Allergenic Content of New Alimentary Pasta Made of Lentils Compared with Lentil Seeds and Analysis of the Impact of Boiling Processing.

There is growing interest in legumes such as lentil as healthy ingredients in gluten-free products. In that respect, foods based on lentils, like alimentary pasta, have been produced and successfully commercialized in recent years. Lentils are also known for inducing severe allergic reactions; however, it is currently unknown if novel alimentary pasta based on lentil retains the same allergenic potential as lentil seeds. In this study, the allergenic content of alimentary lentil pasta compared with lentil seeds was analyzed by immunoassays using sera from patients with allergic sensitization to lentil or with specific antibodies that recognize major lentil allergens. The effect of boiling processing was also analyzed. Results showed that alimentary lentil pasta has a significant allergenic content close to the general allergenic content observed for lentil seeds. Both alimentary lentil pasta and lentil seeds were similarly affected by boiling, with an important transfer of allergens from the food to the boiling water. This study shows that alimentary pasta made of lentils has a significant allergenic potential and highlights the necessity to analyze the allergenic content of new foods and novel ingredients introduced in traditional food products.

Boiling and Pressure Cooking Impact on IgE Reactivity of Soybean Allergens.

BACKGROUND: Soybean is one of the 8 foods that causes the most significant rate of food allergies in the USA and Europe. Thermal processing may impact on the allergenic potential of certain foods. We aimed to investigate modifications of the IgE-binding properties of soybean proteins due to processing methods that have been previously found to impact on the allergenicity of legumes such as peanut. METHODS: Soybean seeds were subjected to different thermal processing treatments. To evaluate their impact on the IgE-binding capacity of soybean proteins, individual sera from 25 patients sensitized to soybean were used in in vitro immunoassays. Detection of specific soybean allergens in untreated and treated samples was carried out with specific monoclonal and polyclonal antibodies. In vivo studies of skin prick testing (SPT) were also performed. RESULTS: The IgE reactivity of soybean was resistant to boiling up to 30 min, and this treatment had a higher impact when applied for 60 min. Treatment that combined heat and pressure produced a fragmentation of proteins in both soluble and insoluble fractions that went along with a decreased capacity to bind IgE and reduced the SPT wheal size. However, allergens such as 7S globulins survived this treatment. CONCLUSIONS: Thermal-processing methods able to attenuate the capacity of soybean proteins to bind IgE may contribute to the improvement of food safety and could constitute a potential strategy for the induction of tolerance to soybean.

Immunoproteomic tools are used to identify masked allergens: Ole e 12, an allergenic isoflavone reductase from olive (Olea europaea) pollen.

Proteins performing important biochemical activities in the olive tree (Olea europaea) pollen have been identified as allergens. One novel 37-kDa protein seems to be associated to the IgE-binding profile of a group of patients suffering allergy to peach and olive pollen. Three previously described olive pollen allergens exhibit very similar molecular mass. Our objective was to identify this allergen by using immunoproteomic approaches. After 2D-electrophoresis and mass spectrometry, peptide sequences from several IgE-binding spots, allowed identifying this new allergen, as well as cloning and DNA sequencing of the corresponding gene. The allergen, named Ole e 12, is a polymorphic isoflavone reductase-like protein of 308 amino acids showing 80% and 74% identity with birch and pear allergens, Bet v 6 and Pyr c 5, respectively. A prevalence of 33% in the selected population is in contrast to 4%-10% in groups of subjects suffering from pollinosis. Recombinant allergen was produced in Escherichia coli, and deeply characterised. Immunoblotting and ELISA detection as well as inhibition experiments were performed with polyclonal antisera and allergic patients' sera. The recombinant allergen retains the IgE reactivity of its natural counterpart. Close structural and immunological relationships between members of this protein family were supported by their IgG recognition in vegetable species. In summary, Ole e 12 is a minor olive pollen allergen, which gains relevance in patients allergic to peach with olive pollinosis. Proteomic approaches used to analyse this allergen provide useful tools to identify hidden allergens, relevant for several allergic populations and thus complete allergenic panels.

Recent advances in cellular and molecular mechanisms of IgE-mediated food allergy.

