泸型酒酒醅细菌群落的发酵演替规律
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摘要
【目的】考察泸型酒发酵过程中酒醅细菌群落的演替规律,探讨菌群演替与环境因素变化的相关性。【方法】采用高通量测序技术分析泸型酒酒醅细菌群落的演替规律,并运用Mantel test分析不同发酵阶段的细菌群落演替与环境因素变化的相关性。【结果】酒醅发酵过程中有397个属的微生物,其中Lactobacillus、Bacillus、Weissella、Dysgonomonas、Comamonas以及Ruminococcaceae为优势属(相对丰度>1.0%)。通过聚类分析可将酒醅发酵过程划分为3个阶段:阶段I (0–5 d),阶段II (6–17 d)和阶段III(18–40d),且3个阶段的酒醅菌群结构差异显著(P<0.05)。Metastats分析结果表明,与阶段I相比,阶段II酒醅细菌群落中Lactobacillus和unclassifiedLactobacillaceae相对丰度显著升高(P<0.05),而unclassifiedBacillaceae、 Staphylococcus、 Bacillus、 unclassified Enterobacteriaceae、 Lactococcus、Pseudomonas、Thermoactinomyces、Leuconostoc、Staphylococcus相对丰度显著降低(P<0.05)。与阶段II相比,阶段III酒醅细菌群落中Lactobacillus相对丰度显著增长(P<0.05),Comamonas、Acetobacter、unclassified Bacilli、Clostridium、Bacillus、Ruminococcus、unclassified Porphyromonadaceae和unclassified Streptophyta相对丰度显著下降(P<0.05)。结果表明,阶段I的细菌菌群演替与酒醅温度、水分和乙醇浓度变化线性相关(P<0.05);阶段II和阶段III的细菌菌群演替与酒醅温度、水分、酸度、乙醇浓度均没有相关性(P>0.05)。【结论】泸型酒酒醅中细菌群落在不同发酵阶段结构差异显著,且温度、水分以及乙醇浓度对酒醅发酵前期(0–5 d)细菌群落演替具有重要作用。
[Objective] This study aimed to elucidate the bacterial community succession and its underlying relevance to the environmental factors in fermented grains of Luzhou-flavor baijiu. [Methods] The 16 S rRNA gene amplicon sequencing analysis was applied to reveal the succession of bacterial community in the fermented grains, and Mantel test was used to correlate bacterial community succession with environmental factors. [Results] A total of 397 bacterial genera were identified in fermented grains, including Lactobacillus, Bacillus, Weissella, Dysgonomonas, Comamonas and Ruminococcaceae(relative abundance>1.0%). Fermentation process was divided into three stages: stage I(0–5 d), stage II(6–17 d) and stage III(18–40 d) according to clustering analysis, and the bacterial community structure was significantly different among these three stages. Metastats analysis demonstrated that the relative abundance of Lactobacillus and unclassified Lactobacillales in stage II was significantly higher than that in stage I(P<0.05), whereas the situation of unclassified Bacillaceae, Staphylococcus, Bacillus, unclassified Enterobacteriaceae, Lactococcus, Pseudomonas, Thermoactinomyces, Leuconostoc and Staphylococcus quite the reverse(P<0.05). Compared with stage II, the relative abundance of Lactobacillus increased in stage III(P<0.05), conversely, downregulation dominated Comamonas, Acetobacter, unclassified Bacilli, Clostridium, Bacillus, Ruminococcus, unclassified Porphyromonadaceae and unclassified Streptophyta(P<0.05). Results of Mantel test indicated that the bacterial community succession in the stage I was correlated with the dynamics of temperature, moisture, and alcohol concentration(P<0.05), whereas no significant correlation was observed between environmental factors and bacterial community succession in the stage II and III(P>0.05). [Conclusion] Our work demonstrated the dramatically divergent bacterial community structure in the fermented grains of Luzhou-flavor baijiu from different brewing stages. Temperature, moisture and alcohol concentration significantly correlated with the bacterial community succession in early stage of fermentation(0–5 d).
