Phage 3Determination of Phage/Probiotic Synergistic Effects on Gastrointestinal Health

Study Overview
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Brief Summary
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The goal of this double blinded clinical trial is to determine whether consumption of the PreforPro product, when co-consumed with Bacillus subtilis DE111 probiotic, synergistically improves bowel regularity, perceived physical symptoms of gastrointestinal distress and other aspects of gastrointestinal health over probiotic use alone. Therefore, the primary goal of this study is to see if PreforPro consumption concurrent with B. subtilis DE111 usage improves probiotic activity. The secondary goal of this study is to assess non-gastrointestinal physiologic parameters to determine whether consumption of PreforPro combined with the probiotic offers any additional health benefits (ie. reduced inflammation, improved gut microbiota profiles) beyond those of consuming a probiotic alone. Participants will be asked to track daily bowel movements for 7 days prior to beginning capsule consumption and record their diet for a total 3 of days (two weekdays and one weekend day). They will then be asked to consume the provided capsules daily for a period of 45 days. Researchers will compare three parallel arms; (1) PreforPro+B. subtilis DE111 probiotic, (2) B. subtilis DE111 alone, or (3) a maltodextrin placebo to establish their impact on gastrointestinal symptoms and other indicators of health.

Detailed Description
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This is a continuation of previous intervention studies exploring the impacts of a bacteriophage formulation, PreforPro, on gastrointestinal health. The purpose of this study is to determine if the PreforPro product acts synergistically with a spore-based probiotic to influence bowel habits, perceptions of gastrointestinal symptoms, microbiota composition and inflammatory and immune parameters. The proposed study will also explore a longer intervention duration than the previous studies. A secondary outcome will include measuring plasma lipids, as the proposed probiotics (B. subtilis DE111) was shown to improve total and LDL cholesterol levels in our previous study. Bacteriophages may directly influence the microbiota and intestinal environment by selectively infecting host species- in this case E. coli. Additionally, they may have indirect effects as infection of target species can open up ecological niches and/or result in assimilation of released nutrients by other commensal organisms. These phages are generally regarded as safe for human consumption and specifically infecting several strains of E. coli, including enterohemorrhagic strains and Shiga-toxin producing strains. The removal of these E. coli alters the gut environment to allow growth of more favorable bacteria. We have previously shown that PreforPro is both safe and tolerable in a human population and does not broadly disrupt the gut microbiota as would be seen with antibiotic treatment. It did not improve the survival of Bifidobacterium lactis probiotic, but did appear to amplify some of its impacts on perceived functional gastrointestinal health. A phone screening will be conducted of all interested individuals to evaluate their eligibility. Those meeting the initial eligibility criteria will be scheduled for a clinic visit to obtain informed consent and to confirm eligibility. Consent will be obtained at the Colorado State University Food and Nutrition Clinical Research Lab (FNCRL) by a screening questionnaire and interview/assessment by the clinical coordinator. After securing consent, eligibility will be confirmed by taking anthropometric measures and participants falling within the BMI range will randomly be assigned to 1 of 3 treatment groups: (1) PreforPro+B. subtilis DE111 probiotic, (2) B. subtilis DE111 alone, or (3) maltodextrin placebo. Visit 1 (baseline): Eligible individuals will be asked to visit the clinic at visit 1 (baseline) to provide consent and confirm eligibility, undergo sample collections (blood and stool) and analysis procedures (weight/height, hip:waist ratio, gastrointestinal symptoms/ quality of life questionnaires) and receive their stool collection container, a stool log and 3-day diet log. Participants will be schedule for their drop off visit, in which they will return to the clinic 7 days after their first visit (Visit 2, Day 0) to return their stool sample, stool log and 3-day diet record. In return, they will be provided with another stool collection kit, their treatment capsules, another stool log and 3-day diet log. At the end of the 45-day treatment period (Visit 3-Final), final blood and stool samples will be collected as well as additional analyses (such as GI questionnaires, stool logs and 3-day diet record). This means that participants will undergo screening (by phone) and make a total of three (3) visits to the clinic during the study (baseline, Day 0, Final). All blood samples will be collected at Colorado State University by trained personnel. Fecal sample collection will be performed by the study participant with collection materials provided by Colorado State University.

