The Different Ways Multi-Strain Probiotics with Different Ratios of Bifidobacterium and Lactobacillus Relieve Constipation Induced by Loperamide in Mice

Abstract

Constipation is currently one of the most common gastrointestinal disorders, and its causes are diverse. Multi-strain probiotics are often considered a more effective treatment than single-strain probiotics. In this study, a constipation model was constructed using loperamide hydrochloride to evaluate the ability of a multi-strain probiotic combination of four different ratios of Bifidobacterium and Lactobacillus to regulate intestinal flora, relieve constipation, and explore the initial mechanism in mice. After four weeks of probiotic intervention, BM1, BM2, and PB2 effectively relieved constipation; however, the pathways involved were different. The Bifidobacteria-dominated formulations BM1 and BM2 mainly changed the composition and structure of the intestinal flora and significantly decreased the relative abundance of Tyzzerella, Enterorhabdus, Faecalibaculum, Gordonibacter, and Mucispirillum in stool; increased the relative abundance of Parabacteroides and the content of short-chain fatty acids (SCFAs) in stool; restored motilin (MTL) and vasoactive intestinal peptide (VIP) levels; and downregulated interleukin 6 (IL-6) and IL-8 levels in serum. This repaired the inflammatory response caused by constipation. Finally, it promoted peristalsis of the gastrointestinal tract, increasing stool water content, and relieving constipation. While Lactobacillus-dominated formula PB2 mainly restored the levels of serum neurotransmitters (MTL, SP (substance P), VIP and PYY (Peptide YY)) and inflammatory factors (IL-1, IL-6 and IL-8), it significantly decreased the relative abundance of Tyzzerella, Enterorhabdus, Faecalibaculum, Gordonibacter and Mucispirillum in stool; it then increased acetic acid content, thereby reducing the level of inflammation and changing stool properties and gastrointestinal motility.

Keywords: constipation; gastrointestinal regulatory transmitters; probiotic supplements; short-chain fatty acids.

Figure 1

The schedule of the animal experiment.

Figure 2

The effects of different probiotic supplements on the intestinal microflora of mice. (A) Bifidobacterium, (B) Lactobacillus, (C) Enterococcus, (D) Enterobacteriaceae, and (E) Clostridium perfringens. Data are shown as mean ± standard deviation. One-way ANOVA followed by Fisher’s LSD test for control and strain groups compared before and after the intervention, n = 10, **** p < 0.0001; and compared with the model group, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001.

Figure 3

The effects of different probiotic supplements on gastrointestinal indicators in constipated mice. (A) Faecal water content, (B) Time of the first black stool defecation, and (C) Small intestine propulsion rate. Data are shown as mean ± standard deviation. One-way ANOVA followed by Fisher’s LSD test for the control and strain groups compared with the model group; n = 10, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

The effects of different probiotic supplements on serum neurotransmitters in mice. (A) mouse motilin, MTL; (B) gastrin, Gas; (C) substance p, SP; (D) vasoactive intestinal peptide, VIP; (E) somatostatin, SS; (F) Peptide YY, PYY; (G) acetylcholine, Ach; (H) 5-hydroxytryptamine, 5-HT. Data are shown as mean ± standard deviation. One-way ANOVA followed by Fisher’s LSD test for the control and strain groups compared with the model group; n = 10, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

The effects of different probiotic supplements on serum inflammatory factors in mice. (A) Interleukin-1, IL-1; (B) Interleukin-6, IL-6; (C) Interleukin-8, IL-8; (D) Interleukin-10, IL-10; and (E) transforming growth factor-β, TGF-β. Data are shown as mean ± standard deviation. One-way ANOVA followed by Fisher’s LSD test for the control and strain groups compared with the model group; n = 10, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

The effects of different probiotic supplements on the content of SCFAs in the faeces of constipated mice. (A) acetic acid, AA; (B) propionic acid, PA; (C) butyric acid, BA; (D) valeric acid, VA; (E) isobutyric acid, IBA; and (F) isovaleric acid, IVA. Data are shown as mean ± standard deviation. One-way ANOVA followed by Fisher’s LSD test for the control and strain groups compared with the model group; n = 10, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7

The effects of different probiotic supplements on the diversity and structure of the gut microbiota of constipated mice at the phylum level. (A) The Chao1 index of α-diversity; (B) The Simpson index of α−diversity; (C) An unweighted−UniFrac analysis based on the relative abundance of operational taxonomic units (OTUs) was performed to evaluate β−diversity; (D) Phyla of the gut microbiota; (E) The Relative abundance of Firmicutes; (F) The Relative abundance of Bacteroidetes; (G) The Relative abundance of Actinobacteria; (H) The Relative abundance of Verrucomicrobia; and (I) The Relative abundance of Proteobacteria. Data are shown as mean ± standard deviation. One-way ANOVA followed by Fisher’s LSD test for the control and strain groups compared with the model group; * p < 0.05, ** p < 0.01.

Figure 8

The effects of different probiotic supplements on the gut microbiota of constipated mice. (A) Distribution histogram based on LDA (LDA score > 3.0). (B) Differential gut microbiota between the model group and control group. The relative abundances of (C) Tyzzerella, (D) Enterorhabdus, (E) Faecalibaculum, (F) Gordonibacter, (G) Mucispirillum, and (H) Parabacteroides. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 9

A correlation analysis of intestinal microbiota and physicochemical indices in mice. (A) A correlation analysis of gastrointestinal indicators and constipation apparent indices. (B) A network diagram of the Pearson correlation analysis. (C) A Pearson correlation analysis of the correlation between intestinal bacterial abundance and constipation−related biomarkers. A solid line represents a positive correlation, and a dotted line represents a negative correlation. The thickness of the arrow represents the size of the correlation coefficient. (* p < 0.05, ** p < 0.01).