Milk allergen quantification in rice flour: An experimental investigation of sampling and testing procedures

Unintended allergen presence in foods may cause severe allergic reactions, thus posing a health risk for food allergic consumers. Consequently, accurate allergen quantification in foods is relevant for food allergic individuals, the food industry and authorities. However, regulation regarding sampling methods for food allergen analysis is lacking. The aim of the present study was to investigate the influence of the laboratory sample size and test portion size on food allergen analytical determinations by ELISA in an amorphous homogeneous milk contamination scenario.

A sampling plan was designed where seven lots of 1000 g rice flour contaminated with increasing amounts of skimmed milk powder (from 0 to 500 ppm milk protein contamination) were produced, and five 100 g as well as five 200 g laboratory samples were collected. From each laboratory sample, two test portions of 0.5 g and two test portions of 5 g were extracted, and two aliquots from each test portion were analysed for detection of milk allergens using a commercial ELISA kit. To evaluate the sampling plan, the measured concentration variability was estimated using a hierarchical mixed effects model, and the statistical significance of each sampling step was validated using permutation tests.

The experimental data indicated that the variance of the measured concentrations increased as a function of the spiked concentration. While increasing the test portion size from 0.5 to 5 g did not improve test precision, increasing the laboratory sample size from 100 to 200 g improved the test precision by reducing the variance of the laboratory sample by 88% and the total variance by an average of 63%. Thus, when estimating producer's and buyer's risks, increasing laboratory sample size from 100 to 200 g greatly improved the precision compared to increasing sample number from one to two 100 g samples, which reduced the total variance by 50%.

The laboratory sample size was critical for the analytical accuracy of the sampling plan for detection of milk protein contamination in rice flour, which was used in this study as an example of an allergen contamination scenario, and for reducing the risks associated with sample misclassification.