【作者】 李敏；

【导师】 范晓军；

【作者基本信息】 太原理工大学 ， 化学工程与技术， 2019， 硕士

【摘要】 近年来夏季高温气候条件下我国食醋酿造企业在生产过程中时常出现过程控制难的问题,且经常伴随着成品醋的返浑、发粘、胀气等异常现象,严重地影响了食醋的营养成分和品质。食醋的品质与发酵过程中的微生物代谢密切相关,为了深入了解食醋高温稳态酿造机制,本课题利用分子生态学技术对夏冬两季食醋酿造过程中的微生物群落结构及其演替特征做了深入研究,并对腐败醋样中的特征微生物的生长与代谢特征进行了初步研究,得到以下研究结果:1、经Illumina高通量测序分析,夏冬两季的细菌菌群结构具有显著的统计学差异（p<0.05）。整体上,夏季细菌菌群的丰富度在醋酸发酵阶段更高,而冬季细菌菌群的丰富度在酒精阶段更高,且冬夏两季醋酸发酵期内各阶段的菌群构成具有较高的相似性。冬季细菌菌群的丰富度（除酒精发酵第6天）及多样性（除酒精发酵第3天及第6天）大于夏季。夏季细菌菌群来源于8个门及51个属,有12个菌属（相对丰度>0.1%）首次在山西老陈醋发酵过程中检测到;冬季细菌菌群共归属于22个门及226个属,有6个属（相对丰度>0.1%）是首次发现。夏冬季的优势菌门均为厚壁菌门,优势菌属为乳酸菌属,季节变化对低丰度菌属的影响远大于优势菌属。2、经Illumina高通量测序分析,夏季醋酸发酵期的真菌菌群丰富度大于酒精发酵期,且各发酵阶段的真菌菌群结构相似性较低。冬季醋酸发酵期的真菌菌群丰富度小于酒精发酵期,且醋酸发酵期内各阶段的真菌菌群构成具有较高的相似性。冬季酒精发酵期的菌群丰富度及多样性大于夏季,而醋酸发酵期的小于夏季。夏季真菌菌群归属于3个门及35个属,有7个属（相对丰度>0.1%）首次发现;冬季菌群归属于4个门及46个菌属,有15个属（相对丰度>0.1%）首次发现。夏冬季的优势真菌菌门均为子囊菌门,优势属为酵母菌属,冬季发酵过程中主要霉菌及酵母菌的多样性远大于夏季。3、从5个不同批次的腐败醋样中共分离得到4个稳定菌群及46株细菌,其中44株细菌分别来自鲁梅利杆菌属、肠球菌属、漫游球菌属、乳酸杆菌属、芽孢杆菌属、葡萄球菌属、短波单胞菌属和类芽孢杆菌属,另外2株分离菌株尚无已知定义属。其中强产气菌群TYF-LIM-L09主要由六个细菌菌属组成,其中优势菌属为乳酸杆菌属（45.77%）。TYF-LIM-L09所产气体为CO₂,浓度可高达71.43%,产气量随pH值的减小及乙醇浓度的增加而减小。该菌群可以在50℃下快速生长,最适温度为45℃;能耐受的最低pH值为5.5;能耐受的乙醇及NaCl浓度分别为8%（v/v）及13%（w/v）。菌株Rummeliibacillus sp.TYF-05可以在55℃下快速增长,最适温度为45℃,能耐受的最低pH值为5.5,耐受的乙醇及NaCl浓度分别为7.5%（v/v）及13%（w/v）;菌株Rummeliibacillus sp.TYK-07可以在55℃下快速增长,最适温度为45℃,能耐受的最低pH值为5.0,耐受的乙醇及NaCl浓度分别为7.5%（v/v）和13%（w/v）。4、分别以葡萄糖和天然玉米秸秆为唯一碳源时,菌群TYF-LIM-L09、TYF-05以及TYK-07所对应的含量最高的代谢物分别为乙醇和丙酮、乙醇和乙醇以及乙酸乙酯和乙偶姻。其中菌株TYK-07可生成食醋中重要香味物质。3个特征菌株在6种不同碳源上的主要代谢产物含量检测结果显示,菌群TYF-LIM-L09在以淀粉为唯一碳源时,乙醇产量最高,为9.3 mM;菌株TYF-05在天然玉米秸秆上的乙醇产量最高,为5.6 mM;菌株TYK-07以天然玉米秸秆为唯一碳源时乙偶姻产量最高,浓度可高达150.8 mM。更多还原

