Bert Popping

First screening method for the simultaneous detection of seven allergens by liquid chromatography mass spectrometry.

The development of a multi-method for the detection of seven allergens based on liquid chromatography and triple-quadrupole tandem mass spectrometry in multiple reaction mode is described. It is based on extraction of the allergenic proteins from a food matrix, followed by enzymatic digestion with trypsin. The chosen marker peptides were implemented into one method that is capable of the simultaneous detection of milk, egg, soy, hazelnut, peanut, walnut and almond. This method has been used to detect all seven allergenic commodities from incurred reference bread material, which was baked according to a standard recipe from the baking industry. Detected concentrations ranged from 10 to 1000 μg/g, demonstrating that the mass spectrometric based method is a useful tool for allergen screening.

Rapid determination of melamine and cyanuric acid in milk powder using direct analysis in real time-time-of-flight mass spectrometry

The use of fast semi-automated method employing direct analysis in real time (DART) ion source coupled to time-of-flight mass spectrometry (TOFMS) for analysis of melamine (MEL) and cyanuric acid (CYA) in milk powder and milk based products has been demonstrated in this study. Simple sample extraction procedure employing methanol-5% aqueous formic acid mixture, which enabled disruption of melamine-cyanurate complex, was followed by direct, high-throughput (30s per run) examination of sample extracts spread on a glass rod by mass spectrometry under ambient conditions, without any prior chromatographic separation. After optimization of instrument parameter settings, limits of detection (LODs) 170 and 450microgkg(-1) were achieved for MEL and CYA, respectively. In the final phase of study, the possibility of minimizing spectral interference, thus improving method performance characteristics through the use of ultrahigh resolving power offered by Orbitrap based mass analyzer is demonstrated.

Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction

The design of real-time polymerase chain reaction (PCR) assays for the detection of meat in processed products has focused on using small amplicons, often to the detriment of specificity. However, the relationship between amplification rates and the amplicon size for processed meat products has yet to be determined. To investigate this relationship, real-time PCR assays were designed to give a series of amplicons of increasing size. These assays were then used to assess amplification rates, in relation to amplicon size, in processed meat matrices. Although the most sensitive assays were those that used the smallest amplicons, amplification was still observed using amplicons of 351 base pairs for highly processed samples. It was found, therefore, that although in general, amplicons should be as small as possible, larger amplicons give efficient amplification and that small amplicons should not be chosen if they compromise assay specificity.

Accuracy of ELISA Detection Methods for Gluten and Reference Materials: A Realistic Assessment

The determination of prolamins by ELISA and subsequent conversion of the resulting concentration to gluten content in food appears to be a comparatively simple and straightforward process with which many laboratories have years-long experience. At the end of the process, a value of gluten, expressed in mg/kg or ppm, is obtained. This value often is the basis for the decision if a product can be labeled gluten-free or not. On the basis of currently available scientific information, the accuracy of the obtained values with commonly used commercial ELISA kits has to be questioned. Although recently several multilaboratory studies have been conducted in an attempt to emphasize and ensure the accuracy of the results, data suggest that it was the precision of these assays, not the accuracy, that was confirmed because some of the underlying assumptions for calculating the gluten content lack scientific data support as well as appropriate reference materials for comparison. This paper discusses the issues of gluten determination and quantification with respect to antibody specificity, extraction procedures, reference materials, and their commutability.

Comparative study of commercially available gluten ELISA kits using an incurred reference material

Handling major hypersensitivity reactions (e.g. celiac disease) triggered by proteins of wheat and other cereals is a challenging task for healthcare systems, legislative forces and the related fields of food analysis as well. In spite of the fact that there are available official threshold levels for labelling the absence of gluten, which is considered to be the toxic protein fraction of wheat, barley and rye, validation of the analytical methodology supporting regulatory requirements is currently problematic. The main limiting factors of method validation are the lack of reference methods and reference materials. The objective of this study was to provide a solution to this problem. An incurred reference material in a model food matrix was developed and studied by commercially available ELISA test kits as a part of the activity of the Food Allergen Working Group within the FP6 funded EU project MoniQA. After successful completion of the reference material development process, the incurred material was u...

Truncation of oligonucleotide primers confers specificity on real-time polymerase chain reaction assays for food authentication.

The advent of real-time polymerase chain reaction has revolutionized the field of molecular biology, but the design and optimization of these assays has been largely overlooked in the literature. This dearth of information is probably in response to the provision of assay design software and robust guidelines issued by the leading manufacturer. However, many applications require highly specific assays with no cross-amplification of non-target DNA and it has been found that the software and guidelines, whilst producing assays of great sensitivity, do not necessarily produce specific assays. Two complementary strategies were used to confer specificity on a real-time assay, first by placing the 3′ end of the primers on a point of sequence heterogeneity and, second, by truncating the primers at the 5′ end to lower the calculated melting temperature. Using these strategies in concert, a specific assay for a conserved region of the mitochondrial cytochrome b gene was developed that can be used for the unambiguous detection of the target species in a meat mixture. This approach can be used for any real-time polymerase chain reaction assay to increase assay selectivity and specificity.

