微生物学通报 Microbiology China tongbao@im.ac.cn 研究报告 Dec. 20, 2014, 41(12): 2466 2473 2014 by Institute of Microbiology, CAS DOI: 10.13344/j.microbiol.china.140210 酱香型白酒发酵中酵母群落结构及其对风味组分的影响 * 邵明凯王海燕徐岩 聂尧 ( 214122) 摘要 目的 研究酱香型白酒发酵过程中酵母群落结构, 及其对酱香型白酒生产的影响 方法 通过变性梯度凝胶电泳 (DGGE) 和实时荧光定量 PCR (RT-qPCR) 技术分别对产量 酒质较高的第五轮次和较低的第七轮次发酵过程中的酵母群落结构进行解析, 同时利用顶空固相微萃取气质联用 (HS-SPME-GC-MS) 以及高效液相色谱 (HPLC) 技术分析酒醅中的代谢组分, 初步分析了酵母群落对酱香型白酒产量及品质的影响 结果 用 DGGE 方法从酒醅中的检测到 Issatchenkia orientalis Torulaspora delbrueckii Pichia galeiformis Schizosaccharomyces pombe Galactomyces geotrichum Trichosporon asahii Zygosaccharomyces bailii Saccharomyces cerevisiae 和 Pichia fabianii 等 9 种含量丰富的酵母, 其中 G. geotrichum 和 S. cerevisiae 是第五轮的优势酵母, 而第七轮的优势酵母增加了 I. orientalis 和 Z. bailii 两种酵母, 且 Sc. pombe 未检测到 第七轮发酵过程中 5 种酵母在发酵后期衰亡明显, 第五轮酵母群落结构相对更稳定 ; 发酵前期第五轮酵母总量是第七轮的 2 5 倍, 而发酵结束时第五轮乙醇含量达到第七轮次的 2.45 倍 酒醅中挥发性风味组分种类丰富, 第五轮中酯类等白酒中重要的风味物质含量明显高于第七轮, 有机酸和杂醇油含量低于第七轮 结论 酱香型白酒酿造过程中酵母菌群多样性较丰富, 酵母菌群结构变化对白酒产量及品质影响明显, 这为酱香型白酒酿造机制研究奠定了基础 关键词 酱香型白酒, 酵母群落, 风味组分 Yeast community structure and its impact on flavor components during the fermentation process of Chinese Maotai-flavor liquor SHAO Ming-Kai WANG Hai-Yan XU Yan * NIE Yao (The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan Uinversity, Center for Brewing Science and Enzyme Technology, Wuxi, Jiangsu 214122, China) Abstract: [Objective] To analyze yeast community structure and its influence on brewing during the fermentation process of Chinese Maotai-flavour liquor. [Methods] The yeast community in the fifth round (higher yield and quality of the liquor) and the seventh round of fermentation process (lower yield and quality of the liquor) were analyzed using denaturing gradient gel electrophoresis (DGGE) 基金项目 : 863 (No. 2012AA021301 2013AA102108) (No. 31000806 31371822 31271921) 2011 * 通讯作者 :Tel:86-510-85918201;Fax:86-510-85864112; :yxu@jiangnan.edu.cn 收稿日期 :2014-03-13 接受日期 :2014-04-17 优先数字出版日期 (www.cnki.net) 2014-05-13
: 2467 and real-time quantitative PCR (RT-qPCR). The metabolic components in the fermented grains were investigated through headspace-solid phase microextraction gas chromatograph-mass apectrometer (HS-SPME-GC-MS) and high performance liquid chromatography (HPLC). Finally, the effects of yeast communities on the yield and quality of Maotai-flavor liquor were analyzed. [Results] At least nine yeasts were detected in the fermented grains, including Issatchenkia orientalis, Torulaspora delbrueckii, Pichia galeiformis, Schizosaccharomyces pombe, Galactomyces geotrichum, Trichosporon asahii, Zygosaccharomyces bailii, Saccharomyces cerevisiae and Pichia fabianii. Among them, G. geotrichum and S. cerevisiae was dominant yeasts of the fifth round, while the other two yeast, I. orientalis and Z. bailii, were also advantage in seventh round and Sc. pombe was not detected. In addition, five kinds of yeasts declined significantly during latter period of the seventh round fermentation process, but the yeast community structure during the fifth round was relatively more stable. The total yeasts during the early stage of the fifth round fermentation is 2 5 times more than