Official Title
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PHAGE 3: Determination of Phage and Probiotic Synergistic Effects on Gastrointestinal Health

Conditions
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Gastrointestinal Dysfunction

Intervention / Treatment
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* Dietary Supplement: PreforPro+B. subtilis DE111 probiotics
* Dietary Supplement: Placebo
* Dietary Supplement: B. subtilis DE111


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Equal numbers of healthy male and female volunteers between 18-75 years old with BMI scores of 18.5 to 34.9. Exclusion Criteria: Individuals less than 18years of age or greater than 75years of age. If an individual's BMI is outside of 18.5-34.9. History of taking antibiotics and/or probiotics/prebiotics supplementation within 2months of starting antibiotics. If individual is on any medications and dietary supplements that would influence the endpoints of the study, such as statins, metformin, NSAIDs and MAO inhibitors. If individuals with diagnosis with cancer, liver or kidney disease, gastrointestinal diseases and metabolic disorders. Pregnant and breastfeeding women. Additionally inclusion and exclusion in the study will be determined case by case based on self reported alcohol and supplement use. If individual is unable to adhere to study protocol such as consuming capsules for a total 45days, providing stool or blood samples and attending scheduled clinic visits.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 75 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Prevention
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Triple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: PreforPro+B. subtilis DE111 probiotics<br>PreforPro is a prebiotic which is a bacteriophage-based product. This prebiotic was used in combination with Bacillus subtilis probiotics. | Dietary Supplement: PreforPro+B. subtilis DE111 probiotics<br>* PreforPro is bacteriophage-based product which was used in combination with Bacillus subtilis probiotics.<br>|
| Placebo Comparator: Placebo(maltodextrin)<br>Maltodextrin was used as a placebo. | Dietary Supplement: Placebo<br>* Maltodextrin is rice based powder which was used as placebo.<br>|
| Active Comparator: B. subtilis DE111 alone<br>Bacillus subtilis probiotics was used in this arm alone to compare with the experimental arm. | Dietary Supplement: B. subtilis DE111<br>* Bacillus subtilis probiotics.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Gastrointestinal Health Questionnaire | Self-assessment of functional measures of gastrointestinal health, including colon and small intestinal pain, gastric function, and gastrointestinal inflammation. | Evaluated at Baseline and Final (52 days apart) with questions designed to capture function from the prior 4 weeks. | 
| Bowel Movement Diary | Daily recording of the number and type (based on Bristol Stool chart) of bowel movements. | 52 days | 
| Quality of Life Assessment | Questionnaire that evaluates the impact of gastrointestinal health on daily activities as well as social and psychological impacts. | Evaluated at Baseline and Final (52 days apart) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Gut microbiota changes (beta-diversity) | 16s amplicon sequencing of stool samples will be conducted and analyzed for beta-diversity (differences between samples) using principle coordinate analysis of Bray Curtis distances. | 2 samples per person, collected  7 weeks apart | 
| Gut microbiota changes (alpha diversity) | 16s amplicon sequencing of stool samples will be conducted and analyzed for alpha diversity differences by applying actual and boot-strapped species number estimates, and Shannon and Simpson diversity indices. | 2 samples per person, collected  7 weeks apart | 
| Intestinal inflammation | Intestinal inflammation will be assessed by levels of fecal calprotectin measured by ELISA | 2 samples per person, collected  7 weeks apart | 
| Intestinal inflammation | Intestinal inflammation will be assessed by levels of secretory immunoglobin A measured by ELISA | 2 samples per person, collected  7 weeks apart | 
| Systemic inflammation | Human T-cell associated markers of inflammation in the blood will be measured using a multi-plex Luminex panel for 13 analytes. | 2 blood samples per person, collected  7 weeks apart | 
| Systemic inflammation | Cultured PBMCs will be stimulated with bacterial LPS and the supernatants will be analyzed for TNF-alpha, Il-6, 1l-10 and IFN-gamma using ELISA. | 2 blood samples per person, collected  7 weeks apart | 
| Lipid panels | The Piccolo Xpress lipid panel will be used with a 200ul blood sample to measure total cholesterol and other blood lipid parameters. | 2 blood samples per person, collected  7 weeks apart | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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PreforPro, Probiotics, Bacillus subtilis, Prebiotics, Gut health, Gut microbiota, Synergistic effects