Variation in Seed Allergen Content From Three Varieties of Soybean Cultivated in Nine Different Locations in Iowa, Illinois, and Indiana.

Soybean (Glycine max) is an important food stock, and also considered an allergenic food with at least eight well characterized allergens. However, it is a less prevalent allergen source than many other foods and is rarely life-threatening. Soybean is incorporated into commonly consumed foods, and therefore, the allergens pose a potential concern for individuals already sensitized. The protein profile of soybean can be affected by several factors including genetic and environmental. To investigate how soybean allergen content may be affected by genetics and/or environment, nine soy allergens were quantified from three commercial soybean varieties grown at nine locations in three states within a single climate zone in North America; Iowa, Illinois, and Indiana, United States. Quantitation was achieved using liquid chromatography-selected reaction monitoring (LC-SRM) tandem mass spectrometry with AQUA peptide standards specific to the nine target allergens. Quantitation of allergen concentration indicated that both genetics and location affected specific allergen content. Seven of the nine allergens were significantly influenced by genetics, with the exceptions of glycinin G4 and KTI 3. The allergens P34, Gly m Bd 28k, glycinin G3, and KTI 1 showed statistically significant impact from location as well, but at a lower threshold of significance compared with genetics (cultivar/variety). This dataset contributes to our understanding of the natural variation of endogenous allergens, as it represents a sampling of soybeans grown in a controlled, distributed plot design under agronomic conditions common for commercial soybean food and feed production. The aim was to build upon our recent understanding of how allergens are expressed as part of the overall soybean proteome.

Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens.

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52-63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14-16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.

Assessment of potential adjuvanticity of Cry proteins.

Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cry proteins are expressed at very low levels in GM crops and are unlikely to function as adjuvants. This conclusion is based on critical review of the published literature on the effects of immunomodulation by Cry proteins, the history of safe use of Cry proteins in foods, safety of the Bt donor organisms, and pre-market weight-of-evidence-based safety assessments for GM crops.

Food processing and allergenicity.

Food processing can have many beneficial effects. However, processing may also alter the allergenic properties of food proteins. A wide variety of processing methods is available and their use depends largely on the food to be processed. In this review the impact of processing (heat and non-heat treatment) on the allergenic potential of proteins, and on the antigenic (IgG-binding) and allergenic (IgE-binding) properties of proteins has been considered. A variety of allergenic foods (peanuts, tree nuts, cows' milk, hens' eggs, soy, wheat and mustard) have been reviewed. The overall conclusion drawn is that processing does not completely abolish the allergenic potential of allergens. Currently, only fermentation and hydrolysis may have potential to reduce allergenicity to such an extent that symptoms will not be elicited, while other methods might be promising but need more data. Literature on the effect of processing on allergenic potential and the ability to induce sensitisation is scarce. This is an important issue since processing may impact on the ability of proteins to cause the acquisition of allergic sensitisation, and the subject should be a focus of future research. Also, there remains a need to develop robust and integrated methods for the risk assessment of food allergenicity.

Measurement of endogenous allergens in genetically modified soybeans--short communication.

The measurement of endogenous allergens is required by the European Commission (EC) as part of the compositional analysis for GM products from host plants that are common causes of food allergy, such as soybean (EC Implementing Regulation No. 503/2013). In each case, the EC Implementing Regulation indicates that analysis be conducted on identified allergens as specified in the Organization of Economic Cooperation and Development (OECD) consensus documents on compositional considerations for new plant varieties. This communication discusses the methods available to measure endogenous allergens as well as the endogenous soybean allergens that should be analyzed. It is suggested herein that in conjunction with the 2012 OECD consensus document on soybean, any list of soybean allergens should be based on clinically relevant data among publicly available allergen databases and peer-reviewed scientific publications, and the ability to measure the identified allergen. Based on a detailed analysis of the scientific literature, the following key points are recommended: (1) the acceptance of serum-free, quantitative analytical method data as an alternative to traditional IgE reactivity qualitative or semi-quantitative data for evaluation of endogenous soybean allergen content; (2) eight of the 15 potential allergens listed in the OECD soybean consensus document (Gly m 3, Gly m 4, Gly m Bd28K, Gly m Bd30K, Gly m 5, Gly m 6, Gly m 8, and Kunitz trypsin inhibitor) have both appropriate supporting clinical data and sufficient sequence information to be evaluated in comparative endogenous soybean allergen studies; and (3) the remaining seven proteins (Gly m 1, Gly m 2, unknown 50kDa protein, unknown 39kDa protein, P-22-25, lipoxygenase and lectin) lack sufficient data for clear classification as confirmed allergens and/or available sequence information and should not be currently included in the measurement of endogenous soybean allergens in the compositional analysis for the EU.

