Recipient Organization
NUTRABERRY
2020 MALTBY RD
BOTHELL,WA 98021
Performing Department
(N/A)
Non Technical Summary
Defatted raspberry seeds (DFRS) are rich in phytochemicals which can be metabolized by the gut microbiota into bioactive compounds. These compounds are associated with a wide variety of beneficial health effects that may combat risk of obesity-related chronic disease. Moreover, there may be additive, or even synergistic, effects between these compounds and fiber to enhance the gut microbiota and the microbial production of the bioactive compounds.Nutraberry has developed a method of grinding DFRS to allow incorporation as an ingredient in a variety of foods, thus creating a functional food that may confer health benefits beyond the nutritional value of the food itself. We propose to test the ability of the products from this process to 1) increase the abundance of beneficial gut microbes in human feces and 2) enhance the production of bioactive compounds by the gut microbiota, utilizing the anaerobic chamber at the Nutrition and Gut Microbiome Lab at Washington State University in Spokane. The process will include five test breads including: defatted raspberry seed ground to 5 microns in a refined grain bread, defatted raspberry seed ground to 10 microns in a refined grain bread, coarsely ground defatted raspberry seeds (210 microns) in a refined grain bread, a refined grain bread without defatted raspberry seeds, and a fiber-enriched wholewheat bread. The breads will undergo a model digestion and absorption process before being combined with human fecal samples and fermented in the anaerobic chamber. We will analyze the microbial genes and taxonomy of the fecal samples before and after fermentation to determine what changes took place from fecal fermentation of our experimental breads. In addition, we will determine changes that have occurred in the bioactive compounds of the ground seeds.This work will inform on potential functional nutritional benefits that can be derived from DFRS, and validate the utilization of an upcycled food that reduces production food waste and addresses food security by highlighting an inexpensive, healthy, and pleasant tasting food ingredient.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
The goal of this project is to utilize a food waste byproduct to develop a pleasant tasting functional food ingredient, improving the food's nutritional value and reducing food waste. In this Phase I study, we will investigate the effect of Defatted Raspberry Seed Powder (DRSP) on human gut microbiota and explore the impact of the microbiome on the availability of beneficial polyphenols from the DRSP.Functional foods contain ingredients that confer health benefits beyond the nutritional value of the food. For instance, fruit pomace and seeds are rich in polyphenols and fiber, both of which confer health benefits that address chronic diseases, like those associated with the 73% of the US population with overweight and obesity. Raspberry seeds are abundant in fiber and phenolic compounds, in particular, ellagitannins and ellagic acid, which are metabolized by the gut microbiota into compounds with antioxidant and anti-inflammatory properties, and which have been demonstrated to mitigate risks of various metabolic diseases. However, raspberry seeds are a food waste byproduct from the production of raspberry juice and purees used in products like yogurts, beverages, and energy bars. Nutraberry has developed a proprietary micronized defatted raspberry seed powder (DRSP) rich in the polyphenols, ellagic acid and ellagitannin, which are metabolized by the gut microbiota into urolithins which are associated with a wide variety of beneficial health effects. We hypothesize micronization facilitates the bioavailability of beneficial phenolic compounds and fiber from the seeds to the gut microbiota We will explore possible effects between ellagic acid and fiber that enhance microbial production of urolithins. Micronized defatted raspberry seeds (DFRS) can be incorporated as an ingredient into a variety of foods to create functional foods. We will test the ability of microbes treated with micronized DRSP to 1) increase representation of beneficial human fecal gut microbes, and 2) enhance the production of urolithins.DRSP will be tested