酒酒球菌胁迫诱导抗冷冻干燥机制研究

发布时间：2018-01-31 01:21

本文关键词： 酒酒球菌胁迫冷冻干燥膜脂肪酸蛋白质组　出处：《西北农林科技大学》2013年博士论文　论文类型：学位论文

【摘要】：我国是葡萄酒消费和生产大国，但葡萄酒行业所使用的苹果酸-乳酸发酵剂主要依赖进口，目前没有我国自主知识产权的葡萄酒乳酸菌发酵剂。酒酒球菌SD-2a是一株分离自山东烟台地区自然MLF葡萄酒中的具有优良特性的菌株，已获得国家专利保护（专利号：ZL02123444.2）。深入研究酒酒球菌SD-2a的胁迫诱导抗冷冻干燥机制，开发拥有我国自主知识产权的葡萄酒乳酸菌发酵剂，具有重要的理论和实践意义。本文以酒酒球菌SD-2a为研究对象，通过对酒酒球菌不同胁迫处理进行研究，评价菌体胁迫适应性反应能否有效的应用于冷冻干燥发酵剂的制备。研究建立酒酒球菌膜脂肪酸甲酯化方法，分析膜脂肪酸与抗冷冻干燥特性的相关性，从膜脂肪酸水平探讨酒酒球菌胁迫诱导抗冷冻干燥机制。通过优化双向电泳条件，构建酒酒球菌高质量的双向电泳图谱，利用蛋白质组学方法与技术，比较酒酒球菌不同胁迫诱导条件下蛋白质差异表达图谱，利用质谱和生物信息学技术对其中差异表达蛋白质进行鉴定、功能分类和代谢途径分析等，从蛋白质组学水平探讨酒酒球菌胁迫诱导抗冷冻干燥机制。克隆和分析与酒酒球菌抗冷冻干燥特性相关蛋白或酶的基因，通过一系列生物信息学分析，系统探讨酒酒球菌胁迫诱导抗冷冻干燥机制。取得的主要研究结果如下： 1.菌体不同生长期、冻干保护剂和复水剂是影响酒酒球菌冻干存活率的重要因素。与对数期菌体相比，稳定前期菌体具有较高的冻干存活率；分别以2.5%谷氨酸钠作为冻干保护剂和ATB液体培养基作为复水剂时，获得了最高的冻干存活率（69.5%）。 2.菌体经适当的胁迫处理后均能不同程度的提高酒酒球菌冻干存活率，其冻干粉接种模拟酒培养基后也具有明显的降酸效果。特别是pH3.5和8%乙醇胁迫处理，其冻干存活率分别为80.5%和82%，与对照相比，分别提高了17.7%和19.2%；其冻干粉在模拟酒培养基中的降酸均在8天内完成，与对照相比，平均降酸效果分别提高了28%和29%。 3.从GC/MS图谱、脂肪酸种类与相对含量、方法学考察对五种甲酯化方法进行比较分析，方法2（先提脂肪酸后用甲醇钠甲酯化法）和方法4（甲醇钠一步甲酯化法）均可以得到良好的脂肪酸检出效果。方法2检测准确、完整，但费时费力，，而方法4检测快速，但完整性稍差。 4.酒酒球菌经不同胁迫处理后，膜脂肪酸的变化主要体现在U/Scyc比例、环丙烷脂肪酸C19cyc11相对含量和不饱和脂肪酸C18:lcis11相对含量上。相关性分析表明，在所有胁迫处理下，C19cyc11与C18:lcis11相对含量具有显著负相关性；在菌体自我酸胁迫、冷胁迫和酸胁迫处理下，冻干存活率与C19cyc11相对含量均具有显著正相关性；在乙醇胁迫处理下，冻干存活率与C19cyc11相对含量、U/Scyc比例均具有较高的相关系数，但不存在显著相关性；各主要膜脂肪酸与降酸效果不存在显著相关性，只有在乙醇胁迫条件下，C14:0与降酸效果（MA）存在显著相关性。 5.从不同细胞破碎方法、不同蛋白裂解液、不同IPG胶条、不同蛋白上样量等方面优化酒酒球菌全蛋白双向电泳条件。利用超声波法破碎菌体，尿素-硫脲裂解液提取蛋白，苯酚/氯仿/异戊醇法纯化蛋白，蛋白上样量为400μg，在pI5-8胶条上聚焦时，获得高质量酒酒球菌全蛋白参考双向电泳图谱。 6.利用优化的双向电泳条件分别构建酒酒球菌不同生长期、不同酸胁迫和不同乙醇胁迫条件下全蛋白双向电泳图谱，经质谱鉴定技术和生物信息学分析，推断热休克蛋白Hsp20为酒酒球菌胁迫诱导抗冷冻干燥机制中关键性蛋白。 7.成功克隆环丙烷脂肪酸合酶基因（cfa）和热休克蛋白基因（hsp），并对基因编码的氨基酸序列进行生物信息学分析，结合前期膜脂肪酸分析和蛋白质组学研究，推断环丙烷脂肪酸C19cyc11在酒酒球菌胁迫诱导抗冷冻干燥机制中起到积极作用，而热休克蛋白Hsp20可能是酒酒球菌胁迫诱导抗冷冻干燥机制中关键性蛋白，起重要作用。
[Abstract]:China is Wine production and consumption country, but the use of malic acid - lactic acid fermentation agent Wine industry mainly rely on imports, there is no Wine lactic acid bacteria fermentation agent of China's intellectual property rights. Oenococcusoeni SD-2a is a strain isolated from strain MLF with excellent characteristics of natural grape wine of Shandong in the Yantai area, has been obtained the protection of national patent (Patent No.: ZL02123444.2). Stress study of Oenococcus oeni SD-2a induced anti freeze drying mechanism, Wine lactic acid bacteria starter development with independent intellectual property rights in our country, it has important theoretical and practical significance.
In this paper, oenococcusoeni SD-2a as the research object, based on the different treatment of oenococcusoeni stress, strain stress evaluation can effectively application of adaptive response on the freeze-drying preparation of starter. The establishment of fatty acid methyl ester oenococcusoeni membrane method, correlation analysis of membrane fatty acid and anti freeze drying characteristics, to explore the induction of anti freezing the drying mechanism of stress from the level of oenococcusoeni membrane fatty acid. By optimizing the conditions of two-dimensional gel electrophoresis, electrophoresis oenococcusoeni construction of high quality, using proteomics methods and technology, comparison of wine from different stress proteins induced by ball under the condition of expression profiling, using mass spectrometry and bioinformatics technology of these differentially expressed proteins were identified. Functional classification and pathway analysis, discussion of Oenococcus oeni stress induced anti freeze drying mechanism from proteomics level. G Long and analysis of drying characteristics of related proteins or enzymes and Oenococcus oeni anti freeze gene, through a series of bioinformatics analysis, the system of oenococcusoeni stress induced by anti freeze drying mechanism. The main results are as follows:
