EFFECTS OF TEMPERATURE ON THE SOLUTION PROPERTIES OF ARABINOGALACTAN-PROTEINS FROM ACACIA GUMS EXUDATES

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Structure of AGPs obtained using Hydrophobic Interaction Chromatography (HIC) fractionation of A. senegal

As already indicated, A. gum can be seen as a continuum of macromolecules differing in sugar, protein and mineral composition, charge density, molar mass, size, shape and anisotropy [4,7,23-25,28,32,40,41]. Using, as others before, hydrophobic interaction chromatography, A. gum, and more specifically A. senegal, can be separated into three main fractions, Arabinogalactan (AG) or F1, Arabinogalactan-protein complex (AGP) or F2, and Glycoproteins (GP) or F3 [4,7,24]. These fractions were formerly named based on their protein content. However, studies have shown that all these fractions react to Yariv’s reactant and have carbohydrate chains of arabinogalactan type II. Therefore, all macromolecular fractions can be strictly considered as AGP’s [2,17]. In order to elude confusions, these fractions will be named in the following, based on their elution order, as HIC-F1, HIC-F2 and HIC-F3, then in theory from the most polar AGP (HIC-F1) to the less one (HIC-F3).

Structure of the HIC-F1 fraction

According to this separation methodology, A. gum is formed mainly by the HIC-F1 fraction. It represents around 85-90% of the total gum, has a low protein content (around 1%), a low molar mass (Mw) around 3×105 g.mol-1 and a hydrodynamic radius, RH, of about 9 nm [2,4,24-26,38]. In addition, an intrinsic viscosity in the range from 16 – 18 mL.g-1 has been reported [4,24,26,39]. This fraction is characterized by a higher amount of arabinose (Ara) as compared to the other fractions. Due to its content in uronic acids, it has been described as a weak polyelectrolyte [4]. Furthermore, it has been suggested that the high carbohydrate moiety of this fraction is responsible for the water sorption capacity of the gum [57]. Its hyperbranched and compact structure is typically at the origin of the low viscosity of A. gum dispersions [26].
Using small angle neutron scattering (SANS) and ab initio calculations, Sanchez et al (2008) proposed an original model for the structure of HIC-F1 fraction. A highly organized open disk structure with an inner-branched structure composed of sugars with a diameter of 20 nm and less than 2 nm of thickness was suggested (Figure I-2) [2,4,26]. In addition, semiaxes of 9.6×9.6×0.7 nm were obtained for the ellipsoid using a dummy atom model (DAM) [26].

Structure of the HIC-F3 fraction

The HIC-F3 fraction is the minor component of A. gum and comprises around 1-2% of the total gum. This fraction is rich in proteins (25-50%) [28,34] and it is formed of at least three different macromolecular populations. It has a weight-molar mass, Mw, ranging between 2×105 and 3×106 g.mol-1 and hydrodynamic radius in the range of 15-30 nm [2,4,24,28]. In addition, it has a lower content of hydroxyproline and serine than HIC-F1 and HIC-F2 [2,4].
The structure of HIC-F3 was analyzed using SAXS and TEM (Figure I.6). Micrographs revealed a mixture of a great number of monomers with ring-like shapes and long branches, and supramolecular assemblies. Performing microscopic imaging with HIC-F3, and with all gums and fractions, is a highly difficult because of the surface effects of AGPs that adsorb easily onto the use microscopy grids and form a macromolecular layer. SAXS highlighted the triaxial ellipsoidal morphology of AGPs and thin objects with 7-9 nm diameters were thus identified. The most remarkable feature of all these micrographs was the presence of ring-like objects. These ring-like objects were also observed with HIC-F2. Finally, the molecular structure of the protein moiety was composed by E-sheets, E-turns, polyproline II and D-helices. We then noted a difference in the averaged distribution of secondary structures between HIC-F2 and HIC-F3 [28].
The increased amount of protein and hydrophobic amino acids (glycine, valine, leucine and phenylalanine) contributes to a great extent to the self-assembly properties of HIC-F3 [2,4]. In addition, this fraction does not rehydrate easily, due to a low water affinity which contributes to its increased non polar characteristic [2,41].

