Kitti Török

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...

Identification of the factors affecting the analytical results of food allergen ELISA methods

In order to support patients’ safety, the analytical methods should be able to quantify the allergenic proteins in food products. Analytical performance of the currently used ELISA methods is not always appropriate, particularly in case of processed foods. A possible way to investigate the sources of analytical errors is the utilisation of model food matrices that mimic the technology and behaviour of real food products. Consequently, factors affecting the analytical results were identified, their contribution to the whole analytical error was determined, and the underlying phenomena were interpreted in this study. Heat-treated model matrices incurred with gliadin, milk, egg, or soy proteins were produced and analysed with commercially available ELISA test kits. The data were evaluated with statistical tools. Results clearly show that the type of the food matrix, the level of processing, and the analytical methods (i.e. the type of the ELISA kit) are the three main factors that play a significant role in the uncertainty. The developed laboratory methodology seems to be suitable for generalising the investigation of other allergens, methods, matrices, and the scientific background of the phenomena.

Investigation of incurred single‑ and multi‑component model food matrices for determination of food proteins triggering allergy and coeliac disease

For detection and quantification of food proteins triggering hypersensitivity reactions, reliable, validated analytical methods are necessary. At present, the most commonly used methodology for this purpose is ELISA. The lack of incurred reference materials (RM) complicates the fulfilling of method validation. Today, a well identifiable research direction is the development of methods (immunoanalytical, PCR and MS methods) suitable for multi-component analysis. As a consequence of these R+D efforts, the development of multi-component RMs is reasonable. Our work presented here is focusing on the development and investigation of multi-component model food matrices that can be suitable for supporting the validation processes. On the basis of the relative incidence of hypersensitivity reactions, four components—wheat, milk, egg and soy—were selected for our preliminary work. The target proteins were determined in single- and multi-component matrices with different ELISA methods, and the results were compared by statistical evaluation. The main direction of our investigation was to identify the effects of the potential interactions of these proteins on the analytical results, and the influence of food processing was also investigated. The results showed that the presence of multiple allergenic components caused much less uncertainty in the analytical results than analytical and technological steps, such as sample preparation, repetition or even heat treatment. However, probably due to the relatively complex matrices, some discrepancies from the expected outcome could also be observed.

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.

Towards development of incurred materials for quality assurance purposes in the analysis of food allergens

Food allergy and intolerance became very important problems in food safety and healthcare during the last few decades. Beside the pharmaceutical treatment of the symptoms, effective cure of these illnesses is the avoidance of the problematic food proteins. According to this reason, proper legislation is crucial for protecting sensitive people. In the European Union 14 allergenic components must be labelled which requires introduction of properly validated analytical methods for the appropriate quantification of allergenic proteins. The aim of our work is studying such parameters which may affect the analytical results, therefore have to be taken into account during the validation process. For investigating these issues, an incurred sample matrix was produced, namely a wheat flour based cookie, which contains allergenic proteins (milk or egg) in a dedicated amount. Using these samples the effects of food processing steps and the analytical performance of the applied Enzyme-Linked Immunosorbent Assay (ELISA) methods were studied. A major finding of our work is that heat treatment caused a large-scale decrease in the amount of measurable allergen content of the samples. The background of this phenomenon has not been clarified yet. Besides, the gathered data indicates that the performance of the ELISA method is highly related to the state of the sample matrix. These problems altogether must be taken into consideration for making a proper validation protocol and revealing their background also has a great importance in further evaluation of the analytical methods.

