Maire-Caroline Michalski

Frédéric Carrière

Claire Berton-Carabin

Gildas Breton

Françoise Nau Thanks

Olivia Ménard

Stefano Nebbia

Léa Salelles

Rima Hachfi-Soussi

François Martin

Manon Hiolle

Xingchen Zhou

Ming Yu

Xiaoxi Yu

Wenfan Cao

Jian Kuang

CD36 cluster of differentiation 36 Control Omelet without DHA supplementation COX

Time flies, this is my third year in France.

List of tables

• Table 1-1. The consumption ratio between the precursors of n-6 and n-3 PUFAs in different western countries.

• Table 12. The recommended intake of n-3 PUFAs.

• Table 123. Major dietary sources of n-3 PUFAs.

• Table 1-4. The possible mechanisms of n-3 PUFAs for improving cognitive functions.

• Table 1-5. The possible mechanisms of n-3 PUFAs in the prevention of mental disorders.

• Table 1-6. The possible mechanisms of n-3 PUFAs in the prevention of heart diseases.

• Table 1-7. Some of n-3 FA enriched products on the market.

• Table 1-8. Different kinds of delivery systems for encapsulating n-3 PUFAs.

• Table 1-9. Particles prepared from food materials are used in Pickering emulsion.

• Table 1-10. The application of WPI in encapsulation.

• Table 3-1. Effect of emulsion and cooking on the DHA concentrations and proportions in omelets.

• Table 4-1. The composition of diets (A) and oil mix (B).

• Table 4-2. The fatty acid composition of the diets and the omelets.

• Table 4-3. The fatty acid composition of the daily intake of T-diet and omelets.

• Table 4-4. The dimethylacetal profile of RBC, heart, brain and eyes.

• Table 4-5. The oxidized derivatives of FA from DHA oil and omelets.

• Table 4-6. The oxidized derivatives of FA from plasma.

• Table 4-7. The oxidized derivatives of FA from heart.

• Table 4-8. The oxidized derivatives of FA from brain.

Introduction gé né rale, objectif et straté gie

Les acides gras polyinsaturés sont des acides gras qui contiennent plus d'une double liaison C=C sur leur squelette. George et Mildred Burr ont été les premiers chercheurs à montrer l'importance des acides gras polyinsaturés dans l'alimentation. En 1929, ils ont découvert que l'élimination des lipides de l'alimentation des animaux induisait plusieurs maladies , notamment des problèmes de peau et autres troubles pouvant entraîner la mort. Cependant, après administration alimentaire d'acides gras polyinsaturés (acide linoléique (AL) et acide α-linolénique (ALA)), ces maladies pouvaient être évitées ou guéries [START_REF] Burr | A New Deficiency Disease Produced By the Rigid Exclusion of Fat From the Diet[END_REF][START_REF] Liput | Effects of dietary n-3 and n-6 polyunsaturated fatty acids in inflammation and cancerogenesis[END_REF][START_REF] Burr | On the Nature and Rôle of the Fatty Acids Essential in Nutrition[END_REF].

Dans les années 1970, Dyerberg et Bang ont découvert, par rapport aux Danois, que les décès cardiovasculaires étaient rares chez les Esquimaux. En effet, les Esquimaux consomment beaucoup de lipides marins riches en acides gras polyinsaturés n-3 dans leur alimentation quotidienne [START_REF] Dyerberg | Haemostatic Function and Platelet Polyunsaturated Fatty Acids in Eskimos[END_REF]. Les recherches sur les acides gras polyinsaturés notamment au niveau de la santé ont été étendues.

La classification des acides gras polyinsaturés est réalisée par famille en distinguant les acides gras polyinsaturés n-3 des acides gras polyinsaturés n-6, qui se repère en fonction de la position de la dernière double liaison à l'extrémité de la fonction méthylique. Les acides gras polyinsaturés n-3

et n-6 sont nécessaires aux cellules pour maintenir une structure, une fonction et une transduction du signal normales, mais ils ont un effet opposé dans la modulation de l'inflammation. En effet, les acides gras polyinsaturés n-3 ont des propriétés anti-inflammatoires, alors que les acides gras polyinsaturés n-6 favorisent l'inflammation. Étant donné que les acides gras polyinsaturés n-3 et n-6 sont en compétition pour les mêmes enzymes dans la synthèse et le métabolisme, leur rapport dans les tissus corporels détermine le profil des médiateurs lipidiques impliqués dans la réponse inflammatoire [START_REF] Kaliannan | A host-microbiome interaction mediates the opposing effects of omega-6 and omega-3 fatty acids on metabolic endotoxemia[END_REF]. Des quantités excessives d'acides gras polyinsaturés n-6 et une carence en acides gras polyinsaturés n-3 entraine un ratio n-6/n-3 de 10-20/1 pour un régime riche (type western diet), alors que le rapport dans un régime classique est d'environ 1/1 [START_REF] Leclercq | Why and how meet n-3 PUFA dietary recommendations?[END_REF]. Ce changement contribue à la prévalence actuelle des maladies chroniques [START_REF] Mariamenatu | Overconsumption of Omega-6 Polyunsaturated Fatty Acids (PUFAs) versus Deficiency of Omega-3 PUFAs in Modern-Day Diets: The Disturbing Factor for Their "Balanced Antagonistic Metabolic Functions" in the Human Body[END_REF], comme notamment les maladies cardiovasculaires, la maladie d'Alzheimer, le diabète de type 2, l'asthme, etc. En effet, de nombreuses recherches ont prouvé que les acides gras polyinsaturés n-3 ont des effets préventifs sur ces maladies [START_REF] Barberger-Gateau | Dietary omega 3 polyunsaturated fatty acids and Alzheimer's disease: Interaction with apolipoprotein E genotype[END_REF][START_REF] Mozaffarian | Omega-3 fatty acids and cardiovascular disease: Effects on risk factors, molecular pathways, and clinical events[END_REF][START_REF] Schubert | Effect of n-3 polyunsaturated fatty acids in asthma after low-dose allergen challenge[END_REF]. Par conséquent, de nombreux pays ont émis des recommandations pour les acides gras polyinsaturés n-3, en préconisant un équilibre avec les acides gras n-6. Ainsi chez l'homme, il est préconisé d'augmenter l'apport en acides gras polyinsaturés n-3 tout en limitant l'apport excessif en acides gras polyinsaturés n-6 pour atteindre un rapport optimal entre acides gras polyinsaturés n-6 et n-3.

Le poisson et les produits dérivés des fruits de mer sont les sources les plus riches en acides gras polyinsaturés n-3. Par conséquent, l'enrichissement des aliments en acides gras polyinsaturés n-3 est considéré comme une stratégie efficace pour atteindre le seuil d'apport recommandé.

Cependant les ressources halieutiques riches en acides gras polyinsaturés n-3 sont confrontées à de nombreux problèmes. Par exemple, des toxines marines et des agents infectieux ont été trouvés dans les produits de la mer, y compris des parasites (par exemple, les nématodes, les ténias et les douves)

et des bactéries [START_REF] Gil | Fish, a Mediterranean source of n-3 PUFA: benefits do not justify limiting consumption[END_REF]. Il est également important de souligner que la surpêche détruit l'équilibre de l'écosystème et entraîne une diminution des ressources marines.

De nos jours, il existe de nombreuses stratégies pour réaliser un enrichissement en acides gras polyinsaturés n-3 dans les aliments, en se concentrant principalement sur les aspects suivants (Saini & Keum, 2018): 1) fortifier les acides gras polyinsaturés n-3 dans les cultures d'oléagineux en utilisant le génie génétique ; 2) enrichir les acides gras polyinsaturés n-3 chez les animaux et les produits d'origine animale en donnant aux animaux des régimes enrichis en acides gras polyinsaturés n-3 ; 3) apporter des acides gras polyinsaturés n-3 dans les aliments et améliorer leurs propriétés sensorielles en utilisant des techniques d'encapsulation.

Ces différentes stratégies ont leurs avantages et leurs inconvénients. Pour ma thèse, la technique d'encapsulation pour réaliser la supplémentation en acides gras polyinsaturés n-3 a été choisie. Les propriétés physiques et chimiques des acides gras polyinsaturés n-3 (tels que les acides gras hautement insaturés) induisent de nombreux défis à relever lors de la production, le transport, le stockage, l'acceptabilité sensorielle et la biodisponibilité des aliments [START_REF] Liu | Fabrication, characterization and properties of DHA-loaded nanoparticles based on zein and PLGA[END_REF][START_REF] Ma | Enhancing the physicochemical stability and digestibility of DHA emulsions by encapsulation of DHA droplets in caseinate/alginate honeycomb-shaped microparticles[END_REF][START_REF] Singh | Nanoencapsulation of docosahexanoic acid (DHA) using combination of food grade polymeric wall materials and its application for improvement in bioavailability and oxidative stability[END_REF]. Pour pallier à ces problèmes, l'encapsulation s'avère être une bonne stratégie.

Différents types de techniques ont été utilisées pour encapsuler les acides gras polyinsaturés n-3, notamment les émulsions [START_REF] Ghosh | Comparative study of gastrointestinal absorption of EPA & DHA rich fi sh oil from nano and conventional emulsion formulation in rats[END_REF][START_REF] Ma | Enhancing the physicochemical stability and digestibility of DHA emulsions by encapsulation of DHA droplets in caseinate/alginate honeycomb-shaped microparticles[END_REF]Ruiz & Perez, 2020), les nanoliposomes (Rasti et al., 2017), les nanoparticules [START_REF] Serini | Omega-3 PUFA loaded in resveratrol-based solid lipid nanoparticles: Physicochemical properties and antineoplastic activities in human colorectal cancer cells in vitro[END_REF], la microencapsulation, les particules d'hydrogel [START_REF] Zhang | Encapsulation , protection , and release of polyunsaturated lipids using biopolymer-based hydrogel particles[END_REF], les complexes d'inclusion (Ruiz et al., 2017), etc.

Parmi ces tecniques, les émulsions sont largement utilisées. Cependant, les émulsions classiques sont thermodynamiquement instables et ont tendance à se décomposer avec le temps par agrégation et/ou coalescence de la phase disperse [START_REF] Mcclements | Advances in fabrication of emulsions with enhanced functionality using structural design principles[END_REF]. Par rapport à l'émulsion traditionnelle, l'émulsification des huiles alimentaires de Pickering a suscité un grand intérêt car elle repose sur l'adsorption (irréversible) de (bio)particules plutôt que sur des tensioactifs (synthétiques) à l'interface huile-eau, ce qui confère une résistance à la coalescence des gouttelettes et améliore la stabilité de l'émulsion [START_REF] Chevalier | Emulsions stabilized with solid nanoparticles: Pickering emulsions[END_REF][START_REF] Low | Recent advances of characterization techniques for the formation, physical properties and stability of Pickering emulsion[END_REF][START_REF] Wu | Recent Studies of Pickering Emulsions: Particles Make the Difference[END_REF].

L'isolat de protéines de lactosérum est couramment utilisé dans les formulations alimentaires et il est utilisé comme molécules à l'interface pour l'encapsulation pour ses fonctions nutritionnelles, telles que l'anti-vieillissement [START_REF] Liu | Retard of D-Galactose-Induced Aging in Mice by Whey Hydrolysates[END_REF], l'anticancéreux [START_REF] Shariatikia | Anticancer activity of cow, sheep, goat, mare, donkey and camel milks and their caseins and whey proteins and in silico comparison of the caseins[END_REF], mais aussi ses fonctions fonctionnelles telles que sa capacité à former un film (Schmid & Müller, 2019), à émulsifier [START_REF] Masson | A study of oil-in-water emulsions stabilized by whey proteins[END_REF] et sa capacité à gelifier [START_REF] Singh | Thermal denaturation, aggregation and gelation of whey proteins[END_REF]. Mais de part ces protéines sensibles traitement thermique, elles se dénaturent ce qui provoque une instabilité de l'émulsion [START_REF] Chen | Food-grade Pickering emulsions: Preparation, stabilization and applications[END_REF]. Cependant, cette susceptibilité de la protéine de lactosérum peut être contrée via une modification physique ou chimique, ce qui offre plus de possibilités pour son application dans l'émulsion de Pickering. Sur la base de nos recherches précédentes, des particules de protéines de lactosérum rendues hydrophobes ont été préparées avec succès par des techniques successives d'acétylation et de traitement thermique [START_REF] Madadlou | Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins[END_REF]. Les protéines de lactosérum après traitement thermique ont été utilisées comme stabilisant Pickering des mousses [START_REF] Destribats | Emulsions stabilised by whey protein microgel particles: Towards food-grade Pickering emulsions[END_REF]. Cependant, son application en tant qu'émulsifiant de type Pickering n'a pas été reportée. Par conséquent, zau cours de cette thèse, deux émulsions Pickering stabilisées par des particules ont été préparées, et après analyse de leurs propriétés, un système d'émulsion a été sélectionné pour les expériences ultérieures.

Des recherches approfondies ont été conduites sur le développement et la caractérisation des émulsions de Pickering mais peu d'études ont portées sur leurs applications potentielles en alimentation. Il est bien démontré que la biodisponibilité des composés sensibles est impactée par la matrice alimentaire [START_REF] Aguilera | The food matrix: Implications in processing, nutrition and health[END_REF][START_REF] Dima | Bioavailability of nutraceuticals: Role of the food matrix, processing conditions, the gastrointestinal tract, and nanodelivery systems[END_REF]. Les produits laitiers fermentés ont été utilisés comme vecteurs de nutriments encapsulés, mais notre étude précédente a montré que les oeufs enrichis en DHA, via l'alimentation de la poule, présentent une également une biodisponibilité intéressante en DHA selon la structure de l'aliment considéré (omelette, oeuf dur ou mousse).

L'omelette a fourni 56 % et 120 % de plus de DHA dans le plasma des porcs que l'oeuf dur et la mousse après 48h d'alimentation, respectivement [START_REF] Pineda-Vadillo | In Vivo Digestion of Egg Products Enriched with DHA: Effect of the Food Matrix on DHA Bioavailability[END_REF]. Par conséquent, afin de maximiser l'efficacité de l'absorption du DHA in vivo, dans la présente étude, l' omelette a été retenue comme matrice alimentaire.

Lorsque de nouveaux composés lipidiques bioactifs sont développés, leur stabilité gastrointestinale, leur bioaccessibilité et lbiodisponibilité doivent être évaluées. L'objectif est de déterminer la part disponible pour l'absorption de la fraction du composé ingéré (bioaccessibilité) [START_REF] Solomando | Lipid digestion products in meat derivatives enriched with fish oil microcapsules[END_REF] ou d'évaluer la fraction de nutriment ou de composé bioactif ingéré qui atteint la circulation systémique et finalement utilisée par l'organisme (biodisponibilité) (C. [START_REF] Galanakis | What is the difference between bioavailability bioaccessibility and bioactivity of food components[END_REF]. Cependant, sur la base de nos connaissances, il n'y a pas de données publiées sur la libération gastro-intestinale et la bioaccessibilité d'omelette enrichie en émulsion de Pickering enrichie en huile de DHA, ni même de recherches axées sur l'effet de l'émulsion Pickering sur le métabolisme du DHA in vivo.

Afin de répondre à cette question de recherche, , la stratégie adoptée se divise en trois étapes (Figure 1):

1. La première partie de la thèse a eu pour objectif d'encapsuler de l'huile de DHA. Dans ce but, deux étapes ont été réalisées: tout d'abord, la préparation et l'analyse des propriétés de particules de protéines à base de protéines de lactosérum comme matière première pour l'émulsion de Pickering (taille des particules, potentiel zéta , hydrophobicité de surface et tension interfaciale); deuxièmement, la préparation de l'émulsion Pickering enrichie en huile de DHA (composée de triacylglycérols (TAG) enrichis en DHA) et l'analyse des caractéristiques (taille des gouttelettes, microscopie optique, stabilité de crémage, comportement rhéologique et stabilité à l'oxydation).

Les résultats de cette partie sont présentés dans le chapitre 2: Supplémentation en CaCl2 des protéines de lactosérum rendues hydrophobes: évaluation des particules de protéines et des émulsions qui en résultent.

2. La deuxième partie de la thèse vise à déterminer l'impact de l'encapsulation sur la libération de DHA lors de la digestion in vitro. Afin d'atteindre cet objectif, de l'huile de DHA native ou une émulsion de Pickering enrichie en huile de DHA a été ajoutée dans un liquide d'oeuf homogénéisé pour obtenir des omelettes enrichies en DHA. L'effet de l'encapsulation sur la bioaccessibilité du DHA a été déterminé en utilisant le modèle de digestion in vitro statique INFOGEST adapté à l'adulte. Pour une meilleure compréhension de la distribution de l'huile de DHA pendant la digestion, la microstructure du digesta a été observée par microscopie confocale à balayage laser. Afin de connaître l'impact de l'encapsulation sur l'évolution du DHA, différentes espèces lipidiques formées lors de la digestion ont été séparées par chromatographie sur couche mince. Ensuite, le profil des acides gras des différentes espèces lipidiques a été déterminé par chromatographie en phase gazeuse couplée à la spectrométrie de masse. Enfin, l'évolution des profils d'acides gras et spécifiquement de DHA, estérifiés dans les différentes espèces lipidiques formées au cours de la digestion, a été caractérisée.

Les résultats de cette partie sont présentés dans le chapitre 3 : L'encapsulation d'huile de DHA via des protéines de lactosérum dénaturées par traitement thermique dans l'émulsion de Pickering améliore la bioaccessibilité du DHA. Les résultats de cette partie sont présentés au chapitre 4: L'encapsulation d'huile de DHA améliore de manière notable le profil d'oxylipines des tissus chez le rat.

Outre les chapitres déjà mentionnés, un premier chapitre comprenant une revue bibliographique est également inclu, et une discussion générale ainsi qu'une section perspective sont présentées à la fin du manuscrit.

General introduction, objective and strategy

Polyunsaturated fatty acids (PUFAs) are fatty acids that contain more than one double bond in their backbone. George and Mildred Burr were the first researchers who showed the importance of PUFAs in the diet. In 1929, they found that the elimination of fat from the diet of animals induced several deficiency illnesses, including skin problems and disorders leading to death. However, after dietary administration of PUFAs (linoleic acid (LA) and α-linolenic acid (ALA)), these illnesses could be prevented or cured [START_REF] Burr | A New Deficiency Disease Produced By the Rigid Exclusion of Fat From the Diet[END_REF][START_REF] Liput | Effects of dietary n-3 and n-6 polyunsaturated fatty acids in inflammation and cancerogenesis[END_REF][START_REF] Burr | On the Nature and Rôle of the Fatty Acids Essential in Nutrition[END_REF].

In the 1970s, Dyerberg and Bang discovered that death from cardiovascular disease (CVD)

was rare among Eskimos due to the large consumption of marine lipids rich in long-chain n-3 PUFAs in their native lifestyle in comparison with Danish who consumed much less of these lipids [START_REF] Dyerberg | Haemostatic Function and Platelet Polyunsaturated Fatty Acids in Eskimos[END_REF]. Since then, more and more interest in PUFAs in health benefits has grown into a large research area.

In general, PUFAs can be classified into n-3 PUFAs and n-6 PUFAs according to the position of the last double bond on the methyl end. Both n-3 and n-6 PUFAs are necessary for cells to maintain normal structure, function, and signal transduction, although they play opposite roles in the modulation of inflammation. Generally, n-3 PUFAs have anti-inflammatory properties, whereas n-6 PUFAs promote inflammation. Since n-3 and n-6 PUFAs compete for the same enzymes in synthesis and metabolism, their ratio in body tissues determines the profile of lipid mediators involved in the inflammatory response [START_REF] Kaliannan | A host-microbiome interaction mediates the opposing effects of omega-6 and omega-3 fatty acids on metabolic endotoxemia[END_REF]. Excessive amounts of n-6 PUFAs and deficiency in n-3 PUFAs have resulted in a n-6/n-3 ratio of 10-20/1 in the Western diet for a ratio around 1/1 in the diet of human evolution [START_REF] Leclercq | Why and how meet n-3 PUFA dietary recommendations?[END_REF]. This change is thought to contribute to today's prevalence of chronic diseases [START_REF] Mariamenatu | Overconsumption of Omega-6 Polyunsaturated Fatty Acids (PUFAs) versus Deficiency of Omega-3 PUFAs in Modern-Day Diets: The Disturbing Factor for Their "Balanced Antagonistic Metabolic Functions" in the Human Body[END_REF], including CVD, Alzheimer's disease (AD), type 2 diabetes, asthma, etc. Indeed, a number of researches have proved that n-3 PUFAs have preventive effects on these diseases [START_REF] Barberger-Gateau | Dietary omega 3 polyunsaturated fatty acids and Alzheimer's disease: Interaction with apolipoprotein E genotype[END_REF][START_REF] Mozaffarian | Omega-3 fatty acids and cardiovascular disease: Effects on risk factors, molecular pathways, and clinical events[END_REF][START_REF] Schubert | Effect of n-3 polyunsaturated fatty acids in asthma after low-dose allergen challenge[END_REF]. Therefore, many countries have formulated the recommended intake value of n-3 PUFAs and recommended intake ratio between n-6 and n-3 FAs.

Individuals should focus on increasing n-3 PUFAs intake while limiting excessive n-6 PUFAs intake to meet the recommended range for the consumption ratio of n-6 and n-3.

Fish and products derived from seafood are the richest sources of dietary n-3 PUFAs. However, there are many problems faced by fish resources rich in n-3 PUFAs. For instance, marine toxins and infectious agents have been found in seafood products, including parasites (e.g., nematodes, tapeworms and flukes) and bacteria [START_REF] Gil | Fish, a Mediterranean source of n-3 PUFA: benefits do not justify limiting consumption[END_REF]. Overfishing destroys the balance of the ecosystem and leads to a decrease in marine resources. Nevertheless, fortification of food with n-3

PUFAs is considered as an efficient strategy to meet the recommended intake threshold. Nowadays, there are many strategies to realize enrichment of n-3 PUFAs in food, mainly focusing on the following aspects (Saini & Keum, 2018): 1) fortify n-3 PUFAs in oilseed crops by using genetic engineering; 2) enrich n-3 PUFAs in animal and animal products by feeding animals n-3 PUFAs enriched diets; 3) supply n-3 PUFAs in foods and improve their sensory properties by using encapsulation techniques.

Different strategies have their advantages and disadvantages. In the present research, the encapsulation technique to realize the supplementation in n-3 PUFAs has been chosen. Due to the physical and chemical characteristics of n-3 PUFAs (e.g., highly unsaturated) there are many challenges associated with the use of n-3 PUFAs in food products on the production, transportation, storage, sensory acceptability and bioavailability [START_REF] Liu | Fabrication, characterization and properties of DHA-loaded nanoparticles based on zein and PLGA[END_REF][START_REF] Ma | Enhancing the physicochemical stability and digestibility of DHA emulsions by encapsulation of DHA droplets in caseinate/alginate honeycomb-shaped microparticles[END_REF][START_REF] Singh | Nanoencapsulation of docosahexanoic acid (DHA) using combination of food grade polymeric wall materials and its application for improvement in bioavailability and oxidative stability[END_REF]. However, many problems have been solved due to the development of encapsulation techniques.

Different kinds of encapsulation techniques have been used to encapsulate n-3 PUFAs, including emulsion [START_REF] Ghosh | Comparative study of gastrointestinal absorption of EPA & DHA rich fi sh oil from nano and conventional emulsion formulation in rats[END_REF][START_REF] Ma | Enhancing the physicochemical stability and digestibility of DHA emulsions by encapsulation of DHA droplets in caseinate/alginate honeycomb-shaped microparticles[END_REF]Ruiz & Perez, 2020), nanoliposome (Rasti et al., 2017), nanoparticles [START_REF] Serini | Omega-3 PUFA loaded in resveratrol-based solid lipid nanoparticles: Physicochemical properties and antineoplastic activities in human colorectal cancer cells in vitro[END_REF], microencapsulation (Ruiz et al., 2017), hydrogel particles [START_REF] Zhang | Encapsulation , protection , and release of polyunsaturated lipids using biopolymer-based hydrogel particles[END_REF], inclusion complexes [START_REF] Paul | Chapter 7-Nanoformulation and administration of PUFA-rich systems for applications in modern healthcare[END_REF], etc. Among them, emulsions are widely accepted methods. However, classical emulsions are thermodynamically unstable and tend to breakdown over time through aggregation and coalescence of the dispersed phase [START_REF] Mcclements | Advances in fabrication of emulsions with enhanced functionality using structural design principles[END_REF]. Compared with traditional emulsion, Pickering emulsification of food oils has received significant interest as it relies on (irreversible) adsorption of (bio)particles rather than (synthetic) surfactants to the oil-water interface, which confers resistance to droplet coalescence and improve the stability of emulsion [START_REF] Chevalier | Emulsions stabilized with solid nanoparticles: Pickering emulsions[END_REF][START_REF] Low | Recent advances of characterization techniques for the formation, physical properties and stability of Pickering emulsion[END_REF][START_REF] Wu | Recent Studies of Pickering Emulsions: Particles Make the Difference[END_REF].

Whey protein isolate (WPI) is commonly used in food formulations and also commonly used as wall material for encapsulation due to its various nutritional values and functions, such as antiaging [START_REF] Liu | Retard of D-Galactose-Induced Aging in Mice by Whey Hydrolysates[END_REF], anticancer [START_REF] Shariatikia | Anticancer activity of cow, sheep, goat, mare, donkey and camel milks and their caseins and whey proteins and in silico comparison of the caseins[END_REF], film formation (Schmid & Müller, 2019), emulsification [START_REF] Masson | A study of oil-in-water emulsions stabilized by whey proteins[END_REF], and gelation [START_REF] Singh | Thermal denaturation, aggregation and gelation of whey proteins[END_REF]. Due to the susceptibility of proteins in heat treatment, which is necessary during food procedures, WPI will denature and cause emulsion instability [START_REF] Chen | Food-grade Pickering emulsions: Preparation, stabilization and applications[END_REF]. However, the functionality of WPI can be modified via physical or chemical modification, which provides more possibilities for the application of WPI in Pickering emulsion. Based on our previous research, hydrophobised whey protein particles were successfully prepared through successive acetylation and heat treatment practices [START_REF] Madadlou | Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins[END_REF]. Whey proteins after heat-treatment have been exploited as a

Pickering stabilizer of foams [START_REF] Destribats | Emulsions stabilised by whey protein microgel particles: Towards food-grade Pickering emulsions[END_REF]. However, the applicability of the hydrophobised whey protein particles as Pickering emulsifiers was not reported. Therefore, in the present research, two different particle-stabilized (hydrophobised and heat-denatured WPI) Pickering emulsions were prepared, and after analyzing their properties, an emulsion system (Pickering emulsion stabilized with heat-denatured WPI particles) was selected for subsequent experiments.

There has been extensive research on the development and characterization of Pickering emulsions, however, there is less information about their potential applications in real food products.

It is well recognized that the bioavailability of sensitive compounds is impacted by the food matrix [START_REF] Aguilera | The food matrix: Implications in processing, nutrition and health[END_REF][START_REF] Dima | Bioavailability of nutraceuticals: Role of the food matrix, processing conditions, the gastrointestinal tract, and nanodelivery systems[END_REF]. Although fermented milk products are widely used as a vector of encapsulated nutrients, our previous study showed that eggs present an interesting bioavailability of docosahexaenoic acid (DHA) according to the recipe (omelet, hard-boiled egg or mousse). The omelet supplied 56% and 120% more DHA in plasma of pigs than the hard-boiled egg and the mousse after 48h of feeding, respectively [START_REF] Pineda-Vadillo | In Vivo Digestion of Egg Products Enriched with DHA: Effect of the Food Matrix on DHA Bioavailability[END_REF]. Therefore, in order to maximize the efficiency of DHA absorption in vivo, in the present study, omelets were selected as the food matrix.

When new vehicles of bioactive lipid compounds are developed, the gastrointestinal stability, bioaccessibility, and bioavailability of the bioactive compounds added to the products should be one of the major concerns. Because this information can allow evaluating the fraction of the ingested compound available to be absorbed (bioaccessibility) [START_REF] Solomando | Lipid digestion products in meat derivatives enriched with fish oil microcapsules[END_REF]; or allow evaluating the fraction of ingested nutrient or bioactive compound that reaches the systemic circulation and ultimately utilized (bioavailability) [START_REF] Galanakis | What is the difference between bioavailability bioaccessibility and bioactivity of food components[END_REF]. However, based on our knowledge, there is no published data on the gastrointestinal release and bioaccessibility of omelets enriched with DHA oil enriched Pickering emulsion, nor even of researches focused on the effect of Pickering emulsion on the metabolism of DHA in vivo.

In order to achieve this objective, the following three-step strategy was adopted (Figure 1):

1. The first part of the thesis aims at realizing the encapsulation of DHA oil. In order to achieve this objective, the following two aspects were studied: firstly, preparation and properties analysis (particle size, ζ-potential, surface hydrophobicity, and interfacial tension) of protein particles based on whey protein as raw material for Pickering emulsion;

secondly, DHA oil (composed of DHA-enriched triacylglycerols (TAG)) enriched

Pickering emulsion preparation and characteristics analysis (droplet size, optical microscopy, creaming stability, rheological behavior, and oxidative stability).

The results of this part are present in chapter 2: CaCl2 supplementation of hydrophobised whey proteins: Assessment of protein particles and consequent emulsions. 3. The third part of the thesis aims at determining the effect of encapsulation on the metabolism of DHA in vivo. In order to achieve this objective, 24 rats (3-weeks-old) were equally divided into three groups. They were acclimated for one week with the habituation diet and were then fed with the treatment diet for four weeks. During this period, rats were daily fed with omelets containing encapsulated DHA oil (EN-DHA-O), DHA oil (UN-DHA-O) or without DHA oil (Control). In the omelets containing DHA, DHA was provided as TAG to achieve 2.3% over total FAs. Finally, different tissues (plasma, red blood cell, liver, heart, brain, retina) were collected and the FA profiles from different tissues were determined by GC-MS. At the same time, the profiles of oxidized metabolites of fatty acids from plasma, brain, and heart were also analyzed by LC-QQQ.

The results of this part are present in chapter 4: Encapsulation of DHA oil substantially improves the oxylipin profile of rat tissues.

Apart from the already mentioned chapters, the first chapter including a bibliographic review is also included, as well as a final general conclusion and perspective section at the end of the present manuscript. at the other end. They are an important part of lipids and are also the important energy substrates.

Some FAs play an important part in the structure and function of cellular membranes and are precursors of lipid mediators which play a key role in cardiovascular and inflammatory diseases [START_REF] Tvrzicka | Fatty acids as biocompounds: Their role in human metabolism, health and disease-A review. Part 1: Classification, dietary sources and biological functions[END_REF]. Although in recent years public health and medical authorities have highlighted many "bad" health effects of dietary FAs, for instance, leading to coronary heart disease [START_REF] Reis | Dietary fatty acids and coronary heart disease[END_REF], high blood sugar, high blood pressure, and dyslipidemia (Russo, 2009), the intake of Therefore, covering the needs in functional FA as derivatives requires precursors from vegetable food or directly derivatives from animal food and in particular fish. The biosynthetic pathway of n-3 PUFAs takes place predominately in the liver, despite the brain possessing the enzyme equipment necessary for their synthesis [START_REF] Layé | Anti-inflammatory effects of omega-3 fatty acids in the brain: Physiological mechanisms and relevance to pharmacology[END_REF]. It starts in the endoplasmic reticulum and the initial step is the desaturation of ALA to stearidonic acid (SDA, 18:4n-3) by Δ6-desaturase, which is generally considered to be the rate-limiting reaction in the pathway (Figure 1-2). Besides, the synthesis of ALA to DHA is inefficient, generally <1% conversion [START_REF] Domenichiello | Is docosahexaenoic acid synthesis from α-linolenic acid sufficient to supply the adult brain?[END_REF].

Since the conversion of n-3 and n-6 PUFAs shares the same enzymes, there is metabolic competition between them. The rate-limiting enzyme Δ6-desaturase has a higher affinity for ALA in the n-3 pathway than for LA in the n-6 pathway [START_REF] Rodriguez | Δ6-and Δ5-desaturase activities in the human fetal liver: Kinetic aspects[END_REF]Sayanova et al., 2003).

However, due to the relative excess of LA in Western diets, the metabolism of n-6 PUFAs is favoured.

In addition to the availability of the essential FA substrates and the competition between n-3

and n-6 PUFAs, a number of other factors have been demonstrated to regulate the pathway of n-3

PUFAs. For instance, DHA metabolism is impacted by gender and hormones. [START_REF] Burdge | α-Linolenic acid metabolism in adult humans: The effects of gender and age on conversion to longer-chain polyunsaturated fatty acids[END_REF] reported that conversion of ALA to EPA occurs but was limited in men and that further transformation to DHA was very low. A lower proportion of ALA was used for β-oxidation in women compared with men, while the fractional conversion to the longer-chain n-3 PUFAs was greater, possibly due to the regulatory effects of oestrogen [START_REF] Burdge | α-Linolenic acid metabolism in men and women: Nutritional and biological implications[END_REF]. Both n-6 and n-3 PUFAs are necessary for cells to maintain normal structure, function, and signal transduction. There is increasing evidence that the ratio between the precursor of n-6 and n-3 PUFAs is more important than the absolute value of PUFAs intake for human health [START_REF] Giles | Omega-3 fatty acids and cognitive behavior[END_REF]. For instance, the metabolism of n-6 PUFAs mainly produces pro-inflammatory eicosanoids (e.g., prostaglandin, leukotriene, thromboxane), while the metabolism of n-3 PUFAs produces antiinflammatory derivatives (eicosanoids or docosanoids e.g., maresins, resolvins and protectins).

Recommendation for the consumption of n-3 polyunsaturated fatty acids

Therefore, the ratio of n-6 to n-3 influences the inflammation status in tissues. Nowadays the imbalance of the n-6 and n-3 intake ratio caused by the change of the diet habits has become an important dietary characteristic in modern society. Typical Western diets tend to be much higher in n-6 than in n-3 PUFAs. The consumption ratio between the precursors of n-6 and n-3 PUFAs in different Western countries is higher than 4:1 (

Sources of n-3 polyunsaturated fatty acids

As already stated in 1.2, human cannot synthesize de novo n-3 PUFA precursors or produce n-3 PUFA derivatives from their precursors in large enough quantities. They have thus to obtain them from their diet. In daily diet, the richest food sources of n-3 PUFAs are fish and seafood, especially marine fish, which are richer in n-3 PUFA derivatives than freshwater fish. A previous study showed that marine fish species have a higher level of DHA (12 to 36% of total FAs) than freshwater fish species (7 and 25% of total FAs) from Turkey (Özogul et al., 2007). Besides, according to the lipid contents, fish can be classified into lean fish (less than 5% fat), mid-fat fish (5 to10% fat), and fatty fish (10 to 25% fat) [START_REF] Taşbozan | Fatty acids in fish[END_REF]. In general, fatty fish contains more n-3 PUFAs than lean fish or mid-fat fish (Table 1- 3). For instance, fatty fish, such as mackerel, herring and salmon contain an average of 2 g long-chain n-3 PUFA per 100g of fish, while lean fish, such as cod, haddock and plaice, contain an average of 0.3 g long-chain n-3 PUFA per 100 g of fish (Ruxton et al., 2005). Microalgae is also an important source of n-3 PUFAs. For instance, Dinophytes and

Haptophytes, are well-known DHA-producers, which can produce high amounts of DHA up to 40% and 30% of the total FAs, respectively [START_REF] Da Costa | Effects of growth phase and nitrogen limitation on biochemical composition of two strains of Tisochrysis lutea[END_REF][START_REF] Mendes | Crypthecodinium cohnii with emphasis on DHA production: A review[END_REF]. At present, numerous algal species have been used in food industries. For instance, cultivated microalgae, such as Cryptehecodinium cohnii, its oil contains around 40% of DHA. The formula generated by this cultivated microalgae has been recommended for infants and young children by the United States Food and Drug Administration [START_REF] Wiktorowska-Owczarek | PUFAs: Structures, metabolism and functions[END_REF]. Other microalgae, such as Arthrospira sp., and Nannochloropsis oculate have also been authorized as a fermenting agent or as an ingredient in food processing to prepare lactose-free beverages [START_REF] Niccolai | Lactic acid fermentation of Arthrospira platensis (spirulina) in a vegetal soybean drink for developing new functional lactose-free beverages[END_REF] or functional cookies [START_REF] Babuskin | Functional foods enriched with marine microalga Nannochloropsis oculata as a source of ω-3 fatty acids[END_REF] in France, Europe, and USA. Besides, other microalgae which biosynthesize DHA, have been successfully cultivated, but this approach is costly and technically demanding, and affected by many factors as well (e.g., light intensity, and temperature) [START_REF] Remize | Microalgae n-3 PUFAs Production and Use in Food and Feed Industries[END_REF]. In addition to fish and microalgae, eggs, chicken breast, beef, pork, also contain a small amount of n-3 PUFAs.

PUFAs derived metabolites

Oxylipins are lipid mediators produced from the metabolism of PUFAs and play important roles in human health and disease (including hyperlipidemia, hypertension, thrombosis, hemostasis, and diabetes) by regulating inflammation and cellular homeostasis [START_REF] Nayeem | Role of oxylipins in cardiovascular diseases review-article[END_REF].

Endocannabinoids are also lipid mediators metabolized from PUFAs, they have a homeostatic role by controlling several metabolic functions, such as energy storage and nutrient transport [START_REF] Sharkey | Endocannabinoids: Biology, mechanism of action and functions[END_REF]. Because of their multiple physiological functions, oxylipins and endocannabinoids are receiving more and more attention.

Oxylipins

Oxylipins represent a group of FA metabolites generated via enzymatic and non-enzymatic oxygenation of PUFAs, including prostaglandins, leukotrienes, thromboxanes, lipoxins, maresins, resolvins and protectins. These compounds are involved in inflammation, immunity, pain, vascular tone, and coagulation [START_REF] Caligiuri | Dietary modulation of oxylipins in cardiovascular disease and aging[END_REF]. Oxylipins can be found throughout the body in all tissues, urine and blood [START_REF] Gabbs | Advances in our understanding of oxylipins derived from dietary PUFAs 1,2[END_REF]. Many studies have reported that oxylipins have a short half-time (minutes to hours) and cannot be stored in the body [START_REF] Caligiuri | Elevated levels of proinflammatory oxylipins in older subjects are normalized by flaxseed consumption[END_REF][START_REF] Nayeem | Role of oxylipins in cardiovascular diseases review-article[END_REF]Wang et al., 2021a). In contrast with other's opinions, [START_REF] Gabbs | Advances in our understanding of oxylipins derived from dietary PUFAs 1,2[END_REF] reported that not all oxylipins have a short half-time considering the steady-state concentration of both free and esterified oxylipins in tissues such as the liver, adipose tissue, the kidney and ileum. Besides, the free forms are presumably the biologically active oxylipins, but the functions of those that are found esterified to phospholipid are not known. According to the family of PUFAs, oxylipins can be classified depending on the original double bonds of the FA they derived from (Figure 1-3). Oxylipins can be produced via enzymatic and non-enzymatic pathways. Among them, the enzymatic pathway mainly involves cyclooxygenases (COX), lipoxygenases (LOX) and cytochrome P450 (CYP). COX, which catalyze the formation of prostaglandins, mainly have two different isoforms COX-1 and COX-2, both of them are expressed in mammalian tissues [START_REF] Markworth | Emerging roles of pro-resolving lipid mediators in immunological and adaptive responses to exercise-induced muscle injury[END_REF]. COX-1 is a ubiquitous enzyme expressed constitutively in normal tissues;

COX-2 is a predominantly inducible enzyme, considered to be mainly responsible for the production of prostanoids in inflammation [START_REF] Rouzer | Cyclooxygenases: Structural and functional insights[END_REF]. Although COX-2 has a major role, COX-1 also contributes in the initial stage of inflammation [START_REF] Tomić | Clinical uses of nonsteroidal anti-inflammatory drugs (NSAIDs) and potential benefits of NSAIDs modified-release preparations[END_REF]. LOX catalyze the formation of hydroperoxide derivatives which can be further converted to leukotrienes, lipoxins, hepoxilins, etc [START_REF] Qian | Lipids in the transcriptional regulation of adipocyte differentiation and metabolism[END_REF]. In the humans, six LOX isoforms (including 15-LOX, 15-LOX-2, 12-LOX, 12R-LOX, eLOX3 and 5-LOX) [START_REF] Mashima | The role of lipoxygenases in pathophysiology; new insights and future perspectives[END_REF] have been known, among them, the most widely studied are 15-LOX, 12-LOX and 5-LOX. Different PUFAs can generate different oxylipins under the action of these enzymes. For instance, 15-LOX is involved in the generation of specialized pro-resolving lipid mediators (SPMs) that play essential roles in resolution and inflammatory responses [START_REF] Kim | Anti-inflammatory role of 15-lipoxygenase contributes to the maintenance of skin integrity in mice[END_REF]; 12-LOX is involved in the generation of hepoxilins which regulate physiological processes such as inflammation, insulin secretion and pain perception in human (An et al., 2018); 5-LOX is involved in the formation of leukotrienes which are a group of lipid mediators of inflammation derived from ARA (Rå dmark & Samuelsson, 2009).

