Littérature scientifique sur le sujet « Proteine allergeniche »

Abstract Current models of digestibility utilize pepsin stability to assess the safety of allergenic versus non-allergenic food proteins. Dietary protein digestion in vivo, however, requires acid denaturation and protease cleavage by pepsin, trypsin, and/or chymotrypsin. The ability of this approach to identify food protein stability in the mammalian gut may be limited. We determined the temporal stability and immunoreactivity of almond, pine nut, and peanut allergenic proteins using simulated physiologic gastric and intestinal digestive conditions in vitro. Gel electrophoresis and immunoblot analyses were used to determine protein stability and immunoreactivity, respectively. Peanut, almond, and pine nut proteins were pepsin- and pancreatin-stable, and immunoreactive for up to 1 h after initiating digestion. Moreover, successive acid denaturation, and pepsin and pancreatin cleavage were necessary to hydrolyze these allergenic proteins and reduce their IgG and IgE-binding capacity, which suggests that digestibility models must be improved for more accurate safety assessment of food allergens.

Edible insects have garnered increased interest as alternative protein sources due to the world’s growing population. However, the allergenicity of specific insect proteins is a major concern for both industry and consumers. This preliminary study investigated the capacity of high hydrostatic pressure (HHP) coupled to enzymatic hydrolysis by Alcalase® or pepsin in order to improve the in vitro digestion of mealworm proteins, specifically allergenic proteins. Pressurization was applied as pretreatment before in vitro digestion or, simultaneously, during hydrolysis. The degree of hydrolysis was compared between the different treatments and a mass spectrometry-based proteomic method was used to determine the efficiency of allergenic protein hydrolysis. Only the Alcalase® hydrolysis under pressure improved the degree of hydrolysis of mealworm proteins. Moreover, the in vitro digestion of the main allergenic proteins was increased by pressurization conditions that were specifically coupled to pepsin hydrolysis. Consequently, HHP-assisted enzymatic hydrolysis represents an alternative strategy to conventional hydrolysis for generating a large amount of peptide originating from allergenic mealworm proteins, and for lowering their immunoreactivity, for food, nutraceutical, and pharmaceutical applications.

Abstract Predicting allergenic potential of novel proteins present in foods such as genetically modified foods is a major challenge facing regulatory agencies and biotech industries. Here we tested the hypothesis that the adjuvant-free mouse model that we have previously reported (Birmingham et al Int Arch Allergy Immunol 2007;144(3):203-10) will be useful to distinguish human food proteins with high vs. low/non allergenic potential. Three readouts were used for testing food allergenicity: 1) systemic specific IgE antibody response to transdermal food protein exposure; 2) clinical symptoms of systemic anaphylaxis to oral food protein challenge; and 3) drop in rectal temperature (hypothermia) to oral food protein challenge. The following food proteins were tested for allergenicity: food proteins with high allergenic potential (cashew nut, milk), food proteins with low/non allergenic potential (kidney bean, Pinto bean); and a food protein with unknown history of allergenicity (Amaranth seed protein). We found that whereas, both cashew nut and milk tested positive for all three readouts of allergenicity, Pinto bean and kidney bean although induced specific IgE antibodies, did not cause systemic anaphylaxis and hypothermia. Interestingly, Amaranth seed protein induced not only a robust IgE response but also elicited clinical symptoms of systemic anaphylaxis as well as hypothermia. These data demonstrate that: 1) further testing with additional food proteins is necessary to evaluate the predictive utility of this mouse model for allergenicity testing; and 2) further studies are needed to evaluate the relationship between circulating food specific IgE antibodies and clinical sensitization for oral food induced systemic anaphylaxis. Funding: US EPA STAR Grant#R833133

Pollen forms large amount of aeroallergens in the atmosphere that spread through winds and insects. Allergenic pollen grains are water-soluble glycoprotein or protein bioparticles and its weight is approximately 5-80 kDa. There are many factors that cause allergies and considering the recently changing climatic conditions and their ability to spread over a wide geographical areas, pollens are among the major factors causing allergies. In this study, it was aimed to detect potential allergen pollen proteins of Tilia cordata which is a potent allergen. The pollen samples belonging to T. cordata were dried and then they were separated with sieve. Acetone washing and dialysis processes were applied to purificate pollen. Afterwards, the amount of protein in the pollens of T. cordata was determined by the bicinchoninic acid (BCA) method. In order to detect allergen proteins, gel runing was performed by SDS-PAGE. Silver staining method was applied to make visible the bands of T. cordata pollen proteins obtained from the gel. Five different allergenic pollen proteins, weights 10, 23, 40, 50 and 80 kDa, were detected as a result of SDS-PAGE. This is the second study in the literature related with the identification of T. cordata allergenic proteins and we showed two different protein bands compared with the former study result. Besides, our study is original in this perspective. We think that this study contributes to the literature on allergenic proteins of T. cordata and should be supported by future studies.

Pollen forms large amount of aeroallergens in the atmosphere that spread through winds and insects. Allergenic pollen grains are water-soluble glycoprotein or protein bioparticles and its weight is approximately 5-80 kDa. There are many factors that cause allergies and considering the recently changing climatic conditions and their ability to spread over a wide geographical areas, pollens are among the major factors causing allergies. In this study, it was aimed to detect potential allergen pollen proteins of Tilia cordata which is a potent allergen. The pollen samples belonging to T. cordata were dried and then they were separated with sieve. Acetone washing and dialysis processes were applied to purificate pollen. Afterwards, the amount of protein in the pollens of T. cordata was determined by the bicinchoninic acid (BCA) method. In order to detect allergen proteins, gel runing was performed by SDS-PAGE. Silver staining method was applied to make visible the bands of T. cordata pollen proteins obtained from the gel. Five different allergenic pollen proteins, weights 10, 23, 40, 50 and 80 kDa, were detected as a result of SDS-PAGE. This is the second study in the literature related with the identification of T. cordata allergenic proteins and we showed two different protein bands compared with the former study result. Besides, our study is original in this perspective. We think that this study contributes to the literature on allergenic proteins of T. cordata and should be supported by future studies.

