Effect of food matrix and processing on release of almond protein during simulated digestion

The aims of the present work were to assess digestibility of almond protein in the upper gastrointestinal tract, evaluate the effects of food matrix on protein release and assess the persistence of immunoreactive polypeptides generated during simulated digestion. Prunin, the most abundant protein in almond flour, was sensitive to pepsin, with complete digestion after 20 min in the gastric phase. Addition of the surfactant phosphatidylcholine did not affect the rate and kinetic of digestion, as observed by SDS-PAGE analysis and HPLC, in the stomach and the small intestine of either natural or blanched almond flour. However, incorporation of almond flour into a food matrix, such as chocolate mousse and Victorian sponge cake, decreased the rate of almond protein degradation by pepsin and immunoreactivity of almond polypeptides detected by dot blots and sandwich ELISA retained better. Most of the almond protein identified by in-gel tryptic digestion and MALDI-TOF analysis corresponded to prunin, with pI values of 5–7.     

Comparative resistance of food proteins to adult and infant in vitro digestion models

IgE‐mediated allergy to milk and egg is widespread in industrialised countries and mainly affects infants and young children. It may be connected to an incomplete digestion of dietary proteins causing an inappropriate immune response in the gut. In order to study this, a biochemical model of infant gastroduodenal digestion has been developed, which has reduced levels of protease (eightfold for pepsin and tenfold for trypsin and chymotrypsin), phosphatidylcholine and bile salts, compared with the adult model. This model has been used to study the behaviour of three characterised food‐relevant proteins (bovine β‐lactoglobulin (β‐Lg), β‐casein (β‐CN) and hen's egg ovalbumin), all of which are relevant cows' milk and hens' egg allergens. Digestion products were characterised using electrophoresis, immunochemical techniques and MS. These showed that ovalbumin and β‐CN were digested more slowly using the infant model compared with the adult conditions. Resistant fragments of β‐CN were found in the infant model, which correspond to previously identified IgE epitopes. Surprisingly, β‐Lg was more extensively degraded in the infant model compared with the adult one. This difference was attributed to the tenfold reduction in phosphatidylcholine concentration in the infant model limiting the protective effect of this phospholipid on β‐Lg digestion.     

Immunoreactivity of hen egg allergens: Influence on in vitro gastrointestinal digestion of the presence of other egg white proteins and of egg yolk

Hen egg white comprises of a complex mixture of proteins, which greatly differ in their physicochemical characteristics and relative abundance. We aimed to identify potential undiscovered egg allergens within the egg white proteome and investigated the existence of matrix effects on the proteolytic stability and resultant IgE-binding of the allergenic proteins. In addition to the main egg allergens: ovalbumin (OVA), ovomucoid (OM) and lysozyme (LYS), two minor egg white proteins, tentatively identified as ovoinhibitor and clusterin, were found to react with serum IgE from egg-allergic patients. Egg white exhibited residual immunoreactivity after gastrointestinal digestion due to the presence of intact OVA and LYS, as well as of several IgE-binding peptides derived from OVA. The presence of egg yolk slightly increased the susceptibility to hydrolysis of egg white proteins and abrogated bile salt-induced precipitation of LYS in the duodenal medium. However, the resultant immunoreactivity against IgE of egg white proteins after in vitro digestion was not significantly modified by the presence of yolk components.     

Vicilin and the basic subunit of legumin are putative chickpea allergens

IgE-mediated reactions to food allergens constitute a major health problem in industrialized countries. Chickpea is consumed in Mediterranean countries, and reportedly associated with IgE-mediated hypersensitivity reactions. However, the nature of allergic reactions to chickpea has not been characterized.     

Molecular fragmentation of wheat-germ agglutinin induced by food irradiation reduces its allergenicity in sensitised mice

WGA, an agglutinin from wheat germ which is largely responsible for many of wheat’s allergies, was used as a model to investigate the action of ionising radiation on WGA’s anti-nutritive effects in sensitised mice. Based on the molecular structure, the present study also examined the structural modification of WGA in relation to the range of dose. Structural integrity was monitored using HPLC, fluorescence spectrometry and circular dichroism. Results showed a loss of intrinsic activity and the formation of insoluble amorphous aggregates with a lack of native conformational structures after irradiation. Current findings suggest that the allergenic epitopes of WGA became less active and antigenic after high-dose radiation. The reduction of cytokines typical of allergic reactions, with decreased lymphocytic infiltrate, was observed in the gut of mice given irradiated versus native WGA. Food irradiation proved effective and safe in combating immunological and allergic effects of WGA.     

