A food matrix reduces digestion and absorption of food allergens in vivo

Scope: Food allergy is caused by primary (class 1) food allergens, e.g. Bos d 5 (cow's milk) and Cor a 8 (hazelnut) or secondary (class 2) food allergens, e.g. Mal d 1 (apple). The latter cannot sensitize susceptible individuals but can cause allergy due to immunological cross‐reactivity with homologous respiratory allergens. Here, we studied the effects of food matrix on gastrointestinal proteolysis, epithelial transport and in vivo absorption of class 1 and class 2 food allergens. Methods and results: Mal d 1 lost its IgE‐reactivity immediately after simulated gastric digestion whereas Bos d 5 and Cor a 8 did not. Only Cor a 8 maintained IgE‐binding capacity after simulated intestinal proteolysis. The presence of hazelnut and peanut extracts, which served as protein‐rich model food matrices, delayed gastrointestinal degradation and reduced epithelial transport rates of all allergens through CaCo‐2 monolayers. Finally, IgE‐reactive allergens were assessed at different time points in sera from rats fed with all three allergens with or without hazelnut extract. The levels of all allergens peaked 2 h after animals were fed without matrix and increased over 8 h after feeding. Conclusions: A protein‐rich food matrix delays gastrointestinal digestion and epithelial transport of food allergens and thereby may affect their sensitizing capacity and clinical symptoms.     

MALDI-based identification of stable hazelnut protein derived tryptic marker peptides

Food allergy is an important health problem especially in industrialised countries. Tree nuts, among which are hazelnuts (Corylus avellana), are typically causing serious and life-threatening symptoms in sensitive subjects. Hazelnut is used as a food ingredient in pastry, confectionary products, ice cream and meat products, therefore undeclared hazelnut can be often present as a cross-contaminant representing a threat for allergic consumers. Mass spectrometric techniques are used for the detection of food allergens in processed foods, but limited information regarding stable tryptic peptide markers for hazelnut is available. The aim of this study was to detect stable peptide markers from modified hazelnut protein through the Maillard reaction and oxidation in a buffered solution. Peptides ³⁹⁵Gly-Arg⁴⁰³ from Cor a 11 and ²⁰⁹Gln-Arg²¹⁷, ³⁵¹Ile-Arg³⁶³, ⁴⁶⁴Ala-Arg⁴⁷⁸ and ⁴⁰¹Val-Arg⁴¹⁷ from Cor a 9 hazelnut allergens proved to be the most stable and could be detected and confirmed with high scores in most of the modified samples. The identified peptides can be further used as analytical targets for the development of more robust quantitative methods for hazelnut detection in processed foods.     

Validating allergen coding genes (Cor a 1, Cor a 8, Cor a 14) as target sequences for hazelnut detection via Real-Time PCR

Hazelnuts have been shown to contain different allergenic proteins. Amongst these, major allergens Cor a 1 and Cor a 8 and the 2S albumin Cor a 14 were selected as targets to comparatively validate three Real-Time PCR protocols. We investigated both on the choice of the amplification target, and on the matrix effect on different sample foods. Applying statistics on the validation parameters obtained from the three protocols, we showed a significant difference in Ct values. This could turn critical when a high sensitivity method is required for the detection of hazelnut traces, confirming how fundamental the choice of the template during primer design phase is. Concluding, statistical approach represents a useful tool for the identification of the best performing primer pairs in Real-Time PCR. Cor a 8 gene permitted the identification of hazelnut based ingredients in complex foods, providing a significantly higher sensitivity in the PCR amplification, when compared to Cor a 1 and Cor a 14.     