Allergic sensitization: screening methods.

Experimental in silico, in vitro, and rodent models for screening and predicting protein sensitizing potential are discussed, including whether there is evidence of new sensitizations and allergies since the introduction of genetically modified crops in 1996, the importance of linear versus conformational epitopes, and protein families that become allergens. Some common challenges for predicting protein sensitization are addressed: (a) exposure routes; (b) frequency and dose of exposure; (c) dose-response relationships; (d) role of digestion, food processing, and the food matrix; (e) role of infection; (f) role of the gut microbiota; (g) influence of the structure and physicochemical properties of the protein; and (h) the genetic background and physiology of consumers. The consensus view is that sensitization screening models are not yet validated to definitively predict the de novo sensitizing potential of a novel protein. However, they would be extremely useful in the discovery and research phases of understanding the mechanisms of food allergy development, and may prove fruitful to provide information regarding potential allergenicity risk assessment of future products on a case by case basis. These data and findings were presented at a 2012 international symposium in Prague organized by the Protein Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute.

Technical variability of 2D gel electrophoresis - Application to soybean allergens.

Two-dimensional gel electrophoresis (2-DE) technique is used as a performing technique to assess the variability of protein expression in crops, and especially soybean endogenous food allergens, which are a subset of proteins of interest for assessing whether genetically modified (GM) soybean has a different allergenic profile compared to its non-GM counterpart. On top of the biological variability of the 2-DE, which has already been studied by several laboratories, technical variability has to be evaluated. In this study, several sources of variability (number of gel replicates, protein extracts, study timings and operators) were assessed qualitatively and quantitatively on all detectable polypeptide spots as well as on food allergen spots. Results showed that the major source of variability was the number of gel replicates. Other sources were minor. This has a direct practical impact on the laboratory work as this supports the utilization of three or four gel replicates to get robust results. Furthermore, this implies that the study can be run over several days, and be performed by several trained operators, without impacting its reproducibility. Furthermore, 2-DE could detect a 2-fold change between two samples with an acceptable rate of false positives (below 7%). This level of sensitivity is acceptable in the context of safety assessment of GM soybean as the biological variability of proteins in soybean is higher than the technical variability shown in this study. Overall, the 2-DE technique is suitable for investigating endogenous food allergen variability between several soybean seeds, including GM and non-GM counterpart.

Toxicological evaluation of proteins introduced into food crops.

This manuscript focuses on the toxicological evaluation of proteins introduced into GM crops to impart desired traits. In many cases, introduced proteins can be shown to have a history of safe use. Where modifications have been made to proteins, experience has shown that it is highly unlikely that modification of amino acid sequences can make a non-toxic protein toxic. Moreover, if the modified protein still retains its biological function, and this function is found in related proteins that have a history of safe use (HOSU) in food, and the exposure level is similar to functionally related proteins, then the modified protein could also be considered to be "as-safe-as" those that have a HOSU. Within nature, there can be considerable evolutionary changes in the amino acid sequence of proteins within the same family, yet these proteins share the same biological function. In general, food crops such as maize, soy, rice, canola etc. are subjected to a variety of processing conditions to generate different food products. Processing conditions such as cooking, modification of pH conditions, and mechanical shearing can often denature proteins in these crops resulting in a loss of functional activity. These same processing conditions can also markedly lower human dietary exposure to (functionally active) proteins. Safety testing of an introduced protein could be indicated if its biological function was not adequately characterized and/or it was shown to be structurally/functionally related to proteins that are known to be toxic to mammals.

