Keywords: protéines végétales et animales, isolat de protéines de pois, protéine de blanc d'oeuf, propriétés thermiques, propriétés de gélification, interactions

Etude des interactions et des propriétés physico-chimiques de mélanges de protéines de pois et de blanc d'oeuf : de l'état colloidal à l'état gélifié.

Résumé: Ces dernières années, en raison de la croissance de la population mondiale, la consommation de protéines animales a augmenté, entraînant une augmentation des émissions de gaz à effet de serre et de l'occupation des sols. Il est donc nécessaire de remplacer partiellement les protéines animales par des protéines végétales pour augmenter la part de protéines végétales dans la consommation quotidienne, et développer des systèmes alimentaires mixtes semble être une solution adéquate. Les protéines de pois en tant que source de protéines végétales ont de bonnes propriétés nutritionnelles mais avec des propriétés fonctionnelles limitées tandis que le blanc d'oeuf (EW) a de bonnes propriétés fonctionnelles telles que la gélification et le foisonnement. Ainsi, les interactions physicochimiques des mélanges des deux types de protéines doivent être comprises pour développer des ingrédients protéiques hybrides. Dans le présent travail, des protéines de pois sous forme d'isolat de protéines de pois (PPI) et d'EW ont été préparées à une concentration totale de protéines de 10 % w/w. Les interactions colloïdales en solution, les propriétés thermiques et de gélification des mélanges ont été étudiées en fonction de différents rapports pondéraux (PPI/EW, 0/100, 25/75, 50/50, 72/25. 100/0) et de différents pH (7.5 et 9). Pour comprendre les interactions dans les mélanges PPI-EW, le PPI en mélange avec différentes fractions protéiques EW (ovalbumine (OVA), ovotransferrine (OVT) et lysozyme (LYS)) a été analysé par des méthodes combinées telles que la calorimétrie de titrage isotherme (ITC), la diffusion dynamique de la lumière (DLS), la granulométrie laser, la microscopie De fortes interactions exothermiques entre PPI et LYS conduisent à une agrégation via des interactions électrostatiques. Aucune ou de très faibles interactions n'ont été détectées entre OVT ou OVA et PPI quel que soit le pH. Les propriétés thermiques et de gélification des systèmes mixtes ont été caractérisées par le profil de solubilité avec le pH, la SDS-PAGE, la calorimétrie différentielle à balayage (DSC), la rhéologie dynamique (25-95-25 °C), les analyses de texture, le CLSM et l'ajout d'agents dissociants après gélification. La température de dénaturation thermique (Td) et l'enthalpie sont peu ou pas influencées en fonction du pH. La Td d'OVT, de LYS et de légumineuses a changé en raison des interactions entre les protéines. La formation de gel était régie par les protéines EW et le module élastique (G ') diminuait avec la teneur en EW dans les mélanges. Le point de gélification de l'OVT (~ 59 °C) a augmenté d'environ 3 ℃, ce qui donne une indication intéressante pour une application ultérieure du traitement de pasteurisation. Parallèlement, des gels obtenus à partir de protéines mixtes (contenant au moins 50 % de blanc d'oeuf) étaient constitués d'un réseau de protéines de blanc d'oeuf, incluant des agrégats de protéines de pois. Des interactions fortes incluant des ponts disulfures et des interactions hydrophobes ont été à l'origine de la structure du réseau du blanc d'oeuf. Des interactions faibles, y compris plus de liaisons hydrogène, pourraient expliquer les propriétés viscoélastiques inférieures des gels à base de PPI. Dans les gels mixtes, les protéines du blanc d'oeuf constituent l'architecture de base du réseau protéique. La gélification de l'EW s'accompagne de la formation confocale à balayage laser (CLSM) et la microscopie optique. d'agrégats de protéines qui peuvent être des agrégats de PPI purs ou des agrégats mixtes constitués de globulines de pois et de certaines protéines de blanc d'oeuf. D'autres investigations sur la fonctionnalité des protéines (foisonnement) et l'effet des traitements de fonctionnalisation tels que l'étuvage de la poudre sont attendues.

Résumé

Title : Study of the interactions and physicochemical properties of pea and egg white protein mixtures: from the colloidal to the gelled state Keywords : Pea protein isolate, Egg white protein, Functionality, Thermal properties, Gelation properties, Interaction Abstract : In recent years, due to the growth of world population, people's consumption of animal protein has increased, leading to the increase of greenhouse gas emissions and land occupation. Therefore, it is necessary to partially replace animal proteins with plant proteins to increase their proportion in daily consumption and developing mixed food systems seems to be one adequate solution. Pea proteins as a source of plant proteins have good nutritional properties but with limited functional properties while egg white (EW) has good functional properties such as gelling and foaming. Thereby, the physicochemical interactions from mixtures of both types of protein needs to be understood to develop hybrid protein products. In the present work, pea proteins as pea protein isolate (PPI) and EW were prepared at total protein concentration of 10% w/w. The colloidal interactions in solution, thermal and gelation properties of the mixtures were studied as a function of different weight ratios (PPI/EW, 0/100, 25/75, 50/50, 72/25. 100/0) and different pH (7.5 and 9). To understand the interactions in PPI-EW mixtures, PPI in admixture with different EW protein fractions (ovalbumin (OVA), ovotransferrin (OVT), and lysozyme (LYS)) was analyzed by combined methods such as isothermal titration calorimetry Thermal and gelation properties of the mixed systems were characterized through solubility profile with pH, SDS-PAGE, Differential Scanning Calorimetry (DSC), dynamic rheology (25-95-25 °C), texture analyses, CLSM, and the addition of dissociating agents after gelation. Thermal denaturation temperature (Td) and enthalpy were slightly or not influenced depending on the pH. Td of OVT, LYS and legumin changed due to the interactions between proteins. Gel formation was governed by EW proteins and elastic modulus (G') decreased with EW content in mixtures. Gelation point of OVT (~59 °C) increased around 3 ℃ giving an interesting indication for further pasteurization treatment application. Meanwhile, gels obtained from mixed proteins (containing at least 50 % egg white) were constituted of a network of egg white proteins, including aggregates of pea proteins. Strong interactions including disulfide bonds and hydrophobic interactions were at the origin of the structure of the egg white network. Weak interactions including more hydrogen bonds could explain the lower viscoelastic properties of PPI-based gels. In mixed gels, the egg white proteins constitute the basic architecture of the protein network. The gelation of EW is accompanied by the formation of protein aggregates which may be pure PPI aggregates or mixed aggregates (ITC), dynamic light scattering (DLS), laser granulometry, confocal laser scanning microscopy (CLSM) and optical microscopy. Strong exothermic interactions between PPI and LYS lead to aggregation via electrostatic interactions. No or very weak interactions were detected between OVT or OVA and PPI whatever the pH.

consisting of pea globulins and some egg white proteins. Further investigations on protein functionality (foaming) and effect of functionalization treatments such as dryheating of powder are expected.

Accomplishments

This thesis was carried out in cooperation between L'institut Agro Dijon, within the PCAV team (physico chemistry of food and wine) belonging to the UMR PAM (mixed research unit for food and microbiological processes) and L'institut Agro Rennes-Angers within the UMR STLO (Science and Technology of milk and egg) research unit. Xiong, as well. It is a kind of fate that we can meet together abroad. We all live, work and play together during the four years making me don't feel lonely.

I would like to thank to the China Scholarship Council, especially for the funding for my academic life in France making me concentrate more on my studies without any worries.

Finally, I want to thank my family members and friends in China, thank you very much for your support during my Ph.D., especially the encouragement and comfort during the period of the thesis manuscript. It's my honor to have all of you in my life.

Jian KUANG, Valérie LECHEVALIER, Rémi SAUREL. Interactions between isolated pea globulins and purified egg white proteins

Protein interactions and assemblies Intra-INRAE Rennes 20 th June 2022

Table of Contents

List of Figures

Figure 1-1 Model of the bipyramidal trigonal quaternary structure possible for dicotyledon 11S globulins. Each sphere includes an acidic and basic polypeptide that makes up the legume components (Marcone, Kakuda & Yada, 1998a).

Figure 1-2: Assembly of 7S vicilin fragments, and potential cleavage sites according to [START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF] Figure 1-3 Assembly of constituent 2S Albumin according to [START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF] Figure 1-4: Main steps in the extraction of pea protein materials, according to [START_REF] Boukid | Pea protein ingredients: A mainstream ingredient to (re) formulate innovative foods and beverages[END_REF][START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF][START_REF] Saurel | Protéines de pois : structure, extraction et fonctionnalités[END_REF] Figure 12345The structure of different components in an egg [START_REF] Froning | Encyclopedia Britannica[END_REF]. [START_REF] Zhu | Relationship between Molecular Structure and Heat-Induced Gel Properties of Duck Myofibrillar Proteins Affected by the Addition of Pea Protein Isolate[END_REF].

Figure 1-8 Increase in the critical gelling temperature (Tc) of casein micelles in the presence of globular proteins (whey, pea or soy) explained by the difference in protein affinity for calcium ions (from [START_REF] Silva | Heatinduced gelation of aqueous micellar casein suspensions as affected by globular protein addition[END_REF].

Figure 1-9 Storage modulus at 0.1 Hz after 1 h heating at 90 °C for (a) individual and mixed MC-SP and (b) individual and mixed MC-PP systems at 6% w/w total protein as a function of MC and plant protein content in the mixtures at pH 5.8 [START_REF] Silva | Heatinduced gelation of mixtures of micellar caseins and plant proteins in aqueous solution[END_REF].

Figure 1-10 Scanning electron micrographs of the SPI/EW protein gels at 0.06 g mL -1 protein concentration. The pictures marked with a-g represent the gels at different SPI/EW ratios. a 1:0, b 9:1, c 3:1, d 1:1, e 1:3, f 1:9, g 0:1. (Su et al., 2015) Figure 1-11 Production diagram of processing egg products (according to [START_REF] Anton | Les ovoproduits: des ingrédients fonctionnels pour des matrices complexes[END_REF]Suman et al., 2018). Figure 3-1 SDS-PAGE profile of extracted pea protein isolate and egg white when prepared at pH 7.5 and 9. The samples on lanes 2, 4, 6, and 8 were treated under reducing conditions with SDS+DTT reagents. Circles in red and blue were the aggregates of PPI and egg white, respectively at the top of the bands. Lane 1-4 at pH 7.5, Lane 5-8 at pH 9; Lane M: molecular weight (Mw) markers; lanes 1-2, and 5-6: PPI; lanes 3-4, and 7-8: EW; LP, lipoxygenase; L (α, β), legume; CV, convicilin; Lα, legume acid polypeptide; Lβ, legume basic polypeptide; V, vicilin; OVA, ovalbumin; OVT, ovotransferrin; LYS, lysozyme. Figure 4-1: Thermograms for the titration of PPI (0.0078 mM) with OVA (1.6 mM) in HEPES buffer pH 7.5 (A1), with OVA (1.7 mM) in Tris-HCl buffer pH 9 (A2), with OVT (0.64 mM) in HEPES buffer pH 7.5 (B1), with OVT (0.68 mM) in Tris-HCl buffer pH 9 (B2), with LYS (0.92 mM) in HEPES buffer pH 7.5 (C1), with LYS (1.02 mM) in Tris-HCl buffer pH 9 (C2), with LYS (0.89 mM) in Tris buffer pH 7.5 (C3). All the titration experiments were performed at 25 °C. Figure 5-2: SDS-PAGE profile of PPI-EW suspensions prepared at pH 7.5 (Lanes 1-4) and 9 (Lanes 5-8), at the weight ratio 50/50 with and without centrifugation. The samples on lanes 2, 4, 5, and 7 were treated under reducing conditions with SDS+DTT reagents. Lane M: molecular weight (Mw) markers; Lanes 1-2, 7-8: PPI-EW at the weight ratio 50/50 without centrifugation; Lanes 3-4, 5-6: PPI-EW at the weight ratio 50/50 with centrifugation; LP, lipoxygenase; L (α, β), legumin; CV, convicilin; Lα, legumin acid polypeptide; Lβ, legumin basic polypeptide; V, vicilin; OVA, ovalbumin; OVT, ovotransferrin; LYS, lysozyme. 2 Emmert & Baker, 1995; 3 Gueguen, 1991; 4 Savage & Deo, 1989; 5 Jackson, Boulter, & Thurman, 1969; 6 O'Kane et al., 2004b; 7 Riblett et al., 2001; 8 Helmick et al., 2021).

List of tables

Table 1-3 Physio-chemical and structural properties of proteins from egg white.

Table 1-4 Amino acid composition of major egg white proteins.

Table 1-5: Summary of the main works on the solubility of pea protein isolate (from [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF][START_REF] Ge | The health benefits, functional properties, modifications, and applications of pea (Pisum sativum L.) protein: Current status, challenges, and perspectives[END_REF].

Table 1-6: Main research work on the heat-induced gelation of pea proteins.

Table 1-7 Research on the techno-functional characteristics of mixed systems between plant protein and animal protein (According to [START_REF] Alves | Mixing animal and plant proteins: Is this a way to improve protein techno-functionalities?[END_REF][START_REF] Schmitt | Heatinduced and acid-induced gelation of dairy/plant protein dispersions and emulsions[END_REF]Hinderink et al., 2021a;[START_REF] Wu | Advancement of food-derived mixed protein systems: Interactions, aggregations, and functional properties[END_REF].

Table 2-1 Procedure of injection and elution of samples. Table 3-1 Thermal denaturation temperature (Td) and enthalpy (ΔH) of PPI at pH 7.5 and 9. Table 3-2 Thermal denaturation temperature (Td) and enthalpy (ΔH) of EW at pH 7.5 and 9.

Table 4-1: The D (4,3) results of PPI-LYS mixtures in TRIS buffer at pH 7.5 and 9.

Table 5- Table 5-2: Thermal denaturation temperatures of EW, PPI, and PPI-EW mixtures at different weight ratios at pH 7.5.

Table 5-3: Thermal denaturation temperatures of EW, PPI, and their mixtures (PPI-EW) at different weight ratios at pH 9.

Table 5-4: Denaturation enthalpy of (∆H) of EW, PPI, and their mixtures (PPI-EW) at different weight ratios at pH 7.5 and 9.

Table 5-5: Gelling point temperature of PPI, EW suspensions, and their mixtures at 10 % protein at pH 7.5 and 9.

Table 5-6: Final G' and G'' values at 95 °C of PPI, EW, and PPI-EW mixtures at different weight ratios at pH 7.5 and 9.

Table 6-1 Effect of various reagents on molecular forces existing in protein Table 6-2: Rheological analysis comparing the storage modulus (G') values for pea protein isolate (PPI), egg white protein (EW), and their composite gels at 95 °C (end of heating cycle), and 25 °C (end of cooling cycle).

Table 6-3 Loss factor of PPI-EW mixtures at different weight ratios, at 1% strain, 1Hz frequency, and frequency slope of G' of PPI-EW mixtures at different weight ratios at pH 7.5 and 9.

Table 6-4 Yield point (%) of PPI, EW, and PPI-EW gels at different weight ratios in strain sweep at pH 7.5 and 9.

Table 6-5: Parameters of texture profile analysis (TPA) of EW 100 and PPI-EW mixture gels at different weight ratios at pH 7.5 and 9.

Main abbreviations

General introduction

As a result of predicted socioeconomic development, the world population will increase by nearly one-third until 2050 (from 6.9 billion in 2010 to 8.5-10 billion in 2050) and global income will triple (from US$68 trillion in 2010 to US$180-290 trillion in 2050) [START_REF] Godfray | Meat consumption, health, and the environment[END_REF]. This results in a twofold need for animal protein among humans. This situation of growing animal protein consumption is a ticking time bomb in terms of sustainability and food security, as noted by various United Nations assessments (FAO, 2011, United Nations, 2015). However, raw animal materials like milk, eggs, meat, and seafood continue to be the most important sources of protein recently employed by food companies, followed by plant sources like legumes and nuts [START_REF] Alves | Mixing animal and plant proteins: Is this a way to improve protein techno-functionalities?[END_REF]. Meanwhile, animal protein production is connected with high greenhouse gas emissions and increased land requirements, whereas plant proteins have a lower economic cost and lower ecological footprint (Davis, Sonesson, Baumgartner & Nemecek, 2009;[START_REF] Alves | Mixing animal and plant proteins: Is this a way to improve protein techno-functionalities?[END_REF]. Legumes proteins, on the other hand, are produced for animal feed yet having physicochemical features that make them valuable for human consumption [START_REF] Boye | Pulse proteins: Processing, characterization, functional properties and applications in food and feed[END_REF]. Furthermore, excessive intake of animal proteins can have a severe influence on human health, including the development of illnesses such as obesity, cardiovascular disease, neurological disorders, allergies, and so on (Hertzler et al., 2020). As a result, the partial substitution of animal protein with plant protein is gaining popularity in designed goods. They are frequently sold as "healthier" (than meat) and sustainable new foods as "substitutes" for traditional animal-derived food items (Godfray, 2019).

Soy-based protein food businesses now provide a wide range of food items. Over the last decade, worldwide soybean output has expanded dramatically (Qin, [START_REF] Wang | Ovomucin may be the key protein involved in the early formation of egg-white thermal gel[END_REF]. This vegetable raw material's extensive use in many culinary items has eclipsed other kinds of plant proteins, such as pea proteins. However, food manufacturers' concerns are shifting in favor of other diverse plant sources, like pea protein, to counteract soy protein's monopoly. Recently, there has been a lot of attention in pea proteins (Pisum Sativum L), which have a lot of promises in the food supply because of their high yields and low pricing (Munialo et al., 2014a;O'Kane et al., 2004b). Peas are one of the world's most frequently farmed and consumed legumes, namely in Canada, France, China, Russia, and the United States (Boy et al., 2010;[START_REF] Burger | Recent progress in the utilization of pea protein as an emulsifier for food applications[END_REF]. Pea proteins have comparable functional qualities as soy proteins, such as emulsification, however it is non-allergenic [START_REF] Aluko | Emulsifying and foaming properties of commercial yellow pea (Pisum sativum L.) seed flours[END_REF]. This protein source is thought to be a viable alternative to animal and soy proteins [START_REF] Liang | pH-dependent emulsifying properties of pea [Pisum sativum (L.)] proteins[END_REF][START_REF] Burger | Recent progress in the utilization of pea protein as an emulsifier for food applications[END_REF]. However, there are some limits for pea proteins to be used as an ingredient, primarily due to a lack of understanding of their structure and functional features [START_REF] Adebiyi | Functional properties of protein fractions obtained from commercial yellow field pea (Pisum sativum L.) seed protein isolate[END_REF][START_REF] Liang | pH-dependent emulsifying properties of pea [Pisum sativum (L.)] proteins[END_REF].

Egg is well-known for its high nutritional content, great digestibility, and full essential amino acid supply. Moreover, egg proteins are the most sustainable animal proteins. The application of egg is thus wild in food industry: for example, whole egg is used in bakeries; egg yolk in mayonnaise, salad dressing, noodles or ice creams, egg white in bakeries, confectioneries, meringue. Different egg products used as food ingredients can be found on the market such as whole egg, egg yolk and egg white, either in pasteurized liquid, frozen or powder state.

Egg white, especially is widely used for its foaming and gelling properties.

Proteins indeed account for more than 90% of the dry substance in egg white, giving it its single functional properties. The water-holding capacity or gel strength of food items can be increased by using egg white as an additive. Because of its low lipid content, egg white is frequently favored over egg yolk for its gelling capabilities [START_REF] Mine | Egg proteins[END_REF].

Many food items' textural and rheological features, such as meringues and angel food cakes, are dependent on the heat coagulation or gelation capabilities of egg proteins, particularly their irreversible heat coagulation [START_REF] Ren | Nutritional and health attributes of eggs[END_REF]. A thermally irreversible gel is a viscoelastic solid created by heat that does not reheat to a viscous liquid.

Studies dealing with partial substitution of animal protein by plant protein mainly deals with milk or meat proteins as animal sources. There is thus currently a lack of research on egg white protein as an animal source of protein blended with plant protein.

Few studies, however, were dedicated to the study of gelation and thermal aggregation of soy protein mixed with egg white protein (Su et al., 2015;[START_REF] Zhang | Heat stability and rheological properties of concentrated soy protein/egg white protein composite microparticle dispersions[END_REF], or hemp seed protein and egg protein cold gel (Alavi, Emam-Djomeh, & Chen, 2020. Moreover, traditional process for ovoproducts such as egg white which requires a liquid pasteurized form or dried form to produce the products, leading us to think use a process similar to ovoproduct by using a mixed ingredient. Therefore, the knowledge of thermal properties of the mixture systems is required, which will provide more information for further pasteurization. Furthermore, there are some products on the market that using egg white as glue in meat analogue applications, such as vegetable steaks and burgers, meat balls….; that justifies the gelling properties study. In addition, no studies have been reported on mixtures of pea and egg white proteins. Pea proteins doesn't have nice gelling properties as good as soy proteins, and it seem interesting to use mixture with egg white to improve this property. As a result, the usage of pea proteins combined with egg white proteins as an ingredient requires thorough investigations of the nature of the interactions between these proteins of different nature. Our research is centered on determining the nature of intermolecular interactions involved in the probable connection of pea proteins and egg white, thermal properties of such mixtures, as well as their effects on the meso-structural and textural aspects of mixed gels. Pea globulins was preferred in this thesis as it higher content in pea flours and lack of the studies of albumins regarding gelling and thermal behaviour. This thesis has the primary objective to study the interactions between egg white and isolated pea globulins under different experimental conditions. Secondly, we will be interested in the thermal properties of mixed systems and the formation of heatinduced gels from egg white and pea globulins. To have a better understanding of the intermolecular interactions of the egg white and pea globulins, we used purified ovalbumin, ovotransferrin, and lysozyme, as well as pea globulins obtained by alkalineisoelectric extraction. Subsequently, egg white proteins and pea globulins were mixed at different ratios and pHs to study the thermal properties and gelation properties through various scales (molecular, microscopic, and macroscopic), in order to better understand the mechanisms involved during this gelation.

In the following sections, we will firstly introduce the primary data to understand the materials in the bibliographic review (Chapter 1), including a full description of the technique employed in the objective of the study section. The second section of the thesis will describe the materials and methods employed in the execution of this work (Chapter 2 Materials and methods). The experimental section of the manuscript will be separated into four chapters (Chapter 3: Preparation and characterization of protein materials; Chapter 4: Interactions between isolated pea globulins and purified egg white proteins in solution; Chapter 5: Thermal behavior of pea and egg white protein mixtures;

Chapter 6: Nature of protein-protein interactions during gelation of mixtures between pea protein isolates and egg white proteins). A final chapter of the manuscript will be the conclusions on the thermal and gelation properties of the mixed system, as well as the perspective of this study.

Chapter 1 Literature review

Pea protein

Peas, notably the yellow or green cotyledon variants known as dry, smooth, or field peas, are naturally dried seeds of Pisum sativum L. and are one of the world's oldest domesticated crops, which are farmed for human and animal nutrition all over the world [START_REF] Smýkal | Pea (Pisum sativum L.) in the genomic era[END_REF]. It belongs to the Leguminosae family (Genus: Pisum, subfamily: Papilionoideae tribe: Fabaceae), and covers more than 34.2 % of the total area under dry pulse [START_REF] Smýkal | Pea (Pisum sativum L.) in the genomic era[END_REF]Eurostat, 2020).

The yellow pea seeds contain approximately 60-65 % of carbohydrates of which 35-40% of starch (24-49 % amylose), 23-30 % of proteins, 1-2 % of lipids, and minor elements such as minerals, vitamins, polyphenols, phytic acid and so on, depending on the difference of cultivar, culture conditions and the stage of maturity of the grain at the time of harvest [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF][START_REF] Bogahawaththa | Impact of selected process parameters on solubility and heat stability of pea protein isolate[END_REF][START_REF] Lu | Composition, physicochemical properties of pea protein and its application in functional foods[END_REF]Eurostat, 2020;[START_REF] Saurel | Protéines de pois : structure, extraction et fonctionnalités[END_REF]. In addition to starch, its carbohydrate also comprises dietary fiber (10-15 % insoluble part and 2-9 % soluble part) as well as non-starch carbohydrates for instance cellulose, sucrose, and oligosaccharides [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF][START_REF] Hoover | Composition, molecular structure, properties, and modification of pulse starches: A review[END_REF].

Pea globulins

Pea globulins (Glob) are so-called storage proteins that provide nutrients (nitrogen, sulfur, and carbon) for seedlings during germination [START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF]. In most dicotyledonous plants, the nitrogen-containing portion of the protein origin is dominated by globulins (7S / 11S), which account for 65 % to 80 % of total protein nitrogen, while the remainder is attributable to albumin (2S) and other insoluble proteins [START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF][START_REF] Kimura | Comparison of Physicochemical Properties of 7S and 11S Globulins from Pea, Fava Bean, Cowpea, and French Bean with Those of Soybean-French Bean 7S Globulin Exhibits Excellent Properties[END_REF]. According to [START_REF] Mertens | Agronomical factors influencing the legumin/vicilin ratio in pea (Pisum sativum L.) seeds[END_REF] found that the ratio of legumin to vicilin is close to 2:1 and legumin contains more sulfur-containing amino acids than vicilin per unit of protein. Differences in content, composition, and structure between legumin and vicilin show up in both nutritional and functional features, with their association dissociation properties and surface structures being the most critical elements in understanding pea protein functioning [START_REF] Barać | Profile and functional properties of seed proteins from six pea (Pisum sativum) genotypes[END_REF]). It should be mentioned that pea protein has a comparable protein structure to soybean proteins in three-dimensional structures and protein sequences, resulting in similar functional properties, except for pea protein having fewer allergenic aspects, a lower amount of sulfur amino acids, and being less digestible [START_REF] Fischer | Pisum sativum vs Glycine max, a comparative review of nutritional, physicochemical, and sensory properties for food uses[END_REF].

11S legumin

Pea legumin has a compact hexametric quaternary structure that is supported by electrostatic and hydrophobic interactions and has a molecular weight of 360 to 410 kDa (Marcone, Kakuda, & Yada, 1998b;[START_REF] Boye | Pulse proteins: Processing, characterization, functional properties and applications in food and feed[END_REF]. They are composed of 60 kDa monomers at pH levels ranging from 7 to 9. Each legume monomer is made up of two acid and basic polypeptides of 38-40 kDa and 19-24 kDa, respectively, that are covalently connected by a disulfide bond [START_REF] Croy | The purification and characterization of a third storage protein (convicilin) from the seeds of pea (Pisum sativum L.)[END_REF][START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF] (Figure 1-1). [START_REF] Plietz | The Structure of 11-S Globulins from Sunflower and Rape Seed: A Small-Angle X-Ray Scattering Study[END_REF] proposed that all 11S globulins from legumes had highly similar quaternary structures, using a bipyramidal trigonal structural model. Meanwhile, previous literature conducted by [START_REF] Gueguen | Quantitative and qualitative variability of pea (Pisum sativum L.) protein composition[END_REF], [START_REF] Karaca | Emulsifying properties of chickpea, faba bean, lentil and pea proteins produced by isoelectric precipitation and salt extraction[END_REF][START_REF] Karaca | Emulsifying properties of chickpea, faba bean, lentil and pea proteins produced by isoelectric precipitation and salt extraction[END_REF]Marcone et al. (1998b), proved that basic polypeptides with a hydrophobic nature were found in the protein's core, whereas acidic polypeptides are found on the protein's surface. Therefore, this arrangement would clarify the orientation of the α polypeptides, which are more hydrophilic on the exterior, and β polypeptides, which are more hydrophobic on the inside of the oligomeric protein (Figure 1-1). The monomers' secondary structure is composed of 45% β-sheets and 15% α-helices [START_REF] Matta | Molecular and subunit heterogeneity of legumin of Pisum sativum L.(garden pea)-a multidimensional gel electrophoretic study[END_REF].

Figure 1-1 Model of the bipyramidal trigonal quaternary structure possible for dicotyledon 11S globulins. Each sphere includes an acidic and basic polypeptide that makes up the legume components (Marcone, Kakuda, & Yada, 1998a).

Acidic polypeptides have an isoelectric point (pI) between 4.5 and 5.8, whereas basic polypeptides have a pI between 6.2 and 8.8 [START_REF] Matta | Molecular and subunit heterogeneity of legumin of Pisum sativum L.(garden pea)-a multidimensional gel electrophoretic study[END_REF][START_REF] Heng | Protein-flavour interactions in relation to development of novel protein foods[END_REF].

Thus, the typical pI value of legumes is between 4.8 and 4.9 [START_REF] Chihi | Etude des interactions dans la formation d'agrégats thermiques mixtes entre globulines de pois et béta-lactoglobuline: application à l'élaboration de gels acides[END_REF]. Ionic strength and pH have a deep influence on legumin structure, according to the report of Subirade, Gueguen, & Schwenke (1992), Gueguen, Chevalier, Barbot, & Schaeffer (1988) and [START_REF] Gueguen | Relation between conformation and surface hydrophobicity of pea (Pisum sativum L.) globulins[END_REF], legume dissociated into a mixture of trimers, dimers, and monomers even though it was in the form of non-aggregated at pH 7 with an ionic strength higher than 0.1 M, and extreme acidic or basic pHs depending on ionic strength.

Thus, this kind of change in the state increased the surface hydrophobicity of these proteins.

7S vicilin

Vicilin is a glycosylated trimeric protein with a molecular weight of 150-200 kDa, nevertheless, although having lower molar weights, the group of 7S vicilins looks more diverse, and its structure is not well known (O'Kane et al., 2004b;[START_REF] Saurel | Protéines de pois : structure, extraction et fonctionnalités[END_REF]. Vicilins are abundant in glutamic acid, aspartic acid, and lysine with a low methionine concentration but are devoid of cysteine, resulting in the non-formation of intramolecular or intermolecular disulfide bonds [START_REF] Gatehouse | Characterisation and subunit structures of the vicilin storage proteins of pea (Pisum sativum L.)[END_REF][START_REF] Shewry | Seed storage proteins: structures and biosynthesis[END_REF]. Therefore, [START_REF] Sikorski | Functional properties of proteins in food systems[END_REF] reported that vicilin is bound together by hydrophobic contacts rather than covalent disulfide bonds in contrast to legumin.

The vicilins are primarily composed of three subunits of ~50 kDa, each monomer has a more hydrophilic surface than legumin and can be cleaved into a variety of low molecular weight fragments (α,β, and γ), as illustrated in Figure 1-2 [START_REF] Gatehouse | The posttranslational proteolysis of the subunits of vicilin from pea (Pisum sativum L.)[END_REF]O'Kane et al. 2004aO'Kane et al. , 2004b;;[START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF]. In detail, the 50 kDa precursor has two potential cleavage sites, denoted by the letters α: β and β:

γ [START_REF] Matta | Molecular and subunit heterogeneity of legumin of Pisum sativum L.(garden pea)-a multidimensional gel electrophoretic study[END_REF][START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF]. Cleavages at both locations result in fragments of 20 kDa (α), 13 kDa (β), and 12-16 kDa (γ). The cleavage between α and β results in pieces of 20 kDa (α) and 25-30 kDa (β+γ), whereas cleavage between β and γ results in fragments of 30-36 kDa (α+β) and 12-16 kDa (γ) [START_REF] Gatehouse | The posttranslational proteolysis of the subunits of vicilin from pea (Pisum sativum L.)[END_REF].

In addition, the γ-subunit is occasionally N-glycosylated around the C terminus [START_REF] Spencer | Sequence interrelationships of the subunits of vicilin from pea seeds[END_REF]. Serine, glutamic acid, and aspartic acid are the most common Nterminal amino groups [START_REF] Sikorski | Functional properties of proteins in food systems[END_REF]. While amino acid fingerprinting revealed 70-80 % commonality between legumin and vicilin from the Vicieae (or Fabaceae) tribe, N-terminal analysis revealed more variability [START_REF] Jackson | A comparison of some properties of vicilin and legumin isolated from seeds of Pisum sativum, Vicia faba and Cicer arietinum[END_REF].

Secondary structures of the legumin and vicilin proteins are dominated by β-sheets [START_REF] Sikorski | Functional properties of proteins in food systems[END_REF]. Despite its structural diversity, vicilin's pI ranged between 5.4 and 5.5 [START_REF] Derbyshire | Legumin and vicilin, storage proteins of legume seeds[END_REF].

7S convicilin

Convicilin is a third-storage protein that may be found in peas and other pulses.

The convicilins are made up of subunits of around 71 kDa that are most likely connected in trimers with a molar mass of 210-280 kDa [START_REF] Barać | Profile and functional properties of seed proteins from six pea (Pisum sativum) genotypes[END_REF][START_REF] Boye | Pulse proteins: Processing, characterization, functional properties and applications in food and feed[END_REF][START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF]. According to the report of [START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF], vicilin had an amino acid content that is generally 80 % similar to that of vicilins, as well as a particularly prolonged and charged N-terminal chain of 122 to 166 amino acid residues, except for one cysteine and one methionine residues per convicilin subunits.

2S albumins

Albumin (2S), accounting for 18-25% of total protein, is water soluble metabolic and enzymatic protein including plenty of important amino acids such as tryptophan, lysine, threonine, and methionine [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF]. According to [START_REF] Klupšaitė | Legume: Composition, protein extraction and functional properties. A review[END_REF], albumin comprises components, for instance, enzymes, protease inhibitors, amylase inhibitors, and lectins. Nevertheless, albumins are particularly susceptible to oxidation due to their high cysteine content. The 2S albumin fraction is exceedingly variable in composition and has a low molecular mass, ranging from 5 to 55 kDa. Within this protein group, two primary fractions have been found: a bigger albumin protein called PA2 (5-10 % of total protein) composed of two polypeptides (molecular mass of ~ 26 kDa) and a smaller one called PA1 (4-7 % of total protein) with a molecular mass of 4-6 kDa [START_REF] Boye | Pulse proteins: Processing, characterization, functional properties and applications in food and feed[END_REF]. Notable in the soluble fraction is the presence of lipoxygenase, an enzyme responsible for the oxidation of fatty acids with a molecular mass of 93 kDa; this protein is frequently associated with lipids due to its affinity for fat [START_REF] Saurel | Protéines de pois : structure, extraction et fonctionnalités[END_REF].

PA1 albumins (10-11 kDa) are composed of two different polypeptides of 6 kDa (PA1a, pI 5. 3-5.4) and 4 kDa (PA1b, Pi = 9.8) connected in dimers through non-covalent interactions [START_REF] Bérot | Centrifugal partition chromatography as a tool for preparative purification of pea albumin with enhanced yields[END_REF]. Both PA1a and PA1b contain extremely high cysteine levels (7.5 and 16.2 %, respectively), suggesting that PA1b might be utilized as an insecticide in biocontrol [START_REF] Gressent | Pea Albumin 1 subunit b (PA1b), a promising bioinsecticide of plant origin[END_REF][START_REF] Eyraud | Expression and biological activity of the cystine knot bioinsecticide PA1b (Pea Albumin 1 Subunit b)[END_REF]. PA2 albumins have a molecular mass of (a) 25-26 kDa or (b) 24-25 kDa, linked by non-covalent bonds, with an isoelectric point of 5.2

(O' Kane et al., 2004b). Each subunit includes three cysteine residues in the chain with an intra-disulfide bond and a free sulfhydryl group.

Amino acids of pea proteins

Table 1-2 displays the mean amino acid content of separated pea legumin, vicilin, and convicilin fractions, as well as soybean 11S (glycinin) and 7S (conglycinin)

fractions. The protein composition of legumes is high in essential amino acids (lysine, isoleucine, glutamic and aspartic acids, and arginine) [START_REF] Savage | The nutritional value of peas (Pisum sativum). A literature review[END_REF].

The amino acid contents of pea globulins appear to be very comparable to those of soy globulins. According to O' Kane et al. (2004b), the concentration of sulfur amino acids in soy globulins is low and considerably lower in pea globulins. It has been found

that the amino acid sequences of legume globulins (7S and 11S) are identical; however, the content of these amino acids differs from species to species [START_REF] Kimura | Comparison of Physicochemical Properties of 7S and 11S Globulins from Pea, Fava Bean, Cowpea, and French Bean with Those of Soybean-French Bean 7S Globulin Exhibits Excellent Properties[END_REF] and relies on cultivar and environmental conditions, as well as the process of protein extraction (Stone et al. 2015b).

Extraction processes of pea protein

According to [START_REF] Grandviewresearch | Grandviewresearch Pea protein market size & share |industry growth report[END_REF], the pea global market is expected to grow at a compound annual growth rate (CAGR) of 7.6% between 2020 and 2027. In order to facilitate pea proteins application in products, the structural and functional properties of peas after different extraction methods must first be explored [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF]. Protein isolates of varying quality can be obtained using various procedures, on which their future physicochemical characteristics will depend, for example, pH, ionic strength, number of washes, temperature, extraction equipment, duration of solubilization, filtering or purifying process [START_REF] Feyzi | Grass pea (Lathyrus sativus L.) protein isolate: The effect of extraction optimization and drying methods on the structure and functional properties[END_REF][START_REF] Shanthakumar | The Current Situation of Pea Protein and Its Application in the Food Industry[END_REF].

Dry Fractionation: air classification and size reduction

As shown in Figure 1-4, after pretreatment, pea seeds will follow the dry fractionation which contains two keyways: milling to reduce the particle size and air classification to separate the particle based on their sizes. Thus, pea seeds will be milled through different methods (roller, pin milling, hammer, and stone) to form smaller structures to facilitate the separation of starch granules from the protein matrix, where roller miller is the most utilized technique [START_REF] Boukid | Pea protein ingredients: A mainstream ingredient to (re) formulate innovative foods and beverages[END_REF].

However, different milling parameters can result in two possible consequences: the obtained flour can be too small to separate protein and starch, or the granules can be coarse to not be separated as it is attached to protein and starch. Hence, optimal milling parameters should be chosen to produce consistent size while avoiding disruption of starch granule structure, which might impair starch gelatinization capabilities. In an air separation process, pea flour can be separated into two parts by turboseparation: small particles (protein-rich particles with a size of 1 -3 μm) and coarse particles (starch particles with a size of 2 -40 μm), based on size, shape, and density [START_REF] Boukid | Pea protein ingredients: A mainstream ingredient to (re) formulate innovative foods and beverages[END_REF]. At the end of the dry extraction process, around 50-55 % of pea protein concentrate (fine fraction) based on dry matter, and around 67 % of pea starch (coarse fraction) based on the dry matter can be obtained [START_REF] Pelgrom | Functional analysis of mildly refined fractions from yellow pea[END_REF][START_REF] Saurel | Protéines de pois : structure, extraction et fonctionnalités[END_REF]. Nonetheless, the purity of protein concentrate (50-55 %, dry basis)

obtained from dry extraction is lower than protein isolates (over 80 %, dry basis)

obtained by wet extraction [START_REF] Pelgrom | Functional analysis of mildly refined fractions from yellow pea[END_REF][START_REF] Rempel | Industrial scale preparation of pea flour fractions with enhanced nutritive composition by dry fractionation[END_REF]. The fundamental benefit of dry fractionation over wet extraction is that it preserves the intrinsic quality, nutritional and functional properties of the protein while consuming less energy and water, making it more cost-effective and hence more sustainable [START_REF] Rempel | Industrial scale preparation of pea flour fractions with enhanced nutritive composition by dry fractionation[END_REF][START_REF] Saurel | Protéines de pois : structure, extraction et fonctionnalités[END_REF][START_REF] Boukid | Pea protein ingredients: A mainstream ingredient to (re) formulate innovative foods and beverages[END_REF].

Wet extraction

The most commonly used typical method for producing pea protein isolates (highly concentrated protein fractions) is alkaline extraction/isoelectric precipitation (AE/IEP), which takes advantage of the high solubility of legumin proteins under alkaline conditions and the minimal solubility at isoelectric point (pI) between 4 and 5 using the same solubility for legumin and vicilin [START_REF] Boye | Pulse proteins: Processing, characterization, functional properties and applications in food and feed[END_REF]Qiaoyun et al., 2017;[START_REF] Shanthakumar | The Current Situation of Pea Protein and Its Application in the Food Industry[END_REF]. Some factors during the extraction process, such as the solvent employed, pH, extraction duration, particle size, and flour/water ratio, impact the isolate yield, purity, and functionality of proteins, even though wet extraction may provide high isolation yields of 80-94 % [START_REF] Hoang | Evaluation of pea protein and modified pea protein as egg replacers[END_REF][START_REF] Feyzi | Grass pea (Lathyrus sativus L.) protein isolate: The effect of extraction optimization and drying methods on the structure and functional properties[END_REF]. The water/flour ratio (v/w or w/v) and extraction pH were determined as the most critical factors by [START_REF] Hoang | Evaluation of pea protein and modified pea protein as egg replacers[END_REF]. [START_REF] Boukid | Pea protein ingredients: A mainstream ingredient to (re) formulate innovative foods and beverages[END_REF] obtained the highest protein yield around 80 % at pH 9.96 and water/flour ratio at 15 v/w. However, at high extraction pH (> 10), pea protein extraction has been linked

to increased starch swelling, leading to starch contamination in pea protein isolate products as reported by [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF] and [START_REF] Hoang | Evaluation of pea protein and modified pea protein as egg replacers[END_REF]. Furthermore, while the procedure is under high alkaline pH, high temperature, or longer holding durations, the isolate is more prone to protein denaturation, decreasing its functionality and solubility, despite these conditions could lead to an increase in isolate production [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF]. The size of the flour particles and the type of solubilizing agent used also have an impact on the isolated yield. The ideal AE flour particle size is 100-150 μm, while alkali-like NaOH and KOH are commonly used to enhance protein recovery and yield [START_REF] Owusu-Ansah | Pea proteins: a review of chemistry, technology of production, and utilization[END_REF][START_REF] Del Mar Contreras | Protein extraction from agri-food residues for integration in biorefinery: Potential techniques and current status[END_REF].

As shown in Figure 1-4, defatted pea flour is dispersed in water, the pH is adjusted to an alkaline range using NaOH or KOH and stood for 30-180 min to maximize the protein solubility [START_REF] Boye | Pulse proteins: Processing, characterization, functional properties and applications in food and feed[END_REF][START_REF] Shanthakumar | The Current Situation of Pea Protein and Its Application in the Food Industry[END_REF]. Afterwards, the supernatant is collected by a centrifugal separator to remove the insoluble residues and adjusted to pH close to the pea protein pI (4.5 -5. [START_REF] Vogelsang-O'dwyer | Production of pulse protein ingredients and their application in plant-based milk alternatives[END_REF]. This approach allows to produce more native protein extracts with superior functional characteristics and greater yields than isoelectric precipitation (Mondor, Tuyishime, & Drolet, 2012). Furthermore, this extraction approach has been demonstrated to be beneficial in eliminating antinutrient chemicals from isolate proteins [START_REF] Boye | Pulse proteins: Processing, characterization, functional properties and applications in food and feed[END_REF]. However, one disadvantage of this technique was that it could not modify the amino acid composition of PPI [START_REF] Hadidi | Pea proteins as emerging biopolymers for the emulsification and encapsulation of food bioactives[END_REF].

Salt extraction (SE) extracts proteins from seed materials in salt solutions at neutral Ph, using the advantage of salting-in and salting-out phenomena of proteins [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF][START_REF] Shanthakumar | The Current Situation of Pea Protein and Its Application in the Food Industry[END_REF]. It is generally followed by a desalting progress to reduce the ionic strength of protein [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF]. Pea flour is mixed in a salt solution with a predetermined ionic strength for 10-60 minutes at a ratio of 1:10 (w/v).

By settling, pouring, screening, and centrifuging, insoluble matter is removed. Since proteins precipitate at a variety of ionic strengths, the supernatant is desalted and dried before, subsequently, the concentration and salt combination are determined depending on the salting-in features of the protein to be extracted as well as the salting-out qualities of any undesirable proteins (Stone et al., 2015b). In general, protein salting-in occurs at low ionic strength, between 0.1 and 1 M. SE has the advantage of not requiring excessive alkaline or acidic pH or high temperatures. The extraction occurs at a natural pH level of 5.5-6.5. SE is more suitable for extracting vicilin and convicilin, because legumin is less soluble in diluted salt solution than vicilin, while alkaline extraction tends to extract a slightly higher legumin content [START_REF] Hadidi | Pea proteins as emerging biopolymers for the emulsification and encapsulation of food bioactives[END_REF][START_REF] Shanthakumar | The Current Situation of Pea Protein and Its Application in the Food Industry[END_REF].

The micellization technique causes protein precipitation by adding cold water in a 1:3 to 1:10 (v/v) ratio of high salt protein extract to water [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF]. Dilution of the protein solution causes solubilized proteins to regulate the low ionic strength via a sequence of dissociation events, resulting in the formation of reduced molecular aggregates. When the aggregates reach a critical protein concentration (CPC), they unite to form micelles, which are comparably low molecular weight entities that precipitate from liquids. The diluted solution is permitted to stand for some time to enhance micelle production [START_REF] Shanthakumar | The Current Situation of Pea Protein and Its Application in the Food Industry[END_REF]. The precipitated protein can then be retrieved by centrifugation, washing, resuscitation, and spray drying. This method has the benefit of less denatured protein during extraction due to less dramatic pH swings, however, the poor protein recovery owing to a lack of protein solubilization is a drawback of the micellization approach (Stone et al., 2015b;Muranyi et la., 2016).

Egg white proteins

Eggs are abundant in nutrients that are needed daily for body tissues growth and maintenance and are also one of the few foods that are consumed worldwide, regardless of religion or ethnic group [START_REF] Abeyrathne | Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents-A review[END_REF][START_REF] Stadelman | Egg science and technology[END_REF][START_REF] Zhu | Impact of food processing on the structural and allergenic properties of egg white[END_REF]. The nutritional content in eggs comprises not only proteins and lipids, but also vitamins such as thiamin, riboflavin, vitamins A, B, D, E, and minerals such as Ca, P, K, Na, Mg, Fe, and Zn [START_REF] Zhu | Impact of food processing on the structural and allergenic properties of egg white[END_REF]. Eggs also offer various functional features that are significant in a variety of culinary items, such as foaming and emulsifying, as well as a distinct color and flavor.

Eggs are made up of three primary parts: eggshell (9 -11 %, containing shell and shell membrane), egg white (60 -63 %), and yolk (28 -29 %), the introduction of each structure is demonstrated in Figure 1-5 [START_REF] Li-Chan | Structure and chemical composition of eggs[END_REF][START_REF] Abeyrathne | Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents-A review[END_REF][START_REF] Mine | Egg proteins[END_REF]. In brief, the eggshell is the egg's outermost layer, and it is made up of a frothy cuticle layer, a calcium carbonate layer, and two flexible membranes (inner and outer membrane) [START_REF] Guha | Egg proteins. Encyclopedia of Food Chemistry[END_REF]). This whole system keeps the egg white or albumen and egg yolk inside the egg, while also preventing dangerous microorganisms from invading [START_REF] Burley | The egg shell and shell membranes: properties and synthesis[END_REF]. The organic matter of eggshell and eggshell membranes is a complex mixture of proteins and polysaccharides, with proteins accounting for over 70% of the total organic matter [START_REF] Tullet | Egg shell formation and quality[END_REF]. Many bacteriolytic enzymes, including N acetylglucosaminidase and lysozyme, have been discovered in the eggshell membrane, as well as other components that may play a role in inhibiting Gram-negative and Gram-positive bacterial invasion [START_REF] Guha | Egg proteins. Encyclopedia of Food Chemistry[END_REF].

Egg yolk is made up of plasma and granules that are suspended between the thin and thick albumen and supported by the chalazae. It is a rich source of vitamins and minerals. Despite containing a high level of cholesterol (11 mg/g of edible part) and lipids, serum yolk serves as a reservoir for huge amounts of hen's immunoglobulin (IgY), which might be deployed as an alternate material of antibodies for infectious disease prevention and therapy [START_REF] Mine | Chicken egg yolk antibodies as therapeutics in enteric infectious disease: a review[END_REF][START_REF] Puertas | Advances in techniques for reducing cholesterol in egg yolk: A review[END_REF].

Egg white has been one of the well-known and wide-used protein sources all over the world since the dawn of recorded history. For human beings, egg white is an important reference protein source because it is rich in essential amino acids, has excellent functional properties, and remarkable nutritional values [START_REF] Mine | Recent advances in the understanding of egg white protein functionality[END_REF].

