Green Banana Flour Contributes to Gut Microbiota Recovery and Improves Colonic Barrier Integrity in Mice Following Antibiotic Perturbation

Abstract

Green banana flour (GBF) is rich in resistant starch that has been used as a prebiotic to exert beneficial effects on gut microbiota. In this study, GBF was evaluated for its capacity to restore gut microbiota and intestinal barrier integrity from antibiotics (Abx) perturbation by comparing it to natural recovery (NR) treatment. C57B/L 6 J mice were exposed to 3 mg ciprofloxacin and 3.5 mg metronidazole once a day for 2 weeks to induce gut microbiota dysbiosis model. Then, GBF intervention at the dose of 400 mg/kg body weight was conducted for 2 weeks. The results showed that mice treated with Abx displayed increased gut permeability and intestinal barrier disruption, which were restored more quickly with GBF than NR treatment by increasing the secretion of mucin. Moreover, GBF treatment enriched beneficial Bacteroidales S24-7, Lachnospiraceae, Bacteroidaceae, and Porphyromonadaceae that accelerated the imbalanced gut microbiota restoration to its original state. This study puts forward novel insights into the application of GBF as a functional food ingredient to repair gut microbiota from Abx perturbation.

Keywords: antibiotic; green banana flour; gut microbiota; intestinal barrier; recovery.

Figure 1

Experimental scheme. After 1 week of acclimation, the mice were treated with Abx and recovered naturally or with GBF as shown in the scheme. 16S ribosomal DNA (rDNA) sequencing analysis of the feces was performed on the 0, 15, and 30th days. Abx, antibiotics; NR, natural recovery; GBF, green banana flour.

Figure 2

Bacteria localization analysis. (A) Distances of closest bacteria to intestinal epithelial cells per condition. (B–D) Representative pictures of microbiota localization. (B) Antibiotics (Abx) treatment. (C) Natural recovery (NR) group after recovery treatment. (D) Green banana flour (GBF) group after recovery treatment. Mucin-2, green; bacteria, red; and DNA, blue. Significance was determined using a one-way ANOVA corrected for multiple comparisons with the Tukey's test. The values were expressed as the means ± SEM, n = 6 per group, 6 fields per mouse. ***P < 0.001, ****P < 0.0001. ns indicated comparisions that were not significant.

Figure 3

GBF treatment improved the integrity of the intestinal barrier in the colon. (A) Intestinal permeability was measured in vivo with the FITC-dextran method. (B) Reduced intestinal permeability between the NR and GBF groups was normalized and compared. (C) Representative images of the expression of mucin-2 and tight-junction proteins with western blot analysis. (D–G) The statistical analysis of the expression of mucin-2 and tight junction proteins. (H) The relative quantity of mucin-2 messenger RNA (mRNA) was measured with quantitative PCR (qPCR). Significance was determined using the two-tailed unpaired t-tests. The values are expressed as the means ± SEM., n = 6 per group. *P < 0.05, **P < 0.01.

Figure 4

Alpha and beta diversity analysis of gut microbiota in the NR and GBF groups. (A) Alpha diversity analysis of Shannon index from both the groups. (B) Alpha diversity analysis of Pielou's evenness index from both the groups. (C) Weighted UniFrac principal coordinates analysis (PCoA) plot of the microbiota composition from both the groups. (D) Group significance plots (weighted UniFrac distance between the NR and GBF groups). Significance was determined using the two-tailed unpaired t-tests. The values are expressed as the means ± SEM, n = 5 per group. *P < 0.05.

Figure 5

Gut microbiota composition differences at the phylum level. (A) Microbiota composition differences. (B) Only the 4 more abundant phyla were represented and compared. Significance was determined using one-way analysis of variance corrected for multiple comparisons with the Tukey's test, n = 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C–E) The relative abundance of Firmicute, Bacteroidetes, and the Firmicutes/Bacteroidetes ratio from both groups were compared throughout the experiment. Significance was determined using two-tailed unpaired t-tests. The values are expressed as the means ± SEM, n = 5 per group. **P < 0.01.

Figure 6

Gut microbiota composition differences at the family level. (A) Microbiota composition differences. (B) Only the 10 more abundant families were represented and compared. Significance was determined using one-way analysis of variance corrected for multiple comparisons with a Tukey's test, n = 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C–F) The relative abundance of S24-7, Lachnospiraceae, Bacteroidaceae, and Porphyromonadaceae from both groups were compared throughout the experiment. Significance was determined using two-tailed unpaired t-tests. The values are expressed as the means ± SEM., n = 5 per group. *P < 0.05.

Figure 7

Gut microbiota composition differences at the genus level. (A) Microbiota composition differences. (B) Only the 10 more abundant genera were represented and compared. Significance was determined using one-way analysis of variance corrected for multiple comparisons with a Tukey's test, n = 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C–F) The relative abundance of S24-7 unknown genus, Clostridium, Bacteroides, and Parabacteroides from both groups were compared throughout the experiment. Significance was determined using two-tailed unpaired t-tests. The values are expressed as the means ± SEM, n = 5 per group. *P < 0.05.