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

2023 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
In support of Objective 1, ARS researchers at Albany, California, in collaboration with scientists from the University of Tennessee, investigated the health benefits of yellow mustard mucilage in an animal model of obesity and metabolic disease. The mucilage is a byproduct of mustard production and consists primarily of soluble polysaccharides. In support of Objective 1, ARS researchers at Albany, California, in collaboration with scientists from the University of California, Davis, investigated the health benefits of Aspergillus fungi grown in a media enriched with extracts of almond hull. Almond hulls are a waste byproduct of almond seed production. The fungi were fed to an animal model of obesity and metabolic disease. Fungi grown in almond hull extracts improved metabolic health. In support of Objective 1, ARS researchers at Albany, California, in collaboration with scientists from the University of Florida, investigated the health benefits of orange peels in an animal model of obesity and metabolic disease. Orange peels are the waste byproduct of orange juice production. Florida Valencia peels had similar properties to bitter orange peels used in traditional medicine. In support of Objective 2, ARS researchers at Albany, California, applied olive extract to melon surfaces. The olive extract was shown to be effective against Salmonella, Listeria monocytogenes and Escherichia coli (E. coli). The antimicrobial activity of olive extract exceeded that of mushroom extracts, apple peels, or 50 parts-per-million (ppm) chlorine treatments. The honeydew melon showed greater reductions of Salmonella and L. monocytogenes than cantaloupe melon. This study demonstrates the potential of olive extract to inactivate foodborne pathogens on melon. In further support of Objective 2, ARS researchers at Albany, California, in a recently published study showed that the commercial tomato glycoalkaloid tomatine and tomatine extracted from green tomatoes actually consists of a mixture of four compounds, a-tomatine, dehydrotomatine, an a-tomatine isomer, and a dehydrotomatine isomer in an approximate ratio of 90:9:1:1, respectively. Our published studies show that the mixture known as tomatine has the potential to serve as a health-promoting functional food against several diseases, including cancer, cardiovascular disease, and trichomoniasis. Also in support of Objective 2, ARS researchers at Albany, California, recently published a study showing that a new functional food created by bioprocessing (fermenting) black rice bran with shiitake mushroom mycelia inhibited pro-inflammatory effects and other biomarkers seen in ovalbumin-induced asthma cell and mice models. The histology of lung tissues revealed that the treatment also reversed the thickening of the airway wall, and the contraction and infiltration of bronchial and blood vessels. The novel food products may have useful therapeutic applications in humans. It would be of interest to investigate if these new food products can protect humans against allergic asthma, and possibly also ameliorate the acute respiratory syndromes associated with other diseases or conditions, including the viral infections such as COVID-19, In support of Sub-objective 3A, ARS researchers at Albany, California, cloned all 60 of the designed milk allergen ß-Lactoglobulin reverse mutants in an expression vector and expressed in E. coli with an express tag containing maltose binding protein and a small ubiquitin-like modifier protein. They have been purified. In support of Sub-objective 3B, ARS researchers at Albany, California, tested protein extracts of macadamia nut, peanut, Brazil nut, hazelnut, walnut, and blackberry. Protein bands that reacted with a polyclonal antibody specific to almond allergen Pru du 8 were identified in macadamia nut exact. Ongoing work includes determining the sequence of the macadamia nut proteins and investigating the physical, chemical, and immunological properties.

Accomplishments
1. Antimicrobial effects of plant extracts against foodborne pathogens on melons. Melons are diverse group of desert fruits including orange fresh cantaloupe, green fresh cantaloupe, green fresh honeydew, and mixed hybrid varieties. Melon surfaces are easily contaminated by Salmonella, Listeria, and Escherichia coli (E. coli) during the growing process. These foodborne pathogens may be harbored in the outer rind of the melon and transferred to the edible portions during cutting and dividing processes. It is difficult to effectively wash, clean and sanitize melons. In the present study, olive extract applied to melon surfaces was shown by ARS researchers in Albany, California, to be effective against Salmonella, L. monocytogenes and E. coli. The antimicrobial activity of olive extract exceeded that of mushroom extracts, apple peels, or 50 parts-per-million (ppm) chlorine treatments. The honeydew melon showed greater reductions of Salmonella and Listeria monocytogenes than cantaloupe melon. This study demonstrates the potential of olive extract to inactivate foodborne pathogens on melon.

2. Commercial tomatine is a mixture of isomers. Interest in tomato compounds called glycoalkaloids arises from the fact that they seem to have important functions in host-plant interactions and have been reported to exhibit several beneficial health-promoting properties. Analytical high-performance liquid chromatography and mass spectral analytical data in a recently published study show that the commercial tomato glycoalkaloid tomatine and tomatine extracted from green tomatoes actually consists of a mixture of four compounds, a-tomatine, dehydrotomatine, an a-tomatine isomer, and a dehydrotomatine isomer in an approximate ratio of 90:9:1:1, respectively. Nuclear magnetic resonance spectroscopy studies are in progress designed to elucidate the stereochemical structures of isomeric forms. The published studies by ARS researchers in Albany, California, show that the mixture known as tomatine has the potential to serve as a health-promoting functional food against several diseases, including cancer, cardiovascular disease, and trichomoniasis.