CYP can be divided into ω-hydroxylase and epoxygenase enzymes according to their activity.

Hydroxy-eicosatetraenoic acids produced by ARA under the activity of ω-hydroxylase can cause inflammation, vasoconstriction and vascular remodeling, while epoxy-eicosatrienoic acids produced by ARA under the activity of epoxygenase can resolve inflammation and cause vasodilation, and have the effect of protecting heart function [START_REF] Tacconelli | Inside epoxyeicosatrienoic acids and cardiovascular disease[END_REF].

1. Alzheimer's disease [START_REF] Wang | Role of 2-series prostaglandins in the pathogenesis of type 2 diabetes mellitus and non-alcoholic fatty liver disease[END_REF]. Among them, prostaglandin E2, is one of the most abundant prostaglandins and exhibits versatile biological activities in the body. For instance, when prostaglandin E2 binds to different receptors, it can regulate the function of many cell types including macrophages, dendritic cells and T and B lymphocytes leading to both pro-and antiinflammatory effects [START_REF] Ricciotti | Prostaglandins and inflammation[END_REF]. In addition to prostaglandins, thromboxanes and leukotrienes are also pro-inflammatory lipid mediators. Studies indicated that leukotrienes may interact with a variety of tissue cells, contributing to the low-grade inflammation of cardiovascular, neurodegenerative, and metabolic conditions, as well as that of cancer [START_REF] Tian | Leukotrienes in tumor-associated inflammation[END_REF].

Similar to ARA, DGLA can be metabolized to form 1-series prostaglandins with the COX pathway, including prostaglandin E1 and D1. Besides, DGLA can also be converted into hydroxy

FAs (e.g., 15-hydroxy-eicosatrienoic acid (15-HETrE)) with the LOX pathway (Figure 1234).

Prostaglandins from the 1-series are also pro-inflammatory and play a vital role in a number of key areas, although compared to 2-series prostaglandins, they received less attention. For instance, prostaglandin H1 has been identified as a potent activator of the pro-inflammatory receptor CRTH2

(chemoattractant receptor homologous molecule expressed on T helper type 2 cells) and strengthens the role of this receptor as an important player in allergic inflammation (Schröder et al., 2012). Oxylipins derived from LA can be synthesized by the LOX and CYP pathways. With the LOX pathway, LA can be metabolized into 9-hydroxyoctadecadienoic acid (HODE) and 13-HODE under the activity of 5-LOX and 15-LOX, respectively. With the CYP pathway, LA can be converted into dihydroxy FAs (e.g., 9,10-dihydroxy-octadecenoic acid (9,10-DiHOME)) (Figure 1234). In the last two decades, most studies indicated 9-HODE and 13-HODE are related to the development of inflammatory diseases, particularly atherosclerosis [START_REF] Pecorelli | 13-HODE, 9-HODE and ALOX15 as potential players in Rett syndrome oxInflammation[END_REF][START_REF] Vangaveti | Hydroxyoctadecadienoic acids: Novel regulators of macrophage differentiation and atherogenesis[END_REF].

For instance, 13-HODE is the predominant HODE in early atherosclerosis, while in advanced disease 9-HODE is at least as abundant [START_REF] Vangaveti | Hydroxyoctadecadienoic acids: Novel regulators of macrophage differentiation and atherogenesis[END_REF]. However, different from the result in atherosclerosis, 9-HODE and 13-HODE showed opposing effects in skin. In keratinocytes, 9-HODE has pro-inflammatory actions mediated through G protein-coupled receptor 132 (GPR132) [START_REF] Hattori | G2A plays proinflammatory roles in human keratinocytes under oxidative stress as a receptor for 9-hydroxyoctadecadienoic acid[END_REF], while 13-HODE is not a ligand for GPR132 and accumulates in the ceramides and phospholipids in the skin as an anti-inflammatory actor [START_REF] Cho | Incorporation of 13-hydroxyoctadecadienoic acid (13-HODE) into epidermal ceramides and phospholipids: Phospholipase C-catalyzed release of novel 13-HODE-containing diacylglycerol[END_REF].

Oxylipins derived from n-3 polyunsaturated fatty acids

Researches on oxylipins of n-3 PUFAs mainly focus on EPA and DHA. EPA is also a 20-carbon PUFA, which is metabolized by 5-LOX and COX to generate 5-series leukotrienes and 3-series prostaglandins, respectively. However, these EPA derived eicosanoids have a different structure from the ARA derived eicosanoids, and this influences their potency [START_REF] Calder | Omega-3 fatty acids and inflammatory processes[END_REF]. For instance, compared with leukotriene B4, which is derived from ARA, EPA derived leukotriene B5 is 10-to 100-fold less potent as a neutrophil chemoattractant [START_REF] Goldman | Human neutrophil chemotactic and degranulating activities of leukotriene B5 (LTB5) derived from eicosapentaenoic acid[END_REF][START_REF] Lee | Characterization and biologic properties of 5, 12-dihydroxy derivatives of eicosapentaenoic acid, including leukotriene B5 and the double lipoxygenase product[END_REF].

Moreover, eicosanoids derived from EPA may antagonize the action of those produced from ARA [START_REF] Tull | Omega-3 Fatty acids and inflammation: Novel interactions reveal a new step in neutrophil recruitment[END_REF].

In addition to eicosanoids, EPA can also be metabolized to resolvins, which belongs to SPMs, whereas, DHA can be metabolized either to resolvins or maresins and protectins, all of them belonging to SPMs as well. These SPMs have as main activity the resolution of inflammation. The synthesis way of SPMs is shown in Figure 12345. EPA is metabolized into E-series resolvins by COX-2/aspirin and CYP via 18-HEPE by 5-or 15-LOX. DHA is converted into 17-HDoHE by COX-2/aspirin, 5-LOX and 15-LOX and then into D-series resolvins by 5-LOX. During this step, protectins also can be produced. Besides, DHA can also be metabolized into 14-HDoHE and then into maresins. SPMs actively orchestrate and finely tune the inflammatory response. They can accelerate the phagocytosis of cellular debris and dead cells without immune suppression [START_REF] Joffre | Polyunsaturated fatty acid metabolism in the brain and brain cells. In Feed your mind-How does nutrition modulate brain function throughout life? IntechOpen[END_REF]. There have been many reports on the physiological functions of SPMs, particularly in brain and heart. D-series and E-series resolvins can promote functional recovery and neuroprotection by reducing the activation of microglia and astrocytes [START_REF] Hamlett | RvE1 treatment prevents memory loss and neuroinflammation in the Ts65Dn mouse model of Down syndrome[END_REF][START_REF] Luo | Enriched endogenous omega-3 fatty acids in mice protect against global ischemia injury[END_REF], reducing inflammatory-induced neuronal loss [START_REF] Bisicchia | Resolvin D1 halts remote neuroinflammation and improves functional recovery after focal brain damage via ALX/FPR2 receptor-regulated microRNAs[END_REF], preventing mitochondrial dysfunction and rescuing the dysregulation of mitophagy [START_REF] Kang | Resolvin D1 attenuates liver ischaemia/reperfusion injury through modulating thioredoxin 2-mediated mitochondrial quality control[END_REF]. In addition, they also show great anti-inflammatory effects in CVD. They can reduce inflammatory state associated with atherosclerosis, inhibit neutrophil tissue infiltration and platelet aggregation, induce neutrophil apoptosis and decrease pro-inflammatory cytokines production [START_REF] Capó | Resolvins as proresolving inflammatory mediators in cardiovascular disease[END_REF]. Except resolvins, protectins and maresins also display both anti-inflammatory and pro-resolving activities in acute or chronic inflammatory-related diseases. For instance, protectins synthesized in white adipose tissue have anti-inflammatory effects in obesity and diabetes [START_REF] Hansen | The protectin family of specialized pro-resolving mediators: Potent immunoresolvents enabling innovative approaches to target obesity and diabetes[END_REF]. Maresin can be antiinflammatory and reduce pain by stimulating tissue regeneration and resolution [START_REF] Serhan | Macrophage proresolving mediator maresin 1 stimulates tissue regeneration and controls pain[END_REF].

Endocannabinoids

Endocannabinoids are derivatives of FA or more complex lipids that are synthesized ubiquitously in the body. They act as lipid mediators through two different cannabinoid receptors 1 (CB1) or 2 (CB2) expressed throughout the body. After cell stimulation, they are inactivated by degradation into the target cell via a two-step process that begins with their transport from the extracellular to the intracellular space and culminates in their intracellular hydrolysis by the FA amide hydrolase [START_REF] Cascio | PUFA-derived endocannabinoids: An overview[END_REF]. The two endogen cannabinoids are anandamide (arachidonoyl ethanolamide or AEA) and 2-arachidonoylglycerol (2-AG), the others are considered as endocannabinoid-like compounds. They are all ligands of CB1 and/or CB2 as agonists or antagonists, depending on the lipid mediators and the tissue of the receptor expression. CB1 receptors are ubiquitous but mostly found in the central nervous system [START_REF] Zou | Cannabinoid receptors and the endocannabinoid system: Signaling and function in the central nervous system[END_REF] whereas CB2 are mainly expressed in periphery, especially in blood cells and in blood-cell producing organs [START_REF] Navarro | Targeting cannabinoid CB2 receptors in the central nervous system. Medicinal chemistry approaches with focus on neurodegenerative disorders[END_REF]. If most of studies have concerned endocannabinoids from n-6 PUFAs, few cannabimimetic compounds have been found derived from n-3 PUFAs such as 2eicosapentaenoylglycerol (EPG), 2-docosahexaenoylglycerol (2-DHG) and Ndocosahexaenoylethanolamine (DHEA), which are primarily known for their anti-inflammatory effects [START_REF] Simopoulos | Omega-6 and omega-3 fatty acids: Endocannabinoids, genetics and obesity[END_REF].

Research on AEA and 2-AG is currently focused on their role in regulating appetite, food intake and energy metabolism [START_REF] Naughton | Fatty acid modulation of the endocannabinoid system and the effect on food intake and metabolism[END_REF][START_REF] Simopoulos | Omega-6 and omega-3 fatty acids: Endocannabinoids, genetics and obesity[END_REF][START_REF] Watkins | The endocannabinoid system: Directing eating behavior and macronutrient metabolism[END_REF]. AEA and 2-AG have been shown to promote inflammation and increase appetite and food intake. Since the supply of dietary lipids may influence the synthesis of AEA and 2-AG, the food ingested may be able to regulate levels of AEA and 2-AG, thereby affecting appetite and food intake. Previous studies revealed that long-term supplementation of EPA and DHA not only reduced the levels of AEA and 2-AG in many tissues (e.g., brain and liver) or modulated the levels of adipocyte phospholipids that act as endocannabinoid precursors, but also increased the level of 2-EPG, 2-DHG and DHEA [START_REF] Artmann | Influence of dietary fatty acids on endocannabinoid and Nacylethanolamine levels in rat brain, liver and small intestine[END_REF][START_REF] Matias | Effect of polyunsaturated fatty acids on endocannabinoid and N-acyl-ethanolamine levels in mouse adipocytes[END_REF][START_REF] Watanabe | N-3 Polyunsaturated fatty acid (PUFA) deficiency elevates and n-3 PUFA enrichment reduces brain 2-arachidonoylglycerol level in mice[END_REF]. Therefore, the metabolism of endocannabinoids can be regulated by increasing the intake of n-3PUFAs, thereby controlling food intake to help patients with obesity.

Altogether, metabolites derived from PUFAs, whether oxylipins or endocannabinoids, play an important role in regulating inflammation. Their metabolism in vivo can be adjusted through diet, thereby affecting health. However, the health benefits of n-3 PUFAs are not only regulated by oxylipins and endocannabinoids, but also involve other mechanisms.

Health benefits and mechanisms of n-3 polyunsaturated fatty acids

n-3 PUFAs have various health benefits, particularly in brain and heart, including preventing cognitive decline, preventing AD, antihypertensive, anticancer, antioxidant, anti-depression, and anti-arthritis effects [START_REF] Siriwardhana | Health benefits of n-3 polyunsaturated fatty acids: Eicosapentaenoic acid and docosahexaenoic acid[END_REF]. Besides, n-3 PUFA is also very important in retina.

Many studies reported a protective role of DHA in retina. For instance, many sight-threatening diseases have two key stages, vessel loss followed by hypoxia-driven destructive neovascularization [START_REF] Querques | Retina and omega-3[END_REF]. [START_REF] Connor | Increased dietary intake of ω-3polyunsaturated fatty acids reduces pathological retinal angiogenesis[END_REF] found that increasing omega-3 PUFA tissue levels through dietary or genetic means could decrease the avascular area of the retina by increasing the generation of vessel after injury, thereby reducing the hypoxic stimulation of neovascularization. [START_REF] Calder | Mechanisms of action of (n-3) fatty acids[END_REF].

Effect on brain

Nearly 60% of the dry-weight of the human brain consists of lipids and about 35% of the lipids in the grey matter are long-chain PUFA (Schipper et al., 2020). In the last decades, there are increasing evidence showing that the n-3 long-chain PUFAs (especially DHA) are involved in a wide range of physiological functions in brain. These functions are related to signal transduction pathway, neurogenesis, neurotransmission, neuroinflammation, myelination, membrane receptor function, synaptic plasticity, membrane integrity, and membrane organization [START_REF] Weiser | Docosahexaenoic Acid and Cognition throughout the Lifespan[END_REF].

The deficiency of n-3 PUFAs in the brain may lead to central nervous system dysfunction, such as cognitive impairment and mental disorder. Epidemiological and intervention studies have shown that infant, maternal, or cord blood DHA status was found to be positively correlated to cognitive outcomes [START_REF] Dunstan | Cognitive assessment of children at age 2 1/2 years after maternal fish oil supplementation in pregnancy: A randomised controlled trial[END_REF][START_REF] Helland | Maternal supplementation with very-long-chain n-3 fatty acids during pregnancy and lactation augments children's IQ at 4 years of age[END_REF][START_REF] Helland | Effect of supplementing pregnant and lactating mothers with n-3 very-long-chain fatty acids on children's iq and body mass index at 7 years of age[END_REF]. A similar result was also found in children and adults for whom higher plasma and red blood cell DHA and EPA levels showed higher cognitive performance or an improvement in attentional [START_REF] Dalton | A randomised control trial in schoolchildren showed improvement in cognitive function after consuming a bread spread, containing fish flour from a marine source[END_REF][START_REF] Fontani | Cognitive and physiological effects of Omega-3 polyunsaturated fatty acid supplementation in healthy subjects[END_REF]. Different from the previous results, for the elderly, the intake of n-3 PUFAs did not improve their cognitive performance but reduced the risk of cognitive decline [START_REF] Dangour | Fish consumption and cognitive function among older people in the UK: Baseline data from the OPAL study[END_REF][START_REF] Morris | Fish consumption and cognitive decline with age in a large community study[END_REF][START_REF] Nurk | Cognitive performance among the elderly and dietary fish intake: The Hordaland Health Study[END_REF]. The decrease of DHA in plasma can result in an increase in the production or accumulation of a specific protein, ßamyloid protein (Aß) in brain, leading to the death of nerve cells. This is the main reason for the cognitive decline in healthy elderly and Alzheimer's disease. It should be noted that there are also some results that did not show a positive relationship between n-3 PUFAs and cognitive performance [START_REF] Kennedy | Cognitive and mood effects of 8 weeks' supplementation with 400 mg or 1000 mg of the omega-3 essential fatty acid docosahexaenoic acid (DHA) in healthy children aged 10-12 years[END_REF][START_REF] Rogers | No effect of n-3 longchain polyunsaturated fatty acid (EPA and DHA) supplementation on depressed mood and cognitive function: A randomised controlled trial[END_REF] due to a number of factors, such as age [START_REF] De | The Groningen LCPUFA study: No effect of postnatal long-chain polyunsaturated fatty acids in healthy term infants on neurological condition at 9 years[END_REF], the cognitive baseline of the participant, the dosage of n-3 PUFAs administration, duration of experiments [START_REF] Martí | Omega-3 fatty acids and cognitive decline: A systematic review[END_REF], and gender [START_REF] Lassek | Sex differences in the relationship of dietary fatty acids to cognitive measures in American children[END_REF]. The possible mechanisms of n-3 PUFAs for improving cognitive functions are numerous and mainly result in the reduction of neuroinflammation and promotion of neuron functionalities (Table 1- 4).

Mechanism Reference

Synthesis of neuroprotectin D1 (derived from DHA) to modulated cell death by regulating anti-or pro-apoptotic mediators [START_REF] Bazan | Neuroprotectin D1 (NPD1): A DHA-derived mediator that protects brain and retina against cell injury-induced oxidative stress[END_REF][START_REF] Martí | Omega-3 fatty acids and cognitive decline: A systematic review[END_REF] Protectin Dx (PDx) exerts neuroprotective activity in vitro by reducing staurosporine-induced apoptosis of human SH-SY5Y neuroblastoma cells [START_REF] Zhu | Pro-resolving lipid mediators improve neuronal survival and increase Aβ 42 phagocytosis[END_REF] Decrease ARA and its metabolites availability in brain compartments, which contributes to glial and neuronal hyperactivation [START_REF] Calder | Omega-3 fatty acids and inflammatory processes[END_REF][START_REF] Cole | Omega-3 fatty acids and dementia[END_REF][START_REF] Martí | Omega-3 fatty acids and cognitive decline: A systematic review[END_REF] Increase of the antioxidant defenses: reduction of in vivo oxidant stress by either direct or indirect pathways [START_REF] Cole | Omega-3 fatty acids and dementia[END_REF][START_REF] Martí | Omega-3 fatty acids and cognitive decline: A systematic review[END_REF][START_REF] Mori | Effect of ω3 fatty acids on oxidative stress in humans: GC-MS measurement of urinary F2-isoprostane excretion[END_REF] Enhancement of the brain-derived neurotrophic factor levels [START_REF] Cole | Omega-3 fatty acids and dementia[END_REF][START_REF] Martí | Omega-3 fatty acids and cognitive decline: A systematic review[END_REF] Increase of phosphatidylserine concentration in the brain: faster translocation and phosphorylation of protein kinase B, which increases neuronal survival [START_REF] Akbar | Docosahexaenoic acid: A positive modulator of Akt signaling in neuronal survival[END_REF][START_REF] Martí | Omega-3 fatty acids and cognitive decline: A systematic review[END_REF] Promotion of neurogenesis and improvement in the fluidity in synaptic membranes [START_REF] Hashimoto | Docosahexaenoic acid-induced protective effect against impaired learning in amyloid β-infused rats is associated with increased synaptosomal membrane fluidity[END_REF][START_REF] Innis | Dietary (n-3) fatty acids and brain development[END_REF][START_REF] Martí | Omega-3 fatty acids and cognitive decline: A systematic review[END_REF] Increase of a glucose transporter which improves brain glucose transport [START_REF] Martí | Omega-3 fatty acids and cognitive decline: A systematic review[END_REF][START_REF] Pifferi | N-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood-brain barrier[END_REF] Improvement of G-protein coupling involved in signal transduction pathways whose deficiency is associated with cognitive deficits [START_REF] Litman | The role of docosahexaenoic acid containing phospholipids in modulating G protein-coupled signaling pathways: Visual transduction[END_REF][START_REF] Martí | Omega-3 fatty acids and cognitive decline: A systematic review[END_REF] Decrease C-reactive protein and increased glutathione concentrations [START_REF] Taghizadeh | Neurochemistry International The effects of omega-3 fatty acids and vitamin E co-supplementation on clinical and metabolic status in patients with Parkinson ' s disease: A[END_REF] Enhance dopaminergic signal transduction (Role et al., 2019) Anti-inflammatory, anti-amyloid, and anti-tau effects [START_REF] Belkouch | ScienceDirect The pleiotropic effects of omega-3 docosahexaenoic acid on the hallmarks of Alzheimer ' s disease[END_REF][START_REF] Huang | Clinical trials of new drugs for Alzheimer disease[END_REF]

Mental disorders

Deficiency of n-3 PUFAs in brain may lead to some mental disorders, including schizophrenia, depression disorder, bipolar disorder, anxiety disorders, obsessive-compulsive disorder, posttraumatic stress disorder, autism spectrum disorders, and borderline personality disorder [START_REF] Bozzatello | Mental health in childhood and adolescence: The role of polyunsaturated fatty acids[END_REF]. Among them, depression disorder is the most common psychiatric disorder affecting about 300 million people worldwide (4.4% of the global population) (World Health Organization, 2017). The possible mechanisms of n-3 PUFAs in the prevention of mental disorders are varied and aim at reducing neuroinflammation and oxidative stress (Table 1- [START_REF]Reference list[END_REF].

Mechanism Reference

Modulation of G-protein signaling via G-protein coupled receptors and effects on lipid raft formation [START_REF] Senese | The role of G-proteins and G-protein regulating proteins in depressive disorders[END_REF] Modulation of pro-inflammatory mediators, increase in telomerase levels as well as a reduction in oxidative stress [START_REF] Su | Progress in Neuropsychopharmacology & Biological Psychiatry Eicosapentaenoic and docosahexaenoic acids have di ff erent e ff ects on peripheral phospholipase A2 gene expressions in acute depressed patients[END_REF] Modulation of the membrane FA homeostasis in cortical [START_REF] Horrobin | The membrane phospholipid hypothesis as a biochemical basis for the neurodevelopmental concept of schizophrenia[END_REF][START_REF] Horrobin | The membrane hypothesis of schizophrenia[END_REF] Modulation of dopaminergic pathways in the mesolimbic system (Role et al., 2019) Increase in brain-derived neurotrophic factor [START_REF] Dong | Dietary eicosapentaenoic acid normalizes hippocampal omega -3 and 6 polyunsaturated fatty acid profile , attenuates glial activation and regulates BDNF function in a rodent model of neuroinflammation induced by central interleukin -1 β administration[END_REF][START_REF] Matsuoka | Factor in Omega -3 Fatty Acid Supplementation to Prevent Posttraumatic Distress after Accidental Injury: An Open-Label Pilot Study[END_REF][START_REF] Okubo | Prostaglandins , Leukotrienes and Essential Fatty Acids Mechanisms underlying the e ff ects of n-3 polyunsaturated fatty acids on fear memory processing and their hypothetical e ff ects on fear of cancer recurrence in cancer survivors[END_REF] Mitigate neuroinflammation and anti-oxidation effects [START_REF] Giacobbe | The Anti-Inflammatory Role of Omega-3 Polyunsaturated Fatty Acids Metabolites in Pre-Clinical Models of Psychiatric, Neurodegenerative, and Neurological Disorders[END_REF] Promote hippocampal neurogenesis [START_REF] Beltz | Omega-3 fatty acids upregulate adult neurogenesis[END_REF][START_REF] Kawakita | Docosahexaenoic acid promotes neurogenesis in vitro and in vivo[END_REF] 1.6.2 Effect on heart

Mechanism Reference

Alterations in plasma lipoprotein profiles, particularly by reducing triglyceride concentrations and reducing the ratio between low-density lipoprotein and high-density lipoprotein [START_REF] Roche | Long-chain n-3 polyunsaturated fatty acids and triacylglycerol metabolism in the postprandial state[END_REF] Reduce endothelial activation [START_REF] Yamagata | Docosahexaenoic acid regulates vascular endothelial cell function and prevents cardiovascular disease[END_REF] Anti-arrhythmic effects [START_REF] Leaf | Clinical prevention of sudden cardiac death by n-3 polyunsaturated fatty acids and mechanism of prevention of arrhythmias by n-3 fish oils[END_REF][START_REF] Tribulova | Omega-3 index and anti-arrhythmic potential of omega-3 PUFAs[END_REF] Anti-atherosclerotic effects [START_REF] Sekikawa | Marine-Derived n-3 Fatty Acids and Atherosclerosis in Japanese, Japanese-American, and White Men. A Cross-Sectional Study[END_REF] Improve plaque stability and anti-thrombotic effects [START_REF] Colussi | Impact of omega-3 polyunsaturated fatty acids on vascular function and blood pressure: Relevance for cardiovascular outcomes[END_REF][START_REF] Robinson | Antiatherosclerotic and Antithrombotic Effects of Omega-3 Fatty Acids[END_REF] Improve cardiac filling and myocardial efficiency [START_REF] Mozaffarian | Omega-3 fatty acids and cardiovascular disease: Effects on risk factors, molecular pathways, and clinical events[END_REF] Anti-inflammatory effects mediated by nuclear receptor, G-protein coupled receptors [START_REF] Endo | Cardioprotective mechanism of omega-3 polyunsaturated fatty acids[END_REF] Reduce resting heart rate, produces vasodilation, reduce blood pressure (Sagara et al., 2011) CVD is described as the leading cause of morbidity and mortality in modern societies [START_REF] Joseph | Reducing the global burden of cardiovascular disease, part 1: The epidemiology and risk factors[END_REF]. There are many CVD risk factors, such as plasma triglycerides and cholesterol, heart rate, blood pressure, thrombosis, arrhythmia, etc [START_REF] Mozaffarian | Omega-3 fatty acids and cardiovascular disease: Effects on risk factors, molecular pathways, and clinical events[END_REF]. Epidemiological researches have shown the beneficial effects of dietary n-3 PUFAs in the prevention and treatment of CVD. For instance, [START_REF] Kris-Etherton | Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease[END_REF] reported that supplementation with 2 to 4 g/day EPA and DHA can lower the level of triglyceride in plasma by approximately 25% to 30% in patients with hypertriglyceridemia. Supplementation with 4g/day EPA decreased triglyceride levels by 12% in healthy subjects [START_REF] Grimsgaard | Highly purified eicosapentaenoic acid and docosahexaenoic acid in humans have similar triacylglycerol-lowering effects but divergent effects on serum fatty acids[END_REF]. Therefore, therapeutics for CVD patients are now completed with DHA-rich oil to achieve a better health status considering the multiple mechanisms (e.g., anti-arrhythmic and anti-atherosclerotic effects) triggered by n-3 PUFAs (Table 1- [START_REF] Watkins | The endocannabinoid system: Directing eating behavior and macronutrient metabolism[END_REF].

Although there are multiple mechanisms to explain the role of n-3 PUFA in both neuroprotection and cardioprotection, it remains unclear which mechanism contributes the most to the protective effects of n-3 PUFAs. In order to better understand the action mechanism and the evolution of n-3 PUFAs involved in vivo, it is necessary to understand their digestion, absorption and metabolism process.

Digestion, absorption and tissue distribution of dietary lipids

Dietary lipids are mainly composed of triacylglycerols (TAG) (90-95%) [START_REF] Iqbal | Intestinal lipid absorption[END_REF] and phospholipids (PL) (1-10%) [START_REF] Cohn | Dietary phospholipids and intestinal cholesterol absorption[END_REF]. The remainder of the dietary lipids consists primarily of cholesterol (CL), cholesterol esters (CE), and free fatty acids (FFA). They are also transporters for fat-soluble vitamins. Dietary lipids digestion, absorption and tissue distribution is a multi-step process, mainly including the following aspects (Figure 123456): 1) the dispersion and rearrangement of dietary lipids, 2) the hydrolysis of dietary lipids by gastric and pancreatic lipases,

3) the desorption and dispersion of insoluble lipid products into an absorbable form (micelles); 4)

the re-synthesis of lipids in the endoplasmic reticulum to form chylomicrons, which are released into the lymphatic and thereafter the blood vessels; 5) the uptake of lipids by tissues mainly transported by lipoproteins in blood [START_REF] Carey | Lipid digestion and absorption[END_REF][START_REF] Michalski | Bioavailability and metabolism of dietary lipids[END_REF].

Digestion of dietary lipids

TAG are the most abundant dietary lipids, which are composed of one glycerol molecule acylated with three FAs (Figure 1234567). TAG are hydrolyzed in the gastrointestinal tract (GIT) by lipase adsorbed on the surface of the oil droplets. In the GIT, there are various lipases, including gastric lipase, pancreatic lipase, phospholipase A2 (PLA2), pancreatic lipase related protein-2, etc.

Among them, gastric lipase and pancreatic lipase are the most important, and their relative contribution to the hydrolysis of ingested TAG are around 10-25% and 40-70% [START_REF] Bauer | Principles of physiology of lipid digestion[END_REF][START_REF] Carriere | Secretion and contribution to lipolysis of gastric and pancreatic lipases during a test meal in humans[END_REF], respectively. [START_REF] Armand | Digestion and absorption of 2 fat emulsions with different droplet sizes in the human digestive tract[END_REF] reported higher hydrolysis of ingested TAG (around 40%) in gastric phase. However, their study corresponded to given time points in the gastric phase at the end of the gastric emptying but does not reflect the overall contribution of gastric lipolysis during a meal. The first step in the digestion of TAG takes place in the stomach and is performed by gastric lipase or lingual lipase depending on the species considered. Rodents (eg., mice and rats) only have lingual lipase and no gastric lipase (Moreau et al., 1988a), whereas primates have a high activity of gastric lipase [START_REF] Denigris | Lingual and gastric lipases: Species differences in the origin of prepancreatic digestive lipases and in the localization of gastric lipase[END_REF][START_REF] Mu | The digestion of dietary triacylglycerols[END_REF]. Therefore, the first step in the digestion of TAG in the human body occurs mainly under the catalysis of gastric lipase which is released from the gastric mucosa and can act in a wide range of pH from 3.0 to 6.0 [START_REF] Meynier | Molecular and structural organization of lipids in foods: Their fate during digestion and impact in nutrition[END_REF]. Gastric lipase hydrolyzes preferentially the sn-3 ester bond of TAG [START_REF] Rogalska | Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases[END_REF] resulting in the formation of 1,2-diacylglycerol (1,2-DAG) and FFA. As the pre-digested food goes (containing 1,2-DAG, FFA as well as unhydrolyzed TAG) further into the small intestine, it can cause bladder emptying, pancreatic lipase secretion and cholecystokinin releasing (J. H. [START_REF] Meyer | Canine pancreatic responses to intestinally perfused fat and products of fat digestion[END_REF][START_REF] Watanabe | Role of pancreatic enzymes on release of cholecystokinin-pancreozymin in response to fat[END_REF]. During this process, pre-digested food is mixed with pancreatic lipase and bile in the duodenum. Bile is made in liver, stored in the gallbladder, and released into the duodenum [START_REF] Di Gregorio | Physiology and Physical Chemistry of Bile Acids[END_REF]. Bile salt is the main surfactant in bile and it has both a hydrophobic and a hydrophilic side, so bile salts are able to be attracted to both fats and water. This feature makes them as effective emulsifiers, they can break large fat globules into smaller droplets, which could increase the interaction area between oil and pancreatic lipase and improve the lipolytic action of pancreatic lipase (Chen & Cassaro, 2019).

Pancreatic lipase is released from the pancreatic tissue into the duodenum upon the hormonal response of cholecystokinin. Then, pancreatic lipase acts on the surface of water and lipid with the co-lipase as a mediator to overcome the expulsion of the lipase into the water phase caused by bile salts and digests the TAG [START_REF] Ben Bacha | Biochemical properties of pancreatic colipase from the common stingray Dasyatis pastinaca[END_REF][START_REF] Mu | The digestion of dietary triacylglycerols[END_REF][START_REF] Van Tilbeurgh | Colipase: Structure and interaction with pancreatic lipase[END_REF].

Pancreatic lipase hydrolyzes specifically the sn-1 and sn-3 ester bonds and subsequently results in the formation of 2-monoacylglycerol (2-MAG) and two FFA (Figure 12345678). During this step, pancreatic lipase could be responsible for hydrolyzing about 98 % of the remaining dietary TAG in healthy humans [START_REF] Porsgaard | Differences in the intramolecular structure of structured oils do not affect pancreatic lipase activity in vitro or the absorption by rats of (n-3) fatty acids[END_REF]. PL are the second abundant dietary lipids. They are a class of lipids with a hydrophilic "head" containing a phosphate group, and two hydrophobic "tails" composed of FA, esterified to the glycerol backbone (Figure 123456789). According to different headgroups, PL can be mainly divided into PC (phosphatidylcholine), PE (phosphatidylethanolamine), PS (phosphatidylserine), PI (phosphatidylinositol), PG (phosphatidylglycerol) and DPG (diphosphatidylglycerol). For instance, PC contains a choline head-group, whereas PE contains an ethanolamine head-group.

Due to their polar head-group, PL are hydrophilic compounds, whereas TAG are highly hydrophobic compounds. Thus, these two groups of lipids have different physical-chemical properties due to the different structures and only PL are able to directly interact with water through hydration of their polar heads to form micelles, liposomes, and other structured assemblies in the presence of water [START_REF] Genot | Bioavailability of Lipids in Fish and Fish Oils[END_REF]. Since gastric lipase is incapable of hydrolyzing PL, the digestion of PL takes place in the small intestinal by PLA2. PLA2 is secreted from the pancreas in response to food intake. It catalyzes the hydrolysis of PL at sn-2 position, which generates one FFA and a 2-lysophospholipid (2-lyso-PL) (Figure 123456789). In addition to TAG and PL, a very small amount of dietary lipids composed of FA is derived from cholesterol as CE. CE can be hydrolyzed into CL and FFA under the catalysis of cholesterol esterase which is found primarily in the pancreas (Figure 1-10). The digestion of dietary lipids is affected by the molecular structures of lipid, including the type of FAs (carbon length and unsaturation) and the positions (sn-1, sn-2 or sn-3) of esterification in dietary lipids [START_REF] Guo | Modulating fat digestion through food structure design[END_REF][START_REF] Michalski | Bioavailability and metabolism of dietary lipids[END_REF][START_REF] Ramırez | Absorption and distribution of dietary fatty acids from different sources[END_REF]. Previous researches have proven that lipids (focus on TAG) enriched in short-chain FAs (< C6) exhibit a higher degree of hydrolysis of these FAs, than those bearing long-chain FAs (> C12); lipids rich in unsaturated FAs exhibit a higher degree of hydrolysis of these FAs, than those bearing saturated FAs [START_REF] Na | In Vitro and In Vivo Digestibility of Soybean, Fish, and Microalgal Oils, and Their Influences on Fatty Acid Distribution in Tissue Lipid of Mice[END_REF][START_REF] Ramırez | Absorption and distribution of dietary fatty acids from different sources[END_REF]. In particular, PUFAs, which have a double bond close to the carboxyl group, have a lower hydrolysis rate, because of their steric hindrance [START_REF] Bottino | Resistance of certain long-chain polyunsaturated fatty acids of marine oils to pancreatic lipase hydrolysis[END_REF][START_REF] Lawson | Human absorption of fish oil fatty acids as triacylglycerols, free acids, or ethyl esters[END_REF].

In general, molecular diversity of dietary lipids is not a brake of hydrolysis by different lipases in the GIT, even if the process of catalysis is affected by the molecular lipid structures. The main hydrolyzed products of dietary lipids are FFA and 2-MAG, with a small amount of lyso-PL and CL.

Absorption and metabolism of dietary lipids

The hydrolyzed products of lipid digestion are water-insoluble, therefore they need to be incorporated into mixed micelles to become solubilized in the intestinal lumen before being absorbed by the enterocytes. Bile salts cluster around the products of lipid digestion to form micelles, which help the lipids get close enough to the microvilli of intestinal cells so that they can be absorbed.

When FAs are composed of a short-chain (< C6) or a medium-chain (C6-C10), they do not need to form micelles, because they are relatively water-soluble and can enter the intestinal epithelial cells from the intestinal lumen. These FAs can be then secreted through the basement membrane in the form of albumin complexes into the portal vein which carries blood from the digestive organs to the liver [START_REF] Ramırez | Absorption and distribution of dietary fatty acids from different sources[END_REF]. When FAs are composed of a long-chain (> C12), they need to form micelles to help their solubilization. When micelles are dissociated in contact with the brush border of the small intestine, they release the lipid hydrolysis products (FFA, 2-MAG, lyso-PL and CL). In enterocytes, the apical uptake of long-chain FA mainly is likely mediated by both diffusion and protein-mediated transfer (Figure 1-11), including plasma membrane fatty acid binding protein (FABPpm), FA transport protein (FATP) 4 and caveolin 1 [START_REF] Michalski | Bioavailability and metabolism of dietary lipids[END_REF][START_REF] Ratnayake | Fat and fatty acid terminology, methods of analysis and fat digestion and metabolism[END_REF][START_REF] Wahbeh | Basic aspects of digestion and absorption[END_REF]. Besides, lipid binding proteins are also involved during the absorption process. For instance, cluster of differentiation 36 (CD36), which is a member of a superfamily of scavenger receptor class B designated as SR-B2, appears to be the predominant membrane protein facilitating FA transport, although the mechanism is not well understood. For CL, Niemann-Pick C1-like 1 (NPC1L1) and scavenger receptor class B1 (SR-B1) are their transports and help them across the brush border membrane [START_REF] Feingold | Introduction to lipids and lipoproteins[END_REF][START_REF] Michalski | Bioavailability and metabolism of dietary lipids[END_REF].

In the endoplasmic reticulum of intestinal epithelial cells, FAs and 2-MAG are re-esterified sequentially by MAG acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT) to form TAG; lyso-PL are esterified to form PL by acyltransferase; CL are esterified as CE by acyl-CoA:

cholesterol acyltransferase (ACAT) [START_REF] Shi | Lipid metabolic enzymes: Emerging drug targets for the treatment of obesity[END_REF]. After re-synthesis, intestinal lipids are mainly packaged into chylomicrons. Chylomicron assembly begins in the endoplasmic reticulum and mainly involves two steps, the formation of primordial chylomicron and pre-chylomicron [START_REF] Mansbach | The biogenesis of chylomicrons[END_REF]. Primordial chylomicrons are formed in the endoplasmic reticulum by lipidation of newly synthesized apolipoprotein (Apo) B48 with TAG via the microsomal triglyceride transfer protein (MTTP). In the absence of lipidation, Apo B48 is degraded. Apo A-IV then combines with CL, CE, and PL to form pre-chylomicrons. Pre-chylomicrons are exported from the endoplasmic reticulum to the Golgi apparatus thanks to the pre-chylomicron transport vesicles (PCTV). During this process, secretion-associated Ras-related GTPase 1B (SAR1b) is essential for the transit of PCTV to the Golgi and thus for the ultimate ability of enterocytes to secrete chylomicrons into the mesenteric lymph. Then, in the Golgi, the pre-chylomicrons acquire Apo A-I to become mature chylomicrons, which consist of TAG and CE in the core and consist of PL, CL and apolipoproteins on the surface (Figure 1-12) [START_REF] Galanakis | Lipids and Edible Oils: Properties, processing and applications[END_REF]. Mature chylomicrons are ultimately exported to the basolateral pole of the enterocyte where they are released into the lymphatic circulation [START_REF] Michalski | Bioavailability and metabolism of dietary lipids[END_REF]. protein (LRP) [START_REF] Burri | Marine omega-3 phospholipids: Metabolism and biological activities[END_REF][START_REF] Feingold | Introduction to lipids and lipoproteins[END_REF][START_REF] Ramırez | Absorption and distribution of dietary fatty acids from different sources[END_REF].

Then, the metabolism of lipids from chylomicron remnants is mainly through the formation of a series of lipoproteins (Figure 1-13), which are identified by their lipid content and the specific apolipoprotein on the surface. The first step is the synthesis of very low-density lipoprotein (VLDL) in liver, which contains TAG and CE in its core. TAG carried in VLDL are hydrolyzed by LPL releasing FA and forming intermediate-density lipoprotein (IDL). Then, the IDL is further catabolized by hepatic lipase (HL) to low-density lipoprotein (LDL), which is taken up by the LDL-R in numerous tissues (particularly in liver) [START_REF] Feingold | Introduction to lipids and lipoproteins[END_REF][START_REF] Zhyvotovska | Introductory Chapter: Overview of Lipoprotein Metabolism[END_REF].