Food allergy is a public health issue the prevalence of which is steadily increasing. New discoveries have contributed to the understanding of the molecular and cellular mechanisms that lead to IgE-mediated food allergy. Novel scientific findings have defined roles for specific cell types, such as T follicular helper cells, in induction of high-affinity IgE by B cells. Also, not only mast cells and basophils contribute to food anaphylaxis, but also other cell types, such as neutrophils and macrophages. Elucidation of mechanisms involved in sensitization to food allergens through organs including the skin is key to deepening our understanding of the "dual exposure" hypothesis, which suggests that allergic sensitization is mainly acquired through inflamed skin while the oral route induces tolerance. This review considers the latest scientific knowledge about the molecular and cellular mechanisms of IgE-mediated food allergy. It reveals crucial components involved in the sensitization and elicitation phases and emerging approaches in anaphylaxis pathophysiology.

Influence of enzymatic hydrolysis on the allergenicity of roasted peanut protein extract.

BACKGROUND: Peanut allergy is recognized as one of the most severe food allergies. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as the soybean, chickpea and lentil. Nevertheless, there are only a few studies carried out with sera from patients with a well-documented allergy. METHODS: Roasted peanut protein extract was hydrolyzed by the sequential and individual action of 2 food-grade enzymes, an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to roasted peanut extract and hydrolyzed samples was evaluated by means of IgE immunoblot, ELISA and 2-dimensional electrophoresis using sera from 5 patients with a clinical allergy to peanuts and anti-Ara h 1, anti-Ara h 2 and anti-Ara h 3 immunoblots. RESULTS: Immunoblot and ELISA assays showed an important decrease of IgE reactivity and Ara h 1, Ara h 2 and Ara h 3 levels in the first 30 min of hydrolyzation with Alcalase. In contrast, individual treatment with Flavourzyme caused an increase in IgE reactivity detected by ELISA at 30 min and led to a 65% inhibition of IgE reactivity at the end of the assay (300 min). Ara h 1 and the basic subunit of Ara h 3 were still recognized after treatment with Flavourzyme for 300 min. CONCLUSION: Hydrolysis with the endoprotease Alcalase decreases IgE reactivity in the soluble protein fraction of roasted peanut better than hydrolysis with the exoprotease Flavourzyme.

Novel alimentary pasta made of chickpeas has an important allergenic content that is altered by boiling in a different manner than chickpea seeds.

Alimentary pasta made of chickpeas has been recently introduced in the market. The novelty and presentation of this food can have a confounding effect on chickpea allergic patients and can pose a risk to them. The allergenic content of novel alimentary chickpea pasta in comparison with regular chickpea seeds has not been analyzed so far. Protein extracts were obtained, and the allergenic content was analyzed with sera from chickpea allergic patients and antibodies against major allergens by western blot, ELISA, dot blot, and cellular assays. Alimentary chickpea pasta showed an important content in IgE-binding proteins and chickpea allergens: 7S globulin, 2S albumin, LTP, and PR-10, similar to hydrated and boiled chickpea seeds. During boiling, more allergens from alimentary chickpea pasta were transferred to the boiling water than chickpea seeds. Novel alimentary chickpea pasta retains an important allergenic content which is affected by boiling by transferring allergens to the cooking water.

IgE epitopes of Ara h 9, Jug r 3, and Pru p 3 in peanut-allergic individuals from Spain and the US.

