Prediction of Dietary Intervention Efficacy in Mild Ulcerative Colitis Patients Based on Fecal Microbiome Signatures
Phase: NA
Conditions: Ulcerative Colitis
Sponsor: Wageningen University

NL79442.091.22                                     PREDUCTOME study 
 
 
 
 
 
 
 
 
 
 
 
Prediction of dietary intervention efficacy  
in mild ulcerative colitis patients based on fecal 
microbiome signatures  
(PREDUCTOME study) 
 
 
 
RESEARCH PROTOCOL 
Version 4 (26 July 2022) 
 
 
 

NL79442.091.22                                     PREDUCTOME study 
 
 
 
PROTOCOL TITLE  
Prediction of dietary intervention efficacy in mild ulcerative colitis patients based on fecal 
microbiome signatures 
Protocol ID 
NL79442.091.22 
Short title 
PREDUCTOME study 
EudraCT number  
Not applicable 
Version 
4 
Date 
26 July 2022 
Coordinating 
investigator/project leader 
 
Zhuang Liu, PhD candidate 
Laboratory of Microbiology 
Wageningen University & Research (WUR) 
Stippeneng 4, 6708 WE, Wageningen 
Phone: +31 (0)617 659 164 
E-mail: zhuang.liu@wur.nl 
Principal investigator(s) (in 
Dutch: hoofdonderzoeker/ 
uitvoerder) 
 
Erwin G. Zoetendal, PhD 
Laboratory of Microbiology 
Wageningen University & Research 
Stippeneng 4, 6708 WE, Wageningen 
Phone: 0317- 483111 
E-mail: erwin.zoetendal@wur.nl 
Sponsor (in Dutch: 
verrichter/opdrachtgever) 
Wageningen University (WU) 
Laboratory of Microbiology 
 
P.O. Box 8033 
6700 EH Wageningen 
Phone: +31 317 483116 
Subsidising party 
Winclove Probiotics B.V., The Netherlands 
China Scholarship Council 
Independent expert (s) 
Dr. Remy H.H. Bemelmans 
Phone: 0318-435614 
E-mail: bemelmansr@zgv.nl 
 

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Medical supervision (in 
Dutch: onderzoeksarts)  
 
Prof. Ben Witteman PhD MD  
Endowed Professor in Nutrition and Intestinal Health 
Transitional Care and Gastro-enterologist at Gelderse 
Vallei hospital, Ede, the Netherlands 
Wageningen University & Research (WUR)  
Division of Human Nutrition  
Stippeneng 4, 6708 WE Wageningen  
E-mail: ben.witteman@wur.nl  
Phone: +31 (0)317 48 51 86  
Laboratory sites  
Wageningen University & Research (WUR) 
Laboratory of Microbiology  
Stippeneng 4  
6708 WE Wageningen  
Pharmacy  
Not applicable 

NL79442.091.22                                     PREDUCTOME study 
 
 
 
PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Sponsor or legal representative: 
Dr. Sjoukje Heimovaara, PhD 
Managing director 
Agrotechnology & Food Sciences 
Group  
Wageningen University & Research 
 
 
09-02-2022 
Coordinating Investigator/Project 
leader 
Zhuang Liu, MSc 
PhD candidate 
Laboratory of Microbiology 
Wageningen University & Research 
 
 
06-12-2021 
Principal Investigator 
Dr. Erwin G. Zoetendal, PhD 
Associate professor 
Laboratory of Microbiology 
Wageningen University & Research 
 
 
21-12-2021 
Medical supervisor 
Prof. Ben Witteman, PhD MD 
Division of Human Nutrition & 
Health 
Wageningen University & Research 
 
28-11-2021 
 
 

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TABLE OF CONTENTS 
 
1. 
INTRODUCTION AND RATIONALE .............................................................................11 
2. 
OBJECTIVES ................................................................................................................14 
3. 
STUDY DESIGN ...........................................................................................................15 
4. 
STUDY POPULATION ..................................................................................................18 
4.1 
Population (base) ...................................................................................................18 
4.2 
Inclusion criteria .....................................................................................................18 
4.3 
Exclusion criteria ....................................................................................................18 
4.4 
Sample size calculation ..........................................................................................19 
5. 
TREATMENT OF SUBJECTS .......................................................................................20 
5.1 
Investigational product/treatment ............................................................................20 
5.2 
Use of co-intervention (if applicable) ......................................................................20 
5.3 
Escape medication .................................................................................................20 
6. 
INVESTIGATIONAL PRODUCT ....................................................................................21 
6.1 
Name and description of investigational product(s) ................................................21 
6.2 
Summary of findings from non-clinical studies ........................................................22 
6.3 
Summary of findings from clinical studies ...............................................................22 
6.4 
Summary of known and potential risks and benefits ...............................................23 
6.5 
Description and justification of route of administration and dosage .........................23 
6.6 
Dosages, dosage modifications and method of administration ...............................24 
6.7 
Preparation and labelling of Investigational Product ...............................................24 
6.8 
Investigational Product accountability .....................................................................24 
7. 
NON-INVESTIGATIONAL PRODUCT ...........................................................................25 
8. 
METHODS ....................................................................................................................26 
8.1 
Study parameters/endpoints ...................................................................................26 
8.1.1 
Main study parameter/endpoint .......................................................................26 
8.1.2 
Secondary study parameters/endpoints ..........................................................26 
8.1.3 
Other study parameters ...................................................................................27 
8.2 
Randomisation, blinding and treatment allocation ..................................................28 
8.3 
Study procedures ...................................................................................................28 
8.4 
Withdrawal of individual subjects ............................................................................31 
8.4.1 
Specific criteria for withdrawal .........................................................................31 
8.5 
Replacement of individual subjects after withdrawal ...............................................31 
8.6 
Follow-up of subjects withdrawn from treatment .....................................................32 
8.7 
Premature termination of the study .........................................................................32 
9. 
SAFETY REPORTING ..................................................................................................33 
9.1 
Temporary halt for reasons of subject safety ..........................................................33 
9.2 
AEs, SAEs and SUSARs ........................................................................................33 
9.2.1 
Adverse events (AEs) ......................................................................................33 
9.2.2 
Serious adverse events (SAEs) .......................................................................33 
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) .........................34 
9.3 
Annual safety report ...............................................................................................34 

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9.4 
Follow-up of adverse events ...................................................................................34 
9.5 
[Data Safety Monitoring Board (DSMB) / Safety Committee] ..................................34 
10. 
STATISTICAL ANALYSIS ..........................................................................................35 
10.1 
Primary study parameter(s) ....................................................................................35 
10.2 
Secondary study parameter(s) ...............................................................................35 
10.3 
Other study parameters ..........................................................................................37 
10.4 
Interim analysis (if applicable) ................................................................................37 
11. 
ETHICAL CONSIDERATIONS ...................................................................................38 
11.1 
Regulation statement .............................................................................................38 
11.2 
Recruitment and consent........................................................................................38 
11.3 
Objection by minors or incapacitated subjects (if applicable) ..................................39 
11.4 
Benefits and risks assessment, group relatedness .................................................39 
11.5 
Compensation for injury .........................................................................................39 
11.6 
Incentives ...............................................................................................................40 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .........................41 
12.1 
Handling and storage of data and documents ........................................................41 
12.2 
Monitoring and Quality Assurance ..........................................................................41 
12.3 
Amendments ..........................................................................................................41 
12.4 
Annual progress report ...........................................................................................42 
12.5 
Temporary halt and (prematurely) end of study report ............................................42 
12.6 
Public disclosure and publication policy ..................................................................42 
13. 
STRUCTURED RISK ANALYSIS...............................................................................44 
13.1 
Potential issues of concern .....................................................................................44 
13.2 
Synthesis ...............................................................................................................44 
14. 
REFERENCES ..........................................................................................................45 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
General Assessment and Registration form (ABR form), the application 
form that is required for submission to the accredited Ethics Committee; in 
Dutch: Algemeen Beoordelings- en Registratieformulier (ABR-formulier) 
5ASA 
5-Aminosalicylate 
AE 
Adverse Events 
AG 
Acacia Gum 
ANOVA  
Analysis of Variance  
ASV 
Amplicon Sequence Variant 
BMI 
Body Mass Index 
BV 
In Dutch: Besloten Vennootschap 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
COVID-19 
Coronavirus disease 2019 
CV 
Curriculum Vitae 
DNA 
Deoxyribonucleic Acid 
DSMB 
Data Safety Monitoring Board 
ELISA 
Enzyme-linked Immunosorbent assay 
EU 
European Union 
EudraCT 
European drug regulatory affairs Clinical Trials  
FDR 
False Discovery Rate 
FFQ 
Food Frequency Questionnaire 
FOS 
Fructo-OligoSaccharides 
GBF 
Germinated Barley Foodstuff 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
GG 
Guar Gum 
GI 
Gastro-Intestinal 
GMP 
Good Manufacturing Practice 
GP 
General Practitioner 
GSRS 
Gastrointestinal Symptom Rating Scale 
HPLC 
High Performance Liquid Chromatography  
IB 
Investigator’s Brochure 
IBD 
Inflammatory Bowel Disease 

