Food allergen detection by mass spectrometry: From common to novel protein ingredients

Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.     

Prediction of food protein allergenicity: a bioinformatic learning systems approach

Food hypersensitivity is constantly increasing in Western societies with a prevalence of about 1-2% in Europe and in the USA. Among children, the incidence is even higher. Because of the introduction of foods derived from genetically modified crops on the marketplace, the scientific community, regulatory bodies and international associations have intensified discussions on risk assessment procedures to identify potential food allergenicity of the newly introduced proteins. In this work, we present a novel biocomputational methodology for the classification of amino acid sequences with regard to food allergenicity and non-allergenicity. This method relies on a computerised learning system trained using selected excerpts of amino acid sequences. One example of such a successful learning system is presented which consists of feature extraction from sequence alignments performed with the FASTA3 algorithm (employing the BLOSUM50 substitution matrix) combined with the k-Nearest-Neighbour (kNN) classification algorithm. Briefly, the two features extracted are the alignment score and the alignment length and the kNN algorithm assigns the pair of extracted features from an unknown sequence to the prevalent class among its k nearest neighbours in the training (prototype) set available. 91 food allergens from several specialised public repositories of food allergy and the SWALL database were identified, pre-processed, and stored, yielding one of the most extensively characterised repositories of allergenic sequences known today. All allergenic sequences were classified using a standard one-leave-out cross validation procedure yielding about 81% correctly classified allergens and the classification of 367 non-allergens in an independent test set resulted in about 98% correct classifications. The biocomputational approach presented should be regarded as a significant extension and refinement of earlier attempts suggested for in silico food safety assessment. Our results show that the framework described here is powerful enough to become useful as part of a multiple-procedure test scheme that also depicts other evaluation approaches such as solid phase immunoassay and tests for stability to digestions.     

Adverse reactions to foods

Allergic reactions to foods represent a prominent, actual and increasing problem in clinical medicine. Symptoms of food allergy comprise skin reactions (urticaria, angioedema, eczema) respiratory (bronchoconstriction, rhinitis), gastrointestinal (cramping, diarrhea) and cardiovascular symptoms with the maximal manifestation of anaphylactic shock. They can be elicited by minute amounts of allergens. The diagnosis of food allergy is done by history, skin test, in vitro allergy diagnosis and — if necessary — oral provocation tests, if possible placebo-controlled. Avoidance of respective allergens for the allergic patient, however, is often complicated or impossible due to deficits in declaration regulations in many countries. Increasing numbers of cases including fatalities, due to inadvertent intake of food allergens are reported. It is therefore necessary to improve declaration laws and develop methods for allergen detection in foods. Allergens can be detected by serological methods (enzyme immunoassays, in vitro basophil histamine release or in vivo skin test procedures in sensitized individuals). The problem of diagnosis of food allergy is further complicated by cross-reactivity between allergens in foods and aeroallergens (pollen, animal epithelia, latex etc.). Elicitors of pseudo-allergic reactions with similar clinical symptomatology comprise low-molecular-mass chemicals (preservatives, colorings, flavor substances etc.). For some of them (e.g. sulfites) detection assays are available. In some patients classic allergic contact eczema can be elicited systemically after oral intake of low-molecular-mass contact allergens such as nickel sulfate or flavorings such as vanillin in foods. The role of xenobiotic components in foods (e.g. pesticides) is not known at the moment. In order to improve the situation of the food allergic patient, research programs to elucidate the pathophysiology and improve allergen detection strategies have to be implemented together with reinforced declaration regulations on a quantitative basis.     

Isolation of food allergens

The identification of food allergens is a priority in the management of food allergy, because of the need to obtain standardized extracts and pure allergens for diagnosis and therapy. It is thus important to develop methods for purification of allergenic molecules in order to study their biological and immunological characteristics. Protocols for protein extraction from foods and for allergen purification are reviewed in this paper. We report published methods for extraction of allergens from either animal and vegetable foods and detailed purification methodologies including ion-exchange, gel filtration and reversed-phase chromatography of well known allergens.     