Influence of baking time and matrix effects on the detection of milk allergens in cookie model food system by ELISA

Milk allergens are common allergens occurring in foods, therefore raising concern in allergic consumers. Enzyme-linked immunosorbent assay (ELISA) is, to date, the method of choice for the detection of food allergens by the food industry although, the performance of ELISA might be compromised when severe food processing techniques are applied to allergen-containing foods. In this paper we investigated the influence of baking time on the detection of milk allergens by using commercial ELISA kits. Baked cookies were chosen as a model food system and experiments were set up to study the impact of spiking a matrix food either before, or after the baking process. Results revealed clear analytical differences between both spiking methods, which stress the importance of choosing appropriate spiking methodologies for method validation purposes. Finally, since the narrow dynamic range of quantification of ELISA implies that dilution of samples is required, the impact of sample dilution on the quantitative results was investigated. All parameters investigated were shown to impact milk allergen detection by means of ELISA.     

Cow's milk allergens identification by two-dimensional immunoblotting and mass spectrometry

Cow's milk allergy (CMA) has become a common disease in early childhood, its prevalence ranging from 1.6% to 2.8% among children younger than 2 years of age. The role of different cow's milk protein (CMP) in the pathogenesis of CMA is still controversial. Even if the proteins most frequently and most intensively recognized by immunoglobulin E (IgE) seem to be the most abundant in milk (caseins and beta-lactoglobulin), with an although great variability all milk proteins appear to be potential allergens, even those that are present in trace amounts (i.e., lactoferrin, IgG, and BSA). In this work proteomics techniques have been applied for CMP allergens analysis. Allergens have been identified by immunoblotting following resolution of CMP components by two-dimensional electrophoresis. Sera from 20 milk-allergic subjects, as proven by oral provocation test, CAP-RAST and skin prick test, have been used for cow's milk major allergen identification. Cow's milk proteins and their isoforms were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. In our group of patients, the prevalence of CMP allergens, i.e., the total number of subjects sensitized to CMP divided by the total number of the subjects enrolled in the study, was: 55% alpha(s1)-casein, 90% alpha(s2)-casein, 15% beta-casein, 50% kappa-casein, 45% beta-lactoglobulin, 45% BSA, 95% IgG-heavy chain, 50% lactoferrin, and 0% alpha-lactalbumin.     

Influence of food processing on the immunochemical stability of celery allergens

Celery roots were processed by microwave heating, cooking, drying, γ-irradiation, ultra high pressure treatment and high voltage impulse treatment. The immunochemical stabilities of the three known allergenic structures of celery were tested with sera from patients who were sensitised to celery. In addition, rabbit antisera were used to detect the allergens profilin and Api g 1 on celery immunoblots. The specificity and reactivity of IgE from the patients' sera were investigated by immunoblotting, by an enzyme allergosorbent test (EAST) and by dose-related IgE inhibition experiments. The results of all three methods agreed closely and indicated high antigenic and allergenic activity in native celery which was reduced by thermal processing. The heat-stability of the known celery allergens decreased in the following order: carbohydrate epitopes> profilin>Api g 1. In contrast, the allergenicity was only mildly reduced by non-thermal processing. The results obtained with human IgE were confirmed by an in vitro mediator-release assay that is based on rat basophil leukemia cells (RBL cells) which were passively sensitised with celery-specific murine IgE. With sera from mice that had been immunised with native celery, the native sample and non-thermal celery preparations elicited the strongest mediator release, whereas a weak response was obtained with samples from heat-processed celery. These results agreed closely with the data obtained in allergic patients whose IgE antibodies were directed against the major protein allergen Api g 1. Our results may be helpful in risk assessment and in selecting food preparations which can be consumed without symptoms by a subgroup of celery-allergic patients with a known sensitisation pattern. ©1997 SCI     

IgE-binding and in vitro gastrointestinal digestibility of egg allergens in the presence of polysaccharides

