Examining the Gastrointestinal and Immunomodulatory Effects of the Novel Probiotic Bacillus subtilis DE111

Abstract

Probiotics make up a large and growing segment of the commercial market of dietary supplements and are touted as offering a variety of human health benefits. Some of the purported positive impacts of probiotics include, but are not limited to, stabilization of the gut microbiota, prevention of gastrointestinal disorders and modulation of the host immune system. Current research suggests that the immunomodulatory effects of probiotics are strain-specific and vary in mode of action. Here, we examined the immunomodulatory properties of Bacillus subtilis strain DE111 in a healthy human population. In a pilot randomized, double blind, placebo-controlled four-week intervention, we examined peripheral blood mononuclear cells (PBMCs) at basal levels pre- and post-intervention, as well as in response to stimulation with bacterial lipopolysaccharide (LPS). We observed an increase in anti-inflammatory immune cell populations in response to ex vivo LPS stimulation of PBMCs in the DE111 intervention group. Overall perceived gastrointestinal health, microbiota, and circulating and fecal markers of inflammation (Il-6, sIgA) and gut barrier function (plasma zonulin) were largely unaffected by DE111 intervention, although the study may have been underpowered to detect these differences. These pilot data provide information and justification to conduct an appropriately powered clinical study to further examine the immunomodulatory potential of B. subtilis DE111 in human populations.

Keywords: Bacillus subtilis; flow cytometry; gastrointestinal health; immune cells; peripheral blood mononuclear cells (PBMC); probiotic; short chain fatty acid.

Conflict of interest statement

The study was funded by Deerland Enzymes and Probiotics, Kennesaw, GA and TLW became a member of their Scientific Advisory Council in November 2020. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. They did provide the treatment capsules for the study, conducted the treatment blinding, and supplied qPCR primer sequences for the B. subtilis DE111.

Figures

Figure 1

Consort flow diagram of study recruitment, randomization and completion.

Figure 2

Symptom severity scores were based on answers to a GI symptom questionnaire by Metagenics [16] that is designed to provide a functional measure to assess symptoms associated with gastric function (A), gastrointestinal inflammation (B), small intestine/pancreas pain (C), and colon pain (D). Error bars represent ±SEM, # indicates a statistical trend (p = 0.051–0.08).

Figure 3

Levels of B. subtilis DNA detected in fecal samples collected pre- and post-intervention (A) and the change in logDNA from baseline between the two intervention groups (B) indicate that DE111 consumption increases B. subtilis in the GI tract. However, four weeks of DE111 consumption did not significantly alter the composition and structure of the gut microbiota (C). Error bars represent ±SEM. * indicates p-values <0.05 and ** indicate p-values <0.01. Ellipses in the PCoA represent the 95% CI.

Figure 4

The major microbially-produced short chain fatty acids (SCFA), acetate (A), propionate (B), and butyrate (C) quantified in feces at baseline and post-intervention. Error bars represent ±SEM, *denotes p < 0.05.

Figure 5

Basal immune status at baseline and post-intervention in Placebo and DE111 groups: Total CD3+ T cell populations (A), CD4+ T regulatory subpopulations (B), T cell variants (C), and myeloid and B cells (D). Error bars represent ± SEM. * denotes p < 0.05, **denotes p < 0.01, and # denotes a p-value between 0.05–0.08.

Figure 6

Cellular responses to LPS stimulation, calculated as stimulated/basal cell counts for CD25+ (A) and CD25+FoxP3+ (B) regulatory T cell populations, as well as double-positive CD4+CD8+ T cells (C) and CD8+ cytotoxic T cells (D). Error bars represent ± SEM. * denotes p < 0.05 and # denotes a p-value between 0.05–0.08.