【作者】 王鹏；

【导师】 吴群；

【作者基本信息】 江南大学 ， 发酵工程， 2017， 硕士

【摘要】 中国浓香型白酒是典型的多菌种固态发酵食品,其发酵过程中的核心微生物决定了白酒发酵的效率和品质,但是如何寻找多菌种发酵系统中的核心微生物群,以及核心微生物的利用在白酒发酵领域尚不完善。因此揭示发酵过程中的核心微生物群及其在白酒生产中的应用是实现白酒生产定向调控和品质提升的关键。本研究通过高通量测序和多元统计学分析判定了浓香型白酒酿造过程中的核心微生物群。通过不同酿造大曲的对比,对某大曲进行了缺失菌株的强化补充,并应用到酒醅发酵过程中。从微生物组和代谢组的角度,结合生物信息学方法,系统地解析了大曲和窖池内酒醅发酵过程中微生物和代谢物的动态变化。为实现白酒生产技术的升级奠定了理论基础。本论文的主要研究结果如下:（1）利用Illumina Miseq高通量测序分析微生物群落结构,并结合挥发性化合物检测揭示出白酒发酵中的风味关联的微生物为Lactobacillus、Saccharomyces和Candida,这3种菌属主要贡献乳酸、乙酸、乙醇、多种酯类和高级醇类的产生。结合相对丰度>1.0%的优势微生物和相互作用关系较强的共现性微生物,揭示出核心微生物主要包括10个属:Lactobacillus、Saccharomyces、Candida、Rhizopus、Saccharomycopsis、Pichia、Dipodascus、Bacillus、Thermoascus和Lactococcus。Bacillus与其它的微生物属共有24条关联,其在网络中的共现率（incidence of co-occurrence）为4.49%,仅次于Lactobacillus（4.68%）,说明Bacillus在白酒酿造微生物群体中的重要网络地位。不同大曲的比较分析表明其中一种大曲（大曲3）的芽孢杆菌数量较其它两种大曲少约50%。由于Bacillus在白酒酿造过程中具有重要的产酶、产风味的能力和丰度优势性,而地衣芽孢杆菌（Bacillis licheniformis）是芽孢杆菌中产风味最为丰富的菌株,因此选择B.licheniformis进行大曲3的强化补充。（2）B.licheniformis强化后,酸性蛋白酶活力无显著性差异;液化力从1.3 g淀粉·g^-1·h^-1显著提高到1.7 g淀粉·g^-1·h^-1;糖化力从627.6 mg葡萄糖·g^-1·h^-1降低至445.6 mg葡萄糖·g^-1·h^-1;酯化力从28.1 mg己酸乙酯·g^-1·100 h^-1显著降低至17.2 mg己酸乙酯·g^-1·100h^-1。芳香族和吡嗪类化合物含量分别从0.37 mg·kg^-1和0.35 mg·kg^-1显著增加到0.90mg·kg^-1和5.71 mg·kg^-1。分析微生物群落结构的变化发现,大曲中的真菌群落相较于细菌群落对强化更为敏感。大曲发酵前期,显著减少的是Weissella;在发酵后期Acetobacter、Acinetobacter、Brevibacterium、Corynebacterium、Lactobacillus和Staphylococcus显著下降,Bacillus的相对丰度显著增加,说明菌株的添加在强化大曲中具有良好的生长优势。11个真菌属在强化后有显著的变化,其中显著增加的有Aspergillus、Candida、Clavispora、Cyberlindnera、Dipodascus、Wickerhamomyces和Eurotium;显著下降的有Pichia、Rhizopus、Saccharomycopsis和Thermoascus。为探究B.licheniformis在大曲中的强化效果是否能在窖池中持续,因此将其应用到窖池发酵中并跟踪监测。（3）通过研究B.licheniformis与12株不同菌之间的相互作用发现,B.licheniformis能够抑制Staphylococcus lentus,Brevibacterium flavum,Saccharomycopsis fibuligera和Pichia kudriavzevii的生长,而促进Candida parapsilosis,Clavispora lusitaniae和Aspergillus oryzae。这基本解释了大曲中微生物变化的现象。B.licheniformis和Cl.lusitaniae均可以产生吡嗪,而共培养中的吡嗪含量高于每种单培养物中吡嗪的总和。且共培养中的芳香族化合物含量要高于单培养。B.licheniformis与其它微生物之间互作会改变各自的代谢活力,从而改变了微生物之间的代谢轮廓并验证了强化大曲中微生物和风味发生的改变。（4）强化大曲应用到窖池发酵中,显著降低了细菌和真菌群落的Chao1丰度和OTU数并改变了某些分类单元的组成,包括Lactobacillus、Candida、Saccharomycopsis的减少和Bacillus、Lactococcus、Aspergillus的增加。共有10种风味化合物在强化前后具有显著差异,其中辛酸乙酯和己酸异戊酯显著降低,壬酸、乙酸、吡嗪类和某些芳香族化合物含量显著升高,说明强化大曲在窖池发酵中也起到了作用。白酒中的挥发性化合物共检测到36种。单因素方差分析得到显著差异的化合物有6种,增加的有辛酸异戊酯（水果香）、辛酸（奶酪味）、乙酸己酯（水果香、花香）、1-己醇（水果芬芳香）和四甲基吡嗪（烘焙香、坚果）,减少的为辛酸乙酯（水果香）。感官品评表明,强化后酒的香气强度、酸感和焦糊感明显增加,并且空杯留香的得分有所增加。更多还原

