The Effect of Concord Grape Polyphenol-soy Protein Isolate Complex (GP-SPI) on Gut Microbiota
Phase: NA
Conditions: Mechanisms, Defense
Sponsor: Rutgers, The State University of New Jersey

Page 0 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
INTERVENTIONAL 
RESEARCH PROTOCOL TEMPLATE 
(HRP-503a) 
 
 
 
 
STUDY INFORMATION 
 
 
Title of Project  
The Effect of Concord grape polyphenol-soy protein isolate complex (GP-SPI) on Gut 
Microbiota  
 
 
Principal Investigator  
Diana E. Roopchand, PhD 
Assistant Professor 
Department of Food Science, Rutgers University 
Institute for Food, Nutrition, and Health 
61 Dudley Rd, Suite 220 
New Brunswick, NJ 08901 
 
roopchand@sebs.rutgers.edu 
            Tel: 848-932-0248 
 
 
Protocol Version and Date: 
V3 July 5, 2019  
 
 
 
 
IND Exemption – Per attached letter from FDA this study has been granted IND exemption. 
 
Funding:  Rutgers’ internal funding and NIH grant (R01 AT010242-01). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 

Page 1 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
Table of Contents 
Skip To Section: Hold CTRL + Click (Below) To Follow Link in Blue 
 
1.0 
Research Design 
 
 
1.1 
Purpose/Specific Aims 
 
 
1.2 
Research Significance 
 
 
1.3 
Research Design and Methods 
 
 
1.4 
Preliminary Data 
 
 
1.5 
Sample Size Justification 
 
 
1.6 
Study Variables 
 
 
1.7 
Drugs/Devices/Biologics 
 
 
1.8 
Primary Specimen Collection 
 
 
1.9 
Interviews, Focus Groups, or Surveys 
 
 
1.10 
Timetable/Schedule of Events 
 
 
2.0 
Project Management 
 
 
2.1 
Research Staff and Qualifications 
 
 
2.2 
Resources Available 
 
 
2.3 
Research Sites 
 
 
3.0 
Multi-Site Research Communication & Coordination 
 
 
3.1 
Outside Research 
 
 
4.0 
Research Data Source/s 
 
 
4.1 
Primary Data – Subjects and Specimens 
 
 
4.2 
Subject Selection and Enrollment Considerations 
 
 
4.3 
Subject Randomization 
 
 
4.4 
Secondary Subjects 
 
 
4.5 
Number of Subjects 
 
 
4.6 
Consent Procedures 
 
 
4.7 
Special Consent Populations 
 
 
4.8 
Economic Burden and/or Compensation For Subjects 
 
 
4.9 
Risks to Subjects 
 
 
4.10 
Secondary Data – Record/Chart Reviews, Databases, Tissue Banks, Etc. 
 
 
4.11 
Chart/Record Review Selection 
 
 
4.12 
Secondary Specimen Collection 
 
 
5.0 
Special Considerations 
 
 
5.1 
Health Insurance Portability and Accountability Act (HIPAA) 
 
 
5.2 
Family Educational Rights and Privacy Act (FERPA) 
 
 
5.3 
NJ Access to Medical Research Act 
 
 
5.4 
Code of Federal Regulations Title 45 Part 46 (Vulnerable Populations) 
 
 
6.0 
Research Data Protection and Reporting 
 
 
6.1 
Data Management and Confidentiality 
 
 
6.2 
Data Security 
 
 
6.3 
Data Safety And Monitoring 
 
 
6.4 
Reporting Results 
 
 
6.5 
Data Sharing 
 
 
7.0 
Data and/or Specimen Banking 
 
 
8.0 
Other Approvals/Authorizations 
 
 
9.0 
Bibliography 
 
 
 
 
 
 
 

Page 2 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
1.0 Research Design 
 
1.1 Purpose/Specific Aims 
The overall purpose of this study is to evaluate the effect of nutritional supplementation with a well-
characterized preparation of Concord grape polyphenol-soy protein isolate (GP-SPI) on the composition of 
the gut microbiota.  
 
A.  Objectives  
Primary objective:  
• 
To evaluate the effect of nutritional supplementation with GP-SPI on gut microbiota 
composition.  
 
Secondary objectives: 
• 
To evaluate the effect of GP-SPI on kidney and liver health/function via comprehensive 
metabolic panel (CMP).  
• 
Collect fecal, blood, and urine samples and store for a microbiome-wide association study 
(MWAS, funded by R01 AT010242-01).  
• 
Obtain consent to enter de-identified study results into microbiome and metabolome nationwide 
data sharing database. 
 
B.  Hypotheses / Research Question(s) 
We hypothesize that healthy subjects eating a diet supplemented with GP-SPI will demonstrate an 
increase in intestinal abundance of Akkermansia spp. within a short time period as well as changes in 
other genera relevant to promotion of intestinal/metabolic health. Such changes may explain, at least 
in part, how poorly absorbed dietary polyphenols from fruits and vegetables improve human resilience 
against chronic metabolic diseases. 
 
 
1.2 Research Significance (500 words or less) 
 
The proposed study will test the hypothesis that oral supplementation with GP-SPI complex can alter the 
human microbiome in a direction that is consistent with improved health and metabolic resiliency. 
Epidemiological, clinical, and preclinical data indicate that polyphenol compounds derived from food, such 
as berries, fruits, vegetables, and herbs have beneficial health effects in humans (1). The B-type 
proanthocyanidin (PAC) class of polyphenols contained in grape berries, especially skins and seeds, have 
been associated with health benefits (2; 3); however, PACs are poorly absorbed and reach high 
concentration only in the colon (4) raising questions about mechanism(s) of action. We and others have 
shown that dietary PACs from grape and cranberry alter the gut microbiota in association with metabolic 
resilience (5; 6). PACs are also biotransformed by gut bacteria to yield microbial metabolites (MMs) that 
may contribute to health benefits (3). 
 
Sensitive to temperature and pH, polyphenols are often labile and lose effectiveness once extracted. 
Studies in the Raskin lab (7-11) have established methods to increase the stability of extracted dietary 
polyphenols through sorption to protein rich foods, such as SPI. The sorption process does not require any 
non-food additives. Dietary polyphenols extracted from blueberries and green tea were sorbed to SPI and 
this polyphenol-protein complex (dosed at 40 g/day for 17 days and delivering 2136 mg total 
polyphenols/day) was tested in human clinical studies (12; 13).  
 
Biochemical properties of the GP-SPI food ingredient are well-documented (9; 10) and GP-SPI and SPI 
supplements have been tested extensively in mice (6; 9; 10; 14). The proposed study is a logical follow up 
to animal studies, which showed that compared to control mice fed a high-fat diet (HFD) supplemented with 
SPI alone, mice fed an isocaloric HFD supplemented with GP-SPI exhibited greater resistance to weight 
gain, adiposity, and glucose intolerance (6). These effects were accompanied by changes in murine gut 
microbiota composition, including increased abundance of the microbe Akkermansia muciniphila, 

Page 3 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
associated with metabolic resilience (6). Similar gut microbiota changes were observed in lean mice fed 
low-fat diet (LFD) supplemented with GP-SPI (14). 
 
The proposed study will first investigate how short-term supplementation with GP-SPI may modify the gut 
microbial community in healthy participants while monitoring liver and kidney function/health. We will also 
perform a longitudinal, microbiome-wide association study (MWAS) to identify gut bacteria species/strains 
that are positively or negatively associated with GP-SPI supplementation. Metabolomics analysis will be 
performed on collected urine and blood samples to identify/quantify known and unknown metabolites. 
Shotgun metagenomic sequencing will be performed on fecal samples to generate high quality draft 
genomes for species/strain level identification. These dynamic data sets will serve as input for the MWAS 
to correlate increasing/decreasing levels of gut bacterial species/strains to increasing/decreasing 
metabolites in blood and urine. These bacteria-metabolite associations will then be used to infer cause-
effect relationships that can be further tested in vitro. We expect that successful completion of these studies 
will contribute to mechanistic explanations for how dietary polyphenols such as grape PACs alter the gut 
microbiota and resulting MM to promote metabolic health.  
 
 
1.3 Research Design and Methods 
 
Overview 
This is a pilot time course study to characterize the effect of GP-SPI on the gut (i.e. fecal) bacterial 
community of healthy subjects. Following screening, enrolled subjects will washout of PAC-rich foods for 5 
days while also consuming SPI. Subjects will then consume GP-SPI over a 10-day supplementation period. 
Fecal, urine, and blood samples will be collected. Comprehensive metabolic panel (CMP) tests performed 
before and after the GP-SPI supplementation period will monitor liver and kidney health. Using deidentified 
fecal, urine, and blood samples collected from time course study a MWAS study (funded by R01 AT010242-
01) will be performed. 
Successful completion of this pilot study will yield data that will inform the design of a follow-up placebo-
controlled intervention study, which will be described in a separate clinical protocol that will be submitted to 
IRB for approval.   
 
A. Research Procedures 
 
Figure 1. Clinical Study Design 
Location   
Screening and out-patient visits will take place in the Surgery Clinic in the Clinical Academic Building (CAB) 
or Surgery Department (MEB 5) and NJ Institute of Food Nutrition and Health (IFNH).  
 
Recruitment 
Prospective participants will be recruited through flyers posted locally on Rutgers University campuses and 
sent to university email lists (Flyer attached). Interested subjects will call in or email to receive further 
information about the study.  A member of the study team will give a brief summary of the study. If the 
subject requests a screening appointment, the investigator will ask for verbal consent to proceed with a list 

Page 4 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
of questions (Telephone Script attached). Collecting this information at this time assists in expediting the 
paperwork during the visit and spares ineligible subjects the inconvenience of a futile appointment. 
 
Eligibility Screening  
At the screening visit (at IFNH), a member of the study team will explain the study in detail to the subject.  
Full explanation of study procedures and potential risks will be provided. The subject will have ample time 
to read the Consent and ask questions before signing and proceeding with screening procedures. A signed 
copy of Consent form (form attached) will be given to the subject.  
 