Expression and characterization of three important panallergens from hazelnut

Several hazelnut allergens with different clinical relevance and crossreactive properties have been identified and characterized so far. The aim of this study was to develop protocols for producing relatively large amounts of three recombinant hazelnut allergens Cor a 1.04, Cor a 2, and Cor a 8 in a folded and immunologically active form. The availability of well-characterized, pure recombinant allergens will improve diagnostic in vitro tests for food allergy, by allowing a highly sensitive component resolved diagnosis. Depending on the individual hazelnut allergen, protocols for heterologous production - either as fusion or nonfusion protein - were developed to obtain homogenous protein batches. The resulting proteins were purified by a two-step FPLC method and their IgE antibody reactivity was verified. Identity was verified by N-terminal sequencing and MALDI-TOF-MS analysis. Their secondary and tertiary structure was controlled by circular dichroism (CD)-spectroscopy and NMR analysis. Decisions on the strategies for expression and purification of allergens on a large scale were made on a case by case basis: Preparation of rCor a 1.04 and rCor a 2 as fusion proteins in E. coli from inclusion bodies resulted in approximately 10 mg pure protein per liter whereas rCor a 8 expression in yeast as nonfusion protein yielded 30 mg/L.     

Characterization of plant food allergens: An overview on physicochemical and immunological techniques

Allergy to plant‐derived foods is a highly complex disorder with clinical manifestations ranging from mild oral, gastrointestinal, and cutaneous symptoms to life‐threatening systemic conditions. This heterogeneity in clinical manifestations has been attributed to different properties of allergenic molecules. Based on this fact, symptom elicitors were grouped into class I and pollinosis‐associated class II food allergens, but clear distinction is rather ambiguous. Moreover, mechanisms underlying food sensitization are not fully understood yet, and food allergy management most often relies on patient's compliance to avoid suspected foods. Therefore, recent efforts aim at the investigation of plant food allergies at the molecular level. This review provides an overview on currently available techniques for allergen characterization and discusses their application for investigation of plant food allergens. Data obtained by an array of physicochemical analyses, such as allergen structure, integrity, aggregation, and stability, need to be linked to results from immunological methods at the level of IgE and T‐cell reactivity. Such knowledge allows the development of computational algorithms to predict allergenicity of novel foods being introduced by biotechnological industry. Furthermore, molecular characterization is an indispensable tool for molecule‐based diagnosis and future development of safer patient‐tailored specific immunotherapy in plant food allergy.     

Potential efficacy of processing technologies for mitigating crustacean allergenicity

ABSTRACT

Crustacean allergy has become a growing food safety concern at a global scale. In the past decades, various food processing approaches have been employed to develop food products with reduced allergenic potential. Thermal treatment can dramatically influence the allergenicity of crustaceans by either reducing or enhancing their allergenic potential. Maillard reaction, enzymatic and acid treatments have shown to be promising in mitigating crustacean allergenicity. Recently, novel processing technologies, namely high-pressure processing, high-intensity ultrasound, irradiation, pulsed ultraviolet light and hurdle technology have attracted special attention from the researchers and the food industry professionals owing to their benefits over the conventional methods. In this context, this review paper provides an updated overview of the current knowledge on how different food processing methods induce structural changes of crustacean allergens and, subsequently, influence their allergenic potential. Data on prevalence and clinical relevance of crustacean allergy are presented, as well as, the molecular characterization of crustacean allergens and the main analytical methods for their detection in processed foods.     

食物过敏：从致敏机理到控制策略

全球范围内食物过敏的发生率逐年升高，食物过敏现已成为人们日益关注的食品安全和公共卫生问题。本文系统综述了近年来食物过敏领域的研究进展，包括食物过敏的流行病学特征、分子机制与机体发生的风险因素，食物致敏原的识别、评价与检测技术，以及食物过敏的诊断、控制及标识的风险评估等；在此基础上，深入探讨了食物过敏与致敏原相关研究的瓶颈挑战与策略，以期为食物过敏的预防和控制，低/无敏食品的研发，以及保障食物致敏原安全提供参考和借鉴。     

Clinical role of lipid transfer proteins in food allergy

Lipid transfer proteins are widespread plant food allergens, highly resistant to food processing and to the gastrointestinal environment, which have recently been described as true food allergens in the Mediterranean area, where they have been associated with severe allergic reactions to foods in patients without pollen allergy. In this review we analyze their molecular structure, biological function, and clinical relevance in food allergy.     

