Valérie Lechevalier-Datin

Catherine Guérin-Dubiard
email: catherine.guerin@agrocampus-ouest.fr

Marc Anton
email: marc.anton@inra.fr

Valerie Beaumal
email: valerie.beaumal@inra.fr

Elisabeth David Briand

Angélique Gillard

Yann Le Gouar
email: yann.legouar@inra.fr

Nuttinee Musikaphun
email: nuttinee.musikaphun@solina-group.fr

Gaëlle Tanguy-Sai

Maryvonne Pasco
email: maryvonne.pasco@agrocampus-ouest.fr

Valerie Lechevalier
email: valerie.lechevalier@agrocampus-ouest.fr

Valérie Beaumal

Elisabeth David Briand
email: elisabeth.david-briand@inra.fr

Angelique Gillard
email: angelique.gillard@cg84.fr

Gaëlle Tanguy
email: gaelle.tanguy@inra.fr

Didier Dupont
email: didier.dupont@inra.fr

Francoise Nau
email: francoise.nau@agrocampus-ouest.fr

Valérie Beaumal

Angelique Gillard

Pasteurisation of liquid whole egg: Optimal heat treatments in relation to its functional, nutritional and allergenic properties

Keywords: Whole egg, pasteurisation, interfacial property, emulsion, in vitro digestion, antigenicity

come

M A N U S C R I P T A C C E P T E D

1.

IntroductionEgg is a widely used ingredient in many food products. Especially, whole egg is indeed a major source of high quality proteins and essential nutrients and provides many desirable functional attributes such as foaming, emulsifying, gelling, colouring, flavouring [START_REF] Yang | Functional properties of eggs in Foods[END_REF].

However, hen egg is also one of the leading causes of food allergy in childhood, affecting 1.6 to 3.2% of young children [START_REF] Eggesbo | The prevalence of allergy to egg: a populationbased study in young children[END_REF].

In the egg product industry, microbiological safety of liquid products is mainly guaranteed by pasteurisation. The USDA requires that liquid whole egg is at least heated at 60°C for no less than 3.5 min, but in the United Kingdom the recommendations are to pasteurize at least at 64°C for 2.5 min [START_REF] Cunningham | Egg product pasteurization[END_REF]. In France, there is no statutory heat treatment; only microbiological results are determined by regulations. To achieve this, the treatments classically used to pasteurize whole egg vary from 65 to 68°C for 2 to 5 min in order to ensure 5 to 6 decimal reductions of vegetative microorganisms and especially Salmonella Enteritidis and Listeria monocytogenes [START_REF] Baron | Qualité microbiologique des ovoproduits[END_REF].

Pasteurisation temperatures used in the egg industry are limited by the sensitivity of egg proteins to heat treatment. Thus, pasteurisation for 2 to 10 minutes from 60 to 68°C modifies whole egg electrophoretic pattern by especially decreasing ovotransferrin, livetin, ovalbumin, apovitellenin, lysozyme and/or ovomucin band intensity [START_REF] Dixon | Electrophoretic and chromatographic changes in egg yolk proteins due to heat[END_REF][START_REF] Martinez | Effect of ultrapasteurization with and without homogenization on the electrophoretic patterns of aseptically processed liquid whole egg[END_REF][START_REF] Mendes De Souza | Rheological properties and protein quality of UV-C processed liquid egg products[END_REF][START_REF] Scholtyssek | The age of the eggs and the pasteurization and their effects on the quality of whole egg[END_REF][START_REF] Woodward | Electrophoresis and chromatography of heat-treated plain, sugared and salted whole egg[END_REF]. Moreover, an increase of whole egg viscosity is observed at temperature as low as 56°C [START_REF] Cunningham | Egg product pasteurization[END_REF], and whereas pasteurisation has little influence on liquid whole egg functionality when heated up to 60°C [START_REF] Ball | Functionality and microbial stability of ultrapasteurized, aseptically packaged refrigerated whole egg[END_REF][START_REF] Cunningham | Egg product pasteurization[END_REF][START_REF] Herald | Functional properties and composition of liquid whole egg proteins as influenced by pasteurization and frozen storage[END_REF], heating above this temperature decreases foaming and emulsifying properties [START_REF] Janssen | Influence of pasteurization, freezing and storage on properties of egg products made from eggs stored for 7 and for 28 days[END_REF][START_REF] Montfort | Physicochemical and functional properties of liquid whole egg treated by application of pulsed electric fields followed by heat in the presence of triethyl citrate[END_REF], custard height [START_REF] Ball | Functionality and microbial stability of ultrapasteurized, aseptically packaged refrigerated whole egg[END_REF][START_REF] Cunningham | Egg product pasteurization[END_REF] or pie filling expansion [START_REF] Herald | Functional properties and composition of liquid whole egg proteins as influenced by pasteurization and frozen storage[END_REF].

The high content of highly digestible proteins in whole egg is of great benefit to human nutrition.

However, the effects of industrial processing such as pasteurisation on the nutritional quality of whole egg proteins are poorly documented. [START_REF] Van Der Plancken | Effect of thermal treatment on the susceptibility of egg white proteins to enzymatic hydrolysis[END_REF] showed that ovalbumin susceptibility to hydrolysis was increased after heat treatment higher than 10 to 30 minutes at 60°C, whereas the ovomucoid susceptibility was not changed after 30 min at 60°C.

