les coacervats de beta-lactoglobuline et lactoferrine pour

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Les coacervats de beta-lactoglobuline et lactoferrinepour l’encapsulation d’une molécule bioactive modèle, la

vitamine B9Anne Laure Chapeau

To cite this version:Anne Laure Chapeau. Les coacervats de beta-lactoglobuline et lactoferrine pour l’encapsulation d’unemolécule bioactive modèle, la vitamine B9. Alimentation et Nutrition. Agrocampus Ouest, 2017.Français. �NNT : 2017NSARB300�. �tel-01697443�

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Thèse AGROCAMPUS OUESTsous le label de l’Université Bretagne Loire

pour obtenir le grade de DOCTEUR D’AGROCAMPUS OUEST

Spécialité Science de l’aliment

Anne-Laure CHAPEAU 26 octobre 2017•

AGROCAMPUS OUEST • Institut national supérieur des sciences agronomiques, agroalimentaires, horticoles et du paysage65 rue de Saint-Brieuc – CS84215 – F-35042 Rennes CedexTél. : 02 23 48 50 00www.agrocampus-ouest.fr

ÉCOLE DOCTORALE • Écologie, Géosciences, Agronomie et Alimentation (EGAAL)

LABORATOIRE D’ACCUEIL • UMR INRA-AO Science et Technologie du Lait et de l’Œuf (STLO)

Les coacervats de

beta-lactoglobuline et lactoferrine

pour l’encapsulation d’une

molécule bioactive modèle,

la vitamine B9

Françoise NAU

Professeur, AGROCAMPUS OUEST, UMR INRA-AO STLO / présidente

Christian SANCHEZ

Professeur, SupAgro Montpellier / rapporteur

Adem GHARSALLAOUI

Maître de conférences, Université Claude Bernard Lyon 1 / rapporteur

André BRODKORB

Senior Scientist, Moorepark, Teagasc, Ireland / examinateur

Marieke VAN AUDENHAEGE

Chargée de recherche, Société Sodiaal / examinatrice

Saïd BOUHALLAB

Directeur de recherche, UMR INRA-AO STLO / directeur de thèse

Denis PONCELET

Professeur, Oniris, Nantes, France/ co-directeur de thèse

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RÉSUMÉ ABSTRACT

Beta-lactoglobulin and lactoferrin complex coacervates for the

encapsulation of a model bioactive, the vitamin B9.

Encapsulation of bioactives is relevant for the development of

functional foods. Food proteins as encapsulating agents could

match the objective of industries to develop “clean label” products.

Moreover, whey proteins (WP) exhibit good potentialities as

encapsulating agents. Previous works have demonstrated that

the WPs, beta-lactoglobulin (BLG) and lactoferrin (LF), are able to

spontaneously co-assemble by complex coacervation. This study

explores the ability of BLG-LF coacervates as a potential carrier for

the encapsulation of a model bioactive, vitamin B9. Throughout

screening experiments, we determined the domains where B9-WP

coacervation occured according to a tested range of pH, proteins

and vitamin concentrations and molar ratios. Optimal conditions

for coacervation were found in water, at pH 5.5, with LF:B9:BLG

molar ratio of 1:5:10, affording coacervation yields of 45 to 55%

and B9 encapsulation up to 98%. Coacervation was scaled-up from

laboratory to bench scale using commercial-grade protein sources

and static mixing. Final effi ciencies were obtained with coacervates containing 4 mg of B9/g coacervates. Under degradative conditions

(UV light irradiation, oxidation, freeze-drying), WP coacervates

provided good protective properties limiting chemical degradation

of native B9. In vivo oral administration of B9-WP coacervates

in rats enhanced the plasmatic concentrations of B9 compared

to unencapsulated B9. In addition, good physical stability over

time was found after incorporated and resuspension of formed

coacervates in milk. The combined results of this thesis provide

better insight into heteroprotein complex coacervation and its use

for innovative applications.

Keywords: heteroprotein coacervation, whey protein, bioactive,

vitamin, encapsulation.

Les coacervats de beta-lactoglobuline et lactoferrine pour l’encap-

sulation d’une molécule bioactive modèle, la vitamine B9.

Les aliments fonctionnels connaissent aujourd’hui un intérêt gran-

dissant. Pour leur formulation, l’encapsulation de bioactifs repré

sente une voie intéressante et les protéines de lactosérum (PS) pré

sentent de bonnes potentialités en tant qu’agents d’encapsulation.

Des travaux antérieurs ont démontré que deux PS, la beta-lactoglo-

buline (BLG) et la lactoferrine (LF), peuvent former spontanément

des co-assemblages par coacervation complexe une technologie

d’encapsulation connue. Cette thèse étudie la coacervation BLG-

LF pour l’encapsulation d’un bioactif modèle, la vitamine B9. En

testant une gamme de pH et de ratios molaires, les conditions opti-

males de coacervation B9-PS sont obtenues dans l’eau, à pH 5,5,

avec un ratio molaire LF:B9:BLG de 1:5:10, permettant d’atteindre

des rendements de coacervation de 45 à 55%, et d’encapsulation

de B9 de 98 %. L’échelle de production des coacervats est aug-

mentée avec succès du µL au L, avec des solutions protéiques de

qualité commerciale et un mélangeur statique. Les rendements de

coacervation et d’encapsulation sont conservés, avec l’encapsu

lation d’environ 4 mg de B9/g coacervats. Par ailleurs, les coacer

vats montrent un effet protecteur de la forme native de B9 vis-à-vis

des UV, de l’oxydation et pendant la lyophilisation. Une étude in vivo

chez le rat démontre une augmentation de la biodisponibilité de

B9 lorsque administrée sous forme de coacervats. Les coacervats

apparaissent stables lorsqu’ils sont resuspendus en gouttelettes

dans du lait. Ce travail permet d’approfondir les connaissances sur

la coacervation hétéroprotéique et son application en tant que vec-

teur d’encapsulation naturel et innovant.

Mots-clés : coacervation hétéroprotéique protéines sériques,

bioactif, encapsulation.

Thès

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Acknowledgments

2

The present Ph.D. thesis was prepared at the STLO laboratory

(Science and Technology of Milk and Egg),

UMR INRA Agrocampus-Ouest, in Rennes, France,

From November 2014 to October 2017.

Acknowledgments

3

Few words of thanks

La thèse de doctorat constitue un travail scientifique au long cours et pour cette

raison, il est loin d'être un travail solitaire. À l'issue de cette thèse, je souhaite donc remercier

toutes les personnes qui de près ou de loin, ont contribué à l'aboutissement de ce travail de

3 ans, pour moi, et à cette petite gouttelette dans l'univers des sciences, pour les autres.

Je remercie tout d'abord les membres de mon jury, Christian Sanchez, Adem

Gharsallaoui, André Brodkorb, Marieke Van Audenhaege et Françoise Nau, pour avoir

accepté d’évaluer ce travail et me faire profiter ainsi de leur expertise.

Je souhaite ensuite adresser mes remerciements, et au-delà ma reconnaissance, à mes

encadrants de thèse. Denis Poncelet, pour l'ouverture d'esprit qu'il m'a apportée et pour la

rigueur dans l'organisation de mes idées... et de leur synthèse ! Et bien sûr, Saïd Bouhallab.

Saïd, je te remercie pour l'intérêt que tu as porté à ma vision de ce sujet de thèse. Pour ton

encadrement et tes conseils au quotidien, me laissant à la fois une grande autonomie, mais

restant toujours présent pour me faire avancer et progresser dans la bonne direction… tant sur le plan scientifique que dans l’apprentissage à devenir un bon chercheur. Merci.

Je remercie également Joëlle Léonil, coordinatrice scientifique du passionnant projet

PROFIL, et Yves Le Loir, directeur d'unité, pour leur accueil au sein du laboratoire STLO.

Également associé au projet PROFIL, je remercie le Pôle Agronomique Ouest, et ses

coordinateurs Stéphan Rouverand et Morgane Raison, les Régions Bretagne et Pays-de-la-

Loire, l’association BBA et les industriels investis, pour le financement et le pilotage de ce

projet collaboratif, et plus particulièrement ceux du comité technique de l'axe 4.

L'ensemble de ce travail n'aurait bien sûr pas pu être mené à bien sans toutes les

personnes avec qui j'ai travaillé au quotidien durant ces 3 ans. Tout d'abord je remercie tous

les membres de l'équipe ISF-PL, pour leur accueil et les échanges scientifiques lors des

réunions d'équipes. Je remercie Thomas Croguennec, Guilherme Tavares, Valérie Briard-

Bion, Stéphane Pezennec, et Florence Rousseau, pour leurs conseils, leur suivi et leur aide au

cours de ce travail. Merci également à toutes les personnes que j'ai côtoyées de près ou de

loin pendant ces années au STLO. Un merci particulier à Paulette Amet et Jessica Musset pour

le soutien logistique, Laurence Fauvel, Danielle Guilloux et Nathalie Le Marre pour leur aide

administrative, Rachel Boutrou, pour l'apprentissage et l'aide rédactionnelle, et également

Juliane Floury, Anne Thierry et Valérie Gagnaire, les premières personnes à m’avoir ouvert les portes de ce laboratoire durant mon stage de fin d’études d’ingénieur.

Acknowledgments

4

Un travail de thèse c'est aussi et surtout beaucoup d'heures passées en laboratoire...

et si j'ai pu aborder toutes ces heures avec bonne humeur et enthousiasme (presque) chaque

jour, c'est grâce à toi Pascaline Hamon. Pascaline, je te remercie du temps que tu as passé à

me former (méthode des cellules de bouches !), de ton enthousiasme partagé lors des

premières microscopies des microsphères, de ta disponibilité aux moindres soucis d'HPLC et

de tous tes conseils bienveillants. J’ai beaucoup appris à tes côtés et tous ces résultats de thèse n'auraient pas été ce qu'ils sont sans toi. J’espère que tu en es aussi fière que moi ! J’ai également eu la chance de pouvoir partir pour le Grand Nord Québécois durant 3 mois et je

remercie Nicolas Bertrand de l’Université Laval de Québec pour son accueil et sa

disponibilité au sein de son équipe. Du coup Nicolas, un grand merci pour le temps que tu as

pris à me former, à m’aider aux pharmocinétiques sur rats, et merci de ton intérêt et de ton

aide pour aller au bout de toutes mes suggestions de manips !

Un travail de thèse c’est aussi beaucoup de temps passé devant l’ordinateur… et je remercie donc mes colocs de bureau, Jonathan Thévenot, Mathieu Person et Lélia Lacou pour

m’avoir supportée pendant ces 3 années. Je remercie également tous les autres doctorants,

stagiaires, post-docs que j'ai côtoyés au sein du laboratoire et qui contribuent à en faire un

lieu de travail où l’on vient chaque matin avec le sourire.

Aux termes de ce parcours je remercie celles et ceux qui au-delà de cette thèse sont

devenus des amis. Je remercie Clémentine Le Boucher, pour toute son aide d’une ancienne esaïenne à une autre, sur sur tout mon parcours au STLO et au delà. Je remercie les girls :

Linda Le Roux, Marie Chevallier, Eve-Anne Norwood et Mélanie Carpin, pour toutes les

soirées filles et les heures de papotage qui m’ont permis de toujours garder la pêche ! Et

enfin, je remercie Fanny Lazzaro. Fanny mon double, au sens propre comme figuré. Si je suis

fière d’avoir réalisé cette thèse, je suis encore plus fière d’y être allez au bout avec toi. De nos premières sorties randonnée sur la côte bretonne, aux soirées et à nos heures de discussion

au laboratoire, tu as toujours su m’épauler. Tous ces moments resteront aussi parmi les plus

beaux résultats de ma thèse ! Merci également à mes partenaires thésardes esaïennes hors

STLO, Anne-Sophie Valable et Marion Bournazel, qui m’ont permis de relativiser et positiver

ce choix de carrière post diplôme d’ingénieur.

Enfin, un grand merci à celles et ceux qui me sont le plus chers, et qui extérieurs à

cette thèse m’ont soutenue, aidée et supportée, avant, pendant et après ces 3 années. Au-delà

des mots de remerciements que je peux écrire ici, sachez que votre présence m’est essentielle :

Simon, Ludivine, Papi, Mamie et vous Papa, Maman, qui avez su me donner toutes les chances

de réussite professionnelle et également personnelle, pour les années passées et celles à venir.

Table of contents

6

Table of contents

Abstract ............................................................................................................................................................... 8

Résumé ................................................................................................................................................................ 9

Thesis outputs ............................................................................................................................................... 10

Tables of figures ............................................................................................................................................ 13

Tables of tables .............................................................................................................................................. 17

Abbreviations ................................................................................................................................................. 18

I. General introduction ......................................................................................................................... 20

II. Literature review ................................................................................................................................ 28

1. Functional foods and encapsulation ........................................................................................ 29

1.1. What are functional foods? ................................................................................................. 29

1.2. Encapsulation, an essential technology for functional foods ................................ 30

1.1. Trends in functional food development ......................................................................... 35

2. Whey proteins, from gutter to gold ...................................................................................... 37

2.1. General overview of milk proteins................................................................................... 37

2.2. Structure and functionalities of whey proteins .......................................................... 39

2.3. Whey proteins for functional foods development ..................................................... 43

3. Complex coacervation, theory and practical use ................................................................ 46

3.1. Principles of complex coacervation ................................................................................. 46

3.1. Applications of complex coacervation ............................................................................ 48

3.2. Encapsulation by complex coacervation ....................................................................... 49

4. Heteroprotein complex coacervates ........................................................................................ 53

4.1. Specificity of heteroprotein coacervation ..................................................................... 53

4.2. Special case of Lactoferrin and Beta-Lactoglobulin .................................................. 55

4.3. Heteroprotein complex coacervates for encapsulation of bioactives ................ 57

4.4. Research objectives and strategy ..................................................................................... 60

Table of contents

7

III. Results ................................................................................................................................................. 63

1. Chapter 1: Production of vitamin B9-WP coacervates ..................................................... 64

1.1. Part 1: Formation of Lactoferrin and Beta-Lactoglobulin coacervates in

presence of vitamin B9 ..................................................................................................................... 65

1.2. Part 2: Bench scale production of vitamin B9-WP coacervates............................ 86

2. Chapter 2: Properties of vitamin B9-WP coacervates .....................................................103

2.1. Part 1: Protective effect of WP coacervates ................................................................104

2.2. Part 2: Bioavailability of vitamin B9 from WP coacervates .................................117

3. Chapter 3: Generalization of the concept of the formulation of natural and

functional dairy encapsulating agents ...........................................................................................124

3.1. Introduction ............................................................................................................................126

3.2. Material and methods .........................................................................................................128

3.3. Results and discussion ........................................................................................................132

3.4. Final conclusions...................................................................................................................147

IV. General discussion ........................................................................................................................149

1. Formation and characterization of WP coacervates ........................................................150

1.1. LF-BLG coacervation for the encapsulation of targeted molecules ..................150

1.2. Production of bioactive-WP coacervates. ....................................................................151

1.3. Physico-chemical characteristics of bioactive-WP coacervates .........................153

2. Application of WP coacervates .................................................................................................155

2.1. Properties of WP coacervates ..........................................................................................155

2.2. Comparison with WP-polysaccharide coacervates .................................................158

2.3. Nutritional potentiality and benefits of WP coacervates ......................................159

V. General conclusion and perspectives ........................................................................................162

References .....................................................................................................................................................168

8

Abstract

Functional foods are today a subject of growing interest, motivated by their potential to

prevent diseases and contribute to healthier daily lifestyles. Encapsulation of bioactives

is a promising approach for the development of such food products with enhanced health

benefits. Food proteins as encapsulating agents appear to be specially relevant and capable to match the objective of the food industries to develop and promote clean label ,

certifying the quality of their products. In particular, whey proteins (WP), co-products of

the dairy industry, exhibit good potentialities to act as encapsulating agents, and this

would in fine enhance their functional and economic value. Moreover, previous works

have demonstrated that the two WPs, beta-lactoglobulin (BLG) and lactoferrin (LF), are

able to spontaneously co-assemble by complex coacervation, a known encapsulation

technology. In this context, this study aims to explore BLG and LF complex coacervation

for the encapsulation of a model bioactive, vitamin B9. A series of experiments have

determined the domains where B9-LF-BLG coacervation is feasible according to a tested

range of pH, proteins and vitamin molar ratios. In our study, optimal conditions for B9-

WP coacervation were found in water, at pH 5.5, with a LF:B9:BLG molar ratio of 1:5:10,

affording WP coacervation yields of 45 to 55% and B9 encapsulation up to 98%. WP

complex coacervation was then scaled-up from the laboratory to the bench scale using

commercial-grade protein solutions and static mixing. Final efficiencies, similar to the

laboratory scale, were obtained, enabling the production of B9-WP coacervates

containing 4 mg of B9/g WP coacervates. Under degradative conditions in vitro (UV light

irradiation, oxidation by H2O2, freeze-drying), WP coacervates were shown to provide

good protective properties to limit chemical degradations of B9. Moreover, in vivo oral

administration of B9-WP coacervates in rats made it possible to enhance the plasmatic

concentrations of the vitamin compared to oral administration of unencapsulated B9. Also

B9-WP coacervates demonstrated good physical stability over time when incorporated

into milk in the form of small droplets suspended in solution. The combined results of this

thesis provide better insight into heteroprotein complex coacervation, and realize a link

between the theory behind this long-established chemical concept and and its

exploitation for innovative applications in the food science and industries.

Key words: heteroprotein coacervation, whey protein, bioactive, vitamin, encapsulation

9

Résumé

Depuis quelques années, les aliments fonctionnels connaissent un intérêt grandissant, de

par leur potentiel à prévenir certains types de maladies et à contribuer à des modes de

vie quotidiens plus sains. Pour la formulation de ces aliments, l'encapsulation de bioactifs

et leur incorporation dans une matrice alimentaire représentent une voie intéressante.

Avec la volonté actuelle de l’industrie agro-alimentaire de développer des aliments

« clean label » (sans additifs), l'utilisation de protéines alimentaires comme agents d’encapsulation semble pertinente. Ainsi, les protéines de lactosérum (PS), coproduits des

industries laitières, présentent de bonnes potentialités en tant qu'agents d’encapsulation,

ce qui pourrait améliorer in fine leurs valeurs ajoutées. Des travaux antérieurs ont

démontré que deux PS, la beta-lactoglobuline (BLG) et la lactoferrine (LF), sont capables

de former spontanément des co-assemblages par coacervation complexe ; la coacervation

étant une technologie d'encapsulation connue. Dans ce contexte, la présente thèse vise à

étudier la coacervation entre la BLG et la LF pour l'encapsulation d'un bioactif modèle, la

vitamine B9. En testant une gamme de pH, de concentrations et de ratios molaires, les

conditions optimales pour la formation des coacervats B9-PS sont obtenues : dans l'eau,

à pH 5,5, avec un ratio molaire de LF:B :BLG de : : . Cela permet d’atteindre des

rendements de coacervation des PS de 45 à 55%, et d’encapsulation de B9 jusqu'à 98 %. Par la suite, l’échelle de production des coacervats est augmentée avec succès du µL au L,

en utilisant des solutions protéiques de qualité commerciale et un mélangeur statique. Les rendements de coacervation et d’encapsulation sont conservés, permettant de produire des coacervats de PS contenant 4 mg B9 / g de coacervats. En appliquant in vitro des

conditions de dégradations (UV, oxydation, lyophilisation), les coacervats de PS

démontrent un bon effet protecteur, limitant la dégradation de la forme native bioactive

de B9. De plus, une étude in vivo sur des rats met en évidence une augmentation de la biodisponibilité de B lorsqu’elle est administrée par le coacervat de PS plutôt que seule. Enfin, les coacervats de PS apparaissent relativement stables lorsqu’ils sont resuspendus sous formes de gouttelettes dans une matrice laitière. Ainsi, l’ensemble des résultats de cette thèse permet d’approfondir les connaissances sur la coacervation hétéroprotéique,

en essayant de relier ses aspects théoriques à ses applications potentielles pour former des agents d’encapsulations naturels et innovants.

Mots clés : coacervation hétéroprotéique, protéines sériques, bioactif, encapsulation

Thesis outputs

10

Thesis outputs

A – Research papers in peer-reviewed journals

[1] Chapeau A.L., Tavares G.M., Hamon P., Croguennec T., Poncelet D., Bouhallab S. (2016).

"Spontaneous co-assembly of lactoferrin and -lactoglobulin as a promising biocarrier for

vitamin B , Food Hydrocolloids, 57: 280-290.

[2] Chapeau A.L., Hamon P., Rousseau F., Croguennec T., Poncelet D., Bouhallab S. (2017).

"Scale-up production of vitamin loaded heteroprotein coacervates and their protective

property", Journal of Food Engineering, 206: 67-76.

[3] Casanova F, Chapeau A.L., Hamon P., de Carvalho A.F., Croguennec T., Bouhallab S.

(2017). "pH- and ionic strength-dependent interaction between cyanidin-3-O-glucoside

and sodium caseinate", Food Chemistry, (in press)

doi.org/10.1016/j.foodchem.2017.06.081.

[4] Chapeau A.L., Bertrand N., Briard-Bion V., Hamon P., Poncelet D., Bouhallab S. (2017).

"Coacervates of whey proteins to protect and improve the oral delivery of bioactive

molecules", Journal of Functional Foods, (submitted).

B – Review paper in peer-reviewed journal

[1] Boire A., Bouchoux A., Bouhallab S., Chapeau A.L., Croguennec T., Ferraro V.,

Lechevalier V., Menut P., Pézennec S., Renard D., Santé-Lhoutellier V., Laleg K., Micard V.,

Riaublanc A., Anton M. (2017). "Proteins for the future: a soft matter approach to link

basic knowledge and innovative applications", Innovative Food Science & Emerging

Technologies, (in press) doi.org/10.1016/j.ifset.2017.06.012

C – Publication in technological journal

[1] Chapeau A.L., Tavares G.M., Hamon P., Croguennec T., Bouhallab S., Poncelet D. (2015).

"Spontaneous co-assembly of proteins for encapsulation of a hydrophilic vitamin",

Bioencapsulation Innovations, Nov. 2015.

Thesis outputs

11

D – Oral communications

[1] Chapeau A.L., Tavares G.M., Hamon P., Croguennec T., Bouhallab S., Poncelet D. (2015).

"Spontaneous co-assembly of proteins for encapsulation of a hydrophilic vitamin", 23rd

International Conference on Bioencapsulation, Delft, The Netherlands, 2-4 Sept. 2015.

Awarded as Best student contribution.

[2] Chapeau A.L., Hamon P., Croguennec T., Bouhallab S., Poncelet D. (2016). "Complex

coacervation of whey proteins to design biocarriers for bioactives", 7th Meeting of Physics

and Biology in Brittany, Nantes, France, 23-24 June 2016.

[3] Chapeau A.L., Hamon P., Croguennec T., Poncelet D., Bouhallab S. (2016).

"Lactoferrin/ -lactoglobulin heteroprotein system: Interactions, coacervation and

encapsulation potentiality", 1st Food Chemistry Conference, Rotterdam, The Netherlands,

30 Oct. - 1 Nov. 2016.

[4] Chapeau A.L., Hamon P., Rousseau F., Croguennec T., Poncelet D., Bouhallab S. (2017).

"Scale-up production of vitamin-whey proteins coacervates and their protective property

against storage degradations", Innovations in Food Science & Technology, Erding,

Germany, 10-12 May 2017.

[5] Chapeau A.L., Bertrand N., Hamon P., Bouhallab S., Poncelet D. (2017). "Bench

production and in vivo evaluation of vitamin loaded whey protein coacervates", 25th

International Conference on Bioencapsulation, Nantes, France, 3-6 July 2017.

[6] Chapeau A.L., Van Audenhaege M., Bouhallab S. (2017). "Les coacervats de protéines

sériques : des nouveaux agents d’encapsulation naturels et fonctionnels", Journée PROFIL, Rennes, France, 21 September 2017.

E – Poster presentation

[1] Chapeau A.L., Tavares G.M., Hamon P., Croguennec T., Poncelet D., Bouhallab S. (2016).

"Complex coacervation of whey proteins: an innovative pathway to design biocarriers for

bioactive molecules". 13th International Hydrocolloids Conference, Guelph, Canada, 16-20

May 2016.

Thesis outputs

12

F – Progress reports of the PROFIL research project

[1] Chapeau A.L., "Encapsulation de molécules bioactives par les assemblages spontanés

de protéines laitières: Stratégie et premiers résultats", Projet PROFIL, Comité Technique

Axe 4 "Vectorisation, encapsulation", Rennes, France, 10 nov. 2015.

[2] Chapeau A.L., "Coacervation B9-LF-BLG pour l’encapsulation de la vitamine B : scale-

up et propriétés protectrices du coacervat au cours du stockage", Projet PROFIL, Comité

Technique Axe 4 "Vectorisation, encapsulation", Rennes, France, 15 avr. 2016.

[3] Chapeau A.L., "Approche des coûts d’encapsulation d’une molécule bioactive par le coacervat de protéines sériques (Beta-Lactoglobuline-Lactoferrine)", Rapport d’étude, Projet PROFIL, janv. 2017.

[4] Chapeau A.L., "Caractérisation du coacervat B9-LF-BLG : Biodisponibilité in-vivo,

resuspension en matrice laitière et stabilité", Projet PROFIL, Comité Technique Axe 4

"Vectorisation, encapsulation", Rennes, France, 7 fév. 2017.

G – Student supervision

Co-supervision of a graduate student, Laurena Masbernat, to obtain the grade of Engineer

(Master 2) in Food Science and Technology from the École nationale supérieure d'agronomie

et des industries alimentaires, April-September 2017.

H – Previous research works

[1] Chapeau A.L., Silva J.V.C., Schuck P., Thierry A., Floury J., (2015). The influence of cheese

composition and microstructure on the diffusion of macromolecules: a study using

Fluorescence Recovery After Photobleaching (FRAP), Food Chemistry, 192:660-667.

[2] Chapeau A.L., Silva J.V.C., Schuck P., Thierry A., Floury J., (2014). Diffusion of macromolecules in cheeses is mainly related to its free water content of the fat-free cheese:

a Fluorescence Recovery After Photobleaching (FRAP) approach, 9th Cheese Symposium, Cork,

Ireland, 12-13 Nov. 2014. Awarded as 2nd best student poster.

___________________________________________________________

Tables

13

Tables of figures

Figure I-1: Schematic representation of the PROFIL research project: academic and industrial partners, public financial supports and coordinator, articulated to explore four thematic functional properties of whey proteins. ........................................................................... 24

Figure II-1: Schematic representation of encapsulating systems, adapted from (Jan Zuidam & Shimoni, 2009). ........................................................................................................................ 31

Figure II-2: Schematic representation of Beta-Lactoglobulin, (A) Quaternary structure of the monomeric BLG, and (B) Dimeric BLG complexed with retinol. ........................................ 41

Figure II-3: Tertiary structure of Lactoferrin, (A) on its native form or (B) binding with two iron atoms (red spheres). ................................................................................................................. 43

Figure II-4: Schematic representation of complex coacervation, (A) Model of the formation kinetic of coacervates between two oppositely charged biopolymers, (B) Transmission-mode CSLM micrograph of beta-lactoglobulin/acacia gum coacervates, (C) Schematic steps and parameters influencing the complex coacervation of two oppositely charged biopolymers, (D) Coacervates/supernatant after centrifugation of coacervates suspension....................................................................................................................................................... 48

Figure II-5: Scientific publications from 2007 to 2017 in the fields of (Encapsulation + Coacervation) or (Encapsulation + Coacervation + Protein), according to key words research on Web of Science©, June 2017. .......................................................................................... 50

Figure II-6: Procedure of encapsulation by complex coacervation illustrated by schematic diagram (A) or schematic design (B). ................................................................................................... 51

Figure II-7: Proposed mechanism for the formation of LF-BLG coacervates ...................... 56

Figure II-8: Fraction of LF (%) recovered in LF-BLG coacervates depending on the initial BLG:LF molar ratio, at pH 5.50 in 10 mM MES buffer at 25°C .................................................... 56

Figure II-9: Chemical composition and representation of vitamin B9 ................................... 59

Figure II-10: Mechanisms of specific binding of vitamin B9 by LF to form B9-LF nanocomplexes explored through Isothermal Titration Calorimetry ITC signal ΔH for B9-LF interactions in kcal mol-1) and size particles (Dh in nm) according to B9/LF ratio, adapted from (Tavares, Croguennec, Lê, et al., 2015). ................................................................... 60

Figure II-11: Lines of research, thesis chapters and subsequent scientific publications. ............................................................................................................................................................................. 62

Figure III-1: Turbidity kinetics of LF-BLG co-assembly in the presence of B9 at six B9:protein mixing molar ratios spanning from low (0.1 mM) to high total protein concentration (1mM), obtained at pH=5.5 and at 20°C.. ............................................................... 73

Tables

14

Figure III-2: Phase contrast microscopy (x40) of LF-BLG co-assembly in the presence of B9 at six B9:protein mixing molar ratios spanning from low (0.1 mM) to high total protein concentration (1mM), obtained at pH=5.5 and at 20°C. (A) B9:protein=2:2; (B) B9:protein=4:2; (C) B9:protein=2:11; (D) B9:protein=4:11; (E) B9:protein=4:15; (F) B9:protein=4:20. ........................................................................................................................................... 74

Figure III-3: Stability analysis of LF-BLG co-assemblies in the presence of B9 0.25 mM. Delta transmission and delta backscattering signals recorded over 24 hours of formed aggregates (1.1 mM total proteins) (A, B) or coacervates (0.55 mM total proteins) (C, D) obtained at pH=5.5 and at 20°C.. ............................................................................................................ 76

Figure III-4: Co-assembly yield η and B loading of B -LF-BLG co-assembly at three different initial B9 contents and B9:protein mixing molar ratios at two total protein concentrations: (A) 0.15mM: (B) (0.55mM). ..................................................................................... 79

Figure III-5: Screening maps of B9-LF-BLG co-assembly by color projection for large range of B9 and protein concentrations at pH=5.5 and at 20°C. (A) maximum turbidity in cm-1; (B) co-assembly type index; (C) entrapment in mg B9/g Protein. ............................... 80

Figure III-6: Characteristics of B9-LF-BLG coacervates. (A) size distribution; (B) sphericity index; (C) image from phase contrast microscopy. ................................................... 83

Figure III-7: Schematic diagram for the of production of B9-WP coacervates (A) and the device used for batch mixing by mechanic stirring (B) or continuous mixing by static stirring (C). ...................................................................................................................................................... 90

Figure III-8: A: Turbidity of B9-WP coacervates solutions at different mixing volumes for purified █ and commercial proteins samples ░). B: Visual difference between

commercial (a) and purified BLG (b). C: Phase contrast microscopy (×40) of B9-WP coacervates obtained at different mixing volume and WP samples: purified WP solution (a) 0.1 mL, (b) 5 mL, (c) 100 mL; and commercial WP solutions, (d) 5 mL, (e) 100 mL. . 93

Figure III-9: A: B9 and WP concentrations recovered in B9-WP coacervates at different mixing volumes and the type of whey proteins used, purified █ versus commercial protein samples (░). B: Entrapment, coacervation yields and total weight of B9-WP coacervates obtained at different mixing volumes with two types of whey proteins purified (█) versus commercial samples (░). .................................................................................. 95

Figure III-10: Batch coacervation at different mechanic stirring. A: B9 and WP concentrations in B9-WP coacervates. B: Entrapment, coacervation yields and total weight of B9-WP coacervates, C: Particle size distribution obtained by laser light scattering and associated schematic representation of B9-WP coacervatesd solutions after (a) no stirring (0 rpm), (b) low stirring (50 rpm) or (c) high stirring (150 rpm). ... 98

Figure III-11: Continuous coacervation of B9-WP depending on initial flows. A: Turbidity of B9-WP solutions measured at different initial flows (mL/min) through the static mixer, B: Corresponding particle morphology prior centrifugation: (a) flow = 50 mL/min, (b) flow = 100 mL/min, (c) flow > 200 mL/min, C: B9 and WP concentrations in B9-WP coacervates, D: Entrapment of B9 and coacervation yield after centrifugation. ...............100

Tables

15

Figure III-12: Evolution of RP-HPLC profiles of vitamin B9 after exposure to UV light during 24 h. Vitamin B9 was treated with UV either alone in solution (A), pre-complexed with lactoferrin (B) or encapsulated in whey protein coacervates (C) . ...............................111

Figure III-13: Protective effect of WP coacervates against UV light induced degradation of vitamin B9. Ion intensities of B9 and derived products from electrospray mass spectrometry analysis before and after 48 h exposure to UV light. ........................................112

Figure III-14: Degradation kinetics of free, complexed or encapsulated vitamin B9 after exposure to 20% hydrogen peroxide at 37°C for 18h. ..................................................................113

Figure III-15: B9 concentrations before, after freeze-drying, and after a one year storage in uncapsulated B9 or B9-WP coacervates. ......................................................................................114

Figure III-16: Pharmacokinetics of vitamin B9 concentrations in rat plasma vs time after a single oral administration at T0 of B9, B9-WP coacervates, B9-BLG, or B9-LF solutions corresponding to a dose of 500 µg B9 / oral administration.. ..................................................121

Figure III-17: Phase contrast microscopy and macroscopic images of bioactive-WP coacervates solutions obtained with different bioactives : (A) C3G, (B) vitamin E, (C) resveratrol. ....................................................................................................................................................132

Figure III-18: WP coacervation and bioactive encapsulation yields (%) obtained by coacervation between whey proteins (LF and BLG) and four bioactives: vitamin B9, vitamin E, resveratrol and C3G. ............................................................................................................133

Figure III-19: Schematic representation of complex coacervation followed by microphase and macrophase separation of bioactive-WP coacervates for potential use as an ingredient. .....................................................................................................................................................136

Figure III-20: Macroscopic images of (A) B9-WP coacervates recovered after centrifugation, and after resuspension in (B) milliQ water at pH 5.5, (C) ultra-filtrate milk permeate pH 6.7 and (D) semi-skimmed milk pH 6.6, (E) phase contrast microscopy of B9-WP coacervates resuspended in ultra-filtrate milk permeate (obj x100). ...........................138

Figure III-21: Turbidity measurements and microscopic images (obj. x40) of lactoferrin-mineral-beta-lactoglobulin solutions ([proteins]=0.33mM) in the presence of increasing ionic strengths (Mineral concentrations from 0 to 3 mM). ........................................................140

Figure III-22: Turbidity measurements and microscopic images (obj. x40) of lactoferrin-beta- solutions ([proteins] = 0.33mM) and the addition of minerals (Zn, Mg or Ca) at increasing ionic strengths (Mineral concentrations from 0 to 10 mM). ...............................141

Figure III-23: Schematic representation of complex coacervation followed by microphase and macrophase separation of bioactive-WP coacervates and their resuspension in dairy matrix. .............................................................................................................................................................142

Tables

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Figure III-24: Logarithmic representation of kinetic release of B9 (%) as assessed by dialysis approach from three dairy matrices, (A) milk ultra-filtrate permeate, (B) semi-skimmed milk and (C) full-fat milk, enriched with B9 free, B9 free and additional WP coacervates, or B9-WP coacervates. ...................................................................................................144

Figure III-25: Release quantity of B9, LF and BLG (mg) recovered in milk ultra-filtrate permeate, initially after resuspension of B9-WP coacervates (T0), 4 hours (T4h) and 24 hours (T24h after)......................................................................................................................................145

Figure III-26: Phase contrast microscopy of B9-WP coacervates resuspended in ultra-filtrate milk permeate at T0, (A) obj. x40 and (B) obj. x100, and after 48 hours stored at 4°C, (C) obj. x40 and (D) obj. x100. ......................................................................................................146

Figure III-27: Schematic representation of complex coacervation followed by microphase and macrophase separation of bioactive-WP coacervates and their resuspension and stability over 24 hours in a dairy matrix, at 4°C. ............................................................................147

Figure IV-1: B9-WP coacervates recovered after centrifugation (A), its corresponding

composition assessed by RP-HPLC analysis (B), and B9-WP coacervates after freeze-

drying (C). ......................................................................................................................................................155

Tables

17

Tables of tables

Table 1: Overview of encapsulation technologies, adapted from (Jan Zuidam & Shimoni,

2009). ................................................................................................................................................................ 32

Table 2: Selection of actives ingredients and their corresponding encapsulation

technologies to use in functional food and ingredients for potential health benefits

(adapted from (M. A. Augustin & Sanguansri, 2012; D. J. McClements, 2012)) ................... 34

Table 3: Milk composition, adapted from (Gaucheron, 2005). .................................................. 37

Table 4: Main physico-chemical properties of bovine milk proteins. ..................................... 37

Table 5: Type of caseins used as vehicles for different ligands, adapted from (Livney,

2010). ................................................................................................................................................................ 38

Table 6: Comparative characteristics of principles steps of complex coacervation for

encapsulation of traditional vs emerging procedure. .................................................................... 52

Table 7: Characteristic parameters of pharmacokinetics of vitamin B9 concentrations in

rat plasma vs time after a single oral administration at T0 of B9, B9-WP coacervates, B9-

BLG, or B9-LF solutions corresponding to a dose of 500µg B9/oral administration.. ....122

Table 8: Physico chemical characteristics and potential health benefits of bioactives

tested for encapsulation by the WP coacervates............................................................................127

Table 9: Initial concentration, encapsulation yields and concentrations recovered in WP

coacervates for four different bioactives. .........................................................................................134

Table 10: Principal studies existing on vitamin B9 encapsulation using whey proteins

(WP) or beta-lactoglobulin as encapsulating agents ....................................................................156

Table 11: Recommended daily intake of B9 according to age classes (Dietary reference

by Food and Nutrition Board, Institute of Medicine) ...................................................................160

Table 12: Highlights of the main results and perspectives of the present work. .............164

Abbreviations

18

Abbreviations

ΔBS: backscattering variation ΔH: enthalpy variation ΔT: transmission variation

AA: amino acid

AUC: area under the curve

B9: vitamin B9

BLG: beta-lactoglobulin

BSA: bovine serum albumin

C3G: cyanidin-3-O-glucoside

Dh: hydrodynamic diameter

DLS: dynamic light scattering

ELISA: enzyme-linked immunosorbent assay

HPCC: heteroprotein complex coacervation

INRA: Institut National de la Recherche Agronomique

ITC: isothermal titration calorimetry

kDa: kiloDalton

l: light path length

LF: lactoferrin

mM: millimolar (mmol/L)

Mw: molecular weight

Pa: P-aminobenzoyglutamic acid

pI: Isoelectric point

Pr: pteridine residue

PROFIL: Protéines Fonctionnalisées pour l’Industrie Laitière

Q: quantity

RP-HPLC: reverse phase high performance liquid chromatography

Rpm: rotation per minute

T: temperature τ: turbidity

UV: ultra-violet

WP: whey proteins

Abbreviations

19

z: valence

[B9]: vitamin B9 concentration

[BLG]: beta-lactoglobulin concentration

[LF]: lactoferrin concentration

[WP]: total whey proteins concentration

General introduction

20

I. General introduction


21

Food is part of our daily necessities. Food is also our first medicine, as already

recognized by Hippocrates centuries ago. From this point of view, food is not merely

intended to satisfy our daily diet on a quantitative basis but, in addition, to provide a

qualitative source of essential nutrients to prevent deficiency-related diseases and to

maintain the overall physical and psychological well-being of the individual. At a broader

level, food is also related to more societal or sustainability issues, involving accessibility

and adequate management of resources. Such issues motivate rational development over

the whole food chain for the formulation, production and distribution of foodstuffs. In this

context, the food industry has attempted to respond to this dual demand by combining

food both for a healthier individual lifestyle and for societal sustainability. This trend has

induced significant technical and economic evolutions of the entire food supply chain, as

well as of the manufacturing and distribution of the final food products.

