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Alternative and sustainable protein sources (e.g., algae, duckweed, insects) are required to produce (future) foods. However, introduction of new food sources to the market requires a thorough risk assessment of nutritional, microbial and toxicological risks and potential allergic responses. Yet, the risk assessment of allergenic potential of novel proteins is challenging. Currently, guidance for genetically modified proteins relies on a weight-of-evidence approach. Current Codex (2009) and EFSA (2010; 2017) guidance indicates that sequence identity to known allergens is acceptable for predicting the cross-reactive potential of novel proteins and resistance to pepsin digestion and glycosylation status is used for evaluating de novo allergenicity potential. Other physicochemical and biochemical protein properties, however, are not used in the current weight-of-evidence approach. In this study, we have used the Random Forest algorithm for developing an in silico model that yields a prediction of the allergenic potential of a protein based on its physicochemical and biochemical properties. The final model contains twenty-nine variables, which were all calculated using the protein sequence by means of the ProtParam software and the PSIPred Protein Sequence Analysis program. Proteins were assigned as allergenic when present in the COMPARE database. Results show a robust model performance with a sensitivity, specificity and accuracy each greater than ≥85%. As the model only requires the protein sequence for calculations, it can be easily incorporated into the existing risk assessment approach. In conclusion, the model developed in this study improves the predictability of the allergenicity of new or modified food proteins, as demonstrated for insect proteins., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Protein allergens can be related by cross-reactivity. Allergens that share relevant sequence can cross-react, those lacking sufficient similarity in their IgE antibody-binding epitopes do not cross-react. Cross-reactivity is based on shared epitopes that is based on shared sequence and higher level structure (charge and shape). Epitopes are important in predicting cross-reactivity potential and may provide the potential to establish criteria that identify homology among allergens. Selected allergen's IgE-binding epitope sequences were used to determine how the FASTA algorithm could be used to identify a threshold of significance. A statistical measure (expectation value, E-value) was used to identify a threshold specific to identifying cross-reactivity potential. Peanut Ara h 1 and Ara h 2, shrimp tropomyosin Pen a 1, and birch tree pollen allergen, Bet v 1 were sources of known epitopes. Each epitope or set of epitopes was inserted into random amino acid sequence to create hypothetical proteins used as queries to an allergen database. Alignments with allergens were noted for the ability to match the epitope's source allergen as well as any cross-reactive or other homologous allergens. A FASTA expectation value range (1 × 10 -5 -1 × 10 -6 ) was identified that could act as a threshold to help identify cross-reactivity potential., (© 2017 Syngenta Crop Protection, LLC. Molecular Nutrition & Food Research published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Assessment of the potential allergenicity (IgE-inducing properties) of novel proteins is an important challenge in the overall safety assessment of foods. Resistance to digestion with pepsin is commonly measured to characterize allergenicity, although the association is not absolute. We have previously shown that specific IgE antibody production induced by systemic [intraperitoneal (i.p.)] exposure of BALB/c strain mice to a range of proteins correlates with allergenic potential for known allergens. The purpose of the present study was to explore further the utility of these approaches using the food allergen, actinidin. Recently, kiwifruit has become an important allergenic foodstuff, coincident with its increased consumption, particularly as a weaning food. The ability of the kiwifruit allergen actinidin to stimulate antibody responses has been compared with the reference allergen ovalbumin, and with the non-allergen bovine haemoglobin. Haemoglobin was rapidly digested by pepsin whereas actinidin was resistant unless subjected to prior chemical reduction (reflecting intracellular digestion conditions). Haemoglobin stimulated detectable IgG antibody production at relatively high doses (10%), but failed to provoke detectable IgE. In contrast, actinidin was both immunogenic and allergenic at relatively low doses (0.25% to 1%). Vigorous IgG and IgG1 antibody and high titre IgE antibody responses were recorded, similar to those provoked by ovalbumin. Thus, actinidin displays a marked ability to provoke IgE, consistent with allergenic potential. These data provide further encouragement that in tandem with analysis of pepsin stability, the induction of IgE after systemic exposure of BALB/c strain mice provides a useful approach for the prospective identification of protein allergens., (Copyright © 2013 John Wiley & Sons, Ltd.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

The biochemical characterization of protein structures has led to a better understanding of allergens, their structure/function relationship, and can be very powerful in identifying protein sequences with significant structural similarity to known allergens. However, for scientists, regulators and food manufacturers there exists a need for acquiring additional data on potential allergenicity of proteins, particularly, biotechnology derived molecules in food products for which minimal or no prior human exposure information is available. Since human exposure testing, while direct, is unacceptable, understanding allergy in animals has been used to investigate the allergic response on a molecular level as well as test the potential in vivo allergenicity of food proteins. Rodents seem to be the most likely candidate for assessing allergenicity. For development of an animal test system for allergenicity characterization and testing, a number of criteria are required for qualification for a model of human allergy including acceptable immunization protocols, allergic response measurements, and for standardization and validation of materials and procedures. If an animal test system can minimally provide a basis for measuring the relative physiological response to known allergens, this should be enough to establish a model that produces a relative measure of potential allergenicity. Our article will consider development of an adequate animal model for allergenicity determination that can be validated as a tool in safety assessments.

In 2001, the FAO/WHO suggested a procedure for performing FASTA or BLAST searches, and a threshold of greater than 35% identity in 80 or greater amino acids to identify potential allergenic cross-reactivity of transgene encoded proteins in genetically enhanced crops. Transgene encoded proteins meeting or exceeding this threshold would require additional in vitro evaluation for allergy safety. In work described herein, a method to calculate an E-score threshold is proposed for utilizing the full capability of bioinformatics to accurately identify potential cross-reactive allergens. The threshold E-score, 3.9E-07, was produced using a test dataset of 7695 corn protein sequences and a method that entailed FASTA searches of the FARRP 7 allergen database with each of the dataset sequences using a conventional full length and an 80 amino acid sliding window FASTA comparison followed by an evaluation of E-score distribution. The results show that this E-score threshold identifies known corn allergens and it displays a false positive rate for known allergens that is comparable to that obtained from the 2001 FAO/WHO guidance. Furthermore, the E-score threshold is of sufficient stringency that it rejects the majority of false positive, composition-based anomalies and is 100% effective at identifying Bet v 1 cross-reactive allergens.