Acacia gum: History of the future

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century, the Emirate of Trarza and its neighbors in what is today southern Mauritania collected taxes on trade, especially AG that the French were purchasing in ever-increasing quantities for its use in industrial fabric production. West Africa had become the sole supplier of world AG by the 18 th century, and its export from the French colony of Saint-Louis  (Chevalier, 1924).

Uses of Acacia gum
Acacia senegal/seyal trees are important for the ecology of arid and semi-arid areas where they naturally grow. They prevent soil degradation, fix atmospheric nitrogen and maintain soil moisture. Trees are resistant in period of drought. They act as wind barrier and are important for dune fixation. In addition, trees participate to soil fertilization and decomposition of dead leaves reinforces anti-erosive roots of trees (Wickens, Seif El Din, Sita, & Nahal, 1995).
The tree has wide usage: the foliage and seed pods make excellent fodder for livestock, ropes can be made from the bark fibers of the roots, and the thorny branches are often used to make hedges to enclose cattle or protect agricultural farms. The tree can also be used for small-scale carpentry or for making agricultural tools. When it passes its gum-productive age, between 15 and 25 years old, its wood is used for both fuel and charcoal production (Touré, 2008;Wickens, et al., 1995) .
AG is unique among the natural gums because of its properties, including high solubility and it is widely used as a stabilizer, emulsifier, flavoring agent, thickener, or surface-finishing agent. It also activates turbidity or retards sugar crystallization. These properties make it a Version postprint Comment citer ce document : Sanchez, C., Nigen, M., Mejia Tamayo, V., Doco, T., Williams, P., Amine, C., .

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8 very interesting additive in the food industry, including for the production of beverages (including Coca-Cola®), confectionery, emulsions, flavor encapsulations, bakery products and brewing (Touré, 2008;Verbeken, et al., 2003;Wickens, et al., 1995). In wine production, AG prevents color pigment and protein precipitations, confers body and stabilizes the color. As mentioned above, AG has been also used for ages in non-food industries including pharmaceutical, printing, textile, and cosmetic industries (Verbeken, et al., 2003).
The modern industrial era has produced an explosion of manufacturing uses for AG.
According to Cecil (2005), gum was important in the 19 th century in early photography as an ingredient in gum bi-chromate prints. It is now used in lithography, where its ability to emulsify highly uniform thin liquid films makes it desirable as an antioxidant coating for photosensitive plates. The same quality also makes gum useful in sprayed glazes and hightech ceramics and as a flocculating agent. It is used as a binder for color pigments in crayons, a coating for papers and a key ingredient in the micro-encapsulating process for the production of carbonless copy paper, laundry detergents etc. It is used in textile sizing and finishing and for metal corrosion inhibition. Moisture-sensitive postage-stamp adhesives and matchsticks are also made with gum. Touré (2008) adds that AG is used in the cosmetic industry as an adhesive when making face powders and masks and to render also creams and lotions smoother. New uses begin to emerge such as for instance the stabilization of carbon nanotubes (Bandyopadhyaya, Nativ-Roth, Regev, & Yerushalmi-Rozen, 2002).  & Stoddart, 1996;Islam, Phillips, Sljivo, Snowden, & Williams, 1997;Verbeken, et al., 2003). Idris et al. (1998) reported AG to be comprised of 39-42% galactose, 24-27% arabinose, 12-16% rhamnose, 15-16% glucuronic acid, 1.5-2.6% protein, 0.22-0.39% nitrogen, and 12.5-
The second fraction (10.4% of total), an arabinogalactan-protein complex (AGP, Fraction 2 or F2), contained 9% protein and had a molecular weight of 1.910 6 g.mol -1 . The third minor fraction (1.3% of total gum), referred as glycoproteins (GP, fraction 3 or F3), will consist of at least three glycoprotein populations with molecular weight ranging from 2.510 5 to 2.610 6 g.mol -1 . One of the GP had a molecular weight of 2.95 x 10 5 g.mol -1 and the highest protein content 24.6%, (Renard, et al., 2006). These different values may change depending on gum origin, age, storage conditions, etc… (Al-Assaf, Andres-Brull, Cirre, & Phillips, 2012). Ray et al. (1995) fractionated AG by both HIC and gel permeation chromatography (GPC); their results were in broad agreement with those of Randall et al. (1989) and Renard et al. (2006). The main amino acids present in the proteinaceous component of AG and AGP were hydroxyproline, serine and proline, whereas in GP, aspartic acid was the most abundant (Islam, et al., 1997). Osman et al. (1993) fractionated AG by HIC to yield four fractions, all of which had a similar carbohydrate composition, but differed in their content of protein, amino acid composition and molecular weight distribution. All four fractions reacted with an array of anti-arabinogalactan-protein monoclonal antibodies via anti-carbohydrate epitopes and were precipitated by Yariv's reagent, which indicated that all four fractions belonged to AGP's family.
Regarding the structure in solution, globular and close-packed shape of Acacia senegal gum molecules was suggested previously based on the low viscosity of gum solutions (D. M. W. Anderson & Dea, 1971;Swenson, et al., 1968). The globular or not-extended shape of AG molecules was also deduced from the relationships between the intrinsic viscosity [] or the radius of gyration R g or the ratio value between R g and R h and the molecular weight M w , (Idris, et al., 1998). For instance, the Mark-Houwink-Sakurada exponent , i.e. the slope of the log-log plot of [] vs M w , produced a slope of 0.54 (D. M. W. Anderson & Rahman, 1967) or 0.47 (Idris, et al., 1998). These values are mainly due to the AGp fraction as we found for this fraction a slope of 0.49 (Sanchez, et al., 2008
Few studies focused on the structural properties of Acacia gums according to the origin of gums (i.e. the country). This missing information could raise some questions about the influence of the geographical area of harvest and the post-harvested treatment, especially from raw to spray-dried gums, on the conformations of molecules. Our group analysed the

