JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B 2014 Vol.15 No.4 P.333-342

Optimization of the quenching method for metabolomics analysis of Lactobacillus bulgaricus *

Author(s): Ming-ming Chen¹, Ai-li Li¹, Mao-cheng Sun², Zhen Feng¹, Xiang-chen Meng¹, Ying Wang¹
Affiliation(s): 1. MOE Key Laboratory of Dairy Science, College of Food Science, Northeast Agricultural University, Harbin 150030, China; more
Corresponding email(s): aili-mail@163.com
Key Words: Metabolomics, Quenching method, Lactobacillus bulgaricus , Leakage

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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.

保加利亚乳杆菌代谢组学中淬灭方法的优化研究

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。
代谢组学；淬灭处理；保加利亚乳杆菌；泄露

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保加利亚乳杆菌代谢组学中淬灭方法的优化研究

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