A mouse model for food allergy using intraperitoneal sensitization

Food allergy is an important health issue. With the increasing interest in novel foods derived from transgenic crop plants, there is a growing need for the development of approaches for the characterization of the allergenic potential of proteins. Although most foreign proteins are immunogenic (able to induce IgG antibody responses), relatively few are important food allergens with the capacity to provoke IgE antibody production. There is currently no validated animal model for the determination of allergenic potential of food proteins. One approach that appears to show some promise is outlined in the current chapter. BALB/c strain mice are immunized by intraperitoneal injection and the potential to cause allergenicity assessed as a function of the induction of specific IgE antibody, measured by homologous passive cutaneous anaphylaxis. Progress to date with this method is summarized, and comparisons are made with other experimental models, including considerations of route of exposure, use of adjuvants and selection of appropriate end points.     

In vivo and in vitro techniques to determine the biological activity of food allergens

Methods for determination of the biological activity of food allergens comprise both determination of the allergenic potency, i.e. the capability to elicit an allergic reaction in an already sensitized individual, and the allergenic potential, i.e. the risk for sensitizing a hitherto non-allergic individual. Several methods are discussed for determination of potency including the double-blinded placebo-controlled food challenge, skin testing, in vitro effector cell assays such as basophil histamine release, and IgE-based techniques such as RAST and RAST inhibition. No reliable methods have yet been developed which can predict the allergenic potential of a food or a food allergen. The progress in the areas of stability studies and animal models for food allergy are discussed.     

The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.     

Genetic modification and plant food allergens: risks and benefits

Plant genetic engineering has the potential to both introduce new allergenic proteins into foods and remove established allergens. A number of allergenic plant proteins have been characterized, showing that many are related to proteins which have potentially valuable properties for use in nutritional enhancement, food processing and crop protection. It is therefore important to monitor the allergenic potential of proteins used for plant genetic engineering and major biotechnology companies have established systems for this. Current technology allows gene expression to be down-regulated using antisense or co-suppression and future developments may allow targeted gene mutation or gene replacement. However, the application of this technology may be limited at least in the short term by the presence of multiple allergens and their contribution to food processing or other properties. Furthermore, the long-term stability of these systems needs to be established as reversion could have serious consequences.     

Computational analysis of the relationship between allergenicity and digestibility of allergenic proteins in simulated gastric fluid

Background  

Safety assessment of genetically modified (GM) food, with regard to allergenic potential of transgene-encoded xenoproteins, typically involves several different methods, evaluation by digestibility being one thereof. However, there are still debates about whether the allergenicity of food allergens is related to their resistance to digestion by the gastric fluid. The disagreements may in part stem from classification of allergens only by their sources, which we believe is inadequate, and the difficulties in achieving identical experimental conditions for studying digestion by simulated gastric fluid (SGF) so that results can be compared. Here, we reclassify allergenic food allergens into alimentary canal-sensitized (ACS) and non-alimentary canal-sensitized (NACS) allergens and use a computational model that simulates gastric fluid digestion to analyze the digestibilities of these two types.     

Reproducibility of atopy patch tests with food and inhalant allergens

Although atopy patch tests (APT) seem a valuable additional tool in the diagnostic work-up for food allergy in children with atopic eczema/dermatitis syndrome, the immunopathology and some technical aspects of testing remain controversial. Few published data are available on the reproducibility of APT with inhalants and only two studies include fresh food allergens. In this study we therefore investigated the reproducibility of duplicate APT (left versus right side of the back) with native and commercially available food (cow s milk, hen s egg, tomato, wheat flour) and with inhalant allergens (Dermatophagoides pteronyssinus and mixed grasses) in a large unselected population of children. We tested a population of 277 Italian school children with three APT allergens: fresh food (cow s milk, hen s egg, tomato and wheat flour), standardised food allergens in petrolatum (the same four foods) and standardised inhalant allergens routinely used for skin prick testing. For the four food allergens (applied in the natural form or as the standardised commercial preparation) from one- to three quarters of the APT gave positive results on one side and negative reactions on the opposite side (Cohen s K coefficient between 0.38, fresh tomato and 0.81, fresh cow s milk). Conversely, APT with inhalant allergens were invariably reproducible (Cohen s K = 1.00). The possible technical and immunologic reasons explaining why reproducibility of APT differed for the two types of allergens await an answer from extensive controlled studies.     