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Comment citer ce document : Sanchez, C., Nigen, M., Mejia Tamayo, V., Doco, T., Williams, P., Amine, C.,  To summarize AG is composed of a continuum of molecular species differing by their protein to sugar ratio, molecular weight and charges (Renard, et al., 2006), but also by different mesoscopic structures in solutions. The precise conformations of different molecular fractions remain uncertain from studies on total AG. The following section reports on the actual knowledge on the structures of the three molecular fractions of A. senegal gum isolated from HIC, i.e. the arabinogalactan-peptide (AGp), the arabinogalactan-protein (AGP) and glycoproteins (GP) fractions.

Structure of the arabinogalactan-peptide fraction (AGp, fraction 1 or F1)
The first structural model for AGp was recently proposed (Sanchez, et al., 2008). From small angle neutron scattering (SANS) experiments in charge screening conditions and dynamic light scattering, AGp appeared to be a dispersion of two-dimensional structures with a R g of 6.5 nm, a R h of 9.1 nm and an inner dense branched structure (Renard, et al., 2006;Sanchez, et al., 2008). Data analysis and modeling of SANS experiments revealed a disk-like morphology with a diameter of 20 nm, a thickness of less than 2 nm and a central intricated ''network''. The structure of AGp could explain the low viscosity of AG solutions, and its ability to self-assemble and to interact with proteins. At the molecular level, no specific secondary structures could be detected using circular dichroism, which could be explained by the low amino-acid composition of the AGp fraction (Renard, et al., 2006). However, Fourier transform infrared spectroscopy suggested the presence of extended -sheet and -

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13 turn structures but not -helix (Renard, et al., 2006). The absence of -helix could explain partly the absence of secondary structures as revealed by circular dichroism.
At the molecular level, AGP contains various secondary structures, including about 27% of polyproline II structures, -sheets, -turns and unordered structures, but not -helices (Renard, et al., 2006). A wattle-blossom model was proposed to describe the structure of the AGP complex. It was postulated that the high molecular weight fraction of the gum is composed of large carbohydrate blocks with a molecular weight of approximately 210 5 g.mol -1 , these blocks being covalently linked to a polypeptide backbone (Connolly, Fenyo, & Vandevelde, 1987Fincher, et al., 1983). An alternative model was suggested by Qi et al. (1991) in the form of a hairy twisted rope. This model would be comprised of a core rodlike protein (150 nm long) with a highly repetitive amino-acid sequence and the carbohydrate blocks (30 sugar residues) attached to hydroxyproline residues. However, as indicated previously, most studies strongly suggested that the molecules of the AGP complex have a spheroidal structure, which better supports the wattle-blossom model (Connolly, et al., 1988;Idris, et al., 1998;Picton, et al., 2000;Vandevelde, et al., 1987).
From a study on total Acacia gum, a more detailed picture of the wattle-blossom structure of AGP was proposed recently (Mahendran, et al., 2008). Mild alkaline hydrolysis of the gum followed by GPC analysis indicated that AG consists of carbohydrate blocks of ~4.510 4 g.mol -1 , blocks much lower in mass than those previously reported, covalently linked to serine and hydroxyproline residues. Two folded polypeptide chains would be present in AG, one with a M w around 310 4 g.

