Functional attributes of Propionibacterium freudenreichii and phenotypic shift during fermentation in a faba bean-based model medium.

This study characterized fourteen Propionibacterium freudenreichii strains previously isolated from dairy environment for their growth, metabolic performance, enzyme activities, and vitamin B12 biosynthesis in yeast extract-lactate (YEL) and faba bean food-like media (FBM), complemented by genomic and pangenomic analyses to elucidate genetic and functional diversity. In YEL, all strains exhibited mild acidification (pH 6.9-7.1) with significant strain-dependent differences in growth (optical density 0.02-0.70). Eight strains were further selected for FBM fermentation, showing intensive acidification (pH 4.8-5.0) and a 1.9-2.9 log increase in cell density. Fermentation depleted lactate and produced propionate and acetate, while simple sugars (e.g., glucose) were completely utilized and raffinose-family oligosaccharides remained unchanged. Enzyme assays revealed pronounced strain-dependent variation: strain 282 displayed high β-glucosidase and Leu-pNA-hydrolyzing aminopeptidase activity, whereas strain AS9 showed strong Pro-pNA-hydrolyzing but low Leu-pNA-hydrolyzing activity; both lacked detectable phytate-hydrolyzing activity. FBM resulted in higher levels of cyanocobalamin, with 282 producing the highest concentration (131.0 ng/mL), while strains AS9, AS5, and J117 yielded substantial amounts (61.9-65.3 ng/mL). In contrast, YEL was associated with higher levels of pseudovitamin B12. Phenotypic microarray profiling of strains 282 and AS9 indicated broad metabolic capacity in YEL but reduced activity in FBM, with a notable acetate-utilization shift in AS9. Comparative genomics of 282, AS5, AS9, and J117 revealed a large, conserved core genome encoding a redundant intracellular peptidase repertoire, β-glucosidase, and anaerobic cobalamin biosynthesis pathways, while structural variations and accessory genes contributed to strain-specific diversity. Overall, the phenotypic traits of P. freudenreichii strains are governed primarily by quantitative regulatory variation within a conserved genomic framework rather than by the gain or loss of metabolic pathways.