Source: NUTRABERRY submitted to NRP
ANALYZING NUTRITIONAL BENEFITS OF MICRONIZED DEFATTED BERRY SEED POWDERS USING AN IN-VITRO MODEL OF THE HUMAN GUT MICROBIOME
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1030138
Grant No.
2023-51402-39332
Cumulative Award Amt.
$175,000.00
Proposal No.
2023-01137
Multistate No.
(N/A)
Project Start Date
Jul 1, 2023
Project End Date
Jun 30, 2025
Grant Year
2023
Program Code
[8.5]- Food Science & Nutrition
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%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
4031123101040%
5011129101030%
5031123101010%
6011129101010%
6041120101010%
Knowledge Area
601 - Economics of Agricultural Production and Farm Management; 403 - Waste Disposal, Recycling, and Reuse; 501 - New and Improved Food Processing Technologies; 604 - Marketing and Distribution Practices; 503 - Quality Maintenance in Storing and Marketing Food Products;

Subject Of Investigation
1120 - Blueberry; 1129 - Berries and cane fruits, general/other; 1123 - Raspberry;

Field Of Science
1010 - Nutrition and metabolism;
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

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:Nutraberry is a business-to-business provider of upcycled food ingredients targeting food manufacturers, R&D teams, and consumer goods companies prioritizing health, sustainability, and equity in food systems. Our goal is to provide nutritious, affordable ingredients--like micronized defatted raspberry seed powder (DRSP)--to partners committed to diversity and environmentally responsible sourcing. DRSP transforms an agricultural byproduct into a functional ingredient with applications in baked goods, smoothies, chocolate, and other food formats. During the reporting period, we engaged with innovation and procurement teams at leading companies, including Nestlé, Mars Inc., Mondel?z International, and King Arthur Baking Company (KABC). At Nestlé, we worked with David Despain, MSc/MSc, CFS, Senior Principal Nutritionist in Global Nutrition, Technology & Innovation at the Nestlé Product Technology Center. David is exploring the feasibility of incorporating DRSP into a smoothie product currently in early-stage development. Results from this USDA-supported study will help inform DRSP's role in microbiome modulation and urolithin production. At KABC, we collaborated with Sue Gray (She/Her), Director of Research and Development, who led the formulation and baking of biscuits used in our fermentation experiments. These were shipped to Dr. Franck Carbonero at Washington State University for use in in-vitro microbiome modeling. As a 100% employee-owned company with a focus on ingredient transparency and sustainability, KABC's mission closely aligns with Nutraberry's values. They also operate the King Arthur Baking School at the Bread Lab, a WSU Extension site in Burlington, WA. Nutraberry showcased DRSP through the Western United States Agricultural Trade Association (WUSATA) at major industry events, including IFT Expo, SupplySide West, and the Winter Fancy Food Show. At IFIA Japan, our product was featured in WUSATA's 2024 Recipe Development and Ingredient Demo, resulting in contact with 39 organizations expressing interest in DRSP. In our outreach to Mondel?z International, we sent 5, 10, and 15 micron DRSP samples with full particle size distribution data to Clive Norton, Director of Ingredient Research. This led to follow-up engagement with Kieran Spelman, Open Innovation Leader, who requested pricing. We are actively pursuing next steps toward a pilot project. Nutraberry is also a finalist for the Mondel?z CoLab Tech Accelerator, which supports innovative food technologies in the areas of fiber, sustainability, and snacking. Samples were also shared with Will Borchert, Scientist, Product Science, at Mars Wrigley Global Innovation Center in Chicago, IL. While that engagement has not yet progressed, Mars's focus on fiber and microbiome health aligns closely with the goals of our Phase I study. Nutraberry also received a purchase order from Salt & Straw, a nationally recognized artisan ice cream company known for its Upcycled Food menu. The order was for 136 lbs of 5-micron defatted raspberry seed powder, intended for use in a seasonal offering. Due to funding gaps and production limitations at the time, we were unable to fulfill the order. While there has been no recent communication, we remain hopeful about re-engaging with Salt & Straw, whose initial interest highlighted strong commercial demand for upcycled, functional ingredients. Lastly, Nutraberry was selected as a finalist for the HudsonAlpha AgTech Investment Accelerator in Huntsville, Alabama--the largest peanut-producing state in the U.S. HudsonAlpha offers $100,000 in funding and a two-month residency to advance ag-based innovations. This opportunity supports our plan to expand USDA-funded work intomicronized peanut skins, inspired by USDA-ARS research inPeanut Skins: More than Meets the Eye. This next-generation ingredient offers high potential for antioxidant, fiber, and microbiome-supportive functionality Changes/Problems:To generate a more pronounced microbiome response in our Phase I study, we transitioned from bread formulations to a simplified biscuit format. While this reduced immediate commercial relevance, it aligned with the scientific objective of maximizing detectable microbiome shifts during in vitro fermentation. King Arthur Baking Company (KABC) advised that higher inclusion levels of defatted raspberry seed powder (DRSP) affect gluten structure and require reformulation. Specifically, they noted that phenolic compounds--such as ellagic acid in DRSP--interact with gluten, impairing cell structure. Their testing suggested that commercial bread formulations would be limited to ~5% DRSP by flour weight without reformulation. To enable side-by-side comparison across fiber formats, we