Proteins are distributed in all parts of the eggs, but the majority of them are in egg white and egg yolks, containing 50 % and 40 % of egg proteins, respectively. Hence, egg white proteins are introduced in detail in the following sections.

Figure 1-5 The structure of different components in an egg [START_REF] Froning | Encyclopedia Britannica[END_REF].

Composition of egg white (albumen)

Egg white usually contains about 11 % proteins which consist of more than 300 different kinds of proteins [START_REF] Guérin-Dubiard | Proteomic analysis of hen egg white[END_REF][START_REF] Mann | The chicken egg white proteome[END_REF]. Many of them are still uncharacterized because of their low concentration. In general, egg white is composed of four individual components: chalaziferous layer, thin egg white, thick egg white, and chalazae (Figure 1-5). The thin layer comprises about 23.3 % of the egg white, which is further divided into two layers, namely the inner and outer thin layers.

The thick or viscous layer accounting for the majority of egg white, around 57.3 %, separates the outer and inner thin layers [START_REF] Brake | Egg handling and storage[END_REF][START_REF] Li | Structural details at active site of hen egg white lysozyme with di-and trivalent metal ions[END_REF][START_REF] Guha | Egg proteins. Encyclopedia of Food Chemistry[END_REF]. Water is the major component of egg white, accounting for around 84 % -89 % of total egg white weight. According to [START_REF] Li-Chan | Biochemical basis for the properties of egg white[END_REF], carbohydrates constitute 0.9 %, lipids occupy 0.03 %, protein content is around 10 to 11 %, and the rest is vitamins and minerals when talking about the solid part inside egg white. Egg proteins are widely known for their high nutritional quality and availability of all critical amino acids required for human nutrition and growth [START_REF] Alleoni | Albumen protein and functional properties of gelation and foaming[END_REF]. However, among egg white proteins, ovalbumin, ovotransferrin, ovomucoid, ovomucin, and lysozyme have been extensively studied due to their abundant presence in egg albumen. Physicochemical characteristics and structure properties of egg white protein are provided in Table 123.

Egg white proteins are globular (Figure 1-6) The structure and chemical composition of these proteins are presented in detail in the following sections. Ovotransferrin * (2,4,7) Ovomucoid * (2,4) Lysozyme * (2,4) Ovomucin ** ( [START_REF] Lewis | Aminoacid composition of egg proteins[END_REF]3: Smith & Back, 1970;4: Messier, 1991; 5: [START_REF] Adachi | Solubilization and characterization of ovomucin without chemical modification[END_REF]6: ITOH et al., 1987;7: Liu, Liu & Zhang, 2012.

Ovalbumin

Ovalbumin (OVA) occupies around 54 % of the total egg white proteins [START_REF] Abeyrathne | Antioxidant, angiotensinconverting enzyme inhibitory activity and other functional properties of egg white proteins and their derived peptides-A review[END_REF]. Ovalbumin is a monomer, phosphorylated glycoprotein made up of complete three subunits having different phosphate groups along with a carbohydrate group attached to its N-terminal (Li-Chan, Powrie, & Nakai, 2017). Three ovalbumin fractions (A1, A2, and A3) were discovered by electrophoretic techniques according to [START_REF] Vadehra | Eggs as a source of protein[END_REF]; [START_REF] Osuga | Egg proteins[END_REF][START_REF] Osuga | Egg proteins[END_REF][START_REF] Zabik | Eggs and products[END_REF]. Differences between these fractions are the phosphorus content of their molecules, A1, A2, and A3 having 2, 1, or 0 phosphate groups, respectively [START_REF] Vadehra | Eggs as a source of protein[END_REF][START_REF] Zabik | Eggs and products[END_REF]. Ovalbumin has a molecular weight of 45 kDa and 386 amino acids, as well as two genetic polymorphisms at 290 (Glu/Gln) and 312 (Asn/Asp) [START_REF] Mcreynolds | Sequence of chicken ovalbumin mRNA[END_REF]. The isoelectric point (pI) of ovalbumin is 4.5 [START_REF] Nisbet | The complete amino-acid sequence of hen ovalbumin[END_REF]. Ovalbumin is distinct from other egg albumen proteins in that it includes 3.5 % carbohydrates and six cysteine residues, two of which are connected by a disulfide bond, while the remaining four residues have free sulfhydryl groups. According to [START_REF] Fothergill | Thiol and disulphide contents of hen ovalbumin. C-terminal sequence and location of disulphide bond[END_REF], one of the four sulfhydryl groups is only reactive when the protein is denatured, the other three staying in their original states. Also, [START_REF] Vadehra | Eggs as a source of protein[END_REF] said that these kinds of the group could be denatured by heat, surface absorption, in films, agitation, or the action of numerous denaturant agents.

In addition, the N-terminal and C-terminal amino acid end with acetylated glycine and proline, respectively [START_REF] Abeyrathne | Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents-A review[END_REF]. Half of the molecule's residues are hydrophobic, while 30% are acidic and charged amino acid residues, leading to the isoelectric point of 4.5 (Huopalahti et al., 2007). Ovalbumin has a heatstable form, S-ovalbumin, which formation depends on temperature, duration time, and pH [START_REF] Vadehra | Eggs as a source of protein[END_REF][START_REF] Alleoni | Albumen protein and functional properties of gelation and foaming[END_REF]. [START_REF] Huang | Estimation of egg freshness using S-ovalbumin as an indicator[END_REF], demonstrated that the conversion of ovalbumin to S-ovalbumin during storage had been related to an increase in pH and storage time. [START_REF] Donovan | A differential scanning calorimetric study of conversion of ovalbumin to S-ovalbumin in eggs[END_REF] performed a differential scanning calorimeter (DSC) to identify the Td of S-ovalbumin, which showed that it was 92.5 ℃, which is higher than the Td of ovalbumin (84 ℃). Sovalbumin has a slightly lighter molecular weight than ovalbumin and its relative proportion in egg white may rise with the duration of storage, from 5 % in fresh eggs to 81 % after 6 months of storage, despite the eggs being stored at a low temperature, around 4 ℃ [START_REF] Huang | Estimation of egg freshness using S-ovalbumin as an indicator[END_REF]. Furthermore, OVA's structure and features might change during storage. For example, the proportions of α-helix and β-sheet are reduced, while the proportions of β-turns and random coils are increased [START_REF] Sheng | A study of storage impact on ovalbumin structure of chicken egg[END_REF].

Ovalbumin is also the main allergen in egg white, causing immunoglobulin E (IgE)-mediated allergic responses [START_REF] Caubet | Current understanding of egg allergy[END_REF]. The ovalbumin epitopes, which bind IgE are mostly composed of polar, charged, and hydrophobic amino acids and these sequences are generally composed of β-sheet and β-turn structures. The only allergenic epitope which comprises an alpha helix is Asp95-Ala102 [START_REF] Kim | The effects of supplementation of ascidian tunic shell into laying hen diet on egg quality[END_REF].

Ovotransferrin

Ovotransferrin, also known as conalbumin, was shown to have the ability to bind iron [START_REF] Williams | A comparison of glycopeptides from the ovotransferrin and serum transferrin of the hen[END_REF]. It is a monomeric glycoprotein containing 686 amino acids with a molecular weight of 77.9 kDa [START_REF] Wu | Ovotransferrin: Structure, bioactivities, and preparation[END_REF]. It accounts for around 12 % of the total egg white proteins [START_REF] Désert | Comparison of different electrophoretic separations of hen egg white proteins[END_REF][START_REF] Bou Abdallah | Transferrins: Hen ovo-transferrin, interaction with bicarbonate and iron uptake[END_REF]. Ovotransferrin has approximately 15 disulfide bonds (6 in N-lobe and 9 in Clobe) and no free sulfhydryl groups [START_REF] Zabik | Eggs and products[END_REF]. One molecule of ovotransferrin can bind 2 iron molecules and transports iron throughout the body. It was reported that Fe +3 can be successfully linked to ovotransferrin at a pH higher than 7 but easily released at a pH lower than 4.5 [START_REF] Ko | An economic and simple purification procedure for the large-scale production of ovotransferrin from egg white[END_REF][START_REF] Guérin-Dubiard | Proteomic analysis of hen egg white[END_REF]. Ovotransferrin is divided into two globule lobes (N-and C-lobes) linked by an α-helix, with each lobe consisting of two different α-domains and β-domains. These two domains are connected by antiparallel β strands that open and close through a hinge (Huopalahti et al., 2007).

The N and C lobes are linked through noncovalent, mainly hydrophobic interactions [START_REF] Charter | NATURAL ANTI-MICROBIAL SYSTEMS| Lysozyme and Other Proteins in Eggs[END_REF]. According to [START_REF] Lambert | Evolution of the transferrin family: conservation of residues associated with iron and anion binding[END_REF], each lobe may reversibly bind one Fe 3+ ion and one bicarbonate anion but with different iron-binding capabilities, in which the iron-binding constant for the C-terminal lobe is 1.5 × 10 18 and 1.5 × 10 14 for the N-terminal lobe.

The structure stability is governed by disulfide bonds [START_REF] Rabbani | pH-dependent conformational transitions in conalbumin (ovotransferrin), a metalloproteinase from hen egg white[END_REF], and the iron binding capability confers antimicrobial properties to ovotransferrin [START_REF] Alleoni | Albumen protein and functional properties of gelation and foaming[END_REF]. Ovotransferrin is the most heat-sensitive protein, denatured and aggregated between 53 and 65 ℃, and altering egg white viscosity and early gelation [START_REF] Chaiyasit | Comparison of physicochemical and functional properties of chicken and duck egg albumens[END_REF][START_REF] Iwashita | Co-aggregation of ovotransferrin and lysozyme[END_REF][START_REF] Jalili-Firoozinezhad | Chicken egg white: Hatching of a new old biomaterial[END_REF].

Ovalbumin, on the other hand, can suppress the thermal aggregation of ovotransferrin at temperatures lower than its Td [START_REF] Iwashita | Co-aggregation of ovotransferrin and lysozyme[END_REF]. Moreover, [START_REF] Iwashita | Co-aggregation of ovotransferrin and lysozyme[END_REF] proved that non-covalent interactions enabled native lysozyme to precipitate soluble ovotransferrin aggregates and suppressed electrostatic repulsion between soluble ovotransferrin aggregates. Ovotransferrin operates similarly to lactoferrin in milk, and both of them have iron scavenging and iron delivery functions [START_REF] Abdallah | Transferrins, the mechanism of iron release by ovotransferrin[END_REF].

Ovomucoid

Ovomucoid is a glycoprotein, widely recognized as a trypsin inhibitor [START_REF] Abeyrathne | Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents-A review[END_REF]. It constitutes 11 % of the total egg white proteins and is thermally stable [START_REF] Li-Chan | Biochemical basis for the properties of egg white[END_REF]. The protein consists of 186 amino acids with a molecular mass of 28 kDa and a pI of 4.1 [START_REF] Kovacs-Nolan | Immunochemical and structural analysis of pepsin-digested egg white ovomucoid[END_REF][START_REF] Benedé | Influence of the carbohydrate moieties on the immunoreactivity and digestibility of the egg allergen ovomucoid[END_REF]. Ovomucoid structure contains 9 disulfide bonds and three distinct domains (I, II (α-type), and III (β-type)) which are crosslinked only by the intra-domain disulfide bonds and lead to structure stability (Figure 1-6) [START_REF] Stevens | Egg white proteins[END_REF]. Domain II has the active site for the trypsin inhibitory activity inside. A previous study by De Oliveria et al., (2009), found that ovomucoid was classified as a single-headed inhibitor of trypsin, which means that each ovomucoid molecule binds trypsin at a ratio of 1:1, and its 3D structure was held together by three disulfide bonds. Domain III differs from domains I and II in that the disulfide bonds between its first and second cysteine residues are shorter [START_REF] Zhu | Impact of food processing on the structural and allergenic properties of egg white[END_REF].

Chicken ovomucoid is one of the major egg white allergens, which plays an important role in the pathogenesis of IgE-mediated allergic responses [START_REF] Mine | The allergenicity of ovomucoid and the effect of its elimination from hen's egg white[END_REF], 2002a;[START_REF] Mine | Immunological and biochemical properties of egg allergens[END_REF]. According to [START_REF] Kovacs-Nolan | Immunochemical and structural analysis of pepsin-digested egg white ovomucoid[END_REF], peptides produced from ovomucoid utilizing pepsin have IgE binding activity and preserve trypsin inhibitory properties. [START_REF] Besler | Allergenicity of hen's egg-white proteins: IgE binding of native and deglycosylated ovomucoid[END_REF] discovered that the ovomucoid epitopes which were responsible for the IgE binding were exclusively found on the protein backbone and not the carbohydrate groups. It was discovered through epitope mutational research that charged amino acids (lysine, glutamic acid, and aspartic acid), polar amino acids (cysteine, tyrosine, threonine, and serine), and hydrophobic amino acids (glycine, leucine, and phenylalanine) are essential for antibody binding (Mine & Zhang, 2002b). Numerous experiments have been done to change the content and the structure of the allergic ovomucoid epitopes [START_REF] Mine | Genetic attachment of undecane peptides to ovomucoid third domain can suppress the production of specific IgG and IgE antibodies[END_REF], such as heating with wheat flour [START_REF] Kovacs-Nolan | Immunochemical and structural analysis of pepsin-digested egg white ovomucoid[END_REF][START_REF] Kato | Decrease in antigenic and allergenic potentials of ovomucoid by heating in the presence of wheat flour: dependence on wheat variety and intermolecular disulfide bridges[END_REF], gamma irradiation along with heating [START_REF] Lee | Allergenicity of hen's egg ovomucoid gamma irradiated and heated under different pH conditions[END_REF], deglycosylation by endo-beta-N-acetylglucosaminidases [START_REF] Yamamoto | Characterization of Bacillus sp. endo-β-Nacetylglucosaminidase and its application to deglycosylation of hen ovomucoid[END_REF], and genetic modifications [START_REF] Rupa | Ablation of ovomucoid-induced allergic response by desensitization with recombinant ovomucoid third domain in a murine model[END_REF]. Under thermal or enzymatic hydrolysis, the allergenic action of ovomucoid remains constant [START_REF] Julià | Effect of heat treatment on hen's egg ovomucoid: An immunochemical and calorimetric study[END_REF]. This kind of feature, however, may be exploited to identify eggs in food items even after heat processing.

Ovomucin

Ovomucin is another important protein in egg white as it accounts for about 3.5 % of the total egg white proteins [START_REF] Abeyrathne | Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents-A review[END_REF]. It is a sulfated glycoprotein that is responsible for the gel-like structure of egg white. The protein is divided into two parts: the soluble part (the primary component of the inner and outer egg white), and the insoluble part (only in the thick egg white portion) which is accounting for the insoluble gel-like fraction of thick albumen (Huopalahti et al., 2007;[START_REF] Omana | Effect of different concentrations of calcium chloride and potassium chloride on egg white proteins during isoelectric precipitation of ovomucin[END_REF]. According to [START_REF] Tominatsu | Light scattering study of ovomucin[END_REF], the molecular weight of insoluble ovomucin in 6.5 M guanidine hydrochloride was calculated to be 23 × 10 3 kDa. The molecular weight of soluble ovomucin has been reported to be in the range of 5.6 to 8.3 × 10 3 kDa [START_REF] Miller | Molecular weight distribution of ovomucin from thinned egg white[END_REF][START_REF] Hayakawa | Studies on the dissociation of the soluble ovomucin by sonication[END_REF].

These discrepancies in molecular weights are most likely attributable to variances in measurement methods and circumstances, as well as ovomucin heterogeneity.

Both soluble and insoluble fractions are made up of two subunits, α-ovomucin and β-ovomucin, with different carbohydrate levels. β-ovomucin is richer in carbohydrates (60 %) than α-ovomucin (16 %) [START_REF] Omana | Effect of different concentrations of calcium chloride and potassium chloride on egg white proteins during isoelectric precipitation of ovomucin[END_REF][START_REF] Shan | Rheological and structural properties of ovomucin from chicken eggs with different interior quality[END_REF]. Previous research has revealed that ovomucin has at least three types of carbohydrate chains, which are constituted of galactose, galactosamine, sialic acid, and sulfate with a molecular ratio of 1:1:1:1. On average, 33 % of ovomucin is carbohydrates (Mine, 2008). α-ovomucin is homogeneous with an MW of 150-220 kDa, whereas β-ovomucin is heterogeneous with an MW of 5500-8300 kDa [START_REF] Itoh | Studies on the characterization of ovomucin and chalaza of the hen's egg[END_REF][START_REF] Omana | Effect of different concentrations of calcium chloride and potassium chloride on egg white proteins during isoelectric precipitation of ovomucin[END_REF][START_REF] Shan | Rheological and structural properties of ovomucin from chicken eggs with different interior quality[END_REF][START_REF] Jalili-Firoozinezhad | Chicken egg white: Hatching of a new old biomaterial[END_REF]. The soluble fraction contains 40 αsubunits and 3 β-subunits, while the insoluble fraction consists of 84 αand 20 βsubunits [START_REF] Robinson | Studies on the composition of egg-white ovomucin[END_REF][START_REF] Omana | Effect of different concentrations of calcium chloride and potassium chloride on egg white proteins during isoelectric precipitation of ovomucin[END_REF]. There are two distinct subunits of the α-subunit, α1, and α2 [START_REF] Abeyrathne | Antioxidant, angiotensinconverting enzyme inhibitory activity and other functional properties of egg white proteins and their derived peptides-A review[END_REF]. The α-subunit is mostly made of acidic amino acids such as glutamic acid and aspartic acids [START_REF] Omana | Effect of different concentrations of calcium chloride and potassium chloride on egg white proteins during isoelectric precipitation of ovomucin[END_REF], whereas the β-subunit is primarily composed of serine and threonine [START_REF] Robinson | Studies on the composition of egg-white ovomucin[END_REF]. A study by [START_REF] Toussant | Ovomucin content and composition in chicken eggs with different interior quality[END_REF] discovered that the fraction of β-subunit or its glycosylation was positively correlated with the quantity of ovomucin present in the thick albumen. Many of the functional and biological features of egg white are attributed to ovomucin. It has an important function in the thinning of egg white during long-term preservation. Ovomucin has high thermal stability and a proclivity to interact with other proteins [START_REF] Lesnierowski | What's new in chicken egg research and technology for human health promotion? -A review[END_REF]. Egg white thinning can be caused by either the disruption of the ovomucinlysozyme complex or the decrease of disulfide bonds, which leads to the degradation of ovomucin [START_REF] Abeyrathne | Sequential separation of lysozyme, ovomucin, ovotransferrin, and ovalbumin from egg white[END_REF]. It is also well-known for its excellent emulsifying and foaming properties [START_REF] Mann | The chicken egg white proteome[END_REF]. Furthermore, ovomucin's physiologically active glycoproteins make it an appealing candidate for food and nutraceutical research.

Lysozyme

Lysozyme is a highly basic protein and is enriched in egg white compared to other forms of lysozyme found in nature. Egg white lysozyme (3.5 % of total egg white proteins) is made up of a single polypeptide chain of 129 amino acids and has a molecular weight of 14.4 kDa with a pI of 10.7 [START_REF] Guha | Egg proteins. Encyclopedia of Food Chemistry[END_REF].

Lysozyme differs from other proteins found in egg white and other foods due to its high solubility and stability. Although the lysozyme is a monomer in eggs, it is also frequently found as a dimer with thermal stability. Lysozyme contains two domains in its three dimensions: an N-domain containing antiparallel β-sheets and a C-domain composed of 4 α-helices (Figure 1-6) [START_REF] Lesnierowski | Lysozyme. In Bioactive egg compounds[END_REF][START_REF] Tokunaga | Analysis of core region from egg white lysozyme forming amyloid fibrils[END_REF]. Recent studies found that a helix-loop-helix motif located on the top side of the enzyme's active site separated the two domains [START_REF] Ibrahim | A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action[END_REF][START_REF] Mine | Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme[END_REF]. All of its polar groups (hydrophilic) are on the surface, while the majority of its hydrophobic groups are buried in the enzyme particle's core.

Egg lysozyme is cross-linked through four disulfide bonds to stabilize its tertiary structure as well as cohesion [START_REF] Kato | Engineering hen egg-white lysozyme[END_REF].

Lysozyme preferentially binds to negatively charged proteins in the egg albumen for instance ovalbumin, ovomucin, and ovotransferrin [START_REF] Wan | Separation of lysozyme from chicken egg white using ultrafiltration[END_REF][START_REF] Abeyrathne | Sequential separation of lysozyme, ovomucin, ovotransferrin, and ovalbumin from egg white[END_REF][START_REF] Iwashita | Co-aggregation of ovotransferrin and lysozyme[END_REF]. The chalaza and the chalaziferous layer are mainly made up of the lysozyme-ovomucin complex. However, many studies are still being conducted to further investigate its structure and function.

Several studies have also been carried out to explore the structural changes of lysozyme caused by various conditions, such as aqueous-organic solvent combinations [START_REF] Griebenow | On protein denaturation in aqueous-organic mixtures but not in pure organic solvents[END_REF], pH variations [START_REF] Babu | Ionic-strength-dependent transition of hen eggwhite lysozyme at low pH to a compact state and its aggregation on thermal denaturation[END_REF], cocrystallization in presence of different alcohols [START_REF] Deshpande | Effect of alcohols on protein hydration: crystallographic analysis of hen egg-white lysozyme in the presence of alcohols[END_REF], sorbitol addition [START_REF] Petersen | Sorbitol prevents the self-aggregation of unfolded lysozyme leading to an up to 13 C stabilisation of the folded form[END_REF], in presence of thiol reagents [START_REF] Raman | Refolding of denatured and denatured/reduced lysozyme at high concentrations[END_REF], and supercritical CO2 treatment followed by heat treatment (Liu, Hsieh & Liu, 2004). Huopalahti et al. (2007), investigated that action of lysozyme remained unaffected by high temperatures (100 ℃) or acidic solutions (pH 3.0-4.0). It may, however, be readily and quickly deactivated by interacting with thiol compounds.

Furthermore, lysozyme aggregates at temperatures below 80 °C, and neutral pH affects its functional properties [START_REF] Iwashita | Co-aggregation of ovalbumin and lysozyme[END_REF].

Methods to purify egg white

Several methods have been used to separate and purify proteins from egg white.

Initially, salts such as ammonium sulfate, sodium chloride, or potassium chloride were used to separate ovalbumin [START_REF] Warner | The preparation of crystalline conalbumin[END_REF], ovotransferrin (Fraenkel-Conrat values greater than 90%, they cannot be scaled up.

The first purification of lysozyme was done using a high concentration of ammonium sulfate; however, it led to the modification of the characteristics and morphology of the protein due to the high salt concentration used during extraction (Liu et al., 2004;[START_REF] Abeyrathne | Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents-A review[END_REF]. The purification technique which is commonly used nowadays for lysozyme is cation exchange chromatography which makes use of the high pI value of the protein [START_REF] Abeyrathne | Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents-A review[END_REF].

Type of interactions

It is vital and fascinating to investigate the partial substitution of animal proteins in processed goods with plant proteins to minimize the intake of animal proteins while keeping good nutritional qualities. To make this sort of partial replacement possible, animal and plant proteins should be able to interact to generate homogenous molecules with functional qualities. The energy (intensity), type, and specificity of interactions found in protein assemblies might vary depending on the structure of the protein assembly and the physicochemical environment. There are 2 main molecular forces involved in protein-protein interactions, covalent disulfide bonds and non-covalent forces which encompass electrostatic interactions, hydrogen bonds, hydrophobic interactions, and Van der Waals forces [START_REF] Alrosan | Molecular forces governing protein-protein interaction: Structure-function relationship of complexes protein in the food industry[END_REF]. Furthermore, these types of protein interactions are likely to play a role in the formation and stability of protein assemblies, as well as modifying protein functional properties, for instance, solubility [START_REF] Wang | Complexation of rice proteins and whey protein isolates by structural interactions to prepare soluble protein composites[END_REF], interfacial behavior [START_REF] Liang | Effects of globular protein type and concentration on the physical properties and flow behaviors of oil-inwater emulsions stabilized by micellar casein-globular protein mixtures[END_REF][START_REF] Wang | Molecular forces and gelling properties of heat-induced gel from egg white protein glycated with isomalto-oligosaccharide[END_REF], and gelation (Sun, Wang, & Guo 2018). In the following section, we will introduce details information on disulfide bonds, hydrophobic interactions, electrostatic interactions, hydrogen bonds, and Van der Waals forces.

Disulfide bonds

Covalent bonds are strong chemical bonds and are formed by the sharing of electrons between atoms to generate electro pairs. These electron pairs are known as shared pairs or bonding pairs, and covalent bonding is the stable equilibrium of attractive and repulsive forces between atoms when they share electrons. Covalent interactions can occur inside the same protein as well as between proteins [START_REF] Whitten | 7-3 Formation of covalent bonds[END_REF].

In the case of the globular protein, this sort of connection exists between two portions of the polypeptide in the form of disulfide bonds formed between the thiol groups of the cysteine residues to produce inter or intramolecular bonds [START_REF] Kinsella | Relationships Between Structure and Functional[END_REF]. Covalent bonds can be formed between the carboxylic groups and amine groups of amino acids in proteins to produce peptide-type linkages. This kind of connection typically necessitates the use of a catalyst, which can be physical (temperature, pressure), biological (particular enzymes such as transglutaminase), or even chemical (glutaraldehyde). They can also be formed between cysteine residues via their thiol groups, resulting in intra-and/or intermolecular disulfide bonds [START_REF] Visschers | Disulphide bond formation in food protein aggregation and gelation[END_REF]. High temperatures and the presence of catalysts can conduce the creation of covalent bonds [START_REF] Creighton | Disulfide bond formation in proteins[END_REF]. During prolonged heating, intramolecular disulfide bonds gradually convert to intermolecular bonds [START_REF] Chaplin | Irreversible heat denaturation of bovine αlactalbumin[END_REF].

Covalent bonds have an important function in protein structure and stability.

Intramolecular disulfide bonds, for example, provide protein stability by establishing the backbone's stability and structure while leaving flexibility to the side chains. [START_REF] Scharnagl | Stability of proteins: temperature, pressure and the role of the solvent[END_REF]. The protein aggregates of egg white are stabilized due to the formation of disulfide bonds by exposed SH groups caused by high pH, heat, and a combination of heat-and high-pressure treatment (Van der Plancken, [START_REF] Van Der Plancken | Changes in sulfhydryl content of egg white proteins due to heat and pressure treatment[END_REF]. [START_REF] Zhao | Changes of microstructure characteristics and intermolecular interactions of preserved egg white gel during pickling[END_REF] proposed that ionic and disulfide connections might potentially help to stabilize alkali-induced gels. Moreover, according to a previous paper conducted by [START_REF] Chen | Changes in gel characteristics of egg white under strong alkali treatment[END_REF], the stability of alkali-induced egg white gel was shown to be closely linked to protein disulfide links. Therefore, these linkages have a favorable impact on the mechanical characteristics of heat gels and aggregates of egg white proteins [START_REF] Wang | Molecular forces and gelling properties of heat-induced gel from egg white protein glycated with isomalto-oligosaccharide[END_REF]Alavi et al., 2020). Several investigations on pea proteins have demonstrated the importance of these interactions in stabilizing and sustaining the structure of heat aggregation [START_REF] Mession | Thermal Denaturation of Pea Globulins (Pisum sativum L.)-Molecular Interactions Leading to Heat-Induced Protein Aggregation[END_REF], Wu et al., 2020).

Hydrophobic interactions

Hydrophobic interactions allow proteins to bind and assemble to create complexes.

They are endothermic attractive and are formed in aqueous fluids between apolar molecules or molecules with apolar domains. These hydrophobic interactions are entropic in nature and very temperature dependent; they prefer high temperatures [START_REF] Ray | Solvophobic interactions and micelle formation in structure forming nonaqueous solvents[END_REF][START_REF] Chandler | Interfaces and the driving force of hydrophobic assembly[END_REF].

The phrase "hydrophobic interaction" is commonly used in biochemistry to characterize the attractive interaction between hydrophobic elements of a system (i.e., proteins); these interactions are the consequence of the London dispersion [START_REF] Holtrop | Steric attraction: A force to be reckoned with[END_REF]. Although the exclusion of water from hydrophobic pockets is induced by both entropic contributions and water molecules' inclination to associate with one another via favorable hydrogen bond formation, the attraction between the hydrophobic sections is produced by the London dispersion forces. These attractive forces play an important role in protein stability and folding [START_REF] Privalov | Stability of protein structure and hydrophobic interaction[END_REF]. With regard to globular protein, apolar amino acids are found inside it attributing to form hydrophobic interactions between these amino acids. Because of the way proteins are organized and folded, the majority of these amino acids are found buried in the core of proteins, where solvent accessibility is limited (except for membrane proteins whose apolar amino acids are exposed to the protein surface). As a result, the creation of hydrophobic linkages between proteins typically necessitates conformational and structural changes in the latter to expose the apolar amino acids on their surface. In the case of globular proteins, exposing solvent hydrophobic regions might cause aggregation via hydrophobic interactions.

Hydrophobic interactions play an important role in the effects of processing on the aggregation of egg white proteins, such as heat treatment [START_REF] Matsudomi | Heat-induced aggregation of lysozyme with ovotransferrin[END_REF][START_REF] Iwashita | Co-aggregation of ovalbumin and lysozyme[END_REF], alkali treatment [START_REF] Chen | Changes in gel characteristics of egg white under strong alkali treatment[END_REF][START_REF] Zhao | Changes of microstructure characteristics and intermolecular interactions of preserved egg white gel during pickling[END_REF], pulsed electric field [START_REF] Wu | Pulsed electric field (PEF)-induced aggregation between lysozyme, ovalbumin and ovotransferrin in multiprotein system[END_REF][START_REF] Wu | Aggregation of egg white proteins with pulsed electric fields and thermal processes[END_REF], high pressure ( [START_REF] Van Der Plancken | Changes in sulfhydryl content of egg white proteins due to heat and pressure treatment[END_REF][START_REF] Singh | Effect of high pressure treatment on rheological characteristics of egg components[END_REF], high intensity ultrasound (Xiong et al., 2016). Several previous studies have demonstrated that when it comes to the thermal denaturation of soybean and pea proteins, high levels of exposure of hydrophobic groups favor protein aggregation and gelation of these systems at high protein concentrations, regardless of ionic strength or pH [START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF]Sun & Arntfield, 2010;Sun & Arntfield, 2012a;[START_REF] Tang | Thermal denaturation and gelation of vicilin-rich protein isolates from three Phaseolus legumes: a comparative study[END_REF].

Electrostatic interactions

Electrostatic interactions are considered weak bonds. These interactions occur between particles with a constant electric charge, such as dipoles or ions. These interactions can be either attractive or repulsive depending on the charges carried by the molecules or chemical groups; interactions are repulsive when the charges have the same sign, whereas interactions are attractive when the charges have opposite signs.

The implementation and strength of electrostatic contacts are determined by the charges of the proteins, particularly their intensity and distribution at the protein level, as well as the physicochemical parameters of the medium in which the protein complexes develop (pH, ionic strength, nature of the ions and temperature).

According to a previous study by [START_REF] Ben-Harb | The rheological and microstructural properties of pea, milk, mixed pea/milk gels and gelled emulsions designed by thermal, acid, and enzyme treatments[END_REF], protein aggregation dynamics and the electrostatic interactions between protein aggregates have a major impact on the rheological properties of pea and PPI-milk protein gels, prepared at a total protein concentration of 14.8 % at a ratio of 1:1 between pea and milk protein with 1 % NaCl. Salt concentration has a considerable impact on electrostatic interaction during gel formation as the higher the salt concentration, the more pronounced the electrostatic interaction [START_REF] Tolano-Villaverde | Thermal gelation of myofibrillar proteins from aquatic organisms[END_REF]. [START_REF] Archer | The dielectric constant of water and Debye-Hückel limiting law slopes[END_REF] illustrated that at higher temperatures, electrostatic interactions may be expected to have a more favorable contribution to folding stability. Electrostatic repulsions between aggregates are often relatively weak at pH close to the isoelectric point (pI) of proteins and/or at high ionic strength, where aggregation/gelation of serum proteins happens via some other non-covalent interactions [START_REF] Alting | Physical and chemical interactions in cold gelation of food proteins[END_REF].

Hydrogen bonds

The hydrogen bonds are a dipole-dipole attraction in nature. A hydrogen bond is created through the interaction of a hydrogen atom (electron acceptor) with an electronegative atom (N, O) and another electronegative atom (electron donor) [START_REF] Britannica | Editors of Encyclopaedia[END_REF]. This bond is weaker than an ionic or covalent bond, but stronger than van der Waals forces. The energy of these bonds is determined by the electronegativity of the atoms as well as their orientation in the bond: the energy is greatest when the electronegative atom and the hydrogen atom engaged in the bond are aligned [START_REF] Macleod | 02 -Directed Assembly of Nanostructures[END_REF]. [START_REF] Kinsella | Proteins in whey: chemical, physical, and functional properties[END_REF] demonstrated that hydrogen bond was not responsible for globular protein aggregation, instead of playing an important role in stabilizing the formed structure. This kind of stabilization has been observed for pea protein thermal gels (Sun & Arntfield, 2010), elastic pea protein hydrogels [START_REF] Zhu | Strong and elastic pea protein hydrogels formed through pH-shifting method[END_REF], as well as heated egg protein gels [START_REF] Chang | Functional and gel characteristics of liquid whole egg as affected by pH alteration[END_REF].

Van der Waals attraction

Van der Waals interactions are non-specific bonds that exist between permanent dipoles, induced dipoles, and permanent and induced dipoles. It contains attraction and repulsion depending on the distance between the interaction partners. Essentially, when two atoms go closer, their attraction grows stronger until they are separated by the vander-Waals contact distance. When two molecules are too close to each other, the potential energy due to repulsion increases dramatically, causing the assembly to become unstable, and repulsion to occur even when these molecules are neutral [START_REF] Righetti | Low-Abundance Proteome Discovery[END_REF]. Van der Waals interactions contribute to the stability of the tridimensional protein structure. It is also involved in the self-assembly together with hydrogen bonds or covalent interactions between polymer chains during the formation of gels [START_REF] Jaberi | Rheological and structural properties of oleogel base on soluble complex of egg white protein and xanthan gum[END_REF]. At lower temperatures, van der Waals as a shortrange interaction led to the gel reinforcement and continues to enhance the gel network with hydrogen bonding and covalent bonds [START_REF] Ikeda | On solid-like rheological behaviors of globular protein solutions[END_REF][START_REF] Shevkani | Structural and functional characterization of kidney bean and field pea protein isolates: A comparative study[END_REF].

Techno-functional properties

Proteins have physico-chemical functional qualities that contribute to the desired aspects of food as well as its physical behavior during preparation, transformation, and storage [START_REF] Alleoni | Albumen protein and functional properties of gelation and foaming[END_REF]. Proteins' functional qualities are essentially determined by their physical, chemical, and structural features, which include size, shape, amino acid content and sequence, net charge, and charge distribution [START_REF] Mine | Egg proteins[END_REF]. These factors will determine the exposure of hydrophobic groups on the molecular surface and their interactions with oil (emulsion), air (foam), or other protein molecules, such as in gels or coagula (Li-Chan & Nakai, 1989;[START_REF] Alleoni | Albumen protein and functional properties of gelation and foaming[END_REF]; the architecture of their secondary (i.e., -helix, -sheet, and random structures), tertiary and quaternary structures; the presence of inter-or intrasubunit bonds (i.e., disulfide crosslinks), and ultimately reflect the nature of their functional properties [START_REF] Damodaran | Food proteins: an overview[END_REF].

Pea proteins are highlighted for their technological-functional features such as hydration properties (solubility, water, and oil holding capacity), surface properties (emulsifying and foaming capabilities), and protein structure and rheological properties (gelation and viscosity), in addition to their nutritional significance [START_REF] Klupšaitė | Legume: Composition, protein extraction and functional properties. A review[END_REF][START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF]. These various features explain how these proteins work in a variety of food-related applications. The techno-functional qualities and applications in food of pea protein can be significantly influenced by protein concentration, concentration in distinct protein families, extraction procedure used, and processing conditions (pH, temperature, pressure, ionic strength, etc.) [START_REF] Rui | Comparative study of the composition and thermal properties of protein isolates prepared from nine Phaseolus vulgaris legume varieties[END_REF][START_REF] Tang | A comparative study of physicochemical and conformational properties in three vicilins from Phaseolus legumes: implications for the structure-function relationship[END_REF][START_REF] Lu | Composition, physicochemical properties of pea protein and its application in functional foods[END_REF].

In terms of egg white, there are three well-known functions, such as gelation, foaming, and emulsion ability. Therefore, it is necessary to understand the different functional qualities of eggs leading to a significant implication for egg consumption and future use in the food industry. For egg white, it exists excellent research papers or reviews in the literature describing foaming (Li-Chan, & Nakai, 1989;[START_REF] Murray | Foam stability: proteins and nanoparticles[END_REF]Lomakina & Mikova, 2006;Murray, 2007;[START_REF] Lechevalier | Evidence for synergy in the denaturation at the air-water interface of ovalbumin, ovotransferrin and lysozyme in ternary mixture[END_REF]Lomakina, & Mikova, 2006;Raikos, Campbell, & Euston, 2007a;[START_REF] Lechevalier | Effect of dry heat treatment of egg white powder on its functional, nutritional and allergenic properties[END_REF][START_REF] Chen | Impact of ultrasound treatment on the foaming and physicochemical properties of egg white during cold storage[END_REF][START_REF] Duan | Effect of oxidative modification on structural and foaming properties of egg white protein[END_REF]Li et al., 2018a;[START_REF] Sheng | Molecular characteristics and foaming properties of ovalbumin-pullulan conjugates through the Maillard reaction[END_REF][START_REF] He | Effect of polysaccharides on the functional properties of egg white protein: A review[END_REF] and emulsion properties [START_REF] Drakos | Stability of acidic egg white protein emulsions containing xanthan gum[END_REF][START_REF] Niu | Ovalbumin/gum arabic-stabilized emulsion: Rheology, emulsion characteristics, and Raman spectroscopic study[END_REF][START_REF] Xiong | Emulsifying properties of ovalbumin: Improvement and mechanism by phosphorylation in the presence of sodium tripolyphosphate[END_REF][START_REF] Chang | Effect of enzymatic hydrolysis on characteristics and synergistic efficiency of pectin on emulsifying properties of egg white protein[END_REF][START_REF] Zhao | Changes in physicochemical properties, microstructures, molecular forces and gastric digestive properties of preserved egg white during pickling with the regulation of different metal compounds[END_REF] of egg white.

However, in this section, we will introduce the techno-functional properties of pea protein isolate and egg white, especially in terms of solubility (PPI) and gelation (PPI and EW).

Solubility of pea protein

Solubility is one of the most widely evaluated functional qualities of food proteins as it affects their functional properties such as foaming, emulsification, and gelation [START_REF] Bogahawaththa | Impact of selected process parameters on solubility and heat stability of pea protein isolate[END_REF]. Protein solubility can be described as Protein-Solvent ↔ Protein-Protein + Solvent-Solvent, which is the equilibrium between protein-protein (hydrophobic) and protein-solvent (hydrophilic) interactions [START_REF] Hall | Methods of testing protein functionality[END_REF][START_REF] Heredia-Leza | Impact of Hydrolysis, Acetylation or Succinylation on Functional Properties of Plant-Based Proteins: Patents, Regulations, and Future Trends[END_REF]. Water or buffer is the most widely used solvent in solubility. Several parameters can impact solubility including pH, ionic strength, temperature, solvent type, and protein concentration [START_REF] Klupšaitė | Legume: Composition, protein extraction and functional properties. A review[END_REF][START_REF] Mccarthy | Emulsification properties of pea protein isolate using homogenization, microfluidization and ultrasonication[END_REF]. Protein surface properties, especially the amount and distribution of hydrophilic and hydrophobic amino acid residues on the surface, can influence how a protein behaves in solution [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF]. Hydrophilic amino acid residues are more orientated toward the solvent interface in water. In contrast, most of the hydrophobic residues are buried in the inner of the protein to reduce free energy.

Hydrophobic patches are formed on the protein surface by the remaining hydrophobic residues, which hinder solubility (Stone et al., 2015b).

The solubility of PPI is strongly pH-dependent with a minimal solubility between pH 4 and 6 regardless of the extraction method or pea cultivar, and the solubility profile performs a classic "U" shape [START_REF] Boye | Comparison of the functional properties of pea, chickpea and lentil protein concentrates processed using ultrafiltration and isoelectric precipitation techniques[END_REF][START_REF] Taherian | Comparative study of functional properties of commercial and membrane processed yellow pea protein isolates[END_REF][START_REF] Withana-Gamage | Physicochemical, thermal and functional characterisation of protein isolates from Kabuli and Desi chickpea (Cicer arietinum L.): A comparative study with soy (Glycine max) and pea (Pisum sativum L.)[END_REF][START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF][START_REF] Zhao | Comparison of wheat, soybean, rice, and pea protein properties for effective applications in food products[END_REF]. Solubility increases at pH levels above and below the pI due to electrostatic repulsion caused by positive and negative net charges on the protein surface [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF][START_REF] Shanthakumar | The Current Situation of Pea Protein and Its Application in the Food Industry[END_REF]. A protein has the lowest solubility at its isoelectric pH because it carries a zero net charge, thereby decreasing electrostatic repulsive forces [START_REF] Vihinen | Solubility of proteins[END_REF]. Under (2010) demonstrated that a substantial difference in the solubility of pea protein isolates extracted by AE/IEP and UF, with AE/IEP resulting in solubility of 90 % at pH 1, but 29 % at pH 3, whereas UF isolates displayed solubilities of 60 % and 56 %, respectively.

In addition, a study conducted by Stone et al. (2015b) highlighted that those isolates created by SE had the maximum solubility (86-91 %), followed by AE/IEP (63-64 %), and by micellization (43-49%). The authors hypothesized that the lower solubility was caused by protein-protein hydrophobic interactions in the AE/IEP and micellization samples. Commercial pea protein may have decreased solubility in wet extraction due to heat-induced denaturation (and possible aggregation) during spray-drying (Chao & Aluko, 2018). Salts in solution can operate to screen the electric double-layer (i.e., diffuse, and stern layers) around the protein, thereby lowering the zeta potential and the quantity of electrostatic repulsive forces that occur. As a result, proteins assemble via hydrophobic interactions, hydrogen bonding, and Van der Waals attractive forces as if they had decreased or low net charges [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF]. In the case of salting-in, thiocyanate, perchlorate, barium, and calcium salts promote protein-water interactions and ordering of hydration layers surrounding the protein to improve solubility [START_REF] Hall | Methods of testing protein functionality[END_REF][START_REF] Walstra | Physical chemistry of foods[END_REF]Damodaran, Parkin, & Fennema, 2007). In the case of salting-out, sulfate, hydrogen phosphate, ammonium, and potassium salts increase ion-water interactions, which operate to destabilize the hydration layers around the proteins, allowing the exposure of hydrophobic moieties to be exposed [START_REF] Hall | Methods of testing protein functionality[END_REF][START_REF] Walstra | Physical chemistry of foods[END_REF]Damodaran, Parkin, & Fennema, 2007). Table 1-5 describes the main published studies on the solubility of pea proteins based on the extraction method used and the experimental physicochemical conditions applied to measure the solubility.

Several researcheres have used solubility comparisons to compare pea protein isolates to other plant protein isolates. In general, soy protein isolates were more soluble than pea protein isolates [START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF][START_REF] Kimura | Comparison of Physicochemical Properties of 7S and 11S Globulins from Pea, Fava Bean, Cowpea, and French Bean with Those of Soybean-French Bean 7S Globulin Exhibits Excellent Properties[END_REF][START_REF] Zhao | Comparison of wheat, soybean, rice, and pea protein properties for effective applications in food products[END_REF]. The study by [START_REF] Ladjal-Ettoumi | Pea, chickpea and lentil protein isolates: Physicochemical characterization and emulsifying properties[END_REF] highlighted that pea protein isolates had similar solubility to lentil protein or chickpea protein at pHs ranging from 2 to 8.

As a result, it is extremely difficult to position the solubility of pea proteins concerning the large range of legume species that exist, depending on a variety of criteria (cultivar, technique of extraction, experimental settings, etc.). This techno-functional feature can be optimized by using different extraction methods that are more respectful of protein structure such as gentle fractionation (Figure 1-4) (Kornet et al., 2021b) or using high hydrostatic pressure, associated with solubility enhancement techniques, for instance,

Gel properties

Gelling properties of Pea protein

Gelation is an essential functional property of globular proteins because it is used to alter the texture of food and plays an important role in the sensory of many food products as well [START_REF] Ge | The health benefits, functional properties, modifications, and applications of pea (Pisum sativum L.) protein: Current status, challenges, and perspectives[END_REF][START_REF] Shanthakumar | The Current Situation of Pea Protein and Its Application in the Food Industry[END_REF]. Protein gelation is the formation of a three-dimensional network of molecular structures by protein molecules immersed in an aqueous solvent [START_REF] Tomé | Protein gels and emulsions from mixtures of Cape hake and pea proteins[END_REF][START_REF] Shanthakumar | The Current Situation of Pea Protein and Its Application in the Food Industry[END_REF]. The mechanism of globular protein gelation is a complicated process that involves partial denaturation of protein molecules, progressive association or aggregation, and network creation [START_REF] Mession | Effect of globular pea proteins fractionation on their heat-induced aggregation and acid cold-set gelation[END_REF][START_REF] Ge | The health benefits, functional properties, modifications, and applications of pea (Pisum sativum L.) protein: Current status, challenges, and perspectives[END_REF].

