Solid-state co-fermentation enables exceptionally high vitamin B12 enrichment but limited bioaccessibility in faba...

Plant-based diets require sustainable strategies to ensure adequate vitamin B12 (B12) intake, as this micronutrient is naturally absent from plant foods. This study evaluated solid-state co-fermentation of faba beans with Propionibacterium freudenreichii and the filamentous fungi Rhizopus oligosporus or Aspergillus oryzae as a natural B12 fortification approach for developing legume-based foods with improved micronutrient profiles. Co-fermentation increased biologically active B12 to 2.1-2.2 μg/g dry weight after 66 h, representing a 170-200-fold increase compared with bacterial monoculture and among the highest levels reported for plant-based fermentations. Thiamin, riboflavin, niacin and folate contents also increased. Thiamin, riboflavin, and niacin showed high apparent bioaccessibility during in vitro digestion, whereas folate bioaccessibility remained relatively low (39-41%), consistent with previous reports for legume matrices. B12 bioaccessibility was moderate (42-54%) after fermentation despite the high total B12 content, consistent with possible retention within microbial cells and/or association with the fermented matrix. Organic acid profiles indicated distinct species-dependent metabolic interactions. These findings demonstrate that fungal-bacterial co-fermentation enables substantial B12 enrichment of legumes while highlighting the importance of coupling efficient biosynthesis with enhanced vitamin release. The resulting high B12 concentrations support the potential of faba bean-based products to contribute nutritionally meaningful amounts of B12 in increasingly plant-based diets. Further development should focus on improving B12 release and optimizing sensory and rheological properties while reducing antinutritional factors to enhance nutritional quality and consumer acceptance.