Metabolic Regulation Role Of Microbe Based On Chromatography-mass Spectrometry

Microbial metabolomics is a functional genomics technology of vital importance for understanding cellular function, because metabolome is a direct reflection of cellular physiological status. According to the different distribution of metabolites, it may divide into metabolic fingerprinting (intracellular) and metabolic footprinting analysis (extracellular). Compared to extracellular metabolites, intracellular metabolites is relatively more abundant and can reflect microbial metabolic characteristic more profound, so the study of intracellular metabolites is even more important, its main steps contain metabolic quenching, metabolites extraction and instrumental analysis three parts. Nuclear Magnetic Resonance (NMR) technology and Chromatography-mass Spectrometry technology (LC-MS、GC-MS、CE-MS) are the main methods in the research of metabolomics at present, while Chromatography coupled to mass Spectrometry technology has been widely used in the field of metabolomics research, for its strong chemical information representation ability to the complex biological system, such as high resolution separation, high sensitive detection.This paper used Exiguobacterium aurantiacum ATCC49676as the main object of study, investigated metabolites affecting the production of lactic acid. Firstly, Minitab software was employed to design the experiment of culture medium optimization, glucose was considered as the key factor in culture medium (p=0.001), other factors had not significant influence to the production of lactic acid (p>0.05). Then single factor experiment was used to confirm the optimal concentration of glucose, when the concentration of glucose was0.1g/mL, production of lactic acid reaches a maximum. Gas chromatography coupled to mass Spectrometry technique was adopted to analyze the intracellular metabolites of the bacterium cultured in the basic medium and optimized medium, identified33metabolites by standard samples. MEV software was employed to conduct SAM analysis, glutamate was regarded to have the greatest impact on the production of lactic acid. In order to study the mechanism of glutamate influence on lactic acid production, Coomassie brilliant blue Elisa kit and lactic dehydrogenase Elisa kit were utilized to assay the relative content of lactic dehydrogenase, the final results verified the hypothesis of that glutamate regulated the production of lactic acid by reducing the relative content of lactic dehydrogenase.On that basis, established the analysis system of studying microbial key regulated metabolites by metabolomics, and then builded a GC-MS library of180common intracellular metabolites in microbe. For the strain ATCC49676is still an extremophile, its basophilic and salt tolerance mechanism may be researched by the newly-built microbial metabolomics analysis platform. Different microbial intracellular metabolites samples were obtained by changing the pH and salinity of optimal culture medium respectively, then by means of newly-built analysis platform and the library of the common microbial intracellular metabolites,16differential metabolites related to alkali-resistant and14differential metabolites related to salt tolerance were acquired with the help of PCA model and PLS-DA model by SIMICA-P software.