# Weakening of hen egg vitelline membrane during egg storage at room temperature is associated with modifications of its mass, thickness and proteomic profile

Océane Hervé, Magali Chessé, Daniel Tomas, Valérie Labas, Justine Jimenez, Nelly Bernardet, Sonia Georgeault, Yves Nys, Sophie Réhault-Godbert, Thierry Moreau, et al.

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Océane Hervé, Magali Chessé, Daniel Tomas, Valérie Labas, Justine Jimenez, et al. Weakening of hen egg vitelline membrane during egg storage at room temperature is associated with modifications of its mass, thickness and proteomic profile. Food Research International, 2026, 228, pp.118291. 10.1016/j.foodres.2025.118291. hal-05457779

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# Weakening of hen egg vitelline membrane during egg storage at room temperature is associated with modifications of its mass, thickness and proteomic profile

Océane Hervé<sup>a</sup>, Magali Chessé<sup>a</sup>, Daniel Tomas<sup>b,c</sup>, Valérie Labas<sup>b,c</sup>, Justine Jimenez<sup>a</sup>, Nelly Bernardet<sup>a</sup>, Sonia Georgeault<sup>d</sup>, Yves Nys<sup>a</sup>, Sophie Réhault-Godbert<sup>a</sup>, Thierry Moreau<sup>a</sup>, Nicolas Guyot<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

- Chicken egg

- Vitelline membrane

- Storage

- Egg quality

- Egg proteins

- Electron microscopy

- Mass spectrometry

## ABSTRACT

The hen egg vitelline membrane is a protein layer surrounding the egg yolk. The integrity of this membrane is important for both the shell egg and the egg products industries; however, its physical properties are altered during prolonged storage, depending on storage temperature. This study aimed to identify the structural and molecular changes of the hen egg vitelline membrane during a 28-day storage at +4 °C and +20 °C using a combination of physicochemical, ultrastructural and proteomic approaches. Our results demonstrated that the degradation of the vitelline membrane during storage at +20 °C was associated with a significant loss of dry matter, a decrease in the thickness of the outer vitelline membrane layer, and alterations in its proteome. Indeed, the abundance of two main components of the outer layer, namely ovomucin and VMO1, decreased during storage at room temperature. VMO1 actually dissociates from the vitelline membrane during egg storage and accumulates in the egg white. Taken together, these results contribute to a better understanding of the mechanisms of alterations of the vitelline membrane during the storage of eggs at room temperature, and provide evidence that VMO1 is a major biomarker of the integrity of the vitelline membrane.

## 1. Introduction

The chicken egg vitelline membrane (VM), also known as yolk membrane or perivitelline layer, is a thin acellular protein layer separating the egg albumen from the yolk content. In fertilized eggs, this egg structure plays critical roles in avian reproduction, being involved in fertilization, development and antimicrobial protection of the avian embryo. In table eggs (unfertilized eggs), the natural physical and antimicrobial properties of the VM can prevent penetration of the yolk by contaminating bacteria potentially present in egg white or on the external surface of the VM. Its physical characteristics are also essential in the egg product manufacturing process to avoid or limit mixing of albumen and yolk during the separation process. VM integrity is

therefore a major parameter that ensures both the hygienic quality and technological properties of eggs.

VM is a complex protein structure with a thickness of about 11 µm, consisting of two distinct layers of protein fibers organized into a network and separated by a very thin ‘continuous membrane’ (Bellairs et al., 1963). Hundreds of proteins have been identified in VM by mass spectrometry (Bregeon et al., 2022; Mann, 2008; Zhou et al., 2020).

The inner vitelline membrane (IVM), in contact with the plasma membrane of the oocyte (i.e. the egg yolk), has an average thickness of 2.7 µm (1.0–3.5 µm) and consists of an entangled network of fibers ranging from 200 to 600 nm in diameter (Bellairs et al., 1963). The major proteins composing IVM are glycoproteins named zona pellucida proteins (ZP), including ZP1, ZP3 and ZPD (Bregeon et al., 2022;

Abbreviations: DAPs, differentially-abundant proteins; VM, vitelline membrane; IVM, inner vitelline membrane; OVM, outer vitelline membrane; VMO1, vitelline membrane outer layer protein 1.

* Corresponding author.

E-mail address: nicolas.guyot@inrae.fr (N. Guyot).

<footnote><sup>a</sup> INRAE, Université de Tours, BOA, Nouzilly, France</footnote>

<footnote><sup>b</sup> INRAE, CNRS, Université de Tours, PRC, Nouzilly, France</footnote>

<footnote><sup>c</sup> PIXANIM, INRAE, Université de Tours, CHRU de Tours, Nouzilly 37380, France</footnote>

<footnote><sup>d</sup> Plateforme IBiSA des Microscopies, Université de Tours, CHRU de Tours, Tours, France</footnote>
Okumura et al., 2004; Takeuchi et al., 1999). These proteins presumably correspond to the GP glycoproteins (GP-I, GP-II, GP-III, GP-IV) initially identified in the IVM by Kido and colleagues (Kido et al., 1975, 1976, 1977; Kido & Doi, 1988). ZP proteins are involved in the primary structure of the inner perivitelline layer. ZP1 and ZP3 proteins are linked to each other to form protein fibrils that are assembled into fibers via covalent (intermolecular disulfide bridges) and non-covalent interactions, while ZPD forms homopolymers and binds to ZP1-ZP3 fibrils to form aggregates (Okumura et al., 2015). This IVM corresponds to the zona pellucida of mammalian oocytes and, as such, is involved in the fertilization process. In addition to this specific role, IVM is also implicated in early embryonic development (Bregeon et al., 2022).

Facing the egg albumen, the outer vitelline membrane (OVM) has a thickness of 3.0–8.5 µm and consists of a varying number of superimposed sublayers composed of thin fibrils joined by swellings (Bellairs et al., 1963). The most abundant proteins of OVM include ovomucin, lysozyme, vitelline membrane outer layer protein 1 (VMO1) and vitelline membrane outer layer protein 2 (VMO2, now known as avian beta-defensin 11 or AvBD11) (Kido et al., 1992; Kido & Doi, 1988). The latter three molecules are globular proteins thought to bind ovomucin fibrils via electrostatic interactions, as previously shown for lysozyme (De Boeck & Stockx, 1986). OVM has functions related to immunity due to its high content of antimicrobial proteins and mucins, and plays roles in embryogenesis (Bregeon et al., 2022).

Both IVM and OVM contribute to the physical strength of the whole VM (Kido & Doi, 1988). Its physical and mechanical properties, assessed by measuring the yolk index, the VM breaking strength or the VM deformation, are optimal on the day of lay and can be strongly influenced by the duration and the temperature of egg storage (Chen et al., 2005; Feeney et al., 1956; Fromm & Matrone, 1962; Jones et al., 2018). As time and temperature increase, these quality parameters deteriorate more quickly. VM weakening likely results from both structural and biochemical changes occurring in the VM during egg storage. Indeed, a dissipation of fibers on the surface of VM could be observed in eggs conserved for one week at 25 °C (Kirunda & McKee, 2000) or for three days at 35 °C (Fromm, 1967). Recent studies revealed numerous quantitative changes in the proteome (Zhou et al., 2020) and N-glycoproteome (Zhou et al., 2021) of VM after 10 days of storage at 30 °C, a high-temperature condition reflecting egg storage environments in some hot climate developing countries. VM weakening might arise from a decrease in the levels of certain IVM or OVM proteins, as previously observed for GP-II after 25 days at 30 °C (Kido et al., 1975, 1977), or for VMO1 and AvBD11 in eggs stored at 20 °C (Back, 1984; Schäfer et al., 1998). However, the fate of these proteins, namely whether they are degraded or whether they dissociate from the VM during egg storage, remains uncertain to date. Given the differences in storage conditions used in all these studies, in terms of time and temperature, it is difficult to obtain an accurate overview of the structural and biochemical changes that occur in VM during egg storage. Furthermore, no proteomic analyses of the VM were performed on eggs stored under typical refrigerated or room temperature conditions, as the only available proteomic data concern eggs stored at high temperature (30 °C).

