High-Efficiency Layer-by-Layer Microencapsulation of <i>Lactobacillus sakei</i> J15 Using Milk Protein–Hyaluronic Acid Conjugates and Chitosan to Enhance Probiotic Protection

To enhance the stability and intestinal delivery of the probiotic <i>Lactobacillus sakei</i> J15, a multilayer microencapsulation system was developed using layer-by-layer (LBL) technology, using milk protein-hyaluronic acid (HA) conjugates (casein-HA or β-lactoglobulin-HA) and chitosan (CHI). The microcapsules exhibited high encapsulation efficiency (up to 98.65%) and significantly improved probiotic viability under harsh conditions, including acid, bile salt, simulated gastrointestinal digestion, and heat. Electrostatic interaction was confirmed as the main force binding the amino groups in the conjugate to the carboxyl groups of CHI, and the surface charge of the samples reversed as a zigzag pattern whenever another outermost layer was added. The (CHI/CN-HA/CHI)₃ and (CHI/β-Lg-HA/CHI)₃ microcapsules exhibited the best adhesion to Caco-2 cells, reaching 24.47 and 26.71 CFU/cell, respectively. Microcapsules with two or four layers (milk protein-HA as the outer layer) showed superior resistance to environmental stresses, while three-layer capsules (chitosan as the outer layer) exhibited the strongest adhesion to Caco-2 cells.