Excess CE is exported from the cells and delivered to HDL to be returned to the liver, where it will be redistributed to other tissues or removed from the body by the gallbladder [START_REF] Marques | Reverse cholesterol transport: Molecular mechanisms and the non-medical approach to enhance HDL cholesterol[END_REF].

In general, under healthy conditions, around 95% of ingested lipids are absorbed by the GIT and transferred to the target organs, and the rest is excreted from the body in the form of feces [START_REF] Zhang | Dietary bioactive lipids: A review on absorption, metabolism, and health properties[END_REF]. Similar to the uptake of FAs in enterocytes, the uptake of FAs in different tissue cells is also mainly by diffusion and protein-mediated transport. During the past years, considerable data have demonstrated that protein-mediated transport accounts for the majority of FA uptake by tissues with high FA metabolism and storage such as skeletal muscle, adipose tissue, liver, and heart [START_REF] Doege | Protein-mediated fatty acid uptake: Novel insights from in vivo models[END_REF]. Particularly, a previous report that at physiological concentrations and serum-toalbumin ratios, the concentration of unbound FAs is low (7.5 nmol/L) and more than 90% of longchain FAs uptake occurs via the protein-mediated pathway [START_REF] Richieri | Unbound free fatty acid levels in human serum[END_REF]. At present, there are several types of proteins involved in the transport of FAs, including CD36, FABPs, FTAPs, long-fatty acyl-CoA synthetases (ACSL), and acyl-CoA-binding protein (ACBP). These proteins differ widely in their expression pattern in the body as well as in their subcellular localization. For instance, the family FATPs is comprised of six related members in humans (FATP1 to FATP6).

FATP1 is the major FATP family member expressed in adipose tissue, FATP1 protein is also found in skeletal muscle and, to a lesser extent, in heart; FATP2 is predominantly expressed in liver and kidney; FATP4 is the only FATP family member in the small intestine where it is localized to the apical brush border of epithelial cells; FATP5 is expressed solely in liver [START_REF] Doege | Protein-mediated fatty acid uptake: Novel insights from in vivo models[END_REF].

For n-3 PUFAs, the important target tissues are brain, retina and heart [START_REF] Schuchardt | Bioavailability of long-chain omega-3 fatty acids[END_REF]. However, n-3 PUFAs transport into the brain is more complicated due to the existence of blood-brain barrier (BBB), which is a physical protective barrier between the cerebral blood vessels and parts of the central nervous system to prevent the entry of potentially harmful substances from the blood. FAs pass into the brain entail traversing first the luminal and abluminal membranes of the endothelial cells that comprise the BBB, and then the plasma membrane of the neural cells [START_REF] Liu | Pathways of polyunsaturated fatty acid utilization: Implications for brain function in neuropsychiatric health and disease[END_REF]. There are still controversies about whether the main form of PUFA entering the brain is esterified or non-esterified, and the relative importance of passive diffusion and protein facilitated transport [START_REF] Bazinet | Polyunsaturated fatty acids and their metabolites in brain function and disease[END_REF][START_REF] Layé | Anti-inflammatory effects of omega-3 fatty acids in the brain: Physiological mechanisms and relevance to pharmacology[END_REF][START_REF] Liu | Pathways of polyunsaturated fatty acid utilization: Implications for brain function in neuropsychiatric health and disease[END_REF]. For instance, [START_REF] Ouellet | Diffusion of docosahexaenoic and eicosapentaenoic acids through the blood-brain barrier: An in situ cerebral perfusion study[END_REF] reported that the main route of DHA transport is passive diffusion. Their in site trans-cardiac perfusion studies have indicated that the BBB transport of 14 C-DHA is unaffected by increasing concentrations of unlabelled DHA, consistent with a non-saturable process. However, others have demonstrated the involvement of membrane transporters (e.g., FATP1, FATP4 and CD36) in the BBB transport of PUFAs [START_REF] Mitchell | Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells[END_REF][START_REF] Nguyen | Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid[END_REF][START_REF] Ochiai | The bloodbrain barrier fatty acid transport protein 1 (FATP1/SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport[END_REF], because they found that the transport of DHA from lysophosphatidylcholine was significantly reduced after knocking out the Mfsd2a transport protein [START_REF] Nguyen | Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid[END_REF]. Besides, the bioavailability of DHA is also controversial depending on the intake form of DHA. Some studies have attempted to compare DHA esterified to PL and TAG and concluded that bioavailability of DHA was similar when dietary DHA was esterified to PL or TAG [START_REF] Schuchardt | Incorporation of EPA and DHA into plasma phospholipids in response to different omega-3 fatty acid formulations-A comparative bioavailability study of fish oil vs. Krill oil[END_REF][START_REF] Vidal | Bioavailability and spatial distribution of fatty acids in the rat retina after dietary omega-3 supplementation[END_REF]. On the contrary, other studies indicated that dietary DHA esterified to PL promoted a higher bioavailability of DHA than dietary DHA esterified to TAG [START_REF] Destaillats | Comparison of the Incorporation of DHA in Circulatory and Neural Tissue When Provided as Triacylglycerol (TAG), Monoacylglycerol (MAG) or Phospholipids (PL) Provides New Insight into Fatty Acid Bioavailability[END_REF][START_REF] Ramprasath | Enhanced increase of omega-3 index in healthy individuals with response to 4-week n-3 fatty acid supplementation from krill oil versus fish oil[END_REF]. For instance, [START_REF] Ramprasath | Enhanced increase of omega-3 index in healthy individuals with response to 4-week n-3 fatty acid supplementation from krill oil versus fish oil[END_REF] compared the concentration of n-3 PUFA in plasma by giving the healthy volunteers krill oil (a phospholipid based oil) or fish oil (a triglyceride-based oil) for 4 weeks, both of them containing 600 mg of n-3 PUFA per day. Their result showed that krill oil consumption increased plasma n-3 PUFA concentrations, including EPA and DHA, and reduced the ratio of n-6

and n-3 PUFA compared with fish oil consumption. In addition to the bioavailability, there is also evidence that DHA esterified to PL may target the tissue (especially in brain) more efficiently than DHA esterified to TAG. [START_REF] Graf | Age dependent incorporation of 14C-DHA into rat brain and body tissues after dosing various 14C-DHA-esters[END_REF] reported that rats were orally administered 14 C-DHA esterified to sn-2 of PC or TAG, after 24h, they found higher radioactivity in brain regions of 10 weeks old rats gavaged with 14 C-DHA-PC, but not in 2 weeks or 4 weeks old rats. Similarly, [START_REF] Liu | Higher efficacy of dietary DHA provided as a phospholipid than as a triglyceride for brain DHA accretion in neonatal piglets[END_REF] orally administered 13 C-DHA esterified in the sn-2 position of TAG or PC to neonatal piglets (on day 16 of life), after receiving the labelled DHA for 6 days, they found approximately twofold greater labelled DHA in grey matter and synaptosomes of 13 C-DHA-PC gavaged piglets than 13 C-DHA-TAG gavaged piglets. Moreover, [START_REF] Kitson | Effect of dietary docosahexaenoic acid (DHA) in phospholipids or triglycerides on brain DHA uptake and accretion[END_REF] reported that a higher percentage of ingested [U-3 H]PC-DHA radioactivity was found in brain lipids compared with [U-

3 H]TAG-DHA. However, the higher brain radiotracer uptake in [U-3 H]PC-DHA gavage did not translate into higher brain DHA concentrations in PL-DHA fed rats compared with TAG-DHA fed rats in the 4-week supplementation study. The possible reasons for the discrepancies in the results of these studies may imply several factors such as the level of n-3 PUFA in diet, the animal model and the animal ages. For instance, 2% of DHA in diet is suggested to be the dietary DHA content at which brain DHA is maximal in mouse [START_REF] Orr | The Fat-1 Mouse has Brain Docosahexaenoic Acid Levels Achievable Through Fish Oil Feeding[END_REF]. Therefore, when the content of DHA in diet exceeds 2%, it may be difficult to find a difference between diets composed of PL-DHA and TAG-DHA. Altogether, the bioavailability of DHA and the further accretion of DHA in brain may be affected by the amount of DHA in diet and the intake form, PL-DHA being supposedly more effective than TAG-DHA in very specific nutritional conditions.

Moreover, as mentioned before, for n-3 PUFAs, the important target tissues are the brain, retina and heart. In these tissues, n-3 PUFAs are primarily present in cell membranes and are incorporated into phospholipids, as well as sphingolipids and plasmalogens [START_REF] Arterburn | Distribution, interconversion, and dose response of n-3 fatty acids in humans[END_REF]. The presence of n-3 PUFAs modulates the biophysical properties (e.g., fluidity, thickness and deformability) of cell membranes [START_REF] Stillwell | Docosahexaenoic acid affects cell signaling by altering lipid rafts[END_REF], which in turn affects the activity of transmembrane proteins [START_REF] Rajamoorthi | Packing and viscoelasticity of polyunsaturated ω-3 and ω-6 lipid bilayers as seen by 2H NMR and X-ray diffraction[END_REF], leading to significant effects on cell and organ function as well as a wide variety of biological processes. For instance, plasmalogens are a class of membrane glycerophospholipids with unique properties, which constitute 20% of the total phospholipid mass in humans [START_REF] Braverman | Functions of plasmalogen lipids in health and disease[END_REF]. Their structure contains fatty alcohol with a vinyl-ether bond at the sn-1 position and is enriched in PUFAs at the sn-2 position of the glycerol backbone [START_REF] Braverman | Functions of plasmalogen lipids in health and disease[END_REF]. The sn-1 position is primarily derived from either 16:0, 18:0 or 18:1 carbon chain, and the head group is usually ethanolamine or choline. Therefore, based on the head group, plasmalogens can be mainly divided into two groups, ethanolamine plasmalogens (PlsEtn)

and choline plasmalogens (PlsCho). The sn-2 position is predominately occupied by ARA or DHA carbon chain [START_REF] Su | Plasmalogens and Alzheimer's disease: A review[END_REF]. Although the role of plasmalogens has not yet been fully elucidated, studies suggest that they have unique functions in the cells, and these functions are directly related to the bonds of sn-1 vinyl ether and sn-2 positions of PUFAs [START_REF] Braverman | Functions of plasmalogen lipids in health and disease[END_REF]. A previous report indicated that plasmalogens can protect other PL, lipid and lipoprotein particles from oxidative stress by oxidizing the vinyl ether bond and protecting the PUFAs present in the sn-2 oxidation position [START_REF] André | Plasmalogen metabolism-related enzymes in rat brain during aging: Influence of n-3 fatty acid intake[END_REF]. Reduced levels of plasmalogens in plasma are connected with various diseases, including CVD [START_REF] Paul | Plasmalogens: A potential therapeutic target for neurodegenerative and cardiometabolic disease[END_REF], AD [START_REF] Igarashi | Disturbed choline plasmalogen and phospholipid fatty acid concentrations in Alzheimer's disease prefrontal cortex[END_REF], hypertension [START_REF] Maeba | Plasmalogens in human serum positively correlate with high-density lipoprotein and decrease with aging[END_REF], cancer [START_REF] Messias | Plasmalogen lipids: Functional mechanism and their involvement in gastrointestinal cancer[END_REF], respiratory disease (Rüdiger et al., 2000), etc.

The possible mechanisms related to the action of plasmalogens include helping maintain the membrane physical bilayer properties to facilitate membrane fusion and signal transduction processes, including cholesterol efflux, preventing oxidative stress and reducing inflammatory response [START_REF] Su | Plasmalogens and Alzheimer's disease: A review[END_REF].

Altogether, lipid digestion, absorption, transport and metabolism are complex processes, that can be affected by many factors (e.g., lipid structure, different lipase, different metabolism pathways) [START_REF] Na | In Vitro and In Vivo Digestibility of Soybean, Fish, and Microalgal Oils, and Their Influences on Fatty Acid Distribution in Tissue Lipid of Mice[END_REF][START_REF] Ramırez | Absorption and distribution of dietary fatty acids from different sources[END_REF]. The lipid digestion process is the first step that will highly affect lipid absorption and metabolism, thus it is very important to consider it in nutrition when diets are designed. In addition to the effects of different lipid molecules on the digestion process mentioned before, there are also some factors, such as the food matrix and digestion model that can play a significant role on the results obtained.

Food matrix and digestion model

Food matrix

In the past, the nutritional evaluation of food used to be largely based on its nutrient profile:

protein, fat, carbohydrates, vitamins, and minerals [START_REF] Turgeon | Food matrix impact on macronutrients nutritional properties[END_REF]. With the improvement of people's quality of life and the emergence of various chronic diseases, people are paying more attention to the importance of diet on health. The actual contribution of each food portion to the recommended daily nutrient intake has received more and more attention. However, increasing data suggest that health benefits from foods result not only from the amount of a particular nutrient we eat but also the mix of nutrients and how they interact within a more complex structure. This involves the notion of the food matrix, which consists of the organization of food components at multiple spatial scales and their interactions [START_REF] Pineda-Vadillo | Développement de produits des laitiers et ovoproduits enrichis en bioactifs contre le syndrome métabolique: Effet de la matrice alimentaire sur la bioaccessiblité et la biodisponibilité des polyphénols et de l'acide docosahexaénoïque[END_REF].

For instance, a study [START_REF] Shen | Food Matrix Effects on in Vitro Digestion of Microencapsulated Tuna Oil Powder[END_REF] compared the lipolysis rate of microencapsulated tuna oil and different foods with microencapsulated tuna oil (yogurt, orange juice, or cereal bar) in vitro.

The results showed that a higher level of lipolysis of n-3 PUFAs enriched-TAG was observed in microencapsulated tuna oil and fortified yogurt (73.2-78.6%) than in the cereal bars (60.3-64.0%) after sequential exposure to simulated gastric and intestinal fluids. Moreover, [START_REF] Lakkis | Encapsulation and controlled release technologies in food systems[END_REF] compared the bioavailability of some n-3 FA enriched products on the market (Table 1234567). Their results revealed that the bioavailability of n-3 PUFA was influenced by the different food matrices, including the quality of fish oil, the type of product as well as the impact of other nutrients that can act as bioavailability enhancers or inhibitors in the enriched foods. Intake of n-3 enriched tomato juice was found to lower serum levels of the cardiovascular risk factor homocysteine but did not alter blood lipids.

Bread and pasta

• Tip top® the one TM n-3 DHA sandwich (tip top, Australia)

• multi grain n-3 (Alpine valley bread, USA) • n-3 bread (Slice of health n-3, India) • wholewheat n-3 (Pasta toscana, Italy) • BARILLA PLUS® (Barilla, Italy)
Ingestion of bread containing microencapsulated tuna oil, both in an acute and a chronic study, resulted in more n-3 FA in plasma lipids, and reduced the level of blood lipids. (From Lakkis, 2016) At present, many DHA enriched foods based on the addition of DHA oil have been developed, including cheese (Bermúdez-Aguirre & Barbosa-Cá novas, 2011;[START_REF] Nguyen | Enhancement of dairy sheep cheese eating quality with increased n-3 long-chain polyunsaturated fatty acids[END_REF]), yogurt (Dal Bello et al., 2015;[START_REF] Estrada | Developing a strawberry yogurt fortified with marine fish oil[END_REF][START_REF] Gumus | Yogurts supplemented with lipid emulsions rich in omega-3 fatty acids: New insights into the fortification, microencapsulation, quality properties, and health-promoting effects[END_REF] and sausage (Pavlí k et al., 2014;[START_REF] Solomando | Lipid digestion products in meat derivatives enriched with fish oil microcapsules[END_REF]. However, most of these studies focused on studying the effect of encapsulation on the bioaccessibility or bioavailability of n-3 PUFAs (Lane et al., 2014;[START_REF] Raatz | Enhanced Absorption of n-3 Fatty Acids from Emulsified Compared with Encapsulated Fish Oil[END_REF] or comparing the effect of different delivery systems on the bioaccessibility or bioavailability of n-3 PUFAs (Sanguansri et al., 2015;[START_REF] Solomando | Lipid digestion products in meat derivatives enriched with fish oil microcapsules[END_REF], but they did not study the impact of food matrix on the bioaccessibility or bioavailability of n-3 PUFAs. Based on our previous research, we found that eggs can lead to a high bioavailability of DHA depending on the type of products [START_REF] Pineda-Vadillo | In Vivo Digestion of Egg Products Enriched with DHA: Effect of the Food Matrix on DHA Bioavailability[END_REF]. In this research, pigs were fed with the three DHA-enriched egg matrices (omelet, hard-boiled egg, and mousse) and DHA bioavailability was estimated from the DHA concentration in plasma over a period of 48 h postprandial. The results revealed that the omelet was the most efficient vector for delivering DHA into the systemic circulation. It supplied 56% and 120% more DHA than the hard-boiled egg and the mousse, respectively. Therefore, in order to maximize the efficiency of DHA absorption in the body, the choice of vector food should be considered.

Digestion model

In vivo model

In order to know the food matrix effect on the bioaccessibility or bioavailability of nutrients, various digestion models have been developed. Different digestion models can be mainly classified into two categories: first is in vivo models, including human models and animal models; second is in vitro models, including static models and dynamic models.

In vivo models are physiologically-relevant but they are usually time-consuming, laborintensive, expensive, lead to high inter-individual variations and involve ethical and practical reasons. There are not many studies using in vivo digestion models available in the literature, especially for human models.

A direct method for measuring digestibility in humans consists in using probes in gastric or intestinal compartments for collecting the gastric and intestinal effluents [START_REF] Fardet | Structure des aliments et effets nutritionnels[END_REF].

However, there are many disadvantages associated with this method. For instance, the impossibility of aspirating relatively big solid food particles and the existence of few adequate digestion markers to quantify the digestive secretions and fluxes. In addition to direct methods, there are also some indirect methods based on the ingestion of a marker with the meal and the measurement of its excretion in the expired air can be also used. Nevertheless, these indirect methods are usually used to investigate both digestive and metabolic processes, and they are more appropriate to compare different modalities than to determine absolute kinetic measurement [START_REF] Pineda-Vadillo | Développement de produits des laitiers et ovoproduits enrichis en bioactifs contre le syndrome métabolique: Effet de la matrice alimentaire sur la bioaccessiblité et la biodisponibilité des polyphénols et de l'acide docosahexaénoïque[END_REF].

Animal models are commonly used in vivo digestion models. Rats and pigs are the two most common animal models in literature for the measurement of macronutrient digestibility, no matter in alone or in complex regimes [START_REF] Pineda-Vadillo | Développement de produits des laitiers et ovoproduits enrichis en bioactifs contre le syndrome métabolique: Effet de la matrice alimentaire sur la bioaccessiblité et la biodisponibilité des polyphénols et de l'acide docosahexaénoïque[END_REF][START_REF] Steingoetter | A Rat Model of Human Lipid Emulsion Digestion[END_REF]. Rats are an ideal choice for many labs due to their physiological similarity to humans. They are easier to feed and smaller in size than other complex animals. Similar to human beings, rats are mono-gastric animals, their digestive systems exhibit many similarities and very few discrepancies [START_REF] Pineda-Vadillo | Développement de produits des laitiers et ovoproduits enrichis en bioactifs contre le syndrome métabolique: Effet de la matrice alimentaire sur la bioaccessiblité et la biodisponibilité des polyphénols et de l'acide docosahexaénoïque[END_REF]. Therefore, they are frequently used in nutrition for total digestibility measurements. However, different from human beings, the rat digestive system has an enlarged large intestine but does not have a gallbladder that exists in the human digestive system [START_REF] Turumin | The role of the gallbladder in humans[END_REF]. Bile from the rat liver flows directly through the (hepatic) bile duct into the small intestine [START_REF] Fitzgerald | Anatomy and embryology of the laboratory rat[END_REF].

Another model that is often used in in vivo experiments is the pig. The pig model has been utilized by researchers to study human developmental aspects, diseases impacts, surgical procedures, and nutritional interventions for over 60 years [START_REF] Sciascia | The pig as a model for humans: Effects of nutritional factors on intestinal function and health[END_REF]. Previous research reported that the GIT of pigs is highly similar to that of human beings anatomically and physiologically [START_REF] Guilloteau | Nutritional programming of gastrointestinal tract development. Is the pig a good model for man?[END_REF]. In addition, the functions of the pig GIT, such as how nutrients and metabolites produced by digestion or metabolism by resident microbiota are absorbed by the GIT, are closer to human beings than any other non-primate animal models [START_REF] Mario | Splanchnic Vasoregulation After Major Abdominal Surgery in Pigs[END_REF]. The pig model is generally used after the surgical installation of cannulas and/or vascular catheters; therefore, the same animal can be used in order to test different diets and products or to determine digestive kinetics without having to sacrifice animals for each point/treatment [START_REF] Pineda-Vadillo | Développement de produits des laitiers et ovoproduits enrichis en bioactifs contre le syndrome métabolique: Effet de la matrice alimentaire sur la bioaccessiblité et la biodisponibilité des polyphénols et de l'acide docosahexaénoïque[END_REF].

Ideally, food digestion should be studied with in vivo models, but this is not always ethically and financially possible. In order to solve this problem, more and more studies have focused on in vitro models.

In vitro model

In vitro models are common and widespread tools for rapid analyses because they are less expensive, less labor-intensive, and do not have the ethical restriction that in vivo studies have [START_REF] Porter | In vitro assessment of oral lipid based formulations[END_REF]. There are two main types of in vitro digestion models: static models and dynamic models.

In vitro static digestion models are getting more and more popular to study food digestion.

They are called static since there is no flow of the food between the different compartments, one step must be completed before the next one starts and the enzymatic and physicochemical conditions are kept constant [START_REF] Bohn | Correlation between in vitro and in vivo data on food digestion. What can we predict with static in vitro digestion models?[END_REF]. They have been applied to homogenized foods or isolated compounds in a closed system, whose products remain largely immobile in a single static bioreactor [START_REF] Mackie | Simulating human digestion: Developing our knowledge to create healthier and more sustainable foods[END_REF]. Static models usually include one or more portions of the GIT (gastric, intestinal) with a limited number of parameters of physiological digestion, but they do have mimic physical processes such as mixing, shearing, hydration, changes in conditions over time, or peristalsis [START_REF] Nguyen | A comprehensive review on in vitro digestion of infant formula[END_REF][START_REF] Verhoeckx | The impact of food bioactives on health: In vitro and Ex Vivo models[END_REF].

Dynamic in vitro digestion models can better simulate the physiological reality as long as relevant parameters are known [START_REF] Dupont | Can dynamic in vitro digestion systems mimic the physiological reality?[END_REF]. They can be used to simulate a series of variable factors during digestion, including the movement of the bolus, the variation of pH, the concentration of enzymes, the appropriate mixing, substrate-enzyme ratio, transport of digested products, etc [START_REF] Dupont | Can dynamic in vitro digestion systems mimic the physiological reality?[END_REF][START_REF] Sensoy | A review on the food digestion in the digestive tract and the used in vitro models[END_REF].

However, in vitro models also have their drawbacks. For instance, there are still differences between the lipases used in vitro digestion models and the lipases of the human digestive system, so sometimes it cannot accurately reflect the digestion conditions of the human body. At present, lipases used for the gastric phase of in vitro digestion experiments mainly come from animals and microorganisms. For instance, a number of microbial lipases, such as Rhizopus oryzae lipase (ROL) and Candida antarctica lipase A (CALA), have been used to simulate gastric digestion in vitro [START_REF] Barker | Application and validation of an advanced gastrointestinal in vitro model for the evaluation of drug product performance in pharmaceutical development[END_REF][START_REF] Christophersen | Fed and fasted state gastro-intestinal in vitro lipolysis: In vitro in vivo relations of a conventional tablet, a SNEDDS and a solidified SNEDDS[END_REF]. ROL and CALA have a preference towards the sn-1 and sn-2 position of TAG [START_REF] Pfeffer | The lipases from Candida antarctica: Cloning, expression and their application in the synthesis of structured lipids[END_REF][START_REF] Rogalska | Stereoselective hydrolysis of triglycerides by animal and microbial lipases[END_REF], respectively, while human gastric lipase (HGL) has a preference towards the sn-3 position [START_REF] Helbig | Digestion of dietary fat: Gastrointestinal behaviour of emulsions and human physiological responses[END_REF]. Animal derived lipases, such as dog gastric lipase (DGL) and rabbit gastric lipase (RGL) have also been used for in vitro gastric digestion. However, DGL showed higher activity on long-chain TAG than short-chain TAG [START_REF] Carriere | Purification and biochemical characterization of dog gastric lipase[END_REF], which is different from HGL that preferentially hydrolyzes short-and medium-chain TAG [START_REF] Koziolek | Lipids in the stomach-implications for the evaluation of food effects on oral drug absorption[END_REF]. For RGL, Moreau et al. (1988b) found that RGL is very similar to HGL in structure and kinetics by analyzing interfacial denaturation and catalytic properties of lipase on TAG emulsions. Besides, both HGL and RGL have their recommended pH is 3.0; at pH 4~5.4, around 50% of their maximum activity measured [START_REF] Gargouri | Kinetic assay of human gastric lipase on short-and long-chain triacylglycerol emulsions[END_REF]Moreau et al., 1988b). Therefore, RGL seems like a suitable replacement for HGL. However, there is still some difference between RGL and HGL. RGL is more stereoselective for TAG with shorter acyl chains. For instance, [START_REF] Rogalska | Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases[END_REF] studied the stereospecificity of HGL and RGL toward chemically alike, but sterically nonequivalent ester groups within one single TAG molecule. Their results showed that the stereoselectivity of human and rabbit gastric lipases, expressed as the enantiomeric excess percentage, was 54% and 70% for trioctanoin and 74% and 47% for triolein, respectively. This is one of the most striking catalytic differences observed between these two gastric lipases [START_REF] Rogalska | Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases[END_REF]. In addition to the impact of lipase, in vitro models also cannot take into account the influence of the intestinal microorganisms on the digestion process, so it is still different from the real in vivo situation.

In general, although there are clear weaknesses in in vitro models, they have obvious advantages over complex methods. The main strengths of static in vitro models are the good intraand inter-laboratory reproducibility, robustness, simplicity, and easy assessment of each digestion phase [START_REF] Brodkorb | INFOGEST static in vitro simulation of gastrointestinal food digestion[END_REF].

Strategy to enhance n-3 polyunsaturated fatty acids in food and improve the bioaccessibility

The insufficient intake of n-3 PUFAs increases the risk for chronic diseases. In order to improve the intake of essential nutrients, one option is to encourage the consumption of n-3 PUFAs enriched foods. Fish and products derived from seafood are the richest dietary source of n-3 long-chain PUFAs. Nevertheless, there are many side effects faced by fish resources rich in n-3 PUFAs, such as pollution of marine ecosystems that results in a high accumulation of toxic dioxins and heavy metals in fish chair. Excessive fishing destroys the balance of the ecosystem and leads to the reduction of marine resources as well. Furthermore, with the increasing number of unsaturated bonds, n-3 PUFAs are prone to oxidize and thus produce very volatile components (ketones, aldehydes and carboxylic acids) with a 'fishy' smell. Meanwhile, oxidation can reduce the nutritional value by causing the destruction of essential FA and the lipid-soluble vitamins A, D, E, and K as well as the decrease in caloric content. Therefore, based on these inconveniences, together with the price and the fishy taste, fish resources are not always the privileged choice for consumers.

It is necessary to develop different kinds of foods rich in n-3 PUFAs that are more easily accepted by some consumers [START_REF] Tao | Oxidation of polyunsaturated fatty acids and its impact on food quality and human health[END_REF].

Nowadays, there are many strategies to realize enrichment of n-3 PUFAs in food, mainly focusing on the following aspects (Saini & Keum, 2018): 1) using genetic engineering to fortify n-3 PUFAs in oilseed crops; 2) using n-3 PUFAs as precursors (ALA) in enriched diets to feed animals which are able to generate n-3 PUFAs as derivatives (for instance DHA) found then in animal products; 3) using encapsulation techniques to realize the supplement of n-3 PUFAs in food while improving the sensory properties. However, these different strategies have their advantages and disadvantages. For instance, [START_REF] Betancor | A nutritionally-enhanced oil from transgenic Camelina sativa effectively replaces fish oil as a source of eicosapentaenoic acid for fish[END_REF] have already produced EPA enriched seeds to substitute fish oil in aquafeeds by using the transgenic technique, whereas, the public is questioning the safety and the ethical production of organisms genetically modified. In addition, n-3 PUFAs enriched eggs produced by feeding laying hens with an ALA enriched oil (e.g., canola and linseed oil) or fish oil are considered as a good source of n-3 PUFAs [START_REF] Kanakri | A reduced cost strategy for enriching chicken meat with omega-3 long chain polyunsaturated fatty acids using dietary flaxseed oil[END_REF]. Nevertheless, concerns over cholesterol present in the yolk cannot be ignored.

Direct supplement of n-3 PUFAs into commonly consumed foods presents a very attractive opportunity but there are many challenges associated with the use of n-3 PUFAs in food products.

For instance, undesirable flavors and short product shelf-life are restricted the wide application of n-3 PUFAs in foods; low water solubility reduces the bioavailability of n-3 PUFAs in the GIT; easy oxidation which further leads to the development of rancid odor and taste, affecting the sensory characteristics of n-3 PUFAs [START_REF] Liu | Fabrication, characterization and properties of DHA-loaded nanoparticles based on zein and PLGA[END_REF][START_REF] Ma | Enhancing the physicochemical stability and digestibility of DHA emulsions by encapsulation of DHA droplets in caseinate/alginate honeycomb-shaped microparticles[END_REF][START_REF] Singh | Nanoencapsulation of docosahexanoic acid (DHA) using combination of food grade polymeric wall materials and its application for improvement in bioavailability and oxidative stability[END_REF]. Due to the development of encapsulation techniques, many problems have been solved. Nowadays, different kinds of delivery systems have been used to encapsulate n-3 PUFAs (Table 12345678). Among them, emulsions are widely accepted methods that play a significant role in the formulation of food, pharmaceuticals, and cosmetics [START_REF] Chatterjee | Encapsulation of fish oil with N-stearoyl O-butylglyceryl chitosan using membrane and ultrasonic emulsification processes[END_REF][START_REF] Mcclements | Advances in fabrication of emulsions with enhanced functionality using structural design principles[END_REF]. For instance, a number of emulsion-based delivery systems have been reported for encapsulating fish oil (n-3 PUFAs rich oil) to improve its water solubility, physicochemical stability, and bioavailability [START_REF] Haug | Bioavailability of EPA and DHA delivered by gelled emulsions and soft gel capsules[END_REF][START_REF] Lin | Pectin and gastric pH interactively affect DHA-rich emulsion in vitro digestion microstructure, digestibility and bioaccessibility[END_REF][START_REF] Solomando | Lipid digestion products in meat derivatives enriched with fish oil microcapsules[END_REF][START_REF] Viciano | Antioxidant activity and lipid peroxidation in Artemia nauplii enriched with DHA-rich oil emulsion and the effect of adding an external antioxidant based on hydroxytyrosol[END_REF]. θ). In this regard, many studies have been carried out to modify these particles with different molecules or to different degrees to make them more amphiphilic [START_REF] Yang | An overview of Pickering emulsions: Solid-particle materials, classification, morphology, and applications[END_REF]. Pickering emulsion is not susceptible to external environment, including changing the pH, ionic strength, temperature, and oil phase composition of the system. Pickering emulsions require fewer amounts of stabilizers during the food preparation in comparison with emulsions stabilized with classical surfactants. In addition, Pickering emulsions have good biocompatibility and can be used as carriers for the delivery of bioactive substances because solid particle materials used as stabilizers are mostly edible natural substances [START_REF] Chen | Food-grade Pickering emulsions: Preparation, stabilization and applications[END_REF]. The stabilizing mechanism of Pickering emulsion that is commonly accepted is based on the formation of a steric barrier by solid particles adsorbing at the oil-water interface. WPI is a well-known functional group of dairy ingredients from an industrial point of view that is widely used in various food applications. Based on its unique functional properties, it can be used as ingredients in food such as gelation, emulsification, foaming, thickening, flavour, and fat binding capacity [START_REF] Sharma | Multifaceted Whey Protein: Its Applications in Food Industry[END_REF]. For instance, WPI can be used for the production of films for food packaging based on its foaming/whipping property [START_REF] Kokoszka | Water vapour permeability, thermal and wetting properties of whey protein isolate based edible films[END_REF]. Besides, WPI can also be used as a good wall material and emulsifier for encapsulation due to its excellent surface-active properties. A number of encapsulation systems, including hydrogel, capsule, nanoparticle, and emulsion (Table 1-10), are prepared by using WPI as the material to encapsulate bioactive compounds (e.g., caffeine, fish oil and curcumin). However, WPI can easily denature under heating conditions, causing the encapsulation systems to lose stability, which greatly affects its application in the food industry. The functionality of WPI can be modified via physical or chemical modification, which provides more possibilities for the application of WPI in encapsulation. Whey proteins after heat-treatment have been exploited as a

Pickering stabilizer of foams [START_REF] Destribats | Emulsions stabilised by whey protein microgel particles: Towards food-grade Pickering emulsions[END_REF]. Based on our previous research, hydrophobised whey protein particles were successfully made up through successive acetylation and heat treatment practices [START_REF] Madadlou | Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins[END_REF]. However, whether hydrophobised whey protein can be used to stabilize Pickering emulsion has not been studied yet. Pickering stabilization of oil does not rely on interfacial tension reduction. Compared to heatdenatured whey protein, hydrophobised whey protein afforded a lower protection to oil against oxidation; the peroxide value of the oil emulsified using hydrophobised protein was higher during storage.

Introduction

Whey proteins contain substantial amounts of branched chain amino acids and are physiologically beneficial; for instance consumption of whey proteins combined with resistance training accelerates fat loss in humans [START_REF] Lockwood | Effects of Hydrolyzed Whey versus Other Whey Protein Supplements on the Physiological Response to 8 Weeks of Resistance Exercise in College-Aged Males[END_REF] and causes increased muscle gain in untrained rats [START_REF] Wróblewska | The effects of whey and soy proteins on growth performance, gastrointestinal digestion, and selected physiological responses in rats[END_REF]. Moreover, whey proteins are widely used for technological purposes such as formation of cold-set and heat-induced gels [START_REF] Egan | Cold-set whey protein microgels as pH modulated immobilisation matrices for charged bioactives[END_REF][START_REF] Oztop | Monitoring the effects of divalent ions (Mn+2 and Ca+2) in heat-set whey protein gels[END_REF], modification of viscosity [START_REF] Patocka | Rheological behaviour of dairy products as affected by soluble whey protein isolate[END_REF] and as fat replacer [START_REF] Akalın | Rheological properties of reduced-fat and low-fat ice cream containing whey protein isolate and inulin[END_REF]. Heat treatment of whey proteins at non-gelling condition produces protein particles, which have been exploited as Pickering stabilizer of foams [START_REF] Destribats | Emulsions stabilised by whey protein microgel particles: Towards food-grade Pickering emulsions[END_REF], oil-in-water (O/W) [START_REF] Destribats | Emulsions stabilised by whey protein microgel particles: Towards food-grade Pickering emulsions[END_REF] and water-in-water [START_REF] Nguyen | Stabilization of water-in-water emulsions by addition of protein particles[END_REF] emulsions.

The functionality of whey protein particles is further tailored via complexation with appositely charged polysaccharides and ions. Monovalent and multivalent salts including NaCl, MgCl2 and AlCl3 (Ince-Coskun & Ozdestan-Ocak, 2020), as well as ZnCl2, MnCl2, and CaCl2 [START_REF] Mohammadian | Cold-set hydrogels made of whey protein nanofibrils with different divalent cations[END_REF] are used to tune the technological functionality of whey protein particles. CaCl2

has also been used to produce soy protein particles through a non-thermal process, consisting of two crosslinking steps: ionic crosslinking by CaCl2 and subsequent amine-aldehyde covalent crosslinking by glutaraldehyde. The produced protein particles were successfully used for Pickering emulsification of soy oil in water [START_REF] Liu | Ca2+-induced soy protein nanoparticles as pickering stabilizers: Fabrication and characterization[END_REF]. However, glutaraldehyde is irritant, and cytotoxic. Therefore, the utilization of glutaraldehyde and other hazardous chemical crosslinkers is not favored by consumers.

Although food-grade amine-aldehyde crosslinkers such as citric acid can be alternatively applied [START_REF] Abaee | The formation of non-heat-treated whey protein cold-set hydrogels via non-toxic chemical cross-linking[END_REF][START_REF] Reddy | Alkali-catalyzed low temperature wet crosslinking of plant proteins using carboxylic acids[END_REF] in combination with ionic crosslinking to particulate plant and whey proteins, other opportunities to produce and tune whey proteins particles are available. For example, recently, hydrophobised whey protein particles were produced through a food-grade, simple and inexpensive method, which includes whey proteins pre-acetylation at room temperature and succeeding acetylation-heat treatment [START_REF] Madadlou | Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins[END_REF]. Acetylation is known to append ester carbonyl groups to proteins [START_REF] Zhao | Study of acetylated food proteins by Raman spectroscopy[END_REF], increase their hydrophobicity and cause conformational changes [START_REF] Lakkis | The bioavailability of an omega-3-rich algal oil is improved by nanoemulsion technology using yogurt as a food vehicle[END_REF][START_REF] Shah | Hydrophobically modified pea proteins: Synthesis, characterization and evaluation as emulsifiers in eggless cake[END_REF]. The subsequent heat treatment resulted in formation of protein particles.

Surface tension measurements at the air-water interface indicated that the hydrophobised whey protein particles are adsorbed at the interfacial layer and can be used for stabilization of water-inwater emulsions [START_REF] Madadlou | Development of an aqueous two-phase emulsion using hydrophobized whey proteins and erythritol[END_REF]. However, the particles hold many more premises. A combinatory application of hydrophobised whey protein particles and ionic crosslinking by CaCl2 may be advantageous to tune the protein particles functionality as O/W Pickering emulsifiers. In the present communication, we report the influence of CaCl2 supplementation on the hydrodynamic size, electric charge, and oil-water interfacial tension of the protein particles. The role of Cl¯ anions was particularly taken into consideration when discussing the observations. The importance of halide anions in development and modification of salt-supplemented protein networks (gels and particles) has been scarcely considered, whereas in addition to Ca 2+ cations, Cl¯ anions are capable to interacting with proteins, thereby influencing protein network characteristics [START_REF] Farjami | Modulating the textural characteristics of whey protein nanofibril gels with different concentrations of calcium chloride[END_REF]. Subsequently, the applicability of the hydrophobised whey protein particles as Pickering emulsifiers for a DHA oil was assessed. To the best of our knowledge, there is no report in the literature about the interfacial and emulsification properties of hydrophobised (acetylated-heat denatured) whey proteins.

Materials and Methods

Materials

WPI was a gift from Lactalis Ingredients (Lactalis Group, Bourgbarré, France). It had 90% protein, 5.1% moisture, 3.0% lactose, and 1.97% ash contents [START_REF] Madadlou | Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins[END_REF]. DHA oil was purchased from Polaris (Polaris, Quimper, France); it comprised 678 mg/g DHA and 23 mg/g EPA as TAG. Deionized water was used throughout the research, and all the chemicals used were of analytical grade.

Fabrication of hydrophobised and heat-denatured WPI particles

Hydrophobised WPI particles were prepared according to the process described by [START_REF] Madadlou | Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins[END_REF], with minor modifications. At first, a WPI solution (65 mg/mL) was prepared by adding deionized water to WPI powder and stirring for 4 h at 20 °C; sodium azide was added (1000 ppm for peroxide value and creaming index determination, 100 ppm for other experiments) to inhibit microbial growth. Then the solution was left overnight at 4 °C to ensure full hydration of whey proteins. WPI hydrophobization was accomplished by the following steps: 9.5 mL of native WPI solution (65 mg/mL) was charged with acetic anhydride (100 µL) and then gradually with 7 M NaOH (a total of 280 µL) with continue stirring. Subsequently, the solution was stirred for an additional period of 30 min; during this time, the WPI solution was occasionally supplemented with 1 M NaOH to maintain pH value in the range of 8.1 to 8.5, facilitating acetylation. Later, the solution (pH = 8.5) was heated at 80 °C while being stirred at 800 rpm for 20 min. After heat treatment, the solution was cooled using an ice bath and pH was adjusted to 8.0 by adding 10-20 µL of 1 M NaOH.