In general, gelation can be generated by a physical process (heat-induced, high pressure) or a (bio)chemical process (chemical acidification or microorganisms, enzymatic crosslinking, salt addition) [START_REF] Totosaus | A review of physical and chemical protein-gel induction[END_REF][START_REF] Nicolai | Gelation of food protein-protein mixtures[END_REF]. For heatinduced gel, when a protein is heated above its denaturation temperature, at a higher concentration than its minimum gelling concentration (MGC), this causes partial unfolding of the protein and exposes the interaction sites, leading to intermolecular interactions, and eventually resulting in clustering of protein aggregates to form a spatial gel network. In terms of cold-induced gel, it performs a specific pre-heating treatment for a low-concentrated protein suspension at pH far from protein pI to prepare soluble protein aggregates in the absence of salts, and then cooled, the cold-set gelation is carried out by adding salts, acidifying agents, or enzymes, allowing it to assemble into network structure [START_REF] Ge | The health benefits, functional properties, modifications, and applications of pea (Pisum sativum L.) protein: Current status, challenges, and perspectives[END_REF][START_REF] Shanthakumar | The Current Situation of Pea Protein and Its Application in the Food Industry[END_REF]. Regarding cold gelation of pea protein, non-covalent and new disulfide linkages were involved in gelation [START_REF] Mession | Effect of globular pea proteins fractionation on their heat-induced aggregation and acid cold-set gelation[END_REF]. Several parameters have a great impact on gelation, such as extrinsic factors related to gelling process, containing pH, temperature, ionic strength, and intrinsic factors related to protein including protein conformation, concentration, the cultivar, and extracted procedures [START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF]Mession et al Heat-induced gels are the most common kind of pea protein gelation, with just a few cold-set gels and enzymatic gels described (Table 123456). According to Table 123456, numerous conclusions may be derived from the study on the various approaches to the gelation of pea proteins, which can allow for the optimization of the different gelation parameters to form well-defined gel structures with desirable rheological and textural features. The commercial pea protein isolate (PPIc) displayed poor gelling capabilities after applying multiple gelling procedures, for instance, an increase in the minimum gelation concentration (MGC) and a decrease in the modulus of elasticity (G'), as compared to the pea protein isolate (PPI) prepared by laboratory methods. This was related to pea protein denaturation during large-scale manufacture [START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF][START_REF] Adebiyi | Functional properties of protein fractions obtained from commercial yellow field pea (Pisum sativum L.) seed protein isolate[END_REF][START_REF] Mession | Effect of globular pea proteins fractionation on their heat-induced aggregation and acid cold-set gelation[END_REF][START_REF] Ben-Harb | The rheological and microstructural properties of pea, milk, mixed pea/milk gels and gelled emulsions designed by thermal, acid, and enzyme treatments[END_REF]. However, Sun & Arntfield (2010) obtained the opposite result, that adding NaCl led to an MGC of PPIc around 14.5 % which was much lower than other reports. NaCl might have promoted gel formation by increasing intermolecular hydrophobic interactions and decreasing electrostatic repulsion and changing the water structure surrounding PPIc proteins, therefore increasing protein hydration and reducing the MGC. [START_REF] Zhao | Comparison of wheat, soybean, rice, and pea protein properties for effective applications in food products[END_REF] found similar results, with the MGC of PPIc being 14 % (w/v). On the other hand, several studies focused on the comparison between PPI and soy protein isolate (SPI), that, SPI, generally, showed greater gelling ability than PPI, with harder, stiffer gels and more organized structures [START_REF] Batista | Accessing gelling ability of vegetable proteins using rheological and fluorescence techniques[END_REF]Munialo et al., 2014a;[START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF]. This was related to the makeup of fractions from these proteins. For instance, the composition of the fractions of pea protein can vary from cultivar to cultivar, resulting in significant differences in gelation capabilities [START_REF] O'kane | Gelation behavior of protein isolates extracted from 5 cultivars of Pisum sativum L[END_REF]. This observation was also corroborated by [START_REF] Shevkani | Structural and functional characterization of kidney bean and field pea protein isolates: A comparative study[END_REF], who discovered that various pea cultivars exhibited variable gels and rheological properties because of differences in their physicochemical and structural composition even though utilizing the same extraction procedures. Pea protein is a mixed protein that may be fractionated into vicilin (7S) and legumin (11S) fractions, as well as water-soluble, salt-soluble, ethanol-soluble, and alkaline-soluble fractions, using various classification techniques [START_REF] Ge | The health benefits, functional properties, modifications, and applications of pea (Pisum sativum L.) protein: Current status, challenges, and perspectives[END_REF]. Thus, O'kane et al. (2004a et 2005) showed that the isolate from five pea protein isolates recovered at the laboratory scale and from various cultivars, with the greatest legumin content formed stiffer gels. However, [START_REF] Bora | Heat induced gelation of pea (Pisum sativum) mixed globulins, vicilin and legumin[END_REF] found that no gel was formed for legumin under the same condition as pea globulin. Thus, it could be the difference between the cultivars [START_REF] Barać | Techno-functional properties of pea (Pisum sativum) protein isolates: A review[END_REF]. In parallel, it was highlighted that the formation of disulfide bonds was a factor that inhibited the gel network from strengthening. These contradictory findings are in complete agreement with other studies that have concluded that vicilin is the main fraction that allows the gel network of pea proteins to be reinforced due to the predominance of hydrophobic, electrostatic, and hydrogen bonding interactions [START_REF] Mession | Effect of globular pea proteins fractionation on their heat-induced aggregation and acid cold-set gelation[END_REF]O'Kane et al 2004a;Sun & Arntfield, 2012a). As a result, pea protein isolates with increased vicilin content may be more suited to gel formation [START_REF] Barać | Techno-functional properties of pea (Pisum sativum) protein isolates: A review[END_REF][START_REF] O'kane | Gelation behavior of protein isolates extracted from 5 cultivars of Pisum sativum L[END_REF].

It was also discovered that the inclusion of starch (high water retention capacity), sugars, and ash in the composition contributed to the gels' hardness (Kornet et al., 2021b;Pelgrom et al., 2015b). (2021b) demonstrated that it was necessary to use restricted extractions and fractionations from pea concentrates to produce a greater "degree of the nativity" of pea proteins and hence improved gel firmness and homogeneity. Thus, with the goal of improving the extrinsic elements that can significantly impact the gelling properties of pea proteins, various research has concentrated on identifying the perfect conditions for pea protein gelation, involving heat-treating temperatures, times and the rate of heating and cooling, ionic strength, pH, and the addition of cross-linking enzymes, to produce final features of improved pea protein gels [START_REF] Yang | Extraction methods significantly impact pea protein composition, structure and gelling properties[END_REF][START_REF] Zhu | Strong and elastic pea protein hydrogels formed through pH-shifting method[END_REF][START_REF] Zhang | Improvement of pea protein gelation at reduced temperature by atmospheric cold plasma and the gelling mechanism study[END_REF][START_REF] Tanger | Influence of pH and ionic strength on the thermal gelation behaviour of pea protein[END_REF]. In general, pea protein formed a weak, heat-induced gel [START_REF] Barać | Techno-functional properties of pea (Pisum sativum) protein isolates: A review[END_REF]. Pea protein gelation is temperature dependent and is largely regulated by the degree of protein denaturation. A stronger gel is created when the degree of denaturation is lower. Protein concentration is also essential in gelation characteristics [START_REF] Sim | High pressure structuring of pea protein concentrates[END_REF][START_REF] Guldiken | Heat induced gelation of pulse protein networks[END_REF]. Gels are often stronger at higher concentrations. Sun & Arntfield (2010) found that the gelling point was concentrationindependent. Heating and cooling speeds have a small impact on the gelation characteristics of pea protein. The gelling point was impacted by the heating rate, with greater heating rates resulting in delayed gelling (higher gelling temperatures). Higher heating and cooling rates resulted in a loss of gel elasticity. According to the previous studies conducted by [START_REF] Felix | Development of thermally processed bioactive pea protein gels: Evaluation of mechanical and antioxidant properties[END_REF] and Munialo et al. (2014b), thermal gelation capability has been demonstrated to be superior at neutral and alkaline pHs and moderate ionic strengths. Furthermore, all the heat-treating temperatures used ranged between 85 and 100 °C, which surpassed the thermal denaturation temperature of the pea proteins. However, when the heating time was increased, so did the gel strength [START_REF] Bora | Heat induced gelation of pea (Pisum sativum) mixed globulins, vicilin and legumin[END_REF]. On the other hand, the heating rate did not affect the strength of the gels (O' Kane et al., 2005), although a slower cooling rate permitted in many studies to increase the stiffness of the gels due to the lengthening of the gelation duration, making it feasible to facilitate and promote the intermolecular contacts and structural rearrangement that occurs during cooling (O' Kane et al., 2004b;Sun & Arntfield, 2011a). Furthermore, cross-linking enzymes such as MTG, have been found to be particularly successful in strengthening the network and enhancing the strength and elasticity of gels (Djoullah et al., 2018;[START_REF] Shand | Transglutaminase treatment of pea proteins: Effect on physicochemical and rheological properties of heat-induced protein gels[END_REF]Sun & Arntfield, 2011b).

Finally, [START_REF] Ben-Harb | The rheological and microstructural properties of pea, milk, mixed pea/milk gels and gelled emulsions designed by thermal, acid, and enzyme treatments[END_REF] examined the rheological parameters of the three gelling procedures listed above on a commercial pea protein isolate to find the differences. The results showed that the acidified gels were substantially stiffer due to electrostatic interactions between the pea protein aggregates, which had a considerable influence on the gel's characteristics.

Gelling properties of egg white

Egg white is frequently used as an additive to improve the water-holding capacity or gel strength of food items. Because of its low lipid content, egg white is frequently favored over egg yolk for its gelling capabilities [START_REF] Mine | Egg proteins[END_REF]. Many food items' textural and rheological features, such as meringues and angel food cakes, are dependent on the heat coagulation or gelation capabilities of egg proteins, particularly their irreversible heat coagulation [START_REF] Ren | Nutritional and health attributes of eggs[END_REF]. A thermally irreversible gel is a viscoelastic solid created by heat that does not reheat to a viscous liquid [START_REF] Mine | Egg proteins[END_REF].

Mechanism of gelation

A gel is a transitional state between liquid and solid. Cross-linking between polymeric molecules forms an intermolecular network inside a liquid medium [START_REF] Alleoni | Albumen protein and functional properties of gelation and foaming[END_REF]. Protein gel production may be separated into two steps, which is depicted schematically in Figure 1234567. The first step involves conformational changes or partial denaturation of the protein molecule (typically by heat), leading to the unfolding of proteins and an elastic solid occurs. The second step shows a slow association or molecular aggregation of denatured proteins resulting in an exponential rise in viscosity and the creation of a continuous network. Depending on the physicochemical conditions, this stage of the process creation is slower than the first one and concludes when an orderly network is developed. When the second stage is quicker, a disorderly protein cluster with limited water holding capacity might develop, resulting in a greater degree of syneresis. [START_REF] Alleoni | Albumen protein and functional properties of gelation and foaming[END_REF].

In this gelation process, non-covalent interactions (electrostatic, hydrophobic, and hydrogen bonds) and intermolecular disulfide cross-linking induced protein aggregation are prevalent [START_REF] Razi | An overview of the functional properties of egg white proteins and their application in the food industry[END_REF]. A significant increase in effective hydrophobicity during heating indicates protein unfolding, and when too many hydrophobic sites are exposed, interactions between the exposed hydrophobic sites are unavoidable, resulting in protein molecule aggregation [START_REF] Totosaus | A review of physical and chemical protein-gel induction[END_REF].

Meanwhile, soluble aggregates of high molecular weight form with intermolecular βsheet interactions. During cooling, unfolded proteins can refold into a refolded configuration, the gel strength increases due to the creation of hydrogen bonds. What should be paid attention to is that if low molecular weight and low protein concentration circumstances generate an aggregation, the protein solution becomes opaque, whereas high molecular weight and high protein concentration conditions produce a coagulum (due to protein chain entanglement) (thermo-irreversible gel) [START_REF] Totosaus | A review of physical and chemical protein-gel induction[END_REF].

By joining the polymers in the case of EWP gels, a 3-dimensional (3D) network arises.

At greater repulsive values, the 3D network cannot develop, but at higher attractive strengths, water is forced off the matrix [START_REF] Alleoni | Albumen protein and functional properties of gelation and foaming[END_REF][START_REF] Razi | An overview of the functional properties of egg white proteins and their application in the food industry[END_REF]. (Adapted from [START_REF] Mine | Recent advances in the understanding of egg white protein functionality[END_REF][START_REF] Campbell | Modification of functional properties of egg-white proteins[END_REF][START_REF] Razi | An overview of the functional properties of egg white proteins and their application in the food industry[END_REF]

Factors affecting the heat-induced gelation of egg white

Because of the interactions between protein chains, egg white creates a threedimensional gel network. The physicochemical parameters of the medium, such as ionic strength, salt type, pH, protein content, and interactions with other components, all affect the gel's quality [START_REF] Croguennec | Influence of pH and salts on egg white gelation[END_REF][START_REF] Raikos | Rheology and texture of hen's egg protein heat-set gels as affected by pH and the addition of sugar and/or salt[END_REF].

Effect of protein concentration

The minimum gel concentration is the starting point for protein cross-linking to create a gel; once this concentration is reached, the protein may form a gel with a threedimensional network structure [START_REF] Lv | Modification methods and applications of egg protein gel properties: A review[END_REF]. The concentration mostly influences the hydrophobic contacts and ionic connections formed between protein molecules.

Increasing protein concentration can enhance protein gel strength and gel hardness [START_REF] Hongsprabhas | Protein and salt effects on Ca2+-induced cold gelation of whey protein isolate[END_REF][START_REF] Quan | Duck egg albumen: physicochemical and functional properties as affected by storage and processing[END_REF]. The protein gel structure created at high concentrations is more compact because a large number of surface groups of protein molecules can contribute to the development of gel networks. [START_REF] Iwashita | Thermal aggregation of hen egg white proteins in the presence of salts[END_REF] discovered that heat treatment readily gelled at a high content of albumen. These authors found that strings of beads, characterized as organized soluble linear aggregates of denatured ovalbumin molecule, are more likely to form via constrained attractive hydrophobic contact but substantial electrostatic repulsion, making it easier to form 3D gel networks by the interconnections of these soluble linear aggregates. In contrast, according to the work of [START_REF] Ren | Nutritional and health attributes of eggs[END_REF], low protein concentrations preferred to form a viscous transparent solution rather than a gel network. [START_REF] Campbell | Modification of functional properties of egg-white proteins[END_REF] obtained the creation of a 3D gel network with sufficiently high protein content, high ionic strength, and pH levels near its isoelectric point. Furthermore, the protein content is known to influence the rheological properties and gelling temperature of egg white gels [START_REF] Quan | Duck egg albumen: physicochemical and functional properties as affected by storage and processing[END_REF]. The temperature of gelation and storage modulus (G') were both found to be concentration dependent, with G' increasing as egg white concentrations rose. This rise is commonly attributed to the consolidation of attractive forces such as van der Waals and hydrogen bonding between protein particles inside the gel network. Furthermore, when the protein content increased, so did the gelation temperature [START_REF] Eleya | Gelling properties of egg white produced using a conventional and a low-shear reverse osmosis process[END_REF].

Effect of pH

The pH has a major influence on the gelling properties of egg white proteins, by influencing the protein net charge and the reactivity of the SH group at higher pH [START_REF] Quan | Duck egg albumen: physicochemical and functional properties as affected by storage and processing[END_REF][START_REF] Lv | Modification methods and applications of egg protein gel properties: A review[END_REF][START_REF] Razi | An overview of the functional properties of egg white proteins and their application in the food industry[END_REF]. The charge distribution of amino acid side chains is affected by the pH of the protein environment. When the pH approaches the protein's isoelectric point, the electrostatic repulsion between protein molecules reduces, and the protein quickly aggregates and solidifies [START_REF] Campbell | Modification of functional properties of egg-white proteins[END_REF][START_REF] Quan | Duck egg albumen: physicochemical and functional properties as affected by storage and processing[END_REF]. The pH of egg white rises during egg storage typically from 7.6 (time of lay) to around 9.5 [START_REF] Sharp | Increase in the pH of the white and yolk of hens' eggs[END_REF]. [START_REF] Hickson | Changes in heat-induced rheological properties during cold storage of egg albumen[END_REF] and [START_REF] Hammershøj | Storage of shell eggs influences the albumen gelling properties[END_REF] found that gel elasticity, viscosity index, and penetrating force increased with increasing pH of egg white during storage. Moreover, [START_REF] Raikos | Rheology and texture of hen's egg protein heat-set gels as affected by pH and the addition of sugar and/or salt[END_REF] illustrated that the gelation temperature of egg proteins increased with increasing pH and/or adding NaCl. [START_REF] Croguennec | Influence of pH and salts on egg white gelation[END_REF] investigated the gelation of egg white in the presence of salts at different pH. These authors found that protein denaturation at pH 5.0 (the pI of most egg white proteins) produced a coarse aggregation and coagulum with little water holding capacity and viscoelastic characteristics. Lower protein net charge was found at this pH, as well as the least hydrophilicity near the pH close to its isoelectric point. More viscoelastic gels were formed at pH 7 and 9, whereas thermo-coagulation was slowed at both pHs, because of an increase in protein net charge. Because of the unfolding of proteins before aggregation, the gel formed at these pHs exhibited a high water-holding capacity as well. [START_REF] Nyemb | The structural properties of egg white gels impact the extent of in vitro protein digestion and the nature of peptides generated[END_REF] have studied the heat-induced gel of EW influenced by four different combinations of pH and ionic strength (IS) (pH 2/IS 0.05 M, pH 5/IS 1 M; pH 7/IS 0.05 M; pH 9/IS 0.05 M). It was found that the Granular-spongy EW gel was formed at pH 5/IS 1 M, the intermediate gel was formed at pH 7/IS 0.05 M, the smoothrigid gel was formed at pH 9/IS 0.05 M, the fracturable gel was formed at pH 2/IS 0.05 M. Minimal elasticity was also observed at pH 5/IS 1 M, as well as a lower cohesiveness than that observed at pH 9 resulted from the heterogeneity of the microstructure, and from the nature of protein interactions which are mainly weak (hydrophobic) in these conditions. The EW gels formed at pH 7 (intermediate gel) have a variety of aggregate structures: ovotransferrin spherical aggregates scattered in an ovalbumin linear-branched aggregate protein network, due to i) random and spherical ovotransferrin aggregates are predominated as pH 7 closed to pI of ovotransferrin (~ 6.5), and ii) development of linear-branched OVA aggregates as pH 7 far way from ovalbumin's pI (approximately 4.5). Gelation is delayed at pH 7 as compared to pH 5 because electrostatic repulsions are higher at pH 7 as compared to pH 5. Gels formed at pH 9 had greater elasticity and cohesiveness values as compared to pH 7 and pH 5 gels and has a smooth-rigid network due to increased net charge and electrostatic repulsions (pH 9 far from pI of ovalbumin and ovotransferrin). Gels formed at pH 2 had a filamentous gel made of linear aggregates due to high net charge and electrostatic repulsions leading to proteins tending to denature rather than aggregate. [START_REF] Khemakhem | The effect of pH, sucrose, salt and hydrocolloid gums on the gelling properties and water holding capacity of egg white gel[END_REF] investigated the influence of pH (4.5 and 6.5) on the gelling characteristics of egg white protein and highlighted that at pH 4.5, gels had the lowest hardness and elasticity (near the pI) but cohesiveness and adhesiveness were greater. Protein-protein interactions can increase at pH around pI, but protein-solvent interactions increase at higher pH (above pI). The gel structure was poor at pH 4.5, and the network had big holes [START_REF] Khemakhem | The effect of pH, sucrose, salt and hydrocolloid gums on the gelling properties and water holding capacity of egg white gel[END_REF]. Protein aggregation is a significant process for gel formation at pH 4.5, on the opposite, at pH 7.0, aggregation is restricted by repulsive electrostatic forces, and produced gels are constituted of organized linear polymers. [START_REF] Chen | Changes in gel characteristics of egg white under strong alkali treatment[END_REF] studied the changes in gel properties of egg white treated at strong alkaline and found that gels formed under strong alkaline relied mostly on ionic and disulfide connections, while hydrophobic interactions and hydrogen bonds were rarely involved in gel conformation preservation. According to [START_REF] Li | Combination effects of NaOH and NaCl on the rheology and gel characteristics of hen egg white proteins[END_REF], the opacity and particle turbidity of egg white protein gel reduced with increasing NaOH concentration. According to [START_REF] Babaei | Effects of enriching with gellan gum on the structural, functional, and degradation properties of egg white heat-induced hydrogels[END_REF], the stiffness of egg white protein gels was higher at pH 4 than at pH 7, which was due to stronger electrostatic repulsion between the proteins at pH 7 compared to pH 4. [START_REF] Raikos | Rheology and texture of hen's egg protein heat-set gels as affected by pH and the addition of sugar and/or salt[END_REF] also highlighted that egg samples at pH 5 and 8 had higher gel strength than egg samples at pH 2. Another analysis by [START_REF] Phillips | Egg proteins[END_REF] found that coagula or soft/turbid gels of egg albumen were generated at high ionic strength and around the isoelectric point, because of the existence of these coagula inside the gel network, opaque gels formed.

Effect of salts and types of salts

Salts can favor protein aggregation in solutions and allow the formation of a gel network by egg protein. Sodium chloride can influence gel characteristics by strengthening or decreasing connections between protein molecules. It was found that moderate levels of sodium chloride can improve the hydrophobic interactions between protein molecules, but too much salt can disintegrate ionic and hydrogen connections between protein molecules, making the gel weaker [START_REF] Raikos | Rheology and texture of hen's egg protein heat-set gels as affected by pH and the addition of sugar and/or salt[END_REF]. Ma, Zhao, & Chi. (2012) also found that increasing the concentration of NaCl increased the water release of hen albumen gel. In the presence of 120 mM NaCl, the most water was released. The greater NaCl content was linked to the excessive heat-induced aggregation of albumen proteins, most likely via ionic or hydrophobic contact. The effects of inorganic salts on the thermal aggregation of albumen, including NaCl, Na2SO4, and NaSCN had also been studied by [START_REF] Iwashita | Thermal aggregation of hen egg white proteins in the presence of salts[END_REF]. Na2SO4 and NaSCN are both kosmotropic and chaotropic agents. The kosmotropes enhanced the surface tension of the solution more than the chaotropes did. This might be because chaotropes destabilize protein tertiary structure, resulting in a lower denaturation temperature, whereas kosmotropes stabilize protein structure. The salting out of protein molecules predominated at salt concentrations greater than 0.5 M. Salt anions attach to the positively charged domains of lysozyme at low concentrations, increasing solubility independent of the type of ions [START_REF] Iwashita | Co-aggregation of ovalbumin and lysozyme[END_REF]. Denaturation of heat-treated proteins containing NaCl reduced the ordered secondary structure (α-helix and β-sheet) of egg white proteins gel by disrupting hydrogen bonds [START_REF] Li | Combination effects of NaOH and NaCl on the rheology and gel characteristics of hen egg white proteins[END_REF][START_REF] Li | Molecular forces and gelling properties of heat-set whole chicken egg protein gel as affected by NaCl or pH[END_REF][START_REF] Li | Characteristics of gelling and water holding properties of hen egg white/yolk gel with NaCl addition[END_REF]. The mechanical characteristics of the produced gel are affected by salt ions, which might improve its stability. Salt ions in macromolecules tend to lower protein electrostatic energy and increase protein solubility in aqueous conditions [START_REF] Li | Combination effects of NaOH and NaCl on the rheology and gel characteristics of hen egg white proteins[END_REF][START_REF] Li | Molecular forces and gelling properties of heat-set whole chicken egg protein gel as affected by NaCl or pH[END_REF][START_REF] Li | Characteristics of gelling and water holding properties of hen egg white/yolk gel with NaCl addition[END_REF]. A reversal effect can be noticed at high salt concentrations. This might be due to greater salt concentrations breaking hydrogen bonds and destabilizing protein molecules, weakening gel structure [START_REF] Li | Combination effects of NaOH and NaCl on the rheology and gel characteristics of hen egg white proteins[END_REF][START_REF] Li | Molecular forces and gelling properties of heat-set whole chicken egg protein gel as affected by NaCl or pH[END_REF][START_REF] Li | Characteristics of gelling and water holding properties of hen egg white/yolk gel with NaCl addition[END_REF].

Several studies mentioned that heat-induced and alkaline-induced gelation of egg albumen is also affected by salt type [START_REF] Croguennec | Influence of pH and salts on egg white gelation[END_REF][START_REF] Zhao | Effects of alkaline concentration, temperature, and additives on the strength of alkaline-induced egg white gel[END_REF][START_REF] Deng | Effects of metal ions on the physico-chemical, microstructural and digestion characteristics of alkali-induced egg white gel[END_REF]. [START_REF] Croguennec | Influence of pH and salts on egg white gelation[END_REF] highlighted that Ca 2+ and Mg 2+ altered the viscoelastic characteristics of egg albumen gel, resulting in a less homogenous gel, with particles aggregated in random aggregates and no string of beads formations. The inclusion of cations enhanced the heat stability of albumen proteins, but CuSO4 significantly softened albumen coagulum gel. Gel stiffness was unaffected by FeCl3 or A1C13. These modifications were caused by the negative charge of albumen proteins being shielded [START_REF] Croguennec | Influence of pH and salts on egg white gelation[END_REF]. The alkali provides an environment for egg white protein denaturation and aggregation, and high concentrations of metal ions can promote cross-linking and more effective aggregation of aggregates. Monovalent and multivalent ions both protect negatively charged protein molecules from electrostatic interactions, and differing valence levels of metal ions have varied effects on protein gels [START_REF] Deng | Effects of metal ions on the physico-chemical, microstructural and digestion characteristics of alkali-induced egg white gel[END_REF][START_REF] Li | Comparative Study on Foaming Properties of Egg White with Yolk Fractions and Their Hydrolysates[END_REF]. For example, [START_REF] Deng | Effects of metal ions on the physico-chemical, microstructural and digestion characteristics of alkali-induced egg white gel[END_REF] investigated the effect of different ions on alkaline-induced egg white gels. It was found that multivalent ions (Ca 2+ , Zn 2+ , and Fe 3+ ) had more positive charges and a higher shielding capacity against electrostatic interactions between protein molecules, which might result in more cross-links when gels were formed at the same concentration. Higher concentrations of the salts, on the other hand, were incompatible with the formation of protein gels. The authors also showed that the performance of the gel generated by monovalent ions (K + ) was superior to that of the gel formed by multivalent ions, which was distinguished by increased soluble protein content and water-holding capacity, as well as a denser gel microstructure.

Mixtures of plant proteins and animal proteins

Raw animal materials such as milk, eggs, meat, and seafood continue to be the most important protein sources lately employed by food companies and humans in the world, followed by plant sources such as legumes and nuts [START_REF] Alves | Mixing animal and plant proteins: Is this a way to improve protein techno-functionalities?[END_REF].

However, unlike plant protein production, animal protein production is coupled with high greenhouse gas emissions and a larger requirement for land space [START_REF] Henchion | Future protein supply and demand: strategies and factors influencing a sustainable equilibrium[END_REF][START_REF] Alves | Mixing animal and plant proteins: Is this a way to improve protein techno-functionalities?[END_REF][START_REF] Schmitt | Heatinduced and acid-induced gelation of dairy/plant protein dispersions and emulsions[END_REF][START_REF] Wu | Advancement of food-derived mixed protein systems: Interactions, aggregations, and functional properties[END_REF]. It has been determined that, in order to achieve long-term development, animal proteins should be more commonly at least partly, substituted by plant proteins in food composition keeping good nutritional qualities. For this sort of partial replacement to be possible, animal and plant proteins must be able to interact with one another to generate homogenous products with intriguing functional features.

Few investigations have been conducted to date to characterize the processes of aggregation and association of plant and egg white protein mixtures. Currently, Alves & Tavares (2019) reviewed the main proteins used in the mixed system: plant protein, soybean, pea beans, and wheat are mainly studied, while in terms of animal proteins, milk containing caseins and whey proteins, egg white protein, and gelatin from cattle or fish are studied. Whey protein also can be divided into two main parts: β-lactoglobulin (β-Lg) and α-lactalbumin (α-La) occupying around 50 % and 20 % of total whey protein content, respectively [START_REF] Chatterton | Bioactivity of βlactoglobulin and α-lactalbumin-Technological implications for processing[END_REF]. Soy proteins and dairy proteins are mostly employed in protein combination studies in food items due to excellent nutritional content, capacity to gel formation, increased consumers demand for soy protein [START_REF] Renkema | Gel formation by βconglycinin and glycinin and their mixtures[END_REF], high purity, availability, and high solubility of dairy proteins [START_REF] Croguennec | Heteroprotein complex coacervation: A generic process[END_REF]. Particularly, formulations including soy and milk proteins may generate goods with outstanding nutritional value [START_REF] Ruiz-Henestrosa | Mixed soy globulins and β-lactoglobulin systems behaviour in aqueous solutions and at the air-water interface[END_REF]. However, pea protein has lately gained popularity because of its excellent functional, and nutritional qualities, with a non-allergenic advantage, compared to soy protein [START_REF] Aluko | Emulsifying and foaming properties of commercial yellow pea (Pisum sativum L.) seed flours[END_REF][START_REF] Burger | Recent progress in the utilization of pea protein as an emulsifier for food applications[END_REF].

The primary studies on the techno-functional properties of mixed systems produced from plant proteins (mainly pea protein) and animal proteins (mainly egg white) are summarized in Table I-7. In the following sections, co-aggregation and gel formation will be used to highlight the impact on the mixture systems (pea-based, egg white-based).

Table 1-7 Research on the techno-functional characteristics of mixed systems between plant protein and animal protein (According to [START_REF] Alves | Mixing animal and plant proteins: Is this a way to improve protein techno-functionalities?[END_REF][START_REF] Schmitt | Heatinduced and acid-induced gelation of dairy/plant protein dispersions and emulsions[END_REF]Hinderink et al., 2021a;[START_REF] Wu | Advancement of food-derived mixed protein systems: Interactions, aggregations, and functional properties[END_REF].

Properties

Plant

Animal proteins mixed with pea proteins

According to [START_REF] Alves | Mixing animal and plant proteins: Is this a way to improve protein techno-functionalities?[END_REF], mixture systems containing soy-animal proteins play an important role. Recently, several researchers have focused on the mixtures prepared by animal proteins and pea proteins, due to their excellent technofunctional properties. Many factors influenced the techno-functional properties of the pea protein isolate, as described in Section 1.4. We focused on the heat-induced gelation prepared by pea proteins and animal proteins which will be discussed in the following sections. [START_REF] Kristensen | Co-precipitation of whey and pea protein-indication of interactions[END_REF]Kristensen et al. ( , 2021aKristensen et al. ( & b, 2022) ) have studied the mixtures between pea protein isolate (PPI) and whey protein isolate (WPI). [START_REF] Kristensen | Co-precipitation of whey and pea protein-indication of interactions[END_REF]Kristensen et al. ( , 2021a) ) have defined the processes behind protein-protein interactions in a combination of two commercial isolates of pea protein (PPI) and whey protein (WPI). The previous paper (2020), wanted to optimize the yield and compare the solubility of co-precipitates and protein blends. Samples were prepared at 10 g/L mixed with different volume ratios (WPI: PPI at 20:80, 50:50, 80:20, v/v) then adjusted to pH 4.6, and heated at 60, 80, 98 ℃ for 30 min, respectively. Then the co-precipitates were obtained by centrifuging.

The maximum precipitation yield was obtained at a higher WPI ratio in the mixture, high temperature, and alkaline protein solvation. Solubilities were measured at pH 3, 7, and 11.5. Co-precipitates were far less soluble than protein blends. High WPI content in the mixtures, low precipitation temperature, and high pH showed the greatest solubility, indicating various protein-protein interactions in the mixtures. The goal of Kristensen et al. (2021a) was to see how the creation of a co-precipitate of these two proteins from distinct sources affected the functional aspects of the mixtures. The mixtures were made in various ratios in water at a total concentration of 2 % (w/v). The co-precipitates were then obtained by lowering the pH to 4.6 and then centrifuging to eliminate the supernatant, and freeze-dried. The powders generated from the assembly of these two proteins were then distributed at various pH levels (3, 7, and 11.5). Because of the massive aggregates created by interactions between the two proteins, coprecipitation did not further improve the rise in solubility. Furthermore, this study demonstrated that a simple mixing of these proteins under neutral or alkaline pH conditions can increase the solubility, emulsifying, and foaming capabilities of the proteins in the mixture compared to the proteins alone. Moreover, Kristensen et al. Several literature focused on the gelation properties of the mixed system conducted by pea protein isolate and animal proteins (mainly dairy proteins) (Table 1234567 and Mession, Roustel, & Saurel (2017b).

In addition, some of these researches have also concentrated on the interaction mechanisms and physicochemical factors that govern pea protein-milk protein interactions during gelation. For example, [START_REF] Wong | Synergistic enhancement in the cogelation of salt-soluble pea proteins and whey proteins[END_REF] studied the thermal gelation of the mixture prepared by pea protein concentrate (PPC) and whey protein concentrate (WPC). They prepared the gels by heating up to 92 ℃ for 30 min and investigated the influence of pH and NaCl concentration on the rheological and textural features of thermal gels formed by combining proteins in various volume ratios and total protein concentrations. The addition of whey proteins improved the gelling properties of pea proteins by gradually enhancing the elasticity and stiffness of the produced thermal co-gels. Furthermore, the rheological and textural qualities created at pH 6 were superior to those formed at pH 4 and 8. This discrepancy was related to electrostatic repulsions between convicilin (7S) subunits as the pH shifted away from their isoelectric pH, which was indicated close to 6. These researchers, on the other hand, discovered that the NaCl concentrations of 0.5-3.0 % (w/v) all demonstrated synergistic enhancement on gel hardness at the same PPC/WPC ratio of 1/4, with the lower NaCl concentrations producing larger degrees of synergistic enhancement. It should be highlighted that the processes of heat aggregation as well as the nature of the molecular connections that structure this mixed system were not explored in this study. These findings indicate a considerable variation in the rheological behavior of cogels generated at temperatures over 70 °C depending on the kind of plant proteins added.

When whey proteins were added, the network was reinforced, and the gel's stiffness increases significantly. Whey proteins could form disulfide bonds with caseins on the surface of the micelles according to the work of [START_REF] Corredig | The mechanisms of the heat-induced interaction of whey proteins with casein micelles in milk[END_REF]. The inclusion of the two plant proteins, on the other hand, did not improve these features, demonstrating that, unlike the serum proteins, they did not interact with the casein micelles. [START_REF] Silva | Heatinduced gelation of mixtures of micellar caseins and plant proteins in aqueous solution[END_REF] conducted additional research to further understand how the protein composition influenced the heat-induced gelation of mixes of casein micelles and plant proteins. The samples were prepared by changing the MC/plant protein ratio from 0/100 to 100/0 for total protein concentrations of 4, 6, and 8 % (w/w) at pH 5.8 and 6.0. The critical temperature of MC gelation was shown to rise when MC was substituted with SPI or PPI due to calcium chelation by these plant proteins, and SPI bound Ca 2+ more efficiently than PPI. This finding demonstrated that the amount of calcium required for casein micelle gelation varies depending on the type of plant proteins used, and that pea proteins allow for more casein replacement while complexing less calcium. Interestingly, it was shown that replacing the micelles with 40% soy proteins vs 70% pea proteins resulted in the lowest gel stiffness (Figure 123456789).

As a result, the stiffness of mixed gels at a given total protein content reduced when MC was replaced by plant proteins or vice versa because the stiffness of both MC and plant protein gels reduced with decreasing concentration. Also, it was concluded that after heating, MC and plant proteins did not co-aggregate and that each kind of protein formed networks independently.

Plant proteins mixed with egg white

According to studies of mixing systems on animal proteins and plant proteins, milk proteins are the most common animal proteins, whereas egg white protein was little considered (Table 1234567). Recently, people have paid more attention to using egg white as a source of animal protein because of their unique functional properties [START_REF] Campbell | Modification of functional properties of egg-white proteins[END_REF]. weight ratio of 1:0, 9:1, 3:1, 1:1, 1:3, 1:9, 0:1 (SPI/EW, w/w) at a total protein concentration of 0.03, 0.06, 0.08, 0.10 g/mL, respectively. The results showed that the hardness, springiness, and water-holding capacity of composite gels all increased as protein concentrations rose. When total protein content was more than 0.03 g/mL, a 1:1 combination of SPI and EW improved springiness and water-holding capacity, suggesting that synergistic interactions were produced between SPI and EW. These results were confirmed by Figure 1-10, that the gel mixed in a 1:1 (SPI/EW) ratio was more homogeneous and included fewer big particles in the network, which was associated with the increased springiness and water-holding capacity. However, when the SPI/EW ratios were greater than 1:1, the homogeneity rose and the big particles reduced in the structures with the addition of EW; when the ratio was less than 1:1, the network of gels became more uneven and rough, with more large particles as the EW content grew. The hardness and storage modulus of gels rose steadily as the egg white content of the composite gels increased. The gels' α-helical structure content was increased initially and subsequently dropped, but the β-sheet structure content was continuously raised with the rise in egg white ratio. The α-helical and β-sheet tendencies correlated with the springiness and hardness of gels, respectively.

Egg white manufacture

The chicken egg is referred to as a polyfunctional component because of its numerous techno-functional properties such as emulsifying, foaming, gelling, thickening, coloring, and fragrant capabilities that make it a staple in both home cooking and the food industry. More particular properties are desired for some preparations, which may necessitate the use of egg white and egg yolk separately: egg white is a standard in terms of whipping, while egg yolk is the emulsifying agent par excellence [START_REF] Anton | Les ovoproduits: des ingrédients fonctionnels pour des matrices complexes[END_REF].

Egg products are eggs that have been taken from their shells for processing.

Breaking eggs, filtering, mixing, stabilizing, blending, pasteurizing, chilling, freezing or drying, and packaging are all part of the egg product production process (Suman et al., 2018). Whole eggs, whites, yolks, and different processed and pasteurized mixes with or without non-egg components are examples of egg products. These egg products may come in liquid, frozen, or dried form. The architecture of the manufacturing facilities must be flexible and efficient in order to handle the specialized procedures necessary for creating various egg products. Some factories additionally contain shell egg grading facilities and dual jurisdiction activities in the egg products facility, such as boiling and hard-boiled operations.

Processing of egg products

Products derived from the processing of the egg or its many components or mixes, which may be partially supplemented by other foodstuffs or additives, and can be liquid or concentrated, dried, crystallized, frozen, deep-frozen, or coagulated are referenced as egg products. The food service business commonly uses liquid, frozen, and dry egg products as ingredients in other dishes such as prepared mayonnaise, ice cream, salad dressings, frozen desserts, cream puff, cakes, confections, and so on or transformed egg products such as scrambled eggs or omelets (FSIS, 2013;[START_REF] Wu | Eggs and egg products processing[END_REF] As a result, the egg products business now provides a variety of goods originating from diverse technical schemes, with the goal of reacting as closely as possible to the products' techno-functional applications (Figure 1 -11). This business, like any other agri-food industry, is subject to a variety of regulatory restraints, one of which is the management of cleanliness. In the great majority of situations, the content of an egg deposited by a healthy hen is sterile, and if the shell is unbroken, it will remain so for a long time. To manage the microbiological quality of egg products, it is required to restrict their initial contamination, eradicate all or part of the contaminating flora, and limit or even prevent its growth using different stabilizing methods.

Regarding the washing step, because it is not possible to produce completely clean eggs, eggs are washed and cleaned to remove stains, dirt, and other surface contaminants in order to reduce bacterial contamination and prevent bacteria from penetrating the egg contents, as well as to improve the appearance to the consumer. To minimize bacterial penetration of the shells, eggs should not be left to soak in water while washing. Modern egg washers blast the egg with water that contains a sanitizer as well as detergent (USDA, 2000) The temperature of the washing water should be kept at 32.2 ℃ or higher, and it should be at least 11 ℃ warmer than the internal temperature of the eggs to avoid the wash water from being sucked into the eggs.

According to USDA (2000), eggs should be rinsed in water at a temperature equal to or hotter than wash water to remove any remaining dirt and chemicals, after washing. The rinsing water usually contains a sanitizer, and chlorine-based compounds such as sodium hypochlorite (not less than 50 ppm or more than 200 ppm of available chlorine or its equivalent) (USDA, 2000). To avoid the possibility of microbial penetration into the eggs, shell eggs must be thoroughly dried immediately after rinsing. Eggs are airdried using electric fans before being moved to conveyors for the oiling process, where they are sprayed with mineral oil that should be tasteless and colorless [START_REF] Wu | Eggs and egg products processing[END_REF].

Figure 1-11 Production diagram of processing egg products (according to [START_REF] Anton | Les ovoproduits: des ingrédients fonctionnels pour des matrices complexes[END_REF]Suman et al., 2018).

After being oiled, the eggs are transported to the candling section, where faulty eggs are removed (Vaclavik & Christian, 2008). Candling is a useful technique to observe the shell and interior of eggs without shattering the shell; candling was previously used to examine arriving eggs for freshness by viewing their internal contents by candlelight, where egg contents may be seen when held up to a candle while being swiftly spun [START_REF] Wu | Eggs and egg products processing[END_REF].

Individual eggs must be shattered, either automatically or manually [START_REF] Anton | Les ovoproduits: des ingrédients fonctionnels pour des matrices complexes[END_REF]. Specific equipment has therefore been designed (Figure 1-12), capable of producing up to 180,000 eggs per hour and enabling instantaneous or somewhat delayed separation of the white and yolk in time.

After filtering to remove shell pieces, the products are chilled to 4 °C before being delivered to storage tanks. In the storage tanks, dry extract adjustments, salt, sugar, or allowed additives (guar, xanthan in white egg, for example) can be added. After breaking and separation, the raw goods obtained include egg white with a dry extract of 10 to 11 % and a pH of 8.5 to 9.5, egg yolk with a pH of 6.5 and a dry extract of 42 to 45 %, and whole egg with a dry extract of 20 to 24 % and a pH of 7 to 7.5 [START_REF] Anton | Les ovoproduits: des ingrédients fonctionnels pour des matrices complexes[END_REF]. Variations in dry extract for the yolk and the whole egg are mostly linked to performance material (quality of white-yellow separation). The shells discarded at this stage are a rather high-humidity byproduct.

To limit the likelihood of food-borne pathogen contamination or proliferation, liquid eggs should be pasteurized as soon as possible after breaking. Pasteurization uses a combination of time and temperature to limit the number of live germs, particularly Salmonella. Pasteurization scales are often set around 65 °C for 2 to 6 minutes for the whole egg and yolk, and about 57 °C for 2 to 6 minutes for the egg white [START_REF] Anton | Les ovoproduits: des ingrédients fonctionnels pour des matrices complexes[END_REF][START_REF] Wu | Eggs and egg products processing[END_REF]. There are also some other methods, such as ultra-heat treatment (UHT), which involves the utilization of high temperatures for a brief period. Typically, liquid eggs are heated at 70 °C for 1.5 mins and then packed in aseptic packages, extending the shelf life to up to 24 weeks, though the final product must still be refrigerated [START_REF] Zeidler | Further-processing eggs and egg products[END_REF][START_REF] Wu | Eggs and egg products processing[END_REF]. High-pressure processing (HPP) treatment typically preserves the food product's taste, look, and texture, as well as its nutrients [START_REF] Cruz-Romero | Effects of high pressure treatment on physicochemical characteristics of fresh oysters (Crassostrea gigas)[END_REF]. When applied to liquid whole egg at 350 MPa and 50 °C at 2-min pulses for four cycles, HPP decreased Salmonella enteritidis, indicating that it can be employed as a pasteurization technique [START_REF] Bari | Effect of hydrostatic pressure pulsing on the inactivation of Salmonella enteritidis in liquid whole egg[END_REF]. Its potential for processing egg products has been thoroughly investigated [START_REF] Juliano | Shelf-stable egg-based products processed by high pressure thermal sterilization[END_REF].

The products may be extended by freezing for up to 24 months. Freezing, however, has significant effects on the yolk and the liquid whole egg, resulting in irreversible gelation of the product and a modification of functional qualities (significant rise in viscosity product after thawing) [START_REF] Anton | Les ovoproduits: des ingrédients fonctionnels pour des matrices complexes[END_REF].

Dried egg products are used in the manufacture of baked items, mayonnaise, salad dressing, pasta, and other products. Because dried eggs are easy to handle and prepare, they are ideal for storage and transportation. Spray drying, pan drying, and belt drying are the most common methods of drying, the most popular approach being spray drying.

Liquid egg products can be concentrated before drying to improve thermal efficiency, increase dryer capacity, and change product properties such as lighter bulk density; liquid eggs can be concentrated using vacuum-type evaporation or ultrafiltration. The liquid egg is then atomized into a stream of heated air by high-pressure nozzles (500-6000 psi) in this procedure for fast water elimination [START_REF] Wu | Eggs and egg products processing[END_REF]. In the case of egg white, a "desugaring" step is required to avoid the development of the Maillard reaction throughout the drying process [START_REF] Anton | Les ovoproduits: des ingrédients fonctionnels pour des matrices complexes[END_REF]. This "desugaring" process involves the removal of glucose (about 0.5 g/L in a white liquid egg). It is produced either by controlled fermentation (yeasts or bacteria) or through an enzymatic method that employs the pair of glucose enzymes oxidase-catalase. This drying phase, as well as the subsequent steaming, can commonly increase specific properties functions of egg products (emulsifying properties, foaming properties).

Moreover, the concentration of the entire egg or egg white by ultrafiltration or reverse osmosis, followed by the addition of sugar and/or high concentration salt, allows to produce goods that are stable for several months at room temperature.

Objective of the work

Through the discussion in the previous sections, we can find that: i: Pea proteins have a similar amino acid content to soybean proteins and are low allergenic proteins compared to soybean proteins. However, the extraction method, cultivation method, environment, and some other conditions may affect their protein and amino acid content.

In addition, they have excellent functional qualities, are gluten-free, and are from a nongenetically modified organism. The techno-functional qualities and applications in food of pea proteins can be significantly influenced by protein concentration, concentration in 7S and 11S globulins, extraction procedure used, and processing conditions. Therefore, we should pay attention to the effect of the conditions of extraction and environment on pea proteins and their functional properties. ii: Eggs are an important source of animal proteins due to their high nutritional quality, excellent digestibility, and complete provision of essential amino acids. Egg white has better gelling properties than egg yolk which can be influenced by several conditions like pH, salt content, and protein content. iii: Although there are some researches focused on the mixtures of plant protein and animal proteins, they mainly concern soy and dairy proteins. There is a lack of research regarding the mixture of egg white as an animal protein source, especially on egg white and pea protein.

The context is the preparation of a new ingredient: so the scientific interests were as follows:

-The colloidal interactions in admixture of native proteins (solubility, complex formation…)

-Thermal behavior is very important regarding pasteurization treatment if a process similar to such ovoproduct is used to design a new ingredient -The main functionality expected are foaming and gelling: a study of gelling properties was preferred as it is the most exploited for plant proteins (meat analogues, eggs analogues): could we expect synergistic effects during the gelation of the mixed systems?

The originality of our work lies in the fact that it is a system derived from protein fractions of pea globulins and egg white which has not been studied so far. A complete study of heat-induced gelation and thermal properties will also be described. The different gels obtained will be characterized from a physicochemical point of view. The mechanisms of interactions between the different species present in solution and gels, as well as the gelation data will be confronted.

On the basis of the bibliography analysis and in order to achieve these objectives, the remainder of the document has been organized into five chapters:

Preparation and characterization of raw materials (Chapter 3). In this chapter, the focus will be on the production of raw materials as well as on their chemical and physico-chemical characterizations necessary for the study, such as sample composition (protein concentration, ash, water, etc.), protein composition (SDS-PAGE, SEC-HPLC), protein solubility.

Interactions between pea proteins and egg white proteins (Chapter 4, submitted to Food hydrocolloids). In this chapter, we will investigate the interactions between pea globulins and egg white proteins in aqueous mixtures at neutral and alkaline pH (pH 7.5 and 9), close to that of egg white. The potential interaction of whole pea globulins with purified LYS, OVA, or OVT was first examined by isothermal titration calorimetry (ITC) and ζ-potential measurements. Subsequently, the detected attractive interactions between LYS and pea globulins were further explored at different pH via characterization of formed structures by dynamic light scattering (DLS), laser granulometry, confocal laser scanning microscopy (CLSM), and optical microscopy.

Thermal behavior of pea and egg white protein mixtures (Chapter 5) (submitted to Food research international). In this section, we first prepare the protein mixtures at a total concentration of 10 % (the concentration of a fresh liquid egg white manually broken from eggs), with different mass ratios (PPI-EW: 100/0, 75/25, 50/50, 25/75, and 0/100) at pH 7.5 and 9. Then, the Nitrogen solubility profile of the pure proteins and 50/50 mixture systems, and their polypeptide composition using electrophoresis are also characterized by Kjeldahl and SDS-PAGE respectively. Studies are followed by thermal properties obtained by MicroDSC from (25-105 ℃), the gelling temperature (point) of the 10% protein PPI-EW mixtures was investigated by small amplitude rheology to anticipate possible pasteurization temperature adjustment in future manufacturing.

Nature of protein-protein interactions during gelation of mixtures between pea protein isolate and egg white proteins (Chapter 6) (to be submitted to Food Hydrocolloids). In this section, mixtures will be prepared at different mass ratios (PPI-EW: 100/0, 75/25, 50/50, 25/75, and 0/100) at pH 7.5 and 9 with a total protein content of 10 % (w/w). Gelation properties will be analyzed by small amplitude rheology (mesoscopic level), completed by a texture analysis (macroscopic level), and the observation of the microstructure of gels by confocal laser scanning microscopy (CLSM) (microscopic level). In addition, dissociation agents (Urea, Guanidine hydrochloride (GuHCl), Propylene glycol (PG), dithiothreitol (DTT), and a mixture of the 4 agents) will be used to have a better understanding of the interactions during gelation of the mixture. The dissociation agent is prepared in 100 mM Tris buffer at pH (7.5 and 9), and 100 mM Tris buffer is as a reference.

Conclusion and perspective (Chapter 7). In this section, we will collect the major conclusions on the interactions, thermal, and gelation properties of the mixture system between pea proteins (plant proteins) and egg white (animal proteins). Scientific perspectives are then proposed to broaden the scope of this work.

