DIN CEN/TS 15633-3:2012-10

The hazelnut (Corylus avellana) belongs to a group of foods commonly referred to as tree nuts. Allergic reactions to hazelnut ranging from oral allergy syndrome to severe anaphylaxis have been widely reported. Food labelling legislation requires the presence of hazelnut in food to be declared in numerous nations of the European Union, North America, Asia and Australasia. IgE binding studies from the sera of sensitized patients have revealed a number of allergens, including both pollen related and non-pollen related allergens. Threshold dose studies have reported provocative doses as low as 1 mg of hazelnut protein. Hazelnut material may occur unintentionally in foods for several reasons. It may be present in contaminated ingredients or cross contamination may occur during food manufacture. Consequently there is a need for sensitive and reliable tests for the detection of hazelnut in food samples. DIN SPEC 10700-2 (CEN/TS 15633-3) specifies an ELISA (enzyme-linked immunosorbent assay)-method for the determination of hazelnut concentration in food samples. Spiking experiments with diluted ground hazelnut have been used to validate the method's use on food matrices such as mixed grain cereals, dark chocolate (45 % cocoa) and ice cream. The range of the method is 0,5 mg to 5,0 mg hazelnut protein per kg of food sample. As hazelnut kernels typically contain between 12 % to 15 % protein, this equates to approximately 3,7 mg to 37 mg hazelnut kernel per kg of food sample. The upper limit of the range of quantitation can be extended, if required, by further dilution of sample extracts. The method is commercially available and has been validated in-house by the manufacturer. The data are included in Annex A.2. The method has been successfully validated by a collaborative study. The study was organized by the Working Group established by the Federal Office of Consumer Protection and Food Safety (BVL) for the execution of § 64 of the German Food and Feed Code (LFGB) for the determination of hazelnut content in dark chocolate. 13 German laboratories participated in the collaborative study. The data are included in Annex A.3. A direct sandwich enzyme-linked immunosorbent assay (ELISA) is used for detection of hazelnut protein. A 13 kDa to 14 kDa protein band common to both raw and roasted hazelnuts was identified using polyacrylamide gel electrophoresis. This protein marker was purified by high performance liquid chromatography and used to raise rabbit anti-hazelnut polyclonal anti-sera. The IgG fraction of this antiserum was purified by affinity chromatography then used to develop a direct sandwich ELISA as outlined below. For measurement of the optical density (OD) of every cavity, a spectrophotometer (primary wave length 450 nm and reference wave length between 620 nm and 650 nm) is used. The produced OD is caused by the hazelnut protein concentration. Hazelnut concentration of a sample can be calculated by comparison of the OD produced by the sample with the OD produced by standards. The standards are calibrated in mg hazelnut protein per kg. They have been produced from an aqueous extract of commercially available hazelnuts (Corylus avellana). The protein concentration has been determined using the Lowry method. The committee responsible for this document is NA 057-01-05 AA "Lebensmittelallergene" ("Food allergens") at DIN.