In the EU, although there are no requirements regarding time and temperature conditions during egg storage and transport, the date of minimum durability for hen eggs and the maximum time of delivery to consumers are set at 28 days after laying (Commission Delegated Regulation (EU) 2022/2258). In addition, consumer practices in terms of egg storage are very variable; some store eggs in the fridge or in cooled environments (cellars), while others keep eggs at room temperature somewhere in the kitchen. Poor egg storage can compromise VM integrity and strength, causing the yolk to weaken and appear visually unattractive upon breaking. These VM alterations can be accompanied by deterioration in both the hygienic quality and technological properties of eggs. Beyond its importance for shell egg consumption, VM deterioration is a major concern for the egg product industry, as it can result in both technological and economic issues. Efficient separation of

yolk and white with automated egg-breaking machines relies on yolk integrity; however, when the VM ruptures during this process, product quality can be compromised due to cross-contamination between the two egg compartments. Previous studies have shown that even trace amounts of yolk contamination (as low as 0.022 %) can significantly impair the foaming properties of egg white (Wang & Wang, 2009). Therefore, investigating the mechanisms underlying VM deterioration during egg storage is of significant interest for both the shell egg and egg products industries.

A deeper understanding of the mechanisms responsible for the VM deterioration during storage at refrigerated or room temperature could enable the egg and egg product industry to develop strategies to optimize or reinforce the quality of eggs destined for consumers and egg breaking facilities. In this context, our study aimed to characterize and integrate the physical and biochemical changes during 28-day storage at 4 °C and 20 °C, to reveal potential quality biomarkers and proteins involved in the structural and mechanical properties of VM, and to explore the mechanisms of proteome modification. For this purpose, a combination of various approaches and techniques was used to investigate storage-induced modifications of VM physical and mechanical properties, ultrastructure, and protein composition.

## 2. Materials and methods

### 2.1. Egg production and storage

Eggs were produced from cage-free 71–73-week-old laying hens (Lohmann Tradition) reared on the floor with no outdoor access, and fed a commercial layer diet (Sanders Ouest, Etrelles, France). All animals were raised in experimental facilities (INRAE, PEAT, Nouzilly, France, authorization # D37-175-1) in compliance with French decree no. 2013-118 of February 1, 2013 and EU directive no. 2010/63 relating to the protection of animals used for scientific purposes, and with the ethical guidelines of the French National Research Institute for Agriculture, Food and Environment (INRAE). The animal handling reported in the protocol fell below the minimum threshold required for ethical consideration by the French and European animal experimentation regulations. Eggs were collected over nine days and candled. Eggs with eggshell cracks were eliminated. Normal eggs were either directly used on the day of lay (D0) or stored on plastic egg trays for 7, 14 or 28 days (D7, D14, D28, respectively) in climate chambers (ICH260, Memmert, Schwabach, Germany) at +4 °C (75–85 % relative humidity) or +20 °C (50 % relative humidity). All eggs used in the study were weighed on the day of lay, before egg storage. Groups of eggs with similar average initial masses were allocated to each storage condition (Fig. S1) to ensure balanced distribution and minimize potential bias related to variations in initial egg quality.

### 2.2. Egg quality measurements

Egg quality parameters including yolk index, yolk mass, and VM deformation and breaking strength, were measured on 40 freshly-laid eggs and 120 stored eggs ($n = 20$ for each time × temperature condition). Yolk index was measured using a digital egg tester (DET6000, Nabel, Japan). The yolks, separated from egg white with an egg separator, were then carefully placed in a disposable plastic container (Ref. 045105, 89 × 89 × 25.4 mm, Dutscher, Bernolsheim, France) and weighed. VM deformation and breaking strength were measured on these isolated yolks using a universal testing machine (Model 5543, Instron, Elancourt, France) equipped with a 50 N force transducer and a 6 cm diameter flat circular sensor. The displacement speed was set at 10 mm/min. This procedure for the determination of VM deformation and breaking strength was adapted from Woodward and colleagues (Woodward et al., 1987). The point of contact between the probe and the yolk was located within the equatorial area, while the poles where chalazae are anchored were avoided. Due to the destructive effect of the
method, only a single measurement per egg was recorded.

## 2.3. VM sampling and solubilization

Freshly-laid eggs (n = 48) and stored eggs (n = 24 for each time × temperature condition) were broken and yolks were separated from egg white using an egg separator. Chalazae were removed and yolks were weighed and placed on a flat surface to measure yolk index using a digital caliper (Mitutoyo 500–162-30, Otelo, Saint-Ouen-l'Aumône, France). Then, yolks were rolled on a paper towel to remove adhering egg white, and immersed into refrigerated water. VM was incised with scissors and carefully removed from the vitellus using pliers, as previously published (Bregeon et al., 2021). VM was vigorously washed three times in demineralized water to remove adherent yolk, and spread on a blotting paper (Whatman Gel Blot Paper GB003, Sigma-Aldrich, Saint-Quentin-Fallavier, France) to remove any remaining gelatinous egg white and chalazae. Individual VM samples were placed in microtubes, frozen in liquid nitrogen, and freeze-dried. Each dried sample was weighed and entirely solubilized in 1 mL of 63 mM Tris-HCl pH 6.8, 2 % (w/v) SDS, 2.5 % (v/v) β-mercaptoethanol for 10 min under vigorous shaking, and then for 24 h at room temperature under gentle agitation using a rotating wheel (Stuart rotator SB3, Dutscher, Bernolsheim, France) set to 40 rpm. Although VM samples appeared fully solubilized, a centrifugation was performed at 21000g for 10 min (room temperature) to remove traces of any potential insoluble material. Protein concentration was determined on samples diluted 10 times in high-purity water, using the Pierce Detergent Compatible Bradford Assay kit (Fisher Scientific, Illkirch, France) and bovine serum albumin as the standard. Final concentrations of SDS and β-mercaptoethanol in protein assays were within the compatible range specified by the manufacturer for these two chemicals. Samples were stored at -80 °C before SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis.

## 2.4. SDS-PAGE analysis

Pools of individual VM samples (n = 6 pools per condition, n = 4 individual VM protein samples per pool) were prepared, diluted at 1.5 µg/µL in reducing Laemmli buffer (final concentration in each sample adjusted to 63 mM Tris-HCl pH 6.8, 2 % (w/v) SDS, 10 % (v/v) glycerol, 0.01 % (w/v) bromophenol blue, 2.5 % (v/v) β-mercaptoethanol) and heated at 100 °C for 5 min. A volume of 10 µL (15 µg) per well was loaded on 4–20 % gradient SDS-PAGE gels (Mini PROTEAN TGX Gels, Bio-Rad, Marnes-la-Coquette, France). Migration was carried out for 40 min at 220 V in Mini PROTEAN Tetra Cell device (Bio-Rad). Gels were stained with Coomassie blue solution (40 % (v/v) ethanol, 10 % (v/v) acetic acid, 0.25 % (w/v) Coomassie brilliant blue R250) for 1 h, and destained in 40 % (v/v) ethanol, 10 % (v/v) acetic acid for approximately 4.5 h. Coomassie blue stained gels were scanned using an Odyssey CLx infrared imaging system (LICORbio GmbH, Bad Homburg, Germany).