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14 1991). These values are much lower than the 2250 amino acid residues determined by Renard et al. (2006) for the AGP fraction. Such a large discrepancy is difficult to explain solely based on different experimental approaches. Rather we think that it is probably symptomatic of different assembly states of the AGP fraction, due to different origins and maturation history. The 45 amino acids peptide is probably associated with sugars in the AGp fraction, as already demonstrated by Renard et al. (2006). It was then assumed that carbohydrate blocks may have a thin oblate ellipsoid structure (Mahendran, et al., 2008;Sanchez, et al., 2008). The model is interesting since it gives a clearer view of the possible spatial configuration of AGP. The steric arrangement of carbohydrate blocks in such a configuration is questionable and merits much more investigation, as well as the fine structure of macromolecules. One can imagine that AGP is a two-dimensional object with a folded protein network and interacting massive sugar blocks or an assembly of sugar blocks linked (covalently or not) between them by several polypeptide backbones.
Regarding the possible morphology of AGP in solution, HPSEC-MALLS provided some informative insights. AGP in solution has a weight average molecular weight of 1.8610 6 g.mol -1 and a radius of gyration of 30 nm (Renard, et al., 2006). In addition, two exponent values are identified in the R g , [], R h vs M w relationships highlighting two types of conformations depending on the molecular weight range considered . AGP would behave in solution as a branched or hyper-branched polymer with conformations ranging from globular to elongated shape depending on the size of the carbohydrate branches. SANS form factor revealed an elongated average conformation corresponding to a triaxial ellipsoid while inverse Fourier transform of the scattering form factor gave a maximum dimension for AGP of 64 nm .
TEM highlighted the existence of isolated spheroidal particles (diameters ranging from about 10 to 40 nm) or more anisotropic morphologies (lengths from 20 up to about 60 nm) (Renard, Garnier, Lapp, Schmitt, & Sanchez, 2013). Remarkably, all the particles were porous supramolecular assemblies of smaller structural subunits with dimensions of about 2-10 nm.
These building structural subunits were mainly branched chains and ring-like structures with diameters of about 1-5 nm.
It was recently suggested that AGP would be in fact a molecular association resulting from an aggregated fraction of AGp units stabilized by low molecular weight proteinaceous

