Results

Screening and Identification of FAEb

MRS‐ethyl ferulate (EFA) opaque solid medium was adopted to screen FAE‐producing strains. Briefly, mouse fecal samples were gradient diluted and spread on MRS‐EFA plates, followed by 72 h anaerobic incubation at 37°C. Strains with distinct transparent hydrolysis zones around colonies were preliminarily selected, as the clear circle diameter directly reflected extracellular feruloyl esterase activity. All candidate single colonies were inoculated into MRS liquid medium for subculture, and the fermentation supernatant of each strain was further verified by the transparent circle method to exclude false‐positive strains. The isolate with the largest transparent zone in both colony and supernatant re‐screening was identified via 16S rRNA gene sequencing. Sequence alignment against the NCBI database confirmed the strain belonged to Lactobacillus johnsonii , which was designated as L. johnsonii 23 for subsequent animal experiments.

Synergistic Effect of RB and FAE‐Producing Bacteria on Disease Symptoms in Colitis Mice

A colitis model was established in mice by inducing with 2% DSS to analyze the synergistic alleviating effects of RB and FAE‐producing bacteria on colitis mice. As shown in Figure 1B, compared with the MD group, the RB, L23, and RL groups all significantly alleviated the decrease in body weight in mice. The DAI of mice in the RB, L23, and RL groups was significantly reduced (p < 0.05), with the most significant decrease observed in the RJ group (Figure 1C). Shortening of colon length is a critical pathological event in DSS‐induced colitis. As can be seen from Figure 1D, compared with the MD group, the colon lengths of the RB, L23, and RL treatment groups were significantly increased (p < 0.05), with a more pronounced increase in the RL group.

Synergistic Effect of RB and FAE‐Producing Bacteria on Organ Indices in Colitis Mice

As shown in Table 1, DSS‐induced colitis led to spleen enlargement in mice. Compared with the MD group, the RB, L23, and RL groups all significantly reduced the spleen index of mice (p < 0.05). However, there were no significant differences in liver and kidney indices between the treatment groups and the control group.

Synergistic Effect of RB and FAE‐Producing Bacteria on Histopathology in Colitis Mice

H&E‐stained colon histological analysis clearly revealed severe damage to the colon of mice with DSS‐induced colitis, characterized by necrosis of epithelial cells, loss of crypt structure, inflammatory cell infiltration, decreased goblet cells, and mucosal erosion and edema (Figure 2A). After treatment, colon tissue recovery was observed in the RB and L23 groups, with reduced epithelial cell necrosis, normal crypt structure, fewer inflammatory cell infiltrates, and increased goblet cell numbers. The RL group exhibited even better improvement, with only minor structural abnormalities in the intestinal tissue, limited epithelial cell necrosis and crypt structure damage, and almost undetectable inflammatory infiltration.

FIGURE 2: Effect of combined intervention of RB and L. johnsonii 23 on colonic histopathology in mice. (A) Colon HE staining. (B) Colonic AB‐PAS staining. Images of colon tissue stained by HE and AB‐PAS were observed at 200× magnification.

Figure 2B shows that the mucus layer of colon tissue in the MD group was severely damaged, and goblet cells and mucin were significantly reduced. The number of goblet cells containing acidic mucopolysaccharides was restored in the RL group. The results indicate that the combined application of rice bran and L. johnsonii 23 can prevent tissue damage induced by DSS by increasing goblet cells in colon tissue and improving the mucus layer.

Synergistic Effect of RB and FAE‐Producing Bacteria on Oxidative Stress in Colitis Mice

The oxidative damage in the colon of mice is shown in Figure 3. Compared with CN, the level of MDA in the MD group was significantly elevated (p < 0.05), while the levels of SOD, GSH, and GSH‐Px were significantly reduced (p < 0.05), indicating that DSS induction led to oxidative stress and disrupted redox homeostasis in the body. After intervention, compared with the MD group, the RB, L23, and RL treatment groups all significantly reduced the level of MDA and significantly increased the levels of SOD, GSH, and GSH‐Px (p < 0.05). Furthermore, compared with the RB and L23 groups, the RL group exhibited stronger resistance to oxidative stress.

FIGURE 3: Effects of combined intervention of RB and L. johnsonii 23 on inflammatory factors. (A) IL‐6. (B) IL‐1β. (C) IL‐10. (D) TNF‐α. The data were expressed as mean ± SEM (n = 8). Mean values with different superscript letters are significantly different (p < 0.05).

The Collaborative Effect of RB and Feruloyl Esterase‐Producing Bacteria on Inflammatory Factors in Mice With Colitis

As shown in the Figure 4, the levels of inflammatory factors in the serum of mice were measured. Compared with the CN group, DSS induction led to a significant increase (p < 0.05) in the expression of proinflammatory factors IL‐6, IL‐1β, and TNF‐α, and a significant decrease (p < 0.05) in the expression of the anti‐inflammatory factor IL‐10, indicating inflammatory damage to the colon. After intervention, compared with the MD group, the RB, L23, and RL treatment groups all significantly reduced the levels of IL‐6, IL‐1β, and TNF‐α (p < 0.05) and increased the level of the anti‐inflammatory factor IL‐10 (p < 0.05). Moreover, the RL group exhibited stronger anti‐inflammatory ability compared to the RB and L23 groups.

FIGURE 4: Effects of combined intervention of RB and L. johnsonii 23 on oxidative stress parameters. (A) MDA. (B) SOD. (C) GSH. (D) GSH‐Px. The data were expressed as mean ± SEM (n = 8). Mean values with different superscript letters are significantly different (p < 0.05).

The Collaborative Effect of RB and Feruloyl Esterase‐Producing Bacteria on SCFAs in the Cecal Contents of Mice With Colitis

Changes in the six SCFAs are depicted in the Figure 5. Compared with the CN group, DSS induction resulted in a consumption of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid (p < 0.05). After intervention, compared with the MD group, the content of acetic acid, propionic acid, and butyric acid significantly increased in the RB group (p < 0.05), while the content of acetic acid and isovaleric acid significantly increased in the L23 group (p < 0.05). Additionally, the content of acetic acid, propionic acid, butyric acid, and valeric acid significantly increased in the RL group (p < 0.05). These results indicate that the collaboration of RB and feruloyl esterase‐producing bacteria can effectively counter the consumption of SCFAs in mice with colitis.

FIGURE 5: Effects of the combined intervention of RB and L. johnsonii 23 on SCFA: (A) Acetic acid; (B) Propionic acid; (C) Isobutyric acid; (D) Isobutyric acid; (E) Isovaleric acid; (F) Valeric acid. The data were expressed as mean ± SEM (n = 8). Mean values with different superscript letters are significantly different (p < 0.05).