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Folic acid, or vitamin B9, is involved in appropriate regulation of DNA replication, synthesis of purines and deoxythymidine (dTMP), conversion of homocysteine to methionine, histidine catabolism, and correct differentiation of the neural tube during fetal organogenesis. Folic acid from food sources is almost completely absorbed in the small intestine, mostly in the jejunum, and does not reach the large intestine. The administration of probiotic strains able to synthesize folates de novo and release them in the extracellular space may provide an additional, constant endogenous source of this important vitamin in the intestinal lumen of humans.A pilot study involving 23 healthy volunteers was conducted to evaluate the ability of 3 probiotic strains, Bifidobacterium adolescentis DSM 18350, B. adolescentis DSM 18352, and Bifidobacterium pseudocatenulatum DSM 18353, to produce folates in the human intestine. Volunteers were randomly assigned to 1 of 3 groups for treatment with a specific probiotic strain (5×109 colony forming units/d). Strain effectiveness was evaluated by determination of the folate concentration in feces evacuated within 48 hours before and after administration of the probiotics. Quantification of microorganisms belonging to the genus Bifidobacterium was performed in parallel to folate analysis.Ingestion of these probiotic strains resulted in a significant increase of folic acid concentration in human feces in all treated groups. Analysis of the fecal Bifidobacteria confirmed the potential of all strains, especially B. adolescentis DSM 18352, to colonize the intestinal environment.The demonstrated ability of the probiotic microorganisms B. adolescentis DSM 18350, B. adolescentis DSM 18352, and B. pseudocatenulatum DSM 18353 to synthesize and secrete folates in the human intestinal environment may provide a complementary endogenous source of such molecules, which is especially useful for the homeostasis of mucosal enterocytes of the colon and, unlike oral administration of the vitamin, ensures its constant bioavailability.