Static in vitro digestion tests to assess allergenic risk of novel proteins
Résumé
Introduction
Evaluating the gastrointestinal fate of proteins is paramount to assess whether they are safe to
consume. The resistance of proteins to digestion may play a role in determining their allergenic
potential since incomplete digestion may cause undesired immune responses via sensitisation in
the duodenum. In order to evaluate the digestibility of proteins, in vitro protocols seem appropriate
when ethical constraints hinder in vivo studies. The current in vitro digestion model used by the
European Food Safety Authority (EFSA Journal 2017, 15, 4862) to assess allergenicity of proteins
is the pepsin resistant test. This uses harsh gastric conditions of acidity and enzyme concentration
that would mimic the end of gastric emptying (late phase) or fasted state in human adults. A more
realistic approach considering other relevant conditions in healthy adults (early phase or fed state),
or impaired digestion (e.g. infants) may provide useful information on how the combined effect of pH
and enzyme concentration affects protein digestibility.
Objective
Our current project contracted by EFSA aims looking at the combination of three different static in
vitro digestion models to evaluate the resistance of proteins to digestion by gastrointestinal enzymes
that could sensitise and eventually trigger an allergic reaction. The main objective is the development
and validation of a robust in vitro digestion protocol for purified proteins consistently reproducible
across different laboratories.
Methodology
Comparison of an infant, early phase adult, and late phase adult model is being applied on a panel
of allergen/non-allergen proteins from animal and vegetal origin. The proteolysis rate and extent is
determined with SDS-PAGE and LCMS in order to detect persistent intact protein and hydrolysis
products larger 9 amino acids.
Main findings
Differences in the kinetics of proteolysis may be found across models for proteins that are not pepsin
resistant.
Conclusion
This project highlights the importance of a multi-test protocol to assess protein digestibility.