芝麻香型酒醅堆积过程中微生物结构变化分析
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摘要
【背景】高温堆积发酵过程是芝麻香型白酒生产过程中的一个重要环节,是芝麻香型白酒典型风格形成的关键工序。【目的】解析堆积发酵过程中原核和真核微生物群落的演替规律。【方法】分别对高温堆积酒醅样本的细菌16S rDNA V3-V4区和真菌ITS2区进行Illumina高通量测序,解析高温堆积过程中酒醅的真菌和细菌菌群结构变化规律,同时解析发酵过程中温度、水分、酸度、淀粉、还原糖的变化规律。【结果】酒醅堆积初期0-6 h,占优势的原核微生物菌属是Bacillus、Weissella和Acetobacter。堆积20 h后微生物群落结构变化显著,Weissella的比例降低,Acetobacter的比例显著增加。堆积初期,占优势的真核微生物菌属是Aspergillus、Saccharomyces和Pichia。堆积6 h后,Aspergillus比例下降,而Pichia和Saccharomyces则逐步增多,其绝对优势地位一直保持至堆积结束。酒醅微生物群落结构受翻堆操作的短暂影响(堆积20 h时),最终恢复至平衡状态。高温堆积36 h以后,酒醅中的原核和真核微生物群落均趋于稳定。还原糖数据显示堆积前期微生物糖化作用迅速,36 h后糖化趋于稳定,温度、酸度、淀粉含量参数基本平稳。【结论】酒醅高温堆积发酵过程中,原核微生物和真核微生物有其不同的变化规律。原核微生物的变化是以20 h时的翻堆为结点,翻堆前的优势菌属是Bacillus、Weissella和Acetobacter;堆积后的优势菌属是Weissella和Acetobacter,其中后者占绝对优势。与原核微生物相比,真菌微生物的结构逐步集中,到发酵结束时毕赤酵母和酵母两者占真菌总量的93%以上。芝麻香型白酒发酵过程的高温堆积环节,原核和真核微生物的菌种繁殖以及相应的菌群结构变化为稍后的高温发酵奠定了微生物基础。
[Background] High-temperature stacking is a key step among the whole sesame-flavor Baijiu fermentation process, and vital for the formation of typical flavor of sesame-flavor Baijiu. [Objective]This study aimed to analyze prokaryotic and eukaryotic microbial communities during the high-temperature stacking process. [Methods] Illumina high-throughput sequencing was performed on the bacterial 16 S rDNA V3–V4 region and the fungal ITS2 region of the high-temperature stacked Jiupei samples. The change pattern of prokaryotic and eukaryotic microbial community structure were analyzed.The changes in temperature, moisture, acidity, starch and reduced-sugar during stacking were also analyzed. [Results] At the beginning of 0–6 hours, the dominant bacteria were Bacillus, Weissella, and Acetobacter. After 20 h of stacking, the flora changed significantly. Weissella decreased and the Acetobacter increased. At the beginning, the dominant fungi were Aspergillus, Saccharomyces, and Pichia. After 6 hours, the proportion of Aspergillus dropped sharply, while Pichia and Saccharomyces gradually increased, and their absolute dominance maintained until the end of the stacking. The overall structure of the microbial community was temporarily affected by the turn-over operation(when it was piled up for 20 h), eventually returning to equilibrium. After 36 h of stacking, both prokaryotic and eukaryotic microbial community stabilized. Reduced-sugar data showed that the saccharification was rapid in the early stage, later became stable after 36 h. Temperature, acidity and starch content were also stable at this stage. [Conclusion] During the high-temperature stacking-fermentation process,fluctuation patterns of the prokaryotic and eukaryotic microbial community structure were different. The prokaryotic community changes were based on the turn-up at 20 h. The dominant species before the turn-up were Bacillus, Weissella and Acetobacte, the dominant strains after the turn-up were Weissella and Acetobacter, the latter was absolute dominant. Compared to prokaryotic community changes, the eukaryotic microbial community concentration gradually, by the end of the stacking, Pichia and Saccharomyces accounted for more than 93% of the total eukaryotic microbial. During the high-temperature stacking of the sesame-flavor Baijiu, the propagation of various prokaryotic/eukaryotic microbes and the corresponding changes of microbial communities laid the microbial basis for later high-temperature fermentation.
