The in situ production of active vitamin B12 was investigated in aqueous cereal‐based matrices with three strains of food‐grade Propionibacterium freudenreichii. Matrices prepared from malted barley flour (33% w/v; BM), barley flour (6%; BF), and wheat aleurone (15%; AM) were fermented. The effect of cobalt and the lower ligand 5,6‐dimethylbenzimidazole (DMBI) or its natural precursors (riboflavin and nicotinamide) on active B12 production was evaluated. Active B12 production was confirmed by UHPLC–UV–MS analysis. A B12 content of 12–37 μg·kg⁻¹ was produced in BM; this content increased 10‐fold with cobalt and reached 940–1,480 μg·kg⁻¹ with both cobalt and DMBI. With riboflavin and nicotinamide, B12 production in cobalt‐supplemented BM increased to 712 μg·kg⁻¹. Approximately, 10 μg·kg⁻¹ was achieved in BF and AM and was increased to 80 μg·kg⁻¹ in BF and 260 μg·kg⁻¹ in AM with cobalt and DMBI. The UHPLC and microbiological assay (MBA) results agreed when both cobalt and DMBI or riboflavin and nicotinamide were supplemented. However, MBA gave ca. 20%–40% higher results in BM and AM supplemented with cobalt, indicating the presence of human inactive analogues, such as pseudovitamin B12. This study demonstrates that cereal products can be naturally fortified with active B12 to a nutritionally relevant level by fermenting with P. freudenreichii.

Propionibacterium freudenreichii is a traditional dairy bacterium and a producer of short chain fatty acids (propionic and acetic acids) as well as vitamin B12. In food applications, it is a promising organism for in situ fortification with B12 vitamin since it is generally recognized as safe (GRAS) and it is able to synthesise biologically active form of the vitamin. In the present study, vitamin B12 and pseudovitamin biosynthesis by P. freudenreichii was monitored by UHPLC as a function of growth in food-like conditions using a medium mimicking cheese environment, without cobalt or 5,6-dimethylbenzimidazole (DMBI) supplementation. Parallel growth experiments were performed in industrial-type medium known to support the biosynthesis of vitamin B12. The production of other key metabolites in the two media were determined by HPLC, while the global protein production was compared by gel-based proteomics to assess the effect of growth conditions on the physiological status of the strain and on the synthesis of different forms of vitamin. The results revealed distinct protein and metabolite production, which reflected the growth conditions and the potential of P. freudenreichii for synthesising nutritionally relevant amounts of active vitamin B12 regardless of the metabolic state of the cells.

Propionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B12. Production of active form of vitamin is a prerequisite for attempts to naturally fortify foods with B12 by microbial fermentation. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Genomic data indicate that P. freudenreichii possesses a fusion gene, bluB/cobT2, coding for a predicted phosphoribosyltransferase/nitroreductase, which is presumably involved in production of vitamin B12. Understanding the mechanisms affecting the synthesis of different vitamin forms is useful for rational strain selection and essential for engineering of strains with improved B12 production properties. Here, we investigated the activity of heterologously expressed and purified fusion enzyme BluB/CobT2. Our results show that BluB/CoBT2 is responsible for the biosynthesis of the DMBI base and its activation into α-ribazole phosphate, preparing it for attachment as the lower ligand of cobalamin. The fusion enzyme was found to be efficient in metabolite channeling and the enzymes’ inability to react with adenine, a lower ligand present in the pseudovitamin, revealed a mechanism favoring the production of the active form of the vitamin. P. freudenreichii did not produce cobalamin under strictly anaerobic conditions, confirming the requirement of oxygen for DMBI synthesis. In vivo experiments also revealed a clear preference for incorporating DMBI over adenine into cobamide under both microaerobic and anaerobic conditions. The herein described BluB/CobT2 is responsible for the production and activation of DMBI. Fusing those two activities results in high pressure towards production of the true vitamin B12 by efficiently activating DMBI formed within the same enzymatic complex. This indicates that BluB/CobT2 is the crucial enzyme in the B12 biosynthetic pathway of P. freudenreichii. The GRAS organism status and the preference for synthesizing active vitamin form make P. freudenreichii a unique candidate for the in situ production of vitamin B12 within food products.