Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Food Science & Human Nutrition
Non Technical Summary
Epidemiological and clinical studies indicate that diets rich in fruits and vegetables are associated with decreased risk for chronic diseases.Polyphenols are compounds found naturally in berries that have demonstrated beneficial health effects, however, their low bioavailability is a limiting factor in their usefulness as a functional food.Among the factors driving the low bioavailability of polyphenols are chemical instability and degradation in the neutral pH environment of the intestines and poor transcellular transport.With respect to berries, a major limitation to research demonstrating their health benefits is the amount that needs to be consumed to achieve those effects. For instance, it has been shown that ~1 cup/day fresh blueberries is needed to observe cardiovascular-protective effects, which may not be feasible for all consumers.Modification of the food product matrix by which berry-derived polyphenolic compounds are delivered to the body can increase their stability and transcellular uptake to increase bioavailability, and presents a novel approach for delivery of berry-derived polyphenols.? Protein-rich foods are an appealing target for enhancing polyphenol bioavailability due to their natural affinity to polyphenols.Increasing consumer demand for non-dairy alternatives for products such as milk, yogurt and protein powders has launched a market for plant-based protein sources valued at $18.5 billion in 2019 and projected to reach $40.6 billion by 2025.19 Despite their increasing share of the global food supply, the functional capabilities of plant proteins are understudied. The goal of this proposal is to delineate structural and functional characteristics of proteins from novel, plant-based sources and investigate their impact on the delivery and bioavailability of blueberry polyphenols when combined in a food matrix.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overarching goal of this project is to characterize the effects of novel, plant-based protein rich matrices on the stability, delivery and bioavailability of dietary polyphenols. This will be investigated by defining physicochemical interactions between blueberry polyphenols and protein isolates from peas and hemp in comparison to whey protein isolate. The effects of these interactions on polyphenol absorption and bioavailability will be assessed in vitro and in vivo. The central hypothesis is that complexation with plant proteins within the context of a food matrix will increase the stability and bioavailability of blueberry polyphenols. We will approach this hypothesis by pursing the following aims:Aim 1: Characterize interactions between blueberry polyphenols and protein isolates in a food matrix.Aim 2: Elucidate matrix effects of protein complexation on the digestion and delivery of blueberry polyphenols in vitro.Aim 3: Assess the influence of protein source on bioavailability of blueberry metabolites in vivo.
Project Methods
Specific Aim 1: Characterize interactions between blueberry polyphenols and protein isolates from dairy- and plant-based sources in a food matrix.SubTask 1.1Calculate binding affinities for protein-polyphenol interactions. Binding affinities for interactions between protein isolates and blueberry polyphenols will be calculated using fluorescence spectroscopy based on the quenching of the intrinsic fluorescence of tryptophan in each protein. The fluorescence quenching rate constant (kq) can be calculated using the Stern-Volmer equation, F0/F = 1+ KSV x [Q], where F0/F are fluorescence intensities of the protein isolate before and after the addition of BPE ([Q]) and KSV is the Stern-Volmer constant. After taking into account any fluorescence from BPE, KSV can be used to calculate the fluorescence quenching rate constant (kq) for each protein isolate based on the fluorescence lifetime of tryptophan (t0) using the equation KSV = kq x t0. Double log transformation of the Stern-Volmer plot will provide a binding constant for each protein-BPE complex.It is expected that a protein having a high affinity for polyphenol binding and thus, enhanced polyphenol delivery capabilities.SubTask 1.2Determine the influence of protein structure on polyphenolic interaction. Circular dichroism (CD) will be used to characterize the relative frequency of secondary structures within each protein isolate studied before and after interaction with blueberry polyphenols. A reference spectrum of each protein isolate will be prepared by analyzing the far-UV region of the CD spectrum on a Jasco J-1100 CD spectropolarimeter. The approximate frequencies of a-helices, b-sheets, b-turns and random coils will be determined based on the shape of the spectrum, yielding average signals for each protein, quantified by Spectra Manager CD processing software. The average signal will be compared to the signals recorded upon titration of BPE, providing information about protein regions modified by polyphenolic binding. Used in conjunction with the findings from SubTasks 1.1, information regarding the structure of each protein at neutral pH in the presence and absence of BPE will help underpin the reasoning for any observed differences in precipitation and binding affinity for each protein.Specific Aim 2: Elucidate the matrix effects of protein adsorption on digestion and delivery of blueberry polyphenols in vitro.SubTask 