[Objective] This study aimed to elucidate the bacterial community succession and its underlying relevance to the environmental factors in fermented grains of Luzhou-flavor baijiu. [Methods] The 16 S rRNA gene amplicon sequencing analysis was applied to reveal the succession of bacterial community in the fermented grains, and Mantel test was used to correlate bacterial community succession with environmental factors. [Results] A total of 397 bacterial genera were identified in fermented grains, including Lactobacillus, Bacillus, Weissella, Dysgonomonas, Comamonas and Ruminococcaceae(relative abundance>1.0%). Fermentation process was divided into three stages: stage I(0–5 d), stage II(6–17 d) and stage III(18–40 d) according to clustering analysis, and the bacterial community structure was significantly different among these three stages. Metastats analysis demonstrated that the relative abundance of Lactobacillus and unclassified Lactobacillales in stage II was significantly higher than that in stage I(P<0.05), whereas the situation of unclassified Bacillaceae, Staphylococcus, Bacillus, unclassified Enterobacteriaceae, Lactococcus, Pseudomonas, Thermoactinomyces, Leuconostoc and Staphylococcus quite the reverse(P<0.05). Compared with stage II, the relative abundance of Lactobacillus increased in stage III(P<0.05), conversely, downregulation dominated Comamonas, Acetobacter, unclassified Bacilli, Clostridium, Bacillus, Ruminococcus, unclassified Porphyromonadaceae and unclassified Streptophyta(P<0.05). Results of Mantel test indicated that the bacterial community succession in the stage I was correlated with the dynamics of temperature, moisture, and alcohol concentration(P<0.05), whereas no significant correlation was observed between environmental factors and bacterial community succession in the stage II and III(P>0.05). [Conclusion] Our work demonstrated the dramatically divergent bacterial community structure in the fermented grains of Luzhou-flavor baijiu from different brewing stages. Temperature, moisture and alcohol concentration significantly correlated with the bacterial community succession in early stage of fermentation(0–5 d).
引文
[1]沈怡方.白酒生产技术全书.北京:中国轻工业出版社,2007.
[2]Tao Y,Li JB,Rui JP,Xu ZC,Zhou Y,Hu XH,Wang X,Liu MH,Li DP,Li XZ.Prokaryotic communities in pit mud from different-aged cellars used for the production of Chinese strong-flavored liquor.Applied and Environmental Microbiology,2014,80(7):2254-2260.
[3]Hou XG,Wang JY,Li XS,Hu BY,Li SL,Gao YY.The research progress on functional aroma-producing microorganisms in Zaopei and pit mud of Chinese strong-flavor liquor.Microbiology China,2013,40(7):1257-1265.(in Chinese)侯小歌,王俊英,李学思,胡炳义,李绍亮,高应运.浓香型白酒糟醅及窖泥产香功能菌的研究进展.微生物学通报,2013,40(7):1257-1265.
[4]Wu Q,Peng SQ,Yu Y,Li YX,Xu Y.Genome sequence of Bacillus licheniformis CGMCC3963,a stress-resistant strain isolated in a Chinese traditional solid-state liquor-making process.Genome Announcements,2013,1(1):e00060-12.
[5]Hu XL,Du H,Xu Y.Identification and quantification of the caproic acid-producing bacterium Clostridium kluyveri in the fermentation of pit mud used for Chinese strong-aroma type liquor production.International Journal of Food Microbiology,2015,214:116-122.
[6]Yao WC,Tang YM,Ren DQ.White wine cellar mud key functions of Luzhou-flavor liquor cultivating the optimization.Liquor Making,2010,37(6):18-21.(in Chinese)姚万春,唐玉明,任道群.浓香型白酒窖泥关键功能菌的优化培养.酿酒,2010,37(6):18-21.
[7]Ye GB,Luo HB,Yang XD,Li DY,Wang Y,Ni B.Community structure of prokaryotes in pit mud of Lu-flavor liquor from Luzhou prefecture based on culture-independent approach.Food Science,2013,34(17):176-181.(in Chinese)叶光斌,罗惠波,杨晓东,李丹宇,王毅,倪斌.基于免培养法研究泸州地区浓香型白酒窖泥原核微生物群落结构.食品科学,2013,34(17):176-181.
