Extensive boiling strongly reduces egg white allergenicity due to ovomucoid aggregation, as determined by high-resolution mass spectrometry

Abstract

Introduction (research context)

Hen's egg allergy is the second most frequent food allergy found in children. The major allergens involved in hen's egg allergy are found in egg white (EW); ovalbumin (OVA) and ovomucoid (OVM). Current treatment involves strict dietary avoidance or minimized contact with egg. Egg ladders, which are tools used to step-wise introduce increasingly allergenic forms of egg at home, have been investigated as an alternative. Despite their increasing use, an in-depth understanding of the impact of heating on the structure and allergenicity of the major EW allergens is lacking.

Objective

We aimed to investigate the structural changes of the major EW allergens upon the moderate and extensive boiling of EW and their impact on EW allergenicity.

Methods

Eggs were boiled for 10 or 45 minutes. EW was separated from egg yolk and subjected or not to gastric digestion. Structural changes of major EW proteins were assessed using SDS-PAGE and mass spectrometry (MS). Specific attention was paid to inter- and intramolecular bonds and chemical modifications during MS analyses. EW allergenicity was assessed using Western-blot, Dot blot and rat basophil leukemia (RBL) cells with a serum pool of egg-allergic patients.

Results

Initial aggregation of OVA was observed after 10 minutes, followed by strong aggregation of OVM after 45 minutes of boiling, as demonstrated by the apparition of intra- and intermolecular disulfide bonds in known epitopes of OVM. These structural modifications translated into a minor reduction in the allergenicity upon 10 minutes of boiling, whereas a strong reduction was seen upon 45 minutes of boiling. Lastly, gastric digestion modestly lowered the allergenicity of raw and 10 minute-boiled EW, but strongly lowered the allergenicity of 45 minute-boiled EW.

Conclusions

We generated heated EW samples with a gradual reduction in allergenicity and describe the associated structural changes of the main EW allergens. Our study highlights the importance of conformational OVM epitopes in EW allergenicity and provides a scientific underpinning for egg ladders. Different times of egg boiling might be exploited in egg ladder-based treatment strategies.