Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Current Topics on Food Authentication, 2011: 173-210 ISBN: 978-81-7895-510-0 Editors: M. Beatriz P.P. Oliveira, Isabel Mafra and Joana S. Amaral

9. Detection of allergens in food Askild L. Holck1, Carmen Diaz-Amigo2, Sandra Kerbach3, Bert Popping4 Stina Mustorp5 and Charlotta Engdahl Axelsson6 1

Nofima mat AS, Osloveien 1, N-1430 Aas, Norway; 2Eurofins CTC, Am Neulaender Gewerbepark 1, D-21079 Hamburg, Germany; 3Eurofins Analytik GmbH Grossmoorbogen 25, D-21079 Hamburg, Germany; 4Eurofins Scientific Group 69a Kilnwick Road, Pocklington, Yorkshire, YO42 2JY, UK; 5Eurofins Norsk Matanalyse Postboks 3055, 1506 Moss, Norway; 6Eurofins Food & Agro Sweden AB, P.O. Box 887 SE53118 Lidköping, Sweden

Abstract. The present chapter gives an overview about the different methods employed for detection of allergens in foods. They include protein based immunoassays relying on antibodies for detection, such as enzyme-linked immunosorbent assays (ELISA), immunoblotting and biosensors, and rapid methods like lateral flow devices and dipsticks. In addition mass spectrometry in combination with liquid chromatography is emerging as a confirmatory analytical technique. An alternative approach is the use of DNA based detection methods involving polymerase chain reaction (PCR) to amplify selected target DNA. PCR may be performed in both qualitative and quantitative (real-time PCR) assays. DNA based methods may be necessary for the detection of food allergens for which other tests are not available or show low sensitivity. The influence of food processing, sampling and sample preparation on the various assays will be discussed. The management of food Correspondence/Reprint request: Dr. Askild L. Holck, Nofima mat AS, Osloveien 1, N-1430 Aas, Norway E-mail: [email protected]

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allergens in the food supply chain, the challenges faced by industry, legislative and regulatory considerations and labelling issues, are also discussed. Finally we examine efforts to harmonising allergen detection and look into some future perspectives.

Introduction Several different adverse reactions to food exist. Some involve metabolic intolerances such as lactose intolerance. Others involve immunological hypersensitivity, of which there are two predominant types: the gluten intolerance syndrome commonly known as coeliac disease, and immunoglobulin E (IgE) mediated food allergies. Food allergies are a major health concern in industrialised countries and may affect up to 2% of the adult population and 6-8% of the children in Europe [1,2]. The number of food allergen-induced life-threatening syndromes is increasing. The immune system of food-allergic patients has developed hypersensitivity to an allergenic food during sensitisation. Subsequent consumption of allergens can then trigger a variety of immunological reactions ranging from hives, pruritus, atopic dermatitis, swelling of the throat or facial tissues, vomiting, diarrhoea, asthmatic wheeze, difficulty in breathing and hypotension to life threatening anaphylaxis [3]. In USA about 30,000 anaphylactic reactions to food are treated in emergency departments each year, and it is estimated that food allergy causes 150-200 deaths annually [4]. The level of exposure to provoke a reaction varies from food to food and from person to person. Most often, reactions are elicited after exposure of 1-100 ppm of an allergen, but sometimes only minute amounts are required. Treatment of food allergy is difficult and avoiding the allergen containing food is often the only option. This may sometimes be difficult, especially for processed foods, which may contain allergens either added deliberately, for example when using spices containing celery powder, or unintentionally when foods are contaminated during shipping and storage and from food production lines, etc. In addition, labelling errors may occur before the product leaves the manufacturing facility. The specific reasons for food allergy development are complicated and not well understood. Several factors that affect the development of IgEmediated food allergy have been identified. The most important determinant seems to be genetic predisposition. An atopic phenotype in one or both parents substantially increases the chances of atopy in the offspring [5,6]. Other important environmental factors are the age at which the food antigen is introduced, formula feeding versus breast feeding, dietary composition, composition of the gut microflora and the gastrointestinal infection status [7]. Induction of tolerance instead of avoiding sensitisation may be a strategy to

