A robust method for monitoring the growth and metabolism of probiotic bacteria in vitro

Lactic acid bacteria are involved in many food, nutritional, and biotechnological applications, where carbohydrates are their main source of energy. Although the study of bacterial growth and metabolism in vitro offers several advantages, protocols detailing optimum conditions for a particular bacterium are often not well-established. The objective of this work was to develop a protocol with optimized bacterial growth and sampling conditions for a specific probiotic bacterium ( Lactiplantibacillus plantarum ) used a model system and apply this protocol to study the interaction between a substrate and this bacterium in vitro for downstream microbiological and metabolomic analyses. Here we detail an optimized medium composition (mCFBM 3) for controlled L. plantarum growth. Use of this medium allowed us to assess growth promotion, effects of treatments and select optimal time points during fermentative growth for downstream broad-spectrum (NMR, LC-MS) and targeted (fatty acid; LC-MS/MS) metabolomic analyses. While untargeted LC-MS analyses allowed for the putative identification of a wider range of products, NMR-based metabolomics proved an efficient and rapid tool for the analysis of major bacterial metabolites, allowing putative identification of the metabolic products formed per colony forming unit, and enabling monitoring of the production and degradation of compounds during fermentation. • Developed a protocol to study probiotic bacteria's responses to growth substrates • Optimized conditions for precise metabolomics of bacterial growth • Employed NMR for rapid analysis of major metabolites • Combined targeted LC-MS/MS and broad-spectrum LC-MS for full metabolic profiling • Provided comprehensive metabolic profiles through integrated analyses