the seventh round s, while ethanol content of fifth round fermented grains was 2.45 times as higher as the seventh s at the end of fermentation. Besides, volatile metabolic component of fermented grains was rich in species. The content of esters of the fifth round was remarkably higher than the seventh round, while organic acids and fusel oils were lower than the seventh round. [Conclusion] During Maotai-flvour liquor brewing process, there is rich diversity in yeast species, and the dynamics of yeast community structure affect liquor s yield and quality obviously, which provides the basis for the study of Maotai-flavour liquor brewing mechanism. Keywords: Maotai-flavour liquor, Yeast community structure, Flavor components [1-2] [3-6] [7-10] Duthoit F. [4] Van-Diep L. [11] 19 [12] ( ) ( )
2468 微生物学通报 Microbiol. China 2014, Vol.41, No.12 1 材料与方法 1.1 试验材料 1.1.1 样品来源 : 2012 5 7 30 d 5 d 5 50 g 70 C 1.1.2 试剂 : EDTA 2 Taq PCR MasterMix PCR PCR DNA pmd19-t Vector Plasmid Mini Kit I SYBR Green Supermix 1.2 试验方法 1.2.1 酒醅基因组提取 : [13] 1.2.2 酒醅中酵母菌群结构分析 : NL1 LS2 [14-15] ( 1) PCR 26S rrna D1 [14] 250 bp DGGE DGGE 24% 45% 100 V 200 min SYBR-Green 1 TAE PCR pmd19-t Escherichia coli JM109 LB DGGE ( ) GenBank 1.2.3 统计分析 : Quantity One DGGE DGGE (PCA) SPSS 20.0 1.2.4 荧光定量 PCR (RT-qPCR) 测定酒醅中酵母生物量 : YeastR YeastF [16] ( 1) 26S rrna D1 DNA PCR [16] 126 bp T 10 Real-time PCR [16] 3 C t y= 3.137 1x+43.736 0 R 2 =0.998 6 108.3% 5.82 10 4 5.82 10 11 copies/g DNA RT-qPCR 3 表 1 Table 1 DGGE 和荧光定量所用引物 Primers for DGGE and RT-qPCR Primers name NL1* LS2 YeastR Primer sequences (5 3 ) GCATATCAATAAGCGGAGGAAAAG ATTCCCAAACAACTCGACTC TCTCTTTTCCAAAGTTCTTTTCATCTTT YeastF GAGTCGAGTTGTTTGGGAATGC * GC (5 -GCGGGCCGCGCGGCCGCCGGGCCGCGCGGCCGGCGGCGG-3 ) 5. Note: *: Add GC clamp (5 -GCGGGCCGCGCGGCCGCCGGGCCGCGCGGCCGGCGGCGG-3 ) to the 5 end of primer.
: 2469 1.2.5 酒醅中乙醇含量测定 : 1 g 3 ml 5 ml 12 000 r/min 5 min 700 μl 700 μl 10% 30 min 0.22 μm Bio-Rad Aminex HPX-87H (300 mm 7.8 mm 9 μm) 60 C 3 mmol/l H 2 SO 4 0.6 ml/min 10 μl (RID) y=0.000 014 487 6x 0.007 96 R 2 =0.999 16 x y (mg/l) 1.2.6 酒醅中风味代谢组分的检测 : [17-18] 2 结果与分析 2.1 发酵过程中酵母群落多样性分析 DGGE ( 1) DGGE 14 DNA ( 2) Issatchenkia orientalis Torulaspora delbrueckii Pichia galeiformis Schizosaccharomyces pombe Galactomyces geotrichum Trichosporon asahii Zygosaccharomyces bailii Saccharomyces cerevisiae S. cerevisiae G. geotrichum S. cerevisiae G. geotrichum I. orientalis Z. bailii Sc. pombe Pichia fabianii I. orientalis T. delbrueckii P. galeiformis Z. bailii S. cerevisiae DNA Figure 1 图 1 不同轮次发酵过程酵母 DGGE 图谱 DGGE dynamic changes of yeasts in different round fermentation A DGGE w1 w9 DNA B DGGE q1 q9 DNA. 0 5 10 15 20 25 30 0 5 10 15 20 25 30 d. Note: A: DGGE dynamic changes of yeasts in the fifth round fermentation, the positions of obvious bands sequenced are indicated by letters from w1 to w9; B: DGGE dynamic changes of yeasts in the seventh round fermentation, the positions of obvious bands sequenced are indicated by letters from q1 to q9. The lane of 0, 5, 10, 15, 20, 25, 30 corresponding to the culture sample of the 0, 5, 10, 15, 20, 25, 30 d fermentation.
