A reference method for determining the total allergenic protein content in a processed food: the case of milk in cookies as proof of concept

Abstract

The establishment of a reference method for the determination of the allergen protein content in a processed food material has been explored. An analytical approach was developed to enable the comparability of food allergen measurement results expressed in a decision-relevant manner. A proof of concept is here presented, resulting in quantity values for the common measurand, namely ‘mass of total allergen protein per mass of food’. The quantities are determined with SI traceability to enable the comparability of reported results. A method for the quantification of total milk protein content in an incurred baked food at a concentration level clinically relevant is presented. The strategy on how to obtain the final analytical result is outlined. Challenges associated with this method are discussed, in particular the optimal extraction of the marker proteins, the complete digestion and release of the peptides in an equimolar fashion, the use of conversion factors to translate the amount of measured proteins into total milk protein and the estimation of the uncertainty contributions as well as of the combined uncertainty of the final result. The implementation of such a reference method for the determination of the total allergen content in a processed food is an important step, which will provide comparable measurement data of relevance to risk assessors.

Total cow’s milk protein in cookies: the first interlaboratory comparison with a well-defined measurand fit for food allergen risk assessment

“…The indicative assigned value (x ILC ) of the mass fraction of total cow's milk protein (TCMP) in the baked cookie was determined by applying the single-laboratory validated LC-MS method described by Martinez-Esteso et al [6], and providing results traceable to the International System of Units (SI). It is based on the measurement of selected marker peptides by mass spectrometry and the conversion of the corresponding MS signals to the not-directly measurable TCMP mass fraction.…”

“…Thus, a compositional factor (f s ) taking into account the part of TCMP, which was not covered by the five marker proteins, was set to f s = 1.087 (=1/0.92). Finally, a value of 11.8 mg kg −1 was obtained by combining measurements for the five proteins into a single result (approach A4 described in [6]). This value was set as the indicative assigned value (x ILC ).…”

“…A relative measurement uncertainty associated with the LC-MS measurement (u char , rel ) of 4 % was estimated by Martinez-Esteso et al applying the approach outlined above [6] (which includes an uncertainty component associated with the compositional factor f s ). A conservative relative uncertainty contribution due to inhomogeneity (u hom , rel ) of 2.4 % was derived from the homogeneity study, while u st was set to zero.…”

“…Since no adequate certified reference material (CRM) was available, a tailor-made test material was prepared and characterised. The corresponding "indicative" assigned value was established using the reference method developed by Martinez-Esteso et al [6]. This value did not rely on the results reported by the participants.…”

Whey allergens: Influence of nonthermal processing treatments and their detection methods

“…Due to more reliable number of copies of cells of various tissues, single-copy nuclear DNA targets are known as the best choice for reliable quantitative approaches (Prado et al, 2016). Most of the data reported utilizing DNA-based approaches involves extending PCR to the initial target DNA sequences, using particular primers, which provide high precision assessments (Martinez-Esteso et al, 2020 Multiplex real-time PCR enables the concurrent amplification of numerous DNA fragments by incorporating a variety of primers. This approach was used to determine the DNA of major eight allergens: milk, hazelnut, peanut, celery, soya, egg, almond, and sesame.…”