Improving diagnostic tests for food allergy with recombinant allergens

Food allergy is one important manifestation of atopic allergy. Primary food allergy mainly affects young children (class I), whereas adults frequently develop food allergy as a consequence of an inhalant sensitization (class II). At present, the diagnostic instrument for proving class II food allergy is not satisfactory. Skin tests as well as serological tests are in general neither very specific nor highly sensitive because they depend on food extracts, which differ in their content of individual allergens, vary between manufacturers, and even between different batches. Since the presence of food allergen-specific IgE antibodies does not always correlate with clinical symptoms against the respective food, oral provocation tests (ideally double-blind placebo-controlled food challenges) have to be performed to validate serological diagnosis or skin tests. However, oral provocation tests are connected to several practical problems and include a specific risk for the allergic patient. Applying DNA technology, up to 40 food allergens have been produced in recombinant form, which implies standardized quality and unlimited quantity of the respective proteins. Hence, such molecules might be used to solve problems of clinical and molecular allergology in diagnosis, research, and therapy of class II food allergies. First experiments with recombinant food allergens in this respect appear very promising.     

Lebensmittelallergie — eine individuelle Gefahr: Wenn das Immunsystem falsch reagiert

Problems with food allergy and other adverse reactions to food are still a challenge for science. Both, in case of diagnostic and therapy and in characterization of allergens there is still a lot to do in research to help the continuously increasing number of allergic patients. But an exact elucidation of allergen structure, the development of hypoallergenic food by gentechnological processing of allergenic proteins, a better therapy, the knowledge of the pathogenetic effect or a complete declaration of ingredients in prepacked foods will never lead to absolutely sure food for allergic patients. As the pathogenesis, the symptoms, and sensibilization individually differ, at least for every allergic patient there will remain a risk.     

Specific conformational epitope features of pathogenesis-related proteins mediating cross-reactivity between pollen and food allergens

Selected members of plant pathogenesis-related and seed storage proteins represent specific groups of proteins with potential characteristics of allergens. Efforts to understand the mechanism by which pathogenesis-related proteins mediate a broad cross-reactivity in pollen-plant food allergens are still limited. In this study, computational biology approach was used to reveal specific structural implications and conservation of different epitopes from members of Bet v 1 and nsLTP protein families mediating cross-reactivity between pollen and food (fruits, vegetables, legumes, and nut/seeds) allergens. A commonly shared epitope conservation was found among all pollen and food Bet v 1 and nsLTP protein families, respectively. However, other allergenic epitopes were also specifically detected in each family. The implication of these conserved epitopes in a broad cross-reactivity for allergy clinical trials is here discussed.     

Yeast Thioredoxin-Enriched Extracts for Mitigating the Allergenicity of Foods

Thioredoxin (TRX) catalyzes the reduction of disulfide bonds in proteins via the NADPH-dependent thioredoxin reductase system. Reducing the disulfide bonds of allergenic proteins in food by TRX lowers the allergenicity. We established in this study a method to prepare TRX-enriched extracts from the edible yeast, Saccharomyces cerevisiae, on a large and practical scale, with the objective of developing TRX-containing functional foods to mitigate food allergy. Treating with the yeast TRX-enriched extracts together with NADPH and yeast thioredoxin reductase enhanced the pepsin cleavage of β-lactoglobulin and ovomucoid (OM). We also examined whether yeast TRX can mitigate the allergenicity of OM by conducting immediate allergy tests on guinea pigs. The treatment with TRX reduced the anaphylactic symptoms induced by OM in these tests. These results indicate that yeast TRX was beneficial against food allergy, raising the possibility that yeast TRX-enriched extracts can be applied to food materials for mitigating food allergy.     

