Extractability of the peanut allergen Ara h 2 from a model food matrix

The extractability of the peanut allergen Ara h 2 from model food matrix, namely cookie dough containing different amount of peanut flour (1%, 5% and 10 % w:w) was assessed through two-dimensional differential in gel electrophoresis (2D DIGE) and an Ara h 2 specific ELISA assay. Protein recovery int he range 97-120% was found using the 2D DIGE method. ELISA results confirmed our findings with total Ara h 2 recoveries within the 85-95 % range.JRC.D.5-Standards for Food Bioscienc

Presence of Peanut and Hazelnut in Cookies and Chocolates: the Relationship between Analytical Results and the Declaration of Food Allergens on Product Labels

Accidental exposure to hazelnut or peanut constitutes a real threat to the health of allergic consumers. Correct information regarding the ingredients of food products is of paramount importance to inform the consumer and thereby reducing the exposure to food allergens. For this study we have purchased 569 cookies and chocolates on the European market. All products were analysed to determine their peanut and hazelnut content allowing a comparison of the analytical results with the information provided on the label of those food products. Compared to cookies, chocolates are more likely to contain undeclared allergens, while in both food categories hazelnut traces were detected at higher frequencies than peanut. The presence of a precautionary label was found to be related to a higher frequency of positive test results. The majority of chocolates carrying a precautionary label tested positive for hazelnut, whereas in three quarters of the cookies carrying a precautionary label peanut traces could not be detected.JRC.D.8-Food safety and qualit

Inter-laboratory Validation Study of Two Commercial Lateral Flow Devices for the Detection of Peanut Proteins in Cookies

The results of an inter-laboratory study with two commercially available lateral flow devices (dipstick tests) designed to detect peanut residues in food matrices are reported. The test samples used in this study were cookies containing peanut at seven different concentrations in the range of 0 – 30 mg peanut per kg food matrix. The test samples with sufficient and proven homogeneity were prepared in our laboratory. The analyses of the samples (5 times per level and each laboratory) were performed by 18 laboratories worldwide that submitted in total 1 260 analytical results. One laboratory was found to be an outlier for one of the test kits. In general, both test kits performed well. However, there were some false-negative results in all matrices below 21 mg peanut/kg cookie. It must be stressed that the test kits were challenged beyond their cut-off limits (5 mg/kg or more depending on the food matrix). One test kit showed less false-negative results whereas it led to some false-positives in the blank materials. The sensitivity of the dipstick tests is comparable to what can be achieved with enzyme-linked immunosorbent assays (ELISA).JRC.D.8-Food safety and qualit

The Sffect of Heat Treatment on the Detection of Peanut Allergens as Determined by ELISA and Real-time PCR

Peanut allergic reactions can result from the ingestion of even very small quantities of peanut and represent a severe threat to the health of sensitised individuals. The detection of peanut traces in food products is therefore of prime importance. Peanut traces which can be (unintentionally) present in food products have usually undergone one or more processing steps like roasting and baking. Therefore, the methods designed to detect such traces have to be capable of detecting heat treated peanut. Commonly used methodologies designed to detect peanut traces in food products are enzyme-linked immunosorbent assays (ELISAs) that detect peanut specific proteins, and polymerase chain reaction (PCR) based methods targeting peanut specific DNA. A comparative analysis of such methods was performed and the impact of heat treatment on peanut kernels as well as the impact on a peanut-containing food matrix was investigated. Our results show that heat treatments have a detrimental effect on the detection of peanut with either type of method and that both types of methods are affected in a similar manner.JRC.D.8-Food safety and qualit

Influence of Baking Time and Matrix Effects on the Detection of Milk Allergens in Cookie Model Food System by ELISA

Milk allergens are common allergens occurring in foods, therefore raising concern in allergic consumers. Enzyme-linked immunosorbent assay (ELISA) is, to date, the method of choice for the detection of food allergens by the food industry although, the performance of ELISA might be compromised when severe food processing techniques are applied to allergen-containing foods. In this paper we investigated the influence of baking time on the detection of milk allergens by using commercial ELISA kits. Baked cookies were chosen as a model food system and experiments were set up to study the impact of spiking a matrix food either before, or after the baking process. Results revealed clear analytical differences between both spiking methods, which stress the importance of choosing appropriate spiking methodologies for method validation purposes. Finally, since the narrow dynamic range of quantification of ELISA implies that dilution of samples is required, the impact of sample dilution on the quantitative results was investigated. All parameters investigated were shown to impact milk allergen detection by means of ELISA.JRC.DG.D.6-Food Safety and Qualit

