Alginate Microcapsules Incorporating Recombinant Lactococcus lactis-Montmorillonite Complexes Enhance Bacterial...

Enterotoxigenic Escherichia coli (ETEC) K88 is a primary pathogen causing bacterial diarrhea in livestock, necessitating the development of efficient antibiotic alternatives. We developed a novel recombinant Lactococcus lactis (rLc) -montmorillonite@sodium alginate (rLc-Mt@SA) system using electrospray ionotropic gelation to overcome the low gastrointestinal survival of oral probiotics. To address the high porosity of conventional alginate, montmorillonite (Mt) was incorporated as a nanostructural skeleton to create a tortuous path for acid diffusion, thereby enhancing the barrier properties of the matrix. Microscopic analysis confirmed a stable composite structure, demonstrating a successful encapsulation process with a high encapsulation efficiency of 73.6%. In vitro digestive simulations showed that the rLc-Mt@SA system significantly preserved bacterial viability, yielding a 47.7% survival rate under gastric acid stress, which represents a 3.3-fold increase compared to the 14.5% survival of free bacteria. This enhanced survival ensured efficient intestinal release, leading to significant therapeutic effects in an ETEC K88-challenged mouse model, where rLc-Mt@SA pretreatment effectively attenuated intestinal injury. This protection was attributed to the upregulation of tight junction proteins and the activation of the Nrf2-Keap1 pathway, which played a crucial role in mitigating oxidative stress and restoring intestinal homeostasis. Furthermore, 16S rRNA sequencing and functional prediction revealed that the treatment remodeled the gut microbial landscape, enriching beneficial taxa such as Ligilactobacillus and Lachnospiraceae. This microbial modulation potentially contributed to colonization resistance and was associated with a shift in microbial metabolic networks toward enhanced glycan and energy metabolism. Overall, the Mt-reinforced alginate matrix serves as a high-performance, biocompatible delivery platform that maintains the bioactivity of recombinant strains and restores intestinal homeostasis, offering a promising strategy for managing enteric dysbiosis and a viable alternative to antibiotics in livestock production.