B. subtilis Probiotic Supplements: Strains, Trial Doses, and Spore Stability

Spore-Based vs. Vegetative Cell Products

Most familiar probiotics, such as the cultures in yogurt, kefir and standard Lactobacillus capsules, are vegetative cells: actively living, growing bacteria. Bacillus subtilis can instead form an endospore, a dormant cell inside a protective coat, and it is mainly in this form that B. subtilis supplements are made.

What the research reports about spores:

• Heat stability and survival of the stomach. A 2011 review describes the heat stability of bacterial spore formers and their ability to survive the gastric barrier as the properties that make them attractive as food additives; the same review argues that Bacilli, often thought of as soil organisms, should be considered gut commensals (PubMed PMID: 21315976).
• Germination in the small intestine. In a randomized, crossover, placebo-controlled study of 11 people with an ileostomy, spores of the strain DE111 began to appear in the ileal fluid 3 hours after they were swallowed, and germinated (vegetative) cells were measured by 7 hours. The authors concluded the spores stayed viable through the stomach and germinated in the human small intestine within 3 hours; four of the seven authors were employees of a probiotic manufacturer (PubMed PMID: 34408741).
• Passage through the gut. In a 4-week trial of strain R0179 in 81 healthy adults, live B. subtilis counts in stool rose with the dose taken, and the authors concluded the strain survives passage through the human gastrointestinal tract (PubMed PMID: 25062611).
• Colonization is individual. A 2018 study of an 11-strain probiotic combination (not a B. subtilis product) found that the probiotics stayed viable through the gut, but that colonization of the gut lining in humans was person-, region- and strain-specific, and the effect on the mucosal community was transient (PubMed PMID: 30193112).

Strain Designations Used in Research

Strains of one species can differ in behavior and safety, and the clinical studies below each tested one specific strain, identified by a code. Results from one strain do not automatically apply to another. Strains studied in published human trials include:

• B. subtilis DE111. A 105-day double-blind randomized trial in 50 people with occasional constipation or diarrhea (1 × 10⁹ CFU per day) reported that the DE111 group moved to a healthier Bristol stool index while the placebo group stayed the same; all four authors were employees of a probiotic and enzyme manufacturer (doi:10.4172/2329-8901.1000189). In a 4-week randomized trial in healthy adults, DE111 lowered total cholesterol by 8 mg/dl and non-HDL cholesterol by 11 mg/dl relative to baseline; changes in endothelial function and LDL cholesterol were trends that did not reach significance (PubMed PMID: 33161737). A 4-week pilot trial found more anti-inflammatory immune cells when blood cells were stimulated in the laboratory, while perceived gastrointestinal health, the microbiota, markers of inflammation and the gut-barrier marker zonulin were largely unaffected; the authors note the study may have been underpowered (PubMed PMID: 33671071).
• B. subtilis R0179. In the 4-week trial of 81 healthy adults (0.1, 1 or 10 × 10⁹ CFU per day, or placebo), general wellness and gastrointestinal function were not affected at any dose, and the strain was well tolerated (PubMed PMID: 25062611). The same paper notes that a formulation of R0179 with Enterococcus faecium R0026 had been tested in earlier trials.
• B. subtilis CU1. In a 4-month randomized trial in 100 healthy people aged 60–74, CU1 (2 × 10⁹ spores per day in four 10-day courses) did not significantly reduce the number of days with symptoms of common infections (5.1 vs 6.6 days, P = 0.2015). In a subset of 44 people who gave samples, CU1 raised secretory IgA in stool and saliva compared with placebo, and a post-hoc analysis of that subset showed fewer respiratory infections (PubMed PMID: 26640504).
• B. subtilis HU58. In a randomized trial in 68 children aged 1–12 with antibiotic-associated diarrhea (see the antibiotics section below), the strain was given at 2 × 10⁹ CFU per day for seven days (PubMed PMID: 40716758).
• PB6 (now Bacillus velezensis). The European Food Safety Authority's feed-additive panel reports that the strain sold as B. subtilis PB6 was originally identified as B. subtilis and was redesignated Bacillus velezensis ATCC PTA-6737 during its 2020 assessment, which concerned its use as a feed additive for poultry (PubMed PMID: 33204311). The “ATCC” number refers to the strain's deposit in a culture collection.

B. subtilis also appears in multi-strain products. One example studied in many trials is a combination of live B. subtilis and Enterococcus faecium, discussed in the antibiotics section below (PubMed PMID: 41125358).

