濃香型白酒窖泥微生物群落結(jié)構(gòu)分析及其對(duì)原酒風(fēng)味物質(zhì)的影響
發(fā)布時(shí)間：2018-08-04 21:04
【摘要】：濃香型白酒以固態(tài)窖泥發(fā)酵為特征,窖泥中微生物的生長(zhǎng)代謝活動(dòng)對(duì)白酒風(fēng)味物質(zhì)的形成具有重要影響。目前關(guān)于窖泥環(huán)境中復(fù)雜的微生物群落結(jié)構(gòu)對(duì)風(fēng)味物質(zhì)構(gòu)成影響的研究鮮有報(bào)道。本研究以河南省宋河酒業(yè)股份有限公司不同窖齡的窖底泥為研究對(duì)象,通過(guò)傳統(tǒng)的微生物分離培養(yǎng)技術(shù)獲得不同窖齡窖泥可培養(yǎng)的好氧和厭氧細(xì)菌;基于16S rRNA高通量測(cè)序技術(shù)研究在窖泥老熟過(guò)程中整體原核微生物的群落結(jié)構(gòu)變化規(guī)律;采用氣相色譜-質(zhì)譜聯(lián)用儀(GC-MS)分析不同窖齡窖池原酒及不同菌株發(fā)酵液的風(fēng)味物質(zhì)構(gòu)成,進(jìn)而在窖泥微生物與原酒風(fēng)味物質(zhì)之間建立聯(lián)系,為改善窖泥質(zhì)量、提高白酒品質(zhì)等提供了理論依據(jù)。主要研究結(jié)果如下:(1)采用傳統(tǒng)的微生物分離培養(yǎng)方法,分離純化了不同窖齡窖泥中的好氧和厭氧細(xì)菌,并對(duì)分離菌株采用16S rRNA基因序列分析進(jìn)行初步鑒定和系統(tǒng)發(fā)育分析。結(jié)果表明,分離得到的45株好氧細(xì)菌屬于9個(gè)不同的種,分別為米氏解硫胺素芽孢桿菌(Aneurinibacillus migulanus)、枯草芽孢桿菌(Bacillus subtilis)、菠茨坦短芽孢桿菌(Brevibacillus borstelensis)、地衣芽孢桿菌(Ba.licheniformis)、側(cè)孢短芽孢桿菌(Br.laterosporus)、短小芽孢桿菌(Ba.pumilus)、Paenibacullus lautus、耐寒短桿菌(Brevibacterium frigoritolerans)、P.cellulositrophicus,其中波茨坦短芽孢桿菌、米氏解硫胺素芽孢桿菌和P.lautus等鮮見(jiàn)研究報(bào)道。隨著窖齡的增長(zhǎng),窖底泥可培養(yǎng)好氧芽孢桿菌的數(shù)量逐漸減少。(2)采用RCM和ES兩種培養(yǎng)基對(duì)窖泥中的厭氧梭菌屬(Clostridium)進(jìn)行分離篩選并進(jìn)行16SrRNA序列分析。結(jié)果表明,分離得到的100株厭氧菌株中,除少數(shù)菌株屬于芽孢桿菌屬(Bacillus)以外,其余78株分別與GenBank中8種梭菌屬(Clostridium)細(xì)菌的序列同源性91%的分別為C.celerecrescens、C.carboxidivorans、C.cochlearium、C.sporogenes、C.sartagoforme、C.thermopalmarium、C.aurantibutyricum、C.butyricum。分布于10年窖池窖泥樣品中可培養(yǎng)梭菌種類(lèi)與6年和1年窖齡窖泥樣品有所不同,主要原因可能是窖泥的理化性質(zhì)隨著窖齡的增加呈現(xiàn)動(dòng)態(tài)變化,進(jìn)而促使其中微生物群落結(jié)構(gòu)發(fā)生相應(yīng)地改變。(3)通過(guò)高通量測(cè)序技術(shù),對(duì)宋河酒廠不同窖齡(1年、6年、10年)窖泥中的原核微生物群落結(jié)構(gòu)變化規(guī)律進(jìn)行研究。結(jié)果表明,隨著窖齡的增長(zhǎng),原核微生物的群落結(jié)構(gòu)發(fā)生了巨大的變化,尤其是不同窖齡中的主要微生物屬的變化尤其顯著。隨著窖齡的增長(zhǎng),厚壁菌門(mén)中的乳酸菌(Lactobacillus)和喜熱菌屬(Caloramator)的相對(duì)豐度呈減小趨勢(shì),與1年窖齡相比,10年窖齡窖泥中乳酸菌和喜熱菌屬的相對(duì)豐度分別減少了5.73倍和6.17倍。但是,厚壁菌門(mén)中的其它屬的相對(duì)豐度在1年窖齡窖泥中普遍偏低,如梭菌屬(Clostridium)、互營(yíng)單胞菌屬(Syntrophomonas)、Sedimentibacter、Unclassified Clostridia等。10年窖齡和1年窖齡相比,廣古菌門(mén)中的甲烷八疊球菌屬(Methanosarcina)、甲烷桿菌屬(Methanobacterium)和短桿菌屬(Methanobrevibacter)的相對(duì)豐度分別增加了5.31、3.26和3.99倍。隨著窖齡的增長(zhǎng),互養(yǎng)菌門(mén)門(mén)下的Aminobacterium和放線菌門(mén)門(mén)下Unclassfied_Coriobacteriaceae的相對(duì)豐度具有相同的增長(zhǎng)趨勢(shì)。從1年窖齡到6年窖齡,擬桿菌門(mén)門(mén)中的Unclassfied_Porphyromonadaceae、Blvii28和Unclassfied_BA008相對(duì)豐度呈增長(zhǎng)趨勢(shì),但在10年窖齡中又逐漸降低了。綠彎菌門(mén)下的T78從1年窖齡中的相對(duì)豐度為0.82%增長(zhǎng)到6年窖齡中的3.43%,到10年窖齡時(shí)又降到0.21%。其次,冗余分析(RDA)表明pH、NH4+、有效磷(AP)、有效鉀(AK)、和Ca2+等環(huán)境因子對(duì)原核微生物的群落結(jié)構(gòu)有顯著性的影響。(4)采用液液萃取和GC—MS對(duì)宋河酒廠1年、6年和10年窖齡原酒中的香味物質(zhì)進(jìn)行分析;采用頂空固相微萃取和GC—MS對(duì)菌株發(fā)酵液揮發(fā)性物質(zhì)進(jìn)行分析,并與原酒中的香味物質(zhì)進(jìn)行比對(duì)。結(jié)果表明,分離的好氧與厭氧菌株均可產(chǎn)生多種濃香型白酒的重要風(fēng)味物質(zhì)或其前體物質(zhì)。好氧菌株O2.4(Bacillus subtilis)、O2.10(Bacillus licheniformis)、O2.11(Brevibacillus laterosporus)、O2.15(Bacillus pumilus)、O2.17(Paenibacillus lautus)、O2.18(Bre.frigoritolerans)、O2.19(Paenibacillus cellulositrophicus)的菌株發(fā)酵液中含有較高比例的己酸乙酯。該物質(zhì)是濃香型白酒的主體香。厭氧菌株R50D1(C.celerecrescens)、R50D4(C.cochlearium)、R50zhong7(C.sporogenes)、R50zhong10(C.sartagoforme)和R70D1(C.aurantibutyricum)在RCM液體培養(yǎng)基中的代謝物主要為丁酸。作為白酒四大酸之一,丁酸在協(xié)調(diào)白酒風(fēng)味的同時(shí),還可以作為形成白酒酯類(lèi)物質(zhì)的前體,促使白酒特征香味物質(zhì)的合成。