[Background] High-temperature stacking is a key step among the whole sesame-flavor Baijiu fermentation process, and vital for the formation of typical flavor of sesame-flavor Baijiu. [Objective]This study aimed to analyze prokaryotic and eukaryotic microbial communities during the high-temperature stacking process. [Methods] Illumina high-throughput sequencing was performed on the bacterial 16 S rDNA V3–V4 region and the fungal ITS2 region of the high-temperature stacked Jiupei samples. The change pattern of prokaryotic and eukaryotic microbial community structure were analyzed.The changes in temperature, moisture, acidity, starch and reduced-sugar during stacking were also analyzed. [Results] At the beginning of 0–6 hours, the dominant bacteria were Bacillus, Weissella, and Acetobacter. After 20 h of stacking, the flora changed significantly. Weissella decreased and the Acetobacter increased. At the beginning, the dominant fungi were Aspergillus, Saccharomyces, and Pichia. After 6 hours, the proportion of Aspergillus dropped sharply, while Pichia and Saccharomyces gradually increased, and their absolute dominance maintained until the end of the stacking. The overall structure of the microbial community was temporarily affected by the turn-over operation(when it was piled up for 20 h), eventually returning to equilibrium. After 36 h of stacking, both prokaryotic and eukaryotic microbial community stabilized. Reduced-sugar data showed that the saccharification was rapid in the early stage, later became stable after 36 h. Temperature, acidity and starch content were also stable at this stage. [Conclusion] During the high-temperature stacking-fermentation process,fluctuation patterns of the prokaryotic and eukaryotic microbial community structure were different. The prokaryotic community changes were based on the turn-up at 20 h. The dominant species before the turn-up were Bacillus, Weissella and Acetobacte, the dominant strains after the turn-up were Weissella and Acetobacter, the latter was absolute dominant. Compared to prokaryotic community changes, the eukaryotic microbial community concentration gradually, by the end of the stacking, Pichia and Saccharomyces accounted for more than 93% of the total eukaryotic microbial. During the high-temperature stacking of the sesame-flavor Baijiu, the propagation of various prokaryotic/eukaryotic microbes and the corresponding changes of microbial communities laid the microbial basis for later high-temperature fermentation.
引文
[1]Huang YL,Zhang B,Wu JH.Discussion on Sesame-flavor liquor[J].Liquor-Making Science&Technology,2007(10):116-119(in Chinese)黄业立,张彬,武金华.试论芝麻香型白酒[J].酿酒科技,2007(10):116-119
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[3]Xu Y.Scientific heritage and integrated innovation for sustainable development of liquor-making techniquesviewpoints of Sesame-flavor liquor[J].Liquor-Making Science&Technology,2013(4):17-20(in Chinese)徐岩.科学传承、集成创新走中国白酒技术持续发展的道路-对芝麻香酒的看法和认识[J].酿酒科技,2013(4):17-20
[4]Liu MM,Wang JG,Sun PP,et al.Application of bran starter in the production of Sesame-flavor liquor[J].Liquor-Making Science&Technology,2013(3):69-70,74(in Chinese)刘明明,王君高,孙朋朋,等.麸曲在芝麻香型白酒生产中的应用[J].酿酒科技,2013(3):69-70,74
[5]Zhang B.Production and application of distiller’s yeast production of Sesame-flavor used especially for the liquor[J].Liquor Making,2012,39(6):38-41(in Chinese)张彬.芝麻香型白酒专用曲的生产及应用[J].酿酒,2012,39(6):38-41
