Research Project: Prevention of Obesity Related Metabolic Diseases by Bioactive Components of Food Processing Waste Byproducts and Mitigation of Food Allergies

Location: Healthy Processed Foods Research

2025 Annual Report

Objectives
Objective 1: Resolve how novel single or in combination bioactive phytochemicals may enable the prevention of obesity and/or obesity related metabolic dysfunction in animal models. • Sub-objective 1A: Investigate the interactions of polyphenols on bile acids, proteins, and enzymes in intestinal lumen and their effect on intestinal permeability and FXR activation. • Sub-objective 1B: Evaluate ingredients containing two or more bioactive compounds such as polyphenols encapsulated for synergism in obese animal models. • Sub-objective 1C: Evaluate polyphenols encapsulated and/or immobilized in plant or microbial cell ghosts or gel bioactive fiber matrices such as HPMC, alginate, or glucomannan. Objective 2: Following gut fermentation of phytochemicals, determine the metabolites and/or peptide products that prevent inflammation in cell culture or animal models. • Sub-objective 2A: Identify major metabolites and proteins in extracts from food processing byproducts fermented by gut bacteria. • Sub-objective 2B: Culture 3T3-L1 adipocytes and lipopolysaccharide-activated RAW 264.7 macrophages with metabolites and/or peptides identified in 2A to assess anti-inflammatory response and lipid accumulation. Objective 3: Integrate measurable allergenic properties with methods to mitigate food allergens in nuts and dairy. • Sub-objective 3A: Develop methods for investigating conformational allergenic determinants of food allergens and assess their prevalence. • Sub-objective 3B: Define, characterize, and develop methods to detect novel nut allergens.

Approach
Hypothesis 1A: Polyphenols complexed by proteins modulate bile acid interactions with gut microbe. Specific research procedures: A high fat diet induced obesity (DIO) mouse (C57BL/6J) will be used. Polyphenols, conjugated and free bile acids in the intestinal lumen, colon, and feces will be measured by HPLC and intestinal permeability by FITC dextran. The expression of FGF15 and FXR expression will be measured by RT-PCR. Inflammatory and diabetic biomarkers by BioPlex multiplex ELISA. Hypothesis 1B: Synergistic interactions exist between polyphenols and other phytochemicals. Specific research procedures: The DIO mice will be fed single compounds or combinations. Polyvinylalcohol removal of polyphenolics and loss of activity would suggest synergistic compound is a polyphenol. Efflux pump inhibitors will be fed with grape seed proanthocyanidins and bile acids. Gut microbiome will be analyzed. Hypothesis 1C: Polyphenols beneficial health effects may be due to reducing the rapid influx of sugar, fat and other nutrients from rapidly digested foods that overload organs by binding to and inhibiting digestive enzymes. Specific research procedures: Yeast, bacteria, and plant cell ghosts infused with grape seed polyphenols, resveratrol or other bioactive phytochemicals will be provided by UC Davis. The encapsulated polyphenols will be tested in the DIO model described in Objective 1A. Hypothesis 2A: Fermentation produces absorbable phytochemicals from plant materials and bacterial protein/peptides. Specific research procedures: Extracts of food processing byproducts from apples, red beets, eggplants, grapes, olives, and cereal brans will be fermented with B fragilis, L plantarum, C perfringens, Bifidobacterium longum. The extract will be analyzed for total protein content, nucleic acids, and total polyphenolics. Specific phytochemicals will be analyzed by LC/MS. Hypothesis 2B: Products of fermentation and/ or bacterial derived peptides inhibit inflammation or lipid accumulation in adipose tissue. Specific research procedures: Glucose metabolism and lipid accumulation of 3T3-L1adipocytes and inflammatory responses in RAW264.7 macrophages will be measured with or without additions of substances from 2A. Hypothesis 3A: Structural information can be used to develop a new method that is applicable to map conformational IGE epitopes. Specific research procedures: An alternative ELISA format would use His-tagged allergens immobilized to N2+ coated microtiter plates. Final reverse mutant will have a His-tag. The pTNS2 vector will be used to construct plasmids for making the C-terminal His-tagged ßLG and its mutant by omitting the stop codon. To map the conformational IgE epitopes in ßLG, sera from 20 subjects with IgE antibodies against milk allergens will be used. Hypothesis 3B: PRU DU 8 orthologs in other plants are also food allergens. Specific research procedures: To characterize Pru du 8, we will use the recombinant allergen as antigen and make polyclonal antibodies in rabbits commercially. We will isolate the coding sequence for the new protein from the corresponding tree nuts to deduce the translated protein sequence.