[8]Li XR,Ma EB,Yan LZ,Meng H,Du XW,Zhang SW,Quan ZX.Bacterial and fungal diversity in the traditional Chinese liquor fermentation process.International Journal of Food Microbiology,2011,146(1):31-37.
[9]Wang XS,Du H,Xu Y.Source tracking of prokaryotic communities in fermented grain of Chinese strong-flavor liquor.International Journal of Food Microbiology,2017,244:27-35.
[10]Huang S,Yang F,Zeng XW,Chen J,Li R,Wen T,Li C,Wei W,Liu JQ,Chen L,Davis C,Xu J.Preliminary characterization of the oral microbiota of Chinese adults with and without gingivitis.BMC Oral Health,2011,11:33.
[11]Schloss PD,Westcott SL,Ryabin T,Hall JR,Hartmann M,Hollister EB,Lesniewski RA,Oakley BB,Parks DH,Robinson CJ,Sahl JW,Stres B,Thallinger GG,van Horn DJ,Weber C.Introducing mothur:open-source,platform-independent,community-supported software for describing and comparing microbial communities.Applied and Environmental Microbiology,2009,75(23):7537-7541.
[12]Hollister EB,Engledow AS,Hammett AJM,Provin TL,Wilkinson HH,Gentry TJ.Shifts in microbial community structure along an ecological gradient of hypersaline soils and sediments.The ISME Journal,2010,4(6):829-838.
[13]Dou X,Han PJ,Liu L,Zhang YH,He JM,Zhuo XX,Wu YY,Bai FY,Yang JG.Study on isolation and identification and population succession law of bacterial in fermented grains during the brewing of Luzhou-flavour liquor.Science and Technology of Food Industry,2017,38(1):169-174.(in Chinese)窦晓,韩培杰,刘莉,张雨涵,贺佳美,卓晓轩,吴依阳,白逢彦,杨建刚.泸型酒酿造期间酒醅中可培养细菌的分离鉴定及其种群演替规律研究.食品工业科技,2017,38(1):169-174.
[14]Wang T,Zhao D,Tian SP,You L,Wang S,Feng RZ,Feng XY,Zhang Y,Cui XL.Phylogenetic diversity of cultivable bacteria during the brewing process of the Luzhou-flavor liquor in Yibin,Sichuan province,China.Acta Microbiologica Sinica,2011,51(10):1351-1357.(in Chinese)王涛,赵东,田时平,游玲,王松,冯瑞章,冯学愚,张云,崔晓龙.宜宾浓香型白酒酿造过程中可培养细菌的系统发育多样性.微生物学报,2011,51(10):1351-1357.
[15]Qiao ZW,Zhang WX,Zhang LY,Zhang QS,Yue YY.Analysis on microbes in brewing mass of strong aromatic spirits during fermentation.Liquor Making,2005,32(1):18-22.(in Chinese)乔宗伟,张文学,张丽莺,张其圣,岳元媛.浓香型白酒发酵过程中酒醅的微生物区系分析.酿酒,2005,32(1):18-22.
[16]Zhang WX,Qiao ZW,Shigematsu T,Tang YQ,Hu C,Morimura S,Kida K.Analysis of the bacterial community in Zaopei during production of Chinese Luzhou-flavor liquor.Journal of the Institute of Brewing,2005,111(2):215-222.
[17]Huang YL,Huang YG,Hu JF,Hu F,Zhong FD.Microbial diversity of the second rounds liquid of Moutai-flavor Baijiu during fermentation process.China Brewing,2017,36(9):30-35.(in Chinese)黄蕴利,黄永光,胡建峰,胡峰,钟方达.酱香型白酒第二轮次酒发酵过程微生物多样性研究.中国酿造,2017,36(9):30-35.