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15 components found in the GP fraction of AG ). More accurately, maturation process would promote interactions between AGP, AGp and GP, inducing a molecular reorganization of the gum and the appearance of composite AGP architectures. In addition, spray-drying was found to increase the molecular weight of AGP due to selfaggregation . In summary, the so called AGP is in fact a heterogeneous population varying in anisotropy, chain density and porosity, and of all possible molecular combinations between AGP, AGp and GP. This structural heterogeneity likely depends on chemical composition of gum sample and its maturation process, natural or induced by processing.
Very recently, AGP and AGp molecular fractions were degraded enzymatically using acidic and alkaline proteases in order to probe the conformation and structure of the two main fractions of Acacia senegal gum. While AGp fraction kept intact whatever the enzymes and conditions used, AGP was found to be degraded only in alkaline conditions. The absence of degradation in acidic conditions questioned about the potential modification of the structure of AGP molecular fraction at low pHs. The decrease in molecular weight of AGP after enzymatic treatment confirmed the accessibility of enzymes toward polypeptide cleavages and papain was found to be the most efficient protease with a decrease of M w from 1.79 × 10 6 to 1.68 × 10 5 g mol −1 . The molecular structure of control and enzyme-treated AGPs surprisingly predicted similar secondary structures content. The similar conformations adopted by control and enzyme-cleaved AGPs probed at the molecular and mesoscopic scale (by SANS) would be in favor of a high flexibility of the polypeptide backbone before and after enzymatic treatment in accordance with the repetitive and palindromic nature of peptide sequence and the overall symmetry of the carbohydrate moieties along the protein backbone (Goodrum, Patel, Leykam, & Kieliszewski, 2000;Kieliszewski, 2001;Kieliszewski & Lamport, 1994). It was finally suggested that a self-similarity driven-process would be at the origin of the assembly of AGP from a consensus glycopeptide building block with a symmetrical distribution of arabinosides and polysaccharide substituents ( Figure 2) (Renard, Lavenant-Gourgeon, Lapp, Nigen, & Sanchez, 2014).
Regarding the amino acid composition, GP fraction is less rich in hydroxyproline and serine but richer in asparagine and aspartic acid, but also in tyrosine and phenylalanine residues (Renard, et al., 2006). Using HIC, at least three different fractions were identified in GP with M W ranging from 310 5 to 310 6 g.mol -1 (Renard, et al., 2006). Following HIC and SEC, the three fractions were purified and displayed each three molecular populations, with a clear continuum of species. It appears obvious that a deeper study of GP is needed to better understand the complexity of this minor fraction. Like AGP, the different glycoproteins are characterized by the presence of polyproline II conformation (9%), -helix (9%), -sheet (38%), -turns (23%) and unordered structures (18%) (Renard, Lepvrier, et al., 2014).
However, no mesoscopic models have been proposed to date for these glycoproteins. As well, physical chemical properties of GP fraction are almost unknown, except its very active surface properties (Castellani, Gaillard, et al., 2010).
Very recently,  studied the structure of one glycoprotein (GP) fraction of AG isolated from HIC and SEC, which revealed a mixture of spheroidal monomers and more anisotropic oligomers in GP solution as suggested by the two exponent values found in the R g vs. M w relationship and TEM observations (Renard, Lepvrier, et al., 2014). The GP conformation probed by SAXS was ascribed to a thin object with a triaxial ellipsoid morphology, certainly attributed to GP oligomers. A 9 nm diameter particle was also identified by SAXS in agreement with the dimensions (diameters of 8 to 11 nm) identified by TEM on single isolated ring-like structures. All the identified isolated particles had a spheroidal shape while slight anisotropy appeared when ring-like structures self-associated.
Contrary to what was previously observed on AGp and AGP, no outer structure combined to an inner porous network of interspersed chains was observed in the spheroidal particles morphology. These spheroidal particles were structurally made of an inhomogeneous outer thick shell and a central hole giving rise to the particles a typical ring-like morphology with a 8 to 11 nm diameters (Renard, Lepvrier, et al., 2014).
In summary, the GP fraction from AG would be an assembly of ring-like glycoproteins modules. These ring-like structures were certainly due to hydroxyproline (Hyp)arabinogalactan (AG) subunits, as suggested by the secondary structures content of GP (Renard, et al., 2013). GP monomer, with a rather globular shape and homogeneous long-

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17 chain branches, would be able to self-associate giving rise to small oligomers with a rather compact conformation and bigger oligomers with a more extended conformation closely related to the self-association mode.
3. Physico-chemical properties of Acacia senegal gum

Solubility in polar and non polar solvents
The ability of AG to easily dissolve in water is not new (Diderot, et al., 1777;Pomet, 1735), and it is said that AG is soluble in cold and hot water up to concentrations of about 50-55% and insoluble in alcohol (Erni, et al., 2007;Ewart & Chapman, 1952;Izydorczyk, Cui, & Wang, 2005;Turner, 1832;Verbeken, et al., 2003;Waters & Tuttle, 1916). However it is not so easy to prepare a dispersion of gum at 50% concentration and for instance a gum solubility of 37% was experimentally determined at 25°C (Taft & Malm, 1929). Despite its high solubility in water, the mineral composition of the bulk can induce the precipitation of AG. The aqueous solution of AG forms a white jelly with basic acetate while it is soluble with neutral lead acetate. The AG solution also precipitates using potassium or sodium silicate, borax, ammonium oxalate, mercuric chloride and ferric salts (Parry, 1918). In addition, it was actually demonstrated using a great number of solvents that AG is poorly soluble in solvents other than water (Taft, et al., 1929;Taft & Malm, 1931).
The nature of interactions and mechanism of demixing in AG-ethanol-water ternary system have not been studied in great details. However the ability of alcohols (and some salts) to induce phase separation/precipitation in polysaccharides, and more generally biopolymers
It would be useful to determine phase diagrams of AG-alcohol-water systems and to characterize in each phase the macromolecular composition, i.e. the relative compositions of AGp, AGP and GP. As these three fractions display or are supposed to display different physical chemical properties, it is likely that simple ways to obtain AG enriched in one or another of the three fractions could be identified.

