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.     

Qualitative and Quantitative Detection of Protein and Genetic Traits in Genetically Modified Food

Due to the market introduction of genetically modified organisms (GMOs) in crops, foods, and ingredients, legislation worldwide came face to face with the question of the use and labeling requirements on GMO crops and their derivatives. In this review, protein- and DNA-based methods, such as enzyme-linked immunosorbent assay, western blots, and qualitative and quantitative polymerase chain reaction PCR (Q-PCR) are reviewed. Qualitative detection methods for genetically modified (GM) sequences in foods have evolved rapidly during the past years. The sensitivity of these systems is extremely high, even for processed foodstuffs. However, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. The recently introduced labeling threshold for GMOs in food ingredients by the European Union has forced official food control laboratories to apply quantitative PCR methods. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are discussed. As quantitative GMO detection methods measure GMO contents of samples in relation to reference material, priority must be given to international agreements and standardization on certified reference materials. The rapidly increasing number of GM foods on the market demands the development of more advanced multidetection systems, such as microarray technology. Challenges and problems arising from the inability to detect GM foods for which the modified sequence is unknown, the lengthy standardization procedures, and the need to continuously update databases comprising commercially available GM foods and the respective detection strategies are also discussed.     

Considerations on Developing Criteria to Determine Whether a Food Allergen is of Public Health Importance

Food allergy is the result of a particular type of immune response against one or more food components, usually proteins. The food allergy reactions that are the focus of regulatory measures are mediated by antibodies of the IgE class. Importantly, food allergy is a two-step process. The first step, the sensitisation phase, consists of an immune response resulting in production of IgE antibodies specific for the allergen. The second step, the provocation phase, is the triggering of a symptomatic allergic reaction as a result of exposure to the allergen after sensitisation has been established. For reasons not well understood, a majority of sensitised individuals never will experience clinical reactions. Determining the number of sensitised individuals (test positives) in a population will therefore grossly overestimate the prevalence of food allergy. The term ‘allergy’ should be used only if clinical reactions occur. For triggering a food allergic reaction in a sensitised individual, only the ‘acute’ food intake and not the intake over time is of importance, and food allergy in this aspect resembles acute poisoning. Because of the cultural, agricultural, economic and nutritional importance of the foods, society accepts that a small fraction of the population develops allergies to traditional food products. Prevention of allergic reactions is then sought by means of education and by labelling of the most important allergenic foods. However, some 200 allergenic foods have been described, but only a small minority of these appears to be of importance in terms of frequency and severity of reactions triggered in the population. When it comes to management of the allergens, however, there is a lack of clear criteria at two levels: at the level of defining what documentation should be required to enter a proposed new food allergen into one of the large allergen databases, as well as at the level of determining which food allergens are of sufficient public health importance to require special regulatory attention in terms of labelling. With the aim of increasing transparency and predictability of decision-making processes and obtaining more consistency between labelling of different allergens as well as between labelling of allergens in different food regulatory jurisdictions, ILSI Europe has taken an initiative to establish clear criteria and a framework to determine the public health importance of a given food allergen. These criteria will consist of factors relating to food properties, population factors, and exposure factors.     

日本食物过敏原的管理及对我国的启示

随着我国食物过敏问题对消费者和食品企业的影响越来越严重,食物过敏原的管理和控制问题亟待解决。日本是世界上第一个建立强制性食物过敏标识并加以立法保护的国家,现已形成完善的食物过敏原标签管理系统。日本食物过敏原法规的形成过程、约束范围及食物过敏原标签的标注方式、不规范标注的处罚措施和过敏原成分的检测方法,对中国制定符合本国国情的食物过敏原强制性管理法规和建立相关的检测技术具有启示作用。     

Food and nutrition labelling in the European Union

This review presents the principles of food labelling, such as the consumers’ right to non misleading information and informed choice, and the harmonisation of national laws to ensure free circulation of goods and fair trade in the Community. The relevant European Regulations and Directives are detailed, and references and websites given for access to full texts. Mandatory label information rules are presented, both “horizontal” rules applying to all foods (mainly pre-packaged foodstuffs for the ultimate consumer or for supply to caterers), and “vertical” rules defining the name and composition of specific products. Ingredient listing is discussed with regards to the recent amendment concerning food allergens. Voluntary label information is also presented, including quality and origin labelling, and organic production. The rules for novel foods and for genetically modified foods are analysed. The nutrition labelling Directive is detailed, together with texts ruling food supplements and foods for particular nutritional uses. Recent and still pending proposals for nutrition and health claims, and for nutrient fortification of foods are discussed. The results of critical surveys on the present implementation of food and nutrition labelling are summarised, together with the corresponding recommendations for improvement.     

