本文選題：植物乳桿菌 + 微生物代謝組學��； 參考：《江南大學》2017年碩士論文

【摘要】：代謝組學是研究細胞和生物體的所有代謝中間體和終產(chǎn)物的一門新興學科,在微生物基礎研究和生產(chǎn)實際中都能發(fā)揮良好作用。植物乳桿菌是一種與人類健康密切相關的,且一般認為對人體安全的食品添加物,在各種傳統(tǒng)食物中都有廣泛應用。本文建立了植物乳桿菌的代謝組學分析方法,并利用這一方法研究植物乳桿菌ZS2058及其三株亞油酸異構酶系基因敲除株和植物乳桿菌ST-III的代謝特征,從極性小分子和脂肪酸這兩類代謝產(chǎn)物變化規(guī)律著手,進一步闡明其生物轉(zhuǎn)化共軛亞油酸(CLA)差異的原因。首先,本文建立了一種以植物乳桿菌ZS2058為研究對象的微生物代謝組學研究的方法。方法系統(tǒng)考察了微生代謝組學分析時樣品制備中的不同淬滅和提取條件,并對提取方法的回收率進行驗證。通過測定淬滅處理后菌體的碘化丙啶(PI)染色率評估淬滅條件對細胞膜完整性的影響,同時以淬滅處理后菌體的系統(tǒng)能荷(EC)值比較8種淬滅條件的淬滅及時性效果,選取PI染色率最低(3.0±0.2%)且EC值最高(0.849±0.003)的20%甲醇/0.9%氯化鈉溶液(-4oC)作為最佳淬滅條件。隨后采用甲基叔丁基醚/甲醇/水、甲醇/水、乙腈/水和乙腈/甲醇/水這4種不同溶劑體系提取胞內(nèi)化合物,通過比較各條件下所得化合物的平均峰面積(90323625)、代謝輪廓圖和數(shù)據(jù)重復性(平均相對標準偏差為6%),確定乙腈/甲醇/水為本實驗的最優(yōu)提取條件。最后,選取6種代表性化合物對方法的線性范圍(0.00128~100μg/mL)、相關系數(shù)(0.9953~0.9999)和回收率(90~110%)進行驗證,結果表明該方法用于植物乳桿菌代謝組學分析時可以得到可靠的信息。其次,利用建立的植物乳桿菌代謝組學分析方法,本文詳細研究了植物乳桿菌ZS2058、植物乳桿菌ZS2058Δmcra、植物乳桿菌ZS2058Δdh、植物乳桿菌ZS2058Δdc和植物乳桿菌ST-III在代謝亞油酸(LA)過程的產(chǎn)物特征及變化規(guī)律。代謝組學分析結果顯示,植物乳桿菌ZS2058和植物乳桿菌ST-III胞內(nèi)代謝產(chǎn)物的輪廓圖存在明顯差異,且通過多元統(tǒng)計分析,篩選出分別在MRS和LA+MRS培養(yǎng)基條件下的差異性代謝產(chǎn)物28種和46種,主要包括糖類、有機酸和堿基類等。最后,為了進一步研究上述五株植物乳桿菌的脂肪酸代謝變化,分析了其在MRS和LA+MRS培養(yǎng)基中,0 h、5 h、10 h、14 h、18 h和24 h這6個時間點的脂肪酸組成。脂肪酸分析結果顯示,植物乳桿菌ZS2058不會在胞內(nèi)明顯的積累LA和10-羥基-順12-十八碳烯酸(10-HOE),而植物乳桿菌ZS2058Δdh、植物乳桿菌ZS2058Δdc和植物乳桿菌ST-III胞內(nèi)外LA和10-HOE含量都相對較高。當植物乳桿菌胞內(nèi)LA積累較多時,會引起胞內(nèi)油酸(OA)等脂肪酸含量的增加。且對于植物乳桿菌ZS2058,其胞內(nèi)10-HOE產(chǎn)生的時間點要早于胞外的,對于植物乳桿菌ST-III,其胞內(nèi)10-HOE產(chǎn)生的時間點和胞外的一樣。
[Abstract]:Metabonomics is a new discipline for the study of all metabolic intermediates and end products of cells and organisms. It can play a good role in the basic research and production of microorganism. Lactobacillus plantarum is a kind of food additive which is closely related to human health and is generally considered to be safe for human body. It is widely used in all kinds of traditional foods. The metabolic characteristics of Lactobacillus plantarum ZS2058 and its three linoleic isomerase gene knockout strains and Lactobacillus plantarum ST-III were studied. The reason of the difference of conjugated linoleic acid (CLA) of polar small molecule and fatty acid was discussed. Firstly, a method of microbial metabonomics based on Lactobacillus plantarum ZS2058 was established. Methods the different quenching and extraction conditions in the preparation of micrometabolites were systematically investigated, and the recovery rate of the extraction method was verified. The effects of quenching conditions on cell membrane integrity were evaluated by measuring the Pi staining rate of the quenched bacteria, and the timeliness of 8 quenching conditions was compared with the system energy charge (EC) values of the quenched cells. The optimum quenching conditions were 20% methanol / 0.9% sodium chloride solution (-4oC) with the lowest Pi staining rate (3.0 鹵0.2%) and the highest EC value (0.849 鹵0.003). Four different solvent systems, methyl tert Ding Ji ether / methanol / water, acetonitrile / water and acetonitrile / methanol / water, were used to extract intracellular compounds. By comparing the average peak area (90323625), metabolic profile and data repeatability (average relative standard deviation of 6%), the optimum extraction conditions of acetonitrile / methanol / water were determined. Finally, six representative compounds were selected to verify the linear range (0.00128 渭 g / mL), correlation coefficient (0.9953 ~ 0.9999) and recovery rate (90 ~ 110%) of the method. The results showed that the method could obtain reliable information when it was applied to the metabolic analysis of Lactobacillus plantarum. Secondly, using the established metabolic analysis method of Lactobacillus plantarum, The product characteristics and changes of Lactobacillus plantarum ZS2058, Lactobacillus plantarum ZS2058 螖 mcrah, Lactobacillus plantarum ZS2058 螖 dh, Lactobacillus plantarum ZS2058 螖 DC and Lactobacillus plantarum ST-III in the metabolic process of linoleic acid (LA) were studied in detail. The results of metabonomics analysis showed that the profiles of intracellular metabolites of Lactobacillus plantarum ZS2058 and Lactobacillus plantarum ST-III were significantly different, and were analyzed by multivariate statistical analysis. 28 and 46 different metabolites, including carbohydrate, organic acids and bases, were screened respectively in Mrs and LA Mrs medium. Finally, in order to further study the changes of fatty acid metabolism of the five strains of Lactobacillus plantarum, the fatty acid composition of the five strains were analyzed in Mrs and LA Mrs media at 6 time points: 0 h, 10 h, 14 h, 18 h and 24 h. Fatty acid analysis showed that Lactobacillus plantarum ZS2058 did not accumulate LA and 10-hydroxy-cis 12-octadecenoic acid (10-HOE) in the cells, while Lactobacillus plantarum ZS2058 螖 dh, Lactobacillus plantarum ZS2058 螖 DC and Lactobacillus plantarum ST-III contained relatively high contents of LA and 10-HOE. When Lactobacillus plantarum accumulated more LA, the content of fatty acids such as oleic acid (OA) increased. For Lactobacillus plantarum ZS2058, the production of 10-HOE was earlier than that of extracellular. For Lactobacillus plantarum ST-III, the time point of intracellular 10-HOE production was the same as that of extracellular.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q936;O657.63

