Probiotics play a critical role in maintaining human health homeostasis, yet their oral delivery faces challenges due to poor gastrointestinal survival, uncontrolled release, and inefficient targeted colonization. To address these limitations, we developed colon-targeted mucoadhesive (sCS-BC)/OKGM-SA microspheres using a W1/O/W2 double emulsion technique combined with ionic crosslinking, employing oxidized konjac glucomannan (OKGM), thiolated chitosan (sCS), and bacterial cellulose (BC). In vitro digestion assays revealed that the microspheres effectively shielded probiotics under simulated gastric and bile salt, while enabling pH- and enzyme-responsive release in the intestinal, achieving a viable probiotic count of 1.5 × 10 CFU/mL. Rheological characterization and in vivo gastrointestinal transit studies demonstrated that the microspheres enhanced colonic colonization through interactions with the intestinal mucus layer. Histological analysis further indicated that the microspheres stimulated colonic goblet cell proliferation and mucus layer formation. Metagenomic and metabolomic profiling confirmed that oral administration of the probiotic-loaded microspheres markedly enriched gut microbial diversity and helped preserve intestinal barrier integrity, showing potential in modulating gut immune function. The (sCS-BC)/OKGM-SA system integrates upper gastrointestinal protection, colon-targeted delivery, mucus adhesion, and probiotic proliferation, offering a novel strategy for targeted probiotic delivery. This work establishes a foundational framework for designing next-generation colon-targeted probiotic carriers and underscores their therapeutic promise in modulating intestinal ecosystems. 8
