Alginate-based biofilm-assisted encapsulation of a Co-culture of Bifidobacterium longum DSM20219 and Bacillus subtilis SOM8 enhances resistance to gastrointestinal stress.

Oral probiotic formulations are increasingly explored for supporting gastrointestinal health, restoring gut microbiome balance, and providing non-invasive alternatives to injections or transplants. However, delivering oxygen-sensitive probiotics orally remains challenging because gastric acidity, bile salts, digestive enzymes, and aerobic storage can substantially reduce bacterial viability. Sodium alginate is widely used for probiotic encapsulation because it forms mild, cell-compatible hydrogel beads, but alginate-only matrices often provide limited protection for highly sensitive anaerobes due to their hydrated and permeable network structure. Here, we developed an alginate-based, biofilm-assisted encapsulation system for the co-culture and co-delivery of Bacillus subtilis SOM8 and Bifidobacterium longum DSM20219. This strategy enhanced probiotic tolerance to simulated gastric fluids (SGF) and simulated intestinal fluids (SIF), and extended shelf life under aerobic conditions. Biofilm-assisted encapsulation improved B. longum DSM20219 viability, maintaining survival above 7-log CFU/g after 2 h in SGF and SIF, representing an approximately 3-log improvement over alginate-only encapsulation. It also maintained over 6-log CFU/g after 28 days of aerobic storage. Imaging and time-resolved co-culture analyses suggested that the enhanced protection was associated with biofilm formation, cell aggregation, extracellular matrix development, and dynamic interspecies interactions within the alginate system. While further in vivo and omics-based validation will be required, these findings support biofilm-assisted co-encapsulation as a promising biologically integrated strategy for polymer-based probiotic delivery.