].Applied and Environmental Microbiology,2009,75(23):7537-7541
[24]Whittaker R H.Evolution and measurement of species diversity[J].Taxon,1972:213-251
[25]王明跃,张文学.浓香型白酒两个产区窖泥微生物群落结构分析[J].微生物学通报,2014,41(8):1498-1506WANG Ming-yue,ZHANG Wen-xue.Analysis of microbial community structure in pit mud from two Chinese Luzhou-flavor liquor producing areas[J].Microbiology,2014,41(8):1498-1506
[26]孟镇,熊正河,钟其顶,等.应用PCR-DGGE技术解析白酒大曲细菌群落结构[J].食品与发酵工业,2010,10:159-162MENG Zhen,XIONG Zheng-he,ZHONG Qi-ding,et al.Application of PCR-DGGE to analysis of bacteria community structure of Daqu[J].Food and Fermentation Industries,2010,10:159-162
[27]张明春,曹敬华,向苇,等.白云边酿酒大曲微生物分析研究[J].酿酒科技,2010,2:65-67ZHANG Ming-chun,CAO Jing-hua,XIANG Wei,et al.Analysis of microbes in Baiyunbian Daqu[J].Liquor-Making Science and Technology,2010,2:65-67
[28]高亦豹,王海燕,徐岩.利用PCR-DGGE未培养技术对中国白酒高温和中温大曲细菌群落结构的分析[J].微生物学通报,2010,37(7):999-1004GAO Yi-bao,WANG Hai-yan,XU Yan.PCR-DGGE analysis of the bacterial community of Chinese liquor high and medium temperature Daqu[J].Microbiology,2010,37(7):999-1004
[29]汤斌,刘金英,周庆武,等.免培养技术对浓香型白酒大曲中细菌多样性的影响[J].食品与发酵工业,2011,37(9):36-40TANG Bin,LIU Jin-ying,ZHOU Qing-wu,et al.Phylogenetic diversity analyse of bacteria in Gujing-flavor liquor Daqu using culture independent method[J].Food and Fermentation Industries,2011,37(9):36-40
[30]明红梅,董瑞丽,许德富,等.浓香型大曲中优势菌的分离及初步鉴定[J].酿酒科技,2013,12:63-66MING Hong-mei,DONG Rui-li,XU De-fu,et al.Separation and preliminary identification of dominant microbes in Nong-flavor Daqu[J].Liquor-Making Science and Technology,2013,12:63-66
[31]熊小毛,严楠峰,黄莹娜,等.兼香型白云边酒不同工艺高温大曲差异性分析[J].酿酒科技,2014,1:006XIONG Xiao-mao,YAN Nan-feng,HUANG Ying-na,et al.Analysis of the difference in physiochemical indexes and microbial quantity of Baiyunbian high-temperature Daqu produced by different techniques[J].Liquor-Making Science and Technology,2014,1:006
[32]李德林,张宿义,毛振宇,等.PCR-DGGE对浓香型白酒糟醅微生物群落结构解析[J].酿酒科技,2014,3:25-27LI De-lin,ZHANG Su-yi,MAO Zhen-yu,et al.Analysis of microbial communities in fermented grains of Nong-xiang type Baijiu(liquor)by PCR-DGGE[J].Liquor-Making Science and Technology,2014,3:25-27
[33]黄永光,黄平,涂华彬.窖泥微生物总DNA的提取纯化研究[J].酿酒科技,2004,3:41-42HUANG Yong-guang,HUANG Ping,TU Hua-bin.Research on extraction and purification of total DNA of microbes in pit mud[J].Liquor-Making Science and Technology,2004,3:41-42
[34]Lonvaud-Funel A.Lactic acid bacteria in the quality improvement and depreciation of wine[M].Lactic Acid Bacteria:Genetics,Metabolism and Applications.Springer Netherlands,1999
[35]Yoon J H,Park Y H.Phylogenetic analysis of the genus Thermoactinomyces based on 16S r DNA sequences[J].International Journal of Systematic and Evolutionary Microbiology,2000,50(3):1081-1086
[36]姚粟,葛媛媛,李辉,等.利用非培养技术研究芝麻香型白酒高温大曲的细菌群落多样性[J].食品与发酵工业,2012,38(6):1-6YAO Su,GE Yuan-yuan,LI Hui,et al.Analysis on bacterial communities in high temperature Daqu of sesame flavor liquor through culture-tree Approach[J].Food and Fermentation Industries,2012,38(6):1-6
[37]孙西玉,潘春梅.张弓酒典型风格及其成因研究[J].酿酒科技,2006,9:94-95SUN Xi-yu,PAN Chun-mei.Studies on the typical style of Zhanggong liquor and its origin[J].Liquor-Making Science and Technology,2006,9:94-95