AbstractAs a result of increased healthcare requirements and the introduction of genetically modified foods, the problem of allergies is becoming a growing health problem. The concept of allergies has prompted the use of new methods such as genomics and proteomics to uncover the nature of allergies. In the present study, a selection of 1400 food proteins was analysed by PLS-DA (Partial Least Square-based Discriminant Analysis) after suitable transformation of structural parameters into uniform vectors. Then, the resulting strings of different length were converted into vectors with equal length by Auto and Cross-Covariance (ACC) analysis. Hierarchical and non-hierarchical (K-means) Cluster Analysis (CA) was also performed in order to reach a certain level of separation within a small training set of plant proteins (16 allergenic and 16 non-allergenic) using a new three-dimensional descriptor based on surface protein properties in combination with amino acid hydrophobicity scales. The novelty of the approach in protein differentiation into allergenic and non-allergenic classes is described in the article.The general goal of the present study was to show the effectiveness of a traditional chemometric method for classification (PLS–DA) and the options of Cluster Analysis (CA) to separate by multivariate statistical methods allergenic from non-allergenic proteins.

The aim of this paper was to focus on proteins present in some food products, like hazelnuts and to investigate their allergenic potential. Several techniques were used to characterize these extracted proteins, with respect to their composition, degradability by digestive proteolytic enzymes and their reactivity with specific antibodies. It was important to analyse which proteins were present in the hazelnuts, to see if there were proteins present to trigger an allergic reaction and if the digestion enzymes trypsin and pepsin influence the presence of the (allergic) protein compounds. Allergies to tree nuts and seeds can cause life-threatening and sometimes fatal reactions. To examine the properties of Hazelnut protein it was important to solubilize it by extraction. After extraction, it was investigated how hazelnut protein can be modified by proteases and what the effect was on the immune reaction. The Bradford method is a fast and sensitive method to determine the concentration of soluble protein. When the Bradford reagent (Coomassie Brilliant Blue) binds to the protein, the colour changes from red to purple and the absorption maximum changes from 495 to 595 nm. The value obtained as the final concentration of proteins was 7.3495. SDS-PAGE is a method to separate mixtures of proteins by electrophoresis. Protein molecules are negatively charged by binding of SDS molecules; subsequently they are separated in an electric field. Their differences in size (molecular weight) leads to separation. In this case the method is used to follow proteolytic degradation of hazelnut proteins (allergens) by intestinal proteases (trypsin, pepsin). A different, more specific and sensitive method is immunoblotting (Western Blot) in which the SDS-PAGE separated proteins are transferred from the gel to a membrane and specific antibodies are used in a series of reactions to visualize specific allergens on this membrane. The remarked spots represented a positive identification of allergenic proteins. This means that peptide fragments of various size, produced during the digestion of a protein can still be immunological active. As it was shown there was still reactivity between proteins and specific antibodies. The Dot Blot is a simple immunoblotting technique used to detected specific proteins in a mixture of different proteins and/or other molecules. No separation technique prior to the actual immuno-detection is necessary. Also, Dot Blot confirmed the presence of allergenic proteins made visible through the light spots on the membrane.

Abstract Most drugs contain pharmaceutical excipients which are pharmacologically inactive substances used as vehicles to deliver the active ingredients of a medication. Some of these pharmaceutical excipients are produced from foods containing allergenic proteins (e.g., milk, egg, peanut, soybean, and sesame) and removing these completely from the excipients is difficult. Therefore, if individuals with food allergies consume drugs containing allergenic food-derived excipients, they are at risk for developing specific allergic symptoms. We determined the levels of total protein in lactose found in pharmaceutical excipients and ethical drugs known to contain lactose by spectrophotometric analyses. The level of total protein in lactose was approximately 1 mg/g. We also determined the levels of allergenic proteins in lactose using commercial enzyme-linked immunosorbent assay systems. The milk proteins in lactose were detected in the range of 1.4-13.1 μg/g. Furthermore, we performed a luciferase-based in vitro elicitation test (EXiLE test) in addition to western-blot analysis using the sera from patients with milk allergies in order to examine the allergenicity of protein residues in lactose. The result of the EXiLE test indicated that the protein residues in lactose have the ability to crosslink human FcϵRI on mast cells.

There is considerable interest in the development and evaluation of approaches for the safety assessment of novel foods, and in particular in methods for characterisation of allergenic potential. One strategy that has found favour is a tiered approach in which the potential of novel proteins to induce allergic sensitisation is assessed based on considerations of stability of the protein in a simulated gastric juice and homology with, or structural similarity to, known allergens. Linked to such an approach may be evaluation of serological identity with proteins known to cause allergic disease. With the aim of supplementing such approaches with a more direct measurement of potential allergenic activity, attempts have been made to characterise the quality of immune responses elicited in BALB/c strain mice. Such evaluations comprise measurement of IgG and IgE antibody production and (to a lesser extent) of induced cytokine expression patterns. Investigations to date suggest that in mice proteins provoke variable immune responses, those with the potential to cause allergic sensitisation stimulating IgE (and IgG) antibody production. In contrast, non-allergenic, but nevertheless immunogenic, proteins are associated with IgG antibody responses in the absence of marked IgE production. Consistent with the selective activation of selective type 2 T lymphocyte responses, exposure of mice to allergenic protein is associated with preferential expression of IL-4, -5, -10 and -13. Collectively these data suggest that characterisation of the nature of immune response induced in mice by proteins may provide a useful adjunct or alternative to current strategies for the assessment of allergenic potential.

The NMR solution structures at different levels of refinement of three different 2 S albumin seed proteins, the recombinant pronapin precursor from Brassica napus, the recombinant RicC3 from Ricinus communis and the methionine-rich protein from sunflower (Helianthus annuus), are described. The resulting common structure consists of a bundle of five α-helices, folded in a right-handed superhelix. The structure is very similar to that of other plant proteins: the hydrophobic protein from soybean, non-specific lipid transfer proteins and amylase/trypsin inhibitors. Analogies and differences in the structures of these families, as well as their possible relationship to allergenicity, are discussed.

