本文關(guān)鍵詞： 酒酒球菌 脅迫 冷凍干燥 膜脂肪酸 蛋白質(zhì)組　出處：《西北農(nóng)林科技大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：我國是葡萄酒消費和生產(chǎn)大國，但葡萄酒行業(yè)所使用的蘋果酸-乳酸發(fā)酵劑主要依賴進(jìn)口，目前沒有我國自主知識產(chǎn)權(quán)的葡萄酒乳酸菌發(fā)酵劑。酒酒球菌SD-2a是一株分離自山東煙臺地區(qū)自然MLF葡萄酒中的具有優(yōu)良特性的菌株，已獲得國家專利保護(hù)（專利號：ZL02123444.2）。深入研究酒酒球菌SD-2a的脅迫誘導(dǎo)抗冷凍干燥機(jī)制，開發(fā)擁有我國自主知識產(chǎn)權(quán)的葡萄酒乳酸菌發(fā)酵劑，具有重要的理論和實踐意義。 本文以酒酒球菌SD-2a為研究對象，通過對酒酒球菌不同脅迫處理進(jìn)行研究，評價菌體脅迫適應(yīng)性反應(yīng)能否有效的應(yīng)用于冷凍干燥發(fā)酵劑的制備。研究建立酒酒球菌膜脂肪酸甲酯化方法，分析膜脂肪酸與抗冷凍干燥特性的相關(guān)性，從膜脂肪酸水平探討酒酒球菌脅迫誘導(dǎo)抗冷凍干燥機(jī)制。通過優(yōu)化雙向電泳條件，構(gòu)建酒酒球菌高質(zhì)量的雙向電泳圖譜，利用蛋白質(zhì)組學(xué)方法與技術(shù)，比較酒酒球菌不同脅迫誘導(dǎo)條件下蛋白質(zhì)差異表達(dá)圖譜，利用質(zhì)譜和生物信息學(xué)技術(shù)對其中差異表達(dá)蛋白質(zhì)進(jìn)行鑒定、功能分類和代謝途徑分析等，從蛋白質(zhì)組學(xué)水平探討酒酒球菌脅迫誘導(dǎo)抗冷凍干燥機(jī)制�？寺『头治雠c酒酒球菌抗冷凍干燥特性相關(guān)蛋白或酶的基因，通過一系列生物信息學(xué)分析，系統(tǒng)探討酒酒球菌脅迫誘導(dǎo)抗冷凍干燥機(jī)制。取得的主要研究結(jié)果如下： 1.菌體不同生長期、凍干保護(hù)劑和復(fù)水劑是影響酒酒球菌凍干存活率的重要因素。與對數(shù)期菌體相比，穩(wěn)定前期菌體具有較高的凍干存活率；分別以2.5%谷氨酸鈉作為凍干保護(hù)劑和ATB液體培養(yǎng)基作為復(fù)水劑時，獲得了最高的凍干存活率（69.5%）。 2.菌體經(jīng)適當(dāng)?shù)拿{迫處理后均能不同程度的提高酒酒球菌凍干存活率，其凍干粉接種模擬酒培養(yǎng)基后也具有明顯的降酸效果。特別是pH3.5和8%乙醇脅迫處理，其凍干存活率分別為80.5%和82%，與對照相比，分別提高了17.7%和19.2%；其凍干粉在模擬酒培養(yǎng)基中的降酸均在8天內(nèi)完成，與對照相比，平均降酸效果分別提高了28%和29%。 3.從GC/MS圖譜、脂肪酸種類與相對含量、方法學(xué)考察對五種甲酯化方法進(jìn)行比較分析，方法2（先提脂肪酸后用甲醇鈉甲酯化法）和方法4（甲醇鈉一步甲酯化法）均可以得到良好的脂肪酸檢出效果。方法2檢測準(zhǔn)確、完整，但費時費力，，而方法4檢測快速，但完整性稍差。 4.酒酒球菌經(jīng)不同脅迫處理后，膜脂肪酸的變化主要體現(xiàn)在U/Scyc比例、環(huán)丙烷脂肪酸C19cyc11相對含量和不飽和脂肪酸C18:lcis11相對含量上。相關(guān)性分析表明，在所有脅迫處理下，C19cyc11與C18:lcis11相對含量具有顯著負(fù)相關(guān)性；在菌體自我酸脅迫、冷脅迫和酸脅迫處理下，凍干存活率與C19cyc11相對含量均具有顯著正相關(guān)性；在乙醇脅迫處理下，凍干存活率與C19cyc11相對含量、U/Scyc比例均具有較高的相關(guān)系數(shù)，但不存在顯著相關(guān)性；各主要膜脂肪酸與降酸效果不存在顯著相關(guān)性，只有在乙醇脅迫條件下，C14:0與降酸效果（MA）存在顯著相關(guān)性。 5.從不同細(xì)胞破碎方法、不同蛋白裂解液、不同IPG膠條、不同蛋白上樣量等方面優(yōu)化酒酒球菌全蛋白雙向電泳條件。利用超聲波法破碎菌體，尿素-硫脲裂解液提取蛋白，苯酚/氯仿/異戊醇法純化蛋白，蛋白上樣量為400μg，在pI5-8膠條上聚焦時，獲得高質(zhì)量酒酒球菌全蛋白參考雙向電泳圖譜。 6.利用優(yōu)化的雙向電泳條件分別構(gòu)建酒酒球菌不同生長期、不同酸脅迫和不同乙醇脅迫條件下全蛋白雙向電泳圖譜，經(jīng)質(zhì)譜鑒定技術(shù)和生物信息學(xué)分析，推斷熱休克蛋白Hsp20為酒酒球菌脅迫誘導(dǎo)抗冷凍干燥機(jī)制中關(guān)鍵性蛋白。 7.成功克隆環(huán)丙烷脂肪酸合酶基因（cfa）和熱休克蛋白基因（hsp），并對基因編碼的氨基酸序列進(jìn)行生物信息學(xué)分析，結(jié)合前期膜脂肪酸分析和蛋白質(zhì)組學(xué)研究，推斷環(huán)丙烷脂肪酸C19cyc11在酒酒球菌脅迫誘導(dǎo)抗冷凍干燥機(jī)制中起到積極作用，而熱休克蛋白Hsp20可能是酒酒球菌脅迫誘導(dǎo)抗冷凍干燥機(jī)制中關(guān)鍵性蛋白，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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：TS262.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張英華;霍貴成;郭

本文編號：1477779