Location: Food Processing and Sensory Quality Research
2020 Annual Report
Objectives
Obective 1: Enable new commercial methods to reduce or eliminate the allergenic properties of peanut and tree nut products.
Objective 2: Integrate overall oral and food allergenic properties of native and recombinant allergens with cross-reactivity among nuts, between nuts and pollens, and with pre and postharvest processing.
Objective 3: Integrate allergenic properties and molecular changes with commercial tree nut development.
Objective 4: Enable new commercial immunoassays for detection of allergens in processed foods.
Approach
The immunoglobulin E (IgE) binding sites 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 epitopes will be modeled on the surface of allergen structures to identify location and common or cross-reactive sequences and structural regions of allergens among nuts and pollens. Simultaneously, peanuts, tree nuts or purified allergens thereof (recombinant or native) will be subjected to existing and novel processing techniques (i.e. heat, chemical and enzymatic treatment). New allergens or changes in allergenic properties of existing allergens due to the processing methods will be identified by immunoassays with serum (containing IgE antibodies) from peanut and or tree nut allergic individuals. Proteins found to be immunologically altered by processing will be assessed within the food matrix or they will be purified and analyzed for alterations in size, structure, digestibility, binding to various antibodies, including, serum IgE, known anti-processing reaction products, and allergen specific antibodies. The specific amino acid residues, or peptides thought to be modified during different processing events, and to contribute to altered allergenic properties will be identified by mass spectrometry. Understanding the molecular basis of processing-induced alterations of allergens with respect to the IgE binding sights will guide the development of processing technologies towards reduced allergenicity of nuts and products thereof. This knowledge will also contribute to the development of better detection tools and labeling practices for industry and regulatory agencies resulting in better protection of consumers. As a possible early interventional method to reduce the allergenic potential of nuts, the expression and accumulation patterns of allergens in a model tree-nut (pecan) will be studied under various conditions, which may allow interference with their accumulation in the future.
Progress Report
This is the final report for project 6054-43440-046-00D, which is being replaced by 6054-43440-052-00D. Progress was made by ARS researchers at New Orleans, Louisiana, on all 4 objectives, all of which fall under National Program 306, “Plant Product Development, Quality, and Marketability.” The project will address NP 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. The following summarizes the progress/accomplishments over the 5 year lifespan of the project:
2015-Objective 1. IgE antibodies are immunological molecules derived from blood that upon binding to allergens can cause the symptoms of allergy. It was shown that a combination amino acids inhibited IgE antibodies binding to peanut allergens. Roasting lead to chemical changes to the most potent peanut allergen, Ara h 2. These modifications were identified by ARS researchers at New Orleans, Louisiana, and shown to change allergenic properties, such as IgE binding and digestion in the stomach. Treatment of peanut and cashew allergens with oleic acid and pulsed light were also explored for reducing the allergenic potency. These results also address Objective 2 to correlate processing induced-modification of allergens with overall allergenic potential. Multiple allergens from peanut and tree nut were produced in bacteria and methods were developed to purify them under Objectives 2 & 4. Purified allergens were used to produce antibodies in order to develop and perform immunological studies, and to study allergen structure. This is important for understanding the allergens and effects of food processing on how they interact with the human immune and digestive systems. These projects involved development of sophisticated biomedical techniques for the evaluation of various antibodies and immunoassays addressing Objectives 1, 2, 3 and 4.
2016 - To meet Objective 1, heat treatment of cashew proteins was shown to make small allergens more soluble and relatively larger allergens less soluble. Novel sources, such as Aspergillus, a fungus typically used in fermentation, were explored for their ability to chop up cashew proteins to reduce the allergenic potential. Under of Objective 2, small quantities of allergenic proteins from hazelnut, cashew, pecan and peanut were purified by ARS researchers at New Orleans, Louisiana, and used to generate antibodies. IgE antibody binding sites on different allergens were identified by experimentation in the lab and compared to IgE binding sites predicted by a computer. Under Objective 3, pecan nut genetic material from different pecan varieties was compared to identify sequence differences. Under Objective 4, select allergens from processed nuts were used in antibody production to study the effects of food processing on allergenicity.
