Location: Food Processing and Sensory Quality Research
2022 Annual Report
Objectives
Objective 1: Decipher the molecular, structural and immunological properties of purified native and recombinant allergens that contribute to allergenic potency towards development of therapeutic products. [NP306, C1, PS1C]
Objective 2: Use serum from verified nut allergic and non-allergic individuals to identify and compare IgE and IgG binding sites (or epitopes) of known nut allergens with peptide microarrays to understand cross-reactivity between multiple allergens and improve diagnosis of nut allergy. [NP306, C1, PS1A]
Objective 3: Characterize, quantify and monitor allergen characteristics and levels pre- and post-harvest, and pre- and post-processing of commercial nuts and nut-containing foods, and during nut seed development to produce hypoallergenic, prophylactic or therapeutic food products. [NP306, C1, PS1B]
The immunoglobulin E (IgE) binding sites (epitopes) that are responsible for the symptoms of allergic disease and cross-reactivity among peanut, tree nut and pollen allergens will be identified with peptide microarray technology. The IgE and immunoglobulin G4 (IgG4, thought to act as an IgE-blocking antibody) epitopes will be identified for the most potent nut allergens. These will be modeled on the surface of allergen structures to identify location and common or cross-reactive (or potentially blocked) sequences and structures of allergens among nuts and pollens. Simultaneously, the changes in peanut and tree nut extracts or purified allergens thereof (recombinant or native) will be assessed before and after processing treatments for changes in allergenic properties. Proteins found to be immunologically altered by processing will be assessed within a total nut extract or they will be purified and analyzed for alterations in size, structure, digestibility, binding to various serum IgE and allergen-specific antibodies. The specific amino acid residues, or peptides thought to be modified during different processing events, and thought to contribute to altered allergenic properties, will be identified by mass spectrometry. The studies above will be combined to identify the influence of the processing-induced alteration in relationship to the immunoglobulin binding sites of nut allergens. This will guide the development of better diagnostics and therapeutics as well as processing technologies to reduce allergenicity of nuts and products thereof. Early intervention methods to reduce the allergenic potential of nuts, the natural variation in allergen gene sequence, expression, post-translational modification, stability and accumulation patterns in a model tree nut (pecan) will be studied. Less allergenic variants and factors that can be used to interfere with allergen accumulation in plants will also be characterized in detail. Collectively our studies will also contribute to the development of better detection tools and labeling practices for industry and regulatory agencies resulting in better protection of consumers.
Approach
The immunoglobulin E (IgE) binding sites (epitopes) that are responsible for the symptoms of allergic disease and cross-reactivity among peanut, tree nut and pollen allergens will be identified with peptide microarray technology. The IgE and immunoglobulin G4 (IgG4, thought to act as an IgE-blocking antibody) epitopes will be identified for the most potent nut allergens. These will be modeled on the surface of allergen structures to identify location and common or cross-reactive (or potentially blocked) sequences and structures of allergens among nuts and pollens. Simultaneously, the changes in peanut and tree nut extracts or purified allergens thereof (recombinant or native) will be assessed before and after processing treatments for changes in allergenic properties. Proteins found to be immunologically altered by processing will be assessed within a total nut extract or they will be purified and analyzed for alterations in size, structure, digestibility, binding to various serum IgE and allergen-specific antibodies. The specific amino acid residues, or peptides thought to be modified during different processing events, and thought to contribute to altered allergenic properties, will be identified by mass spectrometry. The studies above will be combined to identify the influence of the processing-induced alteration in relationship to the immunoglobulin binding sites of nut allergens. This will guide the development of better diagnostics and therapeutics as well as processing technologies to reduce allergenicity of nuts and products thereof. Early intervention methods to reduce the allergenic potential of nuts, the natural variation in allergen gene sequence, expression, post-translational modification, stability and accumulation patterns in a model tree nut (pecan) will be studied. Less allergenic variants and factors that can be used to interfere with allergen accumulation in plants will also be characterized in detail. Collectively our studies will also contribute to the development of better detection tools and labeling practices for industry and regulatory agencies resulting in better protection of consumers.
Progress Report
ARS researchers at New Orleans, Louisiana, made progress on all 3 National Program 306 objectives under, “Plant Product Development, Quality, and Marketability.” The project addresses National Program 306 Action Plan Component 1 Food: Problem Statement 1.A Define, Measure, Preserve/Enhance/Reduce Attributes that Impact Quality and Marketability, 1.B New Bioactive Ingredients and Functional Foods and 1.C New and Improved Food Processing Technologies.
