Project : USDA ARS

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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

2022 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, a long-term feeding study of the effects of high fat diets compared to low fat diets on memory in mice was started. The duration of the feeding phase is 10 months and will be completed in October 2022. In addition to comparing the effects of high fat to low fat diets, high fat diets supplemented with grapeseed polyphenolic extracts and grapeseed flour are being fed to compare effects of grapeseed supplementation to high fat alone. In support of Objective 2, Ecklonia cava, an edible marine brown alga, and shiitake mushrooms were fermented. The treatment inhibited the degranulation of RBL-2H3 cells and immunoglobulin E (IgE) production, suggesting anti-asthma effects in vitro. The in vitro effects in cells were confirmed in mice following the induction of asthma by alumina and chicken egg ovalbumin (OVA). Oral administration of the bioprocessed product and purified fractions suppressed induction of asthma and was accompanied by inhibition of inflammation- and immune-related substances associated with asthma. The in vitro cell and in vivo mouse assays demonstrate the potential value of the food formulation as an anti-inflammatory and anti-allergic formulation against allergic asthma and might also ameliorate allergic manifestations of foods, drugs, and viral infections. In support of Objective 3, researchers in Albany, California, Hilo, Hawaii, and Corvallis, Oregon, initiated a collaborative project to evaluate the effectiveness of pre- and post-harvest treatments to modify protein content of hazelnuts. Efforts for this year included establishment of in-house chemical and near-infrared spectroscopic methods to measure protein, moisture, lipid and oxidized lipid levels. The in-house methods were applied to an initial sample set of nine cultivars.

Accomplishments
1. Tomato leaf powders inhibit pathogenic disease organisms. Trichomonads are parasitic protozoa that cause sexually transmitted disease in cattle and humans. ARS researchers in Albany, California, prepared powders from various parts of tomato plants to determine their efficacy in inhibiting the growth of three pathogenic trichomonads. The powders inhibited pathogen growth to varying degrees without affecting beneficial lactobacilli found in the normal flora of humans and, or, cattle. The results suggest that these powders derived from tomato offer a natural, alternative treatment against infections caused by pathogenic protozoa, bacteria, and fungi. Tomato leaf, a waste material, may be a useful antiparasitic feed ingredient for ranches susceptible to trichomonads.

2. An edible alga has anti-asthma potential. About seven percent of the U.S. population suffers from asthma. ARS researchers in Albany, California, fermented Ecklonia cava, an edible marine brown alga, with shiitake mushroom Lentinula edodes mycelia and isolated fractions. The treatments inhibited the degranulation of RBL-2H3 cells and immunoglobulin E (IgE) production, suggesting anti-asthma effects in vitro. The in vitro effects in cells were confirmed in mice following induction of asthma by alumina and chicken egg ovalbumin (OVA). Histology of lung tissues revealed that the treatment also reversed thickening of the airway wall and contraction and infiltration of bronchial and blood vessels and perialveolar inflammatory cells. This research may result in the development of fermented products with anti-asthma properties.

3. Plant-based antimicrobials from olive and oregano are effective on melons. Melons are often contaminated with pathogenic microbes in the field. ARS researchers in Albany, California, applied plant-based antimicrobials that reduced Salmonella Newport and Listeria monocytogenes populations on melon rind samples, regardless of the melon types, varieties or growing locations. Compared to the control, antimicrobial treatments caused up to 3.6 and 4.0 log reductions in populations of the pathogens, respectively. In most cases, the treatments reduced pathogen populations to below the detection limit (1 log CFU/g) at day three. Producers, restaurants, wholesalers, and consumers may prefer organic sources of disinfectants for food applications.

U.S. DEPARTMENT OF AGRICULTURE

Healthy Processed Foods Research: Albany, CA