Bilayer Nanoencapsulation of Riboflavin-Producing Lactobacillus plantarum B2 Improves Gastrointestinal Survival, Mucoadhesion, and Therapeutic Efficacy in Colitis

Oral administration of probiotic bacteria represents a promising strategy for the prevention and alleviation of colitis; however, its translational potential is frequently constrained by low bacterial viability under gastrointestinal conditions, inadequate mucosal adhesion, oxidative stress, and suboptimal host–microbe interactions at inflamed mucosal sites. To address these limitations, we developed a bilayer nanoencapsulation system for riboflavin-producing L. plantarum B2, consisting of an inner gallic acid–Ca²⁺ metal–polyphenol network and an outer coating of either hyaluronic acid (HA) or bovine serum albumin (BSA). Successful formation of uniform, conformal nanocoatings on the bacterial surface was confirmed by transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), zeta potential and particle size analyses, and laser scanning confocal microscopy (LSCM). Among the coated formulations, HA-coated L. plantarum B2 (H-G- L. plantarum B2) exhibited markedly improved storage stability, enhanced resistance to simulated gastrointestinal fluids, and greater tolerance to hydrogen peroxide-induced oxidative stress, while largely preserving bacterial growth kinetics, acidification capacity, riboflavin production, and isoflavone biotransformation activity during soymilk fermentation. Furthermore, HA-based nanoencapsulation significantly enhanced bacterial adhesion to Caco-2 intestinal epithelial monolayers in vitro and prolonged gastrointestinal retention in mice. In a dextran sulfate sodium (DSS)-induced colitis model, oral administration of nanoencapsulated L. plantarum B2 attenuated body weight loss, colon shortening, histopathological injury, and colonic oxidative stress. Mechanistically, the treatment suppressed the levels of TNF-α, IL-1β, calprotectin, MPO, and MDA; elevated IL-10, CAT, and reduced GSH; restored the expression of tight junction proteins ZO-1 and occludin; and modulated gut microbiota composition. These findings establish bilayer nanoencapsulation as an effective and biocompatible strategy to enhance probiotic oral delivery and highlight the therapeutic potential of riboflavin-producing L. plantarum B2 as a microbiome-targeted intervention for colitis management.