Linking antioxidant activity to strain-specific exometabolomic profiles in probiotic Lactobacillus paracasei and Lactobacillus plantarum fermentation supernatants.

Probiotic microorganisms are increasingly exploited for their capacity to generate bioactive metabolites with potential health-promoting properties. In this study, we investigated the in vitro antioxidant activity of sixteen probiotic bacteria and characterized the extracellular metabolomic profiles of the spent fermentation broths (cell-free supernatants) derived from two strains showing markedly different antioxidant behaviors. Antioxidant potential was quantified using the DPPH radical scavenging assay, and total phenolic content was assessed by the Folin-Ciocalteu method. Among the tested strains, Lactiplantibacillus plantarum 8VEG3C displayed the most favorable antioxidant profile within the tested panel, with the lowest IC50 value (2.47% v/v) and the highest phenolic content, whereas Lacticaseibacillus paracasei LC01 showed a substantially weaker antioxidant activity. To explore the molecular basis of these functional differences, untargeted extracellular metabolomic analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), coupled with multivariate statistical analysis and putative, database-supported annotation. The two strains generated clearly distinguishable extracellular signatures. The L. plantarum 8VEG3C supernatant was enriched in fatty acyl-related metabolites, including oleic acid, 7-oxooctadecanoic acid and γ-dodecalactone, together with aromatic amino acid-derived compounds such as indole lactate. In contrast, L. paracasei LC01 was enriched in amino acid- and small peptide-related metabolites including the putatively annotated 2-(6-hydroxyhexanoylamino)-3-phenylpropanoic acid, compatible with a more prominent involvement of the proteolytic system of lactic acid bacteria. These findings support the use of untargeted metabolomics as a functional screening platform for the rational selection of probiotic strains with postbiotic potential.