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BMBF project AllerGen: DNA test to protect consumers against food allergens

Food allergies are hyperactive responses of the immune system to generally innocuous substances. People who are hypersensitive to a particular food protein can develop an adverse reaction to the smallest amount of the allergenic protein that can range from mild to severe. Although many allergic people are aware of their hypersensitivity to certain food proteins, they can have allergic reactions because they are not always aware of the presence of allergenic proteins in certain food products. This is why the reliable, sensitive and quantitative detection of the smallest quantities of allergenic food constituents has received increasing attention over the last few years. In a BMBF-funded project, Prof. Dr. Jörg Bergemann and his team at the Albstadt-Sigmaringen University of Applied Sciences are working with food control and scientific and industrial partners on the analysis and prevention of allergic cross-contaminants in spices using innovative analytical methods. The project is specifically focused on the development of sensitive and quantitative methods to identify food allergens, test and assess existing methods and detect cross-contamination in the factories of the companies involved in the project.

The production of comparative spice materials consisting of homogeneously distributed allergenic raw materials is a must in the standardization, evaluation and optimization of allergen analytics. The major challenge is the homogeneous addition of smallest amounts (e.g. 1, 5, or 10 mg) of allergens to spice mixtures and the subsequent sensitive detection of these allergens. © Marion Kenk/Silvia Panter, HS Albstadt-Sigmaringen

Clinically manifest food allergies are present in one to three per cent of adults and even higher percentages of children and young adults [1]. Recent figures from the USA show that around 8 per cent of children and young adults have food allergies of whom 38.7% report severe reactions to food allergens and 30.4% have developed allergies to more than just one food component [2]. It is worth noting that many food components that have been found to cause allergic reactions are not synthetic products, but natural foods such as nuts, milk, soy, celery, mustard, etc. Hyperactive responses of the immune system of allergy sufferers can occur when the body’s immune system mistakenly identifies a protein (glycoprotein) as harmful. Even the smallest quantities of allergenic foods can lead to severe allergic reactions and, in the worst cases, to life-threatening anaphylactic shock. This is why highly allergic people need to avoid foods which may carry a risk of trace contamination.

The industrial manufacture of foods might lead to the unintentional or technically unavoidable presence of allergenic components in food, for example when a company uses its manufacturing lines for the production of allergen-containing and allergen-free products. The tainting of food that does not contain any allergens with an allergen during food manufacturing is referred to as cross-contamination. The 2003/89/EC directive on ingredient labelling requires all allergenic substances to be labelled as ingredients if they are present in a certain product. However, as industrially manufactured foods could contain trace contaminations and do not fall under the aforementioned directive, manufacturers tend to label such foods “may contain traces of…” to protect themselves against liability claims. The rather widespread use of this type of labelling increasingly reduces the foods allergic consumers can eat. This therefore calls for the early introduction of legal limits to help prevent life-threatening reactions in people suffering from allergies. However, secure and reliable analytical methods are a prerequisite for the effective control of such limits.

Cross-contamination with potentially allergenic components has become a major issue particularly in the processing of spices, which involves the use of many hundreds of individual substances and mixtures and practically all allergens that fall under the food labelling regulation. The different mixtures are produced in the same manufacturing plants in a process that often creates dust. The AllerGen research group at the Albstadt-Sigmaringen University of Applied Sciences is working on the identification of hidden allergens and the development of sensitive, quantitative and reliable analytical methods for the identification of genomic and extrachromosomal DNA of allergenic spice constituents in a project funded by the German Ministry of Education and Research.

Standardization, testing and optimization of allergen analysis

The project focuses on three major research areas. “In addition to assessing the suitability of existing methods for identifying allergens in spices, we are also focussing on the development of sensitive polymerase chain reactions (PCR) in real-time using novel DNA targets as well as on the development of quantitative methods. When we started the project, little was known about the suitability of existing methods for the analysis of spices. Nor did we have any suitable control material that we could use for comparative studies,” said the professor from Sigmaringen. Control materials are required for the standardization of allergen analyses, the evaluation and assessment of analytical methods and for the development of quantitative methods. One of the AllerGen subprojects related to the production of control spice material that contained defined concentrations of nine allergenic food components.

“We added defined quantities of allergen (10000, 1000, 100, 50, 20, 10, 5, 1 mg/kg) to an allergen-free model spice mixture and mixed it homogeneously,” said Jörg Bergemann. This control spice mixture has already been used for the matrix-independent quantification of authentic spice samples. The researchers checked the samples for the presence of soy, celery and mustard and found that 50% of them were contaminated with mustard and celery. This clearly shows how important the research project is. Some of the samples contained mustard and celery concentrations that were far higher than trace amounts. The observed concentrations of 100 to 1000 ppm represent a potential danger for people who are allergic to these food components.

Efficient DNA isolation is key in reliable real-time PCR analyses

The researchers analysed the control material with classical DNA isolation methods (isolation of total DNA) and found that not all methods are equally well suited to the analysis of spices. “However, the efficient isolation of DNA is essential for analysing spice samples using real-time PCR. We therefore decided to optimize these methods in order to be able to cover a wider range of food matrices and test a broader range of different product groups,” said Jörg Bergemann.

