TRACES OF HAZELNUT

Traces Of Hazelnut

Abstract

Hazelnut seeds (Corylus spp.) are source of allergens and could cause severe adverse reactions in sensitized subjects, even if consumed in traces. This work presents two sensitive real-time PCR methods to quantify hazelnut in foods, one using the Sybr green dye and one based on hazelnut-specific Taqman probe designed on Cor a1.04 gene (specific amplicon: 82 bp). The sensibility and the robustness of the method were estimated analyzing spiked samples and some commercial hazelnut-containing creams. The lowest detection limit was 0.1 ng of genomic DNA. A qualitative specific PCR and a comparison of different DNA extraction protocols are also discussed.

Table of contents

Abstract2

Introduction4

Experimental7

Chemicals7

Sera8

Patient sera8

Polyclonal antisera8

Equipment8

Sample Preparation9

Extraction of hazelnuts and almonds9

Extraction of chocolate samples9

SDS-Electrophoresis10

Electrophoretic transfer (blotting)10

Immunodetection of hazelnut and almond allergens/antigens11

Densitometric evaluation12

Results and discussion12

Identification of IgE-binding hazelnut and almond proteins12

Analysis of consumer products13

Detection of trace amounts of hazelnut and almond proteins in chocolate by immunoprobing with rabbit polyclonal antiserum14

Specifity of antisera15

Detection limit of hazelnut and almond proteins in chocolate15

Screening of chocolates for traces of hazelnut and almond proteins16

Optimization of cleaning steps to minimize cross contamination16

Detection of hazelnut protein using rabbit antibodies17

Conclusions18

Traces Of Hazelnut

Introduction

The Food Standards Agency commissioned this work to see if a test could be developed that would detect very small traces of peanuts in commercial food products. It is part of the Agency's ongoing research programme that aims to increase our knowledge and understanding of food allergy and intolerance and protect allergic consumers. The test shows that it is possible to detect very small traces of peanut (down to 100ng or 1 part in 10 million) in some commercial foods. The PCR technique is not new. However the new Peanut PCR test has further developed the technique to detect foods causing allergic reactions. PCR (Polymerase Chain Reaction) is a sensitive test that can be used to detect very small amounts of DNA. The technique has been used to detect GM material in foods or to determine food authenticity such as which species meat samples come from. (Holzhauser, 2000, 363)

True food allergies are immediate type allergic reactions mediated by allergen-specific IgE antibodies. The vast majority of food allergens are proteins. Besides animal products (milk, egg, fish), vegetable foods such as legume seeds, tree nuts and fruits are important sources of allergens. While fruits (e.g. apples) mainly cause oral symptoms, legume seeds and nuts also induce acute generalized symptoms and even anaphylactic shock [4 and 5].

Food products often contain non-declared ingredients. The reason for this varies from illegal substitution for higher priced components to unintended (cross) contamination during processing. Unexpected contact with milligram quantities of these ingredients can cause severe allergic reactions in people with hypersensitivity to these particular food components.

In contrast to inhalant allergies, food allergies cannot be treated successfully by specific desensitization. Up to now, the only treatment is avoidance of the causative agent. Consequently, it is of importance for food allergic persons to be able to obtain food products which are free of certain allergenic ingredients.

Although there are no generally accepted limits for allergenic compounds in ...