Progress 09/01/21 to 08/31/24

Outputs
Target Audience:According to ReFED, in 2019 35% of food in the United States went unsold or uneaten, contributing 4% of the total U.S. greenhouse gas emissions and lost revenue of $408 billion (2% of the U.S. GDP). This represents both an environmental issue and a missed opportunity for increasing revenue for fruit and vegetable producers. After an extensive analysis of the issue, ReFED identified 'Maximizing Product Utilization' as one of their top 10 Action Areas for reducing food waste. They have estimated that Upcycling these ingredients has the potential to reduce greenhouse gas emissions by 4.8 million metric tons of CO2. There is critical need for a solution that can address this problem by using a sustainable solution that can transform food waste into a nutritionally-relevant consumable. In this SBIR project, our focus wasto reduce the harmful effects of food waste by transforming a current waste stream (fruit pomace) or commodity product into a value-added ingredient that delivers concentrated nutrition to consumers. Our patented VITERO™ technology capitalizes on an energy-efficient, economical and sustainable technology (continuous microwave-assisted extraction), which is able to extract high concentrations of residual biologically-active health protective compounds from pomaces without the need for any solvent besides water. These valuable polyphenolic compounds are then complexed with high quality edible proteins in an efficient process to produce stably bound protein-polyphenol colloidal aggregate particles (ingredients) which have versatility for multiple applications in soft-solid foods and beverages. The VITERO™ ingredients are a strengthened and stabilized version of their individual parts, helping to protect these compounds from degradation through environmental factors such as oxidation. This technology allows for valuable health-promoting and disease-preventing compounds derived from nature to be concentrated into a convenient, bioavailable protein matrix. This represents an entirely new approach to handling this market, by recognizing the value in what is normally wasted. Changes/Problems:The only major problem we encountered was that the outcome from the clinical trial was not as expected (similar earlier clinical trials were conducted). However, because of thiswe were able to conduct a series of investigations to shed light into what had happened and how we would approach a potentialfuture clinical trial. Any additional smaller deviations from the original work plan are also highlighted in the main body of this report (see Accomlishments section). There was also adelay in receiving the final report/findings from the clinical trial as some additional work was done by another lab to further evaluate the samples and data received for statistical signficance. This part took several months longer than originally discussed. Further, scaling up within the equipment framework of large processors is still considered to be a gap without first creating a compelling market demand. To create this, we've developed two value-added functional smoothie products: an Athletic Recovery Smoothie and a Beauty from Within Smoothie, both containing a pea protein-blackcurrant VITERO™ ingredient. Based on the learnings from the clinical trial, these have been specifically formulated to promote anthocyanin composition. Sales samples (packaged in 4 oz aseptic spouted pouches) will be generated to test the stability of anthocyanins and total phenolics in the finished products over time. These samples will be used to engage potential commercialization partners for the VITERO™ technology, including retail brands. What opportunities for training and professional development has the project provided?We were able to gain further insights into fruit processing,pomace generation and processing infrastructures. The lead scientist was able to attend all third party trials and was able to not only lead the trials but also gain valuable new knowledge from those trials. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objectives 1 and 2: We visited a large fruit manufacturer partner facility to evaluate their production process and to fully understand the scope of their operation and where waste materials are generated. We received and evaluated apple, blueberry, concord grape, and cherry pomaces for extraction efficiency. Based on seasonal availability and polyphenol contents found, the large-scale extraction trial with our deployed MAE system focused on blueberry pomace. Due to our partner'sfacility size, we moved forward with bringing in a second extraction partner that we could use as a medium-sized deployment partner to more effectively complete the scale-up validation. We were able to produce enough concentrated extract to move forward with the other pieces of the project plan. We further evaluated both, radiant- and spray drying techniques, to effectively dry the protein-polyphenol aggregates. Due to local spray drier availability and powder quality, we chose spray drying as the means to create powdered VITEROTMingredients. Three spray-dried VITEROTMingredients were generated. The VITEROTMpowders were further evaluated for total phenolic content, and overall spray-dried powder yield. The highest amount of total phenolics were found in the pea/rice VITEROTMfollowed by the pea VITEROTM. However, we encountered issues dialing in the spray drying process for the pea/rice VITEROTMand could only secure a few pounds of spray dried material. On the other hand, we had no to little issues spray drying the pea VITEROTMand were able to obtain around 30 lbs of powdered material. Enough to develop and generate the intervention sports gel required for Technical Objective 4. Based on this, we