The efficacy of postbiotics vary significantly comparing different strains and preparation processes. We aimed at evaluating the effect of an innovative postbiotic (iPB) generated through the sequential fermentation of Lacticaseibacillus rhamnosus GG and Lacticaseibacillus paracasei NPB-01, compared to single-strain postbiotics, on epithelial barrier integrity and innate immunity in human enterocytes. Comparative evaluation of the effects elicited by iPB or by single-strain postbiotics on gut epithelial barrier using a Caco-2 cells-based experimental model by measuring cell growth proliferation, tight junction proteins (occludin and zonula occludens 1, ZO-1), mucus protein Mucin-2 (Muc-2), and lactase expression. The modulatory action on the production of innate immunity peptide Human Beta-Defensin 2 (HBD-2) via RT-qPCR and ELISA was also comparatively assessed. iPB exposure resulted in a higher up-regulation of occludin, ZO-1, MUC2 and lactase expression, if compared with the single-strain postbiotics, suggesting a beneficial synergistic action in modulating epithelial gut barrier. Furthermore, iPB induced a significantly higher production of HBD-2, suggesting a synergistic enhancement of innate immune response. Our findings suggested that the sequential fermentation process acts as a biotechnological catalyst, optimizing the immunomodulatory action and gut barrier-protective properties of Lacticaseibacillus strains. This study introduces iPB as a high-performance postbiotic candidate for the prevention and management of conditions characterized by alterations of epithelial gut barrier and innate immunity.

The efficacy of postbiotics varies significantly between different strains and preparation processes. We aimed at evaluating the effect of an innovative postbiotic product (iPB) generated through the sequential fermentation of Lacticaseibacillus rhamnosus GG and Lacticaseibacillus paracasei NPB-01, compared to single-strain postbiotics, on epithelial barrier integrity and innate immunity in human enterocytes using a Caco-2-cell-based experimental model by measuring human enterocyte proliferation and differentiation (lactase expression), tight junction proteins (occludin and zonula occludens 1, ZO-1), and mucus protein Mucin-2 (Muc-2) expression. The modulatory action on the major innate immunity peptide, Human Beta-Defensin 2 (HBD-2), production was also assessed. The iPB exposure resulted in a higher up-regulation of human enterocyte proliferation and differentiation, as suggested by higher lactase expression, and of occludin, ZO-1, and MUC2 expression compared with the single-strain postbiotics, suggesting a beneficial synergistic action in modulating the epithelial gut barrier. Furthermore, iPB induced a higher production of HBD-2, suggesting a synergistic enhancement of innate immune response. Our findings suggested that the sequential fermentation process could act as a biotechnological catalyst, optimizing the gut-barrier-protective properties and the immunomodulatory action of Lacticaseibacillus strains. This study introduces iPB as a high-performance postbiotic candidate for the prevention and management of conditions characterized by alterations in epithelial gut barrier and innate immunity.