【Abstract】 In recent years,vinegar makers in China had suffered from controlling difficultly of the brewing process during high temperature seasons and the abnormal phenomena such as turbidity,stickiness and flatulence also appeared in the finished vinegar,thereby seriously affecting the nutritional composition and quality of vinegar.The quality of vinegar is closely related to the microbial metabolism in the fermentation process.In order to further understand the steady-state brewing mechanism of vinegar in high-temperature condition,the microbial community in vinegar brewing process during summer and winter and characteristic spoilage microbes in spoilage vinegar were comprehensively and thoroughly investigated using molecular ecology technology.This study had obtained the following research results:1.The structure of bacterial community in vinegar brewing process during summer and winter was investigated by Illumina high-throughput sequencing and the results showed that the structure of bacterial community in summer and winter had significant statistical differences（p<0.05）.In summer,as a whole the richness of bacterial community in acetic acid fermentation period was greater than that in alcohol fermentation period,while the richness of bacterial community in alcohol fermentation period was greater than that in acetic acid fermentation period in winter,and the composition of bacterial community in each stage of acetic acid fermentation period had high similarity in both seasons.The richness（except the 6th day of alcohol fermentation）and diversity the（except the 3rd and 6th day of alcohol fermentation）of bacterial community in winter was higher than that in summer.The bacterial community in summer came from 8 phyla and 51 genera,in which 12 genera（relative abundance>0.1%）were first detected during the fermentation of Shanxi mature vinegar.In winter,the bacterial community belonged to 22 phyla and 226 genera,in which6 genera（relative abundance>0.1%）were first detected.The dominant bacterial phylum and genus were Firmicutes and Lactobacillus,respectively,in both seasons.The effects of seasonality on the low-abundance genera were much higher than that on the dominant genera.2.The structure of fungal community in vinegar brewing process during summer and winter was investigated by Illumina high-throughput sequencing.In summer,the richness of fungal community in acetic acid fermentation period were higher than that in alcohol fermentation,and the similarity of fungal flora composition among different fermentation stages was low.In winter,the richness of fungal community in acetic acid fermentation period were less than that in alcohol fermentation period,and the composition of fungal community in acetic acid fermentation period showed a higher similarity than that in alcohol fermentation period.In winter the richness and diversity of fungal community during alcohol fermentation were greater than those in summer,while in winter the richness and diversity of fungal community during acetic acid fermentation were lower than those in summer.In summer,the fungal community belonged to3 phyla and 35 genera,in which 7 genera（relative abundance>0.1%）were first detected.In winter,the fungal community came from 4 phyla and 46 genera,in which 15 genera（relative abundance>0.1%）were first detected.The dominant bacterial phylum and genus were Ascomycota and Saccharomyces,respectively,in both seasons.In winter the diversity of molds and yeasts with relative abundance over 0.1%were much higher than that in summer.3.4 stable bacterial consortium and 46 bacterial strains were isolated from5 different batches of spoilage vinegar samples and 44 strains of them belonged to the genera Rummeliibacillus,Enterococcus,Vagococcus,Lactobacillus,Bacillus,Staphylococcus,Brevundimonas,Paenibacillus,respectively,while the other two isolated strains came from unknown genera.The strong gas-producing bacterial consortium TYF-LIM-L09 was mainly composed of six bacterial genera,in which the dominant one was Lactobacillus（45.77%）.The gas produced by TYF-LIM-L09 was CO₂ and the highest volume percentage concentration could reach 71.43%.The gas production decreased with the decrease of pH value and the increase of ethanol concentration.TYF-LIM-L09,with high ethanol（8%v/v）and salt（13%w/v）tolerance,could tolerate the pH value decreased to 5.5 and grow rapidly at 50℃,with an optimum temperature of 45℃.Rummeliibacillus sp.TYF-05,with high ethanol（7.5%v/v）and salt（13%w/v）tolerance,could tolerate the pH value decreased to 5.5 and grow rapidly at55℃,with an optimum temperature of 45℃.Rummeliibacillus sp.TYK-07,with high ethanol（7.5%v/v）and salt（13%w/v）tolerance,could tolerate the pH value decreased to 5.0 and grow rapidly at 55℃,with an optimum temperature of 45℃.4.When glucose and natural corn stover were used as a sole carbon source,respectively,the highest metabolites of TYF-LIM-L09,TYF-05 and TYK-07were ethanol and acetone,ethanol and ethanol,ethyl acetate and acetoin respectively.The strain TYK-07 could produce important aroma substances of vinegar.The main product contents of TYF-LIM-L09,TYF-05 and TYK-07 on6 different carbon sources showed that the ethanol yield of TYF-LIM-L09 was the highest when starch was used as the sole carbon source,with a concentration of 9.3 mM;strain TYF-05 had the highest ethanol yield on natural corn straw,with a concentration of 5.6 mM;strain TYK-07 also had the highest acetoin yield when natural corn straw was used as the sole carbon source,with the concentration of 150.8 mM.更多还原