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deal with food allergy [8]. Some success has been achieved using oral immunotherapy [9-11]. In the USA the most food allergy causing foods in young children are cow’s milk (2.5%), egg (1.3%), peanut (0.8%), wheat (approx. 0.4%), soy (approx 0.4%), tree nuts (0.2%), fish (0.1%) and shellfish (0.1%). Early childhood allergies to milk, egg, soy and wheat are usually resolved by school age (80%) [12]. Although peanut, tree nut and seafood allergies are generally considered permanent, 20% of young children with peanut allergy experience resolution by the age of 5 years [13,14]. Adults are therefore more likely to have allergies to shellfish (2%), peanut (0.6 %), tree nuts (0.5%) and fish (0.4%). Reactions to fruits and vegetables are common (approx. 5%) but usually not severe. Foods are composed of proteins, lipids, carbohydrates and micronutrients such as minerals and vitamins. Only few of the plethora of proteins that we eat show allergenic potential, even in atopic individuals predisposed to mounting allergic disease [6]. Most food allergens are water-soluble glycoproteins ranging from 10-70 kDa that are abundant in the food and possess multiple IgE binding sites. So far, no common structural characteristics of IgE binding epitopes have been identified. Allergens are usually very stable proteins that are resistant to food processing and digestion. Many food allergens contain intramolecular disulphide bonds that are important for their allergenicity [15]. In addition, many allergens bind various types of ligands and interact with lipids. Thermal and other processing of food proteins may either increase or decrease their allergenicity [16]. The composition of allergenic foods may also play a role in their allergenic nature (reviewed in [17]). It is thus clear that several features will collectively determine whether a protein will have the characteristics required to induce allergenic sensitisation and elicit an allergenic response. Allergenic foods are used as ingredients in many food products due to their nutritional properties, or as food additives, processing aids (e.g. casein and egg white protein for wine fining), and edible films (e.g. wheat gluten, casein and soy). Many allergenic foods have numerous applications and allergens like egg, milk and soybean can be found in the majority of food products [18-20]. For example, soy may be found as ingredient or food additive in vegetarian products, ice cream, sauces, cream, desserts, chocolate, meat products, milk products, cakes, peanut butter, tuna, muesli, infant food, soups and convenience food. Allergic consumers rely on accurate food labels to make informed choices to be able to avoid offending allergens. Labelling regulations have, therefore, been implemented for foods containing allergens. To minimise or eliminate the risk of cross-contamination the industry needs to have in place an allergen control program, which is usually integrated as

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part of its Hazard Analysis and Critical Control Point (HACCP) program. Analytical techniques can be used as tools to monitor potential errors before, during and after manufacturing activities. Detection methods for allergens are used for screening, routine and confirmatory analysis [21]. Several analytical techniques for detection of allergens exist, being grouped according to the target they recognise. Protein-based assays, including immunoassays and mass spectrometry usually detect an allergenic protein, while DNA-based methods such as PCR and real-time PCR detect DNA specific for an allergenic component. In this chapter, we describe the common techniques for allergen detection and discuss how to manage allergens in the food industry.

Legislation Legislation has been implemented in many countries aiming to achieve a high level of health protection for allergic consumers. The Food Allergen Labelling & Consumer Protection Act (FALCPA) came into effect in the USA at the beginning of 2006 [22]. The FALCPA addresses the most commonly allergenic foods in the USA and requires the identification on the food label of ingredients derived from commonly allergenic sources. This implies mandatory labelling of foods containing milk, eggs, fish, crustacean shell fish, peanuts, soybeans, wheat and tree nuts (hazelnut, walnut, almond etc.) [3]. Except for the lack of labelling of sulphites, this list is in accordance with the recommendations of the Codex Alimentarius Commission. Canada, Australia and New Zealand, in addition to the allergens listed in FALCPA, require labelling of sesame seeds and sulphites. EU Regulation No 852/2004 [23] states that the primary responsibility for food safety stays with the food business operator. In addition to this general regulation, Directives 2000/13/EC [24] and 2003/89/EC [25] require mandatory declaration of allergenic foods, namely those listed by the FALCPA and in addition to these celery, mustard, sesame seeds and sulphites. Commission Directive 2006/142/EC has also included lupine and molluscs in the list [26]. The EU allergen list is intended to be dynamic and more allergens may be included over time. In the USA and Canada crustaceans are grouped with shellfish and therefore include several types of molluscs. In Australia, New Zealand and EU crustaceans do not include molluscs. Japan and South Korea require mandatory labelling for egg, milk, peanut, buckwheat and cereals containing gluten. Legislation only concerns allergenic ingredients that are knowingly and deliberately introduced into food products. Unlabelled unintentionally

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contaminated foods can pose a threat to the allergic consumer. Such presence of non-declared allergens could be interpreted as a non-compliance with legislation and could in turn result in liabilities of the food manufacturer [27]. Many manufacturers have introduced precautionary labelling to voluntarily inform consumers about possible presence of unintended allergens [28]. This happens because in real life it may be difficult to guarantee that foods are not contaminated due to the fact that many different foods are stored in the same facilities and are produced using the same equipment. Ingredients that are exempted from allergen declaration are listed in Directive 2007/68/EC [29] and include certain refined oils and polydextrins, which have been shown by analytical and clinical studies not to present a risk for allergic consumers. Threshold levels of allergens are levels which only pose small risks to few consumers [30]. Trace amounts of protein from allergenic foods have caused allergenic reactions in some allergic individuals [31,32]. Amounts of 1 to 3 mg of peanut, milk or egg protein has elicited reactions in the most sensitive individuals. The lowest observed adverse effect levels for proteins from different allergens were 0.13 to 1.0 mg for egg protein, 0.25 to 10 mg for peanut protein, 0.36 to 3.6 mg for milk protein, 0.02 to 7.5 mg for tree nut protein, 88 to 522 mg for soy protein and 1 to 100 mg for fish protein [33]. The EU regulations do not address threshold levels and require allergens to be labelled regardless of their concentration in the food. The limit of detection of commercial kits is thus regarded to be safe enough to protect the majority of the allergic population. Exceptions are the presence of sulphites and sulphur dioxide (threshold 10 mg/kg) and gluten in food products. The EU Regulation No 41/2009 [34] has adopted “gluten-reduced” products when gluten is