in the context of a bread food product. Samples will include DRSP ground to 5um, 15um, non-micronized DRSP (~210um), a refined grain bread control without DRSP, and a fiber-enriched whole wheat bread. Breads will undergo an in vitro digestion and absorption process before being inoculated with fecal samples (n=24) and fermented in the anaerobic chamber. High-throughput sequencing will be used to analyze the DNA and 16S rRNA of the samples before and after fermentation, and bioinformatics used to determine changes in fecal metagenome and the taxonomy of the microbiota. Lastly, we will utilize LC-MS to determine the ellagitannin, ellagic acid, and urolithin content of the test breads before and after fermentation.Objective 1: Prepare microbial and test materials to ensure consistent and standard testing under controlled lab conditions, including generation of fecal microbiome library, optimization of test recipes for bread product testing, and production of defatted raspberry seed powder (DRSP) through a proprietary micronization process.Objective 2: Determine the effect of micronization on the human fecal microbiome, utilizing high throughput DNA sequencing after fermentation in the anaerobic chamber with human fecal samples. We further will examine the effect of micronization on the human gut microbiota utilizing 16S rRNA sequencing to determine changes that take place in the specific taxonomy, α-diversity and β-diversity of human fecal donors in the in vitro model.Objective 3: Assess the effect of micronization on the urolithin content of the fermentate, utilizing HPLC to measure urolithin content of feces pre- and post-fermentation.Our plan involves three elements: 1) To develop a microbial-functional food test system with minimal bioactive compounds that could otherwise confound our results. This includes the generation of a fecal microbiome library that will enhance the microbial diversity to be tested and therefore the probability of identifying a sample with strong responses to the micronized berry seed powder. 2) To conduct in vitro experiments that mimic the digestion, absorption, and fermentation that occur within the human digestive tract, and utilize high throughput DNA and RNA sequencing to correlate microbial consortia with their respective urolithin responses. 3) UPLC-Q-TOF MS to evaluate the effects of our interventions on the gut microbiome/microbiota and the production of urolithins respectively.
Project Methods
Objective 1: To determine the effect of micronization on the human fecal microbiomeTask 1: Develop and optimize standardized bread recipes for fiber, nutrient testing (Carbonero, WSU).Recipes for the test foods will be developed at the WSU Breadlab to determine optimum time and temperatures for fermentation, proofing, and baking using the specified ingredients. DRSP will be added to foods containing refined wheat flour, yeast, vegetable oil, salt, sugar, and water (Table S1) so that the fiber content is 4g/oz (one serving) for the micronized products and 1 g fiber for the control refined grain bread.Task 2: Fecal sample library collection and pre-processing (Carbonero, WSU).We are currently recruiting 24 stool donors to be used in our in vitro model.[AJ1] Whole, fresh feces are collected from qualifying applicants with BMI > 18.5 kg/m2, aged 18-50 and without metabolic or digestive disease. Stool is combined with 1:1 w/v of PBS with pH 7.2 with 5% sulfoxide ulfoxide added as cryoprotectant and mixed in a Stomacher ® 400. Following preprocessing, stool samples are aliquoted into 2 mL cryotubes and frozen at -20°C.Task 3: Micronization of DFRS (Wishnick and MOORE-TEC, Nutraberry).DFRS powder is typically 70 mesh, or 210mm in size before processing. To reach single-digit micron size, 5mm for this study, the powder is passed through SuperFine Vortex Millsand the final particle size confirmed with Beckman Coulter LS 13320 laser diffraction particle size analyzer. We will develop powders with 5mm and 10mm median particle size, respectively.Objective 2: Determine the effect of micronization on the human fecal microbiomeTask 4: Fecal sample library collection and pre-processing (Nutrition and Gut Microbiome Lab/Carbonero, WSU).Digestion and Absorption:Twenty-eight g of each test bread will be suspended and homogenized with 300 mL of