The Effect of Concord Grape Polyphenol-soy Protein Isolate Complex (GP-SPI) on Gut Microbiota

• Sponsor: Rutgers, The State University of New Jersey (Other)
• Overall Status: Completed
• CT.gov ID: NCT04018066
• Collaborator: (none)
• Enrollment: 34
• Location: 1
• Arm: 1
• Actual Duration (Months): 5.7
• Patients Per Site Per Month: 5.9

Study Details

Study Description

Brief Summary

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.

Condition or Disease	Intervention/Treatment	Phase
Mechanisms, Defense	Dietary Supplement: GP-SPI	N/A

Detailed Description

The proposed study will investigate how short-term supplementation with GP-SPI may modify the gut microbial community in healthy participants while monitoring liver and kidney function/health.

Significance:

Biochemical properties of the GP-SPI food ingredient are well-documented and GP-SPI and SPI supplements have been tested extensively in mice. 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. These effects were accompanied by changes in murine gut microbiota composition, including increased abundance of the microbe Akkermansia muciniphila, associated with metabolic resilience. Similar gut microbiota changes were observed in lean mice fed low-fat diet (LFD) supplemented with GP-SPI.

The B-type proanthocyanidin (PAC) class of polyphenols contained in grape berries, especially skins and seeds, have been associated with health benefits; however, PACs are poorly absorbed and reach high concentration only in the colon raising questions about mechanism(s) of action. Prior studies showed that dietary PACs from grape and cranberry alter the gut microbiota in association with metabolic resilience. PACs are also biotransformed by gut bacteria to yield microbial metabolites (MMs) that may contribute to health benefits.

Research Design and Methods

Prospective participants will be recruited through flyers posted locally on Rutgers University campuses and sent to university email lists. Interested persons will be screened according to inclusion and exclusion criteria.

Enrolled subjects will begin the 17-day study.

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. They will have a blood sample 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.

• 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 created for this study ([e-mail]). 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 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. Fecal samples will be aliquoted and stored at -80 °C until extraction. Collection of fecal samples in 50% glycerol and immediate 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. 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 laboratory to maintain metabolite stability. Subjects will be asked to bring their samples to the 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 have a final blood sample drawn. 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.

Samples will be used for 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, fecal, 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. These bacteria-metabolite associations will then be used to infer cause-effect relationships that can be further tested in vitro and in mouse models. 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.

Study Design

• Study Type: Interventional
• Actual Enrollment: 34 participants
• Allocation: N/A
• Intervention Model: Single Group Assignment
• Intervention Model Description: Longitudinal studyLongitudinal study
• Masking: None (Open Label)
• Primary Purpose: Basic Science
• Official Title: The Effect of Concord Grape Polyphenol-soy Protein Isolate Complex (GP-SPI) on Gut Microbiota
• Actual Study Start Date: Jun 28, 2019
• Actual Primary Completion Date: Dec 20, 2019
• Actual Study Completion Date: Dec 20, 2019

Arms and Interventions

Arm	Intervention/Treatment
Experimental: GP-SPI intervention 20 g of GP-SPI taken twice per day for 10 days	Dietary Supplement: GP-SPI; grape polyphenol-soy protein isolate complex (GP-SPI)

Outcome Measures

Primary Outcome Measures

1. Gut Microbiota Composition [16S rRNA amplicon sequencing will be performed on samples collected at baseline (before intervention, day -5), after 5 days of SPI supplementation (day 0), and after 10 days of GP-SPI supplementation (day 10). Total time frame is 17 days.]

   Evaluate the effect of nutritional supplementation with GP-SPI on gut microbiota composition by 16S rRNA amplicon sequencing and analysis

Secondary Outcome Measures

1. Comprehensive Metabolic Panel (CMP) Blood Test (ALP, AST, ALT) [Samples will be analyzed within one week of blood collection]

   Evaluate the effect of GP-SPI on kidney and liver health/function via CMP blood tests ALP, AST, ALT

2. Comprehensive Metabolic Panel (CMP) Blood Test (Glucose, Blood Urea Nitrogen, Creatine, Calcium, Bilirubin Total) [Samples will be analyzed within one week of blood collection]

   Evaluate the effect of GP-SPI on kidney and liver health/function via CMP blood tests Measure glucose, blood urea nitrogen, creatine, calcium, bilirubin total

3. Comprehensive Metabolic Panel (CMP) Blood Test (Anion Gap) [Samples will be analyzed within one week of blood collection]

   Anion gap was measured for all 27 participants

4. Comprehensive Metabolic Panel (CMP) Blood Test (Sodium, Potassium, Chloride, Total Carbon Dioxide) [Samples will be analyzed within one week of blood collection]

   CMP data was measured for all 27 participants (sodium, potassium, chloride, total carbon dioxide)

5. Comprehensive Metabolic Panel (CMP) Blood Test (Protein, Albumin) [Samples will be analyzed within one week of blood collection]

   Evaluate the effect of GP-SPI on kidney and liver health/function via CMP blood tests

6. Microbiome-wide Association Study [De-identified samples will be processed and analyzed after all subjects complete the study and within 1 year.]

