# Exploring the molecular modifications and allergenicity of the egg white protein matrix during boiling

Mehdi Cherkaoui, Elysa Le Corre, Aché Ahmat-Sougoudi, Emilie Perrin, Véronique Solé-Jamault, Hanitra Rabesona, Sandra Denery-Papini, Martine Morisset, Hélène Rogniaux, Wieneke Dijk

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Mehdi Cherkaoui, Elysa Le Corre, Aché Ahmat-Sougoudi, Emilie Perrin, Véronique Solé-Jamault, et al. Exploring the molecular modifications and allergenicity of the egg white protein matrix during boiling. Food Chemistry, 2025, 483, pp.144304. 10.1016/j.foodchem.2025.144304. hal-05198894

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# Exploring the molecular modifications and allergenicity of the egg white protein matrix during boiling

Mehdi Cherkaoui<sup>a,b</sup>, Elysa Le Corre<sup>a</sup>, Aché Ahmat-Sougoudi<sup>a</sup>, Emilie Perrin<sup>a</sup>, Véronique Solé-Jamault<sup>a</sup>, Hanitra Rabesona<sup>a</sup>, Sandra Denery-Papini<sup>a</sup>, Martine Morisset<sup>c</sup>, Hélène Rogniaux<sup>a,b</sup>, Wieneke Dijk<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Egg allergy

Heat

Boiling

Aggregation

Allergenicity

## ABSTRACT

Hen's egg allergy is the second most common food allergy in young children, with the major allergens ovalbumin and ovomucoid found in egg white. While many egg-allergic children can tolerate baked or hard-boiled eggs, there is limited understanding of how heating affects allergen structure and allergenicity within the egg white protein matrix. This study investigated the impact of egg white boiling for 10 or 45 min on the structure and allergenicity of the main egg white allergens. Our results showed that 45 min of boiling led to significant structural changes and a strong reduction in egg white allergenicity, while 10 min of boiling had a minor effect. Gastric digestion further reduced allergenicity, especially in 45 min boiled eggs. These findings highlight how increasing the boiling time of egg white can reduce allergenicity through structural changes in the main egg white allergens.

## 1. Introduction

Food allergies pose a significant public health problem, due to their increasing prevalence and severity worldwide. Hen's egg allergy is one of the most prevalent food allergies, with self-reported overall lifetime and point prevalence being 2.4 % and 1.8 %, and typically emerges within the first year of life (Spolidoro et al., 2023). The main allergens of hen's egg white are ovalbumin (OVA), which constitutes 54 % of egg white, and ovomucoid (OVM) (Leau et al., 2023). Research indicates that 50 to 85 % of individuals with egg allergies can tolerate eggs that are cooked or extensively heated, highlighting a connection between heat-induced structural changes in egg allergens and their allergenicity (Bartnikas et al., 2013; Lemon-Mulé et al., 2008).

Thermal treatment alters protein structure by disrupting tertiary and secondary structures, breaking native disulfide bonds, and forming new disulfide bonds upon cooling. The characteristic white, gel-like texture that forms when eggs are heated results from this gradual denaturation

and aggregation of egg white proteins (Mine, 1995; Urisu et al., 2015). The progressive unfolding of egg proteins during heating is influenced by (i) the duration and temperature of heating, (ii) the properties of the egg white proteins, and (iii) environmental factors such as pH, ionic strength, and the presence of other proteins like wheat gluten (Farjami et al., 2021). Egg white aggregation typically begins at 60 °C, the denaturation temperature of ovotransferrin, and accelerates at 70 °C, the denaturation temperature of OVA (Mine, 1995). Unlike the heat-sensitive OVA, OVM is highly resistant to heat due to its structure of three tandem domains with intra-domain, but not inter-domain, disulfide bonds (Mine, 1995). Only prolonged heating at temperatures above 90 °C (e.g. boiling >30 min) or the presence of wheat results in the formation of an irreversible denatured state, suggesting that OVM remains in its natural state in more briefly heated forms of egg (Gu et al., 1986; Matsuda et al., 1981).

Several studies have investigated the impact of heating on OVA and OVM immunoreactivity or allergenicity. Studies using

**Abbreviations:** HRMS, High-Resolution Mass Spectrometry; HCD, Higher-energy Collisional Dissociation; IgE, Immunoglobulin E; MW, Molecular weight; RP, Reversed phase; OVA, Ovalbumin; OVM, Ovomucoid; PBS, Phosphate Buffered Saline; PBS-T, PBS containing 0,1 % Tween; PBS-T-G, PBS containing 0,1 % Tween and 0,5 % Gelatin; SEC, Size-exclusion chromatography.

* Corresponding author.

E-mail address: wieneke.dijk@inrae.fr (W. Dijk).

<footnote><sup>a</sup> INRAE, UR1268 BIA, F-44316, Nantes, France</footnote>

<footnote><sup>b</sup> INRAE, PROBE Research Infrastructure, BIBS Facility, F-44316, Nantes, France</footnote>

<footnote><sup>c</sup> CHU d'Angers, Unité D'allergologie, Angers, France</footnote>
using glacial acetic acid and ethanol and rinsed with distilled water before scanning in order to eliminate background coloration (Cytiva, Amersham ImageQuant 800).

## 2.5. Chromatography

### 2.5.1. Size-exclusion chromatography

Size exclusion chromatography was performed to determine the purity and molar mass of the different samples. Two hundred µgrams of samples were loaded on a Superdex 75 Increase 10/300 GL column (10/300 mm, 24 mL) equilibrated in PBS buffer. Elution was performed during 46 min at 0.65 mL/min flow rate. The column was coupled to a pump, Infinity HP 1260 Bio-Inert (Agilent technologies, Santa Clara, USA), controlled by software OpenLab CDS (Agilent technologies, Santa Clara, USA) and an autosampler Infinity HP 1260 Bio-Inert with a cooling module (Agilent technologies, Santa Clara, USA). The online detectors were coupled to the column, a refractometer 1260 RID (Agilent technologies, Santa Clara, USA) and a multiple wavelength detector MWD RL (Agilent technologies, Santa Clara, USA). The Openlab CDS software was used for the data acquisition and processing. Peak integration at 214 nm was used to determine the purity.

