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

【摘要】：代謝組學(xué)是研究細(xì)胞和生物體的所有代謝中間體和終產(chǎn)物的一門新興學(xué)科,在微生物基礎(chǔ)研究和生產(chǎn)實(shí)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(guān)的,且一般認(rèn)為對(duì)人體安全的食品添加物,在各種傳統(tǒng)食物中都有廣泛應(yīng)用。本文建立了植物乳桿菌的代謝組學(xué)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(gòu)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(jìn)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(duì)象的微生物代謝組學(xué)研究的方法。方法系統(tǒng)考察了微生代謝組學(xué)分析時(shí)樣品制備中的不同淬滅和提取條件,并對(duì)提取方法的回收率進(jìn)行驗(yàn)證。通過測(cè)定淬滅處理后菌體的碘化丙啶(PI)染色率評(píng)估淬滅條件對(duì)細(xì)胞膜完整性的影響,同時(shí)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(shí)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(fù)性(平均相對(duì)標(biāo)準(zhǔn)偏差為6%),確定乙腈/甲醇/水為本實(shí)驗(yàn)的最優(yōu)提取條件。最后,選取6種代表性化合物對(duì)方法的線性范圍(0.00128~100μg/mL)、相關(guān)系數(shù)(0.9953~0.9999)和回收率(90~110%)進(jìn)行驗(yàn)證,結(jié)果表明該方法用于植物乳桿菌代謝組學(xué)分析時(shí)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(xué)分析方法,本文詳細(xì)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(xué)分析結(jié)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(jì)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(jī)酸和堿基類等。最后,為了進(jìn)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(gè)時(shí)間點(diǎn)的脂肪酸組成。脂肪酸分析結(jié)果顯示,植物乳桿菌ZS2058不會(huì)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(duì)較高。當(dāng)植物乳桿菌胞內(nèi)LA積累較多時(shí),會(huì)引起胞內(nèi)油酸(OA)等脂肪酸含量的增加。且對(duì)于植物乳桿菌ZS2058,其胞內(nèi)10-HOE產(chǎn)生的時(shí)間點(diǎn)要早于胞外的,對(duì)于植物乳桿菌ST-III,其胞內(nèi)10-HOE產(chǎn)生的時(shí)間點(diǎn)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(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q936;O657.63

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