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ID 
Identity Document 
IC 
Informed Consent 
IMP 
Investigational Medicinal Product  
IMPD 
Investigational Medicinal Product Dossier  
ISO 
International Organization for Standardization 
METC  
Medical research ethics committee (MREC); in Dutch: medisch-ethische 
toetsingscommissie (METC) 
NSF 
National Sanitation Foundation 
PCoA 
Principle Coordinate Analysis 
PERMANOVA Permutational Multivariate Analysis Of Variance 
PHGG 
Partially Hydrolyzed Guar Gum 
P-SCCAI 
Patient Simple Clinical Colitis Activity Index 
qPCR 
quantitative Polymerase Chain Reaction 
QQ 
Quantile-Quantile 
RNA 
Ribonucleic Acid 
RS 
Resistant Starch 
(S)AE 
(Serious) Adverse Event  
SCFAs 
Short Chain Fatty Acids 
SD 
Standard Deviation 
SDL 
Security Development Lifecycle 
SIBDQ 
Short Inflammatory Bowel Disease Questionnaire 
SPC 
Summary of Product Characteristics; in Dutch: officiële productinformatie 
IB1-tekst 
Sponsor 
The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical company, 
academic hospital, scientific organisation or investigator. A party that 
provides funding for a study but does not commission it is not regarded as 
the sponsor, but referred to as a subsidising party. 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 
TNF- α 
Tumour Necrosis Factor alpha  
UAVG 
 
UC 
Dutch Act on Implementation of the General Data Protection Regulation; 
in Dutch: Uitvoeringswet AVG 
Ulcerative Colitis 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 
 
 

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SUMMARY 
Rationale: Ulcerative colitis (UC) patients respond differently to treatments/interventions 
(e.g. diet/fecal microbiota transplantation), but the reason for this individual specificity 
remains unknown. We hypothesize that the baseline fecal microbiota composition 
determines the efficacy of a treatment/intervention, and potential responders, i.e. patients 
showing symptoms improvement after treatment, can be predicted based on fecal microbiota 
composition. 
Objective: The primary objective is to validate the prediction that prebiotics intervention 
boosts butyrate production and thereby induces a higher response (lower mean Patient 
Simple Clinical Colitis Activity Index (P-SCCAI) score) in mild UC patients with low intestinal 
Bacteroidetes levels (predicted responders), but not in those with high intestinal 
Bacteroidetes levels (predicted non-responders) at T = 8 weeks. The secondary objectives 
are to study the effects of prebiotics intervention on response over time (T = 0, 4, 8, 12 and 
60 weeks), mucosal inflammation, gastro-intestinal (GI) complaints, stool consistency, stool 
frequency, fecal microbiota composition, fecal short-chain fatty acids concentrations, quality 
of life, medication use, and safety parameters in mild UC patients. 
Study design: This study is a four-arm double-blind randomized placebo-controlled parallel 
trial. It consists of a screening stage in which mild UC patients will be assigned to be 
predicted responders or predicted non-responders based on their fecal Bacteroidetes levels. 
Afterwards the predicted responders and non-responders will be assigned to either the 
prebiotics group (arm 1 and 3) or placebo group (arm 2 and 4).  
Study population: Adult subjects aged 18-65 years and body mass index 18-30 kg/m2 with 
mild UC defined by P-SCCAI (3-5 points in a 19-point scale), with at least one relapse in the 
last two years. 
Intervention: An 8-week intervention period with four parallel arms: 1) predicted responders 
with prebiotics treatment (acacia gum, partially hydrolyzed guar gum, and resistant starch), 
2) predicted responders with placebo (maltodextrin and corn starch), 3) predicted non-
responders with prebiotics treatment, 4) predicted non-responders with placebo, during 
which the study participants consume the respective supplement (3 grams, twice daily). 
Main study parameters/endpoints: The main parameter is the response (mean P-SCCAI 
score) between arms at T = 8 weeks. The secondary parameters are the response over time 
at T = 0, 4, 8, 12, and 60 weeks, mucosal inflammation (fecal calprotectin), gastro-intestinal 
(GI) complaints, stool consistency, stool frequency, fecal microbiota composition, fecal short-
chain fatty acids concentrations, health-related quality of life, medication use, and safety 
parameters. 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: This study has a relatively low burden for the participants. They have to 

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consume the commercially available supplement twice daily for eight weeks. Besides, 
several questionnaires (one long FFQ questionnaire on one occasion, two short ones on five 
occasions, five short ones on three occasions) have to be completed, which is all possible 
from home. On five occasions they will collect fecal samples (which will be collected from 
their homes by us). There are no physical risks for the study participants, although some 
participants may experience minor bloating complaints at the start of using the prebiotics. 

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1. INTRODUCTION AND RATIONALE 
 
The incidence and prevalence of ulcerative colitis (UC) are increasing worldwide [1]. It is a 
disease that causes inflammation extending from rectum to colon, and it is one of the two 
major types of inflammatory bowel disease (IBD) associated with abdominal pain, bloody 
diarrhea, and urgency to defecate [2]. Compared with the general population, patients with 
longstanding UC, regardless of clinical activity, have an increased risk for colorectal cancer 
[2, 3]. Unfortunately, there is no cure for UC and hence, treatments focus on dampening the 
severity of symptoms. Besides medical or surgical treatments, about half of patients with UC 
have mild symptoms in which a dietary recommendation, including increasing dietary fiber 
intake, could help reduce inflammation [4-6]. Although several successes for inducing 
remission in UC have been reported with treatments including dietary modification and fecal 
microbiota transplantation, there is no golden grail treatment for all subjects. Some 
individuals respond well to a certain treatment strategy while others do not. The reason for 
these differences between individual responses remains speculative. Therefore, it is 
important to find biomarkers that enable clinicians to identify patients more likely to respond 
to specific dietary treatments beforehand, and push them back to remission as fast as 
possible. As a consequence, most therapeutic approaches of UC start with classifying 
patients according to the severity of the disease [7].  
 
Diet could extensively affect the environment of human intestine, which harbors trillions of 
microbes, commonly referred to as the “intestinal microbiota”, and “microbiome” (the 
collection of microbes and all the genes and functionalities they encode [8]). Numerous 
studies have suggested that the intestinal microbiota is closely associated with overall 
human health [9, 10]. More importantly, the human gut microbiome is a rich pool of potential 
biomarkers that have been previously shown to be associated with disease states including 
IBD [11, 12]. Microbiome-based stratification strategies of patients groups have been widely 
employed to guide dietary intervention studies [13]. Recently, a low-fat, high-fiber diet has 
been found to reduce inflammatory markers and alter the microbiome in patients with mild 
UC or UC in remission [14]. Another similar study demonstrated that a fiber-based prebiotic 
resulted in less abdominal pain and cramping in UC patients [15]. This indicates that 
increasing dietary fiber intake could be beneficial for maintaining periods of remission or 
improving clinical outcomes in patients with UC. Furthermore, short chain fatty acids 
(SCFAs) such as butyrate are produced by intestinal bacteria when they break down dietary 
fibers and these are considered beneficial for gut health. In a pilot study, it was demonstrated 
that UC patients could take a fiber-rich diet to increase the fecal butyrate level [5]. However, 
response to a treatment differs drastically between subjects, with some patients entering 
remission (responder), while others fall or remain into relapse (non-responders). The reasons 
for this inconsistent response between subjects remain speculative but may be the result of 
differences in the personal gut microbiota composition [16]. 
 

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Looking from an ecological perspective, 
ecosystems are generally consisting of 
alternative stable states that are resilient to 
(minor) perturbations. Changing from one 
stable state into another stable state needs a 
strong perturbation in which a tipping point 
needs to be crossed (Figure 1) [17, 18]. For 
example, both deserts and rainforests are 
stable states, and only a drastic perturbation, 
like deforestation, allows a rainforest to cross 
a tipping point to become a desert, and 
similarly, it will need a lot of effort to have it 
return again from a desert into a rainforest in 
which a tipping point needs to be crossed as well. We believe that similar ecological 
principles exist for intestinal microbial ecosystems. Recently, we have published a review in 
which groups of microbiomes containing certain characteristics can be considered as 
alternative ecosystems states that are generally resilient to perturbations [19]. These stable 
states can both be homeostatic microbiomes in healthy subjects as well as microbiomes 
associated with disease, such as UC, and switching from one to the other needs strong 
perturbations (such as diseases/infections causing diarrhea) or treatments (such as 
antibiotics use or prebiotic consumption) that result in crossing the tipping point.  
 