### 2.6. Reverse phase chromatography

Reverse phase chromatography was conducted to analyze the peptide profiles of digested egg white. The soluble phase was used for analysis and samples were diluted to 2.5 mg/mL. Analysis of the digesta fractions was performed on a RP-HPLC using a Dionex Ultimate 3000 system (Thermo Fisher Scientific, Courtaboeuf Cedex, France) using linear gradient from solvent A (water/acetonitrile 98/2 (v/v) with trifluoracetic acid (0.05 % TFA) to solvent B (acetonitrile/water 98/2 (v/v) with (0.04 % TFA). A symmetry RP C8 column 3.5 µm, 4.6 mm × 50 mm, (Waters, Milford, MA, USA) was equilibrated with solvent A at a flow rate of 1 mL/min. Elution was carried out at 20 °C by applying solvent gradient from 0 to 50 % of solvent B over 10 min and finally 100 % B until 13 min. Detection of peptides was monitored by UV detection at 216 nm using RS variable wavelength detector (Dionex, VND3400R5). Samples were centrifuged at 10,000g, 10 °C, 30 min before loading 4 µL on the column.

## 2.7. Mass spectrometry

### 2.7.1. Preparations of samples prior to LC-MS analysis

50 µg of protein from each sample, except for gastro-intestinal digestes, was collected and added to 10 µL of trypsin (200 ng/µL) and ammonium bicarbonate 50 mM, pH 8 to reach a final protease-to-protein ratio of 1:25. In order to increase the release of tryptic peptides, 2 µL of 1 % (v/v) ProteaseMAX (Promega, Madison, United States) was added to reach a final concentration of 0.01 % (v/v). Samples were then incubated at 37 °C for three hours and digestion was stopped by cooling samples to -20 °C. Prior to MS analysis, all digested samples were filtered on ZebaSpin 7 kDa columns (ThermoFisher Scientific, #89882) and Affinisep AttractSPE Tips C8-10 µL in order to remove residual salts or large polypeptides and undigested proteins.

### 2.7.2. LC-MS (and MS/MS) analysis

Four µL of digests of each sample was injected on the nanoLC-MS/MS system composed of a hybrid Quadrupole-Orbitrap mass spectrometer (Q Exactive HF, Thermo Fisher Scientific™, Bremen, Germany) coupled to a nanoscale LC system (Ultimate U3000 RSLC system, Thermo-Fisher Scientific™, Bremen, Germany). Peptides were separated on a reversed-phase capillary column (Acclaim PepMap™ C18, 2 µm, 100 Å, 75 µm i. d. x 25 cm long, Thermo-Fisher Scientific™, Bremen, Germany) using a linear gradient of: (A) 99.9 % water, 0.1 % trifluoroacetic acid and (B) 90 % acetonitrile, 9.92 % water and 0.08 % formic acid. After 15 min of column equilibration at 4 % of B, the gradient consisted of a linear

increase from 4 % to 50 % of B in 45 min, followed by a rapid increase to 90 % of B within three minutes, which was maintained for two minutes and then decreased to 4 % of B for re-equilibration of the column during 10 min. Peptides were eluted at a flowrate of 0.3 µL/min and ionized in nanoESI positive mode. Acquisitions were done using a data-dependent acquisition method. Full MS scans (m/z 400–2000) were acquired at 60 K resolution and the fifteen most intense ions (with charges of 2–6) were fragmented in the HCD cell (NCE = 26). Fragments were analysed in the Orbitrap at 30 K resolution. After each run a wash program was engaged to reduce peptide carry-over on the column.

### 2.7.3. Unmodified peptide identification

Identification of peptides was done against a databank restricted to proteins belonging to Taxon 9031 (Gallus gallus) extracted from SwissProt (updated on 2024/04/03) by using MaxQuant (Cox & Mann, 2008). Methionine oxidation was set as a variable modification. Trypsin enzymatic cleavage was specified for non-digested samples and unspecific cleavage was specified for digested samples, with up to 2 miss-cleavage tolerance. Mass tolerance for first and main search peptide mass was set at ±20 ppm or 4.5 ppm, respectively. A FDR cut-off of 0.01 was applied. The “peptides” output file of MaxQuant was used to analyze peptide profiles using Peptigram (Manguy et al., 2017).

### 2.7.4. Linked peptides identification

The LC-MS/MS raw data were processed into mgf and mzXML formats using msConvert (https://proteowizard.sourceforge.io/tools/msconvert.html) prior to several databank searches. Cross-linked peptide identifications were done against a databank containing the sequences of OVA and OVM from SwissProt (P01012, OVAL-CHICK and P01005, IOVO_CHICK) using a dedicated databank search algorithms: pLink v.2.3.9 (Chen et al., 2019). Briefly, mass tolerances for precursors and fragments were set at 3 ppm. Inbuilt FDR evaluation was used to filter identified peptides under a threshold of 1 %. A manual examination of identified MS/MS spectra was performed on all peptide sequences identified by the software, and only unambiguous spectra (for linker positions on sequences) were conserved after this step. To be considered as unambiguous, a MS/MS spectrum needed to present at least one signature fragment of the cross-link position on both peptides (e.g. presence of several b- or y-ion containing the residues implicated in the bridge). An example of a curated and annotated cross-linked peptide spectrum can be found in (Cherkaoui et al., 2022). Cross-links were visualized using xiNET (Combe et al., 2015).