Development of Three Real-Time PCR Assays to Detect Peanut Allergen Residues in Processed Food Products

Hypersensitivity to peanut is a public health problem, since the ingestion of even low amounts of peanut can trigger severe allergic reactions. Allergic consumers rely on the information provided on the label of foodstuffs to identify products that might endanger their health. In order to protect the allergic consumer methods are required for the detection of allergenic ingredients. For this purpose we have developed three real-time polymerase chain reaction (PCR) assays, based on TaqMan chemistry, that are capable of detecting peanut specific DNA sequences in food products. The peanut specific sequence targeted for detection is located within the gene family of the allergen Ara h 3. The occurrence of multiple Ara h 3 sequences in the peanut genome increases the chance to achieve a good sensitivity. DNA extraction is also known to affect detection by PCR, therefore the efficiency of several different DNA extraction methods was compared. The methods reported here are capable of detecting 2.5 pg peanut DNA (less than one copy of peanut genome equivalent) and all three assays were successfully applied to detect peanut traces in a model food product where they could detect 10 mg kg-1 peanut.JRC.D.8-Food safety and qualit

Label-Free Proteomic Analysis of Wheat Gluten Proteins and Their Immunoreactivity to ELISA Antibodies

ELISA (enzyme linked immunosorbent assay) methodology is currently the mainstay for gluten quantification. However, the lack of comparable measurements among commercial kits has caused a great deal of concern. Here, we studied the immunoreactivity of five commercial ELISA kits to wheat gluten fractionated by reversed phase high-performance liquid chromatography (RP-HPLC) and identified the proteins and peptides in the resulting fractions by mass spectrometry to understand the extent by which these may be contributing to the lack of comparability.JRC.F.5-Food and Feed Complianc

Immunofluorescence Detection of Advanced Glycation end Products (AGEs) in Cookies and Its Correlation with Acrylamide Content and Antioxidant Activity

Food processing induces Maillard reactions, which result in the formation of advanced glycation end products (AGEs). AGEs present in food products can affect human health as either enhancers of food and age related diseases, or promoters of beneficial antioxidant activity. In this study an immunodetection method was employed which revealed a correlation between the autofluorescence of the Maillard products and acrylamide and antioxidant activity. But, the immunodection of AGEs did not show such a correlation, suggesting that at higher baking times the increased autofluorescence results from (antioxidant) compounds of non-proteineious origin, whereas the reduction of immunofluorescence is likely to result from the transient character of the AGE epitopes.JRC.D.8-Food safety and qualit

Development of Real-Time PCR Assays for the Detection of Lupin Residues in Food Products

Lupin is a legume that is used for human nutrition. Unfortunately lupin is known to trigger allergic reactions and therefore a mandatory declaration on the label of foodstuffs is required (Directive 2007/68/EC). To protect the allergic consumer lupin detection methods are needed. Here we present the development of two real-time polymerase chain reaction (PCR) methods that allow the detection of lupin-specific DNA. In cookies lupin could be detected at a level of only 5 mg per kg food matrix ELISA analyses were performed to compare the detection of lupin DNA with that of lupin protein, and the real-time PCR method targeting d-conglutin was shown to yield results comparable to those obtained by ELISA.JRC.D.5-Food Safety and Qualit

The feasibility of harmonising gluten ELISA measurements

Many publications have highlighted that routine ELISA methods do not give rise to equivalent gluten content measurement results. In this study we assess this variation between results and its likely impact on the enforcement of the EU gluten-free legislation. This study systematically examines the feasibility of harmonization of gluten ELISA assays by the introduction of: a common extraction procedure; a common calibrator such as a pure gluten extract and an incurred matrix material. The comparability of measurements is limited by a weak correlation between kit results caused by differences in the selectivity of methods. This lack of correlation produces bias that cannot be corrected by using reference materials alone. The use of a common calibrator reduced the between-assay variability to some extent but variation due to differences in selectivity of the assays was unaffected. Consensus on robust markers and their conversion to “gluten content” are required.JRC.F.5-Food and Feed Complianc