Bach T Nguyen

Taco Nicolai
email: taco.nicolai@univ-lemans.fr

Christophe Chassenieux

Lazhar Benyahia

Trong Bach Nguyen

The effect of protein aggregate morphology on phase separation in mixtures with polysaccharides

Keywords: protein, polysaccharide, phase separation, β-lactoglobulin, carrageenan, aggregate structure, rheology, -lactoglobulin, κ-carrageenan, calcium

The morphology of aggregates formed by heating the globular protein β-lactoglobulin ( β-lg) changes with the addition of a small amount of CaCl 2, from small strands to larger spherical aggregates (microgels). We investigated the effect of this morphological transition on the structure of mixtures of β-lg aggregates with the polysaccharide κ-carrageenan (κ-car), using confocal laser scanning microscopy and dynamic light scattering. The change in the morphology of the β-lg aggregates strongly reduced the κ-car concentration at which the system phase separated. As a consequence a dramatic change in the structure of the mixtures occurred over a narrow range of the CaCl 2 concentration. Phase separation leads to the formation of micron-sized protein rich domains that have a tendency to stick together in large locs. There is a big difference between the protein concentrations in the two phases, but the κ-car concentration is only weakly lower in the protein rich phase. A comparison is made between mixtures prepared at room temperature, after separately heating β-lg, and heated mixtures of native β-lg and κ-car. The micro-phase separated structure of the two systems is similar, but the aggregates disperse upon dilution in the former case, while they are covalently bound within the domains in the latter case. Other, more subtle, differences were also observed. The results explain the very high sensitivity of the structure of β-lg/κ-car mixtures to calcium ions.

ACKNOWLEDGMENT

There are many, whom I would like to acknowledge here, that have helped me during my thesis.

In the first place I would like to thank Doctor Dominique Durand, the first person I contacted and he gave me the chance to work with the 'dream team', my supervisors. I am grateful to Doctor Taco Nicolai, Professor Christophe Chassenieux and Professor Lazhar Benyahia for all their help and advice throughout my PhD time at University of Le Mans, I have been really lucky to be able to work with them. Especially, the daily supervision by Taco helped me to improve my research skills and to resolve difficulties. It is the main reason that I have been able to finish my thesis successfully.

I also thank to the Ministry of Education and Training of Vietnam for financial support during 3 study-years.

I am also grateful to Professor Camille Michon and Professor Sylvie Turgeon as members in my academic committee who gave a lot of useful advices.

I should not forget thank the whole staff at PCI, they always supported me and assured the best working conditions. A special thanks to Magali Martin, Jean-Luc Moneger, Frederick Niepceron, Boris Jacquette and Cyrille Dechance for helping with the analysis of SEC, TGA, FRAP and the assistance with the rheometers and the confocal microscopy, and Danielle Choplin who helped me with my official documents. Of course, I also thank all my fun friends at PCI who made my stay a pleasure. I thank all of Vietnamese students and Vietnamese families who accompanied during my stay in France.

Finally, I am indebted to my family, especially, my mother, wife and son, who had a difficult time in my absence that they had overcome and have always fully supported me throughout my long stay in France. It is really wonderful that my wife gave me the biggest present -a new family member, my daughter.

Table of Contents

General Introduction

The main ingredients of foods are proteins, polysaccharides and lipids, which procure both nutrition and texture. The relatively recent recognition that processed foods need to be healthier, has led to an increasing need to develop novel products that contain less fat and salt.

In addition, there is a tendency to add other functional ingredients in a way that retains their functionality during storage and digestion. Finally, there is a drive to replace relatively expensive proteins by less expensive polysaccharides. Obviously, for a rational development of such food products it is essential to understand the physical chemical properties of aqueous solutions and gels containing proteins and polysaccharides by themselves and in mixtures. This explains why these systems are currently intensively investigated.

Carrageenans are an important class of hydrophilic sulfated polysaccharides widely used as thickening, gelling and stabilizing agents in food products such as sauces, meats and dairy products. Especially, in frozen foods its high stability to freeze-thawing cycles is very important. They are also helpful for the smoothness, creaminess, and body of the products to which they are added. In combination with proteins such as β-lactoglobulin (β-lg), casein, etc… their presence allows different textures to be obtained and to reduce the fat content of food.

Many food formulations yield complex microstructures composed of water, proteins, carbohydrates, fats, lipids and minor components. Protein-polysaccharide interactions are of outmost importance in these structures, and play an essential role in the stability and the rheological behavior of the final product. Understanding of the interactions between these macromolecules will therefore facilitate development of new products.

An example is the use of kappa carrageenan (κ-car) in dairy products (Trius et al., 1996). Milk protein/κ-car interaction improves the functional properties of dairy products under controlled conditions of pH, ionic strength, κ-car concentration, -lg/κ-car ratio, temperature, and processing. In industrial applications, κ-car is used to stabilize and prevent whey separation in processing of dairy products such as milk shakes, ice cream, chocolate milk, and creams. κ-car interacts with dairy proteins to form a weak stabilizing network that is able to keep chocolate particles in suspension in chocolate milks. The network also prevents protein-protein interactions and aggregation during storage, inhibits whey separation in fluid products and decreases shrinkage in ice cream.

The texture of many food products is a consequence of gelation of either the proteins or the polysaccharides, or both. Gelation of one type of macromolecules will be influenced by the presence of the other type, when both are present. When both the polysaccharides and the proteins gel, synergy between the two interpenetrating networks may be a useful property that can be exploited in product development.

Objectives

The objective of the present investigation was to study the influence of aggregation and gelation of -lg on the structure and the rheology of κ-car solutions and gels. Protein particles were formed by heating native -lg, which caused their denaturation and aggregation. For this study, protein particles were either formed separately and subsequently mixed with κ-car or formed directly in mixtures of κ-car and native -lg. Systems with the same composition prepared by these two different methods, were compared. Heating mixtures can also lead to gelation of the proteins. In this case interpenetrated networks may be formed by subsequent gelation of the polysaccharides. The research presented in this thesis is essentially experimental using scattering techniques and confocal laser scanning microscopy (CLSM) to study the structure and shear rheology to study the dynamic mechanical properties.

The thesis consists of four chapters and a general conclusion:

Chapter 1 gives a review of the literature on the biopolymers used in this study separately and in mixtures Chapter 2 presents the materials and methods used in the research Chapter 3 describes the investigation of κ-carrageenan gelation in the presence of single or mixed salts Chapter 4 describes the investigation of the structure and rheology of mixtures of κcar and -lg aggregates or gels The research has resulted in 4 publications in scientific journals in which more details can be found. They are included as an appendix to the thesis.

Chapter 1: BACKGROUND 1.1. Beta lactoglobulin β-lactoglobulin (β-lg) is the major whey protein (~50%) in the milk of ruminants and its properties have been regularly reviewed (Tilley, 1960;[START_REF] Lyster | Reviews of the progress of dairy science. Section C. Chemistry of milk proteins[END_REF][START_REF] Kinsella | Proteins in Whey: Chemical, Physical, and Functional Properties[END_REF][START_REF] Hambling | β-Lactoglobulin[END_REF][START_REF] Sawyer | β-Lactoglobulin[END_REF]. 10 different genetic variants of β-lg have been identified. The most important genetic variants A and B differ at positions 64 (Asp/Gly) and 118 (Val/Ala). β-lg has been the subject of a wide range of biophysical studies because of its abundance and ease of isolation from milk. Its biological function is not clear, but it is a member of the lipocalin family of proteins [START_REF] Banaszak | Lipidbinding Proteins: A family of fatty acid and retinoid transport proteins[END_REF][START_REF] Flower | The lipocalin protein family: structure and function[END_REF] known for its ability to bind small hydrophobic molecules into a hydrophobic cavity. This led to the proposal that β-lg functions as a transport protein for retinoid species, such as vitamin A (Papiz et al., 1986). However, according to [START_REF] Flower | The lipocalin protein family: structural and sequence overview[END_REF] β-lg has a wide range of functions, which explains the significant quantities of β-lg found in milk.

Molecular structure

β-lactoglobulin is a small globular protein that is soluble in water over broad range of the pH (2)(3)(4)(5)(6)(7)(8)(9). Its isoelectric point (pI) is about 5.2. The primary structure consists of 162 amino acid residues with a molecular weight M w ~ 18.4 kg/mol. The secondary structure of βlg was found to contain 15% α-helix, 50% β-sheet and 15-20% reversed turn --strands [START_REF] Creamer | Secondary structure of bovine βlactoglobulin B[END_REF]. β-lg contains two disulphide bridges and one free cysteine group (McKenzie and Sawer, 1967;[START_REF] Hambling | β-Lactoglobulin[END_REF].

The 3D tertiary structure of native β-lg is shown in figure 1.1. It shows an eightstranded β-barrel (calyx) formed by β-sheets, flanked by a three-turn α-helix. In aqueous solution the proteins can associate into dimers and oligomers depending on the pH, temperature and ionic strength, with the dimer being the prevalent form under physiological conditions [START_REF] Kumosinski | Molecular Interactions in β-Lactoglobulin. X. The Stoichiometry of the β-Lactoglobulin Mixed Tetramerization[END_REF][START_REF] Mckenzie | Optical rotatory dispersion and sedimentation in the study of association-dissociation: Bovine β-lactoglobulins near pH5[END_REF][START_REF] Gottschalk | Protein self-association in solution: The bovine β -lactoglobulin dimer and octamer[END_REF]. A ninth β-sheet strand forms the greater part of dimer interface at neutral pH (Papiz et al., 1986;[START_REF] Bewley | III.1 -Bovine β-lactoglobulin and its variants : A three-dimensional structural perspective[END_REF]. [START_REF] Brownlow | Bovine [beta]-lactoglobulin at 1.8 A resolution-still an enigmatic lipocalin[END_REF]

Aggregation and gelation of β-lactoglobulin

The well-defined structure of β-lg can be perturbed by heating leading to denaturation of the native proteins, which generally causes their aggregation. Different types of interaction are involved in this process such as hydrogen bonding, Van de Waals and hydrophobic interactions. Close to and above pI, disulfide bonds are exchanged leading to the formation of covalent disulfide bridges between different proteins [START_REF] Bauer | Detection of Intermediate Oligomers, Important for the Formation of Heat Aggregates of β-Lactoglobulin[END_REF][START_REF] Carrotta | Small-angle X-ray scattering studies of metastable intermediates of β-lactoglobulin isolated after heat-induced aggregation[END_REF][START_REF] Croguennec | Stable monomeric intermediate with exposed Cys-119 is formed during heat denaturation of βlactoglobulin[END_REF][START_REF] Surroca | Towards the understanding of molecular mechanisms in the early stages of heat-induced aggregation of β-lactoglobulin AB[END_REF]Otte et al., 2000).

The aggregation process and the resulting structures depend strongly on the temperature, pH, type and concentration of salt and the protein concentration [START_REF] Foegeding | Specific divalent cation-induced changes during gelation of -lactoglobulin[END_REF][START_REF] Iametti | Modifications of High-Order Structures upon Heating of -Lactoglobulin: Dependence on the Protein Concentration[END_REF]Foegeding, 2006;[START_REF] Mehalebi | Light scattering study of heat-denatured globular protein aggregates[END_REF][START_REF] Ako | Micro-phase separation explains the abrupt structural change of denatured globular protein gels on varying the ionic strength or the pH[END_REF][START_REF] Ako | Salt-Induced Gelation of Globular Protein Aggregates: Structure and Kinetics[END_REF]Schmitt et al., 2010;Nicolai et al., 2011;[START_REF] Ryan | Stability and mechanism of whey protein soluble aggregates thermally treated with salts[END_REF][START_REF] Leksrisompong | Effects of Heating Rate and pH on Fracture and Water-Holding Properties of Globular Protein Gels as Explained by Micro-Phase Separation[END_REF]Ruhs et al., 2012;Phan-Xuan et al., 2013;Phan-Xuan et al., 2014). Scattering and microscopy techniques have been used to study the effect of these parameters on the size, mass, and density of the aggregates. In salt free solutions aggregates with three different morphologies are formed during heating, depending on the pH, see figure 1.2: spherical particles around pI in the pH range 4.0-6.1, short curved strands at higher and lower pH, and long rigid strands at low pH (1.5-2.5). The rigid strands can be very long, but are formed only when the proteins are partially hydrolyzed into shorter peptides. The hydrodynamic radius (R h ) of the short curved strands formed above pI was found to increase with decreasing pH from about 12nm at pH 8.0 to about 20nm at pH 6.1 [START_REF] Mehalebi | Light scattering study of heat-denatured globular protein aggregates[END_REF]. [START_REF] Jung | Structure of Heat-Induced β-Lactoglobulin Aggregates and their Complexes with Sodium-Dodecyl Sulfate[END_REF].

During heating the concentration of aggregates increases progressively until all native proteins are transformed and steady state is reached. However, at higher protein concentrations the strands or spheres have a tendency to associate randomly into larger aggregates. The size of the secondary aggregates at steady state increases with increasing protein concentration until above a critical value (C gel ) a gel is formed or macroscopic flocs that precipitate. pH=2.0 pH=7.0 pH=5.8 [START_REF] Mehalebi | Light scattering study of heat-denatured globular protein aggregates[END_REF] have reported the sol-gel/precipitate state diagram of -lg in salt free water at steady state as a function of the protein concentration and the pH between 2 and 9, see figure 1.3. C gel is low close to pI and increases with increasing or decreasing pH to reach about 90g/L for pH  7. In a very narrow range around pI secondary aggregation of the spherical particles leading to precipitation occurs at all concentrations. For this reason stable suspensions of spherical particles were found only in very narrow pH range (5.75-6.1) [START_REF] Phan-Xuan | On the crucial importance of the pH for the formation and self-stabilization of protein microgels and strands[END_REF]. In this range the hydrodynamic radius increased with decreasing pH from about 45nm to about 200nm. The spherical particles consist of a network of crosslinked proteins with a density of about 0.2 g/mL and are therefore called microgels.

The presence of salt influences significantly the aggregation process. At neutral pH, addition of NaCl induces secondary aggregation of the short strands at lower protein concentrations [START_REF] Baussay | Influence of the ionic strength on the heat-induced aggregation of the globular protein β-lactoglobulin at pH 7[END_REF]. As a consequence C gel decreases with increasing NaCl concentration. However, the overall structure of the aggregates is independent of the NaCl concentration. Addition of salt also leads to an increase of the aggregation rate.

The effect of adding CaCl 2 is more dramatic as it influences not only the secondary aggregation, but can also drive a change in the morphology from small strands to microgels.

In the presence of calcium ions, stable suspensions of microgels can also form at pH > 6.1 (Phan-Xuan et al., 2013). The effect is not determined by the total amount of salt, but by the ratio (R) between the molar concentration of CaCl 2 and -lg (Phan-Xuan et al., 2014). The critical ratio at which the transition between the formation of strands and microgels occurs increases with increasing pH from R = 0 at pH < 6.2 to R ≈ 2.5 at pH = 7.5 via R ≈ 1.5 at pH = 7.0. At a given pH, the size and the density of the microgels increases with increasing R.

Stable suspensions of microgels with sizes between 100 to 400 nm and densities between 0.2 -0.4 g/ml can be formed in a narrow range of R. At R > 3 or at -lg concentrations above 60g/L, the microgels associate.

The aggregation rate increases sharply with increasing temperature as it is controlled by the protein denaturizing step (Durand et al., 2002;[START_REF] Baussay | Influence of the ionic strength on the heat-induced aggregation of the globular protein β-lactoglobulin at pH 7[END_REF]Nicolai et al., 2011;Phan-Xuan et al., 2013). The structure and size of aggregates formed at pH > 6.2 in the absence of CaCl 2 were not influenced significantly by the heating temperature (Phan-Xuan et al., 2013). The structure and size of the microgels was not influenced either by the heating temperature when aggregation was fast, i.e. between 75 and 85 0 C (Phan- Xuan et al., 2013). However, at lower heating temperatures when aggregation is slow an influence of the heating temperature on the microgel formation was reported [START_REF] Bromley | Mechanisms of structure formation in particulate gels of β-lactoglobulin formed near the isoelectric point[END_REF]Phan-Xuan et al., 2013).