## 2.8. Immuno-assays

### 2.8.1. Dot blot

Increasing amounts of protein from each sample were spotted on a nitrocellulose membrane (0.2 mm, Sartorius, Germany) corresponding to 0.5, 1 and 2 µg of protein. The membrane was then dried for 30 min at 37 °C and saturated with either 4 % polyvinylpyrrolidone (PVP) in Phosphate Buffered Saline (PBS; 137 mM sodium chloride, 10 mM phosphate, 2.7 mM potassium chloride; pH 7.4), Tween 0.1 % (PBS-T) (for antibodies) or EveryBlot blocking buffer (Bio-Rad, #12010020) (for human serums) for 4 h at room temperature before incubation with antibodies or human sera as previously described (Lupi et al., 2013). Antibodies were prepared at either a 1:5000 dilution (anti-egg white), 1:1000 dilution (anti-OVA) or a 1:200 to 1:500 dilution (anti-OVM) in 1 % PVP in PBS-T and incubated overnight at 4 °C. An anti-rabbit (anti-egg white or anti-OVA, Bio-Rad, #1706515, 1:30000 dilution) or anti-mouse (anti-OVM, Bio-Rad, #1706516, 1:20000 dilution) secondary antibody was used diluted in 1 % PVP in PBS-T for 2 h at room temperature. Pooled sera were prepared at a 1:25 dilution in EveryBlot blocking buffer and incubated overnight at room temperature. An anti-human IgE secondary antibody (Southern Biotech, 9205–05) was used at 1:500000 dilution in EveryBlot blocking buffer for 2 h at room temperature. After each incubation with antibodies, the membranes were washed 6 times
using PBS-0.05 % Tween20 (primary antibodies) or PBS-0.1 % Tween20 (patient sera), three times short and three times 10 min. The chemiluminescent substrate used for revelation was the Western Bright™ Quantum chemiluminescence HRP substrate (ADVANSTA-K12042-D20, Menlo Park, USA), using a Cytiva, Amersham ImageQuant 800 machine.

### 2.8.2. *Western blot*

SDS-PAGE gels were migrated as described in section 2.4.1. Gels were transferred to a nitrocellulose membrane using semi-dry transferring (Bio-Rad, TransBlot Turbo, 10 min 25 V) and the RTA transfer Midi kit (Bio-Rad, # 1704271). A ponceau red staining (Sigma, #P7170) was performed to confirm correct protein transfer. Membranes were saturated using EveryBlot blocking buffer (Bio-Rad, #12010020) for 2 h at room temperature. Primary antibodies were used at the following dilutions in EveryBlot blocking buffer: anti-egg white (in-house) at 1:5000, anti-OVA (in-house) at 1:1000, anti-OVM at 1:200 or 1:500. A pool of serum from 9 patients was diluted 1:25 (Supplemental Table 1). Primary antibodies were incubated overnight at 4 °C, whereas patient sera were incubated overnight at room temperature. An anti-rabbit (anti-egg white or anti-OVA, Bio-Rad, #1706515, 1:30000 dilution) or anti-mouse (anti-OVM, Bio-Rad, #1706516, 1:20000 dilution) secondary antibody was used diluted in EveryBlot blocking buffer for 2 h at room temperature. An anti-human IgE secondary antibody (Southern Biotech, 9205-05) was used for experiments using patient sera at 1:500,000 dilution in EveryBlot blocking buffer for 2 h at room temperature. After each incubation with antibodies, the membranes were washed 6 times using PBS-0.05 % Tween20 (primary antibodies) or PBS-0.1 % Tween20 (patient sera), three times short and three times 10 min. The chemiluminescent substrate used for revelation was the Western Bright™ Quantum chemiluminescence HRP substrate (ADVANSTA-K12042-D20, Menlo Park, USA), using a Cytiva, Amersham ImageQuant 800 machine.

### 2.8.3. *Rat basophil leukemia*

G418-selected rat basophil leukemia-SX38 cells expressing human FcεRI were kindly provided by Pr Kinet (Harvard Medical School, New York, USA). Cells were seeded at 1.2×10⁴ cells/ml in 96-wells plates and cultured at 37 °C in a humidified atmosphere with 5 % CO₂. A pool of patient sera was heated for 45 min at 56 °C in the presence of 4 M glucose to reduce cytotoxicity and used at a dilution of 1:100. Cells were incubated for 2 days with diluted sera. After three washing steps with PBS, IgE-sensitized cells were stimulated in duplicate for 45 min at 37 °C by non-digested and gastric-digested soluble egg fractions, diluted in Tyrode buffer containing 50 % deuterium oxide. The release of β-hexosaminidase was measured as described previously (Bodinier et al., 2008). The spontaneous release was obtained using cells stimulated with the used buffers (without egg) and total specific release was obtained by activation of cells with human anti-IgE (Fc Region, Le27-NBS01, NBS-C Bioscience, #0908-0-050). The percentage of degranulation was expressed as the ratio between sample values and the specific IgE values, corrected by the spontaneous release.

## 2.9. *Statistics*

Non-linear curve fitting was used to assess statistical differences in EC50 and maximal degranulation capacity using GraphPad Prism 10.3.1. Differences were considered statistically significant when *p* < 0.05.

# 3. Results & discussion

## 3.1. *Egg white proteins gradually aggregate with increasing boiling times*

Our first objective was to produce egg white samples with a controlled and gradual increase in protein denaturation and aggregation of the main egg allergens. Based on literature describing the heating

times required to denature and aggregate OVA and OVM (Gu et al., 1986; Matsuda et al., 1981), we decided to boil eggs for 10 or 45 min and to compare these samples to unboiled, raw egg (Fig. 1A). Although it is uncommon to boil eggs for 45 min, this boiling time was intended to mimic the OVM aggregation observed in egg baked in the presence of wheat (gluten) (Kato et al., 2000; Kato et al., 2001; Shin, Han, & Ahn, 2013). The egg white was then manually separated from the egg yolk, and the boiled egg white was grinded mechanically into small pieces. Raw, 10 min or 45 min boiled egg whites were then combined with SSF, heated to 37 °C for 2 min, and centrifuged to obtain a soluble and an insoluble fraction. SSF was used for these analyses as i) this fluid models the solubilisation of proteins in the mouth following ingestion and ii) the analyses can subsequently be pursued to include the gastric and gastrointestinal phases (section 3.4).

We next evaluated the impact of boiling for 10 or 45 min on the molecular masses of the main egg white proteins in the soluble fraction using SDS-PAGE under non-reducing and reducing conditions (Fig. 1B). Under non-reducing conditions, we observed the gradual appearance of high-molecular weight species (> 100 kDa), and a gradual decrease of the main OVA band, ovotransferrin and lysozyme with increasing boiling times (Fig. 1B – left panel). These observations suggest a gradual aggregation of the main egg white proteins with increasing boiling times (Fig. 1B – left panel). This aggregation seemed to be principally driven by the formation of disulphide bonds, as the high molecular weight species mostly disappeared under reducing conditions (Fig. 1B – middle panel). Nonetheless, the presence of bands in the high-molecular weight range in the reduced egg white samples, and notably in the 45 min boiled egg white, suggested that other amino acid cross-links, such as dehydroproteins and lysinoalanine bonds are also formed. The insoluble fraction was analysed only under reducing conditions, due to use of a reducing buffer to resolubilise this fraction (Fig. 1B – right panel). Proteins in the insoluble fraction globally recapitulated the migratory pattern of the soluble fraction, with a gradual appearance of high-molecular weight bands that indicates progressive protein aggregation and modification (Fig. 1B – right panel).