[6]Wu Q,Ling J,Xu Y.Starter culture selection for making Chinese sesame-flavored liquor based on microbial metabolic activity in mixed-culture fermentation[J].Applied and Environmental Microbiology,2014,80(14):4450-4459
[7]Tao Y,Rui JP,Li JB,et al.Microbial community compositions and diversity in pit mud of Chinese Luzhou-flavor liquor[J].CIESC Journal,2014,65(5):1800-1807(in Chinese)陶勇,芮俊鹏,李家宝,等.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,2014,65(5):1800-1807
[8]Deng J,Huang ZG,Wei CH,et al.High-throughput sequencing reveals bacterial structure in the mud pits of heavy-fragrance Baijiu[J].Modern Food Science and Technology,2015,31(7):50-55(in Chinese)邓杰,黄治国,卫春会,等.基于高通量测序的浓香型白酒窖池细菌群落结构分析[J].现代食品科技,2015,31(7):50-55
[9]Li KY,Wen Z,Deng B,et al.High-throughput sequencing using in research for community diversity of prokaryotes in pit mud of different ages[J].The Food Industry,2016(6):121-125(in Chinese)李克亚,文章,邓斌,等.不同窖龄窖泥原核生物多样性的高通量测序研究[J].食品工业,2016(6):121-125
[10]Zhao D,Zheng J,Peng ZY,et al.Prokaryotic microbial community in Wuliangye pit mud using NextSeq high-throughput sequencing technology[J].Food and Fermentation Industries,2017,43(9):1-8(in Chinese)赵东,郑佳,彭志云,等.高通量测序技术解析五粮液窖泥原核微生物群落结构[J].食品与发酵工业,2017,43(9):1-8
[11]Xu ZJ,Meng Z,Zhong QD,et al.Microbial community changes during stacking fermentation in the production of Sesame-flavor liquor using PLFA[J].Food and Fermentation Industries,2012,38(4):20-24(in Chinese)徐泽江,孟镇,钟其顶,等.芝麻香型白酒堆积过程微生物群落结构特征的PLFA分析[J].食品与发酵工业,2012,38(4):20-24
[12]Chen SJ,Wang JW.Influence of stacking conditions on temperature and liquor quality of Sesame-flavor liquor[J].Qingdao University of Science and Technology(Natural Science Edition),2015,36(S2):116-117,120(in Chinese)陈燊君,王京伟.芝麻香型白酒堆积条件对升温及酒质的影响[J].青岛科技大学学报:自然科学版,2015,36(S2):116-117,120
[13]Guo M,Huang YG,Qiu SY,et al.Application of high-throughput sequencing in microbial diversity of Moutai-flavor Baijiu[J].China Brewing,2017,36(5):146-151(in Chinese)郭敏,黄永光,邱树毅,等.高通量测序在酱香白酒微生态多样性研究中的应用[J].中国酿造,2017,36(5):146-151
[14]Wan QH,Xie SK,Gao DY,et al.Effects of two kinds of accumulated grains on fermentation characteristics and aroma quality of sesame-flavor liquor[J].Food and Fermentation Industries,2017,43(11):9-15(in Chinese)万清徽,谢圣凯,高大禹,等.两种堆积醅对芝麻香型白酒发酵特性和香气品质的影响[J].食品与发酵工业,2017,43(11):9-15
[15]Li RQ,Zhu HM,Ruan J,et al.De novo assembly of human genomes with massively parallel short read sequencing[J].Genome Research,2010,20(2):265-272
[16]Yang F,Zeng XW,Ning K,et al.Saliva microbiomes distinguish caries-active from healthy human populations[J].The ISME Journal,2012,6(1):1-10
[17]de Pasquale I,Di Cagno R,Buchin S,et al.Microbial ecology dynamics reveal a succession in the core microbiota involved in the ripening of pasta filata Caciocavallo Pugliese cheese[J].Applied and Environmental Microbiology,2014,80(19):6243-6255
[18]Michel E,Monfort C,Deffrasnes M,et al.Characterization of relative abundance of lactic acid bacteria species in French organic sourdough by cultural,qPCR and MiSeq high-throughput sequencing methods[J].International Journal of Food Microbiology,2016,239:35-43
[19]Wang XD,Zhang XL,Chen MQ.Study on the change trend of the bacteria and the physicochemical indexes in fermented grains in Maotai-flaver liquor pits[J].Liquor Making,2013,41(3):21-24(in Chinese)王晓丹,张小龙,陈孟强.酱香型白酒窖内发酵过程中酒醅理化指标与细菌的变化趋势研究[J].酿酒,2013,41(3):21-24
[20]Yang GH,Qiu SY,Huang YG.Microbiology research of liquor production[J].China Brewing,2011(4):24-27(in Chinese)杨国华,邱树毅,黄永光.酱香白酒生产中产香微生物研究[J].中国酿造,2011(4):24-27
