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Most probiotics are difficult to resist the invasion of gastrointestinal physiological and pathological environments, which limits their beneficial effects. The design of a pH-responsive and adhesive double-layer carrier (Carboxymethyl chitosan polyaldehyde polysaccharide, CMCS-DHG/PDA) aims to safeguard the activity of probiotics and enhance their intestinal colonization. The results obtained from UV-vis spectroscopy and XPS analysis revealed the formation of a polydopamine nanocoating surrounding Bacillus subtilis, and the outer carrier formed a Schiff base covalent bond, providing sufficient mechanical properties for the carrier. The carrier exhibited a significantly higher degree of swelling under pH 1.2 compared to pH 7.4, indicating its pronounced pH responsiveness. The CMCS-DHG/PDA carrier not only provided protection for B. subtilis against simulated digestive fluids, but also improved its tolerance to bile and antibiotics. In addition, carrier-protected probiotics showed extraordinary mucosal adhesion, which could significantly improve oral bioavailability and intestinal colonization. Finally, the impact of carrier-protected B. subtilis on gut microbiota was explored, revealing that the carrier protected B. subtilis could significantly improve the diversity, richness, and composition of gut microbiota. Concurrently, it promoted the formation of short chain fatty acids, creating a more beneficial environment for intestinal health., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)