developed biscuits using: Refined wheat flour + 5 µm DRSP (~4g fiber/serving) Refined wheat flour + 15 µm DRSP (~4g fiber/serving) Refined wheat flour + 70 mesh DRSP (~4g fiber/serving) Whole wheat flour + cellulose (~4g fiber/serving) Refined flour control (no added fiber) These were used in fermentation assays at WSU. Biscuits provided ~24-50 µg of ellagic acid per serving, with polyphenol and fiber values confirmed by third-party labs. Although REEport does not support embedded images, we are providing visual documentation through the following publicly accessible Google Drive resources: Photos of Dough Behavior and Baked Biscuits: https://docs.google.com/document/d/1vCQ5rYM463fEccdxlys8IXd-pXC9NWXw/edit?usp=drive_link Full USDA Phase I Project Folder (includes fiber density tables, COAs, and visuals): https://drive.google.com/drive/folders/18-wWJoxARZFIAuKuASS_n4uY_OzPHbmX?usp=drive_link These comparative images, captured by King Arthur Baking Company, illustrate functional differences between 5 µm and 70 mesh DRSP at identical load levels (~13% of dry mix). Texture, structure, and water absorption varied significantly, informing future product development for Phase II. What opportunities for training and professional development has the project provided?Nutraberry leveraged this USDA-supported project to pursue a wide range of professional development opportunities that advanced our technical, commercial, and strategic competencies. As the lead implementer, Nutraberry received the majority of training benefits. In preparing for and participating in key trade shows and industry events, we developed essential skills in selling to R&D teams, navigating food innovation ecosystems, and adapting technical communication to commercial audiences. Events like Life Science Innovation Northwest, IFT First Look, and regional IFT Suppliers Expos challenged us to articulate the functional value of DRSP (defatted raspberry seed powder) to procurement and innovation teams in real time. Importantly, we learned directly from interested buyers and distribution partners. When companies were intrigued, they told us exactly how to present our ingredient in a way that met their internal criteria. This iterative feedback helped us refine our messaging, application focus, and documentation--especially with formulation support, certificates of analysis, and nutritional claims. Conversations with partners like B&D Nutritional Ingredients and DKSH gave us critical insight into how DRSP fits into functional food portfolios, including which applications (e.g., bakery, smoothies, bars) offer the best product-market fit, and what technical and regulatory data are needed to support purchase decisions. In addition, Nutraberry was selected by WUSATA to participate in global programming like IFIA Japan and the Winter Fancy Food Show, where we developed and demonstrated DRSP-based recipes. These opportunities built our capacity in international marketing, live product demos, and cross-cultural ingredient education. Each event served as hands-on training--deepening our expertise in food science translation, market development, and the commercialization of upcycled, microbiome-targeted ingredients. How have the results been disseminated to communities of interest? Dissemination of Results to Communities of Interest Nutraberry has actively disseminated findings and engaged with industry and research stakeholders through participation in key trade shows and innovation forums, both domestic and international. These efforts enabled us to highlight our work on micronized defatted raspberry seed powder (DRSP) and its relevance to upcycling, microbiome health, and functional food innovation: VERGE, The Climate Tech Event - October 24-26, 2023, San Jose Convention Center Engaged climate-focused investors and sustainability professionals with Nutraberry's upcycled ingredient platform. Institute of Food Technologists (IFT) First Look - July 2023 & upcoming July 2024, McCormick Place, Chicago Exhibited in the innovation showcase. Table secured for 2024 to deepen engagement with ingredient scouts and R&D leads. Life Science Innovation Northwest 2024 - April 17-18, 2024, Seattle Convention Center Presented a poster and delivered a startup pitch focused on DRSP's microbiome-targeted functionality. Chicago Section IFT Suppliers Expo - 2023 & upcoming 2024 Demonstrated DRSP-based food applications; Nutraberry reserved a table for the November 2024 expo. Southern California Section IFT Suppliers Expo - 2023 & 2024 Shared technical data and product samples with formulation teams and regional food technologists. Western United States Agricultural Trade Association (WUSATA) - IFIA Japan, May 22-24, 2024, Tokyo, Japan Selected to develop DRSP-based recipes and conduct ingredient demonstrations at this international showcase. WUSATA-Sponsored Booth at the Specialty Food Association (SFA) Winter Fancy Food Show - January 21-23, 2024, Las Vegas Convention Center Displayed DRSP product innovations and met with buyers and distributors interested in sustainable ingredients. These events allowed Nutraberry to communicate research results, collect feedback, and explore commercialization pathways. Each engagement contributed to a growing network of stakeholders committed to upcycled nutrition and gut health innovation. . What do you plan to do during the next reporting period to accomplish the goals?Once final data have been collected, analyzed, and validated, we plan to disseminate the findings through peer-reviewed publications and presentations at relevant scientific and industry conferences. We will also share results with established collaborators, project cooperators, and businesses whose values and missions align with our own. These efforts will support both the broader scientific understanding of DRSP efficacy and the development of new commercial partnerships.