The general aim of the activities conducted in the framework of my PhD were to learn modern techniques for analyzing proteins by mass spectrometry (MS) and then to apply these techniques and approaches to the analysis of allergenic proteins contained in foods. My research activities were conducted at the Laboratory of Protein Chemistry of CRIBI, University of Padua, where previously I have conducted the research for my Thesis for the Doctor degree in Pharmaceutical Biotechnologies. During the fist year of my PhD I have concluded the Thesis project on the amyloid aggregation of -lactalbumin, a model protein utilized for investigating molecular aspects of protein amyloidogenesis. The results of this research were quite interesting and indeed they have been published in an international journal. During the first two years I acquired a solid knowledge on several aspects of the MS methodology and I was able to learn the theory and practice of several modern techniques and approaches in this ambit. The specific aim was to analyze allergenic proteins contained in complex matrices as foods and to this aim several proteins were extracted and purified from several food samples. The research has been focused mostly on the allergenic proteins from milk and eggs, known to cause widespread allergies. The proteins of interest were analyzed by using several chromatographic and electrophoretic techniques and also by means of HPLC connected to a tandem MS electrospray instrument. I was able to show that MS techniques can be used to identify allergenic proteins even when contained in very complex mixtures. Therefore, these MS techniques perhaps can be used as an alternative to the immunochemical methods nowadays in use for detecting allergens. I have also analyzed the chemical modifications that allergenic proteins suffer during several industrial treatments of foods, including heat treatment. During the third year of my PhD I spent a six months period at the Biochemistry Laboratory of the Imperial College in London, being involved in a project aimed to study in a large scale the proteins of the mosquito Anopheles gambiae. The MS analyses were focused on the proteins responsible of the mating behaviour of A. gambiae, hoping to identify a target for controlling the behaviour of this vector of the malaria disease. Summing up, besides the publication dealing with amyloid aggregates of beta- lactalbumin, this PhD Thesis is composed by a major part dealing with MS analysis of allergenic proteins and by a minor one dealing with MS analysis of proteins from A. gambiae.

