Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens

Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.     

Methods for allergen analysis in food: a review

Food allergies represent an important health problem in industrialized countries. Undeclared allergens as contaminants in food products pose a major risk for sensitized persons. A proposal to amend the European Food Labelling Directive requires that all ingredients intentionally added to food products will have to be included on the label. Reliable detection and quantification methods for food allergens are necessary to ensure compliance with food labelling and to improve consumer protection. Methods available so far are based on protein or DNA detection. This review presents an up-to-date picture of the characteristics of the major food allergens and collects published methods for the determination of food allergens or the presence of potentially allergenic constituents in food products. A summary of the current availability of commercial allergen detection kits is given. One part of the paper describes various methods that have been generally employed in the detection of allergens in food; their advantages and drawbacks are discussed in brief. The main part of this review, however, focuses on specific food allergens and appropriate methods for their detection in food products. Special emphasis is given to allergenic foods explicitly mentioned in the Amendment to the European Food Labelling Directive that pose a potential risk for allergic individuals, namely celery, cereals containing gluten (including wheat, rye and barley) crustaceans, eggs, fish, peanuts, soybeans, milk and dairy products, mustard, tree-nuts, sesame seeds, and sulphite at concentrations of at least 10 mg kg^-1. Sulphites, however, are not discussed.     

Whey allergens: Influence of nonthermal processing treatments and their detection methods

Whey and its components are recognized as value-added ingredients in infant formulas, beverages, sports nutritious foods, and other food products. Whey offers opportunities for the food industrial sector to develop functional foods with potential health benefits due to its unique physiological and functional attributes. Despite all the above importance, the consumption of whey protein (WP) can trigger hypersensitive reactions and is a constant threat for sensitive individuals. Although avoiding such food products is the most successful approach, there is still a chance of incorrect labeling and cross-contamination during food processing. As whey allergens in food products are cross-reactive, the phenomenon of homologous milk proteins of various species may escalate to a more serious problem. In this review, nonthermal processing technologies used to prevent and eliminate WP allergies are presented and discussed in detail. These processing technologies can either enhance or mitigate the impact of potential allergenicity. Therefore, the development of highly precise analytical technologies to detect and quantify the existence of whey allergens is of considerable importance. The present review is an attempt to cover all the updated approaches used for the detection of whey allergens in processed food products. Immunological and DNA-based assays are generally used for detecting allergenic proteins in processed food products. In addition, mass spectrometry is also employed as a preliminary technique for detection. We also highlighted the latest improvements in allergen detection toward biosensing strategies particularly immunosensors and aptasensors.     

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.     

基于蛋白质和DNA的食品过敏原检测技术的研究进展

目前,食品过敏问题已成为全球性的健康问题。在食品加工过程中产生的食品过敏成分或微量过敏原对敏感机体都是巨大的健康威胁。因此,可靠的分析方法是鉴别和检测食品中过敏成分所必需的。该文综述了基于蛋白质和脱氧核糖核酸(DNA)的食品过敏原检测技术的应用及发展,并展望了其未来发展趋势。     

芝麻过敏原分子特征与检测方法研究进展

食物过敏已成为全世界范围存在的公共健康问题,芝麻是一种常见的食物过敏原,对芝麻过敏原的研究日渐深入。目前,芝麻中已确定的过敏原蛋白有7 种（Ses i 1～Ses i 7）。本文综述了近几年来各国对芝麻过敏原的管理规定、芝麻过敏原蛋白的结构特征、加工过程对其结构及致敏性影响、检测方法等方面的研究进展,以期为采用不同工艺、方法消除或降低芝麻过敏原的致敏性提供理论参考,也为过敏原标签标识的实施提供理论依据。最后,本文总结了芝麻过敏原的研究现状并对未来研究趋势进行了展望。     

Cleaning and other control and validation strategies to prevent allergen cross-contact in food-processing operations

Food allergies affect an estimated 10 to 12 million people in the United States. Some of these individuals can develop life-threatening allergic reactions when exposed to allergenic proteins. At present, the only successful method to manage food allergies is to avoid foods containing allergens. Consumers with food allergies rely on food labels to disclose the presence of allergenic ingredients. However, undeclared allergens can be inadvertently introduced into a food via cross-contact during manufacturing. Although allergen removal through cleaning of shared equipment or processing lines has been identified as one of the critical points for effective allergen control, there is little published information on the effectiveness of cleaning procedures for removing allergenic materials from processing equipment. There also is no consensus on how to validate or verify the efficacy of cleaning procedures. The objectives of this review were (i) to study the incidence and cause of allergen cross-contact, (ii) to assess the science upon which the cleaning of food contact surfaces is based, (iii) to identify best practices for cleaning allergenic foods from food contact surfaces in wet and dry manufacturing environments, and (iv) to present best practices for validating and verifying the efficacy of allergen cleaning protocols.     

