Development of incurred reference material for improving conditions of gluten quantification.

Celiac disease and wheat allergy are the most common adverse reactions triggered by cereal proteins, mainly gluten, which is one of the 14 allergenic food ingredients that must be labeled on food products in the European Union (EU). To meet the requirements of this regulation, reliable analytical methodology for proper quantification of gluten is necessary. However, validation of presently used methods (ELISA and lateral flow device) is limited partly due to the lack of reference methods and incurred reference materials. To solve this problem, the goal of our work was to develop an incurred reference material for the quantification of gluten under the auspices of EU-FP6 funded Network of Excellence MoniQA. During this work, we produced a processed model product (cookie) containing gliadin (major allergenic fraction of gluten) in a defined amount. This paper addresses the development process of this material together with the associated problems (insufficient homogeneity and low recovery) and their solutions. As a result, an incurred food matrix was produced on a laboratory-scale with a potential use as a reference material. The model product was tested by an ELISA method followed by a comparative study of commercially available ELISA kits to investigate the applicability of the product. Preliminary results of this study are also presented.

Current perspectives and recommendations for the development of mass spectrometry methods for the determination of allergens in foods.

Allergen detection and quantification is an essential part of allergen management as practiced by food manufacturers. Recently, protein MS methods (in particular, multiple reaction monitoring experiments) have begun to be adopted by the allergen detection community to provide an alternative technique to ELISA and PCR methods. MS analysis of proteins in foods provides additional challenges to the analyst, both in terms of experimental design and methodology: (1) choice of analyte, including multiplexing to simultaneously detect several biologically relevant molecules able to trigger allergic reactions; (2) choice of processing stable peptide markers for different target analytes that should be placed in publicly available databases; (3) markers allowing quantification (e.g., through standard addition or isotopically labeled peptide standards); (4) optimization of protease digestion protocols to ensure reproducible and robust method development; and (5) effective validation of methods and harmonization of results through the use of naturally incurred reference materials spanning several types of food matrix.

Validation procedures for quantitative food allergen ELISA methods: community guidance and best practices.

This document provides supplemental guidance on specifications for the development and implementation of studies to validate the performance characteristics of quantitative ELISA methods for the determination of food allergens. It is intended as a companion document to other existing publications on method validation. The guidance is divided into two sections: information to be provided by the method developer on various characteristics of the method, and implementation of a multilaboratory validation study. Certain criteria included in the guidance are allergen-specific. Two food allergens, egg and milk, are used to demonstrate the criteria guidance. These recommendations will be the basis of the harmonized validation protocol for any food allergen ELISA method, whether proprietary or nonproprietary, that will be submitted to AOAC and/or regulatory authorities or other bodies for status recognition. Regulatory authorities may have their own particular requirements for data packages in addition to the guidance in this document. Future work planned for the implementation and validation of this guidance will include guidance specific to other priority allergens.

Characterisation and comparison of selected wheat (Triticum aestivum L.) cultivars and their blends to develop a gluten reference material.

The reliability and comparability of gluten analytical results in gluten-free foods is hampered by the lack of reference materials (RM). This is partly caused by the yet incomplete knowledge of the effect of genetic and environmental variability of wheat proteins on immunochemical analyses, which affects the choice of gluten source to be applied for RM production. We investigated the genetic variability and the effect of harvest year on the protein composition of five previously selected wheat cultivars. We also compared the magnitude of these effects on ELISA results to get closer to the question of choosing individual cultivar or a mixture as an RM. Our results proved that the application of a blend for this purpose is advantageous. The candidates were also produced on pilot scale to investigate the feasibility of upscaling. The results of comparison studies showed that the pilot scale blended flour can also be suitable for RM.

Variation in protein composition among wheat (Triticum aestivum L.) cultivars to identify cultivars suitable as reference material for wheat gluten analysis.

Gluten proteins of certain cereals (wheat, rye and barley) can trigger hypersensitivity reactions. In special dietary products for people intolerant to gluten, their amount must not exceed the regulatory threshold levels. The source of gluten can influence gluten quantitation due to variability in protein profile of grain cultivars and species. A proper reference material is crucial for accurate measurement of gluten and evaluating assay performance. It should be as representative of the commodity as possible. In this study, protein content and composition of a set of 23 common wheat cultivars grown around the world were determined. According to qualitative and quantitative selection criteria, cultivars that possessed a typical gluten composition were identified. Five cultivars were selected for subsequent experiments to confirm their suitability as a basis for reference material production.

Analysis and critical comparison of food allergen recalls from the European Union, USA, Canada, Hong Kong, Australia and New Zealand.

As part of a European Union-funded project (FP7) developing 'Integrated approaches to food allergen and allergy management', a database was constructed based on publicly available information on food allergen recalls in Europe, North America, Hong Kong, Australia and New Zealand. Over 2000 entries were made into the database. The database covers a 4-year period from 2011 to 2014 and each entry is categorised into food type (two different classifications), identified allergen and cause where indicated by the authorities. Across different authorities, by far the biggest incidence of undeclared allergens occurred in the food categories of prepared dishes and snacks (range = 12-53%), and cereals and bakery products (range = 14-25% of all recalls and/or alerts). The biggest incidence of undeclared allergens, according to the information from most authorities, occurred for milk and milk products (16-31% of all products with recall or alert), followed by cereals containing gluten (9-19%), soy (5-45%), and egg and egg products (5-17%). Although 42-90% of the products with recalls/alerts were explained as being 'Not indicated on the label', this is a generic explanation of cause and does not provide much insight into the causes of the recall/alerts. However, 0-17% of products with recalls/alerts could be coded as caused by the unintended presence of an allergen as the probable result of cross-contact in production. Construction of the database of allergen recalls has provided some important lessons and recommendations to the authorities are made in this paper in terms of the harmonisation of the reporting of allergen recalls into a more standardised format.

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.

Effects of chemical, physical, and technological processes on the nature of food allergens.

A review is presented of studies of different processing techniques and their effect on the allergenicity and antigenicity of certain allergenic foods. An overview of investigated technologies is given with regard to their impact on the protein structure and their potential application in the production of hypoallergenic foods. The use of physical processes (such as heating, high pressure, microparticulation, ultrafiltration, and irradiation), chemical processes (such as proteolysis, fermentation, and refining by extraction), and biotechnological approaches, as well as the effects of these processes on individual allergenic foods, are included. Additionally, the implications of food processing for food allergen analysis with respect to food safety assessment and industrial quality control are briefly discussed.

Polymerase chain reaction techniques for food allergen detection.

Food allergies represent an important health problem in industrialized countries. Undeclared allergenic foods as contaminants in food products pose a major risk for sensitized persons. Reliable detection and quantification methods for food allergens are necessary to ensure compliance with food labeling and improve consumer protection. The methods currently used for the detection of potential allergens in foods are to target either the allergen itself or a marker that indicates the presence of the offending food. As markers for the presence of potentially allergenic foods or ingredients, specific proteins or DNA fragments are targeted. In routine food analysis, the enzyme-linked immunosorbent assay (ELISA) and the polymerase chain reaction (PCR) in the form of a real-time PCR or in combination with an ELISA have been used. The availability, the characteristics, and some future aspects of DNA-based methods in the rapid and sensitive detection of potentially allergenic food constituents or contaminations are discussed in this review.