Classification of hydration water

In a general way, water in biopolymer systems can be categorized into two or three types depending on the definition used. For instance, hydration obtained via densitometry, ultrasound measurements and viscometry can be categorized like bound water if its properties such as partial specific volume (vs°) and partial adiabatic compressibility (Es°) are lower than that of bulk water [78]. However, it depends on the distance of the water molecule from the solute surface, then on the strength of water-molecule interactions. For instance, water molecules present in the first hydration layer have a slower orientational/translational dynamic than bulk water. Therefore, they are strongly bound to the molecule (strongly bound water). Water molecules present in the second hydrating layer do not have direct contact with the solute molecule, thus they do not form hydrogen bonds with the molecule, but their properties are still perturbed by the solute presence (weakly bound water) [78]. In this two state-water system, the physical properties of weakly bound water are close to those of bulk water [77]. Therefore, only two types of water are practically considered, bound water from the first hydration shell (hydrating water) and bulk water. For instance, for the case of the glucose molecule, approximately 8-10 water molecules are considered as strongly bound water, meanwhile up to 40 water molecules can exist in the glucose matrix as weakly hydrated molecules [78,79]. As already explained, the bound water can be described using the hydration number. In the case of monosaccharides such as arabinose and galactose, which are the main sugars found in A. gums, the nh is 13.0 and 16.2 mol H2O/mol sugar, respectively. For globular proteins, e. g. hemoglobin and trypsin, the nh is 1.4 and 2.5 mol H2O/mol protein, respectively [77].
On the other hand, when hydration is estimated via calorimetric methods (e. g. DSC and TGA), water can be categorized into three types: free water, bound freezing water and bound non-freezing water [78,80]. In this case, free water behaves as bulk water and is not bound to the solute molecule. The freezing water is bound water with weak interactions with the molecule. The non-freezing water is tightly bound to the molecule [66,67,71,81] and remains unfrozen at lower temperatures than the freezing temperature of bulk water [78,82].
In this study we will use a two-state model, where the bound water refers to all water molecules physicochemically perturbed by the presence of the solute, and bulk water as all water molecules whose physicochemical properties have not been perturbed by the solute.

Acacia gums

In spite of the importance of hydration on the functional properties, information regarding the hydration properties of A. gums is scarce. The observed hydrophilic nature of AG has probably not motivated many studies on the hydration properties. Studies have been performed mostly in A. senegal using differential scanning calorimetry (DSC). They highlight the capacity of A. gums to retain a larger amount of non-freezing water as compared to other polysaccharides [71]. The total amount of water at saturation was about 3-4 g H2O/g AGP with very low amount of free water [57]. Total freezing bound water was around 2.5 g H2O/g AGP and non-freezing water was around 0.5 g H2O/g AGP bound water [2,71,83]. Obviously, the water retaining capacity is enhanced by the presence of ionic groups which are able to fix water molecules in their matrix [71,84]. In addition, the DSC curves showed that non-freezing water is first bounded to the hydrophilic groups in the carbohydrate moiety. Therefore, this moiety is likely to be responsible for the good sorption capacity of A. gums [85]. However, a possible effect of the protein content on water restraining capacity of A. gums was suggested [66]. In a subsequent study, a hydration of 0.9 g water/g for Ca gum and 1.1 g water/g for Na gum was measured using a membrane equilibrium method [86]. A minimal value of 0.6-0.7 g water/g of gum was found by a cryoscopic method.

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Translational diffusion coefficient

The translational diffusion coefficient (DT) is used to evaluate the mean square displacement of a macromolecule from its center of mass ( R େ ) in function of the time (t) caused by the Brownian motion of the macromolecule in solution [124]: ο 〈(οRେ ) 〉 = 6D t (I.9).
D T is commonly determined using dynamic light scattering (DLS) methods by measuring the temporal evolution of scattered light intensity in particle dispersions. It can be also obtained using diffusion ordered nuclear magnetic spectroscopy (DOSY-NMR). It depends on the particle size, since smaller particles diffuse faster. Then, the hydrodynamic radius (RH) can be obtained from the Stokes-Einstein relation [122,125,126]: ୖౄ D = ୩ా (I.10).
where kB is the Boltzman’s constant (1.38×10-23 m2.kg.s-2.K-1), K is the dynamic viscosity of the solvent (mPa.s) and T is the absolute temperature (K).

Sedimentation coefficient

The sedimentation coefficient (So, S) is a hydrodynamic property characteristic of a macromolecule [127]. It is defined as the rate of migration of a molecule in function of an applied centrifugal force [128]. The sedimentation coefficient is commonly obtained using analytical ultracentrifugation by measuring the displacement of the sedimentation boundary (r) caused by the depletion of the biopolymer. The rate of migration and diffusion of a molecule in dilute conditions is measured by the Lamm equation [128,129]: பେப = െ ୰ ப୰ப r C S୭ ɘ r െ D பେப୰ (I.11).
where C (g.cm-3) is the concentration at the radial distance r (cm), t is the centrifugation time (s) and Z is the velocity of the rotor (rpm). The sedimentation coefficient depends of the hydrodynamic volume of the molecule, since large molecules will exhibit a faster sedimentation velocity [129-131]. Then, the hydrodynamic radius can be obtained.
Finally, using the Svedberg equation, the sedimentation and translational diffusion coefficients, the weight-averaged molar mass (Mw) of the biopolymer can be estimated [132]: M = ୈబ ( బୖ ) (I.12)