proceeded with the pea-blueberry VITEROTM. Objective 3: We accomplished to compare the pea/rice - blueberry VITEROTMthat has been used in a line of gummies (wet version) with the dried pea/rice - blueberry VITEROTMin a gummy in a descriptive sensory trial. The results from the trained sensory panel testing showed that the Wet and Dry VITEROTMGummy samples were noticeably different from one another. Overall, neither gummy was unpalatable. For the Wet VITEROTMformula, the firmer texture, slowness of the flavor release, lower complex flavor impact, and low grainy/chalky feel may be consumer negatives. The Dry VITEROTMformula on the other hand was well blended and had a complex flavor. Objective 4: The purpose of the clinical trial was to investigate the influence of 2-weeks of blueberry, blueberry-protein, versus placebo gel supplementation (randomized, crossover) on oxinflammation markers and blueberry gut-derived phenolics after intensive cycling exercise in 20 subjects. The hypothesis was that 2-weeks of blueberry or blueberry-protein polyphenol versus placebo gel supplementation would attenuate post-exercise increases in inflammation and oxidative stress biomarkers. The clinical trial was successfully conducted as designed and samples and data were analyzed. The urine metabolite analysis and untargeted metabolomics analysis both showed a weak blueberry gut-derived metabolite response to taking the blueberry supplements. The data were not consistent with data from a previous study. Investigations revealed that the pouches containing blueberry extract or the pea protein-blueberry polyphenol VITEROTMingredient contained low concentrations of total anthocyanins (approximately 10 mg of total ANC/pouch). The different interventions were standardized to the total phenolic content and not the total anthocyanin content. Furthermore, we found that the accidental lengthy blueberry pomace extraction and concentration process resulted in some level of extract fermentation (very low level of sugars found in blueberry extract concentrate, in retrospect) which likely also resulted in degradation of phenolic compounds such as anthocyanins. In contrast, similar previously published intervention studies with strong metabolite responses contained 345 mg total anthocyanins (Front Nutr. 2020 Aug 7;7:121.) and 280 mg of total anthocyanin (Sci Rep.2023;13(1):11976) per serving of freeze-dried blueberry powder. Based on our findings the data obtained from our clinical trial cannot be published. It appears that the total anthocyanin content delivered during the intervention in fact plays a critical role. Interestingly, during the time when the clinical trial samples and data were still going through the analysis process (and we did not know about the issues concerning the low anthocyanin content), we had decided to create additional quantities of pea protein-blueberry VITEROTM, this time more tightly controlling the process from start to finish, in the efforts to avoid potential fermentation and to create additional powder material for further R&D and sales samples.Despite our efforts, we had little luck to produce any meaningful amounts of high-quality powder. The material was hard to spray dry.This was surprising to us since we were able to successfully spray dry the same formula and material before. The only difference was that we used a different spray drying system and location. We sent out both blueberry pomace extract concentrates that were used to create the powdered pea protein-blueberry VITEROTMs for sugar analysis, and found that the original extract concentrate contained negligible amounts of sugars due to fermentation, while the second round of prepared blueberry pomace extract concentrate contained relatively high amounts of sugars. It is general knowledge that sugars, especially sugar monomers such as glucose and fructose, can interfere with effective spray drying (i.e. high glass transition temperature) and result in rubbery/sticky particles (hygroscopic). We further explored the potential sugar contribution due to enzymes used to help pre-process the pomace prior to extraction. We confirmed with the enzyme manufacturer that the pectinase enzyme blend will not generate any sugar monomers, while the cellulase enzyme blend in fact will break down cellulose into sugar monomers. A small-scale extraction trial omitting the cellulase enzyme from one batch while adding it together with the pectinase enzyme in another batch and quantifying the sugar content in each resulting extract confirmed this hypothesis. Both extracts were at around 19 Brix but the extract that was generated using pectinase only contained 6.61% total sugars while the extract that was prepared with the use of pectinase and cellulase enzymes contained 8.84% total sugars. Our conclusion is that we either a) need to remove sugars from the polyphenol-rich extract (which would also help with concentrating the existing phenolic compounds), b) need to find the sweet spot of sugars present that still allow for efficient spray drying and quality powder or c) adjust our VITEROTMformula.There are existing filtration methods on the market to remove sugars from liquids, alternatively fermentation is an option but would need to be further investigated because the flavor profile of the extract and therefore the resulting VITEROTMingredient would significantly change. Testing a variety of different extracts with different sugar levels is not a scalable path. Adjusting the formula to include more carrier would defeat the purpose of our technology. The ultimate goal is to extract polyphenols from underutilized plant sources (such as pomace byproducts from the juicing and wine industries) and use these extracts in the VITEROTMtechnology. This hurdle still needs to be overcome.