## 2.5. VMO1 quantification by western blot

Fixed amounts of VM protein samples (500 ng/well, loaded twice on the same gel) and various amounts of purified chicken VMO1 (115, 100, 85, 70, 55, 40, 25, 10 ng/well) (Guyot et al., 2016) were heated at 100 °C for 5 min in reducing Laemmli buffer and separated by SDS-PAGE, as described above. After migration, proteins were transferred onto a nitrocellulose membrane (Amersham Protran premium 0.2 µm NC, GE Healthcare) in a transfer buffer (Pierce Western Blot Transfer Buffer, methanol-free, Thermo Fisher Scientific, Illkirch, France) using a Mini Trans Blot Cell device (Bio-Rad) for 1.5 h at 400 mA (two gels). The membrane was saturated with 5 % skimmed milk in PBS with 0.1 % (v/v) Tween 20 (PBS-T) for 1 h at room temperature, incubated with rabbit anti-chicken VMO1 antibodies (custom polyclonal antibodies obtained after rabbit immunization with native mature VMO1 protein purified

from hen eggs, Proteogenix, Schiltigheim, France) diluted 1:5000 in blocking buffer overnight at 4 °C with gentle agitation. After washing (3 × 10 min in PBS-T), the membrane was incubated with goat anti-rabbit IgG (Alexa Fluor Plus 680, Thermo Fisher Scientific) diluted 1:10000 in blocking buffer for 1 h at room temperature, washed (3 × 10 min in PBS-T), and scanned using an Odyssey CLx infrared imaging system (LICORbio GmbH). VMO1 bands in VM samples and in standards were quantified using Image Studio software v5.2 (LICORbio GmbH). The linear standard curve determined with VMO1 standards was used to determine VMO1 amounts in VM samples.

## 2.6. Mass spectrometry analysis

Mass spectrometry analysis was performed on protein samples prepared in reducing Laemmli buffer. Equal amounts of protein of the previously prepared pooled VM samples (25 µg/pool/well), or equal amounts of protein of egg white samples (n = 4–5 pools per condition, n = 4 individual samples per pool, 25 µg/pool/well), were loaded on 10 % SDS-polyacrylamide separating gel. Migration was carried out for 20 min at 50 V to include and concentrate proteins in the gel. Gels were stained for 5 min with 0.25 % (w/v) Coomassie brilliant blue R250 in 40 % (v/v) ethanol, 10 % (v/v) acetic acid, and destained for approximately 5 h. Each protein band (containing the whole protein sample) was cut into pieces of about 1 mm³ and stored in a 1.5 mL microtube at -20 °C until use.

Gel pieces were washed in water:acetonitrile solution (1:1, 5 min) followed by 100 % acetonitrile (10 min). Reduction and cysteine alkylation was performed by successive incubation with 10 mM dithiothreitol in 50 mM NH₄HCO₃ (30 min, 56 °C), then 55 mM iodoacetamide in 50 mM NH₄HCO₃ (20 min, room temperature, in dark). Pieces were then incubated with 50 mM NH₄HCO₃ and acetonitrile (1:1, 10 min) followed by acetonitrile (15 min). Proteolytic digestion was carried out overnight using 25 mM NH₄HCO₃ with 12.5 ng/µl trypsin (Sequencing grade, Roche diagnostics, Paris, France). For each sample (gel pieces containing 25 µg protein), 25 µL of the trypsin solution was used. Resulting peptides were extracted by incubation in 5 % formic acid (sonicated) with the supernatant removed and saved, followed by incubation in water:acetonitrile and 1 % formic acid (1:1, 10 min) and a final incubation with acetonitrile (5 min), again supernatant was removed and saved. These two peptide extractions were pooled and dried using a SPD1010 speedvac system (Thermosavant, Thermofisher Scientific, Bremen, Germany). The resulting peptide mixture was desalted and enriched with SPIN Columns C18 (Millipore) and dried again before being analyzed by nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS).

Peptide mixtures were analyzed by nanoLC-MS/MS, in triplicate, using a dual linear ion trap Fourier Transform Mass Spectrometer (FT-MS) LTQ Orbitrap Velos Pro (Thermo Fisher Scientific, Bremen, Germany) coupled to an Ultimate® 3000 RSLC Ultra High Pressure Liquid Chromatographer (Thermo Fisher Scientific, Bremen, Germany) controlled by Chromeleon Software (version 6.80 SR13). Samples were desalted and concentrated for 10 min at 5 µL/min on a trap column (Acclaim PepMap 100 C18, 75 µm inner diameter × 2 cm long, 3 µm particles, 100 Å pores). The peptide separation was conducted using a nano-column (Acclaim PepMap C18, 75 µm inner diameter × 50 cm long, 2 µm particles, 100 Å pores) at 300 nL/min by applying gradient of 2–45 % B during 120 min. Mobile phases consisted of (A) 0.1 % formic acid, 97.9 % water, 2 % acetonitrile (v/v/v) and (B) 0.1 % formic acid, 19.9 % water, 80 % acetonitrile (v/v/v).

Data were acquired using Xcalibur version 3.0.63 software (Thermo Fisher Scientific, San Jose, CA, USA), in positive data-dependent mode in the 300–1800 m/z mass range. Resolution in the Orbitrap was set at R = 60,000. The 20 most intense peptide ions with charge states ≥2 were sequentially isolated (isolation width 2 m/z, 1 microscan) and fragmented in the high pressure linear ion trap using CID (collision induced dissociation) mode (collision energy 35 %, activation time 10 ms, Qz
0.25). Dynamic exclusion was activated during 30 s with a repeat count of 1. The lock mass was enabled for accurate mass measurements. Pol-ydimethylcyclosiloxane (m/z, 445.1200025, (Si(CH₃)₂O)₆) ion was used for internal recalibration of the mass spectra MS/MS ion searches were performed using Mascot search engine version 2.7.0.1 (Matrix Science, London, UK) via Proteome Discoverer 2.5 software (ThermoFisher Scientific, Bremen, Germany) against NCBIr *Gallus gallus* protein database (2023/01). The sequence of the chicken beta-microseminoprotein-like protein (LOC101750704 or MSMB3, gene ID: 101750704, protein accession number: XP_004942176.1) was added manually for the search, since this egg protein is absent from *G. gallus* genome assemblies bGalGal1.mat.broiler.GRCg7b and bGalGal1.pat.whiteleghornlayer. GRCg7w (protein sequence: MKFLLVFCILFSRTLCDARCYFRTSSKYGCISNRNLYVFGAVWKTEDCYQCKCKMNAMVCCSLVSIP-KNYDRVNCVGLFHKKSCSIRVVKKTDPDISCKVYNGVG), although it was unambiguously identified in chicken egg, as demonstrated in previous studies (Bregeon et al., 2022; Coste et al., 2021; Guyot et al., 2016). The search parameters included trypsin as a protease with two allowed missed cleavages and carbamidomethylcysteine, methionine oxidation and acetylation of N-term protein as variable modifications. The tolerance of the ions was set to 5 ppm for parent and 0.8 Da for fragment ion matches. Mascot results obtained from the target and decoy databases searches were subjected to Scaffold Q + S v5.2.2 and Scaffold Quant v5.0.3 software (Proteome Software, Portland, OR, USA) using the protein cluster analysis option (assemblage of proteins into clusters based on shared peptide evidence). Peptide and protein identifications were validated using the Peptide Prophet and Protein Prophet algorithms with a probability of 95 % for proteins and 95 % (egg white samples) or 99 % (VM samples) for peptides. Protein identifications were accepted if they contained at least two identified peptides.