Aim and Scientific Approach

A. gums are widely used in food (and non-food) industry, mainly in the beverage industry as emulsifier and in confectionaries as thickening agent. These gums are studied since more than two centuries and despite these efforts, there is still a lack of information on their structure and determinants of their physicochemical properties. More specifically, the architecture of AGPs and their conformation, flexibility and hydration properties remain to be confirmed or determined. Indeed, its intrinsic structural properties and hydration, in relation to chemical composition and solvent affinity, determine its functional properties.
The main objective of this thesis was to study the volumetric properties of AGPs in solution, at different ionic strengths and temperature, so as to provide some new insights about the relationship between the composition, structure and physicochemical properties of Acacia gums exudates. The study was performed using the two commercially available Acacia gum species, A. senegal and A. seyal, but also AGP fractions of the former obtained via hydrophobic interaction and ionic exchange chromatographies. These fractions encompass AGPs differing in their Mw, affinity for the solvent and level of aggregation.
The first specific objective was to study the volumetric hydrostatic properties of A. gums and AGP fractions, more specifically the partial specific volume (vs°) and partial specific adiabatic compressibility (Es°). These thermodynamic parameters were calculated from sound rate and density measurements of gums and AGP dispersions. They give an idea about the balance between unhydrated volume, hydration and flexibility of macromolecules.
The second specific objective was to study the main hydrodynamic properties, intrinsic viscosity ([K]), translational diffusion coefficient DT and sedimentation coefficient (So) of A gums. Measurements were carried out using viscometry, dynamic light scattering and NMR, and analytical ultracentrifugation. The different approaches will give in particular a more precise view of AGP hydrodynamic radius, which is a useful parameter while difficult to measure on polydisperse polyelectrolyte systems. In addition, a dynamic hydration number can be estimated and compared to the static parameter extracted from volumetric measurements.

Table of contents :

CHAPTER I: GENERAL INTRODUCTION 
1. Background Information
1.1. Acacia gum
1.2. Composition and chemical structure of Acacia gums
1.3. Structure of AGPs obtained using Hydrophobic interaction chromatography (HIC) fractionation of A. senegal
1.4. Hydration properties of biopolymers
1.5. Volumetric Properties .
1.6. Hydrodynamic Properties
2. Aim and Scientific Approach
3. Outline of the thesis
4. References
CHAPTER II: VOLUMETRIC PROPERTIES OF ACACIA GUMS 
Abstract
1. Introduction
2. Materials and Methods
2.1. Materials
2.2 Method
2.3 Theoretical treatment of density and sound velocity parameters
3. Results
3.1. Biochemical and structural characteristics of AGPs
3.2. Volumetric properties
4. Discussion
4.1. Microscopic description of AGP volumetric experimental data
4.2. Partial molar volumes of AGPs
4.3. Partial molar adiabatic compressibility of AGPs
4.4. Additional comments on the hydration properties of AGPs
5. Concluding Remarks.
6. Acknowledgements
7.1. Biochemical properties and structural properties
7.2. Volumetric properties
8. Complementary Information
8.1. Branching degree of A. gums
8.2 Amino acid composition of A. gums and macromolecular fractions
9. References
CHAPTER III: HYDRODYNAMIC PROPERTIES OF ARABINOGALACTAN-PROTEINS FROM ACACIA GUMS
1. Introduction
2. Materials and Methods
2.1. Materials
2.2 Methods
3. Theoretical treatment of data
3.1. Intrinsic viscosity
3.2. Translational diffusion coefficient
3.3. Sedimentation coefficient
4. Results
4.1. Refractive index increment
4.2. Structural properties
4.3. Dynamic viscosity
4.4. Translational diffusion coefficient
4.5. Sedimentation coefficient
5. Discussion
5.1. Hydrodynamic radius
5.2. Gyration radius
5.3. Conformational analysis
5.4. Hydration
6. Conclusions
7. Complementary data
8. References
CHAPTER IV: EFFECTS OF TEMPERATURE ON THE SOLUTION PROPERTIES OF ARABINOGALACTAN-PROTEINS FROM ACACIA GUMS EXUDATES
1. Introduction
2. Materials and Methods
2.1. Materials
2.2 Methods
3. Results and Discussion
3.1. Thermogravimetric analysis
3.2. Structural and hydrodynamic properties as determined by HPSEC-MALS
3.3. Temperature dependence of dynamic viscosity as determined by capillary viscometry
3.4. Effect of temperature on volumetric (hydrostatic) properties
4. Conclusions
5. Acknowledgements
6. References
CHAPTER V: GENERAL CONCLUSIONS AND PERSPECTIVES 
1. General conclusions
2. Perspectives
3. References
ANNEXES

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