Heat stability, its measurement, and its lack of utility in the assessment of the potential allergenicity of novel proteins.

Thermal stability has been reported as a shared characteristic among some of the major food allergens and appears to have originated from the observation that some cooked foods retain their ability to cause allergic reactions by Immunoglobulin E (IgE) binding and the subsequent cascade of events that mediate allergic reactions. Based on this observation, the thermal stability of novel food proteins, like those in transgenic crops, is considered correlative with allergenic risk and has prompted requests from some regulatory agencies for additional testing to address safety concerns. Because human testing and serum IgE screening are not feasible nor are they necessarily useful for evaluating the thermal stability of a novel food protein, a protein function assay is often used to assess the thermal stability in the context of an allergenicity risk assessment. Some regulatory authorities also require immunodetection using polyclonal IgG antibodies and gel based methods. Here we review why heat stability as measured by these functional and immunodetection assays does not correlate with allergenicity and provides no useful safety information in assessing the allergenic potential of novel food proteins.

Bioinformatics and the allergy assessment of agricultural biotechnology products: industry practices and recommendations.

Bioinformatic tools are being increasingly utilized to evaluate the degree of similarity between a novel protein and known allergens within the context of a larger allergy safety assessment process. Importantly, bioinformatics is not a predictive analysis that can determine if a novel protein will ''become" an allergen, but rather a tool to assess whether the protein is a known allergen or is potentially cross-reactive with an existing allergen. Bioinformatic tools are key components of the 2009 CodexAlimentarius Commission's weight-of-evidence approach, which encompasses a variety of experimental approaches for an overall assessment of the allergenic potential of a novel protein. Bioinformatic search comparisons between novel protein sequences, as well as potential novel fusion sequences derived from the genome and transgene, and known allergens are required by all regulatory agencies that assess the safety of genetically modified (GM) products. The objective of this paper is to identify opportunities for consensus in the methods of applying bioinformatics and to outline differences that impact a consistent and reliable allergy safety assessment. The bioinformatic comparison process has some critical features, which are outlined in this paper. One of them is a curated, publicly available and well-managed database with known allergenic sequences. In this paper, the best practices, scientific value, and food safety implications of bioinformatic analyses, as they are applied to GM food crops are discussed. Recommendations for conducting bioinformatic analysis on novel food proteins for potential cross-reactivity to known allergens are also put forth.

Quantitation of soybean allergens using tandem mass spectrometry.

Soybean (Glycine max) seed contain some proteins that are allergenic to humans and animals. However, the concentration of these allergens and their expression variability among germplasms is presently unknown. To address this problem, 10 allergens were quantified from 20 nongenetically modified commercial soybean varieties using parallel, label-free mass spectrometry approaches. Relative quantitation was performed by spectral counting and absolute quantitation was performed using multiple reaction monitoring (MRM) with synthetic, isotope-labeled peptides as internal standards. During relative quantitation analysis, 10 target allergens were identified, and five of these allergens showed expression levels higher than technical variation observed for bovine serum albumin (BSA) internal standard (∼11%), suggesting expression differences among the varieties. To confirm this observation, absolute quantitation of these allergens from each variety was performed using MRM. Eight of the 10 allergens were quantified for their concentration in seed and ranged from approximately 0.5 to 5.7 µg/mg of soy protein. MRM analysis reduced technical variance of BSA internal standards to approximately 7%, and confirmed differential expression for four allergens across the 20 varieties. This is the first quantitative assessment of all major soybean allergens. The results show the total quantity of allergens measured among the 20 soy varieties was mostly similar.

Investigation of endogenous soybean food allergens by using a 2-dimensional gel electrophoresis approach.