Assuming that pasteurisation results in egg protein denaturation and/or aggregation, whole egg pasteurisation may hide, destroy or unmask protein allergenic epitopes. Moreover, the potential changes in protein digestion can also change the ability to sensitize and elicit the immune response [START_REF] Jimenez-Saiz | Human immunoglobulin E (IgE) binding to heated and glycated ovalbumin and ovomucoid before and after in vitro digestion[END_REF]. The effect of heat treatment on the allergenic properties of egg proteins has been focused on cooking since some allergic children tolerate cooked eggs [START_REF] Lemon-Mulé | Immunologic changes in children with egg allergy ingesting extensively heated egg[END_REF].

Moreover, egg white proteins have been mainly studied since they are considered more allergenic than yolk proteins [START_REF] Kosti | Food allergen selective thermal processing regimens may change oral tolerance in infancy[END_REF], despite apovitellenins I & VI, α-livetine and phosvitin were reported as allergens by [START_REF] Mine | Recent advances in the understanding of egg allergens: basic, industrial, and clinical perspectives[END_REF]. In most cases, physicochemical changes caused by cooking either enhance a decrease or have no significant effect on allergenicity, depending on the susceptibility of the proteins to unfold and to lose their conformational epitopes. But in some cases, aggregates which are more allergenic and more resistant to digestion can be generated [START_REF] Thomas | Evaluating the effect of food processing on the potential human allergenicity of novel proteins: international workshop report[END_REF]. Up to the authors' knowledge, no study has been performed to test the effect of pasteurisation on whole egg antigenicity.

Consequently, if the effects of pasteurisation on whole egg functional properties have been well

documented, there is a lack of knowledge on the effect of this unit operation on whole egg protein digestibility and antigenicity. The present study aimed at investigating the effect of a wide range of pasteurisation rates on the interfacial properties, in vitro digestibility and antigenicity of whole egg proteins, and to determine the best conditions to optimize these different functionalities.

Material and methods

Experimental design

Raw liquid whole egg was supplied by Ovoteam (Plaintel, France). Dry matter was adjusted to 22% by adding raw egg white (Ovoteam, Plaintel, France). Homogenised whole egg was obtained from raw values of these bacteria in whole egg [START_REF] Jin | Thermal resistance of Salmonella enteritidis and Escherichia coli K12 in liquid egg determined by thermal-death-time disks[END_REF].

Interfacial properties

2.2.1.

Langmuir isotherms at air-water interface

Measurements of the surface pressure (Π) -surface area (A) isotherms have been performed by compression -expansion cycles using the Wilhelmy plate method as described by Lechevalier et al.

(submitted) except that egg white solutions was replaced by liquid whole egg diluted in 50 mM phosphate buffer pH 7, NaCl 300 mM to a final concentration of 1 mg.ml -1 after measurement of protein content according to [START_REF] Markwell | A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples[END_REF].

The surface corresponding to an interfacial pressure of 10 mN.m -1 (S10) was selected as the indicator of the sample behaviour at the air-water interface.

2.2.2.

Emulsifying properties

Preparation of oil-water emulsions

Emulsions were prepared as described by [START_REF] Rannou | Effect of spray-drying and storage conditions on the physical and functional properties of standard and n-3 enriched egg yolk powders[END_REF] with the aqueous phase being 1.5%

(w/v) whole egg protein solutions in phosphate buffer pH7, NaCl 300 mM.

Droplet size distribution

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The droplet size distribution (DSD) was determined as described by Lechevalier et al. (submitted). The volumetric mean diameter (d4,3) was selected to measure droplet size distribution.

Emulsion stability

Emulsion stability towards creaming was determined as described by Lechevalier et al. (submitted). The creaming index (Cri) was calculated as follows:

φ 100 * Vt Va Cr i = eq.1
where Va is the volume of separated aqueous phase, Vt is the total volume of sample in the tube after centrifugation, and Φ is the volumic fraction of the aqueous phase used to prepare the emulsion (i.e. 0.7). The higher the creaming index, the lower the emulsion stability, since it assumes a high release of aqueous phase due to a high compaction of droplets with centrifugation.

2.3. Protein in vitro digestion and characterization of digested samples 2.3.1.

In vitro digestion

Each pasteurised whole egg sample and the control sample were subjected to an in vitro digestion model which simulates successive gastric and intestinal stages of digestion in the adult human as proposed by [START_REF] Dupont | Comparative resistance of food proteins to adult and infant in vitro digestion models[END_REF] and detailed by [START_REF] Lechevalier | Effect of dry-heating on the progression of in vitro digestion of egg white proteins: contribution of multifactorial data analysis[END_REF]. One ml of protein solution was aliquoted before digestion (gast ti), at the end of the gastric stage (gast tf) and at the end of the intestinal stage (int tf).

2.3.2.