The data were normalized and quantified using the eExtracted Ion Chromatogram (XIC) method in Scaffold Quant. The XIC or normalized Average Precursor Intensity, is based on the normalized intensity values of chromatographic peaks corresponding to protein's peptides. Coefficients of variation (CVs) calculated from "precursor intensity" data of VM samples were used as a quality-control metric to assess the repeatability of peptide analysis (including in-gel digestion and peptide extraction steps) at the technical replicate level and the reproducibility at the biological replicate level. For the 50 most abundant proteins (Top 50), technical mean CVs were around 15 % (range: 11–25 %) and biological mean CVs were 43.76 %, 41.39 %, 42.16 % for the conditions D0, D28+4 °C and D28+20 °C, respectively. At the study level, the CVs obtained on the Top 50 proteins revealed a mean variability of 95 %. Based on this variability, a fold-change threshold of 2 was applied for comparative analysis. The quantitative data were transformed using the logarithm (Log₁₀), and statistical analyses, including ANOVA and t-tests, were performed. A Fold Change (FC) was also calculated (Log₂ Fold Change of Precursor Intensity) between two conditions to determine the relative abundance level of each protein. The data were subsequently exported to Excel. The protein list was filtered based on the following criteria: *p* ≤ 0.05 and a fold change of 2 (Log₂(FC) ≥ 1 or ≤ −1) to identify differentially-abundant proteins (DAPs). Gene identifiers and gene symbols were manually retrieved from NCBI gene database and associated to each protein identified. A heatmap representation of Log₂(FC) of the precursor intensity of differential proteins with significant ANOVA (*p* ≤ 0.01) was done using Scaffold Quant.

## 2.7. Transmission electron microscopy (TEM) observations and thickness measurements

VMs were sampled from freshly-laid eggs (n = 8) or stored eggs (n = 4 eggs for each storage condition), as previously described. After washing in demineralized water, VMs were laid flat in a petri dish. Pieces of the membranes, cut away from the chalazae and the germinal disc regions, were sandwiched between two rings of absorbent paper which were then pinned to a cork disc and placed at the bottom of a 24-

well microplate. Samples were fixed by incubation for 24 h in 4 % paraformaldehyde, 1 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). They were then washed in phosphate-buffer and post-fixed by incubation with 2 % osmium tetroxide for 1 h. Samples were then fully dehydrated in a graded series of ethanol solutions and embedded in Epon resin, which was allowed to polymerize from 37 °C to 60 °C. Ultra-thin sections of these blocks were obtained with an ultramicrotome. Sections were stained with 5 % uranyl acetate, 5 % lead citrate, and observations were made with a transmission electron microscope (JEOL 1011, Tokyo, Japan). The thickness of VM, OVM and IVM was measured from TEM pictures, using ImageJ 1.54d software (NIH, Bethesda, MD, USA). For each picture, at least seven measurements were performed and averaged to give a mean value. This procedure was applied to four different pictures per egg. Hence, for each thickness type (VM, OVM, IVM), a total of 32 values for control eggs (fresh) and 16 values for each storage condition were obtained.

## 2.8. Statistical analyses

Statistical analysis of yolk mass, yolk index, VM deformation at rupture, VM breaking strength, VM dry mass, VM thickness, OVM thickness, IVM thickness, VMO1 precursor intensity and VMO1 relative quantity was performed using XLSTAT software (Addinsoft, Paris, France). Due to the low sample size of each experimental group (n < 30) for all these parameters, significant differences between groups were tested using a non-parametric Kruskal-Wallis test (alpha level of 0.05), followed by a Conover-Iman's test for multiple pairwise comparisons. Conover-Iman's test is more powerful than Dunn's test, as it reduces the risk of type II errors. The correlations between variables were analyzed using a Spearman correlation test. Sample size determination using an a priori power analysis (G*Power 3.1.9.7) and assuming a medium effect size (Cohen's d = 0.50), α = 0.05, and power = 0.80, indicated that n ≥ 20 per group were sufficient to detect significant differences of egg quality parameters among storage conditions.

# 3. Results and discussion

To study the structural and molecular changes responsible for VM alteration during egg storage, eggs were stored for up to 28 days at 4 °C or 20 °C, thereby mimicking egg conservation under refrigerated or room temperature conditions. Freshly-laid (unstored) and stored eggs used in this study were analyzed and compared to assess the effects of both duration and temperature on VM physical and biochemical characteristics. In addition to measurements of general egg and yolk characteristics, VMs collected from these eggs were investigated for their dry mass, ultrastructure and thickness, and protein content. Of note, VM quality (i.e. mechanical properties assessed using yolk index, yolk deformation and breaking strength), electron microscopy observation and biochemical composition (dry mass, proteomic analyses) could not be analyzed simultaneously on the same egg sample, so for this reason, three different batches of eggs laid by the same flock of laying hens and stored in the same conditions were used for these measurements. It should also be noted that all experimental groups corresponding to storage conditions contained eggs with similar average initial masses (on the day of lay) (Fig. S1A, S1B, S1C) to avoid any bias resulting from imbalanced egg distribution between groups. In addition, no statistical difference was observed in the average initial egg masses between the three batches (Fig. S1D).

### 3.1. Physical properties of yolk and VM during egg storage

As expected, the physical properties of egg yolk and its associated VM were influenced by the duration and/or the temperature of storage (Fig. 1). Compared with the day of lay, yolk mass increased in all stored eggs (Fig. 1A). Mean values increased by 4–5 % and 5–9 % at 4 °C and 20 °C, respectively, although no significant difference was observed
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<figure><img src="image_8.png" /><figcaption>**Fig. 1.** Changes in egg yolk and VM quality parameters during egg storage at 4 °C or 20 °C. (A) Yolk mass; (B) Yolk index; (C) VM deformation at rupture; (D) VM breaking strength; (E) VM deformation at rupture relative to yolk height (%); (F) VM breaking strength relative to yolk mass. Horizontal bars and crosses in the box plot represent medians and means, respectively. Conditions with different letters indicate statistically significant differences with *p* < 0.05 (Kruskal-Wallis test, Conover-Iman post hoc test).</figcaption></figure>

between the two temperatures. This increase in yolk mass is likely to result from the transfer of water from the egg white to the egg yolk, through the semipermeable VM (Fromm, 1966). This phenomenon is driven by the osmotic pressure difference between the two egg compartments since the concentration of solutes (particularly proteins and lipids) is higher in the yolk than in the egg white. The physical properties of VM were assessed using yolk index (yolk height/width ratio measured

on a flat surface), VM deformation and breaking strength. The use of yolk index as a measure of VM strength has been demonstrated in previous studies (Fromm, 1964; Fromm & Matrone, 1962; Kirunda & McKee, 2000). As shown in Fig. 1B, the yolk index remained relatively stable over time during storage at 4 °C (mean values of about 0.40) and was not significantly different from the day of lay at this temperature (except after 28 days of storage), while it significantly decreased by 9.8
%, 19.5 % and 31.7 % after 7, 14 and 28 days of storage, respectively, at 20 °C (mean values of 0.37, 0.33 and 0.28, respectively). VM deformation at yolk rupture was significantly impaired from 7 to 28 days of storage at both temperatures, but to a much greater extent at 20 °C (Fig. 1C). In parallel, VM breaking strength of eggs stored at 4 °C remained comparable to the day of lay, except after 28 days of storage where it was significantly lower compared to freshly-laid eggs (Fig. 1D). In contrast, storage of eggs at 20 °C has a dramatic impact on this parameter (Fig. 1D). Interestingly, VM mechanical properties at 4 °C were significantly altered as early as 7 days of storage, when these two parameters (deformation at rupture and breaking strength) were expressed relative to total yolk height and yolk mass, respectively, but the degradation rate still remained higher at 20 °C than at 4 °C (Fig. 1E and 1F). After 28 days of storage, the percentage of deformation at yolk rupture (i.e. percentage of the distance to rupture related to total yolk height) dropped by 12 % of its initial mean value at 4 °C, and by 29 % at 20 °C (Fig. 1E). During the same period, a decrease of 24.6 % and 58 % was observed for the VM breaking strength per gram of yolk at 4 °C and 20 °C, respectively (Fig. 1F). Yolk index, VM deformation and breaking strength are indicators of egg freshness and more precisely of the physical properties of VM. In this respect, significant correlations were observed between these three parameters during storage at 20 °C, varying from moderate (yolk index × VM breaking strength) to strong (yolk index × VM deformation, and VM deformation × VM breaking strength) (Table S1). Taken together, our data demonstrate that the VM physical properties weakened during egg storage, with a faster rate at 20 °C when compared to the refrigerated temperature. Our results are consistent with previous studies showing effects of storage time and temperature on the yolk index, VM strength and VM deformation at rupture (Kirunda & McKee, 2000; Wolk et al., 1952; Woodward et al., 1987; Yimenu et al., 2017). These results suggest that water transfer across the VM during egg storage may contribute to the deterioration of its mechanical properties, possibly through swelling or relaxation of the VM protein fiber network. Moreover, the VM is directly exposed to the egg white and its particular pH environment, which progressively increases during storage. This alkalinization process arises from the diffusion of carbon dioxide, initially dissolved in the egg white, through the eggshell pores. In our study, egg white pH was found to be 7.86 ± 0.14 (mean ± SD) at lay, and it increased up to 8.94 ± 0.11 at 4 °C, and 9.23 ± 0.12 at 20 °C, after 28 days of storage. Previous work indicated that egg white alkalinization, rather than water movement per se, is primarily responsible for the deterioration of VM physical properties

