Abstract
This study proposed a quenching protocol for metabolite analysis of Lactobacillus delbrueckii subsp. bulgaricus. Microbial cells were quenched with 60% methanol/water, 80% methanol/glycerol, or 80% methanol/water. The effect of the quenching process was assessed by the optical density (OD)-based method, flow cytometry, and gas chromatography-mass spectrometry (GC-MS). The principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were employed for metabolite identification. The results indicated that quenching with 80% methanol/water solution led to less damage to the L. bulgaricus cells, characterized by the lower relative fraction of prodium iodide (PI)-labeled cells and the higher OD recovery ratio. Through GC-MS analysis, higher levels of intracellular metabolites (including focal glutamic acid, aspartic acid, alanine, and AMP) and a lower leakage rate were detected in the sample quenched with 80% methanol/water compared with the others. In conclusion, we suggested a higher concentration of cold methanol quenching for L. bulgaricus metabolomics due to its decreasing metabolite leakage.
概要
研究目的
为保加利亚乳杆菌的代谢组学研究提供一种简单有效的淬灭方案。
创新要点
采用适当的淬灭技术是获得微生物真实代谢物组数据的必要条件。 本研究首次建立了适用于保加利亚乳杆菌代谢组学研究的淬灭方法, 有助于完善乳酸菌代谢的生理学和遗传学概貌。
研究方法
分别应用 −40 °C 的60%甲醇/水、 80%甲醇/水和80%甲醇/甘油三种方式淬灭保加利亚乳杆菌。 利用光密度(OD)回收率实验、 流式细胞术和气质联用(GC-MS)分析菌体细胞完整性和细胞内外代谢物, 以及主成分分析法(PCA)和正交偏最小二乘法-判别分析法(OPLS-DA)分析代谢物泄露程度。
重要结论
80%冷甲醇/水更适用于淬灭保加利亚乳杆菌, 能有效减少代谢物泄露程度和增加胞内代谢物水平。
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Project supported by the Open Research Fund for the MOE Key Laboratory of Dairy Science (No. 2012KLDSOF-07), the National Natural Science Foundation of China (No. 31000808), the Synergetic Innovation Center of Food Safety and Nutrition, and the Open Research Fund of Northeast Agricultural University, China
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Chen, Mm., Li, Al., Sun, Mc. et al. Optimization of the quenching method for metabolomics analysis of Lactobacillus bulgaricus . J. Zhejiang Univ. Sci. B 15, 333–342 (2014). https://doi.org/10.1631/jzus.B1300149
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DOI: https://doi.org/10.1631/jzus.B1300149