2017 - Under Objective 1, generally recognized as safe (GRAS or food safe) compounds, such as food-grade fermenting agents continued to be tested for reducing IgE binding to cashew allergens and degrading of cashew proteins to reduce their allergic potential. Under Objective 2, rapid purification methods were developed for allergic proteins from hazelnut, cashew, walnut, almond and peanut for further immunological and molecular studies and to develop better detection methods. Immunological methods were used to identify IgE binding locations on the surface of key peanut and tree nut allergens and compared with computer-based predicted ones to enhance and test the accuracy of the prediction methods. Under Objective 3, new proteins contained within pecan nuts were identified and a large library listing all these proteins was updated. Certain metabolism genes were identified as important during pecan nut development. To meet Objective 4, it was determined by ARS researchers at New Orleans, Louisiana, that antibodies made against allergens from raw nuts were able to detect allergens from roasted or processed nuts and that most likely the difficulty in detecting allergens in processed food is due to reduced solubility of proteins in processed nuts, rather than recognition by the antibody.
2018 - Under Objective 1, the allergenic potential of pistachio and cashew was shown to be reduced by digestion with food-grade enzymes under ultrasound treatment. Also, naturally occurring polyphenolic compounds in pomegranate, blueberry, and Concord grape juice were evaluated for allergen reduction in cashew extract. Under Objective 2, IgE-binding sites continued to be identified for multiple nut allergens and computationally predicted immunogenic peptides were tested for IgE binding nut allergic sera. IgE binding sites that were similar between multiple allergens in different nuts were identifies. For Objectives 2 & 4, antibodies were produced against the major cashew allergens, and newly purified minor peanut allergens to develop better detection methods. Under Objective 3, the genes discovered in a library of expressed pecan nut genes from pecan varieties such as the Sumner cultivar continued to be updated and compared to other cultivars to identify significant differences in allergen sequences. Gene sequence updates and a pecan protein library were used to characterize the timing and accumulation of allergens during pecan development.
2019 - Under Objective 1, the effect of different cold plasma processing conditions were assessed by ARS researchers at New Orleans, Louisiana, on cashew nut allergens. Food grade Lactic acid bacterial fermentation of cashew extract caused two of the main cashew allergens to be more susceptible to degradation. Aspergillus niger, a food-grade fungus, was grown in peanut or cashew nut flour containing media and used to identify previously unknown food-grade enzymes (i.e. oryzin and the aspartic endopeptidase aspergillopepsin) that degrade allergens. Under Objective 2, IgE-binding sites were identified for multiple nut allergens and compared to computer predicted immunogenic peptides. Similar IgE binding sites between different protein families (vicilin and 2S albumin), which are important for food allergy were determined by the combination of computer and laboratory-based assays. For Objectives 2 & 4, major allergenic proteins of walnut and cashew were purified on a large scale and used to generate antibodies in rabbits, chickens and mice, which were used for immune-based assay development for detection and characterization of allergens in foods. A new peanut allergen, Ara h 1 leader sequence, was identified and characterized. Under Objective 3, characterization and description of genes from a library of genes in a developing pecan variety continued to be updated and compared to other varieties to find differences in pecan allergen sequences. Preliminary differences in the timing of allergen accumulation in the Sumner and Desirable pecan varieties were identified.
2020 - For Objective 1 the interaction of raw and roasted allergens with immune system cells demonstrated that internalization of these allergens into the cell was higher for the roasted allergens. The mechanism was described. For Objective 2, two new allergens from peanut and walnut, that were thought to be degraded within the nut, were identified, submitted and accepted as new allergens by the Allergen Nomenclature sub-committee as new allergens. They were shown to be similar to ancient, highly conserved proteins that seem to be repeated in many plants and can contribute to the allergic reaction to multiple nuts. IgE binding segments of a potent peanut allergen were found to enhance the diagnostic ability, which improves upon current diagnostics. Under Objective 3, characterizing the genetic diversity of pecan allergens has revealed potential differences in the allergens. Twenty six new proteins were identified that accumulate differentially during nut development in different pecan varieties. For Objective 4, antibodies to the main cashew nut allergen have been developed and used to improve cashew detection in food.