At the same time, foods and beverages, especially those that are part of a normal

diet, were identified as good vehicles for the controlled delivery of bioactive molecules

and nutrients with specific nutritive properties and, in this way, as a means to respond to the consumer’s demand for food products with enhanced health benefits. This triggered

the development of novel ranges of foods known as functional foods, initiated more than

20 years ago (Hasler, 2002). Functional foods represent one of the most interesting and

promising areas of research and innovation in the food sector today. In this context,

researchers have identified and isolated many bioactive molecules that provide a wide

spectrum of health benefits. Some important examples include probiotics for improving

intestinal health, polyphenols to prevent cardiovascular disease, cancer and

neurodegenerative diseases, and vitamins that play a key role in DNA synthesis and cell

regeneration (Augustin & Sanguansri, 2015). However, surprisingly few of these

bioactives have been successfully incorporated into functional foods despite significant

research and developmental efforts in this direction (Annunziata & Vecchio, 2011). Many

scientific and technological challenges still need to be addressed for the successful

development of functional foods and the concretization of their whole potential range of

benefits.

Milk is often described as the most nearly perfect food since it contains a majority of the nutrients and energy required for healthy human nutrition and development

throughout life. Milk and dairy products are consumed throughout the world but are

particularly important in Europe, North America, and Oceania (Muehlhoff, Bennett,


22

MacMahon, & Food and Agriculture Organization of the United Nations, 2013). Social and

technological developments over the past few decades have significantly influenced the

variety of dairy products available: milk liquids and beverages, fermented milks, yogurts,

cheeses, milk powders, creams, butters, etc. Although processes such as fermentation

have been traditionally used, the dairy sector has developed several specific techniques

over time to produce these diverse ranges of milk-based products and dairy ingredients.

These process developments have been possible thanks to the accumulated scientific

knowledge and research on milk, from a macro to a microscopic scale, from empiric

observations to theoretical modeling. The chemistry and physicochemical properties of

milk have been studied for over 200 years and are now well described in the scientific

literature. Among milk constituents, milk proteins have been extensively studied and are

probably the most well-characterized food protein system. Research on milk proteins

dates from the early 19th century. Pioneering work was undertaken by H. Schubler in 1818

on the physicochemical status of milk proteins, and by H. Braconnot in 1830, who

published the first paper in which the word casein was used. Some years ago, the term casein was universally adopted as the word for the pH 4.6-insoluble protein in raw

milk (Boland, Singh, & Thompson, 2014). This casein fraction is the one responsible for

milk coagulation and gelation during the manufacturing of cheeses and yogurts. Whey, a

by-product of this process that contains the remaining soluble whey proteins, had long

been considered as waste by the dairy industry. In the late 20th century, the recognition

of the value of whey components accelerated. Based on several research efforts that

focused on the nutritional and functional properties of whey proteins, whey was gradually

transformed from a waste material to a valuable dairy stream containing a multitude of

components available for use in various dairy, agri-food and feed, biotechnology

industries, and even the medical sector (Smithers, 2008).

Dairy products are universal: milk producing animals are raised in almost every

country across the world, and up to one billion people live on dairy farms. The dairy sector

is a vital part of the global food system and plays a key role in the sustainability of rural

areas worldwide. In many countries, the dairy industry actively contributes to the

economy and provides employment not only to people who work on dairy farms and in

dairy plants, but also to the whole sector, from upstream (inputs and services providers,

etc.) to downstream (marketing of finished products, etc.) (IDF, 2013). In France, with an


23

annual 27 billion euro turnover and 250,000 direct jobs, the French dairy sector is one of

the leaders of our economy. It is responsible for 1/5 of the global turnover of the French

agri-food sector, in second position after the meat industry, and five French dairy

companies are among the top 25 dairy companies in the world (CNIEL, 2016).

Nonetheless, the dairy market is constantly evolving and remains very competitive. The

end of the milk quota policy in Europe, the decrease in milk powder imports by China, the

competition with milk and milk protein substitutes (plant derivatives), and many other

economic and political decisions have been the source of the considerable volatility of

milk prices over the last two years, thus impacting the overall sustainability and

profitability of the dairy sector. As a result of this tight market and its high level of

competition worldwide, French dairy companies are constantly looking for an increased

valorization of their dairy products in order to ensure the sustainability and economic

development of the entire dairy chain.

The West of France is the heart of France’s major milk-producing region,

responsible for half of the French dairy production and more than 10% of the European

one. To maintain this leadership, it is of great interest for the public authorities, both at

the state and regional levels, to encourage the development of this sector through

research and innovation. In that context, public research laboratories in Western France

(Brittany and the Loire Valley) and the top ten dairy companies have decided to work

together within the framework of a project known as PROFIL (PROtéines Fonctionnalisées pour l’Industrie Laitière, i.e., functionalized proteins for the dairy industry), presented in

Figure I-1. Through scientific research results and advances implemented at the industrial

level, the finality of the PROFIL project is to provide added value to dairy products by

means of innovative functionalities from milk proteins. Among milk proteins, particular

focus is placed on whey proteins. Many of their physicochemical properties and

functionalities are not yet completely understood and explored, especially when these

proteins are used in specific supramolecular assemblies such as fibrils, strands, hydrogels

and microspheres. Four types of properties of these whey protein assemblies are

addressed in this study:

- antimicrobial and antifungal properties,

- emulsifying and foaming properties,

- texturing properties,

- encapsulating properties.


24

Figure I-1: Schematic representation of the PROFIL research project: academic and industrial partners, public financial supports and coordinator, articulated to explore four thematic functional properties of whey proteins.


25

The present study focuses on this latter property to explore the potential of using

whey protein assembly for the encapsulation and vectorization of a specific compound.

Several encapsulation technologies using whey proteins, such as spray-drying and

hydrogel formation, have been applied at the industrial scale for decades (C. Onwulata &

Huth, 2009). Globally, the food encapsulation market was estimated at over $3 billion in

2014 and is expected to reach $5 billion by 2020 (Research and Markets, 2015a),

representing great opportunities to confer added value to whey proteins used as

encapsulating materials. Several molecules can benefit from encapsulation. Among them,

bioactive molecules appear to be an essential type of molecule to be encapsulated. A

characteristic of bioactive molecules is that they are very sensitive to their

physicochemical environment and are subject to rapid inactivation or degradation in

ordinary environments. These degradations could lower their biological activities and

their nutritional and potential health benefits. Yet it is essential to maintain their

bioactivity effect, especially when these bioactives are incorporated into food products to

formulate functional foods. Many bioactive food components would therefore benefit

from encapsulation to facilitate their incorporation into a food matrix, protect them

during food processing, reduce their degradation during storage, and preserve them until

their controlled delivery when and where the absorption of the bioactive is desired

(Augustin & Sanguansri, 2015). In this regard, innovative whey protein assemblies to be

used as encapsulating materials have a great application potential.

Of all the types of whey protein (WP) assemblies, WP coacervates resulting from

the complex coacervation of two WP have recently received attention and may constitute

promising encapsulating materials. Complex coacervation is an encapsulation technology

based on the balance of electrostatic interactions between encapsulating materials to

create a supramolecular assembly capable of carrying a specific molecule. Complex

coacervation has been recognized for quite a few years now as an efficient encapsulation

technology (Gouin, 2004). Still, its implementation using a two WP system remains

challenging but promising with regard to its intrinsic advantages and unique properties,

compared to other common encapsulation technologies. WP coacervates are tailored

from natural and edible polymers, without the need to resort to exogenous additives. In

this way, they provide several associated innovations and advantages:


26

There is no need for extended specific research to prove the safety and non-

cytotoxicity of co-assembled milk proteins;

They constitute a relevant alternative for encapsulating materials composed of

lipids, which require the use of organic solvents or surfactants for their fabrication.

They constitute a relevant response to meet the growing demand for additive-free

foods. For example, they can be used as encapsulating materials in infant formulas,

a market with spectacular global growth.

Their reversibility allows controlled disassembly, a fundamental step for targeted

release (Tavares, Croguennec, Carvalho, & Bouhallab, 2014).

Further research is still required for the complete control of WP coacervates as

encapsulating materials for bioactives and the development of their potential use for

natural functional dairy foods and ingredients. The study that is the object of the present

PhD dissertation is a contribution in this direction.

The present dissertation is divided into three sections:

The aim of the Literature Review Section is to present and link the key concepts

of this study, beginning with functional foods and encapsulation, and followed by the

presentation of WP and complex coacervation, in order to highlight the potential of WP

coacervates as new natural encapsulating materials for bioactives that could be further

used for functional food development.

The Results Section is presented in three chapters, each one divided into two

parts.

The first chapter covers the formation of WP coacervates for the encapsulation of

a model bioactive, vitamin B9. The first part focuses on the optimization of beta-

lactoglobulin and lactoferrin complex coacervation in the presence of B9, at the laboratory

scale. This part was published in Food Hydrocolloids (Chapeau et al., 2016). The second

part then explores the scaling-up of the production of WP coacervates for B9

encapsulation at the bench scale. Part of these results were published in the Journal of

Food Engineering (Chapeau et al., 2017)

The second chapter explores the properties of WP coacervates. The first part deals

with the protective properties of WP coacervates to preserve B9 against various

degradations (storage treatments, UV-light radiation and oxidation). Part of these results

were also published in the Journal of Food Engineering (Chapeau et al., 2017). The second


27

part of this chapter then investigates the release property of the coacervates in in vivo

conditions or when incorporated into a dairy matrix. This study is currently under

submission for publication in the Journal of Functional Foods.

The third chapter presents different results in order to promote the use of WP

coacervates as interesting encapsulating agents for further developments.

Finally, the third section presents a General Discussion of the present study,

Conclusions and opens Perspectives.

Literature review

28

II. Literature review

Literature review

29

Milk is by nature our first and most complete food. Through all forms of dairy

products, milk is an important source of dietary energy, protein and fat, providing an

average of 134 kcal of energy/capita per day, 8 g of protein/capita per day and 7.3 g of

fat/capita per day (world averages determined by Food and Agriculture Organization of

the United Nations, FAOSTAT, 2012). In recent decades, technological innovations in the

dairy industry have led to an increasing variety of dairy products with various sensory

and nutritional properties and services for the consumer. Heightened awareness of the

connection between diet and health has led to an increasing demand for certain types of

dairy products such as products enriched with vitamins, minerals and essential fatty

acids. At the same time, the dairy industry is facing new challenges to improve its

profitability and to guarantee the sustainability of the whole dairy chain. One of the

challenges is to create more added-value products using all the various fractions of

processed milk while responding to consumers’ demands for dairy products with enhanced health benefits.

1. Functional foods and encapsulation

1.1. What are functional foods?

To some extent, all foods can be considered as functional foods because all foods

intrinsically provide taste, aroma and nutritive value. Nevertheless, foods are intensively

being examined today for additional physiological benefits such as the reduction of the

risk of chronic disease or the enhancement of overall health (Hasler, 2002). The concept of functional food was first used in in Japan and defined as any food or ingredient that has a positive impact on an individual’s health, physical performance, or state of mind, in addition to its nutritive value. (Hardy, 2000). From this point of view, several

definitions have been added to the concept, with either more emphasis on how the

functional food is developed (addition of a bioactive molecule, selection of specific

nutrients, etc.), or on what the functional food is useful for (providing health benefits,

promoting a specific bioactive action, etc.) (Hasler, 2002). Another term often used in

conjunction with functional foods is nutraceuticals. However, this word seems to be less

favored by consumers since it tends to associate a food with a pharmaceutical treatment

rather than with a daily part of our diet (Annunziata & Vecchio, 2011). Overall, to remain

simple but accurate, a functional food can be defined as any food product providing

Literature review

30

additional or enhanced health benefits beyond its basic nutritional values (Jones & Jew,

2007). It should be noted that this definition applies for the development of a functional

food product as well as for a functional ingredient that can then be incorporated into a

food product or used on its own or in other fields such as pharmaceuticals or cosmetics.

To formulate functional foods or ingredients, a feasible approach is to incorporate

useful bioactive molecules within the food matrix (de Boer, Urlings, & Bast, 2016). These

bioactive molecules include fatty acids, vitamins, peptides, antioxidants and minerals, as

well as living cells such as probiotics. A characteristic of these bioactives is that they are

subject to rapid inactivation or degradation during food processing and storage. In

addition, it might be difficult to directly incorporate some bioactives into a food matrix

due to poor solubility, stability or dispersability issues (de Vos, Faas, Spasojevic, &

Sikkema, 2010). To overcome these limitations, encapsulation is an interesting

technology to make it possible to develop a carrying agent or a delivery system for the

incorporation, protection and controlled release of bioactives into foods (Shimoni, 2009).

1.2. Encapsulation, an essential technology for functional foods

Encapsulation is a process designed to entrap a specific compound (the core

material/bioactive molecule) within a carrier material (the shell material/entrapping

agent or carrier) (Lakkis, 2016). A large variety of encapsulation technologies exists and

in order to define the most suitable for a specific compound, five objectives must be

addressed according to the encapsulation purpose(s) desired (Poncelet, 2006).

Immobilization or entrapment: To limit contact between the core material and

its environment. For example, the encapsulation of bacteria or enzymes can ensure

the preservation of their active property without altering the matrix they are

incorporated into.

Protection: To protect the core material against degradation during processing

and storage. For example, vitamins and polyunsaturated fatty acids can be

degraded by a heat treatment applied during processing or by lengthy contact with

oxygen during storage. In addition, many biological cells are sensitive to shear and

probiotics are very sensitive to extreme gastric conditions.

Controlled release: To create a sustained effect and to control the release at a

specific time for a specific purpose, e.g., a bioactive delivered with well-defined

Literature review

31

kinetics to ensure that it maintains its properties, or the release of a flavor at a

specific time such as during mastication.

Structuration: To allow the best possible handling of the core material. For

example, the conversion of a liquid bioactive ingredient into a powder that might

be dust-free and free-flowing would facilitate its incorporation during the final

stage of production.

Two main types of encapsulating systems must be distinguished: the reservoir

type and the matrix type (Figure II-1). The reservoir type presents a shell around the

encapsulated compound. This type can be referred to as the capsule, single-core, mono-

core or core-shell type. Poly- or multiple-core types with several reservoir chambers in

one particle also exist. In the matrix type, the encapsulated compound is much more

dispersed over the carrier material. It can be in the form of relatively small droplets or

more homogenously distributed over the encapsulating agent. The entrapped compound

can also be present at the surface unless an additional coating is present, creating a

combinative encapsulating system between the reservoir type and the matrix type (Jan

Zuidam & Shimoni, 2009).

Figure II-1 : Schematic representation of encapsulating systems, adapted from (Jan Zuidam & Shimoni, 2009).

Only the spherical shape of an encapsulating system is represented, but other forms exist.

To obtain an encapsulating system, numerous encapsulation technologies exist and the

main ones are presented in Table 1. The choice of a specific encapsulation technology is

determined by several factors such as the expected property of the encapsulating systems,

the property of the compound to be encapsulated, and also depends on the desired

applications, the required cost and production volumes.

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32

Table 1: Overview of encapsulation technologies, adapted from (Jan Zuidam & Shimoni, 2009).

Technology Process steps Morphology Load

(%)

Particle size

(µm)

Spray drying

1. Disperse or dissolve active

in aqueous coating solution

2. Atomize

3. Dehydrate

Matrix 5-50 10-400

Fluid bed coating

1. Fluidize active powder

2. Spray coating

3. Dehydrate or cool

Reservoir 5-50 5-5000

Liposome

entrapment

1. Disperse lipid molecules in

water,

with active agent in lipid or

water

phase

2. Reduce size by high shear

or

extrusion

3. Remove free active (option)

Various 5-50 10-1000

Rapid expansion

of supercritical

fluid (RESS)

1. Create a dispersion of active

and dissolved or swollen shell

material in supercritical fluid

2. Release the fluid to

precipitate

the shell onto the active

Matrix 20-50 10-400

Coacervation

1. Emulsification/ dispersion

of the core in shell material

solution

2. Coacervation by change in

physicochemical conditions or

addition of a second shell

material solution,

3. Gelation / stabilization of

the system

4. Recovering of the

coacervates

Reservoir 40-95 10-800

1. Prepare o/w emulsions

with lipophilic active in oil

phase and

Reservoir 1-90 0.2-5000

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33

Multilayers

emulsions

ionic emulsifiers

2. Mix with aqueous solution

containing oppositely charged

polyelectrolytes

3. Remove excess of free

polyelectrolytes (option)

4. Repeat steps 2 and 3

Co-extrusion

1. Dissolve or disperse active

in oil

2. Prepare aqueous or fat

coating

3. Use an concentric nozzle,

and

press simultaneously the oil

phase through the inner

nozzle

and the water phase through

the

outer one

4. Drop into gelling or cooling

bath

Reservoir 50-60 150 - 8000

For food applications, numerous encapsulating systems with their corresponding

technologies have been developed and an extremely wide variety of functionalities can

now be achieved through encapsulation (Gouin, 2004). Some selected examples of active

compounds that may need to be encapsulated for their use in a functional food or as a

functional ingredient are presented in Table 2.

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34

Table 2: Selection of actives ingredients and their corresponding encapsulation technologies to use in functional food and ingredients for potential health benefits (adapted from ( Augustin & Sanguansri, 2012; D. J. McClements, 2012))

Active

molecules Examples

Possible

encapsulation

technologies

Main advantages

of encapsulation

Functionality into

foods and potential

health benefits

Flavors Citrus oils

Liposome (Taylor,

Davidson, Bruce, &

Weiss, 2005),

Nanoemulsion (David

Julian McClements &

Rao, 2011),

Coacervation (Curt

Thies, 2007).

Facilitate

incorporation in

aqueous medium,

Reduce volatility

and chemical

degradation, Control

flavor release.

Give/enhance

flavors, Increase

flavors shelf life

during process and

storage.

Antimicrobials Essential

oils

Spray drying

(Gharsallaoui,

Roudaut, Chambin,

Voilley, & Saurel,

2007), High pressure

homogenization

(Donsi, Annunziata,

Sessa, & Ferrari, 2011)

Improve matrix

compatibility, Delay

chemical

degradation, Mask

off flavors

Antimocrobial

activity against

bacteria, mould,

yeast, Reducing risk

of cardiovascular

disease (Liu, 2003)

Antioxidants Carotenoids

Oil in water emulsion,

High pressure

homogenization

(Ribeiro, Schuchmann,

Engel, Walz, & Briviba,

2010)

Increase efficacy,

Facilitate

incorporation in

aqueous medium

Protecting

unsaturated lipids

from oxidation,

Natural colorant,

Preventing cell

degradations and

eye diseases

(Krinsky & Johnson,

2005)

Vitamins

Vitamin E /

tocopherol

(lipophilic)

Vitamin B9

/ folic acid

(hydrophili

c)

Liposome,

nanoemuslion

(Gonnet, Lethuaut, &

Boury, 2010),

Hydrogel,

Coacervation (David

Julian McClements,

2015)

Facilitate

incorporation in

aqueous medium,

Prevent degradation

during food process

and storage,

Enhance

bioavailability

Food fortification to

promote a range of

health benefit:

prevent bone

degradation, reduce

cardiovascular

diseases.

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35

Enzymes and

bioactive

peptides

Chole-

cystokinin

Spray drying, Freeze

drying, nanoparticles

by extrusion

(Meesters, 2010).

Prevent degradation

during food process

and storage, Retard

stomach


release profile,

Enhance

bioavailability

Antimocrobial

activity,

Prevent bone

degradation, reduce

cardiovascular

disease.

Probiotics,

Prebiotics

Bacteria

Lactobacilli

Spray drying, Fluid

bed coating,

Coacervation Manojlović, Nedović, Kailasapathy, &

Zuidam, 2010)

Prevent degradation

during food process

and storage, Retard

stomach


release profile,

Enhance

bioavailability

Promoting gut

health, modulation

of gut microflora

(Watson & Preedy,

2015)

For a specific type of bioactive, several encapsulation technologies could be used.

Some of these technologies are relatively straightforward (spray-drying for example),

whereas others require more specific equipment (electric spinning), high energy input

(nano-emulsion by high-pressure homogenization) or the use of a solvent not compatible

with food applications (microencapsulation by acetonitril solvent evaporation) (D. J.

McClements, 2012). Emerging encapsulation technologies attempt to find the best

combination between a simple approach with minimal energy input or equipment

requirements, and the use of a reliable food-grade carrier that provides and enhances

functionality for the protection and controlled release of a specific bioactive like the

development of a particle-filled hydrogel or capsules obtained by complex coacervation

(de Vos et al., 2010; Renard et al., 2002).

1.1. Trends in functional food development

The global market for functional foods is driven by increased awareness among

consumers of the health and nutritional benefits of foods to enhance health and prevent

disease. Modern-day consumers are aware of the importance of a healthy diet and look

for a wider range of food and beverage choices in the market to help them fulfill these

needs (C. I. Onwulata, 2012). An aging population with significant disposable incomes,

mainly present in the developed countries (North America, Europe, South-East Asia, the

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36

Pacific), are the main target for functional foods and nutraceutical product development.

The global functional food market revenue was over $230 billion in 2015, with an annual

average growth of 15% (Research and Markets, 2015b). The global nutraceutical

ingredient market is expected to grow from $23.8 billion in 2013 to $33.6 billion in 2018,

representing a compound annual growth rate of 7.2% (MarketsandMarkets, 2014).

Regarding regulation, functional foods can be regulated as conventional foods,

food additives or ingredients, dietary supplements, nutraceuticals, novel foods, etc. The

classification is generally linked to the final product use and the health claims and

bioactive properties associated to it. Regulation of functional foods and claims is

controlled by several government agencies. For example, the European Food Safety

Authority (EFSA) evaluates proposed health claims for foods or active ingredients, guided

by several food laws related to recommended daily intake requirements and scientific

studies provided by national research institutes. For example, the EFSA recently validated

health claims related to the consumption of walnuts for improved blood circulation, the

antioxidant properties of olive oil for reducing LDL cholesterol, and caffeine for increased

alertness and physical endurance (Augustin & Sanguansri, 2015).

The daily nutritional requirements of a person vary with several factors such as

age, sex, diet, physical activity, etc. The nutraceutical and functional food industry thus

attempts to offer products according to different types of population groups (Augustin &

Sanguansri, 2015). For aging consumers, key products of interest are functional foods that

impact vascular health (e.g., omega-3 fatty acids), immune health (e.g., probiotics), the

aging process (e.g., resveratrol, antioxidants), digestive health (e.g., probiotics, fiber), eye

health (e.g., lutein, zeaxanthin), and bone and skeletal health (e.g., calcium, vitamin D3).

For infants and very young children, products that support brain development (e.g.,

omega-3 fatty acids) and immune health (e.g., probiotics, oligosaccharides) are of the

most interest. For pregnant women, products that ensure fetus development are required

(e.g., vitamin B9, iron). Of the different functional food and beverage products, infant formula is the world’s fastest growing market, followed by energy drinks, pre- and

probiotic yogurts and fortified dairy products (MarketsandMarkets, 2014). These new

trends and market opportunities are of great interest for innovation in the dairy industry

and especially to discover enhanced properties and functionalities for milk components

like whey proteins that have been neglected so far.

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2. Whey proteins, from gutter to gold

2.1. General overview of milk proteins

The overall composition of milk is presented in Table 3. This global composition

can vary depending on the species (cow, goat, sheep), breed (Holstein, Jersey), the

animal's feed, and the stage of lactation. Proteins in milk are divided into two main

categories: caseins and whey proteins, as presented in Table 4. In the present study, whey proteins from cow’s milk were especially studied and used.

Table 3: Milk composition, adapted from (Gaucheron, 2005).

Composition

Water 87.7 %

Lactose 4.9 %

Fat 3.4 %

Protein 3.3 %

Mineral 0.7 %

Concentration (mg/kg) Concentration (mM)

Calcium 1043-1283 26-32

Magnesium 97-146 4-6

Phosphor 930-992 30-32

Citrate 1323-2079 7-11

Sodium 391-644 17-28

Potassium 1212-1681 31-43

Table 4: Main physico-chemical properties of bovine milk proteins.

Proteins Concentration

in milk (g/kg)

Molecular

weight (kDa)

Isoelectric

point

Casein micelle 26 4.6

Caseins

Fractions

s1 10.7 23.6 4.9

s2 2.8 25.2 5.2

8.6 24 5.4

3.1 19 5.6

Whey proteins

-Lactoglobulin 3.2 18.3 5.1

-Lactalbumin 1.2 14.2 4.3

Bovine serum albumin 0.4 66.3 5.0

Lactoferrin 0.1 83 8.5

Lactoperoxidase 78 8

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2.1.1. Caseins

Caseins are the fraction of proteins that precipitate at pH 4.6. They represent 80%

of the total milk protein fraction. These proteins are exclusively synthesized in the

mammary glands, suggesting that one of their biological functions is to provide amino

acids (AA) for the development of the neonate (Muehlhoff et al., 2013). The casein fraction

is composed of four main caseins: s1, s2, , and . These four caseins are unstructured

proteins that can associate in micelles and interact with the calcium phosphate present in

milk due to their capacity to bind divalent and multivalent ions. As a result, caseins allow

milk to be supersaturated in calcium phosphate (Farrell Jr., Malin, Brown, & Qi, 2006) and

are therefore natural carrier materials for these essential nutrients (de Kruif & Holt,

2003). In addition, the use of casein micelles as encapsulating agents for other bioactives

such as vitamins or active peptides has been investigated.

Due to their sponge-like structure consisting of internal cavities connected to each

other and to the porous surface by channels, it has been assumed for a long time that

casein micelles are able to protect and transport molecules of interest (Tavares et al.,

2014). The property of native casein micelles, caseinates and casein fractions to interact with hydrophobic molecules and minerals has been quite thoroughly investigated, as

presented in Table 5.

Table 5: Type of caseins used as vehicles for different ligands, adapted from (Livney, 2010).

Type of caseins Ligands Reference

Casein micelles Flavonoids

Vitamin D2

Lactoferrin

(Sahlan & Pramadewi, 2012)

(Semo, Kesselman, Danino, & Livney, 2007)

(Anema & de Kruif, 2011)

Sodium caseinate Fish oil

Iron

(Day, Xu, Hoobin, Burgar, & Augustin, 2007)

(Sugiarto, Ye, & Singh, 2009)

β-casein Curcumin

Chemotherapic drug (Dimethyl sulfoxide)

(Esmaili et al., 2011)

(Shapira, Assaraf, & Livney, 2010)

Moreover, caseins can be used to encapsulate specific molecules using a bottom-

up approach. In that case, it is the re-assembly of caseinate particles (30-50 nm) that is

used to trigger and encapsulate a bioactive such as vitamin D2 (Semo, Kesselman, Danino,

& Livney, 2007). The re-assembly process was controlled by the addition of potassium

Literature review

39

hydrogen phosphate and calcium chloride, dropped into a solution of sodium caseinate

and tri-potassium phosphate, to obtain casein micelle-like structures with an

encapsulation efficiency for vitamin D2 of approximately 27%. Additionally, caseins can

be used for encapsulation using a top-down approach. In that case, the gelling properties

and the interfacial activity of the caseins are used. For example, the probiotic bacteria,

Bifidobacterium lactis and Lactobacillus paracasei, were encapsulated in a casein gel

obtained either by rennet (Heidebach, Först, & Kulozik, 2009a) or the action of

transglutaminase (Heidebach, Först, & Kulozik, 2009b). More emerging encapsulation

technologies like electro-spraying are also used to create casein nanoparticles to

encapsulate bioactives like vitamin B9 (Pérez-Masiá et al., 2015).

Caseins are mainly responsible for acid and rennet milk gelation during various

dairy processes such as the manufacture of cheese or yogurt. These processes often result

in the separation of the whey from the casein curd. Whey proteins were considered for

decades as a by-product, even a waste, of the dairy industry. It is now relevant to look for

a new way to turn this by-product into a valuable raw dairy material

2.1.1. Whey proteins

Whey proteins represents 20% of the total milk protein fraction (Table 4). They

are mostly globular proteins with a variety of biological functions: the source of essential

amino acids, transport of molecules, co-factors for enzymes, etc. O’Mahony & Fox, .

In contrast to caseins, whey proteins are still soluble after acidification of a raw skim milk

at pH 4.6. Due to the variety that exists among whey proteins, they display several specific

functional and biological properties and their investigation is relatively recent compared

to the quantity of literature that already exists for caseins.

2.2. Structure and functionalities of whey proteins

Whey was prescribed for therapeutic purposes starting with Hippocrates in 430

BC and continuing through the Middle Ages (Tunick, 2008). It has been shown that the

exceptional biological value of whey, especially compared to other common edible

proteins (meat, plants), comes from its amino acid composition, which contains a full

spectrum of the branched-chain amino acids (leucine, isoleucine and valine) required for

tissue growth and repair and metabolic regulation. Whey proteins are also a rich source

of sulfur containing amino acids (methionine, cysteine), playing a crucial role as anti-

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oxidants and in cancer regulation (McIntosh et al., 1998). These high nutritional and

health benefits have made whey proteins the protein of choice for elite athletes and

populations with compromised health, leading the dairy sector to use them as a proper

food ingredient (Smithers, 2008).

A greater understanding of the behavior of whey proteins and their interactions

with other food components (lipids, minerals), and in response to physicochemical

parameters (temperature, pH), has facilitated the development of value-added outlets for

whey, using their large techno-functional properties. The gelling, foaming and emulsifying

properties of whey have been extensively studied in order to use these proteins as agents

for the stabilization and texturization of a numerous variety of food products such as dairy

emulsions (ice cream, cheese spreads), processed baked products (pastries, crackers),

and even soups, salads dressing and meat sausages (C. Onwulata & Huth, 2009). In recent

years, attention has focused on the ability of whey proteins to form edible films or coatings

and to assemble into nanoparticles. This particular functionality is of great interest for the

development of encapsulation technology for the protection and controlled delivery of

specific compounds (Gunasekaran, Ko, & Xiao, 2007), resulting in increasingly

sophisticated food products with enhanced sensory and nutritional qualities to satisfy

consumers and to generate products with high market added value for industry

(Smithers, 2008).

2.2.1. Beta-lactoglobulin

Beta-lactoglobulin (BLG) is the major whey protein in milk, reaching

approximately 50% of the total protein mass of whey. Its concentration in cow’s milk is

approximately 3.2 g/kg. BLG is composed of 162 amino acids, for a total molecular weight

of 18.3 kDa. More than ten genetic variants have been identified for this whey protein.

Variants A and B are the most commonly found variants in cow's milk and are present in

equal proportions. BLG-A and BLG-B differ only by the substitution of two amino acids:

AA64 is an aspartic acid in BLG-A and a glycine in BLG-B, AA118 is a valine in BLG-A and

an alanine in BLG-B. BLG is an acidic protein with an isoelectric point of 5.1/5.2 (for

isoforms A and B, respectively) (Farrell et al., 2004). The denaturation temperature of this

protein is around 75°C and the free cysteine presented in the structure plays a crucial part

in the thermo-stability of the protein.

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BLG belongs to the lipocalin family due to its ability to bind a variety of small

hydrophobic ligands. The quaternary structure of the protein shifts from monomers

(Figure II-2A), dimers and oligomers, depending on the pH, temperature and ionic

strength. The dimer is the prevalent form under physiological conditions (Boland et al.,

2014). While dissociation into monomers is enhanced by low protein concentration, low

ionic strength, extreme pH and temperature increases, octamer formation occurs around

pH 4.6, for temperatures lower than 20ºC (Singh & Havea, 2003). These different states of

association results from a delicate balance among hydrophobic and electrostatic

interactions and hydrogen bonding interactions.

By tuning the state of association of BLG, different functionalities can be achieved

with this versatile whey protein, especially concerning gelling and binding properties (

Onwulata & Huth, 2009). The gelling properties of aggregated BLG by heat denaturation

have been extensively studied with various methods (rheology, infra-red spectroscopy,

electronic microscopy) and for different applications such as dairy product texturization

(Mulvihill & Kinsella, 1987; Schmitt et al., 2009; Vittayanont, Steffe, Flegler, & Smith,

2002). As for binding properties, the 3D structure of the dimeric BLG shows several

potential binding sites for hydrophobic ligands such as fatty acids, vitamins and

polyphenols (Figure II-2B), demonstrating the potential functionality of this whey protein

for a wide range of biological functions (Kontopidis, Holt, & Sawyer, 2004).

Figure II-2: Schematic representation of Beta-Lactoglobulin, (A) Quaternary structure of the monomeric BLG, and (B) Dimeric BLG complexed with retinol. Images created using the data from Protein Data Bank, on line.

