浓香型白酒窖泥中细菌和古菌的组成与多样性

doi:10.3969/j.issn.0438-1157.2014.05.034

摘要/Abstract

摘要： 窖池是中国浓香型白酒的根本，其中窖泥微生物则是决定白酒风味与品质的核心。通过454焦磷酸测序法系统研究了不同窖龄窖泥的细菌和古菌的群落组成及多样性。共获得494293个有效序列，可归为796个可操作分类单元（OTUs），分别属于21个门（phylum）。优势分类门（≥1%）分别为厚壁菌门（Firmicutes）66.8%、拟杆菌门（Bacteroidetes）16.0%、广古菌门（Euryarchaeota）9.0%、螺旋体门（Spirochaetes）2.2%、放线菌门（Actinobacteria）1.8% 和绿弯菌门（Chloroflexi）1.0%。窖泥微生物多样性在1～25年间随着窖龄的增加而呈升高趋势，但在25～50年间保持相对稳定。不同窖龄的窖泥中优势种群存在明显差异，其中乳杆菌（Lactobacillus）在1年窖泥中为绝对优势种群（62.3%），而在10年窖泥中，Lactobacillus丰度显著下降（16.1%），Petrimonas，Clostridium IV和甲烷菌等丰度则显著增加。在25～50年窖泥中，这一趋势更加明显，并且一些未培养菌丰度在这一阶段也显著增加。此外，窖泥中古菌主要为氢型产甲烷菌（Methanobrevibacter、Methanoculleus、Methanobacterium），可同时利用氢气和乙酸的甲烷八叠球菌属（Methanosarcina）在25～50年窖龄的窖泥中也有较高的丰度（3.1%～4.5%）。本研究从微生物群落构成角度为“老窖产好酒”的实践经验提供了理论依据。

关键词: 酿酒, 窖龄, 窖泥, 微生物群落, 焦磷酸测序

Abstract: Pit mud (PM) plays a key role in controlling the quality of Chinese Luzhou-flavor liquor in anaerobic fermentation. Here, microbial community composition and diversity in PM samples using pyrosequencing technique were investigated. A total of 494293 valid sequences were obtained. The reads fell into 796 operational taxonomic units (OTUs) affiliated to 21 phyla. The predominant groups were Firmicutes (66.8% of total reads), Bacteroidetes (16.0%), Euryarchaeota (9.0%), Spirochaetes (2.2%), Actinobacteria (1.8%) and Chloroflexi (1.0%). Microbial diversity increased with cellar age until 25 years old, but kept relatively constant from 25 to 50 years. A large difference between microbial communities was observed in the pit muds with different cellar ages. Lactobacillus predominanted in 1-year pit mud (62.3%), but its abundance decreased remarkably in 10-year and older pit muds. In contrast, the abundances of Petrimonas, Clostridium IV and methanogens increased dramatically. In addition, Archaea in PM were mainly composed of hydrogenotrophic methanogens such as Methanobrevibacter, Methanoculleus, Methanobacterium, while the H₂/acetate-utilizing Methanosarcina was more abundant in PM with cellar age of 25—50 years (3.1%—4.5%). This research supports the practical experience that old fermentation pits produce high quality Luzhou-flavor liquor.

Key words: brewing, cellar age, pit mud, microbial community, pyrosequencing

中图分类号:

陶勇, 芮俊鹏, 李家宝, 徐占成, 李大平, 唐清兰, 魏勇, 李香真. 浓香型白酒窖泥中细菌和古菌的组成与多样性[J]. 化工学报, DOI: 10.3969/j.issn.0438-1157.2014.05.034.

TAO Yong, RUI Junpeng, LI Jiabao, XU Zhancheng, LI Daping, TANG Qinglan, WEI Yong, LI Xiangzhen. Microbial community compositions and diversity in pit mud of Chinese Luzhou-flavor liquor[J]. CIESC Journal, DOI: 10.3969/j.issn.0438-1157.2014.05.034.