(Fromm, 1966). Therefore, it is hypothesized that the progressive rise in egg white pH during storage possibly alters protein-protein interactions within the VM, destabilizing its protein network and thereby reducing its mechanical strength.

## 3.2. Dry mass of VM in fresh and stored eggs

The chicken egg VM is mainly composed of water and proteins (Bellairs et al., 1963). Proteins are an integral part of VM structure and, as such, some alterations in these structural components may be responsible for VM weakening in stored eggs. Therefore, the evolution of the whole protein content of VM during egg storage was monitored by measuring its dry mass. In fresh (unstored) eggs, the average VM dry mass of eggs used in this study was 7.0 ± 0.1 mg (mean ± SD) (Fig. 2A). This value is near or within the range of values previously determined in eggs, even for different hen strains (Bellairs et al., 1963; Chung et al., 2010; Kido & Doi, 1988; Schäfer et al., 1998). As shown in Fig. 2A, VM dry mass remained relatively stable during storage at 4 °C, while it progressively decreased at 20 °C, losing approximately 13.2 % and 25 % of its initial dry mass after 14 and 28 days of storage, respectively. Since egg yolk mass slightly increased during egg storage, it made sense to standardize our data and express the results as a ratio of VM dry mass to whole yolk mass. In this case, a significant decrease of this parameter was observed not only at 20 °C but also at 4 °C after 28 days of storage, although the magnitude of alteration still remained higher at 20 °C (Fig. 2B). Previous studies also demonstrated that the VM mass decreased significantly as storage time and temperature increased (Fromm, 1964, 1967; Zhou et al., 2020). Interestingly, the determination of the correlation coefficients in Table S2 indicated that VM dry mass was positively and significantly correlated with yolk index, in both fresh and stored eggs. However, the highest statistical significance and correlation coefficient between these two parameters were found for eggs stored at 20 °C (Table S2). Positive correlations between VM mass and yolk index, and between VM mass and VM strength, were also observed previously in eggs stored at 25 °C (Fromm, 1964). Therefore, our data strongly suggest that the amount of matter composing the VM (i.e., proteins) is a driving factor for the mechanical properties of VM, and that VM alteration during storage results from a loss and/or a degradation of VM proteins, leading to a loss of the structural integrity of VM.

<figure><img src="image_9.png" /><figcaption>**Fig. 2.** Changes in the mass of VM dry matter during egg storage at 4 °C or 20 °C. (A) VM dry mass; (B) VM dry mass relative to yolk mass. Horizontal bars and crosses in the box plot represent medians and means, respectively. Conditions with different letters indicate statistically significant differences with *p* < 0.05 (Kruskal-Wallis test, Conover-Iman post hoc test).</figcaption></figure>
### 3.3. Ultrastructure and thickness of VM, OVM and IVM

Potential changes in the structural integrity of VM during egg storage were investigated by electron microscopy using TEM. As shown in Fig. 3A and by previous studies (Bain & Hall, 1969; Bellairs et al., 1963; Kido & Doi, 1988), VM is composed of two distinct superimposed layers, OVM and IVM, separated by a thin continuous membrane. OVM and IVM have been shown to contribute almost equally to the strength of the whole VM (Kido & Doi, 1988). In our study, the three structural components (i.e. OVM, IVM and the continuous membrane) initially observed in freshly-laid eggs remained detectable in stored eggs. TEM observations of VM cross-sections were used to determine the thickness of the whole VM and each sublayer (OVM and IVM). On the day of lay, the average thickness measured for VM, OVM and IVM was 11.8 µm, 8.3 µm, and 3.4 µm, respectively (Fig. 3B, 3C, 3D). These values are in the range of those determined previously (Bellairs et al., 1963). During storage at 20 °C, VM and OVM thickness significantly decreased to 7.8 µm and 4.5 µm, respectively, after 28 days (Fig. 3B and 3C) while no significant changes were observed for IVM (Fig. 3D, Kruskal-Wallis $p = 0.12$). Thus, VM lost about 34 % of the initial thickness, mainly via a decrease in the thickness of OVM (–46 %). In contrast, storage at 4 °C had no or a very weak effect on VM, OVM and IVM thicknesses (Fig. 3B, 3C, 3D). All these observations can be paralleled with the change in VM dry matter during egg storage (Fig. 2) and are consistent with other studies showing a dissipation of the fibrous layer at the outer surface of VM, demonstrated either by SEM in eggs stored one week at 25 °C (Kirunda & McKee, 2000), or by light microscopy in eggs stored for three days at 35 °C (Fromm, 1967).

<figure><img src="image_10.png" /></figure>

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

<figure><img src="image_12.png" /></figure>

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

**Fig. 3.** Changes in the thickness of whole VM and its inner and outer layers during egg storage at 4 °C or 20 °C. (A) TEM observation of egg VM in a freshly-laid eggs showing the outer layer (OVM), the continuous membrane (CM) and the inner layer (IVM); (B) VM thickness (OVM + IVM); (C) OVM thickness; (D) IVM thickness. Horizontal bars and crosses in the box plot represent medians and means, respectively. Conditions with different letters indicate statistically significant differences with $p < 0.05$ (Kruskal-Wallis test, Conover-Iman post hoc test).

### 3.4. Protein patterns of VM samples

Protein patterns of VM sampled from fresh eggs and 28-day-old eggs stored at 4 °C and 20 °C were then examined by SDS-PAGE to visualize molecular modifications (at the protein level) induced by egg storage. As illustrated in Fig. 4, VM contains at least four highly abundant proteins, including two OVM proteins, namely lysozyme C (≈14 kDa) and VMO1 (≈18 kDa) (Back et al., 1982), and two IVM proteins, ZP3 (≈34 kDa) and ZP1 (≈95 kDa) (Bausek et al., 2000). No major change in the pattern of corresponding protein bands was observed during egg storage (Fig. 4); however, for VMO1, band intensity appeared to be lower in stored eggs compared to fresh eggs. The apparent decrease in VMO1 band was visibly more pronounced at 20 °C than at 4 °C (Fig. 4). This observation is in agreement with previously published studies demonstrating a decrease in the abundance of VMO1 protein during storage at 20 °C for up to 30 days (Schäfer et al., 1998) or eight weeks (Back, 1984). Of note, a decrease in the intensity of two faint bands with high molecular weights (>150 kDa) and one faint band with a low molecular weight (<10 kDa) was also observed after 28 days at 20 °C (Fig. 4). The appearance of a faint band at approximately 28 kDa in eggs stored at 20 °C (Fig. 4) might correspond to the formation of a lysozyme dimer (2 × 14 kDa), previously described by Back (Back, 1984).

