Strain-resolved metabolomic chemotyping identifies tryptophan and γ-glutamyl peptides as MGO-AGEs breaking postbiotic compounds for carbonyl stress mitigation.

Carbonyl stress, characterized by pathological accumulation of reactive dicarbonyl species such as methylglyoxal (MGO), disrupts mitochondrial function and drives formation of advanced glycation end-products (AGEs), and is implicated in chronic neurobehavioral and metabolic disorders. Here, we established a strain-resolved activity metabolomics framework to rationally select postbiotic extracts with MGO-AGEs breaking activity. We screened a postbiotic library of 177 intracellular metabolite extracts from 113 bacterial strains by untargeted metabolite profiling and in vitro MGO-AGEs breaking activity. Comparative analysis revealed that strain-level chemotypes within Limosilactobacillus fermentum showed marked differences in functional activity that were not apparent at the species level. Using this framework, we selected the postbiotic extract LFEAN031 based on strain-level enrichment of MGO-AGEs breaking compounds, including tryptophan, asparagine, and three γ-glutamyl peptides (GGPs), and evaluated its efficacy in an MGO-induced carbonyl stress mouse model. Oral LFEAN031 administration restored MGO-induced colon length shortening and increased colonic expression of the tight junction protein ZO-1, while lowering serum interleukin-6 (IL-6), cortisol, and reducing hypothalamic glucocorticoid receptor expression, indicating attenuation of hypothalamic-pituitary-adrenal (HPA) axis hyperactivation and inflammatory responses. Depressive- and anxiety-like behaviors also improved. These phenotypic improvements were accompanied by altered cecal GGP-related pathways, with increased levels of the three bioactive GGPs and redox-related metabolites, alongside a shift in tryptophan metabolism toward protective indole derivatives and 5-hydroxytryptophan, with reduced levels of the neurotoxic metabolites 3-indoxyl sulfate and xanthurenic acid. These findings provide proof-of-concept for a metabolite-guided strategy for postbiotic discovery and demonstrate the capacity of microbial metabolites to modulate gut-brain axis function under carbonyl stress.