After consent a participant ID will be assigned to subject. Demographics, medical history, height, and weight 
(Demographics and Medical History forms attached) will be recorded for each subject. Females will be 
given a urine-based pregnancy test onsite and result will be recorded. If inclusion criteria are met at this 
time, subject will be asked to schedule a visit at CAB with study nurses for a blood draw to perform CMP 
screening test. If CMP results are within normal range the subject will be enrolled and return to IFNH to 
receive study materials needed for participation in the study. If any CMP values are outside normal ranges 
subject will be advised that they do not fulfill all eligibility criteria, they will be given a copy of their CMP 
results, and if necessary, advised to see their primary care provider. All screened subjects will be 
documented on Screening and Enrollment Log (attached).   
 
The final selection will be contingent upon the individuals meeting all eligibility criteria and deemed capable 
of complying with the study protocol.  
 
 
Study Procedures 
• 
Thirty subjects will be enrolled. 
 
• 
Food List: Participants will maintain their usual diet except for a provided list of PAC-rich foods 
that they will be asked to abstain from for a 5-day wash-out period and during the 10-day 
intervention. The Food List is provided below. The goal is to have GP-SPI as the main/only source 
of PAC in the diet for the study period.  
  
• 
Wash-out and SPI (Day -5 through -1): On day -5 (pre-baseline) before any supplementation, 
each subject will collect their fecal and urine sample. Each subject will then consume SPI twice a 
day (provided as pre-weighed packets of 20 g) during a 5-day wash-out period (days -5, -4, -3, -2, 
and -1). Subjects will be instructed to mix each packet of SPI in 250 mL of water or in a smoothie 
(recipe example below will be provided) using allowed foods as detailed in instructions and 
consume once in the morning before breakfast and once in the evening before dinner.  
 
• 
Day 0: On the following day each subject will collect their baseline (day 0) stool and urine samples 
(Fig. 1). They will visit the CAB building to have a blood sample (2 tablespoons or 30 mL) drawn 
by a study nurse. Blood will be used for CMP test and prepared serum will be aliquoted and stored 
at -80 °C until processing for marker analysis and metabolomics. Analysis of day 0 samples should 
help isolate any effects due to SPI alone from subsequent samples collected during GP-SPI 
intervention. On day 0 subjects will drop off urine and stool samples at IFNH and collect GP-SPI 
packets. On Day 0 participants will also take a break from consuming SPI and will start GP-SPI 
supplementation on morning of Day 1. 
  
• 
GP-SPI (Day 1 – 10): Each subject will receive twenty pre-weighed 20 g packets of GP-SPI. On 
day 1 subjects will be instructed to mix each packet of GP-SPI in 250 mL of water or in smoothie 
(recipe example will be provided, please see below) using allowed foods as detailed in instructions 
and consume the GP-SPI mix once in the morning before breakfast and once in the evening before 
dinner for 10 consecutive days (Fig. 1).  
 

Page 5 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
• 
Subjects will be provided with a personal blender for smoothie preparation (value ~ $25) that they 
can keep.  
 
• 
Digital food diary: Participants will be asked to take photos of all their food and drink (except 
water) including the study supplements with their personal mobile computing device (e.g. smart 
phone, iPad, or similar). Participants will be required to download the free mobile app WhatsApp to 
send photos with a brief description of the food items. Photos and food description may also be 
sent to an email address that will be created for this study (food4microbes@gmail.com). The food 
diary must be kept over the 5-day SPI and wash-out period, day 0 (break day), the 10-day GP-SPI 
intervention period, and up until the final blood draw on day 11 (17 days total).   
 
• 
Stool samples: Subjects will be provided with Omnigene®Gut OMR-200 kits (DNA genotek, 
Ottawa, Canada)  or tubes containing 95% ethanol and/or tubes containing 50% glycerol (50% 
water) along with paper toilet accessories for easy self-collection of fecal samples; each participant 
will be instructed on use of stool collection materials. The Omnigene®Gut OMR-200 kit allows 
transport and storage of stabilized DNA at ambient temperature for 60 days. Fecal samples 
collected in 95% ethanol are stable at ambient temperature for 8 weeks (15). Subjects will collect 
samples on days 2, 4, 6, 8, and 10 and be asked to bring their samples to the IFNH laboratory as 
soon as possible, within 1-3 days of collection, for processing. Fecal samples will be aliquoted and 
stored at -80 °C until extraction. Extraction of fecal samples collected with the OMR-200 kit or 95% 
ethanol yields high quality DNA (as tested in Roopchand lab) suitable for 16S rRNA 
microbiome profiling, shotgun metagenomic sequencing, qPCR, and arrays. Collection of fecal 
samples in 95% ethanol has additional benefit that sample may be used for metabolomics analysis 
(16). Collection of fecal samples in 50% glycerol and freezing will allow culturing of gut bacteria for 
in vitro experiments.  
 
• 
Bristol stool scale form: Subjects will be provided with the Bristol stool scale (BSS) form and 
asked to complete it for stool samples they collect on days -5, 0, 2, 4, 6, 8, and 10 of the study. 
Stool consistency has been shown to strongly correlate with gut microbiota richness and 
composition, enterotypes, and bacterial growth rates (17). Self-assessed stool consistency as 
categorized by the BSS is used in clinical studies as a proxy for colonic transit rate. The BSS 
classifies human feces into 7 consistency categories. The highest scores (6 - 7) correspond to 
loose stools and fast transit time (i.e. diarrhea), while lower scores (1 - 2) correspond to hard stools 
and longer colon transit time (i.e. constipation). Scores between 3 – 5 are reflective of easily passed 
stools (i.e. normal bowel movement) (18; 19). Each consistency category reflects differences in 
moisture. Decreased water is associated with longer transit time and less microbial growth due to 
lower nutrient mobility and enzyme activity (17). Bowel movement frequency in healthy adults (n= 
124) was reported to range from 3 times per week to 3 times per day (19). Subjects that report at 
least one bowel movement per day will be recruited for ease of compliance with study protocol. In 
addition, the BSS form contains an extra column to capture information about menstruation during 
time of sample collections as this variable could impact metabolite or bacteria profiles .  
 
• 
Urine samples: Subjects will be provided with sterile collection containers for collection of urine 
samples on days 2, 4, 6, 8, 10 and asked to keep samples in 4 °C fridge until transport to the IFNH 
laboratory to maintain metabolite stability (20). Subjects will be asked to bring their samples to the 
IFNH laboratory as soon as possible, within 2-3 days of collection, for processing. Urine samples 
will be aliquoted and stored at -80 °C until processing for metabolomics studies.      
 
• 
Blood sample: On day 11 subjects will go to CAB to visit study nurses for a final blood sample 
draw (2 tablespoons or 30 mL). Blood will be used for CMP test and prepared serum will be 
aliquoted and stored at -80 °C until processing for marker analysis and metabolomics. 
 
 
 

Page 6 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
Table 1. Schedule of events and sample collections 
Days 
Screen 
-5 
0 
2 
4 
6 
8 
10 
11 
Informed Consent 
X 
 
  
  
  
  
  
  
 
Incl/Excl Criteria 
X 
 
  
  
  
  
  
  
 
Med. History 
X 
 
  
  
  
  
  
  
 
Height &Weight 
 X 
 
 
  
  
  
  
  
 
Clinic Visit 
 X 
 
X 
 
 
 
 
 
X 
Blood Draw 
 X 
 
X 
  
  
  
  
 
X 
Urine Sample 
 
X 
X 
X 
X 
X 
X 
X 
 
Stool Sample 
  
1 
2 
1 
1 
1 
1 
2 
 
Urine Pregnancy 
 X 
 
 
  
  
  
  
  
 
SPI 
 
Twice 
a day 
for 5 
days 
Break 
 
 
 
 
 
 
GP-SPI 
  
 
Twice a day on days 1 - 10  
 
Food Diary 
  
Daily up until blood draw on day 11 
 
 
Food List  
To ensure that GP-SPI ingredient is the main source of PAC consumed, participants will be given the below 
list of PAC-rich foods that they will be asked to abstain from during the washout and study period as well 
as a list of allowed foods. The food list is based on published quantification of PACs in commonly consumed 
food items (21; 22). In addition, participants will be provided with the following example smoothie recipe. 
Smoothie Recipe:  
• 
1 banana (or another fruit from the allowed list of foods) 
• 
175 - 200 mL water or milk (add more or less liquid depending on preference; may also 
use milk substitutes like soy, almond, or rice milk) 
• 
1 - 2 tablespoons ice cream or yogurt (may also use frozen yogurt or non-dairy ice cream)  
• 
1 packet of GP-SPI   
• 
1 or more cubes of ice - optional 
• 
Combine ingredients and blend 
 
Plant Foods to abstain from: Study participants should abstain from the below list of plant foods for 5 
days before (i.e. washout period), during the 10-day study intervention period, and until after the final blood 
draw on day 11.  These foods contain high levels of naturally occurring proanthocyanidin compounds, major 
bioactives in GP-SPI ingredient, and will interfere with interpretation of results. 
• 
Berry fruits (green grapes, red grapes, blueberries, cranberries, choke berries, strawberries, black 
berries, bilberry, black currant, aronia, hawthorn, rosehip, sea buckthorn) and all berry products 
(i.e. juices, preserves, jams, etc.)  
• 
Plums 
• 
Apples 

Page 7 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
• 
Apricots 
• 
Cherries 
 
• 
Sorghum 
• 
Barley 
• 
Pinto beans 
• 
Red kidney beans 
• 
Small red beans 
• 
Black-eyed peas 
 
• 
Almonds  
• 
Hazel nuts 
• 
Pecans 
• 
Pistachios 
• 
Walnuts 
• 
Peanuts with skins 
 
• 
Olives 
• 
Olive oil 
• 
All vinegars 
 
• 
Dark chocolate  
• 
Milk chocolate 
 
• 
Red wine 
• 
White wine  
• 
Green tea 
• 
Black tea 
 
• 
Cinnamon 
• 
Curry powder 
 
Plant Foods allowed during study period: The below list of plant foods have undetectable levels of 
proanthocyanidin compounds and may be freely consumed. Vegetables are not a significant source of 
proanthocyanidins and those not listed below may also be freely consumed.  
• 
Cantaloupe 
• 
Medjool dates 
• 
Grape fruit 
• 
Orange 
• 
Tangerine 
• 
Honeydew 
• 
Watermelon 
• 
Pineapple 
• 
Figs 
• 
Tomato 
 