Degree of hydrolysis (DH)

The DH was calculated from the measurement of amino groups released during in vitro digestion using OPA (orthophtaldialdéhyde) according to [START_REF] Church | Spectrophotometric assay using ophtaldialdehyde for determination of proteolysis in milk and isolated milk proteins[END_REF], modified as described by Lechevalier et al. (submitted). The hydrolysis degree (DH) was calculated using the following formula:

% DH = (Lt -L0)/Ltot x 100 eq. 2

where Lt is the amount of free NH2 after t min hydrolysis, L0 is the amount of initial free NH2 (before digestion), and Ltot is the maximum amount of free NH2, i.e. after total acid hydrolysis. Free NH2 was

Electrophoresis

The digested samples were analysed by SDS-PAGE on 12.5% polyacrylamide gels, according to [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the bacteriophage T4[END_REF], modified as explained by [START_REF] Lechevalier | Effect of dry-heating on the progression of in vitro digestion of egg white proteins: contribution of multifactorial data analysis[END_REF].

Samples were distributed onto 26 gels. For each pasteurised whole egg (one temperature, one duration) and the control, one gel was prepared with the samples of the gastric stage, and another gel was prepared with the samples of the intestinal stage (Figure 1).

The gels were Coomassie Blue stained, and digitised using the Image Scanner III device (GE Healthcare Europe GmbH). ImageQuant software (GE Healthcare) was used to quantify the band by Gaussian fitting, and to match the gels. All matches were checked manually and corrected if necessary.

The band volume was used as the response variable in the statistical analysis. Up to 22 bands per sample could be identified (Figure 1).

The final data set was composed of 39 different samples: 13 samples x 3 digestion times (gast ti, gast tf, int tf). The band volumes measured at gast ti were used to characterize sample protein composition after pasteurisation and before digestion; they were analysed separately by principal component analysis as described in section 2.5.2.

2.3.4.

Pre-processing of the data As explained by [START_REF] Lechevalier | Effect of dry-heating on the progression of in vitro digestion of egg white proteins: contribution of multifactorial data analysis[END_REF], raw electrophoresis data are not suitable for processing by statistics: pre-processing of the data was thus necessary. Firstly, the values that were missing in the final dataset, were manually assigned to zero after checking the absence of the corresponding band on the gel.

Then, since the 39 samples were not randomized on the 26 gels, band volumes were normalized using the 30 kDa band of the LMW kit to avoid any differences in staining from one gel to another. The volume of this band on the gel prepared with the digested gastric samples of the control sample (raw whole

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 8
egg) was taken as the reference value (GelrefBV). The volume of the 30 kDa band for each of the other 25 gels was called GelnBV (1<n<25). The correction coefficient (CC) for the gel n was then calculated as follows:

BV Gel BV Gel CC Gel ref n n = eq. 3
All the band volumes of the Geln were then multiplied by the GelnCC coefficient.

The band volumes at the three digestion stages characterized hydrolysates protein composition and were analysed by multiple factor analysis as described in section 2.5.4 to highlight pasteurisation effect on the progress of the in vitro digestion.

Protein antigenicity

Inhibition ELISA using ovomucoïd, ovotransferrin or lysozyme-specific monoclonal antibodies was performed in order to determine the residual immunoreactivity of each protein after whole egg pasteurisation, according to [START_REF] Dupont | Comparative resistance of food proteins to adult and infant in vitro digestion models[END_REF] and as described by Lechevalier et al. (submitted).

Results were expressed as ovomucoïd, ovotransferrin or lysozyme concentration in samples using a four-parameter logistic (sigmoidal) function. The measurements were performed in duplicate.

Statistical analysis

2.5.1.

ANOVA

ANOVA was used to test the effect of pasteurisation temperature and time on each measurement. The normal distribution of the data was first tested using a Shapiro Wilk normality test, and the homoscedasticity of the samples was tested using the Levene's test for homogeneity of variance of R language (R development core team, 2007). Since all variables fitted these ANOVA hypotheses, the following linear model procedure of R was applied:

Yij = Ti + tj + T.tij eq.4 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 9
where, T is the temperature i (20, 60, 62, 64, 66°C); t the treatment time j (0, 4, 7, 10 min); and T.t the interaction. Differences were regarded significant at minimum level of 95% (p-value<0.05). The LS-Means were calculated and compared using multiple comparisons by means of LSD with the "agricolae" package of R software (R development core team, 2007).

2.5.2.

Principal component analysis (PCA)

To analyse the effect of pasteurisation on all egg proteins at once, PCA was applied to the matrix with electrophoresis band volumes at gast ti stage as active variables, and the 13 samples as individuals (Supplementary data 2). Pasteurisation temperature and time were added as supplementary variables.

PCA transformed the observed variables into a new set of independent variables called principal components (PCs) which were uncorrelated linear combinations of the original variables. The first component was the one which explained most of the variance in the data. A large part of the information in the data could thus be plotted in 2-D or 3-D graphs defined by the PCs. PCA was performed using the FactoMineR package of the R software [START_REF] Husson | FactoMineR: An R Package for Multivariate Analysis[END_REF]. The variables were automatically standardized (mean centred and scaled) by the software to give them all the same importance.

2.5.3.

Multiple factor analysis (MFA)

MFA deals with a multiple table, composed of groups of either continuous, categorical variables or frequency tables [START_REF] Escofier | Aspects théoriques et techniques de l'analyse factorielle multiple[END_REF] In the present study, two MFA were performed. The first one ("digestion MFA") was performed on the table containing the quantified band volumes at three digestion sampling times (gast ti, gast tf and int tf), with the row representing the 13 samples (the control and the 12 pasteurised samples) (Supplementary data 3). It provided a representation of the effect of pasteurisation treatment by taking into account the digestion sampling time in a balance way. Moreover, it also provided a superimposed representation of pasteurised samples described by each of the groups of variables onto the subspace of the global analysis generated by PCs [START_REF] Lechevalier | Effect of dry-heating on the progression of in vitro digestion of egg white proteins: contribution of multifactorial data analysis[END_REF].