花生主要致敏物质及其脱敏方法研究进展

花生作为影响部分人群生活质量的主要过敏食物之一,其中含有的过敏原会引起人体强烈的过敏反应,严重时甚至会导致人的死亡。花生过敏反应因其潜在的危险性、长期性以及不断增加的发病率而日益受到各国的广泛关注。研究花生致敏机理及脱敏方法已成为热点,探索有效脱敏方法,对保证花生食品的安全性具有重要的现实意义。本文对花生致敏机制,过敏原致敏组分分析和脱敏方法进行阐述。     

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.     

The consumption of genetically modified foods in Italian high school students

Genetically modified organisms (GMOs) can be defined as organisms in which the DNA has been altered in a way that does not occur naturally. Such methods are used to create GM plants – which are then used to grow (GM) food crops. GM foods have the potential to solve many of the world’s hunger and malnutrition problems, and to help protect and preserve the environment by increasing yield and reducing reliance upon chemical pesticides and herbicides. Nevertheless, the consumption of GM foods provokes doubts and hesitations among consumers, especially in Italy. This paper has two aims, the first is to investigate genetically modified (GM) foods consumption in Italian high school students through a large sample size survey on 2122 students randomly selected in 39 schools of a metropolitan area (Naples, South-Italy). The second, by examining the behavioural process that drives individual’s perceptions of GM food taking advantage of an empirical choice methodology that corrects for endogeneity in decision making relationships, namely structural equation model (SEM). The results show that a very large percentage of students never or rarely eat GM food and a lot of them do not suggest the consumption of GM food. The proposed SEM is a full formative measurement model and shows that GM foods consumption in Italian students depends on the knowledge of GMO and on the impact of the GMO on the men’s health and on the environment. Therefore, in order to orient population it could be realized a standardized evaluation systems relative to human health and environment consequences produced by GM organisms and GM 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.     

花生过敏原及其检测方法研究进展

花生过敏可导致某些人群严重的食品安全问题。过敏患者只能通过避免食用含有花生过敏原成分的食物来避免过敏。但是,食品在生产加工、储存、运输、销售过程中有可能被过敏原污染。因此,确定各类加工食品中是否含有花生过敏原成分,对于预防食用者发生花生过敏反应具有重要意义。花生中已确定的过敏原蛋白有13种(Ara h 1~Ara h 13)。本文综述了花生中过敏原蛋白的结构信息,当前流行的提取方法,以及各种定性定量的检测方法,总结了各种方法的优缺点。同时对建立一种具有特异性强、灵敏度高、定量准确的花生致敏蛋白检测方法的发展趋势进行了展望。     

加工食品中虾蟹类过敏原ELISA检测前处理方法的研究

目的建立了一种可应用于加工食品中虾蟹类过敏原检测的前处理方法。方法针对提取液和提取步骤2个部分选取条件先后进行优化,通过对相应样品提取后的蛋白溶出量等进行评估,选取最适合本ELISA检测的条件。结果采用含有0.5%SDS和0.5%DTT的磷酸盐缓冲液(pH 8.5),按照食物样品与提取液的1:9 (m/V)进行混合,在室温下均质30 min,3020×g离心15 min,取上清液进行检测效果最佳,此方法与PBS(pH7.4)溶液提取方法相比,蛋白质溶出量增加了约5倍。结论在已建立的检测虾蟹类过敏原夹心ELISA的基础上,建立优化前处理方法进一步提高了加工食品中过敏原的提取率。     

Determination of unprocessed genetically modified soybean in foods using simplex and duplex real-time PCR with an internal standard

We developed a method using an internal standard to determine the amount of unprocessed genetically modified organisms (GMO) in foods as GMO weight per weight of total food (w/w_tf) using real-time PCR. Food samples were spiked with an internal standard, a ColE1 plasmid, and DNA was extracted from the samples using a silica membrane-type kit and analysed using real-time PCR. The relationship between the GMO amount and the copy number ratio of the transgene to ColE1 in 0.1–5% w/w_tf GM soybean powders was found to have a correlation coefficient ( r ) of 0.97. GMO was quantified in food samples spiked with GM soybean (final amount 0.5% w/w_tf GMO). The average GMO amount ranged from 0.35% to 0.63% w/w_tf. The results show that our method should be useful for quantifying unprocessed GMO in foods. We also developed a duplex assay, which is simpler and more accurate than the simplex assay.     

