Exopolysaccharide is the potential effector of Lactobacillus fermentum PS150, a hypnotic psychobiotic strain

Abstract

Psychobiotics are a class of probiotics that confer beneficial effects on the mental health of the host. We have previously reported hypnotic effects of a psychobiotic strain, Lactobacillus fermentum PS150 (PS150), which significantly shortens sleep latency in experimental mice, and effectively ameliorate sleep disturbances caused by either caffeine consumption or a novel environment. In the present study, we discovered a L. fermentum strain, GR1009, isolated from the same source of PS150, and found that GR1009 is phenotypically distinct but genetically similar to PS150. Compared with PS150, GR1009 have no significant hypnotic effects in the pentobarbital-induced sleep test in mice. In addition, we found that heat-killed PS150 exhibited hypnotic effects and altered the gut microbiota in a manner similar to live bacteria, suggesting that a heat-stable effector, such as exopolysaccharide (EPS), could be responsible for these effects. Our comparative genomics analysis also revealed distinct genetic characteristics in EPS biosynthesis between GR1009 and PS150. Furthermore, scanning electron microscopy imaging showed a sheet-like EPS structure in PS150, while GR1009 displayed no apparent EPS structure. Using the phenol-sulfate assay, we found that the sugar content value of the crude extract containing EPS (C-EPS) from PS150 was approximately five times higher than that of GR1009, indicating that GR1009 has a lower EPS production activity than PS150. Through the pentobarbital-induced sleep test, we confirmed the hypnotic effects of the C-EPS isolated from PS150, as evidenced by a significant reduction in sleep latency and recovery time following oral administration in mice. In summary, we utilized a comparative approach to delineate differences between PS150 and GR1009 and proposed that EPS may serve as a key factor that mediates the observed hypnotic effect.

Keywords: EPS; Lactobacillus fermentum; PS150; exopolysaccharide; heat-killed; hypnotic; postbiotic.

Figure 1

Strain-specific morphotypes and genomic DNA fingerprinting of L. fermentum PS150 and GR1009. (A) Colony morphologies of PS150 and GR1009. Both strains were spread on MRS agar plates and incubated anaerobically for 48 h. (B) Liquid culture of the two strains after centrifugation at 3,000 × g for 10 min. (C) Random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) PCR fingerprinting profiles of PS150 and GR1009. 1, PS150; 2, GR1009; M, DNA molecular size marker.

Figure 2

The hypnotic properties of L. fermentum PS150, GR1009, and HK-PS150 strains. (A) Schematic view of the experimental design. PS150, GR1009, HK-PS150, and PBS were administered to the mice by oral gavage for 14 days. On day 14, pentobarbital was injected to the mice to evaluate sleep parameters. Time required for losing righting reflex after pentobarbital injection, recovering righting reflex, and restoring mobility are defined as sleep latency, sleep duration, and recovery time, respectively. (B–D) Effects of PS150, GR1009, or HK-PS150 (n = 10 in each group) interventions on the sleep latency, duration, and recovery. Different letters (a, b, or c) above the columns indicate significant differences (p < 0.05) between the five groups. Any two treatments are assigned by the same letter at the top of the graphs, it indicates that there is no statistically significant difference. The comparisons were performed using one-way ANOVA with Tukey’s post hoc test (p < 0.05). The data are expressed as mean ± SEM.

Figure 3

Investigating the effect of PS150 and HK-PS150 supplementation on the microbiota composition. Histogram of relative abundance at the levels of (A) phylum, (B) genus, (C) Lactobacillus fermentum, and (D) Clostridium sp. Culture 54.

Figure 4

Comparison of the eps1 gene region in L. fermentum PS150 and GR1009. (A) Schematic view of the EPS producing cluster in PS150 (112,500–128,000) and GR1009 (112,500–115,400). Detailed description of each locus tag is listed in Table 3. The deletion region of the EPS producing cluster in GR1009 was plotted with a dashed line. (B) PCR amplification of the eps1 cluster region from PS150 or GR1009 with Eps1F/EPS1R primers. M, DNA molecular size marker.

Figure 5

Comparative analysis of EPS levels produced by L. fermentum PS150 and GR1009. (A) The scanning electron microscopy (SEM) images of L. fermentum PS150 and GR1009 at different magnifications (3,000× and 20,000×). Yellow arrows indicate extracellular matrix material. (B) Sugar content in EPS matrix were quantified using carbohydrate assay with optical density (OD) 490 nm in 96-well plates. Data were expressed as mean ± standard error of the mean (SEM). ***p < 0.001 indicates statistical significance verified using an unpaired t-test.

Figure 6

The hypnotic effect of exopolysaccharide-containing crude extracts from L. fermentum PS150 on pentobarbital-induced sleep in mice. (A) Schematic view of the experimental design. The unfermented Mann Rogosa Sharp (MRS) broth was used as the negative control. PBS, diphenhydramine (DIPH), MRS, and exopolysaccharide-containing crude extracts (C-EPS) were administered to the mice by oral gavage for 14 days, followed by the pentobarbital injection to evaluate sleep parameters. (B–D) Effects of C-EPS (n = 5 in each group) intervention on sleep latency, duration, and recovery. Different letters (a and b) above the columns indicate significant differences (p < 0.05) between the four groups. Any two treatments are assigned by the same letter at the top of the graphs, it indicates that there is no statistically significant difference. The comparisons were performed using one-way ANOVA with Tukey’s post hoc test (p < 0.05). The data are expressed as mean ± SEM.