Complementary to SDS-PAGE analyses, size-exclusion chromatography (SEC) was performed on the soluble protein fraction to capture the step-wise aggregation of the main proteins in egg white (Fig. 1C). In correspondence with the SDS-PAGE analyses, a gradual appearance of a peak in the first minutes of SEC was observed with increasing boiling times, indicating the gradual aggregation of the egg white proteins (Fig. 1C). Notably, an almost complete loss of native ovotransferrin (peak B) and OVA (peak C) upon 10 min of boiling could be observed (Fig. 1C). Since OVM and OVA have a similar molecular weight, OVM could not be clearly detected in raw egg white as it has a lower abundance.

## 3.2. *OVA and OVM behave differently upon egg white boiling*

To more specifically assess the behaviour of the main egg white allergens in the boiled egg white samples, we used Western blot and OVA and OVM-specific antibodies. Using SEC fractions collected from the main peaks (fractions A, B and C) (Fig. 1C), we observed that the presence of non-aggregated OVA in fraction C was gradually decreased upon 10 min and 45 min of boiling (Fig. 1D). Interestingly, non-aggregated OVM became visible in fraction C upon 10 min of boiling, suggesting that OVM moved towards the soluble phase upon 10 min of boiling (Fig. 1D). OVM re-disappeared from fraction C upon 45 min of boiling (Fig. 1D).

These results were largely confirmed when studying the soluble and insoluble phases of our different egg white samples. OVA disappeared from the soluble phase upon 10 min of boiling and appeared in greater quantity in the insoluble phase upon 10 min of boiling (Fig. 2A). The movement of OVM from the insoluble to the soluble phase upon 10 min of boiling was confirmed, as OVM was mainly found in the insoluble phase in raw egg white and in the soluble phase in 10 min boiled egg
A

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /></figure>

C

<figure><img src="image_7.png" /></figure>

D

<figure><img src="image_8.png" /><figcaption>**Fig. 1.** Egg white allergens gradually aggregate with increasing boiling times. A) Schematic overview of the egg white cooking protocol and associated analyses. Figure created with images from Servier Smart Art, NIAID BioArt, and Pixabay under CC-BY licences. B) SDS-PAGE of soluble and insoluble proteins in the simulated salivary fluid (SSF) following different times of egg white cooking (0, 10, 45 min). The soluble fractions are shown under non-reducing and reducing conditions, whereas the insoluble fractions are only shown under reducing conditions. C) Size-exclusion chromatography (SEC) of the soluble fractions of 0, 10 and 45 min boiled egg in SSF. The fractions that were collected for SDS-PAGE and Western blot analyses are shown with capital letters A, B and C. D) SDS-PAGE (upper panel) and Western blot (lower two panels) analyses of the SEC fractions that were collected in Fig. 1C under reducing conditions. A polyclonal anti-ovalbumin (OVA) and a monoclonal anti-ovomucoid (OVM) antibody were used to detect these specific proteins using Western blot. "M" signifies molecular weight ladder.</figcaption></figure>

white (Fig. 2A). No OVM was seen at 45 min of boiling in either the insoluble or soluble phases, which might be due to degradation or the fact that the anti-OVM antibody is a monoclonal antibody that does not recognize aggregated or heat-modified OVM. Together, our results show that OVA aggregates upon 10 min of egg boiling, whereas OVM solubilizes upon 10 min of boiling and only aggregates upon 45 min of boiling (see Table 1 for an overview). These results are in accordance with previous studies (Gu et al., 1986; Matsuda et al., 1981), which showed that prolonged boiling times (>30 min) are required to induce OVM aggregation. It should be noted, though, that the aggregation dynamics of OVA and OVM upon heating are dependent on multiple extrinsic factors, including temperature and pH (Gharbi & Labbafi, 2018). We deliberately chose here to used aged eggs from the supermarket (pH 9.15) and not freshly laid eggs (~pH 7.5), to be as close as possible to real egg consumption. However, this choice of aged eggs and not fresh eggs might have impacted the aggregation dynamics of OVA and OVM (Gharbi & Labbafi, 2018; Silversides & Scott, 2001).

### 3.3. Heat-induced structural modifications lower the IgE immunoreactivity and allergenicity of egg white

We next determined the impact of these different structural modifications on egg white IgE immunoreactivity and allergenicity using complementary immunochemical methods; Western blot, dot blot, and humanized rat basophil leukemia cells (RBL SX-38). Using pooled sera from egg-allergic patients and Western blotting (Supplemental Table 1), we observed that the IgE immunoreactivity was modestly reduced in 10 min boiled egg white compared to raw egg, in both the soluble and insoluble phases (Fig. 2A-right panels). A much stronger reduction in IgE immunoreactivity was seen for 45 min boiled egg white (Fig. 2A-right panels). As Western blotting includes an SDS-PAGE under reducing conditions, Western blotting does not always capture the full impact of heat aggregation on protein immunoreactivity. For this reason, we conducted dot blot analyses in parallel, which consist of spotting proteins directly onto nitrocellulose membranes, without
<figure><img src="image_9.png" /><figcaption>**Fig. 2.** Structural behaviour of ovomucoid (OVM) is the main determinant of IgE reactivity in the soluble phase. A) Western blot analyses after SDS-PAGE using reducing conditions of the soluble (upper panels) and insoluble fractions (lower panels) of 0, 10 and 45 min boiled egg white in simulated salivary fluid (SSF), using multiple antibodies (anti-egg white, anti-ovalbumin (OVA), anti-OVM and a pool of sera of egg-allergic patients. Ponceau red panels show loading of the different fractions. B) Dot blot analyses of the soluble (upper panels) and insoluble fractions (lower panels) of 0, 10 and 45 min boiled egg white in simulated salivary fluid (SSF), using different antibodies (anti-egg white, anti-OVA, anti-OVM and a pool of sera of egg-allergic patients. C) Degranulation percentages of rat basophil leukemia (RBL) cells that were stimulated with increasing doses of the soluble SSF fractions of 0, 10 and 45 min boiled egg white. Statistical information indicates differences in EC50 values of the non-linear fits between curves. D) Overview of the cross-link and loop-link proteomics data of disulphide bonds of OVM in 0, 10 and 45 min boiled egg white. E) Localization of native, cross-link and loop-link disulphide bonds as annotated on the amino acid (AA) sequences of OVM in 45 min boiled egg white. Locations of native disulphide bonds, cross-links and loop-links are indicated using coloured stripes. The parts of the OVM AA sequences that contain known epitopic regions are coloured in yellow. Visualization was done using XiNet (Combe et al., 2015). \* = p-value between 0.01 and 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<table><caption>**Table 1**