The second MFA ("global MFA") was performed on the table containing all the previously described measurements dispatched in four groups (Supplementary data 4): the volumes of each SDS PAGE band before hydrolysis in "protein profiles" group; the surface pressure, the droplet size distribution and the emulsion stability in "protein interfacial properties" group; the degree of hydrolysis at the end of gastric and intestinal stages and the coordinates of the samples on the first 2 PCs of the digestion MFA in the "protein digestion" group; the ovomucoïd, ovotransferrin and lysozyme concentrations determined by inhibition ELISA in the "protein antigenicity" group. The rows represented the control and the 12 pasteurised samples. Pasteurisation temperature and time were added as supplementary variables. The "global MFA" provided a representation of the diversity of pasteurisation treatment by taking into account the protein profile before digestion, the protein interfacial properties, their susceptibility to in vitro digestion and their antigenicity. The superimposed representation of pasteurised samples described by each one of the four groups of variables on the subspace of the global analysis generated by PCs highlighted the similarities and discrepancies between samples according to the properties considered. Interfacial properties were measured both on 2D through surface pressure-area isotherms and 3D through emulsifying properties. Surface pressure-area isotherms of films made with pasteurised whole egg proteins are shifted towards higher pressures and areas than the control, at least for the lowest pasteurisation temperatures (Figure 3). Low pasteurisation thus favours the spreading of whole egg proteins at the air-water interface, making pasteurised proteins more surface-active than control proteins. The best interfacial properties are obtained for whole egg pasteurised at 60°C, whatever the time. When pasteurisation temperature increases, surface pressure-area isotherms are shifted towards lower pressures and areas, so that the sample pasteurised 7 min at 66°C is not significantly different from the control one (Figure 3).

Results and discussion

The interfacial properties of whole egg proteins were also investigated for emulsifying properties, as measured by droplet size (d4,3) and creaming index (Cri). Samples pasteurised at 60°C have significantly lower d4,3 and Cri than the control whatever pasteurisation time (Figures 4 & 5). When pasteurisation temperature increases, d4,3 and Cri tend to increase to achieve the control values. A significant pasteurisation time effect is also observed for samples pasteurised at 66°C, d4,3 and Cri increasing with pasteurisation time.

Then, whole egg pasteurisation at 60°C increases protein interfacial properties thus generating more stable emulsions with lower droplet size. Mc Clements (2004) already mentioned this relationship between protein surface activity and emulsion properties. But further increase in pasteurisation temperature decreases protein surface activity and emulsion properties getting back to the control values. The effect of pasteurisation temperature outweighs that of pasteurisation time as suggested by the analysis of variance (Table 1). Pasteurisation time is only significant for samples pasteurised at 66°C, emulsifying properties decreasing with pasteurisation time increase. Up to the authors' knowledge, the effect of pasteurisation rate has never been tested on whole egg interfacial properties.

However, Le Denmat et al. (1999) highlighted that below 69°C pasteurisation had no effect on egg yolk emulsifying properties. The increase in protein interfacial properties after pasteurisation at 60°C may

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13 thus be attributed to egg white proteins, consistently with [START_REF] Hagolle | Study of the stability of egg white protein-based foams : effect of heating protein solution[END_REF] who showed that preheating treatment of ovalbumin solution enhanced its interfacial properties for temperature as low as 62°C. On the contrary, [START_REF] Wang | Effects of yolk contamination, shearing, and heating on foaming properties of fresh egg white[END_REF] noticed that egg white foam expansion and foam stability decreased after pasteurisation at higher temperature (62 or 64°C for 4 min).The increase of whole egg interfacial properties after pasteurisation at 60°C is thus probably due to a moderate denaturation of the proteins that make them more surface active. And the decrease of protein interfacial properties with the increase of pasteurisation rate above 60°C suggests protein aggregation, consistently with the previous results of SDS-PAGE analysis and the literature [START_REF] Campbell | Heat stability of whole egg and egg yolk as related to heat treatment[END_REF][START_REF] Montfort | Physicochemical and functional properties of liquid whole egg treated by application of pulsed electric fields followed by heat in the presence of triethyl citrate[END_REF][START_REF] Niewiarowicz | Application of anticoagulants during pasteurisation of egg white and liquid whole eggs[END_REF]. A pasteurisation at 60°C maximum is thus advised to optimize the emulsifying properties of whole egg.

Low pasteurisation treatments increase the in vitro digestibility of whole egg proteins

Regarding the degree of hydrolysis at the end of both gastric and intestinal in vitro digestion, pasteurisation time and time x temperature interaction have a significant effect on the degree of hydrolysis at the end of both gastric and intestinal stages (Table 1). At the end of gastric stage, samples pasteurised 4 minutes at 60, 62 or 64°C and 7 minutes at 60°C have higher or equal degree of hydrolysis than the control whereas higher pasteurised samples have lower degree of hydrolysis than the control (Figure 6). At the end of intestinal stage, samples pasteurised at 60°C (4, 7 and 10min), 4 minutes at 62 and 64°C and 10 minutes at 66°C have significantly higher degree of hydrolysis than the control. On the opposite, samples pasteurised 10 minutes at 62°C, 7 and 10 minutes at 64°C and 4 and 7 minutes at 66°C have a significantly lower degree of hydrolysis than the control (Figure 6).