Hydration properties of Acacia gum macromolecules
The observed hydrophilic nature of AG has probably not motivated many studies on the hydration properties. Few papers can be found in literature. In one example, it was shown that AG is not hydrated to a great extent as has been claimed by many authors (Grollman, 1931). Using vapor pressure measurements, no hydration of the gum was measured with NaCl (0.05M) or KCl (0.07M), which would indicate that ions were preferentially hydrated.
With 0.18M sucrose, 0.7g water/g of gum was measured. In a subsequent study, a hydration of 0.9g water/g for Ca gum and 1.1g water/g for Na gum was measured using a membrane equilibrium method (Oakley, 1937). A minimal value of 0.6-0.7 g water/g of gum was found by a cryoscopic method and no hydration of the gum in presence of KCl or KBr was found, which seems to confirm previous results (Gortner & Gortner, 1934). An interesting point was that when AG concentration in studied dispersions increased from 3% to 10%, the amount of bound water decreased from 1.2g to 0.6g/g of gum (Newton & Gortner, 1922). It is possible that some self-association (i.e. aggregation) of AG macromolecules occurred with increasing concentration, leading to the decrease of water accessibility towards AG macromolecules and to the release of bound water from AG macromolecules during self-association process.
When analyzing interactions of biopolymers with water, one can thus distinguish free and bound water (Chandler, 1941;Gortner, et al., 1934). Free or freezing water is the water where melting/crystallization temperature and enthalpy of melting/crystallization are not significantly different from those of bulk water (Hatakeyama & Hatakeyama, 1998). Bound

