The Effects of Oral Fructanase Administration on Gastrointestinal Symptoms After Inulin Challenge in Healthy Adults

The carbohydrate inulin (from chicory root) is a healthy prebiotic ingredient found in dietary supplements and fortified foods (Nagy et al). Inulin is representative of a broader class of typically health-associated, yet fermentable carbohydrates called fructans that occur naturally in many vegetables, fruits, and wheat. Fructans, or long chains of fructose units, are resistant to human digestive enzyme hydrolysis and transit intact to the small intestine and colon where they undergo rapid fermentation by intestinal microbes. This microbial metabolism of fructans produces gas and other fermentation byproducts that can lead to excess gastrointestinal (GI) symptoms like abdominal bloating, cramping, stomach rumbling, and flatulence (Bonnema et al; Briet et al; Bruhwyler et al), especially in individuals with irritable bowel syndrome (Van den Houte et al). A new digestive enzyme called fructanase was developed to help with GI symptoms associated with fructan consumption. Positive findings from in vitro digestion simulations (Guice et al) and a first-in-human safety trial (Garvey et al) helped define the fructanase dose for this clinical trial-the primary objective of which is to investigate the effect of oral fructanase administration on GI symptoms in healthy adults after consuming oatmeal with added inulin (25 grams). Secondary outcomes include breath hydrogen and methane levels, which serve as biomarkers of intestinal microbial fermentation. The investigators hypothesize that fructanase administration will lower the severity of GI symptoms after inulin consumption, as well as lower breath biomarkers of intestinal microbial fermentation, compared to placebo.

Fructanase (also known as inulinase, fructan hydrolase, beta-fructosidase, and beta-fructofuranosidase) represents an innovative enzymatic approach to hydrolyze dietary fructans and improve fructan-type fermentable oligo-, di-, monosaccharides, and polyols (FODMAP) tolerance. We have previously shown in vitro that a microbial fructanase (obtained by fermentation of Aspergillus tubingensis) effectively hydrolyzes a variety of dietary inulins and fructans following simulated oro-gastric digestion in the static INFOGEST model \[Guice et al\]. We have also completed a Phase I clinical trial in healthy adults demonstrating the safety and tolerability of twice daily fructanase supplementation (total 2,000 inulinase activity units (INU) per day) for 4 weeks \[Garvey et al\]. Despite its safety and promising application in nutrition and GI health, rigorous clinical evaluation of fructanase's efficacy in improving GI tolerance to fructans is warranted.

This is a randomized, double-blind, two-period crossover study with one screening visit (Visit 1, Day -7) and two test visits (Visits 2 and 3; Days 0 and 7). Participants will arrive for their screening visit on Day -7. After participants provide voluntary informed consent, participants will undergo medical history, prior and current medication/supplement use, inclusion and exclusion criteria assessments, and last menses query (if applicable). Additionally, height, body weight, and vital signs will be measured, and theoretical body mass index (BMI) will be calculated. An in-clinic urine pregnancy test will be performed on all female participants. Participants will be instructed to maintain physical activity and habitual diet as much as possible while abstaining from supplements, beverages, or food products with digestive enzymes, prebiotic ingredients, live probiotics (e.g., yogurt, kombucha), or postbiotic ingredients prior to Visit 2 (Day 0). Those who are taking exclusionary supplements, foods, and beverages will be instructed to stop taking these products, and their Visit 2 (Day 0) will be scheduled to allow for the appropriate pre-randomization washout period. Additionally, pre-menopausal female participants will be scheduled to allow their Visit 2 (Day 0) to occur during the menstrual cycle follicular phase (defined as days 1-14 of the menstrual cycle, with day 1 as the first day of menses). To all participants, a standardized low FODMAP meal will be dispensed with instructions to consume the meal in its entirety the evening prior to Visit 2 (Day 0). The instructions will include timing of meal consumption in order to maintain the appropriate fast. Participants will also be instructed to follow a low FODMAP diet the day prior to Visit 2, while also being reminded to abstain from alcohol consumption for 48 h, to abstain from vigorous exercise for 24 h, to fast (≥10 h; water only), and to brush their teeth in the morning prior to Visit 2 (Day 0).