Impacts
What was accomplished under these goals? Objective 1 - Completed We completed all tasks outlined in Objective 1. Nutraberry supplied Morre-Tec with stone-milled defatted raspberry seed powder (DRSP), which was micronized to median particle sizes of 5 µm and 15 µm. These samples, along with Nutraberry's 70-mesh (210 µm) reference powder, were shipped to King Arthur Baking Company (KABC), which formulated standardized biscuit prototypes for fermentation studies. Using Medallion Labs protocols, we confirmed that DRSP-enriched biscuits achieved post-bake fiber densities of 4.1-4.4 g per 55 g biscuit, compared to 3.8 g in a whole wheat control and significantly less in refined flour controls. These results validate that DRSP maintains its functional fiber properties in a baked matrix. Objective 2 - Near Completion We completed all 24 planned in vitro fermentation experiments. Fecal samples from healthy adult donors were collected and fermented using DRSP biscuit formulations as substrate. We are currently analyzing pre- and post-fermentation samples using 16S rRNA sequencing to evaluate microbiome shifts and taxonomic changes. These data will guide future Phase II clinical trials focused on gut health and urolithin production. Polyphenol Enrichment Confirmed Biscuit formulations were also analyzed for ellagic acid and ellagitannin content. DRSP-enriched biscuits contained 34.7 times more ellagic acid than whole wheat controls and 86.5 times more than refined flour products. A single 56 g biscuit delivers 24-50 µg of ellagic acid, confirming sufficient precursor concentration for microbiota-mediated urolithin production. Additional Key Outcomes • Developed a Technical Data Sheet for 5 µm DRSP, confirming standardized particle size distribution, fiber content (NLT 60%), polyphenol levels (>2100 mg/100g), and FDA compliance on microbial and heavy metal limits. • Created Certificates of Analysis, pesticide screening results, and third-party fiber quantification reports. • Generated visualized post-bake fiber density data and ellagic acid enrichment outcomes to support scientific and regulatory communication. • Conducted high-resolution particle size analysis for DRSP and other micronized seed powders (blueberry, blackberry). • Shared samples and technical data with Nestlé (David Despain, Senior Principal Nutritionist), Mars (Will Borchert, Scientist), and Mondel?z International (Clive Norton, Director of Ingredient Research; Kieran Spelman, Open Innovation Leader). • Selected as a finalist in the HudsonAlpha AgTech Investment Accelerator based in Alabama--the largest peanut-producing state--positioning DRSP and future peanut skin powders for commercial integration. • Received a 136 lb purchase order from Salt & Straw for use in their Upcycled Food menu. Order not fulfilled due to funding gaps, but it confirmed strong commercial demand. • Advanced commercialization strategy in smoothie, bakery, and snack applications with industry partners.

Publications