The aggregation and gelation process is schematically represented in figure 1.4. In aqueous solution, when native β-lg is heated the monomer-dimer equilibrium is shifted towards the monomers (step 1). The protein structure is modified and becomes more mobile.

Irreversible bonds are formed leading to the formation of strand-like or spherical aggregates (step 2, 3) depending on the pH and the type and concentration of added salt [START_REF] Mehalebi | Light scattering study of heat-denatured globular protein aggregates[END_REF][START_REF] Ako | Micro-phase separation explains the abrupt structural change of denatured globular protein gels on varying the ionic strength or the pH[END_REF]Phan-Xuan et al., 2013 and2014). These primary aggregates can further assemble into larger aggregates (step 4) or even a gel at higher protein or salt concentrations.

Kappa carrageenan

Carrageenans are sulfated linear polysaccharides of D-galactose and 3,6-anhydro-Dgalactose extracted from certain genera of red seaweeds. There are different types of carrageenan that differ from one to another in their content of 3,6-anhydro-D-galactose and the number and position of ester sulfate groups (Trius et al., 1996). Κappa carrageenan (κ-car) is the most commonly type used in applications (figure 1.5), because it can form thermoreversible gels in the presence of specific monovalent cations like K + . For this reason, it is frequently employed as a thickener and gelling agent in the food industry, often in milk products.

*

In aqueous solution, -car has a random coil conformation above a critical temperature (T c ) and a helical conformation below this temperature (Rees et al., 1969, McKinnon et al., 1969). The coil-helix transition temperature depends sensitively on the type and the concentration of the cations that are present in the solution (Morris et al., 1980;Rochas & Rinaudo, 1980;Viebke et al., 1994;Kara et al., 2003;[START_REF] Piculell | Food Polysaccharides and Their Applications[END_REF] (Rochas & Rinaudo, 1980).

κ-car is particularly sensitive to potassium and a concentration of 0.01M is enough to induce the transition at room temperature, whereas 0.2M sodium would be needed. Often the transition temperature is higher on heating (T m ) than on cooling.

Aggregation and gelation of kappa carrageenan

κ-car chains with the helical conformation aggregate and if their concentration is sufficiently high they form a percolating network, see figure 1.7. rad.s -1 and 2% strain (Doyle et al., 2002). As a consequence κ-car solutions form a self supporting gel below T c if the polymer concentration is not too low [START_REF] Hermansson | Rheological and Microstructural Evidence for Transient States During Gelation of Kappa-Carrageenan in the Presence of Potassium[END_REF][START_REF] Piculell | Effects of ions on the disorder-order transitions of gel-forming polysaccharides[END_REF]Hermansson et al., 1991;[START_REF] Borgström | On the structure of aggregated kappa-carrageenan helices. A study by cryo-TEM, optical rotation and viscometry[END_REF]Meunier et al., 1999;[START_REF] Chronakis | Viscoelastic properties for kappa-and iota-carrageenan in aqueous NaI from the liquid-like to the solid-like behaviour[END_REF]Doyle et al., 2002).

Gelation is very slow close to T c , but the gelation rate increases with decreasing temperature (Meunier et al., 1999). Since the coil-helix transition is reversible, κ-car gelation can be reversed by heating, but the melting temperature is often higher than the gelling temperature, see figure 1.8.

The coil-helix temperature and thus the gelling temperature depend on the concentration and type of salt. Gelation also depends on the polymer concentration via the counterion concentration if the latter are effective in inducing the coil-helix transition. Meunier et al. (1999) showed that in the range of 0.2 to 2g/L of sodium κ-car, gels were formed at the same temperature in the presence of 0.01M KCl and 0.1M NaCl, see figure 1.9.

However, the gelation rate increased with increasing polymer concentration. Meunier et al., 1999).

The effect of the salt concentration on the elastic modulus of κ-car gels has been studied in some detail for potassium and calcium that are most commonly used to induce gelation. Generally, it is found that the elastic modulus increases with increasing potassium concentration (Doyle et al., 2002), while it reaches a maximum when the calcium concentration is increased (Doyle et al., 2002;MacArtain et al., 2003;Thrimawithana et al., 2010). Another difference between gels induced by potassium and those induced by calcium is that the latter are increasingly turbid with increasing ion concentration (Doyle et al., 2002;MacArtain et al., 2003), while the former remain transparent.

The effect of mixed salts on the gelation of κ-car has been studied relatively little even though in applications often more than one type of salt is present. The most extensive study was reported by Hermansson et al. who found that adding NaCl to a κ-car solution containing 20mM potassium led to an increase of the elastic modulus, whereas in the absence of potassium these solutions did not gel (Hermansson et al., 1991). An even stronger synergistic effect was found when CaCl 2 was added. Addition of as little as 2mM CaCl 2 was found to increase the elastic modulus significantly. Mangione et al. reported that addition of 100mM

NaCl to a κ-car solution containing 20mM KCl did not influence T c , but led to a significant increase of the elastic shear modulus (Mangione et al., 2005). These results clearly show that gelation of κ-car in mixed salt solutions cannot be deduced from that of the pure salt solutions.

Mixtures of -lactoglobulin and κ-carrageenan

In general, mixtures of two different polymers in solution show three types of behaviour: co-solubility, segregative phase separation, and complex coacervation, see figure 1.10. When interaction between the polymers is weak or when the system is very dilute, mixing entropy dominates and homogeneous mixtures are formed. When the interaction is attractive soluble or insoluble complexes may be formed depending on the strength of the interactions, the molecular weight and flexibility of the polymers, and the distribution of negative and positive charges on the polymer. When the interaction is repulsive, contact between the same polymers is more favorable than contact between different polymers. In this case, phase separation occurs into two phases each enriched with one of the two polymers.

The behavior of polymer mixtures may depend on the conditions used such as pH, ionic strength, and temperature. For instance, in mixtures of anionic polysaccharides and proteins, complex coacervation is generally observed below the isoelectric point of the proteins (Tolstoguzov, 1991[START_REF] Tolstoguzov | Some thermodynamic considerations in food formulation[END_REF], while homogeneous mixing or segregative phase

Complex coacervation Co-soluble

Segregative phase separation Mixing separation is often observed above the isoelectric point [START_REF] Grinberg | Thermodynamic incompatibility of proteins and polysaccharides in solutions[END_REF][START_REF] Benichou | Protein-Polysaccharide Interactions for Stabilization of Food Emulsions[END_REF]. In the case of charged polysaccharides mixing is favored by the contribution of counterions to the mixing entropy. For the investigation reported in this thesis we only studied mixtures at pH 7 where both κ-car and -lg are negatively charged. Therefore we limit our review of the literature on κ-car/-lg mixtures to this situation. Native -lg and κ-car form homogeneous mixtures in aqueous solution at least in the range of -lg (up to C b = 100 g/L) and κ-car (up to C k = 20g/L) covered in the investigation. However, -lg aggregates can be incompatible with κ-car depending on the concentration and the size of the aggregates. The effect of phase separation has been studied for mixtures with separately formed -lg aggregates and for mixtures in which the -lg aggregates were formed in-situ by heating.

Mixtures of -lg aggregates and κ-car have been studied extensively in the past. The effect of the pH on the behavior of mixtures was studied by a number of authors (Mleko et al., 1997;Turgeon & Beaulieu, 2001;[START_REF] Gustaw | The effect of pH and carrageenan concentration on the rheological properties of whey protein gels[END_REF]de Jong et al., 2009;[START_REF] Stone | Formation and functionality of whey protein isolate-(kappa-, iota-, and lambda-type) carrageenan electrostatic complexes[END_REF][START_REF] Çakır | The effect of microstructure on the sensory perception and textural characteristics of whey protein/κ-carrageenan mixed gels[END_REF][START_REF] Hosseini | Complex coacervation of β-lactoglobulin -κ-Carrageenan aqueous mixtures as affected by polysaccharide sonication[END_REF]Ould Eleya et al. 2000b). However, as mentioned above, here we focus on the situation at neutral pH. We will distinguish two situations: heated mixtures of native -lg and κ-car (mixing before heating), and mixtures of preheated -lg and κ-car (mixing after heating). In both cases the mixtures contain -lg aggregates and κ-car, but the aggregates were formed in different circumstances. Of course, only mixing before heating allows the formation of -lg gels mixed with κ-car.

Mixing after heating

A number of authors studied mixtures of κ-car with protein aggregates prepared separately by heating native protein in the absence or presence of salt (Tziboula & Horne, 1999;Croguennoc et al., 2001a;Baussay et al., 2006a,b;Gaaloul et al., 2010;Ako et al., 2011). The mixtures were found to phase separate above a critical κ-car concentration leading to the formation of spherical protein rich micro-domains that tend to cluster and slowly precipitate under gravity. The critical κ-car concentration decreased with increasing aggregate size from more than above 10g/L if the radius of gyration was R g = 20nm to less than 2g/L when R g > 300nm (Baussay et al., 2006a). The extent of phase separation increased with increasing κ-car concentration, and in the case of polydisperse -lg aggregates the larger aggregates phase separate preferentially (Croguennoc et al., 2001a).

The effect of κ-car gelation on phase separation in mixtures at neutral pH in the presence of salt was reported by Baussay et al. (2006a) and Ako et al. (2011). Salt induced gelation of κ-car, coil-helix transition occurred at its critical concentration that depends on concentration of polymers. The turbidity induced by micro phase separation was found to drop dramatically during cooling below T c when the κ-car gelled and to increase again when the gel melted at a higher temperature (Baussay et al., 2006b). It was shown that the protein aggregates were expelled from the protein rich domains, which explained the reduction of the turbidity, but the domains remained visible in microscopy. In another study, microphase separation was observed during cooling a mixture of -lg aggregates and κ-car in the presence of 0.2M NaCl after heating at 60 o C, but the domains dispersed again below T c when κ-car gelled (Ako et al., 2011). Inducing only the coil-helix transition in the presence of 0.2M NaI, without gelation, did not lead to dispersion of the domains. The strength of -lg network will increase at low concentration of κ-car (0-1.7g/L) but drop at higher κ-car concentration (2.55 and 3.4g/L).

Mixing before heating

The behavior of heated mixtures of native -lg and κ-car has been studied more often.

The presence of κ-car coils in the mixtures does not influence the denaturation of -lg and the aggregate structure, but it accelerates the aggregate growth (Capron et al., 1999a). Micro phase separation of the mixtures was observed when above a critical polymer concentration [START_REF] Croguennoc | Phase separation and association of globular protein aggregates in the presence of polysaccharides: 2. Heated mixtures of native β-lactoglobulin and -carrageenan[END_REF]Zhang & Foegeding, 2003;[START_REF] Gustaw | The effect of pH and carrageenan concentration on the rheological properties of whey protein gels[END_REF][START_REF] De La Fuente | Influence of κ-carrageenan on the aggregation behaviour of proteins in heated whey protein isolate solutions[END_REF]Gaaloul et al., 2009b;[START_REF] Flett | Whey protein aggregate formation during heating in the presence of κ-carrageenan[END_REF]Gaaloul et al., 2010;Ako et al., 2011). At lower -lg concentrations the protein rich domains form clusters that precipitate, but at higher -lg concentrations they can form a space spanning network that can support its own weight [START_REF] Croguennoc | Phase separation and association of globular protein aggregates in the presence of polysaccharides: 2. Heated mixtures of native β-lactoglobulin and -carrageenan[END_REF]Zhang & Foegeding, 2003). The structure also depends on the heating time, because more and larger aggregates are formed with increasing heating time. de la Fuente et al., 2004 found that mixtures containing 2wt % WPI and 0.1wt% κ-car at 0.1M NaCl were homogeneous after heating at 75 o C for 5 min, but showed micro phase separation when heated longer (15min). (Gaaloul et al., 2009a).

Gelation of -lg in heated mixtures containing -car coils was studied at high concentration of protein either in the absence or presence of NaCl (Mleko et al., 1997;Capron et al., 1999a,b;[START_REF] Gustaw | The effect of pH and carrageenan concentration on the rheological properties of whey protein gels[END_REF]Gaaloul et al., 2009a;[START_REF] Cakir | Combining protein micro-phase separation and protein-polysaccharide segregative phase separation to produce gel structures Food[END_REF]. Gelation is faster if more κ-car is added (Capron et al., 1999a;Gaaloul et al., 2009a). Gaaloul et al. (2009a) found that the elastic modulus of 8% WPI heated at 80°C increased with increasing κ-car up to 0.6% (figure 1.11), while [START_REF] Cakir | Combining protein micro-phase separation and protein-polysaccharide segregative phase separation to produce gel structures Food[END_REF] reported a maximum around 0.2% κcar for 13% WPI heated at 80°C. Capron et al. (1999a) also reported a maximum at about 0.1% κ-car for 5% -lg heated at 75°C.

In the presence of salt, κ-car can gel after cooling in the heated mixtures leading to an increase of the elastic modulus [START_REF] Capron | Effect of addition of κ-carrageenan on the mechanical and structural properties of β-lactoglobulin gels[END_REF]Ould Eleya et al., 2000a,b;Turgeon & Beaulieu, 2001;Harrington et al., 2009;Gaaloul et al., 2009a;Çakır & Foegeding, 2011;Ako et al., 2011). Ako et al. (2011) found that at 250mM NaCl the -car formed in the WPI gel was stronger than without the proteins. However, Harrington et al. (2009) found that in 8mM

CaCl 2 the -car gels was weaker in the WPI gel. They attributed this to competition for Ca 2+

between the proteins and the -car. At higher -car concentrations the gel properties are dominated by those of the polysaccharide (Turgeon & Beaulieu, 2001, Ako et al., 2011).

Meunier, V., Nicolai, T., Durand, D., & Parker, A. (1999) Phan-Xuan, T., [START_REF] Phan-Xuan | On the crucial importance of the pH for the formation and self-stabilization of protein microgels and strands[END_REF]. On the crucial importance of the pH for the formation and self-stabilazation of protein microgels and strands. Langmuir, 27, 15092-15101.

Phan-Xuan, T., Durand, D., Nicolai, T., Donato, L., [START_REF] Phan-Xuan | Tuning the structure of protein particles and gels with calcium or sodium ions[END_REF].

Tuning the Structure of Protein Particles and Gels with Calcium or Sodium Ions. , 14(6), 1980-1989. Phan-Xuan, T., Durand, D., Nicolai, T., Donato, L., Schmitt, C., & Bovetto, L. (2014). Heat induced formation of beta-lactoglobulin microgels driven by addition of calcium ions. Food , 34, 227-235. Tilley, J. M. A. (1960). The chemical and physical properties of bovine -lactoglobulin.

Biomacromolecules

Hydrocolloids

Dairy. Sci. Abstr., 22, 11-25. Tolstoguzov, V. B. (1991). Functional properties of food proteins and role of proteinpolysaccharide interaction. Food Hydrocolloids, 4(6), 429-468. Chapter 2:

MATERIALS AND METHODS

Materials

For the research presented in this thesis I used mainly -lactoglobulin (Chapter 4) and sodium κ-carrageenan (Chapter 3 and 4).

