To improve survival during exposure to adverse conditions, probiotic Bifidobacterium adolescentis 15703T cells were encapsulated in novel mono-core and multi-core phase-separated gelatine-maltodextrin (GMD) microspheres where the gelatine (G) phase was cross-linked with genipin (GP). Microscopy showed that encapsulated cells were exclusively associated with maltodextrin (MD) core(s). Small (average diameter 37 pm) and large (70 mu m) GMD and G microspheres were produced by modulating factors (e.g. mixing speed, surfactant, GP and G concentrations) affecting the size, structural stability and phase-separation. In vitro sequential gastro-intestinal (GI) juice challenge experiments revealed increased survival of cells encapsulated in GMD (similar to 10(6-7) cfu mL(-1)) and G (similar to 10(5) cfu mL-1) microspheres as compared to free cells (similar to 10(4) cfu mL-1). In GMD microspheres, the bacteria derive energy from MD to survive during exposure to acid and bile salts. In conclusion, the novel food grade GMD microencapsulation formulation was shown to protect probiotic bifidobacteria from adverse conditions.