A heat-denatured WPI sample was also made by simply heating the native WPI solution (adjusted to pH 8.2 with 1 M NaOH) at 80 °C for 20 min [START_REF] Law | Effect of pH on the thermal denaturation of whey proteins in milk[END_REF]. Then, the solution was cooled using an ice bath and pH was adjusted to 8.0 by adding 0-10 µL 1 M NaOH. The hydrophobised (i.e. acetylated and heat-denatured) and heat-denatured WPI dispersions were diluted by adding deionized water to a final concentration of 10 mg/mL. Finally, CaCl2 (500 mM) was gradually added into the various samples to be investigated, to reach a final Ca 2+ concentration of 0-7 mM.

Protein particle characterization

Particle size and ζ-potential measurement

The particle size and ζ-potential of hydrophobised WPI and heat-denatured WPI were measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK). A laser wavelength of 633 nm at a backscattering angle of 173° was applied for size measurements at 20 °C.

Surface hydrophobicity measurement

The surface hydrophobicity of heat-denatured and hydrophobised WPIs were measured according to the method published by Alizadeh-Pasdar and [START_REF] Alizadeh-Pasdar | Comparison of protein surface hydrophobicity measured at various pH values using three different fluorescent probes[END_REF] with some modifications.

A stock solution of 1.41 × 10 -3 M 6-propionyl-2-(N-N-dimethylamino)-naphthalene (PRODAN) was prepared in methanol, transferred to screw-capped vials, and wrapped in aluminum foil to avoid exposure to light. The solution was stored in a freezer (≤ -10℃) until the day of experiment, when it was held in ice throughout the experiment. The excitation/emission slits and wavelengths of the spectrofluorometer (FLX-Xenius, SAFAS, Monaco) were set at 5 nm/5 nm and 500 nm/600 nm, respectively. To successive samples containing 4 mL of diluted WPI was added 10 µL of PRODAN stock solution, which was mixed well by vortexing. After 30 min in the dark, the relative fluorescence intensity (RFI) of each solution was measured, starting from blank (deionized water plus probe) and then the lowest to the highest protein concentration (0-2 mg/mL); the fluorometer quartz cell was rinsed between samples with a small volume of the solution to be measured. RFI values of buffer and protein dilution blanks (no PRODAN) were also measured. The RFI of each protein dilution blank was subtracted from that of corresponding protein solution with PRODAN to obtain net RFI. The initial slope (S0) of the net RFI versus protein concentration (percent) plot was calculated by linear regression analysis with Microsoft Excel and used as an index of the protein surface hydrophobicity.

Interfacial tension (γ) measurement

The tension at the interface between DHA oil and WPI dispersion was measured by the pendent drop method using a pendent drop tensiometer ('Tracker' from Teclis-Scientific, Civrieux d'Azergues, France). After formation of a fresh WPI drop (15 µL) at the tip of a syringe, the time evolution of γ was studied for 720 s. The drop profile was determined by image analysis, from which γ was derived. Under mechanical equilibrium of capillary and gravity forces, the Laplace equation relates the pressure difference across the interface, the γ and the interface curvature. These experiments were carried out at 20 °C. After experiments, the following kinetic model was used to describe the variation of γ at the oil/water interface:

In[(γt -γc)/(γw -γc)] = -kt (Eq. 1)

where γw and γc are values of γ at times 0 and 120 sec after droplet formation, γt is the value of γ at time t and k is the rate constant [START_REF] Panizzolo | A Kinetic Model for Describing the Effect of Proteins on the Air-Water Interface Tension[END_REF].

Emulsion preparation

Emulsion samples were prepared by a two-step homogenization process. DHA oil was gradually added into the WPI dispersion (10 mg/mL) while being stirred (IKA T 10 basic Ultra154 turrax, IKA ® -Werke GmbH & Co. KG, Staufen, Germany) at 20,000 rpm. Then, mixing was continued at 20,000 RPM for 6 minutes. The volume ratio of oil-to-WPI dispersion was 0.6:0.4. The resulting coarse emulsion (30 mL) was homogenized within an ice bath using an ultrasonic homogenizer (Q700 Sonicator, Qsonica sonicators, USA) with a standard tip (3.2 mm diameter) immersed 1/2 in a beaker of 40 mm diameter (50 mL volume). The emulsion was simultaneously stirred with a magnetic stirrer to ensure homogeneity. Each sample was sonicated at 20 kHz and an amplitude of 40% for 5 min. The O/W emulsions were subjected to analysis.

Characterization of emulsions 2.3.5.1 Droplet size determination

Droplet size (d4,3) of emulsions was determined using a Malvern Mastersizer 2000 (Malvern Instruments Ltd., Malvern, UK). The refractive index of oil phase and water phase used in the mathematical analysis were 1.45 and 1.33, respectively. The equipment works with two different wavelengths (He/Ne laser: 633nm; electroluminescent diode: 466 nm). This wavelength combination considers the effect of post-diffusion and enhances sensitivity to small particles.

Optical microscopy

Freshly formed emulsions were observed under an optical microscope (Olympus BX51TF, Hamburg, Germany) with 40 × objective. The imaging was carried out at non-fluorescent mode (bright field microscopy) and using the Archimed software (version 7.1.1, Microvision Instruments, Evry cedex, France) at 20 °C.

Creaming stability measurements

The creaming stability of prepared emulsions was studied following the method of [START_REF] Onsaard | Comparison of properties of oil-in-water emulsions stabilized by coconut cream proteins with those stabilized by whey protein isolate[END_REF]. Briefly, 27 g of emulsion samples were transferred into a test glass (with 24 mm internal diameter and 72 mm height), tightly sealed with a plastic cap to prevent evaporation, and then left to stand at 20 ℃ for 8 days. The emulsions separated into a top "cream layer" and a bottom "serum layer" (Hs). The extent of creaming was characterized by the creaming index (CI) = 100 × (Hs/He), where Hs and He are the height of the serum layer and total height of the emulsions in the tubes, respectively.

Rheological behavior

Rheological assessment of the emulsions was performed immediately after emulsion preparation using a MCR 301 rheometer (Anton Paar, Graz, Austria) equipped with a cone-plate geometry (50 mm diameter, cone angle = 1.996º) at 20 ℃. After equilibration, flow curves were determined at an increasing shear rate from 0.1 to 100 s -1 . Herschel-Bulkley model was used to describe the flow behavior of emulsions:

τ = τ0 + K(γ) n (Eq. 2)
where τ and τ0 were shear stress and yield stress, respectively, and γ was the shear rate; n and K were the flow behavior index and the consistency index, respectively.

Oxidative stability

After formation, all emulsions were stored at 20 ℃ for 8 days. A control sample containing only DHA oil was also analyzed. At first, the oil was extracted from emulsions by adding 0.3 mL of emulsion to 6 mL of chloroform/methanol (2:1, v/v). The mixture was mixed 15 min at 30 rpm and then was supplemented with 1.26 mL of 0.9% NaCl solution. The solution was mixed again for 15 min and rested for 30 min. Then the supernatant was removed and the subnatant was collected, filtered and treated with nitrogen to volatilize the solvent. Subsequently, 0.2 mL of absolute ethanol was added to the sample and gassed under nitrogen ensure that all solvent and water were removed from the sample.

The peroxide (PV) of emulsions was measured by an ISO standard method with some modifications [START_REF] Shantha | Rapid, Sensitive, Iron-Based Spectrophotometric Methods for Determination of Peroxide Values of Food Lipids[END_REF]. Briefly, a volume of 100 mL of a chloroform/methanol mixture (7:3, v/v) was added to both the extracted oil and non-emulsified oil (0.100-0.110 g). After

resting for a short time, 9.9 mL of the oil-solvent mixture was supplemented with 50 µL of 3.94 M ammonium thiocyanate solution, the mixture was vortexed for 4 s, then 50 µL of 0.072 M iron (II) solution was added and the mixture was vortexed again for 4 s. After incubation for 10 min at 20 ℃, the absorbance of the samples was read at 500 nm using a UV-Vis spectrophotometer (Thermo Fisher, Waltham, MA, USA).

Statistical analysis

Samples were produced at least three times, and the experiments were performed in triplicate.

The results were analyzed by one-way ANOVA with SPSS software version 17 (International Business Machines, Armonk, NY, USA) using Duncan's test at a significance level of P < 0.05.

Results and discussion

Characterization of protein particles

Particle size

The hydrophobization process resulted in larger protein particles compared with obtained with only heat denaturation (Figure . 2-1). Grafting acetyl moieties onto the surface of native whey proteins before heat treatment could enhance hydrophobic interactions between proteins, resulting in larger particles during the subsequent heat treatment. It is already indicated that roughly 90% of available amino groups on whey proteins undergo acetylation by the method used in the current study [START_REF] Madadlou | Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins[END_REF].

The particle size of the heat-denatured WPI did not significantly change by CaCl2 addition up to 5 mM. However, the particle size increased nearly 20% when CaCl2 concentration increased from 5 to 7 mM (Figure 2-1). The small change of particle size suggests that protein particles did not aggregate; rather the hydration extent of the particles increased by CaCl2 addition at 7 mM.

Kosmotropic cations such as Ca 2+ have a stronger affinity to anions than water; this is valid not only for anions in bulk solvent but also for aspartate and glutamate residues on the surface of a protein.

Protein-Ca 2+ interactions neutralize the favorable structure-making effect of protein anions on water [START_REF] Eggers | Crowding and hydration effects on protein conformation: A study with sol-gel encapsulated proteins[END_REF] and cause disruption of the hydrogen bonds between protein and water, i.e., protein partially dehydrates. Concomitantly, the attached Ca 2+ to protein binds to the water in immediate vicinity, which opposed (at low Ca 2+ concentrations) and eventually dominated the dehydration effect of Ca 2+ binding to protein, leading to a higher hydrodynamic diameter at the highest CaCl2 concentration (7 mM). It is noteworthy that at CaCl2 concentrations greater than 7 mM the heat-denatured protein particles extensively aggregated and WPI gelled (results not shown). In contrast to the results obtained for the heat-denatured WPI, the particle size of the hydrophobised WPI gradually (though not consecuteively) decreased with increasing CaCl2 concentration throughout the whole concentration range between 0 and 7 mM (Figure 2-1). A total size reduction of approximately 20% is observed for hydrophobised WPI particles when the added CaCl2 concentration increased from 0 mM to 7 mM. The small size change indicates that the protein particles did not disaggregate; rather they progressively dehydrated (i.e., became less hydrated) with increasing the CaCl2 concentration.

The dehydration of the hydrophobised WPI particles as a consequence of CaCl2 suppplementation and comparison with changes in the hydrodynamic size of heat-denatured WPI particles indicates that acetylation of whey proteins influenced the protein-salt interaction. It is possible to argue that the counter ions of Ca 2+ , i.e. Cl¯ anions, must be taken into account. It has been reported that at a given ion normality of 50 mM, Cl¯ is among the weakest anions that can associate with protein surface [START_REF] Zhao | Protein stabilization and enzyme activation in ionic liquids: Specific ion effects[END_REF]. Nonetheless, grafting acetyl moieties onto whey proteins could strongly increase Cl¯ binding to proteins via a phenomenon called hydrophobic concavity, i.e., hydrophobic interactions within a nonpolar microcavity. Being a low-charge-density ion, Cl¯ can stick to hydrophobic protein ensembles [START_REF] Farjami | Modulating the textural characteristics of whey protein nanofibril gels with different concentrations of calcium chloride[END_REF]. Hydrophobic concavity accounts for the selective binding of Cl¯ anions over phosphates and carboxylates (that rank far above Cl¯ in the Hofmeister series) to macrocyclic hosts. Likewise, interactions between Cl¯ and hydrophobic amino acids play a crucial role in functionality of chloride-selective protein channels of cell membranes. In such channels, a Cl¯ ion may be bound by just a few H-bond donors but it is surrounded by hydrophobic amino acid residues and contacts with hydrophobic CH2 groups [START_REF] Dabrowa | Catching the chloride: Searching for non-Hofmeister selectivity behavior in systematically varied polyamide macrocyclic receptors[END_REF]. The binding of Cl¯ to acetylated protein particles most likely occurred through complexation of ions within the hydrophobic pockets made of neighboring acetyl groups.

The anions that bind to hydrophobic concavities completely or at least partially lose their solvation shell water molecules [START_REF] Sokkalingam | Binding Hydrated Anions with Hydrophobic Pockets[END_REF], which explains the decrease of WPI particles hydration due to Cl¯ binding at CaCl2 concentrations between 0-7 mM.

ζ-potential

Acetylation of proteins with acetic anhydride involves covalent attachment of acetyl moities to free amino groups of proteins [START_REF] Lakkis | The bioavailability of an omega-3-rich algal oil is improved by nanoemulsion technology using yogurt as a food vehicle[END_REF]. As expected, the hydrophobised WPI had a more negative ζ-potential value compared with the heat-denatured WPI (Figure 2-1), which is attributed to consumption of positively charged free amino groups during the acetylation process.

Addition of CaCl2 reduced the abosulte value of ζ-potential of both heat-denatured and hydrophobised protein particles (Figure 2-1). Negatively charged amino acid residues, namely aspartate and glutamate bind to Ca 2+ ions, which decreases the ζ-potential. It is noteworthy that the maximum alteration of ζ-potential due to CaCl2 supplementation was much more significant for the heat-denatured WPI (nearly 3 fold) than the hydrophobised WPI (nearly 1.7 fold) when the concentration of CaCl2 was increased from 0 to 7 mM. This is in accordance with our hypothesis on the concavity pocketing of Cl¯ anions into the acetylated (hydrophobised) protein particles.

Surface hydrophobicity

As expected, grafting acetyl moieties onto whey proteins increased the surface hydrophobicity from ~119 for the heat-denatured WPI to ~128 for the hydrophobised WPI at 0 mM CaCl2 concentration. The addition of CaCl2 did not significantly influence the surface hydrophobicity of the samples (results not shown). PRODAN is an uncharged aromatic probe and is advantageous for measuring protein hydrophobicity over ionic probes such as cis-parinaric acid (CPA) and 1anilinonaphthalene-8-sulfonic acid (ANS) (Alizadeh-Pasdar & [START_REF] Alizadeh-Pasdar | Comparison of protein surface hydrophobicity measured at various pH values using three different fluorescent probes[END_REF]. Similar to our observation, increasing ionic strength did not influence the surface hydrophobicity of ovalbumin measured by PRODAN but increased that of ovalbumin measured by ANS [START_REF] Haskard | Hydrophobicity of bovine serum albumin and ovalbumin determined using uncharged (PRODAN) and anionic (ANS-) fluorescent probes[END_REF], which is negatively charged.

Interfacial tension

The time-dependent variation of γ at the interface between DHA oil and WPI dispersions is presented in Figure 2-2A. For the freshly formed pendant drop (at time ≈ 0) and in the absence of added CaCl2, there was not a statistically significant difference between the γ values of the heatdenatured and hydrophobised WPI samples; both WPIs had a γ value of approximately 21.1 mN/m, which subsequently decreased with droplet ageing. Supplementation with CaCl2 decreased γ at any given time for both WPIs. We presume that ζ-potential reduction of protein particles due to CaCl2 addition (Figure 2-1) caused a more efficient packing of protein particles at the interface, reducing the γ. Besides, it is known that cations such as Ca 2+ increase the contact angle of adsorbing particles at the oil-water interface, thus the interfacial tension decreases more substantially. The influence of cations on contact angle is associated with decrease of particles hydration level [START_REF] Wen | Nanoparticle-induced ion-sensitive reduction in decane-water interfacial tension[END_REF], which may explain the smaller differences between the γ values of heat-denatured WPI (for which the hydrodynamic size did not decrease between 0-5 mM CaCl2 and slightly increased at 7 mM CaCl2) compared with the greater differences between the γ values of hydrophobised WPI (for which hydrodynamic size decreased between 0-7 mM CaCl2) as a function of CaCl2 concentration (Figure 2-2A). This is in accordance with the results of [START_REF] Fan | Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion: Impact of protein and calcium concentrations[END_REF] who reported that the tension at the oil-WPI interface decreased with increasing CaCl2 concentration.

Analysis of γ values using Eq. ( 1) resulted in two γ reduction constants: k1 which refers to the rapidly falling period of γ in the initial 20 s and k2 which refers to the slowly falling period of γ over the rest of interface ageing (Figure 2-2B). Following Panizzolo, Mussio and Añón (2014) Eq. ( 1)

was applied to time periods between 0 and 120 s of droplet ageing. Although the model was suitable to characterize γ reduction rates of the heat-denatured WPI, i.e., it had high R 2 values especially for k1, the hydrophobised WPI did not properly fit to the model (Figure 2-2B). Nonetheless, the following main results can be concluded from the modelling practice: As expected, k1 was always higher than k2 for both WPI samples; in general, k1 values of the heat-denatured WPI were higher

A B

Whereas small molecule surfactants can cause roughly a 10-fold reduction of γ at the oil-water interface [START_REF] Posocco | Interfacial tension of oil/water emulsions with mixed non-ionic surfactants: Comparison between experiments and molecular simulations[END_REF], the highest extent of γ reduction in the current study was merely ≈0.25 fold compared with the corresponding initial value (i.e., at time≈0). Actually, Pickering-type stabilization of emulsions does not rely on γ reduction. In fact, particle trapping at the oil-water interface does not cause appreciable changes in the interfacial tension. Rather particles position at the continuous side of the droplet interface and stabilize emulsions [START_REF] Vignati | Pickering Emulsions: Interfacial Tension, Colloidal Layer Morphology, and Trapped-Particle Motion[END_REF]. However, the stability of Pickering emulsions is proportional to γ. According to the following equation ( 3), the higher the γ, the higher the energy required to desorb Pickering particles from the interface:

𝐸 = 𝜋𝑅 2 𝛾 𝑜𝑤 (1 + 𝑐𝑜𝑠𝜃) 2 (Eq. 3)
where 𝐸 is the energy required to remove particle, 𝑅 is particle radius, γ is tension at the oil-water interface, and θ is the three-phase contact angle [START_REF] Binks | Influence of particle wettability on the type and stability of surfactant-free emulsions[END_REF]. The reduction of γ with increasing CaCl2 concentration in the present study is therefore expected to cause a lower emulsion stability, and result in destabilization phenomena such as creaming.

Characterization of Pickering emulsions

Bulk and microscopic appearance, and droplet size

Figure 2-3A shows the bulk appearance of oil-WPI dispersion mixes after emulsification practices, i.e., mechanical stirring and ultrasonic homogenization. WPI concentration in the aqueous phase of mixes was 10 mg/mL and the proportion of the DHA oil in mixes was 60% (v/v). The mixture of oil and heat-denatured WPI supplemented with CaCl2 at 3 mM transformed to a soft gel during ultrasonic homogenization, failing to make an emulsion. As well, the mixture of oil and hydrophobised WPI supplemented with CaCl2 at 6.5 mM was highly viscous and oil was not readily dispersed in the aqueous phase, therefore, after preparation a portion of oil did not emulsify and remained at the surface (Figure 2-3A). These samples were not subjected to additional analysis.

The emulsion samples made using heat-denatured WPI without added CaCl2 and hydrophobised WPI supplemented with 0, 3 and 5.5 mM CaCl2 were stored for a period of 8 days.

The sample made using the hydrophobised WPI with 0 mM added CaCl2 did not show any extent of creaming; whereas, the rest of the samples slightly creamed, i.e., those prepared using the hydrophobised WPI with 3 or 5.5 mM CaCl2 and the heat-denatured WPI with 0 mM CaCl2. The creaming index of the phase separated emulsions reached 1.7% (Figure 2 A weaker electrostatic repulsion between the oil droplets can rationally lead to droplet flocculation and emulsion creaming, as observed in the current study. However, electrostatic repulsion between oil droplets was not most probably the sole factor affecting the flocculation/creaming. Heat-denatured WPI without added CaCl2 had an absolute ζ-potential value of ≈32 mV, which is higher than those of the hydrophobised WPI with added CaCl2 at 3-7 mM (Figure 2-1). Yet the emulsion samples made using the heat-denatured WPI without added CaCl2 for particles [START_REF] Binks | Influence of particle wettability on the type and stability of surfactant-free emulsions[END_REF].

Flow behavior

Oxidative stability

The oxidative stability of emulsions was investigated for 8 days. All emulsions were kept at 20 ℃ immediately after preparation and the emulsified oil was extracted at fixed time intervals for PV measurements. Non-emulsified DHA oil was also analyzed for comparison purposes. The DHA oil emulsified using the heat-denatured WPI had the lowest PV among all samples throughout the whole storage period (Figure 2-5), which is attributed to the antioxidative effect of exposed thiol groups in the heat-denatured protein. Heat denaturation exposes the previously hidden sulfhydryl groups, which contribute to protein aggregation by -SH/S-S exchange reactions and have a central role in protein-based redox systems [START_REF] Ulrich | The role of thiols in antioxidant systems[END_REF].

The highest PV belonged to the non-emulsified oil at the end of the storage time. However, the oil emulsified using the hydrophobised WPI without added CaCl2 (i.e., 0 mM CaCl2) had a higher PV than the non-emulsified oil in the first storage days (Figure 2345). The underlying mechanism for this observation is not clear and further systematic studies are required to reveal the effect of hydrophobised WPI on oil oxidation. Nevertheless, it is worth mentioning that the two-step mechanical-ultrasonic emulsification process which was applied in the current study could enhance oil oxidation, leading to higher PV at the early days of storage in comparison to the non-emulsified oil (i.e., no mechanical-ultrasonic treatments). Mechanical agitation of oil-water mixtures causes air incorporation into the emulsion, and ultrasonic irradiation proceeds by formation of acoustic cavities, as well formation of highly reactive radical species [START_REF] Madadlou | Sonodisruption of re-assembled casein micelles at different pH values[END_REF], which could enhance oil oxidation. The prooxidant effect of the emulsification process was most probably masked by the thiol groups present in the heat-denatured WPI, but the hydrophobised WPI which likely had lower exposed thiol groups (due to extensive hydrophobic interactions before heat treatment) could not prevent early oil oxidation. A difference between the thiol groups contents of hydrophobised and heat-denatured WPIs has previously been noticed [START_REF] Madadlou | Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins[END_REF].

When CaCl2 concentration increased from 0 to 5.5 mM, the hydrophobised WPI conferred a better protective effect on the emulsified oil, associated with the more compact packing of protein particles at the oil-water interface (Figure 2-2). Besides, CaCl2 supplementation increased the droplet size of the emulsion stabilized by the hydrophobised WPI. The surface area-to-volume ratio of larger droplets is lower than smaller droplets. [START_REF] Lethuaut | Effect of droplet size on lipid oxidation rates of oil-inwater emulsions stabilized by protein[END_REF] observed that higher interfacial areas of smaller droplets resulted in higher oxidation rates.

Conclusion

Supplementation of WPI with CaCl2 after heat-denaturation or hydrophobization had significant influences on protein ζ-potential and the characteristics of resulting emulsions. We hypothesized that in addition to the expected charge interactions between Ca 2+ ions and negatively charged proteins, Cl -interacted with hydrophobised protein particles through grafted acetyl moieties.

This hydrophobic concavity of Cl -might be exploited at selective binding of Cl -in the presence of other anions when designating artificial cells membranes; Cl -is the sole inorganic anion used by cells at transmembrane gradients.

CaCl2 supplementation of both heat-denatured and hydrophobised WPIs resulted in slightly lower γ values at the oil-water interface at any given time and did not affect γ reduction rate.

Therefore, we conclude that the effects of CaCl2 on emulsion properties (creaming, droplet size, rheology, and oxidative stability) are rather associated with changes at particles size and ζ-potential than γ.

CaCl2 supplementation of hydrophobised WPI decreased the susceptibility of DHA oil to oxidation during emulsification process. Further studies are required to address why hydrophobised WPI (without added CaCl2) had higher peroxide value in early storage than non-emulsified oil, whereas, the oil stabilized using heat-denatured WPI had the lowest peroxide value throughout storage. At present, we attribute this observation to the less count of (surface) thiol groups in hydrophobised WPI particles and the prooxidant effect of mechanical-ultrasonic emulsification on oil.

Chapitre 3 : L'encapsulation de l'huile DHA avec de la proté ine de lactosé rum dé naturé e par la chaleur dans l'é mulsion de Pickering amé liore la bioaccessibilité du DHA Chapter 3: Encapsulation of DHA oil with heatdenatured whey protein in Pickering emulsion improves the bioaccessibility of DHA

Abstract

This study focused on the bioaccessibility of DHA according to the dietary form as encapsulated or unencapsulated food formulation. DHA oil was composed of DHA-enriched triacylglycerols prepared as Pickering emulsion by encapsulation with heat-denatured whey protein isolate particles and then incorporated into homogenized egg liquid to get omelets. The effect of encapsulation was analyzed by using an in vitro model of static digestion for adults. First, size of lipid droplets was smaller and uniformly dispersed in omelets with encapsulated-DHA oil compared to unencapsulated-DHA oil. Distribution of droplets was more regular as well with encapsulated-DHA oil. As a consequence, we showed that encapsulating DHA oil promoted the lipase hydrolysis during the intestinal phase. A larger proportion of DHA enriched-triacylglycerols was digested after two hours of catalysis, leading to a greater release in free DHA in digesta. Therefore, DHA bioaccessibility was significantly improved by encapsulation of DHA oil in omelets.

Introduction

DHA is one of the most important PUFA, mainly known for its health benefits on cognitive development and cardiovascular functions [START_REF] Bercea | Omega-3 polyunsaturated fatty acids and hypertension: A review of vasodilatory mechanisms of docosahexaenoic acid and eicosapentaenoic acid[END_REF][START_REF] Joffre | N-3 polyunsaturated fatty acids and their derivates reduce neuroinflammation during aging[END_REF][START_REF] O'connell | Mechanistic insights into cardiovascular protection for omega-3 fatty acids and their bioactive lipid metabolites[END_REF]. Although the recommended dietary intake is consensually estimated at 250 mg per day of DHA, only 14.6 % of the adult population in France appeared to meet the required level [START_REF] Tressou | Fatty acid dietary intake in the general French population: Are the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) national recommendations met?[END_REF]. DHA may be either de novo synthesized from its essential precursor, the α-linolenic acid, or brought as such by food. In the first case, the rate of synthesis is limited in human [START_REF] Burns-Whitmore | Alpha-linolenic and linoleic fatty acids in the vegan diet: Do they require dietary reference intake/adequate intake special consideration?[END_REF][START_REF] Calder | Docosahexaenoic acid[END_REF]Ruxton et al., 2005) so that the food intake of DHA is necessary to compensate. While marine fish represents the richest dietary source of DHA, a large part of the population consumes little or no fish. Therefore, fortification of food with DHA is considered as an efficient strategy to reach the recommended intake threshold. However, many challenges are associated with the use of DHA oil in food products such as undesirable fishy flavors and short product shelf-life, a low water solubility reducing the bioaccessibility of DHA in the gastrointestinal tract, and a high risk of oxidation which further leads to the development of rancid odor and taste [START_REF] Liu | Fabrication, characterization and properties of DHA-loaded nanoparticles based on zein and PLGA[END_REF][START_REF] Ma | Enhancing the physicochemical stability and digestibility of DHA emulsions by encapsulation of DHA droplets in caseinate/alginate honeycomb-shaped microparticles[END_REF][START_REF] Singh | Nanoencapsulation of docosahexanoic acid (DHA) using combination of food grade polymeric wall materials and its application for improvement in bioavailability and oxidative stability[END_REF]. As each side effect may finally reduce the potential of DHA on the body functions, integrating DHA oil into food products requires appropriate delivery systems to encapsulate, protect, and incorporate DHA oil into the diet. Numerous emulsionbased delivery systems were reported for encapsulating fish oil (DHA rich oil) to improve its water solubility, its physicochemical stability and its bioaccessibility as well [START_REF] Haug | Bioavailability of EPA and DHA delivered by gelled emulsions and soft gel capsules[END_REF][START_REF] Lin | Pectin and gastric pH interactively affect DHA-rich emulsion in vitro digestion microstructure, digestibility and bioaccessibility[END_REF][START_REF] Viciano | Antioxidant activity and lipid peroxidation in Artemia nauplii enriched with DHA-rich oil emulsion and the effect of adding an external antioxidant based on hydroxytyrosol[END_REF]. However, traditional emulsions are easily affected by environmental conditions such as pH, temperature and ionic strengths. Compared with traditional emulsion, Pickering emulsification of alimentary oils has received significant interest as it relies on irreversible adsorption of particles rather than on synthetic surfactants to the oil-water interface, which confers resistance to droplet coalescence and improves the stability of emulsion [START_REF] Chevalier | Emulsions stabilized with solid nanoparticles: Pickering emulsions[END_REF][START_REF] Li | Chitosan hydrochloride/carboxymethyl starch complex nanogels as novel Pickering stabilizers: Physical stability and rheological properties[END_REF][START_REF] Low | Recent advances of characterization techniques for the formation, physical properties and stability of Pickering emulsion[END_REF][START_REF] Wu | Recent Studies of Pickering Emulsions: Particles Make the Difference[END_REF]. Based on our previous research, Pickering emulsions were here prepared by encapsulation of DHA oil with heat-denatured WPI as particles [START_REF] Wang | CaCl2 supplementation of hydrophobised whey proteins: Assessment of protein particles and consequent emulsions[END_REF]. This emulsion showed a better stability against oxidation than the unencapsulated DHA oil, the characteristic of which is very important during the food process.

Encapsulation of the DHA oil was also performed with natural encapsulant materials, allowing a food-grade preparation for further analyses. In this regard, egg products were selected as the target food, as an inexpensive source of high-quality proteins, essential vitamins and minerals. Our previous research showed moreover that the highest bioavailability of DHA was reached with omelet, as compared to various other egg recipes (hard-boiled egg and mousse) [START_REF] Pineda-Vadillo | In Vivo Digestion of Egg Products Enriched with DHA: Effect of the Food Matrix on DHA Bioavailability[END_REF]. Consequently, omelet was selected as the food model to study the impact of encapsulation of DHA oil on the digestion process.

Several studies have reviewed the importance of the dietary supply of DHA as a strategy to improve DHA accretion in tissues, and especially in brain. Some authors suggest that dietary interventions may be effective in targeting serum lipid pools important for brain DHA uptake [START_REF] Lacombe | Brain docosahexaenoic acid uptake and metabolism[END_REF]. Thereby different works have shown a higher bioavailability of DHA esterified to phospholipids (PL-DHA) as compared to DHA esterified to triacylglycerols (DHA-TAG) [START_REF] Maki | Krill oil supplementation increases plasma concentrations of eicosapentaenoic and docosahexaenoic acids in overweight and obese men and women[END_REF][START_REF] Ulven | Metabolic Effects of Krill Oil are Essentially Similar to Those of Fish Oil but at Lower Dose of EPA and DHA, in Healthy Volunteers[END_REF][START_REF] Ramprasath | Enhanced increase of omega-3 index in healthy individuals with response to 4-week n-3 fatty acid supplementation from krill oil versus fish oil[END_REF]. Other studies contradicted however this finding in showing an equivalent bioavailability between PL-DHA and TAG-DHA [START_REF] Carnielli | Intestinal absorption of long-chain polyunsaturated fatty acids in preterm infants fed breast milk or formula[END_REF][START_REF] Schuchardt | Incorporation of EPA and DHA into plasma phospholipids in response to different omega-3 fatty acid formulations-A comparative bioavailability study of fish oil vs. Krill oil[END_REF][START_REF] Yurko-Mauro | Similar eicosapentaenoic acid and docosahexaenoic acid plasma levels achieved with fish oil or krill oil in a randomized double-blind four-week bioavailability study[END_REF]. And still others were more controversial [START_REF] Ghasemifard | Omega-3 long chain fatty acid "bioavailability": A review of evidence and methodological considerations[END_REF][START_REF] Gázquez | Towards an Optimized Fetal DHA Accretion: Differences on Maternal DHA Supplementation Using Phospholipids vs. Triglycerides during Pregnancy in Different Models[END_REF], insomuch as a higher concentration of circulating DHA would not predict brain accretion of DHA [START_REF] Gázquez | DHA supplementation during pregnancy as phospholipids or TAG produces different placental uptake but similar fetal brain accretion in neonatal piglets[END_REF][START_REF] Adkins | Accretion of Dietary Docosahexaenoic Acid in Mouse Tissues Did Not Differ between Its Purified Phospholipid and Triacylglycerol Forms[END_REF][START_REF] Destaillats | Comparison of the Incorporation of DHA in Circulatory and Neural Tissue When Provided as Triacylglycerol (TAG), Monoacylglycerol (MAG) or Phospholipids (PL) Provides New Insight into Fatty Acid Bioavailability[END_REF]. In parallel, similar digestion and intestinal absorption were exhibited in vivo between DHA-PL and DHA-TAG [START_REF] Sehl | In vitro lipolysis and lymphatic absorption of n -3 long-chain PUFA in the rat: Influence of the molecular lipid species as carrier[END_REF]. Based on this knowledge, the DHA oil used in this study was enzymatically prepared from fish oil as DHAenriched TAG. The DHA oil was then encapsulated with heat-denatured WPI particles as Pickering emulsion, and finally incorporated into homogenized egg liquid to get omelets. Bioaccessibility of encapsulated DHA oil as compared to unencapsulated DHA oil was then assessed by using an in vitro model of static digestion for adults according to the INFOGEST protocol.

Materials and Methods

Ingredients and reagents

WPI (Lactalis Ingredients, Bourgbarré, France) contained 90% protein, 5.1 % moisture, 3.0% lactose and 1.97% ash [START_REF] Madadlou | Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins[END_REF]. DHA oil was prepared from fish oil by Polaris (Quimper, France). Eggs were from Moisan aviculture (Plestan, France). All reagents were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France) and solvents were purchased from Fisher Scientific (Elancourt, France).

Preparation of Pickering emulsion and omelets

DHA oil were encapsulated with heat-denatured WPI as described previously [START_REF] Wang | CaCl2 supplementation of hydrophobised whey proteins: Assessment of protein particles and consequent emulsions[END_REF]. Omelet was prepared with a whole egg homogenized by ultraturrax (10000 rpm, 30 sec).

Then encapsulated DHA oil (EN-DHA-O), heat-denatured whey protein dispersion alone (Control)

or completed with the DHA oil (UN-DHA-O) were added to liquid eggs and mixed by stirring (500 rpm, 5 min). Eggs were finally molded and cooked in a water bath (80 °C, 10 min).

In vitro model for static digestion

The in vitro digestion was carried out according to the standardized INFOGEST protocol for adults [START_REF] Minekus | A standardised static in vitro digestion method suitable for food-an international consensus[END_REF]. Briefly, the oral phase was prepared by mixing 5 g of mortar-destroyed omelet with 4 mL simulated salivary fluid (SSF) and 1 mL of 7.5 mmol/L CaCl2. The bolus was incubated for 2 min at 37 °C in a water bath under constant stirring at 170 rpm before the beginning of the digestion process. Then, the gastric phase was prepared by mixing the oral bolus with 8 mL simulated gastric fluid (SGF), 5 µL of 0.3 mol/L CaCl2, 290 µL of 3 mol/L HCl to adjust the pH to 2, and 705 µL of distilled water. After the addition of 0.5 mL porcine pepsin solution (2000 U/mL, Sigma-Aldrich) and 0.5 mL rabbit gastric extracts (RGE, 60 U/mL, Lipolytech®, Marseille, France)(RGE, containing both RGL and pepsin), the gastric phase was incubated for 2 h at 37 °C under stirring at 170 rpm. Finally, the intestinal phase started thereafter by adding 8.5 mL simulated intestinal fluid (SIF), 40 µL of 0.3 mol/L CaCl2, 960 µL of 1 mol/L NaOH to adjust the pH to 7, 3 mL of distilled water and 2.5 mL bile extract solution (Sigma-Aldrich) to obtain 10 mmol/L of bile salts in the final digestion volume. After the addition of 5 mL pancreatin solution made up in SIF (100 U/mL of trypsin activity, Sigma-Aldrich), the solution was incubated for 2 h at 37 °C under stirring at 170 rpm. For each digestion point, two 500 µL samplings, one for microscopy observation and another one for lipid analyses, were collected and stabilized by adding enzyme inhibitors as described elsewhere [START_REF] Brodkorb | INFOGEST static in vitro simulation of gastrointestinal food digestion[END_REF].

Confocal microscopy

Distribution of DHA oil in omelets during the digestion process was followed by confocal laser scanning microscopy by using a ZEISS LSM 880 microscope (Zeiss, Germany) with a 63X magnification (Plan Apochromat objective, oil immersion, NA1.4). DHA oil was stained by Nile Red (120 mg/g oil) before the preparation of Pickering emulsion. Omelet slices and digesta samplings were then dyed with Fast Green (50 µL of a 1% aqueous solution, 10 min at 20°C).

Fluorescence of DHA oil was obtained by a 488 nm excitation wavelength coupled with a GaasP detector between 550 and 590 nm. Detection of egg proteins was performed by excitation at 633 nm coupled with a PMT detector between 635 and 735 nm. The ZEN lite black software (Zeiss, Germany) was used to process the images produced. Each digestion points were analyzed twice with at least 8 repetitions.

Lipid analyses

Lipids were extracted according to the Folch's method (1957b) and lipid classes were separated by thin layer chromatography on silica gel-60 plate (Merck, Darmstadt, Germany). Neutral lipids were primarily separated from polar lipids by using a mixture of diethyl ether: acetone 60: 20 (v:v) and were subsequently separated by species with the mixture hexane: diethyl ether: acetic acid 60: 40: 1 (v:v:v). Each lipid species was identified by authentic standards after spraying with primulin and then scrapped off to perform the fatty acid profile. Briefly, collected silica gel was saponified with 0.5 mol/L NaOH in methanol at 70℃ for 20 min and methylated with BF3 (14% in methanol) at 70 ℃ for 15 min [START_REF] Pédrono | Impact of a Standard Rodent Chow Diet on Tissue n-6 Fatty Acids, Δ9-Desaturation Index, and Plasmalogen Mass in Rats Fed for One Year[END_REF]. FA methyl esters were extracted with pentane and then were expressed as a mass percentage of the total FA.

Statistical analysis

All experiments were performed three times on 3 independent samples. Results are expressed in mean ± SD. The DHA content in omelets was firstly analyzed by using a one-way analysis of variance followed by a post-hoc test depending on the normality of the data distribution. Lipid analyses on digested omelets were then assessed by a two-way analysis of variance followed by a Sidak test. Significant differences were mentioned by an asterisk or different letters when p < 0.05.

Analysis was performed with the R software (version 4.0.5) and figures were made with Graph Pad Prism 7.

Results

DHA content in omelets

The DHA oil used in this study was prepared from fish oil and was further characterized (Table 3-1). It was mainly composed of DHA esterified on TAG (96.7%), but 3.3% of the total DHA was found in minor lipid species as well, such as DAG. DHA was the major FA while EPA and DPAn-3 were also measured at 36 µg/mg and 31 µg/mg respectively (not shown). When the DHA oil was encapsulated as Pickering emulsion, DHA represented 344 µg/mg without modification of the lipid species profile. Then omelet was used as a food matrix, the composition of which was composed of a very small amount of DHA (0.6% of total FA) esterified in PL. When omelets were cooked with DHA oil or with encapsulated DHA oil, DHA reached 9.0% and 10.3% of total FA respectively.

It was mainly supplied as TAG representing 86% of the total DHA in DHA-enriched omelets.

However, heating eggs to get omelets had an effect on the lipid species, as TAG were partially hydrolyzed into 1,2-DAG during the food processing. Therefore, we found an increased proportion of DHA esterified in 1,2-DAG in DHA enriched-omelets as compared to the original DHA oil, whereas the other lipid species were not impacted by the cooking. Nd: Not detected. Lipids were extracted by the Folch's method and separated by thin layer chromatography. The FA profile from different lipid species was determined by gas chromatography coupled with mass spectrometer. The DHA content was measured in total lipids and in different lipid species. Significant difference between omelets was indicated by different letters when p < 0.05. The DHA oil was incorporated as such in omelets or encapsulated with heat-denatured WPI.