【參考文獻】

相關期刊論文 前7條

1 李寧;范雪梅;王義明;王玉;羅國安;;代謝組學及其分析技術的研究進展[J];中南藥學;2014年07期

2 高振;熊強;徐晴;宋萍;李霜;;代謝通量分析在酶合成過程中的應用研究進展[J];化工進展;2013年07期

3 周秋香;余曉斌;涂國全;王強;胡文軍;李漢廣;;代謝組學研究進展及其應用[J];生物技術通報;2013年01期

4 趙強;孫曉紅;盧瑛;潘迎捷;趙勇;;應用代謝輪廓區(qū)分不同致病性副溶血性弧菌[J];高等學�；瘜W學報;2012年08期

5 趙維薇;許文濤;王煈;彭曉麗;黃昆侖;;代謝組學研究技術及其應用[J];生物技術通報;2011年12期

6 郭強;肖麗英;周學東;李明云;魯維希;熊萍;賈向明;李偉;;口腔常見鏈球菌代謝組學鑒定的研究[J];華西口腔醫(yī)學雜志;2009年05期

7 任洪燦;王廣基;阿基業(yè);郝海平;孫建國;查偉斌;嚴蓓;;代謝組學分析技術平臺和數(shù)據(jù)處理的新進展[J];中國臨床藥理學與治療學;2007年12期

相關博士學位論文 前2條

1 楊波;乳酸菌生物轉(zhuǎn)化共軛亞油酸的研究[D];江南大學;2014年

2 張小麗;基于多種分析技術的代謝組學方法研究與應用[D];蘭州大學;2013年

相關碩士學位論文 前6條

1 馬麗娜;基于代謝產(chǎn)物的食源性致病菌快速檢測技術研究[D];中南林業(yè)科技大學;2014年

2 王添琦;白念珠菌中核苷酸類物質(zhì)分析方法的建立及其在代謝組學應用研究[D];第二軍醫(yī)大學;2013年

3 夏夢雷;代謝物輪廓分析提高筑波鏈霉菌FK506產(chǎn)量[D];天津大學;2013年

4 王龍;基于~(13)C同位素標記實驗龜裂鏈霉菌代謝通量分析方法的建立與應用[D];華東理工大學;2013年

5 李兵;基于~1H-NMR的代謝組學方法對四種口腔念珠菌菌種鑒定及代謝產(chǎn)物初步分析[D];蘭州大學;2013年

6 梅輝;基于LC-MS/MS法的色氨酸合成代謝組學分析平臺的構建[D];江南大學;2010年

，

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