Protein and peptide profiles of whole egg digests were characterized by SDS-PAGE, and quantified by the band volumes at the three digestion stages (gast ti, gast tf and int tf). To highlight the effect of pasteurisation on the progress of protein in vitro digestion, these data were submitted to MFA according

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

14 to a method previously reported by [START_REF] Lechevalier | Effect of dry-heating on the progression of in vitro digestion of egg white proteins: contribution of multifactorial data analysis[END_REF]. The coordinates of the samples on the first two PCs were used to test the effect of pasteurisation rate on the progress of protein digestion.

Pasteurisation time and time x temperature interaction have a significant effect on the coordinates of the samples along "digestion MFA" PC1 and only pasteurisation time x temperature interaction has a significant effect on the coordinates of the samples along "digestion MFA" PC2 (Table 1). These PCs explain 20% and 18% of the dataset variability, respectively (Figure 7A). Electrophoretic band at 68 and 55 kDa were attributed to apo-LDL + γ-livetin and phosvitin + α-livetin + apo-LDL, respectively, by [START_REF] Freschi | Effect of ageing on different egg yolk fractions on surface properties at the air-water interface[END_REF] and [START_REF] Guilmineau | Efficient analysis of egg yolk proteins and their thermal sensitivity using sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing and non-reducing conditions[END_REF]. These proteins seemed thus more hydrolysed in control and low pasteurised samples than in mid and high pasteurised samples.

The reduced hydrolysis observed for mid and high pasteurised samples during the gastric phase may be attributed to aggregation induced by heat treatment that can mask enzyme cleavage sites as suggested by [START_REF] Nyemb | The extent of ovalbumin in vitro digestion and the nature of generated peptides are modulated by the morphology of protein aggregates[END_REF].

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 16

The effect of intestinal stage is given by blue arrows. For most samples (except control, 7 and 10 min at 60°C and 7 min at 64°C), the partial points at the end of intestinal stage (blue points) get closer to the centre of the plot i.e. the volumes of the bands correlated to PC1 and PC2 are closer to the mean. The intestinal stage thus reduces the electrophoretic pattern heterogeneity. At the end of digestion (Figure 7B, blue points), the 40, 31, 23, 22 and 14.5 kDa components are in higher quantity in low pasteurised samples (4, 7 and 10 min at 60°C, 10 min at 62°C, 4 min at 64 °C) than in the control one. Higher pasteurised samples are not significantly different from the control one. The 40, 23 and 22 kDa components appeared with hydrolysis, whereas the 44, 31 and 14.5kDa components corresponded to ovalbumin, apo-HDL and lysozyme, respectively [START_REF] Freschi | Effect of ageing on different egg yolk fractions on surface properties at the air-water interface[END_REF][START_REF] Guérin-Dubiard | Proteomic analysis of hen egg white[END_REF].

However, some peptides coming from the hydrolysis of higher molecular weight proteins or aggregates can also correspond to these molecular weights so that it is not possible to conclude to a lower degree of hydrolysis of ovalbumin, apo-HDL and lysozyme in low pasteurized samples. Nevertheless, [START_REF] Van Der Plancken | Effect of thermal treatment on the susceptibility of egg white proteins to enzymatic hydrolysis[END_REF] showed that heat treatment below 78°C had negligible effects on ovalbumin susceptibility to enzymatic hydrolysis even if heat treatment had more effect when it was performed on egg white rather than on purified ovalbumin.

Few data are available in the literature on the effect of heat treatment on egg protein susceptibility to in vitro digestion and most of them have been obtained on purified proteins and for much more drastic treatments [START_REF] Jimenez-Saiz | Human immunoglobulin E (IgE) binding to heated and glycated ovalbumin and ovomucoid before and after in vitro digestion[END_REF][START_REF] Martos | Mechanisms underlying differential food allergy response to heated egg[END_REF][START_REF] Nyemb | The extent of ovalbumin in vitro digestion and the nature of generated peptides are modulated by the morphology of protein aggregates[END_REF]. Only, [START_REF] Schmidt | The nutritive value of egg by-products and their potential bactericidal activity: in vitro and in vivo studies[END_REF] showed that whole egg spray drying increased its sensitivity to digestion (expressed as the fraction of peptide smaller than 12-14 kDa). However, spray drying conditions are hardly comparable to pasteurisation ones.

Regarding digestion, rather low pasteurisation intensity (up to 10 min at 60°C, 10 min at 62°C and 4 min at 64°C) should finally be recommended to improve protein digestion.

3.4.