Distribution of encapsulated DHA oil in omelets

The microstructure of Pickering emulsion emphasized the protein layer on the surface of the DHA oil droplets (Figure 3-1). However, protein particles were not clearly identified by using confocal microscopy since the particle size of heat-denatured WPI was too small, around 42 nm as measured in a previous work [START_REF] Wang | CaCl2 supplementation of hydrophobised whey proteins: Assessment of protein particles and consequent emulsions[END_REF]. Then when incorporated in omelets, the DHA oil was differently distributed according to the delivery form (Figure 3-2). Droplets of encapsulated DHA oil were uniformly dispersed in omelets with an equivalent repartition of DHA oil in terms of size of droplets. On the contrary, unencapsulated DHA oil exhibited a more random dispersion in omelets as bigger droplets, but with a wide range of droplet sizes. At first, the control omelet showed a higher proportion of FA in PL to the detriment of TAG, as compared to the global FA profile from DHA-enriched omelets. The gap averaged 10% of total FA due to the addition of the DHA oil as TAG and was noticeable during the entire digestion process.

Then, concerning more specifically DHA-enriched omelets, the main lipolysis occurred on TAG during the gastric phase and was completed thereafter during the intestinal phase together with the phospholipid hydrolysis.

In the gastric phase, TAG were hydrolyzed to release 1,2-DAG and FFA. When all data were further considered in the statistical analysis, we drew a group representation for lipid species monitored through the digestion process (Figure 3-5A). TAG and PL were positioned at the opposite side in the second dimension whereas products of the digestion i.e. FFA, DAG and MAG, were more centered between both substrates. The first dimension of the representation was not very discriminant. When groups of omelets were further considered, we confirmed the high discrimination between DHA-enriched omelets and the control omelet, the expression of which was opposed in the first dimension of the individual factor map (Figure 3-5B).

The discrepancy was specifically marked with TAG as visualized on the multifactorial analysis (Figure 3-5C). It was provided by FA specific from the DHA oil, mainly EPA and DHA, but also 22:5n-6 and 24:1n-9, DHA and 22:5n-6 being minorly present in eggs on the contrary to 24:1n-9 absent from eggs (not shown). Otherwise the second dimension showed no prominent difference between samples of omelets, this axis being represented by monounsaturated FA as 16:1n-7 and 18:1n-7, and saturates like 16:0 and 18:0 (not shown). In conclusion, the in vitro model of digestion used in this study showed a difference between samples throughout the hydrolysis process. We further studied the specific digestion of DHA oil supplied as such or encapsulated with whey protein isolate in omelets.

Hydrolysis of DHA oil and release of DHA during the digestion of omelets

Release of DHA from DHA oil was followed during the digestion process (Figure 3456). Firstly, DHA was not released during the gastric phase but was only freed during the intestinal phase, whether esterified from TAG or from PL. This contrasts with the hydrolysis of total TAG present in the samples, as the lipase digestion began in the gastric phase and released half of the total FA esterified in TAG (Figure 34). Secondly, digestion of DHA-TAG was more important with EN-long and very long chain FA from DHA oil (24:1n-9, 20:5n-3 and 22:5n-3). Firstly, precursors in C16 and C18 esterified to TAG showed the same profile of digestion. After two hours of gastric phase, more than half of the TAG was hydrolyzed. The considered FA was released to the four fifth as FFA and one fifth as esterified to 1,2-DAG (Figure 3-7 A1, A2, A3). Linoleic acid followed the same profile but the proportions of products were slightly different as 18:2n-6 esterified to 1,2-DAG achieved almost the two fifth of the 18:2n-6 from TAG (Figure 3-7 A4). Then during the intestinal phase, 2-MAG were produced and the FFA proportions kept increasing. Secondly, derivatives of FA with 20 carbons were less present in omelets except ARA, the digestion profile of which was different. Indeed, TAG esterified with 20:4n-6 was hydrolyzed during the gastric phase but without an equivalent rise in 20:4n-6 as FFA (around 7% as FFA of the total 20:4n-6 after 2 hours) (Figure 3-7 A5). Production of 1,2-DAG containing ARA was observed as well. Lipolysis achieved around 10% ARA esterified on TAG and 2% of ARA in 1,2-DAG. 20:4n-6 was also esterified to PL (accounting for around 80% of total 20:4n-6, but it was not released during the gastric phase). ARA from PL was only hydrolyzed in the intestinal phase. Overall hydrolysis of ARA esterified to TAG reached around 60% during the gastric phase and 40% during the intestinal phase, but without being complete for DHA enriched-omelet as around 4% of ARA esterified on TAG still remained at the end of the intestinal phase. RGL was active on TAG composed of ARA but with much less efficacy than on TAG composed of other C16 and C18 FA considering the higher proportions of these other FA in omelets. Thirdly, we further focused on long and very long chain FA esterified on TAG and minorly present in DHA oil. We had three main FA in C20, C22 and C24 (20:5n-3, 22:6n-3 and 24:1n-9).

We observed no reduction of 24:1n-9 esterified on TAG, no increase in FFA, 1,2-DAG and 2-MAG during the gastric phase (Figure 3-7 B1). 24:1n-9 was only released after TAG hydrolysis during the intestinal phase. EPA and DPAn-3 were very slightly released during the gastric phase as FFA (around 2.5% of total EPA as FFA and 2.5% of total DPAn-3 as FFA at 2 hours) after TAG hydrolysis . No significant increase in 1,2-DAG or 2-MAG was observed during the first phase of digestion, on the contrary to the second phase where EPA and DPAn-3 were released.

Overall, these results suggest that the gastric lipase was able to hydrolyze TAG containing long and very long chain FA such as ARA, EPA or DPAn-3 but with a very low efficiency as compared to the pancreatic lipase. The activity yield of RGL may be considered as negligible during the first phase of digestion regarding long and very long chain FA from DHA oil. The enzyme seemed nevertheless more efficient on ARA of TAG from eggs.

FA are considered as precursors when they are synthesized in vegetables by sequential desaturations performed by Δ9-, Δ12-and Δ15-desaturases. They are then usually qualified as derivatives when they are alternatively elongated and Δ6-or Δ5-desaturated. In the present work, we observed that the gastric lipase did not hydrolyze or hydrolyzed not efficiently TAG when esterified with derivatives or other very long chain FA such as 24:1n-9. It has to be noticed that these FA, other than DHA, were present in low proportions in our samples. Nevertheless, this pattern was not obtained when TAG were composed of FA in C18 or less. This result may be inherent to the origin of the enzyme as RGL comes from a lagomorphic animal which is herbivorous and doesn't usually know what a derivative is. Derivatives are found in the animal kingdom, with rare exceptions in the plant kingdom. We can thus postulate that RGL may be less specific for some long and very long chain FA. In the literature, lipolysis by using this enzyme showed that RGL was more efficient on FA with a shorter carbon chain. For instance, [START_REF] Acquistapace | Effects of interesterified lipid design on the short/medium chain fatty acid hydrolysis rate and extent (in vitro)[END_REF] found that the extent of gastric lipolysis was much lower when TAG were esterified with longer chain FA as compared to TAG esterified with three C4 FA under the activity of RGL. Similarly, [START_REF] Borel | Hydrolysis of emulsions with different triglycerides and droplet sizes by gastric lipase in vitro. Effect on pancreatic lipase activity[END_REF] found that

the lipolytic activity of pure RGL towards medium-chain TAG (containing (g/100g fatty acids) C8

(48.5) and C10 (39.7)) was three times higher than that towards long-chain TAG (containing (g/100g fatty acids) C16 (11.5), C18 (82.6), C20 (2.9) and C22 (2.5)). In addition to the length of the carbon chain, hydrolysis of FA by RGL is also impacted by the saturation of FA. For instance, [START_REF] Tormási | Assessment of Fatty Acid-Specific Lipolysis by In Vitro Digestion and GC-FID[END_REF] compared the effects of the addition of RGL on the hydrolysis of lipids in a baked fish meal, their results show that the addition of RGL during the digestion process can increase 9.5% of released FA in comparison with only using pancreatin. More interestingly, they found that the contribution of RGL to the release of saturated FA was more than double (27%) that of the unsaturated species (+12%). Moreover, hydrolysis of FA by RGL can also be affected by proteins and emulsifiers in the food matrix. Sams et al. (2018) reported that the lipolysis levels of β-caseinstabilized rapeseed oil emulsions were extremely low after 60 minutes of digestion with RGL and they thought the reason was that β-casein as the emulsifier lowers the rate of gastric lipolysis of rapeseed oil emulsions. In the present work, during the gastric phase, we found the same result between UN-DHA-O and EN-DHA-O, without distinction with DHA oil encapsulation.

Discussion

The present study demonstrates that encapsulating DHA oil into Pickering emulsion allows to increase the bioaccessibility of DHA in the small intestine. Indeed, a 12% increase of DHA released as FFA was observed in EN-DHA-O compared to that of UN-DHA-O. This increase was in line with the 11% decrease of DHA in TAG in EN-DHA-O compared to UN-DHA-O.

Very often, oil encapsulation is a way of limiting the hydrolysis and oxidation of compounds in the stomach in order to make them available in the small intestine for absorption. The present study showed that DHA was not released in the stomach at all in both EN-DHA-O and UN-DHA-O. Therefore, the beneficial effect of encapsulation mainly occurred in the small intestine where encapsulation tended to exacerbate the release of DHA mainly at the end of the intestinal phase. It looks like that the heat-denatured whey protein shell that was surrounding the DHA oil was not an obstacle to the hydrolysis action of the pancreatic lipase. It could be interesting to investigate at which stage this protein shell was hydrolyzed allowing the digestive lipase to anchor at the interface of the lipid droplet. One possible explanation for the difference observed in the kinetics of release of DHA in the small intestine during in vitro digestion might be the difference observed in the lipid droplet size between the two omelets. Indeed, in UN-DHA-O, lipids appeared as non-homogenous large droplets that were visible by CLSM throughout the gastric phase. In contrast, encapsulation of DHA oil led to the formation of a high number of small droplets of around 4.34 µm in diameter that were not visible by CLSM and that increased the specific surface available for digestive lipases. It has previously been shown that increasing the specific surface of lipids, by homogenization for instance, accelerates the kinetics of lipid digestion in vitro [START_REF] Bourlieu | The structure of infant formulas impacts their lipolysis, proteolysis and disintegration during in vitro gastric digestion[END_REF] and in vivo [START_REF] De Oliveira | Impact of homogenization of pasteurized human milk on gastric digestion in the preterm infant: A randomized controlled trial[END_REF]. The effect of oil encapsulation on DHA bioaccessibility has been investigated

previously. Indeed, DHA encapsulation in caseinate/alginate microparticles enhanced its bioaccessibility [START_REF] Ma | Enhancing the physicochemical stability and digestibility of DHA emulsions by encapsulation of DHA droplets in caseinate/alginate honeycomb-shaped microparticles[END_REF]. It was shown that the digestion of DHA microparticles led to more open loose structures than the large aggregated lipid droplets that were observed with DHA emulsion droplets. Consequently, the rate of lipid digestion was function of the surface area of lipids exposed to the lipase molecules as well as the capability of these enzymes to adhere and further contact the underlying TAG molecules. Similarly, the lipid hydrolysis, oxidative stability and bioaccessibility of algae oil (42% of DHA) was compared for three lipid delivery systems, i.e bulk oil, soy protein stabilized O/W emulsion and carrageenan gelled emulsion [START_REF] Gayoso | DHA rich algae oil delivered by O/W or gelled emulsions: Strategies to increase its bioaccessibility[END_REF]. Emulsification techniques were linked to a slight 3% increase in DHA released during in vitro gastrointestinal digestion. Finally, the addition of pectin to an emulsified algal oil rich in DHA was shown to limit lipid digestion and DHA bioaccessibility [START_REF] Lin | Pectin and gastric pH interactively affect DHA-rich emulsion in vitro digestion microstructure, digestibility and bioaccessibility[END_REF]. The effect of pectin was attributed to its interaction with lipids and other digestive molecules during simulated gastrointestinal digestion that limited the action of pancreatic lipase.

Oil encapsulation with milk proteins has also been shown to limit DHA oxidation [START_REF] Hosseini | Encapsulation of EPA and DHA concentrate from Kilka fish oil by milk proteins and evaluation of its oxidative stability[END_REF]. DHA can be oxidized either by enzymes, such as lipoxygenases or cyclooxygenases, or by non-enzymatic chain process mediated by free radicals and enhanced by autocatalytic or photocatalytic reactions, propagating via the classical mechanism of lipid peroxidation chain reaction [START_REF] Yakubenko | Biological and pathophysiological roles of end-products of DHA oxidation[END_REF]. Oxidation of DHA leads to the generation of end-products exemplified by carboxyethylpyrrole (CEP) modifications of proteins and lipids. Numerous studies from multiple laboratories have demonstrated the presence of CEP modifications in blood and tissues in a variety of pathological conditions including, but not limited to, hyperlipidemia, atherosclerosis, macular degeneration, and tumor progression [START_REF] Yakubenko | Biological and pathophysiological roles of end-products of DHA oxidation[END_REF].

At the beginning of the digestion process, TAG and PL were the main lipid species, accounting for approximately 73% and 24% of total FA, respectively. In addition, there were a small amount of 1,2-DAG, 1,3-DAG, FFA, MAG, and CE. This result is in concordance with previous egg studies, which have shown that TAG were about two-thirds and PL were about one-third of the total lipids in eggs [START_REF] Wang | Effects of yolk contamination, shearing, and heating on foaming properties of fresh egg white[END_REF]. When looking at the evolution of the different classes of lipids over digestion, the present study has also shown that while TAG started to be hydrolysed in the gastric phase, phospholipids were not affected and only digested in the intestinal phase. This is in agreement with the fact that the gastric lipase exhibits a high hydrolytic activity towards TAG, lower towards DAG, is only slightly active on DAG and has no action on PL [START_REF] Lengsfeld | Physiology of gastrointestinal lipolysis and therapeutical use of lipases and digestive lipase inhibitors[END_REF]. In contrast, PL are the favourite substrates of the pancreatic phospholipase A2 [START_REF] Borgström | Phosphatidylcholine as Substrate for Human Pancreatic Phospholipase A2 Importance of the Physical State of the Substrate[END_REF] which catalyses the hydrolysis of the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids) but PL can be hydrolysed as well by other pancreatic enzymes such as the carboxylester hydrolase and pancreatic lipase-related protein-2 [START_REF] Whitcomb | Human pancreatic digestive enzymes[END_REF]. In the gastric and intestinal phase, the released FFA accounted for approximately 29% and 46% of the total FA in all groups, respectively. Therefore, the addition of DHA oil to the omelet did not affect the lipolysis of PL during the digestion.

Oil encapsulation did not affect the proportion of DHA in the oil indicating that the quality of the oil was maintained during the encapsulation process and that DHA oxidation was avoided.

In the present study, the control omelet contained a low amount of DHA (0.5 µg/mg) exclusively brought by the PL. In DHA enriched groups, the enrichment of DHA oil greatly increased DHA concentration, DHA concentration reaching 7.7 and 9.1 µg/mg in UN-DHA-O and EN-DHA-O, respectively. Besides, the proportion of DHA in different lipid species was no different between DHA enriched groups. In addition, the theoretical DHA concentration in omelets should be 9.1 µg/mg of sample, which was the same as the DHA concentration in EN-DHA-O and higher than in UN-DHA-O. It can be attributed to the loss of unencapsulated DHA oil that can easily adhere to the containers and castings during the omelet preparation. In both UN-DHA-O and EN-DHA-O, the proportion of DHA in TAG was around 86%, which was significantly (p<0.05) lower than in DHA oil (97%). At the same time, the proportion of DHA in 1,2-DAG was around 8%, which was significantly higher (p<0.05) than in DHA oil (2%). Therefore, the preparation of omelets affects the DHA oil quality.

The microstructure of Pickering emulsion exhibited a protein layer on the surface of the DHA oil droplet. However, the heat-denatured WPI particles could not be clearly seen from the microstructure of Pickering emulsion. This was due to the particle size of the heat-denatured WPI that was too small (around 42 nm) to be clearly observed by a CLSM. In contrast, for UN-DHA-O, the droplet size and distribution of DHA oil droplets were not uniform and large patches of lipids were observed.

Conclusion

The purpose of the study was to investigate the effect of encapsulation on the bioaccessibility of DHA in DHA enriched omelets through an in vitro digestion model for adults. Firstly, the impact of emulsion and omelet preparation on DHA oil quality was studied by comparing the proportion of different lipid species in total FA. The results revealed that emulsion preparation preserved DHA oil quality; however, omelet preparation decreased DHA oil quality. Then, the addition of unencapsulated DHA oil (pure DHA oil) or encapsulated DHA oil (Pickering emulsion) to omelets achieved the DHA enrichment. However, during this process, unencapsulated DHA oil could easily attach containers and castings, which made some loss of DHA oil. DHA oil can only be digested in the intestinal phase by the pancreatic lipase, which may be because RGL is more stereoselective for TAG with shorter acyl chains. The hydrolysis of DHA-TAG and the release of DHA-FFA in encapsulation groups were significantly higher than those in unencapsulation group at the end of the digestion, which can be attributed to the larger interaction area between DHA oil and pancreatic lipase made by emulsification during the Pickering emulsion preparation. Therefore, encapsulation DHA oil by heat-denatured WPI stabilized Pickering emulsion improved the bioaccessibility of DHA. Future research should focus not only on the application of delivery systems to foods, but also on in vivo studies, because in vivo is a more complex system than in vitro. Chapter 4: Encapsulation of DHA oil substantially improves the oxylipin profile of rat tissues

Abstract

DHA is a major n-3 polyunsaturated fatty acid particularly involved in cognitive and cardiovascular functions. Due to the high unsaturation index, its dietary intake form has been considered to improve oxidation status and to favor as well bioaccessibility and bioavailability. This study aimed at investigating the effect of DHA encapsulated with natural whey protein. DHA was dietary provided as triacylglycerols to achieve 2.3% over total fatty acids. It was daily supplied to weanling rats for four weeks in omelet as food matrix, consecutively to a 6-hour fasting. Firstly, when DHA oil was encapsulated, consumption of chow diet was enhanced leading to promote animal growth. Secondly, brain exhibited a high accretion of 22.8% DHA, which was not improved by dietary supplementation of DHA. Encapsulation of DHA oil did not greatly affect the fatty acid proportions in tissues, but remarkably modified the profile of oxidized metabolites of fatty acids in plasma, heart and even brain. Specific oxylipins derived from DHA were upgraded, such as Protectin Dx in heart and 14-HDoHE in brain, whereas those generated from n-6 polyunsaturated fatty acids were mainly mitigated. This effect did not result from oxylipins measured in DHA oil since DHA and EPA derivatives were undetected after food processing. Collectively, these data suggested that dietary encapsulation of DHA oil triggered a more efficient absorption of DHA, the metabolism of which was enhanced more than its own accretion in our experimental conditions.

Incorporating DHA oil in functional food may finally improve the global health status by generating precursors of protectins and maresins.

Introduction

DHA is an essential n-3 polyunsaturated fatty acid (PUFA), mainly known for its health benefits on brain and heart. This bioactive compound presents a large range of beneficial outcomes inherent to the modulation of both membrane structure and cell functions. Therefore, DHA improves neurological activities by enhancing neurogenesis and synaptic plasticity, or by reducing neuroinflammation [START_REF] Weiser | Docosahexaenoic Acid and Cognition throughout the Lifespan[END_REF]. Its high body level is related as well to the lower risk of developing cardiovascular disease [START_REF] Ajith | Omega-3 fatty acids in coronary heart disease: Recent updates and future perspectives[END_REF][START_REF] Holub | Docosahexaenoic acid (DHA) and cardiovascular disease risk factors[END_REF] and myocardial infarction [START_REF] Richard | Infusion of docosahexaenoic acid protects against myocardial infarction[END_REF]. The functionality of DHA is likely to be mostly implemented through the synthesis of derivatives such as oxygenated metabolites of FA (oxylipins) or endocannabinoid-like mediators. These derivatives may be generated either from DHA itself or from other FA, such as arachidonic acid (ARA, 20:4n-6), dihomo--linolenic acid (DGLA, 20:3n-6), linoleic acid (LA, 18:2n-6) and eicosapentaenoic acid (EPA, 20:5n-3). DHA accumulates indeed in cell membranes concomitantly with the reduction of n-6 FA, principally ARA, or is retroconverted into EPA. Therefore, the effect of DHA may be attributed both to the rise of its own derivatives and the decline of derivatives synthesized from other FA. Many studies have reviewed the importance of the dietary supply of DHA as a strategy to increase tissue DHA concentrations [START_REF] Aghaei | Blood and tissue fatty acid compositions, lipoprotein levels, performance and meat flavor of broilers fed fish oil: Changes in the pre-and post-withdrawal design[END_REF][START_REF] Gerbi | Dose dependent accretion of docosahexaenoic acid (DHA) in cardiac membranes of rats fed egg yolk powder enriched in DHA[END_REF][START_REF] Hajjaji | Determinants of DHA incorporation into tumor tissue during dietary DHA supplementation[END_REF]. Although modulating the FA content in brain still represents a challenge comparatively to other tissues, dietary interventions may be effective in targeting serum lipid pools important for brain DHA uptake [START_REF] Lacombe | Brain docosahexaenoic acid uptake and metabolism[END_REF]. Thereby several works have shown a higher [START_REF] Maki | Krill oil supplementation increases plasma concentrations of eicosapentaenoic and docosahexaenoic acids in overweight and obese men and women[END_REF][START_REF] Ulven | Metabolic Effects of Krill Oil are Essentially Similar to Those of Fish Oil but at Lower Dose of EPA and DHA, in Healthy Volunteers[END_REF][START_REF] Ramprasath | Enhanced increase of omega-3 index in healthy individuals with response to 4-week n-3 fatty acid supplementation from krill oil versus fish oil[END_REF] or equal [START_REF] Carnielli | Intestinal absorption of long-chain polyunsaturated fatty acids in preterm infants fed breast milk or formula[END_REF][START_REF] Schuchardt | Incorporation of EPA and DHA into plasma phospholipids in response to different omega-3 fatty acid formulations-A comparative bioavailability study of fish oil vs. Krill oil[END_REF][START_REF] Yurko-Mauro | Similar eicosapentaenoic acid and docosahexaenoic acid plasma levels achieved with fish oil or krill oil in a randomized double-blind four-week bioavailability study[END_REF] bioavailability of DHA esterified to phospholipids (PL) in comparison to DHA esterified to triacylglycerols (TAG). Other works are more controversial [START_REF] Ghasemifard | Omega-3 long chain fatty acid "bioavailability": A review of evidence and methodological considerations[END_REF]Gá zquez & Larqué , 2021), insomuch as a higher concentration of circulating DHA would not predict brain accretion of DHA (Gá zquez et al., 2017;[START_REF] Adkins | Accretion of Dietary Docosahexaenoic Acid in Mouse Tissues Did Not Differ between Its Purified Phospholipid and Triacylglycerol Forms[END_REF][START_REF] Destaillats | Comparison of the Incorporation of DHA in Circulatory and Neural Tissue When Provided as Triacylglycerol (TAG), Monoacylglycerol (MAG) or Phospholipids (PL) Provides New Insight into Fatty Acid Bioavailability[END_REF]. Furthermore, dietary supplementation of DHA was shown to generate higher levels of its oxygenated derivatives as oxylipins [START_REF] Gabbs | Time Course and Sex Effects of α-Linolenic Acid-Rich and DHA-Rich Supplements on Human Plasma Oxylipins: A Randomized Double-Blind Crossover Trial[END_REF]Ostermann & Schebb, 2017). These lipid mediators are synthesized from PUFA by cyclooxygenase, lipoxygenase, or cytochrome P450 ω-hydroxylase or epoxygenase enzymes, or by nonenzymatic auto-oxidation [START_REF] Gabbs | Advances in Our Understanding of Oxylipins Derived from Dietary PUFAs[END_REF]. The oxylipin pattern depends both on the dietary intake of n-6

or n-3 PUFA, and on the endogenous synthesis of long-chain PUFA considering enzyme competition between n-6 and n-3 families in tissues. The structural diversity of oxylipins is notably high for most of PUFA and foreshadows a diversity of biological functions as well. Hence, these essential signalling messengers are usually involved in physiological responses to maintain homeostasis, contain infection or restrain inflammation process by acting through specific membrane receptors (An et al., 2021;[START_REF] Chiang | Specialized pro-resolving mediator network: An update on production and actions[END_REF]. More particularly, oxygenation of DHA forms docosanoids, including specialized pro-resolving mediators (SPM) such as maresins, D-resolvins and protectins [START_REF] Hajeyah | The Biosynthesis of Enzymatically Oxidized Lipids[END_REF]. The literature reports their bioactive implying on pathophysiological process as they improve immune response, resolution of neuroinflammation or other metabolic disorders including diabetes, atherosclerosis, non-alcoholic fatty liver disease or hepatic steatosis [START_REF] Zaloga | Narrative Review of n-3 Polyunsaturated Fatty Acid Supplementation upon Immune Functions, Resolution Molecules and Lipid Peroxidation[END_REF][START_REF] Devassy | Omega-3 Polyunsaturated Fatty Acids and Oxylipins in Neuroinflammation and Management of Alzheimer Disease[END_REF][START_REF] Li | Role of Resolvins in the Inflammatory Resolution of Neurological Diseases[END_REF][START_REF] Duan | Effect of ω-3 Polyunsaturated Fatty Acids-Derived Bioactive Lipids on Metabolic Disorders[END_REF][START_REF] Maciejewska-Markiewicz | The Role of Resolvins, Protectins and Marensins in Non-Alcoholic Fatty Liver Disease (NAFLD)[END_REF]. These pleiotropic properties justify the interest for DHA, and its food intake other than based on PL or TAG esterification has to be considered as well.

An alternative approach to optimize dietary DHA intake is the use of functional food based on encapsulation. The food-grade delivery system was originally developed to protect bioactive ingredients notably from degradation such as oxidation. Many techniques using different encapsulant materials were processed in diverse food matrices for this purpose [START_REF] Gruskiene | Microencapsulation of Bioactive Ingredients for Their Delivery into Fermented Milk Products: A Review[END_REF]. Nevertheless, controlling the colloidal state of lipophilic molecules underlies the control of composition, size and surface characteristics of particles, to ensure the required bioavailability.

Pickering emulsification of food oil has recently received substantial interest as it relies more specifically on adsorption of bioparticles rather than synthetic surfactants to the oil-water interface, which confers resistance to droplet coalescence and improves the stability of emulsion [START_REF] Chevalier | Emulsions stabilized with solid nanoparticles: Pickering emulsions[END_REF][START_REF] Low | Recent advances of characterization techniques for the formation, physical properties and stability of Pickering emulsion[END_REF][START_REF] Wu | Recent Studies of Pickering Emulsions: Particles Make the Difference[END_REF]. In this study, Pickering emulsion was prepared with natural whey protein to encapsulate DHA enriched-TAG, as previously described [START_REF] Wang | CaCl2 supplementation of hydrophobised whey proteins: Assessment of protein particles and consequent emulsions[END_REF]. Although fermented milk products are widely used as a vector of encapsulated nutrients, our previous study showed that eggs present an interesting bioavailability of DHA according to the recipe (omelet, hard-boiled egg or mousse) [START_REF] Pineda-Vadillo | In Vivo Digestion of Egg Products Enriched with DHA: Effect of the Food Matrix on DHA Bioavailability[END_REF]. In the present study, DHA was therefore encapsulated in heat-denatured whey proteins. Whey protein isolate is commonly used in food industry due to a variety of functionalities such as emulsification or gelation properties. The heat-denatured form was here favored considering the cooking process that needed to be applied during the food manufacture. For this reason, whey protein-based microcapsules of DHA oil were incorporated into eggs and baked thereafter as omelets. Then rats were daily fed with DHA enriched-omelet for four weeks. Effect of encapsulation was finally assessed on DHA accretion, specifically in brain and heart, and subsequently on FA derivative formation such as oxylipins.

Materials and Methods

Animal experiments

All handling protocols performed complied with the European Union Guideline for animal care

Diets

Diets were rodent chows prepared according to the AIN-93-G formulation and made at the Unité de Préparation des Aliments Expérimentaux of INRAE (Jouy en Josas, France) [START_REF] Reeves | AIN-93 Purified Diets for Laboratory Rodents: Final Report of the American Institute of Nutrition Ad Hoc Writing Committee on the Reformulation of the AIN-76A Rodent Diet[END_REF]. The H-diet was composed of 20.6% proteins, 64.8% carbohydrates and 5% lipids. The T-diet was composed of 20.8% proteins, 65.1% carbohydrates and 4.5% lipids. Both diets contained 5.1% fibers and 4.5% mineral and vitamin mix (Table 4-1A) (Potier de [START_REF] Potier De Courcy | Recommandations sur les conditions d'alimentation des animaux de laboratoire (rats et souris)[END_REF]. Pellets were formulated by adding a mix of vegetable oils specific for each diet (Table 4-1B), considering the consumption of eggs during the 4-week period of treatment.

The T-diet was designed similarly to the H-diet to achieve the same final FA profile per day.

Food consumption was previously estimated at 20 g per day of rodent chow combined with 3 g omelet serving over this growth period. In that condition, egg was supposed to contribute to the height of 0.5% lipids and T-diet of 4.5% lipids. The mixture of vegetable oils was adjusted in T-diet as a consequence in considering the FA profile of egg. The portion of linseed was thus increased between H-diet and T-diet to compensate the low level of ALA in omelet, in order to maintain the ratio n-6/n-3 to 5 with 3.4% of 18:3n-3 in average. The composition of dry diets based on the AIN-93-G is presented for the habituation and the treatment diets. Both diets contained a mixture of vegetable oils formulated according to the daily consumption of eggs.

DHA encapsulation and omelets

DHA oil was prepared enzymatically from fish oil and was composed of DHA-enriched TAG containing 615 µg/mg DHA, 31 µg/mg DPAn-3 and 36 µg/mg EPA, accounting for 76.5%, 3.9% and 4.5% of total FA respectively (Polaris, Quimper, France). DHA oil was daily encapsulated with heat-denatured whey protein isolate as described previously [START_REF] Wang | CaCl2 supplementation of hydrophobised whey proteins: Assessment of protein particles and consequent emulsions[END_REF]. The supplementation of proteins was therefore 300 µg per day, corresponding to less than 0.1% of proteins from eggs.

Omelet was daily prepared with a whole egg (Moisan aviculture, Plestan, France) homogenized by ultraturrax (10 000 rpm, 30 sec). Then encapsulated-DHA oil (EN-DHA-O), heat-denatured whey protein isolate dispersion alone (Control) or completed with the DHA oil (UN-DHA-O) were added and mixed by stirring (500 rpm, 5 min). Eggs were finally molded and cooked in a water bath (80 °C, 10 min).

Fatty acid and dimethylacetal analysis

Lipids from omelets and tissues were extracted according to the Folch's method (1957). DHA oil, vegetable oils and lipids from omelets and tissues were saponified with 0.5 mol/L NaOH in methanol at 70 °C for 20 min and methylated with BF3 (14 % in methanol) at 70 °C for 15 min (Supplemental Figure 4-1). Fatty acid methyl esters (FAME) and dimethylacetals (DMA) were extracted with pentane and then separated by a QP 2010-SE gas chromatograph coupled to a mass spectrometer (Shimadzu, Marne-La-Vallée, France) equipped with a BPX70 capillary column (120 m, 0.25 mm i.d., 0.25 µm film, SGE Trajan, Chromoptic, Paris). Helium was used as carrier gas at a constant velocity of 27,5 cm/sec. The temperature of injector was adjusted to 250°C. The column temperature ramped from 50 °C to 175 °C at 20 °C/min and then from 175 °C to 240 °C at 2 °C/min.

The mass spectrometer was operated under electron ionization at 0.2 keV and 200 °C source temperature. Analyses were performed in scan mode over the m/z range of 30 -450 amu.

Components were identified by using the National Institute of Standards and Technology (NIST) mass spectral library (version 2.01) in addition to commercial standards. Concentrations were determined by using 17:0 as the internal standard and were calculated by standard curves of either FAME or DMA. Results were expressed as a mass percentage of the total FA or total DMA, and as concentrations for total FA and total DMA as well.

Oxylipin analysis

Oxidized metabolites of PUFA were quantified in DHA oil, omelets as well as plasma, heart and brain by liquid chromatography combined with tandem mass (LC-QQQ) by the MetaToul lipidomic core facility (Justine Bertrand-Michel and Pauline Le Faouder, I2MC, Inserm 1048, MetaboHUB-ANR-11-INBS-0010 Toulouse, France). Briefly, frozen tissues (250 mg) were crushed with a FastPrep ®-24 Instrument (MP Biomedical, Illkirch-Graffenstaden, France) in 500 µL of Hank's balanced salt solution (Thermo Fisher Scientific, Illkirch-Graffenstaden, France). After 2 crush cycles (6.5 m/sec, 30 sec), cold methanol (300µL) and internal standard (5 µL of LXA4-d5, LTB4-d4 and 5HETE-d8) were added to homogenates. After centrifugation (15 min at 2000 g at 4°C), supernatants were diluted in 2 mL H2O and then submitted to solid phase extraction using OASIS HLB 96-well plate (30 mg/well, Waters, Saint-Quentin-en-Yvelines, France) pretreated with methanol (1 mL), equilibrated with 10% methanol (1 mL) and washed after sample application with 10% methanol (1 mL). Lipids mediators were finally eluted with 1 mL of methanol and reconstituted in 10 µL methanol prior to LC-QQQ analysis (Le [START_REF] Faouder | LC-MS/MS method for rapid and concomitant quantification of pro-inflammatory and proresolving polyunsaturated fatty acid metabolites[END_REF]. They were separated on a ZorBAX SB-C18 column (50 mm, 2.1 mm i.d., 1.8 µm film) using Agilent 1290 Infinity HPLC system coupled to an ESI-triple quadruple G6460 mass spectrometer (Agilent Technologies, Les Ulis, France). Data were acquired in multiple reaction monitoring mode with optimized conditions (ion optics and collision energy). Peak detection, integration and quantitative analysis were done using Mass Hunter Quantitative analysis software (Agilent Technologies) based on calibration lines built with eicosanoid standards (Interchim, Montluçon, France).

Statistics

Results are expressed as the mean ± SEM of 8 animal samples per group. Data analysis was performed using R software. Correlations were evaluated by Pearson correlation coefficients and the analysis of variance was done by ANOVA followed by a post-hoc test depending on the normality of the data distribution. The significance of the effect observed with the DHA oil or the encapsulated-DHA oil was marked by an asterisk or different letters when p < 0.05. Lipids from croquettes and omelets were extracted according to the Folch's method. The fatty acid profile performed by GC-MS was determined for the habituation and the treatment diets, and the different omelets prepared with unencapsulated-DHA oil or encapsulated-DHA oil.

Food intake and animal growth

Animals were fasted for 6 hours before receiving individually the portion of omelets, the consumption of which was complete for each group of rats during the entire experiment. The T-diet intake was followed as well and a significant increase in the food consumption was observed from the first week with EN-DHA-O (Figure 4-2A). with body weights of animals, no significant differences were obtained between groups, which subtends that the raise in organ weights resulted from rat growth. The weight gain of animals resulted presumably from an increased energy intake, since the FA proportion was similar between groups. The FA profile was indeed estimated in the view of the measured consumption of the T-diet per group on the one hand, and the complementary omelet on the other (Table 4-3). The fatty acid profile was calculated per group of omelets on the base of the treatment diet consumption, and was averaged per day on the four-week experimental period. This profile corresponds to the combination between FA consumed from the T-diet and FA supplied by the 3g omelet.

The FA profile combining T-diet and omelet was similar to the FA profile of the H-diet and no major difference in FA proportions or n-6/n-3 ratio was observed between groups. In addition, both diets enriched in DHA oil accounted for a supplementation of TAG-DHA of 2.3% since the FA was supplied equivalently between groups through the distribution of 3g omelet per day.

The first ascertainment showed an effect of the EN-DHA-O diet on food intake and rat growth, so we further investigated the effect of encapsulation of DHA on lipid metabolism.

Effects of diets on blood lipids

The effect of diets was first analyzed on lipids in plasma. respectively. Unlike cholesterol, the encapsulation group had a significantly higher level of TAG in plasma as compared to the two other groups. This result contrasts with the literature, which usually shows a decrease in plasma TAG when DHA is given as a supplement. This observation has to be clarified, as no difference in the concentrations of total FA in plasma was found among the three groups.

Effects of diets on the metabolism of FA

The FA metabolism and particularly the accretion of DHA in tissues were then explored to assess the importance of encapsulation of the DHA oil in the diet. The analysis focused on blood, liver, heart, brain and eyes. Firstly, the diet supplementation of DHA induced a significant increase in DHA in tissues, except for the brain and the eyes (Figure 4-3). Encapsulation of the DHA oil globally tends to increase the DHA accretion in blood and heart (Supplemental Tables 4-1 ~ 4-3).

In liver, a decrease in the DHA proportion was observed with EN-DHA-O but coupled with a slight increase in the FA content, the DHA concentration was not changed. The impact of the diets was also estimated on precursors of DHA, minorly present in the DHA oil. Thus, EPA was significantly increased by the DHA supplementation in plasma, RBC and liver without any significant effect of the encapsulation of the DHA oil. The same result was observed with DPA n-3 only in plasma . Secondly, the proportion of n-6 FA was significantly decreased with the DHA-enriched diets in all tissues, depending on the considered FA. The main effect was obtained in heart on the entire n-6 family (Supplemental Table 4-2) and in eyes especially on ARA (Supplemental Table 4- 3). Only eyes exhibited a significant effect of the encapsulation of the DHA oil by slightly reducing ARA from 11.8% in UN-DHA-O to 10.9% in EN-DHA-O. Otherwise no major effect of the DHA supplementation was observed on the FA profiles. Analyses were further performed on particular lipids or derivatives.

Effects of diets on the DMA profile

Brain and heart are particularly rich in plasmalogens, which are specific etherlipids known as reservoirs of polyunsaturated FA. DMA compounds derived from the aldehyde chain of plasmalogens present at the sn-1 position of the glycerol and were synthesized through the lipid saponification and methylation steps. They co-eluted with FAME. The DMA profile was thus analyzed to quantify plasmalogens (Table 4- 4). Therefore, DMA were only measured in brain, heart, RBC and eyes. Brain contained an average of 2µg of DMA per mg of tissues, against 0.4µg/mg in heart and 0.1µg/mg in RBC and eyes. Moreover, the alkenyl chain was discriminant between tissues.

For instance, in brain, the major DMA was composed of 18:0 (53% in all groups), then 16:0 (22%), 18:1n-9 (15%) and 18:1n-7 (10%). The profile of DMA was completely different in heart with 63% of 16:0, then 18:1n-9 (23%) and 18:0 (13%). Nonetheless, no effect of the diet was measured on the DMA profile. In eyes, the DMA content was reduced by half with the DHA supplementation passing from 0.12µg/mg to 0.06µg/mg and 0.

EN-DHA-O

35.1 ± 1.3 b 34.8 ± 0.6 15.9 ± 0.8 ab 14.2 ± 1.0 ab 0.07 ± 0.01 b Lipids of tissues were extracted by the Folch's method and the dimethylacetal profile was determined by GC-MS. a, b, c and ab indicate significant differences between the three groups; ab means no difference with a and no difference with b (p<0.05).

Effect of diets on oxidized metabolites of PUFA

Considering the slight effect of diets on the FA profiles in tissues, we further investigated the oxidized metabolites. First of all, oxylipins were quantified in DHA oil and food matrices as well . Eight oxidized metabolites from n-6 and n-3 PUFA were measured, mainly from LA and DHA. Hence DHA oil contained a majority of 13-HODE and 9-HODE derived from LA, followed by 17-HDoHE and 14-HDoHE derived from DHA. Much less present, few oxylipins of ARA and 18-HEPE from EPA were detected as well. When oxylipins were then measured in omelets, 13-HODE, 9-HODE and 5-HETE were the only oxidized metabolites quantified in all groups, even in DHA-enriched omelets where the DHA oil was added. No oxylipins from DHA and EPA were detected in UN-DHA-O and EN-DHA-O whereas a small amount of these compounds was expected considering the supplementation with the DHA oil. We further analyzed the oxylipin pattern of animal tissues after the 4-week experiment. First, in plasma, three oxylipins were measured and derived from DHA but also from ARA and LA (Table 456. Overall, the supplementation of DHA strongly modulated the concentrations of oxylipins, and the encapsulation of the DHA oil greatly emphasized this effect. More specifically, 14-HDoHE derived from DHA was reduced by half with UN-DHA-O and by 6 with EN-DHA-O, as 14-HDoHE was negatively correlated with the DHA proportions in plasma (Supplemental Figure 4-2). In parallel, 17-HDoHE was not affected by the diet, as well as with 9-HODE from LA, whereas 13-HODE was reduced by the DHA supplementation without any influence of its encapsulation. Lastly, the greatest effect was the decrease in ARA derivatives by 76% with UN-DHA-O and by 93% with EN-DHA-O as compared to the control. Some of oxylipins even became undetectable with the DHA encapsulation such as PGE2 or 8-HETE.