Detection of hazelnut proteins in foods by enzyme immunoassay using egg yolk antibodies

An enzyme immunoassay (EIA) was developed for the detection of hazelnut proteins in foods. This assay used inexpensive chicken egg yolk antibodies in a sandwich EIA format for the immunospecific capture and detection of hazelnut proteins present in a variety of different food matrices. The assay was able to detect less than 1 ppm of hazelnut protein in most of the foods tested and did not exhibit any appreciable cross-reactivity with other nuts or food matrices. This assay will be a useful tool for the food industry and regulatory agencies that wish to test foods for the presence of undeclared hazelnut allergens.     

Olive oil adulterated with hazelnut oils: simulation to identify possible risks to allergic consumers

According to European Union Regulation EC 1531/2001, olive oil labelled as “extra-virgin” should be cold-pressed and contain no refined oil or oil from other oleaginous seeds or nuts. Adulteration of extra virgin olive oil (EVOO) with hazelnut oil (HAO) is a serious concern both for oil suppliers and consumers. The high degree of similarity between the two fats complicates the detection of low percentages of HAO in EVOO. Many analytical approaches have been developed in recent years to trace HAO in EVOO, principally based on chromatographic analyses, differential scanning calorimetry or nuclear magnetic resonance. In addition adulteration of EVOO with HAO may introduce hazelnut-derived allergens. The aim of this work was to analyse the protein and allergen content of EVOO intentionally spiked with raw cold-pressed HAO or solvent-extracted HAO. SDS–PAGE analysis confirmed the presence of hazelnut proteins in solvent-extracted HAO with molecular masses ranging 10–60 kDa. In contrast, cold-pressed HAO showed no traces of protein. In spiked EVOO, solvent-extracted HAO was still detectable at a 1% contamination level. Several bands on SDS–PAGE migrated at apparent molecular masses coinciding with known allergens, such as Cor a 1 (～17 kDa), Cor a 2 (～14 kDa), Cor a 8 (～12 kDa), oleosin (～17 kDa) and Cor a 9 (～60 kDa). MALDI–TOF MS analysis confirmed the presence of two oleosin isoforms and of Cor a 9. Immunoblotting demonstrated that an allergic patient with known reactivity to Cor a 1 and Cor a 2 recognized a 17-kDa band in solvent-extracted HAO. In conclusion, we have shown that adulteration of extra virgin olive oil with solvent-extracted hazelnut oil can be traced by simple SDS–PAGE analysis, and that adulteration introduces a potential risk for hazelnut allergic patients.     

Development of a Biological Assay to Determine the Allergenic Potential of Foods

Food allergies represent a risk for many people in industrialized countries. Unrecognizable allergenic proteins of foodstuffs may be present as ingredients that are not labeled or as unknown cross-contamination. Such hidden allergens can cause severe reactions in allergics, even at minute quantities, sometimes with fatal outcome. For the verification of the presence of allergenic food constituents, analytical methods such as ELISA and PCR have been developed. However, these tests cannot measure allergenic potential. For this reason, a test system that measures the biological activity of allergens has been developed. It is based on the cellular mechanisms of the type I allergy. Rat basophilic leukemia cells (RBL-2H3) were transfected with the genes of the human high affinity receptor for IgE. The resulting cell line expressed the human receptor α-chain and could bind allergenspecific IgE from allergic subjects, in contrast to the parent cell line. After cross-linking of receptor-bound, allergen-specific human IgE by allergens, the cells released measurable inflammatory mediators. These cells were used for the analysis of a variety of allergen extracts, including extracts prepared from foods containing allergenic hazelnut and peanut. The comparative validation with existing ELISA and PCR for hazelnut and peanut demonstrated similar sensitivity and specificity. The established cell line will be a novel tool in the detection of allergens in complex mixtures, especially to address the issue of their allergenic potential, which cannot be accomplished by classical analytical methods. This will add valuable information about the allergenic potential of food constituents to the risk assessment of foods.     

