浓香型白酒窖泥微生物群落结构分析及其对原酒风味物质的影响

发布时间：2018-08-04 21:04

【摘要】：浓香型白酒以固态窖泥发酵为特征,窖泥中微生物的生长代谢活动对白酒风味物质的形成具有重要影响。目前关于窖泥环境中复杂的微生物群落结构对风味物质构成影响的研究鲜有报道。本研究以河南省宋河酒业股份有限公司不同窖龄的窖底泥为研究对象,通过传统的微生物分离培养技术获得不同窖龄窖泥可培养的好氧和厌氧细菌;基于16S rRNA高通量测序技术研究在窖泥老熟过程中整体原核微生物的群落结构变化规律;采用气相色谱-质谱联用仪(GC-MS)分析不同窖龄窖池原酒及不同菌株发酵液的风味物质构成,进而在窖泥微生物与原酒风味物质之间建立联系,为改善窖泥质量、提高白酒品质等提供了理论依据。主要研究结果如下:(1)采用传统的微生物分离培养方法,分离纯化了不同窖龄窖泥中的好氧和厌氧细菌,并对分离菌株采用16S rRNA基因序列分析进行初步鉴定和系统发育分析。结果表明,分离得到的45株好氧细菌属于9个不同的种,分别为米氏解硫胺素芽孢杆菌(Aneurinibacillus migulanus)、枯草芽孢杆菌(Bacillus subtilis)、菠茨坦短芽孢杆菌(Brevibacillus borstelensis)、地衣芽孢杆菌(Ba.licheniformis)、侧孢短芽孢杆菌(Br.laterosporus)、短小芽孢杆菌(Ba.pumilus)、Paenibacullus lautus、耐寒短杆菌(Brevibacterium frigoritolerans)、P.cellulositrophicus,其中波茨坦短芽孢杆菌、米氏解硫胺素芽孢杆菌和P.lautus等鲜见研究报道。随着窖龄的增长,窖底泥可培养好氧芽孢杆菌的数量逐渐减少。(2)采用RCM和ES两种培养基对窖泥中的厌氧梭菌属(Clostridium)进行分离筛选并进行16SrRNA序列分析。结果表明,分离得到的100株厌氧菌株中,除少数菌株属于芽孢杆菌属(Bacillus)以外,其余78株分别与GenBank中8种梭菌属(Clostridium)细菌的序列同源性91%的分别为C.celerecrescens、C.carboxidivorans、C.cochlearium、C.sporogenes、C.sartagoforme、C.thermopalmarium、C.aurantibutyricum、C.butyricum。分布于10年窖池窖泥样品中可培养梭菌种类与6年和1年窖龄窖泥样品有所不同,主要原因可能是窖泥的理化性质随着窖龄的增加呈现动态变化,进而促使其中微生物群落结构发生相应地改变。(3)通过高通量测序技术,对宋河酒厂不同窖龄(1年、6年、10年)窖泥中的原核微生物群落结构变化规律进行研究。结果表明,随着窖龄的增长,原核微生物的群落结构发生了巨大的变化,尤其是不同窖龄中的主要微生物属的变化尤其显著。随着窖龄的增长,厚壁菌门中的乳酸菌(Lactobacillus)和喜热菌属(Caloramator)的相对丰度呈减小趋势,与1年窖龄相比,10年窖龄窖泥中乳酸菌和喜热菌属的相对丰度分别减少了5.73倍和6.17倍。但是,厚壁菌门中的其它属的相对丰度在1年窖龄窖泥中普遍偏低,如梭菌属(Clostridium)、互营单胞菌属(Syntrophomonas)、Sedimentibacter、Unclassified Clostridia等。10年窖龄和1年窖龄相比,广古菌门中的甲烷八叠球菌属(Methanosarcina)、甲烷杆菌属(Methanobacterium)和短杆菌属(Methanobrevibacter)的相对丰度分别增加了5.31、3.26和3.99倍。随着窖龄的增长,互养菌门门下的Aminobacterium和放线菌门门下Unclassfied_Coriobacteriaceae的相对丰度具有相同的增长趋势。从1年窖龄到6年窖龄,拟杆菌门门中的Unclassfied_Porphyromonadaceae、Blvii28和Unclassfied_BA008相对丰度呈增长趋势,但在10年窖龄中又逐渐降低了。绿弯菌门下的T78从1年窖龄中的相对丰度为0.82%增长到6年窖龄中的3.43%,到10年窖龄时又降到0.21%。其次,冗余分析(RDA)表明pH、NH4+、有效磷(AP)、有效钾(AK)、和Ca2+等环境因子对原核微生物的群落结构有显著性的影响。(4)采用液液萃取和GC—MS对宋河酒厂1年、6年和10年窖龄原酒中的香味物质进行分析;采用顶空固相微萃取和GC—MS对菌株发酵液挥发性物质进行分析,并与原酒中的香味物质进行比对。结果表明,分离的好氧与厌氧菌株均可产生多种浓香型白酒的重要风味物质或其前体物质。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)的菌株发酵液中含有较高比例的己酸乙酯。该物质是浓香型白酒的主体香。厌氧菌株R50D1(C.celerecrescens)、R50D4(C.cochlearium)、R50zhong7(C.sporogenes)、R50zhong10(C.sartagoforme)和R70D1(C.aurantibutyricum)在RCM液体培养基中的代谢物主要为丁酸。作为白酒四大酸之一,丁酸在协调白酒风味的同时,还可以作为形成白酒酯类物质的前体,促使白酒特征香味物质的合成。
[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.
【学位授予单位】：郑州轻工业学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS262.3

【参考文献】