[Abstract]:Luzhou flavor liquor is characterized by solid pit mud fermentation, and the growth and metabolism of microbes in the pit mud have an important influence on the formation of liquor flavoring substances. At present, there are few reports on the influence of complex microbial community structure on the composition of flavor substances in the pit mud environment. This study is based on the Henan Province Song River wine industry Limited by Share Ltd. The pit mud of the cellar age is the research object. Through the traditional microbiological isolation and culture technology, the aerobic and anaerobic bacteria can be obtained from the cellar mud in different cellar ages. Based on the 16S rRNA high throughput sequencing technology, the changes of the community structure of the whole prokaryotic microorganism in the process of the old pit mud are studied. The gas chromatography-mass spectrometry (GC-MS) analysis is not used. In order to improve the quality of pit mud and improve the quality of liquor, the relationship between the flavor substances of the cellar and the fermentation broth of the cellar age pits and the fermentation broth of different strains was established. The main research results were as follows: (1) the different cellar age cellars were separated and purified by the traditional microbiological separation and culture method. The aerobic and anaerobic bacteria in the mud were preliminarily identified by 16S rRNA gene sequence analysis and phylogenetic analysis. The results showed that 45 strains of aerobic bacteria isolated were 9 different species, which were Aneurinibacillus migulanus, Bacillus subtilis, and spinach, respectively. Bacillus subtilis (Brevibacillus borstelensis), Bacillus licheniformis (Ba.licheniformis), Bacillus spore bacillus (Br.laterosporus), Bacillus subtilis (Ba.pumilus), Paenibacullus lautus, Brevibacterium frigoritolerans (Brevibacterium frigoritolerans), P.cellulositrophicus, including Bacillus subtilis and Michaelis thiamine buds With the growth of cellar age, the number of Bacillus aerobic bacilli was gradually reduced with the growth of cellar age. (2) two kinds of medium of RCM and ES were used to isolate and select the anaerobic Clostridium (Clostridium) in pit mud and to analyze the 16SrRNA sequence. The results showed that 100 strains of anaerobic bacteria isolated from 100 strains were isolated. A few strains belong to the Bacillus genus (Bacillus), and the other 78 strains are respectively C.celerecrescens, C.carboxidivorans, C.cochlearium, C.sporogenes, C.sartagoforme, C.thermopalmarium, C.aurantibutyricum, and C.butyricum. distributed in the pit mud of 10 years, respectively, and 8 species of Clostridium (Clostridium) bacteria in GenBank respectively. The species of Clostridium cultures in the samples were different from those of the cellar mud samples of 6 and 1 years. The main reason was that the physical and chemical properties of the pit mud were dynamically changed with the age of