   Collect fecal, blood, and urine samples for a microbiome-wide association study (MWAS)

7. Data Sharing [Data will be deposited no later than within 1 year of the completion of the funded project period for the parent award or upon acceptance of the data for publication, or public disclosure of a submitted patent application]

   Enter de-identified study results into microbiome and metabolome nationwide data sharing database

Eligibility Criteria

Criteria

• Ages Eligible for Study: 18 Years to 35 Years
• Sexes Eligible for Study: All
• Accepts Healthy Volunteers: Yes

Inclusion Criteria:

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.)

8. Treated with antibiotics during the past 6 months

9. Have an allergy to soy or grapes

Contacts and Locations

Locations

	Site	City	State	Country	Postal Code
1	Rutgers, The State University of New Jersey	New Brunswick	New Jersey	United States	08901

Sponsors and Collaborators

• Rutgers, The State University of New Jersey

Investigators

• Principal Investigator: Diana Roopchand, PhD, Rutgers University

Study Documents (Full-Text)

• Study Protocol and Statistical Analysis Plan - Jul 5, 2019
• Informed Consent Form - Jul 5, 2019

More Information

Publications

None provided.

• Responsible Party: Diana Roopchand, Ph.D., Assistant Professor, Rutgers, The State University of New Jersey
• ClinicalTrials.gov Identifier: NCT04018066
• Other Study ID Numbers: Pro2018002579
• First Posted: Jul 12, 2019
• Last Update Posted: Nov 10, 2021
• Last Verified: Oct 1, 2021

• Individual Participant Data (IPD) Sharing Statement: No
• Plan to Share IPD: No
• Studies a U.S. FDA-regulated Drug Product: No
• Studies a U.S. FDA-regulated Device Product: No
• Keywords provided by Diana Roopchand, Ph.D., Assistant Professor, Rutgers, The State University of New Jersey: gut microbiome; metabolome; dietary polyphenols; grape; nutrition

Study Results

Participant Flow

Recruitment Details	34 participants were enrolled in the study based on inclusion/exclusion criteria. 4 participants withdrawn from the study, due to the following reasons: 3 started taking medications and 1 had scheduling conflicts. Of the remaining 30 participants that completed the study, 3 were excluded due to protocol deviations.

Arm/Group Title	GP-SPI Intervention
Arm/Group Description	Each participant consumed 20 g of soy protein isolate (SPI) twice per day for 5 days. After a one day break, participants then consumed 20 g of GP-SPI taken twice per day for 10 days. During the entire 17 day study period, participants abstained from an extensive list of PAC-rich foods. GP-SPI: grape polyphenol-soy protein isolate complex (GP-SPI)
Period Title: Overall Study
STARTED	34
COMPLETED	30
NOT COMPLETED	4

Baseline Characteristics

Arm/Group Title	GP-SPI Intervention
Arm/Group Description	Each participant consumed 20 g of soy protein isolate (SPI) twice per day for 5 days. After a one day break, participants then consumed 20 g of GP-SPI taken twice per day for 10 days. During the entire 17 day study period, participants abstained from an extensive list of PAC-rich foods. GP-SPI: grape polyphenol-soy protein isolate complex (GP-SPI)
Overall Participants	27
Age, Customized (years) [Mean (Standard Deviation) ]
Age	21.7 (3.5)
Sex: Female, Male (Count of Participants)
Female	16 59.3%
Male	11 40.7%
Ethnicity (NIH/OMB) (Count of Participants)
Hispanic or Latino	4 14.8%
Not Hispanic or Latino	22 81.5%
Unknown or Not Reported	1 3.7%
Race (NIH/OMB) (Count of Participants)
American Indian or Alaska Native	0 0%
Asian	15 55.6%
Native Hawaiian or Other Pacific Islander	0 0%
Black or African American	3 11.1%
White	5 18.5%
More than one race	1 3.7%
Unknown or Not Reported	3 11.1%
Region of Enrollment (participants) [Number]
United States	27 100%
Glucose (mg/dL) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mg/dL]	88.78 (6.25)
Blood urea nitrogen (mg/dL) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mg/dL]	12.81 (4.3)
Creatine (mg/dL) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mg/dL]	0.76 (0.19)
Calcium (mg/dL) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mg/dL]	9.82 (0.38)
Sodium (mmol/L) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mmol/L]	142.7 (2.28)
Potassium (mmol/L) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mmol/L]	4.83 (0.34)
Chloride (mmol/L) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mmol/L]	103.4 (2.8)
Total carbon dioxide (mmol/L) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mmol/L]	26.04 (1.72)
Anion gap (meq/L) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [meq/L]	13.26 (2.19)
Protein (g/dL) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [g/dL]	7.38 (0.37)
Albumin (g/dL) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [g/dL]	4.83 (0.34)
Bilirubin total (mg/dL) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mg/dL]	0.54 (0.26)
Alkaline phosphatase (IU/L) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [IU/L]	68.56 (15.19)
Alkaline transaminase (IU/L) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [IU/L]	21.26 (5.4)
Alanine transaminase (IU/L) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [IU/L]	16.89 (7.44)

