Food producers are increasingly looking to food allergen analysis as a means of emphasising greater transparency, traceability and integrity in the supply chain. While this growing awareness has extended to validation and verification of factory cleaning and investigation of recalls and incidents, producers are also investigating new ways to detect allergens in processed foods. Food allergen expert Adrian Rogers explores how incurred samples might show the way forward.

Most of the food and drink we consume has been processed or modified in some way. This processing brings about many benefits in terms of food safety, preservation and taste. However, processing changes the characteristics of the ingredients used to make the food; of particular interest are the changes that allergenic proteins can undergo. There are many kinds of changes relevant to allergen detection: allergenic proteins can be subjected to heat-accelerated chemical reactions including Maillard reactions and other protein-carbohydrate interactions, protein aggregation with loss of solubility, shear effects on protein structure, emulsion formation, pH effects, and water activity during food production. Recent studies have shown that processing allergens can alter their allergenicity, changing how an allergic individual may react to them. If immunoassay-based food allergen detection methods rely on the use of antibodies to detect allergens present in food, it follows that the ability to detect them may be affected by processing.

Such processing effects must be taken into account when developing new analytical methods, either by improved extraction methods intended to increase the solubility of the aggregated proteins or by going back to basics and raising new sets of antibodies that specifically target processed allergens.

HOW DO WE CREATE REAL-LIFE SAMPLES WITH INCURRED CONTROLS?

To evaluate these improved allergen detection methods, incurred sample controls are needed. These are food samples into which a known amount of the food allergen has been incorporated during processing, mimicking as closely as possible the actual conditions under which the sample matrix would normally be manufactured. Analysing the real-life sample would give the most accurate representation of the recovery and response of a particular method for that particular matrix. Yet each food matrix and processing condition is different and it would not be practical to evaluate all possibilities with naturally incurred standards. However, evaluation of an ELISA with one or more typical combinations of food matrix and processing condition is a useful method in determining whether the ELISA can provide reliable results when applied to processed foods.

Quality control is an important factor when producing incurred reference materials. The allergen to be incorporated into the food matrix must be very well defined. wher official reference materials exist they are not usually affordable in the quantities needed to produce the incurred controls. Yet any official reference materials can be used for comparative purposes to define the nature of the material used. Furthermore, the homogeneity of the allergen added to the food matrix is critical for every step of the process. In order to ensure that the allergen is homogeneously incorporated into the incurred material, multiple analyses of the sub-sampled food at various stages of processing are required to make sure that uniform distribution of the allergen is achieved. Contamination between batches with increasing levels of allergen can be controlled by starting with the lowest concentration and steadily increasing the levels.

Another important consideration is the stability of the incurred material. Any processing effects that the allergen undergoes can usually be determined quickly during analysis. But if the incurred material is to be stored for any length of time and used to generate real world standard curves for an analytical method, then the shelf life of the material must be ascertained. There is a possibility that further reactions between the proteins of the allergen and the components of the food matrix into which it is incorporated could occur during storage. Since the production of well-defined incurred controls is not easy, storage of these controls would allow their future use for evaluating improved analytical methods. Such incurred control materials may also be used to evaluate batch-to-batch variability for commercial ELISA kits.

EARLY SUCCESSES WITH INCURRED SAMPLES

Very slowly, fully validated incurred reference materials for food allergen analysis are coming onto the market. The MoniQA Association has recently released a milk-incurred material that incorporates well-characterised dried skimmed milk powder into a gluten free cookie at two individual protein concentrations. This incurred reference material has been produced to mimic as closely as possible the processing through which an allergen-containing food goes during production. This is a distinct advantage as it more closely reflects the real-world foods that are routinely tested in analytical labs across the world and gives a good indication of the suitability of the analytical methods used to detect processed allergens. Advancing such research even further, work undertaken at the University of Manchester under the guidance of Professor Clare Mills has investigated how an allergen-incurred chocolate dessert material originally developed for the diagnosis of food allergies can be adapted for use as an analytical control material.

Such research is encouraging and could play a vital role in expanding the comprehensiveness of allergen testing in processed foods. The use of a matrix and incurred food ingredients with demonstrable allergenic activity for analytical purposes will help ensure that efforts to standardise calibration materials and harmonise allergen-reporting units are meaningfully aligned with ongoing measures to protect allergic consumers from accidental exposure to problem foods.