Processing effects on tree nut allergens: A review

“Tree nut” is a broad term for classification of nuts that include cashews, almonds, hazelnuts, etc. Reports of mild to adverse immune reactions following the consumption of these nuts has been on a rise in recent years. Currently, about 1.2–2% of the world's population suffer from sensitivity to tree nuts. The only solution is complete abstinence from the allergy causing tree nut which is not feasible in most cases due to issues like cross contamination or their presence in the form of hidden ingredients in processed foods. Various studies have shown that food processing can effectively vary the secondary structures of the allergenic protein which in turn influences their functional properties. But, the impact of these processing methods on tree nuts allergens is mixed. This review gives an update on the recent findings on how conventional and novel processing methods influence the tree nut allergens.     

Food matrix standards for the quantification of allergenic food ingredients using real-time PCR

For the quantification of food allergens by real-time polymerase chain reaction (PCR), food matrix standards with defined levels of spiked allergenic food ingredients can be used. The production and homogeneity testing of selected materials as sausages, cookies and sauce hollandaise powder is described. Except for egg and milk, all relevant allergenic ingredients were spiked to each material. Allergens were spiked and quantified as food ingredients, for example, peanut or lupine flour, at levels of 5–400 mg/kg. Material with sufficient homogeneity could be produced even at low levels of 5–10 mg of the allergenic ingredient per kilogram. The effect of the food matrix on allergen quantification was checked. The bias caused by this effect was in an acceptable range for the tested materials. The materials produced within this study were used as samples and for calibration in inter-laboratory validation studies for the quantification of allergenic food ingredients by real-time PCR. The results of this study are a contribution how to produce such reference materials for allergen analysis in the near future. Before threshold or action values of allergens in food are set, the availability of reference materials is essential.     

食品过敏原生物传感检测技术研究进展

食品过敏原已成为全球性的食品安全隐患。应对食品过敏反应最直接、有效的方法就是避免食用和接触各种含有致敏成分的食品。因此,快速和灵敏地检测食品中过敏原对预防和控制食品过敏反应是十分重要的。食品过敏原检测主要可以分为基于核酸和蛋白质的两大类检测技术。与基于聚合酶链式反应技术、液相色谱-串联质谱法的传统检测手段相比,生物传感技术检测平台因具有操作简单、高通量、灵敏度高、现场便携等优点,在食品过敏原快速检测领域备受关注。本文综述了近年来食品过敏原的生物传感检测技术研究进展,包括电化学传感器、光学传感器和其他新型传感器及其在食品过敏原检测中的应用,并展望了生物传感技术在食品过敏原分析中面临的挑战和发展趋势。     

Development of three real-time PCR assays to detect peanut allergen residue in processed food products

Hypersensitivity to peanut is a public health problem, since the ingestion of even low amounts of peanut can trigger severe allergic reactions. Allergic consumers rely on the information provided on the label of foodstuffs to identify products that might endanger their health. In order to protect the allergic consumer methods are required for the detection of allergenic ingredients. For this purpose we have developed three real-time polymerase chain reaction (PCR) assays, based on TaqMan chemistry, that are capable of detecting peanut specific DNA sequences in food products. The peanut specific sequence targeted for detection is located within the gene family of the allergen Ara h 3. The occurrence of multiple Ara h 3 sequences in the peanut genome increases the chance to achieve a good sensitivity. DNA extraction is also known to affect detection by PCR, therefore the efficiency of several different DNA extraction methods was compared. The methods reported here are capable of detecting 2.5 pg peanut DNA (less than one copy of peanut genome equivalent) and all three assays were successfully applied to detect peanut traces in a model food product where they could detect 10 mg kg⁻¹ peanut.     

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.     

Effect of roasting history and buffer composition on peanut protein extraction efficiency

Peanut is a major allergenic food. Undeclared peanut (allergens) from mis-formulation or contamination during food processing pose a potential risk for sensitized individuals and must be avoided. Reliable detection and quantification methods for food allergens are necessary in order to ensure compliance with food labelling and to improve consumer protection. The extraction of proteins from allergenic foods and complex food products is an important step in any allergen detection method. In this study, the protein extraction efficiency of various buffers prepared in-house and some extraction buffers included in some commercial allergen enzyme-linked immunosorbent assay (ELISA) test kits for peanut determination in food products were tested. In addition, the effect of roasting history on the extractability of peanut protein was investigated by the biuret and the bicinchoninic acid (BCA) assays. Elevated roasting temperatures in food processing were found to have a major impact on protein extraction efficiency by reducing protein yields of oil and dry roasted peanuts by 50-75% and 75-80%, respectively, compared with the raw material. Extraction buffers operating in the higher pH range (pH 8-11) showed best yields.     

SYBRGreenER™ Real-Time PCR to detect almond in traces in processed food

Because food ingredients are sometimes considered as causative factors in IgE mediated food allergies, DNA-based tests may prove to be very useful to establish whether allergenic species have been used in foodstuffs production. The development of two SYBR^®GreenER™ Real-Time PCR assays, targeting Pru 1 and rbcL genes, based on melting curve analysis, to detect allergen species in food has been presented. Applicability of these methods was assessed with several commercial products containing processed almond.     