Analyses were then completed by oxidized metabolites from heart (Table 4567). Oxylipins were altogether more numerous but quantitatively less important than in plasma. At last, in brain, no effect of the DHA oil supplementation was observed on measured oxidized metabolites (Table 45678). But more interestingly, three oxylipins were significantly increased when the DHA oil was encapsulated. Therefore, 14-HDoHE derived from DHA was almost doubled with EN-DHA-O as compared to the control and the UN-DHA-O groups, while 8-HETE and 5,6-EET, positively correlated in brain (R² = 0.5845, p < 0.001, not shown), were more reasonably raised with EN-DHA-O. To conclude, the DHA supplementation globally modulated the oxylipin pattern of tissues, but the modulation was particularly prominent when the DHA oil was encapsulated. a, b, c and ab indicate significant differences between the three groups; ab means no difference with a and no difference with b (p<0.05).

Discussion

In this study, DHA esterified to TAG was encapsulated with natural whey proteins in producing Pickering emulsion. Egg was chosen as a food matrix to favor the bioavailability of DHA [START_REF] Pineda-Vadillo | In Vivo Digestion of Egg Products Enriched with DHA: Effect of the Food Matrix on DHA Bioavailability[END_REF], but we used cooked egg as omelet to determine the tissue accretion of encapsulated DHA in using rats as experimental model. Overall the DHA supplementation increased the DHA concentration in tissues except brain and eyes. EPA was increased as well in plasma, RBC and liver. This FA was possibly synthesized from n-3 precursors such as ALA present in T-diet.

Nevertheless, this raise was inherent to the DHA supplementation in omelets, considering that EPA was minorly present in DHA oil and was potentially generated by retro-conversion of DHA or of DPA n-3. DPA n-3 was also minorly present in DHA oil, to the same extent than EPA. However, its metabolism differed since DPA n-3 was weakly accumulated in tissues. As EPA was directly provided by egg food and easily synthesized from ALA, retro-conversion may not be favoured in our conditions of work, although the importance of retro-conversion to produce EPA is still under discussion [START_REF] Brenna | DHA retroconversion revisited: Dietary DHA spares endogenous EPA[END_REF]Metherel et al., 2019;Metherel & Bazinet, 2019). Now considering more specifically DHA, accretion was measured whereas rats were fed from weaning during 4 weeks of growth with an optimized diet. In these conditions, the DHA supplementation did not improve significantly DHA accretion in brain, as the DHA concentration was already high in the tissue, reaching more than 20% of total FA regardless of the diet. Our result contrasts with other studies where DHA proportion in brain was significantly increased by around 20% when rats fed a DHA enriched-diet as compared to the control [START_REF] Destaillats | Comparison of the Incorporation of DHA in Circulatory and Neural Tissue When Provided as Triacylglycerol (TAG), Monoacylglycerol (MAG) or Phospholipids (PL) Provides New Insight into Fatty Acid Bioavailability[END_REF][START_REF] Lin | Altered essential fatty acid metabolism and composition in rat liver, plasma, heart and brain after microalgal DHA addition to the diet[END_REF][START_REF] Lindquist | Effects of dietaryinduced alterations in rat brain docosahexaenoic acid accrual on phospholipid metabolism and mitochondrial bioenergetics: An in vivo 31 P MRS study[END_REF][START_REF] Ross | Dietary omega-3 polyunsaturated fatty acid supplementation in an animal model of anxiety[END_REF]. First, it has to be mentioned that important differences between studies were also highlighted by other authors concerning mice models, where comparisons were difficult considering strains, brain regions, ages and diet conditions [START_REF] Joffre | Modulation of brain PUFA content in different experimental models of mice[END_REF]. Then, it must be noted that this effect was observed in the literature while the DHA proportion in brain was usually close to 10%-15% of the total FA, which greatly differs from our DHA proportion reaching 22.8% in the control group. Additionally, many of studies on DHA were designed with a low n-3 PUFA diet, favoring DHA half-life in brain and synthesis rate from the liver. In our study, the experimental design favoured a higher concentration of DHA in frontal cortex, which was not significantly improved by the dietary DHA supplementation. This effect may result from ALA supplied from the T-diet in complement to the low level of PL-DHA from eggs. It was indeed shown that DHA synthesis from ALA may be sufficient to maintain brain DHA concentrations in the absence of dietary DHA consumption [START_REF] Domenichiello | Whole body synthesis rates of DHA from α-linolenic acid are greater than brain DHA accretion and uptake rates in adult rats[END_REF]. Here, a high level of DHA in brain was possibly conditioned by an important de novo synthesis from dietary ALA and favoured by the daily fasting as well. Short-term fasting was shown to enhance DHA concentrations in serum without dietary supplementation with DHA [START_REF] Marks | Fasting enriches liver triacylglycerol with n-3 polyunsaturated fatty acids: Implications for understanding the adipose-liver axis in serum docosahexaenoic acid regulation[END_REF]. The authors suggested that an increase in the expression of FA transporters specific for DHA likely mediated an enrichment of PUFA in hepatic TAG. This enrichment may foster secretion of DHA from the liver as 2-DHA-lysoPL for recirculation, subsequently promoting brain DHA accretion. Hepatic lipase was actually shown to play a major role in generating 1-lyso-2-DHA-sn-glycerophosphocholine, one of the main forms of DHA transported through the blood brain barrier for brain DHA enrichment [START_REF] Sugasini | Potential role of hepatic lipase in the accretion of docosahexaenoic acid (DHA) by the brain[END_REF]. However, in our study, encapsulation of dietary DHA was shown to significantly improve neither bioavailability nor brain accretion of DHA. That result contrasts with other works based on whey protein or lipid emulsions. When rats were treated by gavage with encapsulated linseed oil, DHA was enhanced in cardiac sarcoplasmic reticulum by 28% and 94% with protein and phospholipid-based emulsions respectively (Sugasini & Lokesh, 2013b). When DHA was supplied as fish oil, DHA was more accumulated in tissues with encapsulated oil than with native oil (Sugasini & Lokesh, 2013a).

Nevertheless, the effect was high with lipid emulsion and much less important with whey protein emulsion. More specifically in brain, DHA accretion was upgraded from 11.4% with native fish oil to 12.6% with whey protein encapsulation. If the nature of encapsulation plays a major role in the tissue delivery of DHA, methods of administration may impact the bioavailability of DHA as well.

In many studies, animals were fed encapsulated oil by gavage. Here we integrated functional food with a cook processing. The daily preparation was constraining but, in that way, omelets were baked with natural compounds and no additives.

In view of the results on FA profiles, we further analysed DMA derived from plasmalogens.

These etherlipids, and more precisely those derived from phosphatidylethanolamine, are known as carriers of long chain PUFA in brain. Zhao et al showed in mouse cortex that plasmenylethanolamine was composed of 30% DHA, 25% DPAn-3 and 10% EPA [START_REF] Zhao | Recovery of brain DHA-containing phosphatidylserine and ethanolamine plasmalogen after dietary DHA-enriched phosphatidylcholine and phosphatidylserine in SAMP8 mice fed with high-fat diet[END_REF]. In brain, we found the major alkenyl chains described in plasmalogen species, as the authors highlighted that plasmenylethanolamine mainly contained 18:0 and 16:0 at the sn-1 position when DHA was esterified at the sn-2 position. But overall, our diets did not change the DMA profile in brain, heart or RBC, although the DHA proportion was increased in heart and RBC with the DHA supplementation. A previous work showed that the plasmalogen content in brain may be increased by dietary DHA through a potent stimulation of dihydroxyacetone phosphate acyl transferase activity [START_REF] André | Plasmalogen metabolism-related enzymes in rat brain during aging: Influence of n-3 fatty acid intake[END_REF]. This effect was however presumed as relevant at the beginning of the life but not during aging. In our study, DHA supplementation consistently affected the DMA structures and concentrations in eyes, but surprisingly in reducing the DMA content. We also found equivalent proportions between saturated DMA (around 35% for both 16:0-DMA and 18:0-DMA) and between monounsaturated DMA (around 15% for both 18:1n-9-DMA and 18:1n-7-DMA), on the contrary to other studies performed on mice [START_REF] Albouery | Impact of a high-fat diet on the fatty acid composition of the retina[END_REF], where the diet enriched in fat modulated as well the DMA profile. DHA supplementation may shift the balance between plasmalogens since few phospholipid species coexist in retina and other structures in eyes [START_REF] Acar | Plasmalogens in the retina: In situ hybridization of dihydroxyacetone phosphate acyltransferase (DHAP-AT) -the first enzyme involved in their biosynthesisand comparative study of retinal and retinal pigment epithelial lipid composition[END_REF]. Some authors showed in mouse cerebral cortex that DHA-enriched diet reduced the n-6 PUFA content in plasmenylethanolamine, the concentration of which tended to decrease in the tissue whereas a substantial raise in peroxidation products was observed as well [START_REF] Sun | Docosahexaenoic Acid (DHA) Supplementation Alters Phospholipid Species and Lipid Peroxidation Products in Adult Mouse Brain, Heart, and Plasma[END_REF]. Considering all the data, high concentrations of DHA in eye may explain a lower content of plasmalogens considered as scavenger agents to protect against oxidation [START_REF] Almsherqi | Potential Role of Plasmalogens in the Modulation of Biomembrane Morphology[END_REF].

Nevertheless, discrepancy between control and DHA-enriched diets remains difficult to explain.

Regulation of dietary FA on enzymes involved in plasmalogen metabolism has to be further investigated.

Finally, many studies showed that DHA enriched-diets modulate the production of mediators derived from FA such as oxylipins. Oxidized metabolites are miscellaneous and mainly generated by cyclooxygenase, lipoxygenase and cytochrome P450 (An et al., 2021). In this study, we first determined the oxylipin pattern of DHA oil. We found eight compounds primarily derived from LA with 13-and 9-HODE, and from DHA with 17-and 14-HDoHE. When quantification was performed on omelets, half of oxylipins were lost. Only 13-HODE, 9-HODE and 5-HETE were still quantified. DHA supplementation was not discriminant as the oxylipin profile was similar between the three omelets. However, in DHA-enriched omelets, all oxylipins were expected in spite of the small amount of DHA oil added in egg preparation. Thus, this result suggests that food processing destroyed most of oxidized derivatives of PUFA supplied by DHA oil in DHA-enriched omelets, while promoting non-enzymatic oxidation by generating specific hydroxylated FA such as HODE and HETE from egg FA. In such conditions, all groups of animals fed a similar amount of oxylipins derived from omelets. Quantification of oxylipins were performed on one set of food, but we may consider a consumption of 8 to 15 ng oxylipins per day per rat. On this basis, we further evaluated the impact of DHA encapsulation. Our data showed that DHA supplementation modified the oxylipin patterns in plasma and heart, and to a lesser extent in brain. The dietary effect on n-6 metabolites dominated over the effect on docosanoids generated from DHA. But most importantly, we demonstrated a very high influence of DHA oil encapsulation on the oxylipin concentrations in tissues, even in brain. Precisely, 14-HDoHE was drastically reduced in plasma and raised in brain with DHA encapsulation. 14-HDoHE is subsequently generated by 12-lipoxygenase and glutathione peroxidase [START_REF] Guichardant | Omega-3 polyunsaturated fatty acids and oxygenated metabolism in atherothrombosis[END_REF]. Other studies showed an increase in 14-HDoHE consecutive to a n-3 enriched-diet in brain (Ostermann & Schebb, 2017;[START_REF] Rey | Dietary n-3 long chain PUFA supplementation promotes a pro-resolving oxylipin profile in the brain[END_REF] or in plasma [START_REF] Barden | Specialized proresolving lipid mediators in humans with the metabolic syndrome after n-3 fatty acids and aspirin[END_REF][START_REF] Schuchardt | Effect of DHA supplementation on oxylipin levels in plasma and immune cell stimulated blood[END_REF]. Blood concentration was enhanced in pathological conditions such as morbid obesity [START_REF] Schulte | The relationship between specialized pro-resolving lipid mediators, morbid obesity and weight loss after bariatric surgery[END_REF] or metabolic syndrome [START_REF] Barden | Specialized proresolving lipid mediators in humans with the metabolic syndrome after n-3 fatty acids and aspirin[END_REF], whereas the local level of 14-HDoHE was usually reduced in inflammatory conditions such as in arthritis [START_REF] Barden | Specialised pro-resolving mediators of inflammation in inflammatory arthritis[END_REF], periodontitis [START_REF] Wang | Maresin 1 Biosynthesis and Proresolving Antiinfective Functions with Human-Localized Aggressive Periodontitis Leukocytes[END_REF] or pulmonary inflammation [START_REF] Kilburg-Basnyat | Specialized Pro-Resolving Lipid Mediators Regulate Ozone-Induced Pulmonary and Systemic Inflammation[END_REF]. 14-HDoHE was shown to be involved in the resolution of inflammation, to participate in the immune response or the platelet activation in reducing thrombus formation [START_REF] Crouch | Frontline Science: A reduction in DHA-derived mediators in male obesity contributes toward defects in select B cell subsets and circulating antibody[END_REF][START_REF] Yamaguchi | DHA 12-LOX-derived oxylipins regulate platelet activation and thrombus formation through a PKA-dependent signaling pathway[END_REF]. If the mechanism of action of 14-HDoHE still remains unclear, a cerebral increase in 14-HDoHE may be a good marker to prevent potent neuroinflammation. In heart, the level of 14-HDoHE tended to increase but more importantly, the accumulation of PDx was favoured with DHA oil encapsulation. This poxytrin is generated by the action of 15lipoxygenase on DHA and displays a pleiotropic function in inhibiting inflammation, cyclooxygenases 1 and 2, production of reactive oxygen species and RNA virus replication [START_REF] Lagarde | Anti-inflammatory and anti-virus potential of poxytrins, especially protectin DX[END_REF]. PDx also improved the liver steatosis status [START_REF] Jung | Protectin DX Ameliorates Hepatic Steatosis by Suppression of Endoplasmic Reticulum Stress via AMPK-Induced ORP150 Expression[END_REF] and lowered hepatic gluconeogenesis in an obese mouse model [START_REF] Jung | Protectin DX suppresses hepatic gluconeogenesis through AMPK-HO-1-mediated inhibition of ER stress[END_REF], making it a potential therapeutic agent for lipid-induced and obesity-linked insulin resistance and type 2 diabetes [START_REF] White | Protectin DX alleviates insulin resistance by activating a myokine-liver glucoregulatory axis[END_REF]. More specifically, PDx may be a resolution mediator of kidney fibrosis and cardiac failure [START_REF] Perazza | Fish oil replacement prevents, while docosahexaenoic acid-derived protectin DX mitigates end-stage-renal-disease in atherosclerotic diabetic mice[END_REF]. Only healthy functions have been attributed to PDx, which makes the DHA supplementation a valuable approach to improve health tissue before potential injury. DHA oil used in our study was prepared enzymatically by esterification of glycerol with enriched DHA oil extracted from fish. The DHA content was high but other PUFA such as EPA were lowly detected. Although minorly present, EPA from the DHA-enriched diets displayed a remarkable impact on cardiac tissues as we found 18-HEPE with DHA supplementation but only when DHA oil was encapsulated. 18-HEPE is generated from EPA through catalysis of COX-2/aspirin or cytochrome P450 as a precursor of resolvin E1. It accumulated as well in brain [START_REF] Rey | Dietary n-3 long chain PUFA supplementation promotes a pro-resolving oxylipin profile in the brain[END_REF] or plasma [START_REF] Barden | Specialized proresolving lipid mediators in humans with the metabolic syndrome after n-3 fatty acids and aspirin[END_REF][START_REF] Schuchardt | Effect of DHA supplementation on oxylipin levels in plasma and immune cell stimulated blood[END_REF] after a diet enriched in n-3 PUFA. Its blood concentration decreased with the obesity-linked inflammation status [START_REF] Barden | Specialized proresolving lipid mediators in humans with the metabolic syndrome after n-3 fatty acids and aspirin[END_REF][START_REF] Schulte | The relationship between specialized pro-resolving lipid mediators, morbid obesity and weight loss after bariatric surgery[END_REF] whereas it raised in inflammatory arthritis [START_REF] Barden | Specialised pro-resolving mediators of inflammation in inflammatory arthritis[END_REF]. 18-HEPE was also particularly concentrated in plasma of fat-1 mice compared to wild-type mice, and was shown to protect against pulmonary metastasis of melanoma [START_REF] Li | An omega-3 polyunsaturated fatty acid derivative, 18-HEPE, protects against CXCR4-associated melanoma metastasis[END_REF]. More specifically, 18-HEPE was highly correlated with the coronary plaque regression on patients with coronary artery disease supplemented with high dose of n-3 PUFA for 30 months [START_REF] Welty | Regression of human coronary artery plaque is associated with a high ratio of (18-hydroxyeicosapentaenoic acid + resolvin E1) to leukotriene B 4[END_REF]. Collectively in heart, the increase in PDx and 18-HEPE, together with the reduction of n-6 eicosanoids triggered by both DHA supply and oil encapsulation, emphasized the upgraded status on cardiac tissues on a short-term supplementation.

Encapsulation of DHA oil with whey protein as Pickering emulsion showed in our study that the delivery system was efficient to modulate derivatives of PUFA in tissues, as compared to nonencapsulated DHA oil. This effect was presumed to be mediated by a different rate of absorption of DHA oil. Indeed, when the same omelets were digested by using an in vitro model of static digestion for adults, lipolysis of DHA-TAG was enhanced when DHA oil was encapsulated (unpublished result). Emulsions presenting the lowest droplet size prior to digestion exhibit a better rate of lipolysis compared to coarser emulsion [START_REF] Armand | Digestion and absorption of 2 fat emulsions with different droplet sizes in the human digestive tract[END_REF]. The same observation was assigned to nanoemulsion as compared to microemulsion where the release of oil took place either by interfacial transfer or degradation of the vehicle driving the lipids out [START_REF] Dey | Comparative study of gastrointestinal absorption of EPA & DHA rich fish oil from nano and conventional emulsion formulation in rats[END_REF]. The breakdown of encapsulated oil depends as well on differences in emulsion structure and surface area available for catalytic activity. Many encapsulant materials are available [START_REF] Venugopalan | Encapsulation and Protection of Omega-3-Rich Fish Oils Using Food-Grade Delivery Systems[END_REF] but the best delivery system of marine oil has to be developed and experienced. The subsequent lipolysis rate and thus absorption into the intestine greatly influenced the bioavailability of DHA as demonstrated with volunteers submitted to fish oil supplementation [START_REF] Haug | Bioavailability of EPA and DHA delivered by gelled emulsions and soft gel capsules[END_REF][START_REF] Raatz | Enhanced Absorption of n-3 Fatty Acids from Emulsified Compared with Encapsulated Fish Oil[END_REF].

Reducing lipid droplet size affects as well satiety by increasing FA sensing in small intestine or by enhancing peptide secretion like cholecystokinin or peptide YY during digestion (P. W. J. [START_REF] Maljaars | The effect of lipid droplet size on satiety and peptide secretion is intestinal site-specific[END_REF]. In our work, encapsulation of DHA oil heightened growth of animals coupled with a change in eating behaviour. We observed that rats were particularly enthusiastic to eat DHAenriched omelets and especially in the EN-DHA-O group. Yet, a study on volunteers showed that palatability was negatively impacted by the highest concentrations of DHA when fish oil was added to ground beef. Panelists were moreover distinctly sensitive depending on the FA as EPA had a greater impact on off-flavour perception than DHA [START_REF] Jiang | Omega-3 fatty acids affected human perception of ground beef negatively[END_REF]. Nonetheless, contrary to the fishy flavours triggered by bulk fish oil, more sophisticated preparation of oil such as encapsulation may improve palatability of food. Encapsulating DHA oil possibly mitigated the unpleasant taste leading to a better acceptance of DHA-enriched omelet. Now the origin of oil may influence as well food intake. In considering a longer-term supplementation, the eating behaviour was shown to be influenced by the effect of DHA on the reward system regulating appetite [START_REF] Maher | Dietary lipids with potential to affect satiety: Mechanisms and evidence[END_REF].

Chalon et al demonstrated that the mesolimbic dopaminergic pathway was overactivated in rats deficient in n-3 PUFA [START_REF] Chalon | Omega-3 fatty acids and monoamine neurotransmission[END_REF]. Lacking n-3 PUFA increased as well reward sensitivity in intrauterine growth restriction patients. This pathology affects brain responses to palatable food creating an impulsive eating behaviour, reversible with n-3 PUFA supplementation [START_REF] Reis | Impulsivity-based thrifty eating phenotype and the protective role of n-3 PUFAs intake in adolescents[END_REF]. The alimentary impact was partly explained by the leptin pathway involved in the anorexigenic system. Indeed, DHA-TAG enhanced leptin secretion in obese mice, consequently reducing hyperphagia in regulating appetite [START_REF] Cao | Triacylglycerol rich in docosahexaenoic acid regulated appetite via the mediation of leptin and intestinal epithelial functions in high-fat, high-sugar diet-fed mice[END_REF]. Conversely one study counteracted this data on haemodialysis patients suffering from anorexia [START_REF] Friedman | Association between plasma endocannabinoids and appetite in hemodialysis patients: A pilot study[END_REF]. Implying of DHA was suggested to be attributed to its derivatives such as the endocannabinoid docosahexaenoyl ethanolamide (DHEA). Positively correlated to appetite, DHEA was suspected to act as an activator of the orexigenic pathway, possibly by antagonizing the putative anorectic effects of linoleoyl ethanolamide. If data are controversial in presenting DHA or derivatives either as an enhancer or as an inhibitor of appetite, other effects on food reward circuit are still poorly described. Aside from the neuronal implication, DHEA was otherwise shown to improve glucose intake of myoblasts in vitro through the activation of the endocannabinoid system [START_REF] Kim | Docosahexaenoyl ethanolamide improves glucose uptake and alters endocannabinoid system gene expression in proliferating and differentiating C2C12 myoblasts[END_REF]. A similar result was observed on sows fed DHA-enriched fish oil during gestation. Intestinal glucose uptake was therefore increased in offspring, in association with the n-3 PUFA enrichment of jejunum tissues [START_REF] Gabler | Feeding long-chain n-3 polyunsaturated fatty acids during gestation increases intestinal glucose absorption potentially via the acute activation of AMPK[END_REF]. This effect was partly mediated by the raise in brush border membrane glucose transporters. Transposed to our work, effect of DHA encapsulation on animal growth was possibly facilitated as well by the modulation of intestinal transport functions. All together, these data suggest that encapsulation of DHA oil may upgrade the food palatability of omelets, improve appetite for the T-diet and favour growth in promoting energy metabolism. For as much, it remains difficult to interpret the effect of encapsulation observed on derivatives of PUFA, more than on PUFA themselves. The molecular mechanism requires further research for understanding.

To conclude, we used a rat model to assess the efficacy of a food-graded delivery system by encapsulating DHA oil into whey protein. In our experimental conditions, brain displayed a great proportion of 22.8% DHA, which was not enhanced by dietary supplementation of DHA oil, as compared to other tissues. Overall, encapsulation of DHA oil did not modulate the FA profile as compared to the literature, but remarkably modified the oxylipin pattern in plasma, heart and even brain. Specific oxidized metabolites derived from DHA were upgraded while those from n-6 PUFA were essentially mitigated. This effect was independent of oxylipins present in DHA oil as food processing induced the loss of the specific oxidized metabolites from DHA and EPA. Finally, this

Challenges were faced during the realization of this work:

• The first challenge was to select a suitable delivery system, a proper wall material, as well as an easy preparation protocol to encapsulate DHA oil, which is very important for the entire project.

• The second challenge was to separate and determine the lipid species in the samples collected during the in vivo digestion experiment, which is very important for studying the evolution of DHA during the lipolysis process.

• The third challenge was the design of animal diets. This process not only needs to consider the combination of different types of oils, but also the lipids present in eggs, to achieve the essential needs of weaning rodents.

• The final challenge was data processing and analysis. There are many samples in vivo and in vitro experiments, so a lot of analyses and calculations were required to answer each scientific question.

Conclusion and perspectives:

Regarding chapter 2: CaCl2 supplementation of hydrophobised whey proteins:

Assessment of protein particles and consequent emulsions.

The main objective of this part was to realize DHA encapsulation. In this part, two different protein particles stabilized emulsions were analyzed.

Firstly, hydrophobised and heat-denatured WPI particles were prepared through only heat treatment or successive acetylation and heat treatment practices, and the particle characteristics were modulated by CaCl2 supplementation. The addition of CaCl2 to two different protein dispersion had the same impact on the variation of ζ-potential and interfacial tension values. However, the impact of CaCl2 was different on the size of the protein particles. The size of hydrophobised WPI particles decreased as increasing CaCl2 concentration, which was opposite to heat-denatured WPI particles.

Then, Pickering emulsions were prepared respectively based on these two protein particles.

With increasing CaCl2 concentration in hydrophobised WPI particles dispersion, the stability of emulsion decreased. For heat-denatured WPI particles stabilized emulsion, as increasing CaCl2 concentration to 3 mM, the mixture of DHA oil and heat-denatured WPI particles transformed to a soft gel during ultrasonic homogenization, failing to make an emulsion.

Finally, oxidative stability of emulsion stabilized by hydrophobised WPI particles and heatdenatured WPI was compared. Compared to heat-denatured WPI particles, hydrophobised whey protein particles afforded lower protection to oil against oxidation. Further studies are required to address why hydrophobised WPI (without added CaCl2) had higher peroxide value in early storage than non-emulsified oil, whereas, the oil stabilized using heat-denatured WPI had the lowest peroxide value throughout storage. Besides, CaCl2 supplementation of hydrophobised WPI decreased the susceptibility of DHA oil to oxidation during emulsification process. At present, we attribute this observation to the lower number of (surface) thiol groups in hydrophobised WPI particles and the prooxidant effect of mechanical-ultrasonic emulsification on oil. Further mechanisms should be studied.

There are still some controversies about the heat-denatured WPI used in the preparation of Pickering emulsion. Fundamentally, Pickering emulsions are strictly defined as emulsions that are stabilized by an adsorbed layer of solid particles at the emulsion drop surface [START_REF] Murray | Pickering emulsions for food and drinks[END_REF].

Unfortunately, this still leaves open the definition of how non-deformable a material must be to be classed as 'solid'. Therefore, some researchers believe that the emulsion prepared by aggregates similar to heat-denatured WPI is not Pickering emulsion [START_REF] Murray | Pickering emulsions for food and drinks[END_REF]. However, most researchers accept as true that the formation of highly monodisperse spherical particles by inducing a specific pattern of whey protein (or soy protein) aggregation can be used to prepare Pickering emulsions [START_REF] Destribats | Emulsions stabilised by whey protein microgel particles: Towards food-grade Pickering emulsions[END_REF][START_REF] Zamani | Formation of shelf stable Pickering high internal phase emulsions (HIPE) through the inclusion of whey protein microgels[END_REF][START_REF] Destribats | Emulsions stabilised by whey protein microgel particles: Towards food-grade Pickering emulsions[END_REF][START_REF] Liu | Soy protein nanoparticle aggregates as pickering stabilizers for oilin-water emulsions[END_REF]. Besides, based on our results in in vitro digestion, we found that the size of emulsion droplets was very stable as observed during the gastric phase. This observation tends to argue in favour of the characterization of our emulsion as Pickering emulsion. Heat-denatured WPI may be hard enough to protect the DHA oil.

In addition, a previous study reported the oxidative stability of fish oil emulsions stabilized with caseinate glycated by dextran. The results showed that emulsions stored at 24℃ for 7 days have a higher level of peroxide value than the present study, although protocols to determine peroxide value were different between the two works [START_REF] Liu | Fish oil emulsions stabilized with caseinate glycated by dextran: Physicochemical stability and gastrointestinal fate[END_REF]. Therefore, it would be of interest to determine the involvement of whey proteins as particles of Pickering emulsion to mitigate oxidation of DHA oil. More globally, future studies may investigate the role of particles from Pickering emulsions by comparing them with classical emulsions on the preservation of the organoleptic qualities (e.g., oxidative stability and sensory properties) from encapsulated materials.

Regarding chapter 3: Encapsulation of DHA oil with heat-denatured whey protein in

Pickering emulsion improves the bioaccessibility of DHA.

The main objective of this part is to study the effect of encapsulation on the hydrolysis of DHA oil in vitro. In this part, we used INFOGEST in vitro digestion model for adults. In gastric phase and intestinal phase, we used RGL and pancreatin as lipases, respectively. RGL was used by default as some other lipases may be more efficient to be used, but lipases purified from human fluids (HGL), from dog fluids (DGL) or from porcine fluids are not commercially available and too expensive for such an experimental design. Besides, previous research showed that RGL not only has a similar structure to HGL but also has a comparable rate of digestion to HGL [START_REF] Capolino | In vitro gastrointestinal lipolysis: Replacement of human digestive lipases by a combination of rabbit gastric and porcine pancreatic extracts[END_REF]Moreau et al., 1988b). Moreover, [START_REF] Ménard | A first step towards a consensus static in vitro model for simulating full-term infant digestion[END_REF] found consistent results of the in vitro gastric lipolysis of infant formula by RGL and in vivo data. Therefore, RGL can be a good substitute for HGL.

Firstly, the effect of emulsion and omelet preparation on DHA oil quality was studied. The result showed that the emulsion preparation preserved the DHA oil quality; however, heating eggs to get omelets decreased the DHA oil quality. This may be caused by the presence of some endogenous lipases in eggs. Previous research showed that when eggs were stored at 4℃ or 22℃ for 10 days, the content from total lipids decreased, especially at 22℃. Besides, they also found that pH of egg yolk increased from 6.10 in fresh eggs to 6.32 and 6.43 in stored eggs (50 days) at 4℃ or 22℃ respectively, which also proved the hydrolysis of FAs [START_REF] Wang | Lipolysis and oxidation of lipids during egg storage at different temperatures[END_REF]. Usually, it is reported in the literature that hydrolysis of lipids can occur with increasing temperatures, however, this effect is described at high temperature (> 200°C), not at 80°C [START_REF] Nawar | Thermal degradation of lipids[END_REF]. Besides, the addition of unencapsulated DHA oil or encapsulated DHA oil (Pickering emulsion) to omelets achieved the DHA enrichment. Nevertheless, during this process, unencapsulated DHA oil could easily attach containers and castings, which made some loss of DHA oil, introducing a little bias in the final DHA concentration.

Then, the evolution of different lipid species at different phases of the in vitro digestion of omelets containing DHA oil is described in the present study. The results showed that DHA oil influenced the evolution of total lipid species during digestion, but without highlighting any effect of the encapsulation of DHA oil.

Finally, the release of DHA from different lipid species was studied. Our results revealed that the hydrolysis of DHA oil only happened in the intestinal phase under the activity of pancreatic lipase but not under the activity of RGL in the gastric phase, which can be attributed to the preference of RGL for TAG composed of shorter acyl chains [START_REF] Rogalska | Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases[END_REF]. However, the configuration of unsaturation on the carbon chain seems important as well in view of our results on ARA (20:4Δ5,8,11,14) compared to EPA (20:5Δ5,8,11,14,17). Besides, the hydrolysis of DHA-TAG and the release of DHA as FFA in encapsulation groups were significantly higher than those in the unencapsulation group at the end of the digestion. Therefore, encapsulation of DHA oil by heatdenatured WPI particles stabilized Pickering emulsion improved the bioaccessibility of DHA.

In this part, we found DHA oil was not hydrolyzed under the activity of RGE. This may be due to the reduced affinity of gastric lipase for this type of TAG, which is particularly rich in DHA and non-natural. Indeed, DHA is the main FA esterified to the three positions of the glycerol backbone, which confers a singular conformation to the molecule. This particularity is superimposed on the herbivorous pattern of rabbits whereas DHA is an animal FA, with a very long carbon chain and a very high degree of unsaturation, which is uncommon in the plant kingdom and may represent an obstacle to optimal catalysis. Our results may also result from the release of DHA in the stomach, further oxidized in secondary products non-detected with our experimental procedures.

Nevertheless, this hypothesis was discarded as the concentration of total DHA did not vary during the digestion process. Only the distribution of DHA in different lipid species has changed.

Considering this, maybe it is a good way to protect DHA and other precious PUFAs from oxidation in avoiding their release in the stomach. Whether gastric lipase (e.g., RGL) can cross the protein layer into the interior of the emulsion for hydrolysis of DHA oil droplets? Further investigations may focus on the gastric lipase activity to explain the hydrolysis of TAG during the gastric phase.

Besides, a comparison with other emulsion types (e.g., classical emulsion) may enrich as well the enzyme activity according to the diet environment.

We also did not do some sensory analysis. n-3 PUFAs are easy to be oxidized and affect product quality, such as producing a fishy smell and reducing product shelf life, making the product difficult to be accepted by consumers. Therefore, it would be interesting to do some sensory properties.

Besides, previous research showed that in the duodenum, the release of FAs can stimulate the 3 of the T-diet was sufficient to allow a good accretion of DHA in brain, considering the optimum rate of synthesis with the ALA supply from the T-diet. In addition, in this study, the concentration of cholesterol and TAG in plasma was also determined. Encapsulation of DHA oil did not modify the circulating level of cholesterol, but unexpectedly increased the one of TAG. Therefore, the effect showed between the groups was possibly due to a bias generated by the method or just as likely, encapsulation may increase the circulating TAG concomitantly with a decrease in plasma PL or FFA or other lipid species carrying FA, which would explain the comparable level of total FA quantified in plasma by GC-MS. It would be interesting to determine if the increase in TAG was represented by the DHA-TAG or by other species. Side effects on plasma lipids have also to be further clarified.

The next question then turned to the subsequent metabolism of DHA. Usually, to observe an effect of DHA on the synthesis of its metabolites, studies are performed in specific conditions, leading to the mobilization of the DHA and its further metabolism. Lots of works have described the effect of DHA in chronic or acute inflammation diseases or during aging, as the DHA metabolism is easily mobilized in such conditions to counteract the inflammation. Here we have chosen another point of view. We obtained an optimum accretion of DHA in physiological conditions, then we checked the subsequent metabolism of DHA, which is sometimes difficult to observe depending on the observation conditions. In our study, inflammation was not supposed to be enhanced. However, we greatly modified the oxylipin profile when DHA was highly present in tissues, with a remarkable effect of encapsulation of dietary DHA oil. This result underlined the metabolic potentiality of DHA when the dietary supply is adequate. The body may maintain a global status well balanced for either a low-grade inflammation or a high-grade inflammation, and may be more efficient by mobilizing then DHA to maintain an adapted anti-inflammatory response. Now this study concerned a physiological state coupled with an optimal accretion of DHA. This preventive condition may inhibit or reduce the development of inflammation or the onset of inflammatory diseases. At the opposite, the curative condition may be interesting as well, in testing a low-grade or a high-grade inflammation with an under-optimal DHA accretion without dietary supplementation of DHA. In that case, it would be relevant for instance to modify the FA balance between n-6 and n-3 in the T-diet. These reverse conditions of work may exhibit a better accretion of DHA in brain with encapsulation and a good protection against inflammation.

Finally, in addition to oxylipins, DHA can also be derived to generate endocannabinoids. We have planned to further analyse the profile of endocannabinoids as performed for oxidized derivatives of PUFAs. Indeed, we observed a different behaviour on food consumption between groups of animals, leading to a shift in rat growth for the four-week experiment. We can suppose that other metabolites were generated from DHA or less synthesised from n-6 PUFAs. Their potential involvement in the orexigenic circuit may in part explain the gap observed in the food intake of the T-diet. In addition, animal and human studies show that activation of the ileal brake by local perfusion with nutrients increases feelings of satiety and reduces ad libitum food intake [START_REF] Maljaars | Ileal brake: A sensible food target for appetite control. A review[END_REF][START_REF] Meyer | Chemical specificities and intestinal distributions of nutrient-driven satiety[END_REF][START_REF] Welch | Effect of ileal and intravenous infusions of fat emulsions on feeding and satiety in human volunteers[END_REF]. [START_REF] Meyer | Chemical specificities and intestinal distributions of nutrient-driven satiety[END_REF] found FFA can activate the ileal brake but not TAG. In our research, the encapsulated group released more FFA than the unencapsulated group in the intestinal phase. Therefore, we may suggest that by ileal braking, the encapsulation group should initiate satiety more intensely and as a consequence, would consume less food after the omelet intake. This contrasts with our observations and results. The nature of FA released may greatly influence the animal behaviour as well and may prevail over the other biological effects. DHA is a precursor of endogenous cannabinoids, such as DHEA, which is suspected to act as an activator of the orexigenic pathway. Our conditions of work lead to a good accretion of DHA in tissues of growing animals and favoured the further metabolism of DHA in derivatives as shown with oxylipins. We may postulate that other derivatives such as endocannabinoids may be affected as well, and consequently modified behaviours of rats towards the diets. To validate this hypothesis, new analyses are currently performed on plasma, brain and heart. These results will balance the positive effects of DHA particularly described in the case of supplementation to compensate for a deficiency, with the more controversial effects when DHA is in excess or supplemented in specific conditions of food intake as here with the fasting, which was designed to allow digestion of the omelet without rodent chow.

Working in other conditions may help as well to understand the importance of encapsulation in such a result. A long-term effect may be also of interest in measuring the consequences on cognitive capacities, on stress management or on mood changing. We can wonder whether there is a limit to the beneficial effects of DHA when its proportion and the proportion of derivatives are high in the body. This study laid ultimately the foundations for new perspectives in DHA metabolism.

In addition, considering the intake of 3g omelet per day, the daily consumption of DHA per rat achieved a mean of 2.1 mg, 27.6 mg and 30.9 mg in Control, UN-DHA-O and EN-DHA-O groups, respectively. Besides, eggs contain a high content of cholesterol in yolk, thus excessive consumption of eggs can lead to a high blood cholesterol level in the body, which can be linked to many medical conditions, including coronary heart disease, stroke and peripheral vascular disease [START_REF] Iribarren | Calcification of the aortic arch: Risk factors and association with coronary heart disease, stroke, and peripheral vascular disease[END_REF]. One egg per day at breakfast could be good for supplying protein and maintaining one's nutritional balance [START_REF] Taguchi | Regular egg consumption at breakfast by Japanese woman university students improves daily nutrient intakes: Open-labeled observations[END_REF]. Therefore, adult DHA intake calculated on a serving of omelet containing 1 egg (egg weight is around 50 g per egg) could provide 34.5 mg, 460 mg and 515 mg of DHA for Control, UN-DHA-O and EN-DHA-O groups, respectively. As we mentioned before, for general health and nutrition, most Global and European authorities and scientific bodies are recommending 250 mg to 500 mg of EPA and DHA per day. Therefore, the DHA content of DHA enriched omelets may meet this requirement. Now considering the rat model used in this study, the consumption of DHA was relatively high if the DHA content is transposed to the daily recommendations in human, in considering individual weights. At the end of the four-week experiment, animals consumed 0.4 mg DHA / g of rat weight, which is ten times the consumption of human (70 kg with a 500 mg DHA per day). This approach may be puzzling for nutritionists as it was not completely nutritional but more pharmacological to get a better point of view of the DHA metabolism in cell signaling while studying the impact of encapsulation as well. Further researches may be led with a lower supply of DHA in omelet, to understand if a nutritional dose of DHA would be sufficient to counteract the deleterious effect of inflammatory conditions for instance.

In the same way, considering the preparation of emulsion, we daily added heat-denatured WPI in omelets. This reached 300 µg per day, which is less than 0.01% of the daily protein intake, which was 4.8 g in growing rats. Therefore, the impact of heat-denatured WPI was considered as negligible.

