【作者】 李栋；

【导师】 王德良；

【作者基本信息】 新疆农业大学 ， 食品科学， 2014， 硕士

【摘要】 葡萄酒是一种蕴含多种营养物质的酒精饮料，很容易被微生物污染而发生变质。微生物污染葡萄酒可能出现产膜、产气、产酸、二次发酵等不良现象，使其品质下降，严重时会危害人体健康，给企业造成重大经济损失，所以对葡萄酒有害菌进行快速、准确的检测及其实际应用意义重大。以特定葡萄酒企业微生物菌库①为基点，通过大量的接种实验筛选有害菌。研究结果表明，微生物菌库中Y5、Y21、Y25、Y32、Y34、Y35、Y36、Y40、Y44、Y48、Y49、Y50、Y51、B15、B26、B48、B55-63为葡萄酒重点微生物②；其中Y③40为有害微生物，主要危害为产膜及产生异香。利用生理生化及特异性DNA片段对Y40进行菌种鉴定，鉴定结果表明该有害菌为拜耳接合酵母(Zygosaccharomycesbailii)。对Y40接菌葡萄酒进行理化指标、香气成分、生物胺以及氨基甲酸乙酯含量检测，并进行方差分析。结果表明，Y40除对香气成分有影响外，对其余指标均无显著影响。由于先进的快速检测方法需要精密的科学仪器，操作复杂，对人员素质要求较高，不适用于企业生产。而培养基因其价格低廉、操作简便已广泛应用于科研和发酵生产，因此将培养基作为研究对象。通过ZBA、TGYA培养基的对比研究发现，ZBA培养基对于拜耳接合酵母的选择特异性较TGYA强。对ZBA培养基进行单因素实验发现，Y40在沙氏葡萄糖琼脂，45g/L；NaCl，0g/L；pH3；山梨酸钾，0％；冰醋酸，0.1％；台盼蓝，0g/L；25℃培养条件下生长率最高。对培养基中沙氏葡萄糖琼脂、NaCl、台盼蓝进行正交优化，实验结果表明当沙氏葡萄糖琼脂为65g/L；NaCl，0g/L；台盼蓝，0g/L时Y40生长率最高，生长速度最快。与优化前培养基相比，该培养基的生长率由18.79％上升为87.80％，生长时间由139h缩短至39h，优化效果显著；且除Y40外其余菌种均不能在优化培养基中生长，表明培养基特异性强。该优化培养基价格低廉、操作简便，适用于企业对拜耳接合酵母的快速检测。当葡萄酒发生微生物污染而导致其品质下降时，我们一方面需要迅速对其进行处理，防止损失进一步扩大；另一方面我们需要了解有害微生物在葡萄酒酿造过程中的分布情况，从而进行合理防范，避免污染的再次发生。利用ZBA优化培养基对葡萄酒生产过程各个阶段进行检测，由实验结果可知在葡萄原料、酿造各阶段、车间环境、酿造设备中均含有拜耳接合酵母(Zygosaccharomyces bailii)，但其数量随着环境的变化而有所不同。更多还原

【Abstract】 Wine is a kind of nutritional liquor that it is easy to get contaminated by spoilagemicroorganisms.When wine is contaminated by microorganism, it may lead to produce many undesirablephenomenas, such as film production, gas, acid, secondary fermentation. As a result, wine quality willdecrease because of spoilage microorganisms contamination. At the same time, excessive economic losseswill also be brought about. So it is significant to detect and practical application for spoilagemicroorganisms in wine quality control in a fast and, accurate way.We study the spoilage microorganisms on the base of microbial bank from winery enterprise by wayof inoculation experiments. The results showed that the following microbial strains includingY5,Y21,Y25,Y32,Y34,Y35,Y36,Y40,Y44,Y48,Y49,Y50,Y51,B15,B26,B48,B55-63are of potential damage to winequality,and Y40is the spoilage microorganisms which could produce film and isovanillic,which is negativeto wine quality.We make a identification for this strain by physiological,biochemical and as well asmolecular biology method,that is to say using26SrDNA D1/D2. The intergrated consequences show thestrain is Zygosaccharomyces bailii.We detected the content of physical and chemical indicators, aromacomponents, biogenic amines and ethyl carbamate for the wine with Y40.The results showed that it only hasinfluence on aroma components.As advanced method for rapid detection needs sophisticated scientific instruments,complexoperation,and it also require a higher quality of personnel.While medium have been widely used inscientific research and fermentation as it is cheap, easy to operate. So we focused on medium.We comparedTGYA and ZBA,and found that ZBA is more specificity.It found that Zygosaccharomyces bailii has thehighest growth rate in condition of Sabouraud dextrose agar,45g/L; NaCl,0g/L; pH3; potassium sorbate,0%; acetic acid,0.1%; trypan blue,0g/L;25℃when we take a single factor experiment for ZBA. Weimplemented an orthogonal optimization for Sabouraud dextrose agar, NaCl and trypan blue.And the resultshow that Zygosaccharomyces bailii has the highest growth rate and shortest time when the condition isSabouraud dextrose agar,65g/L; NaCl,0g/L; trypan blue,0g/L.Compared to ZBA,the growth rate ofoptimized medium rose from18.79％to87.80％,and the growth time is reduced to39h from139h.Similarly, the specificity of optimized medium is strong.The optimized medium is cheaper and easier tooperate, and it is of great potential to be used for winery applications.When the wine contaminated by spoilage microorgannisms,one side,we need to take action to protect it from loss,and on the other side,we need to know the distribution of spoilage microorganisms in theprocess of wine production so that we coud take steps for pollution prevention.We detected the process ofwine production using optimized medium ZBA,and the result showed that Zygosaccharomyces bailiiexisted in various stages of winemaking process, wine grapes, workshop environment and brewingequipment.However, the quantity is changed with living environment.更多还原