MS Analysis of Allergenic Proteins. Food allergy is a significant worldwide public health issue. Proteins from cow's milk, chicken eggs, soybean and peanuts are the most frequent allergens contained in the complex foods prepared by industrial processes. Allergenic proteins can induce allergic reaction in their native structural state or upon chemical or conformational changes induced by the industrial treatments. Nowadays, the identification of allergenic proteins in foods is conducted by using immunochemical methods such as ELISA tests, but these techniques suffer from several limitations due to cross-reactivity and false negative results. Indeed, alterations in the allergen’s structure or chemical modifications can prevent the interaction with the antibody, thus causing misleading data. Since allergens are toxic even in trace amounts, there is a need for reliable and sensitive analytical methods for allergenic proteins. The purpose of this PhD project was to develop procedures for the identification of these proteins in food samples by using mass spectrometry (MS), likely overcoming some limitations of the immunochemical assays. Indeed, the MS approach for identifying proteins makes use of data pertaining to the amino acid sequence of the protein, while immunochemical methods are linked to the integrity of the three-dimensional structure of proteins. In order to test immunochemical approaches, polyclonal antibodies raised against the main allergenic proteins of milk (α-lactalbumin and β-lactoglobulin) and eggs (ovomucoid, ovalbumin and lysozyme) were purchased. Preliminary studies were performed in order to check the quality of the antibodies, in terms of specificity of recognition and cross-reactivity. Moreover, the responses of the antibodies using as antigens the purified commercial proteins and the same proteins contained in complex food matrices after thermal treatment were checked. Since allergenic proteins usually are contained in complex mixtures of huge amounts of other proteins, the methodology nowadays named “targeted proteomics” was considered very appropriate. By this approach, a protein contained in a complex mixture can be identified by a MS analysis of a peptide fragment that is specific for the protein of interest and contained in the very complex mixture of a tryptic digest of a protein sample. The procedure involves specific labelling and isolation of the specific peptide, named “proteotypic”. To this aim, tryptophan (Trp) residues in proteins were modified by reaction with 2,4-dinitrophenyl-sulfenyl chloride (DNPS-Cl), that leads to a Trp-derivative with the DNPS label attached at 2-position of the indole nucleus. The selection of Trp(DNPS)- peptides from the complex mixture of a tryptic digest of a protein sample was achieved by exploiting the significant change in hydrophobicity and retention time of DNPS-modified peptides in a reverse-phase HPLC column. Moreover, DNPS-labelled Trp-peptides were isolated by hydrophobic interaction chromatography, as well as by immunoaffinity chromatography using a column prepared with anti-DNP antibodies. The “targeted proteomics” procedure was optimised using a mixture of model proteins and then applied to identify a protein allergen contained in a raw bakery product. Overall, it was demonstrated that the novel procedure of selective labelling and isolation of Trp-peptides allows a considerable simplification of the fingerprinting/MS approaches nowadays used for the identification of proteins in proteomics research. Other Research Activities. During the PhD course I had the opportunity to collaborate with other members of the lab in a couple of additional projects, partly as a continuation of previous research conducted for the doctoral thesis. Documentation of this activity is herewith included as an Appendix at the end of this PhD Thesis. The molecular properties of the complex formed by α-lactalbumin with oleic acid were investigated in detail. This complex appears to be very interesting, since it has been shown to display cellular toxicity specifically for cancer cells. It was shown that the protein in the complex is in an oligomeric state, at variance from previous statements that the protein was monomeric. Moreover, it was shown the oleic acid can interact also with other proteins, including apomyoglobin. The main conclusion of this work was that the protein moiety serves as a carrier of the otherwise poorly soluble fatty acid, thus leading to an enhancement of its water solubility and consequently of its intrinsic cytotoxic properties. A manuscript rescrubbing these results is in an advanced state of preparation. Enterocin AS-48 is a 70-residue circular polypeptide produced by Enterococcus faecalis displaying a wide antibacterial activity. Limited proteolysis of AS-48 was used to prepare a linear form of this enterocin, as well as 38- and 55-residue fragments. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that the circularization phenomenon is not required for the antibacterial activity, but it is crucial for the stabilization of the native structural state. This research was published in FEBS Lett. (2008).
Analisi di proteine allergeniche mediante spettrometria di massa. Le allergie alimentari rappresentano ormai una problematica clinica di livello mondiale. Tra i prodotti alimentari considerati pericolosi per il loro elevato contenuto in proteine allergeniche troviamo il latte bovino, le uova, la soia e le arachidi. Le proteine allergeniche possono scatenare reazioni allergiche sia mantenendo la loro struttura nativa, sia in seguito a modifiche chimiche e conformazionali indotte dai processi industriali. I metodi d’elezione applicati per l’identificazione di proteine allergeniche negli alimenti sono rappresentati dai saggi immunochimici come i test ELISA. Tali metodi presentano però numerose limitazioni causate da fenomeni di cross reattività e da falsi positivi. Inoltre, alterazioni nella struttura delle proteine allergeniche o eventuali modifiche chimiche possono modificare l’interazione con gli anticorpi specifici, invalidando i risultati. Dal momento che gli allergeni sono tossici anche in tracce, è necessario sviluppare dei metodi analitici efficaci e affidabili per la loro identificazione. Lo scopo di questo progetto di tesi è stato quello di sviluppare delle procedure per l’identificazione di proteine allergeniche mediante spettrometria di massa (MS) che possano superare i limiti metodici dei saggi immunologici. Oltretutto, l’identificazione delle proteine mediante MS si basa sull’analisi della sequenza amminoacidica di quest’ultime, mentre i saggi immunochimici sono strettamente dipendenti dall’integrità della struttura tridimensionale della proteina antigenica. Al fine di testare la validità dell’approccio immnuchimico, sono stati testati alcuni anticorpi policlonali diretti contro le principali proteine allergeniche di latte α-lattalbumina e β-lattoglobulina) e uova (ovomucoide, ovalbumina e lisozima). Sono stati condotti alcuni studi preliminari per validare la qualità di questi anticorpi, in termini di specificità di riconoscimento della proteina antigenica e della presenza di eventuali fenomeni di cross reattività. Inoltre, è stata valutata la risposta anticorpale usando come antigeni sia le proteine commerciali purificate, sia le stesse proteine contenute in prodotti alimentari prima e dopo trattamento termico. Dato che le proteine allergeniche sono contenute in miscele complesse costituite da altre proteine, è stata considerata estremamente appropriata l’applicazione di una tecnica detta “targeted chromatography”. Secondo questo strategia, è possibile identificare mediante MS una proteina contenuta in una miscela complessa attraverso l’analisi di alcuni frammenti peptidici derivati dalla digestione triptica, che sono specifici della proteina stessa. Questa procedura prevede la modifica chimica e il successivo isolamento di specifici peptidi detti “prototipici”. A tale scopo, i residui di triptofano contenuti nelle proteine sono stati chimicamente modificati mediante una reazione con il composto 2,4- dinitrofenilsulfenil cloruro (DNPS-Cl), che porta alla formazione di un derivato triptofanilico, con il DNPS legato in posizione 2 dell’anello indolico. La selezione dei peptidi modificati con il DNPS-Cl contenuti in una miscela triptica è stata effettuata sfruttando l’aumento di idrofobicità e del tempo di ritenzione di questi peptidi modificati in una colonna HPLC a fase inversa. Inoltre, gli stessi peptidi modificati con DNPS-Cl sono stati isolati mediante cromatografia per immunoaffinità utilizzando una resina derivatizzata con anticorpi monoclonali diretti contro il gruppo DNP. La strategia di ”targeted proteomics” è stata ottimizzata utilizzando una miscela modello di sette proteine e successivamente è stata applicata per l’identificazione di una proteina allergenica contenuta in un prodotto dolciario. È stato inoltre dimostrato che queste nuove procedure di modifica selettiva e di selezione dei peptidi triptofanilici permette di semplificare considerevolmente l’analisi di fingerprinting/MS che è solitamente utilizzata per l’identificazione di proteine nei protocolli di proteomica. Altre attività di ricerca. Durante il periodo di dottorato, ho avuto l’opportunità di collaborare con altri membri del laboratorio in due progetti addizionali come continuazione di un progetto di ricerca precedente. La documentazione relativa a queste attività è riportata in appendice alla tesi di dottorato. Sono state investigate le proprietà molecolari del complesso formato da α-lattalbumina con l’acido oleico. Il complesso appare interessante poiché ha mostrato avere tossicità cellulare diretta selettivamente contro cellule tumorali. È stato dimostrato che la proteina nel complesso ha una struttura oligomerica, diversamente da quanto riportato nelle prime osservazioni, che ipotizzavano fosse in uno stato monometrico. Inoltre, è stato osservato che l’acido oleico interagisce anche con altre proteine, come l’apomioglobina. La principale conclusione di questo lavoro è stata che il motivo oligomerico della proteina veicola l’acido oleico, normalmente poco solubile, favorendo quindi la solubilizzazione dell’acido grasso e conseguentemente della sua proprietà citotossica. È in preparazione un articolo riguardante questi risultati. L’enterocina AS-48, è un polipeptide circolare di 70 residui prodotto da Enterococcus faecalis che mostra a vere attività antibatterica. La proteolisi limitata è stata usata per preparare una forma lineare e due frammenti di questa enterocina. Misure di dicroismo circolare nel lontano ultravioletto hanno dimostrato che la proteina ha una bassa ellitticità e una ridotta stabilità alla denaturazione termica, ma mantiene la sua attività antibatterica., mentre i frammenti presentano un’attività ridotta. Questi risultati indicano che la circolarizzazione è un fenomeno che non è richiesto per l’attività antibatterica, ma è cruciale per la stabilizzazione della struttura nativa. Questa ricerca è stata pubblicata in FEBS Lett. (2008).