Moreover, the study focused on derivatives of PUFAs, while many enzymes are involved in the metabolism of oxylipins (e.g., LOX-5, LOX-12, COX-1, COX-1). We only measured the content of oxylipins in demonstrating the global impact of our diets on these lipid mediators. We did not highlight a main pathway impacted by the encapsulation of DHA oil. Therefore, in the future, it would be interesting to further analyze the specific enzyme activity to better understand the cell pathways affected by the regimen. Then a comparison with other animal models (e.g., aging, chronic or acute inflammation) could be relevant to promote the galenic supply of DHA as encapsulated oil in the diet. A previous study reported that exsanguination allowed discriminating brain oxygenated metabolites from those generated in blood (Jouvè ne et al., 2018). The authors described a different profile of metabolites depending on the preparation of samples, particularly when animals were exsanguinated or when organs were perfused before sampling. In our study, animals were exsanguinated before sampling and heart was perfused with phosphate buffer saline to remove remaining blood of the other organs, but maybe not completely as described in the cited article.

Besides, in the Jouvène's study, they euthanized after urethane anesthesia, whereas here, we asked for the authorization of an alternative method of euthanasia with xylazine and ketamine to allow exsanguination by cardiac puncture, this method being considered as non conventional by the European Union Guideline. Results on oxylipins may be also impaired or changed according to this experimental choice. However, if the basal profile of oxylipins was partially inherent to the sampling, the different effects observed with encapsulation of DHA oil may not be truly attributed to the sample preparation as all samples were obtained in the same conditions. Nevertheless, further researches on lipid mediators deserve a specific attention in the future to avoid any useless bias.

Combining the contents of the three chapters in general, our research mainly had the following findings: 1) emulsion stabilized by hydrophobised WPI particles is more susceptible to oxidation than heat-denatured WPI particles; 2) encapsulation improved the release of DHA in intestinal phase;

3) in general, the intake of n-3 PUFA can not only increase the level of anti-inflammatory oxylipins, but also reduce the level of pro-inflammatory oxylipins from n-6 PUFA; 4) encapsulation of DHA oil is a key factor for DHA metabolism in generating favorable precursors of protectins and maresins, thus influencing the global health status.

In the future, we can associate with food companies to industrially produce DHA enriched omelets. At the same time, it is necessary to conduct research on its toxicology and flavor to make sure that it can be supplied and accepted by consumers.

In addition, we can also conduct some epidemiological studies by giving these DHA enriched omelets to volunteers to determine the importance of encapsulated DHA in improving human health, such as delaying cognitive decline, preventing CVD and depression. However, this kind of epidemiological research is usually a long-term process. Besides, we can also do some research on DHA and EPA or EPA alone. Previous researches reported that DHA/EPA did not yield better results on modulation of the serum lipid profile, oxidative damage, and expression of lipid metabolism-related genes, but was more effective at controlling inflammatory factors as compared to that by DHA [START_REF] Shang | Protective effects of various ratios of DHA/EPA supplementation on high-fat diet-induced liver damage in mice[END_REF]. Moreover, the ratio of EPA and DHA is also very important, [START_REF] Song | The role of omega-3 polyunsaturated fatty acids eicosapentaenoic and docosahexaenoic acids in the treatment of major depression and Alzheimer's disease: Acting separately or synergistically?[END_REF] found that a ratio of EPA to DHA from 2:1 to 3:1 would be most effective for depression.

Based on this information, more and more people will have an interest to consume DHA enriched foods, thereby improving the global health status and reducing the pressure on medical resources caused by the increase in chronic diseases. At the same time, this research can also provide a direction for the development of food companies in the future. Supplemental table 1 Abstract : Encapsulation may affect the digestion and bioaccessibility of the encapsulated bioactive compounds, which in turn affects their metabolism. The purpose of this project was to study the effects of encapsulation on DHA bioaccessibility and metabolism, based on omelet as a food matrix, which contains DHA oil as encapsulated or unencapsulated form.

Supplementary Tables

DHA oil composed of DHA-rich triacylglycerols was prepared as a Pickering emulsion, which is stabilized by heat-denatured whey protein isolates. Pure oil or emulsion was then incorporated into eggs and cooked in an omelet. The effects of encapsulation on the digestion and metabolism of DHA were studied by using INFOGEST static in vitro digestion model for adults and in a weanling rat model, respectively.

The results showed that encapsulation can increase the contact surface between DHA oil and lipase during the in vitro digestion, thereby promoting the hydrolysis of DHA oil and improving DHA bioaccessibility. In vivo, encapsulation of DHA oil did not modulate the fatty acid profile in tissues, but remarkably modified the oxylipin pattern in plasma, heart and even brain. Specific oxidized metabolites derived from DHA were upgraded while those from n-6 fatty acids were essentially mitigated. Therefore, encapsulation of DHA oil could not only improve the bioaccessibility of DHA, but is also a key factor in the metabolism of DHA to produce protectins and maresins precursors, thereby improving global health status.

  (±)8-Hydroxy-5Z,9E,11Z,14Zeicosatetraenoic acid12-HETE12(S)-Hydroxy-5Z,8Z,10E,14Zeicosatetraenoic acid

3 .

 3 La troisième partie de la thèse vise à déterminer l'effet de l'encapsulation sur le métabolisme du DHA in vivo. Pour atteindre cet objectif, 24 rats (âgés de 3 semaines) ont été divisés en trois groupes. Ils ont été préparés pendant une semaine avec le régime d'habituation et ont ensuite été nourris avec le régime ciblé pour notre étude pendant quatre semaines. Pendant cette période, les rats ont été nourris quotidiennement avec des omelettes contenant de l'huile DHA encapsulée (EN-DHA-O), de l'huile DHA (UN-DHA-O) ou sans huile DHA (Contrôle). Dans les omelettes enrichies en DHA, le DHA a été ajouté sous forme d'huile de TAG en supplémentation pour atteindre + 2.3 % des acides gras totaux. Suite à l'expérience in vivo, au moment de l'abattage des animaux, différents tissus ont été collectés et les profils d'acides gras de différents tissus et fluides biologiques (plasma, globule rouge, foie, coeur, cerveau, rétine) ont été déterminés par GC-MS. En parallèle, les profils des métabolites oxygénés des acides gras du plasma, du coeur et du cerveau ont également été analysés par chromatographie liquide couplée à la spectrométrie de masse en triple quadrupole (LC-QQQ).

2 .

 2 The second part of the thesis aims at determining the impact of encapsulation on DHA release during in vitro digestion. In order to achieve this objective, native DHA oil or DHA oil enriched Pickering emulsion was added into homogenized egg liquid respectively to get DHA enriched omelets. The effect of encapsulation on the bioaccessibility of DHA was determined by using the INFOGEST static in vitro digestion model for adults. For an enhanced understanding of the DHA oil distribution during digestion, the digesta microstructure was observed by confocal laser scanning microscopy. In order to know the impact of encapsulation on the evolution of DHA, different lipid species formed during digestion were separated by thin layer chromatography. Then the FA profile from different lipid species was determined by GC-MS. Finally, the variation of the proportion of different lipid species of total FA and the proportion of DHA from different lipid species of total DHA during digestion was studied.The results of this part are present in chapter 3: Encapsulation of DHA oil with heat-denatured whey protein in Pickering emulsion improves the bioaccessibility of DHA.

Figure

  Figure 1. Overall experimental design.

  them is actually essential to proper physiological functions. Based on the number of double bonds present inside chains, FAs can be classified into three categories: saturated FAs (SFAs, no double bond), monounsaturated FAs (MUFAs, a single double bond), and polyunsaturated FAs (PUFAs, ≥2 double bonds). Among them, PUFAs can be divided into cis (hydrogens on the same side of the plane) or trans (hydrogens on opposite side of the plane) configuration FAs according to the different geometric structures of their double bonds. Besides, unsaturated FAs (MUFAs and PUFAs) can also be further classified according to the position of the last double bond on the methyl end.Thus, PUFAs can be classified into several families, the most known being n-3 and n-6 series, all of them belong to cis configuration FAs. The structure of common PUFAs is shown in Figure1-1.

Figure

  Figure 1-1. The structure of common PUFAs.

Figure 1 - 2 .

 12 Figure 1-2. The pathway of PUFAs synthesis.

Figure 1 - 3 .

 13 Figure 1-3. Typical oxylipins are produced by the metabolism of different PUFAs. HODE: hydroxyoctadecadienoic acid, oxoODE: keto-octadecadienoic acid, EOME: epoxy-octadecenoic acid, DiHOME: dihydroxy-octadecaenoic acid, HETrE: hydroxyeicosatrienoic acid, PG: prostaglandin, HETE: hydroxyeicosatetraenoic acid, HpETE: hydroperoxy-eicosatetraenoic acid, LT: leukotriene, TX: thromboxane, LX: lipoxin, oxoETE: keto-eicosatetraenoic, DiHETE: dihydroxy-eicosatetraenoic acid, EET: epoxy-eicosatrienoic acid, HOTrE: hydroxy-octadecatrienoic acid, EpODE: epoxyoctadecadienoic acid, HEPE: hydroxy-eicosapentaenoic acid, Rv: resolvin, MaR: maresin, PT: protectin, HDoHE: hydroxy-docosahexaenoic acid, EpDPE: epoxy-docosapentaenoic acid. (Adapted from[START_REF] Rey | Dietary n-3 long chain PUFA supplementation promotes a pro-resolving oxylipin profile in the brain[END_REF][START_REF] Serhan | Anti-inflammatory and proresolving lipid mediators[END_REF][START_REF] Serhan | Resolvins and protectins in inflammation resolution[END_REF].

Figure 1 - 4 .

 14 Figure 1-4. The pathway of oxylipins derived from some common n-6 PUFAs. COXcyclooxygenase; LOX-lipoxygenase; CYP-cytochrome P450; NE-nonenzymatic; PGFSprostaglandin F synthase; PGES-prostaglandin E synthase; PGDS-prostaglandin D synthase; PGISprostaglandin I synthase; TXAS-thromboxane A2 synthase; LTAH-leukotriene A4 hydrolase; HEDH-hydroxyeicosanoid dehydrogenase; PGDH-hydroxyprostaglandin dehydrogenase; 13-PGR-15-ketoprostaglandin ∆13 reductase; sEH-soluble epoxide hydrolase. A list with all the oxylipins can be found in SupplementaryTable 1-1. (Figure from[START_REF] Coras | Circulating pro-and anti-inflammatory metabolites and its potential role in rheumatoid arthritis pathogenesis[END_REF]

Figure 1 - 5 .

 15 Figure 1-5. Specialized pro-resolving mediator (SPM) synthesis. COX: cyclooxygenase; LOX: lipoxygenase; CYP450: cytochrome P450; 14-HDoHE: 14-hydroxy-docosahexaenoic acid; 17-HDoHE: 17-hydroxy-docosahexaenoic acid; 18-HEPE: 18-hydroxy-eicosapentaenoic acid. (Adapted from Joffre, 2019)

  , especially DHA, are very important for cognitive function throughout the life cycle.

Figure 1 - 6 .

 16 Figure 1-6. Schematic summary of lipid digestion, absorption, and metabolic fate of dietary lipids. Triacylglycerols (TAG), phospholipids (PL), cholesterol (CL), cholesterol esters (CE), and free fatty acids (FFA). (Adapted from[START_REF] Michalski | Bioavailability and metabolism of dietary lipids[END_REF]

Figure 1 - 7 .

 17 Figure 1-7. The hydrolysis of TAG into 1,2-DAG and FFA catalyzed by gastric lipase. R represents the carbon chain of different FAs. TAG: triacylglycerol, 1,2-DAG: 1,2-diacylglycerol, FFA: free fatty acid.

Figure 1 - 8 .

 18 Figure 1-8. The hydrolysis of TAG into 2-MAG and two FFA catalyzed by pancreatic lipase. R represents the carbon chain of different FAs. TAG: triacylglycerol, 1,2-DAG: 1,2-diacylglycerol, 2-MAG: 2-monoacylglycerol, FFA: free fatty acid.

Figure 1 - 9 .

 19 Figure 1-9. The hydrolysis of PL into lyso-PL and FFA catalyzed by PLA2. R represents the carbon chain of different FAs. PL: phospholipid, lyso-PL: lysophospholipid, FFA: free fatty acid.

Figure 1 -

 1 Figure 1-10. The hydrolysis of CE into CL and FFA catalyzed by cholesterol esterase. R represents the carbon chain of different FAs. CE: cholesterol ester, FFA: free fatty acid, CL: cholesterol.

Figure 1 -

 1 Figure 1-11. Intestinal lipid absorption. FFA, MAG, lyso-PL and CL are taken up by enterocytes by passive diffusion and various transporters, transported to the endoplasmic reticulum where TAG, PL, and CE are synthesized and assembled to form primordial chylomicrons (pm-CM), matured into prechylomicrons (pre-CM), and ultimately form mature chylomicrons secreted in the lymph. (Figure from[START_REF] Michalski | Bioavailability and metabolism of dietary lipids[END_REF]

Figure 1 -

 1 Figure 1-12. The structure of mature chylomicron. (Figure from[START_REF] Leduc | Genetic markers of neurodegeneration: A role for paraoconase 1 in sporadic Alzheimer's disease?[END_REF] In the blood circulation, lipids in chylomicrons are catabolized by the endothelial-bound enzyme lipoprotein lipase (LPL), which hydrolyzes TAG, releasing FAs. Then the released FAs can undergo ß-oxidation to be used as energy source or be taken up for storage by tissues (e.g., adipose and muscle) (Figure1-13)[START_REF] Zhyvotovska | Introductory Chapter: Overview of Lipoprotein Metabolism[END_REF]. With the hydrolysis of TAG carried in chylomicrons, the size of chylomicrons decreases markedly, leading to the formation of chylomicron remnants. During this process, excess surface materials, including PL, CL and apolipoproteins, are passed on to other particles, mainly high-density lipoprotein (HDL), by phospholipid transfer protein (PLTP). Furthermore, TAG are transferred to HDL in exchange for CE by the action of cholesterol ester transfer protein (CETP). Then, chylomicron remnants are cleared from the circulation by hepatic uptake via the LDL-receptor (LDL-R) or LDL-receptor related

Figure 1 -

 1 Figure 1-13. Lipid metabolism. (Figure from Schulze et al., 2016)

•

  Tropicana pure premium pealthy heart (Tropicana, USA) • Oasis health break (Iassonde industries inc., Canada) • Minute maid enhanced juice (The coca-cola company, USA)

Figure 1 -

 1 Figure 1-14. Sketch of a Pickering emulsion and a classical (surfactant-based) emulsion. The solid particles, microgels, fibrils, and hollow nanoparticles adsorbed at the oil-water interface stabilize the droplets in place of the surfactant molecules. (From Yan et al., 2020)

Chapitre 2 :

 2 Therefore, in the present research, two different particle-stabilized (hydrophobized and heatdenatured WPI) Pickering emulsions were prepared to encapsulate DHA-TAG rich oil, and after analyzing their properties, an emulsion system (Pickering emulsion stabilized with heat-denatured WPI particles) was selected for subsequent experiments. Considering the effect of food matrix on the bioavailability of DHA, in the present research, we chose omelets as the food model. Therefore, DHA enriched omelets (mentioned as EN-DHA-O and UN-DHA-O in Figure 0, respectively) were prepared by adding encapsulated DHA oil (Pickering emulsion) and unencapsulated DHA oil into homogenized egg liquid. Finally, the digestion behavior and the metabolism of DHA were studied on DHA enriched omelets by in vitro (static digestion) and in vivo (rat) experimental models, respectively. Supplé mentation en CaCl 2 des proté ines de lactosé rum hydrophobes: Évaluation des particules de proté ines et des é mulsions qui en ré sultent 2.1 Ré sumé Des particules de protéine de lactosérum rendues hydrophobes ont été préparées par des pratiques successives d'acétylation et de traitement thermique, et les caractéristiques des particules ont été modulées par une supplémentation en CaCl2. Ensuite, l'utilité des particules de protéine rendues hydrophobes pour l'émulsification d'une huile de DHA a été comparée à celle de la protéine de lactosérum dénaturée par la chaleur. L'ajout de CaCl2 dans la protéine de lactosérum rendue hydrophobe a entraîné des particules de protéine plus petites et des valeurs de potentiel ζ et de tension interfaciale plus faibles. Il a également diminué la stabilité de crémage des émulsions résultantes. Il a été avancé qu'outre les interactions de charge Ca 2+ -protéine, les anions Cl ¯ se lient aux particules rendues hydrophobes et que la stabilisation de Pickering de l'huile ne repose pas sur la réduction de la tension interfaciale. Par rapport à la protéine de lactosérum dénaturée par la chaleur, la protéine de lactosérum rendue hydrophobe offrait une protection moindre à l'huile contre l'oxydation ; l'indice de peroxyde de l'huile émulsionnée à l'aide de protéines rendues hydrophobes était plus élevé pendant le stockage. Chapter 2: CaCl 2 supplementation of hydrophobised whey proteins: Assessment of protein particles and consequent emulsions 2.1 Abstract Hydrophobised whey protein particles were prepared through successive acetylation and heat treatment practices, and the particle characteristics were modulated by CaCl2 supplementation. Then, the usefulness of the hydrophobised protein particles for emulsification of a docosahexaenoic acid oil was compared with that of heat-denatured whey protein. Addition of CaCl2 into hydrophobised whey protein resulted in smaller protein particles, and lower ζ-potential and interfacial tension values. It also decreased the creaming stability of the consequent emulsions. It was argued that besides Ca 2+ -protein charge interactions, Cl¯ anions bind to the hydrophobised particles, and the

Figure 2 - 1 .

 21 Figure 2-1. Particle size and ζ-potential value of hydrophobised and heat-denatured WPI samples as influenced by CaCl2 concentration. Values with different lowercase letters for each sample were significantly different (P < 0.05). Error bars indicate standard deviation.

  than those of the hydrophobised WPI; and k1 values of neither of WPIs were influenced by CaCl2 concentration (Figure 2-2B). The higher k1 compared with k2, and invariable k1 values at different CaCl2 concentrations, indicate fast absorption of protein particles to the interface of freshly formed pendant drops irrespective of the added CaCl2 concentration. Therefore, though γ decreased with increasing CaCl2 concentration at any given time (Figure 2-2), the reduction rate of γ in the rapidly falling period (i.e., k1) was not influenced by CaCl2 concentration. The higher k1 (Figure 2-2B) values of the heat-denatured WPI than the hydrophobised WPI was caused by the lower absolute ζ-potential value (Figure 2-1) of the former, which presumably lead to more rapid packing of the heat-denatured protein particles at the interface, in comparison to the hydrophobised WPI.

Figure 2 - 2 .

 22 Figure 2-2. Oil-water interfacial tension (A) and interfacial tension decay rate (B: k1, the slope of the line) of WPI samples: lefthand panels, heat-denatured WPI; righthand panels, hydrophobised WPI. Concentrations (mM) refer to the added CaCl2 into WPI dispersion.

  -3B) after 2 days and remained unchanged over the rest of the storage period. In accordance to these observations,[START_REF] Fan | Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion: Impact of protein and calcium concentrations[END_REF] reported that CaCl2 supplementation of heat-denatured WPI at concentrations above 0.2 mM caused creaming of the subsequent O/W emulsion.The oil droplets covered by the hydrophobised protein particles without added CaCl2 were fully stable to flocculation/aggregation, and CaCl2 supplementation of the hydrophobised WPI reduced the flocculation stability of the emulsion droplets, which is in line with reduction of γ with increasing CaCl2 concentrations. Electrostatic repulsion between the protein particle-coated oil droplets could be the main cause of the considerable flocculation stability of the emulsion made using the hydrophobised WPI without added CaCl2. The absolute ζ-potential value of the hydrophobised WPI particles was ≈36 mV, which is quite high. It decreased to ≈26 mV upon CaCl2 addition at 3 mM (Figure2-1).

  and hydrophobised WPIs supplemented with 3 and 5.5 mM CaCl2 had comparable extents of creaming (Figure2-3B). It can be argued that protein particle size also influenced the flocculation stability of the Pickering emulsions. The hydrophobised WPI had larger particles than the heatdenatured WPI (Figure2-1). Larger particles could cause a more effective steric hindrance between the particle-coated oil droplets. Hence, a longer-range steric hindrance counterbalanced the weaker electrostatic repulsion in emulsions made using the CaCl2-supplemented hydrophobised WPI when compared with the stronger electrostatic repulsion but shorter-range hindrance in CaCl2-free heatdenatured WPI. Such a counterbalancing of the stabilization mechanisms led to comparable creaming extents between the emulsions. The importance of steric hindrance to prevent extensive flocculation of oil droplets in Pickering emulsions has also been shown for low-charged (ζ-potential ≈ -16 mV) starch nanoparticles[START_REF] Ge | Characterizations of Pickering emulsions stabilized by starch nanoparticles: Influence of starch variety and particle size[END_REF].The droplet size of freshly prepared emulsions analyzed by the light scattering technique lies in the order of heat-denatured WPI> hydrophobised WPI supplemented with CaCl2 at either 3 or 5.5 mM)> hydrophobised WPI supplemented with CaCl2 at 0 mM (Figure2-3B). The same trend is perceived in optical microscopy images of emulsion samples immediately after preparation (Figure2-3C). Obviously, the droplet size order does not follow the order of γ (Figure2-2A). As mentioned earlier, particle trapping at the oil-water interface does not appreciably influence γ at the oil-water interface; vice versa, lower γ values caused by CaCl2 addition resulted in lower desorption energy

Figure 2 - 3 .

 23 Figure 2-3. Photographs (A) of oil-WPI dispersion mixes after emulsification using the heat-denatured WPI (He-E) and the hydrophobised WPI (Hy-E) supplemented with different CaCl2 concentrations (0, 3, 5.5 and 6.5 mM); Creaming index (B: empty symbols mean no creaming, full symbols mean creaming and the creaming index was 1.7%) and droplet size distribution of the emulsions stabilized by either heat-denatured or hydrophobised WPI; Microscopic images (C) of He-E and Hy-E samples with different CaCl2 concentrations. Scale bars show 20 μm.

Figure 2 -

 2  shows the flow behavior of the emulsion samples stabilized by either heat-denatured or hydrophobised WPIs. The viscosity of emulsions decreased with increasing shear rate, indicating that all samples were shear thinning, which is attributed to alignment of droplets in direction of the applied shear field[START_REF] Soltani | Two-step sequential cross-linking of sugar beet pectin for transforming zein nanoparticle-based Pickering emulsions to emulgels[END_REF]. The emulsion samples stabilized by the heatdenatured and hydrophobised WPIs without added CaCl2 (0 mM) had statistically indifferent yield stresses (τ0) and consistency coefficients (K); however, the flow behavior index (n) of the sample stabilized by the hydrophobised WPI was lower than that of the emulsion stabilized by the heatdenatured WPI (Figure2-4B). The latter indicates that the hydrophobised WPI-stabilized oil droplets aligned more easily than the heat-denatured WPI-stabilized droplets in direction of shear field. The difference in shear thinning property could be caused by the smaller droplet size of the emulsion stabilized by the hydrophobised WPI (Figure2-3), as well as dissimilar inter-droplet elctrostatic repulsion. A higher electrostatic repulsion between droplets might render the emulsion stabilized by the hydrophobised particles more easily thinning with increasing shear rate.

Figure 2 - 4 .

 24 Figure 2-4. Viscosity (A) and shear stress (B) of emulsions stabilized by either heat-denatured or hydrophobised WPI as a function of shear rate. Error bars indicate standard deviation.Addition of CaCl2 into WPI at 3 and 5.5 mM did not influence the K value of the emulsion made using the hydrophobised WPI (Figure2-4B). Likewise,[START_REF] Keowmaneechai | Effect of CaCl2 and KCl on physiochemical properties of model nutritional beverages based on whey protein stabilized oil-in-water emulsions[END_REF] reported that supplementation with CaCl2 at 2 mM did not affect the viscosity of model beverages based on whey protein-stabilized O/W emulsions. Nonetheless, the flow behavior index (n) and τ0 of the hydrophobised WPI-stabilized emulsion was higher when WPI was supplemented with 5.5 mM CaCl2 compared to 0 mM (Figure2-4B). We attribute the lower shear-thinning property to reduced electrostatic repulsion between oil droplets (due to lower ζ-potential absolute values, Figure2-1) and the increased τ0 to the presence of larger droplets (Figure2-2B) when the added CaCl2 into WPI increased from 0 mM to 5.5 mM.

Figure 2 - 5 .

 25 Figure 2-5. Peroxide value of DHA oil and its emulsions stabilized by either heat-denatured or hydrophobised WPI during storage at 20℃.

3. 1

 1 Ré suméCette étude a porté sur la bioaccessibilité du DHA selon la forme alimentaire en tant que formulation alimentaire encapsulée ou non encapsulée. L'huile de DHA était composée de triacylglycérols enrichis en DHA préparés sous forme d'émulsion de Pickering par encapsulation avec des particules d'isolat de protéines de lactosérum dénaturées à la chaleur, puis incorporées dans un liquide d'oeuf homogénéisé pour obtenir des omelettes. L'effet de l'encapsulation a été analysé en utilisant un modèle in vitro de digestion statique pour adultes. Premièrement, la taille des gouttelettes lipidiques était plus petite et uniformément dispersée dans les omelettes avec de l'huile de DHA encapsulée par rapport à l'huile de DHA non encapsulée. La distribution des gouttelettes était également plus régulière avec l'huile de DHA encapsulée. De plus, nous avons montré que l'encapsulation d'huile de DHA favorisait l'hydrolyse de la lipase pendant la phase intestinale. Une plus grande proportion de triacylglycérols enrichis en DHA a été digérée après deux heures de catalyse, conduisant à une plus grande libération de DHA libre dans le digesta. Par conséquent, la bioaccessibilité du DHA a été améliorée de manière significative par l'encapsulation d'huile de DHA dans les omelettes.

  separated by a QP 2010-SE gas chromatograph coupled to a mass spectrometer (Shimadzu, Marne-La-Vallée, France) equipped with a BPX70 capillary column (120 m, 0.25 mm i.d., 0.25 µm film) from SGE Trajan (Chromoptic, Paris, France). Helium was used as carrier gas at a constant velocity of 27,5 cm/sec. The temperature of injector was adjusted to 250°C. The column temperature ramped from 50 °C to 175 °C at 20 °C/min and then from 175 °C to 240 °C at 2 °C/min. The mass spectrometer was operated under electron ionization at 0.2 keV and 200 °C source temperature.Analyses were performed in scan mode over the m/z range of 30 -450 amu. Components were identified according to the retention time of authentic FAME standards and by using the National Institute of Standards and Technology (NIST) mass spectral library (version 2.01). Concentrations were determined by using two internal standards (Larodan, Coger SA Paris, France), the first one as TAG-17:0 added before the lipid extraction and the second one as 19:0 in the free FA form added after silica collection. Calibration was performed by standard curves of FA methyl esters. Results

Figure 3 -

 3 Figure 3-1. Encapsulated DHA oil with heat-denatured WPI. DHA oil was encapsulated with heat-denatured WPI as Pickering emulsion. DHA oil was previously stained by Nile red (red color) whereas WPI was dyed with Fast Green (green color). Emulsion was pictured by confocal laser scanning microscopy

Figure 3 - 2 .

 32 Figure 3-2. Distribution of unencapsulated and encapsulated DHA oil in omelets. Unencapsulated or encapsulated DHA oil was incorporated to the whole liquid egg to get omelets. DHA oil was previously stained by Nile red (red color) whereas proteins were dyed with Fast Green (green color). The DHA oil distribution in omelets was then visualized by confocal laser scanning microscopy. Pictures were captured in the middle and at the edge of the samples.

  After 2 hours of lipase catalysis, around half of TAG remained both with UN-DHA-O and EN-DHA-O. Meanwhile total FA from 1,2-DAG increased from 1% at G0 to 9% at G120, whereas FFA raised from 0.7% at G0 to 30% at G120, independently of the DHA oil delivery form. No other lipid species evolved otherwise. In the intestinal phase, less than 10% of TAG remained after one hour of lipase catalysis for both UN-DHA-O and EN-DHA-O, whereas the hydrolysis was complete with Control. In parallel, 1,2-DAG released during the gastric phase decreased to the benefit of the final product 2-MAG during the intestinal phase. PL were hydrolyzed as well under the action of phospholipase A2 or other enzymes. Collectively, complete lipolysis led to the release of FFA, the proportion of which represented 79%, 73% and 75% of the total FA with Control, UN-DHA-O and EN-DHA-O respectively.

Figure 3 - 5 .

 35 Figure 3-5. The digestion process is analyzed by lipid species and by omelets. In A, each point represents all the digestion data; in B, each point represents all lipids; in C, each point corresponds to TAG. This analysis was done on data presented as concentrations of FA.

Figure 3 - 7 .

 37 Figure 3-7. The evolution of the major FA from omelets (A) and long and very long-chain FA markers of DHA oil (B). Lipids were extracted by Folch's method and separated by TLC. The FA profile from TAG or FFA species was determined by GC-MS. Total FA of each different FA was the sum of the FA from different lipid species.

  un acide gras polyinsaturé n-3 majeur particulièrement impliqué dans les fonctions cognitives et cardiovasculaires. En raison de son indice d'insaturation élevé, sa forme d'apport alimentaire est considérée comme améliorant le statut oxydant et favorisant également la bioaccessibilité et la biodisponibilité. Cette étude visait à étudier l'effet du DHA encapsulé avec de la protéine naturelle de lactosérum. Le DHA était fourni par l'alimentation sous forme de triacylglycérols pour atteindre +2.3 % des acides gras totaux. Il a été fourni quotidiennement à des rats sevrés pendant quatre semaines, dans des omelettes en tant que matrice alimentaire, consécutivement à un jeûne de 6 heures. Premièrement, lorsque l'huile de DHA a été encapsulée, la consommation du régime alimentaire a augmenté, ce qui a entraîné un gain de croissance des animaux. Deuxièmement, le cerveau présentait une forte accumulation de DHA à 22.8% dans le groupe témoin, proportion qui n'était pas améliorée par la supplémentation alimentaire en DHA.L'encapsulation de l'huile de DHA a peu affecté les profils d'acides gras dans les tissus, mais a remarquablement modifié celui des métabolites oxygénés des acides gras dans le plasma, le coeur et même le cerveau. Des oxylipines spécifiques dérivées du DHA ont été augmentées, telles que la Protectin Dx dans le coeur et la 14-HDoHE dans le cerveau, tandis que celles générées à partir des acides gras polyinsaturés n-6 ont été principalement atténuées. Collectivement, ces données suggèrent que l'encapsulation alimentaire de l'huile de DHA a induit une absorption plus efficace de DHA, dont le métabolisme a davantage été stimulé que sa propre accrétion tissulaire dans nos conditions expérimentales. L'incorporation de l'huile de DHA dans les aliments fonctionnels peut finallement améliorer l'état de santé global en générant des précurseurs de protectines et de maresines.

  and use(2010/63/CEE; decree 2013-118). The present project was authorized by the French Ministry of Higher Education and Research under the number 27678-2020100615388160 v3. The animal experiment was performed at ARCHE Biosit (University of Rennes I, Campus of Villejean, Rennes, France). Male Wistar rats(3 weeks-old) were supplied by the Janvier Labs Breeding Center (Le Genest-Saint-Isle, France). They were randomly divided into 3 groups of 8 animals and housed in pairs. They were acclimated for one week with the habituation diet (H-diet) and were then fed with the treatment diet (T-diet) for four weeks (Figure4-1). During this period, rats were daily fasted from 9am to 3pm. Each group received then 3g per rat of omelet containing no DHA, DHA oil or encapsulated-DHA oil. Thereby, rats were separated for three hours by a plexiglass plate positioned in the middle of the cage, allowing individual consumption of omelet serving while guaranteeing eye and smell contact between congeners. At 6pm, the T-diet was restored until the next morning.Animals had access to water ad libitum. Rodent chows (H-diet and T-diet) were distributed ad libitum as well at the indicated time slots. After four weeks of experiment, fasted rats were anesthetized with intraperitoneal injections of ketamine (100 mg/kg, Imalgene®1000, Mérial, Lyon, France) and xylazine (10mg/kg, Rompun® 2%, Bayer Animal Health, Puteaux, France). Blood was collected in heparin tubes by cardiac puncture. Plasma was separated from red blood cells (RBC) by centrifugation (2000 rpm, 15 min, 15 °C). Eyes were sampled in phosphate buffer saline whereas liver, heart and brain (frontal cortex was studied here) were snap-frozen and stored at -80 °C until analysis.

Figure 4 - 1 .

 41 Figure 4-1. In vivo experimental design. Rats were acclimated with the habituation diet for one week. They were then fed with the treatment diet for four weeks. During this period, rats were daily fasted for 6 hours before receiving 3g of omelet containing DHA as oil (UN-DHA-O) or as encapsulated oil (EN-DHA-O).

Figure 4 - 2 .

 42 Figure 4-2. The food intake (A), the growth (B), the food efficiency ratio (C) and the tissue weights (D) of rats. Food consumption of T-diet and animal growth were followed during the four-week experiment. Food efficiency ratio averaged over the four-week experiment was calculated with the daily consumption of T-diet and omelets over the weight gain of growing animals. The effect of diets was determined as well on the tissue weight prior to lipid analysis. a, b, c and ab indicate significant differences between the three groups when p<0.05. ab means no difference with a and no difference with b. * indicates significant differences between EN-DHA-O and both Control and UN-DHA-O when p<0.05.More precisely, the cumulative intake of the T-diet over the 4-week experiment was increased by 6% with UN-DHA-O and by 20% with EN-DHA-O compared to the control. This behavior was coupled with the animal growth since the final weight of rats fed EN-DHA-O was significantly higher by 15% than the control group (Figure4-2B). Food efficiency ratio, calculated by the ratio between body weight gain and food intake (T-diet and omelet), was not different between groups and averaged 38.4%, 37.9% and 39.3% for Control, UN-DHA-O and EN-DHA-O respectively, over the four-week experiment (Figure4-2C). Likewise, weights of organs were also significantly augmented in the EN-DHA-O group by 24% for liver, by 19% for heart and by 6% for brain as compared to the control group (Figure4-2D). Nevertheless, when organ weights were normalized

Figure 4 - 3

 43 Figure 4-3 The effect of form of the DHA intake on the FA profile of rat tissues after 4 weeks of treatment. Lipids from rat tissues were extracted according to the Folch's method and the fatty acid profile was then determined by GC-MS. The main results on n-3 PUFA and arachidonic acid are presented. a, b, c and ab indicate significant differences between the three groups; ab means no difference with a and no difference with b (p<0.05).

  07µg/mg with the UN-DHA-O and EN-DHA-O diets respectively. Moreover, a switch in the DMA profile was observed in favoring 16:0-DMA (+13.9% with UN-DHA-O and +8.3% EN-DHA-O) to the detriment of 18:1-DMA, but without impacting the 18:0-DMA. No change of the DMA profile was however observed by encapsulation of the DHA oil.

  1754 .9 ± 383.6 1743 .4 ± 323.6 2961 .9 ± 354.3 17-HDoHE 2486 .1 ± 646.4 2178.8 ± 429.8 3218.1 ± 395.3 .8 ± 429.8 3218.1 ± 395.3 The oxidized metabolites of fatty acids were quantified in heart by LC-QQQ as mentioned in methods. a, b, c and ab indicate significant differences between the three groups; ab means no difference with a and no difference with b (p<0.05).No significant effect of the diets was observed this time on 14-and 17-HDoHE derived from DHA, but another derivative named PDx was remarkably increased with the DHA encapsulation.Absent in the control, PDx reached 2.1 pg/mg with UN-DHA-O and was multiplied by 8 with EN-DHA-O. The same applied to 18-HEPE derived from EPA, since it was absent in the control and the UN-DHA-O groups but reached 20.7 pg/mg with EN-DHA-O. In contrast, the cardiac concentrations of eicosanoids derived from ARA were globally decreased with the DHA supplementation, and particularly with the DHA encapsulation for PGF2 and PGE2. Finally, the most remarkable effect was achieved by an eicosanoid from DGLA termed 6keto-PGF1, which was reduced by half with UN-DHA-O and by five with EN-DHA-O.

  Lipids were extracted by the Folch's method. The fatty acid profile was determined by GC-MS and expressed in mass %. Oral presentation: Young researchers webinar organized by SFEL, online, 25/06/2021.

Table 1 -1. The consumption ratio between the precursors of n-6 and n-3 PUFAs in different western countries in the diet. Country Ratio of n-6 / n-3

 1

	France	12.0
	Austria	12.0
	United States	9.8
	United Kingdom	8.7
	Belgium	8.1
	Norway	7.8
	Germany	4.0
	Finland	3.9
	Data from Leray (2014).

Table 1

 1

-1). Therefore, in order to improve the consumption of n-3 PUFAs in diets and to meet the recommended range for the consumption ratio of n-6 and n-3, different countries have established different recommended intake values of n-3 PUFAs (Table

1-2

). For general health and nutrition, most Global and European authorities and scientific bodies are recommending 250 mg to 500 mg of EPA and DHA per day, depending on the target population.

Table 1 -2. The recommended intake of n-3 PUFAs.

 1

	Recommending organization	Population	Recommendation
	International Society for the Study of Fatty Acids and Lipids (ISSFAL)	General Adult Population for CVD	Minimum of 500 mg EPA+DHA daily
	World Health Organization (WHO)	General Adult Population	1-2% of energy/day
	Chinese Nutrition Society	General Adult	Target of 250-2000 mg EPA+DHA
	(CNS)	Population	daily
	National Heart Foundation of Australia	General Adult risk of CVD Population to lower	Target of 500 mg EPA+DHA daily
	Academy of Nutrition and Dietetics (United States)	General Adult Population	Target of 500 mg EPA+DHA daily
	Dietitians of Canada	General Adult Population	Target of 500 mg n-3 PUFAs daily
	European Food Safety Authority	Adult males and non-pregnant adult females	Adequate intake is 250 mg EPA+DHA daily
	French Food Safety Agency (ANSES)	General Adult Population	Target of 500 mg EPA+DHA daily; DHA intake is 250 mg daily; EPA intake is 250 mg daily
	Health Council (Netherlands)	General Adult Population	Target of 450 mg EPA+DHA daily
	German Society for Nutrition	General Adult Population	0.5% of energy total n-3 PUFA intake
	Superior Health Council of Belgium	General Adult Population	Two servings of tatty fish/wk
	Scientific Advisory Committee on Nutrition (United Kingdom)	General Adult Population	Target of 450 mg n-3 PUFA daily
	Data from Schmidtlein (2017).

Table 1 -3. Major dietary sources of n-3 PUFAs.

 1

		EPA (mg/100g)	DHA (mg/100g)	EPA+DHA (mg/100g)
	Lean fish
	Haddock	70	27	97
	Cod	70	160	230
	European plaice	240	260	500
	Medium fat fish
	Halibut	280	410	690
	Atlantic wolffish	400	200	600
	Rainbow trout	320	1160	1480
	Spotted wolffish	700	400	1100
	Fatty fish
	Greenland Halibut	1000	900	1900
	Salmon	650	1800	2450
	Mackerel	1270	3170	4440
	Herring (summer)	570	1250	1820
	Herring (winter)	2480	2240	4720
	Eel	1270	2070	3340
	Shrimp	50	52	102
	Eggs	0	58	58
	Chicken breast	10	20	30
	Beef	2	1	3
	Pork	0	2	2

(Adapt from

[START_REF] Noc ; Mozaffarian | n-3) fatty acids and cardiovascular health: Are effects of EPA and DHA shared or complementary[END_REF] Taşbozan & Gökç e, 2017)

5.1.1 Oxylipins derived from n-6 polyunsaturated fatty acids

	Researches on oxylipins of n-6 PUFAs mainly focus on ARA, DGLA and LA. ARA and DGLA
	are 20-carbon PUFAs, and their oxidized metabolites, eicosanoids (e.g., prostaglandins,
	thromboxanes, leukotrienes), are key mediators and regulators of inflammation and immunity. With
	the COX pathway, ARA can be converted into 2-series prostaglandins (e.g., prostaglandin I2, D2, E2

and F2α) and thromboxanes (e.g., thromboxane A2 and B2); with the LOX pathway, ARA can be metabolized into 4-series leukotrienes. In addition, ARA can also be converted into hydroxy FAs (e.g.,

) with the CYP ω-hydroxylase activity or into epoxy FAs (e.g., 5,6-epoxy-eicosatrienoic acid

[START_REF]Reference list[END_REF]

with the CYP epoxygenase activity (Figure

1-4

). In general, 2-series prostaglandin biosynthesis is significantly increased in inflamed tissues and contributes to the development of the cardinal signs of acute inflammation. They are widely distributed in almost every tissue and involved in the initiation and progression of numerous diseases, including diabetes mellitus, obesity, hypertension, fatty liver disease, inflammatory bowel disease, vascular diseases, carcinoma, rheumatoid arthritis, asthma and allergic diseases and

Table 1

 1

-1. (Figure from

[START_REF] Coras | Circulating pro-and anti-inflammatory metabolites and its potential role in rheumatoid arthritis pathogenesis[END_REF]

Table 1 -4. The possible mechanisms of n-3 PUFAs for improving cognitive functions.