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2.2.2. Lactoferrin

Lactoferrin (LF) is a glycoprotein of the transferrin family due to its specific iron-

binding property. It is the major basic protein in cow’s milk, with a pI of 8.3. LF is

composed of 689 AA, organized into two homologous lobes (around 40% of similarity),

for a total molecular weight of 83 kDa (Figure II-3A). Its electric charges are unevenly

distributed on its surface and large positive patches are located on the N lobe and in the

inter-lobe region. LF tends to polymerize especially at high concentrations. As a result,

various polymeric forms of LF exist, ranging from monomers to tetramers (Bokkhim,

Bansal, Grøndahl, & Bhandari, 2013). LF is monomeric at low ionic strengths, and with

increasing ionic strength, LF tends to form dimers and/or aggregates (Baker & Baker,

2009).

Each lobe of the protein presents an iron binding site (Figure II-3B), which requires

the fixation of bicarbonate as a synergist ion for iron binding (Sawyer & Holt, 1993).

According to the level of iron saturation, three different forms of LF exist: the apo form

(iron saturation < 5%), the holo form (fully saturated) and the native form (15-20% of

iron saturation). The iron saturation of LF affects its flexibility and, consequently, its

functional properties. At neutral pH, the apo LF denaturation temperature is around 71°C

vs. 91°C for the holo LF. The native LF presents two denaturation peaks, the major one

corresponding to the apo form, and the small one corresponding to the holo form

(Bokkhim et al., 2013).

LF concentration in milks varies considerably depending on the species. In cow’s

milk, LF concentration is around 0.1 g/kg, whereas it reaches 1 g/kg in human milk. This

protein is therefore of great interest for the development of infant formulae that mimic

human breast milk and its biological properties (Blanchard, Zhu, & Schuck, 2013). LF

biological properties are mainly related to its key role in the development of our immune

system and to its useful antimicrobial activity against bacteria and viruses (Farnaud &

Evans, 2003). LF is also a key factor in the regulation of the anti-inflammatory response

and is a good anti-oxidant and anticarcinogenic molecule (Corredig, 2009). LF has also

been investigated for its binding properties to bioactive molecules such as vitamin B9

(Tavares, Croguennec, Lê, et al., 2015). All these interesting biological properties make LF

as well as BLG good candidates for the development of functional foods.

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43

Figure II-3: Tertiary structure of Lactoferrin, (A) on its native form or (B) binding with two iron atoms (red spheres). Images created using the data from Protein Data Bank, on line.

2.3. Whey proteins for functional foods development

2.3.1. Intrinsic bioactive properties of whey proteins

It has long been recognized that several whey proteins confer non-immune

protection to the neonate against disease. Since the early 1990s, whey proteins have also

been investigated more extensively for other biological and physiological effects such as

anti-cancer activity, anti-inflammatory properties and their influence on digestive

functions (McIntosh et al., 1998). It has been reported that various positive biological

effects could be linked to the antioxidant and detoxifying activity of whey proteins.

Cysteine, found in high proportions in the amino acid contents of whey proteins, presents

an antioxidant thiol group. This thiol group is able to combine with glycine and glutamate

to form glutathione (GSH), the major endogenous antioxidant produced by cells,

providing protection for RNA, DNA and protein oxidation via its redox cycling from GSH

(the reduced form) to GSSH (the oxidized form) (Bayford, 2010; Liska & Bland, 2004).

Other beneficial biological effects of whey proteins are related to their binding properties,

especially for BLG and LF, as already briefly presented in Section 2.1.2. The ability of BLG

to bind important bioactives such as curcumin (Sneharani, Karakkat, Singh, & Rao, 2010),

essential fatty acids (Loch et al., 2013) and polyphenols (Zorilla, Liang, Remondetto, &

Subirade, 2011) is of great interest to ensure the delivery of the enhanced health benefits

Literature review

44

provided by these bioactives. In addition, due to its ability to chelate iron, it presents good

anti-microbial properties since the micro-organisms requiring iron to replicate would

probably be vulnerable to lactoferrin’s effects. Lactoferrin also demonstrates an ability to stimulate immune responses by stimulation of macrophage cytotoxicity and may play a

key role in tumor cell regulation (Bayford, 2010; Farnaud & Evans, 2003). As a result,

whey proteins intrinsically present some bioactive properties that make them good

candidates to formulate functional dairy products with interesting health-enhancing

benefits. Additionally, an interesting feature of whey proteins is that they can also be used

as encapsulating agents for the protection and controlled delivery of a wide range of

bioactives, turning them into promising functional ingredients for the development of

novel functional foods (Tavares et al., 2014).

2.3.1. Whey proteins as encapsulating agents

Encapsulation of bioactives using whey proteins as encapsulating agents can be

achieved by top-down or bottom-up approaches. For top-down approaches, three main

strategies exist: protein cold gelation, emulsions or a combination of these two strategies

using extrusion techniques. In the case of emulsions, the whey proteins are often used to

stabilize the encapsulating system formed by the emulsified droplets entrapping the

bioactive, e.g., to stabilize oil-in-water emulsions to produce encapsulating systems

capable of protecting and transporting beta-carotene (Cornacchia & Roos, 2011) or dairy

emulsions for the fortification of cheese with vitamin D3 (Tippetts, Martini, Brothersen, &

McMahon, 2012). The cold-set gelation of BLG has been especially studied for the

encapsulation of iron, either by the protein gel itself (Remondetto, Paquin, & Subirade,

2002) or by the whey protein gel fragmented into nanoparticles (Martin & Jong, 2012).

Cold-set gelation was also used to encapsulate microorganisms of interest like probiotic

bacteria (Ainsley Reid et al., 2005) and recombinant yeast (Hébrard et al., 2006). For the

bottom-up approach, thermal aggregation and desolvation are the main strategies for the

production of whey nanoparticles used as carrying agents for bioactives. Nanoparticles

obtained from heat-induced aggregates of whey proteins or BLG itself have been

developed for the encapsulation of different bioactives like antioxidants (Li, Du, Jin, & Du,

2012), vitamins (Relkin & Shukat, 2012) and hydrophobic aromas (Giroux & Britten,

2011). Desolvation has been investigated on bovine serum albumin for the production of

nanoparticles to encapsulate flavonoids (R. Fang et al., 2011) and on BLG to encapsulate

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45

curcumin (Sneharani et al., 2010). A special effort has also been made to develop this

approach using only food-grade solvents, e.g., the encapsulation of zinc using the

desolvation of whey protein isolate by ethanol (Gülseren, Fang, & Corredig, 2012).

Consistent with these classical approaches to encapsulation, whey proteins have

also been investigated for their ability to spontaneously co-assemble or self-assemble into

supramolecular structures that can potentially be used for the encapsulation of bioactives

(Bouhallab & Croguennec, 2014; Nicolai, Britten, & Schmitt, 2011; Tavares et al., 2014).

The supra-molecular structures are tunable with concentration, pH, temperature or ionic

strength variations, inducing changes in protein conformation. Thus, the final

supramolecular structures obtained differ in geometry, size and porosity. Whey proteins

have been reported to generate fibrils (Loveday, Wang, Rao, Anema, & Singh, 2012),

ribbons (Lara, Adamcik, Jordens, & Mezzenga, 2011) and spherulites (Domike, Hardin,

Armstead, & Donald, 2009), nanotubes (Graveland-Bikker & de Kruif, 2006) and

microspheres (Nigen, Croguennec, & Bouhallab, 2009). These whey protein microspheres

are obtained through the complex coacervation of whey proteins. In that regard, complex

coacervation reveals several interesting characteristics to formulate natural carrying

agents using whey proteins for the encapsulation of bioactives (Bouhallab & Croguennec,

2014; Hosseini, Emam-Djomeh, Sabatino, & Van der Meeren, 2015; Joye & McClements,

2014; Schmitt, Aberkane, & Sanchez, 2009; Turgeon, Schmitt, & Sanchez, 2007).

Finally, the interaction between one whey protein and a ligand leading to the

formation of complexes can be considered as a type of encapsulation. In that context, the

ability of BLG to bind different fatty acids has been quite well investigated. As it has

already been shown, the globular structure of BLG allows the natural binding of

hydrophobic ligands inside its internal calix. The specific interaction between the protein

and some hydrophobic fatty acids such as palmitic acid induces a protective effect against

fatty acid hydrolysis. Additionally, the interaction of BLG with fatty acids can modify the

bioaccessibility of the ligand, generally by enhancing its overall solubility (Le Maux,

Bouhallab, Giblin, Brodkorb, & Croguennec, 2014). Nevertheless, these types of

interactions appear to be rather specific and highly dependent on the ternary and/or

quaternary structure of the protein. For example, it has been observed using fluorescence spectroscopy that the apo form of -lactalbumin is able to bind to oleic and palmitic acids,

whereas no interactions were observed for holo -lactalbumin. This demonstrates that

the conformational change induced in -lactalbumin by the removal of calcium enables

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46

the protein to interact with fatty acids (Barbana et al., 2006). It should be noted that many

of these protein-ligand complexes already preexist in food. In fact, these complexes are

often responsible for the bioactivity and functional health benefits attributed to the food.

For instance, it has been hypothesized that osteopontin, a phosphoprotein present at

concentrations ten times higher in human milk than in bovine milk, transports immune-

modulating and antimicrobial proteins to their sites of activity. This bioactivity could be

linked to its potentiality to form nanocomplexes with lactoferrin (Bouhallab, Lopez, &

Axelos, 2017). As a result, these structures act as natural encapsulating agents or carriers

in foods, demonstrating the interesting potentiality to control their assembly to formulate

natural functional encapsulating agents, based on their intrinsic properties.

3. Complex coacervation, theory and practical use

3.1. Principles of complex coacervation

Complex coacervation is a liquid-liquid phase separation occurring between two

oppositely charged biopolymers and generally driven by electrostatic interactions. It

generates the formation of two separated liquid phases: the dilute phase, rich in solvents

and poor in biopolymers, and the dense phase, concentrated in biopolymers, namely the

complex coacervates (Schmitt et al., 2009). After the pioneering work initiated by

Bungenberg de Jong on the complex coacervation between gelatin and acacia gum

(Bungenberg de Jong, 1949), other theoretical studies have been conducted, with the aim

to model and explain this particular phenomenon.

Firstly, Voorn and Overbeek (Overbeek & Voorn, 1957) explained the complex

coacervation by the competition between attractive electrostatic forces (enthalpic effects)

that tend to bring the two biopolymers closer and the entropic effects that tend to distance

them. In this model, the resulting coacervated phase is liquid and potentially reversible.

Later, Veis and Aranyi (Veis, Bodor, & Mussell, 1967) described the complex coacervation

as a two-step phenomenon rather than a spontaneous one: first, (i) the spontaneous

formation of heterocomplexes by electrostatic interactions of low entropy; and then (ii)

the rearrangement of these heterocomplexes by the conformational entropy gain, leading

to the formation of the coacervates. These two steps are a function of pH, with two critical

pH values: pHc for the formation of soluble heterocomplexes, and pHФ for the formation

of coacervates (Figure II-4A) (de Kruif, Weinbreck, & de Vries, 2004). These two main

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models were further supplemented by other studies that include the effect of the

solute/solvent interaction initially neglected in the model of Voorn and Overbeek (Sato &

Nakajima, 1974) or that take into account the entropic contribution of counterions

(Tainaka, 1980) for the final formation of coacervates into account (Figure II-4B).

Beyond these theoretical models, numerous experimental studies have mainly

focused on specific biopolymer systems (e.g., gelatin with carrageenan, chitosan, alginate,

pectin, etc.) and attempted to determine the optimal parameters of coacervation for each

system studied (Turgeon et al., 2007). Globally, due to the electrostatic nature of the

driving forces, the main parameters influencing complex coacervation are (i) related to

the inherent properties of the two biopolymers (molecular weight, charge density,

biopolymer ratios), and (ii) related to the physicochemical properties of the media (pH,

ionic strength, temperature) (Figure II-4C). After an associative phase separation and the

resulting coacervation, the concentrated dense phase of coacervates can be recovered by

centrifugation (Figure II-4D). By tuning and controlling the optimal parameters for

complex coacervation, the resulting coacervates can be used for various applications.

More recently, the development of new theoretical tools to understand the physical

state of polyelectrolyte coacervates has tended to propose a unified model of

coacervation, bridging the gap between theoretical and experimental observations. A

detailed review of these new tools by Sing (2017) demonstrates that while a patchwork

of several model theories collectively captures the main features observed in complex

coacervation, there is no single theory capable of simultaneously addressing its full

spectrum of features. Ideally, such a theory would be able to predict coacervate properties

over a broad range of parameters and length scales, from a molecular level to a

macroscopic one, in order to completely master complex coacervation phenomena. This

would be very useful for further uses of coacervates and their applications.

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Figure II-4: Schematic representation of complex coacervation, (A) Model of the formation kinetic of coacervates between two oppositely charged biopolymers, (B) Transmission-mode CSLM micrograph of beta-lactoglobulin/acacia gum coacervates undergoing coalescence at pH 4.2,

mixing ratio 2:1 and a total biopolymer concentration of 1 wt.%. Scale bar is m, from (C. Schmitt & Turgeon, 2011), (C) Schematic steps and parameters influencing the complex

coacervation of two oppositely charged biopolymers, (D) Coacervates/supernatant after centrifugation of coacervates suspension, System: BSA-F (bovine serum albumin labeled with fluorescein isothiocyanate) + poly(diallyldimethylammonium chloride), at pH 9.5, I=0.1 M NaCl, from (Kizilay, Kayitmazer, & Dubin, 2011).

3.1. Applications of complex coacervation

One of the interesting features of complex coacervation is that this particular

phenomenon is present in many biological processes without being recognized as such,

including various antibody-antigen reactions, protein transcriptions and or enzymatic

pathways (Turgeon et al., 2007). For example, when tasting a good white wine, the ionic

interactions between the salivary proteins and the wine pectin polysaccharides induce

the formation of complex coacervates between the two biopolymers. This leads to the

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decrease of protein–tannin interactions and could contribute to lowering the astringency

of the wine (Carvalho et al., 2006).

Consistent with these biological aspects, complex coacervation has found

numerous industrial applications in pharmaceuticals, cosmetics, foods and even in the

paint and dying industry. The development of complex coacervation for industrial

applications has been subject to growing interest since 1980. From this moment,

numerous studies have been reported in the scientific literature to better understand the

phenomenon in order to optimize coacervate formation for a specific purpose. Complex

coacervates have been used as additives in food processing (Schmitt & Turgeon, 2011)

and personal care products such as encapsulants (Matalanis, Jones, & McClements, 2011),

emulsifiers (Jourdain, Leser, Schmitt, Michel, & Dickinson, 2008), and viscosity modifiers

(Weinbreck, de Vries, Schrooyen, & de Kruif, 2003). Coacervates can also be used to

immobilize enzymes, drug delivery, the design and the production of new biomaterials,

and protein purification (Kizilay, Kayitmazer, & Dubin, 2011). As proof of the success of

these applications, numerous patents exist involving the production of coacervates. More

than 30,000 have been registered so far, most of them owned by the Procter & Gamble

Company, with 17% of the total patents dealing with coacervation (Google Patents

Library, 2017). Recent research into new applications for complex coacervation tend to

go back to the roots of this biological material, with analogues to coacervation found in

underwater adhesives (Stewart, Wang, & Shao, 2011), membrane-less organelles

(Brangwynne, 2013) and even proto-cells meant to reproduce the fundamental aspects of

living systems (Koga, Williams, Perriman, & Mann, 2011).

Among these various industrial applications, encapsulation using complex

coacervation has received increasing attention since the early 1970s, first from scientists,

to optimize the process (Arshady, 1990), and then from different industries, to adapt it,

e.g., like the pharmaceutical industry for drug delivery, the cosmetics industry for the

addition of specific compounds to a complex matrix, and the food industry for the

stabilization, protection and controlled delivery of bioactives.

3.2. Encapsulation by complex coacervation

The use of complex coacervation for encapsulation purposes was first revealed in

1957 by Green and Schleicher, using a system of pork skin gelatin and gum arabic as

encapsulating agents. Since the concept has been known for decades, this field of research

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has been the object of renewed interest over the past ten years, with the number of

scientific publications multiplied by five for studies on encapsulation and coacervation. It

can also be observed that articles in this field that include protein as their topic of study

are on the rise (Figure II-5).

Figure II-5: Scientific publications from 2007 to 2017 in the fields of (Encapsulation + Coacervation) or (Encapsulation + Coacervation + Protein), according to key words research on Web of Science©, June 2017.

Several of these studies have demonstrated some interesting features for using complex

coacervation as an efficient encapsulation technology (C. Thies, 2012). This technology is

able to produce large ranges of capsules or carriers with different properties depending

on the entrapping/shell material (size, dispersity, stability). The encapsulation efficiency

(payload) achievable has now reached 95% and many controlled release possibilities

exist based on mechanical stress, temperature or sustained release (Gouin, 2004).

The formation of carriers or encapsulating systems using complex coacervation is

based on two strategies: to form a capsule around the targeted molecules (reservoir type)

or to create complexes called coacervates that will fix or entrap the targeted molecule

(matrix type). Many procedures exist in the literature to encapsulate by coacervation. A

general and schematic procedure is presented in Figure II-6. Globally, the encapsulation

using the complex coacervation of two oppositely charged biopolymers generally uses a

four-step process. First, the core molecule to be entrapped (bioactive, for example) is

0

50

100

150

200

250

300

2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017

Pu

bli

cati

on

s

Encapsulation + Coacervation Encapsulation + Coacervation + Protein

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incorporated within a first biopolymer solution (1); next, a second biopolymer solution of

opposite electric charges is added to the system, leading to the formation of the coacervates (2), the encapsulating system formed by complex coacervation between the

carriers and the entrapped core molecules. Further steps can be added to stabilize (3) and

recover (4) the coacervates.

Figure II-6: Procedure of encapsulation by complex coacervation illustrated by schematic diagram (A) or schematic design (B).

This procedure was highly developed using gelatin and/or gum arabic as the main

carrier material to encapsulate various bioactive compounds such as flavors, essential

oils, bioactive peptides or drugs (Arshady, 1990). Over the past 20 years, innovative

research has been initiated to adapt this technology to new carrier materials other than

pork skin gelatin, which tends to be poorly perceived by consumers. This has led to

several studies using the complex coacervation of milk, egg, plant and even silk proteins

for coacervation with various anionic polymers ( de Kruif et al., 2004), resulting in the

adaptation of the traditional procedure of encapsulation by coacervation to new emerging

procedures, more suitable for food industry applications. Table 6 compares the

differences between traditional and emerging procedures for the four main steps of

encapsulation by coacervation. Emerging procedures are designed to control energy

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input (complexation rather than emulsification, spontaneous coacervation rather than

induced), to limit the use of aggressive solvents or non-food-grade material

(glutaraldehyde).

Table 6: Comparative characteristics of principles steps of complex coacervation for encapsulation of traditional vs emerging procedure.

Generic steps of

coacervation for

encapsulation

Traditional procedure Emerging procedure

Preparation Emulsification Complexation

Coacervation Induced / Forced : lowering pH, increased ionic strength,

Spontaneous

Stabilization

Gelation : Thermally induced (cooling, protein heating denaturation) Cross linking with glutaraldehyde

Rather unstable in solution Reversible phenomenon Cross linking with transglutaminase

Separation/

isolation

Washing with solvent, Spray drying Freeze-drying Fluidized bed

Thermodynamic equilibrium, dense phase density Induced by centrifugation Possible spray or freeze drying

To develop these emerging procedures, many systems have been specifically

studied. However, the knowledge of each type of system varies. The complex coacervation

between polyelectrolytes, polysaccharides, proteins-polyelectrolytes, and proteins-

polysaccharides is now well understood and described, both from a theoretical point of

view (Bouhallab & Croguennec, 2014; Kizilay et al., 2011) and from an applicative one

(Schmitt & Turgeon, 2011). On the contrary, coacervation using two different proteins

(heteroprotein system) has been much less studied in the literature as of this time, and

even less for encapsulation purposes.

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4. Heteroprotein complex coacervates

4.1. Specificity of heteroprotein coacervation

Heteroprotein complex coacervation (HPCC) is a special case of complex

coacervation where the two biopolymers used to form the coacervates are two different

proteins with opposite electric charges. Due to the unique physicochemical properties of

proteins, HPCC presents some specificities that need to be highlighted. First, proteins

share common properties with amphiphilic molecules due to the chemical properties of

their elementary building units, the amino acid chains. Consequently, a wide variety of

proteins present a heterogeneous distribution of electric charges at their surface This

induces a specific surface anisotropy at the molecular level, enabling proteins to interact

with molecules of different natures (i.e., hydrophobic, hydrophilic and/or charged

molecules, etc.) (Bouhallab & Croguennec, 2014). Since complex coacervation generally

results from electrostatic interactions, the anisotropy of charges at the protein surface

constitutes the main driving forces for HPCC, and the formation of the coacervates results

from a specific balance between short-range and long-range coulombic interactions of the

heteroprotein system. Otherwise, proteins either form an amorphous precipitate and

aggregates or remain in solution (Croguennec, Tavares, & Bouhallab, 2016). HPCC can

only occur within a very narrow range of pH, generally between the isoelectric point of

the two proteins, and is very sensitive to ionic strength, protein concentration and protein

stoichiometry in order to respect the charge and size compensation principle required for

coacervation (Desfougeres, Croguennec, Lechevalier, Bouhallab, & Nau, 2010). The

dynamics and kinetics of HPCC are still not fully understood but most of authors described

a mechanism by nucleation to form primary units (droplets) of heteroprotein complexes,

followed by the growth and coalescence of these units (droplet collision and fusion) to

form the coacervates (Anema & de Kruif, 2011; Salvatore, Croguennec, Bouhallab, Forge,

& Nicolai, 2011). Determining the conditions that give rise to HPCC and understanding

the key parameters governing the interactions between the proteins is the first step

towards the use of a heteroprotein system as an encapsulating agent, and especially using

a food protein-based system.

Food proteins are generally recognized as safe by the food industry (GRAS),

making them good candidates to be used as encapsulating agents (Chen, Remondetto, &

Subirade, 2006). Over the years, different food protein systems have been investigated for

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HPCC. This requires the use of an acidic protein and a basic protein that can either

originate from animal sources, such as milk proteins (caseins, whey proteins) or egg

proteins (lysozyme or ovalbumin), or plant sources such as soy proteins or pea proteins,

for example. Due to their biological sources, these food proteins can present a wide

diversity and variability in their compositions and structures, representing another

challenge when using these types of proteins for HPCC (Matalanis et al., 2011). Among the

heteroprotein systems investigated, those involving lysozyme as the basic protein have

been quite thoroughly studied, especially to approach the first step of the coacervation

process. For example, it has been observed that equimolar mixing at pH 7.5 of hen egg

white lysozyme and bovine calcium-depleted -lactalbumin leads to the rapid self-

association of the oppositely charged proteins into small aggregates. On larger time scales,

these aggregates rearrange themselves into irregularly shaped flocs at 25°C, or well-defined spherical particles ≈ µm at °C (Salvatore et al., 2011). Furthermore,

turbidity experiments and microscopic observations revealed that the formation of

micro-spherical particles was destabilized by the addition of either NaCl, MgCl2 or CaCl2,

and totally suppressed at 100 mM of ionic strength (Nigen, Croguennec, et al., 2009).

Complementary studies using fluorescence techniques at the molecular level revealed that it was both the contribution of -lactalbumin destabilization and -lactalbumin

interactions with lysozyme to form heterodimers that were required to form the micro-

spherical particles (Nigen, Le Tilly, Croguennec, Drouin-Kucma, & Bouhallab, 2009). By

comparing these results with other protein systems, it seems to appear that regardless of

the acidic protein used (beta-lactoglobulin, lactalbumin, bovine serum albumin, etc.),

protein surface charge compensation and size compensation appeared as the main

mechanisms governing HPCC (Croguennec et al., 2016). This influences the possibility of

using a defined heteroprotein system for the encapsulation of a targeted molecule by

complex coacervation. Nevertheless, due to the specificity of heteroprotein interactions,

the experimental conditions for HPCC remain highly dependent on the heteroprotein

system involved. This represents a challenge for using HPCC as an encapsulating

technology for food applications, and each heteroprotein system should be investigated

individually.

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4.2. Special case of Lactoferrin and Beta-Lactoglobulin

Recently, the two whey proteins Lactoferrin (LF) and Beta-lactoglobulin (BLG)

have been specifically studied for their complex coacervation ability, principally by three

research groups within the scientific community: Dubin, Schmitt et al. (Flanagan et al.,

2015; Kizilay et al., 2014; Yan et al., 2013), Anema and De Kruif (Anema & de Kruif, 2014),

and Bouhallab et al. (De Sa Peixoto et al., 2016; Tavares, Croguennec, Hamon, Carvalho, &

Bouhallab, 2015). These studies explored some general aspects about the conditions of

coacervation between the two proteins, the physicochemical conditions required and the

potential mechanisms for the formation of the final LF-BLG coacervates. As expected, the

formation of LF-BLG coacervates was found to be highly dependent on pH, ionic strength,

total protein concentration and LF:BLG stoichiometry (Yan et al., 2013). LF-BLG

coacervation can only occur within a narrow range of pH, close to the pI of the BLG.

However, the optimal range of pH for coacervation varied according to the studies: 5.7 to

6.2 for Yan et al. (2013), 5.0 to 7.3 for Anema and De Kruif (2014), and 5.5 to 6.0 for

Tavares et al. (2015). This is probably due to the nature and characteristics of the whey

proteins: proportions of isoforms of BLG A and BLG B, iron saturation of LF, presence of

denatured/aggregated species (Tavares, Croguennec, Hamon, et al., 2015). For all studies,

LF-BLG coacervation only occurred at low ionic strengths, I < 25 mM for Yan et al. (2013)

and Tavares et al. (2015), and I < 100 mM for Anema and De Kruif (2014). Regarding whey

protein concentrations and stoichiometry, LF-BLG coacervation occurred at medium

protein concentrations (10 to 50 g/L) and for a LF:BLG molar ratio varying from 1:1 (Yan

et al., 2013) to 1:10 (Tavares, Croguennec, Hamon, et al., 2015). The full formation

mechanism of LF-BLG coacervates is not totally understood yet. However, it appeared that

the formation of a primary entity of LF-(BLG2) seemed to be a prerequisite, either by the

interaction of LF with the dimeric form of BLG (Kizilay et al., 2014; Yan et al., 2013) or by

an initial binding of two BLG monomers on two distinct sites of LF (Tavares, Croguennec,

Hamon, et al., 2015). These LF-(BLG2) units would form the primary unit blocks that

would then reassemble for the formation of the final LF-BLG coacervates (Figure II-7).

It can be observed that the overall formation of LF-BLG coacervates is only possible

with a large excess of BLG compared to LF. An excess of LF would instead lead to aggregate

formation (Tavares, Croguennec, Hamon, et al., 2015). In addition, the initial mixing ratio

of LF and BLG solutions and the protein concentrations greatly influence the final ratio of

LF:BLG recovered in the coacervates, as can be seen in Figure II-8.

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Figure II-7: Proposed mechanism for the formation of LF-BLG coacervates, adapted from (De Sa Peixoto et al., 2016; Tavares, Croguennec, Hamon, et al., 2015b)

Figure II-8: Fraction of LF (%) recovered in LF-BLG coacervates depending on the initial BLG:LF molar ratio, at pH 5.50 in 10 mM MES buffer at 25°C, adapted from (Tavares, Croguennec, Hamon, et al., 2015b).

As a result, all of these studies make it possible to explore and determine the

optimal conditions for complex coacervation between LF and BLG. Nevertheless, the

presence of a third molecule could modify the HPCC process and, thus, the use of LF-BLG

coacervates as encapsulating agents still remains a challenge.

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4.3. Heteroprotein complex coacervates for encapsulation of bioactives

4.3.1. Actual trends

Although many studies have been devoted to the encapsulation of bioactives by

protein-polysaccharide coacervation, very few papers have dealt with the use of

heteroprotein (protein-protein coacervates) to encapsulate a third molecule and, on top

of that, a bioactive. The Canadian research group of Subirade et al. has investigated in

detail BLG-lysozyme coacervates for the encapsulation of the lipophilic vitamin D3, chosen

as a model bioactive. Their work has shown that BLG-lysozyme coacervates can

encapsulate vitamin D3 with an encapsulation efficiency of 90.8 ± 4.8% (Diarrassouba,

Remondetto, et al., 2015). Moreover, they have shown that BLG-lysozyme coacervates

could effectively protect vitamin D3 against degradations during long-term storage at 4°C

and against photodegradations. Finally, in vivo studies on rats have demonstrated that

BLG-lysozyme coacervates improved the bioavailability of vitamin D3 compared to

vitamin D3 administrated alone (Diarrassouba, Garrait, et al., 2015). To our knowledge,

this is the only study that focuses on an applicative use of heteroprotein coacervates for

the purpose of encapsulation. Nevertheless, several authors have indicated that this new

research area of controlled co-assembly between mixed proteins should offer new

opportunities to develop innovative, reversible and versatile encapsulating agents for

bioactive and sensitive ingredients (Tavares et al., 2014)

4.3.1. Vitamin B9 as a model bioactive for encapsulation

Vitamin B9 (B9), also known as folate or folic acid, is one of the eight B vitamins.

This vitamin has been identified as being part of several important biochemical processes

in human metabolism. B9 is involved in DNA synthesis and cell multiplication, and

especially in red and white blood cell production, renewal of skin and intestinal cell

epithelium, as well as in the synthesis of chemicals that modulate brain function

(Herrmann & Obeid, 2011; Lucock, 2000). Moreover, B9 has been recognized to provide

several health benefits such as preventing cardiovascular diseases (Adank, Green, Skeaff,

& Briars, 2003), colon cancer (Sanjoaquin, Allen, Couto, Roddam, & Key, 2005) and

congenital malformations during pregnancy (Lucock, 2000; Moat et al., 2004).

Nevertheless, humans cannot synthesize B9 and therefore must find sufficient sources of

B9 in their daily diet (Basset, Quinlivan, Gregory, & Hanson, 2005). Rich dietary sources

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of B9 include animal offal such as kidney or liver, some leguminous plants like lentils and

sunflower seeds, and green vegetables like spinach. The recommended daily intake of B9

varies with the sex and age of the individual. According to ANSES (French National Agency

for Public Health, Food Safety and Environment) guidelines, recommended daily B9

intake is approximately 30 g for adults, to g for children, and up to g for vulnerable populations such as elderly and pregnant women. Regarding this

recommended intake, it has been found that B9 deficiency is relatively common among

vulnerable populations (Mills & Signore, 2004) and especially among pregnant women,

which may represent a risk for the neonate since B9 plays a key part in the neuronal

development of the fetus As a result, there is the need to develop foods and drinks

enriched in B9 (Gregory, 2001), and a recent INSERM (French National Institute of Health

and Medical Research) study prescribes a mandatory supplementation of B9 in some

staple foods such as dairy products (Khoshnood et al., 2015).

Vitamin B9 is a pterylmonoglutamic acid, formed by a pteridine residue, attached

to a molecule of P-aminobenzoylglutamic acid (Figure II-9). B9 belongs to the water-

soluble family of vitamins, whereas it presents a low water solubility of 1.6 mg/mL at 20°C

(Merck Index) and is poorly soluble in acidic aqueous media (Lucock, 2000). Hence, to

successfully enrich acidic food products with B9, the design of specific carriers or

encapsulating agents is required. Over the past decade, there have been major advances

in the development of encapsulating agents and carriers to encapsulate, protect and

release lipophilic bioactives such as fatty acids, polyphenols, flavor compounds and oil-

soluble vitamins (Champagne & Fustier, 2007; de Vos et al., 2010; Z. Fang & Bhandari,

2010; Matalanis et al., 2011; David Julian McClements, Decker, & Park, 2009; Xiao, Liu,

Zhu, Zhou, & Niu, 2014). Nevertheless, the design of encapsulating agents and carriers for

hydrophilic bioactive compounds like B9 has been the subject of little attention and

remains rather specific (He et al., 2015; Liang & Subirade, 2010; Pérez-Masiá et al., 2015).

This is considered a limitation for new product development since encapsulating agents

for hydrophilic bioactives are required in many foods and other industrial applications

such as masking the off-taste of bitter peptides, protecting water-soluble colors or flavors

during storage, improving the bioavailability of water-soluble vitamins, nutraceuticals

and pharmaceuticals, and delivering hydrophilic bioactives to particular regions of the

gastrointestinal tract (McClements, 2015).

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Figure II-9: Chemical composition and representation of vitamin B9

4.3.2. Interactions between vitamin B9 and beta-lactoglobulin or lactoferrin.

The binding of vitamin B9 has been investigated with some food proteins, mostly

for the formation of carriers for the protection and controlled delivery of B9. These

studies are relatively recent. For example, the ability of egg white proteins to bind vitamin

B9 and protect it in the gastrointestinal tract has been investigated. It has been

demonstrated that the formation of B9-egg white nanoparticles can be beneficial for the

transport and release of vitamin B9 after in vitro digestion (Arzeni, Pérez, LeBlanc, &

Pilosof, 2015). Regarding the binding of vitamin B9 with whey proteins, very few studies

exist. For BLG, it has been demonstrated using fluorescence spectroscopy analysis that B9

can bind to the hydrophobic pocket of BLG, in the groove between the α-helix and the -

barrel (Liang & Subirade, 2010, 2012; Zhang, Liu, Subirade, Zhou, & Liang, 2014).

Moreover, in vitro digestion studies have shown that the proteolysis of pure BLG and that

of BLG-B9 nano-complexes showed no differences during gastric and duodenal digestion,

implying no deleterious effects on the bio-accessibility of the protein-based complex

during digestion (Pérez, David-Birman, Kesselman, Levi-Tal, & Lesmes, 2014). However,

this binding interaction seems only possible at neutral pH and is hindered by acidification

of the media (Liang & Subirade, 2012).

Regarding LF, a recent study by the research team of Bouhallab et al. has

determined that vitamin B9 can interact with LF to form LF-B9 complexes (Tavares,

Croguennec, Lê, et al., 2015). Using isothermal titration calorimetry, this study

demonstrated that B9 interacts with LF at pH 5.5 to form stable, slightly positively-

charged nanoparticles. This study explained the formation of B9-LF complexes by a two-

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step mechanism: first, B9/LF interactions, followed by self-association of the complexes

thus formed. It was calculated that ten B9 molecules bind to LF to form saturated reactive

complexes (B910/LF) that further self-associate into aggregates with a finite size of

approximately 15 nm (Figure II-10).

Figure II-10: Mechanisms of specific binding of vitamin B9 by LF to form B9-LF nanocomplexes explored through Isothermal Titration Calorimetry (ITC) signal ΔH for B -LF interactions in kcal mol-1) and size particles (Dh in nm) according to B9/LF ratio, adapted from (Tavares, Croguennec, Lê, et al., 2015).

4.4. Research objectives and strategy

Based on current knowledge about LF and BLG coacervation, the present study

aims at addressing two research objectives:

- to investigate the complex coacervation of LF and BLG in the presence of a

bioactive molecule for its encapsulation;

- to explore the potentiality of using LF-BLG coacervates as good encapsulating

agents for bioactives regarding different encapsulation properties such as protection and

delivery.

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To carry out that study, vitamin B9 was chosen as the model bioactive for different

reasons. Firstly, vitamin B9 represents an essential bioactive that needs to be delivered

by food. However, its solubility in an acidic food matrix may be a limitation. Secondly,

vitamin B9 is known to interact with LF to form B9-LF complexes. It thus appears to be an

interesting starting point to develop B9-LF-BLG coacervation based on this fact. Finally,

vitamin B9 is a hydrophilic bioactive with interesting laboratory features such as

manageable preparation protocols, various dosage techniques and a relatively low cost,

making the vitamin an interesting model bioactive for that study.

After the choice of the model bioactive, it was decided to test B9 encapsulation by

LF and BLG coacervation according to a specific procedure and order. Indeed, in the

literature, it was reported that for encapsulation by coacervation, the core molecule can

first be incorporated into the first biopolymer solution. Based on previous results of our

research group that established B9/LF ratios for the formation of B9-LF nanoparticles

(Tavares, Croguennec, Lê, et al., 2015), it was decided to first incorporate the B9 solution

into the LF solution to form B9-LF complexes. BLG solution was then added to the system.

