Static in vitro digestion tests to assess allergenic risk of novel proteins

IntroductionEvaluating the gastrointestinal fate of proteins is paramount to assess whether they are safe toconsume. The resistance of proteins to digestion may play a role in determining their allergenicpotential since incomplete digestion may cause undesired immune responses via sensitisation inthe duodenum. In order to evaluate the digestibility of proteins, in vitro protocols seem appropriatewhen ethical constraints hinder in vivo studies. The current in vitro digestion model used by theEuropean Food Safety Authority (EFSA Journal 2017, 15, 4862) to assess allergenicity of proteinsis the pepsin resistant test. This uses harsh gastric conditions of acidity and enzyme concentrationthat would mimic the end of gastric emptying (late phase) or fasted state in human adults. A morerealistic 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 pHand enzyme concentration affects protein digestibility.ObjectiveOur current project contracted by EFSA aims looking at the combination of three different static invitro digestion models to evaluate the resistance of proteins to digestion by gastrointestinal enzymesthat could sensitise and eventually trigger an allergic reaction. The main objective is the developmentand validation of a robust in vitro digestion protocol for purified proteins consistently reproducibleacross different laboratories.MethodologyComparison of an infant, early phase adult, and late phase adult model is being applied on a panelof allergen/non-allergen proteins from animal and vegetal origin. The proteolysis rate and extent isdetermined with SDS-PAGE and LCMS in order to detect persistent intact protein and hydrolysisproducts larger 9 amino acids.Main findingsDifferences in the kinetics of proteolysis may be found across models for proteins that are not pepsinresistant.ConclusionThis project highlights the importance of a multi-test protocol to assess protein digestibility.