the cellar, and then the microbial community structure was changed accordingly. (3) through high throughput sequencing technology, the pits of different cellar ages (1 years, 6 years, 10 years) of the Song River distilleries were made. The microbial community structure of the prokaryotic microorganism was studied. The results showed that the community structure of the prokaryotic microbes changed greatly with the growth of the cellar age, especially the change of the main microbes in the cellar age. With the growth of the cellar age, the Lactobacillus and Calo were found in the door of the pits. The relative abundance of ramator was reduced. Compared with the 1 years' age, the relative abundance of Lactobacillus and thermophilus in the pit mud of 10 years was reduced by 5.73 times and 6.17 times respectively. However, the relative abundance of other genera in the pits was generally low in the pit mud of 1 years, such as Clostridium, Syntrophomonas, Sed. Imentibacter, Unclassified Clostridia and other.10 years' cellar age compared with 1 years' cellar age, Methanococcus (Methanosarcina), Methanobacterium (Methanobacterium) and short bacilli (Methanobrevibacter) increased 5.31,3.26 and 3.99 times respectively. With the growth of cellar age, Aminobacter under the portal of intercultive bacteria The relative abundance of Unclassfied_Coriobacteriaceae under the portal of ium and actinomycetes has the same trend of growth. The relative abundance of Unclassfied_Porphyromonadaceae, Blvii28 and Unclassfied_BA008 in the bacilli gate increased from 1 years to 6 years old, but gradually decreased during the 10 years' age. The T78 under the gate of green Bens was from 1 years. The relative abundance of the cellar age increased from 0.82% to 3.43% in 6 years of age, and then to 0.21%. in 10 years. Redundancy analysis (RDA) showed that pH, NH4+, AP, AK, and Ca2+ had a significant effect on the community structure of the prokaryotic microbes. (4) liquid extraction and GC MS were used for 1 years, 6 years and 10 of the Song River distilleries. The aroma substances in the old wine were analyzed. The volatile substances in the fermentation broth were analyzed by headspace solid phase microextraction (SPME) and GC - MS, and compared with the aroma substances in the original wine. The results showed that the isolated aerobic and anaerobic strains could produce the important flavor substances or their precursors in many kinds of Luzhou flavor liquor. O2.4 (Bacillus subtilis), O2.10 (Bacillus licheniformis), O2.11 (Brevibacillus laterosporus), O2.15 (Bacillus pumilus) contain a high proportion of ethyl hexanoate in the fermentation broth. This substance is the main main of Luzhou flavor liquor. The metabolites of anaerobes R50D1 (C.celerecrescens), R50D4 (C.cochlearium), R50zhong7 (C.sporogenes), R50zhong10 (C.sartagoforme) and R70D1 (C.aurantibutyricum) in the RCM liquid medium are mainly butyric acid. As one of the four major acids in liquor, butyric acid can also be used to form liquor esters as well as liquor esters. The precursor that promotes the synthesis of characteristic flavors of liquor.