This work studies the IgE-binding and in vitro gastrointestinal digestibility of the main egg allergens, ovalbumin (OVA) and ovomucoid (OM), in the presence of pectin (P), gum arabic (G) and xylan (X), functional biopolymers commonly used in the food industry. To this aim, solutions of OVA or OM and P, G or X were digested by using a model that mimics physiological conditions. Gastric and duodenal digests were analysed by SDS-PAGE, RP-HPLC and SEC and the specific human-IgE binding capacity was assessed by immunoblotting and ELISA using sera from egg-sensitized patients. The reactivity towards human IgE of OVA and OM was considerably increased in the presence of the polysaccharides and their susceptibility to digestion was diminished when compared with the isolated proteins. As a result, the duodenal digests obtained in the presence of polysaccharides retained more IgE-binding than the isolated protein digests. Overall, the present results underline the importance of the food matrix in the digestibility of food allergens and in their potential to trigger an immune response.     

Identification of immunoglobulin E epitopes on major allergens from dairy products after digestion and transportation in vitro

Dairy processing can alter the digestion stability and bioavailability of cow milk proteins in the gastrointestinal tract. However, analysis of stable linear epitopes on cow milk allergens that could enter into intestinal mucosal is limited. Thus, this study aimed to investigate the digestion and transportation properties and residual allergen epitopes entering into gastrointestinal mucosa of 3 commercial dairy products, including pasteurized milk (PM), ultra-heat-treated milk (UHTM), and dried skim milk (DSM). In this work, the digestive stability of the 3 kinds of dairy products has been performed in a standard multistep static digestion model in vitro and characterized by Tricine-SDS-polyacrylamide gel electrophoresis and reversed-phase HPLC. With respect to gastrointestinal digestion in vitro, the main allergens including β-lactoglobulin (β-LG), α-lactalbumin (α-LA), and caseins were degraded gradually, and the resistance peptides remained in the PM with a molecular weight of range from 3.4 to 5.0 kDa. Simultaneously, the potential allergenicity of the cow milk proteins was diminished gradually and is basically consistent after 60 min of gastrointestinal digestion. After gastrointestinal digestion, the remaining peptides were transported via an Ussing chamber and identified by liquid chromatography-MS/MS. By alignment, 10 epitopes peptides were identified from 16 stable peptides, including 5 peptides (AA 92–100, 125–135, 125–138, and 149–162) in β-LG, 2 peptides in α-LA (AA 80–93 and 63–79), 2 peptides in α_S1-casein (AA 84–90 and 125–132), and 1 peptide (AA 25–32) in α_S2-casein were identified by dot-blotting mainly exist in UHTM and PM. This study demonstrates dairy processing can affect the digestion and transport characteristics of milk proteins and in turn alter epitope peptides release.     

Lupine,a source of new as well as hidden food allergens

The present review summarizes current knowledge about lupine allergy, potential sensitization routes, cross‐reactions between lupine and other legumes, and the respective IgE‐binding proteins. Since the 1990s, lupine flour is used as a substitute for or additive to other flours, mostly wheat flour, in several countries of the EU. In 1994, the first case of an immediate‐type allergy after ingestion of lupine flour‐containing pasta was reported. Since then, the number of published incidents following ingestion or inhalation of lupine flour is rising. So far, the Lupinus angustifolius β‐conglutin has been designated as the allergen Lup an 1 by the International Union of Immunological Societies Allergen Nomenclature Subcommittee. Initially, publications focussed on the fact that peanut‐allergic patients were at risk to develop anaphylaxis to lupine due to cross‐reactivity between peanut and lupine. At present, however, the ratio between cases of pre‐existing legume allergy (mostly peanut allergy) to de novo sensitization to lupine seed is nearly 1:1. Although in December 2006, lupine and products thereof were included in the EU foodstuff allergen list according to the Commission Directive 2006/142/EC amending Annex IIIA of Directive 2000/13/EC in order to prevent severe reactions caused by “hidden food allergens”, the majority of patients and medical personnel are still not aware of raw lupine seed as potentially dangerous food allergen.     

