Influence of Acacia gum molecular polydispersity on the complex coacervation with β-lactoglobulin

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The hydration or solvation effect

Short-range deviations from the DLVO theory are attributed to solvation/hydration or structural forces. Many experimental studies have termed this interaction a “hydration pressure” or “solvation pressure” because the repulsion results from the partial ordering or polarization of water or solvent molecules by the hydrophilic bilayer surface 38,39,40.
At very short distances (1-2 nm) the structure of the liquid confined between two surfaces does not follow a continuum theory any longer. The overlap between the solvent layers on the surfaces results in changes in liquid density (oscillations), causing oscillating structural forces 41. The properties of the water molecules close to the surface of the particles can be very different from those in the solution. Frequently a monolayer of water is chemically adsorbed Interactions and dynamics in colloidal systems
on the surface of particles (e.g. lipid membranes, proteins, metal oxides, clays). Such colloid-water interactions can retard the aggregation of colloidal systems, due to additional repulsive interaction that is distinct from the repulsion of the electric double layer. This repulsion comes from the reorganization of the surface hydration layers (several nm) so that the contact between the particles cannot take place. This induces an increase of the free energy of the system, unfavorable for colloid interactions. This hydration force is relatively important compared with the repulsion due to the electric double layer. It has an effect on colloidal stability, particularly for high ionic strengths and for small particles.
Short range repulsive interactions between electroneutral lipid bilayers were first measured 30 years ago 42. Similar interactions have been observed in systems such as DNA and polysaccharides indicating an ubiquitous nature of these forces 43. Israelachvilli 41 has described the importance of several non-DLVO forces (solvation, structural, hydration, steric, and fluctuation) in the interaction of surfaces separated by liquid. For example, in milk fat globules (MFG), steric repulsion caused by interpenetration and/or compression of hydrophilic chains that extend from the MFG surface has been implicated 44.
The origin of the hydration force remains the subject of intense study, both in experiment and theory. Unfortunately, there are no theoretical models allowing to calculate the magnitude and range of the hydration pressure. For example, the magnitude of this pressure in lipid bilayers has been shown to be proportional to the square of the dipole potential measured in monolayers in equilibrium with liposomes, suggesting that the “hydration” pressure results, at least in part, from the polarization of interbilayer water by electric fields arising from the lipid and water dipoles 42,43.

Hydrophobic interactions

Longer-range attractive deviations to the DLVO theory are attributed to hydrophobic forces between hydrophobic surfaces, as well as steric and fluctuation forces between diffuse interfaces, when polymer chains are present at the surface 45.
The nor affinity between hydrophobic particles and water may be due to the absence of ionic or polar groups and sites for hydrogen bonds. The properties of the water molecules in contact with the surfaces are thus different from those in the solution. Indeed, hydrogen bonds among water molecules structure ordinary water. The presence of a hydrophobic surface can restrict the natural tendency of water to structure by imposing a barrier, which prevents the organization of aggregates in a direction. If the interstice between particles is narrow, the limitation is important and there is an increase in the free energy of the water compared to that of the solution. In other words, there is an attraction of hydrophobic surfaces due to the migration of water molecules from the interstice to the solution, where the possibilities of creating hydrogen bonds are not restricted.
Accurate measurement of interaction forces between different surfaces is important for a variety of natural phenomena and industrial processes 46,47. From there, hydrophobic force is among the most important nonspecific interactions in biological systems and plays a central role in many surface phenomena. Hydrophobic interactions have been largely investigated to describe a variety of biochemical processes such as the conformational changes of biopolymers, the substrate/enzyme binding, and the association of subunits to form a multisubunit enzyme 48. Also, they are relevant in formation of detergent micelles, protein-amphiphile complexes, and biological membranes 49.
Although a number of studies were carried out over the past 20 years, the nature of the hydrophobic force is still unclear. Moreover, no single theory can account for all observed experimental behavior even the experimental data on similar systems is often contradictory. The magnitude and the range of the force are dependent not only on the hydrophobic surface but also on the method of surface preparation.

Thermodynamics of polymer-polymer miscibility

When a solution of polymer A is added to a solution of polymer B, the main characteristic of the blend will be the thermodynamic compatibility or incompatibility of the components. Thus, two different events might occur: the two solutions will mix or the solutions will not mix and the system separates into two phases. Complete miscibility or stability in a mixture of two polymers requires that the change in Gibbs energy upon mixing (∆Gmix) must be lower than zero 50. However, phase separation take place in the case of ∆Gmix > 0.
The thermodynamic properties of a macromolecular blend, giving rise to miscibility or phase separation, depends on many factors, of which the most important are the chemical structure of the polymers, their molecular mass, temperature (T) and blend composition φ. For example, heating or cooling the sample shifts the blend from the mixed state to a completely demixed one. Therefore, a critical temperature and a critical composition define the boundary between the mixed state and the demixed state. The phase diagram of a polymer-polymer system is a representation of the critical volume fraction of one component (for example φA) as a function of T (see Fig. I.8).