 1

Table 1 -5. The possible mechanisms of n-3 PUFAs in the prevention of mental disorders.

 1

Table 1 -6. The possible mechanisms of n-3 PUFAs in the prevention of heart diseases.

 1

Table 1 -7. Some of n-3 FA enriched products on the market. Product type Examples of market products Clinical studies

 1

		• n-3 FA enriched organic milk
		(Stonyfield, USA)
		• smart balance® fat free milk and	Intake of n-3 FA enriched dairy
		n-3s (Smart balance, USA)	products was found to reduce blood
		• St. ivel fresh milk with n-3 (St.	lipids, a predominant risk factor for
	Dairy products	lvel, UK)	cardiovascular disease. Studies were
		• source cardio n-3 yoghurt (Yoplait,	carried out on health volunteers,
		Canada)	subjects with increased risk factors,
		• smart balance® n-3 buttery spread	and cardiovascular patients.
		(Smart balance, USA)
		• PROMISE® (Unilever, USA)

Table 1 -8. Different kinds of delivery systems for encapsulating n-3 PUFAs.

 1

	Encapsulation Type	Reference
	O/W emulsion	(Horn et al., 2011)
	Gelled emulsion	(Pourashouri et al., 2020)
	Nanoemulsion	(Walker, 2016)
	Multiple emulsion (O/W/O or W/O/W)	(Cournarie et al., 2004; O' Dwyer et al., 2013)
	Pickering emulsion	(Ren et al., 2021)
	Microcapsule	(Jimé nez-Martí n et al., 2016)
	Nanoparticle	(Serini et al., 2018)
	Hydrogel particle	(Zhang et al., 2014)
	Beads	(Olloqui et al., 2018)
	Liposome	(Kim et al., 2019)
	Nanoliposome	(Rasti et al., 2017)
	Microgels	(Yan et al., 2020)
	Inclusion complexes	(Paul, 2017)
	However, classical emulsions, which are stabilized by molecular surfactants (Yang et al., 2017),

are thermodynamically unstable and tend to breakdown over time through aggregation and coalescence of the dispersed phase

[START_REF] Mcclements | Advances in fabrication of emulsions with enhanced functionality using structural design principles[END_REF]

. Compared with the classical emulsion, emulsion stabilized by solid particles (e.g., microgels, fibrils, colloidal lipid particles and hollow nanoparticles) instead of surfactants

[START_REF] Destribats | Emulsions stabilised by whey protein microgel particles: Towards food-grade Pickering emulsions[END_REF] Schröder et al., 2019;[START_REF] Yan | Protein-stabilized Pickering emulsions: Formation, stability, properties, and applications in foods[END_REF]

, which is named Pickering emulsion (Figure

1

-14), has attracted wide attention due to its outstanding characteristics in food, pharmaceutical, chemical, and other fields. For instance, Pickering emulsions use solid particles as emulsifiers to stabilize the emulsion, which can be irreversibly adsorbed at the oil-water interface to form a film to prevent the aggregation of oil droplets. Whether oil-in-water (O/W) Pickering emulsion or water-in-oil (W/O) Pickering emulsion can be formed is determined by the wettability of solid particles at the oil-water interface: if one of the liquids wets solid particles more than the other one, the better wetting liquid becomes the continuous phase and the other becomes the dispersed phase. O/W emulsions will come into being if the three-phase contact angle θ (angle at the three-phase boundary of solid particles, continuous phase and dispersed phase) is less than 90°, and W/O emulsions should form if θ > 90°. However, particles can effectively act as a Pickering stabilizer only when θ is relatively close to 90°, because particles tend to remain dispersed in either phase when they are too hydrophilic (low θ) or too hydrophobic (high

Table 1 -9. Particles prepared from food materials are used in Pickering emulsion.

 1

	Raw material	Core material	Reference
		Octenyl succinic
		anhydride modified	Sunflower oil	(Song et al., 2020)
		starch
		Sweet potato starch	soybean oil	(Ge et al., 2017)
	Polysaccharides	Amaranth starches and lauroylated amaranth starches	canola oil and α-tocopherol	(Leal-Castañeda et al., 2018)
		Rice starch	soybean oil	(Song et al., 2015)
		Sago starch	Corn oil	(Azfaralariff et al., 2020)
		Chitin	Corn oil	(Barkhordari & Fathi, 2018)
		Maize starch	Medium-chain triacylglycerols (MCT)	(Ye et al., 2017)
		Cellulose	Soybean oil	(Winuprasith et al., 2018)
		Gliadin and WPI	Corn oil	(Zhu et al., 2018)
		WPI	Corn oil	(Yi et al., 2020)
		WPI	Corn oil	(Wu et al., 2015)
		WPI	Corn oil	(Zamani et al., 2018)
		WPI	Canola oil	(Lv et al., 2020)
		WPI	Peanut oil	(Yang et al., 2021)
		WPI	Miglyol 812N	(Destribats et al., 2014)
	Proteins	Soybean protein isolate (SPI)	Soybean oil	(Benetti et al., 2019)
		Gliadin	Corn oil	(Zhu et al., 2020)
		Peanut protein	Corn oil	(Ning et al., 2020)
		Zein	Cinnamon essential oil and vegetal oil	(Jiang et al., 2020)
		Gelatin	Fish oil; Soybean oil	(Feng et al., 2020)
		Quinoa protein	Corn oil	(Qin et al., 2018)
		Rapeseed protein	Rapeseed oil	(Xu et al., 2021)
		Okara dietary fiber and SPI	Soy oil	(Ashaolu & Zhao, 2020)
		SPI and chitosan	Corn oil	(Yang et al., 2020)
		Zein and chitin	Soybean oil	(G. Sun et al., 2019)
	Protein-	Zein and gum arabic	MCT; soybean oil	(Dai et al., 2018)
	polysaccharide complexes	Zein and xanthan gum	sunflower oil	(Santos et al., 2020)
		Zein and corn fiber gum	MCT	(Zhu et al., 2019)
		Lupin protein and maltodextrin	Sunflower oil	(Burgos-Dí az et al., 2020)
	For instance, Leal-Castañeda et al. (2018) prepared lauroylated amaranth starches particles by
	the esterification reaction between native amaranth starches dispersion and lauroyl chloride. They
	found that both native and modified starch acted as good stabilizers of Pickering emulsions to
	encapsulate sunflower oil.

Table 1 -10. The application of WPI in encapsulation.

 1

	Encapsulation	Core material	Reference
	Hydrogels	Caffeine	(Gunasekaran et al., 2007)
	Hydrogels	Anthocyanins	(Ersus et al., 2016)
	Microgels	Miglyol 812N	(Destribats et al., 2014)
	Capsules	β-carotene	(López-rubio & Lagaron, 2012)
	Microcapsules	Sensitive probiotic cultures	(Picot & Lacroix, 2004)
	Microcapsules	Fish oil	(Hinnenkamp & Reineccius, 2021)
	Microparticle (with casein)	Lactobacillus rhamnosus GG	(Burgain et al., 2013)
	Spray-dried encapsulation (with polysaccharide)	Chia essential oil	(Rodea-gonzá lez et al., 2012)
	Nanoparticles	Caffeine	(Gunasekaran et al., 2007)
	Nanoparticles	Vitamin D3	(Abbasi et al., 2014)
	Nanoparticles	β-carotene	(Rodrigues et al., 2020)
	Nanoparticles	Olive leaf phenolics	(Soleimanifar et al., 2020)
	Nanoparticles	Curcumin	(Solghi et al., 2020)
	Emulsion	α-tocopherol	(Fang et al., 2019)
	Emulsion	MCT	(Hu et al., 2019)
	Emulsion (with cyclodextrin)	Fish oil	(Na et al., 2011)
	Emulsion (with maltodextrin)	Moringa oleifera	(Premi & Sharma, 2017)
	Nanoemulsion	Lutein	(Zhao et al., 2018)
	Micro-emulsions (with pectin)	Sunflower oil	(Esfanjani et al., 2017)
	Double emulsions (with pectin)	Sunflower oil	(Esfanjani et al., 2017)
	W/O/W emulsions (with maltodextrin)	Canola oil	(Assadpour et al., 2016)
	Pickering emulsion	Canola oil	(Lv et al., 2020)
	Film (with glycerol)	Bacteriophages	(Vonasek et al., 2014)
	Micro-beads	Probiotic	(Doherty et al., 2015)
	Coacervation	Garlic extract	(Tavares et al., 2019)

Table 3 -1. Effect of emulsion and cooking on the DHA concentrations and proportions in omelets.

 3

	Oil	Omelet

Table 4 -1. The composition of diets (A) and oil mix (B).

 4

	(A)
	%	H-diet	T-diet
	Starch	54.6	54.9
	Sucrose	10.2	10.2
	Cellulose	5.1	5.1
	Casein	20.6	20.8
	Oil mix	5.0	4.5
	Mineral mix	3.5	3.5
	Vitamin mix	1.0	1.0
	(B)
	%	H-diet	T-diet
	Linseed	1.4	2.9
	Sunflower	8.6	8.8
	Olive	13.5	12.6
	Rapeseed	25.0	25.2
	Palm	51.5	50.5

Table 4 -2. The fatty acid composition of the diets and the omelets.

 4

		Diets		Omelets
	%
		H-diet T-diet	Control UN-DHA-O EN-DHA-O
	12:0	0.1	0.1	0.0	0.0	0.0
	14:0	0.6	0.6	0.3	0.3	0.3
	16:0	26.2	25.7	25.3	22.5	22.1
	18:0	3.4	3.4	8.0	7.1	7.2
	20:0	0.4	0.4	0.0	0.0	0.0
	22:0	0.2	0.2	0.0	0.0	0.0
	24:0	0.1	0.1	0.0	0.0	0.0
	saturates	31.0	30.5	33.6	29.9	29.6
	14:1n-5	0.0	0.0	0.1	0.0	0.1
	n-5	0.0	0.0	0.1	0.0	0.1
	16:1n-7	0.2	0.2	2.9	2.6	2.6
	18:1n-7	1.4	1.4	2.0	1.8	1.8
	n-7	1.6	1.6	4.9	4.4	4.4
	16:1n-9	0.0	0.0	0.7	0.6	0.6
	18:1n-9	46.2	45.7	42.2	37.9	37.2
	20:1n-9	0.5	0.4	0.1	0.2	0.3
	22:1n-9	0.3	0.3	0.0	0.0	0.0
	24:1n-9	0.0	0.0	0.0	0.2	0.2
	n-9	47.0	46.4	43.0	38.9	38.3
	18:2n-6	17.0	17.2	14.8	13.0	12.9
	20:4n-6	0.0	0.0	2.0	1.9	1.9
	22:4n-6	0.0	0.0	0.0	0.1	0.1
	22:5n-6	0.0	0.0	0.3	0.6	0.6
	n-6	17.0	17.2	17.1	15.6	15.5
	18:3n-3	3.4	4.3	0.5	0.4	0.5
	20:5n-3	0.0	0.0	0.0	0.5	0.5
	22:5n-3	0.0	0.0	0.0	0.5	0.5
	22:6n-3	0.0	0.0	0.8	9.8	10.6
	n-3	3.4	4.3	1.3	11.2	12.1

Table 4 -3. The fatty acid composition of the daily intake of T-diet and omelets.

 4

	%	T-Diet with different omelets Control UN-DHA-O EN-DHA-O
	12:0	0.1	0.1	0.1
	14:0	0.5	0.5	0.5
	16:0	25.6	25.4	25.4
	18:0	4.5	4.4	4.4
	20:0	0.3	0.3	0.3
	22:0	0.1	0.1	0.1
	24:0	0.1	0.1	0.1
	saturates	31.2	30.9	30.9
	16:1n-7	0.8	0.8	0.8
	18:1n-7	1.6	1.5	1.5
	n-7	2.4	2.3	2.3
	16:1n-9	0.2	0.2	0.2
	18:1n-9	44.8	44.8	44.8
	20:1n-9	0.4	0.4	0.4
	22:1n-9	0.2	0.2	0.2
	24:1n-9	0.0	0.1	0.1
	n-9	45.6	45.7	45.7
	18:2n-6	16.6	16.6	16.6
	20:4n-6	0.5	0.5	0.4
	22:5n-6	0.1	0.2	0.2
	n-6	17.2	17.3	17.2
	18:3n-3	3.4	3.4	3.5
	20:5n-3	0.0	0.1	0.1
	22:5n-3	0.0	0.1	0.1
	22:6n-3	0.2	2.5	2.5
	n-3	3.6	6.1	6.2
	n-6/n-3	4.9	4.9	4.8
	Total	100.0	102.3	102.3
			TAG-DHA + 2.3%

Table 4 -4. The dimethylacetal profile of RBC, heart, brain and eyes.

 4

		%	16:0-DMA	18:0-DMA	18:1n-9-DMA	18:1n-7-DMA	DMA sum µg/mg
		Control	29.2 ± 0.3	33.2 ± 0.3	31.6 ± 0.3	6.0 ± 0.1	0.12 ± 0.00
	RBC	UN-DHA-O	30.3 ± 0.3	32.6 ± 0.4	31.8 ± 0.5	5.3 ± 0.2	0.12 ± 0.00
		EN-DHA-O	30.0 ± 0.5	33.0 ± 0.4	31.6 ± 0.7	5.4 ± 0.3	0.13 ± 0.00
		Control	63.2 ± 1.0	13.4 ± 1.0	23.4 ± 1.2	0.0 ± 0.0	0.38 ± 0.01
	Heart	UN-DHA-O	64.2 ± 1.8	13.6 ± 1.5	22.2 ± 1.4	0.0 ± 0.0	0.36 ± 0.02
		EN-DHA-O	62.2 ± 1.3	15.1 ± 0.7	22.7 ± 1.5	0.0 ± 0.0	0.42 ± 0.02
		Control	22.2 ± 0.9	52.6 ± 0.7	14.9 ± 0.4	10.3 ± 0.3	2.17 ± 0.07
	Brain	UN-DHA-O	21.5 ± 0.7	51.7 ± 0.6	16.3 ± 0.7	10.5 ± 0.4	2.49 ± 0.13
		EN-DHA-O	21.6 ± 0.5	52.7 ± 0.6	15.4 ± 0.6	10.3 ± 0.4	2.30 ± 0.11
		Control	26.8 ± 0.3 a 34.3 ± 0.4	20.0 ± 0.4 a 18.9 ± 0.2 a 0.12 ± 0.00 a
	Eyes	UN-DHA-O	40.7 ± 2.1 b 34.8 ± 3.0	11.1 ± 2.4 b 13.4 ± 2.2 b 0.06 ± 0.00 b

Table 4 -5. The oxidized derivatives of fatty acids from DHA oil and omelets.

 4 The oxidized metabolites of fatty acids were quantified by LC-QQQ as mentioned in methods on DHA oil used to prepare omelets. * A column named "expected" results from calculations on oxylipin concentrations expected after supplementation of egg with DHA oil, considering the oxylipin pattern from DHA oil.

	Omelets (ng/3 g omelet)

Table 4 -6. The oxidized derivatives of fatty acids from plasma. ng/mL of plasma Control UN-DHA-O EN-DHA-O

 4 The oxidized metabolites of fatty acids were quantified in plasma by LC-QQQ as mentioned in methods. a, b, c and ab indicate significant differences between the three groups when p<0.05. ab means no difference with a and no difference with b.

	LA	13-HODE 9-HODE	13.47 ± 1.90 a 8.04 ± 1.01 b 7.29 ± 1.21 b 2.56 ± 0.30 1.71 ± 0.13 1.72 ± 0.25
		PGF2α	0.39 ± 0.09 a 0.00 ± 0.00 b 0.00 ± 0.00 b
		PGE2	0.78 ± 0.16 a 0.19 ± 0.02 b 0.00 ± 0.00 c
		PGD2	0.59 ± 0.21 a 0.00 ± 0.00 b 0.00 ± 0.00 b
	ARA	TXB2 15-HETE	7.88 ± 2.76 a 1.59 ± 0.41 b 0.77 ± 0.08 c 2.98 ± 0.51 a 1.15 ± 0.12 b 0.64 ± 0.08 c
		8-HETE	2.03 ± 0.33 a 0.54 ± 0.13 b 0.00 ± 0.00 c
		12-HETE	384.28 ± 76.32 a 90.73 ± 22.97 b 25.05 ± 3.25 c
		5-HETE	4.48 ± 0.28 a 2.30 ± 0.14 b 1.53 ± 0.13 c
	DHA	14-HDoHE 17-HDoHE	42.30 ± 7.53 a 20.41 ± 4.20 b 6.82 ± 0.87 c 3.70 ± 0.73 2.91 ± 0.36 2.06 ± 0.26

Table 4 -7. The oxidized derivatives of fatty acids from heart.

 4

	pg/mg protein	Control		UN-DHA-O		EN-DHA-O
	LA	13-HODE 9-HODE	772.1 ± 93.6 220.4 ± 28.9		709.4 ± 64.9 223.6 ± 17.7		765.3 ± 58.5 248.3 ± 20.0
	DGLA 6kPGF1α	437.7 ± 96.6 a	194.6 ± 28.1 b	88.1 ± 6.7	c
		PGF2α	118.1 ± 20.8 a	51.8 ± 8.8	b	23.6 ± 3.9	c
		PGE2	82.2 ± 11.6 a	39.1 ± 7.2	b	24.0 ± 2.3	c
		PGD2	42.8 ± 6.4	a	20.0 ± 3.1	b	22.5 ± 3.4	b
		TXB2	94.8 ± 18.5		55.3 ± 7.4		44.5 ± 6.1
		15-HETE	494.2 ± 50.6 a	270.8 ± 22.0 b	313.6 ± 30.9 b
		8-HETE	109.4 ± 14.8		88.6 ± 7.9		95.9 ± 10.7
	ARA	12-HETE 3562.0 ± 441.1		3213.5 ± 926.4		4335.8 ± 536.1
		5-HETE	532.0 ± 66.0 a	325.7 ± 19.1 b	274.6 ± 18.2 b
		14,15-EET	55.6 ± 11.4 a	0.0 ± 0.0	b	0.0 ± 0.0	b
		5-oxo-ETE	514.4 ± 60.7 a	306.5 ± 18.9 b	222.5 ± 14.8 b
		11,12-EET	102.2 ± 14.8 a	68.9 ± 5.2	ab	48.0 ± 6.3	b
		8,9-EET	179.3 ± 37.2		110.9 ± 10.9		78.0 ± 9.4
		5,6-EET	248.3 ± 59.7 a	121.1 ± 18.

Table 4 -8. The oxidized derivatives of fatty acids from brain.

 4 The oxidized metabolites of fatty acids were quantified in brain by LC-MS-MS as mentioned in methods.

	pg/mg protein	Control	UN-DHA-O		EN-DHA-O
	LA	13-HODE	103.2 ± 14.2	108.6 ± 12.0		154.0 ± 19.6
	DGLA	6kPGF1α	272.3 ± 39.7	324.4 ± 57.6		428.7 ± 64.2
		PGF2α	3641.1 ± 367.7	4062.1 ± 266.9		4184.5 ± 187.4
		PGE2	401.0 ± 39.8	499.0 ± 40.3		540.3 ± 71.4
		PGD2	3040.4 ± 249.4	3679.7 ± 307.1		3644.2 ± 254.5
		TXB2	577.0 ± 53.4	637.4 ± 53.3		715.8 ± 109.6
		15-HETE	716.0 ± 69.6	782.2 ± 65.2		861.5 ± 52.1
		8-HETE	147.3 ± 15.0 a	151.8 ± 8.3	a	218.9 ± 25.5 b
	ARA	12-HETE	3557.6 ± 438.4	4908.4 ± 679.7		5055.1 ± 472.0
		5-HETE	840.3 ± 85.9	834.8 ± 72.2		1046.5 ± 206.8
		14,15-EET	223.4 ± 20.5	217.3 ± 22.3		272.9 ± 37.8
		5-oxo-ETE	588.5 ± 75.7	569.6 ± 66.6		705.5 ± 55.3
		11,12-EET	279.2 ± 36.6	267.3 ± 26.1		297.5 ± 47.1
		8,9-EET	380.9 ± 44.4	370.4 ± 33.9		554.7 ± 95.2
		5,6-EET	566.1 ± 90.9 ab 446.0 ± 54.2 b	730.1 ± 73.0 a
	DHA	14-HDoHE 17-HDoHE	382.6 ± 47.1 a 375.8 ± 49.1	385.5 ± 63.2 a 581.6 ± 113.7	597.1 ± 61.1 b 645.8 ± 55.4

-1. Oxylipin abbreviations and names.

		Supplemental
	Oxylipin	Name	Oxylipin		Name
	9,10 DiHOME	9,10-dihydroxy-octadecenoic acid	9,10 EpOME	9,10-epoxy-octadecenoic acid
	12,13 DiHOME 12,13-dihydroxy-octadecenoic acid	12,13 EpOME 12,13-epoxy-octadecenoic acid
	9-HODE	9-hydroxy-octadecadienoic acid	9-oxoODE		9-oxo-octadecadienoic acid
	13-HODE	13-hydroxy-octadecadienoic acid	13-oxoODE	13-oxo-octadecadienoic acid
	5-HETrE	5-hydroxy-eicosatrienoic acid	8-HETE		8-hydroxy-eicosatetraenoic acid
	15-HETrE	15-hydroxy-eicosatrienoic acid	PGE1		Prostaglandin E1
	PGD1	Prostaglandin D1	5,6-EET		5,6-epoxy-eicosatrienoic acid
	8,9-EET	8,9-epoxy-eicosatrienoic acid	14,15-EET		14,15-epoxy-eicosatrienoic acid
	5,6-diHETrE	5,6-dihydroxy-eicosatrienoic acid	8,9-diHETrE	8,9-dihydroxy-eicosatrienoic acid
	14,15-diHETrE	14,15-dihidroxy-eicosatrienoic acid	15-HETE		15-hydroxy-eicosatatrenoic acid
	8-HETE	8-hydroxy-eicosatetraenoic acid	15-oxoETE		15-oxo-eicosatetraenoic acid
	LTB4	Leukotriene B4	6RLXA4		6R Lipoxin A4
	6SLXA4	6S Lipoxin A4	8,15-diHETE	8,15 dihydroxy-eicosatetraenoic acid
	5,15-diHETE	5,15 dihydroxy-eicosatetraenoic acid	tetranor	12-	Tetranor 12 hydroxy-eicosatetraenoic
			HETE		acid
	HXB3	Hepoxilin B3	12-HETE		12-hydroxy-eicosatetraenoic acid
	12-oxoETE	12-oxo-eicosatetraenoic acid	5-HETE		5-hydroxy-eicosatetraenoic acid
	5-oxoETE	5-oxo-eicosatetraenoic acid	LTA4		Leukotriene A4
	LTB4	Leukotriene B4	11-HETE		11 Hydroxy-eicosatetraenoic acid
	PGH2	Prostaglandin H2	12-HHTrE		12-hydroxy-heptadecatrienoic acid
	TXB2	Thromboxane B2	PGI2		Prostaglandin I2
	6k PGE1	6-keto Prostaglandin E1	15k PGF1α		15-keto Prostaglandin F1α
	6,15 dk-, dh-	6,15-Diketo-13,14-dihydro-PGF1α	PGD2		Prostaglandin D2
	PGF1α
	PGJ2	Prostaglandin J2	15d PGJ2		15-deoxy-Prostaglandin J2
	dhk PGD2	13,14-dihydro-15-keto-prostaglandin D2	15d PGD2		15-deoxy-Prostaglandin D2
	PGE2	Prostaglandin E2	PGB2		Prostaglandin B2
	bicyclo PGE2	bicyclo Prostaglandin E2	dhk PGE2		13,14-dihydro-15-keto-prostaglandin E2
	PGF2α	Prostaglandin F2α	tetranor PGFM Tetranor-Prostaglandin F Metabolite
	dhk PGF2α	13,14-dihydro-15-keto-prostaglandin F2α	dh PGF2α		13,14-Dihydro-prostaglandin F2α
	11b dhk PGF2α	11beta 13,14-dihydro-15-keto-prostaglandin F2α 9-HETE		9-hydroxy-eicosatetraenoic acid
	8 iso PGF2α Ⅲ	8-iso Prostaglandin F2α III	5 iso PGF2α Ⅵ 5-iso Prostaglandin F2α VI
	16-HETE	16-hydroxy-eicosatetraenoic acid

table 4 -3. The fatty acid profile of brain and eyes.

 4

  Encapsulation d'huile de DHA sous forme d'émulsion de Pickering : effet sur la bioaccessibilité et le métabolisme du DHA Mots clés : huile de DHA; Émulsion de Pickering; Omelette; digestion in vitro; in vivo; oxylipineRésumé : L'encapsulation peut affecter la digestion et la bioaccessibilité des composés encapsulés, ce qui peut ensuite affecter leur métabolisme. Le but de ce projet était d'étudier les effets de l'encapsulation sur la bioaccessibilité et le métabolisme du DHA, en étudiant une huile de DHA non-encapsulée ou encapsulée, apportée sous forme d'omelette comme matrice alimentaire. L'huile de DHA, composée de triacylglycérols riches en DHA, a été préparée sous forme d'émulsion de Pickering, stabilisée par des isolats de protéines de lactosérum dénaturés par la chaleur. De l'huile pure ou sous forme d'émulsion a ensuite été ajoutée à l'oeuf liquide pour obtenir de l'omelette. Les effets de l'encapsulation sur la digestion ont été étudiés à l'aide d'un modèle de digestion statique in vitro pour adulte, puis l'impact sur le métabolisme du DHA a été mesuré sur un modèle rat pris au sevrage.Les résultats ont montré in vitro que l'encapsulation peut augmenter la surface de contact entre l'huile de DHA et les enzymes de digestion, favorisant l'hydrolyse lipasique de l'huile de DHA et améliorant ainsi la bioaccessibilité du DHA. In vivo, l'encapsulation n'a pas impacté le profil global des acides gras, et particulièrement l'accrétion du DHA dans le cerveau. En revanche, le profil des oxylipines, dérivés oxydatifs d'acides gras, a été fortement modifié dans le plasma, le coeur et même le cerveau. Les métabolites dérivés du DHA ont globalement été augmentés tandis que ceux issus des acides gras de la famille n-6 ont été essentiellement atténués. Par conséquent, l'encapsulation de l'huile de DHA pourrait non seulement améliorer la bioaccessibilité du DHA, mais constitue également un facteur clé dans le métabolisme du DHA pour produire des précurseurs de protectines et de maresines, améliorant ainsi le statut de santé global.

Titre :

Title : Encapsulation of DHA oil as Pickering emulsion: effect on DHA bioaccessibility and metabolism Keywords : DHA oil; Pickering emulsion; Omelet; in vitro digestion; in weanling rat; oxylipin

Acknowledgments

thank you for always silently supporting me behind my back. Thank you for listening to my complaints when I am unhappy, and for your encouragement and support when I am sad. Home is always the harbor of my soul and the driving force for progress! Thanks to the China Scholarship Council, thanks for your scholarship funding, so that I can continue to work hard on the road of scientific research carefree. Thank you, my country, you are always my strong backing

of Lipid Research, 50, S29-S34. https://doi.org/10.1194/jlr.R800042-JLR200 Rüdiger, M., Von Baehr, A., Haupt, R., Wauer, R. R., & Rüstow, B. (2000). Preterm infants with high polyunsaturated fatty acid and plasmalogen content in tracheal aspirates develop bronchopulmonary dysplasia less often. Critical Care Medicine, 28 (5), 1572-1577. https://doi.org/10.1097/00003246-200005000-00052 Ruiz Ruiz, J. C., Ortiz Vazquez, E. D. L. L., & Segura Campos, M. R. (2017). Encapsulation of vegetable oils as source of omega-3 fatty acids for enriched functional foods. Critical Reviews in Food Science and Nutrition, 57(7), 1423-1434. https://doi.org/10.1080/10408398.2014.1002906 Ruiz, T. A. J., & Perez, C. T. (2020). Improvement of encapsulation and stability of EPA and DHA from monolayered and multilayered emulsions by high -pressure homogenization. September 2019, 1-13. https://doi.org/10.1111/jfpp.14290 Russo, G. L. (2009). Dietary n-6 and n-3 polyunsaturated fatty acids: From biochemistry to clinical implications in cardiovascular prevention. Biochemical Pharmacology, 77(6), 937-946. https://doi.org/10.1016/j.bcp.2008.10.020 Ruxton, C. H. S., Calder, P. C., Reed, S. C., & Simpson, M. J. A. (2005). The impact of long-chain n -3 polyunsaturated fatty acids on human health . Nutrition Research Reviews, 18(1), 113-129. https://doi.org/10.1079/nrr200497 Sagara, M., Njelekela, M., Teramoto, T., Taguchi, T., Mori, M., Armitage, L., Birt, N., Birt, C., & Yamori, Y. (2011). Effects of docosahexaenoic acid supplementation on blood pressure, heart rate, and serum lipids in scottish men with hypertension and hypercholesterolemia. International Journal of Hypertension, 2011(Cvd). https://doi.org/10.4061/2011/809198 Saini, R. K., & Keum, Y. S. (2018). Omega-3 and omega-6 polyunsaturated fatty acids: Dietary sources, metabolism, and significance-A review. Life Sciences, 203(April), 255-267. https://doi.org/10.1016/j.lfs.2018.04.049 Sams, L., Amara, S., Mansuelle, P., Puppo, R., Lebrun, R., Paume, J., Giallo, J., & Carrière, F. (2018). Characterization of pepsin from rabbit gastric extract, its action on β-casein and the effects of lipids on proteolysis. Food & Function, 9(11), 5975-5988. https://doi.org/10.1039/C8FO01450G Sanguansri, L., Augustin, M. A., Lockett, T. J., Abeywardena, M. Y., Royle, P. J., Mano, M. T., & Patten, G. S. (2015). Bioequivalence of n-3 fatty acids from microencapsulated fish oil formulations in human subjects. British Journal of Nutrition, 113(5), 822-831. https://doi.org/10.1017/S000711451400436X Santos, J., Alcaide-González, M. A., Trujillo-Cayado, L. A., Carrillo, F., & Alfaro-Rodríguez, M. C. (2020). Development of food-grade Pickering emulsions stabilized by a biological macromolecule (xanthan gum) and zein. International Journal of Biological Macromolecules, 153, 747-754. https://doi.org/10.1016/j.ijbiomac.2020.03.078 Sayanova, O. V., Beaudoin, F., Michaelson, L. V., Shewry, P. R., & Napier, J. A. (2003). Identification of Primula fatty acid Δ6-desaturases with n-3 substrate preferences. FEBS Letters, 542(1-3), 100-104. https://doi.org/10.1016/S0014-5793(03)00358-2 Schipper, L., Van Dijk, G., & Van der Beek, E. M. (2020). Milk lipid composition and structure; the relevance for infant brain development. OCL, 27, 5. https://doi.org/10.1051/ocl/2020001 Schmid, M., & Müller, K. (2019). Whey protein-based packaging films and coatings. In Whey proteins (pp. 407-437). Elsevier. Schmidtlein, H. R. K. (2019). Via electronic filing and hand delivery. Schmitt, C., Bovay, C., & Rouvet, M. (2014). Bulk self-aggregation drives foam stabilization properties of whey protein microgels. Food Hydrocolloids, 42, 139-148. https://doi.org/10.1016/j.foodhyd.2014.03.010 Schröder, A., Corstens, M. N., Ho, K. K. H. Y., Schroën, K., & Berton-Carabin, C. C. (2018). Pickering Emulsions. In S. Roohinejad, R. Greiner, I. Oey, & J. Wen (Eds.), Emulsionbased Systems for Delivery of Food Active Compounds (pp. 29-67). John Wiley & Sons, Ltd. https://doi.org/10.1002/9781119247159.ch2 Schröder, A., Sprakel, J., Boerkamp, W., Schroën, K., & Berton-Carabin, C. C. (2019). Can we prevent lipid oxidation in emulsions by using fat-based Pickering particles? Food Research International, 120,[352][353][354][355][356][357][358][359][360][361][362][363]. https://doi.org/10.1016/j.foodres.2019.03.004 Schröder, R., Xue, L., Konya, V., Martini, L., Kampitsch, N., Whistler, J. L., Ulven, T., Heinemann, A., Pettipher, R., & Kostenis, E. (2012). PGH1, the precursor for the anti-inflammatory prostaglandins of the 1-series, is a potent activator of the pro-inflammatory receptor

Abbreviations

List of figures

• Figure 1. Overall experimental design.

• Figure 1-1. The structure of common PUFAs.

• Figure 12. The pathway of PUFAs synthesis.

• Figure 123. Typical oxylipins are produced by the metabolism of different PUFAs.

• Figure 1234. The pathway of oxylipins derived from some common n-6 PUFAs.

• Figure 12345. Specialized pro-resolving mediator (SPM) synthesis.

• Figure 123456. Schematic summary of lipid digestion, absorption, and metabolic fate of dietary lipids.

• Figure 1234567. The hydrolysis of TAG into 1,2-DAG and FFA catalyzed by gastric lipase.

• Figure 12345678. The hydrolysis of TAG into 2-MAG and two FFA catalyzed by pancreatic lipase.

• Figure 123456789. The hydrolysis of PL into lyso-PL and FFA catalyzed by PLA2.

• Figure 1-10. The hydrolysis of CE into CL and FFA catalyzed by cholesterol esterase.

• Figure 1-11. Intestinal lipid absorption.

• Figure 1-12. The structure of mature chylomicron.

• Figure 1-13. Lipid metabolism.

• Figure 1

Supplementary material contents

Supplementary Figures

• Supplemental Figure 4-1. Dimethylacetal formation from plasmalogens after saponification and methylation.

• Supplemental Figure 4-2. Correlation between 14-HDoHE concentrations in plasma and DHA proportions in plasma.

Supplementary Tables

• Supplemental Table 1-1. Oxylipin abbreviations and names.

• Supplemental Table 4-1. The fatty acid profile of plasma and RBC.

• Supplemental Table 4-2. The fatty acid profile of liver and heart.

• Supplemental Table 4-3. The fatty acid profile of brain and eyes. Distribution of DHA oil was followed during digestion of omelets. Firstly, the control omelets deprived of DHA oil showed no red staining (Figure 3-3). The digestion process was tracked with proteins, the hydrolysis of which began during the gastric phase and was particularly emphasized with the reduced size of protein-stained particles in the intestinal phase. Secondly, concerning the DHA-enriched omelets, the size and the dispersion of DHA oil with UN-DHA-O were less uniform in the beginning of the gastric phase than with EN-DHA-O. Moreover, DHA oil repartition seemed stable during the gastric digestion, whereas the hydrolysis of DHA oil occurred in the intestinal phase as visualized by the reduction of the red-stained droplets. However, using this method of analysis, differences between UN-DHA-O and EN-DHA-O were difficult to quantify. We further analyzed the digestion process by lipochemistry.

Distribution of DHA oil droplets during the digestion of omelets

Hydrolysis of lipids and release of fatty acids during the digestion of omelets

Omelets contained initially 92 µg FA per mg, esterified as TAG (73%), PL (24%) and other minor species. Digestion of lipids was followed during the gastric and intestinal phases by the FA profile of the different lipid species present in omelets (Figure 3-4). Altogether, these data showed that encapsulating DHA oil enhanced lipase hydrolysis during the process of digestion. DHA oil can only be digested in the intestinal phase by the pancreatic lipase, probably because RGL was described as more stereoselective for TAG with shorter acyl chains [START_REF] Rogalska | Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases[END_REF].

To better understand this point, we further analyzed the digestion profiles of the major FA from eggs with 16, 18 and 20 carbons (16:0, 18:1n-9, 18:2n-6, 18:3n-3 and 20:4n-6). We also compared with work showed that encapsulation of DHA oil remains a key factor for DHA metabolism in generating precursors of protectins and maresins, thus improving the global health status.

Chapter 5: General conclusion and perspectives

The main purpose of this work is to add DHA oil enriched delivery system into the food matrix, and to study the effect of encapsulation on the digestion of DHA in vitro and the metabolism of DHA in vivo. This research can provide information about the bioavailability of DHA as Pickering emulsion in the food matrix. So as to provide a certain theoretical basis for the development of foods rich in n-3 PUFAs and improve global health status.

The ingenuity of this work relies on the following points:

• Firstly, DHA oil delivery system. In this study, we chose Pickering emulsion as the delivery system to carry DHA oil, because based on our knowledge, there is currently no research on the metabolism of DHA enriched Pickering emulsion in vivo.

• Second, this research is based on a food matrix (omelet) to study digestion and metabolism, rather than directly based on Pickering emulsion. Although there have been many studies on the delivery systems based on food matrices (e.g., liposomes in yogurt, microcapsules in sausages) [START_REF] Ghorbanzade | Nano-encapsulation of fish oil in nano-liposomes and its application in fortification of yogurt[END_REF][START_REF] Solomando | Lipid digestion products in meat derivatives enriched with fish oil microcapsules[END_REF], there are no studies on the digestion and metabolism of DHA, neither on oil enriched Pickering emulsion, nor on egg products.

• Finally, this study also focused on the effect of encapsulation on the lipid oxidized metabolites (oxylipins). Most of the current research on encapsulation strives to compare the bioavailability of bioactive compounds by measuring the content of compounds of interest in blood and thereafter in tissues. To our knowledge, there is no research on the impact of encapsulation of DHA on derivatives of DHA (oxylipins) which have been proved to have various physiological effects.

• Specifically, for brain, DHA was the nutrient studied in this research, while in the literature, EPA was also of interest considering its pleiotropic effects on health. We focused on DHA as the DHA oil was specially prepared as pure as possible in DHA. The use of another oil enriched in EPA was also planned in the beginning of the thesis, but for a reason of time, the study was finally developed only on DHA. secretion of cholecystokinin, thereby inhibiting the muscle contraction of the gastric antrum, enhancing the contraction of the pylorus to slow down gastric emptying [START_REF] Aguilera | Why food microstructure[END_REF][START_REF] Feinle | Effects of duodenal fat, protein or mixed-nutrient infusions on epigastric sensations during sustained gastric distension in healthy humans[END_REF]. Therefore, slow lipolysis might have an effect on the degree of satiety experienced after consuming food, if lipolysis is slowed, may potentially prolong the inter-meal interval by producing a more persistent, but lower elevation of satiety hormones [START_REF] Keogh | Slowly and Rapidly Digested Fat Emulsions Are Equally Satiating but Their Triglycerides Are Differentially Absorbed and Metabolized in Humans[END_REF]. This may affect energy intake, which in turn affects animal weight. It would be interesting to develop researches on that topic so that we may figure out an explanation for the increase of animal weight described in chapter 4.

Regarding chapter 4: Encapsulation of DHA oil substantially improves the oxylipin profile of rat tissues.

The main objective is currently to achieve the best accretion of DHA in brain by improving the nutritional conditions of DHA synthesis or dietary intake.

The first question was to test the effect of encapsulation of DHA oil with Pickering emulsion on the bioavailability of DHA. We demonstrated that bioavailability was increased with the DHA supplementation but without a significant effect when DHA oil was encapsulated.

The second question was to test accretion of DHA in different tissues and especially in brain.

Traditionally to answer this question, a nutritional condition is chosen to achieve a significant effect of the DHA supplementation of the diet, often by reducing the endogenous rate of DHA synthesis from ALA. In that condition, the control shows a relatively low proportion of DHA in brain but this proportion is significantly increased with the DHA supplementation of the diet. Then several forms of dietary DHA are compared. Thus, DHA is presumably better incorporated when supplied in the phospholipid form, although the literature is still discussed. DHA is also well incorporated when emulsified, different types of emulsions on various animal models being tested.

In this study, we studied DHA esterified to TAG and incorporated to a food matrix (omelet) completed by an equilibrate diet for growth of animals. In our condition of work, endogenous synthesis of DHA was considered as optimum for growing animals. Indeed, we obtained a very important accretion of DHA in brain in the control group, as rarely observed in the literature according to our knowledge. It's true that eggs contained a low content of DHA esterified to PL (2.1 mg DHA per day in this study). This form of DHA supply combined with the optimized ratio n-6/n-Annexes 1. Supplementary Figures Supplemental Figure 4