[21]Li X.Screening of high yield ofα-amylase Bacillus licheniformis and construction of a recombinant plasmid[D].Wuhan:Master’s Thesis of Hubei University of Technology,2014(in Chinese)李习.高产α-淀粉酶地衣芽孢杆菌的筛选及重组质粒的构建[D].武汉:湖北工业大学硕士学位论文,2014
[22]Sun JQ,Liu WW,Zang W,et al.Community composition of moulds from fermented grains of Maotai-flavor liquor and their enzyme activities[J].Journal of Chinese Institute of Food Science and Technology,2013,13(8):239-247(in Chinese)孙剑秋,刘雯雯,臧威,等.酱香型白酒酒醅中霉菌群落组成与功能酶活性[J].中国食品学报,2013,13(8):239-247
[23]Yan QM,Shen XB,Song B,et al.Study on flavor compounds in different accumulation position of Sesame fermented grains[J].Liquor Making,2017,44(6):50-53(in Chinese)严启梅,沈晓波,宋宝,等.芝麻香型不同位置堆积酒醅风味成分的研究[J].酿酒,2017,44(6):50-53
[24]Shan QMG,Liang HZ,Zhang CX,et al.Changes of microbial diversity in stacked fermentation for the production of Moutai flavor liquor[J].Journal of Food Science and Biotechnology,2016,35(3):330-335(in Chinese)山其木格,梁慧珍,张长霞,等.糟醅堆积过程中微生物种群的变化规律[J].食品与生物技术学报,2016,35(3):330-335
[25]Li XD,Gao DY,Tian QZ,et al.Effects of Sesame-flavor liquor accumulation on cellar fermentation process and liquor quality[J].Food and Fermentation Industries,2018,44(5):63-69(in Chinese)李小东,高大禹,田庆贞,等.芝麻香型白酒堆积发酵对入窖发酵过程及原酒品质的影响[J].食品与发酵工业,2018,44(5):63-69
[2]Qi YM.Investigation on Sesame-flavor liquor production[J].Liquor-Making Science&Technology,2009(8):140-142(in Chinese)戚元民.对芝麻香型白酒生产的认识[J].酿酒科技,2009(8):140-142
[3]Xu Y.Scientific heritage and integrated innovation for sustainable development of liquor-making techniquesviewpoints of Sesame-flavor liquor[J].Liquor-Making Science&Technology,2013(4):17-20(in Chinese)徐岩.科学传承、集成创新走中国白酒技术持续发展的道路-对芝麻香酒的看法和认识[J].酿酒科技,2013(4):17-20
[4]Liu MM,Wang JG,Sun PP,et al.Application of bran starter in the production of Sesame-flavor liquor[J].Liquor-Making Science&Technology,2013(3):69-70,74(in Chinese)刘明明,王君高,孙朋朋,等.麸曲在芝麻香型白酒生产中的应用[J].酿酒科技,2013(3):69-70,74
[5]Zhang B.Production and application of distiller’s yeast production of Sesame-flavor used especially for the liquor[J].Liquor Making,2012,39(6):38-41(in Chinese)张彬.芝麻香型白酒专用曲的生产及应用[J].酿酒,2012,39(6):38-41
[6]Wu Q,Ling J,Xu Y.Starter culture selection for making Chinese sesame-flavored liquor based on microbial metabolic activity in mixed-culture fermentation[J].Applied and Environmental Microbiology,2014,80(14):4450-4459
[7]Tao Y,Rui JP,Li JB,et al.Microbial community compositions and diversity in pit mud of Chinese Luzhou-flavor liquor[J].CIESC Journal,2014,65(5):1800-1807(in Chinese)陶勇,芮俊鹏,李家宝,等.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,2014,65(5):1800-1807
[8]Deng J,Huang ZG,Wei CH,et al.High-throughput sequencing reveals bacterial structure in the mud pits of heavy-fragrance Baijiu[J].Modern Food Science and Technology,2015,31(7):50-55(in Chinese)邓杰,黄治国,卫春会,等.基于高通量测序的浓香型白酒窖池细菌群落结构分析[J].现代食品科技,2015,31(7):50-55
[9]Li KY,Wen Z,Deng B,et al.High-throughput sequencing using in research for community diversity of prokaryotes in pit mud of different ages[J].The Food Industry,2016(6):121-125(in Chinese)李克亚,文章,邓斌,等.不同窖龄窖泥原核生物多样性的高通量测序研究[J].食品工业,2016(6):121-125
[10]Zhao D,Zheng J,Peng ZY,et al.Prokaryotic microbial community in Wuliangye pit mud using NextSeq high-throughput sequencing technology[J].Food and Fermentation Industries,2017,43(9):1-8(in Chinese)赵东,郑佳,彭志云,等.高通量测序技术解析五粮液窖泥原核微生物群落结构[J].食品与发酵工业,2017,43(9):1-8