Medium pasteurisation treatments prevent an increase of whole egg protein antigenicity

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
Pasteurisation temperature, time and their interaction have a significant effect on ovomucoid antigenicity tested using ELISA inhibition method (Table 1). Ovomucoid apparent concentration clearly decreased with pasteurisation time whatever the temperature (Figure 8A), such as it was lower than that of the control sample for all samples pasteurised for 10 minutes. Such a decrease in protein antigenicity after heat treatment has been described for many allergens since protein conformation and thus conformational epitopes are often modified by heating [START_REF] Thomas | Evaluating the effect of food processing on the potential human allergenicity of novel proteins: international workshop report[END_REF]. However, this result was more surprising in the case of ovomucoid since many studies reported the resistance of ovomucoid antigenicity to heat treatment in solution [START_REF] Fu | Effect of heat treatment on the quantitative detection of egg protein residues by commercial enzyme-linked immunoabsorbent assay test kits[END_REF][START_REF] Jimenez-Saiz | Human immunoglobulin E (IgE) binding to heated and glycated ovalbumin and ovomucoid before and after in vitro digestion[END_REF][START_REF] Kosti | Food allergen selective thermal processing regimens may change oral tolerance in infancy[END_REF][START_REF] Shin | The influence of the time and temperature of heat treatment on the allergenicity of egg white proteins[END_REF][START_REF] Urisu | Allergenic activity of heated and ovomucoid-depleted egg white[END_REF]. Only [START_REF] Mine | Comparative studies on antigenicity and allergenicity of native and denatured egg white proteins[END_REF] observed a decrease of ovomucoid antigenicity after heating 15 min at 95°C in solution. Such discrepancies between studies could be attributed to the different sources of antibodies and techniques used [START_REF] Julia | Effect of heat treatment on hen's egg ovomucoid: an immunochemical and calorimetric study[END_REF]. Especially, the nature of monoclonal antibodies that are used is determining, since such antibodies recognize specific and small sequences of the proteins which can be, or not modified by heat treatments.

Pasteurisation time and temperature x time interaction also have a significant effect on lysozyme and ovotransferrin antigenicity (Table 1). Lysozyme apparent concentration appears higher in sample pasteurised at 60 and 62°C than in the control sample (Figure 8B). However, these concentrations were consistent with the theoretical concentration of lysozyme in whole egg (2.4 g.l -1 ). For higher pasteurisation, ie at 64 and 66°C, lysozyme antigenicity decreased when temperature and/or time increased. This is consistent with the fact that egg cooking reduced egg allergenicity, even if pasteurisation treatment occurred at lower temperatures [START_REF] Lemon-Mulé | Immunologic changes in children with egg allergy ingesting extensively heated egg[END_REF][START_REF] Mine | Recent advances in the understanding of egg allergens: basic, industrial, and clinical perspectives[END_REF].

Ovotransferrin antigenicity clearly increased after pasteurisation at 66°C (Figure 8C), and longer the time at this temperature, higher the antigenicity, consistently with [START_REF] Tong | Effect of heat treatment on the potential allergenicity and conformational structure of egg allergen ovotransferrin[END_REF] who showed that the potential antigenicity of ovotransferrin increased with the unfolding of its structure after heat treatment up to 1 hour at 55-60°C. To consider all together the results of protein interfacial properties, antigenicity and in vitro digestibility, MFA was performed on the dataset that contained all the previously described measurements, with the row representing the 12 pasteurised samples and the control. Pasteurisation temperature and time were added as supplementary variables. Four groups of measurements were constituted: "protein profiles", "protein interfacial properties", "protein digestion" and "protein antigenicity" as described in section 2.5.3.

PC1 and PC2 respectively explain 32% and 18% of the dataset variability. PC1 was significantly positively correlated to ovotransferrin antigenicity, electrophoresis band volume of the 129 and 106 kDa components before digestion, digestion MFA-PC2, d4.3 and creaming index (Cri) (Figure 9A). As shown in Figure 9B, PC1 contrasted the samples pasteurised at 66°C (right side) with the samples pasteurised at 60°C (left side). The control and the samples pasteurised at 62 and 64°C were in the middle. PC2 contrasted the samples pasteurised 7 min at 64°C (upper side), with the samples pasteurised 7 and 10 minutes at 60°C and all the samples pasteurised at 66°C (lower side), other samples being in the middle (Figure 9B).

Samples pasteurised at 60°C had thus the best interfacial properties since they were more surfaceactive (S10 increases) and generated more stable emulsions (Cri decreased) with smaller droplets (d4,3 decreased). They were also the more sensitive to protein in vitro digestion since they had the highest DHgast and DHint and the highest concentration in low molecular weight components at the end of intestinal stage as suggested by the positive correlation with digestion MFA-PC1 (Figure 9A). At least, pasteurisation at 60°C increased slightly lysozyme antigenicity but kept ovotransferrin antigenicity to a low level compared to the other samples. An increase in pasteurisation temperature then increased samples coordinates on global MFA-PC1 (Figure 9B). Increasing pasteurisation temperature thus decreased interfacial and emulsifying properties, protein digestibility, lysozyme antigenicity but increased ovotransferrin antigenicity.

Finally, thanks to multiple factorial analysis that enabled to consider all the dimensions of the study at once, a good compromise to improve the interfacial properties of whole egg, without enhancing too much protein resistance to digestion and/or protein antigenicity could be proposed: low pasteurisation treatment of 4 to 10 min at 60°C. However, for hygienic reasons, pasteurisation can also be performed during 4 to 10 min at 62 and 64°C without excessively damaging functional properties or enhancing protein resistance to digestion and/or protein antigenicity. On the contrary, pasteurisation at 66°C should be avoided to prevent resistance to digestion and ovotransferrin antigenicity increase.