【學(xué)位授予單位】：鄭州輕工業(yè)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TS262.3
本文編號(hào)：2165133
[Abstract]:Luzhou flavor liquor is characterized by solid pit mud fermentation, and the growth and metabolism of microbes in the pit mud have an important influence on the formation of liquor flavoring substances. At present, there are few reports on the influence of complex microbial community structure on the composition of flavor substances in the pit mud environment. This study is based on the Henan Province Song River wine industry Limited by Share Ltd. The pit mud of the cellar age is the research object. Through the traditional microbiological isolation and culture technology, the aerobic and anaerobic bacteria can be obtained from the cellar mud in different cellar ages. Based on the 16S rRNA high throughput sequencing technology, the changes of the community structure of the whole prokaryotic microorganism in the process of the old pit mud are studied. The gas chromatography-mass spectrometry (GC-MS) analysis is not used. In order to improve the quality of pit mud and improve the quality of liquor, the relationship between the flavor substances of the cellar and the fermentation broth of the cellar age pits and the fermentation broth of different strains was established. The main research results were as follows: (1) the different cellar age cellars were separated and purified by the traditional microbiological separation and culture method. The aerobic and anaerobic bacteria in the mud were preliminarily identified by 16S rRNA gene sequence analysis and phylogenetic analysis. The results showed that 45 strains of aerobic bacteria isolated were 9 different species, which were Aneurinibacillus migulanus, Bacillus subtilis, and spinach, respectively. Bacillus subtilis (Brevibacillus borstelensis), Bacillus licheniformis (Ba.licheniformis), Bacillus spore bacillus (Br.laterosporus), Bacillus subtilis (Ba.pumilus), Paenibacullus lautus, Brevibacterium frigoritolerans (Brevibacterium frigoritolerans), P.cellulositrophicus, including Bacillus subtilis and Michaelis thiamine buds With the growth of cellar age, the number of Bacillus aerobic bacilli was gradually reduced with the growth of cellar age. (2) two kinds of medium of RCM and ES were used to isolate and select the anaerobic Clostridium (Clostridium) in pit mud and to analyze the 16SrRNA sequence. The results showed that 100 strains of anaerobic bacteria isolated from 100 strains were isolated. A few strains belong to the Bacillus genus (Bacillus), and the other 78 strains are respectively C.celerecrescens, C.carboxidivorans, C.cochlearium, C.sporogenes, C.sartagoforme, C.thermopalmarium, C.aurantibutyricum, and C.butyricum. distributed in the pit mud of 10 years, respectively, and 8 species of Clostridium (Clostridium) bacteria in GenBank respectively. The species of Clostridium cultures in the samples were different from those of the cellar mud samples of 6 and 1 years. The main reason was that the physical and chemical properties of the pit mud were dynamically changed with the age of the cellar, and then the microbial community structure was changed accordingly. (3) through high throughput sequencing technology, the pits of different cellar ages (1 years, 6 years, 10 years) of the Song River distilleries were made. The microbial community structure of the prokaryotic microorganism was studied. The results showed that the community structure of the prokaryotic microbes changed greatly with the growth of the cellar age, especially the change of the main microbes in the cellar age. With the growth of the cellar age, the Lactobacillus and Calo were found in the door of the pits. The relative abundance of ramator was reduced. Compared with the 1 years' age, the relative abundance of Lactobacillus and thermophilus in the pit mud of 10 years was reduced by 5.73 times and 6.17 times respectively. However, the relative abundance of other genera in the pits was generally low in the pit mud of 1 years, such as Clostridium, Syntrophomonas, Sed. Imentibacter, Unclassified Clostridia and other.10 years' cellar age compared with 1 years' cellar age, Methanococcus (Methanosarcina), Methanobacterium (Methanobacterium) and short bacilli (Methanobrevibacter) increased 5.31,3.26 and 3.99 times respectively. With the growth of cellar age, Aminobacter under the portal of intercultive bacteria The relative abundance of Unclassfied_Coriobacteriaceae under the portal of ium and actinomycetes has the same trend of growth. The relative abundance of Unclassfied_Porphyromonadaceae, Blvii28 and Unclassfied_BA008 in the bacilli gate increased from 1 years to 6 years old, but gradually decreased during the 10 years' age. The T78 under the gate of green Bens was from 1 years. The relative abundance of the cellar age increased from 0.82% to 3.43% in 6 years of age, and then to 0.21%. in 10 years. Redundancy analysis (RDA) showed that pH, NH4+, AP, AK, and Ca2+ had a significant effect on the community structure of the prokaryotic microbes. (4) liquid extraction and GC MS were used for 1 years, 6 years and 10 of the Song River distilleries. The aroma substances in the old wine were analyzed. The volatile substances in the fermentation broth were analyzed by headspace solid phase microextraction (SPME) and GC - MS, and compared with the aroma substances in the original wine. The results showed that the isolated aerobic and anaerobic strains could produce the important flavor substances or their precursors in many kinds of Luzhou flavor liquor. O2.4 (Bacillus subtilis), O2.10 (Bacillus licheniformis), O2.11 (Brevibacillus laterosporus), O2.15 (Bacillus pumilus) contain a high proportion of ethyl hexanoate in the fermentation broth. This substance is the main main of Luzhou flavor liquor. The metabolites of anaerobes R50D1 (C.celerecrescens), R50D4 (C.cochlearium), R50zhong7 (C.sporogenes), R50zhong10 (C.sartagoforme) and R70D1 (C.aurantibutyricum) in the RCM liquid medium are mainly butyric acid. As one of the four major acids in liquor, butyric acid can also be used to form liquor esters as well as liquor esters. The precursor that promotes the synthesis of characteristic flavors of liquor.
【學(xué)位授予單位】：鄭州輕工業(yè)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TS262.3
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，本文編號(hào)：2165133
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