Legumin allergens from peanuts and soybeans: effects of denaturation and aggregation on allergenicity

Legumin proteins Ara h 3 from peanuts and glycinin from soybeans are increasingly described as important allergens. The stability of an allergen's IgE binding capacity towards heating and digestion is considered an important characteristic for food allergens. We investigated the effects of heating and digestion on the IgE binding of Ara h 3 and glycinin. Both proteins are relatively stable to denaturation, having denaturation temperatures ranging from 70 to 92 degrees C, depending on their quaternary structure and the ionic strength. Aggregates were formed upon heating, which were partly soluble for glycinin. Heating slightly decreased the pepsin digestion rate of both allergens. However, heating did not affect the IgE binding capacity of the hydrolyzates, as after only 10 min of hydrolysis no IgE binding could be detected any more in all samples. Peanut allergen Ara h 1, when digested under equal conditions, still showed IgE binding after 2 h of hydrolysis. Our results indicate that the IgE binding capacity of legumin allergens from peanuts and soybeans does not withstand peptic digestion. Consequently, these allergens are likely unable to sensitize via the gastro-intestinal tract and cause systemic food allergy symptoms. These proteins might thus be less important allergens than was previously assumed.     

花生中主要过敏原Ara h1的纯化

为了得到花生中引起过敏反应的主要致敏成分Ara h1,以新鲜花生为材料,通过粉碎、脱脂、硫酸铵分步盐析等方法进行粗提;并用离子交换柱以及凝胶柱等方法来进一步纯化过敏原Ara h1。采用聚丙烯酰胺凝胶电泳（SDS-PAGE）分析了纯化后的过敏原Ara h1的纯度,并用高效液相色谱法测定其纯度达到90%以上。      

Isolation and characterization of natural Ara h 6: evidence for a further peanut allergen with putative clinical relevance based on resistance to pepsin digestion and heat

Peanut allergy is a significant health problem because of its prevalence and the potential severity of the allergic reaction. The characterization of peanut allergens is crucial to the understanding of the mechanism of peanut allergy. Recently, we described cloning of the peanut allergen Ara h 6. The aim of this study was isolation and further characterization of nAra h 6. We purified nAra h 6 from crude peanut extract using gel filtration and anion exchange chromatography. The preparation was further characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with subsequent immunoblotting. Stability of nAra h 6 was studied by an in vitro digestibility assay as well as by resistance against thermal processing. Sequencing of nAra h 6 identified the N-terminal amino acid sequence as MRRERGRQGDSSS. Further results clearly demonstrated stability of nAra h 6 against pepsin digestion and heating. Immunoglobulin G (IgE) binding analysis and its biological activity shown by RBL 25/30-test of natural Ara h 6 supported the importance of this peanut allergen. Investigation of nAra h 6 revealed evidence for a further peanut allergen with putative clinical relevance based on resistance to pepsin digestion and heat.     

电泳法在食品过敏原检测中的应用

本文简要介绍了食品过敏原的检测特点和难点,比较了目前常用的聚合酶链式反应法、环介导等温扩增法、酶联免疫法、液相色谱法和液相色谱质谱法等检测方法的优劣势。主要介绍了电泳技术的特点,总结了经典电泳技术在食品过敏原分析上的应用现状。详细介绍了近年来毛细管电泳技术在食品过敏原检测研究方面取得的进展,列举了区带毛细管电泳法、亲和毛细管电泳法、凝胶毛细管电泳法、动态涂层毛细管电泳法和芯片毛细管电泳法在致敏蛋白分析方面的应用,并对电泳法在食品过敏原分析中的发展趋势进行了展望。     

Comparison of different extraction solutions for the analysis of allergens in hen’s egg

An important requirement for the correct procedure of allergen analysis in hen’s egg is to obtain complete and unaltered protein extracts. Besides the aim of a quantitative extraction of the allergens from the matrix, it is equally important not to alter their allergenic potential during the extraction process. This paper describes and compares six extraction solutions for the analysis of whole-egg proteins and allergens. These requirements were examined via protein determination according to Bradford [Bradford, M. M. (1976). Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein–dye binding. Analytical Biochemistry, 72, 248–254] and Kjeldahl [Meyer, A. H. (2006). Lebensmittelrecht, Verlag C.H. Beck München, Stand: 1. February 2006, § 64, Lebensmittel- und Futtermittelgesetzbuch, Amtliche Sammlung von Untersuchungsmethoden, Nr. L 06.00-7] as well as the EAST-inhibition method. It could be demonstrated that the extraction with a urea solution (8 M) led to significant interferences during the protein determination, and substantially reduced the allergenic potential of egg proteins. With all other extraction solutions adequate protein contents could be extracted. The highest protein content was achieved by the extraction with phosphate buffered saline followed by a Tween 20 solution, physiological saline, water, and acetate buffer. The results show that none of these extracts – except for the urea solution (8 M) – was altered in its’ allergenic potential.