As part of the safety assessment of genetically modified (GM) soybean, 2-dimensional gel electrophoresis analyses were performed with the isoxaflutole and glyphosate tolerant soybean FG72, its non-GM near-isogenic counterpart (Jack) and three commercial non-GM soybean lines. The objective was to compare the known endogenous human food allergens in seeds in the five different soybean lines in order to evaluate any potential unintended effect(s) of the genetic modification. In total, 37 protein spots representing five well known soybean food allergen groups were quantified in each genotype. Qualitatively, all the allergenic proteins were detected in the different genetic backgrounds. Quantitatively, among 37 protein spots, the levels of accumulation of three allergens were slightly lower in the GM soybean than in the non-GM counterparts. Specifically, while the levels of two of these three allergens fell within the normal range of variation observed in the four non-GM varieties, the level of the third allergen was slightly below the normal range. Overall, there was no significant increase in the level of allergens in FG72 soybean seeds. Therefore, the FG72 soybean can be considered as safe as its non-GM counterpart with regards to endogenous allergenicity. Additional research is needed to evaluate the biological variability in the levels of endogenous soybean allergens and the correlation between level of allergens and allergenic potential in order to improve the interpretation of these data in the safety assessment of GM soybean context.

Evaluating biological variation in non-transgenic crops: executive summary from the ILSI Health and Environmental Sciences Institute workshop, November 16-17, 2009, Paris, France.

The International Life Sciences Institute Health and Environmental Sciences Institute Protein Allergenicity Technical Committee hosted an international workshop November 16-17, 2009, in Paris, France, with over 60 participants from academia, government, and industry to review and discuss the potential utility of "-omics" technologies for assessing the variability in plant gene, protein, and metabolite expression. The goal of the workshop was to illustrate how a plant's constituent makeup and phenotypic processes can be surveyed analytically. Presentations on the "-omics" techniques (i.e., genomics, proteomics, and metabolomics) highlighted the workshop, and summaries of these presentations are published separately in this supplemental issue. This paper summarizes key messages, as well as the consensus points reached, in a roundtable discussion on eight specific questions posed during the final session of the workshop. The workshop established some common, though not unique, challenges for all "-omics" techniques, and include (a) standardization of separation/extraction and analytical techniques; (b) difficulty in associating environmental impacts (e.g., planting, soil texture, location, climate, stress) with potential alterations in plants at genomic, proteomic, and metabolomic levels; (c) many independent analytical measurements, but few replicates/subjects--poorly defined accuracy and precision; and (d) bias--a lack of hypothesis-driven science. Information on natural plant variation is critical in establishing the utility of new technologies due to the variability in specific analytes that may result from genetic differences (crop genotype), different crop management practices (conventional high input, low input, organic), interaction between genotype and environment, and the use of different breeding methods. For example, variations of several classes of proteins were reported among different soybean, rice, or wheat varieties or varieties grown at different locations. Data on the variability of allergenic proteins are important in defining the risk of potential allergenicity. Once established as a standardized assay, survey approaches such as the "-omics" techniques can be considered in a hypothesis-driven analysis of plants, such as determining unintended effects in genetically modified (GM) crops. However, the analysis should include both the GM and control varieties that have the same breeding history and exposure to the same environmental conditions. Importantly, the biological relevance and safety significance of changes in "-omic" data are still unknown. Furthermore, the current compositional assessment for evaluating the substantial equivalence of GM crops is robust, comprehensive, and a good tool for food safety assessments. The overall consensus of the workshop participants was that many "-omics" techniques are extremely useful in the discovery and research phases of biotechnology, and are valuable for hypothesis generation. However, there are many methodological shortcomings identified with "-omics" approaches, a paucity of reference materials, and a lack of focused strategy for their use that currently make them not conducive for the safety assessment of GM crops.

Scientific advancement of novel protein allergenicity evaluation: an overview of work from the HESI Protein Allergenicity Technical Committee (2000-2008).

