Location: Healthy Processed Foods Research
2024 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 report documents progress for project 2030-41440-008-000D, titled, “Prevention of Obesity Related Metabolic Diseases by Bioactive Components of Food Processing Waste Byproducts and Mitigation of Food Allergies”, which started in May 2020.
In support of Objective 1, ARS researchers in Albany, California, conducted a 14 month long study that analyzed the effects of mice that were fed a high-fat diets supplemented with grape seed polyphenolics. Grape seed polyphenolic extracts or Chardonnay grape seed flour were supplemented in the high-fat diets fed to male C57Bl/6J mice and comparisons were made to the high-fat diet alone or a low-fat diet. The primary objective of this study was to determine learning and memory in aged mice. ARS researchers determined that grape seed diets improved memory performance in the Morris water maze and novel object tests. They also analyzed gene expression (RNAseq) in the hippocampus and adipose and taxa of the microbiome (16S). A manuscript is in progress.
In additional support of Objective 1, ARS researchers in Albany, California, conducted a study of Valencia orange peel and green bitter orange peel extracts to inhibit the formation of trimethylamine from carnitine in mice on high-fat diets. Trimethylamine is converted into trimethylamine oxide by the liver and is considered a risk factor for cardiovascular disease. ARS researchers did not see a significant decrease in body weight or adipose weight by the orange peel extracts but did see an effect due to carnitine. In a subsequent study of the polar and nonpolar fractions of orange peel extract, they found that the polar fraction reduced the conversion of carnitine to trimethylamine by gut bacteria. ARS researchers identified the active compound to be feruroyl putresceine. A further study of blood and urine collected from mice fed feruroyl putresceine and carnitine confirmed its inhibitor activity to the formation of trimethylamine and trimethylamine oxide. A manuscript has been published. A similar study in female and male mice is in progress.
Also in support of Objective 1, ARS researchers in Albany, California, found that extracts of almond hulls, a waste byproduct of almond seed production, produced bioactivity in fungi grown in the extract. Two types of edible food approved fungi, Aspergillus oryzae and Aspergillus awamori, were grown in either almond hull extract or semisynthetic media. The fungi were dried, ground to a powder and fed in high-fat diets to mice. Awamori grown in the almond hull and synthetic media had lower adipose weight. All fungi diets tended to reduce liver weight and tended to reduce adipose weight in the oryzae groups. Adipose and liver gene expression were analyzed as well as gut microbiome by 16S methods. A manuscript is in preparation.
In further support of Objective 1, ARS researchers in Albany, California, examined the combination of cinnamon polyphenolics and astragalus root extracts in mice on high-fat diets. Cinnamon alone and in combination with astragalus root extract reduced weight gain but there were no differences between the two cinnamon treatments.
In support of Sub-objective 3A, ARS researchers in Albany, California, constructed two vectors to express the designed milk allergen beta-Lactoglobulin reverse mutants in lactic acid bacteria and secreted to the growth media without an express tag. The team also obtained four sera of milk-allergic subjects in large enough quantities for the designed experiments.
Progress on Sub-objective 3B included ARS researchers in Albany, California, continuing their efforts to determine the sequence of the macadamia nut proteins that react to a custom-made antibody. Ongoing work also includes the isolation and characterization of peach proteins that cross-react with almond allergen Pru du 8.
Accomplishments
1. Grape seed polyphenolics prevent age and obesity induced memory loss. Most (90%) of the $4.5 trillion health budget is spent on chronic disease and mental health problems such as Alzheimer’s disease. Inflammation, accompanying obesity, is the root cause of most chronic diseases. Grapeseed polyphenolics reduce weight gain and inflammation in animal obesity-focused study models. ARS scientists in Albany, California, found that mice on high-fat diets supplemented with grapeseed or grapeseed extracts had improved performance in memory tests. There were significant differences in expression of genes in the hippocampus and in the taxonomy of gut bacteria with and without grapeseed treatments. This study suggests that antioxidants such as grapeseed polyphenolics in the diet may help to prevent chronic diseases, including Alzheimer’s.
2. Orange peel extract prevents gut bacteria from producing a compound that increases risk of cardiovascular disease. In 2021, about 700,000 people died from cardiovascular disease (CVD) in the United States. Trimethylamine oxide (TMAO) is a risk factor for CVD. Gut bacteria releases trimethylamine (TMA) from carnitine and other food and TMA is converted to TMAO in the liver. ARS scientists in Albany, California, fed mice high-fat diets supplemented with carnitine or carnitine and orange peel extracts. They found TMA and TMAO in the blood and urine were reduced when fed orange peel extracts. This shows that the formation of TMA, a known risk factor for CVD, is reduced by intake of orange peel extract.
