Metabolomic and functional profiling of Lactiplantibacillus plantarum strains reveals distinct probiotic, immunomodulatory, and antimicrobial signatures

Lactiplantibacillus plantarum strains are increasingly recognized for their combined probiotic and antimicrobial activities, offering potential applications in gut health management and pathogen control. This study characterized the intracellular (Met-Int) and extracellular (Met-Ext) metabolomic profiles of L. plantarum UTNGt2 (Gt2), UTNGt3 (Gt3), and UTNGt28L (Gt28L) isolated from tropical fruits and evaluated their probiotic, antimicrobial, cytotoxic, and immunomodulatory properties in vitro. Metabolomic profiling was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a SWATH (Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) acquisition method. Cytotoxicity and cell viability were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LDH (lactate dehydrogenase) release assays in colon epithelial cells, while cytokine responses (IL-10, IL-1β) were quantified to determine immunomodulatory effects. Antimicrobial mechanisms were examined by scanning and transmission electron microscopy (SEM/TEM) on Staphylococcus aureus ATCC1026. LC-MS/MS identified 117 Met-Int and 32 Met-Ext across the three strains, revealing shared metabolites (e.g., L-tryptophan, adenosine) and distinct strain-specific compounds (e.g., harmine, lincomycin, baicalein) associated with bioactivity. Pathway enrichment analysis indicated four enriched pathways in Gt2, eight in Gt3, and ten in Gt28L, reflecting differential specialization in amino-acid, carbohydrate, and cofactor metabolism. Gt3 exhibited the most diverse antimicrobial metabolite repertoire, whereas Gt28L showed the strongest anti-inflammatory effect, increasing IL-10 secretion by ~ 6.5-fold and reducing IL-1β by ~ 50% compared with control cells. All strains maintained > 85% cell viability with minimal LDH release. SEM/TEM analysis confirmed that Met-Ext fractions caused membrane disruption and intracellular damage in S. aureus. Overall, these results demonstrate strain-specific probiotic, antimicrobial, and immunomodulatory signatures, identifying Gt3 and Gt28L as promising candidates for therapeutic or food-grade applications. KEY POINTS: Distinct L. plantarum strains show unique metabolite profiles with probiotic potential. Gt3 and Gt28L strains exhibit strong antimicrobial and anti-inflammatory activity. Metabolite extracts disrupt pathogens while preserving epithelial cell viability.