Generally, UC patients have a gut microbiota composition that is different from that observed 
in healthy subjects. In UC patients, alterations of the intestinal microbiome have been 
observed in many clinical studies, characterized by a general reduction in fecal microbiota 
diversity, along with phylum-level increases in Bacteroidetes and decreases in Firmicutes, 
and lower levels of butyrate [20-22]. Nevertheless, there is also a clear subject-specific 
impact on the gut microbiota composition. For healthy subjects, different alternative healthy 
states of the gut-microbiome have been identified, and we speculate that different alternative 
states also exist for UC patients. Indeed a recent study on the rat model for UC has indicated 
the existence of health states and disease active states [23]. Similarly, alternative intestinal 
ecosystem states have also been observed in pediatric UC and these could affect remission 
in UC [24]. 
 
The Bacteroidetes phylum is one of the most abundant phyla in the human gut microbiome 
and dramatic differences at this phylum level have been observed [20, 25]. Remarkably, 
induction of sustained remission via fecal microbiota transplantation was associated with a 
baseline microbiota that was more similar to that of healthy individuals which have a 
relatively low Bacteroidetes level (average 5% and 8%, respectively), while non-responders 
have a relatively high baseline Bacteroidetes level (average 19%) [20]. Moreover, the 
induction of sustained remission was associated with an improved butyrate production 
capacity. Hence, we hypothesize that the gut microbiome of UC patients with low intestinal 
Bacteroidetes levels are characterized as an alternative state that has a tipping point that is 
Figure 1 Existent tipping point in 
ecosystem. (adapted from reference 17 
and 18.) 

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easier to be crossed by an intervention compared to those with higher Bacteroidetes levels. 
We speculate that crossing this tipping point can be reached by stimulating intestinal butyrate 
production capacity. 
 
There is a growing recognition that not an individual fiber but a mixture of them are more 
likely to promote the growth of SCFAs producing bacteria, because individual or consortia of 
bacteria play different, sometimes complementary, roles in the fiber degradation process. 
Therefore, by using a fiber mixture, the physiological effects of fiber (e.g., SCFAs production 
and bowel movement modulation) would be stimulated [26]. To identify a potential treatment 
targeting the butyrate-producing capacity that could be used in an intervention study, we 
applied an in vitro screening of different dietary fiber mixtures (so-called “butyrate boosters”) 
on the gut microbiome of UC patients to test our hypothesis. From this pilot study, we 
identified a promising fiber prototype product (mixture of acacia gum, partially hydrolyzed 
guar gum, and resistant starch) that boosted butyrogenic activity in microbiota with low 
Bacteroidetes levels, while it remained unaffected in those with high Bacteroidetes levels 
(unpublished results). To further test our hypothesis, we will conduct a dietary intervention 
study with a mixture of butyrate boosting fibers (hereinafter referred to as prebiotics) of which 
we predict they will induce a higher response in mild UC patients with a low intestinal 
Bacteroidetes level, while mild UC patients with a high intestinal Bacteroidetes level will be 
less affected (Figure 2). 
 
 
 
Figure 2 PREDUCTOME study : hypotheses 

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2. OBJECTIVES 
 
Primary Objective:  
The primary objective is to validate the prediction that prebiotics intervention boosts butyrate 
production and thereby induces a higher response (lower mean Patient Simple Clinical 
Colitis Activity Index (P-SCCAI) score) in mild UC patients with low intestinal Bacteroidetes 
levels (predicted responders), but not in those with high intestinal Bacteroidetes levels 
(predicted non-responders) at T = 8 weeks 
 
Secondary Objective(s):  
1. To study the effects of the prebiotic intervention on response over time at T = 0, 4, 8, 
12, and 60 weeks in mild UC patients 
2. To study the effects of the prebiotic intervention on mucosal inflammation (fecal 
calprotectin levels) in mild UC patients 
3. To study the effects of the prebiotic intervention on gastro-intestinal (GI) complaints 
(e.g., indigestion, abdominal pain) in mild UC patients 
4. To study the effects of the prebiotic intervention on stool consistency and frequency 
in mild UC patients 
5. To study the effects of the prebiotic intervention on the gut microbiota composition 
and SCFAs profile in mild UC patients  
6. To study the effects of the prebiotic intervention on health-related quality of life in mild 
UC patients  
7. To study the effects of the prebiotic intervention on medication use in mild UC 
patients  
8. To study the effects of the prebiotic intervention on safety parameters in mild UC 
patients 
 

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3. STUDY DESIGN 
 
This study is a double-blind randomized placebo-controlled parallel trial. A summary of the 
study set-up and workflow is shown in Figure 3.  
The study consists of the screening, intervention, and the follow-up periods.  
 
During the screening period, approximately one hundred and seventy-seven UC patients will 
be recruited. P-SCCAI score data will be collected to select mild UC patients which is defined 
as 3-5 points on a 19-point scale. A stool sample will also be provided by patients to 
determine the Bacteroidetes level by quantitative polymerase chain reaction (qPCR) 
quantification of the number of total bacteria and Bacteroidetes. Based on the baseline 
microbiota from a subject in a fecal microbiota transplantation trial [20], in which healthy 
donors, sustained responding UC patients, and non-responding UC patients had an average 
relative abundance of 8%, 5%, and 19% Bacteroidetes, respectively, predicted responders 
are defined as UC patients having with a relative abundance of Bacteroidetes <=10% in their 
feces, while predicted non-responders are defined as UC patients with a relative abundance 
of Bacteroidetes >=15% in their feces. A total of sixty screened mild UC patients will continue 
with this study that consists of thirty predicted responders and thirty predicted non-
responders. 
 
To assess the habitual dietary intake of the last month, a validated food frequency 
questionnaire (FFQ, see F1) will be taken at the baseline. The data will also be used for 
potential correlation analysis with other parameters as diet is considered an important 
modulator of the intestinal microbiota. For the intervention, at baseline, selected participants 
Figure 3 PREDUCTOME study: set-up and workflow 

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need to fill out several questionnaires including P-SCCAI (once), gastrointestinal symptom 
rating scale (GSRS, once), stool consistency (Bristol stool scale, daily for 7 consecutive 
days), stool frequency (daily for 7 consecutive days), short inflammatory bowel disease 
questionnaire (SIBDQ, once). Information of medication use and adverse events will be 
extracted from patient record and diary. In addition, they have to collect a stool sample for 
determining calprotectin level, microbiota composition, and SCFAs profile. 
 
Sustained remission in UC patients in the published fecal microbiota transplantation trial was 
associated to an increased fraction of butyrate producers [20]. Our in vitro incubation studies 
with fecal inocula from UC patients showed that a prebiotic mixture of acacia gum, partially 
hydrolyzed guar gum, and resistant starch, showed the highest butyrate boosting capacity in 
subjects with low fecal Bacteroidetes levels. Hence, demonstrating the perfect candidate for 
our study. 
Based on published studies on interventions on inducing clinical remission, an 8-week 
intervention should be sufficient for the prebiotics interventions to result in significant effects 
[4, 16, 27, 28]. An 8-week intervention period with four parallel arms:  
1) predicted responders with prebiotics treatment (acacia gum, partially hydrolyzed guar 
gum, and resistant starch) 
2) predicted responders with placebo (maltodextrin and corn starch) 
3) predicted non-responders with prebiotics treatment (acacia gum, partially hydrolyzed guar 
gum, and resistant starch) 
4) predicted non-responders with placebo (maltodextrin and corn starch) 
 
During the intervention period, the subjects will consume the respective supplement (3 
grams/sachet, 2 sachets per day, one in the morning and one in the evening). To avoid GI 
complaints due to a sudden intake of the prebiotics/placebo supplement, there will be a 
gradual increase in dosage: only 1 sachet will be consumed per day in the first week of the 
intervention period, followed by 2 sachets per day for the remainder of the intervention. At 
week 4 and week 8, they will also complete short questionnaires including P-SCCAI (once a 
week), GSRS (once a week), stool consistency (a 7-day record at each week), stool 
frequency (a 7-day record at each week), SIBDQ (once a week). Information of medication 
use and adverse events will be extracted from patient record and diary (once a week). At the 
end of the intervention period (T= 8 weeks), the participants will provide a second stool 
sample for fecal calprotectin measurements, microbiota composition, and SCFAs profile 
analysis. We will determine response with the P-SCCAI score at T= 8 weeks. 
 
