Strain-associated variation in extracellular chemical profiles of fruit-derived Lactiplantibacillus plantarum under standardized cultivation

Introduction Lactiplantibacillus plantarum exhibits substantial strain-level metabolic diversity, yet comparative information on extracellular chemical profiles among fruit-derived strains remains limited. Methods This study characterized and compared cell-free supernatants from five native fruit-derived L. plantarum strains cultivated under standardized conditions, using three independent biological cultures per strain. Untargeted LC–MS/MS profiling using RP-HSS T3 chromatography in positive- and negative-ion electrospray modes and HILIC-BEH Amide chromatography in negative-ion mode yielded 8,692 analytical detections, of which 6,769 had associated MS/MS information. Following annotation-confidence filtering, quality-control assessment, and consolidation across analytical configurations, 999 higher-confidence metabolite annotations (Levels 1a and 1b) were retained for comparative analysis. Results Multivariate analyses revealed strain-associated differences in extracellular chemical profiles, with the extent of separation varying among strains. Welch's one-way ANOVA followed by Benjamini–Hochberg correction identified 438 of the 999 higher-confidence annotations as significantly different among strains (FDR < 0.05). Pairwise Welch's t -tests with FDR correction and |log 2 FC| ≥ 1 revealed strongly comparison-dependent differentiation; among comparisons centered on G1, 4, 39, 62, and 18 FDR-supported annotations were identified for G1 vs. G2, G1 vs. G3, G1 vs. G4, and G1 vs. G5, respectively. Comparison with an incubated uninoculated MRS control showed that part of the extracellular chemical background was also detectable in the cultivation medium; consequently, compounds detected in the cell-free supernatants were not attributed exclusively to bacterial biosynthesis. Conclusion These findings demonstrate substantial strain-associated variation in the extracellular chemical profiles of fruit-derived L. plantarum under standardized cultivation conditions and show that extracellular differentiation involves comparison-specific combinations of metabolite annotations rather than a uniform discriminatory signature. The higher-confidence candidates identified provide a basis for subsequent targeted quantitative and functional investigation of strain-associated extracellular phenotypes.