[18]王海燕.PCR-DGGE技术对清香型汾酒微生物群落结构演变规律的研究.无锡:江南大学博士学位论文,2014.
[19]Xiang WL,Li K,Liu S,Xing YG,Li MY,Che ZM.Microbial succession in the traditional Chinese Luzhou-flavor liquor fermentation process as evaluated by SSU rRNA profiles.World Journal of Microbiology and Biotechnology,2013,29(3):559-567.
[20]Ren C,Du H,Xu Y.Advances in microbiome study of traditional Chinese fermented foods.Acta Microbiologica Sinica,2017,57(6):885-898.(in Chinese)任聪,杜海,徐岩.中国传统发酵食品微生物组研究进展.微生物学报,2017,57(6):885-898.
[21]Xiao C,Lu ZM,Zhang XJ,Wang ST,Ao L,Shen CH,Shi JS,Xu ZH.Bio-heat is a key environmental driver shaping microbial community of medium-temperature Daqu.Applied and Environmental Microbiology,2017,83(23):e01550-17.
[22]张佩佩.细菌Comamonas serinivorans C35对木质素的降解及其代谢机制初步研究.江苏大学硕士学位论文,2017.
[23]Zhang ML,Liu N,Qian CL,Wang QF,Wang Q,Long YH,Huang YP,Zhou ZH,Yan X.Phylogenetic and functional analysis of gut microbiota of a fungus-growing higher termite:Bacteroidetes from higher termites are a rich source ofβ-glucosidase genes.Microbial Ecology,2014,68(2):416-425.
[24]Ze XL,Duncan SH,Louis P,Flint HJ.Ruminococcus bromii is a keystone species for the degradation of resistant starch in the human colon.The ISME Journal,2012,6(8):1535-1543.
[25]Zhang DF,Li K,Liu S,Liu RC,Xing YG,Li MY,Che ZM,Xiang WL.Microbial community succession of Chinese Luzhou-flavor liquor lees.Food Science,2012,33(15):183-187.(in Chinese)张大凤,李可,刘森,刘若尘,邢亚阁,李明元,车振明,向文良.中国浓香型白酒窖池糟醅中微生物群落演替分析.食品科学,2012,33(15):183-187.
[26]Zhang GS,Hu HY,Feng H.Relations between atmospheric temperature and technical parameters/liquor yield/quality liquor rate in liquor production.Liquor-Making Science&Technology,2017,(7):77-79.(in Chinese)张广松,胡海洋,冯浩.酿酒生产过程气温与工艺参数及两率的关系.酿酒科技,2017,(7):77-79.
[27]Zhao JL,Zhang HX,Zhang XH.Effect of low acid environment on fermentation of fen-flover liquor.China Brewing,2013,32(8):35-38.(in Chinese)赵景龙,张红霞,张秀红.低酸环境对汾酒酿造的影响.中国酿造,2013,32(8):35-38.
[28]Zhang YY,Zhu XY,Li XZ,Tao Y,Jia JJ,He HX.The process-related dynamics of microbial community during a simulated fermentation of Chinese strong-flavored liquor.BMC Microbiology,2017,17:196.
[2]Tao Y,Li JB,Rui JP,Xu ZC,Zhou Y,Hu XH,Wang X,Liu MH,Li DP,Li XZ.Prokaryotic communities in pit mud from different-aged cellars used for the production of Chinese strong-flavored liquor.Applied and Environmental Microbiology,2014,80(7):2254-2260.
[3]Hou XG,Wang JY,Li XS,Hu BY,Li SL,Gao YY.The research progress on functional aroma-producing microorganisms in Zaopei and pit mud of Chinese strong-flavor liquor.Microbiology China,2013,40(7):1257-1265.(in Chinese)侯小歌,王俊英,李学思,胡炳义,李绍亮,高应运.浓香型白酒糟醅及窖泥产香功能菌的研究进展.微生物学通报,2013,40(7):1257-1265.
[4]Wu Q,Peng SQ,Yu Y,Li YX,Xu Y.Genome sequence of Bacillus licheniformis CGMCC3963,a stress-resistant strain isolated in a Chinese traditional solid-state liquor-making process.Genome Announcements,2013,1(1):e00060-12.