• 
Coffee 
 
• 
Artichoke 
• 
Asparagus 
• 
Broccoli 

Page 8 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
• 
Broccoli Raab 
• 
Cabbage 
• 
Carrot 
• 
Celery 
• 
Cucumber 
• 
Eggplant 
• 
Lettuce 
• 
Onion 
• 
Pepper 
• 
Potato 
• 
Radish 
• 
Sweet potato 
• 
Zucchini 
• 
Beets 
 
• 
Corn meal 
• 
Tortilla chips 
• 
Wheat 
• 
Oats 
• 
Rice 
• 
Soybeans 
• 
Navy beans 
• 
Chick peas 
• 
Macadamia nuts 
• 
Pine nuts 
• 
Brazil nuts 
 
• 
White chocolate 
 
• 
Basil 
• 
Oregano 
• 
Parsley 
• 
Paprika 
• 
Black pepper 
• 
Mustard seeds 
• 
Poppy seeds 
• 
Garlic powder 
• 
Ginger 
• 
Onion powder 
• 
Tumeric 
• 
Cloves 
• 
Chili powder 
 
 
The following plant foods have low levels of proanthocyanidin compounds and may be consumed in 
moderation during the study period, meaning just 1 serving per day of any food from the list below.  
• 
Pear (1 fruit) 
• 
Nectarine (1 fruit) 
• 
Cherries (10 cherries) 
• 
Apricot (1 fruit) 

Page 9 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
• 
Kiwi (1 fruit) 
• 
Avocado (1 fruit) 
• 
Mango (1 fruit or 1 cup of sliced fruit) 
• 
Banana (1 fruit) 
• 
Persimmon (1 fruit) 
• 
Pomegranate (1/4 cup kernels) 
 
• 
Indian Squash (1 ounce or 28 g) 
• 
Black beans (1 ounce or 28 g) 
• 
Lentils (1 ounce or 28 g) 
 
• 
Roasted peanuts (40 pieces = 1 ounce or 28 g) 
• 
Peanut butter (1 tablespoon) 
• 
Cashews (18 pieces = 1 ounce or 28 g) 
• 
Chestnuts (8-10 kernels) 
 
• 
Beer (1 can/bottle = 12 ounces) 
• 
Apple juice (1 cup or 230 mL) 
• 
Chocolate milk (1 cup or 230 mL) 
 
 
 
 
 
A. Data Points 
Data elements to be collected include: medical history, demographics, digital and written food diaries, 
adverse events, metabolomics data from blood and urine samples, microbiome data from fecal samples 
 
B. Study Duration 
It will take 2 years to complete this study. Each subject will participate for 17 days.  
 
 
C. Endpoints 
Primary endpoint: 
1) Evaluate the effect of nutritional supplementation with GP-SPI on gut microbiota composition using 
16S rRNA gene sequencing.  
 
Secondary endpoints: 
1) To evaluate the effect of GP-SPI on kidney and liver health/function (safety) via comprehensive 
metabolic panel (CMP).  
2) Collect fecal, blood, and urine samples and store for a microbiome-wide association study (MWAS, 
funded by R01 AT010242-01).  
 
Microbiome wide association study (MWAS) 
Overview: Using the deidentified fecal, urine and blood samples from Part 1 of above-described parent 
study, we will perform a MWAS. Healthy humans have unique gut microbial communities; however, we will 
investigate whether supplementation with GP will promote elements of similarity in the gut microbiota and 
resulting microbial metabolites (MM), including PAC-derived MM and host-derived MM such as secondary 
bile acids (BA), that could be linked to healthy states and metabolic resilience. Such data may reveal 
common ecological or functional rules existing in microbiotas of healthy individuals as well as GP- or host-
derived MM biomarkers with diagnostic potential. Below is an example of a MWAS work flow. 

Page 10 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
Shotgun Metagenomics Workflow  
gDNA will be extracted from deidentified fecal samples. Libraries will be constructed followed by high-
throughput sequencing on an Illumina HiSeq instrument (Genewiz). Cluster generation, template 
hybridization, isothermal amplification, linearization, and blocking, denaturing and hybridization of the 
sequencing primer will be performed according to the workflow specified by service provider. Prinseq (23)  
will be used to trim and de-duplicate raw reads. Reads that align to the murine/human genome (24) will be 
removed.  
De novo non-redundant metagenomic gene-catalogue construction and gene abundance-profile 
calculations: High-quality paired-end reads from each sample will be used for de novo assembly with 
IDBA_UD (25) into contigs of at least 500 bp. Genes will be predicted using MetaGeneMark (26). A non-
redundant gene catalogue of microbial genes will be constructed with CD-HIT. Quality reads will mapped 
onto the gene catalogue using SOAPaligner (27). Aligned results will be sampled and downsized to an 
appropriate number of reads per sample.  
Co-abundance gene groups (CAGs): Individual genes will be binned in to co-abundance gene groups 
(CAGs) with a canopy-based algorithm (28). Raw CAGs will be removed in the subsequent analyses if they 
have fewer than 3 genes or 90% of the total canopy profile is distributed in no more than three samples. 
CAGs with >700 genes will be regarded as bacterial CAGs for further analyses. The principal component 
analyses of the bacterial CAGs based on the Bray-Curtis distance and Procrustes will be performed with 
QIIME (29). Abundance of genes identified per sample will be determined to follow changes in gene 
richness over time course of GP-SPI supplementation. 
Assembly and taxonomic assignment of bacterial CAGs: De novo assembly will be performed for each of 
the prevalent bacterial CAGs which are share by more than 20% of the samples as previously described 
(30). The six criteria for high-quality draft genome assembly will be used from the Human Microbiome 
Project (http://www.hmpdacc.org/reference_genomes/finishing.php) and checkM (31) to assess the quality 
of the assemblies. We will identify phylogenetic taxonomy of CAGs and assign them to species or genus 
level using previously described methods (14).  
Profiling the metagenomics of PAC-active enzymes: Genes encoding PAC-active enzymes will be identified 
using a modified HMMscan pipeline (https://www.ebi.ac.uk/Tools/hmmer/) and the best-hit alignment will 
be retained. Abundances of genes belonging to the same enzyme family will be summed together. 
Functional Annotation: Metagenomic data will be metabolically profiled using HUMAnN (32). All high-quality 
reads will be aligned with Bowtie2 to the KEGG database 2014 (33), from which sequences of eukaryotes 
were excluded. Alignments will be transformed into bam format with SAMtools and entered into HUMAnN 
to obtain the abundance of pathways. The non-redundant gene catalogue and the open reading frames will 
be predicted from high-quality draft genomes with Prodigal and aligned to sequences using BLASTP.  
 
Metabolomics Workflow 
1) Sample preparation 
a) Blood samples will be collected, centrifugated at 3000 g at 4 °C for 10 min, and serum supernatant will 
be aliquoted and stored frozen. Serum will be subjected to deproteinization as follows (34): 20 µL ascorbic 
acid (4 mg/mL) and taxifolin (internal standard; 0.25 µM) will be added to 380 µL of sample (final 
concentration of 1 mM). The samples will be combined with 1 mL hexane, homogenized and centrifuged 
for 10 min at 17 000 g. The aqueous phase will be recovered and added dropwise to 1200 ml acetonitrile 
(ACN), vortexed for 2 min and centrifuged for 10 min at 17 000 g. The supernatant will be recovered, and 
the pellet reconstituted with 400 µL ACN. After centrifugation, ACN supernatants will be combined, dried 

Page 11 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
and stored at - 80°C until analysis. Samples will be reconstituted with 100 µL of water and filtered before 
analysis.  
b) Urine samples (100 µL aliquots) will be centrifuged (10,000 g for 10 min at 4 °C) to remove particulates 
followed by dilution with water (1:1 to 1:3 vol/vol). Samples (10 µl) will be added to 10 ml of ACN and 
centrifuged at 3000 g for 15 min. The supernatant will be collected, dried, reconstituted in 500 µl of ethanol 
(50 % v/v) containing 0.1 % ascorbic acid and (w/v) and transferred to HPLC vials for untargeted analyte 
detection by high resolution MS (20; 35).  
To deplete common matrix interferences such as phospholipids, salts, and proteins and optimize sample 
preparation each sample supernatant produced in a) and b) may also be loaded to Oasis Prime HLB 
Cartridges (Waters). Sample preparation methods will be tested.   
2) Chromatographic separation and high-resolution mass spectrometry (MS): Triplicates (i.e. technical 
replicates) of each serum or urine sample will be separated on a Dionex UltiMate 3000 Binary Rapid 
Separation UHPLC with Diode Array Detector with a reverse phase (RP) C18 column for retention of 
medium polar and non-polar analytes as well as a hydrophilic interaction chromatography (HILIC) column 
for retention of highly polar metabolites (35). Separation of samples on RP and HILIC columns prior to MS 
will provide more comprehensive metabolome coverage. To detect the greatest number of metabolites, 
gradient elution protocols will be optimized separately for serum and urine samples. Chromatographic 
separations will be followed by MS using a Thermo Scientific Q Exactive Plus Orbitrap high resolution mass 
spectrometer. The orbitrap provides high mass accuracy (1–2 p.p.m.), resolution up to 100,000 and 
dynamic range of 5,000. Data will be collected in both positive and negative ESI modes (mass range of 
100–2,000 m/z).  
Quality control (QC): Three kinds of QC samples will be implemented: 1) A standard mixture solution of 
PAC-derived MM and/or bile acids at a concentration of 1 mg/L; 2) A bulk QC sample made by pooling 
equal aliquots of each urine or serum sample; 3) Bulk urine or serum QC sample spiked with standard mix. 
These QC controls will help evaluate retention time stability, peak shape, detector response and mass 
accuracy. To avoid freeze-thaws QC controls will be prepared, aliquoted, and stored at -80 °C.  QC samples 
will be injected 5 times at the beginning of the run to ensure system equilibration, and then once every 10 
samples to monitor stability of analysis. Samples will be randomized to reduce systematic error associated 
with measurement variability (35).  
3) Data processing and metabolite identification: XCMS Online software (https://xcmsonline.scripps.edu) 
will be used to identify and line up MS peaks/metabolites. Background noise will be filtered. A 0.2 min 
retention time and 0.002 Da mass tolerance window will be applied. Metabolites will be identified using 
online databases such as Chemspider, METLIN, (https://metlin.scripps.edu/index.php), Human 
Metabolome 
Database 
(HMDB) 
(http://www.hmdb.ca/), 
MZedDB 
(http://maltese.dbs.aber.ac.uk:8888/hrmet/index.html) and Kyoto Encyclopaedia of Genes and Genomes 
(KEGG) (http://www.genome.jp/kegg/). Metabolite profile and identification will be achieved by comparing 
mass, RT, MS/MS fragmentation patterns in secondary LC-MS/MS analysis, and/or against standard 
compounds for aglycones. We will look for particular signatures of metabolites that are significantly 
increased in blood after GP intake (humans) or PAC intake (mice). As many metabolites are likely to be 
conjugated to glucuronide, sulphate and methyl groups, enzymatic treatments will be applied to release the 
aglycone for quantification purposes. After enzymatic treatment, samples will be profiled and compared to 
untreated samples. After deconjugation an increase in the signal of particular aglycones (previously 
quantified) is expected. The relative increase in these signals coupled with the decrease/disappearance of 
the conjugated compound will be used to quantify the concentration of these metabolites.   
4) Statistical analysis: To find the bacteria that respond to GP either positively or negatively (i.e. GP 
responders) we will omit those strains that are found to change with only SPI supplementation. To further 
understand how the GP responders affect host metabolic health as a group, we will derive a GP Responder 