Publications

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:We have continued to strengthenexisting relationships with potential partners such as Fruitsmart (source of waste pomaces)and protein suppliers (e.g. Puris Foods) as well as made progress towards creating new relationships that aid in the commercialization of this ingredient technology (e.g. Florida Naturals, Louis Dreyfus Company, RXBAR). We recently also connected with a more local source of blueberry pomace (Cardinal Foods) to create more blueberry pomace extract and generate more Vitero for testing and review. Changes/Problems:We did not encounter any major issues during this reporting phase. The clinical trial portion started a bit later than anticipated (recruitment of participants etc) and we are now taking some additional time to do a deeper dive by performing some additonal analyses on the samples obtained from the clinical trial. We are also in the process of repeating a scale up trial of the pomace extraction and subsequent preparation of additonal VITERO material, a more commercial ready version - for further testing and to share with our parterns and potentialcustomers. What opportunities for training and professional development has the project provided?Our team has learned a significant amount about large-scale handling of fruit during juice and juice concentrate production. We have learned significant amounts about other pieces of equipment with which we weren't previously familiar including separators, decanters, centrifuges, and concentrators. We have also learned a significant amount about the drying process and the drying industry including having the opportunity to have hands on experience with spray drying. Our engineering team also was able to gain practice and experience with deployment of one of our extraction systems that will be invaluable toward future installations. Our internal lead investigator on this projectis about to travel to MN to attend aVITERO spray drying trial at Dahmes and she oversaw/managed the decanter and concentration trial at Cornell. How have the results been disseminated to communities of interest?We are in close contact with all of our partners/potential outlets for this technology as findings become available .Within the next week we are also able to share samples of a spray dried blueberry extract polyphenol-pea protein VITERO. What do you plan to do during the next reporting period to accomplish the goals?Our research partners will continue completing the clinical trial portion and findings will be used to generate a publication/report.Findings can be used as science-backed marketing material for VITERO and shared with our protein and pomace supplier/s. We will also be conducting an analysis around the costing for the ingredient, solidifying partnerships toward commercialization, and working to develop our future sales outlets.

Impacts
What was accomplished under these goals? We successfully completed goals 1 through 3 and areon track to completing goal 4of the Work Plan as well. 1) We visited the large fruit manufacturer partner facility to evaluate their production process and to fully undersand the scope of their operation and where waste materials are generated. We evaluated apple, blueberry, concord grape, and cherry pomaces for extraction efficency to be used in the rest of the process. Based on the large throughputs of that partner facility, we identified that we needed to focus on a single fruit extract to move forward to the next stage. Based on extraction efficiency, availability, and maximized polyphenol content, the blueberry was identified as the best target. 2) Based on the large throughputs of our partner facility, we moved forward with bringing in a second extraction partner that we could use as a medium-sized deployment partner in order to more effectvely complete the scale-up. We successfully deployed our extraction system to their facility, using their post-extraction separation and concentration equipment. We were able to produce enough concentrated extract at a high enough extraction yield to move forward with the other pieces of the project plan. During this reporting period, we also worked to investigate the best method of drying for our material. We evaluated both a radiant drying and spray drying techniques. Due to the availability and quality of the spray drier, we moved forward with that method to dry enoguh ingredient to be used in Goals 3 and 4 of this project. We also further investigated protein sources based on the latest developments in the plant based protein space. In addition to a pea/rice blend, we also worked with a high acid stable pea protein and a canola protein. We used the blueberry extract, separately combined with these 3 proteins, to produce the spray dried VITERO ingredient at the North Carolina Food Innovation Lab. The finished ingredients from this production were analyzed, and the VITERO version of pea protein - blueberry extractwas further used to formulate amodel sports gel type productto be used in the clinical trial.The clinical trial was successfully completed in Q2 of 2023 and samples and data are currently still being analyzed. In addition, we are currently also in the process of creating more pea protein-blueberry pomace extract polyphenol VITERO. We have sourced 4500 lbs of local blueberry pomace which we extracted on our larger microwave assisted processing line (new expansion facility FirstWave Middlesex) and have partnered with Cornell's pilot plant to decant and subsequently concentrate the resulting blueberry pomace extract. The lead staff working on this projectis about to travel to Dahmes in MN to attend the spray drying trial (spray drying about 500 lbs of wet VITERO) and to meet with members of Puris Foods to discuss VITERO further and share VITERO samples with. We have also successfully completed a sensory analysis of gummies created with dry and wet versions of VITERO as indicated in our last report. We are also still conducting a shelf life study for these powders (3 different VITERO created at the North Carolina Food Innovation lab)that will allow us to understand their stability over time. In addition, through TABA (Tech Opp) we were able to connect with Florida Naturals and Louis Dreyfus Company who have sent us a variety of different citrus pomaces to test for microwave-assisted extraction and suitability for VITERO. Trials have been completed and reports are currently generated and shared with the potential partners.

