A butyrate-producing synbiotic mitigates intestinal inflammation in a murine colitis model

Abstract

Inflammatory bowel disease (IBD) is a chronic condition characterized by intestinal inflammation and gut dysbiosis, with limited treatment options primarily focused on immune-modulating therapies. Among potential therapeutic agents, butyrate has emerged as a promising candidate due to its anti-inflammatory and gut-restorative properties. However, direct administration of butyrate poses significant challenges, including its rapid absorption, uneven distribution within the intestinal tract, and an unpleasant odor that reduces patient compliance. To address these issues, we evaluated the therapeutic potential of Bacillus subtilis BM107, a strain selected for its superior butyrate-producing capabilities and established bacterial safety. BM107 efficiently hydrolyzed tributyrin (TB), a butyrate prodrug, producing substantial butyrate levels in TB-supplemented media. In a dextran sodium sulfate-induced colitis mouse model, co-administration of BM107 and the TB diet significantly improved inflammatory indices, such as reduced disease activity index scores, increased colon length, and restored body weight. Additionally, this combination treatment markedly improved gut microbiome composition, restoring microbial diversity and balance. Furthermore, butyrate levels in the cecum contents of the TB + BM107 group were restored to levels comparable to those in healthy controls, demonstrating the ability of this approach to promote gut homeostasis and intestinal recovery. These findings highlight the therapeutic potential of BM107 combined with a TB diet as a safe, effective, and innovative strategy for addressing gut dysbiosis and inflammation in IBD, paving the way for the development of microbiome-based bacterial therapeutics to improve patient outcomes.

Keywords: Bacillus subtilis; IBD; butyrate; esterase; tributyrin.

Figure 1

Screening and characterization of tributyrin (TB)‐degrading strains from human feces. (A) Bacillus subtilis strains isolated from human fecal samples grown on TB agar plates showing strong TB‐degrading ability. Colonies showing clear zones indicate TB degrading activity (++ for strong activity and + for moderate activity). Hemolysis activity tests were conducted to evaluate bacterial safety, where γ (gamma) indicates nonhemolytic and β (beta) indicates hemolytic activity. Only the nonhemolytic strain BM107 was selected for further analysis. (B) Esterase activity of BM107 measured after 8 h of incubation with and without TB. Cell lysates (8h_lysate) showed higher esterase activity compared to the supernatant (8h_sup), demonstrating intracellular localization of the TB‐degrading enzyme. (C) Growth curve of BM107 cultured in Tryptic Soy Broth with or without TB supplementation. The presence of TB did not significantly affect the growth rate of BM107. (D) Short‐chain fatty acid production during BM107 growth. Butyric acid concentration increased in a time‐dependent manner, indicating efficient TB utilization by BM107. (E) Clear zone formation on TB agar plates by Escherichia coli DH5α transformed with the pCDF::estB plasmid compared to a negative control with a green fluorescent protein (GFP)‐encoding sequence in place of the estB gene (pCDF::gfp), confirming the functional role of EstB in TB degradation. The antibiotic resistance tests were conducted three time with indepenent trials.

Figure 2

Comparison of therapeutic effects between Bacillus subtilis KCTC 3135 and BM107 in a dextran sodium sulfate (DSS)‐induced colitis model. (A) Schematic representation of the experimental design for DSS‐induced colitis in 12‐week‐old male C57BL/6 mice. Mice were divided into four groups: naïve (no DSS, normal chow [NC] diet, phosphate‐buffered saline [PBS] gavage), NC + PBS (DSS, NC diet, PBS gavage), TB + KCTC 3135 (DSS, TB diet, KCTC 3135 gavage), and TB + BM107 (DSS, TB diet, BM107 gavage). Treatments were administered daily from Day 5 to Day 9 as shown. (B) Disease activity index (DAI) scores assessed on the final day (Day 14). Both of TB + BM107 and TB + KCKC 3135 showed significant lower DAI scores compared to the NC + PBS. (C) Colon length measured after mice were killed on Day 14. The TB + BM107 group showed a significant improvement in colon length compared to the NC + PBS and TB + KCTC 3135 groups. (D) Representative hematoxylin and eosin‐stained images of colon tissues from each group. Black arrows indicate regions of tissue damage and inflammatory infiltration. The TB + BM107 group demonstrated greater recovery of colon architecture compared to other groups. Scale bars, 50 μm. The data are presented as means ± standard deviation (SD). All statistical analyses were performed using GraphPad Prism version 9.4.1 (GraphPad Software Inc.). Differences between groups were assessed using a One‐way analysis of variance or Student's t‐test. A p‐value of <0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 3

Therapeutic effects of BM107 combined with TB in a DSS‐induced colitis model. (A) Schematic representation of the experimental design for DSS‐induced colitis in 9‐week‐old male C57BL/6 mice. Mice were divided into five groups: naïve (no DSS, NC diet, PBS gavage), NC + PBS (DSS, NC diet, PBS gavage), NC + BM107 (DSS, NC diet, BM107 gavage), TB + PBS (DSS, TB diet, PBS gavage), and TB + BM107 (DSS, TB diet, BM107 gavage). Treatments were administered daily from Day 5 to Day 9. (B) DAI scores lower in the TB + BM107 group compared to the other DSS‐treated groups at Day 14. (C) Changes in body weight during the experimental period presented as percentages of initial body weight. The TB + BM107 group showed less body weight loss compared to the other DSS‐treated groups . (D) Colon length measured after mice were killed, showing significantly longer colons in the TB + BM107 group compared to the NC + PBS and TB + PBS groups. (E) Representative images of excised colons from each group demonstrating improved colon morphology in the TB + BM107 group. (F) Representative histological images of colon tissues stained with hematoxylin and eosin and Alcian Blue‐periodic acid‐Schiff staining (scale bars, 50 μm). Black arrows highlight tissue damage and goblet cell depletion. The TB + BM107 group showed the greatest recovery of tissue structure and goblet cell abundance. Statistical significance was determined using one‐way analysis of variance. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

Biochemical assessment of BM107 and TB co‐treatment in a DSS‐induced colitis model. (A) Butyric acid concentrations in the cecum contents of mice. The TB + BM107 group showed significantly restored butyrate levels, comparable to those of the naïve group, highlighting the effectiveness of BM107 and TB co‐treatment (*p < 0.05, t‐test). (B) Epithelial barrier integrity assessed using fluorescein isothiocyanate (FITC)‐dextran in serum. The TB + BM107 group demonstrated significantly reduced fluorescence intensity compared to the NC + PBS group, indicating improved epithelial barrier recovery (****p < 0.0001, one‐way analysis of variance).

Figure 5

Alterations in gut microbiome composition in a DSS‐induced colitis model. (A) Changes in the microbiome composition of cecum contents analyzed at the genus level across naïve, NC + PBS, and TB + BM107 groups. A heatmap generated using GraphPad Prism illustrates the relative abundance of major genera, with red indicating higher abundance and white indicating lower abundance. (B) Relative abundance of Akkermansia muciniphila and Turicibacter bilis at the species level. The TB + BM107 group showed significant differences compared to the NC + PBS group (*p < 0.05, **p < 0.01, one‐way analysis of variance). (C) β diversity analysis visualizing gut microbiota compositional differences among naïve, NC + PBS, and TB + BM107 groups. Distinct clustering of microbiota composition was observed between the groups.