Doses Used in Clinical Trials

CFU stands for colony-forming unit, the standard count of living microorganisms: one CFU is one bacterium (or spore) able to divide and form a visible colony on a laboratory plate.

Doses reported in the human trials cited on this page:

• DE111: 1 × 10⁹ CFU per day for 105 days (doi:10.4172/2329-8901.1000189).
• R0179: 0.1, 1 or 10 × 10⁹ CFU per day for four weeks. The authors note the strain had not previously been tested at doses higher than 0.1 × 10⁹ CFU. Stool counts of the strain rose with dose, but general wellness and gastrointestinal function did not differ at any dose (PubMed PMID: 25062611).
• CU1: 2 × 10⁹ spores per day, in four 10-day courses separated by 18-day breaks over four months (PubMed PMID: 26640504).
• HU58: 2 × 10⁹ CFU per day for seven days, in children who already had antibiotic-associated diarrhea (PubMed PMID: 40716758).

None of these trials compared doses for a clinical outcome, so they do not show whether a higher dose produces a larger effect. The R0179 trial is the only one listed that tested several doses, and it measured tolerance and gut survival rather than a health benefit (PubMed PMID: 25062611).

Meal Timing: What the Research Tested

A 2011 study in Beneficial Microbes used a laboratory model of the human upper gastrointestinal tract to test a capsule containing two lactobacilli, a bifidobacterium and Saccharomyces boulardii; no Bacillus species was tested. Survival of the bacteria was best when the capsule was given with a meal or 30 minutes before a meal (cooked oatmeal with milk), and the bacteria did not survive in high numbers when given 30 minutes after the meal. Milk with 1% fat and an oatmeal-milk gruel gave better survival than apple juice or spring water, the authors judged the fat content of the meal probably more important than its protein content, and S. boulardii was not affected by meal timing (PubMed PMID: 22146689). Because the study was done in a model rather than in people, and did not include spores, it does not show how meal timing affects B. subtilis.

None of the B. subtilis trials cited on this page compared morning with evening intake, or intake with and without food.

B. subtilis and Antibiotics: What Trials Report

Diarrhea is a common adverse effect of antibiotic use (antibiotic-associated diarrhea, AAD) (PubMed PMID: 22570464).

Meta-analyses of probiotics in general. A 2012 systematic review and meta-analysis in JAMA included randomized trials of Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, Enterococcus and Bacillus probiotics. Most of the 82 included trials used Lactobacillus-based products, and strains were poorly documented. Pooling 63 trials with 11,811 participants, probiotics were associated with less AAD (relative risk 0.58, 95% CI 0.50–0.68); the authors found the evidence insufficient to say whether the effect differs by probiotic preparation (PubMed PMID: 22570464). An earlier 2006 meta-analysis in the American Journal of Gastroenterology found, from 25 randomized trials, that probiotics reduced the relative risk of AAD (RR 0.43, 95% CI 0.31–0.58) (PubMed PMID: 16635227).

Bacillus-containing products. A 2026 meta-analysis pooled 33 randomized trials, involving 2,919 people given a combination of live B. subtilis and Enterococcus faecium with symptomatic therapy and 2,781 given symptomatic therapy alone. The combination was associated with a higher treatment response in AAD (odds ratio 6.541) and a lower incidence of diarrhea when used for prevention (odds ratio 0.275) (PubMed PMID: 41125358). In a randomized, placebo-controlled trial in 68 children aged 1–12 who already had AAD, 93.5% of the B. subtilis HU58 group had normal stool types by day 3, against 22.6% on placebo; almost all children in both groups had normal stools by day 7. Abdominal pain scores also fell more in the probiotic group (PubMed PMID: 40716758).

Antibiotic resistance in Bacillus products. A 2000 laboratory analysis of five commercial Bacillus products found high levels of antibiotic resistance in four of them (PubMed PMID: 11097897). A safety assessment of strain CU1 reported that it did not show antibiotic resistance above existing regulatory cutoffs against clinically important antibiotics (PubMed PMID: 27825987). Reviews describe the safety of each Bacillus strain as something to be assessed case by case (PubMed PMID: 28848511).

The trials cited here do not report a tested schedule for spacing probiotic and antibiotic doses, or for how long to continue a probiotic after antibiotics end.