Overview of structural behaviour of ovalbumin (OVA) and ovomucoid (OVM) upon boiling. EW = egg white, nd = not determined.</caption><thead><tr><th rowspan="2">EW</th><th colspan="3">OVA</th><th colspan="3">OVM</th></tr><tr><th>raw</th><th>10 min</th><th>45 min</th><th>raw</th><th>10 min</th><th>45 min</th></tr></thead><tbody><tr><td>Soluble fraction</td><td>++</td><td>+</td><td>(+)</td><td>-</td><td>++</td><td>-</td></tr><tr><td>Insoluble fraction</td><td>+</td><td>++</td><td>++</td><td>++</td><td>-</td><td>-</td></tr><tr><td>Non-aggregated</td><td>++</td><td>+</td><td>-</td><td>+/-</td><td>++</td><td>-</td></tr><tr><td>Aggregated</td><td>-</td><td>+</td><td>++</td><td>nd</td><td>+</td><td>nd</td></tr></tbody></table>

denaturation or reduction. Interestingly, in contrast to the Western blots, these dots blots using patient sera showed that the immunoreactivity of the soluble fraction of 10 min boiled egg white was nearly identical to soluble raw egg white (Fig. 2B – right panels). A significant reduction in immunoreactivity of the soluble fraction was only observed upon 45 min of boiling (Fig. 2B – right panels).

Finally, to assess to what extent these modifications in egg white immunoreactivity translate to changes in egg white allergenicity, we tested the allergenicity of the soluble egg white samples using humanized rat basophils pre-treated with egg-allergic patient sera. These results show no significant reduction in allergenicity between raw egg white and 10 min boiled egg white, but a strong reduction in 45 min boiled egg white compared to raw and 10 min boiled egg (EC50 *p* = 0.01) (Fig. 2D).

To determine how the structural changes of OVM upon 45 min of boiling could explain the reductions in immunoreactivity and allergenicity, we used native cross-link HRMS to study the appearance of inter- and intramolecular cross-links in OVM. Cross-link HRMS is a promising technique that can provide information on protein interactions at the single amino acid residue level. However, it should be noted that HRMS tends to detect cross-links mainly for the most abundant proteins and that inter-protein cross-links are more difficult to detect than intra-protein cross-links (Lee & O'Reilly, 2023). Furthermore, because of the complexity of cross-linked peptide spectra, these spectra should be manually checked to validate the cross-link (Lee & O'Reilly, 2023). Cross-link HRMS on our samples showed a gradual appearance of disulphide cross-links and loop-links for OVM with increasing boiling times (Fig. 2D and Supplemental Table 2). Although there is no consensus on OVM epitopes (Leau et al., 2023), it is interesting to note that many of the detected cross-links and loop-links of OVM are found in or near known epitopic regions of OVM (epitopic regions coloured in yellow), when epitopic regions described in at least 2 articles were considered (Fig. 2E). Especially the epitopic regions ⁹⁰AFNPVCGTGDVTYDNE¹⁰⁵ and ¹¹⁴EQGASVDKRHDGGRKE¹³⁰ have a large number of cross-links and loop-links within their sequences or at close proximity (Fig. 2E, indicated as “hot spot”) (Besler et al., 1999; Cooke & Sampson, 1997; Holen et al., 2001; Järvinen et al., 2007; Leonard et al., 2010; Martínez-Botas et al., 2013; Mine & Zhang, 2002; Suprun et al., 2020). It can be hypothesized that the appearance of cross-links in these epitopic regions might hinder the binding of IgEs and might thus be responsible for the strong reduction in egg white immunoreactivity and allergenicity seen upon 45 min boiling (Gu et al., 1986). On top of this increase in cross-links upon heating, it is likely that the loss of OVM conformational epitopes mediates a significant part of the changes in IgE immunoreactivity, as the changes in IgE immunoreactivity between 10 min and 45 min boiled egg white are more pronounced when using dot blots (non-reduced, non-denatured) than when using Western blots (reduced, denatured). This is in accordance with a previous study that highlighted the importance of OVM conformational epitopes for egg allergy, although the locations of these conformational epitopes remain currently unknown (Zhang & Mine, 1998).

Taken together, despite a relatively modest patient sample size (n = 9), our results corroborate and add to previous studies showing that egg white boiling for 10 min moderately lowered or did not impact immunoreactivity (Bloom et al., 2014; Shin, Han, & Ahn, 2013; De Vlieger

et al., 2022), whereas only a prolonged boiling of 30 min lowered immunoreactivity (Shin, Han, & Ahn, 2013). We notably show that 10 min of egg boiling moderately lowers egg white allergenicity of the soluble egg white proteins, whereas 45 min of boiling more strongly impacts egg white allergenicity. This difference between 10 min and 45 min of boiling can, in part, be attributed to the aggregation of OVM upon 45 min of boiling; OVM is highly immunoreactive in the soluble fraction in 10 min boiled egg white and not reactive in 45 min boiled egg white, whereas OVA immunoreactivity is already diminished upon 10 min of boiling. The disruption of tertiary and secondary protein structures, and the breaking and formation of disulphide bonds upon boiling (Mine, 1995) might also directly render inaccessible linear epitopes or cause the destruction or masking of conformational epitopes, thereby reducing egg white allergenicity (Pi et al., 2024). These heat-induced structural changes impact the capacity of OVA and OVM to cross-link IgE-bound receptors and to cause basophil degranulation, as at least two accessible IgE epitopes are required for basophil degranulation (Dibbern et al., 2003). Future studies could further investigate the immunogenic potential of boiled egg white allergens by studying T-cell differentiation or by using animal models, or could investigate the possible generation of neoepitopes during heating.