The sodium κ-carrageenan (κ-car) used for this study is an alkali treated extract from

Eucheuma cottonii and was a gift from Cargill (Baupte, France). Using NMR it was found that the sample contained less than 5% -carrageenan. A freeze-dried sample of κ-car was dissolved by stirring for a few hours in Milli-Q water (70°C) with 200 ppm sodium azide added as a bacteriostatic agent. The solution was extensively dialysed against Milli-Q water at pH= 7 and subsequently filtered through 0.45 µm pore size Anatop filters. The pH of the solution was adjusted to 7 by adding small amounts of HCl 0.1 M. The required amounts of KCl, CaCl 2 or NaCl were added in the form of concentrated salt solutions. The κ-car concentration (C k ) was determined by measuring the refractive index using refractive index increment 0.145 ml/g. The molar mass (M w ) and radius of gyration (R g ) were determined by light scattering as described elsewhere (Meunier et al., 1999) with M w = 2.1x10 5 g/mol and R g = 52nm.

κ-car was fluorescently labelled by fluorescein isothiocyanate (FITC) to investigate the distribution on mixtures with -lg. The protocol was prepared following the procedure from Heilig et al., 2009. 1g κ-car powder was dissolved in 80ml of dimethyl sulfoxide (DMSO) containing 80l of pyridine and stirred for 1 hour before adding 0.1g FITC and 40l dibutyltin dilaurate. The solution was stirred for 30 min and subsequently heated at 95 0 C for 3 hours. After cooling to room temperature, the excess FITC was removed by washing in isopropanol and centrifugation at 9600g for 45min. The washing step was repeated until FITC could no longer be detected in the isopropanol. Finally, the modified κ-car was freeze dried and kept in the dark. No effect of labeling on rheology and on the structure of the mixtures was observed. (Nicolai, 2007) and was found to range from 17 to 320nm, depending on the concentrations of -lg and CaCl 2 and the pH. In the absence of added salt, small strand-like -lg aggregates were formed, while larger spherical particles were produced after adding controlled amounts of CaCl 2 . A detailed description of the formation of the protein particles and their characterization using light scattering can be found in refs (Phan-Xuan et al., 2013& 2014). The specific concentrations used to prepare lg aggregates will be presented in the results section.

Methods

Light scattering

Light scattering is a common useful technique to measure the average molecular weight (M w ), the z-average size (radius of gyration (R g ) and hydrodynamic radius (R h )) and the size distribution. Depending on the purpose of study, two distinct methods are used: static light scattering and dynamic light scattering. Static Light Scattering (SLS) is a technique in which the average intensity of the scattered light of the sample is measured as a function of the scattering wave vector (q) that depends on the scattering angle ():

   / ) sin( 4 n q 
, where n is the refractive index of the solution and  is the wavelength of the incident light. In dilute solution the radius of gyration (R g ) can be calculated from the initial dependence of the intensity on q in the range between 20 and 100 nm.

    3 / . 1 / ) /( 2 g w r R q M KC I   (2.1)
where I r is the scattering intensity of the sample minus that of the solvent divided by that of a standard (toluene). K is an optical constant that depends on the refractive index increment of the solute (  n/  c) and the Raleigh factor of the standard (R st ):

tol s tol a s R n n c n N n K 1 4 2 2 4 2 2                    (2.2)
with N a Avogadro's number. (n tol /n s ) 2 corrects for the difference in refractive index between the sample and the toluene standard. We have used (

 n/  c) = 0.189mL/g; R tol = 1.495x10 -5
cm -1 at 20°C and  = 632nm.

Dynamic light scattering (DLS) is a technique in which the autocorrelation function of the scattered light intensity fluctuation is measured. It can be used to determine the distribution of hydrodynamic radii in the range between about 1 and 500nm. The normalized electric field autocorrelation functions (g 1 (t)) obtained from the dynamic light scattering measurements [START_REF] Berne | Dynamic Light Scattering with applications to Chemistry[END_REF] were analyzed in terms of a distribution of relaxation times (τ):

   ) ( ) / exp( ) ( ) ( 1    d t A t g (2.3)
In dilute solutions, the relaxation is caused by translational diffusion of the particles and the z-average diffusion coefficient (D) can be calculated from the average relaxation time:

2 1 / q D     (2.4)
The z-average hydrodynamic radius of the solute can be calculated using the Stokes-Einstein equation:

D kT R h  6  (2.5)
with η the viscosity of the solvent, k Boltzman's constant, and T the absolute temperature. For polydisperse samples a distribution of the hydrodynamic radii can be obtained in this way from the distribution of relaxation times. If q.R h >1 one has to consider the effects of internal dynamics and polydispersity. Therefore measurements were done at different angles to ensure that the limiting regime of q.R h <1 was reached.

For this study light scattering measurements were done with a commercial apparatus

Turbidity measurements

Turbidity is a measure of the loss of transparency due to very strong scattering of light by the solute:

l I I 1 ln 0               (2.6)
where I is the intensity of the light transmitted through the sample with path length (l) and I 0 is that for the pure solvent.

Here, turbidity measurements were done in rectangular air tight cells using a UV-Visible spectrometer Varian Cary-50 Bio (Les Ulis, France). Different path lengths were used depending on the turbidity of the samples in order to avoid saturation. Measurements were done at different wavelengths (300; 400; 500; 600; 680; 750 and 800nm) and different temperatures that were controlled within 0.2 °C using a thermostat bath.

Determination of the protein concentration with UV-Visible spectroscopy

Protein concentrations (C) were determined by ultraviolet spectroscopy by measuring the absorbance (A) at the maximum situated at a wavelength of 278 nm using the Beer-Lambert's law: A = ε.l.C, with a molar extinction coefficient ε = 0.96 Lg -1 cm -1 . In order to avoid effects of turbidity and interactions, the samples were diluted to a concentration of about 1g/L.

Confocal Laser Scanning Microscopy (CLSM)

Confocal laser scanning microscopy (CLSM) is a useful method to study the structure of systems that are heterogeneous on microscopic length scales. Contrary to standard optical microscopy the image of a single focal plane is obtained, which necessitates the use of fluorescent probes in order to gather sufficient light. Different components can be distinguished by using different fluorescent labeling. A schematic drawing of a CLSM apparatus is given in figure 2.2. A laser beam is reflected by a dichromatic mirror toward to specimen after through the pinhole. The light emitted by the probes passes through the same dichotic mirror and is detected by a photomultiplier detector after passing through a pinhole that stops out-of-focus light. The objective is to concentrate the energy of light ray to specimen.

In this study, observations were made with a Leica TCS-SP2 (Leica Microsystems Heidelberg, Germany). Images of 512x512 pixels were produced at different magnifications with two different water immersion objectives lenses: HCx PL APO 63x NA=1.2 and 20x NA=0.7. -lg was labelled with the fluorochrome rhodamine B isothiocyanate (Rho. B), by adding a small amount of a concentrated rhodamine solution to reach a final concentration of 5 ppm. κ-car was covalently labeled with fluorescein isothiocyanate isomer I (FITC) as described in ref. (Heilig et al., 2009). The incident light was emitted by a laser beam at 543 nm and/or at 488 nm. The fluorescence intensity was recorded between 560 and 700 nm.

In most cases the solutions were inserted between a concave slide and a cover slip and hermetically sealed, which allowed heating in a thermostat bath. In a few cases the samples were inserted in a Chambered Coverglass which allowed rapid observation.

Rheology

Rheology is the study of the flow or deformation of materials under mechanical stress.

Here we use oscillatory shear measurements to characterize the mechanical properties of the materials as a function of the shear stress () and the oscillation. A sinusoidal shear stress is imposed at a frequency ():  =  0 .sin(.t), and resulting deformation () of the material is measured:  =  0 .sin(.t+), where  is the phase shift. At low stresses in the so-called linear response regime  0   0 and the in phase and out of phase deformation are characterized by the storage (G ' ) and the loss modulus (G"), respectively:

    sin ) ( 0 0   G (2.7)     cos ) ( 0 0    G (2.8)
For elastic solids G' is larger than G" and independent of the frequency, while for Newtonian liquids G' is smaller than G" and both moduli depend on the frequency: G'   -2 and G"   -1 . Viscoelastic liquid (solid) materials show solid-like behavior at high frequencies and liquid (solid) like behavior at low frequencies.

A schematic drawing of the instrument used for the present study (AR2000, TA Instruments) is given in figure 2.4. A plate -plate geometry was used with a diameter of 40 mm. The gap was in most cases set at 700 m, but the effect of varying the gap width was tested. The temperature was controlled by a Peltier system on a wide range of value (5 to 85 o C) and the geometry was covered with paraffin oil to prevent water evaporation. For some systems the gel partially slipped leading to a strong decrease of the shear modulus. In this case sandpaper was glued onto the two surfaces of the geometry and plate. In all cases the dynamic measurements were done in the linear response regime.

Calcium activity measurements

The calcium ion activity in solution was determined by a calcium-specific electrode (Fisher Scientific, USA). A calibration curve was obtained by measuring CaCl 2 solutions in water at concentrations ranging from 0 to 25 mM. The concentration of free calcium ions was calculated by assuming that the activity of bound Ca 2+ was zero and the activity of free Ca 2+ was the same as in pure water. (Rees et al., 1969, McKinnon et al., 1969). Most often -car in the helical conformation will aggregate and can form a gel if the concentration is sufficiently high. The critical gelation temperature (T g ) is close to T c , which depends on the concentration and the type of cations that are present (Rochas & Rinaudo, 1980;[START_REF] Michel | Physico-chemical properties of carrageenan gels in the presence of various cations[END_REF].

Cooled water in-outlet

The effect of both the concentration and type of cation on the elastic modulus of -car gels has been studied in some detail for potassium and calcium that are most commonly used to induce gelation. Generally, it is found that the elastic modulus increases with increasing potassium concentration (Doyle et al., 2002;Nguyen et al., 2014;[START_REF] Nono | Gel formation of mixtures of κ-carrageenan and sodium caseinate[END_REF][START_REF] Núñez-Santiago | Rheological and calorimetric study of the solgel transition of κ-carrageenan[END_REF]2007;Thrimawithana et al., 2010), while it reaches a maximum when the calcium concentration is increased (Doyle et al., 2002;MacArtain et al., 2003;Thrimawithana et al., 2010). Another difference between gels induced by potassium and those induced by calcium is that the latter are increasingly turbid with increasing ion concentration (Doyle et al., 2002;MacArtain et al., 2003), while the former remain transparent.

In most applications of κ-car in food products more than one type of salt is present. As was mentioned in chapter 1, κ-car in mixed salts was studied by Hermansson et al. (1991) and Mangione et al. (2005) who found that either adding NaCl or CaCl 2 led to an increase of the elastic modulus of the potassium induced κ-car gel. However, the critical temperature was not influenced.

In this chapter, we present a systematic investigation of the influence of adding CaCl 2 or NaCl on -car gelation at pH 7 induced by potassium and compare it with gelation induced by pure CaCl 2 and pure KCl on the elastic modulus at steady state, on the gelation kinetics and the turbidity. The results have been published in Carbohydrate Polymer to which we refer for further details, see paper 1 of the Appendix.

Results

Single salt induced κ-carrageenan gelation

Gelation of κ-car induced by K +

κ-car solutions at concentrations between 0.5 to 16g/L were investigated at 10, 20 or 30mM KCl. The gel and melting temperatures increased with increasing KCl concentration in agreement with results reported in the literature (Doyle et al., 2002). At a given temperature below T g , the gelation rate increased with increasing KCl concentration as is illustrated in figure 3.2 where the evolution of G' at 0.1Hz is shown for C k = 2g/L after quickly cooling down to 5 o C from the liquid state at 50 o C. Notice that the shear modulus did not reach a stable value, but continued to increase logarithmically at long times indicating that κ-car gels slowly restructure. The frequency dependence of G' was weak so that the value obtained at 0.1Hz can be considered characteristic for the elastic modulus of the κ-car gel. depend significantly on the κ-car concentration in agreement with literature results (Kara et al., 2003). However, at C k = 1g/L gelation was very slow and the rise of G' is only observed at lower temperatures. At C k = 0.5g/L the concentration was too low to allow gelation. Similar results were seen at 10mM KCl (paper 1) and 30mM KCl (results not shown).

Though T g is independent of the κ-car concentration, the gelation rate and final gel strength at a given temperature increases with the κ-car concentrations as was already discussed in some detail by Meunier et al. (1999). This is demonstrated in figure 3.4 where the evolution of G' at 5°C is shown as a function of time. The gelation rate of κ-car after rapid cooling to 5 o C is shown in figure 3.6.

Remarkably, in the presence of CaCl 2, gelation was much faster than in the presence of KCl at the same low concentration of κ-car used in this experiment even at Ca 2+ concentrations at which T g was lower. At [CaCl 2 ] ≤ 4.7mM, no gelation was observed at 5 o C for at least 24h. At 5.3mM

CaCl 2 , gelation was slowest but the gel reached the highest value of G'. As mentioned in the introduction, this is also similar when authors observed that the elastic modulus of κ-car gel increased with increasing potassium concentration (Doyle et al., 2002), while Young's modulus solution containing 10g/L κ-car reached a maximum when the calcium concentration was increased (Doyle et al., 2002;Harrington et al., 2009). was observed only below 10°C, see figure 3.5. This means that the coil-helix transition was in all cases induced by K + . In pure KCl aggregation and gelation of the helices is very slow. The effect of adding CaCl 2 is not to induce the coil-helix transition at a higher temperature, but to reduce electrostatic repulsion between the chains so that gelation can occur rapidly.

Remarkably, gels became stiffer when more CaCl 2 was added while in pure CaCl 2 they became weaker. This is consistent with the observation that these gels were more homogeneous than pure CaCl 2 gels (paper 1). The observed effects of adding KCl can be explained as follows. In the absence of KCl, the coil-helix transition needs to be induced by CaCl 2 , which does not occur at 5°C below 5mM CaCl 2 . The modulus of Ca 2+ -induced gels decreases with increasing CaCl 2 concentration. When KCl is added, the transition is induced by K + leading to a different type of gel that is more homogeneous than Ca 2+ -induced gels. The K + -induced gels were stronger than the Ca 2+ -induced gels even at very low KCl concentrations except at 5.3mM CaCl 2 , which explains why in this case G' dropped when KCl was added even though gelation was faster. G' subsequently increased as more KCl was added, because the K + -induced gel became stiffer.

Influence of Na + on the K + -induced gelation of κ-car

The strong influence of CaCl 2 on Κ + -induced gelation of -car is caused by effective screening of the electrostatic interactions between the helices. The effect of adding a In the absence of KCl weak turbid gels were formed only for [NaCl] 100mM. Similar results were also reported by Mangione et al. (2005) who showed that the modulus of 4g/L κcar at 20mM KCl was much higher after addition of 100mM NaCl. or NaCl is simply screening of the electrostatic interaction, it should be the same at the same ionic strength. However, quantitative comparison of the elastic modulus at the same ionic strength shows that the influence of CaCl 2 was much stronger, which suggest that Ca 2+ binds specifically to κ-car. Indeed, measurement of the Ca 2+ activity showed a significant reduction in the presence of κ-car (figure 3.17).

Total concentration of CaCl 2 (mM) gels, but the gels became increasingly turbid when more salt was added. We note that the gels formed in pure KCl remained transparent up to at least 50mM. When NaCl or CaCl 2 was added the turbidity of K + -induced gels remained much lower than in the absence of KCl (figure 3.18a), but a small increase of the turbidity with increasing NaCl of CaCl 2 was seen here as well. It is clear that the structure of K + -induced gels is different from that induced by Ca 2+ and Na + . The latter are more heterogeneous probably because they form thicker strands of parallel packed helices.

Conclusions

-car gels are formed below the coil-helix transition temperature in the presence of KCl or CaCl 2 , but with different structures. The gel stiffness increases with increasing KCl concentration and the gels remain transparent, while the gel stiffness decreases with increasing CaCl 2 concentration and they become increasingly turbid.

Addition of NaCl reduces the electrostatic repulsion between the helices which facilitates their association for K + induced gelation. As a consequence the gel stiffness and the gelation rate increase with increasing NaCl concentration. A similar effect on K + induced car gelation was observed when CaCl 2 was added. However, the effect was much stronger than for NaCl at the same ionic strength indicating specific binding of Ca 2+ to -car.

