基于GC-MS的微生物代谢组学在植物乳杆菌生物转化共轭亚油酸中的应用

发布时间：2018-06-23 16:16

本文选题：植物乳杆菌 + 微生物代谢组学　；参考：《江南大学》2017年硕士论文

【摘要】：代谢组学是研究细胞和生物体的所有代谢中间体和终产物的一门新兴学科,在微生物基础研究和生产实际中都能发挥良好作用。植物乳杆菌是一种与人类健康密切相关的,且一般认为对人体安全的食品添加物,在各种传统食物中都有广泛应用。本文建立了植物乳杆菌的代谢组学分析方法,并利用这一方法研究植物乳杆菌ZS2058及其三株亚油酸异构酶系基因敲除株和植物乳杆菌ST-III的代谢特征,从极性小分子和脂肪酸这两类代谢产物变化规律着手,进一步阐明其生物转化共轭亚油酸(CLA)差异的原因。首先,本文建立了一种以植物乳杆菌ZS2058为研究对象的微生物代谢组学研究的方法。方法系统考察了微生代谢组学分析时样品制备中的不同淬灭和提取条件,并对提取方法的回收率进行验证。通过测定淬灭处理后菌体的碘化丙啶(PI)染色率评估淬灭条件对细胞膜完整性的影响,同时以淬灭处理后菌体的系统能荷(EC)值比较8种淬灭条件的淬灭及时性效果,选取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%氯化钠溶液(-4oC)作为最佳淬灭条件。随后采用甲基叔丁基醚/甲醇/水、甲醇/水、乙腈/水和乙腈/甲醇/水这4种不同溶剂体系提取胞内化合物,通过比较各条件下所得化合物的平均峰面积(90323625)、代谢轮廓图和数据重复性(平均相对标准偏差为6%),确定乙腈/甲醇/水为本实验的最优提取条件。最后,选取6种代表性化合物对方法的线性范围(0.00128~100μg/mL)、相关系数(0.9953~0.9999)和回收率(90~110%)进行验证,结果表明该方法用于植物乳杆菌代谢组学分析时可以得到可靠的信息。其次,利用建立的植物乳杆菌代谢组学分析方法,本文详细研究了植物乳杆菌ZS2058、植物乳杆菌ZS2058Δmcra、植物乳杆菌ZS2058Δdh、植物乳杆菌ZS2058Δdc和植物乳杆菌ST-III在代谢亚油酸(LA)过程的产物特征及变化规律。代谢组学分析结果显示,植物乳杆菌ZS2058和植物乳杆菌ST-III胞内代谢产物的轮廓图存在明显差异,且通过多元统计分析,筛选出分别在MRS和LA+MRS培养基条件下的差异性代谢产物28种和46种,主要包括糖类、有机酸和碱基类等。最后,为了进一步研究上述五株植物乳杆菌的脂肪酸代谢变化,分析了其在MRS和LA+MRS培养基中,0 h、5 h、10 h、14 h、18 h和24 h这6个时间点的脂肪酸组成。脂肪酸分析结果显示,植物乳杆菌ZS2058不会在胞内明显的积累LA和10-羟基-顺12-十八碳烯酸(10-HOE),而植物乳杆菌ZS2058Δdh、植物乳杆菌ZS2058Δdc和植物乳杆菌ST-III胞内外LA和10-HOE含量都相对较高。当植物乳杆菌胞内LA积累较多时,会引起胞内油酸(OA)等脂肪酸含量的增加。且对于植物乳杆菌ZS2058,其胞内10-HOE产生的时间点要早于胞外的,对于植物乳杆菌ST-III,其胞内10-HOE产生的时间点和胞外的一样。
[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 卤0.2%) and the highest EC value (0.849 卤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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q936;O657.63

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