### 3.5. Proteome changes of VM during egg storage

A GeLC-MS/MS approach (in gel digestion followed by a nanoLC-MS/MS analysis) was conducted to identify proteins present in each VM sample and to detect VM proteins whose abundance varies during egg storage. The integration of the data from all samples allowed the identification of 191 non-redundant proteins classified in 116 clusters
<figure><img src="image_14.png" /><figcaption>**Fig. 4.** SDS-PAGE analysis of VM proteins from freshly-laid eggs (D0) and from eggs stored for 28 days at 4 °C (D28 +4°C) or 20 °C (D28 +20°C). Proteins (15 µg/lane, reducing conditions) were stained with Coomassie blue. The most abundant vitelline membrane proteins are identified by an arrowhead. The asterisk indicates the presumed lysozyme dimer (28 kDa) in 'D28 +20°C' condition.</figcaption></figure>

(Table S3). A total of 107 unique gene IDs were identified among these 191 proteins (Table S4), while no gene IDs were retrieved for some protein accession numbers (33 protein sequences), all corresponding to immunoglobulins (Table S3). Among the 107 proteins with non-redundant gene IDs listed in Table S4, 30 proteins showed statistical differences among conditions ('D0', 'D28+4°C', 'D28+20°C') in terms of relative protein abundance (ANOVA test on Log₁₀ precursor intensity with significance at *p* ≤ 0.01, Table S4). Variations of their relative abundance within samples and between conditions are illustrated in Fig. 5. Strikingly, the relative abundance of most of these differential proteins decreased during egg storage. In agreement with SDS-PAGE observations (Fig. 4), the most abundant VM protein affected by storage conditions was the vitelline membrane outer layer protein 1 (VMO1), followed by ovalbumin-related protein X (OVALX), alpha-ovomucin (OVOA), alpha-2-macroglobulin-like protein 1 (A2ML1), pleiotrophin (PTN), polymeric immunoglobulin receptor (PIGR), olfactomedin-like protein 3 (OLFML3), and avian beta-defensin 11 (AvBD11), for proteins with Log₁₀ precursor intensity >8 at D0 (Fig. 5). Interestingly, none of the other known major VM components, namely lysozyme C, ZP1 and ZP3 were significantly altered by storage conditions (ANOVA *p*-values of 0.99, 0.96, 0.98, respectively) (Table S4), which corroborates SDS-PAGE observations (Fig. 4). The absence of significant changes of ZP1 and ZP3 - two main components of IVM - was consistent with measurements of IVM thickness, which likewise showed no significant variation during egg storage (Fig. 3D). These results suggest that lysozyme C, ZP1 and ZP3 are relatively stable to the storage conditions used in the study and to the variations of egg white pH. Consequently, the observed VM degradation likely results from alterations in less abundant protein components, most likely those associated with the OVM. The hierarchical clustering of the 30 differential proteins according to their variation of abundance revealed two main clusters, identified as Cluster 1 and Cluster 2 in Fig. 5. Cluster 1 contained only four proteins (OC17, LOC771972/OCX25, PLOD1, CLU), whose relative abundance differed between storage at 4 °C and storage at 20 °C and/or increased during storage. In contrast, Cluster 2 comprised 26 proteins with decreased relative abundance during egg storage at 4 °C and/or

20 °C. Cluster 2 can be obviously divided into two subclusters, named Cluster 2a (13 proteins with decreased abundance at 4 °C or at both 4 °C and 20 °C) and Cluster 2b (11 proteins with decreased abundance at 20 °C) (Fig. 5). Within Cluster 2a, a set of proteins including APOB, APOV1, EEF1A1, GAPDH and OVALX appeared to be particularly sensitive to low temperature, with their relative abundance being significantly lower at 4 °C than at 20 °C (Fig. 5). Although not clustered with these proteins, the relative abundance of OVALY was also significantly reduced at 4 °C while it remained unchanged at 20 °C (Fig. 5). The variation pattern of Cluster 2b proteins (including VMO1, OVOA, A2ML1, PIGR, OLFML3 and AvBD11 among the most abundant proteins) was in line with the variation of yolk index and/or VM mechanical parameters (strength, deformation), which thus suggests that these proteins might be potential biomarkers of VM quality/integrity, and thus egg yolk quality and freshness. Of note, seven proteins of this cluster (A2ML1, AvBD11, CD163L, OLFML3, OVOA, PIGR, RARRES1) emerged as biomarkers of room temperature egg storage, as they showed significantly lower abundance at 20 °C (D28+20°C) compared to both D0 and D28+4°C conditions, while remaining unaffected during storage at 4 °C (Fig. 5).

### 3.6. Differentially-abundant proteins related to VM structure and antibacterial protection

Student's *t*-test comparisons of protein abundances between experimental groups revealed 40 proteins with differential abundance (*p* ≤ 0.05 and Log₂(FC) ≥ 1 or ≤ −1) in at least one comparison pair (Fig. 6). When analyzed separately, 23, 28 and 24 differentially-abundant proteins (DAPs) were identified between fresh and 4 °C-stored eggs (D0 vs D28+4°C, Table S5), between fresh and 20 °C-stored eggs (D0 vs D28+20°C, Table S6), and between 4 °C-stored and 20 °C-stored eggs (D28+4°C vs D28+20°C, Table S7), respectively. Importantly, relative abundances of major VM proteins including lysozyme C (LYZ), ZP1 and ZP3 were not significantly altered during egg storage. DAPs identified in our study comprised at least 10 proteins with previously reported antibacterial properties (AvBD11, AVD, EXFABP, MSMB3/LOC101750704, OC17, OVALX, OVST, PTN, RARRES1/OCX32, VMO1) (Moreau et al., 2022). Among these antibacterial DAPs, C-type lectin ovocleidin-17 (OC17) is the only one to show increased relative abundance in VM during storage (Fig. 5, Tables S5, S6, S7). Increased levels of OC17 were also observed in the VM of eggs stored for 10 days at 30 °C (Zhou et al., 2020), but the cause of such an increase remains unknown. Compared to fresh eggs, the relative abundance of the other antibacterial DAPs decreased in eggs stored at 4 °C (AVD, EXFABP), or 20 °C (AvBD11, OVST, RARRES1/OCX32, VMO1), or at both temperatures (MSMB3/LOC101750704, OVALX, PTN) (Fig. 5, Tables S5, S6, S7). It is noteworthy that Extracellular Fatty Acid Binding Protein (EXFABP) was not identified in VM samples of eggs stored at 20 °C (Fig. 5), which suggests that EXFABP was degraded or dissociated from the VM during egg storage. Similar observations were made with ovodefensin A1/gallin (OvoDA1), a 4.7 kDa cationic antimicrobial peptide of the defensin family (Gong et al., 2010; Herve et al., 2014; Whenham et al., 2015). Although not listed as a DAP in our study, OvoDA1 was identified in five out of six VM samples of 'D0' eggs, but only in one out of the six samples of 'D28+4°C' eggs, and in none of the six samples of 'D28+20°C' eggs (Table S4). Another DAP of interest identified in our study is alpha-ovomucin (OVOA, previously named LOC395381 or MUC5B). Ovomucin is a high molecular weight glycoprotein, composed of two subunits (alpha and beta), and involved both in the viscosity of egg white (Pu et al., 2023) and in the structural integrity and strength of OVM (Kido & Doi, 1988). The relative abundance of OVOA was significantly decreased in the VM of eggs stored at 20 °C, compared to freshly-laid eggs (*p* = 0.004, Log₂(FC) = −1.26) (Table S6) and eggs stored at 4 °C (*p* = 0.0052, Log₂(FC) = −1.08) (Table S7). Interestingly, although beta-ovomucin (OVOB, previously named MUC6) was not identified as a DAP in our study because of weak change (−1 < Log₂(FC) < 1), its relative
<figure><img src="image_15.png" /><figcaption>**Fig. 5.** Variations of abundance and hierarchical clustering of VM proteins with significant abundance differences among conditions ('D0', 'D28+4°C', 'D28+20°C'). Proteins with significant abundance differences were identified using ANOVA test on Log₁₀ precursor intensity with significance at *p* ≤ 0.01. Data of the heatmap represent the Log₂(FC) of precursor intensity, using 'D0' (freshly-laid eggs) as the reference condition. For each protein, information about protein accession number, gene symbol, protein abundance at D0, molecular weight (MW), theoretical pI of mature protein sequences, and pairwise significant differential abundance are given on the right of the heatmap. Mature protein sequences predicted with PrediSi (http://www.predisi.de/) and SignalP6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/) were used to calculate MW and theoretical pI (ProtParam: https://web.expasy.org/protparam/). § Pairwise significant differential abundance indicates proteins with significant Student's *t*-test (*p* ≤ 0.05) and a Log₂(FC) ≥ 1 or ≤ −1, in at least one of the following comparison pairs: D0 vs D28+4°C (cyan), D0 vs D28+20°C (brown) or D28+4°C vs D28+20°C (purple). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