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Dynamics of phase separation

In general, Nucleation and Growth (NG) or Spinodal Decomposition (SD) describe the dynamics of phase separation 57. Nucleation theory predicts that small droplets of a minority phase develop over time in a homogeneous mixture that has been brought into the metastable region of the phase diagram (Fig. I.9). Droplet growth proceeds by diffusion of material from the supersaturated continuum. However, once the composition of the supernatant reaches equilibrium, further increases in droplet size occur by droplet coalescence or Ostwald ripening. In the metastable state, homogeneous mixtures must overcome a free energy barrier in order to nucleate a new phase. When a binary mixture is quenched from the miscible region into the thermodynamically immiscible (unstable) state in the phase diagram, phase separation occurs via mechanism of spinodal decomposition. This process was first described theoretically by Cahn 58. Generally, in the early stage, phase separation is controlled by concentration fluctuation and the decrease of bulk energy. At the later stage, phase separation is controlled by diffusion and convection, and the decrease of surface energy. It is established that the domain size R(t) satisfies a scaling law, R(t) ~ tα, during phase separation, where α is the growth exponent 60.

Main demixing mechanisms in mixed macromolecular solutions

Polymer solutions can show segregative or associative phase separation. In the absence of attractive interaction, a segregative phase separation can occur. In case of a moderate interaction, a complete miscibility may be obtained. In case of a strong attractive interaction, associative phase separation may occur, giving one phase rich in polymer and one phase highly polymer depleted. The molecular parameters that control the polymer self-association or segregation are primarily the weight average molecular weight (Mw), the radius of gyration (RG) or the hydrodynamic radius (Rh) and the second virial coefficient, A2, which is the second coefficient in the expansion of the chemical potentials of both solvent and polymer on a polymer concentration 61,62,63. The sign and the magnitude of the second virial coefficient provide the information on the deviation of a macromolecular solution from the ideal state and reflect the nature and the intensity of the intermolecular pair interactions (polymer-polymer and polymer-solvent) in solutions.

Table of contents :

Chapter I Interactions and dynamics in colloidal systems
I.1. Introduction
I.2. Theoretical description of interaction forces
I.2.1. DLVO interactions
I.2.1.1. Electrostatic interactions
I.2.1.2. Van der Waals interactions
I.2.2. Non DLVO interactions
I.2.2.1. The hydration or solvation effect
I.2.2.2. Hydrophobic interactions
I.3. Interactions in macromolecular solutions
I.3.1. Thermodynamics of polymer-polymer miscibility
I.3.1.1. The Flory-Huggins theory
I.3.1.2. Dynamics of phase separation
I.3.2. Main demixing mechanisms in mixed macromolecular solutions
I.3.2.1. Depletion flocculation
I.3.2.2. Thermodynamic incompatibility: segregative phase separation
I.3.2.3. Thermodynamic compatibility: associative phase separation
I.4. Complex coacervation between β-lactoglobulin and Acacia gum
I.4.1. Characteristics of β-lactoglobulin
I.4.2. Particularity of Acacia gum
I.4.3. Mixtures of β-lactoglobulin and Acacia gum
I.5. Objectives of the thesis
I.6. Outline of the thesis
I.7. References
Chapter II Thermodynamic characterization of interactions between β-lactoglobulin and major molecular fractions of Acacia gum
II.1. Introduction
II.2. Experimental Section
II.2.1. Materials
II.2.2. Preparation of β-Lactoglobulin (BLG) and Acacia gum AG) stock dispersions
II.2.3. Isothermal Titration Calorimetry
II.2.4. Optical density
II.3. Results and Discussion
II.3.1. Thermodynamic characteristics of BLG/AG interactions
II.3.2. Ratio-induced structural transitions during complexation between BLG and AG
II.3.3. Thermodynamic contribution of individual molecular fraction of TAG in interactions with BLG
II.3.4. Parameters influencing the thermodynamic characteristics of the different BLG/AG systems
II.3.4.1. Initial concentration of AG
II.3.4.2. pH
II.3.4.3. Temperature
II.4. Conclusions
II.5. References
Chapter III Influence of Acacia gum molecular polydispersity on the complex coacervation with β-lactoglobulin
III.1. Introduction
III.2. Experimental section
III.2.1. Materials
III.2.2. Samples preparation
III.2.2.1. Stock solutions of β-lactoglobulin (BLG) and Acacia Gum (AG)
III.2.2.2. Mixed dispersions
III.2.3. Dynamic light scattering
III.2.4. Electrophoretic mobility measurements (μE)
III.2.5. Small Angle Static Light Scattering (SALS)
III.3. Results and Discussion
III.3.1. Dynamic light scattering (DLS)
III.3.2. Electrophoretic mobility (μE)
III.3.3. Small angle static light scattering (SALS)
III.4. Conclusions
III.5. References
Chapter IV Control of the strength of interactions between β-lactoglobulin and Acacia gum molecular fractions
IV.1. Introduction
IV.2. Experimental section
IV.2.1. Materials
IV.2.2. Preparation of β-Lactoglobulin (BLG), Acacia gum (AG) stock dispersions and BLG/AG mixed dispersisons
IV.2.3. Dynamic light scattering
IV.2.4. Electrophoretic mobility measurements (μE)
IV.2.5. Small Angle Static Light Scattering (SALS)
IV.2.6. Granulo-Polarimetry
IV.2.7. Epi-fluorescence Microscopy
IV.3. Results and Discussion
IV.3.1. Effect of the Pr:Pol ratio on complexation/coacervation mechanism
IV.3.1.1. Dynamic light scattering
IV.3.1.2. Electrophoretic mobility
IV.3.1.3. Small angle static light scattering
IV.3.2. Effect of the total biopolymer concentration (Cp)
IV.3.2.1. Granulo-Polarimetry
IV.3.2.2. Optical microscopy
IV.4. Conclusions
IV.5. References
Summary

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