### 3.4. Boiling increases gastric digestion but has limited impact on gastrointestinal digestion

Egg white is a food and thus subjected to gastro-intestinal digestion following consumption. Digestion kinetics can impact the nature and quantity of the epitopes of egg white allergens that might be absorbed and subsequently recognized by an allergic patients' immune system. For this reason, we aimed to determine how the different times of egg white boiling (raw, 10 min and 45 min) impact the digestibility of egg white and the main egg white allergens. To do so, we continued to apply the INFOGEST digestion protocol to our boiled egg white samples (Brodkorb et al., 2019), with the gastric (SGF; simulated gastric fluid) and gastro-intestinal (SIF; simulated intestinal fluid) phases. We focused specifically on the soluble phase obtained after gastric and gastro-intestinal digestion as the proteins in the soluble phase are likely to be the most capable of reaching and crossing the intestinal barrier.

Using SDS-PAGE, we observed that 10 min of boiling strongly increased the gastric digestibility (SGF) of the soluble egg white proteins, but only modestly increased gastro-intestinal digestibility (SIF) (Fig. 3A). Forty-five minutes of boiling further increased the gastric digestibility (SGF) of the soluble egg white proteins, but had no additional effect on gastro-intestinal digestibility (SIF) (Fig. 3A). These results were confirmed by RP-HPLC of the soluble gastric and gastro-intestinal phases. In the gastric phase, increasing boiling times gradually reduced the peak of full-length proteins, in favour of an increase in peptides (Fig. 3B). In the gastro-intestinal phase, 10 min and 45 min of boiling strongly reduced the peak of full-length proteins, but no additional impact of 45 min of boiling was observed, in correspondence with SDS-PAGE analyses (Fig. 3C).

We next looked more specifically at the behaviour of the main egg white allergens using Western blot. We observed that a significant amount of OVA remained after the gastric digestion (“SGF”) of raw egg white and 10 min boiled egg white (Fig. 3D). In contrast, no OVA was detected after the gastric digestion of 45 min boiled egg white (Fig. 3D). After gastro-intestinal digestion (“SIF”), only small amounts of OVA are detected in raw egg white, but not in 10 min or 45 min boiled egg white (Fig. 3D). In contrast, OVM is mainly detected in the oral phase and gastric phase (“SGF”) of 10 min boiled egg white, and only to a much lower extent in the gastric phase of raw egg and 45 min boiled egg. No OVM was detected following gastro-intestinal digestion (“SIF”) (Fig. 3D).

In addition to Western blotting, we used dot blots to assess the impact of boiling on the gastric and gastro-intestinal digestion of OVA and OVM. For OVA, these results largely recapitulated the Western blot
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 3.** Gastric digestion of the main egg white allergens is increased with increasing boiling times. A) SDS-PAGE of the soluble phase of 0, 10 and 45 min boiled egg white following salivary (SSF), gastric (SGF) and gastro-intestinal (SIF) digestion under reducing conditions. B) Reversed phase chromatography of the soluble fractions of gastric digested 0, 10 and 45 min boiled egg white. C) Reversed phase chromatography of the soluble fractions of gastro-intestinal digested 0, 10 and 45 min boiled egg white. D) Western blot analyses of the soluble fractions of 0, 10 and 45 min boiled egg white that had been subjected to salivary (SSF), gastric (SGF) and gastro-intestinal (SIF) digestion, under reducing conditions. Different antibodies (anti-egg white, anti-ovalbumin (OVA), anti-ovomucoid (OVM)) and a pool of sera of egg-allergic patients were used. E) Dot blot analyses of the soluble fractions of 0, 10 and 45 min boiled egg white that had been subjected to salivary (SSF), gastric (SGF) and gastro-intestinal (SIF) digestion. Different antibodies (anti-egg white, anti-OVA, anti-OVM) and a pool of sera of egg-allergic patients were used. F) Relative quantification of OVA and OVM in soluble fractions of 0, 10 and 45 min boiled egg white that had been subjected to gastric (SGF) digestion, as assessed using mass spectrometry.</figcaption></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>
results, with a strong reduction in OVA seen in gastric digests ("SGF") of 10 min boiled egg white, compared to raw egg (Fig. 3E). A near absence of signal in gastric digests of 45 min boiled egg white was observed (Fig. 3E). More OVM was found in the gastric digests of 10 min boiled egg than in raw egg, in contrast to the Western blot data (Fig. 3E). No OVM signal was observed in dot blots of gastric digests of 45 min boiled egg white (Fig. 3E). For the gastro-intestinal digests ("SIF"), no signal was observed except for raw egg white (Fig. 3E).

We then used HRMS to obtain a relative quantification of OVA and OVM in the soluble fractions of the gastric digests upon 10 and 45 min of boiling. These results largely recapitulated the data obtained used Western blot, showing a gradual reduction of OVA quantity with increasing boiling times and a larger amount of OVM in the gastric digest of 10 min boiled egg white (Fig. 3F). These results do also indicate that certain OVA and OVM peptides go undetected using Western blot, as OVA and OVM are clearly detected in the gastric digest of 45 min boiled egg white with HRMS, but not Western blot (Fig. 3D).

