Reduced allergenicity of processed foods (containing animal allergens)

The survey on severe allergic reactions (SAR) counted SAR cases in clinics, hospitals and surgeries. 60.4% institutions within the pool of duly completed questionnaires (N=191) report on cases of severe allergic reactions (SAR). 39.6% of all returns mention problems related to a food allergy, whereas 49.7% of the sample confirm other eliciting allergies (e.g. drug allergies). In 53 centres (45.7%) both, food allergy and other allergy cases have been given. For the whole survey area contacted doctors report on a total of 2126 patients with severe allergic reactions, which necessitated hospitalisation (41.2% of them with food induced symptoms).

Improved methods for detection of allergens are urgently required by the food industry in order to provide enhanced risk assessment. Highly sensitive and rapid tests have been developed for the detection of trace amounts of food allergens from animal origin in various food matrices. Such methods include dipstick and biochip sensor (based on resonance-enhanced nanoclusters) technologies, which can be applied for the detection of allergens in the raw materials, intermediate products and final food products during the food manufacturing process. The methods developed are sensitive, cost-effective, easy-to-use, robust and reliable, and can be completed in minutes rather than hours. The rapid DNA extraction procedure for PCR analysis takes about 30 min while the CTAB extraction procedure takes more than 3 hours. The limits of detection were suitable for the detection of meat (beef and chicken meat) and skimmed milk powder in various samples. The limits of detection were one order of magnitude higher than for the CTAB extraction method.

The original idea of this network was to realise the planned telephone survey from several survey points in all involved countries and to disseminate the results trough this instrument. Contrarily to these provisions the project partners of REDALL discussed and agreed to carry out this survey in a centralised form, from one single survey point, because of expected positive effects on the methodological scheme. A centralised approach was chosen due to its scientific benefits. For that reason it was decided to redefine the main task of the network of consumer organisations as a dissemination network of the project results. A Network Registration and Information Platform for REDALL activities has been implemented on the Internet and several organisations have registered for participation in this communication process.

About 600 patients had clinical symptoms, positive skin tests and/or specific IgE antibodies for the foods of interest. They were invited to participate in the study. Because of the high number of patients identified, even though a lot of patients declined participation, finally 405 patients were tested by DBPCFC or even identified as having had anaphylaxis to the food. Positive reactions were observed in 259 patients (101 to milk, 141 to egg and 17 to meat).

For the registration of severe allergic cases in the health sector a questionnaire was developed on the basis of a previous instrument (EU 1998). The registration was announced in the Allergo Journal. Further announcement was made by Melloni Hospital among Italian network participants. The questionnaire records severe allergic reactions, symptoms and elicitors, medication, a detailed number of cases in previous years and number of anaphylaxis cases recorded as well as numbers of unspecified death cases. Due to weak response rates a short registration questionnaire was developed for rapid fax based written surveys. It mainly addresses severe allergic reactions, symptoms and elicitors, medication. The questionnaire has been developed in close co-operation within REDALL and approved by the project group.

ELISA is a common immunological method routinely used in analytical laboratories to detect and quantify allergens in food products. Antibodies are fundamental to ELISA as they are antigen (allergen)-specific and will determine the presence of an allergen in a sample extract. As part of the objective to develop sensitive immunological methods, the main allergens of milk whey (ß-lactoglobulin), egg white (ovomucoid and ovalbumin) and egg yolk (a-livetin; from pasteurised and non-pasteurised yolks) were isolated and purified. Rabbits were immunised with purified proteins and sera was collected before immunisation (pre-bleed), during boost immunisations (test bleed) and after immunisation (terminal bleed). Sera collected from the rabbits were characterised for antibody presence. The competitive direct ELISA technique, which allows binding of antibodies to protein antigens immobilised in wells of the microtitration plate was used for the investigations as well as competitive indirect ELISA. Beside ELISA investigations the extracted proteins were separated electrophoretically, blotted and immuno stained. For the characterization of the patients' sera EAST was performed. EAST inhibition experiments were used for quantification of reduction of allergenic potency.