参考文献 31

[1]	Wang Mingyue(王明跃), Zhang Wenxue(张文学), Wang Haiying(王海英), Liu Chaolan(刘超兰). Analysis of bacterial phylogenetic diversity of pit muds with different cellar ages[J]. Food Science(食品科学), 2013, 34(11): 177-181
[2]	Zhao Jinsong, Zheng Jia, Zhou Rongqing, Shi Bi. Microbial community structure of pit mud in a Chinese strong aeomatic liquor fermentation pit[J]. J. Inst. Brew., 2012, 118: 356-360
[3]	Qiao Zongwei(乔宗伟), Zhang Wenxue(张文学), Zhang Liying(张丽莺), Zhang Qisheng(张其圣), Yue Yuanyuan(岳元媛). Analysis on microbes in brewing mass of strong aromatic spirits during fermentation[J]. Liquor Making (酿酒), 2005, 32(1): 18-22
[4]	Yue Yuanyuan(岳元媛), Zhang Wenxue(张文学), Liu Xia(刘霞), Hu Cheng(胡承), Zhang Suyi(张宿义). Isolation and identification of facultative anaerobes in the pit mud of Chinese Luzhou-flavor liquor[J]. Microbiol. Chin. (微生物学通报), 2007, 34(2): 251-255
[5]	Hou Xiaoge(侯小歌), Wang Junying(王俊英), Li Xuesi(李学思), Hu Bingyi(胡炳义), Li Shaoliang(李绍亮), Gao Yingyun(高应运). Research on progress about functional aroma-producing microorganisms in Zaopei and pit mud of Chinese strong-flavor liquor[J]. Microbiol. Chin.(微生物学通报), 2013, 40(7): 1257-1265
[6]	Hu Cheng(胡承), Ying Hong (应鸿), Xu Defu (许德富), Hu Yongsong (胡永松). Research on microflora in pit mud and its application[J]. Liquor-Making Science & Technology (酿酒科技), 2005, 129(3): 34-38
[7]	Wu Yanyong (吴衍庸), Xue Tangrong (薛堂荣), Chen Zhaorong (陈昭荣), Lu Shiheng (卢世珩), Liu Aihua (刘爱华), Lin Shan(林山). Study on distribution and action of anaerobic bacteria in Wuliangye old fermented-pits[J]. Acta Microbiologica Sinica (微生物学报), 1992, 31(4): 299-307
[8]	Tao Yong (陶勇), Xu Zhancheng (徐占成), Li Dongxun (李东迅), Liu Menghua (刘梦华), Fan Kequan (樊科权), Yao Kai (姚开). Succession of bacterial community in the cellar mud and its relationship with environmental factors[J]. Liquor-making Science & Technology(酿酒科技), 2011, 12: 24-28
[9]	Deng Bo, Shen Caihong, Shan Xiaohu, Ao Zonghua, Zhao Jinsong, Shen Caihong, Huang J H. PCR-DGGE analysis on microbial communities in pit mud of cellars used for different periods of time[J]. J. Inst. Brew., 2012, 118: 120-126
[10]	Wang Mingyue (王明岳), Zhang Wenxue (张文学), Wang Haiying (王海英), Liu Chaolan (刘超兰). Phylogenetic diversity analysis of archaeal in the pit mud with different cellar age[J]. Chin. J. Appl. Environ. Biol. (应用环境生物学报), 2012, 18(6): 1043-1048
[11]	Ferrenberg S, O'Neill S P, Knelman J E, Todd B, Duggan S, Bradley D, Robinson T, Schmidt S K, Townsend A R, Williams M W, Cleveland C C, Melbourne B A, Jiang L, Nemergut D R. Changes in assembly processes in soil bacterial communities following a wildfire disturbance[J]. ISME J., 2013, 7(6): 1102-1111
[12]	Lauber C L, Hamady M, Knight R, Fierer N. Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale[J]. Appl. Environ. Microbiol., 2009, 75(15): 5111-5120
[13]	Huber J A, Mark Welch D B, Morrison H G, Huse S M, Neal P R, Butterfield D A, Sogin M L. Microbial population structures in the deep marine biosphere[J]. Science, 2007, 318(5847): 97-100
[14]	Serkebaeva Y M, Kim Y, Liesack W, Dedysh S N. Pyrosequencing-based assessment of the bacteria diversity in surface and subsurface peat layers of a northern wetland, with focus on poorly studied phyla and candidate divisions[J]. PLoS One, 2013, 8(5): e63994
[15]	Humblot C, Guyot J P. Pyrosequencing of tagged 16S rRNA gene amplicons for rapid deciphering of the microbiomes of fermented foods such as pearl millet slurries[J]. Appl. Environ. Microbiol., 2009, 75(13): 4354-4361