[5]Hu XL,Du H,Xu Y.Identification and quantification of the caproic acid-producing bacterium Clostridium kluyveri in the fermentation of pit mud used for Chinese strong-aroma type liquor production.International Journal of Food Microbiology,2015,214:116-122.
[6]Yao WC,Tang YM,Ren DQ.White wine cellar mud key functions of Luzhou-flavor liquor cultivating the optimization.Liquor Making,2010,37(6):18-21.(in Chinese)姚万春,唐玉明,任道群.浓香型白酒窖泥关键功能菌的优化培养.酿酒,2010,37(6):18-21.
[7]Ye GB,Luo HB,Yang XD,Li DY,Wang Y,Ni B.Community structure of prokaryotes in pit mud of Lu-flavor liquor from Luzhou prefecture based on culture-independent approach.Food Science,2013,34(17):176-181.(in Chinese)叶光斌,罗惠波,杨晓东,李丹宇,王毅,倪斌.基于免培养法研究泸州地区浓香型白酒窖泥原核微生物群落结构.食品科学,2013,34(17):176-181.
[8]Li XR,Ma EB,Yan LZ,Meng H,Du XW,Zhang SW,Quan ZX.Bacterial and fungal diversity in the traditional Chinese liquor fermentation process.International Journal of Food Microbiology,2011,146(1):31-37.
[9]Wang XS,Du H,Xu Y.Source tracking of prokaryotic communities in fermented grain of Chinese strong-flavor liquor.International Journal of Food Microbiology,2017,244:27-35.
[10]Huang S,Yang F,Zeng XW,Chen J,Li R,Wen T,Li C,Wei W,Liu JQ,Chen L,Davis C,Xu J.Preliminary characterization of the oral microbiota of Chinese adults with and without gingivitis.BMC Oral Health,2011,11:33.
[11]Schloss PD,Westcott SL,Ryabin T,Hall JR,Hartmann M,Hollister EB,Lesniewski RA,Oakley BB,Parks DH,Robinson CJ,Sahl JW,Stres B,Thallinger GG,van Horn DJ,Weber C.Introducing mothur:open-source,platform-independent,community-supported software for describing and comparing microbial communities.Applied and Environmental Microbiology,2009,75(23):7537-7541.
[12]Hollister EB,Engledow AS,Hammett AJM,Provin TL,Wilkinson HH,Gentry TJ.Shifts in microbial community structure along an ecological gradient of hypersaline soils and sediments.The ISME Journal,2010,4(6):829-838.
[13]Dou X,Han PJ,Liu L,Zhang YH,He JM,Zhuo XX,Wu YY,Bai FY,Yang JG.Study on isolation and identification and population succession law of bacterial in fermented grains during the brewing of Luzhou-flavour liquor.Science and Technology of Food Industry,2017,38(1):169-174.(in Chinese)窦晓,韩培杰,刘莉,张雨涵,贺佳美,卓晓轩,吴依阳,白逢彦,杨建刚.泸型酒酿造期间酒醅中可培养细菌的分离鉴定及其种群演替规律研究.食品工业科技,2017,38(1):169-174.
[14]Wang T,Zhao D,Tian SP,You L,Wang S,Feng RZ,Feng XY,Zhang Y,Cui XL.Phylogenetic diversity of cultivable bacteria during the brewing process of the Luzhou-flavor liquor in Yibin,Sichuan province,China.Acta Microbiologica Sinica,2011,51(10):1351-1357.(in Chinese)王涛,赵东,田时平,游玲,王松,冯瑞章,冯学愚,张云,崔晓龙.宜宾浓香型白酒酿造过程中可培养细菌的系统发育多样性.微生物学报,2011,51(10):1351-1357.
[15]Qiao ZW,Zhang WX,Zhang LY,Zhang QS,Yue YY.Analysis on microbes in brewing mass of strong aromatic spirits during fermentation.Liquor Making,2005,32(1):18-22.(in Chinese)乔宗伟,张文学,张丽莺,张其圣,岳元媛.浓香型白酒发酵过程中酒醅的微生物区系分析.酿酒,2005,32(1):18-22.
