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.
Ming-ming CHENAi-li LIMao-cheng SUNZhen FENGXiang-chen MENGYing WANG
