Branched fat synthesizing Bacillus subtilis improves gastrointestinal symptoms. A phase 1/2A randomized controlled trial

doi:10.21203/rs.3.rs-2880722/v1

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Branched fat synthesizing Bacillus subtilis improves gastrointestinal symptoms. A phase 1/2A randomized controlled trial

https://doi.org/10.21203/rs.3.rs-2880722/v1

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Background

Functional gastrointestinal disorders (FGIDs) are a common, difficult to manage condition. Branched chain fatty acids are emerging as a dietary component that influence gastrointestinal (GI) health. We conducted a double blinded randomized controlled trial of a proprietary strain of deactivated Bacillus subtilis (BG01-4™) that produce branched change fatty acids (BCFA) in the lower GI tract to treat self-reported FGID.

Methods

Participants (n = 67) completed a four-week intervention of BG01-4™ (n = 34) or placebo (n = 33). The Gastrointestinal Symptom Rating Scale (GSRS) served as the outcome measure, collected prior to, at two-weeks, and at the four-week completion of the intervention.

Results

At four weeks one of three primary outcomes, constipation in the experimental group, was improved compared to placebo; both other primary outcomes Total GCRS and diarrhoea, were significantly improved in both experimental and placebo groups. The pre-planned secondary outcome indigestion was improved at four weeks compared to entry with trend to significance compared to placebo (p = 0.079). Exploratory analysis revealed clusters for constipation (18% improvement, p < 0.001), indigestion (11% improvement, p = 0.04), and dyspepsia (10% improvement, p = 0.04), were significantly improved in the intervention group compared to the placebo.

Conclusions

These initial findings suggest that in people with self-reported FGID, BG01-4™ improves specific symptoms of constipation and related GI dysfunction, potentially mediated via synthesis of BCFA. Longer term confirmatory studies for this intervention are warranted.

Trial registration:

This study was registered prospectively (25/10/2021) at the Australian New Zealand Clinical Trials Registry (ACTRN12621001441808).

probiotics

Bacillus subtilis

functional gastrointestinal disorders

Gastrointestinal Symptom Rating Scale

Functional gastrointestinal disorders (FGIDs) are a heterogeneous group of chronic conditions characterised persistent and recurrent gastrointestinal symptoms without any apparent structural or biochemical abnormalities [1–3]. Affecting between 25–40% of the global population, the most common of these conditions include functional bowel disorders and irritable bowel syndrome, but also functional constipation, functional diarrhoea, and functional bloating [2]. Constipation or diarrhoea can be a burdensome FGID issue that occurs in many individuals and whose treatment remains challenging. Similarly, bloating is a subjective abdominal discomfort that is associated with abdominal inflation due to the accumulation of excessive gas (flatulence); and may not necessarily be accompanied by abdominal distension [4–6]. While constipation or diarrhoea can be classified using the Bristol stool form scale, bloating symptoms are generally subjective in nature and therefore assigning an objective score is fraught with inter- and intra-subject variations. However, the severity of bloating-related abdominal discomfort experienced by individuals can range from mild to severe and the discomfort can negatively impact health and mental health.[2] These conditions carry significant morbidity, often leading to missed work/school, social isolation and is commonly associated with psychological comorbidity. FGID presents a significant health-care burden with over two-thirds of people with FGID having sought medical advice from a doctor in the preceding 12 months, 40% prescribed regular medication and around a third undergoing potentially unnecessary abdominal surgery for their symptoms [7, 8].

The pathophysiology and mechanisms underlying FGID are complex and not well understood. Consequently, though FGID is very common, it is a difficult to manage problem. Several mechanisms have been proposed including abnormal gastrointestinal motility, visceral hypersensitivity, psychosocial disturbances and more recently low-grade intestinal inflammation, mucosal immune dysregulation and altered gut microbiota. Current therapeutic strategies are directed by symptomatology, but established treatments such as antibiotics and anti-depressants tend to be inconsistently successful or potentially contraindicated for long-term use [4, 5, 9]. Dietary restriction (e.g low-FODMAPs) is a well-established therapy for some types of FGID, although there is no single diet that will help resolve symptoms for all patients. A recent population-based cross-sectional study by Stocks et al [10] suggested that one-third of adults with a gastrointestinal condition were restricting their diet which affected their mental health and overall quality of life.