Publications

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The target audience that was reached during this reporting period included our ingredient and supplier partners. Specifically, we worked with 2 large fruit juice manufacturers, and 3 different protein suppliers. These groups are also potential outlets to sell the products of the technology. Changes/Problems:1.In addition to blueberry, concord grape and apple pomace, cherry pomace (another waste stream available through FruitSmart) was also investigated early on in phase II. Scale up efforts focused on blueberry pomace (due to both availability and test results). 2.For protein sources, based on timing and ingredient availability as well as new potential partnering opportunities we decided to use a pea/rice blend (suppliers Puris Foods and Axiom Foods, respectively), a pea protein (Puris Foods), and a canola protein (DSM). 3.The original plan was todeploy a test system to our partner site for a factory trial(FruitSmart), but due to timing and volume requirements, we instead deployed a test system to a smaller facility in South Carolina that already processes fruit and pomaces as well (Muscadine Products Corporation). Blueberry pomace was shipped from FruitSmart to this partner facility for the trial. Leftover extracted biomass was not dried and analyzed as a fiber source as indicated in the proposal. 4.The Ripe Revival gummies referenced in the proposal as a model product are no longer being produced and sold, but we are still planning to compare the blueberry complex that has been used in those gummies with the dried blueberry complex (in a gummy)in a descriptive sensory trial to confirm that the scaled up ingredient matches the small batch ingredient. We have successfully created dried complex powders and are taking next steps towards their analysis as well as towards formulation work and the clinical trial. We are anticipating to recruit individuals for the clinical trial in January 2023. What opportunities for training and professional development has the project provided?Our team has learned a significant amount about large-scale handling of fruit during juice and juice concentrate production. We have learned significant amounts about other pieces of equipment with which we weren't previously familiar including separators, decanters, centrifuges, and concentrators. We have also learned a significant amount about the drying process and the drying industry including having the opportunity to have hands on experience with spray drying. Our engineering team also was able to gain practice and experience with deployment of one of our extraction systems that will be invaluable toward future installations. How have the results been disseminated to communities of interest?At this point, we've given verbal and written feedback to our partners. In the coming days, we will also send samples of the VITERO to our partners that they will be able to use to show their customers in order to generate interest. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will continue our analysis of the VITERO ingredients that we have produced. We will put these into several model products including an aseptic sports gel and gummies that will be used for a clinical trial and for sensory testing. We are also starting a shelf life study for these powders that will allow us to understand their stability over time. The clinical trial will be a large portion of the next reporting period, and will give us invaluable data toward the efficacy of our ingredient. We will be comparing interventions with VITERO, our polyphenol extract, and a placebo in terms of its ability to reduce stress markers after exercise. We will also be conducting an analysis around the costing for the ingredient, solidifying partnerships toward commercialization, and working to develop our future sales outlets.

Impacts
What was accomplished under these goals? During this reporting period, we successfully completed goals 1 and 2 of the Work Plan. 1) We visited the large fruit manufacturer partner facility to evaluate their production process and to fully undersand the scope of their operation and where waste materials are generated. We evaluated apple, blueberry, concord grape, and cherry pomaces for extraction efficency to be used in the rest of the process. Based on the large throughputs of that partner facility, we identified that we needed to focus on a single fruit extract to move forward to the next stage. Based on extraction efficiency, availability, and maximized polyphenol content, the blueberry was identified as the best target. 2) Based on the large throughputs of our partner facility, we moved forward with bringing in a second extraction partner that we could use as a medium-sized deployment partner in order to more effectvely complete the scale-up. We successfully deployed our extraction system to their facility, using their post-extraction separation and concentration equipment. We were able to produce enough concentrated extract at a high enough extraction yield to move forward with the other pieces of the project plan. During this reporting period, we also worked to investigate the best method of drying for our material. We evaluated both a radiant drying and spray drying techniques. Due to the availability and quality of the spray drier, we moved forward with that method to dry enoguh ingredient to be used in Goals 3 and 4 of this project. We also further investigated protein sources based on the latest developments in the plant based protein space. In addition to a pea/rice blend, we also worked with a high acid stable pea protein and a canola protein. We used the blueberry extract, separately combined with these 3 proteins, to produce the spray dried VITERO ingredient at the North Carolina Food Innovation Lab. The finished ingredients from this production are currently being analyzed, and formulated into the model products to be used in the clinical trial. We've also met with our clinical trial team to formulate a robust plan toward that study.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Hoskin et al 2022. Continuous flow microwave-assisted aqueous extraction of pomace phytoactives for production of protein-polyphenol particles and a protein-enriched ready-to-drink beverage. Future Foods. Volume 5, June 2022, 100137