Peppermint oil alters gut microbes and gas production in vitro

Peppermint oil reduced microbial gas production and shifted the composition of human gut bacterial communities in an anaerobic laboratory fermentation system, according to an exploratory study that also confirmed antispasmodic and anti-inflammatory effects in animal tissue and cultured macrophages. The researchers incubated stool samples from six healthy adults with the oil for 48 hours at concentrations of 0.2, 2 and 20 milligrams per milliliter. The two higher concentrations produced the clearest changes: total bacterial cell counts and gas output fell, while particular genera increased or decreased relative to donor-matched untreated controls. Because the experiment did not include people with irritable bowel syndrome, it cannot establish that peppermint oil treats IBS through the microbiome or produces the same changes in patients.

The work combined several non-clinical models to examine complementary effects of peppermint oil, whose main active component is L-menthol. In isolated circular smooth-muscle strips from the proximal colon of five male guinea pigs, the oil relaxed contractions at 10 and 100 micrograms per milliliter by 13% and 24%, respectively, compared with the solvent control. In RAW 264.7 mouse macrophages stimulated with lipopolysaccharide, concentrations of 5 and 10 micrograms per milliliter suppressed tumor necrosis factor secretion by at least 10% and reduced inducible nitric oxide production by as much as about 50%. These supportive experiments reproduced established pharmacological effects but did not test clinical outcomes or demonstrate benefits in patients.

Most of the exploratory work focused on direct interactions between peppermint oil and the gut microbiota. Each healthy donor’s fecal inoculum was placed in a closed, anaerobic Colon-on-a-Plate system containing a carbohydrate-depleted medium designed to represent the colon. Mucus-coated carriers represented a mucosal layer, while gas pressure measured overall fermentative activity during the 48-hour incubation. The tested oil, batch BNO 2910, contained 43.2% menthol, 25.5% menthone, 5.8% 1.8-cineole and other constituents. The middle concentration of 2 milligrams per milliliter was close to a theoretical estimate of 1.4 to 2.2 milligrams per milliliter in the colon after a recommended daily dose of 546 milligrams; 0.2 and 20 milligrams per milliliter extended the range by tenfold below and above that estimate.

Gas production did not change significantly at 0.2 milligrams per milliliter but was lower at 2 and 20 milligrams per milliliter, with p-values of 1.77 × 10⁻⁹ and 1.68 × 10⁻⁹ against the respective untreated controls. Total bacterial cell counts also declined significantly at both higher concentrations, each with a p-value of 7.84 × 10⁻⁶. By contrast, alpha-diversity measures—community richness, evenness and Shannon diversity—were significantly increased at 2 milligrams per milliliter and unchanged at the other two concentrations. The investigators noted that diversity metrics alone cannot establish functional stability or health benefits, particularly because they were measured in a simplified fermentation system rather than in a living gastrointestinal tract.

Community profiling combined 16S ribosomal RNA gene sequencing with flow-cytometric counts of total bacterial cells. This allowed the investigators to estimate absolute abundance rather than relying only on the proportions generated by sequencing. Baseline communities differed considerably among donors, with the first two principal components explaining 58% of the variation. Bacillota, Bacteroidota and Actinomycetota predominated, while commonly detected genera included Subdoligranum, Agathobacter, Blautia, Faecalibacterium, Bacteroides and Bifidobacterium. After 48 hours, profiles treated with 0.2 milligrams per milliliter remained close to donor-specific untreated controls, whereas the 2 and 20 milligram-per-milliliter treatments produced distinct and largely similar shifts. Treated communities at the higher concentrations also clustered closer to their pre-incubation profiles than untreated samples did.

Differential-abundance analysis identified 18 of 37 genera that changed significantly at at least one concentration; 16 changed at both 2 and 20 milligrams per milliliter, while two changed only at the highest concentration. Escherichia-Shigella and Eggerthella had lower absolute abundances at both higher concentrations. Faecalibacterium and Blautia were maintained at higher absolute abundances than in corresponding controls, and donor-paired analyses indicated that the trends were not driven solely by an outlier. Adjusted q-values for Escherichia-Shigella at 2 and 20 milligrams per milliliter were 0.002 and 0.001, while those for Eggerthella were 0.004 and 0.001. Faecalibacterium had adjusted q-values of 0.007 and 0.010, and Blautia had values of 0.008 and 0.006.