3. A new functional food has an anti-asthma effect. The results of a recently published study by ARS researchers in Albany, California, show that a new functional food created by bioprocessing (fermenting) black rice bran with shiitake mushroom mycelia inhibited pro-inflammatory effects and other biomarkers seen in ovalbumin-induced asthma cell and mice models. The histology of lung tissues revealed that the treatment also reversed the thickening of the airway wall, and the contraction and infiltration of bronchial and blood vessels. The novel food products may have useful therapeutic applications in humans, such as protection against allergic asthma, viral infections such as COVID-19, chronic obstructive pulmonary disease (COPD), emphysema, and peanut protein and other food allergies.

4. Edible fungi grown on waste almond hull extract have health benefits. Almond hulls are a byproduct of almond seed for food production. About 4.5 billion pounds of hulls are produced in California each year. The hulls have low value and diminishing use for feed or fuel. ARS researchers in Albany, California, grew Aspergillus oryzae, a fungus used in food production in Asia, on synthetic media or an aqueous extract of almond hulls. The fungi grown on the almond hulls reduced body weight gain and blood cholesterol in mice fed a high-fat diet. The results suggest that waste almond hulls may be used as a growth media for food fungi with beneficial nutritional properties.

5. IgE binding epitopes of peanut allergen Ara h 2 mapped with a new method. Peanut and tree nut allergies negatively impact the utilization of agricultural products. Linear IgE epitopes play essential roles in peanut and tree nut allergies. The overall expense of the approaches and the inherent challenges in available methods for epitope mapping necessitate a cost-effective way of mapping linear IgE binding epitopes. ARS researchers at Albany, California, developed a novel method for mapping linear epitopes and applied it to determine dominant IgE binding epitopes of peanut allergen Ara h 2. The new method with internal positive control offers another approach for obtaining information about IgE binding epitopes of food allergens, which can be used to better diagnose food allergies, predict the prognosis of allergies, better understand the allergenicity of food proteins, and positively impact the agricultural and food industry by reducing recalls and enhancing safe use of products.

Review Publications
Zhang, Y., Bhardwaj, S.R., Lyu, S., Chinthrajah, S., Nadeau, K., Li, C. 2022. Expression, purification, characterization, and patient IgE reactivity of new macadamia nut iso-allergen. Protein Expression and Purification. 203. Article 106211. https://doi.org/10.1016/j.pep.2022.106211.
Ding, N., Meng, H., Wu, C., Yokoyama, W.H., Hong, H., Luo, Y., Tan, Y. 2023. Whey protein hydrolysate renovates age-related and scopolamine-induced cognitive impairment. Nutrients. 15(5). Article 1288. https://doi.org/10.3390/nu15051228.
Kozukue, N., Kim, D., Choi, S., Mizuno, M., Friedman, M. 2023. Isomers of the tomato glycoalkaloids a-Tomatine and dehydrotomatine: Relationship to health benefits. Molecules. 28(8). Article 3621. https://doi.org/10.3390/molecules28083621.
Zhang, Y., Che, H., Li, C., Jin, T. 2023. Food allergens of plant origin. Foods. 12(11). Article 2232. https://doi.org/10.3390/foods12112232.
Seo, K., Lee, H., Eor, J., Jeon, H., Yokoyama, W.H., Kim, H. 2022. Effects of kefir lactic acid bacteria-derived postbiotic components on high fat diet-induced gut microbiota and obesity. Food Research International. 157. Article 111445. https://doi.org/10.1016/j.foodres.2022.111445.
Finley, J.W., Holliday, D., Kim, H., Alves Buongiorno, P.L., Shao, D., Bartley, G., Yokoyama, W.H. 2023. Substitution of cooked kidney beans or ground beef in hypercholesterolemic high fat diets reduces plasma and liver lipids in hamsters. Food Production, Processing, and Nutrition. 5. Article 20. https://doi.org/10.1186/s43014-023-00135-2.
Zhang, Y., Bhardwaj, S.R., Vilches, A.M., Breksa III, A.P., Lyu, S., Chinthrajah, S., Nadeau, K., Jin, T. 2022. IgE binding epitope mapping with TL1A tagged peptides. Molecular Immunology. 153:194-199. https://doi.org/10.1016/j.molimm.2022.12.001.
Zou, X., Yokoyama, W.H., Liu, X., Wang, K., Hong, H., Lou, Y., Tan, Y. 2023. Milk fat globule membrane relieves fatigue via regulation of oxidative stress and gut microbiota in BALB/c mice. Antioxidants. 12(3). Article 712. https://doi.org/10.3390/antiox12030712.
Hughes, A.C., Kirkland, M., Du, W.N., Rasooly, R., Hernlem, B.J., Tam, C.C., Zhang, Y., He, X. 2023. Development of thermally stable nanobodies for detection and neutralization of Staphylococcal enterotoxin B. Toxins. 15(6). Article 400. https://doi.org/10.3390/toxins15060400.