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19 water gathers non-freezing water that is very closely associated with the macromolecules and freezing bound water that is less closely associated but displays different physical properties than free water (Hatakeyama & Hatakeyama, 1998). Non-freezing water is made of a monolayer, possibly multilayers, of water molecules in very close interaction with the biopolymer. The monolayer is generally calculated from sorption isotherms (gravimetric method) following the use of an appropriate model, the most often used being the BET (Brunauer-Emmet-Teller) or the GAB (Guggenheim-Anderson-de Boer) model (Blahovec, 2004). This type of hydration water has to be considered as an integrating part of the native biopolymer structures (Luschermattli & Ruegg, 1982). The total amount of water (W c ) interacting with a biopolymer is then the sum of free and bound water. Another parameter dealing with the interaction of biopolymers and especially fibers with water is the waterholding capacity (WHC). The WHC is a measure of the ability of a fiber source to immobilize water within its matrix (Robertson & Eastwood, 1981). WHC encompasses both adsorbed water and possibly water trapped within macromolecules.
Generally, water (W c or WHC) in AG amounts to about 3-6 g water/g gum, which is a range previously mentioned (Takigami, Takigami, & Phillips, 1995). The saturation W c value is in the range of values found for other polysaccharides such as xanthan and hyaluronan (Phillips, Takigami, & Takigami, 1996). The bound water (W b ) is around 1 g water/g gum and the non-freezing water (W nf ) is within 0.4-0.7 g water/g gum. In addition, the second virial coefficient A 2 (mL.mol.g -2 , also called B2 or B22), an indirect way to define the affinity for solvent, can be calculated from light scattering measurements. Negative A 2 values indicate a bad affinity for the solvent and attractive interactions between biopolymers while positive A 2 values indicate preferential interactions with the solvent and repulsions between macromolecules. Values of 5.10 -5 (Picton, et al., 2000) or 4.2.10 -5 mlmolg -2 (Veis, et al., 1954) were found for AG. The values were low but positive, under the experimental conditions used, indicating a preferential interaction with water. An interesting result concerns the effect of temperature on the water monolayer (X m ). It was found that increasing the temperature from 25 to 45°C resulted to an increase of X m from 0.08 to 0.11 g water/g gum. As an increase in temperature lowers the energy of hydrogen bonding, and as hydrogen bonding is at the basis of polysaccharide hydration (Q.  ACCEPTED MANUSCRIPT 20 but also for soy proteins (Cassini, Marczak, & Norena, 2006). It is also well known that by increasing temperatures there is an increase of energy of adsorbed water molecules, which allow the leaving of some adsorbed water molecules from the active centers of the adsorbent. As a result, the amount of adsorbed moisture decreases. The results reported here for AG are somewhat counterintuitive. However, these results are not unique as similar trends were demonstrated for microcrystalline cellulose (Cadden, 1988) cited by Vernon-Carter et al., 2006) or myosin (Das & Das, 2002) cited by Vernon-Carter et al., 2006).
All these results converge on the same conclusion. The affinity of AG for water provides an extremely favorable environment for binding water, which is probably mainly due to the carbohydrate component of AG and its highly branched characteristic (Phillips, et al., 1996).
The polypeptide component also interacts with water since it contains a significant number of hydrophilic aminoacids (Renard, et al., 2006). The sugars units would first bind water at the hydroxyl (OH) groups associated with the uronic acids, forming the non-freezing water (Phillips, et al., 1996). Freezing-bound water is also tightly associated with carbohydrate chains, which could form intra-molecular hydrogen bonds within the highly cross-linked gum structure (Phillips, et al., 1996). Thereafter, there would be large intra-molecular and intermolecular voids which could be occupied by water in a variety of metastable states, preventing the formation of the ideal ice structure.
It is interesting to note that when AG in solution is dried, either with alcohol or by heating (vacuum distillation), it becomes practically insoluble (Thomas, et al., 1928). When it is thus dried, the gum swells in water to a jelly-like mass which does not dissolve except on long standing. AG in powder form heated above 100°C, and especially at 170 °C, when immersed in water, also swells up to a great extent but does not dissolve and the gel thus formed is non-sticky (Moorjani & Narwani, 1948). This insolubility is explained by the complete dehydration of the gum. However, it is likely that aggregation of AG macromolecules occurs as heating from 100 to 170 °C results in an increase of the viscosity of solutions. Protein degradation also occurs at temperatures above 100 °C that may affect the gum solubility (Cozic, 2007). The insolubilization of the gum by heating at high temperature (150 °C) is known for a long time (Fremy, 1860). Boiling the gum or adding alkali at cold temperature dissolves the gum again (Fremy, 1860). The ability of AG to re-bind water which has been applications (Phillips, et al., 1996).

Viscosity (at zero shear rate) of Acacia gum dispersions
The molecular interactions of AG with the solvent determine in part the viscosity of dispersions. Viscosity is also governed by the shape, molecular size and concentration of molecules and is affected by temperature and pressure. Since AG macromolecules are weak polyelectrolytes, it is expected that pH, ionic strength and type of ions (according to the Hofmeister serie) must have a significant effect on the viscosity (Stephen & Churms, 1995).
This can be clearly observed with arabic acid where a maximum of viscosity was reached at a pH in the range 5.5-6.3 (Thomas, et al., 1928). The lower viscosity at acidic pH can be explained by the fact that, due to neutralization of carboxyl groups at low pH, electrostatic repulsions decrease. This leads to a decrease in the hydrodynamic volume of the carboxylbearing polysaccharides, and hence, in the viscosity of the polysaccharide solution (Vanderreijden, Veerman, & Amerongen, 1994). Acid-induced hydrolysis of polysaccharide can also contribute to the lower measured viscosity. The decrease in viscosity observed at pH larger than 8-10 could be explained by a strong weakening of hydrogen bonds, resulting in less efficient interactions with water or, alternatively, to conformational changes resulting in a reduction of charged groups-induced electrostatic repulsions. It can also be noticed that the viscosity was almost steady between pH 5 and 9. A steady viscosity of AG dispersions was also measured between pH 6.2 and 8.5 by Riddell & Davies (1931).
The increase in NaCl concentration induced a decrease in viscosity. For a salt-free solution, electrostatic repulsions due to the charges on the macromolecule favor a stretched chain conformation as a result of long-range electrostatic effects. This behavior results in higher viscosity. Addition of a simple electrolyte screens these intermolecular electrostatic repulsions and allows the molecules to compact towards the volume of an uncharged polymer with the same number of residues linked in the same way (Giannouli, Richardson, & Morris, 2004), resulting in a lower viscosity (Smidsrod & Haug, 1971;Tinland & Rinaudo, 1989). Similar results were obtained by other authors (Amy, 1934;.