Food authentication by PCR-based methods

Food authenticity is presently a subject of great concern to food authorities, as the incorrect labelling of foodstuffs can represent a commercial fraud. The implication of misleading labelling can be much more important concerning the presence of potentially allergenic foods. The need to support food labelling has provided the development of analytical techniques for the analysis of food ingredients. In the last years, several methods based on polymerase chain reaction (PCR) have been proposed as useful means for identifying species of origin in foods, as well as food allergens and genetically modified organisms (GMO), due to their high specificity and sensitivity, as well as rapid processing time and low cost. This work intends to provide an updated and extensive overview on the PCR-based methods for food authentication, including also methods for allergens and GMO the detection in foods.     

Food Allergens: Is There a Correlation between Stability to Digestion and Allergenicity?

Food allergy is a major health problem in the Western countries, affecting 3–8% of the population. It has not yet been established what makes a dietary protein a food allergen. Several characteristics have been proposed to be shared by food allergens. One of these is resistance to digestion. This paper reviews data from digestibility studies on purified food allergens and evaluates the predictive value of digestibility tests on the allergenic potential. We point out that food allergens do not necessarily resist digestion. We discuss how the choice of in vitro digestibility assay condition and the method used for detection of residual intact protein as well as fragments hereof may greatly influence the outcome as well as the interpretation of results. The finding that digests from food allergens may retain allergenicity, stresses the importance of using immunological assays for evaluating the allergenic potential of food allergen digestion products. Studies assessing the allergenicity of digestion products, by either IgE-binding, elicitation or sensitizing capacity, shows that digestion may abolish, decrease, have no effect, or even increase the allergenicity of food allergens. Therefore, the predictive value of the pepsin resistance test for assessing the allergenic potential of novel proteins can be questioned.     

Detection of celery ( Apium graveolens ), mustard ( Sinapis alba , Brassica juncea , Brassica nigra ) and sesame ( Sesamum indicum ) in food by real-time PCR

Legislation requires labelling of foods containing allergic ingredients, amongst them celery, mustard and sesame. Here we present robust quantitative and sensitive methods for real-time PCR detection of celery, mustard (Sinapis alba and Brassica sp.) and sesame in food. The development of the DNA-based assays was part of an effort to generate alternative detection methods for allergens for which effective protein-based assays are lacking. The celery and sesame methods were specific for the celery mannitol dehydrogenase gene and the sesame allergen encoding 2S albumin gene, respectively, when tested against a range of plant materials. The mustard method was specific for the allergen encoding sinA gene and its homologues present in different Brassica sp. All primer probe pairs gave high amplification efficiency and sensitivities below approximately ten molecules of purified template DNA. These DNA-based detection methods will constitute supplementary and complementary methods to the traditional protein-based methods. Laboratories may choose different analysis formats depending on the food matrix, the availability of specific tests and the performance characteristics of the tests.     

低致敏食品制备技术及其工业化应用研究进展

食物过敏是联合国粮农组织和世界卫生组织认定的全球性食品安全问题之一。在食品加工多元化的背景下,食物过敏患者要完全避免过敏原十分困难,研发低致敏食品对食物过敏患者的安全膳食至关重要。总结了低致敏食品制备技术的加工技术原理;以蒸煮、微波和烘烤为主的热加工技术通过加热诱导蛋白质变性的方式破坏致敏性构象性表位;高压、脉冲电场、脉冲光、低温等离子体、辐照和超声等非热加工技术可以通过过敏原蛋白结构修饰、多肽链断裂、新化学键的产生等方式直接破坏致敏性表位;酶水解、酶交联、糖基化、微生物发酵等其他加工方法则通过改变蛋白质构象或将蛋白质与糖类物质结合,破坏或隐藏过敏原致敏性表位。另外,对工业化低致敏蛋白配料的加工方法和生产现状进行了阐述分析。基于酶法水解的部分水解乳蛋白和深度水解乳蛋白已经可以工业化生产,其他消减食物致敏性的方法以及其他低致敏蛋白配料值得进一步研究。希望可以为工业化生产低致敏食品提供参考。     

Simultaneous detection of DNA from 10 food allergens by ligation-dependent probe amplification

The simultaneous detection of DNA from different allergenic food ingredients by a ligation-dependent probe amplification (LPA) system is described. The approach allows detection of several targets in a one-tube assay. Synthetic oligonucleotides were designed to detect DNA from peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts. The specificity of the system was tested with DNA from more than 50 plant and animal species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg kg^?1 range. The limit of detection (LOD) for single allergens in different food matrices was 5 mg kg^?1. The novel analytical strategy represents a useful tool for the surveillance of established legislation on food allergens within the European Union.