Progress Report
This is the final report for project 2030-41440-008-00D, "Prevention of Obesity Related Metabolic Diseases by Bioactive Components of Food Processing Waste Byproducts and Mitigation of Food Allergies", which was replaced by new project 2030-30600-005-000D, "Novel and Sustainable Processing Technologies and Healthy Food Ingredients". For additional information, see the new project. The overall objectives of the project were to resolve how bioactive phytochemicals prevent obesity and/or obesity related metabolic dysfunction in animal models, determine the metabolites and/or peptide products that prevent inflammation, and integrate measurable allergenic properties with methods to mitigate food allergens in nuts and dairy. Significant progress was achieved for all three objectives. Overall for the life of this entire project, 57 peer-reviewed journal articles were published. In support of Sub-objective 1A, plant ingredients or their extracts were evaluated for their ability to prevent or reduce obesity related chronic metabolic dysfunction in a mouse model. Effectiveness was evaluated by reductions in body weight, abdominal adipose weight, liver weight, blood lipids, and blood glucose. These physiological parameters were related to molecular characteristics such as gene expression in liver, adipose, intestine and brain. Blood was analyzed in order to develop predictive biomarkers for insulin resistance and diabetesby multiplex immunoassays. Gut bacteria were also characterized since many bioactive compounds are not absorbable directly into the body but are activated by or modify gut bacterial composition. Under Sub-objective 1B, the obese, chronic metabolic dysfunction mouse model was used to investigate the potential of synergistic interactions between two food components known to have bioactive properties. For Sub-objective 1C, another layer of complexity of the digestive system was investigated by combining probiotic bacteria with indigestible plant materials to model the effects of colonic fermentation on bioactive phytochemicals. The overall goal of Objective 2 was to ferment bioactive compounds by probiotic bacteria and evaluate their anti-inflammatory properties in cell culture or animal models. In support of Sub-objective 2A, black rice bran was fermented (bioprocessed) by shiitake mushroom mycelia, and its ability to protect mice against alcoholic liver damage and reduce tumor size in a cell culture model were evaluated. The fermented rice bran prevented increases in alcohol induced liver weight and reduced blood levels of hepatic enzymes in mice used as markers of healthy liver function. In addition, a compound in rice was identified that had anti-inflammatory effects in this mouse model. These studies suggest that fermentation improved the anti-inflammatory properties of rice bran and may have anti-cancer and liver health promotion abilities. Under Sub-objective 2B, RAW 264.7 macrophage cells were treated with the fermented bran and the metabolites from the treatment were applied to a colon cancer cell line. The cellular metabolites reduced tumor size and inflammatory biomarkers. This study suggests that fermentation products may act indirectly through the production of secondary metabolites from the host to effect their healthful properties. The overall goal of Objective 3 was to integrate observable and measurable allergenic properties with methods to mitigate the undesirable physiological effects of food allergens in nuts and dairy. In support of Sub-objective 3A, ARS researchers in Albany, California, constructed plasmids for the expression, and purified and stored milk allergen ß-Lactoglobulin reverse mutants. Ongoing work includes completing the storage of the full set of the mutants for assaying the reaction with patients’ sera. For a contingency plan for Sub-objective 3B, ARS researchers at Albany, California, confirmed the express of, and are continuing the work to purify the natural vicilin protein from almond. Their previous work identified almond vicilin as a food allergen using a recombinant form of the protein. Ongoing work includes characterizing the properties of natural almond vicilin in relation to food safety and quality.

Accomplishments
1. Novel means for detecting a peanut allergen. In the United States, 5.8-10% of the population have food allergies and the cost of child food allergy was estimated to be $24.9 billion annually, including direct medical costs, lost labor productivity, and opportunity costs. Cost to the food industry due to recalls because of allergens is also high. It is important to detect possible allergens in food products before they reach the market. ARS researchers at Albany, California, produced a novel recombinant antibody and designed immunological assays for detecting peanut allergen. The result may lead to the commercial production of immunoassay reagents for the detection of many food allergens and foodborne toxins. Easing the development of sensitive methods to detect food contaminants, including food allergens, will enable food processors to efficiently improve food product safety and marketability.

2. Age and obesity related memory loss are reduced by grapeseed in diet. In the United States, 7.3 million people over age 64 have Alzheimer’s disease. Aging and obesity are major risk factors for cognitive impairment. Obesity has been associated with structural and functional changes in the brain. ARS researchers in Albany, California, showed that mice fed high-fat diets developed memory impairment compared to mice on normal diets. However, memory improved when high-fats diets were supplemented with grapeseed powder or extracts from grapeseed. Cognitive scores, combining spatial and recognition memory, of grapeseed supplemented high-fat and low-fat diets were similar. Grapeseed supplementation induced production of oxytocin, a hormone known to improve memory and social behavior. These findings have been the basis for two similar studies in humans, and to the inclusions of grape pomace in chocolate confections to increase antioxidant content.

