DIN CEN/TS 15633-2:2013-06

Hazelnuts (Corylus avellana) have a wide distribution in food industry, especially in chocolate and nougat production. In these cases, the content of hazelnut determines the quality of a product. Hazelnuts are also frequently used in confectionaries, bakery products, biscuits, breakfast cereals and ice-creams. On the other hand, hazelnuts are one of the major causes of food allergy. This is why food contamination by hazelnut is dangerous for patients allergic to hazelnuts. Consumption of the smallest amouts of hazelnut can pose a thread for patients allergic to hazelnuts. The amount of hazelnut which causes an allergic reaction depends on the sensitivity of the individuals. Even consumption of a few milligrams of hazelnut can induce allergic reactions in highly sensitive allergic consumers. Amounts ranging from 0,7 mg/kg to 100 mg/kg can induce reactions in sensitised individuals. Symptoms of an allergic reaction include local itching of the mouth and throat to severe life-threatening anaphylaxis. Thus deliberately added non-declared hazelnuts in food products are particularly dangerous. Also trace amounts of hazelnuts or nougat, as a result of cross contamination, pose a health risk. The allergy is caused among other proteins by glycoproteins like corylin, an 18 kDa storage protein contained in the hazelnut, which is similar to the Cor a1-antigen of hazelnut pollen and homologous to the Bet v1 antigen of birch pollen. Corylin is one of the main allergenic proteins beside Cor a8, Cor a9 and Cor a11 as representatives of seed storage and lipid transfer proteins (LTP-proteins). Corylin is differentiated between pollen associated allergy and non-pollen associated allergy. Thus deliberately added non-declared hazelnuts in food products are particularly dangerous. This DIN SPEC 10700-1 (CEN/TS 15633-2) specifies an enzyme linked immunsorbent assay (ELISA) method for the determination of hazelnut from food samples. This method was developed for verification of hazelnut in food. Matrices like cereals, ice cream, cookies, chocolate, sausage, cottage cheese, yogurt and salad dressing were validated by spiking experiments with a carboxymethylcellulose-suspension containing hazelnut paste. The monoclonal antibodies, raised against the whole aqueous extract of hazelnut, detect proteins with approximate molecular weights of 14 kDa and 18 kDa, and a protein with an apparant molecular weight of 42 kDa. The antibodies detect the major thermostable allergen Cor a9 (11S storage protein). Both antibodies were evaluated by western blots with partially purified hazelnut extracts and purified allergenic proteins. The ELISA test method is commercially available. The performance has been validated by an in house validation performed by the manufacturer. All parameters of interest are indicated. In addition, the ELISA was successfully validated by a collaborative study in order to determine the interlaboratory reproducibility. This ring trial was organised 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. A direct sandwich ELISA is used for detection of hazelnut. The basis of the test is an antigen-antibody reaction. Two hazelnut specific monoclonal antibodies are used to detect the analyte. A microtiter plate is coated with the capture monoclonal antibody mouse anti Cor a9 antibody. Hazelnut standards provided with the kit or sample extracts are incubated for 10 min. After washing, a detector monoclonal antibody mouse anti Cor a9 antibody, labelled with peroxidase, is added as the enzyme conjugate for further 10 min. The conjugate binds to the hazelnut protein antibody complex on the plate. Any unbound enzyme conjugate is then removed by a washing step. Chromogen/substrate is added to the wells and incubated for 10 min. Bound enzyme converts the chromogen into a blue coloured product. The addition of stop reagent inhibits the enzymatic process and causes a shift of the coloured product to yellow. Absorbance measurement is performed at 450 nm (optionally at 600 nm) against air. The resulting absorbance values are proportional to the concentration of hazelnut of a sample. The result is expressed as hazelnut in mg/kg. The standard stock solution used is an aqueous hazelnut extract of six different varieties of hazelnut (Hallesche Riesen, Levantiner, Kerassunder, Piemonteser, round Römer, Barcelona Giants). These six varieties, raw and roasted, are representative for the hazelnuts used in food products world-wide by food industry. The extract from the different hazelnuts has a protein content of approx. 9 % protein, measured by the photometric protein determination method according to BCA (Pierce). The committee responsible for this document is NA 057-01-05 AA "Lebensmittelallergene" ("Food allergens") at DIN.