In support of Objective 1, ARS researchers showed that small fragments of proteins known as leader sequences, thought to be degraded during plant development, are stable and can cause an allergic response. We were able to create the leader sequence of vicilin proteins from peanut, walnut, pistachio, cashew, and tomato (a non-allergic control) using recombinant bacteria. The structure of peanut and walnut vicilin leader sequences were determined by nuclear magnetic resonance (NMR) and tested for allergic response. The methods developed using the recombinant bacteria will be used for future studies.
The goal of Objective 2 is to identify the sections of an allergen (often a protein) that illicit an immune response in allergic individuals. To accomplish this, we identified target binding sites of specific antibodies, called immunoglobulin E (IgE) and immunoglobulin G4 (IgG4) that are involved in food allergic reactions. Most believe that that IgE makes allergic reactions worse, while IgG4 is thought to reduce allergic symptoms, but both of them interact with the allergen; maybe even compete. Hypothetically, increasing the levels of IgG4 through therapeutic treatments could reduce allergic responses. To investigate how the antibodies interact with the allergens or sections of the allergens, ARS researchers attached pieces of known allergens on the surface of glass slides and mixed with allergic patient blood serum. This allowed ARS scientist at the SRRC in New Orleans, LA to identify the specific parts of allergenic proteins recognized by antibodies in the blood of people with allergies to peanut and tree nut proteins. As a result, ARS scientists identified antibody binding sites for 100 allergic proteins using blood serum from 100 volunteers in the U.S. with peanut and tree nut allergies. ARS scientist submitted this data to a ‘big data’ collection. Other scientists will be able to use these data to improve the prediction of the allergic potential of the allergens studied.
In support of Objective 3, we evaluated the genetic diversity of pecans from four locations. The results of the genetic analysis provide an understanding of what genes are important for certain nut characteristics. For example, some pecan nut allergen gene sequences were very similar among the cultivars, but others varied. By using genetically defined trees for breeding, the pecan industry will be able to better select desired traits of pecans. These desired traits include climate resistance, fungal and pest resistance, salinity and drought tolerance, and reduced allergen content. The outcome of this research will benefit the U.S. pecan industry and pecan nut consumers.
Food processing steps can alter how the immune system reacts to nut allergens. Fermentation is used to generate several common foods including tempeh, yogurt, and cheese. ARS researchers at New Orleans, Louisiana, assessed the effects of fermentation on the allergenic properties of peanut and tree nut allergens. While fungal fermentation reduced allergen-IgE binding, a substantial amount of IgE binding activity remained. The results indicate a fermented product would not be safe for consumption by those with peanut or tree nut allergy.
Accomplishments
1. Fragments of major allergens in cashew and pistachio contribute to the allergic reactions observed with nut seeds.(referred to as cross-reactivity). Vicilins are seed storage proteins from which a fragment is cleaved after it serves the purpose of directing the protein to the correct compartment in the seed. The scientific community has assumed the severed fragments, referred to as leader sequences (LS), degrade rapidly. However, ARS researchers at New Orleans, Louisiana, identified the LS of vicilins from cashew (Ana o 1 LS) and pistachio (Pis v LS) and found that they contained binding sites for IgE, a protein that plays a role in allergic reactions. ARS scientists found that both common amino acid sequences and the protein structures containing these IgE binding sites contributed to the allergic reaction of certain individuals to multiple nuts, also known as cross-reactivity. Understanding the interaction of the allergic human’s immune system with the seed molecules that illicit an allergic reaction, allows ARS scientists to develop better detection, diagnostic, and therapeutic tools to alleviate allergies to nuts.
2. Development of antibodies and immunoassays to detect walnut and peanut allergens in foods. Methods were developed for empirical purification of single allergens from peanut and walnut, which are then used for antibody production. The antibodies made against the allergens are extensively tested for specificity, sensitivity and utility. Various types of very useful immune assays were developed for these antibodies to detect and quantify allergens within differently cooked and processed foods (i.e. extruded or roasted nuts) and products. In addition to research, these assays are useful for the food and pharmaceutical industry that need to detect quantify allergens in products.