[16]	Lee S H, Kang H J, Lee Y H, Lee T J, Han K, Choi Y, Park H D. Monitoring bacterial community structure and variability in time scale in full-scale anaerobic digesters[J]. J. Environ. Monit., 2012, 14(7): 1893-1905
[17]	Wang X, Hu M, Xia Y, Wen X, Ding K. Pyrosequencing analysis of bacterial diversity in 14 wastewater treatment systems in China[J]. Appl. Environ. Microbiol., 2012, 78(19): 7042-7047
[18]	Tamaki H, Wright C L, Li X, Lin Q, Hwang C, Wang S, Thimmapuram J, Kamagata Y, Liu W T. Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform[J]. PLoS One, 2011, 6(9): e25263
[19]	Li X, Rui J, Mao Y, Yannarell A, Mackie R. Dynamics of the bacterial community structure in the rhizosphere of a maize cultivar[J]. Soil Biol. Biochem., 2014, 68: 392-401
[20]	Edgar R C, Haas B J, Clemente J C, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection[J]. Bioinformatics, 2011, 27(16): 2194-2200
[21]	Shi Si (施思), Wang Haiying (王海英), Zhang Wenxue(张文学), Deng Yu (邓宇), Lai Dengyi (赖登燡), Fan Ao (范鏖). Analysis of microbial communities characteristics in different pit mud of Luzhou-flavor liquor[J]. Liquor-making Sceience & Technology (酿酒科技), 2011, 203(5): 38-41
[22]	Xie Yuqiu (谢玉球), Zhong Yu (钟雨), Xie Xu (谢旭), Wang Weidong (王卫东). Roles and functions of lactic acid bacteria in the production of liquor by solid fermentation[J]. Liquor-making Science & Technology(酿酒科技), 2008, 173(11): 83-86
[23]	Rattanachaikunsopon P, Phumkhachorn P. Lactic acid bacteria: their antimicrobial compounds and their uses in food production[J]. Ann. Biol. Res., 2010, 1(4): 218-228
[24]	Wang Wei (王葳), Zhao Hui (赵辉), Chen Fengge (陈凤阁). Isolation and identification of lactic acid bacteria in the pitmud of Chinese Luzhou-flavor liquor[J]. Liquor-making Science & Technology(酿酒科技), 2006, 142(4): 29-31
[25]	Sun Y Z, Mao S Y, Zhu W Y. Rumen chemical and bacterial changes during stepwise adaptation to a high-concentrate diet in goats[J]. Animal, 2010, 4(2): 210-217
[26]	Grabowski A, Tindall B J, Bardin V, Blanchet D, Jeanthon C. Petrimonas sulfuriphila gen. nov., sp. nov., a mesophilic fermentative bacterium isolated from a biodegraded oil reservoir[J]. Int. J. Syst. Evol. Microbiol., 2005, 55(3): 1113-1121
[27]	Chen S, Dong X. Proteiniphilum acetatigenes gen. nov., sp. nov., from a UASB reactor treating brewery wastewater[J]. Int. J. Syst. Evol. Microbiol., 2005, 55(6): 2257-2261
[28]	Breitenstein A, Wiegel J, Haertig C, Weiss N, Andreesen J R, Lechner U. Reclassification of Clostridium hydroxybenzoicum as Sedimentibacter hydroxybenzoicus gen. nov., comb. nov., and description of Sedimentibacter saalensis sp. nov. [J]. Int. J. Syst. Evol. Microbiol., 2002, 52(3): 801-807
[29]	Obst M, Krug A, Luftmann H, Steinbuchel A. Degradation of cyanophycin by Sedimentibacter hongkongensis strain KI and Citrobacter amalonaticus strain G isolated from an anaerobic bacterial consortium[J]. Appl. Environ. Microbiol., 2005, 71(7): 3642- 3652
[30]	Madigan M T, Martinko J M, Stahl D A, Clark D P. Brock Biology of Microorganisms[M]. 13th ed. San Francisco, CA: Pearson Education Inc., 2012
[31]	Dollhopf S L, Hashsham S A, Dazzo F B, Hickey R F, Criddle C S, Tiedje J M. The impact of fermentative organisms on carbon flow in methanogenic systems under constant low-substrate conditions[J]. Appl. Microbiol. Biotechnol., 2001, 56(3/4): 531-538