The cells of 1. different growth periods, cryoprotector and complex agent is oenococcusoeni freeze-dried important factors of survival. Compared with the logarithmic phase was stable with high pre cell survival rate of freeze-dried; respectively with 2.5% sodium glutamate as cryoprotector and ATB liquid medium as complex agent, obtained the highest survival rate of freeze-dried (69.5%).
2. bacteria after stress treatment appropriate were improved in different degrees oenococcusoeni survival rate of freeze-dried, the lyophilized culture medium after inoculation model wine also has obvious effect of reducing acid. Especially pH3.5 and 8% ethanol stress treatment, the survival rate of freeze-dried were 80.5% and 82%, compared with the control, respectively. Increased by 17.7% and 19.2%; the freeze-dried powder in simulated acid reducing medium wine culture was completed within 8 days, compared with the control, the average deacidification effect were increased by 28% and 29%.
3. from the GC/MS map, fatty acid composition and relative content, method of study comparative analysis of five methyl esterification methods, method 2 (first mention of fatty acid with methanol sodium methyl ester method) and method (4 step methanol sodium methyl ester method) can get good detection effect of fatty acid. 2 the detection is accurate, complete, but time-consuming, and 4 methods of rapid detection, but the integrity of the poor.
4. oenococcusoeni by different stress treatment, changes of membrane fatty acid is mainly reflected in the proportion of U/Scyc, cyclopropane fatty acid C19cyc11 and the relative content of unsaturated fatty acid relative content of C18:lcis11. The correlation analysis showed that in all treatments, C19cyc11 and relative content of C18:lcis11 has a significant negative correlation; in cell self acid stress, treatment cold stress and acid stress, the relative content of freeze-dried survival rate and C19cyc11 had significant positive correlation; in ethanol stress, survival rate and relative content of freeze-dried C19cyc11, the proportion of U/Scyc has the correlation coefficient is high, but there is no significant correlation between the membrane and the main fatty acid; acid reducing effect is not significant the correlation, only in ethanol under stress conditions, C14:0 and deacidification effect (MA) there is a significant correlation.
From 5. different cell disruption methods, different protein lysate, different IPG strip, optimization of two-dimensional electrophoresis of protein of different oenococcusoeni protein amount etc. lysed by ultrasonic method, the extraction of protein urea thiourea lysate, phenol / chloroform / isoamyl alcohol was purified from egg white protein sample volume 400 g, focusing on pI5-8 glue, to obtain high quality oenococcusoeni protein reference electrophoresis.
6. using the optimized two-dimensional electrophoresis conditions were constructed oenococcusoeni different growth periods, different acid stress and different ethanol stress conditions of protein electrophoresis, by mass spectrometry and bioinformatics analysis, concluded that heat shock protein Hsp20 oenococcusoeni stress induced protein critical anti freeze drying mechanism.
7. the successful cloning of cyclopropane fatty acid synthase gene (CFA) and heat shock protein gene (HSP), and the amino acid sequence of the gene encoding the bioinformatics analysis, combined with the membrane fatty acid analysis and proteomics research, inferred cyclopropane fatty acid C19cyc11 plays a positive role in the mechanism of stress induced anti freeze drying oenococcusoeni, and heat shock protein Hsp20 may be oenococcusoeni stress induced protein critical anti freeze drying mechanism, plays an important role.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：TS262.6

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