The safety assessment of genetically modified crops includes the evaluation for potential allergenicity. The current 'state-of-the-science' utilizes a weight of evidence approach, as outlined by the Codex Alimentarius commission (Alinorm 03/34 A), recognizing no single endpoint is predictive of the allergenic potential of a novel protein. This approach evaluates: whether the gene source is allergenic, sequence similarity to known allergens, and protein resistance to pepsin in vitro. If concerns are identified, serological studies may be necessary to determine if a protein has IgE binding similar to known allergens. Since there was a lack of standardized/validated methods to conduct the allergenicity assessment, a committee was assembled under the International Life Sciences Institute Health and Environmental Sciences Institute to address this issue. Over the last eight years, the Protein Allergenicity Technical Committee has convened workshops and symposia with allergy experts and government authorities to refine methods that underpin the assessment for potential protein allergenicity. This publication outlines this ongoing effort, summarizing workshops and formal meetings, referencing publications, and highlighting outreach activities. The purpose is to (1) outline 'the state-of-the-science' in predicting protein allergenicity in the context of current international recommendations for novel protein safety assessment, and (2) identify approaches that can be improved and future research needs.

Safety evaluation of the double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) from maize that confers tolerance to glyphosate herbicide in transgenic plants.

Glyphosate tolerance can be conferred by decreasing the herbicide's ability to inhibit the enzyme 5-enol pyruvylshikimate-3-phosphate synthase, which is essential for the biosynthesis of aromatic amino acids in all plants, fungi, and bacteria. Glyphosate tolerance is based upon the expression of the double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) protein. The 2mEPSPS protein, with a lower binding affinity for glyphosate, is highly resistant to the inhibition by glyphosate and thus allows sufficient enzyme activity for the plants to grow in the presence of herbicides that contain glyphosate. Based on both a review of published literature and experimental studies, the potential safety concerns related to the transgenic 2mEPSPS protein were assessed. The safety evaluation supports that the expressed protein is innocuous. The 2mEPSPS enzyme does not possess any of the properties associated with known toxins or allergens, including a lack of amino acid sequence similarity to known toxins and allergens, a rapid degradation in simulated gastric and intestinal fluids, and no adverse effects in mice after intravenous or oral administration (at 10 or 2000 mg/kg body weight, respectively). In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the 2mEPSPS protein in human food or in animal feed.

Murine models for evaluating the allergenicity of novel proteins and foods.

Genetically modified crops convey many benefits to world population. However, a rigorous safety assessment procedure, including an evaluation of the allergenic potential, is fundamental before their release into the food chain. As an integral part of the safety assessment process, regulatory authorities worldwide strongly recommend the use of tests that can predict the allergenic potential of the novel proteins. All guidance documents are based on an array of tests that have been proposed in 2003 by the Codex Alimentarius. Although the animal model is not a requirement of the Codex Alimentarius weight of evidence approach, allergenic hazard of novel proteins could only be evaluated by an in vivo model that can potentially identify and distinguish commonly allergenic proteins from rarely allergenic proteins. Therefore, food allergy experts encourage its development. During the 2007 International Life Science Institute (ILSI) workshop (Nice, France), worldwide experts shared their latest research results on rodent models to evaluate the allergenic potential of proteins and foods. This review presents the most promising rodent models for assessing food protein allergenicity that were evaluated during this ILSI workshop.

Current and future methods for evaluating the allergenic potential of proteins: international workshop report 23-25 October 2007.

The International Life Science Institute's Health and Environmental Sciences Institute's Protein Allergenicity Technical Committee hosted an international workshop October 23-25, 2007, in Nice, France, to review and discuss existing and emerging methods and techniques for improving the current weight-of-evidence approach for evaluating the potential allergenicity of novel proteins. The workshop included over 40 international experts from government, industry, and academia. Their expertise represented a range of disciplines including immunology, chemistry, molecular biology, bioinformatics, and toxicology. Among participants, there was consensus that (1) current bioinformatic approaches are highly conservative; (2) advances in bioinformatics using structural comparisons of proteins may be helpful as the availability of structural data increases; (3) proteomics may prove useful for monitoring the natural variability in a plant's proteome and assessing the impact of biotechnology transformations on endogenous levels of allergens, but only when analytical techniques have been standardized and additional data are available on the natural variation of protein expression in non-transgenic bred plants; (4) basophil response assays are promising techniques, but need additional evaluation around specificity, sensitivity, and reproducibility; (5) additional research is required to develop and validate an animal model for the purpose of predicting protein allergenicity.