The presence of a small amount of KCl strongly modified the Ca 2+ induced gelation of -car. Especially at higher CaCl 2 concentrations, it led to a strong increase of the elastic modulus and a reduction of the turbidity. The synergy between Ca 2+ and K + was most striking when the gels were weak in the pure salts.

Chapter 4:

MIXTURES OF -LACTOGLOBULIN AND -CARRAGEENAN

Introduction

Food products often contain aqueous mixtures of proteins and polysaccharides and understanding the behavior such mixtures is important for the use of existing food products or the development of new ones. Complex coacervation, phase separation and gelation can give rise to a wide range of phenomena that are, as yet, far from being fully understood [START_REF] Tolstoguzov | Some thermodynamic considerations in food formulation[END_REF][START_REF] Turgeon | Protein polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects[END_REF]Turgeon et al., 2007).

As mentioned in the introduction of chapter 1, when -lg is heated in aqueous solution it denatures leading to irreversible aggregation and at high concentrations to gelation (Clark, 1999;Foegeding, 2006;Nicolai, 2007). The structure and size of the aggregates depend on the protein concentration, the pH, the type and concentration of added salt (Nicolai et al., 2011).

When -lg is heated at a pH above its isoelectric point (pI = 5.2) in the presence of anionic polysaccharides, usually heterogeneous gels with a variety of different structures are formed (Ako et al., 2011;[START_REF] Cakir | Combining protein micro-phase separation and protein-polysaccharide segregative phase separation to produce gel structures Food[END_REF]de Jong et al., 2009). -car is an anionic polysaccharide isolated from algae and is used as thickening or gelling agent in food products [START_REF] Piculell | Food Polysaccharides and Their Applications[END_REF]. In aqueous solution it has a random coil conformation at high temperatures, but in the presence of salt it takes a helical conformation below a critical temperature (T c ) that depends on the type and concentration of the cations. -car helices have a tendency to aggregate which can lead to the formation of a percolating network. Gelation of -car can be reversed by heating above another critical temperature that is generally higher than the gelling temperature (T g ). The effect of adding single or mixed salt on κ-car gelation was discussed in the chapter 3 and is important for the understanding of the gelation of κ-car in the presence of -lg that will be discussed in this chapter.

Here we report on an investigation of aqueous mixtures of the protein -lg and -car at pH 7, where both biopolymers are negatively charged and do not associate. At this pH native -lg is compatible with κ-car up to high concentrations of both components. However, as was discussed in chapter 1 microphase separation has been observed to occur in mixtures of κ-carand-lg aggregates. The objective of the investigation was to elucidate the effect of Ca 2+

ions on the structure and the rheological properties presence of the mixtures, which important as Ca 2+ ions are present in most food application containing -car and milk proteins.

Mixtures of native -lg and κ-car are considered in section 4.2. In section 4. In the absence of -lg, κ-car gelation was very slow, but the rate increased with increasing native -lg concentration. The elastic modulus obtained after 100 min at 5°C increased weakly with increasing protein concentration between 5 and 60g/L, see figure 4.1a.

The effect may be explained by the presence of sodium counterions that are added together with -lg in the mixtures. Indeed if a small amount of NaCl The effect can clearly be seen when an increasing amount of native -lg is added to κcar solution with a given CaCl 2 concentration. This competition for Ca 2+ was also observed in the absence of KCl when gelation was induced by Ca 2+ . Pure κ-car solutions formed a gel within 3 days in the fridge (4°C) for [CaCl 2 ] > 5mM as was discussed in (chapter 3). However, in the presence of 40g/L native protein no gels were formed even at [CaCl 2 ] = 10mM, the gels were only observed at 12-17mM CaCl 2 but they became weaker with increasing concentration of CaCl 2 (figure 4.4a).

Conclusion

Native -lg is compatible with κ-car and mixtures with 2g/L κ-car remain transparent at least up to C b = 100g/L. In mixtures containing CaCl 2 , -lg competes with κ-car for Ca 2+ leading to a decrease of the effect of stiffening κ-car gels induced by Ca 2+ . The turbidity is a useful qualitative measure of the extent of phase separation for the mixtures. If phase separation is caused by thermodynamic incompatibility of polymers of different chemistry, an increase of the compatibility with increasing temperature is expected, because the contribution of the mixing entropy to the free energy increases. Another potential driving force for phase separation is depletion of κ-car chains from between the protein aggregates (Gaaloul et al., 2010;Tuinier et al., 2003). However, the depletion interaction is itself an entropic effect and therefore no effect of the temperature is expected. Therefore we may conclude that depletion was not the main driving force of phase separation in these mixtures.

Effect of κ-carrageenan gelation on phase separation

The effect of κ-car gelation on phase separation was studied by cooling mixtures in the presence of KCl. The effect of gelation on phase separation was less important at higher κ-car concentrations even though the gels were stiffer. The reason is that the incompatibility increased and this effect dominated the effect of increased elasticity. The effect of κ-car gelation on phase separation is significant only close to the phase boundary.

Conclusion

Segregative phase separation between -lg aggregates and κ-car led to the formation of spherical protein rich domains with a diameter of a few microns. Microphase separation is favored by a reduction of the temperature close to the binodal implying that depletion is not the main driving force for phase separation.

Gelation of the κ-car can reverse phase separation close to the phase boundary. The extent of reversibility is directly correlated to the elastic shear modulus of the polysaccharide gel. Close to the binodal, transient phase separation occurs during cooling and heating caused by the combined effects of temperature and elasticity on the phase separation. The distribution of κ-car in the micro phase separated mixtures is shown in figure 4.16 for a mixtures containing 40g/L -lg microgels (R h = 110nm) and 2g/L κ-car covalently labeled with FITC at 15mM KCl. At the conditions of the experiment the fluorescence intensity was proportional to the polymer concentration. The profile of -lg and κ-car fluorescence intensities showed that the protein concentration was more than a factor 2 higher in the domains, but that the κ-car concentration was reduced by less than 30% in the protein rich domains. This means that κ-car distribution was not much influenced by micro phase separation.

As was shown above, cooling renders mixtures of κ-car and -lg aggregates more compatible. In a few cases we observed that homogeneous mixtures at 20°C became inhomogeneous at 5°C. This is illustrated in figure 4.17 for mixtures of 2g/L κ-car and 40g/L -lg aggregates in 10mM KCl. Figure 4.17 shows that the stronger phase separation at 5°C was preserved at 20°C at which temperature the images were taken, because κ-car gelation inhibited dispersion of the micro domains. This appears in contradiction with the observation discussed above that gelation inverses phase separation. We believe that the κ-car gels formed at these low polymer and KCl concentrations are too weak to drive dispersion. However, when the solutions were heated to 50°C so that the κ-car gel melted, the protein domains dispersed rapidly. Even when phase separation already occurred at 20°C it could be reinforced by further cooling to 5°C.

However, the effect of cooling to 5°C was only significant close to the phase boundary.

Effect of κ-carrageenan gelation on the rheology

The shear modulus of pure κ-car and mixtures containing either 10 or 40g/L microgels (R h = 240nm) at 10mM KCl were investigated at different concentrations of κ-car while cooling from 50 to 10 o C in steps of 5 o C maintaining the temperature for 20min at each step.

The storage shear moduli at 10°C are shown as a function of C k in figure 4.18. In each case G' increased with increasing C k , but the increase was stronger when more microgels were present. We believe that the increase of G' is caused by the fact that the microgels were formed in the presence of CaCl 2 and that together with the microgels Ca 2+ is added to κ-car solution. We showed in chapter 3 that a small amount of Ca 2+ reinforces K + -induced κ-car gels. The effect of adding microgels was largest at about 2g/L κ-car. Therefore, we investigated gelation of mixtures with this κ-car concentration during and after rapid cooling from 50 to 5 o C with different concentrations of microgels (figure 4.19a). The rate of κ-car gelation increased with increasing microgel concentration, because more calcium ions were added together with the microgels. As was mentioned above, G' was already significantly higher than for pure κ-car before gelation due to the adsorption of protein at the water-oil interface. When we compare the evolution of G' in figure 4.19a with that of pure κ-car containing the same amount of calcium ions (figure 4.19b), we find that the gels in the presence of microgels are much weaker. The reason is that the proteins strongly bind Ca 2+ so that less Ca 2+ is available to reinforce the κ-car gel as we discussed in section 4.2 for the case of mixtures with native -lg. Figure 4.20a compares the dependence of G' on the concentration of calcium ions for κ-car gels formed at 5°C without protein, with native proteins, with strands and with microgels. Addition of native -lg or strands strongly reduces the elastic modulus, but the addition of microgels reduces it further. The reason is that microgels bind Ca 2+ more strongly as is shown in figure 4.20b in which the fraction of free Ca 2+ is plotted as a function of the total calcium concentration in pure κ-car and in the presence of native or aggregated -lg.

Conclusion

In mixtures with κ-car, -lg microgels formed by heating in the presence of CaCl 2 phase separated at much lower κ-car concentrations than small -lg strands formed by heating in pure water. At lower κ-car concentrations the effect of CaCl 2 on the structure of the mixtures is directly related to the transition between the formation of -lg strands and microgels. Phase separation led to the formation of spherical protein rich domains with a diameter of a few microns. The concentration of κ-car was decreased by only about 30% within the protein rich domains. The protein rich domains cluster into large flocks that slowly sediment, but the precipitate can be easily dispersed by shaking.

In the mixtures -lg microgels competes with κ-car more strongly for Ca 2+ than native -lg or -lg strands. As a consequence the effect of stiffening κ-car by addition of CaCl 2 is less important in mixtures with -lg microgels.

Heated mixtures of κ-carrageenan and native β-lactoglobulin

In sections 4.3 and 4.4 we investigated systems for which -lg was first heated separately and subsequently mixed with κ-car, i.e. mixing after heating (MAH). In this section we will discuss the situation where κ-car was first mixed with native -lg and subsequently heated, i.e. mixing before heating (MBH). The two procedures are schematized in figure 4.21. likely smaller than those that precipitated as larger aggregates will preferentially enter the dense domains.

A major difference between the two methods is that during heating the mixtures protein aggregates become covalently linked within the phase separated structure, while for mixtures prepared by MAH the dense domains disperse rapidly upon dilution.

Effect of κ-car gelation on the structure and the rheology

The effect of κ-car gelation was studied by heating the mixtures in the presence of 10mM KCl and subsequently cooling. Figure 4.28 shows the elastic modulus of the gels during cooling to 5 o C and subsequent heating back to 85°C at a rate of 2 o C per minute. In the absence of KCl, we observed the weak progressive increased of G' with decreasing temperature that is wellknown for protein gels. However, in the presence of 10mM KCl G' increased sharply below a critical temperature of about 20 o C caused by gelation of κ-car within the -lg gel. The shear modulus decreased again during heating when the κ-car gel melted, but at a higher critical temperature than at which it was formed during cooling.

Conclusion

The presence of κ-car led to the formation of larger -lg strands when heated in pure water or with less than one calcium ion per protein. At higher CaCl 2 concentrations competition for Ca 2+ with κ-car led to formation of smaller microgels than in the absence of κ-car.

The structure of mixtures of -lg aggregates and κ-car coils with the same composition are similar whether prepared by mixing after heating or mixing before heating. However, in the former case phase separation can be reversed by dilution, while in the latter case the proteins become strongly bound within the domains and persist after dilution.

Addition of 10mM KCl to the mixtures favors -lg aggregation during heating, which led to micro phase separation at lower CaCl 2 concentrations than mixtures prepared by mixing after heating.

Heat-induced gelation of native -lg was facilitated by the presence of -car, probably because it favored contact between the growing -lg aggregates that are incompatible with car. Above a critical amount of added CaCl 2 micro phase separation occurs during heating and heterogeneous gels are formed. When the solution contains 10mM KCl, -car gels within the -lg gel upon cooling at a temperature close to that of the equivalent pure -car solutions. The -car gels melted again when heated, but the -lg gel remained intact during cooling and reheating. The mixed gel was somewhat stiffer than the sum of the -car gel in the presence of uncrosslinked native -lg and the -lg gel in the presence of uncrosslinked -car coils, indicating that the structure of the -car network that was formed within the -lg gel was different from that formed in the presence of -lg aggregates.

Turgeon, S. L., Beaulieu, M., [START_REF] Turgeon | Protein polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects[END_REF]. Protein-polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects. Current Opinion in Colloid & Interface Science, 8(4-5), 401-414.

Turgeon, S. L., Schmitt, C., & Sanchez, C. (2007). Protein-polysaccharide complexes and coacervates. Current Opinion in Colloid & Interface Science, 12(4-5), 166-178.

GENERAL CONCLUSION AND OUTLOOK Conclusion

The objective of this thesis was to study the structure and the rheology of mixtures of -lg aggregates or gels with κ-car solutions or gels and in particular the effect of calcium ionsIn first instance, we investigated gelation of pure -car solutions. Addition of of KCl induces the formation of homogeneous gels with a stiffness that increases with increasing salt concentration. A larger amount of CaCl 2 is needed to induce gelation and the gels are more heterogeneous. In addition, the stiffness of Ca 2+ -induced gels decreases with increasing concentrations. An important finding is that the presence of even small quantities of NaCl or CaCl 2 in addition to KCl accelerates K + -induced -car gelation and leads to stronger gels, without influencing the critical gelation temperature. It clear, that the type of ion that induces the coil-helix transition is crucial factor that determines the structure and the elasticity of the gels in mixed salts.

Native -lg and κ-car are compatible in aqueous solution up to high polymer concentrations. Addition of large amounts (60 g/L) of native -lg leads to weak acceleration of K + -induced gelation of κ-car, which can be explained by the effect of the sodium counterions that are added together with the proteins. In the presence of native -lg the effect of CaCl 2 on K + -induced gelation of κ-car is less important than for pure κ-car solutions. The reason is that -lg specifically binds Ca 2+ and competes with κ-car for the calcium ions. The same effect of competition was observed in mixtures with -lg strands formed by heating the proteins in pure water.

Mixtures of κ-car coils and -lg aggregates phase separated above a critical κ-car concentration that decreased with increasing sizes of the aggregates, but was little dependent on the -lg concentration. Phase separation led to the formation of micron sized protein enriched domains that stick together, but do not easily fuse. -car chains are only weakly depleted from the protein rich domains. The domains redisperse when the system is diluted.

Close to the critical concentration the extent of phase separation decreased with increasing temperature and gelation of κ-car drives a fraction of the -lg aggregates back into the continuous phase.

The size and morphology of aggregates formed by heating -lg in the presence of CaCl 2 changed from relatively small strands to larger spherical microgels when the number of

THE LIST OF PUBLICATIONS

This thesis is based on the work contained in the following papers:

1. Synergistic effects of mixed salt on the gelation of -carrageenan.

Introduction

The texture of many food products is determined by the combined presence of proteins and polysaccharides. Thorough understanding of the structures formed by these biopolymers in aqueous solution is a prerequisite for rational design and development of novel food products. For this reason, protein/polysaccharide mixtures have been studied extensively in the past and remain an active area of research.

There exists a range of proteins and polysaccharides that are suitable for use in food products, which means that a large number of combinations can be of potential interest. They can be roughly classiied into three categories, depending on the type of interaction [START_REF] Semenova | Biopolymers in Food Colloids: Thermodynamics and Molecular Interactions[END_REF][START_REF] Turgeon | Protein polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects[END_REF]: no/weak interaction, so the properties of the mixture are essentially equal to the sum of the individual components; strong attractive interaction, leading to complex coacervation; and strong repulsive interaction, resulting in segregative phase separation. Complex coacervation leads to an enrichment of one phase in both components, while segregative phase separation means that each phase is enriched in one of the components.