In the present study, all the coacervation tests were performed in that order. Since it

appears to be more relevant for B9, it can be considered that this order may not be the

optimal one for other bioactives, and the order of addition and mixing of all components

may vary according to different coacervation procedures linked to the physicochemical

properties of the bioactive (water or oil solubility, volatility, etc.) (Garti & McClements,

2012).

In the present study, several aspects of encapsulation by coacervation have been

investigated: first, the intrinsic process of encapsulation by coacervation (conditions and

scale production), then the properties conferred by the coacervates for the protection and

release of the bioactives and, finally, an approach that attempts to generalize and apply

the use of WP coacervates. To clarify how the overall study and the present manuscript

are constructed, Figure II-11 presents the main research lines (purple boxes), how they

have been organized for scientific publications (yellow boxes), and how they were

presented in the former thesis (blue boxes) to build a consistent scientific message

according to the initial research questions and objectives.

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Figure II-11: Lines of research, thesis chapters and subsequent scientific publications.

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III. Results

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1. Chapter 1:

Production of vitamin B9-WP coacervates

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65

1.1. Part 1: Formation of Lactoferrin and Beta-Lactoglobulin coacervates

in presence of vitamin B9

Preamble

Previous works, particularly from our research group, have studied the complex

coacervation between Beta-Lacotglobulin (BLG) and Lactoferrin (LF), respectively the

major acidic and basic protein from whey of cow milk. These studies have established the

phase diagram and the optimal conditions of complex coacervation of the whey proteins

(WP), BLG and LF, e.g, pH, molar ratios and concentrations. Based on these results, the

present work investigated the complex coacervation of BLG and LF in the presence of a

third molecule, i.e. the vitamin B9 (B9). The aim was to determine whether LF-BLG

coacervates could be efficient encapsulating agents for B9.

Main questions:

o Are BLG and LF able to co-assemble into coacervates in the presence of the

vitamin B9?

o Are LF-BLG coacervates able to entrap vitamin B9?

o What are the optimal B9/whey proteins molar ratios to obtain efficient

entrapment of vitamin B9?

A series of experimental tests of coacervation between B9, LF, and BLG were

performed over a large range of B9/WP molar ratios. Using turbidity and microscopic

observations, the type of B9-WP co-assembly obtained were determined and

chromatography analysis allowed to evaluate the coacervation yield and the B9 loading

efficiency. By plotting these results, the present study offered an interesting mapping of

B9-WP co-assembly, the domain of coacervation and the B9 loading of WP coacervates.

Main results:

The hydrophilic vitamin B9 does not hinder the co-assembly of LF and BLG into

complex coacervates.

Following B9/WP ratios, aggregates or heteroprotein coacervates are formed:

- aggregates at low and high whey proteins concentration,

- coacervates at intermediate proteins concentrations (10 to 15 g/L).

WP coacervates are effective in encapsulating B9, with a loading efficiency

ranging from 2 to 8 mg B9/g whey proteins.

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Spontaneous co-assembly of lactoferrin and beta-lactoglobulin

as a promising biocarrier for vitamin B9

The content of this part has been published in:

Chapeau A.L., Tavares G.M., Hamon P., Croguennec T., Poncelet D., Bouhallab S. (2016). Spontaneous co-assembly of lactoferrin and β-lactoglobulin as a promising biocarrier for

vitamin B ”, Food Hydrocolloids, 5 :2 0-290.

Graphical Abstract:

Abstract

The design of biocarriers for protection and controlled delivery of bioactives represents

a challenge for developing functional foods. We investigated the potentiality of

heteroprotein Beta-lactoglobulin (BLG) and Lactoferrin (LF) co-assemblies as biocarriers

for vitamin B9 (B9). Using different B9:protein mixing ratios, B9-LF-BLG co-assemblies

were obtained and assessed by turbidity and phase contrast microscopy. Kinetics of their

formation and stability were monitored. B9 entrapment efficiency was evaluated. Two

types of B9-LF-BLG co-assembly were identified: aggregates (at low and high protein

concentrations), and heteroprotein coacervates (at intermediate protein concentrations),

both exhibiting different kinetics and stability over time. Compiling screening maps of B9-

LF-BLG co-assemblies and B9 entrapment efficiency, we evidenced that B9-LF-BLG

coacervates exhibited higher performance as B9 biocarrier, with an optimal entrapment

of ≈10 mg B9/g protein. Therefore, such co-assembly displays useful potentialities as

vitamin biocarriers for the design of natural functional foods, offering enhanced health

benefits, yet without resorting to non-food additives.

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1.1.1. Introduction

Among food components, food proteins are biopolymers that appear

suitable for the protection of several bioactives due to their capacity to establish various

types of interactions with other compounds (Diarrassouba, Remondetto, et al., 2015). In

addition, food proteins are generally recognized as safe (GRAS), which make them

interesting biopolymers for the conception of biocarriers (Chen et al., 2006). In this

regard, milk proteins have been specifically studied for the design of biocarrier systems,

due to their versatility and excellent functional properties (Tavares et al., 2014). Many

strategies were developed to use milk proteins as biocarriers such as formation of simple

complexes with ligands, formation of gel networks by means of covalent, hydrophobic or

hydrogen interactions, and formation of supra-molecular structures through electrostatic

interactions (Bouhallab & Croguennec, 2014; Diarrassouba, Remondetto, et al., 2015, p.

16; C. Schmitt et al., 2009). Among milk proteins, Beta-lactoglobulin (BLG) and Lactoferrin

(LF) exhibit various characteristics that make them good candidates for the design of

biocarriers for bioactive compounds. Furthermore, recent studies have reported the

ability of BLG and LF to spontaneously co-assemble to form two types of supra-molecular structures: aggregates or heteroprotein complexes called coacervates through a process of complex coacervation (Anema et al., 2014; Tavares et al., 2015). The shape of co-

assembly depends strongly on the total protein concentration and protein stoichiometry

of the system (Anema & de Kruif, 2014; Bouhallab & Croguennec, 2014; Y. Yan et al.,

2013a). Heteroprotein aggregation is often described as a kinetically controlled process.

The resultant aggregates are typically fractal objects, which form non-ordered and often

irreversible supra-molecular structures (Bouhallab & Croguennec, 2014; Y. Yan et al.,

2013a). Complex coacervation is a spontaneous co-assembly occurring between two

oppositely charged biopolymers and leading to phase separation. It generates the

formation of two separated liquid phases: the dilute phase, rich in solvent and poor in

biopolymers and the dense phase, concentrated in biopolymers, namely, complex

coacervates (Schmitt et al., 2009). Complex coacervates are often reported as

microspheres due to their µm size dimension and their spherical shapes. Moreover, they

are often suggested as interesting biocarriers for biocative compounds (de Vos et al.,

2010; Ezhilarasi, Karthik, Chhanwal, & Anandharamakrishnan, 2013; Gouin, 2004;

Tavares et al., 2014). In this regard, BLG and LF spontaneous co-assemblies appear as a

promising technique for the design of such biocarriers.

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Among bioactive compounds, the vitamin B family is of great interest owing to its

vital importance and biological role (Lucock, 2000). Folic acid, currently known as vitamin

B9, is involved in several biochemical processes: DNA synthesis and repair, cell division

and co-enzyme in carbon metabolism pathways, including the biosynthesis of several

amino acids (Araújo et al., 2015). B9 has been recognized to provide several health

benefits such as preventing cardiovascular diseases (Adank et al., 2003), colon cancer

(Sanjoaquin et al., 2005), or congenital malformations during pregnancy (Lucock, 2000;

Moat et al., 2004). Nevertheless, humans cannot synthesize B9 and therefore must find

sufficient sources of B9 in their daily diet (Basset et al., 2005). B9 deficiency is relatively

common among the vulnerable populations such as elderly people or pregnant women

(Mills & Signore, 2004). As a result, there is a need for developing foods and drinks

enriched in B9 (Gregory, 2001). However, this appears as a technological challenge due

to the poor solubility of B9 at acidic pH. Hence, to successfully enrich acidic food products

with B9, the design of specific biocarriers for this vitamin is required.

This part examine in details the potentiality of the spontaneous co-assembly

between LF and BLG as biocarriers for the hydrophilic vitamin B9. Co-assembly between

LF and BLG in the presence of B9 was investigated across a range of physico-chemical

conditions and the co-assembly yields were evaluated. The formation kinetics of B9-LF-

BLG co-assemblies were monitored. The final morphology and the biocarrier efficiency of

formed B9-LF-BLG co-assemblies were determined.

1.1.2. Materials and Methods

Stock solutions

Lactoferrin (LF) from bovine milk, purity 90% and iron saturation level of 10% to % according to the manufacturer’s specifications, was purchased from the Fonterra

Cooperative Group, New Zealand. LF powder was used without modification. Beta-

Lactoglobulin (BLG) was obtained from a confidential industrial source. BLG powder was

dispersed in deionized water (45 g/L), adjusted to pH 4.6 with 1M HCl and kept at 30°C

for 5 min in order to precipitate non-native forms of BLG. The dispersion was centrifuged

at 20 000g at room temperature for 10 min (Heraeus Biofuge Primo, Thermo Scientific,

Waltham, MA, USA). BLG suspension were then freeze-dried and stored at -20 °C until use.

LF and BLG stock solutions were prepared by solubilizing the protein powders in

milli-Q water and their pH were adjusted at pH 5.5 using 1 M HCl solution. The protein

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solutions were filtered through a 0.45 µm and a 0.2 µm membrane (cat. no. 4612, Pall

Corporation, Ann Arbor, MI, USA). The exact proteins concentrations were determined by

absorbance at 280 nm (spectrometer UVmc², Safas, Monaco) using 1.47 L.g-1.cm-1 and

0.96 L.g-1.cm-1 as extinction coefficients for LF and BLG respectively. The stock solutions

were prepared to have a final concentration of LF = 0.5 mM (41.5 g.L-1) and BLG = 1 mM

(18.3 g.L-1).

Vitamin B9 (B9) was purchased from Sigma-Aldrich Folic acid, purity ≥ %, Sigma Aldrich, St. Louis, MO, USA). B9 stock solution was prepared by solubilizing the

vitamin powder in milli-Q water and then adjusted to pH=5.5 using 1 M HCl solution. B9

solution was centrifuged at 28 000g for 30 minutes at room temperature (centrifuge Hereaus Biofuge primo, KENDRO Laboratory products, Courtabœuf, France). The

supernatant was filtered through a 0.2 µm membrane (cat. no. 4612, Pall Corporation, Ann

Arbor, MI, USA). The exact vitamin B9 concentration was determined by absorbance at

283 nm (spectrometer UVmc², Safas, Monaco) using 25100 M-1 cm-1 as extinction

coefficient.

LF, BLG and B9 stock solutions were stored at 4°C and protected from UV light

radiation.

Experimental solutions

A series of solutions of B9-LF-BLG were prepared following a range of B9:protein

mixing ratios, where Protein refers to LF+BLG total content. The tested concentration

ranges were 0 to 0.5 mM for B9 and 0 to 1.1 mM for proteins. To obtain the required

concentrations of B9, LF, and BLG according to each ratio, the stock solutions were diluted

into the proper volume of milli-Q water at pH=5.5. Then for each ratio, the three

corresponding diluted solutions were mixed together, always in the same order. All

solutions were prepared at room temperature (20°C ± 2°C), and no significant

modification of pH was detected after mixing.

Turbidity measurements

Turbidity measurements were performed at 600 nm (A600nm) with the

spectrometer UVmc² (Safas, Monaco) to monitor the co-assembly in B9-LF-BLG solutions.

Absorbance measurements were converted to turbidity τ, cm-1) using the following relationship: τ = . A600nm)/l, where l is the light path length (l= 1 cm). Turbidity was

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monitored over 180 minutes. All solutions were prepared in the same way: LF and B9

were first mixed together before adding BLG after 5 minutes. Under the ratios

investigated, the LF, BLG and vitamin B9 stock solutions alone had no detectable turbidity.

Phase contrast microscopy and co-assembly type index

The presence of aggregates, coacervates, or the absence of supra-molecular

structures was determined using a phase contrast optical microscope (Olympus BX51TF,

Olympus, Hamburg, Germany) set at the magnification 40. A 10µL sample of the B9-LF-

BLG solutions was taken at the time of maximum turbidity and immediately observed. A

notation scale was set up in order to code the type of supra-molecular structures observed

for each B9-LF-BLG solution. The scale, called co-assembly type index , was as followed: 0: no co-assembly, 1: aggregates, 2: few and dispersed coacervates, 3: rather dense

coacervates, 4: very dense and numerous coacervates. The samples and observations

were performed three times on each solution to ensure the representability of the results

Solution stability

The stability of the B9-LF-BLG co-assemblies was monitored using the optical

characterization method with a TurbiScan (Turbiscan Laboratory Formulation,

Ramonville St. Agne, France). The analyzed solution is placed in a cylindrical glass cell of

4 mL (diameter = 12mm, height = 25 mm.). The light source is an electro luminescent diode emitting at = nm. The light source scans the solution at 1 min intervals from

top to bottom. Two synchronous optical sensors receive respectively the light transmitted

through the solution (180° from the incident light, transmission sensor), and the light

backscattered by the solution (45° from the incident radiation, backscattering detector).

Each scan provides the profiles of the transmitted and backscattered light flux (%)

relative to standards (suspension of monodisperse spheres and silicone oil) as a function

of the solution height (mm). These two curves could be considered to be macroscopic fingerprint of each solution at a given time (Kowalska, Zbikowska, & Tarnowska, 2015).

The mixing of the B9-LF-BLG was realized directly in the cylindrical glass cell of

measurements according to the B9:protein ratio tested. For each sample, the TurbiScan

analysis was carried out for 24 hours, at 20 °C.

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Co-assembly yield and entrapment efficiency

B9 and protein contents were quantified in order to determine the B9 and proteins

recovery yield in the co-assemblies. For each B9-LF-BLG solution, the supernatant and the

dense phase of co-assembly were separated by centrifugation (Heraeus Biofuge Primo,

Thermo Scientific, Waltham, MA, USA) at 28 000 g for 30 min. B9 and proteins recovered

in the supernatant were quantified after separation by reversed-phase high-performance

liquid chromatography (RP-HPLC), using a LiChrospher RP-18 column (Art. 1509430001,

Merck, Darmstadt, Germany) connected to a Waters 2695 HPLC system. B9 and total

proteins were eluted at a flow rate of 0.4 mL.min-1 using an isocratic gradient of

Acetonitrile/milli-Q water 40/60 (v/v) containing 100 L.L-1 of 1M NaOH and quantified

by absorbance at 283 nm. The quantities of B9 and proteins in the co-assemblies were

calculated by subtracting the quantities in the supernatant from the initial quantities.

The quantities of B9 and proteins recovered in the co-assemblies were used to

calculate three key parameters for each B9-LF-BLG solution according to the following

equations:

the co-assembly yield η, 𝜂 = [ 𝑖 + 9] − 𝑒 𝑦[ 𝑖 + 9]𝑖 𝑖

the B9 loading, 9 𝐿 𝑎 𝑖 = [ 9] − 𝑒 𝑦[ 𝑖 + 9] − 𝑒 𝑦

and the entrapment efficiency, 𝐸 𝑎 𝑖 𝑖 𝑦 = 𝐵9 − 𝑒 𝑦 𝑃 𝑒𝑖 − 𝑒 𝑦

with [Protein] and [B9] the concentration in mM in the initial solution or recovered in the

co-assembly, and Q the relative quantity of B9 (mg) and Protein (g) in the co-assembly.

Screening maps of B9-LF-BLG co-assembly

Screening maps are a color projection of a specific parameter (maximum turbidity

in cm-1, co-assembly type index, or entrapment efficiency in mg B9 / g Protein) as a

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function of the B9:protein mixing ratios. Each screening map plots the specific value of

one parameter obtained from a specific B9:protein ratio. Screening maps were plotted

using the OriginPro 2015 software. The Renka-cline gridding method was used to convert

the random XYZ data to matrices. Then the screening maps were plotted using the XYZ

contour graph method of the software.

Particle size and sphericity analysis

Particle size and sphericity analysis were measured by dynamic image analysis using a Qicpic™ analyzer Sympatec GmbH, Clausthal-Zellerfeld, Germany). Measures

were performed using the SUCELL wet dispersion unit. Data were analysed using

WINDOX 5 software (Sympatec LTD, Bury, UK). The principle and the validity of

measurements by this method had been detailed previously by (Hamilton, Littlejohn,

Nordon, Sefcik, & Slavin, 2012). Briefly, a fast camera records digital images of the

particles dispersed in solution. Proprietary algorithm of the software WINDOX 5 are then

used to analyze each images and to calculate two parameters: the equivalent projected

circle (EQPC) diameter, which gives the diameter of a sphere that has the same 2-

dimensional area as the projected particle in the recorded image, reported as the particle

size in this study, and the sphericity index which is the ratio of the perimeter of the

equivalent circle PEQPC to the real perimeter of the particle Preal. This value is between 0

and 1. The smaller the value, the more irregular the particle shape is.

For measurement, a volume of 5 mL of the B9-LF-BLG solution was dispersed into the

cell of the wet dispersion unit containing 50 mL of milli-Q water. The detection was set to

1% and the recorded film lasted 60 seconds. Analysis were performed three times for

each solution.

1.1.3. Results and discussion

LF-BLG co-assemblies in the presence of B9

To assess that BLG and LF can co-assemble in the presence of B9, six B9:protein

ratios were tested: two ratios at low protein contents (2:2 and 4:2), two ratios at

intermediate protein contents (2:11 and 4:11), one ratio at high protein content (4:15)

and one ratio at very high protein content (4:20). These ratios were selected according to

the previous results obtained by (Tavares, Croguennec, Hamon, Carvalho, & Bouhallab,

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2015a) who assessed the co-assembly of LF and BLG in the same range of protein ratios

in the absence of B9.

Figure III-1: Turbidity kinetics of LF-BLG co-assembly in the presence of B9 at six B9:protein mixing molar ratios spanning from low (0.1 mM) to high total protein concentration (1mM), obtained at pH=5.5 and at 20°C. In this study, BLG/LF molar ratio of 10 was used.

Figure III-1 shows the turbidity kinetics obtained with the six solutions tested.

Turbidity increased for all the ratios as soon as BLG was added into the B9+LF solutions,

except for the solution with the highest protein content (4:20). These increased

turbidities reflected the co-assembly taking place in the B9-LF-BLG solutions. To correlate

these turbidity profiles with the formation of supra-molecular structures, a study by

phase contrast microscopy was conducted. Figure 2 shows the microscopic images

obtained by the phase contrast microscopy analysis performed on the six solutions, at

maximum turbidity. Two types of turbidity profiles and supra-molecular structures can

be identified. The solutions with B9:protein ratios of low protein contents (2:2 and 4:2)

and high protein content (4:15), exhibited a rather slow and gradual increase of turbidity

toward a maximum asymptotic value (Figure III-1). These profiles corresponded to the

formation of aggregates in the solutions (Figure III-2A, B, E). On the contrary, for the

B9:protein ratios at intermediate protein contents (2:11 and 4:11), a sudden and high

increase of turbidity was observed as soon as BLG was added followed by a slow and

gradual decrease toward a minimum asymptotic value (Figure III-1). These profiles

BLG introduction

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supports a spontaneous process and corresponded to the formation of coacervates in the

solutions (Figure III-2C, D). Finally, for the solution of very high protein content (4:20),

no increased turbidity was observed (Figure III-1) and no supra-molecular structures

were detected (Figure III-2F). Therefore, no significant co-assembly occurred between

B9-LF-BLG at very high protein content.

Figure III-2: Phase contrast microscopy (x40) of LF-BLG co-assembly in the presence of B9 at six B9:protein mixing molar ratios spanning from low (0.1 mM) to high total protein concentration (1mM), obtained at pH=5.5 and at 20°C. (A) B9:protein=2:2; (B) B9:protein=4:2; (C) B9:protein=2:11; (D) B9:protein=4:11; (E) B9:protein=4:15; (F) B9:protein=4:20.

These observations are consistent with literature on heteroprotein association.

Moreover they are in adequacy with the previous results on LF-BLG co-assembly which

have shown that BLG+LF aggregation occurred in solutions of low protein concentrations

(Tavares, Croguennec, Hamon, et al., 2015a). In addition, (Y. Yan et al., 2013a) have

mentioned that the formation of LF-BLG aggregates is a relatively slow and kinetically

controlled process. Hence, it is likely that co-assembly by aggregation occurred into

solutions with B9:protein ratios at low protein contents (2:2 and 4:2). Furthermore,

(Tavares, Croguennec, Hamon, et al., 2015a) have reported that for intermediate protein

content, the co-assembly between BLG and LF is spontaneous and lead to the formation

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of BLG+LF coacervates. It can therefore be supposed that complex coacervation occurred

in the presence of B9 at intermediate protein contents (2:11 and 4:11). Moreover,

(Tavares, Croguennec, Hamon, et al., 2015a) have described that at high total protein

concentration, self-suppression of complex coacervation appears with a transition from

coacervates to aggregates formation. This phenomenon can also be observed here for the

solution with high protein content (4:15). Finally, an absence of co-assembly between BLG

and LF has been previously reported for very high total proteins concentration by (Y. Yan

et al., 2013a). Similarly, the absence of co-assembly can be identified here for the solution

of very high protein content (4:20). As a result, it can be assumed that the presence of

vitamin B9 in the system does not prevent co-assembly between LF and BLG.

Furthermore, it can be considered that similar co-assembly behavior occurs in the B9-LF-

BLG systems than in LF-BLG systems.

Stability of the co-assemblies

The stability of the two types of co-assembly (aggregates and coacervates) was

evaluated in order to give insight into the evolution of the two types of co-assembly in

solution, overtime. Stability of co-assembly solutions were assessed using optical

characterization method with a TurbiScan. Two solutions were tested: one for the

aggregates type of co-assembly (high concentration = 1.1 mM) and one for the coacervates

type of co-assembly (intermediate protein concentration = 0.55 mM). In both solutions,

B9 concentration was set at 0.25 mM. The first scan was realized immediately after mixing

B9, LF and BLG solutions together and considered as the reference scan. Figure III-3

presents the patterns of delta transmission and delta backscattering obtained after

subtraction of the reference scan.

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Figure III-3: Stability analysis of LF-BLG co-assemblies in the presence of B9 0.25 mM. Delta transmission and delta backscattering signals recorded over 24 hours of formed aggregates (1.1 mM total proteins) (A, B) or coacervates (0.55 mM total proteins) (C, D) obtained at pH=5.5 and at 20°C. The backscattering intensity depends on particle size and concentration. It allowed to describe the evolution of the aggregate (B) or coacervates (D) particles as indicated by Roman numerals.

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For the solution of aggregates, delta transmission signal decreased progressively

during the first two hours, throughout the entire height of the tube (Figure III-3A). This

indicated a gradual increase of the size of the aggregates in solution over the first two

hours. After two hours, the delta backscattering signal started to increase between 0 to 2

mm height (Figure III-3B). This indicated a sedimentation of the aggregates at the bottom

of the tube. For the solution of coacervates, delta backscattering signal showed an

important drop over the first one minute, indicating the spontaneous formation of supra-

molecular structures (Figure III-3D). Then, the delta backscattering signal decreased

between the 24 to 28 mm height of the tube (Figure III-3D), as well as an increase of the

delta transmission signal (Figure III-3C). This phenomenon was important during five

hours and then remained relatively constant, demonstrating a rapid clarification at the

top of the tube during the first five hours. At the same time, the delta backscattering signal

in the middle of the tube (2 to 24 mm height) was slightly decreasing (Figure III-3D). This

indicated that the solution of coacervates became more opaque and thick over time. As

the clarification at the top of tube happened at the same time, it can be deduced that the

coacervates tended to coalesce together. From the fifth hour until 24h, the delta

backscattering signal of the microspheres solutions increased between 0 to 2 mm height

(Figure III-3D), indicating a densification of the solution at the bottom of the tube.

These observations are in agreement with those presented on Figure III-1 and

Figure III-2, and give more insights into the kinetics of formation and the stability of the

co-assemblies in solutions. In the case of aggregation (low and high protein contents), the

formation of co-assembly seems to be a relatively slow process. Turbidity (Figure III-1)

as well as the size of the particles increased slowly over two hours (Figure III-3A). Then,

due to their density, aggregates tend to settle, creating a concentrated sediment phase

(Figure III-3A). On the contrary, the formation of coacervates appears as a spontaneous

co-assembly. Turbidity increased rapidly after BLG addition in the solutions of

intermediate protein contents (2:11, 4:11) Figure III-1) and particles around 10 µm can

be observed just 5 minutes after the mixing of the three solutions (Figure III-2C), which

correlates the observations made on Figure 3D. The decrease of turbidity observed for the

solutions of coacervates at intermediate proteins contents (2:11 and 4:11) on Figure III-1

could be linked either to dissociation or coalescence and sedimentation of the

coacervates. Figure III-3D shows that it is more likely that coacervates droplets coalesce

together. This coalescence leads to the formation of larger coacervates droplets, which

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separated by gravity from the dilute phase. This dense phase formation is consistent with

the literature on the theory of complex coacervation in protein systems. (C. G. de Kruif,

Weinbreck, & de Vries, 2004) and more recently (Bouhallab & Croguennec, 2014; Kizilay

et al., 2011) reported that complex coacervation leads to the formation of a very

concentrated phase of biopolymers. This phase results of the setting of attractive

interactions occurring between oppositely charged biopolymers in solution, which lead

to phase separation and to the formation of a concentrated phase of coacervates. (C. G. de

Kruif et al., 2004) pointed out that this coacervates can be seen as a new colloidal entity.

Thus, the coacervates formed here were obtained spontaneously by the electrostatic

interactions occurring between positively charged LF and negatively charged BLG and B9

and their subsequent phase separation. As a result, the two solutions of co-assembly lead

overtime to the formation of a two phase system: the upper phase is depleted in co-

assemblies and the downer phase is concentrated on aggregates or coacervates which

possibly form biocarrier to load B9.

B9-LF-BLG co-assembly yield and B9 loading

To assess the co-assembly yield η of the two types of co-assembly, two protein

contents were chosen: low protein content for aggregates (Figure III-4A), and

intermediate protein content for coacervates (Figure III-4B). At a set protein content,

increased contents of B9 was tested to evaluate the B9 loading of each type of co-

assembly. For the two types of co-assembly, η increased as B9 increased (Figure III-4A

and B). The solutions of B9-LF-BLG aggregates showed a co-assembly yield ranging from

0.85 to 0.90 (Figure III-4A). The solutions of B9-LF-BLG coacervates showed a co-

assembly yield ranging from 0.90 to 0.95 (Figure III-4B). Thus, by comparing the two

types of co-assembly, slightly higher co-assembly yields were obtained in the

coacervation conditions. Correspondingly, B9 loading increased from 0.64 to 0.80 for

aggregates solutions (Figure III-4A), and from 0.60 to 0.90 for coacervates solutions

(Figure III-4B), as the content of B9 increased in the B9-LF-BLG solutions. The highest B9

loading obtained was 0.90 for a co-assembly yield of 0.95, obtained for the B9:protein

ratio of 10:11. Thus, it can be assumed that the increase of the co-assembly yield is

correlated with the increase of B9 in the solutions which leads to an increasing B9 loading

throughout its entrapment within the B9-LF-BLG co-assembly. Optimal loading

conditions were further sought by exploring a wider range of B9:protein ratios.

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Figure III-4: Co-assembly yield η and B loading of B -LF-BLG co-assembly at three different initial B9 contents and B9:protein mixing molar ratios at two total protein concentrations: (A) 0.15mM: (B) (0.55mM). Experiment were performed at pH=5.5 and 20°C.

Screening maps of B9-LF-BLG co-assemblies

Screening maps were plotted in order to identify the domains of B9-LF-BLG co-

assembly (maximum turbidity), their types (aggregates or coacervates) and the

corresponding entrapment efficiency. Figure III-5 presents the screening maps obtained

by color projection of maximum turbidity (Figure III-5A), co-assembly type index (Figure

III-5B) and the entrapment efficiency (Figure III-5C) as a function of the concentration of

B9 and proteins.

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Figure III-5: Screening maps of B9-LF-BLG co-assembly by color projection for large range of B9 and protein concentrations at pH=5.5 and at 20°C. (A) maximum turbidity in cm-1; (B) co-assembly type index; (C) entrapment in mg B9/g Protein.

It can be seen on Figure III-5A that no or few turbidity (dark blue color) was

globally observed at low (0.1 to 0.2 mM) or high protein concentrations (0.7 to 0.85 mM).

At intermediate protein concentrations (0.3 to 0.7 mM), the maximum turbidity increased

(light green to red) and varied with B9 concentration. In this range of medium protein

concentrations, these variations designs two domains of maximum turbidity: the first one

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around 0.05 mM B9 the second one around 0.4 mM B9. In addition, rather similar

observations can be drawn from the co-assembly type index (Figure III-5B). For low and

high protein concentrations, no co-assembly was observed which is consistent with the

low maximum turbidity reached (Figure III-5A). Depending on the B9 and protein

concentrations, three domains of maximum coacervation (yellow to red colors) can be

evidenced: two at 0.1 mM B9 and protein concentrations of 0.25 and 0.6 mM and one at

0.35 mM B9 and protein concentration of 0.55 mM. These domains corresponded to the

domains where co-assembly into coacervates was the densest. In the vicinity of these

domains, the co-assembly type index decreased from 3 to 2, indicating less dense

coacervation. Further, a transition toward co-assembly into aggregates was observed.

These results corroborate those presented in Figure III-1 and Figure III-2. Furthermore,

the two screening maps (Figure III-5A and B) appear to be rather stackable, without being

exactly identical. As a result, the compilation of these screening maps better evidenced

the overall domains of no co-assembly, aggregation or coacervation, depending on B9 and

protein concentrations and therefore on the B9:protein ratios. The domains of no co-

assembly corresponded clearly to the solutions with protein concentration above 0.85

mM). B9-LF-BLG aggregates were favored at low (0.1 to 0.2 mM) and high protein

concentrations (0.7 to 0.85 mM). Preferential domains of coacervation were found at

intermediate protein concentrations between 0.3 to 0.7 mM with either low (0 to 0.1 mM)

or intermediate (0.25 to 0.45 mM) B9 concentrations.

Two domains of high biocarrier efficiency (red color) were identified : one at 0.3

mM B9 and protein concentration of 0.3 mM and one at 0.5 mM B9 and protein

concentration of 0.6 mM (Figure III-5C), with a biocarrier efficiency around 16.7 mg B9 /

g Protein. In the vicinity of these domains, the entrapment efficiency decreased

progressively from 16 to 8 mg B9 / g protein. Above, the entrapment efficiency kept on

decreasing to low values from 6 mg B9 / g proteins (light blue color) to 0 mg B9 / g Protein

(dark blue color). These low encapsulation efficiencies corresponded to low (0.1 to 0.2

mM) and high protein concentrations (0.7 to 0.85 mM) tested.

Compiling the three screening maps of Figure III-5 evidenced the entrapment

efficiency of each type of co-assembly. The domains of absence of co-assembly and of

aggregate formation identified at low and high protein concentrations appeared to be in

the domains of lower entrapment efficiency for B9. On the contrary, the domains of high

entrapment efficiency matched relatively well the domains of coacervation (co-assembly

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type index > 2 and maximum turbidity > 3 cm-1) obtained for intermediate protein

concentrations from 0.3 to 0.7 mM and for B9 concentrations from 0.1 to 0.4 mM.

These screening maps constitute a useful tool to predict the type of B9-LF-BLG co-

assembly and the corresponding entrapment efficiency that can be obtained from the B9-

LF-BLG co-assembly at different initial molar ratios. The compilation of these

observations with the co-assembly type screening map evidences that the best B9 loading

efficiencies are obtained for the B9:protein ratios of coacervates. The B9-LF-BLG

coacervates obtained will lead to B9 loading efficiencies ranging from 6 to 16 mg B9 / g

protein.

(Pérez-Masiá et al., 2015) have reported that the encapsulation of B9 performed

by nanospraydrying or electrospraying of whey protein concentrates lead to an

entrapment efficiency of 12 mg B9 / g total proteins. Therefore, the entrapment efficiency

obtained here by spontaneous co-assembly is in the same range of order. Moreover,

(Diarrassouba, Remondetto, et al., 2015) have reported that the coacervates obtained by

the co-assembly between BLG and lysozyme for the design of vitamin D3 biocarriers lead

to a loading efficiency of ≈ 30 mg.L-1 which corresponds to few µg per mg of proteins

matrix as they prepared a solution at 0.1% of BLG. Moreover, the recommended daily

intake for vitamin B9 is around 200 to 400 µg for adults and 400 to 800 µg for the

vulnerable populations such as elderly or pregnant women. Thus, few milligrams of the

B9-LF-BLG coacervates could cover the daily requirement for the vitamin. As a result, the

spontaneous co-assembly of B9, LF and BLG into coacervates appeared as a viable and

efficient process to design biocarriers for vitamin B9.

For further analysis of these biocarriers, it has been chosen to characterize the B9-

LF-BLG coacervates obtained with 0.25 mM B9 and 0.55 mM total proteins. This couple of

B9 and protein concentrations combined all at once a favorable co-assembly type index

of 3, which corresponded to a quite dense coacervates formation (Figure III-5B), with a

maximum turbidity of 3.5 cm -1 (Figure III-5A) and a good entrapment efficiency of 8 mg

B9 / g Protein.

Characterization of B9-LF-BLG coacervates

Figure III-6 shows the volume size distribution and the sphericity index as a

function of the particles size, for the coacervates obtained with the B9:protein ratio of

5:11. The size of the coacervates ranged from 2 to 20 µm diameter, with a mean diameter

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around 8 µm (Figure III-6A). The sphericity index of the coacervates droplets with 2 to 3

µm diameter was between 0.9 and 0.85 (Figure III-6B.). As the size of the coacervates

droplets increased, sphericity index decreased. For coacervates droplets of 3 to 20 µm

diameter, the sphericity index ranged from 0.85 to 0.60 (Figure III-6B). For coacervates

droplets of 20 µm diameter and more, the sphericity index dropped to 0.50. Hence, small

coacervates up to 5 µm diameter were almost perfect spherical droplets. For coacervates

between 5 and 20 µm the sphericity was altered, suggesting droplets rather ovoid than

spherical.

Figure III-6: Characteristics of B9-LF-BLG coacervates. (A) size distribution; (B) sphericity index; (C) image from phase contrast microscopy. Coacervates were obtained with a B9 concentration of 0.25 mM and protein concentration of 0.55mM), pH=5.5 and at 20°C.

A possible explanation for this result may be the coalescence that has been

previously shown in coacervates solutions (Figure III-3D). The coalescence probably

altered the shape of the coacervates, leading to droplets more ovoid than spherical. The

microscopic image on Figure III-6C obtained by phase contrast microscopy of the same

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B9:protein solution confirmed these observations. Thus, B9-LF-BLG coacervates are

spherical droplets or slightly ovoid if they results from coalescence, with a mean diameter

of 8µm.

According to the experimental conditions, the B9-LF-BLG coacervates sizes were

reported to be in the range of the LF-BLG coacervates sizes reported in the literature: from

2.5 to 7.5 µm (Tavares, Croguennec, Hamon, et al., 2015a) and 10 to 20 µm (Kizilay et al.,

2014). Hence, the presence of B9 does not modify the overall size of LF-BLG coacervates.

Moreover, (Pérez-Masiá et al., 2015) have reported that the encapsulation of B9 into a

whey protein matrix using nanospraydrying or electrospraying technology led to the

formation of spherical submicron and micron particles. Therefore, the size of the

coacervates obtained by the spontaneous co-assembly of B9-LF-BLG are in the same order

of magnitude than particles obtained through other technologies to design B9 biocarriers

from whey protein matrices. Furthermore, (Diarrassouba, Remondetto, et al., 2015) have

reported that the co-assembly between BLG and lysozyme for the design of biocarriers

for vitamin D3 led to the formation of spherical particles of 6 to 8 µm. This D3-BLG-

lysosyme co-assembly can be viewed as similar as the B9-LF-BLG coacervates formed

here, thus it is consistent to obtained particules size in the same range of values.

