Antioxidant-rich Aronia Supplementation Impacts Human Metabolism and Immune Response as Well as Gut Microbiome Metabolism

To meet these objectives, a randomized, double-blind, placebo-controlled clinical trial of Aronia versus placebo treatment for 28-30 days in human adults will be conducted. Pre- and post-intervention assessments will be made for the following variables: makeup of the gut microbiome (microbial species and relative abundance), gut metabolome, postprandial response of TG, inflammatory cytokines, and serum metabolome to a high-fat meal challenge (established inflammation stimulus), fasting serum glucose, lipid, insulin, inflammation markers and metabolome, blood pressure, and anthropometric measures including weight, body composition, waist circumference, and quantity of visceral adipose tissue. Physical activity, sedentary behavior, and habitual diet will be measured so that these variables can be used to characterize participants and aid in analysis and interpretation of data.

Procedures:

Postprandial lipidemic and inflammation responses: High-fat meal challenges with 40 to 100 g of dietary fat are an established laboratory test to measure both postprandial triglyceridemic and inflammation responses. Investigators have used a 50 g dose of fat delivered in the form of butter on toast on > 50 individuals because this particular dose is effective at discriminating between low versus high TG and inflammation responders. In brief, participants will report to the laboratory after an overnight fast, and blood samples will be collected before, and 1, 2, 4, and 6 hours following ingestion of the high-fat meal. Samples will be analyzed in real time for TG (and full lipid panel plus glucose) using a clinical chemistry analyzer (Piccolo xpress), while serum samples will be aliquoted and stored at -80 C until analysis for inflammatory cytokines, metabolomics, and insulin. Investigators will measure inflammatory cytokines (TNF-α, interleukin(IL)-1β, IL-6 IL-17, IL-23, and granulocyte macrophage colony stimulating factor (GM-CSF)) using high-sensitivity Luminex multiplexing technology (Bio-RadBio-Plex® 200 HTS) prepared by Millipore.

Dietary intervention: Participants will be randomized to either experimental (Aronia) or placebo-matched control group. The experimental supplement will consist of a once daily dose of 100 mL of Aronia juice. The placebo-matched control supplement will have no polyphenol content and will consist of 100 mL of the following mixture: black cherry Koolaid, blue and red food coloring, sucrose and sorbitol. This placebo will match the sugar content of the chokeberry juice. The daily dose of 100 mL for both groups is consumed once daily for duration of 28-30 day supplementation period. All participants will be instructed to avoid consumption of foods with polyphenolic content for the duration of the supplementation period. A list of disallowed foods will be provided for participants to reference.

Gut microbiome analysis: Bulk DNA will be extracted from fecal samples using the Powersoil® DNA Isolation Kit (Mo Bio Laboratories Inc.). DNA will be shipped overnight to the University of Michigan, Center for Microbial Systems, for Illumina MiSeq amplicon sequencing of the 16S V4 variable region. Raw sequencing reads will be processed and curated using the mothur (v.1.39.5) software package, following the mothur MiSeq standard operating procedure, potentially chimeric sequences will be identified and removed using the Uchime (v4.2.40) algorithm, and taxonomic classifications will be assigned using the Bayesian classifier of the Ribosomal Database Project, and operational taxonomic units (OTUs) will be assigned in mothur using the VSEARCH distance-based clustering algorithm at the 97% sequence similarity threshold.