A Probiotic Bacillus velezensis Consortium Exhibits Superior Efficacy over Two Alternative Probiotics in Suppressing Swine Pathogens and Modulating Intestinal Barrier Function and Immune Responses In Vitro

Despite increasing interest in probiotics as antibiotic alternatives in swine production, few studies have directly compared the functional efficacy of different commercial probiotic formulations under controlled conditions. We conducted an in vitro study using porcine intestinal epithelial (IPEC-J2) and macrophage-like (3D4/21) cell models to compare the efficacy of three commercial probiotic consortia (C1: three strains of <i>Bacillus velezensis</i>; C2: <i>B. licheniformis</i> + <i>B. subtilis</i>; C3: <i>Clostridium butyricum</i>). Treatments were evaluated for their ability to inhibit pathogenic <i>Escherichia coli</i>, <i>Clostridium perfringens</i>, and <i>Salmonella</i> spp., enhance epithelial barrier integrity, and modulate immune responses. Experimental endpoints included pathogen inhibition assays, adhesion to IPEC-J2 cells, transepithelial electrical resistance (TEER), tight junction protein expression, and cytokine profiling via RT-qPCR and proteomics. Data were analyzed using the Kruskal-Wallis test with false discovery rate (FDR) control at 5%. C1 cell-free supernatant (CFS) strongly inhibited pathogen growth (84.8 ± 5.3% inhibition of ETEC F4⁺F18^- vs. medium control; <i>p</i> < 0.05), whereas C2 had no effect, and C3 inhibited only one isolate. The coculture of IPEC-J2 cells with C1 CFS increased the expression of TJ proteins ZO-1, MUC13, and MUC20 (+12.9-46.6% vs. control; <i>p</i> < 0.001) and anti-inflammatory TGF-β; reduced pro-inflammatory IL-6 in LPS-stimulated 3D4/21 cells. In comparison, C2 and C3 showed minimal impact on epithelial barrier integrity and immune modulation, as indicated by negligible changes in TEER values, tight junction protein expression (ZO-1, MUC13, MUC20), and cytokine profiles relative to the control. In conclusion, C1 demonstrated greater in vitro efficacy than C2 (<i>B. licheniformis</i> + <i>B. subtilis</i>) and C3 (<i>Clostridium butyricum</i>), including pathogen inhibition assays, epithelial adhesion, TEER measurements, and cytokine modulation, suggesting its potential as a leading candidate for functional probiotic applications.