2470 微生物学通报 Microbiol. China 2014, Vol.41, No.12 表 2 切胶条带测序结果 Table 2 Sequencing results of the bands Bands No. ( ) Similar strains (accession No.) Identity (%) w1/q1 Issatchenkia orientalis (FJ770560.1) 100 w2/q2 Torulaspora delbrueckii (HE660079.1) 100 w3/q3 Pichia galeiformis (JX880397.1) 100 w4/q4 Pichia galeiformis (JX880397.1) 100 w5 Schizosaccharomyces pombe (HE964968.1) 100 w6/q5 Galactomyces geotrichum (JN974271.1) 99 q6 Pichia fabianii (U73573.1) 100 w7/q7 Trichosporon asahii (FJ463648.1) 100 w8/q8 Zygosaccharomyces bailii (EU441913.1) 99 w9/q9 Saccharomyces cerevisiae (EU441887.1) 100 2.2 发酵过程中酵母群落统计分析 2 (PC1) 63.9% (PC2) 18.7% 2 82.6% (P<0.05) 2 Sc. pombe S. cerevisiae G. geotrichum I. orientalis Z. bailii DGGE 2.3 酒醅中酵母生物量和乙醇含量的分析 3 2 5 10 20 d 2.0 10 5 copies/g 图 2 酵母群落结构主成分分析 Figure 2 Principal component analysis of yeast community structure *. A1 A7 0 5 10 15 20 25 30 d B1 B7 0 5 10 15 20 25 30 d. Io: I. orientalis; Td: T. delbrueckii; Pg: P. galeiformis; Sp: Sc. pombe; Gc: G. geotrichum; Ta: T. asahii; Pf: P. fabianii; Zb: Z. bailii; Sc: S. cerevisiae. Note: : Samples; *: Yeast species. A1 A7 corresponding to the culture sample of the 0, 5, 10, 15, 20, 25, 30 d during the fifth fermentation; B1 B7 corresponding to the culture sample of the 0, 5, 10, 15, 20, 25, 30 d during the seventh fermentation. Io: I. orientalis; Td: T. delbrueckii; Pg: P. galeiformis; Sp: Sc. pombe; Gc: G. geotrichum; Ta: T. asahii; Pf: P. fabianii; Zb: Z. bailii; Sc: S. cerevisiae.
: 2471 4 5 2.45 10 ( 3) 2.4 发酵酒醅中主要风味代谢组分分析 [19] 10 000 42 20 6 7 7 2 ( 5) 2- [20-21] 3 讨论 图 3 酱香型白酒第五轮和第七轮酵母生物量 Figure 3 Yeast biomass during the fifth and seventh round fermentation of Maotai-flavor liquor 图 4 第五轮和第七轮发酵过程中酒醅乙醇含量 Figure 4 Ethanol concentration in the fifth and seventh round of fermented grains [22] DGGE PCA I. orientalis
2472 微生物学通报 Microbiol. China 2014, Vol.41, No.12 图 5 第五轮和第七轮发酵酒醅中的风味物质含量热图 Figure 5 Heat map of flavor substances in the fermented grains during the fifth and seventh round fermentation Note: A1 A7 corresponding to the culture sample of the 0, 5, 10, 15, 20, 25, 30 d during the fifth fermentation; B1 B7 corresponding to the culture sample of the 0, 5, 10, 15, 20, 25, 30 d during the seventh fermentation. X1: 1-Propanol, 2-methyl-; X2: Linoleic acid ethyl ester; X3: Ethyl oleate; X4: Hexadecanoic acid ethyl ester; X5: Pentadecanoic acid ethyl ester; X6: Octanoic acid; X7: Tetradecanoic acid ethyl ester; X8: Phenylethyl alcohol; X9: Benzenepropanoic acid ethyl ester; X10: Benzyl alcohol; X11: Hexanoic acid; X12: Acetic acid 2-phenylethyl ester; X13: Benzeneacetic acid ethyl ester; X14: Butanedioic acid diethyl ester; X15: 2-Furanmethanol; X16: Butanoic acid; X17: 1-Octanol; X18: Furfural; X19: Acetic acid; X20: Octanoic acid ethyl ester; X21: 1-Hexanol; X22: Propanoic acid, 2-hydroxy-, ethyl ester; X23: Heptanoic acid ethyl ester; X24: Hexanoic acid propyl ester; X25: Acetic acid hexyl ester; X26: Butanoic acid pentyl ester; X27: Hexanoic acid ethyl ester; X28: 1-Butanol, 3-methyl-; X29: 1-Butanol; X30: Pentanoic acid ethyl ester; X31: Propanoic acid, 2-methyl-, pentyl ester; X32: Acetic acid butyl ester; X33: Butanoic acid ethyl ester; X34: Propanoic acid ethyl ester; X35: Ethyl acetate; X36: 4-Methyl phenol; X37: Heptanoic acid; X38: Pentanoic acid; X39: Benzaldehyde; X40: Propanoic acid; X41: 2-Butyl alcohol; X42: n-propyl acetate. Sc. pombe S. cerevisiae Wu [12] I. orientalis Sc. pombe S. cerevisiae Sc. pombe
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