The intervention will be followed by a 4-week and 1-year follow-up period. At the end of 
each follow-up (T= 12, and 60 weeks), the participants will collect a stool sample for the 
same measurements as those at the end of the intervention period (fecal calprotectin level, 
microbiota composition, and SCFAs profile), and complete the questionnaires including P-
SCCAI (once a week). Information of medication use will be extracted from patient record 
and diary (once a week). 
 

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As the intervention will take place at home, the study can be conducted in a merely 
coronavirus (COVID-19)-proof setting. 

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4. STUDY POPULATION 
4.1 Population (base)  
This study will be performed in adult UC patients with mild severity of the disease. UC 
severity will be determined by P-SCCAI score (see F1) [29], with a 3-5 score on a 19-
point scale selected as mild UC inflammatory activity [30, 31]. Call for the participation of 
this study will be first spread among patients base (more than 1200 IBD patients) of 
Ziekenhuis Gelderse Vallei in Ede. In case of under-recruitment, UC patients of Rijnstate 
in Arnhem will be included as well. 
 
In order to be eligible to participate in this study, a subject must meet all of the following 
inclusion criteria and none of the exclusion criteria. 
 
4.2 Inclusion criteria 
1. Male and female subjects aged 18 to 65 years* 
2. Body Mass Index (BMI) between 18 and 30 kg/m2 (self-reported) 
3. Ulcerative Colitis confirmed via previous endoscopy and histology  
4. Mild active UC as defined by P-SCCAI score of 3 to 5 (range 0 to 19)  
5. Frequent relapse (at least one exacerbation in the last two years) 
6. No known allergy to any components of the study product (self-reported) 
7. Signed informed consent  
8. Stable UC medication defined as no switch to other medication or no dose change 
9. Mobile phone on which apps (used for questionnaires) can be downloaded (iOS 
version 9 and newer, Android version 4.4 and newer. Phones manufactured after 
2013 are usually suitable) 
10. Stable dietary pattern during the study 
 
* We only include adults (age 18-65) as previous studies have shown that adolescent 
and elderly microbiota as well as UC presentation in adolescents can be different 
from adults[32, 33]. 
 
4.3 Exclusion criteria 
1. Any other underlying disease of the GI-tract or previous bowel surgery, except 
cholecystectomy and appendectomy 
2. Pregnancy or intending to become pregnant during the study 
3. Use of medication that can interfere with the study outcomes, as judged by the 
medical supervisor 
4. The need for antibiotic use during the intervention period 
5. Systemic antibiotics and proton pump inhibitors (except for omeprazole and 
pantoprazole with dosage <20 mg), prebiotic supplements, probiotic supplements four 
weeks prior to study start 
6. Currently participating in another intervention study 
7. Acquaintances of anyone in the research team  
 

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4.4 Sample size calculation 
The primary objective of the trial is to validate the prediction that a higher response (lower 
mean P-SCCAI score) is induced at T= 8 weeks by the prebiotics intervention in mild UC 
patients with low Bacteroidetes levels (predicted responders), but not in those with high 
Bacteroidetes levels (predicted non-responders) in their gut. Mild UC is defined as a P-
SCCAI score of 3 to 5. According to an extensive study regarding UC disease activity 
conducted in the Netherlands, mild UC patients have a P-SCCAI score of 3.5 ± 0.5 (Mean 
± standard deviation (SD)), and patients in remission score at 1.0 ± 0.33 (Mean ± SD)[34, 
35].  
 
In a series of germinated barley foodstuff (GBF) intervention studies, 10 mild to 
moderately active UC patients received 30 grams of GBF (equals to 10 grams fiber) daily 
for 4 weeks [4], and researchers found a significant decrease in clinical activity index 
scores. In a later study, another group of 11 mildly to moderately active UC patients 
consuming 20-30 grams of GBF (equals to 6.66-10 g fiber) daily for 4 weeks showed 
similar results, with decreases in clinical activity index scores [28].  
 
Based on two-sided statistical testing for unpaired continuous data, an α=0.05 (chance on 
type-I error) and β=0.10 (chance on type-II error), and assuming a 20% difference of 
mean P-SCCAI score between predicted responders with prebiotics and predicted non-
responders with prebiotics, it was calculated that at least 11 subjects per group are 
needed for the primary outcome P-SCCAI (µ1=3.5; SD1=0.5; µ2=2.8). According to 
previous trials on prebiotics on IBD patients, a 20% of drop-out rate is commonly seen. 
Given that the intervention duration is 8 weeks long, followed by a 1 year follow-up, 
higher dropout rate (30%) is expected. To compensate for a 30% drop-out rate, 15 
subjects per group are needed. 
 
To reach inclusion of 30 predicted responders and 30 predicted non-responders, we 
made an estimate how many subjects we need to screen based on the earlier obtained 
microbial composition of stool samples from UC patients in the Ede area. Around 50% of 
these subjects were categorized as low Bacteroidetes subjects (predicted responders) 
and while 17% of them as high Bacteroidetes subjects (predicted non-responders). To 
include these responders we will need to screen 177 mild UC patients. 
 

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5. TREATMENT OF SUBJECTS 
5.1 Investigational product/treatment 
In this study, all 60 participants will undertake an 8-week intervention with either a 
prebiotics supplement (arm 1 and 3) or a placebo supplement (arm 2 and 4), which will be 
consumed once per day (1 sachet in the morning) during the first week, and twice per day 
(1 sachet in the morning and 1 sachet in the evening) for the remainder of the 
intervention. During the entire trial, research subjects will maintain their normal routines 
concerning their diet, medication use, and exercise pattern. 
 
Prebiotics supplement: Prebiotics preparation is composed of three individual fibers (40% 
acacia gum, 20% partially hydrolyzed guar gum, and 40% resistant starch) from Winclove 
Probiotics B.V. (Amsterdam, the Netherlands). 
 
Placebo (control): Placebo preparation is composed of food-grade corn starch and 
maltodextrin from Winclove Probiotics B.V. (Amsterdam, the Netherlands). 
 
5.2 Use of co-intervention (if applicable) 
Not applicable. 
 
5.3 Escape medication 
Not applicable. 

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6. INVESTIGATIONAL PRODUCT  
 
6.1 Name and description of investigational product(s) 
Prebiotics: acacia gum (AG), partially hydrolyzed guar gum (PHGG), and resistant starch 
(RS). 
- 
Acacia gum (AG) also known as gum arabic, is obtained from the stems and 
branches of Acacia senegal and Acacia seyal trees. AG is a nonviscous, soluble, 
highly branched fiber, and its main components are arabinose and galactose [36]. It is 
a safe, natural, and plant-based food additive used for many centuries. For further 
details see product brochure (D6). 
- 
Partially hydrolyzed guar gum (PHGG) is a soluble fiber produced by controlled partial 
enzymatic hydrolysis of GG which is derived from guar seeds, and it consists of 
galactomannan, which is characterized by high viscosity. In the modern food industry, 
GG is used as a food additive in various food products for thickener, emulsion 
stabilizer and as fiber source [37, 38]. For further details see product brochure (D6). 
- 
Resistant starch (RS) refers to the fraction of starch which is not hydrolyzed in the 
small intestine, but is fermented by bacteria in the colon. It exists in many common 
foods, including legumes, cereals, grains and vegetables (especially potatoes) [39]. 
For further details see product brochure (D6). 
- 
 
The prebiotics preparation is composed of three non-digestible carbohydrates (40% 
acacia gum, 20% partially hydrolyzed guar gum, and 40% resistant starch). In total 3 
grams of prebiotic preparation contains 1.2 grams acacia gum, 0.6 gram partially 
hydrolyzed guar gum, and 1.2 grams of resistant starch. 
 
The prebiotics, consisting of a mix of non-digestible fermentable fibers described above 
has been selected on the basis of (1) Total butyrate production, (2) Gradual fermentation 
throughout the entire colon (instead of fast fermentation in the proximal colon), (3) A good 
prebiotic tolerability index (low gas production), and (4) Commercial considerations.  
 
 
Placebo: corn starch and maltodextrin. 
Corn starch and maltodextrin are digestible carbohydrates, that are completely digested 
and absorbed in the proximal small intestine, and thus do not reach the colon, which 
makes them suitable placebo dietary compounds and the reason why they are widely 
used in dietary intervention studies.  
The placebo consists of two digestible carbohydrates (20% maltodextrin and 80% corn 
starch). In total 3 grams of placebo preparation contains 0.6 gram of maltodextrin and 2.4 
grams of corn starch). A Technical Data Sheet has been provided for both prebiotic as 
well as placebo product. For further details see product brochure (D6). 
 