2013 - 2014
The great interest of the research activity in food allergies could be attributed to the increase of allergic reactions all over the world not only in infants but even in adult age. As an alternative to the development of an allergen-free diet, many works have been focused on a novel approach for the treatment of allergens: instead of eliminating the allergens from the diet, the immunoresponse can be reduced or even eliminated by inducing some modifications of their molecular structure. In fact, changes in allergen conformation can modulate its identification by the specific antibody produced by immune system in allergic reactions. Structural modifications in allergens could be induced by conventional thermal treatments as well as by non-thermal technologies, namely High Hydrostatic Pressure (HHP), Pulsed Electric Fields (PEF), Pulsed Light (PL) and -radiations. Non-thermal technologies have been widely used in the last years for food preservation, having the advantage of increasing the shelf-life and freshness of the raw food products. These technologies are able to affect the food nutritional and organoleptic properties only slightly thanks to the use of a non-thermal stress to treat foodstuffs. Among them High Hydrostatic Pressure technology has been successfully used in food pasteurization, but also in processes involving the sol-gel transition such as the production of jams, jellies and dairy products. The ability of High Pressure to determine structural changes in foods was studied in order to assess if proteins unfolding and/or aggregation and gelation can be induced and if the treatment affects the functional properties and digestibility of proteins. These effects were studied on particular proteins, namely the allergens, for which unfolding and structural modification have been proven. However, the effectiveness of the High Pressure processing on the reduction of immunoresponse reduction was not clearly assessed so far. The objective of this PhD thesis was the study of the modifications induced by High Pressure Process on allergenic proteins and the possibility of obtaining hypoallergenic peptides by means of a combined High Hydrostatic Pressure hydrolysis. In particular, the effect of the HHP on the allergens structural modification was investigated in a wide range of operating conditions, including both gelling and ungelling conditions. Rheological behavior and functional properties of HHP processed allergens was also determined. [edited by Author]
XIII n.s.

Pollen grains are ubiquitous triggers of allergic asthma and seasonal rhinitis. Proteolytic enzymes in pollen as well as other sources are capable of disrupting airway epithelial integrity in vivo and in vitro. This provides a plausible mechanism for the initiation of sensitisation of the respiratory immune system to inhaled pollen allergens, comparable to that suggested for Group 1 allergens from house dust mites and cat dander, which are known to possess intrinsic proteolytic activity. This thesis explores the relationship between pollen allergens and proteolytic enzymes. It describes the different strategies used for the characterisation, purification and identification of immunogenic and proteolytic proteins in the complex mixtures of pollen diffusates. The peptidases in the diffusates of Kentucky blue grass, ryegrass and Bermuda grass pollens were characterised by a sensitive fluorescence assay and gelatin zymography. Among these, Bermuda grass pollen demonstrated the presence of a serine peptidase at Mr ~30,000 Da, which corresponded to an intense band by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen, Phl p 1. Purification of this enzyme from Bermuda grass was complicated by the low levels of the enzyme present in the diffusate, as well as by its autohydrolysis. Partial purification of the serine peptidase activity by affinity chromatography using Concanavalin A Sepharose demonstrated that the diffusate contained a trypsin-like peptidase, detected by the fluorescent assay, in addition to the ~30,000 Da serine endopeptidase, detected on gelatin zymography. Proteomic analysis of the ~30,000 Da protein using one- and two-dimensional electrophoresis and mass spectrometry identified it as the major pollen allergen of Bermuda grass, Cyn d 1. The studies reported here provide, for the first time, evidence that a pollen allergen may possess intrinsic proteolytic activity. This activity may play a role in the initiation of airway inflammation and allergic sensitisation.

Lipid transfer proteins (LTPs) are a class of low molecular weight hydrophobic conserved proteins comprising four intramolecular disulphide bonds making the structure very resistant to proteolysis and harsh food processing conditions. These proteins are identified as strong allergens sensitizing through the gut and share epitopes with LTPs from closely related species. Peach LTP, Pru p 3 is the primary sensitizer in the Mediterranean area being the most frequent food allergen. Wheat LTP, Tri a 14 is a relatively weak allergen with a very low prevalence. The study here compares the structural properties of these proteins and their resistance to various digestive and processing processes. Ligand binding experiments showed that Pru p 3 binds to ligands more strongly than Tri a 14. The gastroduodenal digestion of these LTPs revealed that both are stable to gastric digestion and while Pru p 3 is susceptible to duodenal digestion, Tri a 14 digestion is negligible. Ligand binding did not affect the digestibility of Pru p 3 but improved the duodenal digestibility of Tri a 14. The IgE binding studies using sera from peach allergic individuals confirmed that both Pru p 3 and its digestion fragments in the presence and absence of ligand were IgE reactive. Model processing conditions were employed to treat these LTPs. It was found that heat treatment destroys the secondary structure of Pru p 3 at 121°C and slightly affects that of Tri a 14. Heat treatment also increased the susceptibility of Pru p 3 to gastric digestion while Tri a 14 was less affected. The IgE binding studies showed that heat treatment of Pru p 3 appeared to reduce its IgE recognition while its digestion fragments lost all of their IgE reactivity. To investigate the effect of the food matrix on the digestibility of these LTPs, peach peel containing Pru p 3 and wheat flour containing Tri a 14 were digested under simulated conditions. It was found that they were resistant to proteolysis in their native matrices. Effect of heat treatment to the food matrix again confirmed that both of these proteins were more stable to heat in the matrix and were less digestible. In conclusion, this study shows that there are factors in food matrices which enhance structural stability of LTPs to both processing and digestion. Thus factors such as the effect of food matrix and effect of processing should be taken into account in assessing the allergenic risk posed by foods and not simply rely on data from purified proteins.