The objectives and strategy of this thesis have been represented in Figure 1-12. distilled water at 100 g/L, and the pH was adjusted to pH 8 with concentrated NaOH every two hours and stirred overnight at 4 ℃. After adjusting the pH to 8, insoluble materials were removed by centrifugation (10 000 g, 30 min, 25 ℃) and the recovered solution was adjusted to pH 4.8 by 0.5 M HCl. After acidification, the precipitated proteins were recovered by centrifugation (10000 g, 25 min, 4 ℃). Afterwards, the pellets were dissolved in 5 L 0.1 M sodium phosphate buffer at pH 8 overnight at 4 ℃ for complete dissolution. The protein suspension was obtained by centrifugation (10000 g, 20 min, 20 ℃) and then concentrated 5 times by ultrafiltration (from 5 L to 800-900 mL) and desalted by diafiltration against 10 L 5 mM ammonium buffer pH 7.2 and 0.05 % sodium azide on an 1115 cm 2 Kvick lab Cassette (UFELA0010010ST, GE Healthcare, Amersham Biosciences, Uppsala, Sweden) with a molecular weight cut-off of 10 kDa. The pressure of the device was lower than 2.5 bars. The pH of obtained protein solution was adjusted to pH 7.2 and then was frozen. The pea globulin powder was obtained after lyophilization and stored at -18 °C. The process for obtaining pea globulins is shown in Figure 2-1. The pea globulins prepared will be referred to as PPI.

Extraction of protein fractions (Ovalbumin) from fresh eggs

Ovalbumin was extracted from fresh eggs from the local market according to [START_REF] Croguennec | Simple rapid procedure for preparation of large quantities of ovalbumin[END_REF]. Egg white recovered from 12 eggs was diluted with 2 volumes of distilled water, then the pH was adjusted to 6.0 with HCl 1 M to precipitate ovomucin. Subsequently, the solution was stirred at 4 ℃ overnight.

Then the supernatant was recovered after centrifugation (10000 g, 4 ℃, 30 min) and adjusted to pH 8.4 with NaOH 5 M. After centrifugation (10000 g, 25 ℃, 25 min), the supernatant was filtered with a plastic strainer and injected to an anion exchange chromatography Q-Sepharose Fast flow column (Pharmacia Biotech AB, Saclay, France) to separate the OVA from the other egg white proteins. The collected ovalbumin was dialyzed by tube with a cut-off of 12000-14000 MWCO, at 4 ℃, around 3-5 days.

Afterwards, the solutions were lyophilized, and the ovalbumin was obtained. The process for obtaining ovalbumin is shown in Figure 2-2.

Preparation of protein solutions

Pea protein dispersions preparation

For thermal analysis and gelling properties evaluation, to obtain an initial dispersion at the necessary protein content (10 %), a quantity of extracted pea protein isolate (PPI) powder was mixed with distilled water. The dispersion was then agitated at 350-400 rpm for more than 3 hours at 4 °C to allow the proteins to completely hydrate.

Then the pH was adjusted to 7.5 and 9 using 1 M NaOH or 1 M HCl without affecting the concentration of the dispersion.

To study the interactions between PPI and egg white fractions (ovalbumin, lysozyme, and ovotransferrin), a quantity of extracted pea protein isolate (PPI) powder was dissolved in either 10 mM HEPES at pH 7.5 or in 10 mM TRIS buffer at pH 7.5 or pH 9 and stirred mechanically at 400 rpm over 3 hours at room temperature to ensure complete hydration of the protein powders.

Preparations of egg white, ovalbumin, lysozyme and ovotransferrin stock solutions

Egg white was manually broken from fresh eggs (Label rouge, France) which were obtained from a local market in Dijon or Rennes (France), and carefully separated from egg yolks and chalaza. Then the fresh liquid egg white (EW) was agitated at 350-400 rpm for more than 3 hours at 4 °C. Then the pH was adjusted to 7.5 and 9 using 1 M

NaOH or 1 M HCl without affecting the concentration of the dispersion. Lysozyme (LYS), ovalbumin (OVA), and ovotransferrin (OVT) stock solutions were dissolved either in 10 mM HEPES at pH 7.5 or in 10 mM TRIS buffer at pH 7.5 or pH 9 and stirred mechanically at 400 rpm over 3 hours at room temperature to ensure complete hydration of the protein powders. The insoluble protein part was estimated as negligible.

The pH of the protein suspensions was then adjusted by 0.1 M HCl or NaOH before each test.

Preparations of mixture systems 2.2.2.3.1 Preparation of PPI-EW mixture systems

Extracted pea protein isolate powder (PPI) was dissolved in distilled water. The dispersion was then agitated at 350-400 rpm for more than 3 hours at 4°C to allow the proteins to completely hydrate. PPI-EW combinations were then made at various mass ratios of 100/0, 70/30, 50/50, 30/70, and 0/100 with the native pH of PPI and EW dispersions. Subsequently, the different combination systems were adjusted to pH 7.5 and 9 using 1 M NaOH or 1 M HCl without affecting the concentration of the dispersion.

preparation of gel

Protein combinations were prepared as mentioned in Section 2.2.2.3.2. Subsequently, the solutions were heated in a water bath from 25 to 95 °C, and then hold at 95 °C for around 30 min, then, all the samples were cooled down to room temperature with ice and were kept at 4 °C overnight.

Characterization of native protein fractions

Determination of moisture content

The moisture content was determined according to method 925.1 (AOAC 1990).

A 1 g sample is weighed inside an aluminum capsule that has been pre-tared. It is then baked at 105 °C until it reaches a constant mass, after which it is chilled in a desiccator for 2 hours. The dry mass is calculated as the average of three observations. The proportion of moisture (MS) is calculated using the following equation ( 1 K: conversion factor for pea protein (5.44), egg white and pea flour (6.25).

Content of lipids extractable by petroleum ether

The lipid content was characterized by extraction on a Soxhlet apparatus with petroleum ether, based on the AOAC reference method n° 923.03. In cellulose cartridges, 5 g of product was precisely weighed (m2). These cartridges were placed in the Soxhlet apparatus, which was filled with enough petroleum ether to allow the solvent to reflux via the siphon. To compensate for anticipated solvent evaporation, an extra 25 mL of petroleum ether was added. The flasks were heated to reflux, and the extraction was carried out for 5 hours. At the end of the extraction, the solvent remaining in the flasks was evaporated using a rotary evaporator, under partial vacuum, with a water bath at 65 °C. The flasks were dried under a hood for one night, then for at least one hour in a desiccator to remove any remaining solvent and humidity before being weighed (m3). The lipid % was calculated using the following equation ( 4):

%𝑳𝒊𝒑𝒊𝒅 = 𝒎𝟑-𝒎𝟏 𝒎𝟐 × 𝟏𝟎𝟎………………………………….

. Equation (4)

Where: m1 the mass of the balloon and the glass beads alone, m2 the mass of sample initially placed in the cellulose cartridge, m3 the mass of the lipids, the balloon and the glass beads,

Content of carbohydrates

The mass fraction of carbohydrates was determined by the following formula [START_REF] Ganzaroli | Absolute quantification of fatty acids in chia seeds produced in Brazil[END_REF] with some modifications:

%𝑴 𝑪 =

Protein solubility as a function of pH

To study the effect of pH on the solubility of proteins in an aqueous medium, we used the experimental protocol described by [START_REF] Djoullah | Native-state pea albumin and globulin behaviour upon transglutaminase treatment[END_REF]. Protein solutions (PPI and EW) and PPI-EW mixture at a weight ratio of 50/50 were prepared in distilled water to obtain the desired protein content (1 %, w/v). All the solutions were stirred for at least 3 hours to make sure completely hydrate, then were adjusted with a solution of NaOH (0.1 M) or HCl (0.1 M) to widen the range of pH studied varying from 2 to 10, also 0.1M NaCl was used to maintain the ionic strength in EW diluted condition. The mixtures were stirred for one hour at 4 °C. Protein suspensions were centrifuged at 10,000 g for 20 min at 4 °C. The protein content of the supernatants was determined by the Kjeldahl method. Solubility is expressed in grams of soluble protein per 100 grams of total protein or soluble nitrogen (NS) is calculated according to equation (6). where NS is nitrogen solubility and N is nitrogen amount.

𝑵𝑺(%) =

Meanwhile, the expected solubility at each pH was used as a reference for the solubility of PPI-EW (at a weight ratio of 50/50) and calculated by equation 7.

𝒔𝒐𝒍𝒖𝒃𝒊𝒍𝒊𝒕𝒚 𝒆𝒙𝒑𝒆𝒄𝒕𝒆𝒅 = 𝒔𝒐𝒍𝒖𝒃𝒊𝒍𝒊𝒕𝒚 𝒆𝒘 × 𝟎. 𝟓 + 𝒔𝒐𝒍𝒖𝒃𝒊𝒍𝒊𝒕𝒚 𝑷𝑷𝑰 × 𝟎. 𝟓 ……Equation (7)

Polypeptide composition by electrophoresis (SDS-PAGE)

Electrophoresis is an analytical technique that allows for the separation of proteins on gel under the influence of an electric field, resulting in protein migration based on the difference in mobility of the polypeptides and their molecular weights. The electrophoresis gel is prepared by cross-linking acrylamide monomers, which form a three-dimensional network. This kind of method makes it possible to identify and quantify the protein composition of a sample. It also allows for the estimation of denaturation effects and the clarification of relationships amongst aggregated proteins.

The pore size of the gel is determined by the concentration of acrylamide. In a lowmesh gel, proteins migrate at a constant rate, whereas in a high-mesh gel (separating gel), the migration rate varies depending on the protein species; small particles will reach the migration front (low molecular weight) quickly, while large particles will be trapped away from this migration front (high molecular weights). Sodium dodecyl sulfate (SDS) functions as an anionic surfactant during gel formation to raise the total negative charge of proteins. Furthermore, adding SDS to the sample solution dissociates the subunits from the oligomeric protein, allowing the polypeptide chain to completely stretch. Reducing chemicals, such as dithiothreitol (DTT), decrease all S-S covalently bonded proteins, whether native or aggregated. The addition of SDS and DTT demonstrates the entire movement of protein components.

The different samples (pea protein isolate, egg white, and PPI-EW mixtures at a weight ratio 50/50) were treated and prepared at least half in sample buffer: 187.5 mM Tris-HCl, pH 8.9, 10 % (m/v) glycerol, 2 % (m/v) SDS and 0.05 % (m/v) of bromophenol blue, in the presence (reducing conditions) or in the absence (nonreducing conditions) of 2 % (m/v) of dithiothreitol (DTT). Novex™ electrophoresis gels (ThermoFisher, Dardilly, France) at 10 % to 20 % Tris-Glycine were used. It should be noted that samples prepared in reducing conditions should be heated at 90 °C for 10 min to reduce disulfide covalent bonds. Therefore, the samples under reducing conditions were heated in a water bath for 10 min at 95 °C. All the samples were was performed on protein suspensions before analysis.

Thermal properties by differential scanning calorimetry

Differential scanning calorimetry (DSC) is a technique for measuring the absorption and/or release of heat during the thermo-denaturation of a molecule. In other words, the device can determine the temperature and heat flow associated with material transitions as a function of time and temperature. What is known to us is that intense heating causes proteins to lose their spatial conformation permanently. Therefore, this research determines the temperature of thermal denaturation of a molecule as well as the amount of energy required for the latter to change states.

The sample is heated simultaneously with an inert reference (solution in which the protein is solubilized) during the experiment, and the differential heat flow between the sample and the reference is monitored over time. When an exothermic or endothermic reaction occurs, a positive or negative peak with respect to the baseline develops. If a sample does not change state, the flow exchanged with the reference is restricted to the baseline. The average denaturation temperature is indicated at the peak apex (Td). As a result of the DSC analysis, it is feasible to determine not only the temperatures of the transitions of state but also their associated enthalpies, which are represented by the areas beneath their respective peaks (ΔHd). Meanwhile, deconvolution is a method of separating the overlapping peaks, and it can be used in DSC peak separation [START_REF] Miyagawa | Analysis of nonisothermal crystallization of rapeseed oil by deconvolution of differential scanning calorimetry curve[END_REF]. The values at beginning of the endothermic peak (50 ℃) and the end of the heating ramp (105 ℃) were connected by a straight line to draw the baseline.

Deconvoluted curves were generated by subtracting baselines from DSC curves.

Differentiating the original DSC curve with time determined the number of peaks to be separated. The function that represents each peak was chosen from the functions built into the software so that the curve obtained by summing the individual peaks could accurately represent the original curve, i.e., the asymmetric double sigmoid and Lorentz functions shown in equations ( 8) and ( 9), respectively, were chosen to represent the peaks. where y is the heat flux, θ is the Celsius temperature, θc is the Celsius temperature at the center of the distribution, w, w1, w2, and w3 are constants related to peak width, and H1 and H2 are constants.

𝒚 = 𝑯

The study of the thermodynamic properties of the initial dispersions of PPI, EW, and PPI-EW mixtures at different mass ratios (25/75, 50/50, 75/25) at pH 7.5 and 9 with a protein concentration of 10 % (w/w) was carried out using a Micro DSC III differential scanning calorimeter (Setaram, Caluire, France). Approximately 0.5 g of sample was weighed in an aluminum pan, hermetically sealed, and heated from 25 to 105 °C at 0.5 °C/min. A pan with distilled water at room temperature was used as a reference. All experiments were conducted in triplicate. One replicate of each sample was re-heated after cooling to check that denaturation was irreversible. The evolution of the heat flux as a function of temperature (thermogram) was processed using the thermal analysis SETSOFT 2000 software (Setaram) and the following parameters were evaluated for each peak: the denaturation temperature (Td) and the associated denaturation enthalpy (ΔHd). Deconvolution of thermograms was performed by Origin 2019 Pro.

Determination of particle surface charge or Zeta potential (ζ)

The zeta potential is an essential and easily measured indication of the stability of colloidal dispersions that may be used to assess protein-protein and protein-solvent electrostatic interactions. From a theoretical standpoint, zeta potential is the electric potential in the interfacial double layer of a dispersed particle or droplet against a point in the continuous phase distant from the interface (Lu & Gao, 2010). The amount of the zeta potential in a dispersion shows the degree of electrostatic repulsion between nearby, similarly charged particles. A large zeta potential will impart stability on molecules and particles of sufficient size, i.e., the solution or dispersion will resist aggregation as shown in Figure 2-3 [START_REF] Selvamani | Stability studies on nanomaterials used in drugs[END_REF]. When the potential is low, attractive forces may outweigh repulsion, causing the dispersion to break apart and flocculate. Colloids with a high zeta potential (either negative or positive) are electrically stabilized, whereas colloids with a low zeta potential coagulate or flocculate.

The pH of the medium is the most critical element affecting zeta potential. Other parameters include ionic strength, additive concentration, and temperature. The zeta potential of particles can be calculated by the following equation ( 10

………………………………….. Equation (10)

Where: ε: the medium permittivity, η: the viscosity of the dispersion medium, f (ka): a function related to the proportion between the size (a) characteristic of the system and the Debye length (1/κ), V: particle velocity (μm.s -1 ), E: the electric field applied per unit length (V.cm -1 ). [START_REF] Selvamani | Stability studies on nanomaterials used in drugs[END_REF] The measurements were made on 1mL of PPI (2.4 g/L), LYS (14.3 g/L), and their mixtures at different molar ratios (see the ratios in section 4) prepared in TRIS buffer at pH 7.5 and 9 using a Malvern Zetasizer Nano ZS (Nanosizer, Malvern Instruments, UK). The ζ-potential was measured at 25 ℃ using a laser Doppler velocimetry and phase analysis light scattering (M3-PALS0) using disposable electrophoretic mobility cells (DTS1070). The equilibration time was set at 120 s, and at least 11 runs were performed for each measurement. The measurements were carried out at a voltage of 150 volts and were repeated three times for each sample (PPI, LYS, and PPI-LYS mixtures at pH 7.5 and 9). To identify the ζ-potential of PPI and LYS as a function of pH, PPI and LYS stock solutions were prepared as follows. PPI (2.4 g/L) and LYS (14.3 g/L) were dissolved in distilled water until complete protein hydration. Then, 0.1-1 M HCl or NaOH was used to adjust pH from 2-12.

Determination of particle size by dynamic light scattering (DLS) and laser granulometry 2.2.7.1 Study of particle size by DLS

DLS was performed to have a better understanding of the changes in size and distribution of the particles present in various protein mixtures. DLS is a technique for determining the polydispersity of particles in protein suspensions as well as the size of the populations produced by these particles. The basic principle of this equipment is based on measuring the intensity of light dispersed by particles agitated by a Brownian motion and with no interactions between them at a specific detection angle. Small particles disperse more quickly in the medium, resulting in a fast-fluctuating intensity signal when compared to big particles, which diffuse more slowly [START_REF] Hassan | Making sense of Brownian motion: colloid characterization by dynamic light scattering[END_REF]. The primary results of DLS are particle size (hydrodynamic radius or hydrodynamic diameter (Dh)) which is governed by the Stokes-Einstein equation, which is based on Brownian motion of particles as a function of aqueous phase viscosity and temperature. Individually moving things, such as single particles and particle aggregates or agglomerates, are referred to by the hydrodynamic diameter. It usually represents the outside dimensions, but it has nothing to do with the size of the constituent particles within an aggregate or agglomeration. To measure the particle size, it is necessary to calculate the speed of movement of these particles in their solvent by measuring the diffusivity of light (coefficient of translation).

Variations in the intensity of scattered light as a function of time are attributed to the scattering of the entities in solution when the solvent is known, and the temperature is constant. A correlator calculates the adjustment of the cumulative intensity of scattered light following an exponential decline in the case of spherical particles during the measurement. This allows the translational diffusion coefficient to be calculated, which is connected to a single apparent (spherical) particle diameter. The Stokes-Einstein equation ( 11) was used to calculate the distribution of apparent hydrodynamic diameter (Dh) from the mean translational diffusion coefficient distribution:

𝑫𝒉 = 𝒌𝑻 𝟑𝝅𝜼𝑫

……………………………………… Equation (11)

Where: Dh: the particle's hydrodynamic diameter (m) ; T: The absolute temperature (K) ; K: The Boltzmann constant (J.K -1 ) ; η: The dispersing medium's viscosity (m -1 .Kg.s -1 ) ; D: The diffusion coefficient (m 2 .s -1 ).

The size distribution of PPI and LYS was determined by DLS (Nanosizer, Malvern Instruments, UK). The progress was measured according to the previous paper by [START_REF] Schmitt | Whey protein soluble aggregates from heating with NaCl: physicochemical, interfacial, and foaming properties[END_REF] with some modifications. PPI and LYS stock solutions were prepared as mentioned before in section 2.2.2, and first diluted 5 times in 10 mM Tris-HCl buffer at pH 7.5 or 9, before measurement. Solutions were filtered with a 0.22 μm cellulose membrane (Millipore Corp.) as well. 1 mL of the samples was measured at least 3 times at 25 ℃.

Study of particle size by laser granulometry

Laser granulometry as known as laser diffraction is based on the principle of laser diffraction, in which particles diffract light at a specific angle. The diffraction angle is inversely proportional to particle size, and the intensity of the diffracted beam at any angle is a measure of the number of particles in the optical path with a certain crosssectional area [START_REF] Di Stefano | Comparison between grain-size analyses using laser diffraction and sedimentation methods[END_REF].

A parallel beam of monochromatic light is directed onto detectors after passing through a suspension housed in a sample cell. Two diffraction theories are often employed to calculate particle sizes from light intensity perceived by detectors: the Fraunhofer diffraction (Franuhofer, 1817) and the Mie theory [START_REF] Mie | Beiträge zur Optik trüber Medien, speziell kolloidaler Metallösungen[END_REF]. Both theories imply that the particles have a spherical form; in other words, the particle dimension is the optical spherical diameter, which is the diameter of the sphere with the same cross-section area as determined by laser diffraction [START_REF] Di Stefano | Comparison between grain-size analyses using laser diffraction and sedimentation methods[END_REF][START_REF] Magno | A comparison between Laser Granulometer and Sedigraph in grain size analysis of marine sediments[END_REF]. It evaluates grain size as a proportion of volume. The volumetric diameter d [4;3] was calculated by equation ( 12)

𝒅 [𝟒;𝟑] = ∑ 𝒏 𝒊 𝒅 𝒊 𝟒 ∑ 𝒏 𝒊 𝒅 𝒊 𝟑 ………………………………………Equation (12)
Where: ni, di: The total number of particles with particle size di is ni

The size distribution of PPI-LYS mixtures at different ratios was determined by laser granulometry (Mastersizer 2000, Malvern Instruments, UK). PPI and LYS stock solutions were prepared as mentioned before in section 2.2.2. 1 mL of the samples was measured at least 3 times at 25 ℃. The stirring speed was set at 1100 rpm. The refractive index of the continuous phase was set at 1.33, which corresponds to that of water, and at 1.45 for the dispersed phase.

Determination of thermal properties by isothermal titration calorimetry (ITC)

Isothermal titration calorimetry (ITC) is not only advantageous in that it provides thermodynamic parameters, namely variations in Gibbs energy (ΔG), changes in entropy (ΔS), and enthalpy (ΔH), as well as binding constant and stoichiometry (n) from a single titration. What is most important is that ITC is label-free, so it doesn't add artifacts [START_REF] Rajarathnam | Isothermal titration calorimetry of membrane proteins-Progress and challenges[END_REF]Loh, Brinatti, & Tam, 2016). Typical equipment and the progress of ITC are shown in Figure 234. In brief, the sample cell and the other reactant are inserted in the syringe as a ligand. An adiabatic device separates the sample cell and the reference cell (Figure 234). The ligand is continually dripped into the sample pool by the syringe, which also has a stirring function. After a specific amount of time, the instrument monitors the sample cell's heat change and compares it to the reference cell, displaying an endothermic or exothermic peak. To maintain the temperature constant, the exothermic reaction causes the negative feedback of constant temperature power, whereas the endothermic reaction causes the positive feedback of constant temperature power. Observations are shown versus time as the power required to keep the reference and sample cells at the same temperature (Figure 234). As a result, the experimental raw data is a series of heat flow (power) spikes, with each spike corresponding to one ligand injection. These heat flow spikes/pulses are time-integrated to give the total heat exchanged after each injection.

The pattern of these heat effects as a function of the molar ratio

[ligand]/[macromolecule] may then be examined to determine the thermodynamic characteristics of the interaction under consideration. Degassing samples is frequently required in order to achieve accurate measurements since the presence of gas bubbles within the sample cell causes incorrect data plots in the recorded findings. The entire experiment is computer-controlled.

The study provides stoichiometry (n), affinity constant (K), and the binding reaction's enthalpy (ΔH). Therefore, the free energy of binding (ΔG) and the entropy (ΔS) are then calculated using the equation ( 13) and ( 14 To analyze ITC results, the mean molecular weight of globulins in PPI (Mw PPI) was approximated by the following equation ( 15):

Mw PPI = (Mw PPI-11S) × (11S-to-(7S+11S) ratio) + Mw PPI-7S (7S-to-(7S+11S) ratio)

Equation ( 15)

with Mw PPI-11S = 360 kDa, Mw PPI-7S = 150 kDa, and 11S-to-(7S+11S) ratio = 0.59 and 7S-to-(7S+11S) ratio = 0.41 deduced from enthalpy area deconvolution from DSC spectra (chapter).

The Mw PPI value was thus estimated at 273,9 kDa. Then, the molar concentration of PPI (Cppi) was obtained by dividing the protein concentration in the solution by the mean PPI Mw.

High-performance liquid chromatography (HPLC)

Size exclusion chromatography for PPI

High performance size exclusion chromatography (SEC-HPLC) is a type of liquidphase chromatography that is also known as gel chromatography. The basic theory behind this technology is to move molecules or macromolecules at a constant pace through an elution buffer (mobile phase) in a column packed with a material comprising porous polymers (stationary phase). Macromolecules with a high molecular weight are quickly eluted, but those with a low molecular weight have a prolonged residence time and travel a more convoluted course through the stationary phase.

The particle size distributions of the species present in the different samples studied (Shimadzu SPD-20AV) were carried out using a high-pressure liquid chromatography system (HPLC Shimadzu Corporation, Kyoto, Japan) equipped with an isocratic pump (Shimadzu LC-20AT) and a UV-Vis) with a Yarra 3 μ-SEC-S3000 phenomenex size exclusion column (4.6 mm id x 30 cm long, Phenomenex industry, France). The column was equilibrated at 25 °C with a mobile phase consisting of 50 mM phosphate buffer (Na2HPO4) and 0.2 M NaCl, respectively, for samples prepared at low and high ionic strength, pH 7.2, pre-degassed and filtered through a 0.2 μm filter

The column is pre-calibrated using SIGMA ALDRICH and GE Healthcare protein standards. The markers used were glucan blue (MW: 2000 kDa), thyroglobulin (MW: 669 kDa), Ig A (MW: 300 kDa), Ig G (MW: 150 kDa), ovalbumin (MW: 44 kDa), Myoglobulin (MW: 17 kDa), Cytochrome (MW: 12 kDa).

The protein solutions (20 g/L) were prepared in sodium phosphate 50 mM + NaCl 0.2 M buffer solution and adjusted to pH 7.5 and 9, subsequently filtered through a 0.2 μm membrane (Millipore Corp.) according to the procedure cited by [START_REF] Roesch | Heat-induced soy-whey proteins interactions: Formation of soluble and insoluble protein complexes[END_REF]. Samples (25 μL) were manually injected into the system using a 100 μL injection volume sample loop. The speed and total elution time were set at 0.8 ml/min and 20 min respectively. The analyzes were recorded at a wavelength of 214 nm and 280 nm and the elution peaks were analyzed with the LC solution software (V.

1.25, Labsolutions, Shimadzu). All samples were measured in triplicate.

RP-HPLC for ovalbumin

The determination of extracted ovalbumin was carried out using a Reverse phase high-pressure liquid chromatography system (HPLC Shimadzu Corporation, Kyoto, Japan) equipped with an isocratic pump (Shimadzu LC-20AT) and a UV-Vis) with a Vydac® 214TP C4 HPLC Columns (50 x 4.6 mm, Avantor®, France). The column was stored in a 70% acetonitrile solution without trifluoroacetic acid (TFA). Two buffer solutions were prepared: buffer A) Mili Q water with 0.02655 TFA and filtered through 0.2 membranes (Millipore Corp.); buffer B) 100% acetonitrile and 0.025% TFA. For samples prepared at low and high ionic strength, pH 7.2, pre-degassed and filtered through a 0.2 μm filter. The column is pre-calibrated using SIGMA ALDRICH and GE Healthcare protein standards. The markers used were lysozyme (MW: 14 kDa), ovalbumin (MW: 44 kDa), and Ovotransferrin (MW: 77 kDa).

The protein solutions (100 g/L) were prepared and made a 1/200 dilution with buffer A, subsequently filtered through a 0.2 μm membrane (Millipore Corp.)

According to the procedure cited by [START_REF] Roesch | Heat-induced soy-whey proteins interactions: Formation of soluble and insoluble protein complexes[END_REF]. Samples (100 μL)

were manually injected into the system using a 100 μL injection volume sample loop.

The procedure of injection and elution of samples was shown in table 2-1. 100 μL of buffer A was used as a baseline. The speed and total elution time were set at 0.8 mL/min and 32 min respectively. The analyzes were recorded at a wavelength of 280 nm and the elution peaks were analyzed with the LC solution software (V. 1.25, Labsolutions, Shimadzu). Gradients 2 and 3 are used to rinse the column because highly hydrophobic ovalbumin tends to adsorb onto the column. All samples were measured in triplicate. 95 % 5 % 0,8 ml/min. 0-20 min, using gradient 1, 22-25 min using gradient 2, and 25-32 min using gradient 3.

Gel properties determined by small-strain dynamic rheology

The objective of this manipulation is to identify the gelling properties (gel point, loss factor, linear viscoelastic region) of PPI, EW, and PPI-EW at different weight ratios (25/75, 50/50, 75/25) by small-strain dynamic rheology.

Samples were prepared as described in section 2.2.2. Subsequently, the sample was loaded into a rheometer MCR 302e (Anton Paar, Graz, Austria) equipped with a cone-plate geometry (50 mm diameter, 2 angles). Approximately 1 mL of the protein suspension was transferred to the lower plate of the rheometer. The upper cone was lowered to give a gap width of 1.0 mm. A thin layer of light mineral oil was added to the well of the upper cone geometry and a solvent trap cover was used to prevent sample drying during heating. In this way, a water-saturated atmosphere was maintained at the surface of the sample. The following heating protocol was used. The sample was first equilibrated at 25 °C for around 3 min, then heated and then cooled under 1 % of shear strain and 1 Hz of frequency over a temperature range of 25-95-25 °C at a rate of 2 °C/min (heating ramp) and 5 °C/min (cooling ramp) respectively. Rheological data were collected for every degree change during heating and cooling. Subsequently, a frequency sweep at 1 % strain and a strain sweep at 1 Hz were performed at 25 °C. In detail, frequency sweep and strain sweep of the gel were conducted over a range of 0.01-40 Hz and 0.01-100 %, respectively. Values of G' (storage modulus) and G'' (loss modulus) were recorded for temperature, strain and frequency sweeps as has been done

previously [START_REF] Arntfield | Dependence of thermal properties as well as network microstructure and rheology on protein concentration for ovalbumin and vicilin[END_REF][START_REF] Cai | Thermal gelation in relation to binding of bovine serum albumin-polysaccharide systems[END_REF]. The loss factor or tangent delta (tan δ = G''/G') was also calculated, as well as the linear viscoelastic region (LVR).

The gelling point temperatures of protein suspensions were determined as the intersection of two linear parts of curves on either side of the marked inflection point of the elastic modulus (G′). The thermal gelation profiles of EW, i.e., the storage modulus (G') as a function of temperature, obtained at pH 9 was chosen as a typical curve to illustrate how the data were analyzed (Figure 2

Gel solubility by dissociation agents

The goal of this manipulation is to investigate the effect of different dissociating agents (urea, dithiothreitol, guanidine hydrochloride, and propylene glycol) on protein solubilization in order to identify the nature of the interaction forces that stabilize the three-dimensional networks formed by heat-treated protein mixtures after gelation.

Control samples were tested in parallel.

The experimental approach was applied according to the previous methods of Liu & Hsieh ( 2008

Texture analysis of gels

Texture profile analysis (TPA) was developed to link mechanical testing to sensory evaluations of food texture. A sample of standard size and form is put on a base plate and compressed and decompressed twice by an upper plate connected to the drive system in such a test. The force vs. deformation curve is recorded, and seven textural characteristics may be derived from it: hardness, elasticity, adhesiveness, cohesiveness, brittleness, chewiness, and gumminess [START_REF] Bourne | Food texture and viscosity: concept and measurement[END_REF]. textural property according to the method of Bourne [START_REF] Bourne | Texture profile analysis[END_REF]. The parameters were set as follows: pre-test speed: 0.5 mm/s; test speed: 0.5 mm/s; post-test speed: 0.5 mm/s; compression deformation: 37.5 %. A time of 10 s was allowed to elapse between the two compression cycles. All the samples (height of 20 mm and diameter of 40 mm)

were placed on the platform of the TA-XT Plus, fitted with a 5 N load cell and a cylindrical plunger 12 mm in diameter (SMS-P/35). All samples were prepared in duplicate and tested at least 2 times. Hardness was defined as the maximum peak force during the first compression cycle. Springiness was defined as the recovery degree of gels after decompression to their initial shape. Cohesiveness was measured as the ratio of the work of penetration of the second penetration on one of the first penetration as well. Data were calculated using the Texture Expert software version 1.22 (Stable Micro Systems).

Confocal laser scanning microscopy (CLSM)

A Fast Green aqueous solution (1 % w/v, 6 μL). Subsequently, the solutions were kept in dark at 20 °C for at least 10 mins. 20 μL of the mixture was deposited on a glass slide in a spacer and a coverslip was placed on top of all samples. Fast green was excited using a He-Ne laser system at a wavelength of 633 nm at a 1.72 μs pixel dwell scanning rate and detected using a PMT between 635 and 735 nm.

Protein particle formation

Images were observed inside the channel slide system using the high-resolution mode of the confocal microscope equipped with the Airyscan detection unit and a Plan Apochromat 63x with a high numerical aperture (NA = 1.40) oil objective. Airyscan images were acquired with a main beam splitter MBS488/561/633, no additional emission filter, again setting of 700-780, a pixel dwells time of 1.54 μs and no averaging. The zoom was automatically set to 1.8 as requested by the system. Images were processed using confocal acquisition software Zen Black 2.1 (Version 13.0.0.0) to process the acquired datasets using the 2D mode at the default setting of the Airyscan processing function.

Gel structure preparation

The device used in the study of the microstructure of the gels formed is a confocal laser scanning microscopy (CLSM) using a ZEISS LSM 880 (Carl Zeiss AG, Oberkochen, Germany) with the Airyscan detection unit. Samples were prepared as in the previous section 2.2.2. Then, around 400 μL of the pH 7.5 or 9 mixture solution was poured into 1mL Eppendorf tubes. They were mixed with a 12 μL aliquot of 1% (w/v) of Fast Green. Then all the solution was slowly injected into the chamber of an IBIDI μ-Slide 8 well Uncoated system (IBIDI GmBH, Grafelfing, Germany). The system was then covered using an included lid and wrapped tightly with Parafilm (Dispense Parafilm Through This Opening, USA) at the gap of the lid, meanwhile, aluminum foils were used to prevent photo-bleaching of fluorescent molecules. Finally, the systems were put into the IBIDI systems and were heated as the previous gel preparation 2.2.2.3.2. To optimum resolution improvement, a Plan Apochromat 63x with a high numerical aperture (NA = 1.40) oil objective was used. A He/Ne laser with a wavelength of 633 nm was used to excite the Fast Green dye, with appropriate emission in each system. Zen Black 2.1 (version 13.0.0.0) software was used to process the acquired datasets using the 2D mode at the default settings of the Airyscan processing function.

Chapter 3 Preparation and characterization of protein materials

The primary objective of this section will be to describe the extraction of adequate amounts of the most "native" fractions of pea protein isolate (mainly globulins which are simplified as PPI) and ovalbumin. Characterizing and preparing samples demands a substantial number of raw materials (PPI), and most pea protein isolates on the market are denatured globulins that cannot be utilized as substrates for this study, thus enough protein must be obtained at the beginning of the experiment.

This section is followed by an evaluation of the physical chemical properties of pea protein isolate, liquid egg white, and ovalbumin, including the SDS-PAGE, thermal properties, the solubility of pea protein isolate and egg white. In addition, the result of SEC-HPLC regarding PPI and ovalbumin was added to complete the properties of extract samples.

Preparation and characterization of pea protein

isolate, egg white and ovalbumin

Extraction method

We collected enough experimental samples using the procedure described in section 2 and pea powder supplied by the industry. To produce "natural" pea protein powder, the approach employs combined processes: isoelectric-precipitation extraction and ultrafiltration as well as diafiltration. The extracted materials are fully combined before undergoing characterization investigations.

Physicochemical composition

Physicochemical composition of pea protein isolate and pea flour

The composition of the pea flour and purified pea protein isolate (pea globulin) fraction are given in Table 3-1. The concentration of protein, ash, moisture, lipid, carbohydrate, and endosperm color are the quality parameters of a yellow pea. In particular, protein content is the most critical factor in these parameters. Thus, around 24.3 % protein (based on a dry basis) was present in yellow pea flour, which was consistent with the previous value reported to be in the range of 23.1 %-30.9 % [START_REF] Boye | Pulse proteins: Processing, characterization, functional properties and applications in food and feed[END_REF]. Regarding the pea globulin extraction, the protein and lipid concentration (based on a dry basis) was approximately 89.74 % (N factor of 5.44) and 0.95 %, respectively.

According to [START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF], it is critical to note that the presence of a small amount of fat in the globulin isolate reduces lipid-protein interaction, otherwise, large amounts of lipid in fractions would change the solubility in water and increase the turbidity of protein suspensions, as seen in soy protein solution [START_REF] Li | Effect of concentration, ionic strength and freeze-drying on the heat-induced aggregation of soy proteins[END_REF]. Similar protein concentrations (higher than 80 %) have been reported by others for laboratory-produced pea protein isolates [START_REF] Barać | Profile and functional properties of seed proteins from six pea (Pisum sativum) genotypes[END_REF][START_REF] Boye | Comparison of the functional properties of pea, chickpea and lentil protein concentrates processed using ultrafiltration and isoelectric precipitation techniques[END_REF]Stone et al., 2015b).

Some differences in the protein content of extracted isolates may be due to the difference in protein content of cultivars, which was confirmed by [START_REF] Boye | Comparison of the functional properties of pea, chickpea and lentil protein concentrates processed using ultrafiltration and isoelectric precipitation techniques[END_REF], who highlighted that the protein concentration of six pea types studied ranged from 22.3 to 31.8 % and that in isolates ranged from 84-89 %.

prepared at pH 7.5 and 9. The samples on lanes 2, 4, 6, and 8 were treated under reducing conditions with SDS+DTT reagents. Circles in red and blue were the aggregates of PPI and egg white, respectively at the top of the bands. Lane 1-4 at pH 7.5, Lane 5-8 at pH 9; Lane M: molecular weight (Mw) markers; lanes 1-2, and 5-6: PPI; lanes 3-4, and 7-8: EW; LP, lipoxygenase; L (α, β), legume; CV, convicilin; Lα, legume acid polypeptide; Lβ, legume basic polypeptide; V, vicilin; OVA, ovalbumin;

OVT, ovotransferrin; LYS, lysozyme.

Regarding PPI, a high number of component polypeptides of pea globulins is noticeable at first look. Focusing on Lanes 1 and 5 non-reducing part at pH 7.5 and 9, Lanes 2 and 6, the reducing part at pH 7.5 and 9, respectively, a molecular weight of around 88 kDa, was probably lipoxygenase (Sun & Arntfield, 2010;[START_REF] Mession | Thermal Denaturation of Pea Globulins (Pisum sativum L.)-Molecular Interactions Leading to Heat-Induced Protein Aggregation[END_REF]. A strong band at around 60 kDa corresponded to legumin 11S main subunits (Lαβ), which were dissociated into acidic subunits Lα (~38-40 kDa)

and basic subunits Lβ (~20-22 kDa) under reducing conditions as also observed elsewhere [START_REF] Gueguen | Quantitative and qualitative variability of pea (Pisum sativum L.) protein composition[END_REF][START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF][START_REF] Liang | pH-dependent emulsifying properties of pea [Pisum sativum (L.)] proteins[END_REF]Mession, Chihi, Sok, & Saure, 2015). The band around 15 kDa could be vicilin fragments γ (12-16 kDa) [START_REF] Gatehouse | Characterisation and subunit structures of the vicilin storage proteins of pea (Pisum sativum L.)[END_REF][START_REF] Mession | Thermal Denaturation of Pea Globulins (Pisum sativum L.)-Molecular Interactions Leading to Heat-Induced Protein Aggregation[END_REF]. Polypeptides of vicilin were considered to be the majority pea globulin polypeptides with different bands around 16-50 kDa (Figure 3-1), in detail, i: polypeptides in the range of 20-37 could respond to fragments α (≈20 kDa), α:β (~30-36 kDa), and β:γ (~25-30 kDa), ii: the presence of a minority fraction of vicilin of 19 kDa [START_REF] Gatehouse | The posttranslational proteolysis of the subunits of vicilin from pea (Pisum sativum L.)[END_REF], iii: large band around 50 kDa could be assigned to the vicilin monomer [START_REF] Mession | Thermal Denaturation of Pea Globulins (Pisum sativum L.)-Molecular Interactions Leading to Heat-Induced Protein Aggregation[END_REF]. The convicilin fraction, which was considered to be the third storage polypeptide, was present at 71 kDa as expected [START_REF] Croy | The purification and characterization of a third storage protein (convicilin) from the seeds of pea (Pisum sativum L.)[END_REF][START_REF] Liang | pH-dependent emulsifying properties of pea [Pisum sativum (L.)] proteins[END_REF]. Aggregates with molecular weights of more than 250 kDa (Figure 3-1, lanes 1 and 5) were found (indicated by a red solid circle), which were probably produced during the extraction process regarding the study of [START_REF] Karaca | Emulsifying properties of chickpea, faba bean, lentil and pea proteins produced by isoelectric precipitation and salt extraction[END_REF].

Three main polypeptide components were shown in egg white samples (Figure 3-1, lanes 3 and 7 at pH 7.5 and 9, respectively): ovotransferrin (76 kDa), ovalbumin (44 kDa), and lysozyme (14.6 kDa), in agreement with previous works [START_REF] Li-Chan | Stability of bovine immunoglobulins to thermal treatment and processing[END_REF], Raikos, Hansen, Campbell, & Euston, 2006). No band corresponding to ovomucoid (∼28 kDa, 11 %) could be visualized on the gel. As expected, ovalbumin was the largest band on the gel, because it is the most abundant protein in egg white [START_REF] Li-Chan | Stability of bovine immunoglobulins to thermal treatment and processing[END_REF]. A fraction of protein aggregates that did not enter the electrophoresis gel (indicated by blue solid circles in Figures 3 lanes 3 and7)

was presumably formed via covalent interactions such as disulfide bonds, as they disappeared under reducing conditions (Figure 3-1, lanes 4 and 8), as already mentioned by [START_REF] Alavi | Effect of free radical-induced aggregation on physicochemical and interface-related functionality of egg white protein[END_REF]. It can be noticed that the bands of ovotransferrin and ovalbumin appeared with a higher molecular weight under reducing conditions (Figure 3-1, lanes

3 vs 4 at pH 7.5, lanes 7 vs 8 at pH 9), probably due to the rupture of their internal disulfide bonds that expand their structure and thus increase their apparent molecular weight [START_REF] Katekhong | Changes in physical and gelling properties of freeze-dried egg white as a result of temperature and relative humidity[END_REF][START_REF] Chaiyasit | Comparison of physicochemical and functional properties of chicken and duck egg albumens[END_REF].

Solubility of PPI and EW as a function of pH

In the food sector, good protein solubility is a highly desired physicochemical feature. It provides the benefit of homogenous protein distribution in the food matrix, which affects the texture and organoleptic qualities of the complete product. Proteinprotein and protein-solvent interactions influence protein solubility, resulting in either precipitation or protein solubility [START_REF] Damodaran | Functional properties. Food proteins: Properties and characterization[END_REF]. The environment, such as pH and ionic strength [START_REF] Deng | Functional properties of protein isolates, globulin and albumin extracted from Ginkgo biloba seeds[END_REF], the extraction method [START_REF] Boye | Comparison of the functional properties of pea, chickpea and lentil protein concentrates processed using ultrafiltration and isoelectric precipitation techniques[END_REF][START_REF] Papalamprou | Influence of preparation methods on physicochemical and gelation properties of chickpea protein isolates[END_REF], the drying process [START_REF] Gueguen | Legume seed protein extraction, processing, and end product characteristics[END_REF], the amino acid composition (hydrophilic and hydrophobic residues), and their distribution on the protein's surface [START_REF] Kimura | Comparison of Physicochemical Properties of 7S and 11S Globulins from Pea, Fava Bean, Cowpea, and French Bean with Those of Soybean-French Bean 7S Globulin Exhibits Excellent Properties[END_REF] all influence both types of interaction. The solubility of PPI and EW (0.1 M NaCl) was examined as a function of pH in this study. The change in the solubility of PPI and EW as a function of pH is shown in Figure 3-2. The globulin's solubility reaches a minimum of around pH5, corresponding to the region of the isoelectric point (pI~4.8) (Gueguen et al., 1988). Previous paper of [START_REF] Shevkani | Structural and functional characterization of kidney bean and field pea protein isolates: A comparative study[END_REF] reported the solubility of tested pea protein isolate was only 2-4 % at pH 5. Outside this range, the solubility increased and reached values of around 85 % and 89 % for pH values below 3 and above 7, respectively. On the other hand, the solubility of PPI showed a U-shaped solubility profile. Similar pHdependent protein solubility profiles of pea proteins have been observed for commercial or native PPI [START_REF] Adebiyi | Functional properties of protein fractions obtained from commercial yellow field pea (Pisum sativum L.) seed protein isolate[END_REF][START_REF] Burger | Recent progress in the utilization of pea protein as an emulsifier for food applications[END_REF][START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF][START_REF] Taherian | Comparative study of functional properties of commercial and membrane processed yellow pea protein isolates[END_REF][START_REF] Liang | pH-dependent emulsifying properties of pea [Pisum sativum (L.)] proteins[END_REF] or pea protein concentrate (Boye et al., 2010).

This solubility behaviour of globulins is attributed to electrostatic repulsion and hydration of charged residues [START_REF] Damodaran | Amino acids, peptides and proteins[END_REF]. At pI, the protein has as many positive as negative charges, which promotes the affinity and interaction between protein-protein at the expense of protein-solvent, thus minimizing solubility. For acid and basic pH, the protein is positively and negatively charged, respectively. This large net charge increases the repulsion forces, thereby promoting the protein-solvent interaction, which results in better solubility. Furthermore, in an acidic condition, the quaternary structure of the globulins is ruptured by the dissociation of the legumin subunits caused by the protonation of the carboxylic groups [START_REF] Lakemond | Heat denaturation of soy glycinin: influence of pH and ionic strength on molecular structure[END_REF].

When focusing on the solubility of egg white, it was always over 88%. However, a little lower solubility of EW was observed around pH 4, which is close to the isoelectric point of ovalbumin (pH 4.5), the major egg white protein. This result was in good agreement with the previously reported solubility profiles of egg white and attested to the high hydrophilicity of egg white proteins [START_REF] Machado | Solubility and density of egg white proteins: Effect of pH and saline concentration[END_REF]. [START_REF] Abdo | Nutritive sweetener of short-chain xylooligosaccharides improved the foam properties of hen egg white protein via glycosylation[END_REF] reported that the solubility of egg white declined at a pH range of 4-6 due to isoelectric point of egg white protein.

Thermal properties of PPI and EW

Thermal properties of PPI

Thermal characteristics of the acquired pea protein isolate, and egg white were investigated. The DSC measurements allowed us i: to confirm that the extraction and purification steps had limited effect on the protein structure, ii: to identify the ideal temperature for denaturing the proteins alone, and therefore, iii: to pick the proper temperature for creating the mixed soluble aggregates. Thermograms, denaturation temperature (Td), and change of enthalpy (ΔH) acquired by differential scanning calorimetry (DSC) at pH 7.5 and 9 were shown in Figure 3-3, Table 3-1 for pea protein isolate, Figure 3-4, Table 3-2 for egg white, respectively. Arntfield, 2011a). Td and ΔH of faba bean proteins also declined on each side of the isoelectric point [START_REF] Arntfield | The influence of processing parameters on food protein functionality I. Differential scanning calorimetry as an indicator of protein denaturation[END_REF]; enthalpy and Td of red bean globulin decreased under the impact of excessively acidic and alkaline pHs [START_REF] Meng | Thermal properties of Phaseolus angularis (red bean) globulin[END_REF]. This kind of decreased phenomenon on Td suggested that a decrease in thermal stability was caused by growing repulsive negative charges at pH which was far away from pI [START_REF] Arntfield | The influence of processing parameters on food protein functionality I. Differential scanning calorimetry as an indicator of protein denaturation[END_REF][START_REF] Meng | Thermal properties of Phaseolus angularis (red bean) globulin[END_REF]. Table 3-1 shows the enthalpy (∆H) values for suspensions PPI at pH 7.5 and 9, respectively The denaturation enthalpy (∆H) of pea globulins was in the same order of magnitude as those obtained by Sun & Arntfield (2011a), i.e., around 8.3 J/g of salt-extracted pea protein isolate at an increasing temperature rate of 0.5 ℃/ min, or by [START_REF] Mession | Effect of pea proteins extraction and vicilin/legumin fractionation on the phase behaviour in admixture with alginate[END_REF], i.e., 11.4 J/g of acidic precipitated pea protein isolate at 10 ℃/ min at pH 7.5, but these values were higher than those obtained by [START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF] (from 0.725 J/g to 0.992 J/g of native pea protein isolate under NaCl concentration from 0% to 2%).

The ∆H value of the PPI sample at pH 7.5 in our study is an indication that the globulin fractions produced were low-denatured. However, the enthalpy at pH 9 decreased compared to pH 7.5. This could be explained by the high pH, far from pI, causing the partial unfolding of protein molecules due to the increasing intramolecular net charges and repulsive forces, as demonstrated by [START_REF] Meng | Thermal properties of Phaseolus angularis (red bean) globulin[END_REF] on red bean globulins.