1.1.4. Conclusion

This work demonstrated that BLG and LF can form two types of supramolecular

structures in the presence of B9, aggregates or coacervates, showing that the presence of

a hydrophilic compound does not prevent the co-assembly between the two whey

proteins to occur. Moreover, B9-LF-BLG coacervates were formed spontaneously and

showed a relative stability, only prone to a slight coalescence overtime. On the contrary,

B9-LF-BLG aggregation was a rather long and kinetically controlled process and

aggregates were subject to significant sedimentation. Consequently, the spontaneous co-

assembly of BLG and LF into coacervates appears as an efficient process to design

biocarriers for a hydrophilic vitamin. It requires no particular energy input and

constitutes an interesting way to enhance the functionalities of whey proteins, a co-

product of the dairy industry. Screening maps of B9-LF-BLG co-assemblies and

entrapment efficiencies were plotted. They constitute a useful tool to predict the type of

B9-LF-BLG co-assembly and its corresponding entrapment efficiency, obtained for a set

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B9:protein ratio. The compilation of these screening maps identified that the highest

entrapment efficiencies were obtained for B9-LF-BLG coacervates and ranged from 6 to

16 mg B9 / g Protein. This result is consistent with other technologies for the design of B9

biocarriers, and consistent with the recommended daily intake of B9. As a result, B9-LF-

BLG coacervates display useful potentialities for the development of natural and

functional foods. The biocarriers materials are food-grade ingredients and the vitamin

offer several health benefits, thus enhancing the nutritional quality of the food product.

Further studies should be now undertaken to assess the stability of these supramolecular

structures over time and during processing. Also, studies are required to shed light into

how these co-assemblies are able to protect B9 from photodegradation and oxidation and

to assess the bioavailability of B9 loaded in such biocarriers.

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1.2. Part 2: Bench scale production of vitamin B9-WP coacervates

Preamble

In the first part, we established that complex coacervation between beta-

lactoglobulin (BLG) and lactoferrin (LF) in presence of vitamin B9 (B9) leads to the

formation of vitamin B9-WP coacervates, encapsulating the vitamin into the whey

proteins matrix. This proof of concept has been established at a laboratory scale, for a

production of µg of B9-WP coacervates. However, one of the purpose of exploring WP

coacervates as new encapsulating agents is their potentiality to be used for new industrial

development. Scaling up the production of coacervates is challenging as complex

coacervation process is known to be highly sensitive to environmental factors: interface,

working volumes, agitation, shearing.

Main questions:

o Is it possible to produce B9-WP coacervates in larger volume while maintaining

good WP coacervation and B9 encapsulation yields?

o What system(s) can be most appropriate to scale up the production of B9-WP

coacervates?

To investigate the scale up production of B9-WP coacervates, the preparation and

purification steps of initial BLG and LF solutions were investigated first. Then, two mixing

systems were studied for coacervation between B9, LF and BLG solutions: one system in

batch and one in system in continuous, using two different types of mixing devices.

Main results:

Remaining few aggregates of LF and BLG commercial solutions do not alter

complex coacervation process.

Both batch and continuous systems allow high B9 encapsulation yield up to 98%,

similarly to yields obtained at the laboratory scale.

WP coacervation yields varied from 55% for batch system (similar at µL scale)

and up to 65% for continuous system, (higher than yields obtained at µL scale).

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Scale-up production of vitamin loaded heteroprotein coacervates

The content of this part is based on results published in: Chapeau A.L., Hamon P., Rousseau F., Croguennec T., Poncelet D., Bouhallab S. (2017). Scale-up production of vitamin loaded heteroprotein coacervates and their protective

property, Journal of Food Engineering, 206, 67-76;

Abstract

The production of biocarriers for the protection and controlled delivery of bioactives is of

great interest to develop functional foods. The ability of two whey proteins (WP), Beta-

Lactoglobulin (BLG) and Lactoferrin (LF), to efficiently entrap vitamin B9 by complex

coacervation has been reported at laboratory scale. In the present work, we report on the

scaling-up production of B9 loaded LF-BLG coacervates (B9-WP coacervates). Complex

coacervation was performed at bench scale, using commercial protein solutions. Under

optimized conditions, B9-WP coacervates were produced by static mixing at a flow rate

of 300 mL/min. Bench scale efficiency similar to that found for laboratory scale was

reached with a coacervation yield of 65 % and the B9 entrapment of 98 %, demonstrating

an efficient scaling-up.

1.2.1. Introduction

The concept behind the formation of biocarriers by complex coacervation is to first

disperse the bioactive in one of the initial biopolymer solution and then use the phase

separation to newly form the coacervates phase containing the bioactive ingredient

(Sánchez, García, Calvo, Bernalte, & González-Gómez, 2016). The formation of biocarriers

by coacervation presents numerous advantages. It only requires mild preparation

conditions with no use of neither organic solvent, drastic temperature nor high pressure

which is of interest to reduce the industrial cost of biocarriers formation (C. Yan & Zhang,

2014). Coacervation also offers high shell integrity, high encapsulation efficiency and

good controlled-release properties (Gouin, 2004). Moreover, this method has the

advantage that a specialized equipment is not required to complete the coacervation

process. Hence, it appears as potentially scalable to the industrial production of

biocarriers (Shewan & Stokes, 2013). Numerous biopolymers systems have been studied

to use complex coacervation as a technology to form biocarriers. Most of them studied the

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interactions between proteins and polysaccharides (Doublier, Garnier, Renard, &

Sanchez, 2000; C. Schmitt & Turgeon, 2011) to form biocarriers for specific core bioactives

such as essential oils or lipophilic vitamins (Matalanis et al., 2011), flavors (Koupantsis,

Pavlidou, & Paraskevopoulou, 2014; Xiao, Liu, Zhu, Zhou, & Niu, 2014b) or probiotics (de

Vos et al., 2010) for example. Nevertheless, few studies deal with the complex

coacervation of a two proteins systems.

The potentiality of using the complex coacervation of two whey proteins (WP), the

lactoferrin (LF) and the beta-lactoglobulin (BLG), to form a biocarrier for a bioactive, the

vitamin B9 (B9), was previously investigated (Chapeau et al., 2016). Nevertheless, this

work was carried out at a laboratory scale (µL). For a possible industrial application of

B9-WP coacervates, the scale-up production to bench scale (L) has to be considered.

However, coacervation is known to be a process highly dependent of the biomaterials

system involved and each coacervation system operates under a unique set of conditions

(C. Yan & Zhang, 2014). Moreover, the scale-up of the complex coacervation process is

often achieved empirically, leading to possible trial and errors setup and may involve

expense of cost and resources (Lemetter, Meeuse, & Zuidam, 2009; Paul, Atiemo-Obeng,

& Kresta, 2004). That is why the specific conditions of complex coacervation of such

proteins systems have to be specifically investigated, especially when industrial

applications are considered to maximize process yield and minimize loss and batch-to-

batch variations (Curt Thies, 2007). The aim of this part is now to investigate the scale-up

production of B9-WP coacervates from laboratory scale (µL) to bench scale (L) using

batch and continuous mixing systems.

1.2.2. Materials and methods

Materials

Vitamin B9 (B9) was purchased from Sigma Aldrich (Folic acid, purity > 97%,

Sigma Aldrich, St. Louis, MO, USA). Lactoferrin (LF) from bovine milk, (purity 90% and

iron saturation level of 10% to 20%) was purchased from the Fonterra Cooperative

Group, New Zealand. LF powder was used without further modification. Beta-

Lactoglobulin (BLG) powder was obtained from a confidential industrial source. Its

composition (w/w) was: protein 93.5%, moisture 4% and ash < 1.8%. Protein purity was

determined by reversed-phase HPLC and no proteins other than BLG were detected.

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Stock solutions

B9 stock solution was prepared by solubilizing the vitamin powder in milliQ water

and then adjusted to pH 5.5 using 1 M HCl solution. B9 solution was centrifuged at 28,000

g for 30 min at room temperature (centrifuge Hereaus Biofuge primo, KENDRO

Laboratory products, Courtaboeuf, France) to remove the insoluble fraction of B9. The

exact vitamin B9 concentration was determined by absorbance at 283 nm (spectrometer

UVmc2, Safas, Monaco) using 25.1 L g-1 cm-1 as extinction coefficient.

Before use, BLG was purified. BLG powder was dispersed in deionized water (45

g/L), adjusted to pH 4.6 with 1 M HCl and kept at 30 °C for 5 min in order to precipitate

non-native forms of BLG. The dispersion was centrifuged at 20,000 g for 10 min at room

temparature (Heraeus Biofuge Primo, Thermo Scientific, Waltham, MA, USA). BLG

suspension was then freeze-dried and stored at - 20 °C until use. Then, LF and BLG stock

solutions were prepared by solubilizing the protein powders in milliQ water adjusted to

pH 5.5 using 1 M HCl. The protein solutions were filtered through a 0.45 mm and a 0.2

mm membrane (cat. no. 4612, Pall Corporation, Ann Arbor, MI, USA). The exact proteins

concentrations were determined by absorbance at 280 nm (spectrometer UVmc², Safas,

Monaco) using 1.47 L g-1 cm-1 and 0.96 L g-1 cm-1 as extinction coefficients for LF and BLG

respectively. B9, LF, and BLG stock solutions were stored at 4 °C and protected from UV

light radiation.

Production of B9-whey protein coacervates

The production diagram of B9-whey proteins (B9-WP) coacervates is depicted in

Figure III-7A. First, a solution of B9-LF complex was prepared by blending B9, LF and

milliQ water, to have final concentrations of 4.15 g/L for LF and 0.11 g/L for B9. The B9-

LF complex solution was let to equilibrate by itself for 10 min at 20 °C. Then, BLG solution

was added to the B9-LF complex solution to reach a final BLG concentration of 9.15 g/L,

in order to induce complex coacervation. Addition of BLG was performed under either

batch or continuous mixing conditions. The coacervatesd phase recovered by

centrifugation at 38,000 g for 45 min at 20°C (Centrifuge Avanti J-26S XP, Beckman

Coulter Inc., Brea, CA 92821 USA), was weighed and kept in the dark at 4°C.

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Figure III-7: Schematic diagram for the of production of B9-WP coacervates (A) and the device used for batch mixing by mechanic stirring (B) or continuous mixing by static stirring (C).

Batch mixing by mechanic stirring was performed by stirring the solution in a

vessel containing a propeller pale of 2.5 cm diameter, with 3 blades (Figure III-7B). The

propeller was set in rotational motion by the mean of an electric motor set at the desired

stirring speed, during 2 minutes. Unstirred solution was also equilibrated during 2 min

prior centrifugation.

Continuous mixing by static stirring was made using a static mixer (Figure III-7C)

constituted by a series of 10 static mixer units of 0.4*0.6 cm diameter, set in-line, for a

total length of 6 cm. The input pipes for B9-LF and BLG solutions were of 0.4 cm diameter

and 30 cm length. The output pipes for B9-WP coacervates solution was of 0.2 cm

diameter. According to preliminary tests to set the system, permanent regime of the static

mixer was achieved after 20 seconds. Aliquots were withdrawn over time for further

analyses.

Turbidity analysis

Turbidity measurements at 600 nm (A600nm) (spectrometer UVmc2, Safas, Monaco)

was used to monitor complex coacervation and the formation of B9-LF-BLG coacervates

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in the coacervatesd solutions. Absorbance measurements were converted to turbidity (t,

cm-1) using the following relationship: t = (2.303A600nm)/l, where l is the light path length

(l= 1 cm) ( Melik and Fogler, 1983). The stock solutions of vitamin B9, LF and BLG

(purified and filtered) had no detectable turbidity before mixing.

Phase contrast microscopy

The formation of B9-WP coacervates in the coacervatesd solution was observed by

a phase contrast optical microscope (Olympus BX51TF, Olympus, Hamburg, Germany) set

at the magnification x40. A 10 µL sample of the B9-LF-BLG coacervatesd solutions was

taken, deposed on standard microscope slide, recovered by a coverslip (22 mm x 22 mm)

and immediately observed. The samples and observations were performed three times

on each solution to ensure the representability of the results.

Particle size distribution by laser light scattering

The size distribution of B9-WP coacervates in the coacervatesd solution prior to

centrifugation was determined by laser light scattering using a Mastersizer 2000

(Malvern instruments, Malvern, UK), with two different wavelengths (He/Ne laser:

633nm; electroluminescent diode: 466nm). The samples (about 0.2 to 0.5 mL of B9-WP

coacervates solution) were diluted in 100 mL of milliQ water at pH 5.5directly in the

measurement cell of the apparatus in order to reach 10% obscuration. The refractive

indexes used in this study were 1.52 for WP coacervates and 1.33 for water.

Quantification of B9 and proteins in the coacervates

B9 and total whey protein concentrations recovered in the B9-whey protein

coacervates were quantified using a PLRPS column (S/N 1006329-5, Varian Inc., Shropshire, UK connected to a Waters HPLC. MilliQ water containing . ‰ v/v of trifluoroacetic acid and an 80/20 acetonitrile/milliQ water (v/v) mixture containing . ‰ v/v of trifluoroacetic acid were used for elution. The absorbance was monitored

at 280 nm using a Waters 2487 detector. B9-WP coacervates were resuspended and diluted in solution of MilliQ water containing . ‰ v/v of trifluoroacetic acid. According to the dilution factor and the measured density of B9-WP coacervates, i.e. 1.356

g/mL, the B9 and WP concentrations were expressed in g/kg of B9-WP coacervates.

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Biocarrier efficiency: entrapment and coacervation yields

The efficiency of the B9-WP coacervates as biocarrier was characterized by two

yields: the entrapment yield of B9 in the coacervates and the coacervation yield of the WP.

These two yields were calculated according to the following equations:

Entrapment yield for B9 (%), 𝐸 𝑎 𝑦𝑖 = 𝐵9 𝑒 𝑒𝐵9 𝑖 𝑖 𝑖 𝑖 [ ]

cCacervation yield of the whey proteins (%), 𝑎 𝑎 𝑖 𝑦𝑖 = 𝑊𝑃 𝑒 𝑒𝑊𝑃 𝑖 𝑖 𝑖 𝑖 [ ]

where Q is the quantity of B9 (g) and whey proteins (g) recovered in the B9-WP

coacervates or in the initial solution. Q were calculated depending on the B9 whey

proteins concentrations recovered in the B9-WP coacervates and the total quantity of B9-

WP coacervates (g) recovered for each experiment.

1.2.3. Results and discussion

The optimal conditions for coacervation between BLG and LF and entrapment of

vitamin B9 were previously established at laboratory scale (Chapeau et al., 2016). These

conditions constitute the starting point for the present work.

Scaling-up the complex coacervation of whey proteins in the presence of B9

Effect of increasing the volume

The first step of scaling-up was the gradual increase of the mixing volume of

coacervatesd solutions to increase the quantity of B9-WP coacervates recovered after

centrifugation, without hindering the coacervation process. In fact, complex coacervation

process is known to be highly sensitive to volume and biopolymer concentrations (C.

Schmitt & Turgeon, 2011; C. Yan & Zhang, 2014). Firstly, coacervation phenomena were

compared in three solutions with total mixing volume of 0.1, 5 and 100 mL, using purified

and filtered whey proteins solutions as in (Chapeau et al., 2016).

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Figure III-8: A: Turbidity of B9-WP coacervates solutions at different mixing volumes for purified █ and commercial proteins samples ░). B: Visual difference between commercial (a) and purified BLG (b). C: Phase contrast microscopy (×40) of B9-WP coacervates obtained at different mixing volume and WP samples: purified WP solution (a) 0.1 mL, (b) 5 mL, (c) 100 mL; and commercial WP solutions, (d) 5 mL, (e) 100 mL.

Figure III-8A represents the final turbidity of the three coacervatesd solutions prior to

phase separation and centrifugation. For the three mixing volumes using purified and

filtered whey proteins, the turbidity values were high with similar magnitude around 3.3.

This high turbidity is characteristic of complex coacervation between whey proteins in

solution (Chapeau et al., 2016; Y. Yan et al., 2013b). The formation of coacervates in the

three solutions was attested by phase contrast microscopy (Figure III-8C a,b,c). The

formed coacervates appeared spherical, with a mean diameter between 5 to 10 µm,

similarly as previous results (Chapeau et al., 2016). After centrifugation, 0.23 mg of B9-

WP coacervates were recovered for the 0.1mL solution and 1g for the 100 mL solution,

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attesting that increasing the volume increased the recovered dense phase of B9-WP

coacervates. Figure III-9A shows the concentrations of B9 ([B9]) and whey proteins

([WP]) in the B9-WP coacervates recovered after centrifugation of the three coacervates

solutions. For the three mixing volumes, [B9] was between 5.0 and 5.5 g/kg and [WP] was

between 330 and 360 g/kg. So, [B9] and [WP] yields recovered in the B9-WP coacervates

can be considered constant, without significant impact of the mixing volume from 0.1 mL

to 100 mL. As a result, a 1000 fold increase of the mixing volume of B9, LF and BLG

solutions do not affect the complex coacervation between the three molecules. The B9-

WP coacervates recovered after centrifugation presented similar compositions for small

and high studied mixing volume.

Effect of the purity of WP solutions

The production of a high amount of coacervates requires the preparation of

important volume of WP solutions. Subsequent purification and filtration steps usually

performed at laboratory scale to remove residual small protein aggregates may constitute

a limit for a scaling-up purpose. Thus, it would be more efficient to perform heteroprotein

coacervation with commercial WP samples, without any purification and filtration of the

WP solutions. Nevertheless, it has been demonstrated that proteins aggregation may

impair and compete with coacervation in WP systems (Y. Yan et al., 2013b). To explore

this observation here, two types of LF and BLG solutions were tested:

- BLG was purified prior to reconstitution and then the LF and BLG solutions were

filtered to remove any aggregated proteins (as described in 2.2),

- LF and BLG solutions were directly reconstituted from commercial LF and BLG

powders, without any purification or filtration steps.

The presence of small aggregates in commercial BLG is clearly visible to the eye

(Figure III-8B). The turbidity value of non-purified and non-filtered BLG commercial

solution Figure III-8B a) was higher than that measured after purification and filtration

(Figure III-8B b). However, the turbidity of the coacervatesd solutions obtained with

commercial proteins solutions was equivalent to the ones obtained with purified and

filtered proteins solutions at both working volumes of 5 and 100 mL Figure III-8A).

Similar formation of B9-WP coacervates was observed for mixing solutions using

commercial WP, at 5 mL and 100 mL (Figure III-8C d, e) compared to B9-WP coacervates

obtained using purified and filtered WP solutions. Regardless the purity of the used BLG

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solution, similar concentrations of [B9] and [WP] was recovered in the coacervates

(Figure III-9A). Therefore, the presence of small amounts of aggregates in WP solutions

did not hinder the coacervation to occur under the present conditions. As a result,

complex coacervation of B9, LF and BLG can be performed at larger volume using

commercial protein samples without further purification steps.

Figure III-9: A: B9 and WP concentrations recovered in B9-WP coacervates at different mixing volumes and the type of whey proteins used, purified █ versus commercial protein samples ░). B: Entrapment, coacervation yields and total weight of B9-WP coacervates obtained at different mixing volumes with two types of whey proteins purified (█) versus commercial samples (░).

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B9 entrapment efficiency

As expected, the increasing mixing volumes allowed the increase of the recovered

weight of B9-WP coacervates (Figure III-9B). For 0.1 and 5 mL, the entrapment yield of

B9 was between 96 and 99 %. The same trend can be observed for the coacervation yield

of WP, with a high coacervation yield of WP at 50 % for 0.1 and 5 mL. These entrapment

and coacervation yields are in accordance with the previous results obtained by Chapeau

et al., (2016). Nevertheless, for the higher volume tested, the B9 entrapment and

coacervation yields decreased to 66 % and 35 %, respectively. Such yield decrease could

be linked to the processing parameters. It has been reported that shear, pressure or

residence time can impact greatly the complex coacervation process and the coacervation

yield (Lemetter et al., 2009; C. Yan & Zhang, 2014).

Effect of the solutions mixing mode

Mixing is defined as the reduction of inhomogeneity in order to achieve a desired

process result. Secondary effects such as chemical reaction and changes in product

properties are usually the critical objectives (Paul et al., 2004). In the present study, the

step of mixing was defined at the addition of BLG solution and the chemical reaction

required was complex coacervation (Figure III-7). At laboratory scale, the complex

coacervation between B9-LF intermediate solution and BLG occurred instantaneously

after the addition of the BLG solution. This was possible because the step of mixing

(homogeneous equilibration) of the solutions was also instantaneous due to the very

small volumes used. Increasing the mixing volume at fixed speed may impair the mixing

efficiency and consequently the coacervation yield. Thus, the mixing step appeared to be

crucial for the feasibility of a scale-up production of B9-WP coacervates. For this study,

the mixing system was defined as a liquid-liquid mixing system where the two liquids are

miscible together (two aqueous solutions). According to the literature, two types of

mixing can be considered for this system: a batch mixing by mechanic stirring and a

continuous mixing by static stirring (Paul et al., 2004).

Batch mixing by mechanic stirring

The set-up used to produce B9-WP coacervates by batch mixing using mechanic

stirring is described in Fig. 1B. An increasing speed of stirring was applied at the mixing

step between B9-LF and BLG solutions in a total volume of 100 ml: no stirring (0 rpm),

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low stirring (50 rpm) or high stirring (150 rpm). Above this last stirring value, the

formation of foam is observed on the top of the solution. This constitutes a limit for

coacervation scale up as already reported by (Lemetter et al., 2009). Due to a vortex

created by the propeller speed, air was incorporated into the system, inducing the

foaming of the protein solutions and prevented the occurrence of complex coacervation.

Figure III-10A presents the B9 and WP concentrations recovered in the

coacervates obtained at the three stirring rates. The concentration of the two entities and

the overall composition of the B9-WP coacervates were not affected by the applied

stirring rate. Interestingly, while the composition remained constant, increasing the

stirring speed induced an increase of the B9 entrapment and the coacervation yields

(Figure III-10B). Thus, B9 entrapment as well as the coacervation yield doubled when the

solution was stirred at 150 rpm. It has to be noted that stirring at 150 rpm allowed

reaching the maximum yields found at laboratory scale (Chapeau et al., 2016).

Figure III-10C presents the size of the B9-WP coacervates droplets recovered in

the coacervatesd solutions after the stirring step as measured by laser light scattering. For

unstirred solution, the coacervates droplets presented a heterogeneous size distribution

that evolved with post-stirring time. The droplets of the coacervatesd solution are highly

polydisperse but evolved to a rather homogeneous population of particles with a mean

diameter of 10 µm after 15 min (Figure III-10C a). At low stirring rate, the polydispersity

of the particles was lesser, with two main populations of droplets around 2 to 3 µm and

25 µm at T0 (Figure III-10C b). These two populations remained stable over time (Figure

III-10C b). At high stirring rate, the coacervates droplets were rather monodisperse, with

a mean diameter of particles of 2 to 3 µm and stayed stable overtime (Figure III-10C c).

These results can be explained by stirring-induced acceleration of the liquid-liquid phase

separation phenomenon throughout coalescence of the coacervates droplets. With no

stirring, the formation of droplets of B9-WP coacervates was spontaneous as soon as BLG

was added. The formed droplets were rather polydisperse and unstable in solution, as

illustrated by the schematic representation of Figure III-10C a. They tended to coalesce

leading to liquid-liquid phase separation overtime, characterizing the completion of the

complex coacervation.

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Figure III-10: Batch coacervation at different mechanic stirring. A: B9 and WP concentrations in B9-WP coacervates. B: Entrapment, coacervation yields and total weight of B9-WP coacervates, C: Particle size distribution obtained by laser light scattering and associated schematic representation of B9-WP coacervatesd solutions after (a) no stirring (0 rpm), (b) low stirring (50 rpm) or (c) high stirring (150 rpm). Measurements were performed at 0, 5 and 15 min after the end of the mixing step.

These observations were in agreement with other works performed with no

stirring but at small volume scale (Flanagan et al., 2015; Tavares, Croguennec, Hamon, et

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al., 2015b; Y. Yan et al., 2013b). Increasing stirring induced an increase of the coalescence

between the droplets of B9-WP coacervates. The size of resulting droplets of coacervates

became large enough to decant forming the concentrated phase of B9-WP coacervates, as

illustrated by the schematic representation of Figure III-10C b. At high stirring, it can be

supposed that the formation of the concentrated phase of B9-WP coacervates was

complete right after the two minutes of stirring. Only the smaller droplets of B9-WP

coacervates that were stable and in equilibrium with the dense phase remained in

solution, as illustrated by the schematic representation of Figure III-10C c.

As a result, increasing the stirring rate favored the complete phase separation of

the dense phase of B9-WP coacervates. Then, the B9-WP coacervates were fully recovered

by centrifugation. Thus, the B9-WP coacervation yield increased with the increase of the

mechanic stirring speed. The B9-WP coacervates obtained with high level of stirring

showed the highest biocarrier efficiency. This biocarrier efficiency was similar to the

biocarrier efficiency obtained at laboratory scale.

Continuous mixing by static stirring:

The set-up used to produce B9-WP coacervates by continuous mixing using static

stirring is described in Figure III-7C. Four flow rates were set for mixing the B9-LF and

BLG solutions: 50, 100, 200, and 300 mL/min. As stated above for mechanic stirring, the

maximum flow rate used was 300 mL/min to avoid the formation of foam in the solution.

Figure III-11A presents the turbidity of the B9-WP solutions obtained by continuous

mixing by static stirring depending on the flow of the initial solutions (mL/min), and using

commercial WP. At 50 mL/min, turbidity of B9-WP solution was low, around 1.05. Then,

the turbidity increased with increasing the flow rate to reach an optimum value at 200

mL/min. Microscopy analysis indicates the presence of aggregates at low flow rate (Figure

III-11B, image a). At flow rate > 200 mL/min only coacervates are observed (Figure

III-11B, image c). At 100 mL/min, the formation of B9-WP coacervates also occurred

(Figure III-11B, image b) but to a lesser amount than at higher flow rate. The formation of

aggregates rather than coacervates following a change in the processing conditions was

also reported for gelatin and gum Arabic mixture (Lemetter et al., 2009).

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Figure III-11: Continuous coacervation of B9-WP depending on initial flows. A: Turbidity of B9-WP solutions measured at different initial flows (mL/min) through the static mixer, B: Corresponding particle morphology prior centrifugation: (a) flow = 50 mL/min, (b) flow = 100 mL/min, (c) flow > 200 mL/min, C: B9 and WP concentrations in B9-WP coacervates, D: Entrapment of B9 and coacervation yield after centrifugation.

It was attributed to an insufficient level of stirring that induced an incomplete

mixing of the solutions preventing the occurrence of complex coacervation. As a result,

increasing flow rate favored complex coacervation between B9 and WP, with a critical

flow rate for coacervation around 100 mL/min.

Figure III-11C presents the B9 and WP concentrations recovered in the B9-WP

coacervates obtained by continuous mixing by static stirring at flow rates where

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coacervation was favored, and using commercial WP. B9 concentrations were relatively

constant varying from 5.3 to 4.9 g/kg of coacervates. WP concentrations in the

coacervates reached 400 g/kg under flow rates of 100 and 300 mL/min. A higher WP

concentration value of 465 g/kg was recovered under a flow rate of 200 mL/min. Figure

III-11D presents the entrapment and coacervation yields of the B9-WP coacervates. For

100 mL/min, the entrapment yield was around 65 % and the coacervation yield was low,

around 30 %. For 200 mL/min and 300 mL/min, the entrapment yield was around 97 %,

which is in agreement with previous results obtained at laboratory scale (Chapeau et al.,

2016). The coacervation yield increased from 48 to 65 % when the flow rate increased

from 200 to 300 mL/min. This coacervation yield was superior to the coacervation yields

obtained up to now for B9-WP coacervation. Hence, increasing the flow rate favored the

coacervation yield offering the possibility to entrap more B9 molecules throughout an

increase of initial B9 concentration.

Batch versus continuous mixing for scale-up production of B9-WP coacervates

We showed that without mixing, the entrapment yield for B9 in the coacervates

was 46 % and the coacervation yield reached only 24 %. These yields corresponded to a

low biocarrier efficiency, demonstrating that a mixing device is required for the scale-up

production of B9-WP coacervates. For batch mixing by mechanic stirring, the optimum

conditions were found for a stirring at 150 rpm, during 2 min. It lead to the formation of

B9-WP coacervates with a coacervation yield of 55 % and an entrapment yield of 97 %.

These yields demonstrated a satisfying biocarrier efficiency, equivalent to the biocarrier

efficiency obtained at laboratory scale. For the continuous mixing by static stirring, B9

entrapment yields up to 98 % were obtained at flow rates of 200 and 300 mL/min with a

highest coacervation yield of 65 % reached at 300 mL/min. Hence, the continuous mixing

by static stirring seems to be the best mode for LF-BLG coacervation and entrapment of a

hydrophilic bioactive molecule such as the vitamin B9. Moreover, the continuous mixing

by static stirring presents other advantages for coacervation at large scale production. For

the coacervation using polysaccharides or polyelectrolytes systems, a device by

continuous flow such as microfluidic can be successfully use to reach a good yield of

coacervates production and enable to control more precisely the amount of initial

biomaterials required (Simone, 2016; van Swaay, Tang, Mann, & de Mello, 2015). In that

regard, the static stirring that enable the B9-WP coacervates production by continuous

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flow can be viewed as more efficient than the batch mixing by mechanic stirring. Largely,

literature on industrial mixing present’s static stirring as highly engineered for

continuous process or reactions. Static stirring devices are presented as a good choice

when a high degree of homogeneity is required in a very short term (Etchells & Meyer,

2003).This characteristic applies to the coacervation process here as the formation of B9-

WP coacervates is known to be instant at the addition of BLG. As a result, the continuous

mixing by static stirring at a flow rate of 300 mL/min of initial solutions can be viewed as

an efficient device for the large scale production of B9-WP coacervates, offering the

possibility of high scale production for potential industrial application.

This results could open new potentialities for bench scale production of

coacervates. Indeed, until now, most of the coacervates systems used for encapsulation

required the use of particular solvent, even nonfood grade ones. The large scale

production of these systems often induced the use of large volume of these solvents, even

difficult to wash out completely afterwards (Sen & Pathak, 2016). With heteroprotein

complex coacervates this issue can be overcame. Furthermore, the high encapsulation

efficiency obtained here and the coacervation yields appear as very promising for an

encapsulation technology, even if very few comparative studies exist on the scale up

production of such systems (Paliwal, Babu, & Palakurthi, 2014).

1.2.4. Conclusion

This work has demonstrated that WP coacervates with entrapped significant

amount of B9 was produced at a large volume scale, showing the possibility of using the

coacervation of whey proteins to develop biocarrier for bioactives. The preparation of

initial protein solutions for coacervation was optimized for industrial implementation,

using commercially available protein samples. The feasibility of scaling-up the production

of B9-WP coacervates from µL scale to bench scale (1-2 liters) has been achieved. The next

step is to enlarge the volumetric range from bench to pilot scale. Static mixing appeared

to be an efficient device to produce B9-WP coacervates at a large scale and this equipment

is easily available in food plants. This device enabled to reach a coacervation yield of 65

% and a B9 entrapment of 98 %, which was equivalent to the biocarrier efficiency

obtained at laboratory scale, demonstrating an efficient scaling-up.

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2. Chapter 2:

Properties of vitamin B9-WP coacervates

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2.1. Part 1: Protective effect of WP coacervates

Preamble

Once the efficient production of vitamin B9-WP coacervates has been

demonstrated, next step is to explore the potential protective properties of B9-WP

coacervates. When incorporated into foods, bioactive molecules can undergo different

stress induced by drastic temperatures variations, UV light irradiations or oxidation. At

the end; theses degradations could impair the bioactive forms of the molecules resulting

in a loss of their bioactivity and potential health benefits. As hydrosoluble vitamins,

vitamin B9 is known to be quite sensitive towards environmental or process degradations

during food processing or storage. Compared to free B9, we explored the ability of WP

coacervates to reduce or prevent B9 degradations when exposed to a stress.

Main questions:

o Can WP coacervates protect B9 against degradations?

o What type of degradations can be limited by WP coacervates?

o Can WP coacervates prevent the loss of B9 bioactivity induced by degradations?

To explore the potential protective properties of WP coacervates, different

degradative conditions simulating degradations occurring during food processing and

storage have been studied: freezing and freeze-drying processes, UV light radiations,

oxidation, and longtime storage.

Main results:

WP coacervates are able to limit vitamin B9 degradations during freezing, freeze

drying, UV light irradiations, and oxidation.

By limiting the apparition of degradative products of vitamin B9, WP

coacervates maintain the native form of the vitamin, thus limiting the loss of its

overall bioactivity.

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Benefit 1 of heteroprotein complex coacervates: protection of vitamin

B9 against environmental degradations

The content of this part is based on results published in:

Chapeau A.L., Hamon P., Rousseau F., Croguennec T., Poncelet D., Bouhallab S. (2017). Scale-up production of vitamin loaded heteroprotein coacervates and their protective

property, Journal of Food Engineering, 206, 67-76;

Chapeau A.L., Bertrand N., Briard-Bion V., Hamon P., Denis Poncelet, Bouhallab S. (2017). Coacervates of whey proteins to protect and improve the oral delivery of

bioactive molecules, Journal of Functional Foods, (under submission).

Abstract

The potentiality of heteroprotein complex coacervates as efficient encapsulating agents

for bioactives was investigated regarding their potential protective property. Vitamin B9

(B9), was encapsulated by coacervation of the two whey proteins (WP), beta-

lactoglobulin (BLG) and lactoferrin (LF). Under degradative conditions in vitro (UV light

irradiation and accelerated oxidation by H2O2), WP coacervates protected B9 against

chemical degradations, compared to free B9. In addition, WP coacervates were able to

limit the loss of the bioactive form of B9 during a storage treatment (freeze-drying) and

also to limit the loss of bioactive B9 during a long time storage of one year. We thus

demonstrate the efficiency of heteroprotein complex coacervates to act as good

encapsulating agents for the protection of bioactives.

2.1.1. Introduction

To formulate functional foods, bioactive molecules like minerals, antioxidants,

omega-3 fatty acids or vitamins can be incorporated into food matrices (de Boer, Urlings,

& Bast, 2016). Yet, in industrial settings where processes and storage can affect stability,

solubility and bioavailability, the incorporation of these molecules and the preservation

of their biological properties remain challenging. In this context, encapsulating agents can

act as carriers or delivery systems to overcome these limitations (Shimoni, 2009). To

ensure industrial sustainability, such systems should be developed from natural food-

grade ingredients while using economical and reliable raw materials and processes

(McClements, Decker, Park, & Weiss, 2009). The design of natural food-grade carriers, thereafter called biocarriers represents an important source of innovation for current

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food technologies. By evaluating how coacervates prepared from whey proteins can

protect and increase the stability of a model bioactive molecule, the present study

highlights important parameters for a promising technology.

The formation of biocarriers throughout complex coacervation of a protein-

protein system, named heteroprotein complex coacervation, is a two-step process. First,

the bioactive is incorporated in a solution of protein A. Next, a solution of protein B,

carrying opposite electric charges, is added to the system. Driven by the electrostatic

interactions between the two proteins, complex coacervation occurs, resulting in the

formation of supramolecular co-assemblies of the two proteins called coacervates,

entrapping the bioactive. As coarcervation is mainly driven by electrostatic interactions,

the nature of the proteins, their stoichiometric ratios, as well as the pH and ionic strength

of the solutions all strongly affect the coacervation process (Croguennec, Tavares, &

Bouhallab, 2016; C. Schmitt & Turgeon, 2011). Thus, the optimal choice of proteins and

the methods for heteroprotein complex coacervation are still under investigation to

maximize the efficiency and benefits of the encapsulation process.

