Metabolic footprinting for investigation of antifungal properties of Lactobacillus paracasei

Lactic acid bacteria with antifungal properties are applied for biopreservation of food. In order to further our understanding of their antifungal mechanism, there is an ongoing search for bioactive molecules. With a focus on the metabolites formed, bioassay-guided fractionation and comprehensive screening have identified compounds as antifungal. Although these are active, the compounds have been found in concentrations that are too low to account for the observed antifungal effect. It has been hypothesized that the formation of metabolites and consumption of nutrients during bacterial fermentations form the basis for the antifungal effect, i.e., the composition of the exometabolome. To build a more comprehensive view of the chemical changes induced by bacterial fermentation and the effects on mold growth, a strategy for correlating the exometabolomic profiles with mold growth was applied. The antifungal properties were assessed by measuring mold growth of two Penicillium strains on cell-free ferments of three strains of Lactobacillus paracasei pre-fermented in a chemically defined medium. Exometabolomic profiling was performed by reversed-phase liquid chromatography in combination with mass spectrometry in electrospray positive and negative modes. By multivariate data analysis, the three strains of Lb. paracasei were readily distinguished by the relative difference of their exometabolomes. The relative differences correlated with the relative growth of the two Penicillium strains. Metabolic footprinting proved to be a supplement to bioassay-guided fractionation for investigation of antifungal properties of bacterial ferments. Additionally, three previously identified and three novel antifungal metabolites from Lb. paracasei and their potential precursors were detected and assigned using the strategy.