Here, we report on an investigation of aqueous mixtures of β-lactoglobulin (β-lg) and κ-carrageenan (κ-car) at pH 7, in which both biopolymers are negatively charged. β-lg is a globular protein and is the major protein component of whey [START_REF] Brownlow | Bovine [beta]-lactoglobulin at 1.8 A resolution-still an enigmatic lipocalin[END_REF][START_REF] Wit | Nutritional and functional characteristics of whey proteins in food products[END_REF][START_REF] Fox | Advanced Dairy Chemistry: Proteins[END_REF][START_REF] Kinsella | Proteins in Whey: Chemical, Physical and Functional Properties[END_REF]. κ-car is an anionic polysaccharide isolated from algae [START_REF] Morris | Understanding and Controlling the Microstructure of Complex Foods ed D J McClements[END_REF][START_REF] Piculell | Food Polysaccharides and Their Applications[END_REF]). Both biopolymers are widely used in the food industry and their aggregation and gelling properties have been investigated extensively in the past both individually [START_REF] Bryant | Molecular basis of protein functionality with special consideration of cold-set gels derived from heat-denatured whey[END_REF], Clark 1999[START_REF] De La Fuente | Recent advances in the characterization of heat-induced aggregates and inetrmediates of whey proteins[END_REF][START_REF] Gosal | Globular protein gelation[END_REF][START_REF] Morris | Understanding and Controlling the Microstructure of Complex Foods ed D J McClements[END_REF][START_REF] Piculell | Food Polysaccharides and Their Applications[END_REF][START_REF] Totosaus | A review of physical and chemical protein-gel induction[END_REF] and in mixtures [START_REF] Kruif | Polysaccharide protein interations Food[END_REF][START_REF] Morris | Interactions of food biopolymers[END_REF][START_REF] Nishinari | Hydrocolloid gels of polysaccharides and proteins[END_REF][START_REF] Turgeon | Protein polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects[END_REF]. At pH 7, native β-lg is compatible with κ-car up to high concentrations of both components (Croguennoc et al 2001a).

β-lg denatures and aggregates irreversibly when heated above about 60 °C. Depending on the environmental conditions, aggregates with different morphologies are formed, as was recently reviewed [START_REF] Nicolai | Controlled food protein aggregation for new functionality[END_REF], Nicolai et al 2011[START_REF] Mezzenga | The self-assembly, aggregation and phase transitions of food protein systems in one, two and three dimensions Rep[END_REF]. For pH > 6.2 small strand-like aggregates are formed, with a hydrodynamic radius of about 15 nm (Durand et al 2002), while closer to the iso-electric point (pI = 5.2) mainly spherical particles are formed, with hydrodynamic radii ranging between 40 and 200 nm depending on the pH [START_REF] Donato | Mechanism of formation of stable heat-induced beta-lactoglobulin microgels[END_REF], Schmitt et al 2009, 2010). Around pH 6.0 a signiicant fraction of both strands and microgels is formed within the same sample (Phan-Xuan et al 2011). Stable suspensions of the spherical particles, also called microgels, can only be formed in a very narrow range of the pH (5.75-6.1). Closer to the iso-electric point secondary aggregation leads to precipitation. Secondary aggregation of the strands and the microgels into larger selfsimilar aggregates occurs at higher protein concentrations and leads to gelation.

Interestingly, the discontinuous change of the morphology can also be induced at higher pH by adding bivalent cations before heating, but not by adding monovalent salt (Phan-Xuan et al 2014, 2013). The transition is driven by the reduction of the net protein charge due to speciic adsorption of Ca 2+ and is controlled by the molar ratio of calcium to protein (R). At pH 7 the fraction of microgels increases rapidly from less than 20% at R < 1 to more than 80% at R > 2. At R = 1.5, the solution contains an almost equal weight fraction of strands and microgels that can be clearly distinguished in transmission electron microscope images (Phan-Xuan et al 2013). The transition occurs between R = 1 and R = 2 independent of the protein concentration and heating temperature, but a stable suspension of individual microgels can only be formed in a narrow range of R and for protein concentrations less than 60 g L -1 (Phan-Xuan et al 2014).

When κ-car chains are added to a stable suspension of β-lg aggregates at room temperature, the two components phase separate above a critical concentration of κ-car. This phenomenon has been studied in some detail for mixtures of κ-car with small strand-like β-lg aggregates and with larger self-similar aggregates formed by secondary aggregation of the strands (Baussay et al 2006, Croguennoc et al 2001a, de Jong et al 2009, Gaaloul et al 2010, Nguyen et al 2014). It was shown that the critical concentration of κ-car to induce phase separation is about 8 g L -1 for the strands (Nguyen et al 2014) and it decreases strongly with increasing aggregate size (Baussay et al 2006). The critical κ-car concentration decreased weakly with increasing protein concentration. It has been suggested that depletion of the κ-car chains is the origin of the formation of dense domains of protein aggregates (Tuinier et al 2003). However, we have shown that, very close to the critical concentration, microphase separation can be reverted by increasing the temperature (Nguyen et al 2014). Since the depletion interaction is entropic no temperature dependence would be expected if it was the main driving force.

For reasons that are not yet fully elucidated, phase separation was found to halt when the protein rich domains had grown to a diameter of a few microns. In pure water these domains are liquid and fully disperse when diluted. However, in the presence of salt the protein aggregates within the domains irreversibly crosslink at a rate that depends on the amount and valence of the added salt. In either case, the protein rich domains stick to each other and form larger clusters that precipitate, but the bonds between the domains are relatively weak and can be easily broken by shaking the sample.

Micro-phase separation also occurs when mixtures of native β-lg or whey proteins and κ-car are heated [START_REF] Cakir | Combining protein micro-phase separation and protein-polysaccharide segregative phase separation to produce gel structures Food[END_REF], Capron et al 1999[START_REF] Croguennoc | Phase separation and association of globular protein aggregates in the presence of polysaccharides: 2. Heated mixtures of native β-lactoglobulin and -carrageenan[END_REF][START_REF] Eleya | Rheology of kappacarrageenan and beta-lactoglobulin mixed gels Food[END_REF], Gaaloul et al 2009, Harrington et al 2009[START_REF] Roesch | Kappa-carrageenan and beta-lactoglobulin interactions visualized by atomic force microscopy Food[END_REF]. In this case, phase separation is induced in situ by the growth of the aggregates. If the protein concentration is suficiently high, heating of the mixtures leads to the formation of heterogeneous gels, consisting of a network of covalently bound protein rich domains.

In view of the sensitivity of the mixtures to the β-lg aggregate size, one expects that the transition of the aggregate morphology induced by adding a small amount of CaCl 2 , will have a dramatic effect on the structure of heated mixtures. We investigated the effect of the transition on the structure: by mixing at room temperature κ-car with β-lg aggregates formed separately at different CaCl 2 concentrations; or by heating mixtures of native β-lg and κ-car at different CaCl 2 concentrations. We will show that adding small amounts of CaCl 2 has indeed a dramatic effect on the structure of the mixtures that can be explained by the transition in the morphology of the β-lg aggregates. For this study we used sodium κ-car that had in all mixtures the random coil conformation. It is well known that, in solution, a conformational transition of κ-car between a random coil and a helix can be induced by cooling in the presence of speciic salts, which leads to aggregation or gelation [START_REF] Piculell | Food Polysaccharides and Their Applications[END_REF]. The effect of gelling the κ-car chains on the structure and the rheology of the mixtures will be reported elsewhere.

Materials and methods

Materials

The β-lactoglobulin (Biopure, used in this study was purchased from Davisco Foods International, Inc.

(Le Sueur, MN, USA) and consisted of approximately equal quantities of variants A and B. The powder was dissolved in Milli-Q water, which contained 200 ppm NaN 3 to prevent bacterial growth. Solutions were dialyzed extensively against Milli-Q water with 200 ppm sodium azide and subsequently iltered through 0.2 µm pore size Anatope ilters. The pH of the solution was adjusted to seven by adding small amounts of NaOH 0.1 N under vigorous stirring. The protein concentration was determined by UV absorption at 278 nm with a UV-Visible spectrometer Varian Cary-50 Bio (Les Ulis, France) using extinction coeficient 0.96 Lg -1 cm -1 . The protein concentration in the supernatant of the mixtures was determined in the same manner, taking into consideration the effect of the turbidity.

The sodium κ-carrageenan is an alkali treated extract from Eucheuma cottonii and was a gift from Cargill (Baupte, France). Using NMR it was found that the sample contained less than 5% ι-carrageenan. A freeze-dried sample of κ-car was dissolved by stirring for a few hours in Milli-Q water (70 °C) with 200 ppm sodium azide added as a bacteriostatic agent. The solution was extensively dialyzed against Milli-Q water at pH 7 with 200 ppm sodium azide and subsequently iltered through 0.45 µm pore size Anatope ilters. The pH of the solution was adjusted to seven by adding small amounts of HCl 0.1 N under vigorous stirring. The κ-car concentration (C K ) was determined by measuring the refractive index using refractive index increment 0.145 g ml -1 . The molar mass (M w ) and radius of gyration (R g ) were determined by light scattering, as described elsewhere (Meunier et al 1999), with M w = 2.1 × 10 5 g mol -1 and R g = 52 nm.

Preparation of the mixtures

Mixtures of β-lg aggregates and κ-car, with identical compositions, were prepared by two methods. The irst method, called mixing after heating (MAH), consisted of heating β-lg solutions at 85 °C during 10 h at different concentrations of CaCl 2 and subsequently mixing with the κ-car solution at room temperature. The second method, called mixing before heating (MBH), consisted of mixing native β-lg and κ-car at different CaCl 2 concentrations at room temperature and subsequently heating at 85 °C for 10 h. The protein concentration during heating was the same in both methods. After the heat treatment, all proteins were aggregated and a steady state was reached. In order to avoid dilution with method MAH, the concentration of the aggregate solution was increased by slow evaporation of some of the water. Care was taken that the inal concentrations of the biopolymers and the CaCl 2 were the same. The pH was set at 7.0 before heating by adding small amounts of a 0.1 M NaOH solution.

Methods

Confocal Laser Scanning Microscopy (CLSM) was used in the luorescence mode. Observations were made with a Leica TCS-SP2 (Leica Microsystems Heidelberg, Germany). A water immersion objective lens was used HCxPL APO 63x NA = 1.2 with theoretical resolution of 0.3 µm in the x-y plane. β-lg was physically labelled with the luorochrome rhodamine B isothiocyanate, by adding a small amount of a concentrated rhodamine solution (5 ppm) to the β-lg solutions. No effect of labelling on the phase separation was observed. κ-car was covalently labelled with luorescein isothiocyanate (FITC) following Heilig et al (2009).

Light scattering measurements were done with a commercial apparatus (ALV-CGS3, ALV-Langen) operating with a vertically polarized He-Ne laser with wavelength λ = 632 nm. The temperature was controlled at 20 °C with a thermostat bath to within ± 0.2 °C. The normalized electric ield autocorrelation functions (g 1 (t)) obtained from dynamic light scattering (DLS) measurements [START_REF] Berne | Dynamic Light Scattering[END_REF] were analyzed in terms of a distribution of relaxation times (τ):

∫ ττ τ =- g tA td () () exp( /)() .
1

In dilute solutions, the relaxation was caused by translational diffusion of the particles. The diffusion coeficient (D) was calculated from the average relaxation rate:

τ =< > - Dq / 12
with q the scattering wave vector. The z-average hydrodynamic radius (R h ) of solute was calculated using the Stokes-Einstein equation:

πη = R kT D 6 h
with η the viscosity of the solvent, k Boltzman's constant, and T the absolute temperature.

Results

We investigated the effect of the size and the morphology of β-lg aggregates formed at different CaCl 2 concentrations on the structure of mixtures with κ-car. We distinguish between situations in which the aggregates were formed: in the presence of κ-car (MBH); separately, and subsequently mixed with κ-car (MAH). We ix, in the irst instance, the β-lg concentration at 20 g L -1 and discuss the effect of varying the protein concentration later.

β-lg aggregation without κ-car

For the case of MAH, β-lg aggregates with different sizes and morphologies were formed separately by heating during 10 h at 85 °C aqueous solutions containing C b = 20 g L -1 β-lg at pH 7 in the presence of different CaCl 2 concentrations ([CaCl 2 ]). The z-average hydrodynamic radius of the aggregates was determined using dynamic light scattering, as described in the material and methods section. R h increased initially weakly with increasing [CaCl 2 ] from 18 to 24 nm, but above 1 mM it increased sharply, due to the transition from the formation of strands to microgels, see igure 1. We note that the z-average value of R h is strongly weighted towards the largest aggregates in the solution, implying that in mixtures of strands and microgels one measures essentially the size of the latter. It also means that the contribution of κ-car to the scattering intensity can be neglected when it is present in mixtures with protein aggregates.

The fraction of microgels can be estimated by measuring the fraction of proteins that sediment (F sed ) after centrifugation at 5 × 10 4 g (Phan-Xuan et al 2011). The latter increased rapidly for [CaCl 2 ] > 1 mM, see igure 1. A small fraction of aggregates sediment also for [CaCl 2 ] < 1 mM, even though no microgels are observed by TEM or light scattering in that case, indicating that a small fraction of the strands is large enough to sediment at 5 × 10 4 g, as was discussed elsewhere (Phan-Xuan et al 2013). For [CaCl 2 ] > 3 mM secondary aggregation of the microgels occurred, leading eventually to precipitation.

3.2. Structure of the mixtures 20 g L -1 β-lg was mixed with different concentrations of κ-car (C K ) either before (MBH) or after heating (MAH). In both cases the microstructure of the mixtures was investigated at room temperature with CLSM. As an example, igure 2 shows the effect of the CaCl 2 concentration on the protein distribution in mixtures containing 5 g L -1 κ-car. In both situations, phase separation was observed when CaCl 2 was present that caused the formation of spherical protein rich domains. The irst signs of phase separation were seen for heated mixtures (MBH) for [CaCl 2 ] = 0.5 M, while for mixing after heating it was observed irst at 1.0 mM CaCl 2 . The fraction of proteins involved in the formation of the domains (F ps ) increased with increasing [CaCl 2 ], see below.

The size of the protein rich domains decreased with increasing CaCl 2 concentration and they had a tendency to stick together into large clusters or even a space illing network. In some cases partially fused domains can be seen. Similar structures were obtained with either method, though in the case of MAH the domains appeared smaller and less clustered. An important difference between mixing before and after heating was, however, that phase separation was reversible for MAH even a week after preparation, while the protein rich domains that formed by heating the mixture resisted dilution (see supplementary information). The reason that the aggregates within the domains become irreversibly crosslinked during heating is that as long as native proteins are present that can denature, the aggregates continue to grow and form a gel. Growth of the aggregates only stops when all native proteins have been consumed or the temperature is dropped to far below the denaturation temperature. In the heated mixtures, micro-phase separation occurred when the aggregates reached a critical size, but at this point native proteins were still present, which allowed the aggregates in the domains to cross-link irreversibly. The aggregates that were formed separately no longer contained native proteins. In addition, they were mixed with κ-car far below the denaturation temperature. Therefore, no cross-linking of the aggregates occurred within the domains formed by MAH.

When left standing, we observed slow sedimentation of the clusters at a rate that decreased with increasing κ-car concentration, see igure 3. The precipitate could be easily dispersed by shaking but small clusters could not be broken into individual domains in this way. Therefore, sedimentation was signiicantly faster after manual dispersion of the precipitate. Similar results were obtained at other κ-car concentrations between 1 and 5 g L -1 . At lower κ-car concentrations the mixtures remained homogeneous up to 3.0 mM CaCl 2 , while at higher concentrations phase separation was observed even in the absence of CaCl 2 .