Orientadores: Maria Helena Andrade Santana, Ricardo de Lima Zollner
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
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Resumo: Os lipossomas têm sido utilizados como imunoadjuvantes antigênicos, em estudos que enfatizam a sua atuação no sistema imunológico. Extratos peptídicos ou protéicos de várias fontes foram encapsulados em lipossomas ou associados à sua superficie, e aplicados em imunoterapias e vacinas. Apesar disso, poucos são os estudos voltados para a performance do processo de preparação desses lipossomas, eficiência da associação proteína/lipídio e estabilidade das vesículas. Este trabalho trata da avaliação do encapsulamento das proteínas: Albumina de Soro Bovino (BSA), Mioglobina (Mio) e Cito cromo C (Cit C), simulando extratos alergênicos provenientes de fungos e ácaros, no que se refere à faixa de peso molecular das suas proteínas. Os lipossomas foram preparados com os lipídios L-a-distearoilfosfatidi1colina (DSPC) e Colesterol (Col) na razão molar 70:30 respectivamente. As proteínas foram encapsuladas individualmente e em forma de misturas de composições molares BSA:Mio:Cit C de 20:40:40, 40:20:40 e 40:40:20, respectivamente. Além disso, a mistura de proteínas de composição 40:20:40 (BSA:Mio:Cit C) foi também associada covalentemente à superficie das vesículas. Os lipossomas foram caracterizados pelo teor de fósforo e diâmetro médio. O desempenho do encapsulamento foi analisado através dos perfis e eficiências de encapsulamento das proteínas individuais e em misturas, além da estabilidade das vesículas em tensoativo penta etileno glicol mono-n-dodecil éter (C12Es), e de plasma humano. Para as misturas, analisou-se a exclusão de proteínas durante o encapsulamento e a associação à superficie dos lipossomas. Os resultados indicam que a eficiência de encapsulamento depende mais das interações proteína:lipídio que do tamanho das proteínas. As eficiências de encapsulamento variaram entre 0,03% e 3,55% para as proteínas individuais e entre 1,04% e 3,94% para as misturas. Não houve alteração na estabilidade dos lipossomas em C12Es com a presença das proteínas. Em plasma, essas vesículas permaneceram estáveis por aproximadamente 20 horas. Na associação à superficie dos lipossomas, predominou a presença de BSA em relação às outras proteínas
Abstract: Liposomes has been studied as antigenic immunoadjuvants with emphasis on their performance in the imunological system. Peptides or proteins of various sources were encapsulated in liposomes or associated to their surface, and applied in immunotherapy and vaccines. In spite of this, there are few studies about the performance of preparation process ofthese liposomes, efficacy ofthe association proteinllipid and the stability ofvesic1es. This work concems with the evaluation of the encapsulation of the proteins Bovine Serum Albumin (BSA), Myoglobin (Myo) and Cythochrome C (Cyt C), simulating allergen extracts from molds and mites, related to the range of molecular weight of their proteins. Liposomes were prepared with lipid L-a-disteraoylphosphatidylcholine (DSPC) and Cholesterol (Chol) at molar ratio 70:30 respectively. The proteins were encapsulated individualyand in mixtures with molar composition BSA:Myo:Cyt C of20:40:40, 40:20:40 and 40:40:20 respectively. Furthermore, the mixture 40:20:40 (BSA:Myo:Cyt C) was also covalent1y associated to the surface of vesic1es. Liposomes were characterized by their phosphate contents and mean diameter. The performance of the protein encapsulation was evaluated through the profiles and efficiency of the encapsulation and throught the stability of vesic1es in the presence of the surfactant penta ethylene glycol mono-n-dodecyl ether (C12ES) and human plasma. For the mixtures, the exclusion of proteins duringentrapment or association to the liposome surface was also evaluated. The experimental results indicate that the efficacy of encapsulation is more dependent of the interactions proteinllipid than the size of the proteins. The efficiencies of encapsulation changed between 0.03% and 3,55% for individual proteins and were between 1,04% and 3,94% for the mixtures. The presence ofproteins does not altered the stability of liposomes in C12ES . In human plasma the vesic1es remained with stability about 20 hours. For the mixtures, the presence ofthe BSA protein predominated in the vesicles
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestre em Engenharia Química