Evaluation of protein safety in the context of agricultural biotechnology.

One component of the safety assessment of agricultural products produced through biotechnology is evaluation of the safety of newly expressed proteins. The ILSI International Food Biotechnology Committee has developed a scientifically based two-tiered, weight-of-evidence strategy to assess the safety of novel proteins used in the context of agricultural biotechnology. Recommendations draw upon knowledge of the biological and chemical characteristics of proteins and testing methods for evaluating potential intrinsic hazards of chemicals. Tier I (potential hazard identification) includes an assessment of the biological function or mode of action and intended application of the protein, history of safe use, comparison of the amino acid sequence of the protein to other proteins, as well as the biochemical and physico-chemical properties of the proteins. Studies outlined in Tier II (hazard characterization) are conducted when the results from Tier I are not sufficient to allow a determination of safety (reasonable certainty of no harm) on a case-by-case basis. These studies may include acute and repeated dose toxicology studies and hypothesis-based testing. The application of these guidelines is presented using examples of transgenic proteins applied for agricultural input and output traits in genetically modified crops along with recommendations for future research considerations related to protein safety assessment.

The utility of an international sera bank for use in evaluating the potential human allergenicity of novel proteins.

In the safety assessment of novel foods produced through biotechnology, careful consideration is given to determining the allergenic potential of newly introduced proteins. IgE serum screening is one tool for evaluating whether the protein in question has sequence identity to a known allergen or if the source of the gene encoding the protein is a known allergenic food. A "specific" serum screen involves testing a gene product with sera from patients with documented clinical allergy to a specific allergen to confirm that the gene product of interest is not the same protein to which the patient produces IgE antibodies. A "targeted" serum screen involves testing the gene product of interest with sera from patients sensitive to food or aeroallergens from the same broad group. The concept of a global sera bank with accessible, well-characterized sera for use in such assays is an appealing option. This paper summarizes the consensus elements from a workshop to evaluate the potential utility of an international sera bank for evaluating the allergenicity of novel proteins. Areas of agreement following the workshop included the following: (1) specific sera screens are appropriate for exploring potentially cross-reactive proteins that have been identified through bioinformatics analyses; however, additional validation is needed, particularly for targeted sera screens, (2) practical and ethical considerations may preclude the formation of a global sera bank, and therefore, (3) a regional network of clinicians who could serve as sources of patient sera or be approached to conduct sera studies would be the most practical alternative.

Evaluating the effect of food processing on the potential human allergenicity of novel proteins: international workshop report.

The ILSI Health and Environmental Sciences Institute Protein Allergenicity Technical Committee organized an international workshop in June 2006 in Estoril, Portugal, co-sponsored by the ILSI Research Foundation, ILSI International Food Biotechnology Committee and ILSI Europe. The objective was to discuss the effects of food processing on the allergenic potential of proteins and foods. The impact of food processing on the sensitization/induction phases of food allergy, and the bioavailability of allergens to the immune system were presented. Studies evaluating the stability, digestibility, and allergenicity of processed food allergens were identified, and their complexity and limitations discussed. Participants agreed that investigating food allergy mechanisms, validating appropriate methods for identifying allergenic proteins, and refining strategies to assess and manage the risks from food allergy were important before processing considerations are integrated into public-health decision-making for novel proteins. Other factors may also play a role in food allergy and include: food matrix; multiplicity of epitopes; geographic variation in patterns/prevalence of food allergies; and genetic factors, but required further exploration. Food processing may increase or decrease the intrinsic allergenicity of a protein, but current data do not facilitate the identification of specific variables that could be used to reliably determine how processing will influence protein allergenicity.