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All ingredients are non-pathogenic, non-toxigenic and are classified as Generally 
Recognized As Safe (GRAS) as food ingredients. The prebiotics and placebo 
supplements are produced by Winclove Probiotics B.V., Amsterdam, the Netherlands. 
Winclove is a National Sanitation Foundation (NSF) International Certified Good 
Manufacturing Practice (GMP) Facility for manufacturing dietary supplements. Winclove’s 
food safety management system is International Organization for Standardization (ISO) 
22000:2005 certified for the development and production of pre- and probiotics. 
 
6.2 Summary of findings from non-clinical studies 
 
Prebiotics: acacia gum (AG), partially hydrolyzed guar gum (PHGG) and resistant starch 
(RS). 
In vitro and animal models have been employed to study AG, PHGG or RS, with regard to 
their potential beneficial effect on health.  
- 
Using an in vitro colon model inoculated with healthy human fecal material, AG 
showed the potential prebiotic effects, as it promoted the SCFAs production, 
especially butyrate [40].  
- 
In a mice colitis model, rectal administration of AG (30 mg/kg) showed significant 
effects in reducing the inflammatory markers, and the stronger therapeutic effect 
were observed when combining with anti-inflammatory drug 5-Aminosalicylate 
(5ASA) [41]. 
- 
In a mice colitis model, PHGG and RS boosted SCFAs and butyrate production in 
the colon. In addition, they reduced intestinal inflammation [42] and induced 
proliferation of butyrate producing bacteria [43].  
 
As described earlier in sections 1 and 3, we have tested the mix of AG, PHGG and RS 
using in vitro fecal batch fermentation models. In this study, four prebiotics candidates 
were incubated with fresh stool samples from UC patients in remission or from UC 
patients with mild active disease over a 48 hour period. The prebiotic (acacia gum, 
partially hydrolyzed guar gum, and resistant starch), that is selected for this 
PREDUCTOME study showed the most promising effects, namely (unpublished results); 
- 
Increased production of butyrate and other SCFAs 
- 
Promotion of the growth of butyrate producing bacteria 
- 
Higher prebiotics tolerance defined as stimulated SCFAs production while minimal 
gas production 
 
Collectively, these pre-clinical data show that the selected prebiotics could potentially 
have a beneficial effect on the course of UC by modulating butyrate production. 
 
6.3 Summary of findings from clinical studies 
Prebiotics: acacia gum (AG), partially hydrolyzed guar gum (PHGG), and resistant starch 
(RS). 

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Many clinical studies with the supplementation of AG, PHGG, or RS have been 
performed to investigate prebiotics effects (e.g., modulating the gut microbiome and 
boosting butyrate production) and GI tolerance. 
- 
AG is slowly fermented compared with other fermentable fibers [44], therefore it is 
well-tolerated, i.e., less GI symptoms after consumption. Cherbut et al found AG 
did not induce adverse GI effects even when consumed up to a daily dose of 30 
grams in healthy subjects [45]. A study by Marzorati et al also observed that 
blending AG with fructo-oligosaccharides (FOS, a well-researched prebiotic) can 
help to extend the time of fermentation of FOS in the colon [46]. 
- 
Partially hydrolyzed guar gum (PHGG) showed no side effects when consumed in 
20-40 grams per day [47]. Besides of favouring butyrate-producing bacteria [48], 
PHGG also benefits bowel function. Increased stool frequency in constipated 
individuals and improved stool consistency and abdominal pain have previously 
been reported [49, 50]. 
- 
In general, in healthy adults, doses of RS up to 30 grams per day are well 
tolerated [51]. A dose-response study indicated that a daily dose of 20-50 grams 
of RS supplementation for one week resulted in beneficial changes in fecal 
microbiota and fecal metabolites in healthy individuals [52]. 
 
 
6.4 Summary of known and potential risks and benefits 
No serious adverse events are expected from the administration of the 
prebiotics/placebo. If adverse effects occur, they are likely to relate to minor GI 
symptoms, such as bloating or flatulence or a minor change in defecation, especially 
when one uses prebiotics for the first time [53]. In our case, UC patients are already used 
to GI complaints, hence they are expected to report less bloating and flatulence. These 
effects are expected to disappear within a few days. 
 
Furthermore, to minimize GI complaints due to a sudden intake of the prebiotics/placebo 
supplement, there will be a gradual increase in dosage: only 1 sachet will be consumed 
per day in the first week of the intervention period, followed by 2 sachets per day for the 
remainder of the intervention.  
 
Nevertheless, in case of suspected serious adverse events, the coordinating investigator 
will immediately notify the Ethics Committee, all study personnel and the manufacturer of 
the product about the nature of the event. A decision on continuation or discontinuation of 
the trial will be made by the coordinating investigator in agreement with the Ethics 
Committee. All the adverse events also will be noted in the Case Report Forms. 
 
6.5 Description and justification of route of administration and dosage 
The boxes of prebiotics/placebo will be given to subjects by the coordinating investigator. 
Boxes include prebiotics/placebo sachets and Winclove’s user instruction. Subjects 

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should consume prebiotics/placebo twice a day in the morning before breakfast and in the 
evening before dinner during the 8-week intervention period. Prebiotics/placebo will be 
administered orally. The content of the sachet needs to be dissolved in a glass of water or 
juice and should be stirred before consumption. The sachets need to be stored at room 
temperature in their original package. 
 
6.6 Dosages, dosage modifications and method of administration 
  3 grams of prebiotics per time, twice a day for 8 weeks. 
3 grams of placebo per time, twice a day for 8 weeks. 
Prebiotics and placebo and powders are similar in colour, solubility, and taste. 
 
 The dose of non-digestible fermentable fibers has been selected to maximize the potential 
benefits but to limit potential GI side effects and is based on previous studies that have 
been conducted. 
 
6.7 Preparation and labelling of Investigational Product 
Preparation and labelling will be conducted at Winclove Probiotics B.V. All components 
are legally admitted as food additives or food components. Winclove is a NSF 
International Certified GMP Facility for manufacturing dietary supplements. Winclove 
works with the food safety management system ISO 22000:2005 and is certified for the 
development and production of pre- and probiotics.  
 
After block randomization, the sachets will be labelled with: study name, instruction for 
use, expiration date and storing conditions. For further details see the product label in 
product brochure (D6). The products will be dispensed by the coordinating investigator. 
 
6.8 Investigational Product accountability 
The boxes of prebiotics/placebo will be given to subjects by the coordinating investigator 
(and are supplied to the subjects for the entire study period). Boxes include sachets and 
Winclove’s user instruction. The shipment with boxes and sachets will be delivered to 
Laboratory of Microbiology, Wageningen University & Research, Wageningen by courier. 
The food supplements will be safely stored and locked by key at the Laboratory of 
Microbiology and only accessible by the research team (a list of the randomization codes 
is provided, which can only be opened in case of emergency). The product will be kept at 
room temperature. The coordinating investigator will distribute the boxes to participants in 
person. For compliance reasons, the patients will be asked to save all the empty sachets, 
and return everything (used and unused sachets) to the coordinating investigator after the 
end of the intervention. After counting the empty sachets, to get numbers on protocol 
compliance, all study material can be destroyed. As this is a food supplement, there is no 
need for special waste treatment. 
 
 
 

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7. NON-INVESTIGATIONAL PRODUCT 
 
Not applicable. 
 

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8. METHODS 
8.1 Study parameters/endpoints 
8.1.1 Main study parameter/endpoint 
 
 Response (mean P-SCCAI score) between arms at T = 8 weeks: 
 At T = 8 weeks (the end the intervention), the P-SCCAI score will be collected via 
the P-SCCAI questionnaire. As main endpoint, the mean P-SCAAI score for each 
arm will be determined and compared to each other to determine the difference in 
response at T = 8 weeks. 
 
 P-SCCAI is derived from the original SCCAI questionnaire by translating into 
patients' comprehensible language, and has been validated to give substantial 
agreement with the original SCCAI [54]. P-SCCAI refers to disease symptoms 
during the previous week. It contains 13 items clustered into six domains: bowel 
frequency (during the day), bowel frequency (during the night), urgency of 
defecation, blood in stool, general well-being, and a number of defined extracolonic 
features of UC (i.e. arthritis, erythema nodosum, pyoderma angrenosum, uveitis).  
8.1.2 Secondary study parameters/endpoints  
 
Response over time 
In addition to week 8, subjects need to fill out the P-SCCAI questionnaire at T = 0, 4, 
12, and 60 weeks to determine the response over time. Comparisons will be made 
between groups as well as within subjects over time. With P-SCCAI score <= 2 
being regarded as clinical remission, and a decrease in the P-SCCAI score by more 
than two points from baseline being regarded as clinical response [30, 31, 55](see 
F1). We will also determine whether subjects have reached clinical remission and/or 
showed a clinical response over time. 
 