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
This work reports the biochemical characterization, cytotoxic and allergenic effects of a lipid transfer protein isolated from M. citrifolia seeds (McLTP1), with trypsin and alpha-amylase inhibition properties. McLTP1 was purified with a procedure involving trichloroacetic acid precipitation and gel filtration chromatography. This protein showed significant inhibitory activities against trypsin (767,10 Â 8,36 TIU/mgP), chymotrypsin (25,36 Â 0,86 IU/mgP), papain (65,419 Â 0,152 IU/mgP) and alpha-amylase (24,40%). Atomic force microscopy displayed that McLTP1 oligomerized in tetramers showing a central channel. Fluorescence and CD assays revealed that the McLTP1 structure is highly stable, regardless of pH and temperature levels. In vitro, McLTP1 presented a selective cytotoxic effect to human ovarian cancer cells (OVCAR-8; IC50 of 16,6 μg/mL) and demonstrated hemolytic effect against fresh rabbit red blood cels. Similarly to other non-specific lipid transfer protein reported, McLTP1 showed allergenic properties in mice, being considered as a true food allergen since it was able to sensitize the animals via the gastrointestinal tract.
Morinda citrifolia L. à uma espÃcie nativa do Sudeste da Ãsia intensamente investigada em funÃÃo de suas propriedades terapÃuticas reportadas hà mais de 2.000 anos. Recentemente, uma proteÃna transferidora de lipÃdeos denominada McLTP1 (UniProt Accession Number: C0HJH5) foi isolada de sementes de noni pelo nosso grupo de pesquisa. McLTP1 à uma proteÃna termoestÃvel de massa molecular 9,4 kDa, resistente à proteÃlise e dotada de atividades moduladoras da inflamaÃÃo e da dor pela via oral, promissoras e inÃditas para esse grupo de molÃculas. Este trabalho objetivou caracterizar bioquimicamente McLTP1, bem como avaliar o seu potencial alergÃnico em camundongos, como etapas bÃsicas para o seu uso racional e seguro do ponto de vista farmacolÃgico. Em adiÃÃo, as propriedades terapÃuticas de McLTP1 foram tambÃm ampliadas, atravÃs da investigaÃÃo de seu efeito citotÃxico em diferentes linhagens de cÃlulas tumorais. A proteÃna em estudo foi isolada utilizando o protocolo jà estabelecido, envolvendo as etapas de precipitaÃÃo seletiva de proteÃnas do extrato total das sementes de noni com Ãcido tricloroacÃtico 2,5% e cromatografia de exclusÃo molecular. O ensaio de alergenicidade in vivo foi conduzido apÃs prÃvia aprovaÃÃo pelo Comità de Ãtica para Uso de Animais da Universidade Federal do Cearà e utilizou fÃmeas nulÃparas com massa corporal entre 25 e 30 g. McLTP1 apresentou in vitro atividades inibitÃrias de tripsina (767,10  8,36 UIT/mgP), quimotripsina (25,36  0,86 UI/mgP), papaÃna (65,419  0,152 UI/mgP) e alfa-amilase (24,40%). A atividade inibitÃria de tripsina de McLTP1 foi reduzida significativamente em temperaturas superiores a 37 ÂC, apresentando atividade residual de apenas 5,91% quando aquecida a 100 ÂC por 30 min. Essa atividade foi tambÃm influenciada pelo pH, sendo de apenas 30,13% e 39,05% quando a proteÃna foi incubada em tampÃes de pH 3,0 e 12,0. O padrÃo de oligomerizaÃÃo de McLTP1 demonstrou a formaÃÃo de agregados dimÃricos/tetramÃricos delimitando um canal central de diÃmetro de 4,4 nm. As anÃlises espectroscÃpicas mostraram que McLTP1 apresenta espectro de CD similar Ãquele apresentado por outras proteÃnas transferidoras de lipÃdeos e caracterÃstico de proteÃnas ricas em alfa-hÃlice. Espectro de CD de McLTP1 nÃo mostrou alteraÃÃes significativas em diferentes temperaturas e pHs, corroborando com os dados de estabilidade obtidos anteriormente. Diferentemente, em condiÃÃes redutoras (DTT 1 mM) o espectro de CD mostrou alteraÃÃo na estrutura secundÃria da proteÃna e os mÃnimos e mÃximos de elipticidade molar foram tambÃm alterados na presenÃa de micelas iÃnicas de SDS (10 mM). McLTP1 apresentou atividade citotÃxica seletiva contra cÃlulas de cÃncer de ovÃrio (Ovcar-8; CI50: 16,6 μg/mL), nÃo sendo citotÃxica para as cÃlulas tumorais de cÃlon humano (HCT-116), leucemia humano (HL-60) e glioblastoma humano (SF-295) testadas. McLTP1 foi capaz de promover hemÃlise significativa em hemÃcias de coelho a partir da concentraÃÃo de 0,005 mgP/mL. McLTP1 apresentou potencial efeito alergÃnico in silico e em camundongos imunizados pela via oral, induzindo a sÃntese de anticorpos IgG e IgG1. Tal como descrito na literatura para outras LTPs, anticorpos anti-McLTP1 produzidos em coelho foram tambÃm capazes de reconhecer proteÃnas presentes em extratos de Rosaceae, Cucurbitaceae e na polpa do fruto de noni. Os dados obtidos permitiram caracterizar parcialmente a proteÃna em estudo, bem como avaliar o seu potencial imunogÃnico apÃs administraÃÃo oral. Novos testes serÃo conduzidos objetivando avaliar a importÃncia clÃnica dessas respostas, uma vez que testes de toxicidade demonstraram que McLTP1 nÃo foi capaz de promover reaÃÃes adversas em camundongos, mesmo apÃs administraÃÃo da dose de 8 mg/kg por 28 dias.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
ABSTRACT: Two experiments were conduct to evaluate the effect of using soy protein concentrate (SPC) in pre-starter piglet diets over performance, intestinal histomofometria and blood parameters. In the first trial 72 piglets weaned at 21 days of age were used and 54 for the second trial. Both were conducted as a randomized blocks design with six replications and three piglets per unit. The first trial had four diets (0% SPC, 3% SPC, 6% SPC and 9% SPC), which evaluated different levels of SPC inclusion in diets, and the second trial, which evaluated the replacement of spray dried blood plasma (SDP) by SPC, had three diets (0.0%-SPC + 5.0-SDP, 2.5%-SPC + 2.5%-SDP and 5.0%-SPC + 0.0%-SDP). In both trials, diets and water were offered ad libitum in pre-starter l (21-32 days old) and pre-starter ll (33-42 days old) phases, but during the starter phase (43-66 days old) all piglets received a single diet. At 32 days of age, blood was collected from one animal per experimental unit and then these animals were euthanized to collect samples of the small intestine. Linear effect was observed In the first trial, over feed conversion (FC) as the level of CPS in diet was increased in the period between 21 and 32 days of age. Linear effect was also observed on the FC in the period of 33-42 days old, however in a reverse form from the previous period, wherein the increase of the SPC level in diets resulted in the increase of FC. In the total period of this trial (21-66 days old), it was found a quadratic effect of SPC on piglets FC. In the second trial, the replacement of SDP by CPS caused effect on performance variables, except for feed conversion (FC) during 21-32 days of piglets age. The average daily feed intake (ADFI) was higher for pigs fed 2.5%-SPC + 2.5%-SDP and the average daily gain (ADG) and the final body weight (FBW) of the piglets in this period was higher for 2.5%-SPC + 2.5%-SDP compared to 5.0%-SPC + 0.0%-SDP. In the period of 21-42 days old, it was observed best results of ADFI and FBW for piglets fed diet containing 2.5%-SPC + 2.5%-SDP compared to piglets fed 5.0%-SPC + 0.0%-SDP. The ADG showed better results for diets containing 0.0%-SPC + 5,0-SDP and 2.5%-SPC + 2.5%-SDP and FC was higher for the diet with 0.0%-SPC + 5.0%-SDP compared to the diet with 5.0%-SPC + 0.0%-SDP. During total period of the experiment (21-66 days old) ADG and ADFI was influenced by substitution of SDP for SPC, wherein the best results was presented by piglets fed 2.5%-SPC + 2.5%-SDP compared to those fed 5.0%-SPC + 0.0%-SDP. In both trials, no effect was observed upon histomorphometric variables (villus height, crypt depth and villus: crypt). Regarding the blood variables (leukocytes, eosinophils and lymphocytes), no effect of SPC at 32 days of age the piglets was observed in the first trial. But in the second trial, total leukocyte count was higher in animals fed 5.0%-SPC + 0.0%-SDP compared to ones fed 0.0%-SPC + 5.0%-SDP and lymphocyte count was lower in piglets receiving 0.0%-SPC + 5.0%-SDP. In the first trial, the use of SPC in diets of post-weaning piglets during pre-starter period (21-42 days old) did not influence performance from 21 to 66 days of life, or intestinal morphology and leukocyte, lymphocytes and eosinophils count in these animals. In the second, the combined use of SPC and SDP in the diets of pigs between 21 and 42 days of age reduces the activation of the immune system and improves productive performance.
Foram conduzidos dois experimentos com objetivo de avaliar o efeito da utilização de Concentrado proteico de soja (CPS) em dietas pré-inicias de leitões sobre o desempenho, histomofometria intestinal e alguns parâmetros sanguíneos. Foram utilizados 72 leitões machos castrados desmamados aos 21 dias de idade no primeiro experimento e 54 no segundo. Ambos foram delineados em blocos ao acaso com seis repetições e três leitões por unidade experimental, tendo o primeiro quatro tratamentos (0% CPS, 3% CPS, 6% CPS e 9% CPS), o qual avaliou diferentes níveis de inclusão de CPS nas dietas, e o segundo, para avaliar a substituição de plasma sanguíneo (SDP) por CPS, três (0,0%-CPS + 5,0-SDP, 2,5%-CPS + 2,5%-SDP e 5,0%-CPS + 0,0%-SDP). Em ambos as rações experimentais e água foram fornecidas à vontade nas fases pré-inicial l (21 a 32 dias de idade) e pré-inicial ll (33 a 42 dias de idade), sendo que na fase inicial (43 a 66 dias de idade) todos os leitões receberam uma única dieta. Aos 32 dias de idade foi coletado sangue de um animal por unidade experimental e em seguida estes animais foram eutanasiados para coleta de amostras de intestino delgado. No primeiro experimento, foi observado efeito linear decrescente sobre a conversão alimentar (CA) à medida que aumentou o nível de CPS na dieta no período entre 21 e 32 dias de idade. Efeito linear também foi observado sobre a CA no período entre 33 e 42 dias de idade dos leitões, entretanto de forma inversa ao período anterior, ou seja, à medida que se aumentou o nível de CPS na dieta a CA aumentou. Foi observado no período total (21 a 66 dias de idade) do primeiro experimento efeito quadrático do CPS sobre a CA dos leitões. No segundo experimento a substituição de SDP por CPS causou efeito sobre as variáveis de desempenho no período entre 21 e 32 dias de idade, exceto sobre a CA. O CDR foi superior para os leitões alimentados com 2,5%-CPS + 2,5%-SDP e o GDP e o peso final (PF) dos animais neste período foi superior com 2,5%-CPS + 2,5%-SDP comparado a 5,0%-CPS + 0,0%-SDP. No período entre 21 e 42 dias de idade foi observado melhores resultados de CDR e PF para a dieta com 2,5%-CPS + 2,5%-SDP comparado a 5,0%-CPS + 0,0%-SDP. O GDP apresentou melhores resultados para as dietas com 0,0%-CPS + 5,0-SDP e 2,5%-CPS + 2,5%-SDP e a CA foi superior para a dieta com 0,0%-CPS + 5,0%-SDP diante da dieta com 5,0%-CPS + 0,0%-SDP. No período total do experimento (21 a 66 dias de idade) foi observado efeito da substituição dos SDP por CPS sobre o GDP e o CDR, sendo que os animais alimentados com 2,5%-CPS + 2,5%-SDP apresentaram resultados superiores aos alimentados com 5,0%-CPS + 0,0%-SDP. Emambos os experimentos nenhum efeito foi observado sobre as variáveis histomorfométricas (altura de vilosidade, profundidade de cripta e relação vilo:cripta). Em relação às variáveis sanguíneas (leucócitos, linfócitos e eosinófilos) do primeiro experimento, não foi observado nenhum efeito do CPS aos 32 dias de idade dos leitões. No segundo experimento, a contagem total de leucócitos foi superior para os animais alimentados com 5,0%-CPS + 0,0%-SDP sobre os alimentados com 0,0%-CPS + 5,0%-SDP e a contagem de linfócitos foi menor nos leitões que receberam 0,0%-CPS + 5,0%-SDP. No primeiro experimento, a utilização de CPS em dieta de leitões pós-desmame no período préinicial (21 a 42 dias de idade) não influenciou o desempenho produtivo dos leitões no período entre 21 e 66 dias de vida, nem a histomorfometria intestinal e a contagem de leucócitos, linfócitos e eosinófilos desses animais. No segundo, a utilização conjunta de CPS e SDP nas rações reduz a ativação do sistema imune e melhora o desempenho produtivo.