nucleic acid free water using a stomacher 400. Following homogenization pH will be reduced to 2.5 with 1 M HCl. To mimic protein and peptide digestion, 10 mL of pepsin (1 g/10 mL in 50 mM HCl, P-7000) will be added to the mixture and incubated for 30 min at 37°C with orbital shaking at 150 rpm. In order to terminate proteolysis, 50 mL of 0.1 M sodium malate buffer (pH 6, containing 1 mM CaCl2) will be added and the pH will be adjusted to 6.9 using 1 M NaHCO3 and 50 mL of pancreatin (6.2b5 g/50 mL in sodium malate buffer; P-7545) To mimic duodenal hydrolysis of oligosaccharides and starch, 2 mL of amyloglucosidase (3,260 U/ml) will be added and incubated for 6 h 37 ?C with orbital shaking at 150 rpm. Following digestion, the material will be dialyzed at 4°C using dialysis tubing with a molecular weight cutoff of 100 -500 Da to remove digested sugars, fatty acids, and amino acids.Fermentation:Fermentation media, human fecal samples (n=24) and dialyzed test foods will be introduced into a type B vinyl anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) which is supplied with a mixed gas composed of 5% CO2 as a buffer, 10% H2, and 85% N2 to maintain [O2] < 20PPM and [H2] 2.5-3.5%. The chamber is equipped with two palladium catalytic plates that convert excess O2 to water in the presence of H2. Temperature will be maintained at 37°C.Prior to fermentation, one liter of fermentation media will be prepared in nucleic acid free water. The mixture is to be hydrated and mixed for 1 h and inoculated with 0.5 mL of fecal slurry in 15 mL minibioreactors incubated at 37°C with continual flow supplied with a 24-channel peristaltic pump (Darwin Microfluidics, France) at 2μL/min.Task 5: Assessment of Microbial genome and taxonomy (Carbonero, WSU).Microbial DNA will be extracted from fecal samples collected and stored during the in vitro experiments (QIAamp DNA Stool Kit, QIAGEN) and DNA quality and quantity will be verified by gel electrophoresis and NanoDrop spectrophotometer (Thermo Scientific™), respectively. The 16S rRNA gene region from the DNA extracts will be amplified by PCR (KAPA Taq PCR Kit) using universal primers tagged with Illumina adapters and barcodes. Amplicon concentration in purified PCR products will be quantified by Qubit Fluorometer (Invitrogen), or by qPCR for more precise measurements, using universal PCR primers and SYBR Green Real-Time PCR Mix (Applied Biosystems). Our partners at the Environmental Molecular Sciences Laboratory at Pacific Northwest National Laboratory will generate sequencing libraries and sequence the samples on an Illumina MiSeq platform.Resulting sequencing files will be analyzed using well validated pipelines. Mothur will be used for initial quality control; removal of adapters, barcodes, and primers; and de-multiplexing of 16S iTag. Chimeric sequences will be detected and deleted. Operational taxonomic units (OTUs) will then be determined and classified against reference databases (SILVA). Basic and multivariate statistical analyses will be performed in JMP (SAS Inc., Cary, NC). Relevant statistical tests will include non-metric multidimensional scaling, PcoA, UniFrac71, diversity indices, indicator species determination, Mann and Whitney, Kruskal-Wallis, and more.Objective 3: Assess the effect of micronization on the urolithin content via HPLCTask 6: UPLC-Q-TOF MS analysis of UrolithinsDr Rodriguez-Mateos, is a Reader at the Department of Nutritional Sciences of King'sCollege London and will be conducing this task. Her research aims to investigate the health benefits of plant foods and phytochemicals, with a strong focus on understanding the bioavailability, metabolism and cardiovascular health benefits of dietary polyphenols. More recent interests include the investigation of the role of the gut microbiome on the health benefits of phytochemicals, and the development of biomarkers of food intake using metabolomic approaches. Her expertise includes development and validation of analytical methods for the analysis of foods and biological samples using UPLC-Q-TOF MS and performance of randomized controlled trials with cardiovascular outcomes.]This should be sourced from a lab, or collection of defined microbiomes: for instance, this is available:https://interestingengineering.com/science/human-gut-microbiome-built-from-scratch