 
Mucosal inflammation 
At T= 0, 8, 12, and 60 weeks, a fecal sample will be provided by subjects for 
calprotectin measurement. 
 
 
GI complaints: (e.g. abdominal pain, bloating, flatulence) 
At T= 0, 4, and 8 weeks, the GI complaints will be assessed by the GSRS 
questionnaire. The GSRS contains 15 items clustered into five domains (reflux, 
abdominal pain, indigestion, diarrhea and constipation) addressing different 
gastrointestinal symptoms [56]. It has a seven-point graded scale where 1 
represents the absence of troublesome symptoms and 7 represents very 

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troublesome symptoms (see F1). The reliability and validity of the GSRS are well-
documented, and norm values for a general population are available [57].  
 
Stool consistency and frequency 
At T = 0, 4, and 8 weeks, subjects will be asked to keep diaries to record stool 
frequency (bowel movement per day) and consistency of each stool using Bristol 
stool form scale (see F1) on a daily basis for 7 days. 
 
Gut microbiota and SCFAs: (e.g. microbiota composition, SCFAs profile) 
At T = 0, 8, 12, and 60 weeks, a fecal sample will be provided by subjects for 16S 
rRNA gene-based microbiota profiling and SCFAs measurement. Subsequent 
analyses of a variety of microbial ecological characteristics, including microbiota 
composition, α-diversity (diversity within the sample), β-diversity (diversity between 
the samples), population dynamics over time, and group differences will also be 
conducted.  
 
Health-related quality of life 
At T = 0, 4, and 8 weeks, health-related quality of life will be assessed by the SIBDQ 
questionnaire (see F1). SIBDQ is a 10 item shortened version of the original IBDQ 
which was 32 items. It has a seven-point graded scale where 1 represents very 
troublesome symptoms and 7 represents the absence of troublesome symptoms 
Measures quality of life as measured in four domains (bowel symptoms, emotional 
health, systemic systems and social function) [58]. Its reliability and responsiveness 
to change has been validated in UC [59]. 
 
Medication use 
At T= 0, 4, 8, 12 and 60 weeks, information of medication use will be extracted from 
patient record and diary. It consists of the current medication use (e.g., 
aminosalicylates, corticosteroids, immunosuppressive agents, antimicrobial agents, 
and inhibitors of tumour necrosis factor-alpha (TNF- α)). Medication changes at 
week 4, 8, 12, and 60 provide information on disease development, with a 
downgrade means improvement and an upgrade means worsening of UC. 
 
Safety parameters 
At T= 0, 4, and 8 weeks, safety parameters (i.e., incidence of adverse events) will 
be monitored by patient record and diary. In this diary, all relapse-relevant 
information, including the need for systemic steroids, hospitalization, and surgery 
will be collected.  
 
8.1.3 Other study parameters 
Habitual food intake 

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At T= 0 (baseline), habitual dietary intake of the last month will be assessed by a 
validated food frequency questionnaire (FFQ, see F1) via FFQ-tool, a web-based 
interface tool. It provides data that will be used for potential correlation analysis with 
other parameters. 
 
Participants demographics and characteristics 
At T= 0 (baseline), general information (e.g., sex, age, BMI, disease duration, age at 
diagnosis, smoking habit, UC location, type of treatment) will be collected. 
 
 
8.2 Randomisation, blinding and treatment allocation 
During the screening period, participants will provide a fecal sample for microbiome 
signature measurement. This information will be used to assign subjects to four 
intervention arms by block randomization. All subjects screened for the trial will be 
assigned a unique participant ID number. Suitable participants will subsequently be 
randomised to prebiotics or placebo group using an online randomisation system 
accessible via password-protected access. 
 
All sachets will be uniformly packaged to ensure double-blinding. Research teams 
and trial participants will be blinded to the treatment group for the duration of the trial. 
After collection of all laboratory & questionnaire data and performing all analysis of 
the baseline/intervention period the study (up to the 4-week follow-up; T=12 weeks) 
will be unblinded for the investigators. At the end of the study (T=60 weeks), the 
participants will be informed of the kind of intervention they received. (only when 
approved in the informed consent). 
 
8.3 Study procedures 
A detailed description of procedures is shown in Table 1. 

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Informed consent procedure 
One or more information meetings will be organized prior to the study. In this meeting, 
the investigators will explain the background, objectives and study procedures. During 
this meeting, there will be time for the potential research subjects to ask questions. 
After the information event, subjects have two weeks to decide whether they are 
interested in participation or not. Research subjects who are willing to participate will 
receive the informed consent and the screening package which includes the P-SCCAI 
questionnaire and one stool collection kit. Then within one week, subjects will receive 
packages with corresponding products and instructions. 
 
If COVID-19 restrictions are still in place, we would also provide the option of having 
online information meetings via Microsoft Teams (“Teams is designed and developed 
in compliance with the Microsoft Trustworthy Computing Security Development 
Lifecycle (SDL), https://docs.microsoft.com/en-us/microsoftteams/teams-security-
guide). No personal data will be collected or send via this route. If preferred, research 
subjects will always have the option to attend a physical meeting. 
 
Screening 
Table 1 Procedures 

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Subjects who fulfil all inclusion criteria and none of the exclusion 
criteria will be invited to a screening. 
One stool sample will also be provided via the specific stool 
collectors (Figure 4). For female participants, pee gutters will also 
be provided to accurately separate stool from urine. After 
collection, stool material will be immediately frozen at the home 
freezers of the subjects, transported on dry ice (via courier) to the 
laboratory and subsequently frozen at -80 °C until further 
analysis. DNA will be isolated from part of the stool 
samples and subsequently be used for qPCR. Primers 
targeting total bacteria and Bacteroidetes will be used to quantify the total gene 
copies and calculate the relative abundance of Bacteroidetes in the stool. Subjects 
that have a relative abundance of Bacteroidetes <=10% will be assigned to the 
predicted responders group, while those who have a relative abundance of 
Bacteroidetes >= 15% will be assigned to the predicted non-responders group. 
 
Baseline 
At baseline, selected participants need to fill out several questionnaires P-SCCAI, 
FFQ [60], GSRS [61], Bristol stool form scale [62], stool frequency, SIBDQ [58], 
information of medication use and adverse events will be extracted from patient 
record and diary. All questionnaires can be found in F1 Questionnaires. The GSRS 
questionnaire provides baseline GI complaints and tolerance information. P-SCCAI 
provides disease activity information. FFQ provides information regarding the usual 
foods consumed and the frequency of consumption. Bristol stool form scale provides 
stool consistency information. SIBDQ provides information on health-related quality of 
life. 
 
One stool sample will also be provided. The sample will undergo the same collection 
and storage procedures as those in the screening period. DNA will be isolated from 
part of the stool samples and subsequently be used for 16S rRNA gene-based 
microbiota profiling using NG-Tax [63]. The stool sample will be also used for 
measurements of fecal calprotectin using enzyme-linked immunosorbent assay 
(ELISA) methods [64], and SCFAs concentrations using high-performance liquid 
chromatography (HPLC) [65].  
 
Dietary intervention period (8 weeks) 
During the intervention period, the subjects will consume the respective supplement 
(3 grams/sachet) twice per day. To minimize potential GI complaints that might occur 
due to intake of the prebiotics/placebo supplement, there will be a gradual increase in 
dosage that only 1 sachet daily will be consumed in the first week of the intervention 
period, followed by 2 sachets daily for the remainder of the intervention. The patients 
will be asked to save all the empty sachets. 
 
Figure 4 Stool collector 

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At T= 4, and 8 weeks, subjects will also need to complete short questionnaires on 
response (P-SCCAI, once a week), GI complaints and tolerance (GSRS, once a 
week), stool frequency and consistency (Bristol stool form scale, a 7-day record at 
each week), SIBDQ (once a week). Information of medication use and adverse 
events will be extracted from patient record and diary (once a week). 
 
At the end of the intervention period (T= 8 weeks), the participants will provide a 
second stool sample for fecal calprotectin measurements, 16S rRNA gene-based 
microbiota profiling, and SCFAs profile analysis. We will determine response with P-
SCCAI score. 
 
At the end of the intervention period (T= 8 weeks), the participants will be asked to 
return everything (used and unused sachets) to the coordinating investigator to 
determine compliance to the study. 
 
Follow-up period (4 weeks and 52 weeks) 
During the follow-up period, participants will not take supplements anymore. 
 
At T=12, and 60 weeks, P-SCCAI (once a week) and medication use (once a week) 
will be completed. 
 
At T=12, and 60 weeks, one time stool sample will be collected at each time point. 
These samples will be used for fecal calprotectin measurements, 16S rRNA gene-
based microbiota profiling, and SCFAs profile analysis. 
 
We will determine response with P-SCCAI score at T= 12, and 60 weeks. 
 