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Comment citer ce document : Sanchez, C., Nigen, M., Mejia Tamayo, V., Doco, T., Williams, P., Amine, C., Renard Like for other common biopolymers, viscosity increased with AG concentration  and decreased with the increase in temperature (Stephen, et al., 1995). Regarding the effect of AG concentration, we collected a great number of data from literature and the relationship between the relative viscosity / 0 and the AG concentration was exponential over a 0.13-56 wt% concentration range. However, what is important to notice, irrespective of the geographical origin of AG, various chemical compositions and physical chemical conditions of sample preparation for viscosity measurements, practically all data are described by a single exponential (Figure 3). Such an exponential delimitates two regions, one region where the viscosity gently increases with concentration and the other region where viscosity sharply increases with concentration.

Flow and viscoelastic properties of Acacia gum dispersions
Unlike most polysaccharides, used for their thickening or gelling properties (Wand & Cui, 2005), AG dispersions display low viscosity even at quite high concentration and do not gel (except when AG powder is thermally treated at high T then rehydrated, as noted above).
More recently, it has been shown that AG dispersions also display shear-thinning flow behavior, even at AG concentrations as low as 1-4% (Li, et al., 2009;Mothe & Rao, 1999;Sanchez, Renard, Robert, Schmitt, & Lefebvre, 2002;Weinbreck & Wientjes, 2004). It was hypothesized that the presence of AG aggregates could explain such an unusual flow behavior (Li, et al., 2009;Mothe, et al., 1999). Hydrogen bonding could partly explain the formation of these hypothetical aggregates (Li, et al., 2009). In addition, time-dependent or thixotropic flow behavior was also observed (Li, et al., 2009;. It was suggested that the aggregation of AGP component was at the origin of this behavior (Li,
Viscoelastic properties of AG dispersions were also characterized and revealed, as expected, a predominant liquid-like behavior (Goycoolea, Morris, Richardson, & Bell, 1995;Matsumura, Satake, Egami, & Mori, 2000;. Indeed, mechanical spectra obtained at 6wt% AG concentration by oscillatory testing revealed that the viscous or loss modulus (G", N.m -2 ) was higher than the elastic or storage modulus (G', N.m -2 ) throughout a wide frequency range but G' became larger than G" at the highest frequencies . Similar behavior was recorded at 18wt% (Matsumura, et al., 2000) or 50wt% (Goycoolea, et al., 1995) AG concentration. Interestingly, the evolution of G' and G" as a function of frequency followed a power law behavior with exponents of 1.4 and 0.8, respectively, smaller than the exponents 2 and 1 classically found for viscoelastic liquids . It was then concluded that AG dispersions were structured liquids. Surface effects also have an impact on measured viscoelastic properties. Dynamic mechanical spectra after 120 min rest of AG samples at 6wt% gum concentration in the rheometer therefore showed a typical gel-like behavior with G' larger than G" over the entire range of selected frequencies . The building-up with time of a predominantly elastic interfacial structure was demonstrated.
In summary, AG dispersions display newtonian flow behavior at gum concentrations below about 20wt% and shear-thinning above. However, in practical industrial situations where applied shear rates are usually above 100 s -1 , flow behavior of AG can be considered as newtonian. Sometimes, the measured flow behavior is non-newtonian and even thixotropic ACCEPTED MANUSCRIPT 24 at low AG concentrations. This unusual behavior seems to be mainly due to surface effects and reversible shear-induced aggregation of AG macromolecules. In this case, further studies are needed to clarify the situation, by using for instance rheology coupled to small angle xray or neutron scattering.

