【作者】 李西柱；

【导师】 刘树文；

【作者基本信息】 西北农林科技大学 ， 发酵工程， 2009， 硕士

【摘要】 苹果酸-乳酸发酵是酿造优质红葡萄酒的一个重要环节。经酒精发酵后的葡萄酒都会形成一个相对较恶劣的环境(低pH值、高酒精浓度和高SO2浓度、营养物质匮乏等),从而影响苹果酸-乳酸发酵的启动和完成。利用抗性较强的乳酸菌菌株进行苹果酸-乳酸发酵就成为解决这个问题的关键。进行苹果酸-乳酸发酵的乳酸菌经研究发现主要是酒酒球菌。因此,为了筛选优良的发酵菌株,对酒酒球菌抗逆性方面的研究是非常必要的。但目前关于酒酒球菌抗性方面的研究多集中在蛋白质水平,从其一级信息源—基因方面考虑其抗性的研究国内外鲜见报道。本研究旨在从DNA分子水平上了解耐酸性菌株与酸敏性菌株之间的差异,筛选与酒酒球菌耐酸性相关的RAPD分子标记,为优良酒酒球菌的筛选奠定基础。1、从宁夏地区分离得到30株苹果酸-乳酸发酵菌,经过初步鉴定,其生长特征基本上与酒酒球菌的生长特征相同。通过葡萄酒乳酸菌属的鉴定表和明串珠菌属细菌(Leuconostoc)种内鉴定双歧式检索表对所分离的菌种做进一步鉴定,结果表明,所分离得到的菌株均为酒酒球菌。利用这30株菌进行了抗酸性的筛选,结果得到10株抗酸性菌株和9株酸敏性菌株。2、以学院保藏的酒酒球菌SD-2a为试验材料,研究了酒酒球菌基因组DNA提取方法,结果表明,采用改良的传统细菌基因组DNA提取方法,所提取的DNA质量较高,提取的DNA含量在170ng/μl左右,A260/A280比值均介于1.75-1.90之间。经电泳检测条带清晰,无杂带,而且没有蛋白质,糖类的污染,能够满足RAPD反应的需要。3、以提取的酒酒球菌SD-2a基因组DNA和随机引物S333为试验材料,对影响RAPD-PCR反应体系的主要因素进行了优化,最终建立了适合本实验室的RAPD反应体系:25μl反应体积、1.0UTaq酶、160μmol/L dNTP、0.4μmol/L随机引物、3.0mmol/L Mg2+、10ng的DNA模板用量;PCR反应程序为:94℃预变性5min;94℃变性1min,36℃退火1min,72℃延伸1.5min,45个循环;72℃延伸5min。4、在RAPD-PCR反应体系建立的基础上以33株抗酸性酒酒球菌和9株酸敏性菌株为试材。采用BSA法和RAPD相结合的技术,通过对45个随机引物的筛选以及在单菌株中的验证,结果两条随机引物在单菌株中表现稳定的多态性,出现了较为稳定的有规律的三条特异性条带,这两条随机引物分别为:S40、S333。三条特异性条带命名如下:S40—1400;S333—2500;S333—650。这样就标记出了与酒酒球菌抗酸相关基因相连锁的三条特异性条带。证明抗酸性菌株和酸敏性菌株基因组之间确实存在显著的差异性,同时也证明酒酒球菌的抗酸性状是多基因控制的。为建立快速筛选酒酒球菌优良菌株的方法提供了理论依据。更多还原

【Abstract】 Malolactic fermentation (MLF) is an indispensable processing technology for red wine. The poor environmental conditions for lactic acid bacteria growth will be created after wine alcoholic fermentation, such as low pH, high alcohol, SO2 concentration and lack of nutrients. In this poor condition MLF is often sluggish and even bring about fall of inoculation. Using strong resistance lactic acid bacteria strains in Malolactic fermentation have been becoming the key to solve this problem. It is found that Oenococcus oeni is the main lactic acid bacteria in the processing of Malolactic fermentation. Therefore, it is necessary to study the resistance of the Oenococcus oeni to pH, alcohol and SO2, in order to screen excellent to carry out the Malolactic fermentation.However, at present, the research about resistance of Oenococcus oeni concentrates on the protein level. As a source of information, genetic study about its resistance is scarcely at home and abroad. The purpose of this study is to learn the difference between acid-resistant strains and acid-sensitive strains based on DNA of the Oenococcus oeni. RAPD specific molecular markers linked to acid resistance gene in Oenococcus oeni will be discovered in this study. These molecular markers will lay a foundation for screening of the superior Oenococcus oeni.1. With selective culture media, we have isolated 30 strains malolactic bacteria from NingXia region wines of China. Preliminary identification of the isolates was performed. The Growth characteristics of the isolates are the same as Oenococcus oeni. Further identification of the isolates was performed according to the criteria established by Roger and Villani. The results show that 30 isolates are also Oenococcus oeni. The work that selected acid-resistant strains and acid-sensitive strains in 30 Oenococcus oeni strains has been done. As a result, we obtained 10 Oenococcus oeni strains of acid-resistance and 9 Oenococcus oeni strains of acid-sensitive.2. In this study, Oenococcus oeni SD-2a was used as experimental material. The method of genome DNA extraction was studied in Oenococcus oeni. The results showed that high grade genome DNA was obtained by the revised traditional method. The genome DNA extracted by the revised traditional method concentration is about 170ng/μl. The ratios of A260/A280 are from 1.75 to 1.90. With detection of bands by electrophoresis, we obtain a clear, non-hybrid band. These bands have not been polluted by protein or carbohydrate. This genome DNA can accomplish the reaction of RAPD.3. In this study, genome DNA of Oenococcus oeni SD-2a and random primers S333 were used as experimental material. The primary conditions of the RAPD-PCR were optimized and RAPD reacting system that is appropriate for our laboratory was established. The optimal PCR system for RAPD analysis was as follows: 1.0UTaq polymerase, 160μmol/L dNTP, 0.4μmol/L random primer, 3.0mmol/L Mg2+, 10 ng DNA template, in 25μL reaction system; The reaction program of PCR was devised at 94℃for 5minutes and followed by 45 cycles, each with l minute at 94℃, l minute at 36℃, l.5minutes at 72℃, and a final extension at 72℃for 5 minutes.4. Using the optimal PCR system for RAPD analysis, 33 Oenococcus oeni strains of acid-resistance and 9 Oenococcus oeni strains of acid-sensitive were used as experimental material for screening the RAPD markers linked to the gene resistant to acid. The methods of random amplified polymorphic DNA (RAPD) markers and bulked segregate analysis (BSA) were employed in this study. Finally, three markers S40—1400, S333—2500 and S333—650 linked to Acid in Oenococcus oeni were obtained through a selection among 45 random primers and confirmation in single Oenococcus oeni strain. This study proved that there was a significant difference between Oenococcus oeni strains of acid-resistance and Oenococcus oeni strains of acid-sensitive in their genome. At the same time, the research also proved that the trait of acid-resistance is controlled by multiple genes in Oenococcus oeni. This work may lay a theoretical basis for the establishment of the rapid screening of fine Oenococcus oeni strains.更多还原