Accomplishments
1. The first food approved by FDA as a pharmaceutical. Oral immunotherapy is a promising approach to desensitize food allergic individuals, which involves daily ingestion of gradually increasing doses of an allergic food over time. Until this year (2020) no treatment has been available for peanut allergy. Standardized pharmaceutical drug development requires extensive characterization and quality control. ARS scientists in New Orleans, Louisiana have facilitated this by developing and transferring multiple chemical, physical and immunological tools and methods necessary for extensive analysis of the allergens in peanut flour. Peanut flour is the very first food to be characterized as a pharmaceutical and approved by FDA. This drug is now marketed as oral immunotherapy for treatment of peanut allergy.
2. Generation of Big Data on the IgE and IgG4 epitopes of peanut and tree nut allergens. IgE is the antibody mediator of allergic symptoms and IgG4 is thought to block IgE binding to allergens. Due to symptom differences, it is unknown if the IgE and IgG4 binding to allergens is different in various locations around the world. ARS scientists in New Orleans, Louisiana, completed the screening of ~700 patient sera from various locations around the U.S. and the world for IgE and IgG4 binding to intact allergen and fragments of those allergens (peptides) using microarray technology. This data allows ARS to establish a Big Data, searchable database to identify the major reactive allergic proteins and parts of those allergens that are most involved in allergic reactions in different location around the U.S. which in turn, will allow development and use of more targeted and individualized therapeutics and diagnostics for allergic consumers.
3. Identification of peptide combinations that enhance diagnosis of peanut allergy over the existing methods. The only reliable diagnosis for a food allergy is oral food challenge which is time consuming, laborious, costly and dangerous. ARS scientists in New Orleans, Louisiana, hypothesized that an in vitro blood test could replace oral food challenge. Preliminary multivariant analysis of the IgE/IgG4 peptide binding patterns of blood serum from proximately 200 peanut allergic and non-allergic individuals from around the U.S. indicates that this method improves diagnosis of peanut allergy over the existing in vitro marketed methods and may be a real alternative to oral food challenge. Also, IgE epitopes of peanut allergic children in UK identified by microarray showed that addition of Ara h 2-IgE epitopes to intact Ara h 2 enhance the existing diagnostic accuracy of intact Ara h 2-specific IgE measurements alone. Improved diagnostics would reduce the cost, length, and number of physician visits and the difficulties and dangers associated with diagnosis of food allergy by oral food challenge.
4. Discovery of peptide biomarkers that may indicate responsiveness to oral immunotherapy. Oral immunotherapy is a promising treatment for food allergy, but currently the outcomes of this treatment is measured by oral food challenge. ARS scientists in New Orleans, Louisiana, have compared the patient serum IgE/IgG4 peptide recognition patterns from pre and post peanut oral immunotherapy Phase 2 and 3 trials with a collaborator. The data will lead to biomarker (or individual peptides) discovery that will allow physicians to monitor the progress of desensitization and as an endpoint test for oral immunotherapy that doesn’t involve oral food challenge (invention disclosure filed).
5. Proteomic analysis of pecan nut development. The timing and levels of allergen accumulation in pecan nut and its association with weather and other plant traits are not known. Therefore, ARS scientist at New Orleans, Louisiana, have compared the timing and accumulation of proteins in developing pecan nuts. Mass-spectrometric analysis of pecan nut samples allowed the identification of 556 different proteins. Computerized comparisons allowed ARS researchers at New Orleans, Louisiana, to characterize the accumulation of individual proteins and compare this process between different pecan cultivars. This process revealed 26 protein spots that accumulated differentially between the cultivars tested. These results can be used to correlate important characteristics like drought tolerance, pest resistance, and allergen accumulation to specific proteins and may improve breeding strategies resulting in pecan nuts with reduced allergen content and increased quality and yield.