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Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
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Index (GRI) based on the abundance and diversity [Heip evenness] of the identified bacteria (36). Data will 
be subject to two-way repeated measures ANOVA to decipher main effects and interactions between GP 
and SPI. P< 0.05 will be considered significant. 
To detect significant differences in metabolites MetaboAnalyst 3.0 software (http://www.metaboanalyst.ca/) 
will be used. Principal components analysis (PCA) will transform original data variables into a smaller 
number orthogonal variables built from linear combinations to explain most of the data variance and cluster 
samples from each time-point or group to be analyzed. Significant difference (p< 0.05) will be determined 
within subject at different time-points, within group, and between groups. Variable importance in projection 
(VIP) scores will be estimated to select metabolites biomarkers from a partial least squares-discriminant 
analysis (PLS-DA). VIP scores are a weighted sum of PLS weights for each variable and measure the 
contribution of each predictor variable to the PLS-DA model. Compounds with VIP score > 4 represent 
metabolites with greatest change, which will be chosen for proof of identity. Before carrying out PCA and 
PLS-DA analyses, peak intensity will be controlled by a logarithmic transformation, and monitored by Pareto 
scaling. Methods for data preprocessing and statistical analysis will be optimized and additional corrections 
and tools will be applied as needed (37; 38).    
Co-inertia analysis: To aid interpretation of the relationship between the bacterial responders and 
metabolomics datasets, they will be integrated using a multivariate method known as co-inertia analysis 
(CIA) (39). We expect to uncover bacterial species/strain level increases and decreases in GP-SPI-
supplemented subjects that correlate with a concomitant increase and decrease in production of their 
metabolites.  
 
 
1.4 Preliminary Data 
 
The proposed study is a logical follow up to animal studies, which showed that compared to control mice 
fed a high-fat diet (HFD) supplemented with SPI alone, mice fed an isocaloric HFD supplemented with GP-
SPI exhibited greater resistance to weight gain, adiposity, and glucose intolerance (6). Compared to HFD 
control group, mice fed the HFD supplemented with GP-SPl had lower levels of bacteria-derived 
lipopolysaccharide (LPS; endotoxin) in circulation. LPS triggers well-characterized systemic inflammatory 
responses in humans and mice. Coincidentally, analyses of intestinal tissue of mice fed the GP-SPI 
supplemented HFD revealed: i) a decrease in inflammatory mediators (TNFα, IL-6, iNOS), ii) increased 
expression of the tight junction protein occludin, indicating improved intestinal barrier integrity, iii) increased 
expression of Fiaf, indicating reduced peripheral deposition of fatty acids, iv) lower Glut2 expression, 
suggesting decreased glucose absorption, and v) higher expression of proglucagon (6). Collectively, these 
data suggested that GP-SPI supplementation improves metabolic resilience in a gut stressed by HFD. Fecal 
and cecal samples collected from mice fed HFD supplemented with GP-SPI showed higher levels of the 
gut bacterium Akkermansia muciniphila and a lower Firmicutes/Bacteroidetes ratio (6). In other words, 
polyphenols extracted from Concord grape or cranberry fruits and used as supplements triggered dramatic 
shifts in the type of bacteria that live in the gut, referred to as the “gut microbiota”.  
We recently showed that GP-SPI can induce the A. muciniphila bloom within 14 days, independent of 
dietary fat, and that PACs, the major constituent of GP, was sufficient to induce the bloom (14). We also 
found that in addition to promoting intestinal abundance of A. muciniphila in mice, GP-SPI induced changes 
in several genera (i.e. Oscillibacter, Clostridium IV, Intestinimonas, Acetatifactor, Blautia, Gemella, 
Romboutsia, Weissella) in a direction that may also contribute to intestinal health (14). While there are 
differences at the strain/species level, mouse and humans share 89% of bacterial genera (40). Compared 
to controls, mice fed HFD supplemented with GP-SPI, but not LFD supplemented with GP-SPI, showed 
improved oral glucose tolerance after 2 weeks (14).    
 

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Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
A. muciniphila is a Gram-negative obligate anaerobe that colonizes the mucus layer covering the intestinal 
epithelium. A. muciniphila can metabolize mucin as its only source of carbon and nitrogen (41) and was 
shown to modulate mucosal gene expression pathways involved in basal metabolism and immune 
tolerance toward the gut microbiota (42). For these reasons, it is thought that the bacteria species A. 
muciniphila has beneficial effects on carbohydrate metabolism. Consistent with this, treatment of Type 2 
diabetic patients with the commonly used drug, metformin, triggered an increase in A. muciniphila 
population (43; 44). Increases in A. muciniphila were similarly observed in pregnant women with normal 
weight gain as compared to those with excessive weight gain (44; 45), and in normal weight and post-
gastric-bypass subjects (46; 47).  Prebiotic (oligofructose) treatment of ob/ob mice resulted in an improved 
metabolic profile (increased glucose tolerance, L-cell number, intestinal proglucagon gene expression and 
plasma GLP-1 levels alongside reduced adiposity, oxidative stress, and low-grade inflammation) 
concomitant with a ~100 fold increase in A. muciniphila compared to control ob/ob mice (48). Probiotic 
administration of A. muciniphila to HFD-fed mice reduced adiposity, metabolic endotoxemia, adipose 
inflammation and insulin resistance in association with increased intestinal levels of endocannabinoids that 
control inflammation, the gut barrier, and gut peptide secretion (49).  Significantly, although it represents 
only 1 - 4 % of the gut microbiota in healthy subjects, the presence of A. muciniphila in the gut is inversely 
related to intestinal bowel disease (IBD), Crohn’s disease, ulcerative colitis and appendicitis, suggesting its 
utility as a biomarker of intestinal health (41). A recent intervention study of sweetened dried cranberries, 
which are rich in PACs, observed a trend towards increased Akkermansia species after 2 weeks compared 
to baseline (50). 
 
 
1.5 Sample Size Justification 
 
Using conservative estimates for mean relative abundance (RA) of A. muciniphila in human subjects (i.e. 
baseline mean RA of 0.1% increasing to 0.4% on day 10 with standard deviation of 0.5%), a sample size 
of 16 subjects would be required to detect significant difference from baseline levels (target power = 95%, 
actual power = 96.2%; α = 0.05, Hotelling-Lawley Trace test for repeated measures, GLIMMPSE).  
 
16S rRNA gene sequencing data will be compared longitudinally within each subject and in aggregate and 
consists of surveying the relative composition of gut microbiota populations in fecal samples and mine data 
for differences in phyla (e.g. Bacteriodetes vs. Firmicutes), genera, and specific microbes (Akkermansia 
spp.) as far as resolution allows. Power calculations will be performed using data obtained from this pilot 
time course study to assist in finalizing design of a future randomized, placebo-controlled intervention study. 
 
 
1.6 Study Variables 
 
A.   Independent Variables, Interventions, or Predictor Variables  
The independent variables will be supplementation with SPI or GP-SPI.  
 
B.  Dependent Variables or Outcome Measures  
Outcome measures are gut microbial community analyses, CMP results, urine and serum 
metabolites, and bacterial genes, species/strains.  
 
1.7 Drugs/Devices/Biologics  
N/A  
 
 
1.8 Specimen Collection 
 
A. Primary Specimen Collection 
 
 
Types of Specimens:  

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Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
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Blood samples: A blood samples will be collected by study nurse at screening visit for CMP 
test. If CMP is within normal ranges, subject will be enrolled in study. Blood will be collected 
again at baseline (day 0) and at end of study (day 11). Each blood draw will be no more than 
30 mL (2 tablespoons).    
 
Stool samples: Subjects will be provided with Omnigene®Gut OMR-200 kits or collection tubes 
containing 95% ethanol and/or tubes containing 50% glycerol (50% water) along with paper 
toilet accessories (DNA genotek, Ottawa, Canada) for easy self-collection of a pea-sized 
amount of fecal sample. Each participant will be instructed on use of stool collection materials.  
 
Urine samples: Subjects will be provided with sterile collection containers (100 mL) for self-
collection of urine samples on same days fecal samples are collected and asked to keep 
samples in 4 °C fridge until transport to the IFNH laboratory to maintain metabolite stability 
(20). 
 
 
Annotation:  
 
Blood samples: CMP tests will be performed on blood samples collected at screening visit, 
day 0, and day 11. Targeted and untargeted metabolomics analyses will be performed on de-
identified blood samples collected on days 0 and 11 from participants enrolled in study.  
 
Stool samples: Microbial community structure will be analyzed from de-identified fecal 
samples as previously described (51) and used for shot gun metagenomics studies. 
 
Urine samples: De-identified urine samples will subject for targeted and untargeted 
metabolomics analyses.  
 
 
Transport:  
 
Blood samples: Blood samples collected and processed by study nurse at Clinical Academic 
Building will be picked up by a study staff member of Roopchand lab and transported to IFNH 
on dry ice.  
 
Stool samples: The Omnigene®Gut OMR-200 kit allows transport and storage of stabilized 
DNA at ambient temperature for 60 days and 95% ethanol stabilizes samples for 8 weeks. 
Subjects will be asked to bring their stool samples to the IFNH laboratory as soon as possible, 
within 1-3 days of collection, for processing. Subjects will be asked to freeze fecal samples 
collected in 50% glycerol as soon as possible to minimize shifts in the microbiota, which can 
use glycerol as a carbon source.  
 
