Assessing protein digestibility in allergenicity risk assessment: A comparison of in silico and high throughput in vitro gastric digestion assays

The susceptibility of a novel food protein to digestion in the pepsin resistance test is widely used to inform the allergenicity risk assessment process. However, it does not model the variation in the intragastric environment found in vivo. Consequently a 96-well plate format in vitro gastric digestion protocol has been developed with a high and low pepsin activity test executed at pH 1.2, 2.5, 5.5 and 6.5. It was used to analyse seven allergens (from milk, egg, peach and peanut) and two non-allergens (cytochrome c and zein). Digestion was monitored using SDS-PAGE and densitometry. In silico predictions were not confirmed experimentally for most of the proteins studied. Proteins were ranked according to half-life and showed susceptibility to digestion was related to the stability of protein structure and protein solubility rather than allergenicity per se. Highly digestible proteins, such as beta-casein and Ara h 1, generated abundant resistant fragments Mr > 3.5 kDa in the low pepsin activity test which could be immunologically significant within the context of allergenicity risk assessment for susceptible groups such as infants. The high-and low pepsin activity tests used in this study provided complementary data to support allergenicity risk assessment and used only 10 mg protein.