[16]Zhang WX,Qiao ZW,Shigematsu T,Tang YQ,Hu C,Morimura S,Kida K.Analysis of the bacterial community in Zaopei during production of Chinese Luzhou-flavor liquor.Journal of the Institute of Brewing,2005,111(2):215-222.
[17]Huang YL,Huang YG,Hu JF,Hu F,Zhong FD.Microbial diversity of the second rounds liquid of Moutai-flavor Baijiu during fermentation process.China Brewing,2017,36(9):30-35.(in Chinese)黄蕴利,黄永光,胡建峰,胡峰,钟方达.酱香型白酒第二轮次酒发酵过程微生物多样性研究.中国酿造,2017,36(9):30-35.
[18]王海燕.PCR-DGGE技术对清香型汾酒微生物群落结构演变规律的研究.无锡:江南大学博士学位论文,2014.
[19]Xiang WL,Li K,Liu S,Xing YG,Li MY,Che ZM.Microbial succession in the traditional Chinese Luzhou-flavor liquor fermentation process as evaluated by SSU rRNA profiles.World Journal of Microbiology and Biotechnology,2013,29(3):559-567.
[20]Ren C,Du H,Xu Y.Advances in microbiome study of traditional Chinese fermented foods.Acta Microbiologica Sinica,2017,57(6):885-898.(in Chinese)任聪,杜海,徐岩.中国传统发酵食品微生物组研究进展.微生物学报,2017,57(6):885-898.
[21]Xiao C,Lu ZM,Zhang XJ,Wang ST,Ao L,Shen CH,Shi JS,Xu ZH.Bio-heat is a key environmental driver shaping microbial community of medium-temperature Daqu.Applied and Environmental Microbiology,2017,83(23):e01550-17.
[22]张佩佩.细菌Comamonas serinivorans C35对木质素的降解及其代谢机制初步研究.江苏大学硕士学位论文,2017.
[23]Zhang ML,Liu N,Qian CL,Wang QF,Wang Q,Long YH,Huang YP,Zhou ZH,Yan X.Phylogenetic and functional analysis of gut microbiota of a fungus-growing higher termite:Bacteroidetes from higher termites are a rich source ofβ-glucosidase genes.Microbial Ecology,2014,68(2):416-425.
[24]Ze XL,Duncan SH,Louis P,Flint HJ.Ruminococcus bromii is a keystone species for the degradation of resistant starch in the human colon.The ISME Journal,2012,6(8):1535-1543.
[25]Zhang DF,Li K,Liu S,Liu RC,Xing YG,Li MY,Che ZM,Xiang WL.Microbial community succession of Chinese Luzhou-flavor liquor lees.Food Science,2012,33(15):183-187.(in Chinese)张大凤,李可,刘森,刘若尘,邢亚阁,李明元,车振明,向文良.中国浓香型白酒窖池糟醅中微生物群落演替分析.食品科学,2012,33(15):183-187.
[26]Zhang GS,Hu HY,Feng H.Relations between atmospheric temperature and technical parameters/liquor yield/quality liquor rate in liquor production.Liquor-Making Science&Technology,2017,(7):77-79.(in Chinese)张广松,胡海洋,冯浩.酿酒生产过程气温与工艺参数及两率的关系.酿酒科技,2017,(7):77-79.
[27]Zhao JL,Zhang HX,Zhang XH.Effect of low acid environment on fermentation of fen-flover liquor.China Brewing,2013,32(8):35-38.(in Chinese)赵景龙,张红霞,张秀红.低酸环境对汾酒酿造的影响.中国酿造,2013,32(8):35-38.
[28]Zhang YY,Zhu XY,Li XZ,Tao Y,Jia JJ,He HX.The process-related dynamics of microbial community during a simulated fermentation of Chinese strong-flavored liquor.BMC Microbiology,2017,17:196.
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