Branched chain fatty acids (BCFA) were first recognized as nutrients important to gastrointestinal (GI) health in the context of vernix caseosa, the white waxy substance that develops on late term human foetuses. Vernix caseosa sloughs into amniotic fluid as particles that are swallowed by the foetus throughout the last trimester [11]. Vernix is rich in BCFA which constitutes about 30% of all fatty acids. BCFA are rapidly taken up and incorporated into enterocyte membranes where they moderate the inflammatory response [12–14].

Many microorganisms use BCFA in their membranes to modulate biophysical properties[15]. Prominent among these are the gram-positive Bacillus genus which synthesize BCFA de novo[16]. Bacillus subtilis (B. subtilis), the best studied gram-positive microorganism, generally has in excess of 50% BCFA in its membrane lipids [17]. Populations of Bacillus used as probiotics [18, 19] are safe up to 10 × 10⁹ colony forming units per day [20]. Specifically, certain species of Bacillus (e.g., Bacillus subtilis) that are present in baked and fermented food products have a long and safe history of use in humans [5, 21, 22].

B. subtilis produces spores that remains viable in a wide temperature and pH range making it a suitable probiotics supplement for improving gut health. Further B. subtilis has no known contraindications. In concentrated form, safety studies have reported doses of 2 × 10⁹ Bacillus subtilis spores per day administered for a total 40 days (4 consumption periods of 10 days separated by 18-day washouts) was safe and well-tolerated in human participants without undesirable physiological effects on markers of liver and kidney function, complete blood counts, hemodynamic parameters, and vital signs [23]. Apart from shifting the distribution of microbiota in the lower gut, B. subtilis produces abundant BCFA that have numerous putative effects in the GI tract. BCFA are rapidly taken up into enterocytes where they are incorporated into membrane lipids and modulate the inflammatory response to lipopolysaccharide challenge [12, 14]. They are effective in reducing incidence of necrotizing enterocolitis in a neonatal rat model, where they also attenuate the inflammatory response [24]. Our current data are consistent with BCFA as a contributor to the observed efficacy of B subtilis. Because this organism is widely distributed among humans and because BCFA are in foods, notably dairy products, this treatment is expected to be safe [25].

We recently screened numerous B. subtilis strains cultured at lab scale and identified a particularly fast-growing strain with high levels of BCFA (unpublished data), designated “BG01-4™”. We prepared spores of deactivated B. subtilis BG01-4^™ in a maltodextrin carrier after heat deactivation of vegetative cells in a powdered form in sachets that can be mixed into drinks or foods and consumed as a probiotic.

Clinical and interventional B. subtilis studies have previously been shown to control intestinal hydrogen production [26]. Supplementation has also been reported to promote improvements in those with constipation and diarrhoea [4]. A recent clinical trial found that a formulation containing B. subtilis significantly reduced the frequency and severity of abdominal bloating [27]. However, well designed placebo-controlled randomized control trials (RCTs) investigating B subtilis efficacy on mental health and overall quality of life in people with FGID have not yet been undertaken.

The aim of this double-blinded RCT was to test the efficacyof a proprietary strain of deactivated B. subtilis (BG01-4™) in sachet form to improve symptoms of participants with self-reported FGID. We hypothesized that those who receive 5 × 10⁹ colony forming units of probiotic BG01-4™ per day over four weeks would improve self-reported symptoms associated with FGID via the Gastrointestinal Symptom Rating Scale (GSRS) [28].

This study was approved by Bellberry Human Research Ethics Committee Australia (2021-09-1137), conforming to the CONSORT 2010 guidelines, and registered as a prospective clinical trial with Australian and New Zealand Clinical Trials Registry (25/10/2021, ACTRN12621001441808p). All study procedures were conducted in accordance with the ethical principles in Declaration of Helsinki. Prior to participation, the study was adequately explained, and voluntary written and informed consent to participate in the study was obtained prior to any data collection.

