Modified in vitro digestion model for allergenicity risk assessment for novel foods, including GMOs

Abstract

The application of agricultural biotechnology and introduction of alternative, novel protein sources are increasingly being used to provide the human population with sustainable, nutritious food. However, one of the major concerns raised by such novel technologies and foods is their allergenic potential. As a result, a “weight of evidence, case-by-case” approach is widely used by regulators and risk assessors for assessing the allergenic risks posed by novel foods and those derived from genetically modified organisms (GMOs). Data from in vitro digestion tests is used in this strategy as it provides data on the form in which novel proteins may be presented to the gut mucosal immune system. However, the tests currently used can lack physiological relevance and do not take account of intestinal and brush-border digestion. There is also lack of consensus as to what constitutes a resistant fragment of allergenic significance. Initially 96-well plate format gastric digestion test, based on validated protocols, was developed with a high and low pepsin activity test executed at pH 1.2, 2.5, 5.5 and 6.5 using purified allergens from different protein families including five animal sourced allergens bovine milk α-lactalbumin (Bos d 4), β-lactoglobulin (Bos d 5) and β-casein (Bos d 11), hen egg lysozyme (Gal d 4) and ovalbumin (Gal d 2), and two plant sourced allergens peach non-specific lipid transfer protein (Pru p 3) and peanut 7S vicilin-like globulin (Ara h 1). Two non-allergenic comparators were used, cytochrome c and α-zein. Selected gastric digests were then subjected to a 96-well plate intestinal digestion test using either a high or low enzyme activity protocol executed at pH 6.5 and 8.0. A set of novel exopeptidase digestion tests were developed using purified carboxy- and amino-peptidases and a brush border membrane assay using membrane derived from the gut epithelial cell line Caco-2. Based on the availability of purified proteins, the tests were applied to the analysis of selected gastrointestinal digests from α-lactalbumin and Pru p 3. Finally the in vitro digestion model was extended to the analysis of a whole food using a soy-enriched wheat-based pizza base. Digestion time courses were analysed using SDS-PAGE, densitometry analysis with selected digests being analysed by HPLC and/or mass spectrometry (MS). Due to the limited availability of patients’ sera, the immunoreactivity of digests was only investigated on Pru p 3 using animal antibodies and peach allergic patient serum IgE binding studies using immunoblotting and enzyme-linked immunoassay. Overall in this study the modified in vitro gastrointestinal digestion model provides a more comprehensive readout to assess the allergenic potential of digests and potential strategy for quantifying the digestibility of parent proteins and persistent and transient fragments. Based on the half-lives in the gastric digestion, proteins could be ranked from the least (β-lactoglobulin) to most digestible (soluble α-zein), while in the intestinal digestion ovalbumin, lysozyme and cytochrome c were proved to be the most resistant. In general in silico predictions of digestibility by endoproteases pepsin, trypsin and chymotrypsin, were not confirmed experimentally. Bile salts enhanced digestion of β-lactoglobulin, lysozyme and Pru p 3, showing the critical role they play in protein digestion. IgE binding tests and MS analysis of Pru p 3 digests showed that intramolecular disulphide bonds allow peptide fragments to be assembled into larger structures which, retain their IgE-binding capacity although at a much-reduced level. The exopeptidase digestion proved to be highly complementary with the combined amino-and carboxy peptidase assay digesting many of the peptides and protein fragments resulting from prior gastrointestinal digestion. The Caco-2 cell line proved to be a feasible, economic and reproducible source of brush border proteases which digested proteins in a comple

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Elizabeth Clare Mills (Supervisor) & Rene Crevel (Supervisor)