8.4 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without 
providing a reason and without any consequences. The investigator can decide 
(upon consultation with the medical supervisor) to withdraw a subject from the study 
for urgent medical reasons, such as serious GI complaints, negative reactions upon 
supplement intake, etc., or when study participants have to start medication that 
interferes with outcomes during the study period. 
8.4.1 Specific criteria for withdrawal 
A subject will be withdrawn from the study in the case of serious gastro-intestinal 
complaints or an exacerbation of UC reported by the subjects (such as continuous 
diarrhea or constipation, bloating, abdominal pain). 
8.5 Replacement of individual subjects after withdrawal 
Not applicable. 

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8.6 Follow-up of subjects withdrawn from treatment 
After possible withdrawal, no follow-up of research subjects will take place, except in 
case of withdrawal for other medical reasons. 
8.7 Premature termination of the study 
A premature termination of the study is not expected to happen as there are only 
limited safety risks. Adverse events are not expected, since maltodextrin and dietary 
prebiotics have been proved to be safe. In addition, subjects will be checked on the 
remission status, so any complaints will be reported in time. In case of unexpected 
adverse events that harm the subjects, the study will be stopped immediately. In 
case of premature termination, we still intend to analyse and publish the results of 
the part of the study that has been completed. 
 
 

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9. SAFETY REPORTING 
9.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise the subject 
health or safety. The sponsor will notify the accredited METC without undue delay of a 
temporary halt including the reason for such an action. The study will be suspended 
pending a further positive decision by the accredited METC. The investigator will take 
care that all subjects are kept informed. 
  
 
9.2 AEs, SAEs and SUSARs 
9.2.1 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject 
during the study, whether or not considered related to [the investigational 
product/trial procedure/ the experimental intervention]. All adverse events reported 
spontaneously by the subject or observed by the investigator or his staff will be 
recorded. 
 
9.2.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that  
- 
results in death; 
- 
is life-threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
any other important medical event that did not result in any of the outcomes 
listed above due to medical or surgical intervention but could have been based 
upon appropriate judgement by the investigator. 
An elective hospital admission will not be considered as a serious adverse 
event. 
 
The investigator will report all SAEs to the sponsor without undue delay after 
obtaining knowledge of the events. The sponsor will report the SAEs through the web 
portal ToetsingOnline to the accredited METC that approved the protocol, within 7 
days of first knowledge for SAEs that result in death or are life threatening followed by 

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a period of maximum of 8 days to complete the initial preliminary report. All other 
SAEs will be reported within a period of maximum 15 days after the sponsor has first 
knowledge of the serious adverse events. In case of AEs or SAEs, the study may be 
prematurely terminated if the health or safety of the subject is at risk. 
9.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
Not applicable. 
 
9.3 Annual safety report 
 
Not applicable. 
 
9.4 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been 
reached. Depending on the event, follow up may require additional tests or medical 
procedures as indicated, and/or referral to the general physician or a medical specialist. 
SAEs need to be reported till the end of study within the Netherlands, as defined in the 
protocol. 
 
9.5 [Data Safety Monitoring Board (DSMB) / Safety Committee] 
Not applicable. 

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10. STATISTICAL ANALYSIS 
Statistical analyses will be done using the statistical program embedded within R. The 
threshold for significance will be set at p<0.05, with a FDR correction for 16S rRNA gene 
sequence data. In general, the continuous data will be presented as mean ± standard 
deviation or mean and interquartile range when skewed, and categorical data as counts 
and percentages. Normality will be checked by visual inspection and QQ plots, followed 
by the Shapiro Wilk test for testing if data show a normal distribution. 
 
10.1 Primary study parameter(s) 
 
Response (mean P-SCCAI score) between arms at T = 8 weeks 
To assess the response, data of mean P-SCCAI score within each arm at T = 8 weeks 
(after the intervention) will be obtained. 
 
Firstly, the mean P-SCCAI score between the predicted responders with prebiotics and 
predicted non-responders with prebiotics will be compared by using a Student T-test if 
normally distributed, otherwise a Mann-Whitney U test when skewed. 
 
Secondly, the mean P-SCCAI score between prebiotics and placebo groups within 
predicted responders or predicted non-responders will be compared by using a Student 
T-test if normally distributed, otherwise a Mann-Whitney U test when skewed. 
10.2 Secondary study parameter(s)  
 
Response over time 
To assess the response over time, data of mean P-SCCAI score within each arm at T = 
0, 4, 8, 12 and 60 weeks will be obtained. Comparisons will be made between groups (as 
done for T = 8 for the primary study parameter) as well as within subjects over time. With 
P-SCCAI score <= 2 being regarded as clinical remission, and a decrease in the P-
SCCAI score by more than two points from baseline being regarded as clinical response,  
we will determine whether subjects have reached clinical remission and/or showed a 
clinical response over time. 
 
To assess the response within each subject (clinical remission and/or clinical response) 
over time, data of sum P-SCCAI score within each subject at T = 0, 4, 8, 12 and 60 
weeks will be obtained. For comparative analyses between baseline and intervention, the 
parametric paired sample T-test or the non-parametrical Kruskal-Wallis or Mann-Whitney 

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U test will be used, depending on the distribution of the samples. Differences between 
treatments before and after supplement intake will also be analysed by linear mixed 
models for repeated measures, using “treatment” [prebiotics/placebo], “responding” 
[responders/non-responders], “time point” [0, 4, 8, 12 and 60 weeks] and “treatment x 
responding x time point” as fixed effects and subject as random effect. Clinically relevant 
variables such as BMI, age, sex, and medication use will be taken into account. 
 
Mucosal inflammation 
Fecal calprotectin data will be analysed in the same manner as described above 
(Response over time). 
 
GI complaints: 
Data from GSRS will be computed into scores, and the score of each item will be pooled 
together as the sum score, and the score of each item within each domain will be pooled 
as well. All data will be analysed in the same manner as described above (Response 
over time). Differences over time within and between groups will be tested with mixed 
model analysis. Variables such as timepoint, group, age, sex will be taken into account. 
 
Stool consistency and frequency 
Average bowel movement per day and dynamic change on stool consistency will be 
calculated and collected. All data will be analysed in the same manner as described 
above (Response over time).. 
 
Gut microbiota and SCFAs 
Fecal SCFAs data will be analysed in the same manner as described above (Response 
over time 
 
16S rRNA gene sequencing provides sequence read counts data. It will be normalized to 
microbial relative abundance, and microbiota diversity indices (Shannon, Simpson, and 
inverse Simpson) are calculated at amplicon sequence variant (ASV) level, as 
implemented in the Picante [66] and Phyloseq [67] packages in R, respectively. Wilcoxon 
test will be applied to determine whether diversity as well as the relative abundance of 
specific bacterial taxa, were significantly different between groups since the data was 
non-parametric. For microbiota analysis, p-values will be corrected for multiple 
comparisons using the Benjamini–Hochberg procedure. To correct for a false discovery 
rate (FDR), a FDR adjusted p-values will be computed, a so-called q-value [68]. A 
corrected q-value of less than 0.2 was considered significant. Paired tests will be used to 

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compare the effects of prebiotics. Pairwise weighted Unifrac [69] and unweighted UniFrac 
[70] distance-based principle coordinate analysis (PCoA) will be used to visualize 
microbial community variation at the ASV level. Permutational multivariate analysis of 
variance (PERMANOVA) will be used to test for significant differences between groups, 
as implemented in the Vegan package [71]. 
 
Health-related quality of life 
Data from SIBDQ will be computed into scores, and the score of each item will be pooled 
together as the sum score, and the score of each item within each domain will be pooled 
as well. All data will be analysed in the same manner as described above (Response 
over time). 
 
Medication use 
Type, Dose, frequency and duration of each medication use will be collected. All data will 
be analysed in the same manner as described above (Response over time). 
 
Safety parameters 
Incidence rate of each adverse event is recorded and calculated. All data will be analysed 
in the same manner as described above (Response over time). 
 
 
10.3 Other study parameters 
Habitual food intake 
Data from FFQ will be analysed in the same manner as described above (Response over 
time). Also, it will be used for Pearson r correlation coefficient calculation, or a Spearman 
rank when not normally distributed, with other parameters.  
 
Participants demographics and characteristics 
Data will be analysed in the same manner as described above (Response over time). 
Correlational analysis will be used to explore possible associations between symptoms 
and baseline characteristics. 
 
10.4 Interim analysis (if applicable) 
Not applicable. 

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11. ETHICAL CONSIDERATIONS 
11.1 Regulation statement 
This study will be conducted according to the principles of the Declaration of Helsinki 
(64th WMA Assembly, October 2013), and in accordance with the Medical Research 
Involving Human Subjects Act (WMO 1998), and other guidelines, regulations and acts. 
 