Pea globulin denaturation was found to be irreversible, as evidenced by the absence of transition following a re-heating of the same sample (results not shown).

Figure 3-4 Samples of Deconvolution of pea protein isolate 10%.

It is necessary to know the vicilin/ legumin ratio (V/L) in pea as pea proteins can be used in a variety of dietary applications due to the diversity of the V/L ratio [START_REF] Gallardo | Reserve accumulation in legume seeds[END_REF][START_REF] Mertens | Agronomical factors influencing the legumin/vicilin ratio in pea (Pisum sativum L.) seeds[END_REF]. Indeed, this ratio influences both the nutritional value of pea proteins (such as accessible lysine concentration) and their functionality (such as solubility, interfacial, textural, sensory, foaming, and emulsifying capabilities) (Dagorn-Scaviner, [START_REF] Dagorn-Scaviner | A comparison of interfacial behaviours of pea (Pisum sativum L.) legumin and vicilin at air/water interface[END_REF][START_REF] Mujoo | Characterization of storage proteins in different soybean varieties and their relationship to tofu yield and texture[END_REF][START_REF] Rangel | Functional properties of purified vicilins from cowpea (Vigna unguiculata) and pea (Pisum sativum) and cowpea protein isolate[END_REF][START_REF] Martínez-Villaluenga | Assessment of protein fractions of three cultivars of Pisum sativum L.: effect of germination[END_REF]. Vicilin exhibits superior interfacial characteristics than legumin, according to [START_REF] Dagorn-Scaviner | A comparison of interfacial behaviours of pea (Pisum sativum L.) legumin and vicilin at air/water interface[END_REF]. It also makes gels stiffer and improves the stability of foams and emulsions [START_REF] Mujoo | Characterization of storage proteins in different soybean varieties and their relationship to tofu yield and texture[END_REF]. According to the previous papers of [START_REF] Mertens | Agronomical factors influencing the legumin/vicilin ratio in pea (Pisum sativum L.) seeds[END_REF], [START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF], different analysis methods can change the variability of the V/L ratio, such as ultracentrifugation, rocket immunoelectrophoretic, sodium dodecyl sulfatepolyacrylamide gel electrophoresis/densitometry, reverse-phase high-performance liquid chromatography (RP-HPLC), differential scanning calorimetry, and ion exchange chromatography. In this paper, the respective areas of the 2 peaks were calculated from the differential scanning calorimetry curve through deconvolution.

Figure 3-4 shows one of the results of the deconvolution calculation, and the area assigned to 7S (~58,8 %) was much higher compared to the area assigned to 11S (~41,2 %) confirming that 7S was the major fraction of globulins in our PPI sample.

The rest of the deconvolution result is shown in Figure Annex 1.

Thermal properties of EW

Figure 3-5 shows typical DSC thermograms for EW with 4 main peaks. In agreement with literature data [START_REF] Ferreira | A calorimetric study of egg white proteins[END_REF][START_REF] Barhut | Thermal Analysis of Foods[END_REF], the peaks at ~63, ~69, ~76 and ~83 °C could be assigned to ovotransferrin, lysozyme, ovalbumin, and S-ovalbumin (the more heat-stable form of ovalbumin [START_REF] Smith | Studies on ovalbumin II. The formation and properties of S-ovalbumin, a more stable form of ovalbumin[END_REF], respectively. However, at pH 7.5, for the egg white sample, the first peak showed a shoulder probably corresponding to lysozyme, indicating the peak of lysozyme was overlayed by ovotransferrin one (Figure 3-5), maybe due to coaggregation and heteroprotein formation between ovotransferrin and lysozyme as suggested by [START_REF] Wei | Heteroprotein complex formation of ovotransferrin and lysozyme: Fabrication of food-grade particles to stabilize Pickering emulsions[END_REF] and [START_REF] Iwashita | Co-aggregation of ovotransferrin and lysozyme[END_REF]. Many studies performed on liquid egg white around neutral pH indeed mentioned two main denaturation peaks around 65 and 80 °C attributed to ovotransferrin and ovalbumin, respectively [START_REF] Ibanoglu | Thermal denaturation and functional properties of egg proteins in the presence of hydrocolloid gums[END_REF][START_REF] Tóth | HHP treatment of liquid egg at 200-350 MPa[END_REF][START_REF] Renzetti | Amino acids, polyols and soluble fibres as sugar replacers in bakery applications: Egg white proteins denaturation controlled by hydrogen bond density of solutions[END_REF].

Table 3-2 shows the enthalpy (∆H) values for suspensions EW at pH 7.5 and 9, respectively. The ∆H value of pure protein suspensions was influenced by pH. The ∆H value of the EW sample showed a slight but significant increase from pH 7.5 to 9. The denaturation enthalpy (∆H) of the egg white agreed with previous data of 20.6 J/g at pH 7 obtained by [START_REF] Ferreira | A calorimetric study of egg white proteins[END_REF] and 21.0 J/g at pH 8.58 obtained by [START_REF] Rossi | Thermal denaturation and aggregation of egg proteins[END_REF]. Some differences in denaturation enthalpy compared to published values can be explained by the sensitivity of the apparatus (which is higher in our experiments using Micro DSC) and by variable baseline fitting [START_REF] Ferreira | A calorimetric study of egg white proteins[END_REF].

RP-HPLC of EW and SEC-HPLC of PPI

Several batches of purification of ovalbumin were performed, and then the purity was analyzed by RP HPLC as shown in Figure 3-6. Then, ovalbumin fractions with high purity were kept for the following experiments. and 9, respectively. To our knowledge, there was no big difference between pea protein isolates prepared at pH 7.5 and 9. The globulin fraction represented by two peaks was obtained around 9-11 min, which agreed with the results of [START_REF] Mession | Thermal Denaturation of Pea Globulins (Pisum sativum L.)-Molecular Interactions Leading to Heat-Induced Protein Aggregation[END_REF]. In detail, the two main peaks were attributed to legumin 11S and vicilin, with a molecular weight of around 360 and 150 kDa. To be noted that between the two main peaks, it could be the peak of 7S convicilin, which has a molecular weight of around 210-280 kDa [START_REF] Barać | Profile and functional properties of seed proteins from six pea (Pisum sativum) genotypes[END_REF][START_REF] Boye | Pulse proteins: Processing, characterization, functional properties and applications in food and feed[END_REF][START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF]. There are also some minor peaks around 44, 17, or 12 kDa, which could be the vicilin fragments in small amounts. [START_REF] Mession | Thermal Denaturation of Pea Globulins (Pisum sativum L.)-Molecular Interactions Leading to Heat-Induced Protein Aggregation[END_REF] also found this kind of vicilin fragments at a molecular weight of around 54, 30, or over 30 kDa. The SEC-HPCL results also agreed with SDS-PAGE.

Conclusion

In our study, pea globulin fractions were obtained from extraction from delipidated pea flour. These fractions are rich in legumin (41.2 %) and vicilins and convicilins (58.8 %) subunits. Delipidation made it possible to have protein fractions richer in protein and purer.

The solubility of extracted pea protein isolate was over 89 % when pH is over 7, for egg white, the solubility was higher at an alkaline solution, which could help us to have a better soluble protein at selected pH of 7.5 and 9.

The thermograms obtained by MicroDSC confirmed that pea globulins were lowdenatured proteins by the chemical and physico-chemical treatments during the extraction phases and therefore usable for the rest of our study. For egg white, it showed four thermal peaks regarding ovotransferrin, ovalbumin, lysozyme, and s-ovalbumin at pH 9, while at pH 7.5, the peak of lysozyme was overlapped, maybe due to coaggregation and heteroprotein formation between ovotransferrin and lysozyme.

Chapter 4 Interactions between isolated pea globulins and purified egg white proteins in solution

Abstract

In the present work, the interactions and associations between low-denatured pea globulins (PPI) and purified main egg white proteins (ovalbumin (OVA), ovotransferrin (OVT), and lysozyme (LYS)) were studied at pH 7.5 and 9 by using isothermal titration calorimetry (ITC), dynamic light scattering (DLS), laser granulometry and confocal laser scanning microscopy (CLSM). From ITC, we detected strong exothermic interactions between PPI and LYS at both pHs, which led to aggregation. At these pH values, the net positive charge of lysozyme favored electrostatic interactions with negative charges of pea proteins, and oligomers were formed during titration experiments. Furthermore, DLS, laser granulometry, and CLSM data showed that the particle size of the mixture increased with increasing LYS to PPI ratio. Large irregular aggregates up to 20-25 µm were formed at high molar ratios and no complex coacervate was observed. No or very weak interactions were detected between OVT or OVA and PPI whatever the pH. These results suggest a selective association of PPI with LYS when added to egg protein mixtures.

Introduction

With the increase in world population and food transition in emerging countries, the demand for protein is expected to increase by 40 % until 2030 [START_REF] Ozanne | Les filères protéiques végétales sauront elles répondre aux demandes alimentaires mondiales? Proteines 2030: Les demandes à saisir en France et dans le monde[END_REF]. If the demand for animal proteins increases in emerging countries, developed countries have initiated their second food transition to diversify their protein supply to benefit plant proteins [START_REF] Mottet | Cartographie mondiale des demandes en produits animaux dans les 20 prochaines années. Proteines 2030 : Les demandes à saisir en France et dans le monde[END_REF]. However, plant proteins may raise concerns for organoleptic acceptability, functional properties, and nutritional quality compared to animal proteins.

Therefore, interest in food-based design protein mixtures between animal and plant proteins has been increasing over the years [START_REF] Ersch | Modulating fracture properties of mixed protein systems[END_REF]Ainis, Ersch & Ipsen, 2017;[START_REF] Hinderink | Behavior of plant-dairy protein blends at air-water and oil-water interfaces[END_REF][START_REF] Guyomarc'h | Mixing milk, egg and plant resources to obtain safe and tasty foods with environmental and health benefits[END_REF]. One of the issues of mixing animal and plant proteins is to at least maintain, or better increase, their functional properties such as foaming, gelling, or emulsifying. Several studies obtained value-added functionalities of the resulting product [START_REF] Kebary | Functional properties of the coprecipitation of whey and bean proteins[END_REF][START_REF] Alu'datt | Preparation, characterization and properties of whey-soy proteins co-precipitates[END_REF][START_REF] Kristensen | Co-precipitation of whey and pea protein-indication of interactions[END_REF][START_REF] Jarpa-Parra | Quality characteristics of angel food cake and muffin using lentil protein as egg/milk replacer[END_REF].

Alves & Tavares (2019) recently compile the impact of technological treatment on protein structure and techno-functional properties of animal and plant proteins in mixed systems. They noticed that mixing animal and plant proteins enable to obtain gels and /or films to display various microstructure and by a consequence displaying different rheological behavior. All the studies on gelling properties mentioned used milk proteins as animal proteins and either soy or pea proteins as plant proteins.

Few studies concerned foaming and emulsifying properties of mixed systems and among them, very few dealt with egg proteins as animal proteins [START_REF] Wang | Using modified soy protein to enhance foaming of egg white protein[END_REF][START_REF] Jarpa-Parra | Quality characteristics of angel food cake and muffin using lentil protein as egg/milk replacer[END_REF][START_REF] Wouters | Foaming and air-water interfacial characteristics of solutions containing both gluten hydrolysate and egg white protein[END_REF]. [START_REF] Wouters | Foaming and air-water interfacial characteristics of solutions containing both gluten hydrolysate and egg white protein[END_REF] pointed out that in combination with hydrolyzed gluten, egg white proteins formed a second layer by electrostatic and hydrophobic interactions after adsorption of hydrolyzed gluten at the interface, thus increasing the resistance of the foam bubbles against coalescence.

Some other studies focused on protein interactions at a molecular level in the mixed systems [START_REF] Roesch | Heat-induced soy-whey proteins interactions: Formation of soluble and insoluble protein complexes[END_REF][START_REF] Anuradha | Complexation of bovine β-lactoglobulin with 11S protein fractions of soybean (Glycine max) and sesame (Sesamum indicum)[END_REF][START_REF] Chihi | Heat-induced soluble protein aggregates from mixed pea globulins and β-lactoglobulin[END_REF], Mession, Roustel, & Saurel, 2017a). Globular proteins denature when heated and form mixed aggregates depending on the initial protein mixes and the respective properties of the mixed proteins, such as the presence of free sulfhydryl groups and disulfide bonds [START_REF] Guyomarc'h | Mixing milk, egg and plant resources to obtain safe and tasty foods with environmental and health benefits[END_REF]. The presence of milk proteins drives the heat-induced co-aggregation of all disulfide-containing proteins and yields new aggregate forms, while soybean and pea proteins mostly aggregate through hydrophobic and non-covalent interactions.

Egg white is a proteinic solution used in many food applications for its gelling or foaming properties. It is a good candidate for mixing with plant proteins, especially because its basic pH (from 7.5 just after laying to 9.5 a few days later) may help their solubilization. However, the literature is very scarce on the interactions between egg white and plant proteins. The aim of this study is thus to highlight the interactions that may occur between egg white proteins as a model of animal proteins, and pea proteins, as a model of plant proteins.

Egg white usually contains about 11 % proteins which consist of more than 40 different kinds of proteins. Ovalbumin (OVA) is the major protein and represents about 54 % of the total egg white proteins, while ovotransferrin (OVT) and lysozyme (LYS)

constitute about 1 2% and 3.4 %, respectively [START_REF] Belitz | Eggs[END_REF][START_REF] Burley | The egg shell and shell membranes: properties and synthesis[END_REF]. OVA consists of a peptide chain containing 385 amino acid residues and its isoelectric point is estimated at 4.5. It has a molecular weight of 44.5 kDa and contains four thiols and one disulfide group. OVT, unlike OVA, is not denatured at the interface but coagulates at lower temperatures. It consists of one peptide chain of 686 amino acids and contains one oligosaccharide unit made of four mannose and eight N-acetylglucosamine residues. Its molecular weight is around 77.7 kDa. The isoelectric point is 6.1. OVT has 15 disulfide bonds and about 55 % reactive residues [START_REF] Zabik | Eggs and products[END_REF]. LYS is a relatively small secretory glycoprotein, consisting of 129 amino acids linked by four disulfide bonds, and is a 14.4 kDa protein with an isoelectric point of 10.7. LYS is relatively stable, its denaturation temperature being 74 °C (Shih & Kirsch, 1995[START_REF] Shih | Thermal stability determinants of chicken egg-white lysozyme core mutants: Hydrophobicity, packing volume, and conserved buried water molecules[END_REF][START_REF] Ueda | Remarkable thermal stability of doubly intramolecularly crosslinked hen lysozyme[END_REF].

Recently, there is a growing interest in the consumption of pulse proteins, such as yellow pea (Pisum sativum L.) protein isolate (PPI), as a preferable alternative to animal proteins due to its cheaper price, more sustainable source of proteins with a lower carbon footprint, allergen-free and gluten-free claims that can be made on food labels [START_REF] Adebiyi | Functional properties of protein fractions obtained from commercial yellow field pea (Pisum sativum L.) seed protein isolate[END_REF][START_REF] Havemeier | Dietary guidance for pulses: The challenge and opportunity to be part of both the vegetable and protein food groups[END_REF]. Protein accounts for 20-30 % of pea seed, which mainly consists of globulins and albumins. Globulins, known as salt-soluble proteins, represent around 50-60 % of total pea proteins while water-soluble albumins accounted for 15-25 %, according to a study by [START_REF] Gueguen | Legume seed protein extraction, processing, and end product characteristics[END_REF].

Meanwhile, legumin (11S) and vicilin/convicilin (7S) constitute pea globulins.

Legumin is a hexameric homo-oligomer with a molecular weight (Mw) of 360-400 kDa.

Each subunit is around 60 kDa and consists of an acidic (~ 40 kDa) and a basic polypeptide (~20 kDa) linked by a disulfide bond. The acidic chain also has one free thiol [START_REF] Gatehouse | Characterisation and subunit structures of the vicilin storage proteins of pea (Pisum sativum L.)[END_REF]O'Kane, Happe, Vereijken, Gruppen, & van Boekel, 2004a). Vicilin is a trimeric protein with a molecular weight of around 150 kDa, where the main vicilin subunit (~50 kDa) can undergo in vivo proteolysis at two potential cleavage sites. The vicilin-associated protein, convicilin, is a 210-290 kDa protein, consisting of subunits (~71 kDa) associated in trimeric or tetrameric form (O'Kane et al., 2004a).

To better understand the behavior of the two types of proteins in association with food systems, this article proposes a first approach to investigate the interactions between pea globulins and egg white proteins in aqueous mixtures at neutral and alkaline pH (pH 7.5 and 9), close to that of egg white. The potential interaction of whole pea globulins with purified LYS, OVA, or OVT was first examined by isothermal Whatever the egg protein studied, the ITC signal exhibited an exothermic profile.

However, the signal intensity depended on the protein injected and the pH value (Figure 4-1). Weak interactions were observed between OVA or OVT and PPI at both pHs (7.5 and 9). The observed interactions in these mixed systems exhibited a saturating behavior but the signals are too weak to allow access to the thermodynamic parameters (Figure 4-1A, B). These results suggested that when mixed with PPI, OVA or OVT coexist in solution without co-aggregation or complexation at neutral to basic pH values and low ionic strength. In contrast, when LYS was injected into PPI, a large exothermic signal was obtained at pH 7.5 but also at pH 9 (Figure 4-1 C). Meanwhile, to be consistent with the same buffer at both pH, and to avoid the potential buffer/protein interaction already reported by Rabiller-Baudry & Chaufer ( 2001), LYS in TRIS-HCl buffer at pH 7.5 was kept for further analyses.

The strong interaction between LYS and PPI was further explored. Figure 4-2 shows the ITC profiles and corresponding binding isotherms of the injection of LYS into PPI solution at two different pHs. The isotherms resulting from titrating PPI with LYS exhibited a visually obvious biphasic profile. The initially integrated heats of injection show a trend toward increasingly negative enthalpy, while later data trend positively until saturation was reached.

The area under each peak represented the heat exchange within the ITC cell after each injection, after subtraction of the heat of dilution of LYS into the buffer solution.

While the overall ITC profiles were similar at both pH values, the enthalpy of the interaction was higher at pH 7.5 than at pH 9. The observed difference does not seem to be linked to the buffer nature as observed in other protein systems [START_REF] Nigen | Molecular interaction between apo or holo α-lactalbumin and lysozyme: Formation of heterodimers as assessed by fluorescence measurements[END_REF]. Indeed, the same ITC signal was recovered at pH 7.5 when HEPES buffer was substituted by Tris-HCl (Figure 4-2 A, C).

At both pHs studied, a strong biphasic exothermic signal was obtained, underlying at least two distinct events. During the first phase, the height of the exothermic peaks continuously increased with the addition of LYS until a critical value of LYS/PPI molar ratio beyond which the trend was reversed; further addition of LYS decreased the exothermic intensity of the signal (phase 2) until saturation. By comparing the general appearance of the two signals, two major linked differences can be noticed: i) the slope of the two phases is steeper at pH 7.5 than at pH 9; ii) the critical inversion LYS/PPI molar ratio is shifted to a higher value at pH 9, i.e., around 13 against 5 at pH 7.5.

Similar biphasic ITC profiles were reported for other heteroprotein systems involving LYS such as LYS/bovine lactalbumin at 45 °C [START_REF] Nigen | Temperature affects the supramolecular structures resulting from α-lactalbumin-lysozyme interaction[END_REF] and LYS/conglycinin [START_REF] Zheng | Sodium chloride-programmed phase transition of β-conglycinin/lysozyme electrostatic complexes from amorphous precipitates to complex coacervates[END_REF]. Such results were explained by ionic complexation between oppositely charged polymers forming supramolecular structures.

The shift of the molar ratio can be explained by the change of the negative-positive charge balance at the surface of the proteins, in particular, LYS given its high isoelectric point (Ip). At pH 9, a value approaching its Ip (i.e., 10.7), the LYS is less positively charged than at pH 7.5. Consequently, more LYS molecules are required to neutralize the actual number of negative charges on one PPI molecule, which do not vary significantly from pH 7.5 to pH 9. Charge compensation is the main parameter driving electrostatic complexation between oppositely charged proteins [START_REF] Croguennec | Heteroprotein complex coacervation: A generic process[END_REF].

The explanation of what happens during the two phases is not simple since each thermodynamic signal is a result of the contribution of several phenomena: classical interaction, protein conformational change, the release of water, protons, and other ions, complexation, reorganizations, aggregation, etc [START_REF] Doyle | Thermochemistry of binary and ternary protein interactions measured by titration calorimetry: Complex formation of CD4, HIV gp120, and anti-gp120[END_REF]. The measured signal, therefore, comes from endothermic and exothermic reactions whose final absolute value is the result of the dominant energy.

To go further in the exploration of the thermodynamic changes occurring during titration, we tried to fit the binding isotherms using different binding models offered by

LYS-PPI aggregates size depends on protein molar ratio

From the previous study of ITC, two steps in aggregation between PPI and LYS happened. To have a better understanding of the aggregation, DLS and laser granulometry were performed. Due to large particles scattering more light than small particles (according to Rayleigh approximation, the scattering light intensity of particles is proportional to the sixth power of its diameter), intensity distribution and volume distribution were listed together to understand the relative amounts of the proteins with different particle sizes. and 189 nm at pH 7.5 and 9, respectively, could be aggregated protein particles formed during PPI preparation or initially present [START_REF] Li | Effect of concentration, ionic strength and freeze-drying on the heat-induced aggregation of soy proteins[END_REF][START_REF] Chihi | Heat-induced soluble protein aggregates from mixed pea globulins and β-lactoglobulin[END_REF]. The mean size of LYS at pH 7.5 and pH 9 was in the range of 2.5 to 3.0 nm, in line with the LYS monomer [START_REF] Zheng | Heteroprotein complex coacervate based on β-conglycinin and lysozyme: Dynamic protein exchange, thermodynamic mechanism, and lysozyme activity[END_REF]. At pH 9 results also showed a double distribution where particles around 314 nm could originate from the aggregation of LYS resulting from less electrostatic repulsion between protein molecules at this pH closer to the Ip of LYS.

To characterize aggregation for the mixture in a larger range of particle sizes, laser granulometry was used.

(A) (B) Means followed by a different small letter for the same column are significantly different (P<0.05) Figures 4-4 and 4-5 demonstrated the particle size distribution by laser granulometry (A) and visual appearance (B) of PPI-LYS mixtures at pH 7.5 and 9, respectively. The particle size of the mixtures formed by PPI and LYS at different LYS/PPI molar ratios were reported in table 1 for the respective pH. As shown in Table 4-1, the size particle in the PPI-LYS mixture at pH 7.5 showed two distinct situations.

First, it increased with the increasing proportion of LYS, then decreased when the LYS/PPI molar ratio was more than 11.21. Table 4-1 also gives the mean particle size for the pH 9 counterparts, showing similar behavior to the results at pH 7.5 with maximum particle size for an LYS/PPI molar ratio of 17.45. As the particle size decreased from an LYS/PPI molar ratio of ~11 at pH 7.5 and 17 at pH 9, respectively (Table 4-1), it could be hypothesized that mixed aggregates became more and more compact from this threshold, as repulsive forces between aggregates increased with the addition of LYS. This increased the density of the aggregates which led to more precipitation of the aggregates as suggested by the lower quantity of the material on the CLSM picture (Figure 4-7 G and H). Furthermore, Figures 4 B and 5 B showed the visual appearance of PPI-LYS mixtures at different molar ratios at pH 7.5 and 9.0, respectively. Precipitates were observed directly after mixing PPI and LYS as the molar ratio exceeded the inflection point previously revealed for ITC binding isotherms, i.e., > 5 and > 12 at pH 7.5 and 9.0 respectively.

The maximal size of aggregates was obtained for neutral charge

The ζ-Potential of PPI, LYS, and their mixtures were measured in TRIS buffer at At both pHs, the PPI-LYS mixture's charge increases with LYS content, ranging from a negative charge at the smaller LYS/PPI ratio in the mixture to a positive charge at a higher LYS ratio in the mixture. The variation of the ζ-potential showed a typical charge inversion from positive ζ-values when the polycation was in excess (Z < 1) to negative ones when the polyanion was in excess (Z > 1) (Figure 4-6) in line with the recent work of [START_REF] Rodriguez | Emulsion stabilisation by complexes of oppositely charged synthetic polyelectrolytes[END_REF]. We can hypothesize that a positive charge on the LYS interacted with negatively charged segments of pea protein isolate, leading to the formation of electrostatic complexes. This behavior indicated the presence of an interaction between the carboxyl groups of pea protein isolate and the amino group of LYS, featuring electrostatic binding. The charge was null for molar ratios close to 12 and 21 at pH 7.5 and 9, respectively. These results agreed with the previous results of ITC where the enthalpy didn't change anymore with the increasing proportion of LYS from these molar ratios (Figure 4 The white color indicated the protein particles stained by Fast Green. From Figure 4-7 A, the PPI solution showed homogeneous distribution of tiny particles. A similar microstructure was previously reported for soluble PPI [START_REF] Lan | Phase behavior, thermodynamic and microstructure of concentrated pea protein isolate-pectin mixture: Effect of pH, biopolymer ratio and pectin charge density[END_REF].

LYS showed aggregates from Figure 4-7 B, as the solution is very concentrated (14.3 g/L), however, when mixed in the PPI solution, the aggregates dissociated with dilution, and no more aggregates are observed as suggested by DLS results (Figure 4-3). As the concentration of LYS increased, large aggregates with increased size were observed (Figure 4-7 C to H), in agreement with the previous particle size results (Figure 45).

These protein aggregates had heterogeneous forms with irregular shapes. This increased size of protein particles could be attributed to strong attractive interactions between the two oppositely charged proteins (i.e., PPI and LYS) and contributed to forming of larger aggregated complexes which increased with LYS addition. As the particle size decreased from an LYS/PPI molar ratio of ~11 at pH 7.5 (Table 4-1), it could be hypothesized that mixed aggregates became more and more compact and more and more individualized from this threshold. Similar CLSM images of complex aggregation were also previously reported in a PPI-low-methoxyl pectin mixture [START_REF] Lan | Phase behavior, thermodynamic and microstructure of concentrated pea protein isolate-pectin mixture: Effect of pH, biopolymer ratio and pectin charge density[END_REF], whey protein-beet pectin [START_REF] Chen | Formation and microstructural characterization of whey protein isolate/beet pectin coacervations by laccase catalyzed cross-linking[END_REF], and soybean protein-chitosan [START_REF] Yuan | Associative interactions between chitosan and soy protein fractions: Effects of pH, mixing ratio, heat treatment and ionic strength[END_REF]. Obviously, the present results showed that no spherical-shaped aggregates between PPI and LYS were formed excluding the possibility of complex coacervation in the studied conditions. To complete the microstructure observations, phase contrast micrographs obtained at pH 7.5 in Tris-buffer (10 mM) at room temperature for different LYS/PPI molar ratios, ranging from 0.8 to 4.8, are shown in Figure 4-8. At a low LYS/PPI molar ratio, the formed complexes seemed to be no different. Small aggregated structures were observed against numerous larger aggregates obtained for higher molar ratios discussed above. Moreover, the number and size of formed particles seemed to increase with increasing LYS/PPI molar ratios confirming the results of particle size measurements.

Optical microscopy

Interestingly, the overall shape of the particles obtained at a low molar ratio was more C D spherical (Figure 4-8 A-C) than at higher ratios. The spherical nature of these particles corresponds probably to heteroprotein complex coacervation between positively charged LYS and negatively charged PPI. Complex coacervation in heteroprotein mixtures, corresponding to liquid-liquid phase separation that occurs in specific physico-chemical conditions starts to be well documented [START_REF] Croguennec | Heteroprotein complex coacervation: A generic process[END_REF].

The formation of complex coacervates between LYS and a mix of globulins in PPI deserves to be studied in depth.

Conclusion

The interactions and aggregation phenomena of pea proteins with three different egg white proteins were investigated. Only weak interaction was detected between PPI and acidic or neutral proteins from egg-like OVA and OVT, respectively. Special attention was paid to the mixture of PPI and LYS which showed specific interactionaggregation behavior. It was evidenced that non-spherical aggregates were formed from low LYS/PPI molar ratio growing into large irregular aggregated structures that insolubilized at high molar ratio excluding the formation of pure complex coacervates.

By combining the results obtained by the different techniques implemented here, we proposed a simple mechanism for the interaction-aggregation that occurs when LYS is mixed with PPI (Figure 456789). At low ionic strength, LYS interacts with PPI at pH 7.5 and pH 9 according to two major structuring step processes: (i) the first step leads to the spontaneous formation of soluble complexes, and (ii) the second step involves the aggregation of these structures to form large separated aggregates with higher size centered around 20-25 µm. The transition from step 1 to step 2 is governed by pHdependent protein stoichiometry needed to achieve opposite charge compensation. This transition occurs at a lower LYS/PPI ratio at pH 7.5 thanks to the higher surface positive charge of LYS as compared to pH 9. These results suggest that LYS, as an egg basic protein, will play a key interacting role when PPI is mixed with egg white for application purpose that deserves to be studied in depth in such a complex system. plant-based foods and less dependence on meat and other animal-based products will contribute to improving the sustainability of the food system [START_REF] Aiking | The next protein transition[END_REF][START_REF] Alexandratos | World agriculture towards 2030/2050: the 2012 revision[END_REF][START_REF] Clark | Comparative analysis of environmental impacts of agricultural production systems, agricultural input efficiency, and food choice[END_REF][START_REF] Godfray | Meat consumption, health, and the environment[END_REF][START_REF] Poore | Reducing food's environmental impacts through producers and consumers[END_REF][START_REF] Röös | Protein futures for Western Europe: potential land use and climate impacts in 2050[END_REF][START_REF] Shepon | The opportunity cost of animalbased diets exceeds all food losses[END_REF]. Meanwhile, the food industry is increasingly using plant protein components, particularly from legume seeds as an alternative to animal-based sources due to their diversity, nitrogenfixing ability, higher availability, low price, and consumer perception of health and sustainability [START_REF] Stone | Functional attributes of pea protein isolates prepared using different extraction methods and cultivars[END_REF][START_REF] Boye | Pulse proteins: Processing, characterization, functional properties and applications in food and feed[END_REF]. The mixtures of plant and animal proteins have also been considered to address food transition concerns and to explore synergistic effects in terms of consumer acceptance, nutrition, digestibility, and techno-functional properties of such systems [START_REF] Alves | Mixing animal and plant proteins: Is this a way to improve protein techno-functionalities?[END_REF][START_REF] Guyomarc'h | Mixing milk, egg and plant resources to obtain safe and tasty foods with environmental and health benefits[END_REF]. Among the new sources of proteins, pea proteins are increasingly attractive, and several studies have targeted their behaviour when mixed with dairy proteins, to form gels, emulsions, or foams [START_REF] Chihi | Heat-induced soluble protein aggregates from mixed pea globulins and β-lactoglobulin[END_REF]Hinderink et al., 2021a). On the other hand, no study has explored mixtures of pea and egg proteins, which may be an interesting perspective for the development of ovo-vegetarian products.

Pea proteins represent ≈ 23 % (w/w on a dry basis (d.b.)) of the dry seeds [START_REF] Gueguen | Legume seed protein extraction, processing, and end product characteristics[END_REF]Tzitzikas, Vincken, de Groot, Gruppern, & Visser, 2006) and are mainly composed of globulins (≈ 70 %), i.e., legumin 11S and vicilin/convicilin 7S (Sharif, et al., 2018) and the rest corresponds mainly to the 2S albumin fraction (≈20%) and other insoluble proteins. Legumin is a hexameric protein of 360-400 kDa, comprising six subunits of ~60 kDa associated with non-covalent interactions. Each monomer consists of an acidic (~40 kDa) and an alkaline (~20 kDa) subunit linked by a disulfide bond [START_REF] Dziuba | Proteomic analysis of albumin and globulin fractions of pea (pisum sativum L.) seeds[END_REF][START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF]. Vicilin (7S) is a trimeric protein of around 150 kDa. Each monomer ~50 kDa has two cleavage sites possibly generating small fragments during pea seed development: α (~20 kDa), β (~13 kDa), γ (~12-16 kDa), αβ and βγ polypeptides (Gatehouse, Croy, Morton, Tyler, & Boutler, 1981;[START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF][START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF][START_REF] Liang | pH-dependent emulsifying properties of pea [Pisum sativum (L.)] proteins[END_REF]. A third minor globulin, convicilin, is a multimeric protein of 210-290 kDa whose subunit (~70 kDa) has a highly homologous core amino acid sequence with vicilin monomer, yet possesses an extended hydrophilic N terminus (O'Kane, Happe, Vereijken, Gruppen, & van Boekel, 2004a).

Among animal-rich protein products, egg white is a desirable ingredient used in many foods such as bakery products, meringues, and meat products, because of its excellent foaming and gelling properties [START_REF] Lechevalier | Egg white drying: Influence of industrial processing steps on protein structure and functionalities[END_REF]. It usually contains about 11 % proteins which consist of more than 40 different kinds of proteins. Ovalbumin (54 %), ovotransferrin (12 %), ovomucoid (11 %), lysozyme (3.5 %), and ovomucin (1.5-3.5 %) are among the major proteins of egg white [START_REF] Belitz | Eggs[END_REF][START_REF] Guha | Egg proteins. Encyclopedia of Food Chemistry[END_REF].

Ovalbumin (44.5 kDa) consists of a peptide chain of 385 amino acid residues and contains four thiols and one disulfide group. Its isoelectric point is estimated at 4.5.

With high pH and temperature-dependent denaturation, ovalbumin converts into a thermally stable form known as S-ovalbumin [START_REF] Smith | Studies on ovalbumin II. The formation and properties of S-ovalbumin, a more stable form of ovalbumin[END_REF]. Ovotransferrin consists of one peptide chain of 686 amino acids and contains one oligosaccharide unit made of four mannose and eight N-acetylglucosamine residues. Its molecular weight and isoelectric point are around 77.7 kDa and 6.1 respectively. Lysozyme (14.4 kDa) is a relatively small secretory glycoprotein, consisting of 129 amino acids linked by four disulfide bonds with an isoelectric point of 10.7. Recently, [START_REF] Iwashita | Co-aggregation of ovotransferrin and lysozyme[END_REF] highlighted that ovotransferrin can co-aggregate with lysozyme, and [START_REF] Wei | Heteroprotein complex formation of ovotransferrin and lysozyme: Fabrication of food-grade particles to stabilize Pickering emulsions[END_REF] confirmed heteroprotein complex formation between ovotransferrin and lysozyme. Ovomucoid is a heat-stable glycoprotein containing 186 residues with a molecular weight of 28 kDa and pI of 4.1 [START_REF] Julià | Effect of heat treatment on hen's egg ovomucoid: An immunochemical and calorimetric study[END_REF][START_REF] Winiarska-Mieczan | Avian egg's white ovomucoid as foodallergen for human[END_REF]. It contains 9 disulfide bonds and has three different domains which are crosslinked only by the intra-domain disulfide bonds (Huopalahti, Anton, López-Fandiño, & Schade, 2007). Ovomucin is a sulfated glycoprotein that is responsible for the jellylike structure of egg white [START_REF] Abeyrathne | Enzymatic hydrolysis of ovomucin and the functional and structural characteristics of peptides in the hydrolysates[END_REF][START_REF] Strixner | Interactions between whey protein or polymerized whey protein and soybean lecithin in model system[END_REF]. The molecular weight of ovomucin is 1.8-8.3×10 3 kDa [START_REF] Abeyrathne | Sequential separation of lysozyme, ovomucin, ovotransferrin, and ovalbumin from egg white[END_REF][START_REF] Baumgartner | Egg allergens. Chemical and biological properties of food allergens[END_REF]. Based on their composition and characteristics, ovomucin subunits may be classified into two types: α-and βovomucin (Huopalahti et al., 2007).

At an industrial level, egg white is available in pasteurized liquid or frozen form, or powder after spray drying. During processing, the egg white is pasteurized at moderate temperatures between 54 and 57 °C for a few minutes to prevent the coagulation of the heat-sensitive egg white proteins. The egg white can also be subjected to higher temperatures during spray drying operating up to 180 °C. To develop a mixed ingredient with pea proteins undergoing treatments analogous to egg white alone, it appears necessary to evaluate the thermal behaviour of the mixture from native proteins. To our knowledge, egg proteins have been rarely studied in association with plant proteins except for soybean (Su et al., 2015;[START_REF] Zhang | Preparation and characterization of hen egg proteins-soybean protein isolate composite gels[END_REF], and the physicochemical properties of egg white and pea protein mixture have not yet been considered in previous works.

In this paper, we mainly evaluate the thermal properties by differential scanning calorimetry (DSC) of isolated pea globulins (PPI) and egg white (EW) mixtures at different PPI-EW mass ratios (100/0, 75/25, 50/50, 25/75 and 0/100) at pH 7.5 and 9.

The nitrogen solubility profile of the pure proteins and 50/50 mixture systems, and their polypeptide composition using electrophoresis were also characterized. Additionally, the gelling temperature (point) of the 10 % protein PPI-EW mixtures was investigated by small amplitude rheology to anticipate possible pasteurization temperature adjustment in future manufacturing.

Results and discussion

Solubility profile of protein systems

Solubility is a prerequisite for proteins in a variety of high-moisture food applications. It is also a key determinant parameter for many important functional properties including emulsifying, foaming, and gel-forming capabilities [START_REF] Malik | Polyphenol removal from sunflower seed and kernel: Effect on functional and rheological properties of protein isolates[END_REF]. [START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF] reported that the solubility of pea protein is highly dependent Regarding the PPI-EW mixture, the solubility profile showed the same shape compared to PPI with a minimum of solubility at around pH 5. Meanwhile, recalculated NS values data gave a reference (Table 5-1). When pH was smaller than 4, the measured NS values of the 50/50 mixture showed no significant difference from the recalculated ones. However, at pH 5 the measured NS value was significantly higher than the recalculated one while above this pH the measured NS profile showed significantly lower values. The mixture of both proteins could either favor protein solubility (around pH 5) or enhance the formation of insoluble (co-)aggregates that decrease protein solubility above pH 5. This could be explained by hydrophobic or electrostatic interactions between protein molecules of different natures. Close to pH 5, ovalbumin, the main egg white protein, is indeed less charged and may interact with PPI, thus increasing their solubility while at higher pH, PPI could form aggregated complexes with lysozyme via electrostatic interaction that could influence their solubility (Chapter 4). Means followed by a different lowercase letter for the same row are significantly different. The polypeptide profile of PPI-EW mixtures at a weight ratio of 50/50 at pH 7.5

Polypeptide composition

(lanes 1-4) and 9 (lanes 5-8) under reducing and non-reducing conditions with and without centrifugation was shown in Figure 5-2. Regarding the line 1 (at pH 7.5) and 8

(at pH 9) for PPI-EW mixtures under non-reducing conditions, the profile showed the presence of L (α, β) polypeptide around 60 kDa, which is consistent with the result of The thermal denaturation temperature (Td) and enthalpy of PPI and EW were discussed in chapter 3.1.5. In general, PPI thermal curves showed the two endothermic peaks for 7S (~76, 71 °C) and 11S globulins (87, ~85 °C) with an enthalpy of 10.8 and 3.6 J/g at pH 7.5 (former) and 9 (latter), respectively. EW had 4 main peaks, and in order were ovotransferrin, lysozyme, ovalbumin, and S-ovalbumin, while lysozyme was overlayed by ovotransferrin at pH 7.5.

Td values of PPI, EW, and PPI-EW mixtures at different weight ratios were compared at pH 7.5 and 9 in Tables 5-2 and 5-3, respectively. In these tables, the peaks were assigned to the different proteins present in PPI, EW, and their mixtures. For PPI, Td3 and Td5 corresponded to vicilin and legumin, respectively. For EW, Td1, Td2, Td3, and Td4, corresponded to ovotransferrin, lysozyme, ovalbumin, and S-ovalbumin, respectively. For the PPI-EW mixtures, Td3 resulted from the superimposition of ovalbumin and vicilin peaks which could also overlay the peak of lysozyme at pH 9.

Td4 and Td5 corresponded to S-ovalbumin and legumin denaturation, respectively. At pH 7.5 (Table 5-2), no significant difference in Td of ovotransferrin between pure EW and PPI-EW mixtures occurred. However, with the addition of PPI, the peak corresponding to lysozyme appeared. This may be due to either a slight shift of ovotransferrin signal toward lower temperatures thus resulting in a better separation of ovotransferrin and lysozyme signals or to an increase of lysozyme denaturation temperature due to its stabilization through interactions with PPI proteins as evidenced previously (Chapter 4, Section 4.3.1). When PPI was mixed with EW, the pea proteins could indeed form electrostatic interactions with lysozyme since they are strongly oppositely charged at pH 7.5. This result agreed with SDS-PAGE results that suggested the implication of lysozyme in aggregates that precipitated with centrifugation.

Moreover, the Td value of legumin (Td5) decreased by about 2 °C in PPI-EW mixtures compared to pure PPI suspension. This means that legumin proteins were more sensitive to temperature in mixtures. Conformational changes towards a more unfolded state of legumin molecules could thus be hypothesized in the presence of egg white proteins. Modification of the hydration environment of molecules influenced by the new composition in the mixture, and/or interactions with egg white proteins could explain the partial unfolding of legumin to a less stable form.

As vicilin and ovalbumin have close denaturation temperatures, only one mean Td value (Td3 ≈ 76 °C) was recorded for the PPI-EW mixtures. This value was not significantly affected by the PPI-EW weight ratio and corresponded to the mean of ovalbumin and vicilin values in pure EW and PPI systems, respectively. Td of Sovalbumin decreased slightly but not significantly for the mixtures compared to the pure EW sample.

At pH 9,0 (Table 5-3), the Td value of ovotransferrin (Td1) decreased significantly from ~63 °C to ~59 °C with the increase in PPI content in admixture. Ovotransferrin is thus more sensitive to heat denaturation in presence of PPI at pH 9 than at pH 7.5, assuming the unfolding of ovotransferrin in presence of PPI or a decrease in electrostatic interactions with lysozyme due to the competition with PPI proteins as suggested by the increase in Td value of legumin in the mixture at this pH (Td5). No peak corresponding to lysozyme (Td2) was detected for PPI-EW mixtures at pH 9 although it was observed for pure EW samples at this pH. We assumed that the peak of lysozyme may be overlapped by the larger peak of ovalbumin and vicilin. In presence of PPI, the total peak shifted slightly to a lower Td value. As in pure systems, Td values for 7S proteins and lysozyme are lower compared to ovalbumin one, the superimposition of the three denaturation temperatures could explain the resultant average lower Td. It could not be excluded also that the 7S peak and/or lysozyme peak increased to Td values > 71 °C closer to Td of ovalbumin in admixture due to a cooperative denaturation effect or thermo-protective effect of respective proteins. For instance, Mession, Roustel, & Saurel (2017a) reported that when mixing casein with pea legumin and vicilin enriched fractions, the Td of the latter proteins increased by about 4 °C. Otherwise, [START_REF] Zheng | Heteroprotein complex coacervate based on β-conglycinin and lysozyme: Dynamic protein exchange, thermodynamic mechanism, and lysozyme activity[END_REF] found that the Td temperature of the mixture of lysozyme and β-Conglycinin was higher than single lysozyme, which indicates the thermal stability of lysozyme was improved via partial unfolding of β-Conglycinin during complexation. Td of S-ovalbumin showed no significant difference for all the samples at both pHs, which confirmed the high thermostability of this protein. presented measured ∆H values significantly lower than recalculated ones with differences comprised between 0.8 to 2.5 J/g. These differences may reflect the loss of solubility of some proteins as revealed for the 50/50 mixture in Sections 5. 3.1 and 5.3.2 at this pH as the precipitated protein part is less prone to contribute to the total enthalpy. PPI-EW mixture at 25/75 and 75/25 ratios at pH 9, showed recalculated ∆H values similar to measured ones, which could probably indicate that the interactions that structure the different proteins were not significantly modified in the mixture in these conditions. However, the 50/50 ratio at pH 9 showed lower measured values than recalculated ones. This could not be attributed to a decrease in solubility since it was not the case for the mixture at this pH but maybe to a specific ratio effect in a complex In order to consider, the overall contribution of all proteins in the systems upon the thermal treatment, we measured the final values of G' and G'' at the end of heating (Table 56). In a first approach, these values could be representative of the level of association of the proteins undergoing denaturation up to 95 °C. Moreover, it could indicate that only the contribution of hydrophobic interactions and covalent SS bond formation can be considered in thermal protein aggregation upon temperature sweep, as electrostatic interactions such as hydrogen bonds were considerably weakened in such a temperature range. The work of [START_REF] Wang | Changes in chemical interactions and protein conformation during heat-induced wheat gluten gel formation[END_REF] on wheat gluten gel formation and [START_REF] Chronakis | Gelation of edible blue-green algae protein isolate (Spirulina platensis strain pacifica): thermal transitions, rheological properties, and molecular forces involved[END_REF] on the formation of Spirulina protein thermal gels indeed highlighted that disulfide bonds and hydrophobic interactions were dominated during the heating progress while hydrogen bonds and electrostatic interactions did not significantly contribute to gel formation but may reinforce the network rigidity of the protein on cooling.

Except for the pure PPI sample at pH 9 that didn't gel, all the protein systems presented a final G' >> G'' by a factor of about 10. The G' and G'' values decreased significantly and gradually when the proportion of EW decreased in the protein suspensions. Considering that for the pure PPI sample at pH 7.5, the viscoelastic parameters were very low, and no gel was formed at pH 9, it could be deduced that the sol-gel transition upon heating in the EW-containing systems mostly reflects the contribution of EW proteins. Native EW proteins were able to gel at elevated pH as reported in other studies [START_REF] Handa | Heat-induced egg white gels as affected by pH[END_REF][START_REF] Croguennec | Influence of pH and salts on egg white gelation[END_REF]. The decrease in G' values when adding PPI was explained by a lower concentration of EW in the system and a possible steric hindering caused by pea globulin unfolded/aggregated molecules formed all along the temperature sweep. As already indicated, the repulsive force between pea proteins at the pHs used was not favorable for the self-association of globulin molecules or aggregation through hydrophobic interactions, neither in the pure PPI sample nor in PPI-EW mixtures.

Regarding the pH effect, the G' and G'' values were significantly higher and lower at pH 9 (vs pH 7.5) for pure EW samples and PPI-EW mixtures, respectively (Tables 56). It has been already reported that EW gels present higher gel strength when prepared at a pH of around 9 compared to lower pH [START_REF] Handa | Heat-induced egg white gels as affected by pH[END_REF][START_REF] Croguennec | Influence of pH and salts on egg white gelation[END_REF].

The inverted behaviour between pH 9 and pH 7,5 observed in the case of PPI-EW mixtures confirmed the hindering effect of highly repulsive pea protein particles on the EW protein association. This effect was found favorable to delay the sol-gel transition of ovotransferrin at pH 9 as already supposed when considering the first gel point in the system. This result could be considered positive in order to apply higher pasteurization temperature at elevated pH during ingredient processing.

Conclusion

The thermal behaviour of PPI-EW mixtures at pH 7.5 and 9 was evaluated by determining the thermal denaturation parameters by DSC and the sol-gel transitions by dynamic rheology upon heating. The thermal parameters (Td values of each protein and total ∆H) of the protein systems were found slightly or not different compared to those measured for the pure proteins solution of EW and PPI. The slight differences observed for ∆H could be explained by a limited loss (<10 %) of protein solubility in the mixtures.

However, the slight shifts of Td value observed for some proteins in the mixed system could be explained by interactions between some proteins (main lysozyme with ovotransferrin and/or legumin) acting positively or negatively on the thermal stability of the proteins depending on the pH. The pH played indeed a significant role as the result of protein unfolding at pH far from the pI of pea proteins (pH 7.5 and pH 9) could affect structural modification upon heating. The repulsive forces between proteins created at elevated pH could also hinder the self-association of proteins in the system as observed from the thermal sol-gel transition data. The heat-induced gelation behaviour of the PPI-EW mixtures seemed governed by the EW proteins in the systems that were partially hindered by the presence of pea proteins that underwent denaturation but insufficient self-association to contribute to the gel network during heating. In this case, the early sol-gel transition of ovotransferrin around 60 °C was slightly delayed by ~3 °C but only at pH 9, which is the natural pH of egg white. So, the maintenance of pH 9 should be considered to optimize the heat treatment of the PPI-EW mixtures in the production of mixed ingredients. Further investigations into the thermal coagulation properties of these protein mixtures are also expected for adequate applications in food.