Allergenicity of soybean: new developments in identification of allergenic proteins, cross-reactivities and hypoallergenization technologies

Soybean is considered one of the "big eight" foods that are believed to be responsible for 90% of all allergenic reactions. Soy allergy is of particular importance, because soybeans are widely used in processed foods and, therefore, represent a particularly insidious source of hidden allergens. Although significant advances have been made in the identification and characterization of soybean allergens, scientists are not completely certain about which proteins in soy cause allergic reactions. At least 16 allergens have been identified. Most of them, as with other plant food allergens, have a metabolic, storage, or protective function. These allergens belong to protein families which have conserved structural features in relation with their biological activity, which explains the wide immunochemical cross-recognition observed among members of the legume family. Detailed analysis of the structure-allergenicity relationships has been hampered by the complexity and heterogeneity of soybean proteins. A variety of technological approaches have been attempted to decrease soybean allergenicity. This paper provides a comprehensive review of the current body of knowledge on the identification and characterization of soybean allergens, as well as an update on current hypoallergenization techniques.     

Food allergy—towards predictive testing for novel foods

The risks associated with IgE-mediated food allergy highlight the need for methods to screen for potential food allergens. Clinical and immunological tests are available for the diagnosis of food allergy to known food allergens, but this does not extend to the evaluation, or prediction of allergenicity in novel foods. This category includes foods produced using novel processes, genetically modified (GM) foods, and foods that might be used as alternatives to traditional foods. Through the collation and analysis of the protein sequences of known allergens and their epitopes, it is possible to identify related groups which correlate with observed clinical cross-reactivities. 3-D modelling extends the use of sequence data and can be used to display eptiopes on the surface of a molecule. Experimental models support sequence analysis and 3-D modelling. Observed crossreactivities can be examined by Western blots prepared from native 2-D gels of a whole food preparation (e.g. hazelnut, peanut), and common proteins identified. IgEs to novel proteins can be raised in Brown Norway rat (a high IgE responder strain), and the proteins tested in simulated digest to determine epitope stability. Using the CSL serum bank, epitope binding can be examined through the ability of an allergen to cross-link the high affinity IgE receptor and thereby release mediators using in vitro cell-based models. This range of methods, in combination with data mining, provides a variety of screening options for testing the potential of a novel food to be allergenic, which does not involve prior exposure to the consumer.     

Peanut Allergy,Peanut Allergens,and Methods for the Detection of Peanut Contamination in Food Products

ABSTRACT: Attention to peanut allergy has been rising rapidly for the last 5 y, because it accounts for the majority of severe food‐related anaphylaxis, it tends to appear early in life, and it usually is not resolved. Low milligram amounts of peanut allergens can induce severe allergic reactions in highly sensitized individuals, and no cure is available for peanut allergy. This review presents updated information on peanut allergy, peanut allergens (Ara h1 to h8), and available methods for detecting peanuts in foods. These methods are based on the detection of either peanut proteins or a specific DNA fragment of peanut allergens. A summary of published methods for detecting peanut in foods is given with a comparison of assay formats, target analyte, and assay sensitivity. Moreover, a summary of the current availability of commercial peanut allergen kits is presented with information about assay format, target analyte, sensitivity, testing time, company/kit name, and AOAC validation.     

食品过敏原检测技术研究进展

目前食物过敏在人群中的収生率呈明显上升趋势,食物过敏已成为突出的食品安全问题。食物过敏事敀最有敁的预防方式是过敏者避克食用过敏食物,因此检测不同食物中是否含有过敏原其有十分重要的意义。本文比较了食品法具委员会、澳大利亚、加拿大、中国、欧盟、日本、南非、美国对食品过敏原标识管理的情况,综述了基于蛋白水平的酶联克疫(enzyme-linked immuno sorbent assay, ELISA)法、克疫层析技术和基于核酸水平的实时荧光定量PCR技术、环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)检测食物过敏原的方法,探讨了质谱法以及生物芯片、生物传感器等兵他新关检测技术在过敏原检测领域的应用,有利于加强食品质量监管的力度,确保食品安全。     

Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library

The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA library of hazelnut was constructed. The library was screened with serum of six hazelnut allergic patients displaying different IgE-binding patterns on hazelnut immunoblot. Rapid amplification of cDNA ends (RACE) protocols were applied to obtain full-length clones. Expression experiments were carried out in Eschericchia coli. Expression was monitored by SDS-PAGE, protein staining and immunoblotting. A hazelnut cDNA library was constructed. IgE screening resulted in the cloning of two isoforms of a novel putative hazelnut allergen. The clones were identified as oleosins, with theoretical molecular masses of 16.7 and 14.7 kDa and pI of 10.5 and 10.0, respectively. The isoforms demonstrated only 37% amino acid sequence identity but contained the typical hydrophobic stretch in the middle of the protein (53% identity) with the characteristic oleosin proline knot region (11/12 amino acids identical). Expression in E. coli of the longer isoform resulted in a clear band on SDS-PAGE. The expressed protein was recognized on an immunodot blot by IgE from serum that was used for screening the cDNA library. Hazelnut contains multiple isoforms of oleosin. IgE binding of a hazelnut-allergic patient to a recombinant version suggest that hazelnut oleosin is an allergen, as has been described for peanut and sesame.     

食品中过敏原及其检测方法的研究进展

近年来食物过敏作为食品安全的热点问题在全球引起了广泛关注。目前食物过敏仍无有效的根治方法,严格避免摄入致敏食品仍是过敏性疾病防治的最有效方式。因此,食品中过敏原的识别鉴定、性质分析及检测方法的研究至关重要。本文针对八大类过敏食物,从分子结构、免疫学特性等方面介绍了常见食物过敏原的研究进展;归纳了现阶段用于食品中过敏原分析的常规检测方法,以及在此基础上发展而来的新兴检测丰富,并比较分析了不同检测技术方法的优缺点。相比于植物源性过敏原,动物源性过敏原的相关研究有待深入,尤其是蛋白家族的确定及空间结构解析等是未来动物源性过敏原研究的重要方向;目前食品中常见过敏原的常规检测方法及新兴检测方法依然存在检测效率和准确性不高、成本昂贵等局限性,建立高效、准确、低成本的过敏原检测方法仍是该研究领域发展的重要趋势。     

Development and validation of two dipstick type immunoassays for determination of trace amounts of peanut and hazelnut in processed foods

Two dipstick-type sandwich-ELISAs were developed allowing the detection of trace amounts of peanut and hazelnut in processed foods. The detection limit of both assays was about 10 ng/ml of hazelnut or peanut protein, equivalent to 1 ppm protein in a food sample. The dipstick format allows a fast and cost-effective screening of foods because no specific instrumentation, such as microplate reader and washer, is necessary. The tests can be performed within 3 and 4 hours respectively. Various commercial food samples were tested and the results were compared with previously described and validated quantitative ELISAs for peanut and hazelnut. Results of the dipstick assays and the corresponding microplate ELISAs were in complete concordance. Our results indicated that the dipstick format could be a useful tool for fast and convenient monitoring of food production with regard to the labelling of allergens in food products. This would contribute to improve the protection of consumers from severe allergic reactions.     

食品中常见过敏原及检测技术研究进展

近年来，食物过敏发生率呈现上升趋势，由食物过敏引发的食品安全问题引起广泛关注。目前对于食物过敏尚无有效治疗手段，避免摄入含过敏原食物是最有效的预防方式。因此，过敏原检测与标识对过敏人群具有重要的警示意义。本文介绍了8 类常见致敏食品中主要过敏原的结构与致敏特点，综述了现阶段用于过敏原检测的主要技术，包括基于蛋白水平的免疫学检测技术、基因水平的分子生物学检测技术以及质谱技术，分析了各方法的优势与局限性，并对过敏原检测技术的发展方向进行了展望。