Urine samples: Subjects will be instructed to keep urine samples in their 4°C fridge until 
transport to the IFNH laboratory to maintain metabolite stability (20). 
 
 
 
Processing: 
 
Blood samples: Initial processing will be performed by study nurse. Further processing for 
metabolomics analysis will be performed by Roopchand lab research personnel working on 
this study.   
 
Stool and urine samples: Processing will be performed by Roopchand lab research personnel 
working on this study.   
 
 
Storage: 

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Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
 
Blood, stool and urine samples: Deidentified samples will be aliquoted and stored at -80 °C  
 
De-identified specimens will be stored in locked -80°C freezers belonging to the Roopchand laboratory in 
IFNH (61 Dudley Rd, New Brunswick, NJ 08901) until analysis. Specimens may be used for future 
experiments which may include: 1) new sample analyses that are necessary for this study but are not listed 
in the current protocol and sample analyses for future studies. Consent to use stored samples for 
experiments is required for participation in this study. Specimens will be stored in IFNH laboratory freezer 
until they are no longer in a fit state for scientific analyses and may only be accessed by authorized research 
personnel associated with this study.  
 
 
Disposition: 
Blood, stool, and urine samples: Specimens will be stored in IFNH laboratory freezer until 
they are no longer in a fit state for scientific analyses, after which they will be disposed of by 
incineration by REHS. Only de-identified study data will be associated with the specimens 
banked for future use by this PI, removing need for a repository. 
  
 
B. Secondary Specimen Collection    
N/A 
 
 
 
 
1.9 Data Collection (Interviews, Focus Groups, or Surveys)  
N/A 
 
 
 
1.10 Timetable/Schedule of Events 
 
 
Dec 2018 - Feb 
2019 
Mar - Aug 
2019 
Sept 2019 - 
Mar 2020 
April 2020 - 
ongoing 
Staff/student 
training, 
research 
material production 
 
 
 
 
Eligibility screening, recruitment, and 
sample collection 
 
 
 
 
Sample processing, data generation 
and analyses 
 
 
 
 
Publications 
and 
results 
dissemination  
 
 
 
 
 
 
2.0 Project Management 
 
2.1 Research Staff and Qualifications 
 
Diana Roopchand, PI, Rutgers IFNH Dr. Roopchand is an Assistant Professor in the Food Science 
Department with a laboratory located at IFNH. Her research focuses on dietary components and 

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Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
interventions that promote metabolic health by altering the gut microbial community. She pioneered 
development of dietary polyphenol-protein complexes and has experience in phytochemical analysis, 
molecular biology, and gut microbiota community analysis. She will oversee research material production 
and will direct day-to-day research and training activities including participant recruitment, scheduling, 
sample organization, data entry, and analyses. 
 
Susette Coyle, Co-investigator, RWJMS Nurse Ms. Coyle has several years of experience in clinical 
protocol development and performing clinical studies. She will perform blood draws for CMP tests and 
collection of blood samples for metabolomics analyses.  
 
Marie Macor, Co-investigator, RWJMS Nurse Ms. Macor has several years of experience in clinical 
protocol development and performing clinical studies. She will perform blood draws for CMP tests and 
collection of blood samples for metabolomics analyses.   
 
Rocio M. Duran, Study Staff, Research Technician, Rutgers IFNH Ms. Duran is the lead research 
technician in the laboratory of Dr. Roopchand. She brings extensive experience in molecular biology, 
microbial genetics, and biochemical analysis of microbial metabolites. She will assist with participant 
recruitment, scheduling, sample organization, storage and processing, data entry, and analyses.  
 
Esther Mezhibovsky, Study Staff, Graduate Student, Rutgers IFNH 
Ms. Mezhibovsky has experience with prior clinical studies, specifically as a study coordinator at the 
University of Wisconsin for the CAREDS2 Study. She has experience in participant screening, data 
collection, entry, and quality assurance and is trained in handling confidential information as defined by 
HIPAA. She organized research material production and will assist with participant recruitment, scheduling, 
sample organization, data entry, and analyses. 
 
David M. Krol, MD, Study Physician, Medical Director, Rutgers IFNH and RWJMS 
Dr. Krol is the Medical Director at IFNH and Associate Professor and Chair of Pediatrics at RWJMS. He 
serves as Medical Director for the NJ Healthy Kids Initiative, a partnership between the IFNH and Child 
Health Institute of NJ. Dr. Krol will order CMP tests and review CMP data. As onsite study physician, the 
clinical study team and/or study participants will consult with Dr. Krol in case of adverse events or safety 
concerns. 
 
 
2.2 Research Staff Training 
 
RESEARCH TRAINING. All investigators and research personnel will complete the Collaborative 
Institutional Training Initiative (CITI) training and the Lab Safety/Biological Safety/Blood Borne Pathogens 
Training from Rutgers Environmental Health & Safety.  
 
The PI and RWJMS collaborators have had several meetings to develop the study. Prior to starting the 
study, PI will meet with study personnel to review the protocol, procedures, study forms, and each one’s 
responsibilities going forward.  Meetings will be held periodically throughout the study to review progress 
and make changes if necessary. 
 
2.3 Resources Available 
IFNH and CAB are equipped with conference/interview rooms for eligibility screening visits. The GP-
SPI and SPI materials will be stored in freezers of Food Science pilot plant, which has space dedicated 
for production of food-grade study materials.   
 
2.4 Research Sites 
 
Rutgers Robert Wood Johnson Medical School (RWJMS) 
 
Rutgers SEBS, Institute for Food, Nutrition, and Health 

Page 17 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
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IRB Form Version 12.18.18 
 
 
Rutgers RWJMS Clinical Research Center 
 
3.0 Multi-Center Research 
N/A 
 
4.0 Subject Considerations 
 
4.1 Subject Selection and Enrollment Considerations  
 
A. Method to Identify Potential Subjects 
Flyers will be posted locally to recruit volunteers and emails will be sent to Rutgers university email 
lists. 
 
B. Recruitment Details 
Recruitment of the 30 participants will start in March 2019. Prospective participants will be recruited 
through flyers on Rutgers University campuses and email lists. Interested individuals will be asked 
to contact the research team by email or phone to obtain an overview of the study. We expect to 
recruit the majority of our participants from the New Brunswick campus of Rutgers University. We 
conservatively expect a recruitment rate of 3-6 participants per month over 6 months. 
 
   
C. Subject Screening 
Interested subjects will call in to receive further information about the study.  A co-
investigator/coordinator will give a brief summary of the study. If the subject requests a screening 
appointment, the investigator will ask for verbal consent to proceed with a list of questions.  
Collecting this information at this time assists in expediting the paperwork during the visit and 
spares ineligible subjects the inconvenience of a futile appointment. 
 
At the screening visit, a co-investigator/coordinator will explain the study in detail to the subject.  
Full explanation of procedures and potential risks will be provided.  The subject will have ample 
time to read the consent and ask questions before signing and proceeding with screening 
procedures.  A signed copy will be given to the subject.  
 
Inclusion Criteria: 
A subject will be deemed as “healthy” and eligible for inclusion in this study if all of the following criteria are 
met: 
1. Healthy as assessed based on a medical evaluation including a comprehensive metabolic panel 
(CMP) test with values in normal range, medical history, and not presently taking any medication 
2. Adults between 18 and 35 years  
3. BMI  18.5 - 29.9  
4.  Have at least one bowel movement per day 
5. Capable of giving written informed consent, which includes compliance with the requirements and 
restrictions listed in the consent form.  
   
Exclusion criteria: 
1. History/current cancer, rheumatoid arthritis immunologic, renal, hepatic, endocrine, neurologic or 
heart disease, hypertension, diabetes, GI dysfunction, or CMP test results showing values outside 
of normal range. 
2. Cannot provide written informed consent. 
3. Exposure to any experimental agent or procedure within 30 days of study. 
4. Pregnancy or breast-feeding 
5. Taking dietary supplements   
6. Current smoker or have smoked within previous 6 months  
7. Taking medications regularly (prescription, over the counter, supplements etc.) 

Page 18 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
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IRB Form Version 12.18.18 
 
8. Treated with antibiotics during the past 6 months 
9. Have an allergy to soy or grapes 
 
 
4.2 Secondary Subjects 
N/A 
 
 
4.3 Number of Subjects 
 
A. Total Number of Subjects 
30 subjects will be enrolled after successfully passing screening. The anticipated lost to follow-up 
rate for this study will be 20% (i.e. 6 non-completers) 
 
B. Total Number of Subjects If Multicenter Study 
N/A 
 
C. Feasibility 
We expect to recruit the majority of our participants from the New Brunswick campus of Rutgers 
University. With a relatively flexible study schedule and no particularly stringent eligibility criteria, we 
do not anticipate difficulties in engaging interest and accommodating schedules of participants. We 
conservatively expect a recruitment rate of 3-6 participants per month over 6 months. 
 
 
4.4 Consent Procedures 
 
A. Consent Process 
 
Location of Consent Process 
The Consent Process will take place at the New Jersey Institute for Food, Nutrition & Health 
 
 
Ongoing Consent 
N/A 
 
 
Individual Roles for Researchers Involved in Consent 
Principal Investigator Dr. Diana Roopchand, Co-Investigators Susette Coyle or Marie Macor, 
Rocio Duran, or graduate student Esther Mezhibovsky will conduct the consent process to 
explain all aspects of the study and answer any questions the prospective participants may 
have. Dr. Roopchand, Susette Coyle, Marie Macor, Rocio Duran, or Esther Mezhibovsky will 
sign the consent form. 
 
 
Consent Discussion Duration 
It is expected that the consent discussion will last approximately 45 min.  
 
 
Coercion or Undue Influence 
In order to minimize the possibility of coercion or undue influence, the consent process will not 
be conducted by research personnel who have any known relationship or conflict of interest 
with the prospective participants. 
 
 
Subject Understanding 
Throughout the consent process the prospective participants will be encouraged to ask any 
questions should they feel the need to do so. The consent process will be divided into a series 
of small sections. Members of the research team will summarize each section, use probe 
questions to check for sufficient understanding before proceeding to the next section. An 
illustrated Study Summary will be provided to assist subject understanding of study procedures. 
 

Page 19 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
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B. Waiver or Alteration of Consent Process  
N/A 
 
 
C. Documentation of Consent 
 
Documenting Consent 
Please see attached Consent form. 
 