The optimal conditions for optimal encapsulation of a model bioactive, vitamin B9

(B9) by LF-BLG coacervates was previously studied (Chapeau et al., 2016). Building on

previous work which reported the coacervation of whey proteins at the bench scale level

(Chapeau et al., 2017), the results presented here show that the encapsulation step

contributes to the physicochemical stability of B9. To progress toward the complete

formulation of efficient biocarriers, we investigated here the protective properties of

heteroprotein complex coacervates under different stresses encountered during process

and storage of formulated produtcs: UV-light radiations, oxidation and storage in freeze-

drying conditions.

2.1.2. Material and Methods

Stock solutions

B9 stock solutions at 1 g/L were prepared by solubilizing folic acid powder (purity

> 97%, Sigma Aldrich, St. Louis, MO, USA) in aqueous NaOH solution (pH > 8). pH was

adjusted to pH 5.5 using 1 M HCl solution, then the preparation was centrifuged at 28,000

g for 30 min at room temperature (centrifuge Heraeus Biofuge Primo, Thermo Scientific,

Waltham, MA, USA) to remove the insoluble fraction of B9. The exact vitamin B9

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concentration was determined by absorbance at 283 nm (spectrometer UVmc2, Safas,

Monaco) using 25.1 L·g-1·cm-1 as the extinction coefficient.

LF and BLG stock solutions were prepared by solubilizing protein powders ( LF

from bovine milk, purity 90% and iron saturation level of 10% to 20%, Fonterra

Cooperative Group, New Zealand, and BLG from a confidential industrial source) in

ultrapure water adjusted to pH 5.5 using 1 M HCl. The exact protein concentrations of LF

and BLG (16.6 and 18.3 g/L, respectively) were determined by absorbance at 280 nm

(spectrometer UVmc², Safas, Monaco) using 0.96 L·g-1·cm-1 and 1.47 L·g-1·cm-1 as

extinction coefficients, respectively.

Preparation of B9-WP coacervates

Coacervates of B9 and whey proteins (B9-WP) were prepared as described

previously (Chapeau et al., 2017). A solution containing 0.11 g/L of B9 and 4.15 g/L of LF

was prepared, and equilibrated for 10 min, at room temperature. Then, an equivalent

volume of the BLG solution was added (final BLG concentration of 9.15 g/L). Spontaneous

coacervation occurred and the B9-WP coacervates phase was recovered by centrifugation

at 38,000 g for 45 min at 20°C (Centrifuge Avanti J-26S XP, Beckman Coulter Inc., Brea, CA

92821 USA). The recovered pellet had a final protein concentration of 26% w/w, with a

2:1 ratio of BLG to LF, and a final B9 loading of 0.04 % w/w. Coacervates of the two whey

proteins without B9 were also prepared and used as control when required.

Photodegradation

Method

Photodegradation was induced according to a method adapted from (Akhtar, Khan,

& Ahmad, 1999). A Philips 30W TW tube was used to expose experimental solutions to

UV-light radiation, simulating a daylight exposure. The UV tube emits 88.7% of its

radiation energy at 254 nm, corresponding to one of the absorption maxima (256 nm) of

B9. The experimental solutions contained B9 at 0.4 g/L alone, in combination with LF

(4.15 g/L) or as B9-WP coacervates (as described above). Aliquots of 500µL in 1.5 mL

microcentrifuge tubes were irradiated with UV-light, while controls were kept in the dark,

at 20 °C. After 0, 8, 24 or 48 hours, two aliquots of each solutions were withdrawn and

analyzed by reverse-phase high performance liquid chromatography (RP-HPLC), and

mass spectrometry analysis.

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RP-HPLC analysis

B9 and derived products were analyzed by reversed-phase high-performance

liquid chromatography (RP-HPLC). Briefly, FA was separated on a PLRPS column (S/N

1006329-5, Varian Inc., Shropshire, UK) connected to a Waters 2695 HPLC system.

Elution was performed using a gradient of solvent (acetonitrile 80%, water 20%,

trifluoroacetic acid 0.1%) in water containing 0.1% trifluoroacetic acid. Detection was

performed by absorbance at 283 nm. B9 degradation products, P-aminobenzoylglutamic

acid (Pa) and a Pteridine residue (Pr), were identified by mass spectrometry as described

below.

Mass spectrometry analysis

Electrospray mass spectrometer operating in positive ion mode with a voltage of

4kV one-line with RP-HPLC was used for the identification of B9 degradation products.

Spectra were recorded in full MS mode and selected in a mass range 130-2000 m/z for MS

spectra with a resolution of 70 000 at m/z 200. The instrument was externally calibrated

according to the supplier's instructions. Ions recovered in the sample were analyzed from

the MS spectra using Xcalibur 2.2 Software (Thermo Scientific, San Jose, USA). The specific

ions of B9 and its photoproducts, Pr and Pa, were identified by their respective molecular

weights: B9 (Mw = 442 Da), Pr (Mw = 267 Da) and Pa (Mw = 192 Da) (Patring &

Jastrebova, 2007; Santos, Scurachio, & Cardoso, 2014). Experiments were conducted in

triplicates.

Oxidative degradation

Oxidative degradation was induced by exposure to hydrogen peroxide (H2O2).

Experimental solutions containing 1 g/L of B9 alone or as or as B9-WP coacervates were

prepared. Solutions of B9 (1 g/L) with either LF (27.8 g/L) or BLG (55.2 g/L) were also

used. In each solution, H2O2 (30% v/v, Sigma Aldrich, St. Louis, MO, USA) was added to

reach final concentrations of 0.33 g/L B9 and 20% (v/v) H2O2. Samples were incubated

at 37°C in the dark. At 0, 2, 4, 6, and 18 hours after the addition of H2O2, the remaining

residual concentration of B9 in the solution were quantified by the enzyme linked

immune assay (ELISA) method. Experiments were conducted in triplicates.

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Freeze drying and storage

It was chosen to evaluate the effect of a storage treatment on the stability of B9 in

the WP coacervates by a freeze drying method. The samples were freeze-dryed using a

freeze-dryer (RP2V-22304, S.G.D Group, 95100 Argenteuil, France). A 0.5g aliquot of B9-

WP coacervates was placed in plastic tubes and was frozen at -25°C in 50 minutes. The

frozen samples were dried under vacuum conditions for a night. Freeze dryed B9-WP

coacervates were stored for one year at room temperature, in dark conditions. B9

quantifications in freeze dryed B9-WP coacervates were realized by enzyme-linked

immunosorbent assay after resuspension.

B9 quantification by enzyme-linked immunosorbent assay

Concentrations of B9 were determined using a competitive ELISA (Vitamin B9

Elisa Kit 96T, Abbexa, Cambridge, UK). Each sample was analyzed in technical duplicates

(with no dilution or a 0.5 dilution in Abbexa kit diluent buffer). Appropriately diluted

samples (50 µL) were added into each well of the microplate pre-coated with B9. Then,

50 µL of biotinylated anti-B9 antibodies were added into each well. The microplate was

incubated for 45 min à 37 °C, then washed three times with the Abbexa washing solution.

Then, 100 µL of horseradish peroxidase (HRP) streptavidin solution was added into each

well, followed by incubation for 30 min à 37 °C. The microplate was washed five times

with the washing solution. For quantification, 90 µL of tetramethylbenzidine substrate

was added into each well and the microplate was incubated for 15 min in the dark at 37°C.

The reaction was stopped by the addition of 50µL of Abbexa stop solution. Finally, the

absorbance of each well was measured at 450 nm using a spectrophotometer microplate

reader (NanoQuant infinite M200 Pro, Tecan, Switzerland). For each analysis, a

calibration curve was performed over the range of 1000 to 12.5 ng/mL (R² > 0.98). Under

these experimental conditions, the limit of quantification of this method was calculated to

be 7.5 ng/mL.

2.1.3. Results

To investigate the ability of WP coacervates to limit the degradation of B9,

unencapsulated B9, mixtures of B9 with LF and BLG and B9-WP coacervates were

exposed to either UV-light (photodegradation) or H2O2 (oxidative degradation). These

accelerated degradation conditions provided surrogates for processes occurring during

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food transformation or long time storage. Additionally, a standard storage treatment of

freeze drying, followed by a year of storage was applied on samples to followed B9

degradations in this conditions that could potentially be found in industry.

Photodegradation

In aqueous environments, UV-light converts B9 into two main photoproducts, a

pteridine residue (Pr) and a P-aminobenzoylglutamic acid (Pa), with the capture of a

water molecule according to the following chemical scheme [1] (Jamil Akhtar, Ataullah

Khan, & Ahmad, 2003; Off et al., 2005):

Figure III-12 shows the elution profiles of B9 of the three treated samples after

exposure to UV light for 0, 8 or 24 hours. On shown, native B9 had a retention time of 3.3

min (Figure III-12A, B, C). When B9 was not encapsulated (i.e., solutions of B9 alone or in

complex with LF), degradation products were evidenced after 8 hours of irradiation with

the apparition of a peak with a retention time of 2.7 min. The relative abundance of this

peak increased after 24 hours of irradiation for treated B9 alone or in complex with LF

with concomitant decrease of B9 peak (Figure III-12A, B). Mass spectrometry indicated

the presence of ions with molecular masses, m/z of 442, 267, and 192 corresponding to

B9 and its two main degradation products Pr and Pa, respectively. In the absence of UV

light, these two ions were not detected suggesting that they origin from the

photodegradation of B9. The formation of these products from B9 has been already

reported by Santos et al. (Santos et al., 2014). Interestingly, when B9 was encapsulated in

WP coarcervates, the two degradation products were not detected (Figure III-12C),

suggesting the WP coacervates can mitigate the degradation of B9 induced by UV light.

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Figure III-12: Evolution of RP-HPLC profiles of vitamin B9 after exposure to UV light during 24 h. Vitamin B9 was treated with UV either alone in solution (A), pre-complexed with lactoferrin (B) or encapsulated in whey protein coacervates (C) .

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The relative degradations of B9 alone or protected in WP coacervates with and

without UV irradiation was assessed by mass spectrometry analysis and is depicted in

Figure III-13.

Figure III-13: Protective effect of WP coacervates against UV light induced degradation of vitamin B9. Ion intensities of B9 and derived products from electrospray mass spectrometry analysis before and after 48 h exposure to UV light.

Based on the initial ion intensity, about 90% of free B9 disappeared after 48 hours under

UV light with simultaneous appearance of degradation products Pr and Pa (see the

scheme above). Meanwhile, despite a slight decrease in B9 ion intensity, the untreated B9

solution stored in the dark was stable (Figure III-13). Encapsulation into WP coacervates

limited the observed degradation of B9. More than 50% of initial B9 (initial ion intensity)

were still detected after 48h of UV light treatment. This was also confirmed by a lower

detection of the ions corresponding to the degradation products Pr and Pa.

Oxidative degradation

Hydrogen peroxide was used to accelerate oxidative degradation, an efficient

method to study drug decomposition in pharmaceutical sciences (Blessy, Patel, Prajapati,

& Agrawal, 2014). Figure III-14 presents the remaining quantities of B9 in solutions (B9

alone or in complex with LF) and encapsulated into WP coacervates after exposure to 20

% H2O2, at 37 °C, for 18 hours.

0

20

40

60

80

100

B9 unencapsulated B9-WP coacervates B9 unencapsulated

no UV

AU

(m

V %

)

B9 UV-0h B9 UV-48h Pa UV-48h Pr UV-48h

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Figure III-14: Degradation kinetics of free, complexed or encapsulated vitamin B9 after exposure to 20% hydrogen peroxide at 37°C for 18h.

In these experiments, the ELISA assay allowed quantification of only the native form of B9

(without interference from H2O2). For all the samples, native B9 decreased over time, yet

the kinetics were different depending on whether B9 was free, in complex with LF, or

encapsulated in WP coacervates. High degradation rate was found for B9 either in free

form or in complex with LF. In these samples, degradation of about 75 % on the initial

vitamin was found after 18 h exposure to H2O2. Hence, complexation of B9 with LF does

not seems to be enough for efficient protection. In contrast, the encapsulated B9 exhibited

much slower degradation rate underlying a protective effect. Approximately, 80 and 70 %

of the initial B9 into WP coacervates ware quantified after 6 and 18 h of treatment,

respectively.

Storage treatments

Vitamin B9 is known to be sensitive to degradation when frozen (Czarnowska &

Gujska, 2012), and to some extent when freeze-dried, resulting in a loss of the vitamin’s bioactivity (Lucock, 2000). To evaluate the protective effect of B9-WP coacervates, a

freeze-drying treatment representative routinely applied in industry for the

lyophilization of an ingredient was performed. Figure III-15 presents the evolution of B9

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concentrations, of solutions of unencapsulated B9 or B9-WP coacervates after freeze

drying and after one year of freeze-dried storage. It has to be noted that B9 quantifications

was performed by ELISA, guaranteeing the dosage of only B9 in its native bioactive form.

Figure III-15: B9 concentrations before, after freeze-drying, and after a one year storage in uncapsulated B9 or B9-WP coacervates.

Initially, B9 concentrations in unencapsulated B9 solution and in B9-WP coacervates were

similar, at 4.8 g/kg (± 0.15). After freeze-drying, the concentration of B9 in B9

unencapsulated solution decreased to 4.2 g/kg (± 0.11), corresponding to a B9 loss of

12.5 %. In the B9-WP coacervates, the B9 concentration after freezing were at 4.5 g/kg

(± 0.12), corresponding to a B9 loss of 6.25 %. After one year of storage of the freeze dried

samples, 3.5 g/kg of B9 were recovered in the unencapsulated B9 solution corresponding

to a B9 loss of 17 % in one year. For B9-WP coacervates, 4.3 g/kg of B9 was recovered in

the freeze dried coacervates after one year of storage, corresponding to a limited loss of

only 5 %.

2.1.4. Discussion

Vitamin B9 plays a crucial role in various metabolic and biochemical processes

such as the replication of DNA and cell division, notably during the early stages of

embryology (Herrmann & Obeid, 2011; Lucock, 2000). This bioactivity is mostly due to

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

5,0

Native Freeze-drying T0 Freeze-drying 1 year

storage

[B9

] (

g/k

g)

Unencapsulated B9

B9-WP coacervates

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the native form of B9 (Gazzali et al., 2016; Hirakawa, Suzuki, Oikawa, & Kawanishi, 2003;

Patro, Adhikari, Mukherjee, & Chattopadhyay, 2005). To prepare biocarriers that

maintain the bioactivity of the vitamin, it is therefore critical to preserve its chemical

integrity.

Under accelerated degradation conditions, unencapsulated B9 was efficiently

converted into various degradation products. B9 can be photochemically cleaved, yielding

6-formylpterin (Pr) and p-aminobenzoylglutamic acid (Pa) (Araújo et al., 2015; Thomas,

Suárez, Cabrerizo, Martino, & Capparelli, 2000). This photodegradation preferentially

occurs in acidic solutions and in the presence of oxygen (Thomas et al., 2000). Although

B9 is known to remain mostly stable in aqueous solution at 37 °C (Tripet & Kesselring,

1975; Vora, Riga, Dollimore, & Alexander, 2002), addition of H2O2 significantly increases

oxidative degradation via the production of singlet oxygen (Gazzali et al., 2016; Thomas

et al., 2000). These oxidative conditions allowed degradation of native B9 forming

peteridin product that was identified by mass spectrometry. In our conditions,

unencapsulated B9 degraded within few hours. Oppositely, under both UV light

irradiation and oxidative conditions, WP coacervates significantly hindered the

degradation of B9. After up to 48 hours of exposure to UV light, few degradation products

were detected for the encapsulated vitamin, in stark contrast with the degradation

observed for free B9. Although small fluctuations in concentration were observed over

time, these differences were similar in samples that were kept in the dark; therefore, these

changes can hardly be ascribed to photodegradation. The observed protective effect of the

coacervates could be attributed to decreased contact between the vitamin, entrapped in

a hydrophobic environment of the proteins, and water or oxygen molecules, with about

0.4 g B9 being protected in highly concentrated environment with nearly 260 g proteins

per kg of coacervates. Similar protective effects have been reported for B9 bound to BLG

at neutral pH, essentially by decreasing the effective concentration of all reagents

involved in the photodecomposition reaction (Liang, Zhang, Zhou, & Subirade, 2013).

The encapsulation of B9 in WP coacervates also strongly limits its H2O2 induced

oxidation. After 6 hours of oxidative stress, more than twice as much native B9 was still

detectable in the sample containing B9-coacervates compared to free B9. After 18 hours,

more than 60% of the initial vitamin remains detectable in the coacervates (compared to

less than 30% for treated free B9). B9-LF complex showed intermediate protective effect

toward oxidative degradation, underlying that the WP coacervates present the more

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efficient protective effect. Once again, the encapsulation of the vitamin within a

dehydrated protein complex would limit its reactivity toward the solution containing

singlet oxygen. It is also conceivable that the protein could capture singlet oxygen and act

as a buffering antioxidant to preserve the vitamin.

Regarding the application of a freeze drying treatment, comparable protective

effect have been found, limiting the degradation of the native form of B9 during freeze

drying and during a one year storage at room temperature. Similarly, this can be explained

by the presence of WP coacervates inducing higher concentrations of proteins present in

the samples. This observed protective effect was also found for WP capsules formed by

electrospraying for another bioactive compound, the beta-carotene (López-Rubio &

Lagaron, 2012). As a results, WP coacervates were able to maintain a higher amount of B9

in its native form compare to unencapsulated B9 during the storage of freeze dried

samples, thus guaranteeing to maintain a higher amount of B9 in its bioactive form.

2.1.5. Conclusion

Herein, WP coacervates have demonstrated their capacity to protect a model

bioactive molecule, the vitamin B9, from chemical degradations usually occuring during

food storage (UV light radiations, oxidations) or to protect the vitamin during a food

process like freeze drying. By protecting the native chemical form of the vitamin and

limiting the apparition of degradative products, the WP coacervates are able to maintain

the native form of the molecule and thus preserve its bioactivity closely linked to its

bioactive molecular form. As a result, WP coacervates fulfill interesting attribute for an

encapsulating agent, demonstrating good potentiality for further biocarrier development.

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2.2. Part 2: Bioavailability of vitamin B9 from WP coacervates

Preamble

Besides protection, another objective of encapsulation is to better control the

release of the encapsulated molecule. For a bioactive molecule, the need is to ensure its

bioavailability after absorption and digestion. The bioavailability of a compound is

defined by the European Agency for Evaluation of a Medical Product as the rate at which

a substance or its active moiety is delivered from a pharmaceutical/food form and becomes

available in the general circulation”. In the present case, it means the rate of vitamin B9

resulting from the absorption and digestion of the B9-WP coacervates and consequently

the amount of vitamin B9 available in the blood circulation. Moreover, it is necessary to

ensure that vitamin B9 available is the blood circulation has maintained its bioactive form,

essential to maintain its metabolic properties.

Main questions:

o Is the vitamin B9 bioavailable in its bioactive form after the absorption of B9-

WP coacervates?

o Are the B9-WP coacervates able to enhance the bioavailability of B9 compared

to B9 absorbed alone?

The bioavailability of B9 from B9-WP coacervates was explored in vivo. A

pharmacokinetic study was conducted on rats to compare the bioavailability of B9

administrated orally either free in solution or encapsulated in the WP coacervates.

Main results:

WP coacervates are able to enhance the overall bioavailability of B9 compare to

B9 administrated free in solution.

The vitamin B9 bioavailability maintain its overall bioactive form and thus its

metabolic activity.

The enhanced bioavailability from B9-WP coacervates could be explained by the

protective effect of the coacervates and their ability to enhance the solubility of

B9 thus facilitating its absorption towards the gastro intestinal tract.

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Benefit 2 of heteroprotein complex coacervates: increasing the

bioavailability of vitamin B9 during in vivo rats’ digestion.

The content of this part is based on results published in:

Chapeau A.L., Bertrand N., Briard-Bion V., Hamon P., Denis Poncelet, Bouhallab S. (2017). Coacervates of whey proteins to protect and improve the oral delivery of bioactive

molecules, Journal of Functional Foods, (submitted).

Abstract

The potentiality of heteroprotein complex coacervates as efficient encapsulating agents

for bioactives was investigated regarding their delivery property. Vitamin B9 (B9), was

encapsulated by coacervation of the two whey proteins (WP), beta-lactoglobulin (BLG)

and lactoferrin (LF). After an oral administration of B9-WP coacervates to healthy rats an

enhanced plasma level of vitamin B9 was obsersved in rats’ blood samples compared a

free B9 administration. This improved bioavailability can be ascribed to the improvement

in the solubility of B9 throughout the gastro-intestinal tract. We thus demonstrate the

efficiency of heteroprotein complex coacervates to act as good encapsulating agents for

the delivery of bioactives. This constitutes a first conclusive preliminary test to explore

the digestion of loaded heteroprotein coacervates. To go further, in vitro digestions

methods can now be used to better understand the mechanisms involved.

2.2.1. Introduction

In recent years, the interest in functional foods and nutraceuticals has been

motivated by their potential to contribute to healthy lifestyles. Their ability to enhance

the daily intakes of specific nutrients and prevent deficiencies can possibly reduce the

risks of certain diseases. (Annunziata & Vecchio, 2011). To formulate functional foods,

bioactive molecules like minerals, antioxidants, omega-3 fatty acids or vitamins can be

incorporated into food matrices (de Boer et al., 2016). Yet, the incorporation of these

molecules into foods and the preservation of their biological properties remain

challenging. For instance with the vitamin B9, and as many other bioactives, this vitamin

is poorly soluble in acidic aqueous solutions. Moreover the relative instability and

precipitation of B9 in some food matrices and in the gastro-intestinal tract might comprise

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its overall oral bioavailability (McNulty & Pentieva, 2004). The encapsulation of B9 in WP

coacervates might therefore provide an interesting alternative to enrich acidic foods

while preserving the bioavailability of the vitamin. To further progress towards the

complete formulation of an efficient biocarrier, we investigated the ability of WP

coacervates to promote the gastrointestinal absorption of B9 in rats.

2.2.2. Material and Methods

Stock solutions and B9-WP coacervates

Stock solutions of B9, BLG and LF or B9-WP coacervates were prepared as

previously described in the section III.2.1.2. Briefly, B9 stock solutions at 1 g/L were

prepared by solubilizing folic acid powder (purity > 97%, Sigma Aldrich, St. Louis, MO,

USA) in aqueous NaOH solution (pH > 8) and pH was adjusted to pH 5.5 using 1 M HCl

solution. LF and BLG stock solutions were prepared by solubilizing protein powders in

ultrapure water adjusted to pH 5.5 using 1 M HCl. For the fabrication of B9-whey

proteines (B9-WP) coacercates, a solution containing 0.11 g/L of B9 and 4.15 g/L of LF

was prepared, and equilibrated for 10 min, at room temperature. Then, an equivalent

volume of the BLG solution was added (final BLG concentration of 9.15 g/L). Spontaneous

coacervation occurred and the B9-WP coacervates phase was recovered by centrifugation

at 38,000 g for 45 min at 20°C (Centrifuge Avanti J-26S XP, Beckman Coulter Inc., Brea, CA

92821 USA).

In vivo pharmacokinetic studies in male rats

Ethics

All experiments were performed in accordance with the Canadian Council on

Animal Care and were approved by the Institutional Committee of the CHU de Quebec

Research Center (CHUL) (protocol number 2015074-2).

Tested solutions

The 4 solutions used in this experiment were prepared by dissolving B9, BLG and

LF or B9-WP coacervates in PBS. The concentration of B9 in all solutions was set at 1 g/L,

while the concentrations of BLG and LF were 55.2 and 27.8 g/L, respectively. Coacervates

contained both proteins at these concentrations.

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Pharmacokinetic studies

Pharmacokinetic studies were performed in male Sprague Dawley rats (350 g to

450 g) obtained from Charles River (St-Constant, QC, Canada) and housed in 12 h

light/dark cycles at 20 ± 2 °C and 55-60 % relative humidity. Prior to the experiment,

animals were fed ad libitum for a minimum of 10 days with a folic acid deficient diet (TD

95247, Envigo Teklad Diets, Madison Wisconsin, USA) and free access to drinking water.

For the experiment, rats were randomly divided into 4 groups of 5 animals each. Each

group was administrated a specific PBS solution: B9, B9-WP coacervates, B9 and BLG or

B9 and LF. Each animal received a dose of 500 µg of B9. Solutions were administered using

a blunt needle via the esophagus into the stomach. Just before the oral administration (0

min) and at different times post administration (0.5, 1, 2, 4, 6 and 10 h), 200 µL blood

samples were collected via the saphenous vein using capillary tubes (Microvette

CB300K2E, Sarstedt, Germany). Blood samples were centrifuged at 2 000 rcf for 15 min

at 4 °C, and the isolated plasma was kept in microcentrifuge tubes at -20 °C until further

analysis (5-7 days). Concentrations of B9 recovered in rat plasma were determined by

ELISA assay.

Pharmacokinetic parameters

The maximal B9 concentration in rat plasma (Cmax) and the time taken to reach Cmax

(Tmax) were obtained from experimental data. The area under the plasma concentration

vs. time curve from time 0 to 10h (AUC) was calculated using the trapezoidal method.

Statistical analysis

A series of ANOVA followed by post-hoc Tuckey tests were performed to

investigate potential statistical differences between the pharmacokinetic parameters. In

all cases, p < 0.05 was considered to be statistically significant.

2.2.3. Results

Figure III-16 shows the pharmacokinetics of B9 concentrations recovered in

plasma after a single oral gavage of rats with either B9 alone, B9 complexed with

lactoferrin, B9 in the presence of BLG or encapsulated in WP coacervates.

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Figure III-16: Pharmacokinetics of vitamin B9 concentrations in rat plasma vs time after a single oral administration at T0 of B9, B9-WP coacervates, B9-BLG, or B9-LF solutions corresponding to a dose of 500 µg B9 / oral administration. Values represent mean +/- SD, n = 5.

For free B9 group, the B9 level in the plasma of animals increased rapidly during the two

first hours post-administration followed by a phase where the detected vitamin decreased

slowly until the end of the experiment. The others groups displayed similar profiles.

Nevertheless, the plasma level of B9 from B9-WP coacervates was significantly higher

than that found for groups receiving free B9. The plasma level of B9 in the other groups

(B9+LF and B9 + BLG) exhibited intermediate levels. Table 7 summarizes the

pharmacokinetic parameters (Cmax, Tmax, and AUC) obtained for each group.

Maximum concentrations were obtained within 1.8 to 3 hours of gavage. The group

receiving B9-WP coacervates showed the highest Cmax value and the largest total blood

exposure (i.e., AUC). The B9-WP coacervates group provided a blood exposure which was

1.5-fold higher than the free B9 solution (5868 vs. 3804 ng·h·mL-1). Theses parameters

were also slightly higher with WP coacervates compared to proteins taken individually at

the same concentration.

0

200

400

600

800

1000

1200

1400

0 2 4 6 8 10

[B9

] pla

sma

(ng

/mL)

Time (h)

B9

B9-WP coacervate

B9-BLG

B9-LF

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122

Table 7: Characteristic parameters of pharmacokinetics of vitamin B9 concentrations in rat plasma vs time after a single oral administration at T0 of B9, B9-WP coacervates, B9-BLG, or B9-LF solutions corresponding to a dose of 500µg B9/oral administration. Statistical analysis by bilateral T-Test, a, b, c, statistically different at a significance level of p < 0.05.

Cmax

(ng/mL)

SD

(ng/mL)

Tmax

(h)

SD

(h)

AUC

(ng.h/mL)

SD

(ng.h/mL)

B9 872 a ± 49 1.8 a ± 0.3 3804 a ± 190

B9-BLG 942 ab ± 95 2.8 b ± 0.3 5108 ab ± 602

B9-LF 1054 bc ± 77 1.8 a ± 0.3 4516 ab ± 496

B9-WP coacervates

1162 c 94 1.8 a ± 0.3 5868 b ± 574

2.2.4. Discussion

Pharmacokinetic experiments in rats demonstrated that the encapsulation of B9 in

coacervates increased the overall bioavailability of the vitamin, notably by increasing the

maximum concentrations measured in blood after a single oral dose. This could be

explained by the ability of coacervates to increase the apparent solubility of B9, especially

at the acidic pH values found in the stomach. It has already been reported that the binding

between B9 and a whey protein (BLG) can increase the water solubility of the vitamin

(Liang & Subirade, 2010). Since molecules require to be dissolved to be absorbed through

the gastro-intestinal wall, any interactions preventing the precipitation of B9 could be

beneficial to increase bioavailability. Similar results were obtained with egg white

proteins and B9 (Arzeni et al., 2015), as well as with curcumin and BLG (Teng, Li, & Wang,

2014), and more generally with many pharmaceutical formulations (Saha, 2010). The

intrinsic resistance of BLG to the gastric enzyme pepsin (Mandalari et al., 2009; Reddy,

Kella, & Kinsella, 1988), combined with a rapid proteolysis during duodenal digestion, can

increase the bioavailibity of B9 during the digestion of B9-BLG complexes (Pérez, David-

Birman, Kesselman, Levi-Tal, & Lesmes, 2014b). These observations agree with the

present pharmacokinetic data indicating longer times taken to reach maximal

concentrations (Tmax) and potentially delayed adsorption when B9 is administered in

presence of BLG alone.

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Considering the results obtained in vivo, it is conceivable that proteins, including

BLG, LF and coacervates, could protect B9 from gastric degradations. Penalva et al.

suggested that the delayed release of B9 from casein nanoparticles in acidic environment

could prevent degradation of the vitamin and improve its oral bioavailability (Penalva et

al., 2015). Herein, the improved protection observed in vitro, added to the differences in

pharmacokinetic measured between WP coacervates and simple protein solutions,

suggests the WP coacervates architecture provides benefits over plain interactions

between vitamin and proteins. This study constitutes a first conclusive preliminary test

to explore the digestion of loaded heteroprotein coacervates. To better understand the

role of WP coacervates and their kinetics of degradations during digestion, it would be

relevant to undertake in vitro digestions methods.

2.2.5. Conclusion

This overall work studying the properties of B9-WP coacervates evidenced that

WP coacervates could protect a bioactive molecule from chemical degradation in vitro and

enhance its bioavailability in vivo. This is of great interest for the development of natural

and functional ingredient to protect and ensure the controlled delivery of a bioactive

compound. To go further on that matter, it will be interesting to study the feasibility of

encapsulating other types of bioactives within the WP coacervates and to investigate the

incorporation of WP coacervates into a complex food matrix.

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3. Chapter 3:

Generalization of the concept of the formulation of natural and

functional dairy encapsulating agents

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125

WP coacervates as versatile encapsulating agents for food applications.

Preamble

The present study was carried out using vitamin B9 as a model hydrophilic

bioactive to explore the formation and properties of WP coacervates as encapsulating

agents. To validate the proof of concept that WP coacervates can be used as versatile

encapsulating agents, we tested the ability of WP coacervates to encapsulate other types

of bioactives with different physicochemical properties. In view of potential applications,

the WP coacervates could be directly used as an ingredient and, in that case, its freeze-

drying should be optimized; or the WP coacervates could be solubilized in a food matrix

and, in that case, the WP coacervates should remain stable and maintain a relative

entrapment for the bioactive after its incorporation into the matrix.

Main questions:

o Are WP coacervates able to encapsulate different types of chemical compounds?

o Are WP coacervates resuspendable in a food matrix?

o Are WP coacervates stable when resuspended in a food matrix?

The formation of WP coacervates with different chemical compounds was tested.

Giving the context of this study, only molecules with recognized bioactive effects were

considered. Furthermore, the resupension and stability of WP coacervates were tested in

dairy solutions.

Main results:

LF-BLG coacervation is achievable in the presence of different chemical

compounds, with acceptable coacervation yields.

WP coacervates are able to encapsulate various types of bioactive compounds.

The resuspension of WP coacervates in small droplets dispersed in solution is

possible but seems to be facilitated by the ionic strength of the solution.

B9-WP coacervates resupended in dairy solutions are stable and partially retain

their load of B9.

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3.1. Introduction

A large variety of bioactives can be incorporated into foods for the purpose of

developing functional foods. According to their chemical structure, their physicochemical

properties and their health benefits, different classes of bioactives have been identified,

including vitamins, polyphenols, fatty acids and peptides. These bioactives present their

own chemical structure and physicochemical characteristics. In that regard, it appears

interesting to test different compounds for encapsulation by the WP coacervates. Our

hypothesis is that the structure and physicochemical properties of small bioactives may

impact the coacervation process and/or the encapsulation yields by the WP coacervates.

In the present study, the hydrophilic vitamin B9 was used to investigate whey

protein coacervates for encapsulation purposes in detail. This bioactive is soluble in water

at alkaline pH and has a negative electric charge (Table 8). Vitamin E was chosen as the

hydrophobic model molecule. Vitamin E is actually a group of vitamins composed of molecules called tocopherols, the major one of which is -tocopherol. -tocopherol has a

molecular mass of 441.4 g/mol, close to that of vitamin B9 (Table 8). This vitamin is not

soluble in water but is soluble in ethanol and carries no specific electric charges. In

addition, vitamin E has interesting anti-inflammatory and antioxidant properties and is

little synthesized by the body (Somchue, Sermsri, Shiowatana, & Siripinyanond, 2009).

Therefore, its daily intake must be guaranteed through diet or supplementation. To cover

other types of bioactives, it appears interesting to test phenolic compounds such as

bioactive polyphenols. Polyphenols constitute a large family of biomolecules, mainly of

plant origin, characterized by the presence of several phenolic groups associated in more

or less complex structures. These compounds are generally known for their antioxidant

properties and could thus reduce the risk of cardiovascular disease and diabetes (Sabliov,

Chen, & Yada, 2015). Nevertheless, some polyphenols are barely soluble and stable when

incorporated into foods, inducing a poor bioavailability of these compounds (Fang &

Bhandari, 2010). As a result, these compounds could also benefit from encapsulation by

WP coacervates. To investigate this matter, we tested two polyphenols: resveratrol and

an anthocyanin, cyanidin-3-O-glucoside (C3G). Resveratrol is a hydrophobic compound

with a low molecular mass of 228.2 g/mol and carries no electric charges (Table 8). On

the contrary, cyanidin-3-O-glucoside (C3G) has a higher molecular mass of 484.8 g/mol

and is hydrophilic with a positive electric charge (Table 8).

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Table 8: Physico chemical characteristics and potential health benefits of bioactives tested for encapsulation by the WP coacervates.

Bioactive Chemical formula

Molecular Mass

(g/mol)

Water affinity

Solubility Potential

health

benefits

Vitamin B9 (Folic acid)

C19H19N7O6

441.4

Hydrophilic negative charge

water : 1.6 mg/L water at alkaline pH

DNA and cell division

Fetal development

Vitamin E -tocopherol)

C29H50O2

430.7 Hydrophobic ethanol

Antioxidant Prevent

degeneration

Resveratrol

C14H12O3

228.2 Hydrophobic

water : 0.03 mg/mL ethanol : 50 mg/mL

Antioxidant Skin

protection

Cyanidin-3-O-glucoside

(C3G)

C21H21O11

484.8 Hydrophilic

positive charge

water at acidic pH

Antioxidant Prevent cardio-

vascular disease

Additionally, with the aim of using of WP coacervates as versatile encapsulating

agents, we attempted to explore the behavior of WP coacervates once incorporated into a

food matrix, i.e., in interactions with other food chemical compounds. It is interesting to

note that while many studies exist on the formation of coacervates, very few studies have

explored the behavior of the concentrated phase of heteroprotein coacervates (phase

recovered after complete phase separation and/or centrifugation), in interaction with

other chemical food compounds. As a first approach, we proposed to investigate here the

resuspension/or solubilization of the WP coacervates in different food aqueous solutions

and evaluate their overall stability over time regarding the potential release of the

bioactive encapsulated.

As a result, the overall study to generalize the use of WP protein coacervates as

versatile encapsulating agents for food applications is presented in three parts:

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(i) the encapsulation of different bioactives by WP coacervates,

(ii) the resuspension of WP coacervates in solution,

the stability of the WP coacervates resuspended in solution and their ability to maintain

the encapsulated bioactive.