At Visit 2 (Day 0), participants will arrive at the clinic in a fasting state (≥10 h; water only). Eligible participants will undergo clinic visit procedures (concomitant medication/supplement use, assess inclusion/exclusion criteria, and body weight), last menses query (where applicable), and adverse event (AE) assessment. Participants will be queried about compliance with study instructions and changes to habitual lifestyle, diet, and alcohol consumption. At t = -0.25 h (± 5 min), where t = 0 h is the time of study product consumption, participants will provide a breath sample for hydrogen and methane analysis and complete a questionnaire to assess baseline GI symptoms prior to study product consumption. Participants will then be randomized to a study product sequence and will be administered their assigned study product capsule (fructanase or placebo) that will be consumed with oatmeal and 25 g of inulin (t = 0 h). In addition, 8 oz. of water will be provided. Participants will be allowed up to 10 minutes to completely consume the oatmeal and inulin mixture, with fructanase or placebo, with t = 0 h designated as the start of study product consumption. Following study product consumption, participants will complete the GI symptom questionnaire and provide a breath sample for hydrogen and methane analysis at t = 1, 2, 3, 4, 5, 6, 7, and 8 h (± 5 min). Participants will also be provided with 8 oz. of water at t = 2 and 4 h. Participants will be allowed to drink each 8 oz. of water ad libitum over each 2 h period, but the entire 8 oz. of water must be consumed prior to the next water administration (e.g., drink all 8 oz. provided at t = 2 h prior to the next water administration at t = 4 h). A standard low FODMAP lunch with 8 oz. of water will be provided after the t = 6 h assessments.

Prior to departure from the study center, AEs will be assessed, then study instructions will be provided, including: 1) maintaining habitual physical activity, 2) continuing to follow the low FODMAP diet for the remainder of the day, 3) abstaining from alcohol consumption for the remainder of the day, and 4) abstaining from supplements, beverages, or food products with digestive enzymes, prebiotic ingredients, live probiotics (e.g., yogurt, kombucha), or postbiotic ingredients throughout the study. The same standardized low FODMAP meal consumed the evening prior to Visit 2 will be dispensed with instructions to consume the meal in its entirety the evening prior to Visit 3 (Day 7). The instructions will include timing of meal consumption in order to maintain the appropriate fast. Participants will also be instructed to follow a low FODMAP diet the day prior to Visit 3 (Day 7), while also being reminded to abstain from alcohol consumption for 48 h, to abstain from vigorous exercise for 24 h, to fast (≥10 h; water only), and to brush their teeth in the morning prior to Visit 3 (Day 7).

The following morning (Day 1), participants will receive an email link at t = 24 h to complete a series of brief questionnaires, including:

* Modified Single-Item Sleep Quality Scale
* Single-Item Stool Consistency Scale
* GI-VAS assessing symptoms "since participant left the clinic"
* A single question to confirm compliance with study instructions.

Participants will be instructed to complete these questionnaires the morning of Day 1 after their first bowel movement or immediately prior to their usual lunch time, whichever occurs first.

At Visit 3 (Day 7), participants will arrive at the clinic fasted (≥10 h; water only) and undergo clinic visit procedures, including: concomitant medication/supplement use, review inclusion/exclusion criteria, and body weight, last menses query (if applicable), and AE assessment. Participants will be queried about compliance with study instructions and changes to habitual lifestyle, diet, and alcohol consumption. Participants will then complete all study procedures outlined in Visit 2 (except for randomization). The same timing and volume of water consumed at Visit 2 will be replicated at Visit 3. The same lunch (items and quantity) consumed at Visit 2 will be replicated at Visit 3.

Eligibility Criteria:
Inclusion Criteria:

1. Male or female, age ≥20 to ≤50 y at Visit 1 (Day -7).
2. BMI ≥18.5 to less than 30.0 kg/m2 at Visit 1 (Day -7).
3. Non-user (never used, or former user defined as cessation ≥12 months) of tobacco or nicotine products (e.g., cigarette smoking, vaping, chewing tobacco) with no plans to begin use during the study period.
4. Willing to maintain habitual diet, physical activity, and body weight throughout the trial.
5. Willing to refrain from exclusionary medications, supplements, and products throughout the study.
6. No health conditions that would prevent him/her from fulfilling the study requirements as judged by the Clinical Investigator on the basis of medical history and routine laboratory test results.
7. Understands the study procedures and signs forms providing informed consent to participate in the study and authorizes the release of relevant protected health information to the Clinical Investigator.