【Abstract】 Chinese strong flavored liquor is characterized by multi-species solid-state fermentation.The core microbiota determines the efficiency and quality of fermented liquors.However,little is known about how to dig out the core microbiota in the multi-species fermentation system.It is still incomplete for the application of core microbes in the food fermentation.Therefore,it has become increasingly urgent to reveal out the core microbiota and apply it into liquor fermentation,which will be helpful for the directional control and quality improvement of Chinese liquor.This study firstly identified the core microbiota in Chinese liquor fermentation using amplicon sequencing and multivariate statistical analysis.By comparision of different groups of Daqu starters,we inoculated a core microbe into one Daqu fermentation,and systematically traced the whole process of liquor fermentation from the microbial and metabolic point of view.It is expected to be useful for constructing synthetic microbial communities and the further fortification of liquor production.The main results of this work are as follows:（1）The microbial community structure was characterized by Illumina Miseq sequencing.We revealed flavor-associated microbiota by the microbe-metabolite correlation approach,and identified co-occurring microbiota by co-occurrence network analysis.The flavor-associated microbiota consisted of Lactobacillus,Saccharomyces and Candida,which were associated with lactic acid,acetic acid,ethanol,a variety of volatile esters and alcohols.The core microbiota during the liquor fermentation process consisted of Lactobacillus,Saccharomyces,Candida,Rhizopus,Saccharomycopsis,Pichia,Dipodascus,Bacillus,Thermoascus and Lactococcus.In particular,Bacillus had 24 associations with other microbial genera,with a 4.49% incidence of co-occurrence in the network,second only to Lactobacillus（4.68%）,suggesting the importance of Bacillus in the microbial network.Comparison results suggested the amount of Bacillus in one Daqu（Daqu 3）was extremely lower（⁵0%）than the other two Daqu.Bacillus not only dominats the fermentation process,but also has good ability of enzyme and flavor production.While Bacillus licheniformis was the most abundant flavor-producing species among Bacillus strains.Consequently,we inoculated B.licheniformis into Daqu 3 for fortification.（2）Inoculated Daqu exihibited significantly higher amylase activity(1.7 g starch·g^-1·h^-1)than uninoculated Daqu(1.3 g starch·g^-1·h^-1).The glucoamylase activity decreased significantly from 627.6 mg glucose·g^-1·h^-1 to 445.6 mg glucose·g^-1·h^-1 after inoculation.However,no significant difference was observed for the acid protease activity.Inoculaition significantly decreased the esterase activity from 28.1 mg ethyl caproate·g^-1·100 h^-1 to 17.2 mg ethyl caproate·g^-1·100 h^-1.The aromatic compounds and pyrazines were enriched from 0.37 mg·kg^-1 and 0.35 mg·kg^-1 to 0.90 mg·kg^-1and 5.71 mg·kg^-1,respectively.The inoculation of B.licheniformis made the fungal community more sensitive than bacterial community.In the early stage,the inoculation affected only one genus（Weissella;decreased）,but influenced seven genera in the later stage.Among these genera,the growth of Bacillus was significantly enhanced after day 20.The other affected genera（Acetobacter,Acinetobacter,Brevibacterium,Corynebacterium,Lactobacillus and Staphylococcus）indicated their negative interaction with B.licheniformis.Nine of the fungal genera responded to inoculation in both stages,including the significant change in Aspergillus,Candida,Clavispora,Cyberlindnera,Dipodascus,Wickerhamomyces and Eurotium,indicating different interactions occurred when B.licheniformis was involved in Daqu fermentation.（3）The interaction between B.licheniformis and 12 strains（distributed in most of the responsive genera in Daqu）suggested that the inoculated strain inhibited the growth of Staphylococcus lentus,Brevibacterium flavum,Saccharomycopsis fibuligera and Pichia kudriavzeviii,while promoted the Candida parapsilosis,Clavispora lusitaniae and Aspergillus oryzae.This explained the variation of microbial community in Daqu.Interestingly,when B.licheniformis and Cl.lusitaniae were cocultured,the pyrazine content in cocultures was higher than the sum of pyrazines in each pure culture.Similar result was observed for aromatic compounds.Therefore,we concluded that the microbial members shifted their microbial activity and then metabolic status when interacting with B.licheniformis,explaining the variations in microbial communities and flavor components in Daqu fermentation.（4）The application of inoculated Daqu significantly reduced Chao1 richness,OTU number and taxa composition,including the decrease in Lactobacillus,Candida and Saccharomycopsis,and the increase in Bacillus,Lactococcus and Aspergillus.There were 10 significantly different compounds between the two pits,including the decrease in octanoate and isoamyl hexanoate,and the increase in nonanoic acid,acetic acid,pyrazines and some aromatic compounds.This indicates that inoculated Daqu exerted its effectiveness in pits.We identified 36 volatile compounds in final distilled liquor.There were 6 compounds with significant differences obtained by single-factor analysis of variance,including the increased isoamyl octanoate,ethyl octanoate,hexyl acetate,1-hexanol and tetramethylpyrazine,mostly with fruit fragrance,floral and baked fragrance.Whereas octanoic acid decreased.Sensory evaluation indicated that the inoculated liquor had a higher score of fragrant strength,acidity and coke paste,especially the empty cup fragrance score had a significant increase.更多还原