Oral Supplements of Combined Bacillus licheniformis Zhengchangsheng® and Xylooligosaccharides Improve High-Fat Diet-Induced Obesity and Modulate the Gut Microbiota in Rats

Abstract

Gut dysbiosis induced by high-fat diet (HFD) may result in low-grade inflammation leading to diverse inflammatory diseases. The beneficial effects of probiotics and prebiotics on obesity have been reported previously. However, their benefits in promoting human health and the underlying mechanisms still need to be further characterized. This study is aimed at understanding how probiotic Bacillus licheniformis Zhengchangsheng® (BL) and prebiotic xylooligosaccharides (XOS) influence the health of a rat model with HF (60 kcal %) diet-induced obesity. Five groups of male Sprague Dawley (SD) rats were fed a normal fat diet (CON) or an HFD with or without BL and XOS supplementation for 3 weeks. Lipid profiles, inflammatory biomarkers, and microbiota composition were analyzed at the end of the experiment. Rats fed an HFD exhibited increased body weight and disordered lipid metabolism. In contrast, combined BL and XOS supplementation inhibited body weight gain and returned lipid metabolism to normal. Furthermore, BL and XOS administration changed the gut microbiota composition and modulated specific bacteria such as Prevotellaceae, Desulfovibrionaceae, and Ruminococcaceae. In addition, supplements of combined BL and XOS obviously reduced the serum LPS level, which was significantly related to microbial variations. Our findings suggest that modulation of the gut microbiota as a result of probiotic BL and prebiotic XOS supplementation has a positive effect on HFD-induced obesity in rats.

Figure 1

Effects of Bacillus licheniformis (BL) and xylooligosaccharides (XOS) on body composition. (a) Total body weight; (b) total weight gain on days 6 and 21; (c) epididymal and (d) perirenal fat pad weights were normalized against total body weight. The differences were assessed by one-way ANOVA with Tukey's test. ^∗P < 0.05; data represent mean ± SEM of six mice in each group.

Figure 2

Effects of BL and XOS on serum lipid profile and serum and liver lipopolysaccharides. (a) Serum total cholesterol (TC), (b) serum triglyceride (TG), (c) serum low-density lipoprotein cholesterol (LDL-C), (d) serum high-density lipoprotein cholesterol (HDL-C), (e) serum lipopolysaccharides (LPS), and (f) liver LPS levels of rats from different experimental groups were analyzed by ELISA. The differences were assessed by one-way ANOVA with Tukey's test. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗∗P < 0.0001; data represent mean ± SEM of six mice in each group.

Figure 3

Preliminary evaluation of the intestinal microbiota in rats by PCR-denaturing gradient gel electrophoresis (DGGE). (a) DGGE profiles of the V3 region of 16S rRNA gene amplicons derived from fecal DNA of rats in each group. PCR was performed using a F338-GC/R518 set of primers. Lanes indicate the microbial groups in fecal samples from different experimental groups (n = 3) taken at day 21; (b) cluster analysis of the DGGE profiles. The dendrogram was constructed using the UPGMA method.

Figure 4

Evaluation of Illumina HiSeq sequencing data showing that BL and XOS could modulate the overall structure of gut microbiota in HFD-fed rats. (a) Venn diagram of shared and independent bacterial OTUs in different experimental groups (n = 5‐6); (b) comparison of the observed species of different groups; (c) comparison of the Shannon index of different groups; (d) comparison of the Chao1 index of different groups; (e) principal coordinate analysis (PCoA) based on weighted UniFrac distances among different samples. PC1 and PC2 account for 70.78% of the variation; (f) multivariate analysis of variance from PCoA matrix scores using the UPGMA method based on weighted UniFrac distances. ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001.

Figure 5

Composition analysis of gut microbiota at different taxonomic levels among all samples. (a) The average abundance of microbial community in different groups at phylum level; (b) the ratio of Firmicutes to Bacteroidetes; (c) proportion of Bacteroidetes; (d) microbial community bar plot at family level; (e) microbial community bar plot at genus level. All values are mean ± SEM (n = 5‐6). The differences were assessed by one-way ANOVA with Tukey's test. ^∗P < 0.05; ^∗∗P < 0.01.

Figure 6

The analysis of species difference in different groups. The comparison between HF and the other four groups in family (a), genus (b), and species (c) level based on t test. ^∗P < 0.05; ^∗∗P < 0.01.

Figure 7

LEfSe analysis of intestinal microbiota among different groups. (a) LEfSe identified the most differentially abundant bacterial taxons among groups. Group-specific enriched taxa are indicated with a positive LDA score bar with different colors. Only taxa meeting an LDA significant threshold > 4 are shown; (b) taxonomic cladogram obtained from LEfSe analysis of 16S rDNA sequences. The brightness of each dot is proportional to its effect size.

Figure 8

The correlation between intestinal bacterial groups and LPS levels in serum and liver. (a) Canonical correlation analysis (CCA) analysis. Arrows represent the variables serum LPS and liver LPS and indicate the direction and magnitude of the variables associated with bacterial community structure; (b) the correlation between the relative abundance of different microbial groups at genus level and the levels of LPS in the serum and liver was tested by Spearman's correlation method. The positive correlation was displayed as correlation value > 0, and the negative correlation was displayed as correlation value < 0; statistically significant correlation was displayed as ^∗P < 0.05 and ^∗∗P < 0.01.