Glabrous canaryseed, a novel cereal grain, is emerging as a valuable source of plant proteins due to its high content in protein (22%). This true cereal was approved for human consumption in Canada and the United States, and as part of the regulatory safety assessment, its allergenic potential was evaluated. Canaryseed was found to be gluten-free and thus, suitable for individuals with celiac disease, however, possible allergic cross-reactivity between canaryseed and wheat was also revealed. Based on these findings, a cautionary labelling alluding to the potential of allergic reaction is requested on canaryseed food products, and further research to clarify the relationship between canary seed proteins and known wheat allergens was recommended. Therefore, the purpose of this study was to further assess the immunological cross-reactivity risks of canary seed to phylogenetically related grains, including wheat and oat, using wheat-allergic sera IgE based 1D and 2D- immunoblots and ELISA, followed by proteomic/bioinformatics identification of IgE-binding proteins. The results demonstrated extensive serological cross-reactivity between wheat, oat and canaryseed proteins, where the less abundant protein fractions showed the strongest IgE-binding. The in-gel tryptic digestion and LC-MS/MS identification of the IgE-binding canaryseed proteins showed high homology to proteins from wheat, barley, oat and Brachypodium distachyon (also known as stiff brome), which all belong to the Pooideae botanical subfamily. A majority of the IgE-binding proteins were mostly minor metabolic enzymes or uncharacterized proteins. Low sequence homology was observed for the 11-12S globulin storage proteins. Positive serological testing cannot ascertain allergic reaction to canaryseed, it does not rule out, however, the risks for wheat, oat or barley sensitized atopic population. Clinical oral food challenge remains the ultimate tool to conclude on the allergenicity of canaryseed. Until then, these data serve the reinforcement of the regulatory requirement to use allergen precautionary labeling for products containing canaryseed proteins.