Programmed probiotic delivery via chitosan–lipid bilayer coatings toward microenvironmental homeostasis and inflammatory regulation

The limited robustness of probiotics during food processing and gastrointestinal transit remains a major obstacle to their effective formulation and delivery, highlighting the need for coating strategies that integrate complementary protective functions at the bacterial surface. In this study, a hierarchical chitosan–lipid bilayer was constructed on individual Lacticaseibacillus rhamnosus GG cells through sequential interfacial assembly. Unlike matrix-type encapsulation or simple single-layer coatings, this design directly organized an electrostatically anchored chitosan inner layer and a membrane-like lipid outer layer on the bacterial surface, thereby combining hydration buffering with barrier protection within a single-cell interface. Structural characterization supported the sequential formation of a chitosan–lipid coating closely associated with the bacterial surface, while normal growth behavior was maintained. Compared with uncoated and single-layer-coated counterparts, the bilayer-coated probiotics exhibited consistently enhanced resistance to multiple processing- and digestion-relevant stresses, including thermal treatment, freeze–thaw cycling, storage, and simulated gastrointestinal digestion. The bilayer architecture also improved the retention of intracellular enzyme activity after freeze-drying, indicating better maintenance of cellular integrity. In addition, heat-inactivated bilayer-coated bacteria displayed a more favorable anti-inflammatory cytokine profile in macrophage models, as evidenced by reduced TNF-α production and increased IL-10 secretion, suggesting better retention of structure-associated immunomodulatory activity. Overall, these results indicate that a hierarchically organized polysaccharide–lipid interface can improve probiotic stability and functional maintenance under in vitro processing- and digestion-relevant conditions, providing a useful design basis for single-cell probiotic coating strategies.