No studies had so far investigated the impact of egg white boiling on egg white allergen digestion within the egg white matrix. Our results show that an increase in egg boiling times gradually increases the gastric digestion of OVA, but not the gastro-intestinal digestion of OVA. The impact of egg boiling on the gastric digestion of OVM is less clear, with a reduction seen using Western blotting, but not dot blotting. The reason for this apparent discrepancy is not clear, but might be linked to the structural modifications of OVM during gastric digestion (low pH), which might diminish the recognition of OVM by our monoclonal antibody. A previous study did study the impact of gastro- and gastro-intestinal digestion on the main egg allergens, but only in raw egg (Martos et al., 2013). In line with our study, Martos et al. showed that OVA and OVM remain present after a gastric digestion. In contrast to our results, these authors also showed that both OVA and OVM remain after a gastro-intestinal digestion, but this is likely to be explained by the shorter duration of their gastro-intestinal digestion (60 min gastric digestion, 30 min intestinal digestion) (Martos et al., 2013) compared to ours (120 min gastric digestion, 120 min intestinal digestion). The impact of egg white boiling thus seemed to be more pronounced for gastric digestion than for gastro-intestinal digestion. This may in part be due to the highly effective gastro-intestinal digestion of unboiled OVM and OVA, making it difficult to observe an additional effect of boiling. The usage of shorter gastric or gastro-intestinal digestion times, or another type of *in vitro* digestion protocol using different pH and enzyme concentrations could be of interest to further study the impact of boiling on egg white gastro-intestinal digestion. Especially, the usage of an *in vitro* infant digestion protocol could be particularly relevant in the case of egg allergy (Ménard et al., 2018).

### 3.5. Changes in gastric digestion impact the IgE immunoreactivity and allergenicity of raw and boiled egg white

We next determined the impact of gastric and gastro-intestinal digestion on egg white IgE immunoreactivity and allergenicity. Using sera from egg-allergic patients and Western blotting (Supplemental Table 1), we show that IgE immunoreactivity was modestly reduced upon the gastric digestion of raw egg, with the disappearance of a smaller form of OVA of around 40 kDa (Fig. 3D). This smaller form might be a hydrolysed form of OVA (~40.1 kDa) that was previously shown to appear rapidly following the gastric digestion of OVA (Martos et al., 2010). Interestingly, this form is recognized in non-digested raw egg white and boiled egg white, but not in gastric digested egg white despite its apparent presence on the SDS-PAGE (Figs. 3A & 3D). In contrast to raw egg, the IgE immunoreactivity of 10 min boiled egg was more strongly impacted by gastric digestion, whereas no IgE immunoreactivity was detected in the gastric digests of 45 min boiled egg (Fig. 3D). No IgE immunoreactivity was detected in gastro-intestinal digests of raw, 10 min boiled or 45 min boiled egg white using Western blot (Fig. 3D). These results are largely corroborated by dot blotting,

with an absence in IgE immunoreactivity in gastric digests of 45 min boiled egg white and gastro-intestinal digests. However, contrary to Western blot, the IgE immunoreactivity of the gastric digests of 10 min boiled egg white was higher compared to raw egg white (Fig. 3E). These results are similar to the results obtained for OVM using Western blotting and dot blotting (Fig. 3D and Fig. 3E), suggesting that the observed differences are related to a different IgE recognition of OVM with dot blotting and Western blotting. We believe that this could be related to the recognition of OVM conformational epitopes by patient sera when using dot blotting, but not Western blotting.

On top of these changes in immunoreactivity, we hypothesized that the nature of available epitopes could also be affected by boiling and boiling time. We therefore performed HMRS analyses on the gastric and gastro-intestinal digests. These results showed that the quantity and nature of the generated peptides are indeed altered by egg white boiling, as shown by the coverage of the OVA and OVM sequences with the peptides detected using HRMS (Fig. 4A, Supplemental Fig. 1). For OVA, a strong reduction of the highly immunoreactive C-terminal epitope ³⁷⁰HIATNAVLFFGRVSP³⁸⁴ is seen in boiled egg white samples (Fig. 4A, Supplemental Fig. 1) (Benedé et al., 2014). For OVM, a large number of peptides corresponding to epitopes in the N-terminal part of OVM are detected in gastric digests of 10 min boiled egg white samples, but only little peptides from other epitopes are found in the boiled samples (Fig. 4A, Supplemental Fig. 1) (Besler et al., 1999; Cooke & Sampson, 1997; Holen et al., 2001; Järvinen et al., 2007; Leonard et al., 2010; Martínez-Botas et al., 2013; Mine & Zhang, 2002; Suprun et al., 2020).

Lastly, to evaluate the impact of boiling on the allergenicity of gastric digests, we again used humanized RBL and a pool of egg-allergic patient sera. Gastro-intestinal digests could not be tested due to cellular toxicity of these digests. These data show that the EC50 of degranulation by raw egg white was not affected by gastric digestion, but that the maximal degranulation capacity was significantly reduced (Fig. 4B). In contrast, the maximal degranulation capacity was not affected by the gastric digestion of 10 min boiled egg white, but the EC50 significantly increased (Fig. 4B). Lastly, both maximal degranulation capacity and to a lesser extent EC50 were impacted by the gastric digestion of 45 min boiled egg white (Fig. 4B). Together, these results suggest that boiling gradually lowers the allergenicity of egg white and that the impact of boiling is further accentuated when a gastric digestion is applied.

As briefly noted above, differences can be observed between the results of Western blot, dot blot and RBL. These differences can be explained by a different recognition of IgE epitopes in the different models. As previously mentioned, Western blotting requires a denaturing and sometimes a reducing step, whereas dot blotting does consider protein conformation. These differences might impact the accessibility and recognition of IgE epitopes. To induce degranulation in RBL cells, cross-linking of two IgE molecules bound to the FcεRI receptor is necessary, requiring the presence of two epitopes, instead of one for Dot and Western blotting (Dibbern et al., 2003).