Detection of DNA in soybean oil

 The presence of DNA in foodstuffs which are, or contain, genetically modified organisms (GMO) is the basic requirement for labelling of GMO food in Switzerland and is also being discussed as a requirement for labelling in the European Union. The present work presents data indicating that no genetic material can be recovered after the first processing steps of soybean oil, i.e. when crude soybean oil is simply centrifuged. This fact is of some relevance because centrifugation is one of the first steps in industrial oil processing. A nested PCR system utilising a single copy gene (lectin 1) showed that centrifugation purified the oil from the genetic material by at least a factor of 10 000. The same results were observed when industrially processed oil fractions were analysed. Thus, with respect to the presence of DNA, soybean oil from GMO soybeans is identical to traditional oil and does not need to be labelled as a GMO product in Switzerland. Received: 21 January 1998 / Revised version: 30 March 1998     

Detection of DNA in soybean oil

 The presence of DNA in foodstuffs which are, or contain, genetically modified organisms (GMO) is the basic requirement for labelling of GMO food in Switzerland and is also being discussed as a requirement for labelling in the European Union. The present work presents data indicating that no genetic material can be recovered after the first processing steps of soybean oil, i.e. when crude soybean oil is simply centrifuged. This fact is of some relevance because centrifugation is one of the first steps in industrial oil processing. A nested PCR system utilising a single copy gene (lectin 1) showed that centrifugation purified the oil from the genetic material by at least a factor of 10 000. The same results were observed when industrially processed oil fractions were analysed. Thus, with respect to the presence of DNA, soybean oil from GMO soybeans is identical to traditional oil and does not need to be labelled as a GMO product in Switzerland. Received: 21 January 1998 / Revised version: 30 March 1998     

Governing GMOs in the USA: science,law and public health

Controversy surrounds the production and consumption of genetically modified organisms (GMOs). Proponents argue that GMO food sources represent the only viable solution to food shortages in an ever‐growing global population. Science reports no harm from GMO use and consumption so far. Opponents fear the potentially negative impact that GMO development and use could have on the environment and consumers, and are concerned about the lack of data on the long‐term effects of GMO use. We discuss the development of GMO food sources, the history of legislation and policy for the labeling requirements of GMO food products, and the health, environmental, and legal rationale for and against GMO food labeling. The Food and Drug Administration regulates food with GMOs within a coordinated framework of federal agencies. Despite mounting scientific evidence that GMO foods are substantially equivalent to traditionally bred food sources, debate remains over the appropriateness of GMO food labeling. In fact, food manufacturers have mounted a First Amendment challenge against Vermont's passage of a law that requires GMO labeling. Mandatory GMO labeling is not supported by science. Compulsory GMO labels may not only hinder the development of agricultural biotechnology, but may also exacerbate the misconception that GMOs endanger people's health. © 2015 Society of Chemical Industry     

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.     

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.     

Survey of undeclared egg allergen levels in the most frequently recalled food types (including products bearing precautionary labelling)

Since the number of recalls involving undeclared allergens is commonly associated with bakery and snack foods, we aimed to determine the frequency of egg allergens in a large number of these products using two commercial enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no egg identified on the product label or which had an egg precautionary statement. Among all samples, egg protein was detected in 5% of products using a Morinaga (MO) kit and 1% of products using a R-Biopharm (RB) kit. For bakery samples, egg protein was detected in 6% of 363 samples with no precautionary labelling (6% by MO and 1% by RB kit) and 12% of 80 samples which had precautionary labelling. For snack samples, egg protein was detected in 2% of 371 samples with no precautionary labelling (2% by MO and < 1% by RB kit) and 5% of 21 samples which had precautionary labelling. The disagreement rates between two methods were 5.2% for bakery products and 2.6% for snack products. The sample repeatability was at an acceptable level for bakery (< 12.5%) and snack foods (< 7.5%) for each method. The relative standard deviation between test kits was high (103.1%) for bakery foods. Four bakery products without precautionary labelling had a higher level of egg protein per serving compared with the eliciting dose (ED₁₀ of 3.7 mg protein) for egg allergic patients. These results highlight the fact that detection methodology plays a vital role for accurate labelling control and mitigation of risk for egg allergic consumers.     

食品中花生过敏原及其检测方法的研究进展

花生是常见的过敏原之一,能够引起严重的过敏反应。由于缺乏明确的治疗花生过敏的方法,只能让花生过敏患者尽量避免食入含有花生的食物。但在实际的生产过程中,食品加工往往需要经过复杂的生产工艺,会造成食品之间的交叉污染,部分食品难以准确判断是否含有花生过敏原。因此对于食品中花生过敏原的检测方法的开发就显得尤为重要。本文主要对花生中过敏原的种类及其检测方法的研究进展进行了综述,主要对以下几种方法做了介绍,包括酶联免疫吸附法(ELISA)、免疫印迹法、聚合酶链式反应(PCR)等,以及新兴的生物传感器和质谱法,并对检测方法的未来发展趋势进行了展望。