Stakeholders’ Guidance Document for Consumer Analytical Devices with a Focus on Gluten and Food Allergens

Until recently, analytical tests for food were performed primarily in laboratories, but technical developments now enable consumers to use devices to test their food at home or when dining out. Current consumer devices for food can determine nutritional values, freshness, and, most recently, the presence of food allergens and substances that cause food intolerances. The demand for such products is driven by an increase in the incidence of food allergies, as well as consumer desire for more information about what is in their food. The number and complexity of food matrixes creates an important need for properly validated testing devices with comprehensive user instructions (definitions of technical terms can be found in ISO 5725-1:1994 and the International Vocabulary of Metrology). This is especially important with food allergen determinations that can have life-threatening consequences. Stakeholders-including food regulators, food producers, and food testing kit and equipment manufacturers, as well as representatives from consumer advocacy groups-have worked to outline voluntary guidelines for consumer food allergen- and gluten-testing devices. These guidelines cover areas such as kit validation, user sampling instructions, kit performance, and interpretation of results. The recommendations are based on (1) current known technologies, (2) analytical expertise, and (3) standardized AOAC INTERNATIONAL allergen community guidance and best practices on the analysis of food allergens and gluten. The present guidance document is the first in a series of papers intended to provide general guidelines applicable to consumer devices for all food analytes. Future publications will give specific guidance and validation protocols for devices designed to detect individual allergens and gluten, as statistical analysis and review of any validation data, preferably from an independent third party, are necessary to establish a devices fitness-for-purpose. Following the recommendations of these guidance documents will help ensure that consumers are equipped with sufficient information to make an informed decision based on an analytical result from a consumer device. However, the present guidance document emphasizes that consumer devices should not be used in isolation to make a determination as to whether a food is safe to eat. As advances are made in science and technology, these recommendations will be reevaluated and revised as appropriate.

European Regulations for Labeling Requirements for Food Allergens and Substances Causing Intolerances: History and Future

Food allergens and intolerances have been diagnosed by doctors for decades, but have received heightened attention in the last two decades because diagnosis and awareness have increased. Consequently, regulators in many jurisdictions have addressed this topic by introducing labeling requirements for substances causing allergies and intolerance reactions in affected individuals. Mandatory labeling of food allergens allows persons suffering from these to make informed choices. However, regulations in some geographic areas have resulted in significant problems for manufacturers as well as consumers. This has been mainly due to frequent changes and amendments, and it has been difficult for all stakeholders to follow and understand the status quo of legislation. The present paper describes the development of European directives and regulations for the labeling of food allergens and intolerances to substances like gluten over the past decades and provides an outlook of what can reasonably be expected to change in the coming years. It also identifies existing gaps, like a lack of threshold levels for adventitious contamination and consequently a proliferation of precautionary allergen labeling, which neither benefits the consumer nor the food industry in its current form.

Food Allergens: A Regulatory/Labelling Overview Including the VITAL Approach

Food allergens have received increased attention from consumers and regulators around the globe alike. Numerous countries have introduced labelling requirements for food allergies to allow the consumer to make informed choices. However, the regulations of different countries are often not harmonized and have significant gaps. Examples are Europe, requiring the labelling of 14 groups of substances causing food allergies and intolerances and Japan with its seven allergens requiring mandatory labelling and a further 20 which are recommended to be labelled. Both countries deviate from the “big eight” list of food allergens as defined in Codex Alimentarius STAN 1-1985. Beyond inconsistencies in the labelling requirements for food allergens among countries, there are often regulatory gaps in how to deal with unintended allergen contamination of foods. Here, guidelines and labeling thresholds are often missing. This article will highlight regulations and regulatory gaps for food allergens of selected countries.

Molecular Biological and Immunological Techniques and Applications for Food Chemists: Popping/Techniques for Food Chemists

CONTRIBUTORS. PREFACE. PART Ia MOLECULAR BIOLOGICAL METHODS: TECHNIQUES EXPLAINED. 1. Molecular Biology Laboratory Layout (Rainer Schubbert). 2. Polymerase Chain Reaction (Hermann Broll). 3. Quantitative Real-Time PCR (Hermann Broll). 4. Polymerase Chain Reaction Restriction Fragment Length Polymorphism Analysis (Klaus Pietsch and Hans-Ulrich Waiblinger). 5. Single-Stranded Conformation Polymorphism Analysis (Hartmut Rehbein). 6. Sequencing (Rainer Schubbert). PART Ib MOLECULAR BIOLOGICAL METHODS: APPLICATIONS. 7. Meat (Ines Laube). 8. Genetically Modified Organisms (Bert Popping). 9. Detection of Food Allergens (Carmen Diaz-Amigo and Bert Popping). 10. Offal (Neil Harris). 11. Aquatic Food (Hartmut Rehbein). PART IIa IMMUNOLOGICAL METHODS: TECHNIQUES EXPLAINED. 12. Antibody-Based Detection Methods: From Theory to Practice (Carmen Diaz-Amigo). PART IIb IMMUNOLOGICAL METHODS: APPLICATIONS. 13. Animal Specification in Speciation (Bruce W. Ritter and Laura Allred). 14. International Regulatory Environment for Food Allergen Labeling (Samuel Benrejeb Godefroy and Bert Popping). 15. Japanese Regulations and Buckwheat Allergen Detection (Hiroshi Akiyama, Shinobu Sakai, Reiko Adachi, and Reiko Teshima). 16. Egg Allergen Detection (Masahiro Shoji). 17. Soy Allergen Detection (Marcello Gatti and Cristina Ferretti). 18. Milk Allergen Detection (Sabine Baumgartner). 19. Gluten Detection (Ulrike Immer and Sigrid Haas-Lauterbach). 20. Nut Allergen Detection (Richard Fielder, Warren Higgs, and Katie Barden). 21. Fish Allergen Detection (Christiane Kruse Faeste). 22. Lupin Allergen Detection (Christiane Kruse Faeste). 23. Mustard Allergen Detection (Anne E. Ryan and Michael S. Ryan). 24. Celery Allergen Detection (Charlotta Engdahl Axelsson). INDEX.