3.2. Material and methods

Lactoferrin and beta-lactolglobulin stock solutions

LF and BLG stock solutions were prepared by solubilizing protein powders (LF

from bovine milk, 90% purity and iron saturation level of 10% to 20% (Fonterra

Cooperative Group, New Zealand) and BLG from a confidential industrial source) in milliQ

water adjusted to pH 5.5 using 1 M HCl. The exact protein concentrations of LF and BLG

(16.6 and 18.3 g/L, respectively) were determined by absorbance at 280 nm

(spectrometer UVmc², Safas, Monaco) using 0.96 L·g-1·cm-1 and 1.47 L·g-1·cm-1 as

extinction coefficients, respectively.

Tested molecule solutions

Vitamin E: preparation and quantification

Vitamin E solution was prepared by dissolving vitamin E (alpha-tocopherol,

purity > 96 %; Sigma Aldrich St. Louis, MO, USA) in ethanol to obtain a concentration of

4.3 mg/mL (10 mM). This solution was then diluted 10 times in ethanol to obtain a

solution of vitamin E concentrated at 0.4 mg/mL (1 mM). This final solution was used for

the formation of vitamin E-WP coacervates. Quantification of the vitamin E recovered in

the WP coacervates was performed using the HPLC method described by Hoehler,

Frohlich, Marquardt, & Stelsovsky (1998), This method was used after extraction of the

vitamin E contained in the sample using hexane at 4:1 (v/v).

Resveratrol: preparation and quantification

Resveratrol solution was prepared by dissolving resveratrol powder (purity

> 98%; Alfa Aesar, Haverhill, Massachusetts, USA) in ethanol to obtain a concentration of

10 mg/mL (44 mM). It was then diluted ten times in ethanol to obtain a solution

concentrated at 1 mg/mL (4 mM) and further diluted two times in milliQ water to reach

a concentration of 2 mM. This solution was used for the formation of resveratrol-WP

coacervates to obtain a final concentration of resveratrol in the solutions for coacervation

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of 0.25 mM. Quantification of the anthocyanin recovered in the WP coacervates was

performed using the HPLC method described by Zupancic et al. Zupančič, Lavrič, & Kristl, 2015).

Cyanidin-3-O-glucoside: preparation and quantification

Cyanidin-3-O-glucoside (C3G) was prepared by diluting cyanidin-3-O-glucoside purity ˃ %; Extrasynthese, Genay, France) at 2 mM in 25 mmol.L-1 KCl/HCl buffer. This

solution was then diluted two times in milliQ water to obtain a solution of antocyanin

concentrated at 0.48 mg/mL (1 mM). This solution was used for the formation of

antocyanin-WP coacervates to obtain final concentrations of antocyanins in the

coacervated solutions of 0.125, 0.25 and 0.4 mM. Quantification of the antocyanin

recovered in the WP coacervates was performed using the HPLC method reported by

Pineda-Vadillo et al. (2017).

Mineral solutions

Mineral solutions were prepared by solubilizing mineral powders in 10 mM MES

buffer adjusted to pH 5.5. The minerals studied were zinc (ZnCl2), magnesium (MgCl2)

(Merck KGaA, Darmstadt, Germany) and calcium (CaCl2) (VWR Int., Fontenay-sous-Bois,

France). Stock solutions were prepared at a concentration of 20 mM and further diluted

in 10 mM MES buffer at pH 5.5 according to the final concentrations required in LF-BLG

solutions studied.

Formation of WP coacervates in the presence of tested bioactives

The formation of coacervates was performed using the protocol established in this

study for vitamin B9. In place of vitamin B9 solution, the other tested compound solutions

were used. Firstly, the tested solution was added to the LF solution to reach variable

concentrations of the tested bioactive and an LF concentration of 0.05 mM (4.15 g/L). An

equivalent volume of the BLG solution was then added to obtain a final BLG concentration

of 0.5 mM, (9.15 g/L). Spontaneous coacervation occurred and the tested molecule-WP

coacervate phase was recovered by centrifugation at 38,000 g for 45 min at 20°C

(Centrifuge Avanti J-26S XP; Beckman Coulter Inc., Brea, CA, USA). BLG and LF recovered

in the coacervates were quantified by RP-HPLC using a PLRPS column (S/N 1006329-5;

Varian Inc., Shropshire, UK) connected to a Waters 2695 HPLC. Elution was performed

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using a gradient of solvent (80% acetonitrile, 20% water, 0.1% trifluoroacetic acid) in

water containing 0.1% trifluoroacetic acid. Detection was performed by absorbance at

280 nm. Each sample and corresponding analysis were performed three times to ensure

the reproducibility of the results.

WP coacervation and encapsulation yields

The coacervation and the core encapsulation yields obtained with the different

bioactives were calculated according to the following equations:

- coacervation yield of the whey proteins (%): 𝑎 𝑎 𝑖 𝑦𝑖 % = 𝑊𝑃 𝑒 𝑒𝑊𝑃 𝑖 𝑖 𝑖 𝑖 ×

- encapsulation yield of the bioactive (%): 𝑖 𝑎 𝑖 𝑎 𝑎 𝑖 𝑦𝑖 % = 𝑖 𝑖 𝑒 𝑒 𝑒𝑖 𝑖 𝑒 𝑖 𝑖 𝑖 𝑖 × where Q is the quantity of bioactive (g) or whey proteins (g) recovered in the bioactive-

WP coacervates or in the initial solution depending on each experiment.

Resuspension of B9-WP coacervates in solution

The resuspension of B9-WP coacervates recovered after centrifugation was tested

in three solutions: milliQ water at pH 5.5, ultra-filtrate milk permeate and semi-skimmed

milk. A sample of 0.15 g of B9-WP coacervates was added to 1 mL of the tested solution.

Resuspension was then made by ultrasonication using seven repeated cycles of 1 min

(Bransonic 220 Ultrasonic Bath; Branson Ultrasonics, St Louis, MO, USA) followed by 30

seconds of vortex (Topmix-94223; Fisher Scientific Bioblock, Whaltam, MA, USA).

Resuspension was monitored both visually and by phase contrast microscopy.

Stability of re-suspended B9-WP coacervates assessed by the dialysis method

The dialysis method was used to explore the stability and release behavior of B9

from the B9-WP coacervates when incorporated into a dairy product such as milk. Two

processed milk samples were used: Ultra High Temperature (UHT) semi-skimmed milk

(Sodiaal, France) and pasteurized whole milk (Québon, Canada). Each of the two types of

milk was enriched by the addition of free B9, B9-WP coacervates or free B9 and additional

WP coacervates. The addition of coacervates was made following the resuspension

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protocol. The addition of free B9 was made by dilution of B9 stock solution into the milks,

followed by the same cycles of vortex and sonication as in the resuspension protocol.

To ascertain stability in enriched milks, 5 mL of milk samples with added B9 (50

mg/L of B9) were placed in a 5-cm long cellulose-ester dialysis bag with a molecular

weight cut-off of 100 kDa (Spectra/Por product; Spectrum Laboratories, Inc., USA).

Dialysis experiments were performed under constant slow agitation in the dark and at

4°C against two liters of corresponding milk. B9 concentration inside the dialysis bag was

quantified by the ELISA method at various times: 0, 1, 2, 4, 8 and 24 hours. Each

experiment was carried out in triplicate.

The B9 released from the dialysis bag was calculated using the following equation: 9 𝑎 % = − [ 9][ 9] 0 ×

where [B9]t represents the concentration inside the dialysis bag at time t, and [B9]t0, the

initial concentration. The absence of interference between the dairy matrices on the

ELISA quantification was validated prior to the experiments.

B9 and WP recovered in milk ultra-filtrate permeate after B9-WP coacervate resuspension

Three samples of 0.1 g of B9-WP coacervates were resuspended in 1 mL of milk

ultra-filtrate permeate according to the resuspension protocol presented here. Just after

resuspension, 8 hours and 24 hours after, the corresponding sample was centrifuged

(Heraeus Biofuge Primo; Thermo Scientific, Waltham, MA, USA) at 11,000 g for 20 min.

The supernatant was recovered and the LF and BLG contained in it were quantified by RP-

HPLC analysis and B9 by ELISA assay.

Turbidity measurements

Turbidity measurements were performed at 600 nm (A600nm) with the UVmc²

spectrometer (Safas, Monaco) to monitor the co-assembly in B9-LF-BLG solutions. Absorbance measurements were converted to turbidity τ, cm-1) using the following relationship: τ = . A600nm)/l, where l is the light path length (l = 1 cm). Turbidity was

monitored over 30 minutes.

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Phase contrast microscopy

The formation of bioactive-WP coacervates was assessed using a phase contrast

optical microscope (Olympus BX51TF, Olympus, Hamburg, Germany) at a magnification

of x40. A 10-µL sample of the bioactive-WP coacervates was deposited on a standard

microscope slide, covered with a coverslip (22 mm x 22 mm) and immediately observed.

3.3. Results and discussion

3.3.1. Encapsulation of bioactives

Firstly, the formation of WP coacervates in the presence of the three different

bioactives, C3G, vitamin E and resveratrol, was tested. Figure III-17 shows microscopic

and macroscopic images obtained in bioactive-WP solutions with the three different

bioactives. Microphase separation with the formation of microspheres with a mean

diameter of 1 to 10 µm (Figure III-17A, B) was observed in all cases. A separated dense

phase after centrifugation of the solution was clearly identified (Figure III-17C).

Figure III-17: Phase contrast microscopy and macroscopic images of bioactive-WP coacervates solutions obtained with different bioactives : (A) C3G, (B) vitamin E, (C) resveratrol.

These microspheres and the dense phase recovered after centrifugation is

characteristic of complex coacervation, as previously described (Croguennec, Tavares, &

Bouhallab, 2016). Hence, it seems possible to form WP coacervates in the presence of

various chemical compounds. The presence of these compounds does not alter the

coacervation process, with the formation of droplets constituting a microphase

separation, and subsequent macrophase separation (Wang & Schlenoff, 2014). It is now

necessary to evaluate whether the WP coacervates are efficient to encapsulate the

targeted chemical compound.

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For all the bioactives tested here, the initial concentrations of the bioactive

solutions were 0.25 mM. This tested concentration was set according to the previous

results obtained with B9 (Chapeau et al., 2016). It was identically to the optimal initial

concentration of B9 required for its efficient encapsulation by the WP coacervates. Figure

III-18 displays the WP coacervation and bioactive encapsulation yields obtained.

Figure III-18: WP coacervation and bioactive encapsulation yields (%) obtained by coacervation between whey proteins (LF and BLG) and four bioactives: vitamin B9, vitamin E, resveratrol and C3G. NB: data for WP coacervation yield with resveratrol could not be determined here.

Globally, the WP coacervation yields were between 60 to 75%, which is in the same

range of the coacervation yield obtained with B9. On the other hand, the bioactive

encapsulation yields varied with the type of bioactive tested, from 95% for vitamin E and

B9, to 36% for resveratrol and 19% for C3G. Expressed in mass of encapsulated bioactive,

two distinct groups were formed (Table 9).

The high encapsulation yield of 98% obtained for vitamin B9 and vitamin E,

represented a quantity of 10 mg of bioactive/g of WP coacervates. Compared to other

encapsulation technologies, high encapsulation yields of around 80% can also be reached

for the encapsulation of vitamin E, for example, by spray-drying with Octenyl Succinic

Anhydride (OSA) modified starches as the encapsulating matrix (Hategekimana,

Masamba, Ma, & Zhong, 2015).

0

20

40

60

80

100

Vitamin B9 Vitamin E Resveratrol C3G

Yie

ld (

%)

Bioactive

WP coacervation yield (%)

Bioactive encapsulation yield (%)

N.A.

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Table 9: Initial concentration, encapsulation yields and concentrations recovered in WP coacervates for four different bioactives.

Vitamin

B9 Vitamin E Resveratrol C3G

Initial concentration of bioactive in the bioactive-LF-BLG solution mixed for coacervation

Initial concentration (mM) 0.25 0.25 0.25 0.25

Initial concentration (mg/g) 0.11 0.11 0.06 0.12

Coacervation and complete phase separation

Bioactive encapsulation yield (%) 96 97 36 19

Concentration of bioactive recovered

in WP coacervates (mg/g) 10.6 10.4 2.1 2.3

These high encapsulation efficiencies could be explained by the lipophilic nature

of the compound and its affinity for the matrix during the encapsulation process. On the

contrary, only 2 mg of resveratrol or C3G were found per g of WP coacervates. However,

the physicochemical conditions and the bioactive/protein ratios tested here were the

ones used for efficient encapsulation of B9. An optimization of these physicochemical

conditions and bioactive/proteins ratios could improve the encapsulation yields for

resveratrol and C3G. For example, resveratrol is known to bind to BLG at pH 7.4 to form

1:1 resveratrol-BLG complexes without apparent effects on the secondary structure of the

protein (Liang, Tajmir-Riahi, & Subirade, 2008). Thus, after complexation with

resveratrol, it can be supposed that BLG could still interact with LF to form coacervates.

In that case, the optimal resveratrol-BLG-LF ratios for coacervation have to be

investigated, as well as the optimal pH.

The present results demonstrate that high encapsulation yields can be reached

with WP coacervates. In represent a loading of bioactives in the overall co-assembly,

between 0.5 to 1%, that is to say 5 to 10 mg bioactives / g WP coacervates, (depending on

the bioactive and scale of production). Comparisons with other system using coacervation

or with other types of encapsulating systems for bioactive (using also B9 for example)

demonstrate that the results obtained here can be competitive. For example,

encapsulation of curcumin in BLG-alginate coacervates exhibited a high encapsulation

efficiency of 98 % in the BLG-alginate coacervates (Mirpoor, Hosseini, & Yousefi, 2017).

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However, given the concentration of biopolymers and bioactive used, it represented a

loading of 0.05 % of curcumin, that is to say 0.5 mg curcumin / g BLG-alginate coacervates,

which is ten times lower than in WP coacervates here. In addition, another study realizing

the encapsulation of the flavor compound beta-pinene in sodium caseinate-

carboxymethylcellulose coacervates also reported a loading capacity of 0.5 % beta-pinene

in the coacervates (Koupantsis, Pavlidou, & Paraskevopoulou, 2014), equivalent to the

results here. In addition, for B9 encapsulation, the loading efficiencies can vary greatly

depending on the system. For example, an average loading of 80 mg B9/ g carriers was

obtained for encapsulation in mesoporous silica particles (Ruiz-Rico et al., 2017), which

is ten times higher than the loading obtained here for B9. On the other hand, encapsulation

of B9 in alginate-pectin particles exhibited a loading efficiency of 3 µg B9 / g particles

(Madziva, Kailasapathy, & Phillips, 2006), which is thousand time lower than the loading

of B9 in WP coacervates. As a result, the loading efficiency is greatly system dependent

and the choice of the encapsulating system can be made with additional criteria than only

the loading effeciency. In that sense, WP coacervates present other types of advantages to

be considered.

Primary, as mentioned in the context of the present study, the use of milk proteins

as encapsulating agents is interesting for the development of a clean label, natural

enriched ingredients and functional foods. Secondly, coacervation is a mild encapsulation technology, with no use of extreme pressure or temperature conditions as

in the case of spray-drying for example. In that context, other mild encapsulation technologies exist such as micro- or nano-emulsion. They also demonstrate good

encapsulation efficiencies of around 60 to 80% for vitamin E and resveratrol, for example

(Davidov-Pardo & McClements, 2015; Yang & McClements, 2013). However, in that case,

the final loading was not mentioned and the stabilization of the emulsion droplets in

solution could appear to be very challenging. Other encapsulation technologies using milk

proteins have also been investigated. For example, casein micelles can be used as

interesting biocarriers for C3G (Casanova et al., 2017), but the stability of the complexes

also appeared as an issue and cross-linking might be required to ensure their stability,

especially at acidic pH. With regard to these observations, it appeared interesting to study

the overall stability of the WP coacervates.

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Conclusion on different bioactives encapsulation by the WP coacervates

We showed that LF-BLG coacervation was achievable in the presence of

compounds with different chemical structures. Additionally, it seems possible to use the

WP coacervates to encapsulate various types of bioactives such as hydrophobic vitamins

or polyphenols. However, the core encapsulation yields varied among bioactives and each

encapsulation system should be optimized individually. The work presented in this

section constitutes a first approach to further evaluate the potentiality of using WP

coacervates for the encapsulation of various nutrients and small bioactives. Further

research should be undertaken to better understand the impact of the physicochemical

properties of the core molecules on the encapsulation process and to optimize the

encapsulation yields for each core molecule.

Moreover, it has been observed that the encapsulation of a bioactive by the WP

coacervates results first in the formation of spherical droplets of WP coacervates in solution. These droplets can be referred to a microphase separation, according to a

study conducted with polyelectrolyte coacervates (Wang & Schlenoff, 2014). After

microphase separation, the droplets coalesce until complete phase separation, i.e., macrophase separation, forming a concentrated dense phase of WP coacervates enriched in bioactives (Figure III-19). This is this dense phase that can further be used for

potential applications. To investigate these potentialities, it is now interesting to study the

resuspension or solubilization of the bioactive-WP coacervates and their stability in a

food matrix.

Figure III-19: Schematic representation of complex coacervation followed by microphase and macrophase separation of bioactive-WP coacervates for potential use as an ingredient.

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3.3.2. Resuspension of B9-WP coacervates

In order to use B9-WP coacervates as food ingredients, a key factor is to be able to

incorporate them into a food matrix. After macrophase separation, the recovered B9-WP

coacervate recovered is a concentrated protein phase (Figure III-20A). The phase has a

density of around 1.356 (measured here by comparing its weight to a volume of 1 mL, 1 g,

of water). We tested its re-suspension or solubilization in a series of solutions with

increasing complexity:

- milliQ water at pH 5.5, the solution in which coacervates are formed,

- milliQ water at pH 5.5 with the addition of 100 mM of NaCl,

- ultra-filtrate milk permeate at pH 6.7, to simulate a dairy solution, without

proteins or lipids,

- semi-skimmed milk at pH 6.6, an example of a dairy matrix.

In milliQ water, B9-WP coacervates visually appeared to not be homogeneously

resuspended (Figure III-20B). Moreover, a few minutes after the cycles of resuspension,

the B9-WP coacervates had already decanted, reforming the macrophase pellet. As a

result, the resuspension appeared unsuccessful in milliQ water. On the other hand, in

milliQ water at pH 5.5 with 100 mM NaCla and identically in ultra-filtrate milk permeate,

B9-WP coacervates visually appeared to be homogeneously dispersed after the

resuspension cycles, indicating a successful resuspension (Figure III-20C). Microscopic

images confirmed the resuspension of the B9-WP coacervates into small droplets

dispersed in solution, with a mean diameter of 2 to 5 µm. These small droplets also

appeared as a cluster of several droplets forming a bigger structure with a length of

around 10 to 20 µm (Figure III-20E). Homogeneous resuspension was also reached in

semi-skimmed milk (Figure III-20D).

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Figure III-20: Macroscopic images of (A) B9-WP coacervates recovered after centrifugation, and after resuspension in (B) milliQ water at pH 5.5, (C) ultra-filtrate milk permeate pH 6.7 and (D) semi-skimmed milk pH 6.6, (E) phase contrast microscopy of B9-WP coacervates resuspended in ultra-filtrate milk permeate (obj x100).

These results suggest that resuspension of the concentrated phase of B9-WP

coacervates is possible and results in the formation of small droplets, but requires a

minimum of ionic strength at both tested pH, pH 5.5 and pH 6.7. In the literature, few

studies explored the resuspension behavior of the concentrated phase of heteroprotein

coacervates. However an approach developped on protein-polyelectrolyte coacervates

have explored the water composition and the surface tension of this specific coacervates

that could be elements to explain their resuspension behavior. This study reported that

the concentrated phase of protein-polyelectrolyte coacervates exhibited a bicontinous

fluid structure, resulting in a low interfacial tension (Huang, Yoo, Jho, Han, & Hwang,

2016). This was attributed to the presence of unbound water in the concentrated dense

phase with water molecules only weakly disturbed by the polyelectrolyte interface and

network. According to the authors, the dense phase of coacervates is a bicontinuous

network constituted of a water-water phase and a water-polyelectrolyte phase with a

weak cohesive energy between these two phases. To corroborate these explanations, a

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study on LF-BLG coacervates explored the state of water, nonfreezing water (bound to

proteins) vs. freezing water (unbound free water), in the dense phase of coacervates. The

study showed that 9 to 15% of the overall water content of coacervates is nonfreezing

water, thus, water closely bound to the proteins (Du, Seeman, Dubin, & Hoagland, 2015).

As a result, up to 85% of water can remain more or less free in the dense phase of

coacervates. This phase is surely responsible for the low interfacial energy of the overall

dense phase of coacervates, explaining its resuspension behavior. It is interesting to note

that the minimal ionic strength required for the resuspension of the dense phase

constrasts with the effect of the ionic strengh during the initial formation of coacervates.

A previous study on complex coacervation between lysozyme and alpha-lactalbumin

demonstrated that increasing the ionic strength reduced the ability of the lysozyme and

alpha-lactalbumin to interact and to form the coacervates. Moreover, the formation of

coacervates was completely eliminated at an ionic strength of 100 mM (Nigen,

Croguennec, & Bouhallab, 2009). The dependence and sensitivity to ionic strength

observed during the formation of complex coacervates is a general rule well described in

the literature (Bungenberg de Jong, 1949). This sensitivity were also controlled here.

The sensitivity of LF-BLG coacervation to the ionic strength was checked in this

study. Three different minerals, zinc, magnesium and calcium, were tested at increasing

concentrations, and the total protein concentration was 0.33 mM. The results are

presented in Figure III-21. Since all results were similar for the three minerals tested, only

one set of kinetics is presented. With no ionic strength ([Minerals] = 0 mM)), the addition

of BLG induced a high increase of turbidity up to 3.5, and microscopic images clearly

showed the formation of WP coacervates in solution. With increased ionic strength equal

to the molar concentration of proteins ([Minerals] = 0.03 mM and 0.3 mM), a similar

increase in turbidity after BLG addition can be observed. The corresponding microscopic

images also showed the presence of WP coacervates in solution. Conversely, with a

mineral concentration of 3 mM, i.e., quite above the protein concentration, no significant

increase in turbidity was detected after BLG addition, with the formation of only a few

aggregates. As a result, WP coacervation was only possible at very low ionic strength and

mineral concentrations, and increasing the ionic strength hindered the coacervation

process, as previously reported for the lysozyme-alpha-lactalbumin system (Nigen,

Croguennec, et al., 2009). As expected, this suggests the involvement of electrostatic

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interactions as first events of a two-protein assembly and the subsequent formation of

coacervates.

Figure III-21: Turbidity measurements and microscopic images (obj. x40) of lactoferrin-mineral-beta-lactoglobulin solutions ([proteins]=0.33mM) in the presence of increasing ionic strengths (Mineral concentrations from 0 to 3 mM). Addition of BLG solution in the LF-mineral solution is indicated by the black arrow ⬇

Hence, according to the general principles of complex coacervation, the initial

formation of B9-LF-BLG coacervates is highly sensitive to the ionic strength. Above a

critical value, complex coacervation was eliminated. Conversely, once complex

coacervation and complete macrophase separation occurred, the coacervates seemed to

be less impacted by ionic strength. Indeed, the study on alpha-lactalbumin and lysozyme

showed that once the coacervates were formed and started to separate, the addition of

minerals up to 150 mM did not destabilize the formed coacervates (Nigen, Croguennec, et

al., 2009). Additionally, the resuspension here of the dense phase of WP coacervates even requires a relatively high ionic strength, i.e., ≈ mM, to form resuspended small droplets in solution. This led us to assume that these resuspended droplets of coacervates

should not have the same physicochemical nature compared to the small droplets formed

just after coacervation, during microphase separation.

To begin to explore this hypothesis, we carried out a test that consisted of adding

minerals (Zn, Mg or Ca), 10 min after mixing LF and BLG for coacervation, i.e., during the

microphase separation of the coacervate system. Minerals were added at concentrations

ranging from nearly 10 times (2.5 mM) to 30 times (10 mM) greater than the total protein

concentrations, fixed at 0.33 mM. LF and BLG coacervation were observed by microscopy,

with high turbidity values of 4 cm-1 during the first 10 minutes of the experiment (Figure

III-22). When minerals were added to the system after 15 min, a large decrease in

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turbidity occurred, from 4 cm-1 to 1 cm-1 for mineral concentrations of 2.5 mM proteins,

and no turbidity was found for mineral concentrations of 5 mM and above. Microscopic

images clearly identified very few remaining aggregates at mineral concentrations of 2.5

mM, and no co-assembly above that. As a result, the addition of minerals during

microphase separation of coacervates could clearly destabilize the system.

Figure III-22: Turbidity measurements and microscopic images (obj. x40) of lactoferrin-beta- solutions ([proteins] = 0.33mM) and the addition of minerals (Zn, Mg or Ca) at increasing ionic strengths (Mineral concentrations from 0 to 10 mM). Addition of mineral solution in the LF-BLG solution is indicated by the black arrow ⬇

This indicated that for LF-BLG coacervation, an ionic strength 10 times greater

than the total protein concentration can eliminate coacervation during microphase

separation. By comparing these results, with the results on alpha-lactalbumin and

lysozyme where a ionic strength of 150 mM was required to eliminate coacervation

during or after microphase separation (Nigen, Le Tilly, Croguennec, Drouin-Kucma, &

Bouhallab, 2009), two hypothesis can be proposed. First, the ionic strength that affects

microphase separation can be system-dependent; second, the kinetics of microphase

separation plays a key role in terms of the ionic strength sensitivity of the coacervate

system. That lead us to suppose that the physicochemical structure of the small droplets

of coacervates during their formation and the subsequent microphase and macrophase

separation may be different, and the nature of the interactions involved in the coacervate

systems may not be the same. As a result, a comparative study of the properties of the

subsequent resuspended droplets deserves to be undertaken in comparison to the small

droplets observed in the first microphase separation, and the mechanism of the salt effect

as well as the nature of the interactions involved has to be determined.

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Conclusion on resuspension of B9-WP coacervates

The resuspension or solubilization of WP coacervates into a homogeneous solution is possible but depends, among other things, on the ionic strength of the solution.

With no ionic strength, the homogeneous resuspension and solubilization is not possible,

whereas high ionic strength favored the dispersion of the continuous coacervate phase.

Moreover, ionic strength has an antagonist effect on the formation of coacervates

compared to resuspension of the concentrated phase. A high ionic strength during

microphase formation prevents or destabilizes the coacervates, whereas it has less effect

once the coacervates are completely formed and is even necessary for the resuspension

after complete separation of coacervates in the concentrated dense phase. This dense

phase presents a low interfacial tension and a bicontinous fluid structure, explained by

the dynamics of water inside the phase. The resuspension of this concentrated dense

phase leads to the formation of small droplets in solution. Their overall stability is

investigated in the next section (Figure III-23).

Figure III-23: Schematic representation of complex coacervation followed by microphase and macrophase separation of bioactive-WP coacervates and their resuspension in dairy matrix.

3.3.3. Stability of B9-WP coacervates resuspended in dairy matrices

The ability of the B9-WP coacervates to maintain interactions with B9 in conditions

where other components of food matrices can compete and alter the nature of the protein

complexes was investigated. Three dairy matrices of increased composition complexity

were studied: an ultra-filtrate milk permeate and two types of milk commonly consumed

in North America and Europe, semi-skimmed UHT milk and whole full-fat pasteurized

milk. The release of B9 from the coacervates was followed by a dialysis approach. Free B9

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or B9-WP coacervates were added to each dairy matrix by the resuspension protocol and

submitted to dialysis against the same matrices without B9 or coacervates. The free B9

solution was assumed to represent a control sample where B9 was assumed to diffuse

freely through the dialysis bag by osmotic equilibration (Bertrand, Leclair, & Hildgen,

2007). It is assumed that if the protein complexes in the coacervates are totally disrupted,

B9 should be released at a rate comparable to that of the free vitamin solution. On the

other hand, if B9-WP coacervates maintain a relative integrity during dialysis, B9 should

remain inside the dialysis bag since WP coacervates are unable to diffuse freely through

the dialysis bag. An additional control was carried out on semi-skimmed milk to

determine if WP coacervates without B9 do not prevent the diffusion of free B9. B9

dialysis kinetics in the dairy matrices was followed by logarithmic representations of the

proportion of B9 (%) remaining inside the dialysis bag as a function of dialysis time

(Figure III-24).

In milk ultra-filtrate permeate (Figure III-24A), the release of free B9 was nearly

complete over the time of the experiment. The B9 remaining in the dialysis bag was less

than 10% of the initial amount after 24 hours of the experiment and B9 kinetics

represented a classical exponential diffusion behavior. Comparatively, the encapsulated

B9 in B9-WP coacervates appeared to be more retained inside the dialysis bag. Release of

B9 was lower and plateaued at around 45% of the initial B9 concentration, indicating a

relative stability of B9-WP coacervates. Interestingly, similar kinetic rates were observed

for free B9 and B9 encapsulated in B9-WP coacervates in the two other dairy matrices,

semi-skimmed milk (Figure III-24B) and full-fat milk (Figure III-24C). Of note, in the

control experiment with free B9 incubated with WP coacervates prepared in the absence

of vitamins, B9 showed a release kinetics closer to the one found for free B9. Thus, the

effect of the WP coacervates on the diffusion of B9 was not solely due to the presence of

additional WP. Finally, it is also interesting to note that in the first hours of dialysis, B9 in

the B9-WP coacervates-enriched dairy matrices displayed the same kinetics behavior as

the free B9. Thus, it is assumed that this fraction of B9 could have been released from the

B9-WP coacervates by the vortex and sonication applied for the resuspension of the

coacervates.

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Figure III-24: Logarithmic representation of kinetic release of B9 (%) as assessed by dialysis approach from three dairy matrices, (A) milk ultra-filtrate permeate, (B) semi-skimmed milk and (C) full-fat milk, enriched with B9 free, B9 free and additional WP coacervates, or B9-WP coacervates. 5 mL of B9 enriched milk were placed into dialysis bags and submitted to dialysis against 2 L of unenriched dairy matrices for 24 h at 4°C.

To corroborate this hypothesis, B9, LF and BLG were quantified in the milk ultra-

filtrate permeate, just after the resuspension of B9-WP coacervates (T0), and after 4 hours

(T4h) and 24 hours (T24h) of resuspension. Figure III-25 presents the quantity of each

compound recovered in the milk ultra-filtrate permeate. Initially, the resuspension of the

B9-WP coacervates induced a release of 0.11 mg of B9. Compared to the initial quantity of

B9 contained in the B9-WP coacervates before its resuspension (0.4 mg), it represented a

release of 28%. The resuspension also induced a release of 2.4 mg of LF and 6.7 mg of BLG.

Compared to the initial quantity of LF (7.2 mg) and BLG (18mg) before coacervates

resuspension, it represented 33% and 37% of the initial amounts of LF and BLG,

respectively. After 4 hours, 0.19 mg of B9, representing 47% of the initial amount, and B9

were released. Three mg of LF, representing 41% of its initial amount, and 7.8 mg of BLG,

representing 47% of its initial amount, were found in milk permeate after 4 hours. These

proportions were stable after 24 hours. These results support the hypothesis that the

resuspension of B9-WP coacervates by sonication induced a partial rupture in the overall

structure of coacervates and a partial solubilization of the B9, LF and BLG released during

the resuspension. This could therefore explain the kinetic behavior of B9 observed during

the dialysis, as the dialysis membrane used was a 100 kDa membrane, thus enabling

free/released B9 to diffuse freely across it, but not when bound the WP coacervates.

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Figure III-25: Release quantity of B9, LF and BLG (mg) recovered in milk ultra-filtrate permeate, initially after resuspension of B9-WP coacervates (T0), 4 hours (T4h) and 24 hours (T24h after).

The resuspension of B9-WP coacervates into small droplets in milk permeate

induced a solubilization of 28 to 47% of B9 because the B9- WP coacervates is dissociated,

as shown by the simultaneous release of LF and BLG in the permeate after B9-WP

coacervates resuspension. The released B9 could therefore diffuse freely in the media.

Moreover, once the resuspension was complete, B9-WP coacervates droplets remained

stable since no significant increase of B9, LF and BLG proportions were found in the

permeate up to 24 hours after resuspension.

To complement these experiments, B9-WP coacervates resuspended in ultra-

filtrate milk permeate were observed by phase contrast microscopy. Initially, after

resuspension by vortex and sonication, the B9-WP coacervates showed a resuspension of

small droplets with a mean diameter of around 2 to 5 µm dispersed relatively uniformly

in the permeate (Figure III-26A and B). After 48 hours at 4°C, the resuspended B9-WP

coacervates showed similar droplets in the milk permeate (Figure III-26C, D), thus

confirming an overall stability of the dense phase of WP coacervates when resuspended

as small droplets in a dairy matrix.

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Figure III-26: Phase contrast microscopy of B9-WP coacervates resuspended in ultra-filtrate milk permeate at T0, (A) obj. x40 and (B) obj. x100, and after 48 hours stored at 4°C, (C) obj. x40 and (D) obj. x100.

Conclusion on stability of B9-WP coacervates resuspended in dairy matrices

B9-WP coacervates can be successfully resuspended in a dairy matrix in the form

of micro-sized droplets. The resuspension induces a partial release of B9 and WP proteins

from the coacervates. To limit this release, it can be assumed that the sonication protocol

of resuspension could be optimized. However, once resuspended, the small droplets of

B9-WP coacervates showed a good stability over time, maintaining the major part of their

initial B9 loading. Moreover, no sedimentation or coalescence of the resuspended

droplets of coacervates was observed during a two-day storage period (Figure III-27).

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Figure III-27: Schematic representation of complex coacervation followed by microphase and macrophase separation of bioactive-WP coacervates and their resuspension and stability over 24 hours in a dairy matrix, at 4°C.

3.4. Final conclusions

This study represents a first approach toward the generalization of the use of WP

coacervates as encapsulating agents for food applications. It has been demonstrated that

the WP coacervates are able to encapsulate various bioactives that exhibit different

physicochemical characteristics and health benefits. The encapsulation of a bioactive

results in the formation of a dense phase of bioactive-WP coacervates. Moreover, it is

supposed that this dense phase has a low interfacial tension and a bicontinous fluid

structure. To be used as an ingredient, the dense phase can be resuspended or solubilized

in an aqueous food solution. Its conditions of resuspension appear to be linked to the

physicochemical composition of the solution and, in particular, to the ionic strength and

mineral content. The solubilization of the dense phase of the bioactive-WP coacervates

formed small droplets of around 10 µm in suspension in the food solution. Additionally,

the resuspended small droplets showed a relative stability over time. Model dialysis

experiments where B9-WP coacervates were resuspended in dairy solutions showed that

WP complexes retained part of the initial vitamin content up to 24 hours. The release

kinetics of B9 from the coacervates were found to be similar in milk permeate, semi-

skimmed and whole milk. This suggested that B9-WP coacervates are stable in food

matrices such as milk with complex chemical compositions. Together, all these results

support the benefits and potentiality of using WP coacervates as encapsulating agents for

bioactives in complex food products. Now, further studies should be undertaken to better

understand the nature of interactions and their kinetics involved in the solubilization and

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148

stability of the resupended coacervates. This study should provide interesting new results

to better understand the physicochemical nature and behavior of the resupended

droplets of the concentrated phase of coacervates and how these structures can differ

from the initial droplets obtained during microphase separation and that coalesce to

macrophase separation.

General discussion

149

IV. General discussion

General discussion

150

The present study encompassed two complementary objectives:

- to study the coacervation of two whey proteins, lactoferrin and beta lactoglobulin,

by covering different aspects that have been little or not yet studied for

heteroprotein systems,

- to explore the potentiality of whey protein (WP) coacervates as encapsulating

agents, using a model bioactive: vitamin B9.

1. Formation and characterization of WP coacervates

1.1. LF-BLG coacervation for the encapsulation of targeted molecules

1.1.1. LF-BLG coacervation in presence of a third molecule

Previous studies have extensively dealt with the conditions of coacervation

between lactoferrin (LF) and beta-lactoglobulin (BLG) and proposed different

mechanisms of interactions leading to their coacervation (De Sa Peixoto et al., 2016;

Kizilay et al., 2014; Tavares, Croguennec, Hamon, Carvalho, & Bouhallab, 2015b; Yan et

al., 2013). These previous studies established the physicochemical parameters, protein

ratios and concentrations required for optimal coacervation. Starting from this point, our

study first sought to test the possibility of heteroprotein coacervation in the presence of

a third molecule. The objective was to answer a double problematic: (i) whether the

presence of a third chemical entity can affect the mechanism of coacervation, modifying

the optimal protein ratios and the final coacervation yield between LF and BLG; (ii)

whether the third molecule can be encapsulated inside the LF-BLG coacervates, known as

WP coacervates, thus enabling the use of these coacervates as encapsulating agents.