abundance was significantly lower in eggs stored at 20 °C than in fresh eggs (*p* = 0.027, Log₂(FC) = −0.761) (Table S6). The molecular mechanism responsible for this reduction remains unknown; however, it is possible that certain structural changes occurring in ovomucin during egg white thinning, including alkaline hydrolysis of the disulfide bonds (Donovan et al., 1972) and beta-elimination of O-glycosidically linked carbohydrate units (Kato et al., 1979), may also occur in VM and lead to the degradation or dissociation of ovomucin from VM. In this respect, a recent study revealed the removal of OVOA/MUC5B N-glycans in the VM after 10 days of storage at 30 °C (Zhou et al., 2021). Supporting our data, a previous study also demonstrated similar changes of abundance for AvBD11, EXFABP, RARRES1/OCX32, VMO1, and OVOB, after 10 days of storage at 30 °C, but unlike our results, relative abundances of AVD, OVALX, OvoDA1, and OVOA/MUC5B were found to increase in these conditions (Zhou et al., 2020). These discrepancies may be due to differences in storage conditions (10 days at 30 °C vs. 28 days at 20 °C in our study), analytical approaches (in solution vs. in gel in our study), and possibly the strain and age of the hens (42-week-old White Leghorn hens vs. 71–73-week-old Lohmann Tradition hens in our study). Interestingly, most of the proteins, whose abundance was altered in our study (AvBD11, MSMB3/LOC101750704, OVALX, OVOA/MUC5B, OVOB/

MUC6, OvoDA1, PTN, RARRES1/OCX32, VMO1), are synthesized in the upper oviduct and present in OVM (Bregeon et al., 2022). Some of them have been suggested to participate in the physical association of OVM with IVM (Bregeon et al., 2022), as it was previously demonstrated for AvBD11 (previously known as VMO2) in quail eggs (Rahman et al., 2009). Moreover, ovomucin (OVOA, OVOB), VMO1 and AvBD11/VMO2 are among the major components of OVM (Back et al., 1982; Kido et al., 1992; Kido et al., 1995). Consequently, reduced levels of these OVM proteins in eggs stored at 20 °C may be responsible for the decrease in OVM thickness (Fig. 3C) and for the weakening of VM (Fig. 1B, 1C, and 1D). It is also likely that alterations in antibacterial proteins and ovomucin content may impair the protective role of the VM against pathogenic microorganisms, and hence the hygienic quality of eggs.

### 3.7. VMO1 levels in VM and egg white samples

In our study, VMO1 protein was one of the most abundant VM proteins showing a significant change during storage at 20 °C (*p* = 0.025, Log₂(FC) = −1.07). This cationic 18 kDa lectin-like protein (Moreau et al., 2022) is synthesized by the upper oviduct and deposited in the OVM (Bregeon et al., 2022) where it is tightly associated with ovomucin
<figure><img src="image_16.png" /><figcaption>**Fig. 6.** Distribution of the 40 differentially-abundant proteins (DAPs) between pairs of conditions: D0 vs D28+4°C (cyan), D0 vs D28+20°C (brown) and D28+4°C vs D28+20°C (purple). All DAPs identified in the study are proteins with a significant Student's t-test ($p \le 0.05$) and a $Log_2(FC) \ge 1$ or $\le -1$, in at least one comparison pair. In the Venn diagram, proteins are represented by their corresponding gene symbol. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

fibrils in freshly-laid eggs, together with lysozyme and AvBD11/VMO2 (Kido et al., 1995). Even though some carbohydrate-binding and/or antibacterial properties have been previously reported (Guyot et al., 2016; Kido et al., 1995; Shimizu et al., 1994), the biological and structural function of VMO1 in the VM remains largely unknown. Label-free protein quantification of VMO1 revealed that its abundance in VM samples was significantly reduced by approximately twofold in eggs stored for 28 days at 20 °C compared to freshly-laid eggs, while VMO1 levels in eggs stored at 4 °C were not significantly different from unstored eggs and eggs stored at 20 °C (Fig. 7A). From this observation, it can be assumed that the loss of VMO1 during storage is either due to a dissociation and subsequent release from the VM, or to a degradation. Protein degradation can be mediated by proteolytic enzymes and, in this regard, a number of proteases including ovochymase-2, cathepsins (E-A like, D, and V), carboxypeptidases (D and E) and metalloproteases (angiotensin-converting enzyme, aminopeptidase Ey, PLAC domain-containing protein) have been previously identified in VM proteomes (Bregeon et al., 2022; Mann, 2008; Zhou et al., 2020; Zhou et al., 2021). Therefore, the protein level and integrity of VMO1 in VM, and the presence of any potential cleavage products, were investigated by western blot using a polyclonal chicken VMO1 antibody. As shown in Fig. 7B, an immunoreactive band with a molecular weight similar to purified intact VMO1 was detected in all VM samples. The quantification of this band by densitometry revealed that VMO1 concentration was about 0.185 mg/mg of total protein in fresh VM, and it significantly decreased to 0.126 mg/mg (32 % decrease) during storage at 20 °C (Fig. 7C). Similar changes were observed when results are expressed as mg/mg of VM dry matter (0.111 vs. 0.079, respectively) (Fig. 7D). These levels were even found to be significantly different between storage at 4 °C and 20 °C (Fig. 7D). These results are consistent with a previous

study showing that the amount of VMO1 in the salt-soluble fraction of VM, estimated by measuring absorbance peak area in HPLC, decreased over 30 days of storage at 20 °C, while remaining stable at 4 °C (Schäfer et al., 1998). According to the estimates based on our data (Fig. 2A and Fig. 7D), about 20 % of the VM dry matter lost during storage at 20 °C was due to VMO1 loss. As shown by western blot, the band intensity of VMO1 obviously decreased during storage at 20 °C (Fig. 7B). However, no immunoreactive fragment of VMO1 was detected (Fig. 7B), which suggests that VMO1 was not degraded during storage. In this regard, average protein coverage of VMO1 identified by mass spectrometry in VM samples was similar between groups, with values of 76 %, 73 % and 75 % in D0, D28+4°C and D28+20°C groups, respectively (86 %, 83 % and 85 % of the mature protein sequence, respectively). All these observations support the hypothesis that VMO1 rather dissociated from the VM during storage. Consequently, it is expected that upon dissociation, the VMO1 concentration in egg white would increase progressively during storage. This protein has been previously identified in the egg white proteome (Guerin-Dubiard et al., 2006; Mann, 2007; Mann & Mann, 2011; Pu et al., 2023), but at lower relative abundance than in the VM where it is one of the major components of OVM. A GeLC-MS/MS approach combined with label-free MS quantification, similarly to that performed for VM samples, was conducted to quantify VMO1 in egg white samples. Results presented in Fig. 8 showed that VMO1 levels significantly increased in egg whites of eggs stored at 20 °C, compared to unstored eggs and 4 °C-stored eggs. All these observations are consistent with an accelerated dissociation and release of VMO1, from the VM to the egg white, during storage at 20 °C, with no detectable degradation of the protein. The nature of interactions of VMO1 with the other proteins composing VM (type of interaction, identity of partners) is not well defined. However, VMO1, lysozyme and AvBD11/VMO2 are cationic
<figure><img src="image_17.png" /><figcaption>**Fig. 7.** Detection of VMO1 abundance in the vitelline membrane samples of freshly-laid eggs (D0) and eggs stored for 28 days at 4 °C (D28 +4 °C) or 20 °C (D28 +20 °C). (A) Label-free MS quantification of VMO1 using precursor intensity; (B) Western-blot analysis with anti-VMO1 polyclonal antibodies (each lane contains 500 ng of vitelline membrane proteins or 115 ng of purified VMO1); (C) and (D) Western blot-based quantification of VMO1 related to VM total proteins and VM dry mass, respectively. In all scatter dot plots, horizontal bars and crosses represent medians and means, respectively. Conditions with different letters indicate statistically significant differences with *p* < 0.05 (Kruskal-Wallis test, Conover-Iman post hoc test).</figcaption></figure>