[11]Xu ZJ,Meng Z,Zhong QD,et al.Microbial community changes during stacking fermentation in the production of Sesame-flavor liquor using PLFA[J].Food and Fermentation Industries,2012,38(4):20-24(in Chinese)徐泽江,孟镇,钟其顶,等.芝麻香型白酒堆积过程微生物群落结构特征的PLFA分析[J].食品与发酵工业,2012,38(4):20-24
[12]Chen SJ,Wang JW.Influence of stacking conditions on temperature and liquor quality of Sesame-flavor liquor[J].Qingdao University of Science and Technology(Natural Science Edition),2015,36(S2):116-117,120(in Chinese)陈燊君,王京伟.芝麻香型白酒堆积条件对升温及酒质的影响[J].青岛科技大学学报:自然科学版,2015,36(S2):116-117,120
[13]Guo M,Huang YG,Qiu SY,et al.Application of high-throughput sequencing in microbial diversity of Moutai-flavor Baijiu[J].China Brewing,2017,36(5):146-151(in Chinese)郭敏,黄永光,邱树毅,等.高通量测序在酱香白酒微生态多样性研究中的应用[J].中国酿造,2017,36(5):146-151
[14]Wan QH,Xie SK,Gao DY,et al.Effects of two kinds of accumulated grains on fermentation characteristics and aroma quality of sesame-flavor liquor[J].Food and Fermentation Industries,2017,43(11):9-15(in Chinese)万清徽,谢圣凯,高大禹,等.两种堆积醅对芝麻香型白酒发酵特性和香气品质的影响[J].食品与发酵工业,2017,43(11):9-15
[15]Li RQ,Zhu HM,Ruan J,et al.De novo assembly of human genomes with massively parallel short read sequencing[J].Genome Research,2010,20(2):265-272
[16]Yang F,Zeng XW,Ning K,et al.Saliva microbiomes distinguish caries-active from healthy human populations[J].The ISME Journal,2012,6(1):1-10
[17]de Pasquale I,Di Cagno R,Buchin S,et al.Microbial ecology dynamics reveal a succession in the core microbiota involved in the ripening of pasta filata Caciocavallo Pugliese cheese[J].Applied and Environmental Microbiology,2014,80(19):6243-6255
[18]Michel E,Monfort C,Deffrasnes M,et al.Characterization of relative abundance of lactic acid bacteria species in French organic sourdough by cultural,qPCR and MiSeq high-throughput sequencing methods[J].International Journal of Food Microbiology,2016,239:35-43
[19]Wang XD,Zhang XL,Chen MQ.Study on the change trend of the bacteria and the physicochemical indexes in fermented grains in Maotai-flaver liquor pits[J].Liquor Making,2013,41(3):21-24(in Chinese)王晓丹,张小龙,陈孟强.酱香型白酒窖内发酵过程中酒醅理化指标与细菌的变化趋势研究[J].酿酒,2013,41(3):21-24
[20]Yang GH,Qiu SY,Huang YG.Microbiology research of liquor production[J].China Brewing,2011(4):24-27(in Chinese)杨国华,邱树毅,黄永光.酱香白酒生产中产香微生物研究[J].中国酿造,2011(4):24-27
[21]Li X.Screening of high yield ofα-amylase Bacillus licheniformis and construction of a recombinant plasmid[D].Wuhan:Master’s Thesis of Hubei University of Technology,2014(in Chinese)李习.高产α-淀粉酶地衣芽孢杆菌的筛选及重组质粒的构建[D].武汉:湖北工业大学硕士学位论文,2014
[22]Sun JQ,Liu WW,Zang W,et al.Community composition of moulds from fermented grains of Maotai-flavor liquor and their enzyme activities[J].Journal of Chinese Institute of Food Science and Technology,2013,13(8):239-247(in Chinese)孙剑秋,刘雯雯,臧威,等.酱香型白酒酒醅中霉菌群落组成与功能酶活性[J].中国食品学报,2013,13(8):239-247
[23]Yan QM,Shen XB,Song B,et al.Study on flavor compounds in different accumulation position of Sesame fermented grains[J].Liquor Making,2017,44(6):50-53(in Chinese)严启梅,沈晓波,宋宝,等.芝麻香型不同位置堆积酒醅风味成分的研究[J].酿酒,2017,44(6):50-53
[24]Shan QMG,Liang HZ,Zhang CX,et al.Changes of microbial diversity in stacked fermentation for the production of Moutai flavor liquor[J].Journal of Food Science and Biotechnology,2016,35(3):330-335(in Chinese)山其木格,梁慧珍,张长霞,等.糟醅堆积过程中微生物种群的变化规律[J].食品与生物技术学报,2016,35(3):330-335
[25]Li XD,Gao DY,Tian QZ,et al.Effects of Sesame-flavor liquor accumulation on cellar fermentation process and liquor quality[J].Food and Fermentation Industries,2018,44(5):63-69(in Chinese)李小东,高大禹,田庆贞,等.芝麻香型白酒堆积发酵对入窖发酵过程及原酒品质的影响[J].食品与发酵工业,2018,44(5):63-69