古井贡酒微生物群落结构及其与主要风味物质的关联研究
发布时间：2018-07-15 23:06
【摘要】：古井贡酒,中国八大名酒之一,其特有的酿酒微生物群落生产出具有独特风味的浓香型白酒。解析古井贡酒特色发酵菌群结构有助于了解其发酵菌群组成;比较优质发酵菌群和普通发酵菌群的差异有助于确定酿酒质量差异的关键菌属/菌种;优质酒样和普通酒样风味物质的差异,优质窖泥与普通窖泥中代谢组分的差异,有助于从发酵过程的角度解释优质发酵菌群与普通发酵菌群出现差异的原因。本研究为改善普通发酵菌群提高酿酒质量提供理论依据,具有十分重要的现实意义。通过全长16S rDNA/ITS TA克隆测序、16S rDNA V4区高通量测序和宏基因组de novo高通量测序等方法研究了古井贡酒大曲、窖泥和酒醅中的微生物群落结构组成,发现:(1)大曲微生物群落中具有五种相对特有的物种Pantoea agglomerans,Pantoea vagans,Talaromyces thermophiles,Thermomyces lanuginosus,和Thermoascus aurantiacus;(2)窖泥微生物群落组成复杂,其中含有大量未培养菌/未知菌;(3)发酵结束的酒醅微生物群落中乳酸杆菌占垄断地位,含量最多的菌种是Lactobacillus acetotolerans。通过主成分分析,差异显著性分析和相关性分析深入比较古井优质发酵菌群与普通发酵菌群的差异,得到如下结论:(1)优质窖泥菌群与普通窖泥菌群结构在门和属的层面上均有显著差异,差异最显著的是Lactobacillus,普通窖泥中含量偏高约34%;(2)另外,优质窖泥和普通窖泥中六个属的含量差异也很明显:普通窖泥中Klebsiella属的含量相对较高;而优质窖泥中Clostridium,Syntrophomonas,Methanoculleus,Aminobacterium和Sedimentibacter的含量相对较高;(3)发酵后优质酒醅微生物群落和普通酒醅微生物群落组成之间几乎没有差异,但其内部OTU之间的关系有所差异。根据微生物群落中7个物种(Lactobacillus plantarum,Bacillus subtilis,Lactobacillus fermentum,Bacillus licheniformis,Staphylococcus saprophyticus,Enterobacter asburiae,和Pseudomonas putida)的全基因组序列成功设计了7对物种特异性引物,并验证了引物的特异性;并通过绝对定量qPCR方法测定了7个物种在几个酿酒菌群中的组成。将来随着具有全基因组序列的物种越来越多,有望使用该思路实现对酿酒微生物群落随时快速监测,对酿酒质量起到预警作用。采用气相色谱法解析了古井贡酒中典型的56种风味物质在两个发酵排次三个厂区的127个酒样中的含量,通过相关性分析得到了与口感最相关的风味物质、与窖龄最相关的风味物质,并通过CCA(Canomical Correlation Analysis)分析建立了风味物质与微生物群落组成之间关系;请国家级品酒师对酒样质量根据国家标准进行打分,进而建立了酒中风味物质与其口感打分之间的Lasso(the Least Absolute Shrinkage and Selection Operator)线性回归模型,发现对两个排次的酒样数据建立的两个Lasso回归模型中保留的风味物质不同。通过液相色谱-质谱联用方法解析了不同厂区/质量的窖泥中3193种代谢组分,其中30%含量的代谢组分确定了其具体化学名称,其含量最多的是酸类。比较发现,A、B厂区优质窖泥中的代谢组分更相似,C厂区普通窖泥中的代谢组分明显不同于AB厂区,其中很多代谢组分的含量明显多于A、B厂区的优质窖泥。优质窖泥和普通窖泥中代谢组分和酒样中风味物质组成的差异都预示着其微生物群落的代谢关系不同。优质窖池与普通窖池中菌群代谢功能差异有待于进一步深入研究。
[Abstract]:Gujing tribute wine, one of the eight famous liquor in China, produces a unique flavor liquor of Luzhou flavor. It is helpful to understand the composition of the fermented bacteria group in the characteristic fermenting flora of the ancient well, and the difference between the high quality fermenting bacteria group and the common fermenting bacteria group is helpful to determine the key bacteria of the difference in the quality of the wine. The difference between high quality wine samples and ordinary wine flavor substances, the difference in the metabolic components of high quality pit mud and ordinary pit mud, is helpful to explain the difference between high quality fermenting bacteria group and common fermenting bacteria group from the angle of fermentation process. This study provides a theoretical basis for improving the quality of common fermenting bacteria to raise the quality of wine making, which is very important. By means of full length 16S rDNA/ITS TA clone sequencing, high throughput sequencing of 16S rDNA V4 region and high throughput sequencing of macrogenome de novo, the microbial community structure in the Gujing tribute wine Daqu, the pit mud and the fermented grains were studied. (1) there were five species of relatively unique species Pantoea agglomerans, Pant in the Daqu microbial community. OEA vagans, Talaromyces thermophiles, Thermomyces lanuginosus, and Thermoascus aurantiacus; (2) the microbial community composition of the pit mud is complex, which contains a large number of uncultured and unknown bacteria. (3) Lactobacillus in the fermented fermented fermented grains is monopolized by lactic acid bacilli, and the most content of the bacteria is Lactobacillus acetotolerans. through the main formation. Analysis, difference significance analysis and correlation analysis, the difference between high quality fermenting bacteria group and common fermenting bacteria group in ancient wells was compared. The following conclusions were obtained: (1) there were significant differences between high quality pit mud bacteria group and common pit mud flora structure on the level of gate and genus, the most significant difference was Lactobacillus, the content of ordinary pit mud was about 34%; (2) In addition, the content of six genera of six genera in high quality pit mud and ordinary pit mud is also obvious: the content of Klebsiella in ordinary pit mud is relatively high, while the content of Clostridium, Syntrophomonas, Methanoculleus, Aminobacterium and Sedimentibacter in high quality pit mud is relatively high; (3) the microbiological community of high quality fermented grains and the ordinary fermented grains after fermentation. There are almost no differences between the composition of the community, but the relationship between the internal OTU is different. According to the whole genome sequence of 7 species (Lactobacillus plantarum, Bacillus subtilis, Lactobacillus fermentum, Bacillus licheniformis, Staphylococcus saprophyticus, Enterobacter) 7 pairs of species specific primers were successfully designed and the specificity of primers was verified. The composition of 7 species in several species of Saccharomyces was determined by the absolute quantitative qPCR method. In the future, with more and more species with full genome sequence, it is hopeful to use this idea to monitor the microbial community at any time and to make wine quality. Gas chromatography was used to analyze the content of 56 typical flavoring substances in the wine of the ancient well and 127 liquor samples in three plant areas by two fermentation. Through correlation analysis, the most related flavor substances, the most related flavor substances with the age of the cellar age were obtained, and CCA (Canomical Correlation Analysis) was used. The relationship between the flavor substance and the composition of the microbial community was established, and the wine sample quality was graded according to the national standard, and the Lasso (the Least Absolute Shrinkage and Selection Operator) linear regression model was established between the flavor substance and the taste score of the wine, and the number of two rows of wine samples was found. According to the two Lasso regression models, 3193 kinds of metabolic components in the pit mud of different plant areas / quality were analyzed by liquid chromatography mass spectrometry. The metabolic components of 30% of them determined the specific chemical names, the most content was acid. The metabolism of high quality pit mud in the A and B plants was compared. The components of the C plant were more similar. The metabolic components in the ordinary pit mud were obviously different from the AB plant, and many of the metabolic components were more than that of the A. The quality pit mud in the B plant, the metabolic components in the high quality pit mud and the ordinary pit mud and the difference in the flavor composition in the wine samples indicate that the metabolic relationship of the microorganism community is different. The difference of metabolic function of bacteria in pit can be further studied.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS261.1
，
本文编号：2125667
[Abstract]:Gujing tribute wine, one of the eight famous liquor in China, produces a unique flavor liquor of Luzhou flavor. It is helpful to understand the composition of the fermented bacteria group in the characteristic fermenting flora of the ancient well, and the difference between the high quality fermenting bacteria group and the common fermenting bacteria group is helpful to determine the key bacteria of the difference in the quality of the wine. The difference between high quality wine samples and ordinary wine flavor substances, the difference in the metabolic components of high quality pit mud and ordinary pit mud, is helpful to explain the difference between high quality fermenting bacteria group and common fermenting bacteria group from the angle of fermentation process. This study provides a theoretical basis for improving the quality of common fermenting bacteria to raise the quality of wine making, which is very important. By means of full length 16S rDNA/ITS TA clone sequencing, high throughput sequencing of 16S rDNA V4 region and high throughput sequencing of macrogenome de novo, the microbial community structure in the Gujing tribute wine Daqu, the pit mud and the fermented grains