 
Waiver of Documentation Of Consent (i.e., will not obtain subject’s signature) 
N/A  
 
 
4.5 Special Consent/Populations 
 
A. Minors-Subjects Who Are Not Yet Adults  
N/A 
B. Wards of the State 
N/A 
C. Non-English-Speaking Subjects 
             N/A 
D. Adults Unable to Consent / Cognitively Impaired Adults (for interventional studies) 
             N/A 
 
 
4.6 Economic Burden and/or Compensation for Subjects 
 
A. Expenses  
Participants may incur expenses when they travel to our research facilities.  
 
B. Compensation/Incentives 
Subjects will receive parking for each blood draw study visit and compensated $5 for each of 9 
stool, 7 urine, and 2 blood samples delivered on days -5 through 10 and an additional $110 on 
Day 11 visit for completing all requested study samples, completion of all study visits and 
delivery of complete digital and written food diary for a total of $200 paid in cash. One missed 
or incomplete visit will result in only $5 for each blood, urine and stool sample delivered on Days 
-5 through 10. 
 
C.  Compensation Documentation 
Each participant will be asked to sign a form confirming receipt of stated amount of compensation 
at end of their participation in the study. See page 3 of “Log of Study Visits & Receivables” form; 
once signed this page will be moved to separate binder and kept in a separate locked cabinet. 
  
 
4.7 Risks of Harm/Potential for Benefits to Subjects 
 
A. Description of Risks of Harm to Subjects 
 
Reasonably Foreseeable Risks of Harm 
Study Supplement – there are no known risks to ingesting the study supplement. To minimize 
the risk of an allergic reaction the study excludes persons allergic to soy or grapes. 
 
Blood Sampling – may cause a bruise or bleeding at the site. Infection at the site is rare.  
A total of 65 mL (~ 4 Tablespoons) will be drawn during the course of the study. Healthy 
subjects can tolerate this blood loss without any side effects. 
 
Urine and Stool collection – There is risk of discomfort with sample collection and storage. 

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Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
 
These risks are considered minimal and are addressed in the protocol and consent form. To 
minimize expected risk, study staff will communicate with subjects during intervention period 
and do a final follow up by phone or email 2 - 3 weeks after end of intervention.  
  
 
Risk of Harm from an Intervention on a Subject with an Existing Condition 
N/A  
 
 
Other Foreseeable Risks of Harm 
All efforts will be made to keep personal information in subject research record confidential, but 
total confidentiality cannot be guaranteed.   
 
 
Observation and Sensitive Information 
N/A  
 
B. Procedures which Risk Harm to Embryo, Fetus, and/or Pregnant Subjects 
Soy protein and dietary polyphenols are often consumed as part of a normal diet; however, in case 
of pregnancy within the 15-day study period, the risk of GP-SPI to a newly formed embryo/fetus is 
uncertain. This study will not include pregnant subjects and a urine-based pregnancy test will be 
performed to confirm inclusion of only non-pregnant participants. 
 
C. Risks of Harm to Non-Subjects 
N/A 
 
D. Assessment of Social Behavior Considerations 
N/A 
 
 
E. Minimizing Risks of Harm 
All procedures will be explained to subjects at the time of obtaining consent and subjects will be 
encouraged to ask questions about any concerns throughout the study. Every effort will be made 
to maintain subject confidentiality.  
 
 
Certificate of Confidentiality 
This study is partially funded by NIH-NCCIH therefore a Certificate of Confidentiality is 
automatically issued to protect data collected.   
 
 
Provisions to Protect the Privacy Interests of Subjects 
Should any participants desire not to interact with or provide personal information to specific 
members of the research team, they may report such a need to the Principal Investigator and 
the arrangement of the research personnel will be adjusted accordingly. Participants will not 
be required to provide a reason if they desire not to do so. Should changing of the research 
personnel is not possible, the Principal Investigator will discuss with the participants about 
alternative solutions.  
 
In order to ensure the protection of personal health information of the participants, there will be 
no identifiable information on any of the data collection instruments. Each participant will be 
assigned a unique identification code. A master code identifier which links the identification 
code and personal information (name, date of birth and contact information) will be maintained 
separately from the study data. Only the Principal Investigator and approved research 
personnel who have a specific need for identifiable information will have access (e.g. their 
primary physician, research student/staff to schedule appointments). All data and materials 

Page 21 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
collected during this study are for research purposes only, and the data will be kept in strict 
confidence. No information will be given to anyone without permission from the participants.  
 
F. Potential Benefits to Subjects 
There are no direct benefits for the subjects.  Subjects will receive a copy of results from health 
assessment blood tests, if abnormal they will be encouraged to see their doctor.   
 
5.0 Special Considerations 
 
5.1 Health Insurance Portability and Accountability Act (HIPAA) 
HIPPA will be collected with consent. 
 
5.2 Family Educational Rights and Privacy Act (FERPA) 
N/A 
 
5.3 NJ Access to Medical Research Act (Surrogate Consent) 
N/A 
 
5.4 General Data Protection Regulation (GDPR) 
N/A 
 
5.5 Code of Federal Regulations Title 45 Part 46 (Vulnerable Populations) 
N/A 
 
 
6.0 Data Management Plan 
 
6.1 Data Analysis 
Statistical comparisons will focus on individual and aggregate comparisons with respect to baseline to 
follow-up change in gut microbiota populations and biochemistry profile in blood and urine samples.  
 
For each outcome, the equality of the outcome means for GP-SPI versus SPI will be tested against a 
two-directional alternative, controlling the type 1 error at α = 0.05. The analyses will be conducted using 
t-tests for between group analysis and with repeated measures to conduct analysis of variance. Post-
hoc tests will be conducted for pairwise comparisons of GP-SPI and SPI employing the Tukey method 
for controlling the global comparison-wise significance at 0.05 level. Logarithms or other 
transformations will be applied for data seriously deviating from normality; alternatively, non-parametric 
analyses may be conducted. 
 
Handling of missing data: The data will be analyzed using the procedures PROC MIXED and PROC 
GLIMMIX SAS (v 9.3 or later). Thus, mixed effects models for repeated measures will be employed to 
analyze all available data irrespective of missing or incomplete data attributable to dropouts or other 
phenomena. In anticipation of the possibility of missing data, we plan to conduct sensitivity analyses 
that compare our findings against protocol completer analyses and last observation carried forward 
analyses in addition to investigating characteristics of participants who fail to protocol. 
 
6.2 Data Security 
A code will be assigned to every participant and the collected data will be recorded using the 
participant’s unique code for identification purposes. The Principal Investigator, study nurses, and 
research staff/students who schedule appointments will be the only ones with access to the master 
code identifier. Participants’ hard copy files will be kept in locked cabinets only accessible by authorized 
research personnel in IFNH, a secured building. Electronic files will be password-protected and stored 
on the university’s secure server and will only be accessible to authorized research personnel. The 
master code identifier and the data files will be kept in two separate locations. All personnel who will 

Page 22 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
have access to data of any form will be required to complete CITI training and will only have access to 
the data during a specific duration deemed necessary by the Principal Investigator.  
 
6.3 Data and Safety Monitoring 
 
A. Data/Safety Monitoring Plan 
An Independent Monitoring Committee (IMC) was formed, composed of three Rutgers University faculty 
and staff members who are not directly involved with the study. The IMC will monitor recruitment, 
retention, adherence, and review participant safety including adverse events, proposed major protocol 
modifications, and reports of related studies as appropriate. 
 
Study progress and safety will be reviewed monthly (and more frequently if needed) by study team and 
IMC. The IMC will receive monthly email updates (i.e. IMC Report, attached) on study progress and 
meet once every two months or as needed.   
 
Data will be collected on prepared forms for Adverse Event, Serious Adverse Event, Unanticipated 
Problems, and Protocol Deviation Tracking Log.    
 
An Annual Report (i.e. IMC Report attached) will be compiled and will include a list and summary of 
AEs.  In addition, the Annual Report will address (1) whether AE rates are consistent with pre-study 
assumptions; (2) reason for dropouts from the study; (3) whether all participants met entry criteria; (4) 
whether continuation of the study is justified on the basis that additional data are needed to accomplish 
the stated aims of the study; and (5) conditions whereby the study might be terminated prematurely.  
The Annual Report will be sent to the Independent Monitors and will be forwarded to the IRB and 
NCCIH, and FDA. The PI will send copies of signed recommendations and comments from the 
Independent Monitors to the NCCIH Program Officer within 1 month of each monitoring review.  
 
B. Data/Safety Monitoring Board Details 
 
The Independent Monitoring Committee for this study is comprised of Dr. Amy Davidow 
(Biostatistician), Dr. Thomas Gianfagna (expert in plant-derived natural products), and Dr. Vinod Rustgi 
(Clinician, gastroenterologist). Dr. Rustgi has previously served on a monitoring committee. Drs. 
Davidow, Gianfagna, and Rustgi are not associated with this research project and work independently 
of the PI, Dr. Roopchand. They are not part of the key personnel involved in this grant. No member of 
the Committee has collaborated or co-published with the PI within the past three years. They are 
qualified to review the patient safety data generated by this study because of their unique expertise.  
  
Any serious adverse event that is life-threatening or results in death, that is possibly, probably, or 
definitely related to the research protocol, will trigger an immediate suspension of the research.  
 
C. Source and Preparation of Study Supplement 
 
Concord grapes are a rich source of dietary polyphenols. Concord grapes are pressed to produce 
polyphenol-rich Concord grape juice, which also large amounts of fruit sugars, mainly glucose and 
fructose (10).  Concord grape pomace is comprised of the seeds and skins of the grapes that are left 
over after the juicing process. The pomace is also rich in dietary polyphenols, mainly proanthocyanidins 
(PAC), but in contrast with the juice, contains low amounts of sugars (9). 
 
We have previously demonstrated that, due to a natural affinity of polyphenols and proteins, dietary 
polyphenols can be stabilized for at least 24 weeks (and up to one year, unpublished data) at 37 ºC 
when sorbed to a protein-rich food matrix, such as soy protein isolate (SPI), without compromising 
subsequent release from the matrix or efficacy (7; 9; 11). Studies performed in models of the human 
intestine have also demonstrated that protein-polyphenol particles provide an efficient vehicle for 
delivering intact polyphenols, e.g. anthocyanins, to the lower part of the intestine (52). We will therefore 
use this protein sorption process to maintain stability of extracted Concord grape polyphenols.  