Spore Stability and Shelf Life

Reviews of Bacillus probiotics describe spore formers as heat-stable (PubMed PMID: 21315976) and as more stable during the processing and storage of foods and pharmaceutical preparations than other probiotics (PubMed PMID: 28848511). The studies cited on this page do not report a shelf-life figure for B. subtilis products or compare storage temperatures; the expiry date and storage conditions printed on a product are set by its maker.

Label Accuracy: What Product Testing Found

When researchers characterized five commercial Bacillus spore products used for oral bacteriotherapy, biochemical, physiological and genetic analysis showed that four of the five were mislabeled, and four showed high levels of antibiotic resistance (PubMed PMID: 11097897).

A 2017 review notes that debate over whether Bacillus species should be labelled probiotic or pathogen has confused consumers, and stresses the need to evaluate the safety of individual Bacillus strains and species case by case and to identify probiotic candidates more rigorously (PubMed PMID: 28848511).

Independent testing of a product can confirm what organism it contains and how much; it cannot show that a strain produces the effects reported in clinical trials of that strain.

Tolerance and Safety Findings

Tolerance in trials. In the R0179 trial, daily symptom scores and weekly gastrointestinal symptom scores did not differ from baseline at doses from 0.1 to 10 × 10⁹ CFU per day, and the strain was well tolerated by healthy adults (PubMed PMID: 25062611). In the CU1 trial in elderly people, the strain was reported safe and well tolerated, with no undesirable effects on markers of liver and kidney function, blood counts, blood pressure and heart measures, or vital signs (PubMed PMID: 27825987).

Duration of the trials. The trials cited here ran from seven days (HU58) to 105 days (DE111); each reported its outcomes at the end of its own period, and none set out a general timeline for when effects appear.

Infections in vulnerable patients. A 1998 case report describes recurrent septicemia caused by probiotic strains of B. subtilis in an immunocompromised patient (PubMed PMID: 9431982). A 2025 single-hospital study in Japan found that 69 of 70 B. subtilis strains isolated from blood cultures over seven years were B. subtilis var. natto; 25 of these were confirmed as true bacteremia, most often with intra-abdominal infection, pneumonia or urinary tract infection, in patients with a median age of 79, and 4 of the 25 died within 30 days (PubMed PMID: 41018695). Decisions about probiotics for people with weakened immune systems or serious illness belong with a clinician.

Key Research Papers

The following studies document clinical and laboratory evidence on Bacillus probiotics, including Bacillus subtilis supplement safety, gut effects and probiotic properties.