Optimization of Arabinoxylan Isolation from Rye Bran by Adapting Extraction Solvent and Use of Enzymes

Abstract Physicochemical and functional properties of arabinoxylans (AXs) can be significantly influenced by their isolation method. Finding balanced process conditions that allow optimal extraction yields while preserving AXs functionality is a challenge. The aim of this study was to determine the effect of different chemical solvents with neutral and alkaline pH on the intrinsic properties and extraction yield of AXs isolated from rye bran. Additionally, the application of xylanases and other cell wall degrading enzymes (Pentopan Mono BG, Deltazym XL‐VR, Viscoflow BG) to solubilize bound AXs was investigated. Results show that the use of Ca(OH)2 for isolation was superior to water and Na2CO3, as it selectively solubilized AXs and delivered isolates with a purity of up to 43.92% AX and a moderate ferulic acid (FA) content (209.35 ± 16.79 mg FA/100 g AX). Application of xylanases was further able to duplicate these achieved AX yields (7.50 to 9.85g AX/100 g bran). Additionally, isolates displayed highest ferulic acid contents (445.18 to 616.71 mg FA/100 g AX) and lowest impurities in comparison to chemical extracted AXs. Rheological characterization of the isolates showed a pronounced shear thinning behavior which fitted well to the power‐law model (R 2 > 0.989). Differences in pseudoplasticity of the isolates suggested that structural and chemical properties might have been responsible for this behavior.

Protein interactions during flour mixing using wheat flour with altered starch

Wheat grain proteins responses to mixing and thermal treatment were investigated using Mixolab-dough analysis systems with flour from two cultivars, Ventura-26 (normal amylose content) and Ventura-19 (reduced amylose content). Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis revealed that, stress associated and metabolic proteins largely interacted with dough matrix of Ventura-26 after 26min (56°C); gliadins, avenin-like b proteins, LMW-GSs, and partial globulins showed stronger interactions within the dough matrix of Ventura-26 at 32min/C3 (80°C), thereafter, however, stronger protein interactions were observed within the dough matrix of Ventura-19 at 38min/C4 (85°C) and 43min (80°C). Thirty-seven proteins associated with changes in dough matrix due to reduced amylose content were identified by mass spectrometry and mainly annotated to the chromosome group 1, 4, and 6. The findings provide new entry points for modifying final product attributes.

Protein-transitions in and out of the dough matrix in wheat flour mixing

Sequential protein behavior in the wheat dough matrix under continuous mixing and heating treatment has been studied using Mixolab-dough samples from two Australian wheat cultivars, Westonia and Wyalkatchem. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis indicated that 32min (80°C) was a critical time point in forming large protein complexes and loosing extractability of several protein groups like y-type high molecular weight glutenin subunits (HMW-GSs), gamma-gliadins, beta-amylases, serpins, and metabolic proteins with higher mass. Up to 32min (80°C) Westonia showed higher protein extractability compared to Wyalkatchem although it was in the opposite direction thereafter. Twenty differentially expressed proteins could be assigned to chromosomes 1D, 3A, 4A, 4B, 4D, 6A, 6B, 7A and 7B. The results expanded the range of proteins associated with changes in the gluten-complex during processing and provided targets for selecting new genetic variants associated with altered quality attributes of the flour.

Development and characterization of wheat lines with increased levels of arabinoxylan

Improving the nutritional quality and health benefits of food has been of increasing interest globally over the last decade. Staple cereal foods are the major sources of dietary fiber and a recent study identified the Chinese wheat cultivar Yumai-34 as having unusually high levels of water-extractable arabinoxylan (WE-AX) and total arabinoxylan (TOT-AX) in flour. Crosses were therefore made between this variety and three Central European varieties (Lupus, Mv-Mambo, Ukrainka) and the physical properties (test weight, thousand-kernel weight, flour yield, kernel hardness), composition (protein, gluten, WEAX, total AX) and processing quality (gluten index, Zeleny sedimentation, Farinograph parameters) of the grain were compared for thirty-one breeding lines (F7–F9) and the four parents in a 3-year field experiment (2013–2015). Increases of 0.5% in the WE-AX content and 1% in the content of total AX content of the flour were achieved, with an improvement in dough properties. The thousand-kernel weight, protein content, gluten content, Zeleny sedimentation and water absorption of the flour also increased in many lines, while three of the lines had yields that were competitive with the official control varieties, making them suitable for registration.