Outcome Measures

1. Primary Outcome

Title	Gut Microbiota Composition
Description	Evaluate the effect of nutritional supplementation with GP-SPI on gut microbiota composition by 16S rRNA amplicon sequencing and analysis
Time Frame	16S rRNA amplicon sequencing will be performed on samples collected at baseline (before intervention, day -5), after 5 days of SPI supplementation (day 0), and after 10 days of GP-SPI supplementation (day 10). Total time frame is 17 days.

Outcome Measure Data

Analysis Population Description

All 27 participant samples were analyzed.

Arm/Group Title	GP-SPI Intervention
Arm/Group Description	Each participant consumed 20 g of soy protein isolate (SPI) twice per day for 5 days. After a one day break, participants then consumed 20 g of GP-SPI taken twice per day for 10 days. During the entire 17 day study period, participants abstained from an extensive list of PAC-rich foods. GP-SPI: grape polyphenol-soy protein isolate complex (GP-SPI)
Measure Participants	27
Firmicutes (day -5), % relative abundance	58 (10)
Bacteroidetes (day -5), % relative abundance	30 (9.7)
Verrucomicrobia (day -5), % relative abundance	0.04 (0.1)
Actinobacteria (day -5), % relative abundance	7.3 (5.3)
Proteobacteria (day -5), % relative abundance	4 (2.9)
Tenericutes (day -5), % relative abundance	0.4 (0.1)
Firmicutes (day 0), % relative abundance	59.3 (6.9)
Bacteroidetes (day 0), % relative abundance	31.1 (8.06)
Verrucomicrobia (day 0), % relative abundance	0.04 (0.09)
Actinobacteria (day 0), % relative abundance	5.29 (3.7)
Proteobacteria (day 0), % relative abundance	3.66 (2.66)
Tenericutes (day 0), % relative abundance	0.009 (0.05)
Firmicutes (day 10), % relative abundance	54.3 (8.9)
Bacteroidetes (day 10), % relative abundance	33.44 (10.4)
Verrucomicrobia (day 10), % relative abundance	0.07 (0.13)
Actinobacteria (day 10), % relative abundance	8.1 (5.6)
Proteobacteria (day 10), % relative abundance	4.1 (2.6)
Tenericutes (day 10), % relative abundance	0.03 (0.1)

2. Secondary Outcome

Title	Comprehensive Metabolic Panel (CMP) Blood Test (ALP, AST, ALT)
Description	Evaluate the effect of GP-SPI on kidney and liver health/function via CMP blood tests ALP, AST, ALT
Time Frame	Samples will be analyzed within one week of blood collection

Outcome Measure Data

Analysis Population Description

CMP data was measured for all 27 participants

Arm/Group Title	GP-SPI Intervention
Arm/Group Description	20 g of GP-SPI taken twice per day for 10 days GP-SPI: grape polyphenol-soy protein isolate complex (GP-SPI)
Measure Participants	27
Alkaline phosphatase	70.7 (16.79)
Aspartate transaminase (IU/L)	21.41 (5.6)
Alkaline transaminase	17.52 (8.59)

3. Secondary Outcome

Title	Comprehensive Metabolic Panel (CMP) Blood Test (Glucose, Blood Urea Nitrogen, Creatine, Calcium, Bilirubin Total)
Description	Evaluate the effect of GP-SPI on kidney and liver health/function via CMP blood tests Measure glucose, blood urea nitrogen, creatine, calcium, bilirubin total
Time Frame	Samples will be analyzed within one week of blood collection