3. The major peanut allergen content during development of meat alternatives by high-moisture extrusion processing of peanuts in the presence of transglutaminase. High moisture extrusion in the presence of cross-linking chemicals like transglutaminase are being tested to produce plant-based, meat alternatives. In order to monitor allergen content and potency, ARS scientists and collaborators determined that a three-stage sequential protein extraction significantly improved the protein recovery in processed samples, which in turn gave more accurate measurements of allergenic protein content. Extrusion reduced the soluble allergen content significantly with a significant reduction in overall IgE antibody binding, which is the most important allergy associated antibody in humans. In different processing zones of the extruder, the most significant reduction in allergenic proteins was in the melting zone. It was found that the alteration in secondary and tertiary protein structures resulting from transglutaminase-induced crosslinking, shearing, and degradation of proteins is likely to reduce allergenic potency by altering IgE binding sites.
4. Completed pecan genome sequences reveal pecan allergen similarity and provide genetic targets for desirable traits. U.S. pecan production is no longer number one in the world. An increased understanding of the genes controlling beneficial characteristics including increased climate resistance, fungal and pest resistance, salinity and drought tolerance, and reduced allergen content will allow U.S. pecan farmers to regain a competitive edge in the global market. With traditional breeding, it can take more than ten years to identify pecan varieties with improved traits. ARS researchers at New Orleans, Louisiana, sequenced the genomes of four genetically important pecan cultivars from different locations. Several genetic markers associated with important plant characteristics were identified. The genome sequences and genetic markers obtained by this research will allow for more directed strategies and reduce the time needed to create new pecan cultivars with desirable traits.
Review Publications
Mattison, C.P., Vant-Hull, B., de Castro, A., Chial, H.J., Bren-Mattison, Y., Bechtel, P.J., de Brito, E.S. 2021. Characterization of anti-ana o 3 monoclonal antibodies and their application in comparing brazilian cashew cultivars. Antibodies. 10(4):46. https://doi.org/10.3390/antib10040046.
Foo, A.C.Y., Nesbit, J.B., Gipson, S.A.Y., Cheng, H., Buschel, P., Derose, E.F., Schein, C.H., Teuber, S.S., Hurlburt, B.K., Maleki, S.J., Mueller, G.A. 2022. Structure, immunogenicity, and IgE cross-reactivity among walnut and peanut vicilin buried peptides. Journal of Agricultural and Food Chemistry. 70(7):2389-2400. https://doi.org/10.1021/acs.jafc.1c07225.
Faisal, S., Zhang, J., Meng, S., Shi, A., Li, L., Wang, Q., Maleki, S.J., Adhikari, B. 2022. Effect of high-moisture extrusion and addition of transglutaminase on major peanut allergens content extracted by three step sequential method. Food Chemistry. 385:132569. https://doi.org/10.1016/j.foodchem.2022.132569.
Lovell, J.T., Bentley, N.B., Bhattarai, G., Jenkins, J.W., Sreedasyam, A., Alarcon, Y., Bock, C., Boston, L.B., Carlson, J., Cervantes, K., Clermont, K., Duke, S., Krom, N., Kubenka, K., Mamidi, S., Mattison, C.P., Monteros, M.J., Pisani, C., Plott, C., Rajasekar, S., Rhein, H.S., Rohla, C., Song, M., St. Hilaire, R., Shu, S., Wells, L., Webber, J., Heerema, R.J., Klein, P.E., Conner, P., Wang, X., Grauke, L.J., Grimwood, J., Schmutz, J., Randall, J.J. 2021. Four chromosome scale genomes and a pan-genome annotation to accelerate pecan tree breeding. Nature Communications. 12:4125. https://doi.org/10.1038/s41467-021-24328-w.
He, Z., Mattison, C.P., Zhang, D., Grimm, C. 2021. Vicilin and legumin storage proteins are abundant in water and alkali soluble protein fractions of glandless cottonseed. Scientific Reports. 11. Article 9209. https://doi.org/10.1038/s41598-021-88527-7.
Mattison, C.P., Mack, B.M., Cary, J.W. 2021. Comparative transcriptomic analysis of Aspergillus niger cultured in peanut or cashew nut flour based media. Journal of Applied Biology & Biotechnology. 9(05):56-63. https://doi.org/10.7324/JABB.2021.9508.