In the written interview survey questions about needs and attitudes of food allergic consumers have been addressed, including investigations on purchasing and nutritional habits, information requirements and the individual impact of food allergy on everyday life, which is given 'big' or 'very big' by 24% of respondents. According to the data this impact turned out to be rather strong in specific food-related activities like buying food, preparing it or ingesting. Apparently consumers have little confidence in allergen information and ingredient declaration of food and food products. When reading food labels and other information of concerns about allergens more than one third (34%) of consumers in our sample feels 'rather unsure' or 'always unsure'. Additionally 67% of surveyed subjects consider the statement 'may contain traces of..' as being 'very useful' for their purchasing decision. The relevance of broad and transparent information on allergens and allergen impact of food is reflected by ratings for 'information requirements'. List of ingredients (4.1), general allergy information (4.1), indication of allergens (4.0) and FA labelling (4.0) are reportedly of interest for the consumer. When asked about specific information needs of concerned consumers the general allergy indication (2.3) and a complete list of ingredients (3.2) is the most frequently requested information, with the complete ingredients list being the most frequently mentioned item in the 'very much' category (41.7%).

In only one case of milk allergy a mediator release was observed to milk allergen extracts and skin prick test solution. It appeared that not all sera were able to sensitize RBL cells. Thus in this system there are non-responders, which theoretically also may be due to wrong or irrelevant proteins, or because commercially available extracts (from Sigma Aldrich) do differ from naturally occurring proteins. However, non-responders have also been described in other cellular tests, such as the histamine release test. For egg allergic patients, in 11/19 patients a mediator release could be detected. Release was higher for single allergens, such as albumine as compared to collective extracts (e.g. hen's egg allergen) and for skin prick tests. In the 11 positive sera, eight were tested with allergen-reduced egg and a reduction of mediator release of a factor of >1000 was found. In some sera, the flat curve to normal egg allergen compromised detailed analysis of the reduction factor, however, a drastic reduction of allergenicity was found in all sera. The human mast cell line of a patient with leukemia (LAD2) carries a functional human receptor FceR1and was evaluated for its use for food allergy in parallel to the RBL cells. Best results were achieved, when high antibody concentrations of anti-IgE were taken (1µg/ml). As for most patients, a 1:20 dilution of serum gave good results, we tested serum of four patients with milk and/or egg allergy. We were able to degranulate mast cells of this cell line by preincubation of serum of allergic patients and incubation with anti-IgE antibodies, as well as by incubation with cow's milk and hen's egg allergens.

High-pressure processing (HPP) and temperature treatment. HPP is in the meat industry a comparatively new tool to, primarily, enhance the hygienic stability of pieces of meat and of sausages. We applied HPP either before a heat treatment - to F-values between 1 and 12 - or after the heat treatment. Investigations on differently heated and high pressure treated meat products containing whey or egg allergens showed decrease of allergenicity of whey and egg the more heated. Just a small effect of high pressure could be visualized on whey and egg.

Four different PCR primers were chosen for the detection of bovine and chicken DNA in differently treated food samples using optimised DNA extraction protocols. The limits of detection were superior for the detection of meat (beef and chicken meat) and suitable for the detection of skimmed milk powder in various samples. The developed rapid method was suitable for the mentioned analytes too. Limits of detection were one order of magnitude higher. The comparison of the developed PCR methods with ELISA methods showed that the PCR is an important tool for detecting allergens in foods. The method of choice should be ELISA for the detection of milk and egg containing products at trace levels, while for the detection of beef and chicken meat containing products the PCR method is favourable. The result showed a positive correlation between DNA and protein content of food samples. A correlation between the degree of treatment of a food (sausage) and results of different PCR methods was clearly shown.

Threshold level analysis revealed that 53 reactions occurred already to the lowest given allergen level of 0,1ml. Thus, for some patients there are threshold levels corresponding to a large drop of the food of animal origin already. There is virtually no safe amount of undiluted food that can be reproducibly given without reaction. On the other hand, 50 patients reacted only after provocation of 144ml food. These patients may not be endangered by minute amounts and might even profit from contact to minute amounts because of tolerance induction. The reactivity to food allergens of animal origin is very individual.

Networks have been implemented by IFAV and the clinical partners. Main objective was to improve knowledge on the occurrence of severe allergic reactions to food in Europe. The Network is closely related to Deliverable 6 ("Questionnaire on the Occurrence of Severe Allergic Reactions"), since the questionnaire was chosen as a registration tool to initiate contacts for the Network. A registration platform was established on the Internet and further registrations have been carried out during the survey on severe allergic reactions. Clinical partners have implemented clinical networks for communication and their work in medical science.

A collection of guidelines for allergen management including analytical methods for measuring allergen traces in food and a gap analysis in form of a development of a list of issues which need to be addressed and solved in the future to improve current allergen management strategies was done. The investigation instruments were a web search of documents from individual food industry partners, food and drink industry organisations and other relevant organisations such as Public Health and Food Authorities and a literature review on allergen tests for food and cleaning methods in food industry.

Deliverables

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