1. Nyangale et al. Bacillus coagulans GBI-30, 6086 modulates Faecalibacterium prausnitzii in older men and women. J Nutr. 2015. — PubMed PMID: 25948780
2. Lefevre et al. Probiotic strain Bacillus subtilis CU1 stimulates immune system of elderly during common infectious disease period. Immun Ageing. 2015. — PubMed PMID: 26640504
3. Cutting SM. Bacillus probiotics. Food Microbiol. 2011;28(2):214-20. — PubMed PMID: 21315976
4. Hoa NT, Baccigalupi L, Huxham A, et al. Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders. Appl Environ Microbiol. 2000;66(12):5241-7. — PubMed PMID: 11097897
5. Elshaghabee FMF et al. Bacillus as potential probiotics: status, concerns, and future perspectives. Front Microbiol. 2017. — PubMed PMID: 28848511
6. Khalesi S et al. Effect of probiotics on blood pressure: a systematic review and meta-analysis of randomized, controlled trials. Hypertension. 2014;64(4):897-903. — PubMed PMID: 25047574
7. Zmora N, Zilberman-Schapira G, Suez J, et al. Personalized gut mucosal colonization resistance to empiric probiotics is associated with unique host and microbiome features. Cell. 2018. — PubMed PMID: 30193112
8. Sorokulova I. Modern status and perspectives of Bacillus bacteria as probiotics. J Prob Health. 2013;1(4). — doi:10.4172/2329-8901.1000e106
9. Hanifi A, Culpepper T, Mai V, et al. Evaluation of Bacillus subtilis R0179 on gastrointestinal viability and general wellness: a randomised, double-blind, placebo-controlled trial in healthy adults. Benef Microbes. 2015;6(1):19-27. — PubMed PMID: 25062611
10. Colom J, Freitas D, Simon A, et al. Presence and germination of the probiotic Bacillus subtilis DE111 in the human small intestinal tract: a randomized, crossover, double-blind, and placebo-controlled study. Front Microbiol. 2021;12:715863. — PubMed PMID: 34408741
11. Mazza P. The use of Bacillus subtilis as an antidiarrhoeal microorganism. Boll Chim Farm. 1994;133(1):3-18. — PubMed PMID: 8166962
12. Cuentas AM, Deaton J, Davidson J, Ardita C. The effect of Bacillus subtilis DE111 on the daily bowel movement profile for people with occasional gastrointestinal irregularity. J Prob Health. 2017;5(4):189. — doi:10.4172/2329-8901.1000189
13. Trotter RE, Vazquez AR, Grubb DS, et al. Bacillus subtilis DE111 intake may improve blood lipids and endothelial function in healthy adults. Benef Microbes. 2020;11(7):621-630. — PubMed PMID: 33161737
14. Freedman KE, Hill JL, Wei Y, et al. Examining the gastrointestinal and immunomodulatory effects of the novel probiotic Bacillus subtilis DE111. Int J Mol Sci. 2021;22(5):2453. — PubMed PMID: 33671071
15. Sorensen K, Jog P, Mankar S, Holz C, Jadhav S. Effects of spore-forming probiotic Bacillus subtilis HU58 in children with antibiotic-associated diarrhoea: a randomised, double-blind, placebo-controlled trial. Benef Microbes. 2025;17(1):1-10. — PubMed PMID: 40716758
16. Zhang N, Chen Y, Shen Z. Live combined Bacillus subtilis and Enterococcus faecium for the treatment and prevention of antibiotic-associated diarrhea: a RCT-based meta-analysis. Tohoku J Exp Med. 2026;268(4):559-573. — PubMed PMID: 41125358
17. Hempel S, Newberry SJ, Maher AR, et al. Probiotics for the prevention and treatment of antibiotic-associated diarrhea: a systematic review and meta-analysis. JAMA. 2012;307(18):1959-69. — PubMed PMID: 22570464
18. McFarland LV. Meta-analysis of probiotics for the prevention of antibiotic associated diarrhea and the treatment of Clostridium difficile disease. Am J Gastroenterol. 2006;101(4):812-22. — PubMed PMID: 16635227
19. Tompkins TA, Mainville I, Arcand Y. The impact of meals on a probiotic during transit through a model of the human upper gastrointestinal tract. Benef Microbes. 2011;2(4):295-303. — PubMed PMID: 22146689
20. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP). Safety and efficacy of Bacillus subtilis PB6 (Bacillus velezensis ATCC PTA-6737) as a feed additive for chickens for fattening, chickens reared for laying, minor poultry species (except for laying purposes), ornamental, sporting and game birds. EFSA J. 2020;18(11):e06280. — PubMed PMID: 33204311
21. Lefevre M, Racedo SM, Denayrolles M, et al. Safety assessment of Bacillus subtilis CU1 for use as a probiotic in humans. Regul Toxicol Pharmacol. 2017;83:54-65. — PubMed PMID: 27825987
22. Oggioni MR, Pozzi G, Valensin PE, Galieni P, Bigazzi C. Recurrent septicemia in an immunocompromised patient due to probiotic strains of Bacillus subtilis. J Clin Microbiol. 1998;36(1):325-6. — PubMed PMID: 9431982
23. Sada RM, Yamamoto G, Hamaguchi S, et al. The majority of Bacillus subtilis strains isolated from blood cultures were derived from traditional Japanese fermented soybeans natto: a single-center retrospective study. Open Forum Infect Dis. 2025;12(9):ofaf574. — PubMed PMID: 41018695

Editorial note (2026-10-14): three earlier entries in this list could not be found in PubMed or Crossref and were replaced: “Hoa TT et al., Characterization of Bacillus subtilis isolated from Vietnamese fermented soybeans as a potential probiotic, Lett Appl Microbiol 2000” (replaced by the Hoa NT 2000 paper above), “Nithya V et al., Adhesion characteristics and anti-infective property of probiotic Bacillus subtilis VGNB7, LWT 2012” (replaced by Hanifi 2015) and “Marsool MDM et al., Immunomodulatory properties of Bacillus subtilis in the gut, J Immunol Res 2019” (replaced by Colom 2021). The Cell 2018 colonization paper was previously credited to Suez J; its first author is Zmora N.

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Connections

• All Bacteria
• Bacillus subtilis Overview
• B. subtilis Probiotic Uses
• B. subtilis Safety and Side Effects
• Fermented Foods and Natto
• Probiotics in Food
• Benefits & Safety

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