Page 23 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
 
The biochemical composition of the grape pomace extract used in this study and the pilot scale 
production of grape polyphenols (GP) sorbed and stabilized to the SPI matrix (i.e. GP-SPI) have been 
previously described in detail (9). Aliquots of extracted GP will be biochemically characterized using 
LC-MS and quantified for total polyphenols and PAC using previously described colorimetric methods 
(9; 14). 
 
GP-SPI will be manufactured in the Rutgers Food Science Pilot plant. Briefly, Concord grape pomace 
(supplied by Welch’s) will be extracted with 50% ethanol (1:5 solids:solvent; wt./vol. ratio) with pH 
adjusted to 2 (using sulphuric acid) at 80°C for 2 h with agitation and ambient pressure. The cooled 
polyphenol extract is then separated by filtration and the concentration of total polyphenols is quantified 
using the Folin-Ciocalteu assay. A calculated amount of SPI is mixed with the grape polyphenol extract 
and dried until moisture is < 10% to produce the GP-SPI complex containing 5% total grape 
polyphenols.  Since the basic process involves the mixing of two foods and removal of water and 
ethanol, it is equivalent to cooking. The resulting product remains a food, and we feel it can be Generally 
Regarded as Safe (GRAS) http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/.  GP-SPI is 
not intended to cure, prevent or mitigate a disease. Its suggested use is to maintain normal functions 
of the human body.  
 
Safety Monitoring: A representative sample of the GP-SPI product will be provided to the laboratory 
of Prof. Donald Schaffner (Distinguished Professor and Extension Specialist, Food Science 
Department) to perform microbiological testing using same procedures they use for weekly testing of 
Rutgers University dining hall foods (including total aerobic plate count, coliforms, generic E. coli, 
Salmonella, S. aureus, B. cereus, C. perfringens, L. monocytogenes, etc.).  
  
Based on our quantification of total polyphenols in commercial Concord grape juice (e.g., Welch’s), the 
daily dose of GP-SPI we propose to test in this clinical study (40 g per day) is equivalent to the 
concentration of polyphenols that can be found in four-to-six 250 mL glasses of Concord grape juice.  
 
Drinking 4-6 glasses of Concord grape juice would normally deliver 150 g – 225 g sugar along with 
1860 – 2160 mg of total polyphenols while 40 g of GP-SPI product will deliver this level of polyphenols 
without the excess sugar (4g total sugars/100g GP-SPI).  Grape pomace polyphenols are mainly 
catechins and PACs (catechin polymers), which are compounds commonly found in pigmented fruits 
and spices.  Given that subjects will be instructed to consume a diet low in PAC-rich foods, we don’t 
expect that consuming 2000 mg/day of grape polyphenols delivered in GP-SPI will promote side 
effects.  Average daily polyphenol intake has been reported to be 1000 mg/day (53).  Total polyphenol 
intake has been reported to be 3000 mg/day in individuals consuming a Spanish diet, typically higher 
in polyphenols (54). Subjects consuming GP-SPI would be consuming the level of total polyphenols 
within the normal range of a total diet.  
 
6.4 Reporting Results 
A. Individual Subjects’ Results  
Subjects will receive a copy of their CMP blood test results.  Subjects will receive a copy of their 
individual gut microbiota community analysis data after all study data is analyzed.   
 
B. Aggregate Results 
Aggregate results will be accessible to participants after study is published in the peer-reviewed 
literature. 
 
C. Professional Reporting 
Scientific manuscripts reporting study findings will be prepared and submitted for publication in 
peer-reviewed academic journals. The study findings will also be prepared for presentation at 
regional, national, and international research conferences. Key finding will be disseminated to the 
public by press release.  
 

Page 24 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
D. Clinical Trials Registration, Results Reporting and Consent Posting 
This study will be registered on ClinicalTrials.gov. 
 
6.5 Secondary Use of the Data    
N/A 
 
7.0 Research Repositories – Specimens and/or Data 
Data: De-identified electronic data will be stored on the university’s secure server indefinitely. Data banked 
for future use will only be accessible by the Principal Investigator. Release of these data, irrespective of 
whether the Principal Investigator has ownership of the research project(s) that require the banked data, 
will need to obtain separate IRB approvals. When the banked data are transferred or shared, they will not 
be associated with any identifiable data, personal information of the research participants, or the master 
code identifier for deciphering the codes.  
 
Specimens: The stool, urine, and blood samples collected during the study will be coded and stored in a 
locked -80°C laboratory freezers until samples can be processed. With consent of participants, de-identified 
samples from the clinical study will be stored for use by PI only in future studies.  
 
8.0 Approvals/Authorizations 
 
NIH Approval letter 
Roopchand IBC, Foty IBC  
 
 
9.0 Bibliography 
 
1. Visioli F, De La Lastra CA, Andres-Lacueva C, Aviram M, Calhau C, Cassano A, D'Archivio M, Faria A, 
Fave G, Fogliano V, Llorach R, Vitaglione P, Zoratti M, Edeas M: Polyphenols and human health: a 
prospectus. Crit Rev Food Sci Nutr 2011;51:524-546 
2. Zunino S: Type 2 diabetes and glycemic response to grapes or grape products. J Nutr 2009;139:1794S-
1800S 
3. Blade C, Aragones G, Arola-Arnal A, Muguerza B, Bravo FI, Salvado MJ, Arola L, Suarez M: 
Proanthocyanidins in health and disease. Biofactors 2016;42:5-12 
4. Choy YY, Jaggers GK, Oteiza PI, Waterhouse AL: Bioavailability of intact proanthocyanidins in the rat 
colon after ingestion of grape seed extract. J Agric Food Chem 2013;61:121-127 
5. Anhe FF, Roy D, Pilon G, Dudonne S, Matamoros S, Varin TV, Garofalo C, Moine Q, Desjardins Y, Levy 
E, Marette A: A polyphenol-rich cranberry extract protects from diet-induced obesity, insulin resistance 
and intestinal inflammation in association with increased Akkermansia spp. population in the gut 
microbiota of mice. Gut 2015;64:872-883 
6. Roopchand DE, Carmody RN, Kuhn P, Moskal K, Rojas-Silva P, Turnbaugh PJ, Raskin I: Dietary 
Polyphenols Promote Growth of the Gut Bacterium Akkermansia muciniphila and Attenuate High-Fat 
Diet-Induced Metabolic Syndrome. Diabetes 2015;64:2847-2858 
7. Roopchand DE, Grace MH, Kuhn P, Cheng DM, Plundrich N, Pouleva A, Howell A, Fridlender B, Lila MA, 
Raskin I: Efficient sorption of polyphenols to soybean flour enables natural fortification of foods. Food 
Chem 2012;131:1193-1200 
8. Roopchand DE, Krueger CG, Moskal K, Fridlender B, Lila MA, Raskin I: Food-compatible method for the 
efficient extraction and stabilization of cranberry pomace polyphenols. Food Chem 2013;141:3664-3669 
9. Roopchand DE, Kuhn P, Krueger CG, Moskal K, Lila MA, Raskin I: Concord grape pomace polyphenols 
complexed to soy protein isolate are stable and hypoglycemic in diabetic mice. J Agric Food Chem 
2013;61:11428-11433 

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Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
10. Roopchand DE, Kuhn P, Poulev A, Oren A, Lila MA, Fridlender B, Raskin I: Biochemical analysis and in 
vivo hypoglycemic activity of a grape polyphenol-soybean flour complex. J Agric Food Chem 
2012;60:8860-8865 
11. Roopchand DE, Kuhn P, Rojo LE, Lila MA, Raskin I: Blueberry polyphenol-enriched soybean flour 
reduces hyperglycemia, body weight gain and serum cholesterol in mice. Pharmacol Res 2013;68:59-67 
12. Nieman DC, Gillitt ND, Knab AM, Shanely RA, Pappan KL, Jin F, Lila MA: Influence of a polyphenol-
enriched protein powder on exercise-induced inflammation and oxidative stress in athletes: a 
randomized trial using a metabolomics approach. PLoS One 2013;8:e72215 
13. Ahmed M, Henson DA, Sanderson MC, Nieman DC, Gillitt ND, Lila MA: The protective effects of a 
polyphenol-enriched protein powder on exercise-induced susceptibility to virus infection. Phytother Res 
2014;28:1829-1836 
14. Zhang L, Carmody RN, Kalariya HM, Duran RM, Moskal K, Poulev A, Kuhn P, Tveter KM, Turnbaugh P, 
Raskin I, Roopchand DE: Grape proanthocyanidin-induced intestinal bloom of Akkermansia muciniphila 
is dependent on its baseline abundance and precedes activation of host genes related to metabolic 
health. J Nutr Biochem 2018;56 142-151 
15. Song SJ, Amir A, Metcalf JL, Amato KR, Xu ZZ, Humphrey G, Knight R: Preservation Methods Differ in 
Fecal Microbiome Stability, Affecting Suitability for Field Studies. mSystems 2016;1:e00021-00016 
16. Wang Z, Zolnik CP, Qiu Y, Usyk M, Wang T, Strickler HD, Isasi CR, Kaplan RC, Kurland IJ, Qi Q, Burk RD: 
Comparison of Fecal Collection Methods for Microbiome and Metabolomics Studies. Front Cell Infect 
Microbiol 2018;8:301-301 
17. Vandeputte D, Falony G, Vieira-Silva S, Tito RY, Joossens M, Raes J: Stool consistency is strongly 
associated with gut microbiota richness and composition, enterotypes and bacterial growth rates. Gut 
2016;65:57 
18. Heaton KW, Radvan J, Cripps H, Mountford RA, Braddon FE, Hughes AO: Defecation frequency and 
timing, and stool form in the general population: a prospective study. Gut 1992;33:818-824 
19. Walter SA, Kjellström L, Nyhlin H, Talley NJ, Agréus L: Assessment of normal bowel habits in the 
general adult population: the Popcol study. Scandinavian Journal of Gastroenterology 2010;45:556-566 
20. Rotter M, Brandmaier S, Prehn C, Adam J, Rabstein S, Gawrych K, Bruning T, Illig T, Lickert H, 
Adamski J, Wang-Sattler R: Stability of targeted metabolite profiles of urine samples under different 
storage conditions. Metabolomics 2017;13:4 
21. Gu LW, Kelm MA, Hammerstone JF, Beecher G, Holden J, Haytowitz D, Gebhardt S, Prior RL: 
Concentrations of proanthocyanidins in common foods and estimations of normal consumption. Journal 
of Nutrition 2004;134:613-617 
22. Gu LW, Kelm MA, Hammerstone JF, Beecher G, Holden J, Haytowitz D, Prior RL: Screening of foods 
containing proanthocyanidins and their structural characterization using LC-MS/MS and thiolytic 
degradation. J Agr Food Chem 2003;51:7513-7521 
23. Schmieder R, Edwards R: Quality control and preprocessing of metagenomic datasets. Bioinformatics 
2011;27:863-864 
24. Langmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2. Nat Methods 2012;9:357-359 
25. Peng Y, Leung HC, Yiu SM, Chin FY: IDBA-UD: a de novo assembler for single-cell and metagenomic 
sequencing data with highly uneven depth. Bioinformatics 2012;28:1420-1428 
26. Zhu W, Lomsadze A, Borodovsky M: Ab initio gene identification in metagenomic sequences. Nucleic 
Acids Res 2010;38:e132 
27. Li R, Yu C, Li Y, Lam TW, Yiu SM, Kristiansen K, Wang J: SOAP2: an improved ultrafast tool for short 
read alignment. Bioinformatics 2009;25:1966-1967 
28. Nielsen HB, Almeida M, Juncker AS, Rasmussen S, Li J, Sunagawa S, Plichta DR, Gautier L, Pedersen 
AG, Le Chatelier E, Pelletier E, Bonde I, Nielsen T, Manichanh C, Arumugam M, Batto JM, Quintanilha Dos 