The taxonomic shifts carried different implications depending on the organisms involved. Some Escherichia-Shigella species are pathogens, and increases in the group have been associated with inflammatory microbial profiles; Eggerthella species participate in transformations of bile acids, drugs and other compounds. Faecalibacterium, including the species F. prausnitzii, and Blautia have been linked in prior research with anti-inflammatory activity, short-chain-fatty-acid metabolism and intestinal epithelial functions. The new findings are therefore consistent with a shift away from some organisms commonly associated with dysbiosis and toward others often considered health-associated. However, genus-level labels do not reveal species-level functions or metabolic outputs, and the present experiment did not directly measure short-chain fatty acids, bile-acid transformation or host immune responses.

The division between relative and absolute abundance was important because total bacterial biomass declined at the higher peppermint-oil concentrations. If sequencing percentages alone had been used, an apparent increase in a genus might have reflected the loss of other organisms. By multiplying relative 16S rRNA gene abundances by sample-specific total cell counts, the researchers showed that the reduced abundance of Escherichia-Shigella and Eggerthella and the maintained abundance of Faecalibacterium and Blautia were not solely compositional artifacts. The lowest concentration produced no significant change in the abundance of any genus in the filtered analysis, whereas the two higher concentrations produced multiple significant changes. With only three concentrations tested, the experiment did not define a biological threshold or establish the shape of a dose-response relationship beyond the tested range.

Baseline microbial structure remained an important influence even though the direction of peppermint-oil responses was broadly consistent across donors. Multivariate analysis of genus-level fold changes showed that the first two principal components accounted for approximately 72% of the variation. Samples exposed to 2 and 20 milligrams per milliliter moved in similar directions, while the lowest-concentration samples stayed nearer untreated controls. The persistence of donor-specific clustering suggests that peppermint oil modulated each microbiome within its existing ecological context rather than replacing it with a uniform community. The similar responses at the two higher concentrations may be compatible with a maximal response near 2 milligrams per milliliter, but only three concentrations were tested, so plateau behavior cannot be confirmed.

Several limitations sharply constrain translation of these results to people. The microbiome work used only six healthy donors, a closed fermentation environment, a 48-hour incubation and a simplified nutrient medium. Such systems cannot reproduce continuous nutrient flow, intestinal absorption, spatially distinct microbial niches or the release behavior of enteric-coated peppermint-oil preparations. Long-term responses and disease-specific effects also cannot be inferred from healthy samples. The study was explicitly exploratory and hypothesis-generating, with no formal power calculation, and its statistical models accounted for donor variability while correcting for multiple comparisons. Validation in larger cohorts, IBS-specific models and clinical trials would be required. An earlier one-week trial in children with functional abdominal pain found no significant overall microbiome-composition change, although the researchers said this does not rule out effects at other doses, durations or levels of taxonomic detail.

For peppermint oil, the immediate mechanistic implication is that its effects may extend beyond smooth-muscle relaxation and anti-inflammatory signaling to include direct changes in microbial abundance and fermentation. Reduced gas production is consistent with the established use of peppermint oil for flatulence and abdominal discomfort, while the preserved diversity and altered composition add a possible microbiome dimension. Neither observation yet demonstrates a new clinical pathway. The researchers concluded that disease-specific studies are needed to determine whether the shifts in Escherichia-Shigella, Eggerthella, Faecalibacterium and Blautia translate into benefits for patients with IBS. Until then, the study provides controlled laboratory evidence of concentration-dependent microbial effects and a rationale for testing whether peppermint oil’s symptom relief can involve direct modulation of the gut ecosystem.

Subject of Research: In vitro effects of peppermint oil on gut microbial composition, bacterial abundance, gas production, smooth muscle and macrophage inflammation markers

Article Title: Peppermint oil exhibits antispasmodic and anti-inflammatory properties and alters gut microbial community composition and gas production in vitro

Article References: Buettner, C., Peterburs, P., Schulz-Kuhnt, A., Herrmann, L., Soeberdt, M., & Abels, C. (2026). Peppermint oil exhibits antispasmodic and anti-inflammatory properties and alters gut microbial community composition and gas production in vitro. Frontiers in Pharmacology, 17, Article 1963380. https://doi.org/10.3389/fphar.2026.1963380

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1963380

Keywords: peppermint oil, gut microbiome, microbial gas production, irritable bowel syndrome, antispasmodic effects, anti-inflammatory effects, 16S rRNA sequencing, anaerobic fermentation, Faecalibacterium, Blautia, Peppermint, exhibits