Non-specific lipid transfer proteins (LTPs) are well studied allergens that can lead to severe reactions, but often cause oral allergy syndrome in the Mediterranean area and other European countries. However, studies focused on LTP reactivity in allergic individuals from the United States are lacking because they are not considered major allergens. The goal of this study is to determine if differences in immunoglobulin (Ig) E binding patterns to the peanut allergen Ara h 9 and two homologous LTPs (walnut Jug r 3 and peach Pru p 3) between the US and Spain contribute to differences observed in allergic reactivity. Synthetic overlapping 15-amino acid-long peptides offset by five amino acids from Ara h 9, Jug r 3, and Pru p 3 were synthesized, and the intact proteins were attached to microarray slides. Sera from 55 peanut-allergic individuals from the US were tested for IgE binding to the linear peptides and IgE binding to intact proteins using immunofluorescence. For comparison, sera from 17 peanut-allergic individuals from Spain were also tested. Similar IgE binding profiles for Ara h 9, Jug r 3, and Pru p 3 were identified between the US and Spain, with slight differences. Certain regions of the proteins, specifically helices 1 and 2 and the C-terminal coil, were recognized by the majority of the sera more often than other regions of the proteins. While serum IgE from peanut-allergic individuals in the US binds to peptides of Ara h 9 and its homologs, only IgE from the Spanish subjects bound to the intact LTPs. This study identifies Ara h 9, Jug r 3, and Pru p 3 linear epitopes that were previously unidentified using sera from peanut-allergic individuals from the US and Spain. Certain regions of the LTPs are recognized more often in US subjects, indicating that they represent conserved and possible cross-reactive regions. The location of the epitopes in 3D structure models of the LTPs may predict the location of potential conformational epitopes bound by a majority of the Spanish patient sera. These findings are potentially important for development of peptide or protein-targeting diagnostic and therapeutic tools for food allergy.

Effect of instant controlled pressure drop on IgE antibody reactivity to peanut, lentil, chickpea and soybean proteins.

BACKGROUND: The use of legume seeds is being expanded in the food industry due to their excellent nutritional and technological properties. However, legumes have been considered causative agents of allergic reactions through ingestion. Previous studies indicated that processing methods combining heat and steam pressure, such as instant controlled pressure drop (DIC®), could decrease allergenicity. The aim of this study was to investigate the impact of DIC treatment on peanut, lentil, chickpea and soybean IgE antibody reactivity. METHODS: Peanut, lentil, chickpea and soybean seeds were subjected to DIC treatment at different pressure and time conditions (3 and 6 bar for 1 and 3 min). Control (raw) and DIC-treated extracts were analyzed by SDS-PAGE and immunoblotting using a serum pool from sensitized patients. RESULTS: DIC treatment did not affect the total protein content of legume seeds. Nevertheless, modifications of protein profiles after DIC showed a general decrease in IgE binding to legume proteins that was correlated to a higher steam pressure and longer treatment. The immunoreactivity of soybean proteins was almost abolished with treatment at 6 bar for 3 min. CONCLUSIONS: The results demonstrated that DIC treatment produces a reduction in the overall in vitro IgE binding of peanut, lentil and chickpea and a drastic reduction in soybean immunoreactivity.

Epitope mapping of the major allergen 2S albumin from pine nut.

The epitopes of the major allergen of pine nut, Pin p 1, were analyzed using a peptide library and sera from patients with clinical allergy to pine nut in order to deepen into the allergenic characteristics of Pin p 1. Analyses of epitope similarities and epitopes location in a 3D-model were also performed. Results showed that three main regions of Pin p 1 containing 5 epitopes were recognized by patient sera IgE. The epitopes of Pin p 1 had important similarities with epitopes of allergenic 2S albumins from peanut (Ara h 2 and 6) and Brazil nut (Ber e 1). The epitopes of Pin p 1 were found in α-helices and coils in the 3D protein structure. Interestingly, all epitopes were found to be well-exposed in the protein surface, which suggests facile access for IgE-binding to the structure of Pin p 1 which is known to be highly resistant.

Differences among pollen-allergic patients with and without plant food allergy.