Introduction

To match the increasing protein demand, it is now a need to expand the plant-rich protein products. Meanwhile, associations between plant and animal proteins have become increasingly attractive in the formulation of high-protein food products in terms of nutritional, economic advantages, functional and organoleptic properties (Chili, Sok, & Saurel, 2018;[START_REF] Mccann | Rheological properties and microstructure of soy-whey protein[END_REF]. Egg proteins have been widely used in the food industry owing to their capacity to form gels with favorable nutritional and texture properties [START_REF] Mine | Recent advances in the understanding of egg white protein functionality[END_REF][START_REF] Valverde | Egg yolk and egg yolk fractions as key ingredient for the development of a new type of gels[END_REF]Li, Zhang et al., 2018). It is rich in ovalbumin (about 54%), ovotransferrin (about 12%), ovomucoid (about 11%),

and lysozyme (about 3.4%) [START_REF] Mine | Recent advances in egg protein functionality in the food system[END_REF][START_REF] Guha | Egg proteins. Encyclopedia of Food Chemistry[END_REF]. Its gelation is a complex process involving protein denaturation, aggregation, and formation of a gel network [START_REF] Mine | Recent advances in the understanding of egg white protein functionality[END_REF]. The gel characteristics of egg white mainly depend on the medium conditions such as pH, ionic strength, and type of salts (Croguennec, Nau, & Brulé, 2002;[START_REF] Nasabi | Effect of salts and nonionic surfactants on thermal characteristics of egg white proteins[END_REF]. Gelation of egg proteins is a two-step process: the first includes changes in protein structure or partial denaturation; the second involves additional aggregations of denatured proteins, which results in an exponential increase in viscosity and the creation of a continuous network [START_REF] Alleoni | Albumen protein and functional properties of gelation and foaming[END_REF]. During gel formation, non-covalent bonds (i.e., hydrophobic interaction during heating and hydrogen/ionic bonds during cooling) and covalent bonds (disulfide bonds) develop the ordinates aggregation of unfolded chains of polypeptides [START_REF] Campbell | Modification of functional properties of egg-white proteins[END_REF][START_REF] Razi | An overview of the functional properties of egg white proteins and their application in the food industry[END_REF]. In previous studies, [START_REF] Raikos | Effects of sucrose and sodium chloride on foaming properties of egg white proteins[END_REF] reported that increasing pH and the addition of NaCl will result in elevated gelation temperatures of egg white proteins.

As an alternative to animal proteins, pulse proteins such as yellow pea (Pisum sativum L.) proteins are gaining attention due to their cheaper price, allergen-free, gluten-free chain [START_REF] Aluko | Emulsifying and foaming properties of commercial yellow pea (Pisum sativum L.) seed flours[END_REF][START_REF] Havemeier | Dietary guidance for pulses: The challenge and opportunity to be part of both the vegetable and protein food groups[END_REF][START_REF] Alves | Mixing animal and plant proteins: Is this a way to improve protein techno-functionalities?[END_REF][START_REF] Burger | Recent progress in the utilization of pea protein as an emulsifier for food applications[END_REF]. Pea seeds contain four main protein fractions. The major fractions are globulins (55-65 % of total proteins), which are soluble in saline solutions; albumins (18-25 %) which are soluble in water;

prolamins (4-5 %) which are soluble in hydroalcoholic solutions, and glutelins (3-4 %) which are soluble in highly alkaline solutions [START_REF] Lu | Composition, physicochemical properties of pea protein and its application in functional foods[END_REF]. Pea globulins are oligomeric storage proteins, which are composed of legumin (11S) with a hexameric structure of 360 -400 kDa. It contains 6 monomers (~60 kDa), linked by non-covalent interactions. Each monomer consists of an acidic polypeptide subunit of ~40 kDa and a basic subunit of ~20 kDa, connected by a disulfide bond [START_REF] Barać | Profile and functional properties of seed proteins from six pea (Pisum sativum) genotypes[END_REF][START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF]. Vicilin (7S) is a trimeric glycosylated protein of molecular weight of 150 -200 kDa, distinguished by the absence of cysteine, which prevents it from participating in intramolecular or intermolecular disulfide bond formation [START_REF] Shewry | Seed storage proteins: structures and biosynthesis[END_REF]. Each monomer ~50 kDa has two cleavage sites possibly generating small fragments during pea seed development: α (~20 kDa), β (~13 kDa), γ (~12-16 kDa), αβ and βγ polypeptides [START_REF] Liang | pH-dependent emulsifying properties of pea [Pisum sativum (L.)] proteins[END_REF][START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF][START_REF] Tzitzikas | Genetic variation in pea seed globulin composition[END_REF] Arntfield, 2012a;[START_REF] Shand | Physicochemical and textural properties of heat-induced pea protein isolate gels[END_REF]. The contribution of disulfide bonds in these heat-induced phenomena seems limited (O'kane et al, 2004 a & c;[START_REF] O'kane | Gelation behavior of protein isolates extracted from 5 cultivars of Pisum sativum L[END_REF]Sun & Arntfield,2012a;[START_REF] Mession | Effect of globular pea proteins fractionation on their heat-induced aggregation and acid cold-set gelation[END_REF].

Gelation property is one of the important functional properties of proteins, which provide unique texture, senses, and flavor for food products [START_REF] Zhang | Preparation and characterization of hen egg proteins-soybean protein isolate composite gels[END_REF][START_REF] Harfmann | The winning wheys of proteins[END_REF]. The cross-linking of polypeptide chains to form a three-dimensional network is known as protein gelation (Sun & Arntfield, 2012a). Protein cross-linking is induced by several molecular forces, which may include hydrogen bonds, ionic attractions, disulfide bonds, hydrophobic associations, or a mix of the aforementioned [START_REF] Otte | Protease induced gelation of unheated and heated whey proteins: effects of pH, temperature and concentration of proteins, enzyme and salts[END_REF]Sun & Arntfield, 2012a). Some reagents However, fewer studies have been reported about the heat-induced gels formed by egg proteins and plant proteins, such as egg white with soy proteins (Su et al., 2015); whole egg or egg yolk proteins with soy proteins [START_REF] Zhang | Preparation and characterization of hen egg proteins-soybean protein isolate composite gels[END_REF]; egg white with hempseed proteins (Alves, Emam-Djomeh, & Chen, 2020). For pea protein isolate, gelation of PPI and animal proteins concerned mainly milk proteins, and most of them were about acid gels (Mession, Roustel, & Saurel, 2017b;[START_REF] Ben-Harb | The rheological and microstructural properties of pea, milk, mixed pea/milk gels and gelled emulsions designed by thermal, acid, and enzyme treatments[END_REF][START_REF] Chihi | Acid gelation of mixed thermal aggregates of pea globulins and β-lactoglobulin[END_REF][START_REF] Oliveira | Colloidal and Acid Gelling Properties of Mixed Milk and Pea Protein Suspensions[END_REF] and rarely heat-induced gels [START_REF] Wong | Synergistic enhancement in the cogelation of salt-soluble pea proteins and whey proteins[END_REF][START_REF] Silva | Heatinduced gelation of aqueous micellar casein suspensions as affected by globular protein addition[END_REF]. To our knowledge, there is no study regarding the heat-induced gelation of egg white and pea protein mixtures. In previous work, the storage modulus (G') of the mixed protein systems during heating was found to decrease with the EW content, and interactions between proteins changed the thermal denaturation temperature of ovotransferrin, lysozyme, and pea legumin (Chapter 5, Section 5. 3.3). Meanwhile, more experimental data are needed to better understand the importance of various forces in network formation. Thus, the work aimed to prepare and investigate the gelling properties, texture properties, and microstructure of composite gels based on the liquid egg white (EW) and pea protein isolate (PPI) by considering various weight ratios (PPI/EW 100/0, 25/75, 50/50, 75/25, 0/100) at pH 7.5 and 9, respectively. Meanwhile, the characterization of intermolecular interactions during gel formation and rheological properties was also investigated to identify gelling behavior of composite protein systems. As shown in Figure 6-1 A and a, for the pure EW system, the microstructural organization of the gel appeared quite different at pH 7.5 and 9. At pH 7.5, the EW gel presented a more porous, loosely packed, and heterogeneous protein network. At pH 9, the protein network became denser and more homogenous. This finding was consistent with previously published SEM and cryo-TEM data showing granular (pH 7) and smooth (pH 9) EWGs (egg white gels) microstructure [START_REF] Nyemb | The structural properties of egg white gels impact the extent of in vitro protein digestion and the nature of peptides generated[END_REF][START_REF] Clark | Globular protein gelation-theory and experiment[END_REF], and other authors observed by CLSM a more homogenous structure of EW gels at pH 9 than at lower pH 5 (Somaratne et al., 2020a).

Results and discussion

Confocal imaging

The explanation for the different gel structures according to the pH was that the various egg white proteins (EWPs) present (particularly ovalbumin (OVA) and ovotransferrin (OVT)) were predicted to react differently when forming EW gels. These two proteins account for over 70% of total EWP and consequently have a large influence on the EW gelation process [START_REF] Nyemb | The structural properties of egg white gels impact the extent of in vitro protein digestion and the nature of peptides generated[END_REF]. At pH 7.5, OVT was more disposed to form random and spherical aggregates and dominated because it was close to its pI (6.5), whereas OVA began to form linear branching aggregates because it was far from its pI (4.5) [START_REF] Nyemb | The structural properties of egg white gels impact the extent of in vitro protein digestion and the nature of peptides generated[END_REF]. As a result, in this case, egg white gel was made up of a variety of aggregated structures: dispersion of OVT spherical aggregates in the protein network of OVA linear branching aggregates. [START_REF] Van Der Plancken | Effect of heat-treatment on the physico-chemical properties of egg white proteins: A kinetic study[END_REF] highlighted that the net protein charge and the electrostatic repulsions were greatly enhanced at pH 9, and the activation energy barrier required to unfold the protein was lowered. In this case, the proteins tended to unfold to form a homogeneous protein network rather than spherical aggregates [START_REF] Clark | Globular protein gelation-theory and experiment[END_REF].

Figures 6-1 E and e showed the microstructure of heated PPI at pH 7.5 and 9, respectively. It was found that, at pH 9, the particles and small aggregates kept moving (no gel was formed), while a more solid-like gel was found at pH 7.5, which agreed with previous results that protein-rich fraction prepared by alkaline extractionisoelectric precipitation, shown a more heterogenous network for PPI at pH 7 compared to other pea protein fractions prepared by other methods and conditions of extraction (the former one extracted at natural pH, the latter one by only with alkaline extraction) (Kornet et al., 2021a). Meanwhile, larger particles were seen at pH 7.5 (Figure 6-1 E) and a loose network of small particles was apparent compared to the pH 9 condition (Figure 6-1 e), where predominantly smaller individual aggregates were observed. The higher repulsive force between protein particles at high pH (far from the pI of pea globulins (~4.5) could explain the formation of smaller aggregates with insufficient interconnections to form a solid network.

Focused on the pictures from Figure 6-1 B to D and b to d at pH 7.5 and pH 9, respectively, different structures were shown. For the composite gel at pH 7.5 (Figure 6-1 B), it was found that aggregates of irregular shapes (> 10 µm) were formed, surrounded by a white homogeneous protein network when PPI took the proportion of 25 % in PPI-EW mixtures. We hypothesized that this homogenous network could be attributed to egg protein as egg white is predominant in the mixture, surrounding PPI aggregates. When PPI occupied half of the mixtures (Figure 6-1 C), more aggregates with black holes were formed and the area of surrounding egg-white colloids decreased.

With more PPI concentration in PPI-EW mixtures, the gel structure seemed to be more heterogeneous and formed more random clusters of smaller sizes. In contrast, PPI-EW gel at pH 9 (Figure 6-1 b-d) showed some differences. When egg white was the dominant part, the gel looked brain-sharp surrounded by continuous egg white gel.

When PPI took 50 % of the mixtures, the gel contained more clusters with smaller sizes.

When PPI was the dominant part, the random clusters formed spread farther and farther in the gel structure. Similar observations regarding mixed gels were found in previous literature. Kornet et al. (2021a) found that whey protein-PPIc gel formed large clusters at high pea protein concentrations. Silva, Cochereau, Schmitt, Chassenieux, & Nicolai, (2019) showed that mixtures of micellar caseins and PPI at pH 5.8 formed gels with protein clusters, whereas more homogeneous gels were obtained for individual proteins. It was worth mentioning that, as can be seen from the structure of the abovementioned mixed gels, the network structure of these gels was not as tight as that of egg white with the formation of large clustered aggregates which also did not exist for the pure PPI systems. In the mixtures, it is suggested that the egg white proteins could constitute the basic architecture of the protein network, and that gelation is accompanied by the formation of protein aggregates which may be pure PPI aggregates or mixed aggregates consisting of pea globulins and some egg white proteins.

Particularly, LYS can form complexes with pea proteins as shown in chapter 4, section 4.3.1. The total or partial phase separation between EW and pea proteins could be caused by depletion or thermodynamic incompatibility phenomena [START_REF] Tolstoguzov | Some physico-chemical aspects of protein processing in foods. Multicomponent gels[END_REF][START_REF] Tolstoguzov | Some thermodynamic considerations in food formulation[END_REF][START_REF] Turgeon | Proteinpolysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects[END_REF]. Even if thermodynamic incompatibility is widely described between food proteins and polysaccharides, these phase separation phenomena could exist between proteins of different natures with a favorable effect of denaturation [START_REF] Polyakov | Thermodynamic incompatibility of proteins[END_REF]. In our systems, these phenomena would be undoubtedly amplified by the lower temperature coagulation of the OVT. Indeed, we have shown previously (Chapter 5, Section 5. 3.3) that the gel point appeared at a temperature < 59 °C in egg-based systems and this early coagulation was assigned to OVT. The primary gel network that is thus formed would be susceptible to exclude the other protein particles in formation during heating, constituted mainly of the nascent pea protein aggregates that would reassemble into large clusters. This was confirmed by the following section 6.2.2, G' decreased with the decreasing proportion of EW in the mixtures, these aggregates would rather have a passive role in the structure of the gel and the properties of the gel would be dominated by the network created by the egg white proteins, OVT firstly and OVA later, even at high proportions of PPI in the mixture. The differences in gel structure noted at pH 9 would be due to greater difficulty for pea proteins to associate due to the repulsive forces between protein particles at this pH. Indeed, smaller aggregates would be formed in this case, and fewer interconnections in the protein network would be possible. All systems formed gels except PPI at pH 9. In this case, heat treatment led to the aggregation of pea proteins giving just a heterogeneous coagulum that was not forming a solid gel. This can be explained by the repulsive force existing between pea proteins at this pH far from their pI impeding protein particles to form a regular threedimensional network. At pH 7.5, a very weak PPI gel was formed, especially during the cooling phase (G' < 100 Pa). There is indeed less repulsive force at this pH; however, given the low storage modulus, the number of connections between protein aggregates and the inter-particular forces was limited. Sun & Arntfield (2010) studied the minimum gelling concentration (MGC) of heat-induced gel formed by salt-extracted pea protein isolate at pH 5.65 was around 5.5 % (w/v), while commercial pea protein isolate (PPIc) was around 14.5 % (w/v Finally, G' of all gels further increased during the subsequent cooling process. The appearance of growth was called gel reinforcement, a typical characteristic of protein gels, which was generally attributed to the formation of many non-covalent interactions such as hydrogen bonds (O'Kane et al., 2004a;[START_REF] Renkema | Heat-induced gel formation by soy proteins at neutral pH[END_REF] and ionic interactions between proteins (Clark & Lee-tuffnell, 1986). It is noteworthy that the G′ of the mixture and EW samples increased around 3-5 times during cooling than during the heating.

Viscoelastic properties during thermal gelation

The EW samples presented the higher G' values after the heating and cooling stages (Table 6-2). These values decreased by increasing the proportion of PPI in the mixture confirming that PPI is less susceptible or unable to participate in the gel network in these conditions. According to the previous gelling point data (Chapter 5, Section 5.3.4), the first gelling point around 59 ℃ corresponded to ovotransferrin present in EW. This early gelation stage could strengthen the system, then the gel was reinforced by later ovalbumin coagulation; probably in mixed systems, the PPI did not contribute significantly to the gelled network.

It is noteworthy that up to a 50/50 ratio the EW-containing systems presented the same final G' values at both pHs (values not significantly different) at the end of treatment indicating probably that EW proteins governed the gelation of these systems.

The PPI-EW at a ratio of 75/25 wasn't taken into this conclusion, as the standard deviation was too high for this ratio at pH 9.

However, for pure pea protein isolate at pH 7.5, the storage modulus is lower than 100 Pa after temperature sweep as already mentioned in Kornet et al. (2021b) who worked at 10 % of protein content and pH 7. So, it could be concluded that pea protein isolate formed a very weak gel. Table 6-2 showed the same tendency of G' after heating and cooling at pH 9 compared with that at pH 7.5 except for the EWP-PPI mixture at the 25/75 weight ratio. Surprisingly, the G' of this mixture after cooling is higher (not significantly) than that of other mixtures containing less pea protein at pH 9. We noticed that the standard error of the pure PPI and this mixture was large, which could indicate the gelation phenomena of the samples were not repeatable. This effect could be explained by the formation of large protein aggregates that increased the viscosity of the system but without forming a solid gel as was presented before by studying the gel microstructure of the systems at the same weight ratio (Figure 6-1F).

Frequency sweep test

The frequency sweep tests for PPI, EW, and PPI-EW mixtures at different weight ratios at pH 7.5 and 9 are shown in Figures 6-3 A and B, respectively. According to these data, G' and G'' were frequency-dependent and both increased with increasing frequency. In addition, the amount of G' was higher than G'' for all the applied frequencies, revealing a gel-like network structure of the EW-PPI mixtures. Similar findings have previously been reported for soy protein-egg white protein mixtures (Su et al., 2015), egg white-oat globulins [START_REF] Ma | Rheological and Structural Properties of Egg White/Oat Globulin Co-Gels[END_REF], and whey proteinpectin [START_REF] Raei | Rheological and structural characteristics of whey protein-pectin complex coacervates[END_REF] mixtures. Tan (δ) < 0.1 revealed the formation of a strong gel (Clark & Ross-Murphy, 1987).

The loss factor of pure egg white is close to 0.1, which means pure egg white tended to form a strong gel. With the addition of pea protein isolate, the loss factor tended to increase significantly when the proportion of PPI exceeded 50 % which means the mixtures formed weaker gels. According to Tunick (2010), the larger the slope the weaker the gel. Hence, with the addition of PPI, the slope tended to increase but with no significant difference (except for the PPI-EW mixture at 75/25 ratio at pH 9).

Strain sweep

The typical strain sweep curves of G' and G'' presented in In LVR, the gels deformed elastically, with storage modulus (G') larger than loss modulus (G''), indicating the gel-like nature of samples. Beyond that region, G′ decreased due to the breakdown of the network structure. Yield points are given in table 6-4. With the addition of PPI, the LVR first increased, until pea protein isolate took the majority in the mixture (the 50/50 weight ratio presented a maximum at both pHs).

Then, LVR decreased and there was no significant difference with pure PPI. It could be suggested that with the addition of PPI, pea globulins aggregates were formed and disturbed the network of egg white protein giving more weaker gel. On the opposite, at lower concentrations of PPI, egg white proteins were concentrated giving a gel with more elastic properties and higher LVR. Furthermore, the decrease of LVR when PPI exceeded 50 % in the mixture would indicate lower connections in the protein network due to decreased EW content leading to earlier network ruptures.

The region of the linear response also increased with pH values, suggesting that the protein gel network was stronger and more deformable at pH 9 (Wang & Chen, 2011). According to the previous research of [START_REF] Handa | Heat-induced egg white gels as affected by pH[END_REF], it was found that the gel hardness and elasticity were stronger at pH 9 than at pH 7. The same results were found in the study of [START_REF] Alleoni | Texture profile and expressible moisture in albume gels of eggs coated with whey[END_REF], when the pH ranged from 9.0 to 9.45, the hardness in egg white gels was greater than when the pH was between 7.7 and 8.1. These authors thought that it was due to the s-ovalbumin along with other proteins that can improve the hardness of albumen gels, as the increase in the proportion of s-ovalbumin in egg white during storage at pH 9 is higher than at pH 7.0. In our case, it could be likely because egg white gels formed a more compact and microstructurally homogenous gel at pH 9, which was confirmed by the CLSM results (Figure 6-1 A & a). [START_REF] Somaratne | Egg white gel structure determines biochemical digestion with consequences on softening and mechanical disintegration during in vitro gastric digestion[END_REF] found that the hardness of egg white gel at pH 9 was higher than that at pH 5, due to a more homogenous network at pH 9 compared to heterogeneous proteins made of larger aggregates particles at pH 5.

Intermolecular interactions

Typical protein gel can be stabilized by both non-covalent and covalent forces.

Chang & Chen (2000) illustrated that hydrophobic interactions, disulfide bonds and hydrogen bonds stabilized the heated egg protein gels. Yang, Wang, Vasanthan & Chen (2014) confirmed that disulfide bonds and hydrogen bonding contributed to the gel network formation of canola protein gels. To evaluate the role of the types of interactions in PPI-100 % gel, EW-100 % gel, and PPI-EW mixture-based gels at pH 7.5 and 9, respectively, a dissociation approach was investigated and compared with the predicted effects. The utilization of urea, propylene glycol, DTT, and Guanidine-HCl as dissociating agents allowed us to assume interactions between proteins in various gels. Urea, DTT, propylene glycol, and Guanidine-HCl were used to disrupt hydrogen bonds, disulfide bonds, and hydrophobic interactions, respectively. The effect of different dissociating agents on the dissociation of proteins from PPI-100 %, EW-100 % gels, and PPI-EW mixtures gels, at pH 7.5 and pH 9, are reported in Figures 6-5, 6-6, 6-7, and 6-8, respectively. In general, DTT weakens the existing disulfide bonds between cysteine residues and prevents the formation of inter-and intramolecular disulfides [START_REF] Léger | Thermal gelation of the 12S canola globulin[END_REF]Sun & Arntfield, 2012a). According to [START_REF] Zou | Urea effects on protein stability: hydrogen bonding and the hydrophobic effect[END_REF], urea binds to amide groups via hydrogen bonds, decreasing the hydrophobic effect through dehydration of the protein molecule and indicating that both hydrophobic and hydrophilic groups are involved in ureainduced denaturation. [START_REF] Walstra | Physical chemistry of foods[END_REF] pointed out that the denaturing effect of urea was caused by the dehydration of urea-bound peptide bonds, which also weakens hydrophobic interactions. Meanwhile, [START_REF] Tanford | Protein denaturation[END_REF] indicated that urea is a strong denaturing agent which can induce an extensively unfolded state, in which the protein molecule behaves like a random coil. GuHCl is a strong ionic denaturing agent, which weakens hydrophobic interactions and inhibits hydrogen and ionic bonds [START_REF] Tanford | Protein denaturation[END_REF]Sun & Arntfield, 2012a). [START_REF] Tanford | Protein denaturation[END_REF] concluded that GuHCl produced the most extensively unfolded state, in which protein molecules lack their native conformation and behave as random coils. GuHCl is a more effective denaturant than urea, unfolding proteins at two to three times lower concentrations [START_REF] Greene | Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, α-chymotrypsin, and b-lactoglobulin[END_REF], and it is chemically stable. Propylene glycol (PG) disrupts hydrophobic interactions but enhances hydrogen bonds and electrostatic interactions by lowering the dielectric constant of solvent and reducing the energy barrier to protein-protein interaction enough to enable structure formation [START_REF] Ustunol | Forces involved in mixed pork myofibrillar protein and calcium alginate gels[END_REF][START_REF] Utsumi | Forces involved in soy protein gelation: effects of various reagents on the formation, hardness and solubility of heat-induced gels made from 7S, 11S, and soy isolate[END_REF].

Effect of dissociating agent on PPI-100% and EW-100% gel, at pH 7.5 or pH 9

Figures 6-5 and 6-6 show that the percentage of proteins that were solubilized by the dissociating agent varied with both the kind of gels and the pH. In PPI-100 % gel as compared with the control (21.5 ±0.6 %), both urea and guanidine-HCl led to 3 times higher solubilization of protein (Figure 6-5 A). On the contrary, in EW100 %-gel, only DTT and urea have a significant effect on total protein solubilization (2 times increase) (Figure 6-5 B) as compared with the control (3.7 ±0.1 %) or the other agents when used alone. The mixing of the 4 agents (urea + DTT + PG + Gu-HCl) showed an overwhelming increase in protein solubility for all samples, showing the synergistic effect of the four dissociation agents whatever the pH and the type of gel.

Whatever the type of dissociating agent (control including) the amount of total protein dissociated from the PPI-100 % gel is always much higher than that of the EW-100 % gel. The remaining protein in the gel (in particular for the EW sample) represented those that were still interacting despite the presence of dissociating agents.

This means that, in addition to the phenomena described above, there may be other interactions (covalent bonds, ionic interactions) not affected by the dissociating agents or that the intrinsic solubility of the particles released was not complete. What should be kept in mind was that there are some limits to using solubility to identify the interaction during gelation. In the sense of the method, solubility corresponds to the dissolution of the gel under the effect of dissociating agents. While certain chemical reagents were added to the protein gel, the constitutive protein of the gel would dissolve [START_REF] Jiang | Extreme pH treatments enhance the structurereinforcement role of soy protein isolate and its emulsions in pork myofibrillar protein gels in the presence of microbial transglutaminase[END_REF]. Thus, a high solubility indicates that polypeptides or smallsized protein particles have detached from the gel thanks to the breaking of certain interactions and that a part of these is non-sedimentable. The remaining protein in the gel represented those that were still interacting despite dissociating agents suggesting very strong interactions between proteins. Nevertheless, the results must be taken with caution because the chemical agents can break interactions, but the protein particles can remain large and therefore remain insoluble due to their unfavorable surface properties (i.e., lack of hydrophilicity); moreover, new interactions created between released particles could lead to their precipitation. The efficiency of the agent in dissolving the gel is therefore relative. Subsequently, we will consider that the more the gel is dissolved in the presence of a chemical agent, the more the agent can destroy interactions releasing soluble protein particles. This represents the ability of the agent to solubilize the gel considering the interactions it can affect. Dissolution of gels in 100 mM Tris buffer (used as control) allows knowing which fraction of the protein system is dissociated in the absence of any dissociating agent. It could be hypothesized that this solubility corresponds to protein particles not bound to the gel network or that certain interactions were weakened by the buffer-releasing part of the protein material.

Tris (C4H11NO3) is a very polar molecule having 1 amine and 3 hydroxyl groups (a weak base); it is therefore positively charged with a pKa of 8.3 close to the 2 pHs studied.

At a concentration of 100 mM, the properties of the molecule can affect hydrogen and ionic bonds, which would explain the partial protein dissociation from the gels in this buffer. The EW-100% gel was poorly dissociated in this buffer (~4 % at both pHs) and the solubility increased up to ~21 % for PPI-100 % at pH 7.5. The significant difference between the network structure of PPI-100 % and EW-100% gel at pH 7.5 resulted in the majority of the protein interactions. So, based on our results obtained at pH 7.5, we can suggest that disulfide bonds between EW-proteins are significantly involved in the EW-100 % gel (Figure 6-5B). [START_REF] Huang | Effects of NaOH/NaCl pickling on heat-induced gelation behaviour of egg white[END_REF] investigated the intermolecular force of egg white gelation by using 30 Mm dithiothreitol, 2 M Gu-HCl, and 2 M urea. It was found that disulfide bonds involved in the production of the egg gel outnumbered the hydrophobic interaction. Jin, Chen, Zhang, & Sheng (2021) also reported that disulfide bonds play the primary role in gel formation followed by hydrophobic interactions, hydrogen bonds, and ionic bonds no matter how long the heat-induced time. Wang et al. (2020) found the same results, that the primary intermolecular forces in the development of heat-induced native egg white protein gel were disulfide bonds, with hydrophobic interactions playing a minor role. On the contrary, both hydrogen, and hydrophobic interactions would be strongly involved in the PPI-100 % gel formation (Figure 6-5A). These results were consistent with the results of Sun & Arntfield (2012a), that mentioned hydrophobic interactions and hydrogen bonds were involved in heatinduced pea protein gelation with 0.3 M NaCl and pH 5.65, while disulfide bonds were not involved in gel formation. [START_REF] Tanger | Influence of pH and ionic strength on the thermal gelation behaviour of pea protein[END_REF] found that the main protein interactions in pea protein gels were non-covalent bonds regardless of pH and ionic strength, and disulfide bonds only at a lower extent.

At pH 9, the quantity of protein dissociated from both PPI-100 % (rather a coagulum in this case) or EW-100 % gel is generally higher than at pH 7.5 (Figure 6-6

A & 6-6 B), suggesting the greatest part of i) low-energy interactions and/or ii) proteins not associated to the protein network. This hypothesis is consistent with previous microscopy results showing that aggregates of PPI-100% after the heat-induced process kept moving at pH 9. This agreed with the finding of [START_REF] Tanger | Influence of pH and ionic strength on the thermal gelation behaviour of pea protein[END_REF], who reported that pea protein isolates with 15 % protein content formed rather an entangled solution than a continuous gel network at pH 9 and 0.9 M NaCl.

At pH 9, the solubility of protein from EW-100 % gels remained low in all cases (<= 11.5 % in the case of urea at pH 9). This means that even if some interactions are affected by the chemical agents, the gel particles released remain insufficiently soluble, which reveals a combination of strong interactions. The EW gels, therefore, remain particularly insoluble even if the agents are used simultaneously, as already noted since only 35.8 % of proteins are solubilized. These gels are especially sensitive to urea and DTT, which would indicate an important role of hydrogen bonds and disulfide bonds in the structure of the gel. The effect of DTT is present for both types of gel (PPI-100 % and EW 100 % at both pH), but even if disulfide bonds are reduced, few soluble particles are released, because other interactions structure the remaining gel particles.

Finally, we observed that adding all the chemical agents at the same time does not allow to solubilize the whole gel except in the case of PPI at pH 9 (but no gel was formed in this case); the total solubility values varied from 65 % minimum to 97.5 % maximum. Figures 6-7 and 6-8 show that the protein solubility of PPI-EW mixed gel at different weight ratios at pH 7.5 and pH 9, respectively, increased in presence of dissociating agents as the proportion of PPI protein in mixed gels increased. The protein solubility in mixed gels was generally higher at pH 9 than at pH 7.5. It could be hypothesized that this solubility corresponded to protein particles not bound to the gel network or that certain interactions were weakened by the buffer-releasing part of the protein material. Mixed gels rich in EW proteins (such as PPI-EW 50/50 and PPI-EW 25/75) remain particularly insoluble even if the agents were used simultaneously.

Nevertheless, these gels were especially sensitive to urea and DTT, which would indicate an important role of hydrogen bonds, hydrophobic interactions, and disulfide bonds in the structure of the mixed gel. On the contrary, when the mixed gel was enriched in PPI proteins (PPI-EW 75/25), the agents that allow the most protein particles to be solubilized were urea (47.8 % ±0.9) and guanidine-HCl (29 % ±0.5) at pH 7.5 (Figure 67). This result indicates a combination of non-specific and lower energy interactions as in the case of PPI-100% gel with a dominance of hydrogen and hydrophobic bonds. The gels were not very sensitive to propylene glycol; this agent would be ineffective because its effect on hydrogen bonds could be masked by the TRIS-HCl buffer used. Overall, an increase in protein solubility is observed when the proportion of PPI increases in the system regardless of the chemical agent. The mixed gels have intermediate behaviors with increasing solubility values between EW-100 % and PPI-100 % systems. Thus EW-based gels are not very dissociable up to the 50/50 ratio, which shows that EW proteins remained dominant in the structure of the gels; indeed, the solubility values become significantly different from the EW-100 % system from or beyond the 50/50 ratio. In all cases, the gels at pH 9 are more dissociable than at pH 7.5 because, at this higher pH, the charge of the protein particles is greater generating more repulsive forces within proteins during the gel formation.

In conclusion, we can say that EW-based gels are less dissociated by the different chemical agents, indicating that egg white proteins form gels with more numerous and stronger molecular interactions regardless of pH. This conclusion agrees with the data of dynamic rheology where the gels showed increased elastic behaviour with the The textural properties of the gels are quite important in determining the quality of food. It can imitate the actions applied to the gels by the tongue and teeth, therefore this test was performed to compare the eating performance of the gels. Hardness and springiness were typically regarded as more relevant measures of gel performance [START_REF] Li | Effect of microwave-assisted phosphorylation modification on the structural and foaming properties of egg white powder[END_REF]Alavi, Emam-Djomeh & Chen, 2020). The changes in TPA parameters (hardness, springiness, and cohesiveness) of gel samples are presented in Table 6-5, and gel appearance of EW and PPI-EW combination gels at different weight ratios are shown in Figure 6-9. The color of the gels obtained from different PPI-EW mass ratios changed from pale yellow to light brown at pH 7.5 and dark brown at pH 9 with the increasing proportion of PPI. Obviously, the color of PPI-EW mixtures gels at pH 9 was darker than the one at pH 7.5. However, the color appearance of egg white gel between pH 7.5 and 9 was hard to distinguish. Since PPI hardly or even did not gelify at pH 9, it was hard to experiment with 100% PPI gels. Therefore, only TPA parameters of EW 100 % and PPI-EW at different weight ratios were reported in Table 6-5. At both pHs, 100 % EW-gel showed the highest gel hardness, and the hardness of gel decreased significantly with the increasing proportion of PPI content (from 0 to 75 %). This is consistent with the rheological data where elastic modulus and loss factor decreased with increased PPI content (see Section 6. 2.3 & 6.2.4). A similar tendency was found in the literature on egg white-hempseed protein mixtures (Alavi, Emam-Djomeh & Chen, 2020) and egg white-soy protein composite gels at higher protein concentrations (Su et al., 2015).

Springiness is a textural quality that is connected to sample elasticity. Springiness in TPA is related to the height that the food recovers during the time that elapses during the end of the first bite and the start of the second bite [START_REF] Chandra | Texture profile analysis and functional properties of gelatin from the skin of three species of fresh water fish[END_REF].

High springiness is caused by the gel structure splitting into a few big pieces during the initial TPA compression, whereas low springiness is caused by the gel breaking into numerous tiny pieces [START_REF] Lau | Texture profile and turbidity of gellan/gelatin mixed gels[END_REF]. As shown in Table 6-5, springiness decreased with higher PPI concentration in composite gels meaning that compression led to more irreversible deformation.

Cohesiveness is often used as an indicator of the ability of the gels to maintain an intact network structure. Higher values of cohesiveness indicate the strength of more intact network structures [START_REF] Handa | Heat-induced egg white gels as affected by pH[END_REF]Fernandez-Lopez, et al., 2006). In the present study, cohesiveness data showed that cohesiveness had the same tendency as springiness and hardness. 100% EW gel had higher values and it decreased with increasing PPI proportion in composite gels. This result was consistent with microstructure observations in Figure 6-1 (section 6.2.1) which has been discussed in the previous section. 3.10±0.21a 0.73±0.02a 0.95±0.04a 3.90±0.08a 0.75±0.01a 0.93±0.02a PPI-EW 25/75 2.78±0.17a 0.67±0.02a 0.92±0.04ab 3.45±0.16a 0.74±0.01ab 0.91±0.03ab PPI-EW 50/50 1.69±0.03b 0.58±0.02b 0.84±0.00ab 1.84±0.08b 0.71±0.00bc 0.89±0.01ab PPI-EW 75/25 0.79±0.00c 0.59±0.02b 0.80±0.01b 0.51±0.01c 0.67±0.01c 0.81±0.01b

Different superscripts in each column chart represent a significant difference (p < 0.05).

Figure 6-9: Gel appearance of EW, and PPI-EW gels at different weight ratios. A: samples prepared at pH 7.5, B: samples prepared at pH 9.

Mechanism of gelation

Combining the results of dissociating agent tests, texture, microscopy, and dynamic rheology data, a possible mechanism of PPI-EW gel formation can be proposed. With the temperature increasing, egg white proteins started to be partially denatured resulting in the unfolding of the proteins. Ovotransferrin, a protein sensitive to temperature, was the first to be denatured as its thermal denaturation temperature was around 61 and 63 ℃ at pH 7.5 and 9, respectively (Chapter 5), followed by lysozyme and ovalbumin. According to the research of [START_REF] Wang | Ovomucin may be the key protein involved in the early formation of egg-white thermal gel[END_REF], ovotransferrin, lysozyme, and ovomucin were involved in the early formation of egg white gels, moreover, in binary mixtures lysozyme co-aggregated with ovalbumin or ovotransferrin through disulfide bonds and physical interactions [START_REF] Matsudomi | Heat-induced aggregation of lysozyme with ovotransferrin[END_REF][START_REF] Matsudomi | Inhibition against heat coagulation of ovotransferrin by ovalbumin in relation to its molecular structure[END_REF][START_REF] Iwashita | Co-aggregation of ovalbumin and lysozyme[END_REF][START_REF] Iwashita | Co-aggregation of ovotransferrin and lysozyme[END_REF]. Association or molecular aggregation of denatured proteins increased with involved protein-protein interactions, such as hydrophobic interactions, hydrogen bonds, and disulfide bonds as suggested by the results from the dissociation tests. During cooling, gel strength increased due to the creation of hydrogen bonds (as G' increased in the dynamic rheology test), resulting in a continuous network. However, a denser and more homogenous network was formed at pH 9 due to higher net protein charges and the electrostatic repulsions at this pH, rather than a more porous, loosely packed, and heterogeneous protein network as obtained at pH 7.5. For heat-induced PPI gelation, as globular proteins, the proteins were firstly partially denatured leading to their unfolding; aggregates would then be formed between pea globulins, hydrogen bonds, and hydrophobic interactions taking the major role during gel formation. While at pH 7.5, a more solid-like gel was formed, at pH 9, no gel formed, possibly due to high repulsive force at higher pH leading to insufficient interconnections. For the combined PPI-EW heat-induced gel systems, during the heating process, the protein was denatured leading to their unfolding and exposed protein subunits. Protein-protein interactions were involved and led to protein aggregates, such as the aggregate between pea protein and lysozyme or the possible aggregate with ovotransferrin or ovalbumin.

During the cooling step, with the increasing proportion of PPI, hydrogen bonds, and hydrophobic interactions took a major role in the gel network formation, while disulfide bonds and electrostatic interactions took a minor role. When EW prevailed in the mixtures, the network had less irregular shapes leading to higher hardness and springiness compared to lower EW proportions in mixtures. Increasing the PPI proportions, larger clustered aggregates were formed making the structure not as tight as pure egg white gel, leading to the decrease in the texture properties. The different results between the mixtures at pH 7.5 and pH 9 may be due to high repulsive forces and negative charges at higher pH.

Conclusion

In this study, the gelling properties of egg white proteins and pea protein isolate were analyzed by dynamic rheology, texture analysis, dissociation test after gelation, and CLSM. The gels obtained from the mixtures were probably constituted of a primary network of egg white proteins, mainly associated with hydrogen and disulfide bonds, including pea protein aggregates driven by electrostatic and hydrophobic interactions.

However, there was good evidence that hydrophobic interactions and hydrogen bonds took a major influence on the composite gel formation with the increasing proportion of pea protein isolate, by contrast, disulfide bonds had less effect on gel formation. This can be confirmed by the rheological data which indicated that weaker gels were formed with the increase in PPI proportion. Consistently, adding more proportion of PPI to egg white led to a decrease in the hardness, springiness, and cohesiveness of the gels. A more homogeneous network was obtained for pure egg white at pH 9, while PPI-only didn't form a gel at this pH. For the combined gels, it showed a difference in the gel network that was more heterogeneous and formed more random clusters of smaller size at pH 7.5 when the dominant part was PPI. At pH 9, when increasing the proportion of PPI, random clusters were formed and spread farther and farther in the gel structure.

This kind of gel was lack of tight connections in the gel network, resulting in a lower hardness. This study gives us the first time to understand what interaction could happen in the PPI-EW composite gels and give more understanding for further application in food design.

purity, solubility, thermal parameters, and polypeptide composition. It was found that pea globulins were rich in legumin (~40 %) and vicilins and convicilins (~60 %) subunits. Egg whites were made up of ovalbumin, ovotransferrin, and lysozyme from SDS-PAGE. Extracted pea protein isolate showed higher solubility at alkaline pH over 7 (> 89 %), and acidic pH (< 3) (> 85 %) which decreased significantly around the isoelectric point region (pH 4.5-5). Meanwhile, egg white showed high solubility (>88 %) whatever the pH; just a small decrease in solubility was observed around pH 4 close to the major ovalbumin fraction's isoelectric point (~pH 4.5). The pH chosen at pH 7.5 and 9 for further experiments provided a good solubility of proteins and the solubility of the mixtures of both proteins at these pHs seemed to be slightly affected.

The thermograms obtained by MicroDSC confirmed that pea globulins were lowdenatured proteins by the chemical and physico-chemical treatments during the extraction phases and therefore usable for the rest of our study. Two thermal peaks at around 76, 87 ℃ (pH 7.5) and 71, 85 ℃ (pH 9) regarding vicilin and legumin were shown respectively. The thermal enthalpy of PPI was 10.8 J/g at pH 7.5 and 3.6 J/g at pH 9. Both thermal peak and enthalpy decreased at higher pH (pH 9, in our case), it could be due to the partial unfolding of proteins molecules at pH far from pI, leading to increased intramolecular net charges and repulsive forces. For egg white, it showed four thermal peaks regarding ovotransferrin (~63 ℃), ovalbumin (~76 ℃), lysozyme (~ 70 ℃), and s-ovalbumin (~83 ℃) at pH 9, while at pH 7.5, the peak of lysozyme was overlapped, maybe due to co-aggregation and heteroprotein formation between ovotransferrin and lysozyme. The enthalpy of egg white was ~ 22 and ~24 J/g at pH 7.5 and 9, respectively.

To have a better understanding of which fractions of egg white protein interact with pea protein isolate in a mixed system, the first main part of the work focused on the study of the interactions between pea protein isolate and purified egg white proteins (ovalbumin (OVA), ovotransferrin (OVT) and lysozyme (LYS)) in solution. Isothermal titration calorimetry (ITC) was first used to identify which binary solution (PPI-LYS, PPI-OVA, PPI-OVT) could have interactions. The results showed strong exothermic interactions between PPI and LYS at both pHs, while no or very weak interactions were detected between OVT or OVA and PPI whatever the pH. Then, zeta potential was used to determine the nature of interactions in PPI-LYS systems. Electrostatic interaction was involved between lysozyme and pea protein isolate at both pHs. During ITC, we found that the titration thermograms underwent two distinct events, as the height of the exothermic peaks continuously increased with the addition of LYS in the first phase until a critical value of LYS/PPI molar ratio beyond which the trend was reversed;

further addition of LYS decreased the exothermic intensity of the signal (phase 2) until saturation. As a result, we performed dynamic light scattering, laser granulometry, confocal laser scanning microscopy, and optical microscopy to identify the influence of the proportion of lysozyme in PPI-LYS systems on the particle size and structure.

Particle size increased firstly with the increasing LYS concentration until the critical molar ratio of LYS/PPI around 5 (pH 7.5) and 13 (pH 9), leading to a formation of heterogeneous aggregates with irregular shapes due to strong attractive interactions between the two oppositely charged proteins. When more LYS was added, the size decreased. It could be hypothesized that mixed aggregates became more and more compact and more and more individualized from this threshold. This phenomenon was confirmed by the microstructure results. According to the above results of this part, we proposed a possible mechanism for the interaction-aggregation that occurs when LYS is mixed with PPI, which contained two major structuring step processes: (i) the first step leads to the spontaneous formation of soluble complexes, and (ii) the second step involves the aggregation of these structures to form large separated aggregates with higher size centered around 20-25 µm. The transition from step 1 to step 2 is governed by pH-dependent protein stoichiometry needed to achieve opposite charge compensation. This transition occurs at a lower LYS/PPI molar ratio at pH 7.5 (~5) thanks to the higher surface positive charge of LYS as compared to it at pH 9 with a higher LYS/PPI molar ratio of around 13.

After having highlighted the electrostatic interactions and possible repulsive forces in PPI-LYS systems, we turn to the second main part of our study, which was the thus resulting in a better separation of ovotransferrin and lysozyme signals or to an increase of lysozyme denaturation temperature due to its stabilization through interactions with PPI proteins as mentioned before. The latter one was assumed to be overlapped by the larger peak of ovalbumin and vicilin. The slight differences observed for ∆H could be explained by a limited loss (<10 %) of protein solubility in the mixtures.

We conclude that pH played indeed a significant role as the result of protein unfolding at pH far from the pI of pea proteins (pH 7.5 and pH 9) could affect structural modification upon heating.

Gelation properties are one of the most important properties of globular proteins and are one of the principal means to give desirable texture to food products. Meanwhile, a heat-induced gel is the most common gelation process, especially for pea protein isolate and egg white proteins. As a result, we prepared the gel at a total protein concentration of 10%, from the mixtures at different mass ratios, at pH 7.5 and 9. The work presented aimed to study the interactions during gelation, as well as the gel characteristics in terms of viscoelastic properties, microstructure, and textures depending on the conditions, to propose a gel formation mechanism.

First of all, storage modulus (G') as a function of temperature during temperature sweep was measured to understand the sol-gel transition behaviour of the mixtures. Two transition temperatures were obtained for pure egg white and PPI-EW composite mixtures containing at least 50% of EW whatever the pH (1 st gelling point assigned to ovotransferrin, the 2 nd gelling point assigned to ovalbumin and vicilin), and the temperatures were close to the thermal denaturation temperature obtained by DSC experiments. In particular, the first gelling temperature increased by nearly 3 °C with the increase in PPI in the mixture at pH 9.0 rather than no changes occurring at pH 7.5, due to highly negative charges of pea proteins at pH 9.0. This resulted in hampering ovotransferrin molecules/particles association until more advanced denaturation (or aggregation) was achieved at slightly higher temperatures. So, the maintenance of pH 9 should be considered to optimize the heat treatment of the PPI-EW mixtures in the production of mixed ingredients.

The PPI-EW mixtures at the 75/25 weight ratio at pH 7.5 and 9 didn't show any early sol-gel transition previously associated with ovotransferrin in this mixture. It could be hypothesized that even if thermal denaturation of ovotransferrin occurred around 60 °C, the resulting unfolded/aggregated proteins were not numerous enough to interact and form a three-dimensional network, and/or their association was sterically hindered by the presence of the pea globulins in the mixture. G' and G'' at the end of the heating step both decreased with the increase of PPI content. Only the contribution of hydrophobic interactions and covalent SS bond formation can be considered in thermal protein aggregation upon temperature sweep, as electrostatic interactions such as hydrogen bonds were considerably weakened in such a temperature range. This means that both former interactions could have a structuring effect during gelation all the more strongly as the proportion of egg white is high.

At pH 9, PPI only formed a heterogeneous coagulum rather than a gel, due to the high repulsive forces at this pH preventing the protein particles from forming a regular three-dimensional network. This was confirmed by the microstructure study of the systems where the formation of small aggregates was observed at this pH. At pH 7.5, there was a solid-like gel structure formed with larger particles and a loose network of small particles. The application of several dissociating agents indicated that hydrogen bonds and hydrophobic interactions played an important role in PPI heat-induced gel formation while disulfide bonds only have a minor role.

Egg white gels, in general, had the highest hardness, springiness, and cohesiveness, confirmed by the lowest loss factor, with a porous, loosely packed, heterogeneous network at pH 7.5 and a denser, more homogenous network at pH 9. Hydrogen and disulfide bonds played a major role in egg white heat-induced gel formation.

With the increased proportion of PPI in admixture with EW, the loss factor increased, as well as the frequency slope of G', meaning weaker gels were formed. Also, hardness, springiness, and cohesiveness decreased with the increasing proportion of PPI, with a more heterogenous network including more random clusters of smaller size.