11.2 Recruitment and consent 
There are more than 1200 IBD patients in the patients base of Ziekenhuis Gelderse Vallei 
(ZGV). With around half UC patients have mild activity, this guarantees that enough 
potential patients will be reached out. Previous surveys performed in ZGV on dietary 
beliefs and behaviors showed that around 60% of IBD patients believe that diet could 
make a difference on the disease course. This increases the chance to involve enough 
patients who actually support the dietary strategy. 
 
Recruitment will be done by the investigators. During the outpatient clinic visit, the UC 
patients of ZGV will be informed by nurses about this study. For questions, potential 
subjects can contact the investigators through e-mail or phone for further explanation of 
study procedures. Only when the patients agree to participate the study, their contact 
information will be shared with investigators, who will send the invitation for information 
meeting. Interested subjects will be invited for (digital) information events, where 
additional information will be given, and subjects have the chance to ask questions. After 
the information event, subjects have two weeks to decide whether they are interested in 
participating or not. If they wish to enter the study, they are asked to sign the informed 
consent form at home, and then send it to the researcher. The participants will be notified 
in the consent form to give permission to share the information of unexpected new 
findings regarding the (future) health of them with their General Practitioner (GP). The 
subject will receive a copy of the signed informed consent directly after signing. After this, 
subjects will receive the fecal material collection kit and information to provide the first 
fecal sample for pre-screening. Then within one week, subjects will receive packages with 
corresponding products and instructions. 
 
Data will be stored at secured hard drives of Wageningen University, and will be locked 
with a password. Only members of the research team will have access to these data and 
only after they sign a confidentiality agreement. Paper data such as informed consent will 
be stored in a cabinet with a lock. Subjects will be given a study code, and only members 
of the research team will have access to the files decrypting the study code. 

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11.3 Objection by minors or incapacitated subjects (if applicable) 
Not applicable. 
 
11.4 Benefits and risks assessment, group relatedness 
There is negligible risk for the participants. Dietary prebiotics has been proved to help 
with remission in UC patients. Participants may potentially benefit from the dietary 
intervention with improved disease activity. 
 
Intervention products 
The prebiotics used in this study has been in vitro tested to be effective in boosting 
butyrate production in healthy individuals and it also showed the same ability in mild UC 
patients in vitro beforehand. Maltodextrin and corn starch as a placebo has been widely 
used in many clinical trials, no adverse effects has been reported. 
 
Time investment research participants 
Research participants will invest approximately 9 hours during the trial. A schematic 
overview of the time investments is listed in Table 2. 
 
Table 2: Overview of time investment of the research subjects 
Items 
Time (minutes) 
Number 
Time in total 
Information meeting 
60 
1 
60 
Taking supplement 
2 
105 
210 
Long questionnaire 
45 
1 
45 
Short questionnaires 
3-5 
40 
160 
Stool collection 
15 
5 
75 
Total investment 
 
 
550 min ≈ 9 hours 
 
11.5 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of 
the WMO. The sponsor (also) has an insurance which is in accordance with the legal 
requirements in the Netherlands (Article 7 WMO). This insurance provides cover for 
damage to research subjects through injury or death caused by the study. The insurance 
applies to the damage that becomes apparent during the study or within 4 years after the 
end of the study.   

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11.6 Incentives 
Based on burden calculations, after completion of the full study, each participant will 
receive a financial compensation of €105,-. Participants not selected for the study after 
the screening will receive €17. Research participants are free to withdraw from further 
participation for any reason and at any time during the trial and will receive a proportional 
repayment for the effort they made. However, if a participant needs to withdraw because 
of adverse events, or because of medical advice, he or she would receive the full financial 
compensation.   

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12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
 
12.1 Handling and storage of data and documents 
Before the start of the study, subjects will be assigned to a study code that will not 
change during the study. This number is linked with the name, address, date of birth, and 
telephone number of the subject in a password-protected file that is stored separately 
from the study data. Only members of the research team involved in the study logistics 
(e.g. collecting samples) can access this file. For all other purposes, the study code will 
be used for subject identification. The informed consent will be stored separately from all 
other information. All researchers have signed a confidential statement. The handling of 
personal data will be done according to the General Data Protection Regulation (GDPR), 
which is enforced on the 25th of May 2018. According to standard data management 
procedures, all research data are stored for a period of 10 years after collection. 
Thereafter, data and documents will be deleted and destroyed (e.g. by a shedder). 
Biological samples will be stored for a maximum of 10 years after study end, defined as 
the last data collection point, and then disposed of according to biomedical waste 
procedures. In case we want to use the collected material for additional research, which 
includes research in relation to the health of the patients or other research activities that 
are not covered by this study, we will provide our plans to the medical ethics committee 
Oost Nederland for evaluation. 
12.2 Monitoring and Quality Assurance  
Not applicable. 
 
12.3 Amendments  
Amendments are changes made to the research after a favourable opinion by the 
accredited METC has been given. Non-substantial changes (such as typing errors, 
administrative changes like changes in names, telephone numbers and other contact 
details of involved persons mentioned in the submitted study documentation) will not be 
notified to the reviewing METC and the competent authority, but will be recorded and filed 
by the sponsor. 
 
Substantial amendments will be notified to the METC that gave a favourable opinion. The 
documentation that will be included in the submission should cover the following 
information: 

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• 
Covering letter, including the reasons for the amendment in one or two sentences, 
a brief description of the changes that are included in the amendment and the 
name of the documents that are modified; 
• 
An extract of the modified documents, where applicable, showing both the 
previous and new wording, where applicable; 
• 
The new version of the modified documents, where applicable, identified with 
updated number of version and date. 
 
12.4 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the 
accredited METC once a year. Information will be provided on the date of inclusion of the 
first subject, numbers of subjects included and numbers of subjects that have completed 
the trial, serious adverse events/ serious adverse reactions, other problems, and 
amendments. 
 
12.5 Temporary halt and (prematurely) end of study report 
The investigator/sponsor will notify the accredited METC of the end of the study within a 
period of 8 weeks. The end of the study is defined as the collection of the last data.  
The sponsor will notify the METC immediately of a temporary halt of the study, including 
the reason of such an action.  
In case the study is ended prematurely, the sponsor will notify the accredited METC 
within 15 days, including the reasons for the premature termination. 
Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, 
to the accredited METC. 
  
 
12.6 Public disclosure and publication policy 
 
Subjects will be informed about the results of the study. Results will be presented in 
agreement with the CCMO publication statement. Results will be published in scientific 
peer-reviewed papers and possible also in professional journals by the investigators. The 
authorship of the article shall be determined in appropriate consultations based on a 
considerable contribution to the set-up and execution of the study and active participation 
in publication. Outcomes of the study will be presented at conferences and meetings; data 
will be reported anonymously. 

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None of the collaborating parties will withhold the public disclosure of study results. 
Collaborating parties may delay publication for up to three months after analysing the 
research results if it is applying for a patent or for other important reasons. All 
collaborating parties are entitled to examine any form of public disclosure prior to 
submission. 
 
The study protocol will be registered at a public database (www.clinicaltrials.gov).  

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13. STRUCTURED RISK ANALYSIS  
 
13.1 Potential issues of concern 
Not applicable. All ingredients are non-pathogenic, non-toxigenic and are classified as 
Generally Recognized As Safe (GRAS) as food ingredients.  
 
13.2 Synthesis 
 
The following actions will be taken to reduce the small risks of this study: 
• 
The following aspects point towards a safe use of the dietary prebiotic 
supplements: 
o They are commonly consumed throughout the world. 
o All compounds are commercially available and tested safe for human 
consumption. 
o Prebiotic supplements were selected on the basis of expected low GI 
tolerance issues. 
o Prebiotic supplements will be given in a relatively low daily dose.  
o To avoid gastrointestinal complaints, due to a sudden intake of fiber 
supplements during the first week, the study participants will consume only 
one sachet per day, followed by two sachets per day for the remainder of 
the study. 
• 
The following aspects include safety monitoring throughout the study: 
o We will monitor GI complaints via questionnaires throughout the study.  
o A medical doctor has been appointed for this study to check and discuss 
reported AEs with the subjects. 
• 
The following aspects point towards a low-risk patient group: 
o Only UC patients with mild disease activity are included. 
o Subjects with other digestive tract disorders that are expected to interfere 
with this study are excluded from participation.  
• 
The following aspects point towards a non-burdensome sampling procedure 
o The stool sample collection aligns with their routine clinical check and will 
therefore not cause an extra burden. 
o We will take the applicable COVID-19 regulation into account when it 
comes to potential contact with the subjects (e.g., information meeting). 
 
 

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