BACKGROUND: A considerable number of pollen-allergic patients develops allergy to plant foods, which has been attributed to cross-reactivity between food and pollen allergens. The aim of this study was to analyze the differences among pollen-allergic patients with and without plant food allergy. METHODS: Eight hundred and six patients were recruited from 8 different hospitals. Each clinical research group included 100 patients (50 plant food-allergic patients and 50 pollen-allergic patients). Diagnosis of pollen allergy was based on typical case history of pollen allergy and positive skin prick tests. Diagnosis of plant-food allergy was based on clear history of plant-food allergy, skin prick tests and/or plant-food challenge tests. A panel of 28 purified allergens from pollens and/or plant foods was used to quantify specific IgE (ADVIA-Centaur® platform). RESULTS: Six hundred and sixty eight patients (83%) of the 806 evaluated had pollen allergy: 396 patients with pollen allergy alone and 272 patients with associated food and pollen allergies. A comparison of both groups showed a statistically significant increase in the food and pollen allergy subgroup in frequency of: (1) asthma (47 vs. 59%; p < 0.001); (2) positive skin test results to several pollens: Plantago, Platanus, Artemisia, Betula, Parietaria and Salsola (p < 0.001); (3) sensitization to purified allergens: Pru p 3, profilin, Pla a 1 - Pla a 2, Sal k 1, PR-10 proteins and Len c 1. CONCLUSION: Results showed relevant and significant differences between both groups of pollen-allergic patients depending on whether or not they suffered from plant-derived food allergy.

Food allergens: Classification, molecular properties, characterization, and detection in food sources.

Food allergy is a large and growing public health problem in many areas of the world. The prevalence of food allergy has increased in the last decades in a very significant way in many world regions, particularly in developed countries. In that respect, the research field of food allergy has experienced an extensive growth and very relevant progress has been made in recent years regarding the characterization of food allergens, the study of their immunological properties, and their detection in food sources. Furthermore, food labeling policies have also been improved decidedly in recent years. For that immense progress made, it is about time to review the latest progress in the field of food allergy. In this review, we intend to carry out an extensive and profound overview regarding the latest scientific advances and knowledge in the field of food allergen detection, characterization, and in the study of the effects of food processing on the physico-chemical properties of food allergens. The advances in food labeling policies, and methodologies for the characterization of food allergens are also thoroughly reviewed in the present overview.

Interaction of Monocyte-Derived Dendritic Cells with Ara h 2 from Raw and Roasted Peanuts.

Ara h 2 is a relevant peanut allergen linked to severe allergic reactions. The interaction of Ara h 2 with components of the sensitization phase of food allergy (e.g., dendritic cells) has not been investigated, and could be key to understanding the allergenic potential of this allergen. In this study, we aimed to analyze such interactions and the possible mechanism involved. Ara h 2 was purified from two forms of peanut, raw and roasted, and labeled with a fluorescent dye. Human monocyte-derived dendritic cells (MDDCs) were obtained, and experiments of Ara h 2 internalization by MDDCs were carried out. The role of the mannose receptor in the internalization of Ara h 2 from raw and roasted peanuts was also investigated. Results showed that Ara h 2 internalization by MDDCs was both time and dose dependent. Mannose receptors in MDDCs had a greater implication in the internalization of Ara h 2 from roasted peanuts. However, this receptor was also important in the internalization of Ara h 2 from raw peanuts, as opposed to other allergens such as raw Ara h 3.

Anti-inflammatory effects of flavonoids.

Inflammation plays a key role in diseases such as diabetes, asthma, cardiovascular diseases and cancer. Diet can influence different stages of inflammation and can have an important impact on several inflammatory diseases. Increasing scientific evidence has shown that polyphenolic compounds, such as flavonoids, which are found in fruits, vegetables, legumes, or cocoa, can have anti-inflammatory properties. Recent studies have demonstrated that flavonoids can inhibit regulatory enzymes or transcription factors important for controlling mediators involved in inflammation. Flavonoids are also known as potent antioxidants with the potential to attenuate tissue damage or fibrosis. Consequently, numerous studies in vitro and in animal models have found that flavonoids have the potential to inhibit the onset and development of inflammatory diseases. In the present review, we focused in flavonoids, the most abundant polyphenols in the diet, to give an overview of the most recent scientific knowledge about their impact on different inflammatory diseases.

Effect of an instantaneous controlled pressure drop on in vitro allergenicity to lupins (Lupinus albus var Multolupa).