The detailed mapping obtained for coacervation domains between LF, vitamin B9

and BLG, as well as the preliminary tests performed with a lipophilic vitamin and two

antioxidants polyphenols, established the proof of concept that LF-BLG coacervation is

achievable in the presence of a third molecule with different physicochemical properties.

Moreover, the LF:BLG ratios leading to coacervation in the presence of B9 appeared to be

relatively similar to the ones previously found in the absence of B9 (Tavares, Croguennec,

Hamon, Carvalho, & Bouhallab, 2015a). The optimal LF:BLG molar ratio required for

coacervation is 1:10 either for LF-BLG coacervation alone, or with an added molecule.

With that ratio, the LF and BLG proportions recovered in the coacervates are also in the

General discussion

151

same ranges with LF-BLG coacervates or B9-WP coacervates, with 30 to 35% of LF and

65 to 70% of BLG. As a result, we have demonstrated here that the presence of a third

molecule, with a molecular mass that is 50 to 200 times smaller than that of the whey

proteins, does not alter the LF-BLG coacervation phenomenon.

1.1.2. Encapsulation of targeted molecules using WP coacervates

The third molecule added in LF-BLG coacervation can be entrapped in the

heteroprotein coacervates formed, as shown by the mapping of encapsulation efficiencies

obtained here. Furthermore, it can be supposed that interactions between the bioactive

and one of the two whey proteins are also a favorable factor for coacervation and

encapsulation. For instance, in B9-WP coacervates, the ratios between B9 and LF that

induce the formation of B9-LF complexes were determined from a previous study

(Tavares, Croguennec, Lê, et al., 2015). These primary complexes further interact with

BLG to form the B9-WP coacervates. Similarly, resveratrol and vitamin E are known to

bind to BLG (Liang et al., 2008; Liang, Tremblay-Hébert, & Subirade, 2011) and,

potentially, the anthocyanin cyanidin-3-O-glucoside as well (Oliveira, Ruiz-Henestrosa,

von Staszewski, Pilosof, & Pintado, 2015). However, in this case, the final encapsulation

yields reached in the WP coacervates differed according to the tested bioactive. As a

result, it could be supposed that the optimum ratios of complexation between one whey

protein and a bioactive should be investigated prior to optimizing the final encapsulation

yield by the WP coacervates.

1.2. Production of bioactive-WP coacervates.

1.2.1. Scale-up of heteroprotein coacervation

If WP coacervates are considered as potential encapsulating agents, another

element to take into account is their scale production. Indeed, the experiments conducted

here to study bioactive interactions and encapsulation were first carried out at the

laboratory scale (µL). At this scale, drop-wise addition followed by vortex agitation

and/or equilibration are enough to ensure the homogeneous and fast mixing of the

working solutions. However, at a larger scale, this equilibration process may be impaired.

That is why different studies underlined the important challenge of scaling-up

coacervation (He et al., 2017; Lemetter, Meeuse, & Zuidam, 2009). Moreover, the scale-up

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152

production of heteroprotein coacervates is a relatively new approach since the majority

of the studies on heteroprotein systems are generally performed at the laboratory scale.

Our results demonstrated that the scale-up production of WP coacervates is

achievable. A continuous mixing system using a static mixer enabled the production of

hundreds of mL of B9-WP coacervates solutions, with no significant decrease in the

coacervation and encapsulation yields. Both at the laboratory and the bench scale, the WP

coacervation yields obtained are between 50 to 65%, and the encapsulation yields for B9

in the WP coacervates are as high as 98%, also at both scales. This can be viewed as a

successful scale-up since many encapsulation technologies reveal a loss in encapsulation

efficiencies when their production parameters are modified (A. Lamprecht, U. Schäfer, C.-

M. Leh, 2001). It is difficult to further discuss the WP coacervation yield reached here after

scale-up since no similar heteroprotein coacervate production was found in the literature.

However, the overall encapsulation procedure using WP coacervates can be compared to

the procedure already present in the literature at other scales or with other systems of

coacervates.

1.2.2. Procedure of encapsulation using WP coacervates

Compared to existing procedures of encapsulation by complex coacervation (Table

6, Section II. 3.3.3), the coacervation between LF and BLG occurs spontaneously, without

requirements of extreme changes in pH or temperature. This is of great interest for developing mild encapsulation technologies. Moreover, WP coacervates appear suitable for the encapsulation of both hydrophilic and lipophilic compounds. For lipophilic

compounds, this even appeared to be achievable without prior emulsification in oil. For

the WP coacervates, it is the possible complexes formation between the bioactives and

one of the two whey proteins that could substitute for this step. Finally, another difference

between WP coacervates and other biopolymer coacervates is their final available forms.

In traditional encapsulation by coacervation procedures, the small microspheres of

coacervates obtained in solution are cross-linked, guaranteeing that their spherical forms

will be maintained after the recovery step by centrifugation. In this case, the coacervates

present a spherical shape, referred to as a microcapsule. With emerging coacervation procedures, coacervates may refer to different types of objects: electrostatic complexes

(Kurukji, Norton, & Spyropoulos, 2016), micro/nanocapsules (Weinbreck, Minor, & de

General discussion

153

Kruif, 2004) or a dense, concentrated and continuous phase like the one obtained here

with WP coacervates.

1.3. Physico-chemical characteristics of bioactive-WP coacervates

1.3.1. Forms of WP coacervates

When complex coacervation between LF and BLG occurs, it first forms

suprastructures shaped as micrcospheres suspended in solution. In the literature, these structures are characteristic of coacervation and are referred to as coacervates (Priftis,

Farina, & Tirrell, 2012). The formation of these microspherical structures refers to the

stage of coacervation called microphase separation (Wang & Schlenoff, 2014). However,

these microspheres are relatively unstable in solution and form a dense phase, mostly by

coalescence of the microspheres. This stage is referred to as macrophase separation

(Wang & Schlenoff, 2014). The formation of the dense phase subsequent to macrophase

separation is also identified in the literature as coacervates (de Kruif, Weinbreck, & de

Vries, 2004). This diversity in terms could lead to difficulties for comparisons between

studies, especially regarding the final physicochemical properties of the coacervates

obtained.

Regarding the system studied here, the B9-WP coacervate refers to the coalesced

dense pellet recovered after centrifugation. This dense proteic phase entraps vitamin B9, creating a matrix structure for its encapsulation. This matrix structure has also been reported for other heteroprotein coacervates used for encapsulation, e.g., with

coacervates formed by two variants of gelatin (gelatin A and gelatin B) for the

encapsulation of a specific drug (salbutamol sulfate) (Tiwari, Bindal, & Bohidar, 2009).

Moreover, we demonstrated here that the recovered phase of B9-WP coacervates can be

resuspended in dairy solutions by sonication. In that case, a minimal ionic strength of the

solution seemed necessary. The resuspension led to the formation of small droplets of B9-

WP coacervates in solution, also in the shape of microspheres. However, these

microspheres did not seem to present the same stability properties and coalescence

behavior than the coacervates formed during the microphase separation stage, prior to

complete phase separation. As a result, we assumed that the resuspended B9-WP

coacervate droplets do not present the same physicochemical structures as B9-WP

coacervates prior to macrophase separation. A first element to support this hypothesis is

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154

the effect of salt addition to WP coacervates. During microphase separation, the addition

of a relatively low concentration of salt (mineral concentrations of Zn or Ca of around 5 to

10 mM) to formation and suspension of small droplets of WP coacervates during

microphase separation hinders further coacervation and subsequent macrophase

separation. On the contrary, a high ionic strength (around 100 mM or more) is required

to ensure the solubilization and the stability of the small droplets of the resuspended

dense phase of WP coacervates. This difference in the physicochemical behavior of the

small droplets of WP coacervates could be explained by the nature of interactions

involved in the heteroprotein system before and after complete macrophase separation.

Further research should be undertaken to identify and explain these interactions and how

they can affect the overall properties of heteroprotein coacervates in solution.

1.3.2. Composition of B9-WP coacervates

The dense phase of B9-WP coacervates produced here (Figure IV-1A) contained

around 70% of water (Figure IV-1B). However, it has not been elucidated yet how the

water is distributed into the WP coacervates and between its constituents. A study

conducted by Dubin et al. on LF-BLG coacervates revealed that around 15% of the overall

water content of coacervates is nonfreezing water, meaning water closely bound to the

proteins (Du et al., 2015). As a result, up to 85% of water can remain more or less free in

the dense phase of coacervates. This water distribution leads to a bicontinous fluid

structure of the coacervates. In a protein-polyelectrolyte coacervate, it was reported that

the water molecules of the system are only weakly disturbed by the polyelectrolyte

interface and network and that they are responsible for the low interfacial energy of the

concentrated phase of coacervates (Huang et al., 2016). This low interfacial energy could

be a first element to explain the resuspension behavior of WP coacervates into small

droplets when added to a dairy solution. These resuspended droplets of WP coacervates

could constitute an interesting and directly usable form of the WP coacervates.

Moreover, it has been reported that the concentrated WP coacervates can be freeze-dried

to obtain a powder (Figure IV-1C). However, to control the physicochemical

characteristics and their desired properties to be used as an ingredient, the freeze-drying

process should be optimized. Other processes such as the spray-drying of WP coacervates

could also be considered.

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Figure IV-1: B9-WP coacervates recovered after centrifugation (A), its corresponding composition assessed by RP-HPLC analysis (B), and B9-WP coacervates after freeze-drying (C).

Finally, the WP coacervates obtained here for the encapsulation of vitamin B9

present an interesting composition regarding whey proteins and vitamin content (Figure

IV-1). This composition is of great interest for potential applications of the WP

coacervates as encapsulating agents, even compared to other types of coacervate or whey

protein encapsulating systems.

2. Application of WP coacervates

2.1. Properties of WP coacervates

The present study aimed at exploring the encapsulating properties of WP

coacervates. Traditionally, these properties cover aspects concerning the protection

and/or stabilization of the sensitive bioactives as well as their controlled release, enabling

delivery of the encapsulated material at the right time (kinetics) and the right place

(targeted applications).

2.1.1. B9 as a model bioactive

Vitamin B9 appeared to be a convenient bioactive to study all of these aspects,

nevertheless, with some limitations. For instance, vitamin B9 is relatively stable in terms

of heating degradations. However, its laboratory manageability for preparation and

quantification with different methods, its photosensitivity and solubility limitations, as

well as the demand for staple food enrichment with B9, justify this choice.

B9 encapsulation is relatively well covered in the literature. However, only a few

studies have focused on vitamin B9 encapsulation using whey proteins as encapsulating

agents (Table 10). On the basis of these studies, two main strategies for B9 encapsulation

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156

can be demonstrated. The first one is to use the ability of BLG to interact with B9 at neutral

pH to form a complex by protein-ligand interactions: electrostatic interactions followed

by hydrophobic bounds (Hosseini, Emam-Djomeh, Sabatino, & Van der Meeren, 2015;

Liang & Subirade, 2010). B9-BLG complexes can be further complexed with other

molecules such as alginate (Hosseini et al., 2015). In that case, the final B9-BLG-alginate

complexes present good manageability and stability for food and pharmaceutical

applications. The second strategy for B9 encapsulation using WP is to use the WP as a

matrix that coats the bioactive. In that case, WP are mixed with B9 and encapsulation is

then carried out using different methods such as spray-drying, electro-spraying (Pérez-

Masiá et al., 2015) and coacervation (Chapeau et al., 2017). For these strategies, the

loading obtained for B9 in the encapsulating system are similar, around 12 mg/g dry WP

matrix.

Table 10: Principal studies existing on vitamin B9 encapsulation using whey proteins (WP) or beta-lactoglobulin as encapsulating agents

Encapsulating system

Loading Main properties References

B9-BLG-Alginate complexes

30 mg B9 / g complexes

Colloidal stability (Hosseini et al., 2015)

B9-BLG complexes Not available Decreasing B9 photodegradation

(Liang & Subirade, 2010)

B9-BLG complexes 0.5 mg to 1 mg / g BLG Improving B9 solubility during in vitro digestion

(Pérez et al., 2014a)

B9/WP concentrate spray drying

12.6 mg B9 / g dry WP concentrates

Stability during storage (Pérez-Masiá et al., 2015)

B9/WP concentrate elctro spraying

12.1 mg B9 / g dry WP concentrates

Stability during storage (Pérez-Masiá et al., 2015)

B9-WP coacervates 12 mg B9 / g dry WP

coacervates

Decreasaing B9 photodegradation and oxidation

Increasing B9 bioavalibilty during in vivo digestion

(Chapeau et al., 2017)

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157

For complexation, the loading is more difficult to compare since it could only be for

the BLG itself or for the overall BLG-alginate complexes. For all the studies, B9

encapsulation was able to enhance B9 properties either by improving its stability

(Hosseini et al., 2015; Pérez-Masiá et al., 2015), decreasing its degradation (Chapeau et

al., 2017; Liang & Subirade, 2010) or improving its accessibility during digestion

(Chapeau et al., 2017, Journal of Functional Food (under submission); Pérez et al., 2014a).

After comparing all these systems, WP coacervates seem to be good encapsulating agents.

2.1.2. Bioactive protection and bioavailability

In the present thesis, we showed that WP coaceravates are able to protect a

bioactive from different types of degradation: oxidation, UV light radiation, degradations

occurring in extreme temperature and pressure conditions (freeze-drying), etc.

For all these degradations, the presence of WP coacervates limits the appearance of

degradative forms of the encapsulated molecules (appearance of photoproducts, for

example). This protective capacity could be attributed to the very high protein

concentration of the WP coacervates, about 250 mg whey proteins/g coacervates,

entrapping the bioactive. Macroscopically, this highly concentrated media creates a very

dense network, thus limiting the access to vitamin B9 of degradative compounds such as

free oxygen in the case of oxidation, for example. At the molecular scale, it was

demonstrated that BLG can provide a protective effect for a bioactive by means of the

antioxidant activity of the free thiol group of BLG (Diarrassouba et al., 2015; Liu, Chen, &

Mao, 2007). In the case of photodecomposition induced by UV light radiation, other

studies have demonstrated that the binding between a bioactive and another food protein

such as -lactalbumin, bovin serum albumin or BLG can decrease the photosensitivity of

the bioactive, essentially by decreasing the effective concentration of all of the reagents

involved in its photodecomposition reaction (Liang, Zhang, Zhou, & Subirade, 2013). An

overall protective effect of WP encapsulation was also observed for spray-drying and the

electro-spraying of B9-WP concentrates (Pérez-Masiá et al., 2015). In storage conditions

at room temperatures (23°) and for 60 days, the WP concentrates are able to maintain B9

stability, i.e., to maintain its native bioactive form. The same effect of maintaining the

native and bioactive form of B9 has also been demonstrated here with the WP coacervates

regarding degradative conditions occurring during long-term storage (oxygen and UV

exposure). By maintaining the native and bioactive form of its bioactive, the WP

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158

coacervates present good potential to ensure the bioactivity of the bioactives when

absorbed.

An improved B9 bioavailability in vivo constitutes another benefit of WP

coacervates as encapsulating agents. In vivo studies demonstrate that after oral

administration of B9-WP coacervates, the plasmatic concentrations of B9 are higher

compared to an administration of free unencapsulated B9. As a result, the WP coacervates

are able to improve the bioavailability of the encapsulated bioactive. This could be

explained by different factors. Firstly, as mentioned above, the WP coacervates could offer

a certain protective effect for the bioactive against the extreme degradative conditions

found during digestion. The improved bioavailability could also be linked to the better

solubility of encapsulated B9. It has been demonstrated that the binding between a

bioactive and BLG can improve its overall solubility (Diarrassouba et al., 2015; Mandalari

et al., 2009; Pérez et al., 2014a). Thus, by being more soluble, the bioactive could be better

absorbed. As a result of these good protective and enhanced in vivo properties, the WP

coacervates are potentially interesting for use as encapsulating agents. It is now of

relevance to make a comparison with coacervates that are not heteroprotein coacervates

but are based on a WP-polysaccharide systems, which are more commonly used so far.

2.2. Comparison with WP-polysaccharide coacervates

Based on the pioneering work of Bungenberg de Jong (1949), the gelatin-gum

arabic coacervation system was the most widely studied for encapsulation purposes.

However, a recent trend for substituting the gelatin protein by other types of proteins has

appeared. Among them, whey proteins were particularly investigated for the formation of

whey protein-polysaccharide coacervates and for their properties of encapsulation (de

Kruif & Weinbreck, 2005; Schmitt & Turgeon, 2011; Turgeon et al., 2007). Regarding whey

proteins, whey protein isolate can be used (Koupantsis et al., 2014; Weinbreck et al.,

2004), but BLG seems to be the most commonly used. For polysaccharides, a large variety

of molecules have been tested (chitosan, carrageenan cellulose, etc.), conferring different

properties on the coacervates formed (Schmitt & Turgeon, 2011; Turgeon et al., 2007).

The wide variety of WP-polysaccharides coacervates that exist have been mostly

developed for the encapsulation of flavor compounds or essential fatty acids and some

similarities with the system studied here can be underlined. Similarly as WP coacervates,

the formation of WP-polysaccharide coacervates is also very sensitive to pH variations

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159

and presents an optimal pH for coacervation (Turgeon et al., 2007). Once formed, the WP-

polysaccharide coacervates and WP coacervates present the same high loading efficiency

for the encapsulated molecule (Xiao, Liu, Zhu, Zhou, & Niu, 2014). Nevertheless, in the

literature, the WP-polysaccharide coacervates obtained in solution are often cross-linked

prior to centrifugation and recovery, thus producing micro- or nanospheres of

coacervates rather than a concentrated pellet to be redispersed (Devi, Sarmah, Khatun, &

Maji, 2017). The use of coacervates formed as nano- or microspheres could present an

advantage, given their easy incorporation into a food matrix. For example, the

incorporation of whey proteins-gum arabic coacervates encapsulating orange oil was

tested in a model gouda cheese (Weinbreck et al., 2004). This study demonstrated that

large microspheres (50– m allowed an easier release of the flavor from the cheese matrix compared to smaller ones (5– m . In that case, the role of mastication appeared

essential but it can also be supposed that coacervates introduced as hard microcapsules

could modify the sensory characteristics of the product. As a result, it can be supposed

that the use of WP coacervates as resuspended gelled microspheres could present an

advantage. The protective properties of WP-polysaccharide coacervates were also

investigated. Different studies have reported the good protective property of whey

protein isolate–gum arabic coacervates to prevent oxidation of fatty acids such as

essential omega-3-rich tuna oil (Eratte, Wang, Dowling, Barrow, & Adhikari, 2014).

Another study demonstrated that the encapsulation of probiotic cells inside whey protein

isolate–gum arabic coacervates was able to maintain a higher viability of probiotics after

60 days of refrigerated storage at acidic pH, compared to unencapsulated probiotics

(Bosnea, Moschakis, & Biliaderis, 2014). This study also underlines a better protection of

probiotic cells in in vitro digestion conditions, thus enabling an enhanced bioavailability

of the probiotics. Similarly to WP coacervates, these studies did not detail the mechanisms

linked to these protective and enhanced bioavailability properties. Nevertheless, the WP

coacervates seem to present properties equivalent to those of WP-polysaccharide

coacervates in terms of preventing physicochemical degradations and improving the

bioavailability of the encapsulated compound.

2.3. Nutritional potentiality and benefits of WP coacervates

By compiling all the presented results, different elements support the potentiality

of using WP coacervates as natural and functional dairy ingredients. First, the B9-WP

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coacervates present an interesting nutritional composition (Figure IV-1B). One mg of

concentrated B9-WP coacervates provides 4.5 µg of vitamins. Given the concentrations of

total proteins in the hydrated coacervates (Figure IV-1), i.e., 25-30% for a water content

of 70%, this encapsulation ratio is similar to the one reported for spray-dried B9-WP concentrates, ≈ mg B /g WP concentrates (Pérez-Masiá et al., 2015). According to the

B9 recommended daily intake (Table 11), around 50 to 100 mg of B9-WP coacervates

would be able to theoretically cover the dietary requirement of B9 for children and adults,

and 150 mg would be able to cover daily B9 needs of more vulnerable populations like

pregnant women and seniors. Moreover, since it has been demonstrated that WP

coacervates can encapsulate other types of molecules, bioactives with equivalent dietary

requirements could also be sufficiently provided by several hundred mg of B9-WP

coacervates.

Table 11: Recommended daily intake of B9 according to age classes (Dietary reference by Food and Nutrition Board, Institute of Medicine)

Age Recommended daily intake

0 – 1 year 70 µg

1 – 3 years 150 µg

4 – 8 years 200 µg

9 – 13 years 300 µg

Teen-agers/adults 400µg

Pregnant woman 600µg

Senior 500µg

Furthermore, it has been demonstrated that WP coacervates are able to enhance

the bioavailability of their encapsulated bioactive. This constitutes a first proof of concept

about the benefit of heteroprotein coacervates in terms of bioactive delivery. Moreover,

another interesting feature is that the WP intrinsically have a recognized bioactivity. LF

and BLG contain several essential amino acids and different peptides with biological

activities. LF itself also has a recognized bioactivity and is therefore highly desirable,

especially for the development of infant formula, mimicking maternal milk. Finally, the

WP coacervates are also an iron carrier through LF interactions. Given that the LF iron

saturation is 10% (molar ratio), iron is presented at a rate of 0.9 mg/kg WP coacervates,

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161

which is very interesting since this essential mineral also plays a crucial role in several

metabolic pathways.

General conclusion and perspectives

162

V. General conclusion and perspectives


163

Using a multi-scale and a multi-field approach, this study is an attempt to improve

our understanding of heteroprotein complex coacervation and the benefits of using it for

the encapsulation of bioactive molecules. Indeed, the use of whey heteroprotein complex

coacervates as encapsulating agents has good potential for the development of new dairy

products. The formulation of functional dairy products or ingredients without the

requirement of external additives is particularly interesting to meet consumer demands

for clean label products. The versatility of the WP coacervates constitutes an opportunity

to diversify the use of whey proteins and, concomitantly, to develop new products with

enhanced added value.

The present research project was divided into three main parts, for which the main

results and further perspectives are summarized in Table 12. The first part of this

research project was to investigate the complex coacervation between LF and BLG in

order to encapsulate a model hydrophilic bioactive, vitamin B9. By combining a screening

approach at the laboratory scale and an industrial reflection to scale-up the process, this

study demonstrates the feasibility of producing B9-LF-BLG coacervates, identified as B9-

WP coacervates. The formed coacervates present a high encapsulation yield of 98% for

B9, and a coacervation yield of up to 65%. This constitutes a first proof of concept that WP

coacervates can be used for the encapsulation of a specific bioactive. Moreover, the results

obtained at the bench scale show good potential for increasing the production of B9-WP

coacervates at a larger scale. Further research should now be undertaken to optimize the

production of B9-WP coacervates up to pilot and industrial scales. To do so, it will be also

interesting to look for more efficient ways to recover the coacervates and to identify the

forms in which they would be better formulated (concentrated, powder). With that focus,

spray-drying appears to be an interesting technology to be considered.


164

Table 12: Highlights of the main results and perspectives of the present work.

Research

steps Main results Perspectives

Production of

WP

coacervates

for B9

encapsulation

Laboratory scale (µL):

- Mapping of B9-LF-BLG co-assembly by molar ratios

- Optimal B9-WP coacervation for B9-LF-BLG ratios of 5-1-10,

- WP coacervation yield : 60%, - B9 Encapsulation yield: 98%. Bench scale (L):

- best mixing by static mixer device,

- WP coacervation yield : 65 % - B9 encapsulation yield : 98 %

Composition

of B9-WP

coacervates

Optimization:

of the production of WP coacervates : - level of protein’s purity, - mixing device and flow rates, - recovery of WP coacervates.

Properties of

B9-WP

coacervates

Good protection:

- against degradative conditions : UV, accelerated oxidation, process conditions (freeze drying), long time storage; - by preventing / limiting the formation of degradative products from the native bioactive form of B9. Favorable delivery: Enhanced bioactive bioavailability during digestion compared to the bioactive administrated alone (in vivo assay)

Comprehension:

- molecular level mechanisms responsible for the protective effect of coacervates, - in vitro digestion of WP coacervates : degradative products, digestion kinetics, liberation of bioactive peptides, bioactive delivery.

WP

coacervates

for further

functional

food

development

Versatile encapsulating agents:

- for different bioactives with different physico-chemical properties, - but with also different encapsulation yields. Potential incorporation in foods: possible re-suspension and stability in dairy matrix

Implementation:

- screening of bioactives for best coacervation encapsulation yields, - mechanisms of resuspension and stability in foods,

-rheology and sensory characteristics.


165

The second part of this work focused on the characterization of the WP coacervates

in terms of their encapsulation properties. It was decided to investigate these properties

by simulating conditions potentially occurring during the storage of a food ingredient (i.e.,

UV light radiation and accelerated oxidation) and during processing (freeze-drying).

Using methods that made it possible to investigate the potential loss or degradation of the

native bioactive form of B9 (mass spectrometry, ELISA test), we showed that WP

coacervates are able to prevent or limit the appearance of degradative products of B9 under the tested conditions. WP coacervates could potentially act as a shield or covering matrix to limit access of the degradative factors to the bioactive, thus reducing the

probability of degradative reactions. For example, the covering matrix of WP coacervates

limits the contact of the bioactive with free oxygen that could induce an oxidative reaction

of degradation. To better understand this protective effect, it could be interesting to

investigate at the molecular level, the interactions between the WP coacervates and the

bioactive, as has already been done for ligand-protein interactions between BLG and

vitamin E using fluorescence spectroscopy (Liang et al., 2011).

Furthermore, the delivery of B9 has been investigated in rats after oral

administration. The plasmatic concentrations of B9 recovered after injection were higher

with B9-WP coacervates, compared to free B9. This is probably due to a combination of a

protective effect, allowing the bioactive to better resist the extreme physicochemical

conditions of digestion, and the potentiality of WP coacervates to enhance the solubility

of B9, known to be low in acidic environments. As a result, the WP coacervates appear to

be able to enhance the overall absorption and bioavailability of B9. Yet, at this time, the digestion of WP coacervates can be considered as a black box and several questions can be raised such as: what are the degradation kinetics of the B9-WP coacervates? What are

the degradative products or peptides released during the digestion of WP coacervates?

Do the released products still maintain their bioactivity (especially regarding specific

peptides from LF and BLG)? To address these questions, it might be possible to set up in

vitro digestion experiments of the WP coacervates and to investigate the enzymatic

sensitivity of WP coacervates, the kinetics and degradative products obtained through

simulated digestion conditions (enzymes, pH, temperature, etc.). This approach is

relatively new for heteroprotein coacervation and should extend the benefits and overall

applications of the WP coacervates.


166

The third part of this work was dedicated to extending the use of WP coacervates

to various bioactives and in different food environments. Based on a range of preliminary

tests using bioactives with various physicochemical characteristics (lipophilic vitamins,

polyphenols, etc.), it was revealed that the formation of WP coacervates is achievable and

that they can entrap different bioactives. However, it appears that the encapsulation

yields cannot be generalized. Thus, for now, each type of bioactive should be specifically

studied in order to optimize the encapsulation yield that can be obtained through WP

coacervation. By doing this, it can be expected that more global encapsulation yields could

be identified given the physicochemical nature of the bioactives.

Finally, the behavior of WP coacervates was observed after introduction into a

dairy matrix. Interestingly, it was found that high ionic strength (100 mM) and minerals

present in these matrices surely constitutes a key factor to ensure the resuspension of the

dense phase of WP coacervates and to maintain their relative stability in food matrices

over time. On the contrary, a rather low ionic strength (10 mM) added to the system prior

to complete phase separation could prevent coacervation. As a result, it can be assumed

that the protein structures and interactions involved in the formation of heteroprotein

coacervates are probably not the same as the ones obtained in the concentrated dense

phase of coacervates. In that case, it could be assumed that the small droplets of

coacervates obtained prior to complete phase separation, referred to as microphase

separation, and the concentrated WP coacervates recovered after macrophase separation

and resuspended in small droplets are not the same chemical entities. Further

investigations should be undertaken to identify and explain these potential differences.

This would provide a better understanding of the behavior of the WP coacervates inside

a food matrix and in interaction with other food components such as minerals, lipids and

polysaccharides. To characterize the food matrix enriched by the WP coacervates, it

would also be interesting to perform rheology analyses and to combine them with sensory

analysis of the final food products. As a starting point, the intrinsic rheology of WP

coacervates should be investigated.

In the literature, the rheology of protein-polysaccharides has already been

investigated (Schmitt, Aberkane, & Sanchez, 2009; Schmitt & Turgeon, 2011; Turgeon et

al., 2007) but very few studies have focused on the rheological behavior of heteroprotein

coacervates. Recently, Bohidar et al. undertook a first approach to elucidate the

rheological behavior of heteroprotein coacervates based on gelatin A and bovine serum


167

albumin (Pathak, Rawat, Aswal, & Bohidar, 2014) or gelatin B and beta-lactoglobulin

(Pathak, Rawat, & Bohidar, 2015). By comparing gel phases to the coacervate phases

formed by these systems, they attempted to determine the level of interactions between

the proteins and how it affects the overall stability as well as the thermal and rheological

properties of the heteroprotein systems. They were able to observe that the coacervates

had a lower storage modulus G’ and were therefore supposedly more viscoelastic compared to the gels. This was attributed to the considerable heterogeneity of the

coacervate phase, whereas gels were relatively homogeneous. These studies can also be

linked to the study intended to elucidate the dynamics of water in the coacervate phase

(Du et al., 2015; Huang et al., 2016) and, of course, to rheological measurements

performed on WP coacervates. Additionally, the encapsulation of a bioactive has to be

taken into account since it could modify the physicochemical properties and,

consequently, the rheological properties of the WP coacervates.

Overall, the present study represents a contribution to improving current

knowledge about heteroprotein coacervates and, more precisely, WP coacervates. By

demonstrating how this chemical entity can be used as an interesting encapsulating agent,

this study aims at reviving a renewed interest in the old concept of complex coacervation.

This research combines the interesting application of coacervates for encapsulation with

current research into new ways to give added value to industrial co-products and whey

proteins for the ultimate purpose of developing new ingredients or foods. This last part

still remains a challenge but presents interesting research and industrial opportunities

for the development of natural and functional ingredients that can find applications in

various fields, from cosmetics to pharmaceuticals, nutraceuticals and foods.

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Thèse AGROCAMPUS OUESTsous le label de l’Université Bretagne Loire

pour obtenir le grade deDOCTEUR D’AGROCAMPUS OUEST

Spécialité Science de l’aliment

Anne-Laure CHAPEAU 26 octobre 2017•

AGROCAMPUS OUEST • Institut national supérieur des sciences agronomiques, agroalimentaires, horticoles et du paysage65 rue de Saint-Brieuc – CS84215 – F-35042 Rennes CedexTél. : 02 23 48 50 00www.agrocampus-ouest.fr

ÉCOLE DOCTORALE • Écologie, Géosciences, Agronomie et Alimentation (EGAAL)

LABORATOIRE D’ACCUEIL • UMR INRA AO Science etTechnologie du Lait et de l’Œuf (STLO)

Les coacervats de

beta-lactoglobuline et lactoferrine

pour l’encapsulation d’une

molécule bioactive modèle,

la vitamine B9

Françoise NAU

Professeur, AGROCAMPUS OUEST, UMR INRA AO STLO / présidente

Christian SANCHEZ

Professeur, SupAgro Montpellier / rapporteur

Adem GHARSALLAOUI

Maître de conférences, Université Claude Bernard Lyon 1 / rapporteur

André BRODKORB

Senior Scientist, Moorepark, Teagasc, Ireland / examinateur

Marieke VAN AUDENHAEGE

Chargée de recherche, Société Sodiaal / examinatrice

Saïd BOUHALLAB

Directeur de recherche, UMR INRA AO STLO / directeur de thèse

Denis PONCELET

Professeur, Oniris, Nantes, France/ co-directeur de thèse

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9

RÉSUMÉ ABSTRACT

Beta-lactoglobulin and lactoferrin complex coacervates for the

encapsulation of a model bioactive, the vitamin B9.

Encapsulation of bioactives is relevant for the development of

functional foods. Food proteins as encapsulating agents could

match the objective of industries to develop “clean label” products.

Moreover, whey proteins (WP) exhibit good potentialities as

encapsulating agents. Previous works have demonstrated that

the WPs, beta-lactoglobulin (BLG) and lactoferrin (LF), are able to

spontaneously co-assemble by complex coacervation. This study

explores the ability of BLG-LF coacervates as a potential carrier for

the encapsulation of a model bioactive, vitamin B9. Throughout

screening experiments, we determined the domains where B9-WP

coacervation occured according to a tested range of pH, proteins

and vitamin concentrations and molar ratios. Optimal conditions

for coacervation were found in water, at pH 5.5, with LF:B9:BLG

molar ratio of 1:5:10, affording coacervation yields of 45 to 55%

and B9 encapsulation up to 98%. Coacervation was scaled-up from

laboratory to bench scale using commercial-grade protein sources

and static mixing. Final effi ciencies were obtained with coacervates containing 4 mg of B9/g coacervates. Under degradative conditions

(UV light irradiation, oxidation, freeze-drying), WP coacervates

provided good protective properties limiting chemical degradation

of native B9. In vivo oral administration of B9-WP coacervates

in rats enhanced the plasmatic concentrations of B9 compared

to unencapsulated B9. In addition, good physical stability over

time was found after incorporated and resuspension of formed

coacervates in milk. The combined results of this thesis provide

better insight into heteroprotein complex coacervation and its use

for innovative applications.

Keywords: heteroprotein coacervation, whey protein, bioactive,

vitamin, encapsulation.

Les coacervats de beta-lactoglobuline et lactoferrine pour l’encap-

sulation d’une molécule bioactive modèle, la vitamine B9.

Les aliments fonctionnels connaissent aujourd’hui un intérêt gran-

dissant. Pour leur formulation, l’encapsulation de bioactifs repré-

sente une voie intéressante et les protéines de lactosérum (PS) pré-

sentent de bonnes potentialités en tant qu’agents d’encapsulation.

Des travaux antérieurs ont démontré que deux PS, la beta-lactoglo-

buline (BLG) et la lactoferrine (LF), peuvent former spontanément

des co-assemblages par coacervation complexe, une technologie

d’encapsulation connue. Cette thèse étudie la coacervation BLG-

LF pour l’encapsulation d’un bioactif modèle, la vitamine B9. En

testant une gamme de pH et de ratios molaires, les conditions opti-

males de coacervation B9-PS sont obtenues dans l’eau, à pH 5,5,

avec un ratio molaire LF:B9:BLG de 1:5:10, permettant d’atteindre

des rendements de coacervation de 45 à 55%, et d’encapsulation

de B9 de 98 %. L’échelle de production des coacervats est aug-

mentée avec succès du µL au L, avec des solutions protéiques de

qualité commerciale et un mélangeur statique. Les rendements de

coacervation et d’encapsulation sont conservés, avec l’encapsu-

lation d’environ 4 mg de B9/g coacervats. Par ailleurs, les coacer-

vats montrent un effet protecteur de la forme native de B9 vis-à-vis

des UV, de l’oxydation et pendant la lyophilisation. Une étude in vivo

chez le rat démontre une augmentation de la biodisponibilité de

B9 lorsque administrée sous forme de coacervats. Les coacervats

apparaissent stables lorsqu’ils sont resuspendus en gouttelettes

dans du lait. Ce travail permet d’approfondir les connaissances sur

la coacervation hétéroprotéique et son application en tant que vec-

teur d’encapsulation naturel et innovant.

Mots-clés : coacervation hétéroprotéique, protéines sériques,

bioactif, encapsulation.

Thès

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HAP

EAU

Ann

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les coacervats de beta-lactoglobuline et lactoferrine pour

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