  [START_REF] Jimenez-Saiz | Human immunoglobulin E (IgE) binding to heated and glycated ovalbumin and ovomucoid before and after in vitro digestion[END_REF]

  using a two-stage high-pressure homogeniser (Rannie LAB 16/15, APV France, Evreux, France) at 5 Mpa. Heat treatments of homogenised whole egg were performed with an indirect tubular processing system (Microthermics UHT/HTST Lab 25 EDH, Microthermics, Inc., Raleigh, NC 27615, USA) according to the following complete factorial design 4x3: whole egg was heated to 60, 62, 64 or 66°C and held at temperature for 4, 7 or 10 min. The pasteurisation temperature curves are given in Supplementary Data 1. After cooling to 25°C, glass bottles (volume 250 mL) were filled and then frozen. Homogenised raw whole egg was also frozen as a control. The time-temperature rates applied enables to reach from 23 to 1243 decimal reductions of Salmonella enteritidis according to D and z

  per g of protein. The DH at the end of the gastric and the intestinal stages were kept as results.2.3.3.

  . MFA balances the influence of the groups on the first principal dimension by dividing the weights of the variables (columns) of a group by the first eigenvalue of the separated PCA of this group. The highest axial inertia of each group is standardized at 1. MFA provides the classical results of principal component methods. PCA characteristics and interpretation rules are retained. MFA offers tools for comparing the different groups such as the partial representation of the individuals (rows). This representation allows comparison of the typologies provided by each group in a common space. In other words, it allows comparison of the PCA results in a common space. A graph of the groups allows global comparison of the groups and evaluation if the relative positions of the individuals are globally similar from one group to another. It also allows the comparison of partial M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 10 groups and assesses whether they provide the same information. Another graph gives the correlations between the dimensions of the MFA and each separate analysis (PCA dimensions).

  Pasteurisation under 66°C minimizes protein aggregationWhole egg protein profile was analysed by SDS-PAGE for each pasteurised sample and the control.The volume of the protein bands measured on SDS-PAGE gels before digestion (gast ti samples) were submitted to PCA to have a global overview of the effect of pasteurisation on protein profile. The graph of the variables, i.e. of the electrophoretic band volumes, on the first two dimensions is given in Figure1A. PC1 and PC2 explain 28% and 21.6% of the variability of the data set, respectively. PC1 is positively correlated to the volume of the band at 159, 138, 97, 94, 75 and 51 kDa, and negatively correlated to the volume of the band at 106 kDa (Figure2A). PC2 is positively correlated to the volumes of the band at 198, 129, 118 and 14 kDa. Pasteurisation temperature and time are not well correlated with PC1 and PC2 assuming that pasteurisation treatment is not responsible for the heterogeneity of samples electrophoretic patterns. However, the samples pasteurised at 66°C, whatever the time, have especially low and high coordinates on PC1 and PC2, respectively (Figure2B). These samples have thus more components of 198, 129, 118, 106 and 22 kDa and less components of159, 138, 97, 94, 75 and 51 kDa than the other samples. The bands at 198, 118, 106 and 22 kDa are only present in pasteurised samples and specifically in samples pasteurised at 66°C (data not shown). On the opposite, the band at 51 kDa has completely disappeared in samples pasteurised at 66°C and the volume of the bands at 159 and 138 kDa decreases. The occurrence of high molecular weight components at the expense of lower molecular components suggests protein aggregation for the highest pasteurisation treatment, i.e. from a few minutes at 66°C. This is in agreement with[START_REF] Hamid-Samimi | Flow behaviour of liquid whole egg during thermal treatments[END_REF] who observed a rapid decrease of soluble protein in the pasteurisation temperature range 67.5-72.5°C. The analysis of whole egg electrophoretic pattern thus confirms that pasteurisation temperature should stay below 66°C to prevent protein aggregation.3.2.Pasteurisation at 60°C maximizes whole egg interfacial properties

