The Effect of Concord Grape Polyphenol-soy Protein Isolate Complex (GP-SPI) on Gut Microbiota
Condition: Mechanisms, Defense
Study type: Interventional

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.
    

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 (food4microbes@sebs.rutgers.edu). 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.
    

Intervention name(s): GP-SPI
Intervention type(s): Dietary Supplement
Intervention description(s): grape polyphenol-soy protein isolate complex (GP-SPI)
Intervention model: Single Group Assignment
Intervention model description: Longitudinal study
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: gut microbiome; metabolome; dietary polyphenols; grape; nutrition