Participants

Healthy adults between 18–75 years of age were recruited via word of mouth, and social media announcement (see Table 1 for demographic data). Functional gastrointestinal disorders often exist in people with no attribution to formal medical diagnosis, therefore inclusion for participation for into the study was meeting the Rome IV (https://theromefoundation.org/rome-iv/rome-iv-criteria/) criteria,[29] being a self-diagnosis (according to Rome IV criteria) FGID in the last 3 months, and have one or more of the following self-reported symptoms experienced for at least 6 months or longer: 1) recurrent abdominal bloating and/or distension occurring on average at least one day/week; and abdominal bloating and/or distension predominates over other symptoms, such as minor bowel movement abnormalities; 2) Loose or watery stools, without predominant abdominal pain or bothersome bloating, occurring in more than 25% of stools; and 3) Functional constipation: straining during more than 25% of defecation per week; sensation of anorectal obstruction/blockage or incomplete evacuation more than 25% of defecations; needing manual maneuvers (e.g., digital assistance) to help with more than 25% of defecations; and loose stools are rare without the use of laxatives. Exclusion criteria included current use of probiotics, intolerance to maltodextrin, meeting the criteria for opioid-induced constipation (OIC), diagnoses of irritable bowel syndrome (IBS) or including diarrhea-predominant IBS (IBS-D). Sample size determination was based on a-priori power calculation for the primary continuous outcomes variable (total score of GSRS) pre and post intervention (3 time points), for a between groups (experimental vs placebo) repeated measures design suggests effect size f = 0.2, power (1-beta) = 0.95, α ≥ 0.05 for a total minimum sample size of 66.

Table 1

Participant demographics (mean ± SD)
	All participants (n = 67)		Male (N = 25)		Female (N = 42)
	Age	GSRS Score	Age	GSRS Score	Age	GSRS Score
BG01-4™ (n = 34)	47.8 ± 14.8	48.5 ± 9.4	54.0 ± 14.2	42.4 ± 7.7	45.6 ± 14.6	50.7 ± 9.2
Placebo (n = 33)	51.9 ± 13.4	52.6 ± 10.6	53.0 ± 12.6	54.8 ± 14.1	51.6 ± 13.9	50.0 ± 10.2

Study design

We used a randomized, double-blind, placebo-controlled two-arm parallel group study of a four-week supplementation intervention period. Prior to intervention, participants meeting the inclusion criteria, were provided with a password-protected online link to complete a modified GSRS survey[28] (mGSRS). Following completion of the mGSRS, participants were randomized to receive BG01-4™ or a placebo (maltodextrin) once-daily for four-weeks. Follow up surveys were completed by participants at the mid-point (two-week) mark and following the completion of the intervention at four-weeks. Individuals were reminded to maintain their current physical activity levels and to continue on their usual diet throughout the study. Participants were also instructed to record any adverse events and to contact researchers if they stopped the supplement.

A simple randomization schedule was prepared by an unblinded person who facilitated pre-screening and written informed consent. Participants who met the inclusion criteria were assigned a unique code number based on the randomization schedule generated by the unblinded research member who was not involved in conducting the study assessment.

Intervention

Interventional supplements (BG01-4™ and placebo) provided by Vernx Pty Ltd (Melbourne, Victoria, Australia) via mail were 1 g unmarked sachets with the instruction to consume one sachet per day for four-weeks. Participants were instructed that they could either pour the contents of the sachet into a glass of water (250 ml), juice, or to sprinkle over their usual foods such as yogurt or cereal. Experimental and placebo were indistinguishable by visual inspection.

Outcome measures

Three primary outcomes were pre-planned: Total GSRS score, GSRS-constipation, and GSRS-diarrhoea scores were compared for change from entry at four weeks. Pre-planned secondary outcomes were indigestion, dyspepsia, and abdominal pain syndrome were similarly tested for change from entry at four weeks.

The GSRS questionnaire is well-documented and has a good reliability with Cronbach’s alpha ranging from 0.79 to 0.83 across studies across a number of different languages [30–33]. We used a modified version of the original GSRS (mGSRS) containing 15 questions, self-administered online [34–36], evaluated the severity of five symptom clusters: abdominal pain, dyspepsia syndrome, indigestion, and constipation and diarrhea. Participants answered questions using a Likert scale from one (least severe) to seven (most severe) [5, 35].