The researchers from Sigmaringen have also evaluated a modular system that involved the isolation of DNA using magnetic capture hybridization (MCH) followed by real-time PCR. MCH involves the sequence-specific isolation of target analytes using streptavidin-coated magnetic particles and biotynilated capture probes. “MCH is designed to eliminate potential PCR inhibitors, background DNA and associated materials such as fats, proteins and polysaccharides,” said Jörg Bergemann highlighting that this method has not yet been used for food analysis. However, the researchers from Sigmaringen have already been able to establish an MCH system for the sequence-specific isolation of hazelnut DNA, which has recently been submitted for publication.

Innovative approach with high sensitivity

The AllerGen team is also focused on the development of an analytical method that involves the use of mitochondrial DNA (mtDNA), the advantage being that mtDNA is present in higher copy numbers than genomic DNA. It is expected that the higher copy number of mtDNA will enable the more sensitive detection of allergens.

Mitochondrial DNA is present in higher copy numbers than nuclear DNA. It is expected that the higher copy number will enable the more sensitive detection of allergens using real-time PCR.<br />
Mitochondrial DNA is present in higher copy numbers than nuclear DNA. It is expected that the higher copy number will enable the more sensitive detection of allergens using real-time PCR. © Tobias Bauer, HS Albstadt-Sigmaringen [3]

The researchers used soy for their analyses, which is one of several allergens that can be present in food. They used a probe-based real-time PCR system to detect the atpA gene of mitochondrial soy DNA. “This system is around 100 times more sensitive than a commercially available soy detection system and we are able to detect trace levels of soy more accurately than before,” said Jörg Bergemann highlighting the advantages of the mitochondrial DNA detection system. This system detects cross-contaminations in soy-processing companies and helps manufacturers put in place measures to minimize, or ideally, completely eliminate soy in food products. The researchers have already successfully used this system to assess the effectiveness of the purification processes used by their project partners.

Standard line for mitochondrial DNA (solid line) and nuclear DNA (dashed line). The resulting CT values were compared with the total DNA used. The average CT value difference between mitochondrial and nuclear DNA is 7.63 ±0,12 (n=6), which explains why it is almost 100 times more sensitive. Whereas the nuclear system was able to detect a minimal amount of 25 pg total DNA, the mitochondrial system enabled the detection of as little as 0.25 pg total DNA. This experiment confirmed that using mitochondrial DNA leads to an increase in sensitivity.
Standard line for mitochondrial DNA (solid line) and nuclear DNA (dashed line). The resulting CT values were compared with the total DNA used. The average CT value difference between mitochondrial and nuclear DNA is 7.63 ±0,12 (n=6), which explains why it is almost 100 times more sensitive. Whereas the nuclear system was able to detect a minimal amount of 25 pg total DNA, the mitochondrial system enabled the detection of as little as 0.25 pg total DNA. This experiment confirmed that using mitochondrial DNA leads to an increase in sensitivity. © Tobias Bauer, HS Albstadt-Sigmaringen [3]

A paper on the use of the atpA real-time PCR system for the sensitive detection of soy has been accepted for publication by the Journal of AOAC International and will be published in autumn 2011 [3].

Collaborative project involving scientific and industrial partners

The AllerGen project is funded by the BMBF for a period of three years. Partners of the Albstadt-Sigmaringen University of Applied Sciences include: the Eastern Westphalia-Lippe University of Applied Sciences, the Chemical and Veterinary Investigation Office Sigmaringen, the companies Spices and More GmbH (Stockstadt), CONGEN Biotechnologie GmbH (Berlin), Rubinmühle GmbH (Lahr), Schumann & Sohn GmbH (Karlsruhe) and Metro Group Buying International/Real (Düsseldorf). The importance of this project can be seen in other ongoing projects at the Albstadt-Sigmaringen University of Applied Sciences which show that more than 50 per cent of all commercially available foods contain allergenic cross-contaminants (unpublished work). Faced with these statistics, the scientific, food industry and food control partners are working hard to improve the situation for people who are hypersensitive to food allergens.

Sources:

[1] Scientific Panel of Dietetic Products, Nutrition and Allergies (2004), The EFSA Journal 32:1-197

[2] The Prevalence, Severity, and Distribution of Childhood Food Allergy in the United States Ruchi S. Gupta, Elizabeth E. Springston, Manoj R. Warrier, Bridget Smith, Rajesh Kumar, Jacqueline Pongracic, and Jane L. Holl; Pediatrics. 2011 Jul;128(1):e9-17. Epub 2011 Jun 20.

[3] Sensitive Detection of Soy (Glycine max) by Real-time Polymerase Chain Reaction Targeting the Mitochondrial atpA Gene; Bauer, Kirschbaum, Panter, Kenk, Bergemann; J AOAC Int, 2011, ahead of print

Further information:

Prof. Dr. Jörg Bergemann
Albstadt-Sigmaringen University of Applied Sciences, Biomedical Engineering
Anton-Günter-Straße 51
72488 Sigmaringen
Tel.: +49 (0) 7571 732 8273
Fax: +49 (0) 7571 732 8235
E-mail: bergemann(at)hs-albsig.de

 

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/bmbf-project-allergen-dna-test-to-protect-consumers-against-food-allergens