Tethering pH-Induced Structural Modifications of Ovomucin to Stabilize W1/O/W2 Double Emulsions for Enhanced Protection of Companilactobacillus crustorum MN047

Ovomucin (OVM) exhibits pH-responsive conformational changes, aggregating under acidic conditions and extending under alkaline conditions, making it a promising stabilizer for double emulsions (DEs) designed to protect probiotics in the gastrointestinal environment. This study presents an OVM-based DE system for the efficient encapsulation and delivery of Companilactobacillus crustorum MN047. The primary W 1 /O emulsion was formed by homogenizing olive oil with 4% PGPR and sodium chloride solution (8:2 ratio), followed by homogenization with 2.5% pH 9 OVM solution (3:7 ratio) to create the DE. Particle size, zeta potential, rheology, and interfacial tension analyses confirmed the stabilizing role of OVM under alkaline conditions, whereas CLSM and circular dichroism revealed the structural effects of OVM, resulting in increased α-helix and decreased β-sheet contents under acidic conditions. DE achieved an encapsulation efficiency of 93.55%, with protection rates of 82.26% in gastric fluid and 66.82% in intestinal fluid. In a dextran sulfate sodium-induced ulcerative colitis mouse model, OVM-encapsulated C. crustorum MN047 demonstrated enhanced effects in inflammation alleviation, intestinal barrier integrity, and gut microbiota regulation. This study underscores the potential of OVM-based DE systems for probiotic oral delivery and introduces an effective encapsulation approach for gastrointestinal applications.