Statistical analysis

Primary outcomes

Analyses were performed using intention-to-treat analysis. The difference between scores at four-weeks and at entry were tabulated and tested by one way t-test for non-zero, with significance (α ≤ 0.05) adjusted for multiple (three) comparisons (p = 0.0167 = 0.05/3). Differences in improvement between experimental and placebo were tested by pairwise t-test similarly adjusted for multiple comparisons.

Exploratory outcomes

For exploratory analysis, responses (pre-, mid-, post-intervention) were combined within the five cluster areas, plus total score of the mGSRS, averaged between groups. Mean change scores from pre-intervention to mid-intervention, and mid-intervention to post-intervention where calculated and analysed using a mixed-model ANOVA, except for age where independent samples t-test was used. Where ANOVA was found significant, post hoc testing with Bonferroni correction was employed. All data was analysed using Jamovi [37] and significance was set at α ≤ 0.05.

A total of 186 individuals were screened, with 80 eligible participants enrolled and randomized into the study (Fig. 1). Groups were matched for age and sex (BG01-4^™: mean age 47.8 ± 14.8 years; Placebo: mean age 51.9 ± 13.4 years). Compliance in the BG01-4™ group was 81% with two participants (5%) dropping out due to not adhering to the protocol and six participants (14%) dropping out voluntarily. Similar compliance in the placebo group was found (87%) with one participant leaving due to protocol violation (3%) and four participants (10%) leaving voluntarily (Fig. 1) leaving a final combined sample of 67 (see Table 1).

Primary and secondary outcomes

Results of the primary outcomes show that all experimental groups had improvement in scores at four weeks, while placebo improved for Total CSRS and diarrhoea (Fig. 2). Constipation scores improved 35% over the four-week period while the placebo group improvement did not reach significance.

Among secondary outcomes, all treatments including placebo improved significantly over the 4 weeks. Indigestion was trending improvement compared to placebo (p = 0.079).

Exploratory outcomes

Scores from the mGSRS across the five clusters are presented in Table 2. Significant interaction effects were observed between groups for dyspepsia (F_1,53=4.10, p = 0.04), indigestion (F_1,53=4.32, p = 0.04), constipation (F_1,53=12.58, p < 0.001), and GSRS total score (F_1,53=10.40, p = 0.002). Post hoc analysis revealed group differences at end of the intervention for dyspepsia (t₅₃=-3.28, p = 0.002), indigestion (t₅₈=-2.36, p = 0.02), constipation (t₅₈=-3.46, p = 0.001), and GSRS total score (t₅₈=-3.331, p = 0.002). Main effects only were observed for abdominal pain (F_1,53=7.98, p = 0.007).

Table 2

Mean (± SD) mGSRS scores between groups across the five symptom clusters.
Group	Abdominal			Dyspepsia			Indigestion			Constipation			Diarrhoea			Total Score
Group	Pre	2-wk	4-wk	Pre	2-wk	4-wk	Pre	2-wk	4-wk	Pre	2-wk	4-wk	Pre	2-wk	4-wk	Pre	2-wk	4-wk
BG01-4™	7.22 (± 2.01)	5.15 (± 2.25)	4.41 (± 2.15)	6.26 (± 2.65)	5.41 (± 2.66)	4.81 (± 2.57)	15.00 (± 3.55)	11.30 (± 3.61)	10.00 (± 3.41)	11.67 (± 4.88)	9.93 (± 4.58)	7.85 (± 4.46)	8.41 (± 4.28)	7.44 (± 4.26)	5.81 (± 2.87)	48.56 (± 9.45)	39.22 (± 10.56)	32.89 (± 10.60)
Placebo	7.43 (± 1.89)	5.07 (± 2.68)	4.86 (± 2.52)	8.36 (± 4.00)	6.11 (± 3.76)	6.29 (± 3.46)	15.29 (± 4.38)	10.71 (± 5.16)	11.14 (± 4.58)	10.43 (± 4.52)	7.55 (± 4.13)	8.32 (± 4.08)	10.79 (± 5.45)	7.39 (± 4.23)	8.18 (± 5.13)	52.64 (± 10.89)	36.96 (± 14.67)	39.07 (± 14.04)

The investigation of individual bacterial strains to alleviate FGID in people with chronic symptoms is an emerging area of research. In this study, we show constipation improves in four weeks when consuming a proprietary strain of B. subtilis (BG01-4^™) versus placebo, one of three registered primary outcomes. Exploratory analysis showed improvements in participants responses for dyspepsia, and indigestion.