were studied. (1) there were five species of relatively unique species Pantoea agglomerans, Pant in the Daqu microbial community. OEA vagans, Talaromyces thermophiles, Thermomyces lanuginosus, and Thermoascus aurantiacus; (2) the microbial community composition of the pit mud is complex, which contains a large number of uncultured and unknown bacteria. (3) Lactobacillus in the fermented fermented fermented grains is monopolized by lactic acid bacilli, and the most content of the bacteria is Lactobacillus acetotolerans. through the main formation. Analysis, difference significance analysis and correlation analysis, the difference between high quality fermenting bacteria group and common fermenting bacteria group in ancient wells was compared. The following conclusions were obtained: (1) there were significant differences between high quality pit mud bacteria group and common pit mud flora structure on the level of gate and genus, the most significant difference was Lactobacillus, the content of ordinary pit mud was about 34%; (2) In addition, the content of six genera of six genera in high quality pit mud and ordinary pit mud is also obvious: the content of Klebsiella in ordinary pit mud is relatively high, while the content of Clostridium, Syntrophomonas, Methanoculleus, Aminobacterium and Sedimentibacter in high quality pit mud is relatively high; (3) the microbiological community of high quality fermented grains and the ordinary fermented grains after fermentation. There are almost no differences between the composition of the community, but the relationship between the internal OTU is different. According to the whole genome sequence of 7 species (Lactobacillus plantarum, Bacillus subtilis, Lactobacillus fermentum, Bacillus licheniformis, Staphylococcus saprophyticus, Enterobacter) 7 pairs of species specific primers were successfully designed and the specificity of primers was verified. The composition of 7 species in several species of Saccharomyces was determined by the absolute quantitative qPCR method. In the future, with more and more species with full genome sequence, it is hopeful to use this idea to monitor the microbial community at any time and to make wine quality. Gas chromatography was used to analyze the content of 56 typical flavoring substances in the wine of the ancient well and 127 liquor samples in three plant areas by two fermentation. Through correlation analysis, the most related flavor substances, the most related flavor substances with the age of the cellar age were obtained, and CCA (Canomical Correlation Analysis) was used. The relationship between the flavor substance and the composition of the microbial community was established, and the wine sample quality was graded according to the national standard, and the Lasso (the Least Absolute Shrinkage and Selection Operator) linear regression model was established between the flavor substance and the taste score of the wine, and the number of two rows of wine samples was found. According to the two Lasso regression models, 3193 kinds of metabolic components in the pit mud of different plant areas / quality were analyzed by liquid chromatography mass spectrometry. The metabolic components of 30% of them determined the specific chemical names, the most content was acid. The metabolism of high quality pit mud in the A and B plants was compared. The components of the C plant were more similar. The metabolic components in the ordinary pit mud were obviously different from the AB plant, and many of the metabolic components were more than that of the A. The quality pit mud in the B plant, the metabolic components in the high quality pit mud and the ordinary pit mud and the difference in the flavor composition in the wine samples indicate that the metabolic relationship of the microorganism community is different. The difference of metabolic function of bacteria in pit can be further studied.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS261.1
，
本文编号：2125667
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