Partitioning of β-lg and κ-car

The partition of the β-lg aggregates and the κ-car chains between the two phases could be quantiied by measuring the luorescence intensity of β-lg and κ-car labelled with different luorescent probes. Figure 4 compares an image taken with luorescence from κ-car with that from β-lg for a mixture with C b = 20 g L -1 and C K = 5 g L -1 at [CaCl 2 ] = 1.5 mM that was mixed before heating. After heating this mixture, about 50% of the proteins entered the dense domains. Individual protein rich domains can be clearly seen with the protein signal. The concentration of κ-car is less in the protein rich domains, but the individual domains cannot be seen clearly with the κ-car signal, as it appears to also be lower in the space between neighbouring protein domains.

We have quantiied the partitioning of β-lg and κ-car by tracing the luorescence intensity proile along the white lines shown in the images, see igure 5. At the conditions of the experiment, the luorescence intensity is proportional to the polymer concentration. It is clear that phase separation is much more important for the proteins than for the polysaccharides. Quantitative measurements on this and other images conirmed the visual impression that κ-car is also depleted from between the aggregated protein micro-domains. Similar results were obtained when the κ-car was mixed with β-lg after heating and at other κ-car concentrations. shifted progressively to larger values with decreasing κ-car concentrations. We will discuss the origin of this behaviour below. Interestingly, at C K < 4 g L -1 phase separation was less extensive in heated mixtures.

The critical κ-car concentration needed to induce phase separation depended only weakly on the protein concentration as was already reported for β-lg strands (Nguyen et al 2014) and fractal aggregates (Baussay et al 2006, Croguennoc et al 2001a). A systematic investigation with microgels of different sizes (R h = 130-350 nm) conirmed this observation (data not shown). For instance, for microgels with R h = 240 nm, the onset of phase separation occurred between 0.5 and 1.0 g L -1 κ-car over the whole range of protein concentrations investigated (C b = 0.5-40 g L -1 ), see supplementary information.

Size of the soluble β-lg aggregates in heated mixtures

After association of the protein rich domains into larger locs and their precipitation under gravity, no domains remained in the supernatant. The size of the β-lg aggregates that did not phase separate, and therefore remained in the supernatant, was determined using dynamic light scattering. In igure 7 the size of the aggregates in the supernatant is plotted as function of the CaCl 2 concentration at different κ-car concentrations. We distinguish between solutions for which the fraction of phase separated protein aggregates was negligible (open symbols) and those were it was signiicant (F ps > 0.1, illed symbols). The presence of up to 1 g L -1 κ-car did not signiicantly inluence the size of the aggregates, but at higher κ-car concentrations we observed an increase of the size of the strands formed at lower CaCl 2 concentrations. This explains why weak phase separation was observed in the heated mixtures at C K = 5 g L -1 , but not in MAH, see igure 2.

At higher CaCl 2 concentrations, where in the absence of κ-car microgels were formed, we found that the aggregates in the supernatant were smaller than those formed in the absence of κ-car. This may be due to the competition between κ-car and β-lg for Ca 2+ (Harrington et al 2009). If less Ca 2+ is available for the proteins they will form smaller microgels. Notice, however, that when a fraction of the microgels has entered the dense domains the residual aggregates in the supernatant will be smaller since preferentially the larger aggregates will enter the dense protein domains. This explains why the average size of the residual aggregates in the supernatant remains small even at high CaCl 2 concentrations. The effect of varying the protein and polysaccharide concentrations on the structure is shown in igure 8 for mixtures (MAH) of κ-car and β-lg microgels. The microgels were formed by heating β-lg at 40 g L -1 at 5.3 mM CaCl 2 and had a hydrodynamic radius of 240 nm. Phase separation was observed when C K > 0.5 g L -1 independent of the β-lg concentration at least between C b = 2 g L -1 and 40 g L -1 . The number of protein rich domains increased with increasing protein concentration, as might be expected, but their diameter remained approximately the same. The number of protein rich domains also increased with increasing κ-car concentration, but again their diameter remained approximately the same. Similar results were obtained for larger (R h = 320 nm) and smaller (130 nm) microgels (results not shown) the only difference being that the larger microgels already showed clear signs of phase separation at C K = 0.5 g L -1 and the smaller microgels started to phase separae at C K = 2 g L -1 . In all cases the domains agglomerated into large clusters except at very low protein concentrations.

The effect of CaCl 2 on the structure of mixtures at other β-lg concentrations is similar to those shown above for C b = 20 g L -1 when they are compared at the same molar ratio of CaCl 2 to β-lg. The reason is that, as was mentioned above, the transition between the formation of small strands and larger microgels occurs at a critical value of R independent of C b . We illustrate this here for C b = 40 g L -1 for which twice as much CaCl 2 is needed to induce the morphological transition than at C b = 20 g L, see supplementary information. CLSM images of mixtures with C K = 2 g L -1 showed that protein rich domains were formed at [CaCl 2 ] ≥ 4.0 mM both when the microgels are formed by heating native β-lg separately (MAH) and when they are formed by heating the mixtures (MBH), see igure 9. The microstructures of mixtures prepared by the two different methods were very similar, as was the case for C b = 20 g L -1 . Again coarser structures were found close to the critical concentration.

At C b = 40 g L -1 self supporting gels were formed when the protein were heated at [CaCl 2 ] > 6 mM in the absence of κ-car. However, in the presence of 2 g L -1 κ-car gels were formed already at [CaCl 2 ] = 3 mM. Thus the systems formed by MBH shown in igure 9 are gels, but nevertheless, they have the same structure as the liquid systems formed by MAH. It appears that during heating micro-phase separation occurs irst and subsequently the protein rich domains are permanently cross-linked. The effect of CaCl 2 on the elastic modulus of these gels will be discussed elsewhere.

Discussion

We will irst discuss the situation where protein aggregates were formed separately and subsequently mixed with the polysaccharides and then the situation where the aggregates were formed by heating native β-lg after mixing with κ-car.

Mixing after heating

The size of the protein aggregates increased when more CaCl 2 was present during heating. The reason that phase separation was observed at lower κ-car concentrations when more CaCl 2 was present is thus directly related to the increase of the average size of the aggregates. The results of the present study conirm earlier reports (Baussay et al 2006, Croguennoc et al 2001a) that phase separation occurs at increasingly lower κ-car concentrations when the aggregate size is larger. In these earlier studies the aggregates were formed by heating the proteins in the presence of NaCl at neutral pH. In that case, small strands are formed initially and larger aggregates are formed only by secondary aggregation of the strands at higher β-lg or NaCl concentrations. Larger aggregates formed by secondary aggregation have a fractal structure and have a broad size distribution of the aggregates. The β-lg microgels studied here have a much higher density and are more monodisperse. Both effects may explain why phase separation is induced at lower κ-car concentrations for microgels than for fractal aggregates with the same z-average hydrodynamic radius. The effective exclude volume of fractal aggregates is less than that of a homogeneous sphere of the same size so that there is less interaction with κ-car chains. At the same z-average hydrodynamic radius determined by light scattering, the number average hydrodynamic radius of the more polydisperse fractal aggregates is smaller than that of the more monodisperse microgels so that its mixing entropy is higher.

There are two possible driving forces for phase separation: thermodynamic incompatibility due to the different chemistry of proteins and polysaccharides and depletion of κ-car chains from between the protein aggregates. Depletion interaction was suggested to be the driving force for phase separation in mixtures of whey protein aggregates and polysaccharides (Tuinier et al 2003, Gaaloul et al 2010). However, we have two reasons to believe that depletion is not the principal driving force for phase separation in these mixtures. Firstly, we showed recently (Nguyen et al 2014) that phase separation was reduced at higher temperatures, which is expected in the case of thermodynamic incompatibility, but not for depletion interaction, which is purely entropic. Secondly, the κ-car concentration is not strongly reduced within the protein rich domains.

A striking phenomenon is that the domains cease to grow when they have reached a certain size. The size of the domains at steady state decreased with increasing CaCl 2 concentration i.e. when a larger fraction of the protein aggregates phase separated. Interestingly, the domains stick together into large clusters, but do not fuse completely even though partial fusion can be observed. A possible reason for this phenomenon is that the microgels within the domains are bound together by bonds that are strong enough to resist fusion. However, these bonds are too weak to resist dilution in the case of MAH. Within this scenario, smaller protein rich domains are formed when phase separation is stronger, because they are sooner suficiently strongly held together to inhibit fusion. Notice that the interfacial tension that drives fusion is very small in the case of two aqueous phases.

The extent of phase separation as a function of the κ-car concentration can be explained by considering the size distribution of the β-lg aggregates. Even though neither the strands nor the microgels are monodisperse, we may consider that the systems studied here contained two distinct populations of strands and microgels. At κ-car concentrations between 2 and 5 g L -1 , practically none of the strands and all of the microgels phase separated. This explains why the extent of phase separation as a function of [CaCl 2 ] closely mirrored the fraction of microgels as shown by the dashed line in igure 6(a). At C K = 1 g L -1 the smallest microgels did not phase separate, which explains the shift to higher [CaCl 2 ]. At C K = 0.5 g L -1 even the largest microgels formed by heating at [CaCl 2 ] = 3 mM did not phase separate.

Mixing before heating

The main features observed for mixtures of κ-car and β-lg aggregates formed at different CaCl 2 are the same whether the proteins are heated before or after mixing. In both cases the critical κ-car concentration decreased strongly when the CaCl 2 was increased, because microgels were formed instead of small strands. The major difference was that phase separation in heated mixtures could not be reversed by dilution implying that the aggregates were rapidly strongly bound together within the domains. In addition, we found other more subtle differences.

The size of the strands increased when more than 1 g L -1 κ-car was present during heating. A possible reason for the formation of larger strands is that the presence of κ-car inluences the interaction between the growing β-lg aggregates. Notably, the incompatibility of the two polymers could modify locally the distribution of the growing aggregates in the solution even if it does not lead to phase separation. This could modify the growth mechanism of the strands. Increasing the size of the aggregates reduces the critical κ-car concentration needed to induce phase separation. This is why we observed weak phase separation at [CaCl 2 ] = 1 mM for C K = 5 g L -1 even when no microgels were formed. As a consequence, the effect of microgel formation on the evolution of the structure of the mixtures was not obvious at C K = 5 g L -1 .

The evolution of F ps was closest to that of the fraction of microgels for C K = 4 g L -1 suggesting that in this case most of the strands remained in solution and most of the microgels phase separated. At C K = 3 g L -1 and C K = 2 g L -1 , the increase of F ps started at larger [CaCl 2 ] than for MAH, which implies that for a given [CaCl 2 ] smaller or fewer microgels were formed when the systems were heated in the presence of κ-car. Indeed, igure 8 shows that in the presence of 2 and 3 g L -1 κ-car smaller microgels were formed at [CaCl 2 ] = 2 mM than in the absence of κ-car. As we mentioned above, this is probably caused by the competition between κ-car and β-lg for the calcium ions so that amount of calcium available to the proteins is reduced.

We expect that the inluence of CaCl 2 on the structure of the mixtures also inluences the properties of gels that can be formed either by adding KCl or by using higher protein concentrations. In the irst case κ-car gels upon cooling, while in the second case β-lg gels during heating. We will report elsewhere on the effect of CaCl 2 on the structure and rheology of gelled mixtures and we will discuss there the role of competition for calcium ions between β-lg and κ-car in more detail.

Conclusion

Addition of a small fraction of CaCl 2 has a major inluence on the structure of heated mixtures of β-lg and κ-car. When more than about 1.5 calcium ions are present per protein, heating leads to the formation of microgels with R h > 100 nm rather than small strands with R h < 20 nm. In mixtures with κ-car, β-lg microgels phase separate at much lower κ-car concentrations than the small strands. Therefore the appearance and structure of heated mixtures is very sensitive to the addition of CaCl 2 . Phase separation leads to the formation of spherical protein rich domains with a diameter of a few microns. The concentration of κ-car is decreased by only about 30% in the protein rich domains. The protein rich domains cluster into large locs that slowly sediment, but the precipitate can be easily dispersed by shaking. The structures of mixtures with the same composition are similar when separately formed β-lg microgels are mixed with κ-car at room temperature or when mixtures of native β-lg and κ-car are heated. However, in the former case phase separation can be reversed by dilution, while in the latter the proteins become strongly bound within the domains. Therefore, the domains persist after dilution in the latter case.

Structure and rheology of mixtures of the protein -lactoglobulin and the polysaccharide -carrageenan

Trong Bach NGUYEN L'Université Nantes Angers Le Mans Résumé Les protéines et les polysaccharides constituent avec les lipides les principaux ingrédients de l'alimentation et lui confèrent à la fois ses propriétés de nutrition et de texture. Une tendance actuelle de l'industrie agroalimentaire est d'élaborer des aliments plus sains c'est-à-dire moins gras et moins salés. A ce titre, les polysaccharides sont des agents de texturation efficaces lorsqu'ils sont utilisés seuls ou en combinaison avec des protéines. Le développement de nouveaux produits alimentaires nécessite donc de rationaliser et mieux comprendre les propriétés physico-chimiques des solutions et des gels mixtes à base de protéines et de polysaccharides. Au cours de ce travail de thèse, nous avons étudié des mélanges de protéines globulaires (la -lactoglobuline: -lac) et de polysaccharide (le κ-carraghénane: κ-carr). Ce dernier provient d'algues et, en solution, il conduit à des gels au dessous d'une température critique qui dépend de la nature du sel ajouté. Le κ-carr est un additif important dans l'industrie alimentaire et plus particulièrement comme texturants des produits laitiers. Il est donc essentiel de comprendre les interactions qu'il développe avec les protéines du lait comme la -lac. L'objectif de ce travail est d'étudier la structure et les propriétés mécaniques d'agrégats ou de gels de -lac mélangés avec du κ-carr et d'étudier leur influence sur la gélification de ce dernier. Nous nous sommes plus particulièrement intéressés à la sensibilité des mélanges aux ions calcium. Des agrégats protéiques ont été formés soit indépendamment puis mélangés au κ-carr soit directement in situ en dénaturant thermiquement des mélanges κ-carr/-lac native. Les deux méthodes de préparation ont été comparées pour des compositions constantes des mélanges. La diffusion de la lumière, la rhéologie et la microscopie laser confocale ont été mises en oeuvre pour étudier la texture des mélanges. La taille et la morphologie des agrégats protéiques dépendent fortement de la concentration en ions calcium ajoutés qui se lient spécifiquement aux protéines. Nous avons montré que les très grands agrégats protéiques formés en présence de calcium conduisent à une microséparation de phase quand ils sont mélangés avec du κ-carr même à très faible concentration. Ainsi, la structure des systèmes mixtes est très sensible à la quantité de calcium en présence. Les agrégats protéiques renforcent les gels de κ-carr formés en présence de potassium tout comme l'ajout de calcium. Ce renforcement dans le cas des agrégats protéiques est dû au transfert des ions calcium de la -lac vers le κ-carr. De plus, nous avons montré que la gélification du -carr induite par des ions potassium continuait à avoir lieu en refroidissant des mélanges κ-carr/-lac où cette dernière est dénaturée in situ. Cela conduit à des réseaux interpénétrés qui sont plus forts mécaniquement que la somme des deux réseaux pris individuellement. En conclusion, nous avons montré que la compétition entre la -lac et le κ-carr pour les ions calcium était le paramètre de contrôle des propriétés texturales des gels mixtes.