As a result, we proposed a possible mechanism of gelation for PPI, EW, and PPI-EW mixtures. Combining the results of dissociating agent test, texture, microscopy, and dynamic rheology, a possible mechanism of PPI-EW gel formation can be proposed.

For heat-induced EW gelation, egg white proteins were partially denatured resulting in the unfolding of the proteins, with involved protein-protein interactions, such as hydrophobic interactions, hydrogen bonds, and disulfide bonds. During cooling, gel strength increased due to the creation of hydrogen bonds (as G' increased in the dynamic rheology test), resulting in a continuous network. However, a more homogenous network was formed at pH 9 due to the higher net protein charges and the electrostatic repulsions at this pH, rather than a more porous and heterogeneous protein network obtained at pH 7.5. For heat-induced PPI gelation, in our case, it is difficult to form a gel at pH 9, compared to a solid-like gel formed at pH 7.5, it could be due to higher repulsive force at higher pH (9, in this case) leading to an insufficient interconnection. For the combined PPI-EW heat-induced gel systems, proteins were denatured with increased temperature, leading to their unfolding and exposed protein subunits. Protein-protein interactions were involved and led to protein aggregates, possibly containing self-aggregates of PPI, aggregates between lysozyme and ovotransferrin, lysozyme and ovalbumin, or combined aggregates between LYS, OVT, and OVA. In addition, PPI could form aggregates with LYS in a PPI/LYS binary system (Chapter 4), therefore, it could have PPI/LYS aggregates in PPI/EW mixtures. During the cooling step, with the increasing proportion of PPI, hydrogen bonds, and hydrophobic interactions took a major role in the gel network formation, while disulfide bonds took a minor role. When EW prevailed in the mixtures (75% of the mixture), it showed higher hardness and springiness compared to other proportions of mixtures and the network appeared irregular shapes surrounded with homogenous egg white colloids.

Increasing the PPI proportions, larger clustered aggregates were formed making the structure not as tight as pure egg white gel, leading to a decrease in the texture properties.

The different results between the mixtures at pH 7.5 and pH 9 may be due to high repulsive forces and negative charges at higher pH.

Perspectives

The study contributed to the understanding of the mechanisms of interactions in the formation of pea protein isolate and egg white protein systems. The thermal properties of the mixtures depended on pea protein isolate content, and the characteristics of the heat-induced gel formed were markedly improved compared to the pure thermal gel of pea proteins. In solution, except for the evidenced complexation between pea protein and lysozyme, the properties of the mixture seemed relatively driven by the additive effects of each type of protein. In the structured mixed systems as gels, we could not observe any synergy even if a specific structure of the mixed gel was highlighted (i.e., continuous protein network including other protein aggregates in which it was supposed that egg white proteins and pea proteins dominated in each domain respectively). Regarding this specific microstructure, two different dying agents could be added to stain separately PPI and EW, in order to give more detailed information on the gel structure. Nevertheless, there were still many other questions to consider experimentally. As already known, the type and concentration of salts influence the gelation properties and thermal properties of proteins, therefore, adding salts inside the mixture could help us to have a better understanding of the technofunctional properties of composite systems between plant and animal proteins. As fat is another constituent of food products in general, the impact of a lipid-dispersed phase on gelling properties could be also investigated to design new food products.

In addition, regarding the co-precipitate of the proteins between lysozyme and ovalbumin or ovotransferrin, the mechanism of thermal results was not well understood when PPI was added to egg white, it could be a viable solution to have a hybrid ternary protein system, such as PPI/OVT/LYS, PPI/OVT/OVA, PPI/OVA/LYS, to know more information of the aggregate or precipitate between PPI and egg white fractions.

Furthermore, regarding two fractions of legumin and vicilin in pea globulins, it could be useful to obtain purified 11S and 7S globulins first and then mixed with egg white, leading to a more specific understanding of the co-aggregates, or functional properties.

To complete the study on the functional properties of the system, it could be interesting to investigate emulsifying and especially foaming properties of the mixed system, as the formation of foam is one of the sought-after qualities of egg white ingredients.

Moreover, regarding the technological point in the design of new mixed ingredients, the pasteurization treatment could be performed to evaluate the impact of this treatment on the physicochemical and functional properties of egg white and pea protein mixtures. Other treatments such as drying are expected to cause further denaturation/aggregation among proteins likely to change the final functional properties of the powdered ingredients. Moreover, dry heating of egg white powder (40 to 80 °C for a few days) is a classical treatment applied in industry to enhance the functionality of this ovoproduct. Improved foaming and gelling properties are obtained in this way [START_REF] Lechevalier | Effect of dry heat treatment of egg white powder on its functional, nutritional and allergenic properties[END_REF][START_REF] Yuno-Ohta | Characterization of the effect of dry-heat treatment on the gelation of alkaline dried egg whites using dynamic viscoelastic measurement and ultrasound spectroscopy[END_REF]. So, we could take advantage of this treatment to functionalize the present pea and egg white protein mixtures. Indeed, encouraging preliminary results (unpublished) on the application of the method to pea proteins and egg white system have been obtained by the UMR STLO/UMR PAM consortium in the framework of the VeggIn project (2018-2021) supported by the Carnot Qualiment in France.

In addition, one of the limitations of using egg white proteins is their proven allergenicity. Pea proteins are less allergenic than soy proteins for example and are thus interesting plant proteins to attenuate the allergenicity in mixtures. The possible mechanism leading to allergy reduction could be then explored in future studies.

Furthermore, as mentioned in section 1.4, it could be very interesting in using different treatments to modify egg white and pea protein isolate to favor protein interactions and increase the functional properties, such as gelation properties.

B

Annex-3

First

  of all, I would like to sincerely thank Dr. Claire BERTON CARABIN, Prof. Paul MENUT, and Prof. Pascal DEGRAEVE to have kindly accepted to evaluate my work and to be part of my Ph.D. defense. I would like to specially thank my thesis director, Prof. Rémi SAUREL, as well as codirector, Dr. Valerie DATIN LECHEVALIER for providing me the opportunity to study in France, taking me into the field of plant-animal protein admixture research. Thanks to your instruction, assistance, company, and encouragement during my life in France, especially during the lockdown and final months of thesis manuscript. Thanks to Eliane Case as a co-supervisor for the follow-up and suggestion on the thesis work. I also would like to warmly thank you to all the members of PCAV team, and STLO team, particularly to Yanis, Bonastre Oliete, Bernadette Rollin, Adrien Lerbert, Alexandra Da-SILVA, Julie Chanut, François Potin, Elise Charton, Kandi Sridhar, Jeehyun Lee, Said Bouhallab, Pascaline Hamon, Thomas Croguennec and so on, for the help of life, experiment, training, as well as suggestion to the thesis work. Special thanks to Clara, Master student, for her contribution to the advancement of this thesis. I want to thanks to my Chinese friends in France Jian Yang, Yue Ma, Qian Jiang, Ming Yu, Jun Wang, Hang Zhang, Mei Wang, Jian Gao, Jiajun Feng and Beiyao

  Accomplishments ........................................................................................................... 6.2.7 Mechanism of gelation ...................................................................... 6.3 Conclusion ................................................................................................... General conclusion and perspective ........................................................................... General conclusion ..................................................................................................... Perspectives ................................................................................................................ Reference ................................................................................................................... Annex ......................................................................................................................... Annex-1 .............................................................................................................. Annex-2 .............................................................................................................. Annex-3 .............................................................................................................. Annex-4 ..............................................................................................................

Figure 1

 1 Figure 1-6 3D-strucure of five major egg white proteins: ovalbumin, ovotransferrin, lysozyme, ovomucin, ovomucoid. 1: download from Protein Data Bank, 2: Jalili-Firoozinezhad et al., 2020.

Figure 1 - 7

 17 Figure 1-7 Effect of pea protein addition level (0%, 0.5%, 1%, 1.5%, and 2%) on the microstructure (SEM, 2000×) of DMPI-PPI mixed gel. Each treatment was performed in triplicate. (a) DMPI-0%PPI; (b) DMPI-0.5%PPI; (c) DMPI-1.0%PPI; (d) DMPI-1.5%PPI; (e) DMPI-2.0%PPI[START_REF] Zhu | Relationship between Molecular Structure and Heat-Induced Gel Properties of Duck Myofibrillar Proteins Affected by the Addition of Pea Protein Isolate[END_REF].

Figure 1 -

 1 Figure 1-12 Synthesis of the thesis objectives and the sequence of the different phases

Figure 2 Figure 2 . 3 :

 223 Figure 2-1 Pea globulin extraction procedure

Figure 2 -

 2 Figure 2-6: Strain intersection (yield point) of PPI-EW mixtures at a weight ratio of 25/75 at pH 7.5.

Figure 2 - 7 Figure 2 - 8

 2728 Figure 2-7 Typical profile for a texture profile analysis for a gel (Adapted from Wang[START_REF] Wang | Understanding the physical properties of food polysaccharides[END_REF]

Figure 3 - 2

 32 Figure 3-2 Nitrogen solubility of PPI and EW (0.1 M NaCl) as a function of pH in distilled water.

Figure 3 - 3

 33 Figure 3-3 Thermogram of 10% pea protein isolate (m/m) heated at 0.5 ℃/min at pH 7.5 and 9.

Figure 3 - 4

 34 Figure 3-4 Samples of Deconvolution of pea protein isolate 10%.

Figure 3 - 5

 35 Figure 3-5 Thermogram of 10% egg white (m/m) heated at 0.5 ℃/min at pH 7.5 and 9.

Figure 3 - 6

 36 Figure 3-6 Several batches of extracted ovalbumin fractions analyzed by RP-HPLC. (A): markers of different molecular weights: ovotansferrin (75 kDa), lysozyme (14 kDa), ovalbumin (44 kDa); (B): one sample of several batches of extracted ovalbumin.

Figure 3 -

 3 Figure 3-7 SEC-HPLC profile of pea protein isolate studied with UV detection at 280 nm as a function of time.

Figure 4 - 2 :

 42 Figure 4-2: Thermograms (top panels) and binding isotherms (bottom panels) for the titration of PPI (0.0078 mM) with LYS (0.92 mM) in HEPES buffer pH 7.5 (A), with LYS (1.02 mM) in Tris-HCl buffer pH 9 (B), with LYS (0.89 mM) in Tris-HCl buffer pH 7.5 (C). All the titration experiments were performed at 25°C.

Figure 4 - 3 :

 43 Figure 4-3: Particle size distribution measured by DLS of PPI (2.4 g/L) and LYS (14.3 g/L) suspensions in TRIS buffer at pH 7.5 and 9.

Figure 4

 4 Figure 4-4: Particle size distribution by laser granulometry (A) and pictures (B) of PPI-LYS suspensions at different LYS/PPI molar ratios in TRIS buffer pH 7.5

Figure 4 - 6 :

 46 Figure 4-6: The ζ-potential of PPI-LYS mixtures at different LYS/PPI molar ratios in TRIS buffer at pH 7.5 (A) and pH 9 (B), and PPI and LYS solutions alone as a function of pH (C).

Figure 4 - 7 :

 47 Figure 4-7: Microscopic observations by CLSM of mixed PPI-LYS suspensions at 20 °C in TRIS buffer at pH 7.5: PPI (2.4 g/L) (A), LYS (B), and LYS/PPI molar ratio of 0.8 (C), 1.6 (D), 3.2(E), 4.8 (F), 11.2 (G), 20 (H).

Figure 4 - 8 :

 48 Figure 4-8: Phase contrast micrographs obtained by optical microscopy of mixed PPI-LYS systems taken at room temperature in TRIS buffer at pH 7.5 as a function of the ratio between PPI and LYS: LYS to PPI at a ratio of 0.8 (A), 1.6 (B), 3.2(C), and 4.8 (D) respectively

Figure 5 - 1 :

 51 Figure 5-1: Nitrogen solubility of PPI, EW, and PPI-EW mixture at the weight ratio of 50/50 in 0.1 M NaCl.

Figure 5 - 3 :

 53 Figure 5-3: Typical DSC thermograms for PPI, EW, and PPI-EW at different weight ratios at pH 7.5 (A) and 9 (B), respectively.

Figure 6

 6 Figure 6-1: CLSM images visualizing the microstructure of PPI-EW protein mixtures at 63x magnification. A: EW gel at pH 7.5, a: EW gel at pH 9; B: PPI-EW 25/75 at pH 7.5, b: PPI-EW 25/75 at pH 9; C: PPI-EW 50/50 at pH 7.5, c: PPI-EW 50/50 at pH 9; D: PPI-EW 75/25 at pH 7.5, d: PPI-EW 75/25 at pH 9; E: PPI-gel at pH 7.5, e: PPI-gel at pH 9.

Figure 6 - 2 :

 62 Figure 6-2: Temperature sweep G' of egg white protein (red), PPI (blue) and PPI-EW mixtures at different weight ratios (75/25 in orange, 50/50 in yellow, 25/75 in green) at different pH 7.5 (a) and 9 (b).

Figure 6 - 3 :

 63 Figure 6-3: Storage modulus (G') (solid one) and loss modulus (G'') (hollow one) from frequency sweep test of the egg white protein (red), PPI (blue), and PPI-EW mixtures at different weight ratios (75/25 in orange, 50/50 in yellow, 25/75 in green) at different

Figure 6 - 4 :

 64 Figure 6-4: Strain sweep curves of G' and G'' on PPI-EW at a weight ratio of 50/50 at pH 7.5.

Figure 6 - 5 :

 65 Figure 6-5: Effect of different dissociating agents on total protein solubilization from PPI-100% gel (A) or EW-100% gel (B) at pH 7.5. Control: 100 mM Tris-HCl.

Figure 6 - 6 :

 66 Figure 6-6: Effect of different dissociating agents on total protein solubilization from PPI-100% gel (A) or EW-100% gel (B) at pH 9. Control: 100 mM Tris-HCl.

Figure 6 - 7 :

 67 Figure 6-7: Effect of different dissociating agents on total protein solubilization from PPI-EW gel at pH 7.5. Control: 100 mM Tris-HCl.

Figure 6

 6 Figure 6-8: Effect of different dissociating agents on total protein solubilization from PPI-EW gel at pH 9. Control: 100 mM Tris-HCl.

Figure 6

 6 Figure 6-9: Gel appearance of EW, and PPI-EW gels at different weight ratios. A: samples prepared at pH 7.5, B: samples prepared at pH 9.

Figure Annex- 1

 1 Figure Annex-1 The rest deconvolution results of pea protein isolate 10% (A-B).

Figure Annex- 2

 2 Figure Annex-2 Different batches of extracted ovalbumin analyzed by RP-HPLC (A-G).

Figure Annex- 3 - 1

 31 Figure Annex-3-1 Temperature sweep G'' of egg white protein (red), PPI (blue), and PPI-EW mixtures at different weight ratios (75/25 in orange, 50/50 in yellow, 25/75 in green) at different pH 7.5.

Figure Annex- 3 - 2

 32 Figure Annex-3-2 Temperature sweep G'' of egg white protein (red), PPI (blue), and PPI-EW mixtures at different weight ratios (75/25 in orange, 50/50 in yellow, 25/75 in green) at different pH 9.

Figure

  Figure Annex-4-1 Storage modulus (G') (solid one) and loss modulus (G'') (hollow one) from strain sweep test of the egg white protein (red), PPI (blue), and PPI-EW mixtures at different weight ratios (75/25 in orange, 50/50 in yellow, 25/75 in green) at pH 7.5.

  1: Nitrogen solubility (NS) values of the PPI-EW mixture at the ratio of 50/50 at different pH. Recalculated NS values were obtained from experimental NS values of individual PPI and EW suspensions.

Figure 1 - 2 :

 12 Figure 1-2: Assembly of 7S vicilin fragments, and potential cleavage sites according to Tzitzikas et al., 2006.

Figure 1 - 3

 13 Figure 1-3 Assembly of constituent 2S Albumin according to Tzitzikas et al., 2006.

  Figure 1-4 summarizes the main steps in the extraction of pea globulins. The seeds are first treated, and then the proteins are extracted by either a dry or wet process from concentrations acquired by the dry method. According to the previous paper by Stone et al. (2015b), based on the extraction methods, pea proteins are available in a variety of forms, including flours (20-50 % of protein), concentrates (50-65 % of protein), and isolates (70-92 % of protein). In addition, non-protein ingredients such as water, starch, lipids, and mineral salts can be reduced or removed by extraction step to obtain what are known as raw vegetable protein materials (VPP).

Figure 1 - 4 :

 14 Figure 1-4: Main steps in the extraction of pea protein materials, according to Boukid,[START_REF] Boukid | Pea protein ingredients: A mainstream ingredient to (re) formulate innovative foods and beverages[END_REF][START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF][START_REF] Saurel | Protéines de pois : structure, extraction et fonctionnalités[END_REF]

Figure 1

 1 Figure 1-6 3D-structure of five major egg white proteins: ovalbumin, ovotransferrin, lysozyme, ovomucin, and ovomucoid. 1: download from Protein Data Bank, 2: Jalili-Firoozinezhad et al., 2020

  these circumstances, hydrophobic interactions between nearby proteins can cause aggregation, and if the aggregates are large enough in size and number, precipitation is developed. Extraction and dehydration alter protein solubility by changing the hydrophobicity of the protein surface, exposing hydrophobic regions, and strengthening hydrophobic interactions between proteins[START_REF] Mccarthy | Emulsification properties of pea protein isolate using homogenization, microfluidization and ultrasonication[END_REF].Boye, Aksay et al.

  of the gel: PPIa > PPI-c > PPI-d > PPI-b > PPIc-no gel b) Rigidity of the gel: PPIc > PPI-a > PPI-d > PPI-b c) gelling temperature: PPIa > PPI-a > PPI-c > PPI-d > PPI-b > PPIc-no gel

Figure 1

 1 Figure 1-7 Schematic representation of heat-induced gelation of egg-white proteins.

  (2020) investigated the interactions inside WPI-PPI co-precipitate, by adding N-Ethylmaleimide (NEM) to break disulfide bond formation, 0.5 M NaCl to investigate electrostatic interactions and hydrophobic interactions and 0.2 % SDS to identify hydrophobic interactions. The results showed that the impact of NEM and NaCl was strong and additive when combined, but SDS had minimal to little effect, indicating a multi-reaction of disulfide bonds and electrostatic forces was responsible for the mechanism underlying protein-protein interactions between whey and pea protein isolates. The work of[START_REF] Kristensen | Mechanisms behind protein-protein interactions in a β-lg-legumin co-precipitate[END_REF] wanted to identify if the β-lactoglobulin (β-lg) and pea legumin played a substantial part in these interactions. The interactions of β-lg and legumin in both co-precipitates and blends were shown to be a synergy of electrostatic interactions and disulfide bonds. Thus, β-Lg and legumin are the major proteins responsible for previously discovered interactions in whey and pea protein isolates.[START_REF] Chihi | Heat-induced soluble protein aggregates from mixed pea globulins and β-lactoglobulin[END_REF] investigated the formation of heat-induced mixed aggregates (heat treatment at 85 °C for 60 minutes) of serum proteins (β-lactoglobulin obtained by purification of a commercial isolate of whey proteins) and purified pea proteins (pea globulins (Glob) obtained by isoelectric precipitation) mixed at various ratios (30/70, 50/50, and 70/30). The experiment, which was conducted at pH 7.2 in the presence of 5 mM NaCl, indicated the creation of mixed thermal aggregates including both the formation of new covalent connections (disulfide bonds) and non-covalent contacts between the subunits denatured β-lactoglobulin and Glob. The soluble aggregates in the mixes had a larger molecular weight and a smaller diameter than the Globs' thermal aggregates alone. Furthermore, increasing the quantity of -lactoglobulin in the mixture resulted in a decrease in the size of the soluble aggregates formed following heat treatment. Finally, it was postulated that thermal aggregation of these proteins in a mixture is driven first by -lactoglobulin denaturation, followed by the creation of complexes stabilized by disulfide bonds between non-refolded β-lactoglobulin and legume subunits (11S) produced after heat treatment. These complexes can then create non-covalent connections with other pea globulins (legumin and vicilin) to form soluble aggregates with sizes ranging from 90 to 110 nm.Mession et al. (2017a) investigated the aggregation of proteins after heat treatment of a mixed system constituted of casein micelles and pea globulins separated into vicilin and legumin. To be more specific, casein/vicilin micelles or casein/legumin micelles were generated at pH 7.1 in the absence of salts, with total concentrations of 1.8 and 3.6 % (w/w) and a 50/50 ratio for each dispersion before being subjected to an 85 °C heat treatment for 60 minutes. The denaturation temperature of the combined legume and vicilin rose by roughly 4 °C when compared to the denaturation temperature of these fractions alone, according to the authors. This improvement in thermal stability was linked to the influence of the casein micelle's steric hindrance, which inhibits the legumin's molecular mobility and flexibility, hence the unfolding of this protein. The solubility of these protein fractions in the mixes was significantly enhanced after heating. Furthermore, it was shown that the acidic and basic legumin subunits formed soluble and insoluble aggregates with the casein micelles in the mixes due to disulfide bonds. Denatured vicilin, on the other hand, generated soluble mixed aggregates mostly through non-covalent interactions. Finally, caseins were not engaged in pea protein aggregation, even though heat-induced pea protein interactions were changed in the presence of micelles.

  ).[START_REF] Tomé | Protein gels and emulsions from mixtures of Cape hake and pea proteins[END_REF] investigated heat-induced gel prepared by the mixture of PPI and Cape hake protein powder (CHPP, prepared by alkaline extraction) at a total protein content of 20 % with various weight ratios (PPI/CHPP: 100/0, 20/80, 50/50, 80/20, 0/100%, w/w). The results showed that heat-induced gels were also produced for all the samples after 30 minutes of treatment at 90°C. A drop in G′ and G′′ values as PPI concentration increased in the mixes, suggested that pea proteins inhibited the gelation of fish proteins. During heating, the gel network was quickly generated by the CHPP and then reinforced by pea protein. Thus, increasing the CHPP fraction resulted in stronger and more structured gels. Mixture gels prepared by myofibrillar proteins (MPI) from different species (chicken, red sea bream, duck) and PPI were studied by Sun & Arntfield (2012b), Lin et al. (2019), and Zhu et al. (2022), respectively. Sun & Arntfield (2012b) studied the heat-induced gel prepared by Chicken-MPI (C-MPI) and PPI, with a total of 4% protein content and different proportion and NaCl concentration. The results showed that the addition of PPI to C-MPI reduced gel strength; with 0.6 M NaCl, the PPI denaturation temperature was higher than the gel formation temperature, restricting interactionsbetween the two proteins. The authors also studied the influence of transglutaminase (MTG) added to the mixture. It was found that the gel strength of a C-MPI/PPI at a ratio of 3/1 was considerably enhanced in this investigation with the addition of suitable doses of MTG, indicating that partial crosslinking between PPI and C-MPI via glutamyl-lysine bonds with the inclusion of MTG. As a result, MTG improves the gelation characteristics of heat-induced MPI/PPI gels, which was also confirmed by soy-MPI with MTG[START_REF] Jiang | Extreme pH treatments enhance the structurereinforcement role of soy protein isolate and its emulsions in pork myofibrillar protein gels in the presence of microbial transglutaminase[END_REF][START_REF] Han | Microbial transglutaminase catalyzed the cross-linking of myofibrillar/soy protein isolate mixtures[END_REF].[START_REF] Zhu | Relationship between Molecular Structure and Heat-Induced Gel Properties of Duck Myofibrillar Proteins Affected by the Addition of Pea Protein Isolate[END_REF] investigated the heat-induced gel obtained by PPI and duck-MPI (D-MPI) with a total protein content of 40 mg/mL and various amounts of PPI. The results showed that with increased PPI addition, D-MPI's gel-forming capabilities, including water retention and textural qualities, increased. Pea proteins help in the conversion of free water to immobilized water. When heated, the α-helix content dropped while the β-sheet content increased, providing the driving force for the gel formation, and generating a more compact and homogenous gel structure (Figure1-7), which may also have a conductive impact on water entrapment in the gel network. Therefore, Pea proteins may enhance the characteristics and microstructure of duck myofibrillar protein gels, thereby accelerating the transition from a weak to a non-aggregated, rigid form.

Figure 1 - 7

 17 Figure 1-7 Effect of pea protein addition level (0%, 0.5%, 1%, 1.5%, and 2%) on the microstructure (SEM, 2000×) of DMPI-PPI mixed gel. Each treatment was performed

  [START_REF] Silva | Heatinduced gelation of aqueous micellar casein suspensions as affected by globular protein addition[END_REF][START_REF] Silva | Heat-induced gelation of micellar casein/plant protein oil-in-water emulsions[END_REF] focused on the heat-induced gelation of the mixtures prepared by micellar casein (MC) and plant proteins, in which the former (2018) studied the gel prepared by MC and globular protein (GP: soy or pea or whey protein), while the latter (2019 a & b) analyzed the gel prepared by MC and SPI or PPI in aqueous solution or emulsions, respectively.[START_REF] Silva | Heatinduced gelation of aqueous micellar casein suspensions as affected by globular protein addition[END_REF] analyzed the impact of the addition of globule protein (GP: SPI, PPI, or WPI) with parallel concentration (0-6% w/w) at pH 5.6-6 on the heat-induced gelation of MC dispersion at 6 wt%. The critical gelation temperature (Tg) of MC suspensions increased with the addition of globular protein regardless of pH.Surprisingly, this rise varied by GP type and was strongest in the sequence Soy Proteins (SP) > Pea Proteins (P) > Whey Proteins (WP) (Figure1-9 d). For example, at pH 5.8, adding 2 % w/w SP to 6 % w/w MC improved the Tg by 30 °C compared to 15 °C for PP and just 5 °C for WP. This impact was mostly explained by changes in calcium binding effectiveness across the various GP (SP > PP >> WP), as shown in Figure1-8, and/or mineral makeup of the components. In other words, native globular protein influenced MC gelation indirectly by binding calcium, limiting the quantity available for micelles to bind. The addition of CaCl2 counteracted this action, resulting in a decrease in Tg that was no longer dependent on the GP content and source in excess of CaCl2. Tg of the suspensions dropped with lowering pH due to a decrease in MC net charge density as well as Ca 2+ release, which favored heat-induced gelation of MC. The results suggested that globular proteins do not have the same capacity to bind calcium as other proteins. Plant proteins bind more calcium than serum proteins, with soy proteins having a higher affinity than pea proteins. This differential in calcium affinity has a direct influence on the stiffness of acid gels and protein interactions.

Figure 1 - 8

 18 Figure 1-8 Increase in the critical gelling temperature (Tc) of casein micelles in the presence of globular proteins (whey, pea, or soy) explained by the difference in protein affinity for calcium ions (from Silva et al., 2018).

Figure 1 - 9

 19 Figure 1-9 Storage modulus at 0.1 Hz after 1 h heating at 90 °C for (a) individual and mixed MC-SP and (b) individual and mixed MC-PP systems at 6% w/w total protein as a function of MC and plant protein content in the mixtures at pH 5.8 (Silva et al., 2019b).

  [START_REF] Zhang | Heat stability and rheological properties of concentrated soy protein/egg white protein composite microparticle dispersions[END_REF] prepared the composite protein microparticles by mixing soy protein isolate (SPI) and egg white (EW) at a total protein of 15 % w/w, in which, the proportion of SPI was 12 wt%, EW was 3 wt%, with different heating time (95 ℃,5, 10, 15 min). The results showed that EW was engaged in the microparticulate of SPI leading to more intermolecular disulfide bonds. When the duration of heating during preparation was increased, the number of disulfide bonds in SPI/EWP composite particles increased by about 50 % compared to SPI-only. The flexibility of protein conformation decreased significantly as more covalent interactions engaged in creating the network inside the particles. Following heat treatment, the dispersions comprising SPI/EWP composite microparticles with a protein content of 12 wt% still showed poor viscosity and weak gelation ability. Meanwhile, their lubricating characteristics have been enhanced.Su et al. (2015) demonstrated the gel properties of a mixed system of SPI and EW at pH 7 and with 0.05 g GDL. The mixtures were prepared by mixing EW in SPI at a

Figure 1 -

 1 Figure 1-10 Scanning electron micrographs of the SPI/EW protein gels at 0.06 g mL -1 protein concentration. The pictures marked with a-g represent the gels at different SPI/EW ratios. a 1:0, b 9:1, c 3:1, d 1:1, e 1:3, f 1:9, g 0:1. (Su et al., 2015)

Figure 1 -

 1 Figure 1-12 Synthesis of the thesis objectives and the sequence of the different phases of the project.

Figure 2 - 1

 21 Figure 2-1 Pea globulin extraction procedure

Figure 2 - 2

 22 Figure 2-2 Ovalbumin extraction procedure

  : the moisture content (%) Mi: mass of the initial sample (g) Msec: mass of the dry sample (g) after passing through the oven at 105 °C. matrass was installed on BÜCHI's K355 distillation machine (Rungis, France). 32% NaOH was added until the color changed to blue or light brown, indicating the transition from ammonium ion to ammonia. The ammonia condensates were distilled by trapping them in 50 mL of 2 % (m/v) boric acid. The assay was then carried out with a 0.1 M HCl solution until the initial pH of the 2 % (m/v) boric acid solution was obtained. The percentage of nitrogen was determined by the following equation (3): Where: V: volume of HCl used for sample titration (mL), N: normal hydrochloric acid, m: the mass of the sample, % MS: dry matter percentage of the sample,

  prepared and then 10 µg of protein was deposited. Molecular weight protein markers from Sigma-Aldrich® (SigmaMarker™ S8445, wide range, Mw 6.5 to 200 kDa) or Thermo Scientific™ (PageRuler™ Unstained Broad Range Protein Ladder, Mw 5 to 250 kDa) were used. The migration was carried out at 35 mA per gel, with the following migration buffer: 0.3 % (w/v) trizma base, 1.45 % (w/v) glycine, and 0.1 % (w/v) SDS, in a Scientific® Mini Gel Tank of Migration (Thermo Fischer). The gels were then rinsed with distilled water, and the fixation was performed in four successive distilled water baths heated for 1 min in a microwave at 550 W. The staining of the gels was performed with Coomassie blue, Thermo Scientific™ PageBlue™ Protein Staining Solution, overnight. The discoloring was then achieved in several baths of distilled water, until the desired color. The gels were imaged by the ChemiDoc™ XRS+ System from Bio-Rad Lab. To know the difference between the polypeptide of the PPI-EW mixture with or without centrifugation, a centrifugation step (10000 g, 20 min, 4 ℃)

Figure 2 - 3 :

 23 Figure 2-3: Schematic diagram of the electric double layer on the surface of liquid nanoparticles[START_REF] Selvamani | Stability studies on nanomaterials used in drugs[END_REF]

Figure 2

 2 Figure 2-4: Introduction of ITC (Srivastava & Yadav 2019). A: equipment of typical ITC, B: presentation of the progress and result of ITC

  -3). In this case, we observed two inflection points at ~58 °C and ~75 °C respectively where the G' values rose sharply and diverge significantly from the loss modulus values (G'>>G'', data not shown). The points on both sides of one inflection point were fitted by linear models and the intersection of the respective straight lines was considered as the gelling temperature or gelling point as represented in Figure2-5. LVR was calculated from Figure2-6 by using an example of PPI-EW mixtures at a weight ratio of 25/75. The intersection of the two lines on both sides of the inflection point is the maximum strain without causing permanent deformation or called the yield point. Samples were run at least in triplicate.

Figure 2 - 5 :

 25 Figure 2-5: Temperature sweep for EW at pH 9 showing the determination of gelling point.

Figure 2 - 6 :

 26 Figure 2-6: Strain intersection (yield point) of PPI-EW mixtures at weight ratio of 25/75 at pH 7.5.

  ) and[START_REF] Chen | Polyphenols weaken pea protein gel by formation of large aggregates with diminished noncovalent interactions[END_REF] with some modification. Four different extracting reagents were used to analyze protein-protein interactions contributing to gelation. In brief, a 100 mM Tris buffer solution (Tris) (pH 7.5 and pH 9) was used as control (i). Tris containing 8 M Urea (ii) or 2 M Guanidine hydrochloride (GuHCl) (iii) or 20% Propylene glycol (PG) (iv) was used to extract proteins by affecting noncovalent interactions. Urea is more efficient in breaking hydrogen bonds, while its substituted form is mainly targeted for hydrophobic interactions. Tris containing 100 mM dithiothreitol (DTT) was used to extract proteins by reducing disulfide bonds (v). Tris containing 6 M urea, 100 mM DTT, 2 M Guanidine hydrochloride (GuHCl) and 20% Propylene glcol (PG) was used to extract proteins by dissociating all disulfide and noncovalent bonds as a second control (vi).The different gel samples (~2.5 g) were added into individual extractant (~40 mL), stirred at 25 °C (1 h), homogenized at 10000 rpm for 1 min with a T 25 digital ULTRA-TURRAX® (IKA, USA), then centrifugated (16000 rpm, 30 min, 4 ℃). The supernatants were collected, filtered (0.45 μm filter), weighed, and diluted around 6 times with the same extractant. The protein content in the dilutes was measured with a commercial Coomassie protein assay kit (660 nm) using BSA as the standard. The solubilized protein was then calculated by the ratio of the protein content in the supernatant to that of total proteins in gels and expressed as percentages, as shown in the following equations. At least three extractions were conducted and analyzed for each sample, and the average results were shown.

Figure 2 -

 2 7 gave a typical TPA curve. Hardness is determined by the maximum force exerted during the first compression cycle. The springiness of a compressed sample is the amount to which it recovers to its original size when the force of the initial compression cycle is eliminated. It is determined by dividing the original sample height by the distance compressed during the second compression to the peak force. The ratio of the effort required to compress the sample on the second bite (positive force area A2) to the work necessary to compress the sample on the first compression is defined as cohesiveness (positive force area A1).

Figure 2 - 7 :

 27 Figure 2-7: Typical profile for a texture profile analysis for a gel (Adapted from Wang & Cui, 2005).

  confocal microscope's principal purposes are to generate a point source of light and reject out-of-focus light, allowing it to scan deep into tissues with high resolution, and optical sectioning enabling 3D reconstructions of imaging materials. The illumination and detection optics are focused on the same diffraction-limited spot, which is moved across the sample to form the entire picture on the detector, according to the basic idea of confocal microscopy. While the whole field of vision is illuminated during confocal imaging, anything outside the focus plane adds nothing to the picture, reducing haze and enabling optical sectioning. Figure 2-8 depicts a schematic of the core optics of a contemporary confocal microscope.

Figure 2 -

 2 Figure 2-8: Principle of confocal microscopy (Adapt from Elliott, 2020). A: component of confocal microscopy; B: the scanning mirrors used by confocal microscopes to sweep the excitation light across the sample are seen schematically.

Figure 3 - 2

 32 Figure 3-2 Nitrogen solubility of PPI and EW (0.1 M NaCl) as a function of pH in distilled water.

Figure 3 - 3

 33 Figure 3-3 Thermogram of 10% pea protein isolate (m/m) heated at 0.5 ℃/min at pH 7.5 and 9.

Figure 3 - 5

 35 Figure 3-5 Thermogram of 10% egg white (m/m) heated at 0.5 ℃/min at pH 7.5 and 9.

Figure 3 -

 3 Figure 3-6 several batches of extracted ovalbumin fractions analyzed by RP-HPLC. (A): markers of different molecular weights: ovotansferrin (75 kDa), lysozyme (14 kDa), ovalbumin (44 kDa); (B): one sample of several batches of extracted ovalbumin.

Figure 3 -

 3 Figure 3-6 B showed several batches of purification, the purity of extracted ovalbumin, that different batches of extracted ovalbumin of 2-4, 2-5, and 2-6 were not pure compared to the rest of extracted ovalbumin. The rest batches of extracted ovalbumin analyzed by RP-HPLC were in Figure Annex 2. After all the extracted

Figure 3 -

 3 Figure 3-7 SEC-HPLC profile of pea protein isolate studied with UV detection at 280 nm as a function of time.

Figure 3 -

 3 Figure 3-7 shows the SEC-HPLC profile of extracted pea protein isolate at pH 7.5

  titration calorimetry (ITC) and ζ-potential measurements. The detected attractive interactions between LYS and pea globulins were further explored at different pH via the characterization of formed structures by dynamic light scattering (DLS), laser granulometry, and confocal laser scanning microscopy (CLSM).

Figure 4 Figure 4 - 2 :

 442 Figure 4-1: Thermograms for the titration of PPI (0.0078 mM) with OVA (1.6 mM) in HEPES buffer pH 7.5 (A1), with OVA (1.7 mM) in Tris-HCl buffer pH 9 (A2), with OVT (0.64 mM) in HEPES buffer pH 7.5 (B1), with OVT (0.68 mM) in Tris-HCl buffer pH 9 (B2), with LYS (0.92 mM) in HEPES buffer pH 7.5 (C1), with LYS (1.02 mM) in Tris-HCl buffer pH 9 (C2), with LYS (0.89 mM) in Tris buffer pH 7.5 (C3). All the titration experiments were performed at 25 °C.

Figure 4 - 3 :

 43 Figure 4-3: Particle size distribution measured by DLS of PPI (2.4 g/L) and LYS (14.3 g/L) suspensions in TRIS buffer at pH 7.5 and 9.

Figure 4 -

 4 Figure 4-3 showed that the size distribution of pea globulins evidenced a bimodal distribution at pH 7.5 and 9. Particles around 19 nm and 11 nm at pH 7.5 and 9, respectively, may correspond to 7S and 11S oligomers, whereas those around 180 nm

Figure 4 - 4 :

 44 Figure 4-4: Particle size distribution by laser granulometry (A) and pictures (B) of PPI-LYS suspensions at different LYS/PPI molar ratio in TRIS buffer pH 7.5

  pH 7.5 and 9 (Figure4-6 A-B). PPI and LYS solutions alone as a function of pH were also presented in Figure4-6 C, which indicated the Ip of PPI and LYS were around 4.9 and 10.7, respectively. These values are in good agreement with the previously reported Ip values of these proteins[START_REF] Klassen | Effect of pH on the formation of electrostatic complexes within admixtures of partially purified pea proteins (legumin and vicilin) and gum Arabic polysaccharides[END_REF] Helmick, Hartanto, Bhunia, Liceaga, & Kokini, 2021;[START_REF] Rezwan | Change of ζ potential of biocompatible colloidal oxide particles upon adsorption of bovine serum albumin and lysozyme[END_REF][START_REF] Yadav | Structure and interaction in the pH-dependent phase behavior of nanoparticle-protein systems[END_REF]. Therefore, LYS showed a positive charge at pH 7.5 and 9, whereas PPI showed a negative charge respectively.

Figure 4 - 6 :

 46 Figure 4-6: The ζ-potential of PPI-LYS mixtures at different LYS/PPI molar ratios in TRIS buffer at pH 7.5 (A) and pH 9 (B), and PPI and LYS solutions alone as a function of pH (C).

Figure 4

 4 Figure 4-7: Microscopic observations by CLSM of mixed PPI-LYS suspensions at 20 °C in TRIS buffer at pH 7.5: PPI (2.4 g/L) (A), LYS (B), and LYS/PPI molar ratio

Figure 4 - 8 :

 48 Figure 4-8: Phase contrast micrographs obtained by optical microscopy of mixed PPI-LYS systems taken at room temperature in TRIS buffer at pH 7.5 as a function of the ratio between PPI and LYS: LYS to PPI at a ratio of 0.8 (A), 1.6 (B), 3.2(C), and 4.8 (D) respectively

Figure 4 -

 4 Figure 4-9 mechanism of the aggregate process between lysozyme and pea protein isolate.

  on ionic strength and pH. The solubility of PPI, EW, and the PPI-EW mixture at the weight ratio of 50/50 are shown in Figure 5-1. Solubility regarding PPI and EW only was discussed in Chapter 3.1.4. In general, PPI showed a U-shaped nitrogen solubility profile, with a minimum around pH 5, and increased and reached values of around 85 % and 89 % for pH values below 3 and above 7. The nitrogen solubility of egg white was always over 88 %, with a little lower solubility of EW at around pH 4, close to the isoelectric point of ovalbumin (pH 4.5).

Figure 5

 5 Figure 5-1: Nitrogen solubility of PPI, EW, and PPI-EW mixture at the weight ratio of 50/50 in 0.1 M NaCl.

Figure 5 - 2

 52 Figure5-2 shows the electrophoresis profile of the 50/50 weight ratio PPI-EW mixture before and after centrifugation under reducing and non-reducing conditions at pH 7.5 and 9. The electrophoretic profile of the PPI and EW prepared at pH 7.5 and 9 was already discussed in chapter 3.1.3, and showed great similarities, respectively. In general, three main polypeptide components were shown in egg white samples ovotransferrin (76 kDa), ovalbumin (44 kDa), and lysozyme (14.6 kDa). PPI contained a molecular weight of lipoxygenase around 88 kDa, convicilin around 71 kDa, and legumin 11S 60 kDa concluding acidic subunits Lα (~38-40 kDa) and basic subunits Lβ (~20-22 kDa) under reducing conditions, main vicilin fractions around i) 15 kDa, ii) 20-37 kDa responding to fragments α (≈20 kDa), α:β (~30-36 kDa), and β:γ (~25-30 kDa), iii) 19 kDa, iiii): vicilin monomer around 50 kDa.

Figure 5 - 2 :

 52 Figure 5-2: SDS-PAGE profile of PPI-EW suspensions prepared at pH 7.5 (Lanes 1-4) and 9 (Lanes 5-8), at the weight ratio 50/50 with and without centrifugation. The

Figure 3 -

 3  Section 3.1.3). In reducing conditions lane 2 (at pH 7.5) and 7 (at pH 9), L (α, β) was separated into Lα and Lβ because of the rupture of disulfide bonds. Major components of the pea protein isolate and egg white have been found under non-reducing conditions, such as vicilin (around 50 kDa, 20-37 kDa, 19 kDa), convicilin, Legumin (L (α, β)), ovalbumin, ovotransferrin and lysozyme (Figure5-2, lanes 1, 8, 6). When compared to the polypeptides under reducing conditions between Figure5-2 (lanes 2, 7) and Figure3-1 (lanes 2,4, and 6,8 for PPI and EW, respectively), no bands disappeared. However, with centrifugation and regarding supernatant, the bands of vicilin (around 19 kDa) decreased at pH 7.5 (Figure5-2, lane 3) but not at pH 9 (Figure5-2, lane 5). Moreover, the bands at pH 9 were more intense than those at pH 7.5, which could be explained by the solubility that was higher at pH 9.0. At pH 7.5, the bands attributed to lipoxygenase and lysozyme (Figure5-2, lanes 3 & 4) strongly decreased after centrifugation compared to those before centrifugation (Figure5-2, lanes 1 & 2). This would suggest the contribution of these two proteins in the aggregates lost after centrifugation. Interestingly, this did not occur at pH 9.0 (Figure5-2, lane 5-8) when lysozyme was less positively charged.

Figures 5- 3 A

 3 Figures 5-3 A and B show typical DSC thermograms for pea proteins, egg white proteins, and PPI-EW mixture at different mass ratios at pH 7.5 and 9, respectively.

  Recalculated ∆H: ∆𝐇 𝒓𝒆𝒄𝒂𝒍𝒄𝒖𝒍𝒂𝒕𝒆𝒅 = ∆𝐇 𝒆𝒘 × 𝒓𝒂𝒕𝒊𝒐 + ∆𝐇 𝒑𝒑𝒊 × 𝒓𝒂𝒕𝒊𝒐 .Table5-4 shows the specific enthalpy (∆H) values for suspensions of PPI and EW and their mixtures at different weight ratios at pH 7.5 and 9. Concerning the PPI-EW mixtures at different weight ratios, the ∆H value was recalculated by equation (4) to know if the measured values of ∆H resulted from the additive denaturation of PPI and EW proteins considering their relative content in the mixtures. Mixtures at pH 7.5

Figure 6 -

 6 Figure 6-1 shows the network of 10% (w/w) mixed proteins gel systems at the various PPI-EW weight ratios (0/100, 25/75, 50/50, 75/25, 100/0) and different pH.Proteins show gray and white on confocal micrographs due to Fast green labeling, whereas related pores containing aqueous phase look black. It is important to mention that both types of proteins (EW and PPI) were labeled and that one cannot distinguish between them in the images.

  McCann et al. (2018) and[START_REF] Roesch | Heat-induced soy-whey proteins interactions: Formation of soluble and insoluble protein complexes[END_REF] observed a discontinuous network in soy protein-whey protein gel at a total protein concentration of around 6 %, indicating phase separation, while Gómez-Mascaraque, & Pinho (2021) found microgel structure between soy and whey protein gel.

FigureFigure 6 - 2 :

 62 Figure

  were significantly promoted. The formation of a threedimensional network is indeed caused by various molecular forces, including hydrogen bonding, ionic attractions, disulfide bonding, hydrophobic interactions, or a combination of these forces(Sun & Arntfield, 2012a; Zhao, Sun, Li, Liu, & Kong, 2017).

Figure 6 - 3 :

 63 Figure 6-3: Storage modulus (G') (solid one) and loss modulus (G'') (hollow one) from frequency sweep test of the egg white protein (red), PPI (blue), and PPI-EW mixtures at different weight ratios (75/25 in orange, 50/50 in yellow, 25/75 in green) at different pH 7.5 (A) and 9 (B).

Figure 6

 6 Figure 6-4: Strain sweep curves of G' and G'' on PPI-EW at a weight ratio of 50/50 at pH 7.5.

Figure 6 - 5 :

 65 Figure 6-5: Effect of different dissociating agents on total protein solubilization from PPI-100% gel (A) or EW-100% gel (B) at pH 7.5. Control: 100 mM Tris-HCl.

Figure 6 - 6 :

 66 Figure 6-6: Effect of different dissociating agents on total protein solubilization from PPI-100% gel (A) or EW-100% gel (B) at pH 9. Control: 100 mM Tris-HCl.

Figure 6 - 7 :

 67 Figure 6-7: Effect of different dissociating agents on total protein solubilization from PPI-EW gel at pH 7.5. Control: 100 mM Tris-HCl.

Figure 6 - 8 :

 68 Figure 6-8: Effect of different dissociating agents on total protein solubilization from PPI-EW gel at pH 9. Control: 100 mM Tris-HCl.

Figure A- 1

 1 Figure A-1 The rest deconvolution results of pea protein isolate 10% (A-B).

  Figure Annex-3-1 Temperature sweep G'' of egg white protein (red), PPI (blue) and PPI-EW mixtures at different weight ratios (75/25 in orange, 50/50 in yellow, 25/75 in green) at different pH 7.5.

Figure Annex- 3 - 2

 32 Figure Annex-3-2 Temperature sweep G'' of egg white protein (red), PPI (blue) and PPI-EW mixtures at different weight ratios (75/25 in orange, 50/50 in yellow, 25/75 in green) at different pH 9.

Table 1 -

 1 1 Classification and chemical properties of pea protein.

Table 1 -

 1 2: Mean amino acid composition of the protein fractions of peas and soy 100 g of protein (according to 1 Derbshyre,[START_REF] Derbyshire | Legumin and vicilin, storage proteins of legume seeds[END_REF]

Table 1 -

 1 1 Classification and chemical properties of pea protein.

	Types	Content Protein	Svedberg Unit	Molecular weight (MW) kDa	No. of subunit	MW of subunit kDa	Isoelectric point
			legumin	11S	360-410	6	60-65	4.8-4.9
	Globulin 55-65%	vicilin	7S	150-200	3	48-50	4.6-4.8
			convicilin	7S	210-280	3	70-71	4.6
			Albumin
	Albumin 18-25%	PA1 Albumin	2S 2S	11 52	2 2	5-6 25 or 26	9.8 e 5.3 5.2
			PA2
	Prolamin 4-5%	prolamin	/	/	/	/	/
	Glutelin 3-4%	glutelin	/	/	/	/	/
	Cited and summarized from Adebiyi and Aluko (2011); Barać et al. (2010); Beghdadi,
	(2021); Gressent et al. (2011); O'Kane et al. (2004a, 2004b); Reinkensmeier et al.
	(2015); Tzitzikas et al. (2006), etc.
	According to the classification methods in Table 1-1, pea proteins can be
	categorized into 4 types: globulins (salt soluble), albumins (water soluble), gliadins
	(alcohol soluble), and glutenins (insoluble).