<figure><img src="image_18.png" /></figure>

proteins that can be extracted from VM by high-salt buffer (Back et al., 1982; Kido et al., 1992). Interactions of these salt-soluble proteins with ovomucin fibrils are likely to be predominantly electrostatic, and might involve, at least in part, binding of basic amino acids to negatively-charged moieties of ovomucin, as previously demonstrated for lysozyme (De Boeck & Stockx, 1986). We hypothesize that egg white alkalization, which occurs during egg storage, could modify electrostatic potential values at the surface of VMO1 molecule, thus altering its interactions with molecular partners such as ovomucin, and leading to a progressive dissociation of VMO1 from OVM. In that case, storage methods aiming at stabilizing internal egg white pH near the initial value at lay might prevent this dissociation. On the other hand, it could also be assumed that the ovomucin alteration observed in our study leads to the reduction of bound proteins (including VMO1), but this is unlikely to be the predominant hypothesis since the abundance of lysozyme, another molecular partner of ovomucin, was not affected during egg storage. It has previously been suggested that dissociation of VMO1 and AvBD11/VMO2 participates in the disintegration of VM

structure, leading to reduction of VM strength (Schäfer et al., 1998); however, another study revealed that VM strength in freshly-laid eggs would not be attributable to these salt-soluble proteins, but rather to ovomucin itself (Kido & Doi, 1988). Further studies are needed to investigate in more detail the protein-protein interactions within the VM, the dissociation kinetics of VMO1 during storage, and how the loss of VMO1 during storage destabilizes the structural integrity of the VM.

## 4. Conclusions

To conclude, the present study highlights the strong relationship between the weakening of VM mechanical properties, the decrease in VM dry mass and thickness, and the alterations in the VM proteome, especially during storage at 20 °C. Our data indicate that the increase in egg white pH during egg storage alters the abundance of several OVM proteins essential for VM structure and integrity. These changes lead to a reduction in VM thickness and, ultimately, to weakened mechanical properties. All of these modifications strongly depend on storage
<figure><img src="image_19.png" /><figcaption>**Fig. 8.** Label-free MS quantification of VMO1 in egg white of freshly-laid eggs (D0) and eggs stored for 28 days at 4 °C (D28 +4 °C) or 20 °C (D28 +20 °C). Horizontal bars and crosses in the scatter dot plot represent medians and means, respectively. Conditions with different letters indicate statistical differences with *p* < 0.05 (Kruskal-Wallis test, Conover-Iman post hoc test).</figcaption></figure>

temperature, which dictates the rate of pH increase: the higher the storage temperature, the faster the rise in egg white pH and the greater the weakening of the VM. Notably, this alteration may also involve a destabilization of the VM protein fiber network caused by the migration of water from the egg white to the yolk, across the VM. Among the differentially abundant proteins, a subgroup of proteins whose relative abundance correlated with the deterioration of VM was identified as potential biomarkers of egg quality. In relation to VM alteration, the abundance of some OVM structural and antibacterial components was found to be lowered during egg storage. Among these components are ovomucin and VMO1, two of the most abundant proteins of OVM. Moreover, approximately one-fifth of the VM dry mass shed after 28 days at 20 °C was composed of VMO1 protein, which thus suggests that this protein is a critical component for VM integrity and solidity. A key finding of this study is the elucidation of VMO1's fate during storage. Indeed, this abundant protein of OVM was found to dissociate from the membrane and be released into the egg white, presumably as a result of egg white pH increase occurring during egg storage. However, at this stage, it is not known whether this mechanism also occurs for other VM proteins. Interestingly, the VM proteome was altered not only at 20 °C but also at 4 °C; however, the global proteome changes differed when comparing 4 °C to 20 °C. Despite these modifications, VM physical and mechanical properties were maintained or slightly altered at 4 °C, which is likely due at least in part to lower alkalinization of egg white. Overall, this study provides novel findings contributing to a better understanding of the mechanisms of VM alteration occurring during the storage of eggs at room temperature. Further investigations would be needed to define the effects of alkaline pH on protein interactions within the VM. These findings might also be useful for the conception of strategies (improving storage conditions, using VMO1 as a biomarker for genetic selection) to preserve VM during egg storage or to reinforce VM quality. Indeed, the quality of VM is essential for the egg sector, especially the egg-product industry which uses automatic egg breakers and yolk separators and needs to minimize yolk breakage during the yolk-white separation process. As this study focused on 71-73-week-old Lohmann Tradition laying hens, and because VM characteristics might vary with layer age, strain, or housing system, future research should examine these factors to determine the broader applicability of the present findings.

### CRediT authorship contribution statement

**Océane Hervé:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Magali Chessé:** Writing – review & editing, Validation, Project administration, Methodology, Investigation, Data curation. **Daniel Tomas:** Writing – review & editing, Validation, Resources, Methodology, Investigation, Formal analysis. **Valérie Labas:** Writing – review & editing, Validation, Resources, Methodology, Data curation. **Justine Jimenez:** Writing – review & editing, Investigation. **Nelly Bernardet:** Writing – review & editing, Investigation. **Sonia Georgeault:** Writing – review & editing, Methodology, Investigation. **Yves Nys:** Writing – review & editing, Methodology, Conceptualization. **Sophie Réhault-Godbert:** Writing – review & editing, Resources, Methodology, Investigation. **Thierry Moreau:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization. **Nicolas Guyot:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### 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

This research was funded by the Agence Nationale de la Recherche (ANR), grant number ANR-19-CE21-0006. Océane Hervé received a joint doctoral funding from the Centre-Val-de-Loire Region and INRAE PHASE scientific department. The authors thank Christophe Souchet and Marie Bourin (ITAVI) as well as Emmanuel Ledru and Jérémy Bernard (PEAT, INRAE, Nouzilly, Doi: 10.15454/1.5572326250887292E12) for breeding the laying hens, monitoring animal performance and supplying eggs. On the PIXANIM platform (Imaging facility for phenotyping, from animal to molecule, Doi: 10.17180/CQ4D-DW26), the high-resolution mass spectrometer (Orbitrap) was financed (SMHART project, 35069) by the European Regional Development Fund (ERDF), the Conseil Régional du Centre, the French National Institute for Agricultural Research (INRA) and the French National Institute of Health and Medical Research (Inserm).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodres.2025.118291.

### Data availability

The mass spectrometry proteomics data of the vitelline membrane samples have been deposited to the ProteomeXchange (Deutsch et al., 2023) Consortium via the PRIDE (Perez-Riverol et al., 2025) partner repository with the dataset identifier PXD066675 and https://doi.org/10.6019/PXD066675. All other data will be made available on request.

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