PAN, M.; Ye, C.; Song, Y.; Tian, M.; Wang, R.; Chen, P.

Background: Folate deficiency is a global nutritional problem associated with multiple adverse health outcomes, including impaired one-carbon metabolism and elevated homocysteine levels (hyperhomocysteinemia). Gut microbiota-mediated folate biosynthesis has emerged as a promising strategy for improving host folate status. This study aimed to isolate folate-producing probiotic strains, clarify their folate synthesis mechanisms, and evaluate their regulatory effects on folate metabolism and gut microbiota in folate-deficient mice. Methods: High-throughput cultivation and screening were performed to isolate folate-producing probiotics. Whole-genome sequencing, pathway reconstruction, and metabolite profiling in fermented milk were used to explore folate biosynthesis pathways and potential microbial cross-feeding interactions. A folate-deficient mouse model was established to evaluate the effects of a probiotic cocktail on serum folate, homocysteine (Hcy) levels, and gut microbiota composition using microbiological assays, biochemical analyses, qPCR, 16S rRNA gene sequencing, alpha diversity analysis, principal coordinates analysis (PCoA), and Linear discriminant analysis Effect Size (LEfSe) analysis. Results: Over 1,000 bacterial isolates were obtained, and over 10 strains, mainly belonging to Lactobacillus, Bifidobacterium, and Bacillus, showed folate production levels above 100 ng/mL. Genomic analysis revealed that most selected probiotic strains lacked genes involved in para-aminobenzoic acid (pABA) biosynthesis but retained downstream folate synthesis modules, suggesting a potential dependence on pABA-producing gut commensals for precursor supply through microbial cross-feeding. In fermented milk, probiotic strains mainly produced bioactive folates (5-MeTHF and THF), with strain-specific production capacities; L. plantarum, W. coagulans, and B. animalis subsp. lactis significantly increased 5-MeTHF levels in fermented milk. In vivo, high-dose probiotic intervention significantly elevated serum folate (p<0.01) and reduced Hcy (p<0.05) in folate-deficient mice, while medium-dose intervention showed no significant effects. The probiotic strains colonized the mouse gut in a dose-dependent manner: high-dose group exhibited >4,000-fold increase in relative abundance (Bifidobacteriaceae and Bacillaceae enriched), medium-dose group only enriched Bacillaceae, and low-dose group showed no effective colonization. High dose probiotic treatment enhanced gut microbial species diversity (increased Shannon index) and restored folate deficiency-induced gut microbiota dysbiosis (PCoA clustering closer to normal group). Conclusion: This study screened high folate-producing probiotic strains and demonstrated their ability to synthesize active 5-MeTHF, which may rely on microbial cross-feeding in gut microbiota. Furthermore, we demonstrated that folate-producing probiotic intervention significantly improves folate status and Hcy metabolism and restores gut microbiota homeostasis in folate-deficient mice. These findings suggensted that such probiotics could serve as a safer, more physiological intervention for folate deficiency and hyperhomocysteinemia, especially in populations with MTHFR polymorphisms.