The placebo used in this study (maltodextrin) was indistinguishable from the B subtilis treatment. An interesting finding from the exploratory aspect of our study reflected the GSRS findings from Shi et al [38] and Östlund-Lagerström et al [39] in that over the course of the four-week trial, the first two weeks showed self-reported improvements in both groups before the placebo group deteriorated in self-reported symptoms. In our study, both groups improved in the first two-weeks possibly due to expectation bias. However, the separation at four-weeks where the placebo group reported worsening of previously improving symptoms (at two-weeks) compared to the BG01-4^™ arm suggests a physiological response. Expectation bias is a concern, as with rapid improvements, individuals may discontinue supplementation therapy and potentially return to restrictive dieting [10]. As currently there is no cure for FGID, it is important to manage psychological aspects of this condition through continued supplementation and using a holistic approach via the biopsychosocial model [40].

We observed significant improvements for items within the GSRS. Self-reported symptoms for functional dyspepsia showed similar improvements to a recent exploratory study demonstrating the efficacy of B subtilis MY02 versus placebo [41]. Similarly, indigestion symptoms were improved supporting previous work [39] as well as relief from constipation [42]. Taken together, these findings suggest that B subtilis has the potential to significantly relieve FGID symptoms.

Overall the supplement was well tolerated, with the number of dropouts similar to previous B. subtilis research (Fig. 1) [41]. There was one adverse event reported in the placebo group, but the participant was assessed medically and deemed unlikely to be related to the study product as the participant had not taken the sachet for a day prior to the incident.

While we measured GSRS as a proxy for overall quality of life showing general psychometric improvements, we did not specifically measure mood or depression/anxiety which is a limitation of the study. However, it was outside the scope of this study to quantify psychological state the research aim was to address individuals with chronic FGID who would otherwise be psychologically healthy. We do acknowledge however, that future RCTs should include those with FGID who have clinically diagnosed mood disorders resulting from chronic gastrointestinal disorders.

Although we found statistically significant differences between groups, duration of treatment was brief in our study (four-weeks), which has been also acknowledged in previous work [5]. While some probiotic studies report at least 60 days intervention useful for statistical significance,[43] others have suggested this period is not long enough for full effects on gastrointestinal disorders [27]. Future trials extended to longer treatment periods required.

In conclusion, consumption of a proprietary strain of probiotic B. Subtills (BG01-4^™) was well tolerated and resulted in symptoms of constipation and related GI dysfunction in this randomised double-blinded placebo-controlled trial. While longer studies are required, statistically significant improvements in overall GSRS suggest that BG01-4^™ has a potential role in relieving specific symptoms of dyspepsia, indigestion, and constipation after four-weeks.

Ethical approval and consent to participate

The study was conducted following Helsinki Declaration. The Ethical clearance was obtained from Bellberry Human Research Ethics Committee Australia (2021-09-1137), conforming to the CONSORT 2010 guidelines, and registered as a clinical trial with Australian and New Zealand Clinical Trials Registry (ACTRN12621001441808p).. Each study participant was informed about the objective and purpose of the study. Written informed consent was obtained from each selected study participant before prior to data collection. Participants were informed that they have the right to continue or withdraw from the study at any time. The confidentiality of information provided by study participants was protected by making the data collection procedure anonymous and all information of the study subject would be kept confidential.

Consent for publication

Not applicable

Availabilty of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

CP, MC and JTB are directors of Vernx Peribiotics. Other authors declare they have no conflict of interest regarding the publication of this paper.

Funding

For this research paper, we did not receive any grant from any funding agency.

Author contributions

CP, AJP, MC participated in conceptualization, data-collection, formal analysis, methodology, project administration, writing the original draft, editing and reviewing the paper. JTB contributed to the data-analysis, reviewing and writing of subsequent drafts and the final paper. RB contributed to the reviewing and editing of the final paper.

Acknowledgements

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Competing interest reported. CP, MC and JTB are directors of Vernx Peribiotics. Other authors declare they have no conflict of interest regarding the publication of this paper.

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Branched fat synthesizing Bacillus subtilis improves gastrointestinal symptoms. A phase 1/2A randomized controlled trial

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