Mots clés structure, rhéologie, -lactoglobulin, κ-carraghénane, calcium

Abstract

Protein and polysaccharide are together with lipids the main ingredients of food and procure both nutrition and texture. A recent tendency in the food industry is to develop more healthy products that contain less fat and salt. The addition of polysaccharides is recognized as a good way to control the texture of food products. The texture of many food products is determined by gelation of either the proteins or the polysaccharides, or both. When both are present, gelation of the protein or the polysaccharide will be influenced by the presence of the other type. Understanding of the physical chemical properties of aqueous solutions and gels containing protein and polysaccharides by themselves and in mixtures is needed for a rational development of novel food products. This thesis describes an investigation of mixtures of the globular protein lactoglobulin (-lg) and the polysaccharide κ-carrageenan (κ-car). κ-car is a polysaccharide isolated from algae that is often used as an additive in food industry. In solution it forms a gel below a critical temperature that depends on the amount and the type of salt. Addition of κ-car can improve the smoothness, creaminess, and body of food products and is often used modify the texture of dairy products. Therefore it is important to understand the interaction of κ-car with milk proteins such as -lg, which is the main protein component of whey. The objective of the present investigation was to study the structure and the mechanical properties of -lg aggregates or gels when mixed with κ-car and to study the influence of the former on the gelation of κ-car. The focus was on the sensitivity of the system to calcium ions Protein particles were either formed separately and subsequently mixed with κ-car or formed directly in mixtures of κ-car and native -lg by heating. The two different methods of preparation were compared with the same composition of polymers. The research presented in this thesis is essentially experimental using scattering techniques and confocal laser scanning microscopy to study the structure and shear rheology to study the dynamic mechanical properties. The size and morphology of protein aggregates formed by heating -lg is strongly dependent on the concentration of Ca 2+ that binds specifically to the proteins. It is shown that larger aggregates formed in the presence of Ca 2+ micro-phase separate already at low κ-car concentrations. Therefore the structure of mixed systems is extremely sensitive to the amount of Ca 2+ present in the system. The presence of protein aggregates was found to reinforce potassium induced κ-car gels, but it was also found that addition of CaCl2 strengthens potassium induced pure κ-car gels. We show that the reinforcement by addition of protein aggregates is caused by the transfer of a fraction of Ca 2+ from -lg to κ-car. It was shown that potassium induced gelation of κ-car also occurs during cooling heat-set -lg gels formed in mixtures at higher protein concentrations leading to interpenetrated networks that are stronger than the sum of the individual networks. The main conclusion of the investigation reported here is that the competition of κ-car and -lg for calcium ions determines both the structure and the mechanical properties of the mixed systems.

  Introduction ...................................................................................... Chapter 1: Background ................................................................................................ 1.1. Beta lactoglobulin ............................................................................................................. Molecular structure ....................................................................................................... Aggregation and gelation of β-lactoglobulin ................................................................ 1.2. Kappa carrageenan .......................................................................................................... Aggregation and gelation of kappa carrageenan ........................................................ 1.3. Mixtures of -lactoglobulin and κ-carrageenan .......................................................... Mixing after heating .................................................................................................... Mixing before heating ................................................................................................. vi Chapter 4: Mixtures of -lactoglobulin and -carrageenan..................................... 4.1. Introduction .................................................................................................................... 4.2. Mixtures of κ-carrageenan with native β-lactoglobulin ............................................. Conclusion ............................................................................................................................... 4.3. Mixtures of κ-carrageenan with β-lactoglobulin strands ........................................... 4.3.1. Mixtures with κ-car coils ............................................................................................... 4.3.2. Effect of κ-carrageenan gelation on phase separation ................................................... 4.3.3. Conclusion .................................................................................................................... 4.4. Mixtures of κ-carrageenan with β-lactoglobulin microgels ....................................... 4.4.1. Mixtures of -lg microgels with κ-car coils .................................................................. 4.4.1.1. Effect of the concentration of κ-car and -lg microgels ............................................ 4.4.1.2. Effect of the size and morphology of the -lg aggregates .......................................... 4.4.2. Effect of κ-carrageenan gelation on the structure ......................................................... 4.4.3. Effect of κ-carrageenan gelation on the rheology ......................................................... 4.4.4. Conclusion ..................................................................................................................... 4.5. Heated mixtures of κ-carrageenan and native β-lactoglobulin .................................. 4.5.1. Mixtures of -lg and κ-car coils .................................................................................... 4.5.2. Effect of κ-car gelation on the structure and the rheology ............................................ 4.5.3. Conclusion ..................................................................................................................... References ............................................................................................................................... General conclusion and outlook ................................................................................ The list of publications ................................................................................................ iii

Figure 1

 1 Figure 1.1. Schematic drawing of the structure of β-lactoglobulin[START_REF] Brownlow | Bovine [beta]-lactoglobulin at 1.8 A resolution-still an enigmatic lipocalin[END_REF]

Figure 1 . 2 .

 12 Figure 1.2. Negative-staining TEM images of β-lg aggregates formed at different pH: long rigid strands at pH 2.0, spheres at pH 5.8 and small curved strands at pH 7.0. Scale bars are 500 nm[START_REF] Jung | Structure of Heat-Induced β-Lactoglobulin Aggregates and their Complexes with Sodium-Dodecyl Sulfate[END_REF].

Figure 1 . 3 .

 13 Figure 1.3. Sol-gel state diagram of β-lg solutions at steady state. The closed and open symbols indicate, respectively, the critical concentration beyond which the systems no longer flow when tilted or beyond which insoluble material is observed after dilution (Mehalebi et al., 2008).

Figure 1 . 4 .

 14 Figure 1.4. Schematic representation of the aggregation process of -lactoglobulin

Figure 1 . 5 .

 15 Figure 1.5. Idealized repeating unit of κ-carrageenan

  , see figure 1.6. It is important to consider also the activity of the counterions in the calculation of the total ion concentration. For instance, in the case of potassium counterions the effective potassium concentration in salt free κ-car solutions is equal to 0.55*C P /M m where C P is the κ-car concentration and M m is the molar mass of the monomer (M m = 383g/mol).

Figure 1 . 6 .

 16 Figure 1.6. Dependence of the transition midpoint temperature (T m ) of kappa-carrageenan on the total concentration (C T ) of various cations in the systems.(Rochas & Rinaudo, 1980).

Figure 1 . 7 .

 17 Figure 1.7. Schematic representation of the aggregation and gelation of κ-carrageenan

Figure 1

 1 Figure 1.9. (a) Time dependence of the storage shear modulus on cooling at different concentrations of κ-car. The solid line indicates the time dependence of the temperature. The dashed lines indicate the position of the temperature where the coil helix transition occurs. (b) Master curve of the data shown in figure (a) obtained by vertical and horizontal shifts with C ref = 1.0 g/L(Meunier et al., 1999).

Figure 1 . 10 .

 110 Figure 1.10. Behaviour of binary polymer solutions.

Figure 1 .

 1 Figure 1.11. Evolution of shear modulus (G') during heating for mixtures containing 8% WPI and without κ-car (a), 0.2% (b), 0.4% (c), 0.6% (d) and 0.8% κ-car (e). Dashed line shows temperature of mixtures(Gaaloul et al., 2009a).

  The -lactoglobulin (-lg)(Biopure, used in this study was purchased from Davisco Foods International, Inc. (Le Sueur, MN, USA) and consisted of approximately equal quantities of variants A and B. The powder was dissolved in Milli-Q water which contained 200 ppm NaN 3 to prevent bacterial growth. Solutions were dialysed extensively against Milli-Q water with 200 ppm sodium azide and subsequently filtered through 0.2 µm pore size Anatop filters. The pH of the solution was adjusted to 7 by adding small amounts of NaOH 0.1 M. The protein concentration was determined by UV absorption at 278 nm using extinction coefficient 0.96 Lg -1 cm -1 . Stable suspensions of protein particles were prepared by heating aqueous solutions of -lg at different concentrations (10 to 80) g/L in pure water at pH= 7 and (20 and 40) g/L in pure water at pH= 7 with different amounts of CaCl 2 . The solutions were heated in airtight vials in a water bath at 85°C until steady state was reached (overnight) and the residual fraction of native proteins was negligible. The z-average hydrodynamic radius (R h ) of the particles was determined by dynamic light scattering

(

  ALV-CGS3, ALV-Langen) operating with a vertically polarized He-Ne laser with wavelength =632nm. The sample is inserted in a temperature controlled vat and is illuminated by a laser. The fluctuations of the scattered light intensity are detected at different scattering angle by a fast photon diode on a rotating arm (figure 2.1). The range of scattering angles was 12-150 degrees. The temperature was controlled at 20 0 C with a thermostat bath to within  0.2 0 C.

Figure 2 . 1 .

 21 Figure 2.1. Schematic diagram of a light scattering apparatus.

Figure 2 . 2 .

 22 Figure 2.2. Schematic diagram of the optical pathway and principal components in a modern confocal laser scanning microscope.

Figure 2 . 3 .

 23 Figure 2.3. Schematic drawing of the rheometer used in this study.

  s mentioned in the chapter 1, in aqueous solution, -carrageenan has a random coil conformation above a critical temperature (T c ) and a helical conformation below this temperature

Figure 3 . 1 .

 31 Figure 3.1. Storage shear modulus at 0.1 Hz during cooling (open symbols) and subsequent heating (filled symbols) ramps at a rate of 2 o C/min at C k = 8g/L in the presence of 10mM (circles), 20mM (triangles) and 30mM KCl (squares)

Figure 3 . 2 .Figure 3 . 3 .

 3233 Figure 3.2. Evolution of the storage shear modulus at 0.1Hz for 2g/L κ-car at different concentrations of KCl during and after rapid cooling to 5°C. The solid line indicates the temperature of the sample.

Figure 3 .

 3 Figure 3.3 shows that the gel (T g ≈ 24 o C) and melting temperature (T m ≈ 39 o C) did not

Figure 3 . 4 .Figure 3 . 5 .

 3435 Figure 3.4. Evolution of the storage shear modulus at 0.1Hz for different concentrations of car at 10mM KCl during and after rapid cooling to 5°C. The solid line indicates the temperature of the sample.

Figure 3 . 6 .

 36 Figure 3.6. Evolution of the storage shear modulus at 0.1Hz for 2g/L κ-car at different concentrations of CaCl 2 during and after rapid cooling to 5°C. The solid line indicates the temperature of the sample.

Figure 3 .Figure 3 . 7 .

 337 Figure 3.7 shows the effect of the κ-car concentration on gelation during and after rapid cooling to 50°C for [CaCl 2 ]= 10mM. Interestingly, the rate of gelation induced by calcium did not depend on the κ-car concentration at least in the range investigated between 1 and 8g/L, but the elastic moduli of the gels increased with increasing κ-car concentration.

Figure 3 .Figure 3 . 8 .Figure 3 . 9 .

 33839 Figure 3.8 compares the elastic modulus at the quasi-plateau where the increase of G' becomes logarithmic as a function of the salt concentration for CaCl 2 and KCl at C k = 2g/L. Below 5mM CaCl 2 and below 10mM KCl, gelation was not observed at 5 o C. The elastic modulus increased with increasing KCl concentration, but it decreased with increasing CaCl 2 concentration. A possible reason for this behavior is that the gels became increasingly heterogeneous (MacArtain et al., 2003) when more CaCl 2 was added, see paper 1 in the appendix.

Figure 3 .

 3 Figure 3.10. Storage shear modulus at 0.1 Hz during cooling (open symbols) and subsequent heating (filled symbols) ramps at a rate of 2 o C/min of aqueous solutions of 2g/L -car containing 10mM KCl and different concentrations of CaCl 2 .

Figure 3 .Figure 3 .Figure 3 .

 333 Figure 3.11 compares cooling and subsequent heating ramp of κ-car in mixed salt at different κ-car concentrations. As in the pure salts, the critical temperature was independent of the concentration, and the elastic modulus increased with increasing κ-car concentration.

Figure 3 .Figure 3 .

 33 Figure 3.13. Elastic modulus of -car gels at C k = 2g/L as a function of the KCl concentration at 5°C in the presence of different CaCl 2 concentrations

Figure 3 .

 3 Figure 3.15. Evolution of the storage shear modulus at 0.1Hz of 2g/L car with 10mM KCl at different NaCl concentrations during and after rapid cooling to 5°C. The solid line indicates the temperature of the sample.

Figure 3 . 16 .

 316 Figure 3.16. Elastic modulus of 2g/L -car with 10mM KCl at 5°C as a function of the ionic strength of added CaCl 2 (crosses) or added NaCl (circles). The solid lines are guides to the eye.

Figure 3 .

 3 Figure 3.17. Fraction of free calcium ion concentration as a function of the total calcium ion concentration in 2g/L κ-car.

Figure 3 .

 3 Figure 3.18. (a) Turbidity of 2g/L car as a function of the total ionic strength at 5°C in the presence of NaCl (circles) or CaCl 2 (triangles). (b) Turbidity of 2g/L car containing 10mM KCl as a function of the total ionic strength at 5°C in the presence of NaCl (circles) or CaCl 2 (triangles).

Figure 4 . 1 .

 41 Figure 4.1. Evolution of G' at 0.1Hz during and after rapid cooling from 50°C to 5°C for pure -lg (closed symbols) or mixtures with 2 g/L -car at 10mM KCl (open symbols), containing different concentrations of native -lg (a). In figure (b) we show the effect of

  (7mM) was added to the mixture containing 5g/L -lg a similar acceleration of κ-car gelation was found, see figure 4.1b.It was shown in chapter 3 that addition of CaCl 2 increases the elastic modulus of Κ +induced κ-car gels. The effect of adding CaCl 2 on the elastic modulus of κ-car gels at C= 2g/L in the presence of 40g/L native -lg is shown in figure 4.2. It is clear that the presence of native -lg strongly reduces the stiffening by Ca 2+ . The reason is that -lg competes with κcar for Ca 2+ as was already noted byHarrington et al. (2009) for calcium induced gelation of κ-car in the presence of native WPI.

Figure 4 . 2 .

 42 Figure 4.2. Elastic shear modulus of -car gels at 2 g/L after 100 min at 5°C in the presence of 10mM KCl as a function of the concentration of Ca 2+ . The results for pure -car (triangles) are compared with mixtures containing 40 g/L native -lg (circles).

  Figure 4.3a shows the evolution of G' during and after rapid cooling to 5°C for 2g/L κ-car with 2.65mM CaCl 2 and 10mM KCl in the presence of different concentrations of native -lg. With increasing concentration of native protein elastic modulus of the κ-car gel decreased (figure 4.3b), because the fraction of Ca 2+ bound by the proteins increased (figure 4.3c).

Figure 4 . 3 .

 43 Figure 4.3. (a) Evolution of G' at 0.1Hz during and after rapid cooling from 50°C to 5°C for mixtures of 2 g/L -car at 10 mM KCl and 2.65 mM CaCl 2 , containing different concentrations of native -lg. The solid line indicates the temperature as a function of time. (b) Dependence of the elastic shear modulus of -car gels after 100 min at 5°C on the concentration of native -lg. The systems contained 2 g/L -car, 10 mM KCl and 2.65 mM CaCl 2 . (c) Fraction of bound Ca 2+ ions in the mixtures for which the elastic shear modulus is shown in figure a.

Figure 4

 4 Figure 4.4. Mixtures of 2g/L κ-car in the presence (a) and absence (b) of 40g/L native -lg at different concentrations of CaCl 2 after keeping in the fridge for 3 days.

3 .

 3 Mixtures of κ-carrageenan with β-lactoglobulin strands 4.3.1. Mixtures with κ-car coils -lg strands were prepared by heating 50g/L native -lg in pure water at pH = 7 overnight at 85 o C. Using light scattering we found that the strands had a z-average hydrodynamic radius R h = 17nm and a weight average molar mass M w = 1.5x10 6 g/mol. In mixtures with κ-car coils, micro phase separation occurred above a critical concentration of κcar, see figure 4.5. Dense protein domains with a radius of a few microns were observed with CLSM at C k > 8g/L κ-car for C b ≤ 15g/L, but at higher protein concentrations the critical κcar concentration decreased with increasing C b .

Figure 4 . 5 .

 45 Figure 4.5. Phase diagram of mixtures of -lg strands (R h = 17nm) and -car coils. The open and closed symbols correspond to homogeneous and micro phase separated mixtures, respectively. The solid line indicates the phase boundary. CLSM images representing 150x150m are shown for mixtures containing 20 g/L -lg and different -car concentrations: 10, 8 and 7.5 g/L, top to bottom.