Outcome Measure Data

Analysis Population Description

CMP data was measured for all 27 participants

Arm/Group Title	GP-SPI Intervention
Arm/Group Description	20 g of GP-SPI taken twice per day for 10 days GP-SPI: grape polyphenol-soy protein isolate complex (GP-SPI)
Measure Participants	27
Glucose	85.96 (6.79)
Blood urea nitrogen	14.81 (3.28)
Creatine	0.76 (0.18)
Calcium	9.64 (0.53)
Bilirubin total	0.54 (0.25)

4. Secondary Outcome

Title	Comprehensive Metabolic Panel (CMP) Blood Test (Anion Gap)
Description	Anion gap was measured for all 27 participants
Time Frame	Samples will be analyzed within one week of blood collection

Outcome Measure Data

Analysis Population Description

CMP data was measured for all 27 participants

Arm/Group Title	GP-SPI Intervention
Arm/Group Description	20 g of GP-SPI taken twice per day for 10 days GP-SPI: grape polyphenol-soy protein isolate complex (GP-SPI)
Measure Participants	27
Mean (Standard Deviation) [meq/L]	14.41 (1.9)

5. Secondary Outcome

Title	Comprehensive Metabolic Panel (CMP) Blood Test (Sodium, Potassium, Chloride, Total Carbon Dioxide)
Description	CMP data was measured for all 27 participants (sodium, potassium, chloride, total carbon dioxide)
Time Frame	Samples will be analyzed within one week of blood collection

Outcome Measure Data

Analysis Population Description

CMP data was measured for all 27 participants

Arm/Group Title	GP-SPI Intervention
Arm/Group Description	20 g of GP-SPI taken twice per day for 10 days GP-SPI: grape polyphenol-soy protein isolate complex (GP-SPI)
Measure Participants	27
Sodium	142.2 (2.33)
Potassium	4.57 (0.43)
Chloride	102.3 (1.7)
Total carbon dioxide	25.51 (1.69)

6. Secondary Outcome

Title	Comprehensive Metabolic Panel (CMP) Blood Test (Protein, Albumin)
Description	Evaluate the effect of GP-SPI on kidney and liver health/function via CMP blood tests
Time Frame	Samples will be analyzed within one week of blood collection

Outcome Measure Data

Analysis Population Description

CMP data was measured for all 27 participants

Arm/Group Title	GP-SPI Intervention
Arm/Group Description	20 g of GP-SPI taken twice per day for 10 days GP-SPI: grape polyphenol-soy protein isolate complex (GP-SPI)
Measure Participants	27
Protein	7.37 (0.33)
Albumin	4.87 (0.29)

7. Secondary Outcome

Title	Microbiome-wide Association Study
Description	Collect fecal, blood, and urine samples for a microbiome-wide association study (MWAS)
Time Frame	De-identified samples will be processed and analyzed after all subjects complete the study and within 1 year.

Outcome Measure Data

Analysis Population Description

[Not Specified]

Arm/Group Title

Arm/Group Description

8. Secondary Outcome

Title	Data Sharing
Description	Enter de-identified study results into microbiome and metabolome nationwide data sharing database
Time Frame	Data will be deposited no later than within 1 year of the completion of the funded project period for the parent award or upon acceptance of the data for publication, or public disclosure of a submitted patent application

Outcome Measure Data

Analysis Population Description

[Not Specified]

Arm/Group Title

Arm/Group Description

Adverse Events

Time Frame	20 days
Adverse Event Reporting Description	An adverse event (AE) is any untoward medical occurrence in a subject during participation in the clinical study or with use of the experimental agent being studied. An adverse finding can include a sign, symptom, abnormal assessment (laboratory test value, vital signs, electrocardiogram finding, etc.), or any combination of these regardless of relationship to participation in the study.
Arm/Group Title	GP-SPI Intervention
Arm/Group Description	Each participant consumed 20 g of soy protein isolate (SPI) twice per day for 5 days. After a one day break, participants then consumed 20 g of GP-SPI taken twice per day for 10 days. During the entire 17 day study period, participants abstained from an extensive list of PAC-rich foods. GP-SPI: grape polyphenol-soy protein isolate complex (GP-SPI)
All Cause Mortality
	GP-SPI Intervention
	Affected / at Risk (%)	# Events
Total	0/34 (0%)
Serious Adverse Events
	GP-SPI Intervention
	Affected / at Risk (%)	# Events
Total	0/34 (0%)
Other (Not Including Serious) Adverse Events
	GP-SPI Intervention
	Affected / at Risk (%)	# Events
Total	0/34 (0%)

Limitations/Caveats

[Not Specified]

More Information

Certain Agreements

Principal Investigators are NOT employed by the organization sponsoring the study.

There is NOT an agreement between Principal Investigators and the Sponsor (or its agents) that restricts the PI's rights to discuss or publish trial results after the trial is completed.