2.1Assess the stability of protein-polyphenol complexes throughout in vitro digestion. Polyphenol stability and bioaccessibility will be measured throughout the course of an in vitro digestion process by quantifying free versus bound polyphenols during each step of the digestion, and protein-BPE complex digestibility will be monitored using SDS-PAGE. In brief, 10 mL of the protein-BPE beverage will be diluted into 5 mL of 0.9% saline before acidification with hydrochloric acid and introduction to pepsin for gastric digestion. Gastric digestion will be carried out for 1 h at 37°C. The pH of the sample will then be raised to 6.8 with sodium carbonate to simulate the small intestine. Pancreatin will be added prior to another incubation for 2 h at 37°C.Aliquots will be drawn from each sample prior to in vitro digestion and hourly thereafter, then centrifuged to separate insoluble complexes from solution. The free polyphenol content of the supernatant will be analyzed using the Folin-Ciocalteu assay.Bound polyphenols will be measured after extraction of the pellet using an aqueous acetone/ethanol solution and similarly analyzed using the Folin-Ciocalteu assay. Aliquots from the complete in vitro digestion will be stored at -80°C and used in SubTask 2.2 to measure small intestinal transport.Subtask 2.2Evaluate the impact of protein complexation on transepithelial transport and uptake of polyphenols in vitro. To model the human small intestine, Caco-2 cells will be cultured on a semipermeable transwell and grown for 21-28 days into a confluent, polarized monolayer, the integrity of which will be verified using a volt-ohm meter. The transwell system allows cells to fully differentiate to form apical and basolateral domains with functional tight junctions and brush border enzymes, creating a simple, yet robust model for studying bioavailability in vitro. Digested protein-BPE aliquots from Subtask 2.1 will be added to the apical compartment of each transwell system and incubated at 37°C for 4 h. A single aliquot of the sample added to the apical chamber will be stored for analysis, and aliquots will be collected from the basolateral chamber after 30, 60, 120 and 240 min. These aliquots, demonstrating transepithelial transport, will be stored at -80°C until analysis by RP-HPLC. The concentration of total polyphenols at each time point will be used to calculate the apparent permeability coefficient of each of the protein-BPE complexes using the equation Papp = DQ/(Dt*A*C0), where DQ/Dt is the flux of polyphenols across the monolayer, A is the transwell membrane surface area and C0 is the concentration of polyphenols (free and bound) introduced to the apical compartment at time 0 h.This value will allow for direct comparison of the influence of protein source on cellular uptake of polyphenols in vitro.Specific Aim 3: Assess the influence of protein source on bioavailability of blueberry metabolites in vivo.SubTask 3.1Perform bioavailability study in humans. A randomized, double-blind, controlled, crossover study will be performed. Treatments will consist of a single dose of blueberry polyphenols (280 mg) in the presence or absence of 15 g of protein isolate in beverage form (BPE ± whey, pea, or hemp protein). Twenty healthy males and females > 18 years of age will be recruited from the Fort Collins, CO area. After telephone prescreening, participants will attend a screening visit where they will receive verbal and written explanation of the project, provide informed consent, and undergo a health assessment to confirm they meet inclusion or exclusion criteria. Individuals with HbA1c < 6.4%, blood pressure < 140/90 mm Hg, total cholesterol < 240 mg/dL, LDL cholesterol < 90 mg/dL, triglycerides < 350 mg/dL and a body mass index between 18.5 and 29.9 kg/m2 will be included. Individuals taking blood pressure-, lipid- or glucose-lowering or hormone replacement medications, chronic diseases, > 3 days/wk vigorous exercise, weight change > 5% in the past 3 months, history of smoking in the past 12 months, heavy drinkers (>7 drinks/wk for women; >14 drinks/wk for men), or unwillingness to maintain usual diet/physical activity will be excluded. Participants will be asked to not consume high-polyphenolic foods or beverages such as berries or tea for 7 days prior to testing. On testing days, fasted participants will consume 250 mL of a beverage prepared with or without 15 g protein from isolates with matched fiber and 280 mg blueberry polyphenols.Blood samples will be collected through an intravenous catheter at baseline (t = 0 h) and 1, 2, 4, 6, 8 and 24h. A low-protein, low-polyphenol meal will be provided at the 2 h time point after blood collection. Test days will be separated by a 1-week washout period.SubTask 3.2Determine systemic bioavailability of blueberry polyphenols in plasma. Targeted metabolomics will be used to assess bioavailability and biotransformation of blueberry polyphenols upon delivery in conjunction with each protein isolate (see table). In short, metabolites will be extracted from plasma using C18 solid phase extraction cartridges eluted with formic acid in both methanol and acetone, respectively. The extract will then be dried under N2 and resuspended at the time of analysis.Qualitative analysis of phenolic metabolites will be performed by UHPLC-QTOF, while quantitation of each analyte will be done via HPLC-QQQ. Quantitation will be carried out using standard curves of commercial compounds.