BACKGROUND: Lupin seed flour has been reported as a causative agent of allergic reactions, especially in patients with allergy to peanut. Previous studies have demonstrated that autoclave treatment can considerably reduce the allergenicity of lupins. AIMS: The aim of this work was to evaluate the effect of instantaneous controlled pressure drop (détente instantanée contrôlée, DIC) treatment on lupin in vitro allergenicity. METHODS: Lupin cotyledons were subjected to instantaneous controlled pressure drop at several pressure and time conditions (3, 4.5 and 6 bar for 1, 2 and 3 min, respectively). Immunoreactivity to raw and DIC-treated extracts was evaluated by Western blot using a serum pool from 19 sensitized patients. RESULTS: Depending on the operating parameters used during DIC treatment, a reduction in protein solubility of lupin seed was observed. Moreover, drastic modifications in protein profiles were observed after DIC treatment by SDS-PAGE analysis. Western blot experiments showed that the decreases in IgE binding to lupin proteins were associated with the increases in steam pressure and time treatment, and binding was completely abolished by DIC at 6 bar for 3 min. CONCLUSIONS: The results suggest that DIC treatment could produce a reduction in lupin allergenicity.

Thermal processing effects on the IgE-reactivity of cashew and pistachio.

Thermal processing can modify the structure and function of food proteins and may alter their allergenicity. This work aimed to elucidate the influence of moist thermal treatments on the IgE-reactivity of cashew and pistachio. IgE-western blot and IgE-ELISA were complemented by Skin Prick Testing (SPT) and mediator release assay to determine the IgE cross-linking capability of treated and untreated samples. Moist thermal processing diminished the IgE-binding properties of both nuts, especially after heat/pressure treatment. The wheal size in SPT was importantly reduced after application of thermally-treated samples. For cashew, heat/pressure treated-samples still retain some capacity to cross-link IgE and degranulate basophils, however, this capacity was diminished when compared with untreated cashew. For pistachio, the degranulation of basophils after challenge with the harshest heat/pressure treatment was highly decreased. Boiling produced more variable results, however this treatment applied to both nuts for 60 min, led to an important decrease of basophil degranulation.

Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits.

Oranges are clinically relevant allergenic foods. To date, orange allergens have not been characterized in detail. The study is aimed at analyzing the sensitization profile in orange-sensitized subjects with and without clinical allergy, and to identify orange allergens. Fifty-six sensitized subjects with self-reported reactions to orange were grouped into reactors (anaphylaxis or multiple episodes of immediate reactions and/or positive challenge tests) and non-reactors (negative open food challenge tests). Allergens were characterized by IgE immunoblotting, N-terminal sequencing, IgE-inhibition assays, and mediator release assays were performed to determine the allergenic potency of orange profilin. Of 56 subjects, 23 were classified as orange allergic showing mainly an oral allergy syndrome. Of 23 subjects classified as orange allergic, 22 were sensitized to profilin, Cit s 2. In patients with mono-sensitization to profilin in vitro histamine releases up to 75% from basophils were induced using orange extract and purified plant profilins. Of the allergic patients 78% were sensitized to germin-like protein, Cit s 1. Both allergens showed retained IgE reactivity in heat-processed orange juice. Interestingly, subjects with and without clinical allergy showed a comparable sensitization profile. Profilin and germin-like proteins are major orange allergens. The potential clinical relevance of orange profilin was indicated by its strong capacity to release histamine from basophils. However, a predominant sensitization to both allergens in subjects without symptoms also indicates a high frequency of clinically insignificant sensitization.

Pin p 1 is a major allergen in pine nut and the first food allergen described in the plant group of gymnosperms.

This study aimed to report the complete sequence of a 2S albumin purified from pine nut and to analyze its allergenic properties. Individual recognition of this protein by serum IgE from pine nut-allergic patients was assessed. IgE cross-linking capacity was analyzed in a basophil activation test. Inhibition of IgE-binding and stability to heating was also assessed. The complete nucleotide sequence was obtained and a phylogenetic study was carried out. 2S albumin from pine nut (registered as Pin p 1.0101) was recognized by IgE of 75% of sera. The allergen was heat-stable and had a robust capacity to inhibit IgE-binding to whole pine nut extract. The IgE cross-linking capacity of Pin p 1 on basophils was also demonstrated. Despite the low homology of Pin p 1 sequence with other allergenic 2S albumins from angiosperms, Pin p 1 contains the typical skeleton of 8 cysteine residues, important for its α-helixes enriched structure.