Protease resistance of food proteins : a mixed picture for predicting allergenicity but a useful tool for assessing exposure

Background: Susceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios. Aim: To evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability. Methods: Four pairs of established allergens and their related non/weakly-allergenic counterparts (seed albumins, muscle tropomyosins, plant lipid transfer proteins [LTP] and collagens) plus fish parvalbumin, were subjected to nine combinations of pH (1.2-2.5-4.0) and pepsin-to-protein ratio (PPR: 10-1-0.1 U/µg) for pepsin digestion, followed by pancreatin digestion in the presence of bile salts. Digestion was monitored by SDS-PAGE in conjunction with Coomassie staining and immunoblotting using rabbit antisera and human IgE. Results: At pH 4.0 and at PPR 0.1 most proteins, both allergen and non-allergen, were highly resistant to pepsin. Under conditions known to favor pepsin proteolysis, the established major allergens Ara h 2, Pru p 3 and Pen a 1 were highly resistant to proteolysis, while the allergen Cyp c 1 was not. However, this resistance to pepsin digestion only made Ara h 2 and to a lesser extent Pen a 1 and Pru p 3 stand out compared to their non-allergenic counterparts. Largely irrespective of preceding pepsin digestion conditions, pancreatin digestion was very effective for all tested proteins, allergens and non-allergens, except for Cyp c 1 and bovine collagen. Conclusions: Sub-optimal pH, low pepsin-to protein ratio, and sequential pepsin and pancreatin digestion protocols do not improve the predictive value in distinguish allergens from non-allergens. Digestion conditions facilitating such distinction differ per protein pair

Core and Supplementary Studies to Assess the Safety of Genetically Modified (GM) Plants Used for Food and Feed

Genetically modified (GM) plants used for food and feed have an established history of safe use over more than 25 years of their commercialization. Developers and regulatory authorities have accumulated extensive experience in evaluating their safety over time. The studies required for the safety assessment of GM plants used for food and feed should now be re-defined to leverage this experience and increased scientific knowledge. This paper, a companion paper for Waters et al. also published in this issue, presents a systematic approach for the safety assessment of newly expressed proteins (NEPs) in GM plants by evaluating the two components of risk: hazard and exposure. Although the paper focuses on NEPs, the principles presented could also apply to other expression products that do not result in a NEP. A set of core studies is recommended, along with supplementary studies, if needed, to evaluate whether the GM plant poses risk. Core studies include molecular and protein characterization and hazard identification encompassing toxicity and allergenicity. In the absence of hazard, core studies are sufficient to conclude that GM plants are as safe as their conventional counterparts. Depending on the GM trait and intended use, supplementary studies should be performed to characterize hazard and exposure when a hazard is identified. Problem formulation should be used to identify hypothesis-driven supplementary studies. Acute toxicity studies, compositional assessment, and dietary exposure assessment are recommended to be hypothesis-driven supplementary studies. Further discussion on the current food and feed safety assessment landscape for GM plants and the use of problem formulation as a tool for identifying supplementary studies can be found in the companion paper [62]. doi: 10.21423/jrs-v09i1brun