Production and analysis of exopolysaccharides from Bifidobacterium bifidum and Bifidobacterium animalis subsp. lactis: Genomic basis, structure, and prebiotic properties

This study investigates the production and prebiotic properties of exopolysaccharides (EPS) from Bifidobacterium bifidum 3469 (BBF-EPS) and B. animalis subsp. lactis 9749 (BAL-EPS). To eliminate interference from exogenous polysaccharides present in conventional MRS-c broth, EPS were produced using a modified medium (MRS-TY) lacking complex carbohydrate components, enabling accurate structural characterization of the intrinsically synthesized EPS. Whole-genome sequencing revealed fundamentally different eps biosynthetic gene clusters between the strains. The structural analysis indicated that the compact gene cluster of B. bifidum 3469 was associated with a simpler EPS (molecular weight 1.98 ×10 5 Da; consisting of glucose, galactose, rhamnose, and mannose in a 1.00:0.78:0.34:0.25 molar ratio), whereas the large, complex cluster of B. animalis subsp. lactis 9749 was associated with the production of a galactose-rich EPS (molecular weight 2.02 ×10 5 Da; consisting of galactose, rhamnose, glucose, and mannose in a 5.47:1.46:1.00:0.86 molar ratio). Both EPS supported growth of Lactobacillus strains. In vitro fecal fermentation demonstrated that BBF-EPS enriched Megamonas and Faecalibacterium , leading to increased propionic acid production (51.77 mM). In contrast, BAL-EPS enriched Bifidobacterium and Blautia , enhancing butyric acid (28.67 mM) production. These findings highlight the importance of culture medium optimization for reliable characterization of bifidobacterial EPS, supporting their potential application as targeted prebiotics for intestinal health.