Immunological identification and characterization of individual food allergens

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.     

Supervised identification of allergen-representative peptides for in silico detection of potentially allergenic proteins

MOTIVATION: Identification of potentially allergenic proteins is needed for the safety assessment of genetically modified foods, certain pharmaceuticals and various other products on the consumer market. Current methods in bioinformatic allergology exploit common features among allergens for the detection of amino acid sequences of potentially allergenic proteins. Features for identification still unexplored include the motifs occurring commonly in allergens, but rarely in ordinary proteins. In this paper, we present an algorithm for the identification of such motifs with the purpose of biocomputational detection of amino acid sequences of potential allergens. RESULTS: Identification of allergen-representative peptides (ARPs) with low or no occurrence in proteins lacking allergenic properties is the essential component of our new method, designated DASARP (Detection based on Automated Selection of Allergen-Representative Peptide). This approach consistently outperforms the criterion based on identical peptide match for predicting allergenicity recommended by ILSI/IFBC and FAO/WHO and shows results comparable to the alignment-based criterion as outlined by FAO/WHO. AVAILABILITY: The detection software and the ARP set needed for the analysis of a query protein reported here are properties of the Swedish National Food Agency and are available upon request. The protein sequence sets used in this work are publicly available on http://www.slv.se/templatesSLV/SLV_Page____9343.asp. Allergenicity assessment for specific protein sequences of interest is also possible via ulfh@slv.se     

Plant food allergens--structural and functional aspects of allergenicity

The three dominating plant food allergen groups belong to the prolamin and cupin superfamilies and to the family 10 of pathogenesis-related proteins. The prolamin superfamily comprises allergenic 2S albumins, nonspecific lipid transfer proteins and cereal alpha-amylase/trypsin inhibitors. These allergens have related structures and are stable to thermal processing and proteolysis. The cupin superfamily comprises the allergenic 7S and 11S globulin storage proteins from peanuts, soybean and tree nuts which are heat stable and can form immunogenicity enhancing aggregates. The Bet v 1 family of allergens includes tree pollinosis-associated food allergens with low stability which induce the symptoms of the oral allergy syndrome.     

IgE binding to proteins from sesame and assessment of allergenicity: implications for biotechnology?

Successful prediction of the potential allergenicity of a protein may be a key factor in the development of novel, genetically modified foods. The use of the decision tree approach for the prediction of allergenicity is discussed. The methods currently used for identifying allergenic proteins (including use of IgE from patient sera for recognition of proteins) are reviewed. Finally, a specific review of the literature concerning identification of allergens from sesame leads to the conclusion that in the absence of validated animal models, identification of allergenicity (and, consequently, prediction of allergenicity) may be problematic.     

Yeast thioredoxin-enriched extracts for mitigating the allergenicity of foods

Thioredoxin (TRX) catalyzes the reduction of disulfide bonds in proteins via the NADPH-dependent thioredoxin reductase system. Reducing the disulfide bonds of allergenic proteins in food by TRX lowers the allergenicity. We established in this study a method to prepare TRX-enriched extracts from the edible yeast, Saccharomyces cerevisiae, on a large and practical scale, with the objective of developing TRX-containing functional foods to mitigate food allergy. Treating with the yeast TRX-enriched extracts together with NADPH and yeast thioredoxin reductase enhanced the pepsin cleavage of β-lactoglobulin and ovomucoid (OM). We also examined whether yeast TRX can mitigate the allergenicity of OM by conducting immediate allergy tests on guinea pigs. The treatment with TRX reduced the anaphylactic symptoms induced by OM in these tests. These results indicate that yeast TRX was beneficial against food allergy, raising the possibility that yeast TRX-enriched extracts can be applied to food materials for mitigating food allergy.