Taken together, our results show that increasing the boiling time of egg white gradually increases the gastric digestibility, which lowers its immunoreactivity and allergenicity. Boiling strongly affects protein structure and these changes impact the digestibility and the nature of the peptides released following gastric digestion. These results thus provide a detailed molecular explanation for the observation that cooked or extensively heated egg is tolerated by a significant proportion of children allergic to egg (Bartnikas et al., 2013; Lemon-Mulé et al., 2008; Martos et al., 2013). The observation that increasing the boiling time lowers egg white allergenicity and also gradually increases digestibility, suggests that a gradual relationship exists between egg white boiling time and egg white allergenicity (see Table 2 for an overview). This notion can possibly be exploited to improve the treatment of egg-allergic patients, for example in egg ladders. Egg ladders are tools used to progressively introduce increasingly allergenic forms of egg at home, in order to accelerate oral tolerance development in egg-allergic children (Chomyn et al., 2021; Gallagher et al., 2024; De Vlieger et al., 2022).
<figure><img src="image_16.png" /><figcaption>**Fig. 4.** Gastric digestion lowers the allergenicity of 10 or 45 min boiled egg white, but not raw egg white. A) Relative quantity, intensity and localization of peptides of ovalbumin (OVA) and ovomucoid (OVM) detected in samples of 0, 10 and 45 min boiled egg white that had been subjected to gastric (SGF) digestion. AA sequences of OVA and OVM are depicted horizontally, with known epitopic regions highlighted in yellow, while quantity and intensity are depicted by the length of stripes and green colour, respectively. B) EC50 (top) and maximal degranulation percentages of rat basophil leukemia (RBL) cells that were stimulated with increasing doses of 0, 10 and 45 min boiled egg white that had been subjected to salivary (SSF) or gastric (SGF) digestion. Statistical information indicates differences in EC50 and Top of non-linear curve fitting analyses. C) Schematic overview of changes in OVA, OVM and egg white allergenicity upon increasing egg white boiling times. Figure created with images from Servier Smart Art, NIAID BioArt, and Pixabay under CC-BY licences * = p-value between 0.01 and 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

**Table 2**

Overview of changes in immunoreactivity (IR) and allergenicity upon the boiling of egg white (EW). SSF = simulated salivary fluid; SGF = simulated gastric fluid; SIF = simulated intestinal fluid, nd = not determined.

<table><thead><tr><th rowspan="2">EW</th><th colspan="3">Patient sera pool</th><th colspan="3">10 min</th><th colspan="3">45 min</th></tr><tr><th>raw</th><th>raw</th><th>raw</th><th>raw</th><th>raw</th><th>raw</th><th>raw</th><th>raw</th><th>raw</th></tr></thead><tbody><tr><td>IR soluble fraction</td><td>++</td><td>++</td><td>+/-</td><td>++</td><td>++</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>IR insoluble fraction</td><td>++</td><td>nd</td><td>nd</td><td>++</td><td>nd</td><td>nd</td><td>++</td><td>nd</td><td>nd</td></tr><tr><td>Allergenicity</td><td>++</td><td>++</td><td>nd</td><td>+</td><td>+</td><td>nd</td><td>+/-</td><td>-</td><td>nd</td></tr></tbody></table>

Often, these egg ladders consist of subsequent steps going from extensively baked to well-cooked, lightly cooked and almost raw eggs. In extensively baked eggs, egg is often found in the presence of wheat (muffins, for example). It has previously been shown that the presence of wheat renders OVM insoluble due to the formation of high-molecular weight complexes with gluten (Kato et al., 2000; Kato et al., 2001; Shin, Lee, et al., 2013). It is likely that this wheat-induced protein complex formation has a similar effect on egg white allergenicity as the OVM aggregation caused by prolonged boiling. We thus believe that a gradual reduction in egg boiling times (e.g. 45, 30, 15, 10 to 5 min) could

perhaps provide a more standardized manner to re-introduce eggs in egg-allergic patients and thereby limit adverse effects (Chomyn et al., 2021; Gallagher et al., 2024; De Vlieger et al., 2022). To test this hypothesis, further studies at the clinical level should be conducted. Also, on a more molecular level, further studies should formally confirm that a gradual reduction in allergenicity occurs with increasing egg boiling times, as the present study only used 10 and 45 min boiled egg white, and we cannot formally exclude a more step-wise reduction. Lastly, it would be of interest to extend this work and to study the impact of other heating methods such as sautéing, baking and steaming on the
allergenicity and digestibility of egg white proteins.

## 4. Conclusion

In conclusion, in this work we provide a detailed molecular view of the structural changes of the egg allergens OVA and OVM induced by boiling within an egg white matrix. Our results show that short egg white boiling (10 min) modestly lowers egg white immunoreactivity and allergenicity, but that prolonged boiling (45 min) is necessary to achieve a significant reduction. This prolonged boiling is needed to induce OVM aggregation, which impacts the recognition of both OVM conformational and linear epitopes by patient IgE. We show that within the egg white matrix OVM is resistant to aggregation with shorter boiling times and even solubilises better in 10 min boiled egg white than in raw egg white. Despite the need for prolonged boiling to strongly lower egg white allergenicity, shorter boiling times do increase the gastric digestibility of egg white. This increased digestibility, together with a reduction in soluble protein content following boiling, supports a gradual relationship between egg white boiling and egg white allergenicity. This gradual relationship can be exploited for therapeutic purposes, such as egg ladders, where increasingly allergenic forms of egg are given to egg-allergic patients to induce tolerance towards egg white allergens.

## Author contribution

Conceptualization and methodology: M.C., H.R. and W.D.; Formal analysis and investigation: M.C., E.L.C., A.A.S., E.P., V.S-J, H.R., W.D.; Data curation: M.C., W.D.; Writing, original draft preparation: W.D. and H.R.; Writing-review and editing.; all; Supervision: H.R., W.D., S.D-P; Funding acquisition: H.R. and W.D. All authors have read and agreed to the published version of the manuscript.

## CRediT authorship contribution statement

**Mehdi Cherkaoui:** Writing – review & editing, Visualization, Methodology, Formal analysis, Data curation, Conceptualization. **Elysa Le Corre:** Writing – review & editing, Investigation, Formal analysis. **Aché Ahmat-Sougoudi:** Writing – review & editing, Investigation, Formal analysis. **Emilie Perrin:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Véronique Solé-Jamault:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Hanitra Rabesona:** Methodology, Investigation, Formal analysis. **Sandra Denery-Papini:** Writing – review & editing, Validation, Supervision. **Martine Morisset:** Writing – review & editing, Validation, Resources. **Hélène Rogniaux:** Writing – original draft, Supervision, Funding acquisition, Conceptualization. **Wieneke Dijk:** Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Funding

This work was supported by INRAE (SpectALL project) and the Pays de la Loire region (PULSAR).

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

The authors kindly thank the biological resource center (CRB) of Angers University Hospital (BB-0033-00038) for conditioning, storage

and delivery of the sera we have studied. We would also like to express our gratitude to all the patients who offered their serum for the benefit of research. Lastly, we would like to thank Marie Bodinier for scientific discussions and input.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2025.144304.

## Data availability

Data will be made available on request.

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