Exclusion Criteria:

1. Known sensitivity, intolerability, or allergy to any of the study products or their excipients.
2. Clinically important GI condition that would potentially interfere with the evaluation of the study product (e.g., inflammatory bowel disease, irritable bowel syndrome, gastroesophageal reflux disease requiring any medication, dyspepsia, Crohn's disease, celiac disease, history of surgery for weight loss, gastroparesis, and clinically significant lactose or gluten intolerance or other food or ingredient allergies).
3. Recent (within 2 weeks of Visit 1; Day -7) history of an episode of acute GI illness such as nausea/vomiting or diarrhea (defined as ≥3 loose or liquid stools/d).
4. Self-reported history (within 6 weeks of Visit 1; Day -7) of constipation (defined as fewer than 3 bowel movements per week).
5. Uncontrolled and/or clinically important pulmonary (including uncontrolled asthma), cardiac (including, but not limited to, atherosclerotic disease, history of myocardial infarction, peripheral arterial disease, stroke), hepatic, renal, endocrine (including Type 1 and Type 2 diabetes mellitus), hematologic, immunologic, neurologic (such as Alzheimer's or Parkinson's disease), psychiatric (including depression and/or anxiety disorders) or biliary disorders. Conditions which are well-controlled or resolved will be assessed by the Clinical Investigator on a case-by-case basis.
6. Uncontrolled hypertension (systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg) as defined by the blood pressure measured at Visit 1 (Day -7). Stable use of hypertension medication is allowed \[defined as no change in medication regimen within the 3 months prior to Visit 1 (Day -7)\].
7. Have received a COVID-19 vaccine within 2 weeks of randomization or expected to receive a COVID-19 vaccine during the study period.
8. Had a positive SARS-CoV2 test and experienced symptoms for more than 2 months (i.e. "long-haulers").
9. Extreme dietary habits (e.g., vegetarian, vegan, Atkins diet, etc.) at the discretion of the Clinical Investigator.
10. History or presence of cancer in the prior 2 years, except for non-melanoma skin cancer.
11. Major trauma or any other surgical event within 3 months of Visit 1 (Day -7).
12. Signs or symptoms of an active infection of clinical relevance within 5 days of Visit 1 (Day -7). The visit may be rescheduled such that all signs and symptoms have resolved (at the discretion of the Clinical Investigator) at least 5 days prior to Visit 1 (Day -7).
13. Weight loss or gain more than 4.5 kg in the 3 months prior to Visit 1 (Day -7).
14. Currently or planning to be on a weight loss regimen during the study.
15. Use of weight loss medications, incretin mimetics, and/or GLP-1 agonists in the 3 months prior to Visit 1 (Day -7).
16. Consumption of supplemental inulin/chicory root or inulin/chicory root-fortified foods and beverages equivalent to more than 10 g inulin per day within 1 month of Visit 1 (Day -7).
17. Antibiotic use within 3 months of Visit 1 (Day -7) and throughout the study period.
18. Use of steroids within 1 month of Visit 1 (Day -7) and throughout the study period.
19. Habitual use (i.e., daily) of marijuana and hemp products including CBD products within 30 days of Visit 1 (Day -7).
20. Chronic use (i.e., daily on a regular basis) of anti-inflammatory medications (e.g., NSAIDS) within 1 month of Visit 1 (Day -7).
21. Use of medications (over-the-counter or prescription) and/or dietary supplements, known to influence GI function, including but not limited to, digestive enzymes, pre- and probiotic supplements as well as foods or beverages containing live probiotics (e.g., yogurt, kombucha), fiber supplements, laxatives, enemas, suppositories, H2 blockers, proton pump inhibitors, antacids, anti-diarrheal agents, anti-depressants, and/or anti-spasmodics within 2 weeks of Visit 1 (Day -7) and throughout the study period. Standard multivitamin and mineral supplements are allowed.
22. Participated in endoscopy or endoscopy preparation within 3 months prior to Visit 1 (Day -7).
23. Exposure to any non-registered drug product within 4 weeks prior to Visit 1 (Day -7).
24. Female who is pregnant, planning to be pregnant during the study period, lactating, or is of childbearing potential and is unwilling to commit to the use of a medically approved form of contraception throughout the study period. Participants who are pregnant during the study will be discontinued.
25. Recent history (within 12 months of screening; Visit 1; Day -7) of alcohol or substance abuse. Alcohol abuse is defined as more than 14 drinks per week (1 drink = 12 oz beer, 5 oz wine, or 1½ oz distilled spirits).
26. Receipt or use of study products in another research study within 4 weeks prior to Visit 2 (Day 0) or longer if the previous study product is deemed by the Clinical Investigator to have lasting effects that might influence the eligibility criteria or outcomes of current study.
27. Has a condition the Clinical Investigator believes would interfere with the participant's ability to provide informed consent, comply with the study protocol, which might confound the interpretation of the study results, or put the participant at undue risk.