Statistical Evaluation And Application Of Quenching And Extraction Protocols For Quantitative Metabolomics Of Lactobacillus Paracasei

A reliable metabolomics method for investigating intracellular metabolites in Lactobacillus paracasei by gas chromatography-mass spectrometry was developed. The effect of different solvents (pyridine, tetrahydrofuran and dimethylformamide), and derived reagents (MTBDSTFA, BSTFA+TMCS and MSTFA), for peak signal intensity and numbers were investigated. The best condition with pyridine as solvent and BSTFA+TMCS as derived reagent was determined. Total48kinds of intracellular metabolites were identified in the samples of Lactobacillus paracasei. This method showed perfect accuracy, stability and reproducibility on the basis of the methodological analysis.A reliable quenching and metabolite extraction method has been developed for metabolome. The metabolome profiles were generated using gas chromatography mass spectrometry. Univariate and multivariate statistical analysis were employed to define the most appropriate method. The results indicated that60%methanol (-60℃) buffered with0.9%(w/v) NaCl was the most suitable reagent to quench metabolism and reduce the leakage of metabolites. Furthermore, a consecutive extraction of intracellular metabolites using70%methanol followed by acetonitrile/methanol/water extraction was proposed. With this new method, the majority of metabolic contents exhibited not only the greatest efficiency but also high reproducibility.At last, the established GC-MS analysis methods and sample treatment method for metabolomics were successfully applied for process analysis of lactic acid fermentation with Lactobacillus casei. It was observed that lactic acid productivity and yield were significantly reduced while DO level recovered from zero to20%of air saturation when cells entered into stationary phase. PCA was used to analyse the obtained metabolome data indicating the metabolic profiling changed and most of intracellular metabolites appeared to have significant different levels with the recovery of DO level, especially for those intermediates of EMP and TCA circle, which accumulated greatly during this period. So that after DO recovery metabolic pathway shifted from lactic acid formation to byproducts formation, resulting in the decline of lactic acid productivity and yield.