Outperforming Probiotic Rivals: Bacillus Velezensis Consortium Leads in Gut Adhesion and Inflammation Control

The synergistic potential of multi-strain probiotics remains poorly defined. Our previous studies demonstrated that three individual Bacillus velezensis strains enhanced tight junction integrity and promoted epithelial wound healing, suggesting strong immune-modulatory properties. This study investigates whether combining these strains into a consortium (C1) provides superior efficacy compared to other commercial formulations, such as B. licheniformis/B. subtilis mix (C2) and Clostridium butyricum (C3). The objective was to compare the epithelial adhesion capacity and cytokine modulation profiles of C1 with C2 and C3 in porcine epithelial (IPEC-J2) and immune (3D4/21) cell models. Adhesion assays were performed by co-culturing bacterial strains with IPEC-J2 cells for 30 minutes at 37 °C. After washing and cell lysis, adherent bacteria were quantified via serial dilution and plating. For cytokine profiling, cell-free supernatants (CFS) from C1, C2, and C3 were applied to IPEC-J2 and 3D4/21 cells at concentrations equivalent to 1 × 107 CFU/mL. After 24-hour incubation, cytokine expression was analyzed using qRT-PCR and proteomics. Results indicate that C1 strains exhibited significantly higher adhesion to IPEC-J2 cells, with adhesion rates of 0.015 ± 0.006% (strain A), 0.011 ± 0.004% (strain B), and 0.014 ± 0.004% (strain C), compared to control strains CC1 (0.001 ± 0.001%) and CC4 (0.000 ± 0.000%, P < 0.01). C2 and C3 showed lower adhesion (0.001–0.011%), indicating reduced colonization potential. In IPEC-J2 cells treated with C1 CFS, cells significantly expressed reduced IL-6 (94.1 ± 2.5% vs. control: 100 ± 0.0%, P < 0.05) and increased TGF-β (107.6 ± 2.9%, P < 0.05). Proteomics confirmed elevated anti-inflammatory cytokine IL-27α (4839.8 ± 455.3 vs. 3177.0 ± 357.0) and Suppressor of Cytokine Signaling 3 (SOCS3) (14439.1 ± 820.3 vs. 10666.0 ± 435.0, P < 0.05). C2 and C3 did not induce significant changes. In unstimulated 3D4/21 cells, C1 reduced pro-inflammatory cytokines including Interferon-induced protein (35187.5 ± 7280.8 vs. 111538.0 ± 62903.0) and TNF ligand superfamily member (74336.8 ± 1919.6 vs. 87092.0 ± 3306.0), while enhancing Galectin (1399844.0 ± 99284.7 vs. 1160881.0 ± 35049.0), a key regulator of macrophage activation and immune cell recruitment. In LPS-stimulated 3D4/21 cells, C1 suppressed IL-6 (37869.2 ± 1447.9 vs. 42110.9 ± 1456.3) and upregulated SOCS3 (219667.6 ± 24966.6 vs. 160183.7 ± 12770.8) and TGFB-induced factor homeobox 2 (53398.4 ± 5986.8 vs. 37202.4 ± 4548.4, P < 0.05). Neither C2 nor C3 produced comparable anti-inflammatory responses in any model. In conclusion, the Bacillus velezensis consortium (C1) demonstrated superior epithelial adhesion and consistent anti-inflammatory activity across both epithelial and immune cell models. Its ability to suppress pro-inflammatory cytokines and enhance anti-inflammatory mediators confirms strain compatibility and synergistic efficacy. These findings position C1 as a leading candidate for antibiotic-free swine production and intestinal immune regulation.