古井貢酒微生物群落結構及其與主要風味物質的關聯(lián)研究
發(fā)布時間：2018-07-15 23:06
【摘要】：古井貢酒,中國八大名酒之一,其特有的釀酒微生物群落生產出具有獨特風味的濃香型白酒。解析古井貢酒特色發(fā)酵菌群結構有助于了解其發(fā)酵菌群組成;比較優(yōu)質發(fā)酵菌群和普通發(fā)酵菌群的差異有助于確定釀酒質量差異的關鍵菌屬/菌種;優(yōu)質酒樣和普通酒樣風味物質的差異,優(yōu)質窖泥與普通窖泥中代謝組分的差異,有助于從發(fā)酵過程的角度解釋優(yōu)質發(fā)酵菌群與普通發(fā)酵菌群出現差異的原因。本研究為改善普通發(fā)酵菌群提高釀酒質量提供理論依據,具有十分重要的現實意義。通過全長16S rDNA/ITS TA克隆測序、16S rDNA V4區(qū)高通量測序和宏基因組de novo高通量測序等方法研究了古井貢酒大曲、窖泥和酒醅中的微生物群落結構組成,發(fā)現:(1)大曲微生物群落中具有五種相對特有的物種Pantoea agglomerans,Pantoea vagans,Talaromyces thermophiles,Thermomyces lanuginosus,和Thermoascus aurantiacus;(2)窖泥微生物群落組成復雜,其中含有大量未培養(yǎng)菌/未知菌;(3)發(fā)酵結束的酒醅微生物群落中乳酸桿菌占壟斷地位,含量最多的菌種是Lactobacillus acetotolerans。通過主成分分析,差異顯著性分析和相關性分析深入比較古井優(yōu)質發(fā)酵菌群與普通發(fā)酵菌群的差異,得到如下結論:(1)優(yōu)質窖泥菌群與普通窖泥菌群結構在門和屬的層面上均有顯著差異,差異最顯著的是Lactobacillus,普通窖泥中含量偏高約34%;(2)另外,優(yōu)質窖泥和普通窖泥中六個屬的含量差異也很明顯:普通窖泥中Klebsiella屬的含量相對較高;而優(yōu)質窖泥中Clostridium,Syntrophomonas,Methanoculleus,Aminobacterium和Sedimentibacter的含量相對較高;(3)發(fā)酵后優(yōu)質酒醅微生物群落和普通酒醅微生物群落組成之間幾乎沒有差異,但其內部OTU之間的關系有所差異。根據微生物群落中7個物種(Lactobacillus plantarum,Bacillus subtilis,Lactobacillus fermentum,Bacillus licheniformis,Staphylococcus saprophyticus,Enterobacter asburiae,和Pseudomonas putida)的全基因組序列成功設計了7對物種特異性引物,并驗證了引物的特異性;并通過絕對定量qPCR方法測定了7個物種在幾個釀酒菌群中的組成。將來隨著具有全基因組序列的物種越來越多,有望使用該思路實現對釀酒微生物群落隨時快速監(jiān)測,對釀酒質量起到預警作用。采用氣相色譜法解析了古井貢酒中典型的56種風味物質在兩個發(fā)酵排次三個廠區(qū)的127個酒樣中的含量,通過相關性分析得到了與口感最相關的風味物質、與窖齡最相關的風味物質,并通過CCA(Canomical Correlation Analysis)分析建立了風味物質與微生物群落組成之間關系;請國家級品酒師對酒樣質量根據國家標準進行打分,進而建立了酒中風味物質與其口感打分之間的Lasso(the Least Absolute Shrinkage and Selection Operator)線性回歸模型,發(fā)現對兩個排次的酒樣數據建立的兩個Lasso回歸模型中保留的風味物質不同。通過液相色譜-質譜聯(lián)用方法解析了不同廠區(qū)/質量的窖泥中3193種代謝組分,其中30%含量的代謝組分確定了其具體化學名稱,其含量最多的是酸類。比較發(fā)現,A、B廠區(qū)優(yōu)質窖泥中的代謝組分更相似,C廠區(qū)普通窖泥中的代謝組分明顯不同于AB廠區(qū),其中很多代謝組分的含量明顯多于A、B廠區(qū)的優(yōu)質窖泥。優(yōu)質窖泥和普通窖泥中代謝組分和酒樣中風味物質組成的差異都預示著其微生物群落的代謝關系不同。優(yōu)質窖池與普通窖池中菌群代謝功能差異有待于進一步深入研究。
[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.
【學位授予單位】：哈爾濱工業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TS261.1
，
本文編號：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.
【學位授予單位】：哈爾濱工業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TS261.1
，
本文編號：2125667
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