  Table 1-1, globulins are soluble in a salt solution, which can be consumed during seed germination to supply nutrients to plant development. Globulin dissociates into subunits under high pH and ionic strength. In general, the sedimentation coefficient can be used to classify pea

globulins into vicilin / convicilin (7S) and legumin (11S)

[START_REF] Barać | Profile and functional properties of seed proteins from six pea (Pisum sativum) genotypes[END_REF]

. The content of these two primary divisions is influenced mostly by genetic and environmental factors. The legumin/vicilin (Lg/Vn) ratio rises throughout seed development, because of the differing rates of synthesis of the 11S and 7S protein fractions. Several studies have illustrated the variability of these fractions (7S and 11S) by studying different pea genotypes. Tzitzikas et al. (2006) investigated genetic diversity in pea seed globulin protein composition of 59 different distinct varieties, discovering that vicilin proteins were predominated with Lg/ Vn ratios ranging from 0.12 [variety: NGB 102149 Iran] to 0.77 [variety: P arvense (CGN 10193)]. According to Boye, Zare, & Pletch. (2010), Lg/Vn ratios ranged from 0.23 to 0.50 for wrinkled pea cultivars and from 0.31 to 1.67 for smooth pea cultivars. Previous works of literature conducted by O'Kane et al. (2004b) and

Table 1 -

 1 Riblett et al., 2001; 8 Helmick et al., 2021).

			Pea protein		Soy protein
	Amino acids	Legumin 11S 1,4,5	Vicilin 7S 1,4,6	Convicilin 8	Glycine 11S 6,7	Conglycinin 7S

2: Mean amino acid composition of the protein fractions of peas and soy 100 g of protein (according to 1 Derbshyre, Wright, & Boulter, 1976;

2

Emmert & Baker, 1995;

3 Gueguen, 1991; 4 Savage & Deo, 1989; 5 Jackson, Boulter, & Thurman, 1969; 6 O'Kane et al., 2004b; 7

  2) with hydrochloric or sulfuric acid solutions to precipitate globulins. Finally, the precipitate is collected, washed, resuspended in neutralized water at pH 7, and dried to obtain pea protein isolate (PPI).

	Another technique can be used to isolate proteins called ultrafiltration (UF) with

diafiltration (Figure

1-4

). Previous protein extraction and removal procedures for starch are identical to those used in IEP, then, expected protein isolates can be extracted through UF membranes with specific molecular weight cutoffs (Vogelsang-O'Dwyer,

Table 1 -

 1 3 Physio-chemical and structural properties of proteins from egg white 1

	Protein	Content % of egg white	pI	MW / kDa	Td / ℃	characteristics	structures
	Ovalbumin	54	4.5	45	84	Heat-stable phosphoglyco-protein, emulsifying and foaming agent, could change to S-ovalbumin	Serpin-like structure with a three-turn α-helical reactive center loop
	Ovotransferrin	12	6.1 77.9 61	binds metal ions	An α-helix interconnect with two (N-and C-) lobes
	Ovomucoid	11	4.1	28	70	Trypsin inhibitor, thermal stable	Three domains
				α:
				150-
	Ovomucin	3.5	4.5-5.0	220 β:	-	Structural protein maintaining viscosity and structure of egg white	Two (α-and β-) subunits linked by disulfide bonds
				5500-
				8300
	Lysozyme	3.4	10.7 14.4 75	Highly soluble and stable	Monomer, two (N-and C-) domains segregated by a helix-loop-helix motif
	Ovoglobulin G2	4	5.5	36 92.5	Good foam agent
	Ovoglobulin G3	4	5.8	45	-	Good foam agent
	Ovomacroglobulin	0.5	4.5	760-900	-	Strongly antigenic protein, inhibits proteinases serine and cysteine	Four subunits linked by disulfide bonds
	Ovoglycoprotein	1	3.9 24.4	-	An acidic glycoprotein	13.6% hexose, 13.8% hexosamine, and 3% sialic acid
	Flavoprotein	0.8	4	32-	-	binds riboflavin	Two domains: N-terminal with the

, ** Ovalbumin * (1,3,4,7)

1: Ovalbumin Tristram, 1949; 2:

						5,6)
	Alanine	6.4	5.4	2.3	5.6	5.6
	Valine	7.1	7.6	6.0	4.8	6.6
	Leucine	9.5	8.6	5.1	6.9	6.4
	Isoleucine	6.3	4.5	1.4	5.2	5.1
	Proline	3.6	4.7	2.7	1.4	4.3
	Phenylalanine	6.9	5.5	2.9	3.1	4.7
	Tryptophan	1.2	3.4	0.3	10.6	-
	Methionine	4.9	2.0	1.0	2.1	1.8
	Tyrosine	3.5	4.6	3.2	3.6	3.5
	Glycine	3.1	5.2	3.8	5.7	7.6
	Serine	7.8	6.1	4.2	6.7	9.1
	Threonine	4.2	5.1	5.5	5.5	7.7
	Cystine/2*	1.1	3.4	6.7	6.8	6.4
	Arginine (+)	5.7	7.3	3.7	12.7	3.2
	Histidine (+)	2.3	2.4	2.2	1.0	2.1
	Lysine (+)	6.3	9.1	6.0	5.7	6.3
	Aspartic acid (-)	9.3	12.1	13.0	18.2	10.1
	Glutamic acid (-)	15.4	11.7	6.5	4.3	9.7
	*: mean amino acid value calculated from literature measured by g of amino acid per
	100 g of protein,
	**: mean amino acid value calculated from literature measured by the number of
	residues / 100 amino acid residues, -: not determined

Table 1 -

 1 5: Summary of the main works on the solubility of pea protein isolate (from[START_REF] Lam | Pea protein isolates: Structure, extraction, and functionality[END_REF][START_REF] Ge | The health benefits, functional properties, modifications, and applications of pea (Pisum sativum L.) protein: Current status, challenges, and perspectives[END_REF].

				Conditions for
	Comparison	Materials used	Extracted conditions	solubility	Major results	References
				assessment
		CDC Golden peas,			PPC-UF (60%) < PPC-AE/IP
		CDC Grandora Green chickpeas, Common Blaze Red and Green lentils, Mylese desi chickpea and Xena	Ultrafiltration (UF) and alkaline extraction (AE) /isoelectric precipitation (IEP)	10 mg /ml in water, pH 1-10	(90%) at pH 1.0; PPC-UF lentil was similar to red but (56%) > PPC-AE/IP (29%); At pH 3.0 solubility of two chickpea was similar to each other, green	Boye, Aksay et al., 2010
		kabuli chickpea			different at pH 4
	Comparison between different pulses	Chickpea (CDC Frontier, Kabuli), faba bean (CDC SSNS), lentil (CDC Grandora), and pea (CDC Leroy) Defatted soy flour	IEP and salt extraction (SE)	1.0%, w/v 10 mm sodium phosphate buffer (pH 7.00)	IEP: Soy (97 %) > chickpeas process significantly affected PPI (91 %) > lentil (97 %) > field beans (90 %) > pea (61 %); SE: soy (97 %) > lentil (90 %) > field chickpea (29 %); Extraction beans (53 %) > pea (38 %) >	Karaca, Low, & Nickerson, 2011
					At pH 4.5, minimum solubility of
		PPIc, PPI, SPIc, Soy protein isolate (SPI)	AE/IEP	0.5 mg/ml, pH 3-10	PPIc, PPI, SPI and spic; >4.5 or <4.5; Solubility of PPI > PPIc, and SPI> spic; Solubity of SPI >	Shand et al. (2007)
					PPI at all pH
		Pea (Pisum sativum), chickpea (Cicer	AE/IEP	pH 2-8	At pH 4.5, minimum solubility of pea, chickpea and lentil; the	Ladjal-Ettoumi et

  ., Table 1-6: Main research work on the heat-induced gelation of pea proteins.

	Method of gelation Heat-induced gelation	Type of pea protein Three soluble pea extracts prepared in the laboratory A) Extract rich in globulins B) Purified legumin extract C) Purified vicilin extract 2 vicilin fractions: vicilin 1 and vicilin 2, from 2 pea cultivars (Silara and supra) Purified legumin fraction from peas by non-denaturation fractionation progress; glycinin purified from soybean by acid precipitation and Pea protein isolate (PPI) was prepared by alkaline and isoelectric precipitation from 5 pea cultivars (Solara, Supra, Classic, Finale, and Espac) Pea protein isolate, PPI (Pisane HD, Cosucra, Belgium), lupin protein isolate, LPI (lupie, Fraunhofer Inst., Germany), soy protein isolate, SPI (S974, ADM, Netherlands) Native pea protein isolate (PPIn), and soy protein isolate (SPIn) extracted by alkaline (pH 8) and acidic precipitation (pH 4.5) Commercial pea protein isolate (PPIc), soy protein isolate (SPIc) Soy protein isolate (SPI) obtained by alkaline extraction/ isoelectric precipitation; commercial pea protein isolate (PPIc), soy protein isolate (SPIc) Commercial pea protein isolate (PPIc), soy protein isolate (SPIc); pea protein isolate (PPIs) prepared by salt extraction and dialysis Commercial pea protein isolate (PPIc), soy protein isolate (SPIc); pea protein isolate (PPIs) prepared by salt extraction and dialysis Pea protein isolate (PPIs) obtained by salt extraction and dialysis Commercial soy protein isolate (SPIc) pea protein isolates prepared by salt-extraction followed by dialysis	Physico-chemical parameters of the initial dispersion 25-30 μL of protein in 30 mm tris-HCl buffer, pH 7.2. Mixed globulin: 30 mm tris-HCl buffer, at different pH: 5.95-8.6. Mixed globulin: 15%, 30 mm tris-HCl buffer at pH 7.1, with different NaCl concentrations: 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 m A) Minimum gelling concentration (MGC): protein concentration (6-16 %, w/v), pH 7.6, in 75mm potassium phosphate buffer b) Various parameters of samples in 75 mm potassium phosphate buffer: protein concentration 10% w/v, at pH 6.1; protein concentration: 2, 4, 6, 8, 10 %, w/v, at pH 3.8; protein concentration 10% w/v, NaCl (0-1 m), at pH 7.6; mixed of legumin/vicilin: 1/0.22, 1/0.57, 1/1.2, final concentration is 11% w/v, at pH 7.6 A) Minimum gelling concentration (MGC): protein concentration (8-16 %, w/v), pH 7.6, in 75mm potassium phosphate buffer b) legumin (8.4%), glycinin (6.6%), at pH 7.6, in 75 mm potassium phosphate buffer 18% w/v protein concentration, in 75 mm potassium phosphate buffer at pH 7.6 20mm of n-ethylmaleimide (NEM), block thiols-blocking agent PPI (12.5, 16 %, w/w), SPI (10%, w/w), LPI (16% w/w) dispersed in demineralized water at pH 5.5 A) Comparison of PPIc, and PPIn gels: 19.6 % w/w in 1% NaCl deionized water, pH 6.5 B) Optimization of the gelation of PPIc: 19.6 % w/w, PH 7.1 in demineralized water with different NaCl concentrations from 0-2% A) Study the influence of MTG: 1% w/w of NaCl, 19.6 % w/w protein content, pH 6.5; various parameters: 0-0.7% w/w of MTG, incubation temperature (22-78 °C) and incubation time (10-80 min) B) Comparison with PPIc and PPIn gels: with 0.6 % w/w of MTG, deionized water, pH 6.5, 10% w/w protein content A) Minimum gelling concentration: 0.3M NaCl, 4-18% (w/v) PPIs; 0.3M NaCl, 8-20% W/V PPIc B) Gelling point: PPIs 5.5-18% w/v pH 5.65-5.7 0.3 M NaCl C) Comparison of PPIs, PPIc, SPIc, 14.5% w/v, 0.3M NaCl, pH 5.7 A) Influence of heating A) Minimum gel concentration (MGC): 2-5% (w/v) of PPIs, in 0.3 M NaCl buffer with 10 U MTG B) MTG treatment: 0.3 M NaCl pH 5.65, 10.5% w/v, 0-30 U/g C) comparison between PPIs, SPIc: 10.5%w/v, 0.3M NaCl, with or without MTG 10 U/g rate: 14.5% (w/v), 0.3 M NaCl B) Influence of cooling rate: 14.5% (w/v), 0.3 M NaCl C) Comparison of PPIc, PPIs, and SPIc: 10.5% (w/v), 0.3 M NaCl pea protein dispersion (10.5%, w/v) dissolved in solutions with two combined parameters: pH: 3-11 NaCl concentration: 0, 0.1, 0.	Parameters of the applied gelation process Heated at 87 °C at different holding times: 0-45 min. Heated at 87 °C 20 min A) Heated at 100 °C for 30 min, cooled to room temperature, and stored at 4°C overnight b) Heated at 100 °C for 20 min, cooled to room temperature, and stored at 4°C overnight A) Heated around 100 °C for 30 min, cooled to room temperature (1 h), and stored at 4 °C overnight b) Heated from 45 °C to 98 °C, holding at 98 °C for 30 min, cooling to 25 °C, and holding at 25 °C for 30 min, with different heating and cooling rate (0.5, 1 °C/min) Heated from 45 to 98 °C, kept at 98 °C (30 min), cooled to 25 °C, and kept at 25 °C (30 min) with different heating and cooling rate: standard: heating and cooling at 1 °C/min a) 0.5 °C/min heating, 1 °C/min cooling b) 1°C/min heating, 0.2 °C/min cooling Heated to 90 °C for 15 min A) Heating at 82 or 92 °C for 45 min, cooling and incubation at 4°C for 14 h B) Heating at 79, 82, 87, 92, or 95°C for 25 min, cooling and incubation at 4°C for 14 h A) Heated at 92°C for 45 min (storage at 4°C for 14 h) B) MTG heat-treatment at 50 °C (0, 30 min), 35 to 95 °C at a rate of 2 °C A) 95 °C in a water bath for 10 min B) Influence of heating and cooling rate: 25-95-25 °C at a controlled rate (0.5, 1, 2, 4 °C/min 25 °C at 2 °C/min cooling: 25-95-C) Heating and A), B) Influence of A) i: Heated at 40 °C for 30 min; ii: from 40-95 °C, and kept at 95 °C 10 min, storing at 4 °C (for MGC) b) Heated and cooled: from 40-95-25 °C, (0.25, 0.5, 1, 2, 4 °C/min) c) Heated and cooled: from 40-95-25 °C, 2 °C/min heating and cooling rate: 25-95-25 °C at a controlled rate (0.5, 1, 2, 4 °C/min C) Heating and cooling: 25-95-25 °C at 2 °C/min.	Major conclusion A) Vicilin formed gel while legumin didn't gel under the same conditions B)Gel hardness of globulin decreased then the concentration of NaCl was ≥ 0.05 m C) Increase in the strength of the gel with the heating time (maximum strength after 20 min of heating) whatever the protein concentration. D) Formation of a gel for mixed globulin at pH 6.4 (maximum strength at pH 7.1) A) Vicilin 1: MGC 10% w/v, turbid gels; vicilin 2: MGC 14% w/v, transparent gels b) Turbid gels were stronger than transparent ones c) Vicilin 1 (supra) gel was stronger than gel made of vicilin 1 (Silara) d) Concerning NaCl, a gel formed byvicilin2: at 0-0.1 m transparent, 0.2 m turbid, 0.5 m phase separation, and 1 m NaCl smooth brown gel; gels by vicilin 1: turbid and opaque at from 0 to 1 m NaCl e) No gelation after the addition of vicilin2 to the legume dispersion for the ratios 1/0.57 and 1/1.2; gelling not impacted by the addition of vicilin (1) to the legume dispersion A) MGC: 8.4% for pea legumin, 6.6% for soy glycinin b) For both protein gels, a lower heating rate (0.5 1 °C/min) did not affect gel formation compared to a higher rate at 1 °C/min, while a lower cooling rate led to the formation of a stronger gel c) After reheating/recooling, pea legumin gels became stronger due to the rearrangement of gel networks, while glycinin gel was not A) No impact of lower heating rate on gelation of all the cultivars b) With the highest legumin content, Solara formed a firmer gel at a lower cooling rate c) Supra and Classic only formed stronger gels with the presence of NEM at a lower cooling rate, indicating gel strength depended on the disulfide bond d) Strong and self-supporting gels cannot be formed in Finale and Espace due to rich vicilin level A) Gelling ability: SPI > PPI > LPI, although SPI in lower protein concentration b) At 16% protein concentration, both PPI and LPI showed weak gel, with no significant difference on G' and G'' c) In texture, SPI (1.23 N, 10% w/w) and PPI (1.34 N, 16% w/w) had firmer gels than LPI (0.43 N, 16 % w/w) a) Gels stiffness: PPIc > PPIn; gels elasticity: PPIn > PPIc at studied two temperatures b) Firmer PPI c gels for higher heating temperatures c) Under the same conditions, SPIc gels are stiffer and more elastic than PPI c gels a) Increase in stiffness and elasticity of PPIc gels with: -Increase in MTG concentration -The increase in the incubation temperature -Increased incubation time Elasticity and stiffness of gels: PPIc with 0.7% w/w showed a close value to SPIc without MTG b) gelling point for PPIn decreased in the presence of MTG 0.6 % w/w -No significant differences for PPIc gels with or without MTG the heating rate at a high point was independent of 1.0 °C/min); the gelling a) MGC: 5.5% PPIs, 14.5% PPIc b) Gelling point was independent of protein concentration; the gelling point was dependent on the heating rate at a lower heating rate (0.5-heating rate (2-4 °C/min c) Gel strength: PPIs > SPIc > PPIc a) At the same cooling rate (2 °C/min), the gelling a) MGC around 3% w/v, with 10 U MTG b) Increased MTG concentration means increased G* led to stronger gel without changing relative elasticity; at the same concentration of MTG, lower rate of heating and cooling produced stronger PPI gels c) Rigidity of gels: with MTG: PPIs > SPIc, without MTG: SPIc > PPIs temperature increased with increasing heating rate, but no statistical changes on G' b) At the same heating rate (2 °C/min), G' decreased with increasing heating rate, but no changes in gelling temperature c) Gel strength: SPIc > PPIs > PPIc	References Bora, Brekke, & Powers, 1994 O'Kane et al., (2004a) O'Kane et al., (2004b) O'Kane et al., (2005) Batista et al., 2005 Shand et al., 2007 Shand et al., 2008 Sun & Arntfield, 2010 Sun & Arntfield, 2011b Sun & Arntfield, 2011a

Table 1 -

 1 6 summarizes the published work concerning the heat-induced gelation of purified pea proteins or pea protein isolates.

	Taherian et
	al., 2011

Table 1 -

 1 6 shows that several extraction and fractionation processes have been thoroughly explored and compared, with salt extraction (SE), alkaline extraction, and isoelectric precipitation (AE/IEP) being the most used. Furthermore, Kornet et al.

  𝟏𝟎𝟎 -(𝑴 𝑾 + 𝑴 𝑨 + 𝑴 𝑳 + 𝑴 𝑷 )………………. Equation (5)

	Where: MC: carbohydrates content,
	MW: moisture content,
	MA: ash content,

ML: lipid content, MP: protein content

Table 2 -

 2 1 Procedures of injection and elution of samples.

	Time	Buffer A	Buffer B	Rate
	0 min.	95 %	5 %	0,8 ml/min.
	17 min.	30 %	70 %	0,8 ml/min.
	19 min.	30 %	70 %	0,8 ml/min.
	20 min.	95 %	5 %	0,8 ml/min.
	22 min.	95 %	5 %	0,8 ml/min.
	24 min.	30 %	70 %	0,8 ml/min.
	25 min.	95 %	5 %	0,8 ml/min.
	27 min.	30 %	70 %	0,8 ml/min.
	28 min.	95 %	5 %	0,8 ml/min.
	32 min.

Table 3

 3

		-1 Physicochemical composition of flour and the globulin fraction
		Protein
		Moisture			Carbohydrate
	Samples	content	Lipid / %*	Ash / %*
		/ %			/ %**
		/ %*
	Pea flour	24.3±0.12 13.32±0.15 4.55±0.27	2.85±0.15	54.98
	Pea
		89.74±0.02 3.61±0.01	0.95±0.04	1.89±0.07	3.81
	globulin

* %: calculated on a dry matter basis, **: Calculated by the difference of 100 %.

Table 3 -

 3

	and 9.
	samples	Td vicilin (°C)	Td legumin (°C)	∆H(J/g)
	pH 7.5	75.8±0.4*	87.4±0.5*	10.8±0.1*
	pH 9	71.3±0.4**	84.5±0.2**	3.6±0.2**
	Means followed by different numbers of * for the same column are significantly
	different.
	According to Emkani, Oiete, & Saurel (2021), PPI thermal curves showed the two
	characteristic denaturation endothermic peaks for 7S and 11S globulins. Regarding pea
	protein isolate (Figure 3-3, Table 3-1), the first peak at around 75.8 °C and 71 °C
	corresponded to the denaturation of the lower molecular weight fraction (7S vicilin) at

1 Thermal denaturation temperature (Td) and enthalpy (ΔH) of PPI at pH 7.5

Table 4 -

 4 1: The D (4,3) results of PPI-LYS mixtures in TRIS buffer at pH 7.5 and 9.

	pH 7.5	pH 9
	Samples LYS/PPI	D [4, 3] -Volume	Samples LYS/PPI	D [4, 3] -Volume
	molar ratio	weighted mean (μm)	molar ratio	weighted mean (μm)
	3.2	5.2±0.6a	5.2	4.9±0.2a
	4.8	6.2±0.005a	8.7	5.5±0.5a
	6.4	12.8±0.04b	12.2	11.8±0.1b
	8.0	21.7±0.5de	14.0	21.9±0.2c
	9.6	22.7±0.1df	15.7	27.2±0.4e
	11.2	25.3±0.1g	17.5	28.1±0.2e
	12.8	23.8±0.2f	19.2	27.9±0.2e
	14.4	23.1±0.2df	20.9	27.0±0.5e
	20.0	20.5±0.1d	23.6	23.9±0.2d
	23.2	17.2±0.1c	25.3	22.0±0.2c

Table 5

 5

	-1: Nitrogen solubility (NS) values of the PPI-EW mixture at the ratio of 50/50
	at different pH. Recalculated NS values were obtained from experimental NS values of
	individual PPI and EW suspensions.
	pH	NS (%)	Recalculated NS (%)
	2	84.8±1.2a	87.6±0.5a
	3	87.5±1.4a	91.5±1.3a
	4	76.6±0.7a	78.3±1.6a
	5	55.0±0.8a	48.9±0.2b
	6	57.5±0.8a	78.6±1.3b
	7	76.2±0.1a	92.5±0.7b
	8	81.3±1.8a	92.9±0.4b
	9	89.7±1.2a	93.4±0.3b
	10	90.5±1.0a	95.3±0.5b

Table 5

 5

	-2: Thermal denaturation temperatures of EW, PPI, and PPI-EW mixtures at
	different weight ratios at pH 7.5.
	samples	Td1(

°C) Td 2(°C) Td 3(°C) Td 4(°C) Td 5(°C) EW

	100%	61.1±0.1a	-	76.7±0.1a	83.5±0.8a	-
	PPI-EW 25/75	60.1±0.1a 64.4±0.6a 76.0±0.1ab	81.6±0.1a	85.4±0.3a
	PPI-EW 50/50	60.5±0.4a 64.5±0.6a 76.2±0.1ab	81.5±0.1a	85.6±0.1a
	PPI-EW 75/25	61.4±0.1a	-	76.0±0.1ab	81.3±0.2a	85.0±0.7a
	PPI 100%	-	-	75.8±0.4b	-	87.4±0.5b
	All data were given as mean ± SD of triplicate measurements. Means in a column
	bearing the same letter are not significantly different. Td1: ovotransferrin or
	ovotransferrin and lysozyme; Td2: lysozyme; Td3: ovalbumin (EW), vicilin (PPI) or
	ovalbumin and vicilin (mixture case), Td4: s-ovalbumin; Td5: legumin.

Table 5 -

 5 3: Thermal denaturation temperatures of EW, PPI and their mixtures (PPI-EW) at different weight ratios at pH 9.

	samples	Td1(°C) Td 2(°C) Td 3(°C)	Td 4(°C)	Td 5(°C)
	EW 100%	63.2±0.1a 69.5±0.1 76.4±0.1a	83.1±0.7a	-
	PPI-EW 25/75	62.1±0.1b	-	76.1±0.1ab	81.3±0.1a	86.3±0.7a
	PPI-EW 50/50	60.3±0.1c	-	75.3±0.2b	81.4±0.1a	86.1±0.1a
	PPI-EW 75/25	59.5±0.1d	-	75.5±0.1b	81.4±0.1a	86.9±0.2a
	PPI 100%	-	-	71.3±0.4c	-	84.5±0.2b
	All data were given as mean ± SD of triplicate measurements. Means in a column
	bearing the same letter are not significantly different. Td1: ovotransferrin or
	ovotransferrin and lysozyme; Td2: lysozyme; Td3: ovalbumin (EW), vicilin (PPI) or
	ovalbumin, vicilin and lysozyme (mixture case), Td4: s-ovalbumin; Td5: legumin.

Table 5 -

 5 4: Denaturation enthalpy of (∆H) of EW, PPI, and their mixtures (PPI-EW) at All data were given as mean ± SD of triplicate measurements. Means in a row bearing the same letter are not significantly different at the same pH. Means followed by different numbers of * for the same row are significantly different.

	different weight ratios at pH 7.5 and 9.
		pH 7.5		pH 9
	samples	∆H(J/g)	Recalculated ∆H(J/g)	∆H(J/g)	Recalculated ∆H(J/g)
	EW 100%	22.3±0.5*	-	23.8±0.2**	-
	PPI-EW 25/75	18.6±0.2a	19.4±0.4b	18.3±0.3a	18.7±0.1a
	PPI-EW 50/50	14.1±0.2a	16.6±0.2b	12.4±0.1a	13.7±0.1b
	PPI-EW 75/25	12.6±0.1a	13.7±0.1b	8.6±0.1a	8.6±0.2a
	PPI 100%	10.8±0.1*	-	3.6±0.2**	-

Table 5 -

 5 6: Final G' and G'' values at 95 °C of PPI, EW, and PPI-EW mixtures at different weight ratios at pH 7.5 and 9.

	Samples	Heating final G' / Pa pH 7.5 pH 9	Heating final G'' / Pa pH 7.5 pH 9
	EW 100%	4865±156a	5496±131a	425±21a	369±6a
	PPI-EW 25/75 2552±149b	2077±117b	210±17b	167±8b
	PPI-EW 50/50 1189±100c	953±65c	137±26bc	83±1c
	PPI-EW 75/25 742±181c	106±6d	94±29c	17±1d
	PPI 100%	30±26d	2.2±0.4d no gel	2±1d	1±1d no gel
	All data were given as mean ± SD of triplicate measurements. Means in a column
	bearing the same letter are not significantly different.

Table 6 -

 6 1 Effect of various reagents on molecular forces existing in protein.

		Non-covalent bonds	Covalent bond	References
		Ionic effect/ Electrostatic interaction	Hydrophobic interaction	Hydrogen bond	Disulfide bond
						Rüegg &
	Dithiothreitol (DTT)				Disrupt	Rudinger (1977), Léger & Arntfield
						(1993), Sun &
						Arntfield (2012a),
	Guanidine-HCl (GuHCl)	disrupt	weaken	Disrupt		Tanford (1968), Léger & Arntfield (1993), Sun & Arntfield (2012a)
						Tanford (1962),
	Propylene glycol (PG)	Promote	Disrupt	Promote		Ustunol et al. (1992), Utsumi &
						Kinsella (1985)
						Gordon & Jencks
	Urea		Disrupt	Disrupt		(1963), Uruakpa & Arntfield
						(2006b), Ustunol
						et al. (1992)

Table 6 -

 6

	2. The gelling temperatures for the different protein systems were already commented
	in the previous paper (Chapter 5, Section 5.3.4), where two thermal sol-gel transitions
	were observed at around 60 °C and 75 °C in EW-containing systems coinciding with
	Td of ovotransferrin and ovalbumin/S-ovalbumin/7S globulins, respectively. Especially,
	the first transition associated with ovotransferrin shifted by about + 2-3 °C at pH 9.
	According to our previous study on the thermal properties of EW/PPI mixtures
	(Chapter 5, Section 5.3.3), the maximum denaturation temperatures of PPI-EW were
	around 83-87°C, which assumed that complete denaturation of proteins by thermal
	treatment was achieved over 87 °C. Although there was no constant temperature stage
	in this test, compared to Zhang et al. (2019) where the time sweep for hen egg proteins-

soybean mixtures was maintained at 85 °C for 10 min, in this test, the temperature sweep was increased until 95 °C, where we assumed that resulting molecular

Table 6 -

 6 2: Rheological analysis comparing the storage modulus (G') values for pea protein isolate (PPI), egg white protein (EW), and their composite gels at 95 °C (end of heating cycle), and 25 °C (end of cooling cycle).

		pH 7.5		pH 9
	samples	G' (Pa) at 95 °C after heating	G' (Pa) at 25 °C after cooling	G' (Pa) at 95 °C after heating	G' (Pa) at 25 °C after cooling
	EW 100%	4865 ± 156aA	15115 ± 632a*	5496 ± 131aB	14446 ± 413a*
	PPI-EW 25/75	2552 ± 149bA	7284 ± 192b*	2077 ± 117bA	7204 ± 281ab*
	PPI-EW 50/50	1189 ± 100cA	4725 ± 324c*	953 ± 65cA	4182 ± 440b*
	PPI-EW 75/25	742 ± 181cA	3446 ± 331c*	106 ± 6dB	9237 ± 3249ab*
	PPI 100%	30 ± 26 d	97 ± 4 d	no gel	no gel

All data were given as mean ± SD of at least triplicate measurements. Means in a column bearing the same letter are not significantly different. Means at different numbers * and different uppercase letters in a row show a significant difference.

Table 6 -

 6 3 Loss factor of PPI-EW mixtures at different weight ratios, at 1% strain, 1Hz frequency, and frequency slope of G' of PPI-EW mixtures at different weight ratios at

	pH 7.5 and 9.
		pH 7.5	pH 9
	samples	Tan (δ) or Loss	Frequency	Tan (δ) or Loss	Frequency slope
		factor	slope of G'	factor	of G'
	EW 100%	0.135 ± 0.002aA	0.080 ± 0.001a* 0.115±0.001aB 0.076±0.001a**
	PPI-EW 25/75	0.138 ± 0.001abA	0.087 ± 0.001a* 0.118±0.003abB 0.077±0.002a**
	PPI-EW 50/50	0.151 ± 0.002bcA	0.092 ± 0.001a* 0.134±0.004abB 0.086±0.002a*
	PPI-EW 75/25	0.157 ± 0.003cA	0.097 ± 0.003a* 0.158±0.008abA 0.124±0.011b*
	PPI 100%	0.227 ± 0.005dA	0.161 ± 0.015b 0.229±0.060bA	(no gel)

All data were given as mean ± SD of at least triplicate measurements. Means in a column bearing the same letter are not significantly different. Means at different numbers of * and different uppercase letters in row show a significant difference.

Table 6

 6

	-4 Yield point (%) of PPI, EW, and PPI-EW gels at different weight ratios in
	strain sweep at pH 7.5 and 9.
	samples	yield point / %
		pH 7.5	pH 9
	EW 100%	5.5 ± 0.1a	16.6 ± 0.6a
	PPI-EW 25/75	9.7 ± 0.6b	41.6 ± 5.4b
	PPI-EW 50/50	11.4 ± 0.6b	52.3 ± 2.0b
	PPI-EW 75/25	3.9 ± 0.2a	9.7 ± 3.8a
	PPI 100%	5.6 ± 0.3a	no gel
	All data were given as mean ± SD of triplicate measurements. Means in a column
	bearing the same letter are not significantly different.

Table 6 -

 6 5: Parameters of texture profile analysis (TPA) of EW 100 and PPI-EW mixture gels at different weight ratios at pH 7.5 and 9.

	Samples	pH 7.5 Hardness /N Cohesiveness	Springiness	Hardness /N	pH 9 Cohesiveness	Springiness
	EW
	100%

ultrasound, microwaves, germination, enzymatic hydrolysis, and pH-shifting[START_REF] Boukid | Pea protein ingredients: A mainstream ingredient to (re) formulate innovative foods and beverages[END_REF][START_REF] Jiang | Modifying the physicochemical properties of pea protein by pH-shifting and ultrasound combined treatments[END_REF][START_REF] Zhu | Strong and elastic pea protein hydrogels formed through pH-shifting method[END_REF].

Sun& Arntfield, 2011c

Boom, & Schutyser, 2015a

[START_REF] Klost | Structure formation and rheological properties of pea protein-based gels[END_REF]

[START_REF] Sim | High pressure structuring of pea protein concentrates[END_REF]

Scientific publications

Submitted papers

Chapter 2 Materials and methods

Determination of mineral (ash) content

The mineral content of protein isolates was determined according to Method 923.03 (AOAC, 1990). The dehydrated samples were placed in a muffle furnace (MR 260, Heraeus) at 600 °C for 24 hours. The percentage of mineral matter is calculated by equation ( 2 Where: %MM: mineral matter content (%, w/w on a dry basis);

Msec: mass of the sample after dehydration (g);

Mcendres: mass of ashes after calcination at 600°C (g).

Three measurements were performed for each sample.

Determination of protein content by determination of total nitrogen

The determination of total nitrogen was carried out according to method 920.87 (AOAC 1990), also known as the Kjeldahl method, in a digestion unit (Scrubber B414, Büchi, Rungis, France) and a distillation unit (K355, Büchi, Rungis, France). The protein content of the protein isolates and dispersions was then determined using the nitrogen-protein conversion coefficient K, which was found to be 5.44 for pea proteins (Mosse,1990) and 6.25 for egg white proteins (Stitcher, et al., 1969).

A precisely weighed amount of material was put into the matrass, resulting in 10 to 200 mg of protein per matrass. Each matrass received 10 ml of 96 % sulfuric acid, 2 glass balls, and 1 catalyst pellet (Kjeltabs NACT AA44, Thompson and Capper Ltd, Cheshire, England). Mineralization was carried out utilizing a BÜCHI K355 digesting equipment (Rungis, France). The samples were heated at 320°C for 20 mins, then 400 °C for an hour, allowing complete nitrogen conversion to ammonium ion. The acid vapors were eliminated by a B414 purifier from BÜCHI (Rungis, France). After cooling, each matrass received 25 mL of distilled water and 2 drops of phenolphthalein. The

Optical microscopy

The objective of the test is to understand what happened at a small molar ratio of PPI-LYS mixtures.

An optical microscope (Olympus BX51TF, Olympus Andre ORVAIN) equipped with an Olympus DP11 camera was used to obtain the images of the PPI-LYS systems.

Samples were prepared as described before in section 2.2.2. Structures were immediately observed at a magnification of 10× at room temperature.

Statistical analysis

Values were expressed as means ± standard deviations of triplicate determinations.

Statistical analyzes of the experimental data were carried out by analysis of variance (ANOVA). Significant differences (p < 0.05) between samples were determined by Tukey's test using STATISTICA 12 (64 BIT) software. Regarding the linear regressions and the associated statistical parameters, they were obtained with the Excel® software (2020.5.1.1040).

Physicochemical composition of egg white and ovalbumin

The protein content of egg white was 10.4±0.25 % calculated by the Kjeldahl method (N x 6.25) mentioned in section 2.2.3.3, which agreed with the literature that mentioned that egg white consists of 11% of protein (section 1.2).

The protein concentration of ovalbumin was analyzed by the Bradford method (Bradford, 1976) using BSA as a standard. So, the protein content of ovalbumin extract was around 96.6 %.

Polypeptide composition of PPI and EW

The protein isolate was separated into distinct fractions using an SDS-PAGE gel under denaturing (SDS) and reducing (DTT+SDS) conditions. The composition of the PPI and EW at pH 7.5 (left part) and 9 (right part) was shown in Figure 3-1. In general, the electrophoretic profile of the PPI and EW prepared at pH 7.5 and 9 showed great similarities (Figure 3-1).

Figure 3-1 SDS-PAGE profile of extracted pea protein isolate and egg white when

Results and discussions

LYS interacts with PPI through electrostatic interactions

The ITC experiment was used to provide a detailed thermodynamic description and a better understanding of the mechanism of interactions of PPI and egg white proteins in solution. The ITC profiles for PPI with OVA (as acidic protein), OVT (as neutral protein), and LYS (as basic protein) were measured. The heat flow versus time profiles resulting from the titration of the PPI with the three egg proteins at various conditions are shown in Figure 4-1.

Microcal Origin software. The 'two sets of sites' model seems to better match the experimental titration profiles (data not shown). However, as already pointed out by other authors relating to other macromolecular systems [START_REF] Girard | Thermodynamic parameters of βlactoglobulin-pectin complexes assessed by isothermal titration calorimetry[END_REF][START_REF] Aberkane | Thermodynamic characterization of acacia gum-β-lactoglobulin complex coacervation[END_REF], we are convinced that the existence of two independent sets of binding sites has no physical meaning when dealing with interactions involving two macromolecules. In particular because of the simultaneous occurrence of several complex events as mentioned above. Hence, the use of the "2-stages structuring model" expression, underlying the presence of two distinct structuring phases instead of the "2-sites model" is more appropriate.

When using the "2-binding site model" as an approximation to extract the thermodynamic parameters of the interaction (namely Ka and △H) between LYS and PPI at the three experimental conditions, erroneous values with large errors were obtained (data not shown). Consequently, we were unable to quantify the binding parameters using the ITC Microcal-associated origin software because the curves were complex and difficult to fit.

Although the appropriate thermodynamic parameters for the interaction between LYS and PPI could not be calculated, it is clear that the overall process leading to particle formation is enthalpically driven. A contrary situation occurred with the two other egg proteins tested, with no or only small negative heats detected by ITC. From the literature data [START_REF] Leavitt | Direct measurement of protein binding energetics by isothermal titration calorimetry[END_REF][START_REF] Klebe | Applying thermodynamic profiling in lead finding and optimization[END_REF], enthalpy (ΔH) is related to the energy involved in molecular interactions and reflects the contribution of hydrogen bonds, electrostatic interactions, and van der Waals forces, while the change in entropy (TΔS) reflects a change in the order of the system and is related to hydrophobic interactions.

As possible particle formation between PPI and LYS was supposed from ITC data, the aqueous mixture of both proteins was further analyzed in terms of particle size, ζpotential, and microstructure.

Chapter 5 Thermal behaviour of pea and egg white protein mixtures

Abstract

In this work, a laboratory-prepared pea protein isolate (PPI) was used to partially substitute raw egg white (EW), and the thermal behaviour and sol-gel transition of the mixed systems submitted to heat treatment (<100 °C) were studied. The protein systems were investigated at pH 7. associated pea globulins at this pH. These results will serve to develop an adapted heat treatment for this protein mixture used as a food ingredient.

Introduction

Owing to the population growth and diet-related socioeconomic changes over the coming decades, humans are increasingly recognizing that greater consumption of

Thermal properties of the mixtures

The thermal properties of proteins directly reflect their native status and can be evaluated by calorimetry.

(A) (B) manner, as the decrease in enthalpy was also significantly higher for this ratio at pH 7.5.

Gelation temperatures

Temperature sweeps were performed by small amplitude rheology to understand the sol-gel transition behaviour of the different protein suspensions upon thermal treatment. The gelling temperatures were reported in Table 5-5. For EW and the PPI-EW mixtures containing at least 50 % EW, two transition temperatures were measured whatever the pH. For pure EW at pH 7.5 and 9, these gelling temperatures could be attributed preferentially to the denaturation of ovotransferrin and ovalbumin at ~60 and ~75 °C, at pH 7, respectively, as reported by [START_REF] Barhut | Thermal Analysis of Foods[END_REF] and [START_REF] Ferreira | A calorimetric study of egg white proteins[END_REF]. It can be noticed that these temperatures are close to the thermo-denaturation temperature identified for these proteins in DSC thermograms (Tables 5-2 & 5-3). For the mixtures containing at least 50 % of EW, considering that pure PPI suspensions didn't show any transition temperature around 60 °C whatever the pH, the presence of a first gelling point can also be associated mainly with ovotransferrin. This first transition temperature was not affected by the EW/PPI ratio at pH 7.5 whereas it increased by nearly 3 °C with the increase in PPI in the mixture at pH 9.0. At this latter pH, the gelation point assigned to ovotransferrin was therefore delayed even though the DSC results reported in Section 5.3.3 showed a slight decrease in the denaturation temperature of this protein in admixture with PPI. The presence of the pea globulins carrying highly negative charges at this pH, far from their pI, could be prone to hamper ovotransferrin molecules/particles association until more advanced denaturation (or aggregation) was achieved at slightly higher temperatures. Similar results were observed by Watanabe et al. (2002) with dry-heated ovalbumin inhibiting ovotransferrin heat aggregation and coagulation.

Besides, the PPI-EW mixtures at the 75/25 weight ratio at pH 7.5 and 9 didn't

show any sharp G' rise in this temperature range meaning that no early sol-gel transition can be associated with ovotransferrin in this mixture. It could be hypothesized that even if thermal denaturation of ovotransferrin occurred around 60 °C, the resulting unfolded/aggregated proteins were not numerous enough to interact and form a threedimensional network, and/or their association was sterically hindered by the presence of the pea globulins in the mixture.

The second transition temperature was observed in all cases except for the pure PPI sample at pH 9 that didn't reach gelation during the temperature sweep. In this last case, the formation of a three-dimensional network of unfolded/aggregated pea globulins was hindered by high repulsive forces within protein particles because the negative charges dominated at this pH far from the pI of globulins (pI = 4.5-4.8). For all other mixtures, the second sol-gel transition around 75°C was associated with ovalbumin and 7S globulins which denatured in the same range of temperature, as previously evidenced by DSC analysis in Section 5. Chapter 6 Nature of protein-protein interactions during gelation of mixtures between pea protein isolates and egg white proteins have been used to investigate the potential methods and molecular forces responsible for the binding reactions (Table 6-1).

To eliminate the primary molecular forces in protein gels, bond-disrupting chemicals can be utilized. They destabilized the protein's original structure and caused irreversible molecular rearrangements, resulting in variations in protein gel characteristics [START_REF] Yu | Molecular forces involved in heat-induced freshwater surimi gel: Effects of various bond disrupting agents on the gel properties and protein conformation changes[END_REF][START_REF] Wang | Probing the molecular forces involved in binding of selected volatile flavour compounds to salt-extracted pea proteins[END_REF]. The molecular forces involved in the gel network are determined by the protein nature and the protein structure which can be altered by the protein extraction methods used (Sun & Arntfield, 2012a; Shimada & Matsushita,1980;[START_REF] Utsumi | Forces involved in soy protein gelation: effects of various reagents on the formation, hardness and solubility of heat-induced gels made from 7S, 11S, and soy isolate[END_REF].

Urea and guanidine hydrochloride (GuHCl) may disrupt protein hydrogen bonds and hydrophobic interactions. Urea denatures a protein molecule by preferential adsorption with charged protein solutes, dehydrating the molecules and inducing protein repulsion, therefore stabilizing the unfolded state [START_REF] Wallqvist | Hydrophobic interactions in aqueous urea solutions with implications for the mechanism of protein denaturation[END_REF]. Shan et al. (2014) showed that urea inhibited network formation in soy protein gel and significantly reduced gel stiffness by breaking down hydrogen bonds and hydrophobic interactions.

Dithiothreitol (DTT) reacts with sulfhydryl groups to form a stable alkyl derivative, preventing the formation of disulfide bonds between protein molecules. Several studies have illustrated that sulfhydryl/disulfide interchange is involved in soy protein gelation based on the reaction of the gel (a loss of the gel integrity) [START_REF] Mcklem | Investigation of molecular forces involved in gelation of commercially prepared soy protein isolates[END_REF][START_REF] Utsumi | Forces involved in soy protein gelation: effects of various reagents on the formation, hardness and solubility of heat-induced gels made from 7S, 11S, and soy isolate[END_REF]Wolf, 1993). In terms of electrostatic forces, several studies have shown that it can be involved in gel formation by the effect of pH and salts (Sun & Arntfield, 2012a; O'Riordan, [START_REF] O'riordan | The effect of salts on the rheological properties of plasma protein gels[END_REF][START_REF] O'riordan | The effect of salts on the rheological properties of plasma protein gels[END_REF].

General conclusion and perspective

General conclusion

The purpose of the thesis was to partially replace egg white proteins (EW) with pea protein isolate (PPI) under different physico-chemical conditions (total protein concentration at 10% w/w, different weight ratios of PPI/EW, as well as different pHs)

in order to further understand the possible interactions between both types of proteins after mixing. The impact on the functional properties of the mixture such as thermal and gelling properties was also studied.

The study was developed in two main steps. The first part was to know which kind of interactions could happen in the aqueous mixture, to give us an idea for further experiments. The second part was dedicated to the study of the thermal and gelation properties of the samples prepared by mixed systems and single proteins. The mechanical and structural properties of the different composite systems as well as the pure PPI and EW ones were studied by different biochemical and physicochemical analytical tools.

In order to carry out this project, it was essential, first of all, to obtain qualitative raw materials including pea globulins, and ovalbumin in sufficient quantity to cover our needs during this study. Two protocols were used for the extraction and purification of these proteins. Pea globulins were obtained by alkaline extraction (pH 8) followed by isoelectric precipitation (pH 4.8). The purification procedure by ultrafiltration/ diafiltration was carried out using a cassette with a cutoff of 10 kDa. Subsequently, the diafiltered protein solution was then freeze-dried to obtain a "pure" globulin powder containing 89 % protein on a dry basis (N= 5.44). Ovalbumin was extracted by adjusting egg white pH to 6 to precipitate ovomucin and adjusting pH to 8.4 with covered supernatant. After centrifugation, the supernatant was filtered and injected into anion exchange chromatography Q-Sepharose to separate the OVA from the other egg white proteins. Finally, the solutions were lyophilized, and the ovalbumin was obtained.

Several physicochemical analyses on the raw materials were carried out, especially influence of different weight ratios and pHs on the functional properties of the PPI-EW composite system regarding thermal and gelation properties. As the system was based on egg white, the total protein concentration was settled at around 10% w/w protein of egg white and quantified by the Kjeldahl method (N = 6.25). As mentioned before, to have a better quantity of soluble proteins in the mixtures, high pH at 7.5 and 9 were selected. The other reason for choosing these pHs is because the pH of egg white is just after laying (7.5) or a few days after laying (9). Different weight ratios were chosen as PPI/EW 0/100, 25/75, 50/50, 75/25, and 100/0. The solubility of PPI-EW at a weight ratio of 50/50 showed a lower nitrogen solubility profile than recalculated ones, which may be attributed to the formation of aggregates between lysozyme and pea proteins through electrostatic interactions, especially at pH over the isoelectric point of pea protein isolate. From SDS-PAGE data, we could not evidence any new bond was formed for the 50/50 PPI-EW mixture.

For the design of egg products, thermal properties are an important value regarding pasteurization treatment. We performed MicroDSC to obtain the thermal denaturation temperature (Td) and the changes of enthalpy (ΔH) of the proteins. Thermal properties of egg white only and PPI only were discussed in the previous graph. When taking the different protein mixtures into account regarding the Td of PPI-EW mixtures, it was found in general that there were 5 peaks corresponding to Td1 of ovotransferrin, Td2 of lysozyme, Td3 of superimposition of ovalbumin and vicilin peaks which could also overlay the peak of lysozyme at pH 9, Td4 of S-ovalbumin and Td5 of legumin. The Td was slightly or not different compared to those measured for the pure protein solution of EW and PPI. Especially, the Td value of ovotransferrin (Td1) increased significantly at pH 9 from ~59 °C to ~63 °C with the increase in PPI content in admixture, possibly due to OVT being more sensitive to heat at this pH leading to its unfolding in presence of PPI or a decrease in electrostatic interactions with lysozyme due to the competition with PPI proteins. The Td2 of lysozyme performed differently in the PPI-EW mixture at both pH; in detail, it appeared at pH 7.5 but not at pH 9. The former one would be due to either a slight shift of ovotransferrin signal toward lower temperatures at pH 7.5

Research International, 28(1), 9-16 Li-Chan, E., & Nakai, S. (1989)