Page 26 of 28 
 
Version Number: 3 
Protocol Number: 2018002579 
PI Name: Diana E. Roopchand 
Protocol Title: The Effect of Concord 
grape polyphenol-soy protein isolate 
complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
DO NOT MODIFY THIS SPACE 
 
 
 
IRB Form Version 12.18.18 
 
Santos MB, Blom N, Borruel N, Burgdorf KS, Boumezbeur F, Casellas F, Dore J, Dworzynski P, Guarner F, 
Hansen T, Hildebrand F, Kaas RS, Kennedy S, Kristiansen K, Kultima JR, Leonard P, Levenez F, Lund O, 
Moumen B, Le Paslier D, Pons N, Pedersen O, Prifti E, Qin J, Raes J, Sorensen S, Tap J, Tims S, Ussery DW, 
Yamada T, Meta HITC, Renault P, Sicheritz-Ponten T, Bork P, Wang J, Brunak S, Ehrlich SD, Meta HITC: 
Identification and assembly of genomes and genetic elements in complex metagenomic samples without 
using reference genomes. Nat Biotechnol 2014;32:822-828 
29. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, 
Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, 
Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, 
Zaneveld J, Knight R: QIIME allows analysis of high-throughput community sequencing data. Nat 
Methods 2010;7:335-336 
30. Zhang C, Yin A, Li H, Wang R, Wu G, Shen J, Zhang M, Wang L, Hou Y, Ouyang H, Zhang Y, Zheng Y, 
Wang J, Lv X, Wang Y, Zhang F, Zeng B, Li W, Yan F, Zhao Y, Pang X, Zhang X, Fu H, Chen F, Zhao N, 
Hamaker BR, Bridgewater LC, Weinkove D, Clement K, Dore J, Holmes E, Xiao H, Zhao G, Yang S, Bork P, 
Nicholson JK, Wei H, Tang H, Zhang X, Zhao L: Dietary Modulation of Gut Microbiota Contributes to 
Alleviation of Both Genetic and Simple Obesity in Children. EBioMedicine 2015;2:968-984 
31. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW: CheckM: assessing the quality of 
microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 2015;25:1043-
1055 
32. Abubucker S, Segata N, Goll J, Schubert AM, Izard J, Cantarel BL, Rodriguez-Mueller B, Zucker J, 
Thiagarajan M, Henrissat B, White O, Kelley ST, Methe B, Schloss PD, Gevers D, Mitreva M, Huttenhower 
C: Metabolic reconstruction for metagenomic data and its application to the human microbiome. PLoS 
Comput Biol 2012;8:e1002358 
33. Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M: KEGG for integration and interpretation of 
large-scale molecular data sets. Nucleic Acids Res 2012;40:D109-114 
34. Pimpao RC, Ventura MR, Ferreira RB, Williamson G, Santos CN: Phenolic sulfates as new and highly 
abundant metabolites in human plasma after ingestion of a mixed berry fruit puree. Brit J Nutr 
2015;113:454-463 
35. Want EJ, Wilson ID, Gika H, Theodoridis G, Plumb RS, Shockcor J, Holmes E, Nicholson JK: Global 
metabolic profiling procedures for urine using UPLC-MS. Nat Protoc 2010;5:1005-1018 
36. Zhao L, Zhang F, Ding X, Wu G, Lam YY, Wang X, Fu H, Xue X, Lu C, Ma J, Yu L, Xu C, Ren Z, Xu Y, Xu S, 
Shen H, Zhu X, Shi Y, Shen Q, Dong W, Liu R, Ling Y, Zeng Y, Wang X, Zhang Q, Wang J, Wang L, Wu Y, 
Zeng B, Wei H, Zhang M, Peng Y, Zhang C: Gut bacteria selectively promoted by dietary fibers alleviate 
type 2 diabetes. Science 2018;359:1151-1156 
37. Misra BB: New tools and resources in metabolomics: 2016-2017. Electrophoresis 2018;39:909-923 
38. Vinaixa M, Samino S, Saez I, Duran J, Guinovart JJ, Yanes O: A Guideline to Univariate Statistical 
Analysis for LC/MS-Based Untargeted Metabolomics-Derived Data. Metabolites 2012;2:775-795 
39. Fagan A, Culhane AC, Higgins DG: A multivariate analysis approach to the integration of proteomic 
and gene expression data. Proteomics 2007;7:2162-2171 
40. Krych L, Hansen CH, Hansen AK, van den Berg FW, Nielsen DS: Quantitatively different, yet 
qualitatively alike: a meta-analysis of the mouse core gut microbiome with a view towards the human 
gut microbiome. PLoS One 2013;8:e62578 
41. Belzer C, de Vos WM: Microbes inside--from diversity to function: the case of Akkermansia. ISME J 
2012;6:1449-1458 
42. Derrien M, Van Baarlen P, Hooiveld G, Norin E, Muller M, de Vos WM: Modulation of Mucosal 
Immune Response, Tolerance, and Proliferation in Mice Colonized by the Mucin-Degrader Akkermansia 
muciniphila. Front Microbiol 2011;2:166 

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Protocol Title: The Effect of Concord 
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complex (GP-SPI) on Gut Microbiota 
 
 
 
RESERVED FOR IRB STAMP 
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43. Shin NR, Lee JC, Lee HY, Kim MS, Whon TW, Lee MS, Bae JW: An increase in the Akkermansia spp. 
population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice. 
Gut 2014;63:727-735 
44. Santacruz A, Collado MC, Garcia-Valdes L, Segura MT, Martin-Lagos JA, Anjos T, Marti-Romero M, 
Lopez RM, Florido J, Campoy C, Sanz Y: Gut microbiota composition is associated with body weight, 
weight gain and biochemical parameters in pregnant women. Br J Nutr 2010;104:83-92 
45. Santacruz A, Collado MC, Garcia-Valdes L, Segura MT, Martin-Lagos JA, Anjos T, Marti-Romero M, 
Lopez RM, Florido J, Campoy C, Sanz Y: Gut microbiota composition is associated with body weight, 
weight gain and biochemical parameters in pregnant women. Brit J Nutr 2010;104:83-92 
46. Liou AP, Paziuk M, Luevano JM, Jr., Machineni S, Turnbaugh PJ, Kaplan LM: Conserved shifts in the 
gut microbiota due to gastric bypass reduce host weight and adiposity. Sci Transl Med 2013;5:178ra141 
47. Zhang H, DiBaise JK, Zuccolo A, Kudrna D, Braidotti M, Yu Y, Parameswaran P, Crowell MD, Wing R, 
Rittmann BE, Krajmalnik-Brown R: Human gut microbiota in obesity and after gastric bypass. Proc Natl 
Acad Sci U S A 2009;106:2365-2370 
48. Everard A, Lazarevic V, Derrien M, Girard M, Muccioli GG, Neyrinck AM, Possemiers S, Van Holle A, 
Francois P, de Vos WM, Delzenne NM, Schrenzel J, Cani PD: Responses of gut microbiota and glucose 
and lipid metabolism to prebiotics in genetic obese and diet-induced leptin-resistant mice. Diabetes 
2011;60:2775-2786 
49. Everard A, Belzer C, Geurts L, Ouwerkerk JP, Druart C, Bindels LB, Guiot Y, Derrien M, Muccioli GG, 
Delzenne NM, de Vos WM, Cani PD: Cross-talk between Akkermansia muciniphila and intestinal 
epithelium controls diet-induced obesity. Proc Natl Acad Sci U S A 2013;110:9066-9071 
50. Bekiares N, Krueger CG, Meudt JJ, Shanmuganayagam D, Reed JD: Effect of Sweetened Dried 
Cranberry Consumption on Urinary Proteome and Fecal Microbiome in Healthy Human Subjects. OMICS 
2017; 
51. David LA, Maurice CF, Carmody RN, Gootenberg DB, Button JE, Wolfe BE, Ling AV, Devlin AS, Varma 
Y, Fischbach MA, Biddinger SB, Dutton RJ, Turnbaugh PJ: Diet rapidly and reproducibly alters the human 
gut microbiome. Nature 2014;505:559-563 
52. Ribnicky DM, Roopchand DE, Oren A, Grace M, Poulev A, Lila MA, Havenaar R, Raskin I: Effects of a 
high fat meal matrix and protein complexation on the bioaccessibility of blueberry anthocyanins using 
the TNO gastrointestinal model (TIM-1). Food Chem 2014;142:349-357 
53. Scalbert A, Williamson G: Dietary intake and bioavailability of polyphenols. J Nutr 2000;130:2073S-
2085S 
54. Saura-Calixto F, Serrano J, Goni I: Intake and bioaccessibility of total polyphenols in a whole diet. 
Food Chem 2007;101:492-501