Effect of the lower ligand precursors on vitamin B12 production by food-grade Propionibacteria

Abstract

Propionibacterium freudenreichii is the only generally recognized safe (GRAS) bacterium known to synthesize active vitamin B12 and offers previously untapped potential for naturally fortifying foods with vitamin B12. Biosynthesis of the lower ligand 5,6-dimethylbenizimidazole (DMBI) is often a key limiting factor in the production of active vitamin B12 in Propionibacteria. Here, we studied the effect of the natural food-grade precursors of DMBI [riboflavin (RF) and nicotinamide (NAM)] on vitamin B12 production by 27 P. freudenreichii and 3 Propionibacterium acidipropionici strains in whey-based medium. We employed sensitive and selective UHPLC and LC-MS/MS to confirm and quantify the synthesized vitamin B12. In 12 P. freudenreichii strains, co-supplementation with RF (40 µM) and NAM (27 mM) increased the volumetric yield of vitamin B12 up to 4-fold compared to the control cultures. For the majority of these strains, the production level with RF and NAM exceeded the yield obtained with DMBI supplementation (100 µM). The significant positive correlation between RF consumption and vitamin B12 production suggests that RF is proportionally directed towards vitamin B12 biosynthesis. This study shows that the availability of RF and NAM enhances the production of active vitamin B12 by P. freudenreichii in a strain-dependent manner.

In situ production of active vitamin B12 in cereal matrices using Propionibacterium freudenreichii

“…Three dairy‐originated P. freudenreichii strains (designated 256, 263, and 266) were selected based on their differing capabilities to produce B12 (Chamlagain et al., ). First, the cryopreserved cultures (−80°C) were transferred to propionic agar medium (Chamlagain et al., ).…”

“…Three dairy‐originated P. freudenreichii strains (designated 256, 263, and 266) were selected based on their differing capabilities to produce B12 (Chamlagain et al., ). First, the cryopreserved cultures (−80°C) were transferred to propionic agar medium (Chamlagain et al., ). Then, three individual colonies of each strain were subcultured separately three times in whey‐based medium (Chamlagain et al., ) under anaerobic conditions (Anaerocoult; Merck, Germany) at 30°C for 3–4 days.…”

“…First, the cryopreserved cultures (−80°C) were transferred to propionic agar medium (Chamlagain et al., ). Then, three individual colonies of each strain were subcultured separately three times in whey‐based medium (Chamlagain et al., ) under anaerobic conditions (Anaerocoult; Merck, Germany) at 30°C for 3–4 days.…”

“…The matrices were studied for B12 production with and without the addition of Co, Co in combination with DMBI or Co co‐supplemented with RF and NAM (Table ). The supplements Co (5 mg·kg −1 medium), RF (15 mg·kg −1 medium) and NAM (3 g·kg −1 medium) were added on day 0, whereas DMBI (15 mg·kg −1 medium) was provided after 144 hr of incubation (Chamlagain et al., ). Each matrix with or without supplements was studied with three biological replicate cultures of each strain.…”

“…However, DMBI supplementation is a common practice in pharmaceutical B12 production (Martens, Barg, Warren, & Jahn, ). In a recent study, we showed that enhanced B12 production by P. freudenreichii in a whey‐based medium was possible by substituting DMBI with riboflavin (RF) and nicotinamide (NAM) co‐supplementation (Chamlagain et al., ). For natural fortification, avoiding DMBI supplementation for improved B12 production in plant‐based foods by substituting with natural precursors RF and NAM is of significant interest.…”