6. Identification of novel allergens in peanut and walnut that contribute to cross-reactivity. Cross-reaction of allergic individuals with peanut and tree nut allergens is a commonly seen problem and allergists generally recommend that patients with an allergy to one nut avoid all nuts. This poses a serious quality of life and nutritional problem for the patient and their family, as many nut allergic individuals have multiple allergies (i.e. egg, milk, wheat, soy, etc). ARS scientists in New Orleans, Louisiana, identified fragments of Ara h 1 (a major peanut allergen) and Jug r 2 (a major walnut allergen) from peanut and walnut, that were thought to be degraded within the nut by a combination of bioinformatics prediction, immunoassays and shown to contribute to cross-reactions among walnut and peanut allergic individuals. They were submitted and accepted as novel allergens by the World Health Organization-Allergen Nomenclature Sub-Committee. Parts of these new allergens were found to be repeated among major allergens which could to contribute even further cross-reactivity among nuts. Understanding what causes cross-reactivity, will allow allergists to eliminate relevant nuts and foods from a patient’s diet as opposed to blanket eliminations that are commonly practiced today.
Review Publications
Filho, E.G., Silva, L.A., Filho, F., Rodrigues, S., Fernandes, F.A., Gallo, M., Mattison, C.P., De Brito, E.S. 2019. Cold plasma processing effect on cashew cuts composition and allergenicity. Food Research International. 125:108621. https://doi.org/10.1016/j.foodres.2019.108621.
Maleki, S.J., Crespo, J.F., Cabanillas, B. 2019. Anti-inflammatory effects of flavonoids. Journal of Food Chemistry. 299:125124. https://doi.org/10.1016/j.foodchem.2019.125124.
Mattison, C.P., Vant-Hall, B., Bren-Mattison, Y., Grimm, C.C. 2019. A cashew specific monoclonal antibody recognizing the small subunit of Ana o 3. Toxicology Reports. 6:736-744. https://doi.org/10.1016/j.toxrep.2019.06.018.
Ghasemi, A., Falak, R., Mohammadi, M., Maleki, S.J., Assarezadegan, M.A., Jafary, M. 2020. Incorporation of T-cell epitopes from tetanus and diphtheria toxoids into in-silico-designed hypoallergenic vaccine may enhance the protective immune response against allergens. Iranian Journal of Basic Medical Sciences. 23:636-644.
Novak, N., Maleki, S.J., Cuadrado, C., Crespo, J.F., Cabanillas, B. 2020. Interaction of monocyte-dervied dendritic cells with Ara h 2 from raw and roasted peanut. Foods. 9:863. http://dx.doi.org/10.3390/foods9070863.
Santos, A., Nuno, B., Hurlburt, B.K., Sneha, R., Kwok, M., Bahnson, H., Cheng, H., James, L., Maleki, S.J., Lack, G., Gould, H., Sutton, B. 2020. IgE to epitopes of Ara h 2 enhance the diagnostic accuracy of Ara h 2-specific IgE. Allergy. https://doi.org/10.1111/all.14301
Nesbit, J.B., Schein, C.H., Braun, B.A., Gipson, S.A., Cheng, H., Hurlburt, B.K., Maleki, S.J. 2020. Epitopes with similar physicochemical properties contribute to cross reactivity between peanut and tree nuts. Molecular Immunology. https://doi.org/10.1016/j.molimm.2020.03.017.
Xie, M.M., Liu, H., Yang, K., Koh, B., Wu, H., Maleki, S.J., Hurlburt, B.K., Kaplan, M.H., Dent, A.L. 2020. Peanut-specific IgE produced in a mouse food allergy model requires help from T follicular regulatory cells and IL-10 signaling by B cells. Journal of Clinical Investigation. 130(7):3820-3832. https://doi.org/10.1172/JCI132249.
Mcbride, J.K., Cheng, H., Maleki, S.J., Hurlburt, B.K. 2019. Purification and characterization of pathogenesis related class 10 panallergens. Foods. 8:609-623. https://doi.org/10.3390/foods8120609.