3. Binary combinations of functional foods. A study of mice that were fed a high-fat diet shows that adding bioprocessed black rice bran and green tea extract to the diet resulted in a synergistic loss of weight gain during an eight-week high-fat feeding study as compared to the two diets in which only one of the supplements was added. ARS researchers in Albany, California, conducted additional studies on the content of insulin, glucose, cholesterol, triglycerides, HDL, and LDL cholesterols in serum and associated biomarkers, which appear to indicate that, in addition to weight loss, the combination diet has the potential to treat/prevent diabetes and heart disease and to also prevent liver damage. Their results also show that gut hormones and inflammatory cytokines seem to be involved in governing the anti-obesity mechanisms in the mice. These considerations suggest the need for human trials to confirm the rodent data. A manuscript has been submitted for publication.
4. The hazelnut allergen, Cor a 9, is a heterocomplex. In the United States alone, the costs borne by families due to food allergies and the direct medical costs of food allergies were estimated at $24.8 billion annually. Defining all the allergens in an allergenic food is crucial for allergen detection and mitigation. ARS researchers in Albany, California, discovered, for the first time, a food allergen with multiple isoforms exists naturally in food as a complex of different molecules. The new information about food allergens may have a profound impact on the definition of allergens and the understanding of the allergenicity of food proteins. Better definitions of food allergens can be used to improve food allergy diagnosis and prognosis and enable food processors to efficiently develop mitigation methodologies to improve food product marketability.
5. Bioprocessed black rice bran enhances the growth inhibitory effect of an immune checkpoint inhibitor against murine colon tumors. Black rice bran, a waste product produced during the milling process of black rice which removes the bran and germ, contains bioactive phenolic, flavonoid, and phytosteroid compounds that may be responsible for reported antioxidant and anti-inflammatory activities resulting in health-promoting effects including antiproliferative properties. The bioprocessed black rice bran used in this study is a fermentation product of black rice bran using shiitake mushroom, mycelia. Immune checkpoint inhibitors that affect antitumor immune cells are used in medicine for the treatment of a wide range of cancers. ARS researchers in Albany, California, administered this new food product to mice that had colon tumors, and the results showed that this food additive significantly reduced tumor size by approximately 48%, as compared to the control diet. The additional administration of an immune checkpoint inhibitor increased the reduction in tumor size from about 48% to 63%, which was significant. The mechanism that governs the anti-tumor effects is like that reported for medical human chemotherapies.
Review Publications
Lee, K., Kwon, K., Hwang, W., Lee, W., Kim, J., Lee, S., Kim, S., Friedman, M. 2023. Bioprocessed black rice bran potentiates the growth inhibitory activity of an immune checkpoint inhibitor against murine colon carcinoma. Food and Nutrition Sciences. 14(12):1149-1171. https://doi.org/10.4236/fns.2023.1412072.
Lee, H., Koh, G., Lee, H., Alves Buongiorno, P.L., Yokoyama, W.H., Wang, Y. 2024. Discovery of a novel bioactive compound in orange peel polar fraction on the inhibition of trimethylamine and trimethylamine N-oxide through metabolomics approaches and in vitro and vivo assays: Feruloylputrescine inhibits. Journal of Agriculture and Food Chemistry. 72(14):7870-7881. https://doi.org/10.1021/acs.jafc.3c09005.
Guo, F., Zhang, Y., Howard, A., Xu, Y. 2024. Crystal structure of hetero hexameric 11S seed storage protein of hazelnut. Plant Physiology and Biochemistry. 210. Article 108653. https://doi.org/10.1016/j.plaphy.2024.108653.
Garcia-Rocha, K.F., Capaceta-Osuna, A., Ochoa-Acosta, A., Avena Bustillos, R.D., Osuna-Martinez, U., Cárdenas-Torres, F.I., Yokoyama, W.H., McHugh, T.H., Teran-Cabanillas, E. 2023. Upcycled romaine lettuce powder as a dietary supplement for control of metabolic syndrome. ACS Food Science and Technology. 3(8):1393-1402. https://doi.org/10.1021/acsfoodscitech.3c00204.