3. A novel method for mapping the allergy producing areas of food proteins IgE binding epitopes of peanut allergen Ara h 2 mapped with a new method. . Peanut and tree nut allergies negatively impact the consumption of agricultural products. The part of an allergen that interacts with the immune system and triggers allergic reactions is known as the epitope. It is essential to know the structure of epitopes of food allergens. However, the overall expense in available methods for mapping epitopes necessitates the development of new techniques. ARS researchers at Albany, California, developed a novel method for mapping epitopes. They applied the new method to investigate a peanut allergen called Ara h 2. The new method offers a novel approach for obtaining information about the epitope and other immunological properties of food allergens. This information can be used to improve diagnose food allergies, predict the prognosis of allergies, and find solutions to mitigate the problem of food allergies.

Review Publications
Hao, M., Wang, Q., Wang, J., Che, H., Guo, X., Zhang, Y. 2024. Identification, purification, and characterization of Kunitz-type inhibitors from white-fleshed dragon fruit (Selenicereus undatus) and red-fleshed dragon fruit (Selenicereus costaricensis) seeds as novel food allergens. Journal of Agricultural and Food Chemistry. 72(41):22836-22847. https://doi.org/10.1021/acs.jafc.4c04328.
Kwon, K., Lee, E., Lee, K., Hwang, W., Lee, W., Kim, J., Kim, J., Lee, S., Kim, S., Friedman, M. 2024. Anti-obesity and other health benefits of bioprocessed black rice bran in combination with green tea extract in 3T3-L1 preadipocyte cells and in mice on a high fat diet. Food and Function. 15:12083-12100. https://doi.org/10.1039/D4FO03210A.
Guo, X., Zhu, B., Li, J., Guo, P., Niu, Y., Shi, J., Yokoyama, W.H., Huang, Q., Shao, D. 2025. Cholesterol metabolism in tumor immunity: Mechanisms and therapeutic opportunities for cancer. Biochemical Pharmacology. 234. Article 116802. https://doi.org/10.1016/j.bcp.2025.116802.
Zhou, Y., Jiang, Y., Zhang, Y., Yokoyama, W.H., Wu, J., Chang, S., Hong, H., Luo, Y., Li, B., Tan, Y. 2025. Unveiling a new chapter for collagen peptides: Comprehensive insights into oral bioavailability and the enhancement via encapsulation systems. Trends in Food Science and Technology. 156. Article 104849. https://doi.org/10.1016/j.tifs.2024.104849.
Jeffries, K.A., Fan, Z., Mattia, M.R., Stover, E.W., Baldwin, E.A., Manthey, J.A., Breksa Iii, A.P., Bai, J., Plotto, A. Flavonoid contributors to bitterness in juice from Citrus and Citrus hybrids with/without Poncirus trifoliata in their pedigree. Food Chemistry: X. 26:102289. 2025. https://doi.org/10.1016/j.fochx.2025.102289.
Jeffries, K.A., Fan, Z., Sun, X.N., Olmedo, G., Zhao, W., Mattia, M.R., Stover, E., Baldwin, E., Manthey, J., Breksa Iii, A.P., Bai, J., Plotto, A. New insights in the flavor and chemistry of Huanglongbing tolerant citrus hybrids with/without Poncirus trifoliata in their pedigree. Frontiers in Horticulture. 3:1425366. 2024. https://doi.org/10.3389/fhort.2024.1425366.
Satterlee, T.R., Hawkins, J.A., Mitchell, T.R., Wei, Q., Lohmar, J.M., Glenn, A.E., Gold, S.E. 2025. Fungal Chemical Warfare: The role of aflatoxin and fumonisin in governing the interaction between the maize pathogens, Aspergillus flavus and Fusarium verticillioides. Frontiers in Cellular and Infection Microbiology. Front. Cell. Infect. Microbiol. 14:1513134. https://doi.org/10.3389/fcimb.2024.1513134.
Kahlon, T.S., Olson, D.A., Friedman, M., Haff, R.P. 2025. Comparison of infrared heating and hot air frying for creating novel puffed snacks from bengal gram and garbanzo grains. Vegetos: An International Journal of Plant Research. https://doi.org/10.1007/s42535-025-01188-z.
Ponciano, G.P., Dong, N., Dong, C., Breksa III, A.P., Vilches, A.M., Abutokaikah, M.T., McMahan, C.M., Holguin, F.O. 2024. Overexpression of tocopherol biosynthesis genes in guayule (Parthenium argentatum) reduces rubber, resin and argentatins content in stem and leaf tissues. Phytochemistry. 222. Article 114060. https://doi.org/10.1016/j.phytochem.2024.114060.