  PC1 positively correlated to the band volumes of the 51, 31 and 10 kDa bands of gast tf stage, and40, 31, 23, 22, and 14.5 kDa bands of int tf stage (p<0.05). It also significantly negatively correlated to the band volumes of the 129 kDa bands of gast ti stage (Figure7A). PC2 significantly (p<0.05) positively correlated to the band volumes of the 106, 86 and 22 kDa bands of gast ti stage, and 86, 68 and 55 kDa bands of gast tf stage. It also significantly negatively correlated to the band volumes of the 159, 138, 97, 75 and 44 kDa bands of gast ti stage and 40 kDa band of gast tf stage (Figure7A). Pasteurisation time also positively correlated with PC2 (correlation coefficients equal to 0.41). Thus, when the intensity of pasteurisation increased, the band volumes of the proteins positively correlated to PC2 also tended to increase, whereas the band volumes of those negatively correlated rather decreased. PC2 is correlated to the band volume of samples of the gastric stages only; it is thus representative of the effect of the gastric step of the in vitro digestion. Its correlation with pasteurisation time suggests that the progress of protein digestion during the gastric stage depends on pasteurisation time. The band volumes of the samples of the intestinal stage are correlated only to PC1, which is thus representative of the intestinal stage of the in vitro digestion. Partial point positions on the PC1-PC2 plot give information on SDS-PAGE profiles before digestion (red points), at the end of gastric stage (green points), and at the end of intestinal stage (blue points). The way from one point to another is representative of what happens during gastric (red arrows) and intestinal (blue arrows) stages. Figure 7B red points), control sample and mid pasteurised samples (i.e. 7 and 10 min at 60°C and 62°C and 4 and 10 min at 64°C) are close to the centre of the plot whereas low pasteurised samples (i.e. 4 min at 60°C and 62°C and 7 min at 64°C) have negative coordinates along PC2 and high pasteurised samples (i.e. 4, 7 and 10 min at 66°C) have positive coordinates along PC2.Low (respectively high) pasteurised samples have thus higher (respectively lower) concentration of 159, 138, 97, 75 and 44 kDa components than the control group in agreement with the results of the PCA given in the section 3.1.For most samples (except samples pasteurised for 10 min at 60°C and for 7 and 10 min at 66C°), red arrows are mostly vertical suggesting that during the gastric stage, changes occurred according to PC2.For most samples, partial points at the end of gastric stage (Figure7B, green points) are farer from the centre of the plot than the corresponding partial points before digestion. Gastric stage thus increases electrophoretic pattern heterogeneity. At the end of gastric stage (Figure7B, green points), control and low pasteurised samples (i.e. 4 and 7 min at 60°C and 62°C and 7 min at 64°C) have negative coordinates along PC2 whereas mid and high pasteurised samples (4 min at 64°C, 7 min at 66°C and 10 min at 60, 62, 64 and 66°C) have positive coordinates along this PC. Control and low pasteurised samples have thus lower concentration of 86, 68 and 55 kDa components but higher concentration of 40 kDa component than the average whereas it is the opposite for mid and high pasteurised samples.

  pasteurisation intensity (64°C) should be recommended to keep ovomucoïd, lysozyme and ovotransferrin antigenicity as low as possible.3.5. Which conditions of pasteurisation to simultaneously optimize protein interfacial properties, antigenicity and in vitro digestibility ?

  It was also negatively correlated to lysozyme antigenicity, S10, digestion MFA-PC1, DH gast and DH int and electrophoresis band volume of the 51 and 138 kDa components before digestion. PC1 thus contrasted on the right side samples with poor interfacial properties, weak lysozyme antigenicity but strong ovotransferrin antigenicity and poorly hydrolysed during both gastric and intestinal stages of in vitro digestion, with on the left side samples with good interfacial properties, strong lysozyme antigenicity but weak ovotransferrin antigenicity and highly hydrolysed during both gastric and intestinal stages of in vitro digestion. PC2 was significantly positively correlated to the electrophoresis band volume of the 175, 159, 138, 97, 94 and 75 kDa components before digestion and d4.3 and negatively correlated to the electrophoresis band volume of the 198 and 22 kDa components before digestion and digestion MFA-PC2. PC2 thus contrasted on the upper side samples with aggregates after pasteurisation, poor emulsifying properties and highly hydrolysed during the gastric stage of in vitro digestion, with on the lower side samples with pasteurisation, good emulsifying properties and poorly hydrolysed during the gastric stage of in vitro digestion.

Figure 1 :

 1 Figure 1: In vitro digestion of whole egg followed by SDS PAGE analysis for gastric (A) and intestinal

  Figure 2 :

Figure 3 :

 3 Figure 3: Surface pressure -surface area isotherms obtained by the compression of the film after

Figure 4 :

 4 Figure 4: Droplet size distribution of emulsions (O:W / 30:70) prepared with 1.5% whole egg proteins as

Figure 5 :Figure 6 :

 56 Figure 5: Index of creaming of emulsions (O:W / 30:70) prepared with 1.5% whole egg proteins as a

Figure 7 :

 7 Figure 7: A) Projection of variables (electrophoresis band volumes before digestion (red), at the end of

Figure 8 :

 8 Figure 8: Ovomucoïd (A) , lysozyme (B) and ovotransferrin (C) apparent concentration determined by

Figure 9 :

 9 Figure 9: A) Projection of variables onto the plane defined by the first two principal components of the

Table 1 :

 1 Significant levels of variables and their interaction for the different analytical parameters, and model R² and p-value.

		Temperature Time Temperature x Time	R²	p-value
	PCA PC1	NS	NS	NA	0.73	0.12
	PCA PC2	NS	NS	NA	0.22	0.92
	S 10	***	NS	NS	0.77	0.0088
	d4/3	**	***	**	0.91	7 10 -5
	Cri	***	NS	NS	0.92	4 10 -5
	Lysozyme immunoreactivity	***	**	NS	0.94	3 10 -13
	Ovomucoid immunoreactivity	***	***	NS	0.62	3 10 -5
	Ovotransferrin immunoreactivity	***	*	***	0.92	2 10 -11
	DH gast	***	***	***	0.99 < 2 10 -16
	DH duo	***	***	***	0.99 < 2 10 -16
	Digestion MFA PC1	NS	*	*	0.49	0.09
	Digestion MFA PC2	NS	NS	*	0.48	0.10
	* significant effect at P < 0.05; ** significant effect at P < 0.01; *** significant effect at P < 0.001; NS, no
	significant effect; NA, non available

Funding source :

This work was supported by the French National Research Agency (Grant ANR PNRA 2007 OVONUTRIAL)