  Figure 4.6. CLSM images (160x160µm) of mixtures containing 10 (top), 20 (middle) and 30g/L (bottom) -lg strands at different concentrations of κ-car. The -lg strands were labeled (red).

  Figure 4.7a shows the evolution of turbidity for a mixture containing 20g/L -lg strands and 8g/L κ-car during stepwise cooling from 80°C to 20°C and subsequently heating back to 80°C. The turbidity increased for T < 35 o C, due to micro phase separation. Results obtained at other concentrations of κ-car are shown in figure 4.7b. For C k  7g/L no phase separation was observed even at the lowest temperature tested (10°C), while at C k > 8g/L, phase separation occurred already at the highest temperature tested (50°C).

Figure 4

 4 Figure 4.7. (a) Evolution of turbidity for 20g/L strands and 8g/L κ-car on cooling and heating. (b) Temperature dependence of the turbidity of aqueous mixtures of 20 g/L -lg strands and different -car concentrations indicated in the figure after cooling (open symbols) and subsequent heating (closed symbols) in steps of 5 °C.

Figure 4

 4 Figure 4.8. (a) Evolution of turbidity for 20g/L strands and 8g/L κ-car at 20mM KCl on cooling and heating. (b) Correlation between the turbidity and the elastic modulus of κ-car gels in mixtures of 20g/L -lg strands and 10g/L κ-car obtained during cooling (open symbols) and heating (closed symbols) at either 10mM (circles) or 20mM KCl (triangles).

Figure 4 Figure 4 Figure 4 Figure 4 Figure 4

 44444 Figure 4.9. CLSM images (160x160µm) of mixtures containing 2 (top), 10 (middle) and 30g/L (bottom) -lg microgels with R h = 240nm at different concentrations of κ-car

Figure 4 .

 4 Figure 4.14. Mixtures of 10g/L microgels (R h = 240nm) in the absence and presence of 20mM KCl at different κ-car concentrations. The samples were photographed after 3 days.

  Figure 4.15. CLSM images (160x160µm) of mixtures containing 10g/L -lg microgels (R h =240nm) and 20mM KCl at different concentrations of κ-car after preparation (top row) and heating at 50 o C for 15min (bottom row).

Figure 4 .

 4 Figure 4.16. (a) CLMS images (40x40µm) visualizing -lg (red) and κ-car (green) for a mixture containing 40 g/L -lg microgels and 2 g/L κ-car at 15mM KCl. (b) Relative variation of the -lg concentration (red) and the κ-car concentration (green) along the white lines in the CLMS images.

Figure 4

 4 Figure 4.17. CLSM images (160x160µm) of mixtures containing 2g/L κ-car, 40g/L -lg microgels and 10mM KCl at different concentrations of CaCl 2 and with different temperature histories.

Figure 4 . 18 .

 418 Figure 4.18. Dependence of the shear modulus on the κ-car concentration and 10mM KCl without protein (circles), with 10g/L -lg microgels (triangles) and with 40g/L -lg microgels (squares).

Figure 4

 4 Figure 4.19. (a) Evolution of G' at 0.1Hz for mixtures of 2 g/L -car and different concentrations of -lg microgels with R h = 240nm in 10 mM KCl during and after rapid cooling from 50°C to 5°C. The microgels contained 2.5 calcium ions per protein (R=2.5). (b) Evolution of G' at 0.1Hz for solutions of 2 g/L -car in the presence of 10mM KCl at different concentrations of CaCl 2 during and after rapid cooling from 50°C to 5°C. The solid lines indicate the temperature as a function of time for both (a) and (b).

Figure 4

 4 Figure 4.20. (a) Elastic shear modulus of -car gels (C k = 2 g/L) after 100 min at 5°C in the presence of 10mM KCl as a function of the concentration of Ca 2+ . The results for pure -car are compared with mixtures containing native -lg, strands or microgels. The molar ratio of calcium ions to -lg was R = 2.5. (b) Fraction of bound Ca 2+ in mixtures containing 2g/L κ-car and different concentration of native, strands, microgels or pure κ-car solutions as a function of CaCl 2 concentrations.

Figure 4 .

 4 Figure 4.21. Scheme of the two different procedures to prepare mixtures of κ-car and aggregated -lg: (1) mixing before heating (MBH) and (2) mixing after heating (MAH)

Figure 4

 4 Figure 4.22. CLSM images (160x160µm) of mixtures containing 40g/L -lg and 2g/L κ-car at different concentrations of CaCl 2 .

Figure 4 .

 4 Figure 4.23. Phase diagram of mixtures containing 20g/L -lg as a function of the -car and CaCl 2 concentrations prepared by mixing before heating (MBH) or mixing after heating (MAH). The open and closed symbols correspond to homogeneous and phase separated mixtures, respectively. The solid lines indicate the phase boundary.

  Figure 4.25 compares the effect of dilution for mixtures prepared by mixing before heating and mixing after heating. In the case of MAH, dilution by a factor two was sufficient to render the system homogeneous at [CaCl 2 ] = 1.5mM, while at [CaCl 2 ] = 3mM dilution by a factor 20 was needed. In the case of MBH no amount of dilution led to dispersion of the flocculated domains.

Figure 4 .

 4 Figure 4.25. Effect of dilution on the structure of mixtures of -car and partially labeled -lg (C k = 5g/L, C b = 20g/L) in the presence of 1.5mM and 3mM CaCl 2 prepared by heating after mixing or heating before mixing. Dilution factors are from top to bottom: undiluted, x2, x5, x20. The images represent 160x160µm.

  Figure 4.26 compares heated mixtures containing 40g/L native protein, 2g/L κ-car and different amounts of CaCl 2 , with mixtures prepared by MAH with the same composition.

Figure 4 .

 4 Figure 4.26. CLSM images (160x160µm) of mixtures containing 40g/L -lg and 2g/L κ-car at 10mM KCl and different concentrations of CaCl 2 .

Figure 4 . 28 .

 428 Figure 4.28. Temperature dependence of G' at 0.1Hz during cooling (closed symbols) and subsequent heating (open symbols) of -lg gels (40g/L) containing 2g/L -car. The gels were formed at different concentrations of CaCl 2 with (a) and without (b) 10mM KCl.

Figure 4 .Figure 4 . 29 .

 4429 Figure 4.29 shows the elastic moduli of the interpenetrated networks of κ-car and -lg measured 100min after cooling quickly from 50 o C to 5 o C. The contribution of the -lg gels was determined by extrapolation of the linear temperature dependence of G' at T > T c to 5 o C.The difference corresponds to the contribution of the κ-car gel. If use for the latter the values found for the mixtures with native -lg before heating, we find that the mixed gels were somewhat stiffer than the sum of the two individual networks up to [CaCl 2 ] < 5.3mM (figure4.29). This means that either a stronger κ-car gel was formed within the gelled -lg than with native -lg or that the two interpenetrated networks did not deform independently. At [CaCl 2 ] > 5.3mM, the sum is not shown because there was syneresis of strong κ-car gels in the mixtures with 40g/L native -lg. The synergic effect was largest at [CaCl 2 ]  2mM where the

2 .

 2 Combined effects of temperature and elasticity on phase separation in mixtures of κcarrageenan and β-lactoglobulin aggregates. (Food Hydrocolloids 2014, 34, 138-144) 3. The effect of the protein aggregate morphology on phase separation in mixtures with polysaccharides. (Journal of Physics: Condensed Matter 2014, 26, 464102) 4. The effect of the competition for calcium ions between -carrageenan and -lactoglobulin on the rheology and the structure in mixed gels. (Journal of Colloids and Surfaces A: Physicochemical and Engineering Aspects, In press) Related publications not included in the thesis: 5. Stabilization of water-in-water emulsions by addition of protein particles. (Langmuir 2013, 29, 10658-10664)

Figure 1 .Figure 2 .

 12 Figure 1. z-average hydrodynamic radius of the β-lg aggregates (closed symbols) and the fraction of proteins that sedimented at 5 × 10 4 g (open symbols) as a function of the CaCl 2 concentration or the molar ratio (R) between Ca 2+ and β-lg. The aggregates were formed by heating aqueous solutions of β-lg at 20 g L -1 . [CaCl 2 ] (mM) 0.00 .5 1.01 .5 2.02 .5 3.0

Figure 3 .Figure 4 .

 34 Figure 3. Mixtures prepared by MBH or MAH containing 5 g L -1 κ-car and 20 g L -1 β-lg aggregates formed in the presence of 1.5 (a) or 3.0 mM CaCl 2 (b). The photographs were taken 5 days after preparation of the samples.

Figure 5 .Figure 6 .

 56 Figure 5. Relative variation of the β-lg concentration (red) and the κ-car concentration (green) along the white lines in the CLMS images of mixtures formed at 1.5 mM CaCl 2 shown in igure 4. Distance (µm) 0510 15 20 Relative concentration 0.5 0.6 0.7 0.8

Figure 7 .

 7 Figure 7. Dependence of the z-average hydrodynamic radius of the β-lg aggregates in the supernatant as a function of the CaCl 2 concentration for mixtures (MBH) that were heated in the presence of different concentrations of κ-car indicated in the igure. Filled symbols indicate systems for which F ps > 0.1.

Figure 9 .

 9 Figure 9. CLSM images of mixtures containing 40 g L -1 partially labelled β-lg aggregates and 2 g L -1 κ-car at different CaCl 2 concentrations. The protein aggregates were formed by heating either after mixing (MAH) (top row) or before mixing (MBH) (bottom row). The images represent 160 × 160 µm 2 .

Figure 8 .

 8 Figure 8. CLSM images of mixtures (MAH) of κ-car and partially labelled β-lg microgels with R h = 240 nm at different κ-car and β-lg concentrations. The images represent 160 × 160 µm 2 .

  3.2.2. Influence of Ca 2+ on the K + -induced gelation of κ-carWe have seen that both potassium and calcium ions can induce κ-car to form a gel, but in a different manner. We will in the following consider the synergic effects of adding both ions. Figure3.10 compares cooling and subsequent heating ramps of 2g/L κ-car at 10mM KCl and different amounts of CaCl 2 . Addition of CaCl 2 did not influence the gelation temperature (T g  22°C). The apparently lower value at the lowest CaCl 2 concentration was caused by the slow kinetics at low CaCl 2 concentrations (paper 1). No significant gelation was observed during the cooling ramp in the absence of CaCl 2 . However, the melting temperature significantly increased with increasing CaCl 2 concentration. Notice that with pure CaCl 2 at 5.3mM or less, no gelation was observed during the cooling ramps and that at 10mM CaCl 2 it

  The effect of adding ΚCl up to 10mM on κ-car gelation was investigated at different CaCl 2concentrations (4, 5.3, 10, 15.3mM). At 4mM CaCl 2 no gelation was observed at 5°C without KCl, but with increasing KCl concentration gelation was faster and led to stiffer gels (figure3.12). We stress that in the absence of CaCl 2 , gelation of -car was extremely slow at these low KCl concentrations. At 10mM and 15.3mM CaCl 2 gelation was already observed in the absence of KCl (figure3.5), but again addition of KCl led to faster gelation and stiffer gels. The effect of KCl on the elastic modulus was especially important at low KCl concentrations ([KCl] < 2mM, see figure3.13).

		10 4
		10 3
	el (Pa)	10 2
	G	10 1				4mM CaCl 2 5.3mM 10mM 15.3mM
		10 0	0	2	4	6	8	10
					[KCl] (mM)

J. Phys.: Condens. Matter 26 (2014) 464102

Acknowledgments

BTN thanks the Ministry of Education and Training of

Vietnam for inancial support.

between small aggregates and a low concentrations of κ-car chains was a cause to inhibit phase separation, thus it was necessary to phase separate at high C k.

At C k = 1-3g/L, the fraction of protein in the supernatant reduced sharply when protein started forming a microgel (R ~ 2), they were still about 60% and around 10% when large microgels were completely formed. However, at C k ≥ 4g/L the fraction of protein in the supernatant decreased immediately when protein formed small microgels or even strands (C k = 5g/L). The reason was the mixtures phase separated by thermodynamic incompatibility and could form a gel for mixtures containing 20g/L -lg and 5g/L κ-car at [CaCl 2 ] > 2mM. The hydrodynamic radius of the residual aggregates in the supernatant is plotted in figure 4.24b. It appears that larger -lg strands are formed when native -lg is heated in the presence of κ-car, which may explain why phase separation is observed at a lower critical κcar concentration for [CaCl 2 ] ≤ 1mM. On the other hand, smaller microgels are formed in the presence of κ-car as can be clearly seen at C k = 2 or 3g/L and [CaCl 2 ] = 2mM. At these conditions phase separation was still negligible, but the aggregates were clearly smaller than in the absence of CaCl 2 . This may be due to the competition for Ca 2+ with κ-car and can explain why more CaCl 2 was needed to induce phase separation at these κ-car concentrations.

For phase separated samples the size of the residual aggregates in the supernatant is most

We find that the heated mixtures phase separated at significantly lower CaCl 2 concentrations ([CaCl 2 ]  2.5mM) than the mixtures prepared by MAH ([CaCl 2 ]  4.5mM).

In the absence of KCl, we found that the critical CaCl 2 concentrations were the same ([CaCl 2 ]  4mM) (figure 4.22). We believe that the addition of 10mM KCl favored aggregation of the proteins, which in turn favored phase separation. At higher CaCl 2 concentrations the morphology of the systems is similar whether or not the proteins and/or the polysaccharide have gelled.

As was mentioned in chapter 1, in pure water heating 40g/L -lg leads to gelation for calcium ions per protein was increased from 1 to 2. As a consequence, micro phase separation was observed at much lower -car concentrations when the aggregates were prepared in presence of CaCl 2 . The rate of K + -induced gelation of κ-car increased with increasing protein concentration in mixtures with microgels. This effect was caused by the increase of the concentration of Ca 2+ counterions together with the microgels. The effect was less than for equivalent pure κ-car solutions because of the competition for the calcium ions with the microgels. Microgels bind Ca 2+ more strongly than native -lg or -lg strands so that at the same composition of -lg, κ-car and Ca 2+ , κ-car gels are weaker in the presence of microgels than in the presence of native -lg.

Similar effects on the structure are observed when native -lg is mixed with κ-car before heating at different CaCl 2 concentrations. However, in this case the protein rich domains are covalently crosslinked and remain stable upon dilution. Another important difference is that heated -lg gels at lower CaCl 2 concentrations in the presence of κ-car than in pure solutions. In the presence of KCl, κ-car forms a gel within the heat-set -lg gel. The elastic modulus of the mixed gel is close to the sum of the κ-car gel mixed with native -lg and the -lg gel formed with κ-car coils at the same polymer and salt composition.

Calcium ions bind specifically to both -lg and κ-car which strongly modifies their aggregation and gelation. The structure and mechanical properties of mixed -lg and κ-car solutions and gels is determined by the competition of the two polymers for the calcium ions.

Perspective

In this thesis we have studied only mixtures at neutral pH. In many food applications the pH is lower, which will no doubt have a significant effect on the properties of the mixtures. For instance, the strand-microgel transition occurs at lower CaCl 2 concentrations, when the pH is decreased. Therefore, the present investigation of the effect of calcium on the properties of the mixtures needs to be extended to mixtures at lower pH.

We have tested on for a few mixtures the effect of adding Mg 2+ and found that it was similar to the effect of Ca 2+ . Nevertheless, generalisation of the present investigation on the effect of Ca 2+ to other divalent ions will be valuable for food applications using κ-car as a textural additive. In addition, the effect of combining both mono-and divalent salt should be investigated.

Here we have studied only the linear rheological properties of gels formed at static conditions. However, it would be interesting to study also the non-linear rheological properties and the effect of shearing during the gelation process. An investigation by Gaaloul et al. (2009a,b) in the absence of salt showed that interesting textures can be obtained by shearing during gelation of WPI and κ-car, but the effect of Ca 2+ was not investigated.