Assembly properties of Acacia gum
Molecular associations and assembly of biopolymers depend strongly on the extent of macromolecule-solvent and macromolecule-macromolecule interactions. Several factors such as solvent chemical properties, physical chemical treatments and macromolecules physical chemical properties can influence the assembly pathway and of course the functional properties of the assemblies. It is well known that AG can associate with itself or other biopolymers, such as proteins. Its association and assembly properties are often used in several areas (food, pharmaceutical, medicine, etc.) to elaborate AG-based assemblies with specific functional properties. According to physical chemical conditions and the presence or not of other macromolecules as partner of the assembly, AG can assemble following different mechanisms such as aggregation or coacervation (simple or complex).

Self-association and aggregation properties of Acacia gum
Aggregation of AG is apparently a natural mechanism depending on the physiology of trees, and particularly the ageing (Idris, et al., 1998). The characterization of AG harvested on trees of different ages, from 5 to 15 years old, showed both the increase in the mean radius of gyration of AG and the proportion of aggregates in solution. This aggregation mechanism occurs when the trees grow older up to about 15 years.
Aggregation of AG in aqueous solution was also evidenced in several studies using size exclusion chromatography (SEC) that showed an elution peak in the void volume of the column, in addition to the peaks corresponding to the three main fractions of AG (Idris, et al., 1998;Mukherjee, et al., 1949;Ray, et al., 1995;. This peak,

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25 of AG increased from 0.0413 to 5.21 g·L -1 , respectively (Q. Wang, et al., 2008). Aggregates were also evidenced in more concentrated AG solution, ranging from 5 to 300 g·L -1 , by SAXS and SANS measurements, and observed using cryo-TEM (Dror, Cohen, & Yerushalmi-Rozen, 2006). All these experiments confirm the self-association behavior of AG in aqueous solution over a large range of concentration.
The apparent contradiction between the high solubility of AG in water and its propensity to self-associate could origin from the chemical composition of the molecular fractions isolated from AG. Indeed, as described above, AG is composed of arabinogalactan-protein type macromolecules (Akiyama, Eda, & Kato, 1984). In plant kingdom, the association property of AGPs is well established: these macromolecules have specific functions in interaction and recognition cellular mechanism (Showalter, 2001). It is also well known that AGPs can selfassemble and aggregate both in vitro and in vivo (Baldwin, McCann, & Roberts, 1993; Capataz- Tafur Recently, studies devoted to the characterization of the three dimensional structure of isolated AG, AGP and GP fractions, also evidenced the self-assembly behavior of these isolated macromolecules (Renard, et al., , 2013Renard, Lavenant-Gourgeon, et al., 2014;Sanchez, et al., 2008). These studies highlighted some differences between the three main fractions towards their affinity for aqueous solvent and of course their ability to selfassemble and aggregate in aqueous solution. The study focusing on GP fraction showed first of all that it was not easy to rehydrate GP powder. A significant proportion of GP macromolecules aggregated with the formation of a substantial undissolved material after centrifugation (Ray, et al., 1995;Renard, et al., 2013). On the contrary, the rehydration of AGp and AGP powders were complete in aqueous solution without the formation of undissolved material Sanchez, et al., 2008). Hence, GP fraction contains some macromolecules with a lowest affinity towards aqueous solvent compared to those included in AGp and AGP fractions, in agreement with the delayed elution of this fraction by HIC. The self-aggregation behavior of GP fraction during its rehydration was attributed to the

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26 more pronounced hydrophobic nature of the GP macromolecules (Renard, et al., 2013;Renard, et al., 2006). Transmission Electron Microscopy (TEM) experiments performed on each fraction also revealed that the self-aggregation behavior and the morphology of formed aggregates depend clearly on the fraction studied. It is likely that drying of samples for TEM experiments could impact the observed morphologies. Self-assembled or aggregated macromolecules were observed with AGP and GP fractions (Renard, et al., , 2013, but not with AGp fraction (Sanchez, et al., 2008). The aggregates evidenced with AGP and GP macromolecules in aqueous solution could be promoted by several intermolecular attractive weak forces such as hydrogen bond between saccharidic residues as suggested by Mahendran et al. (2008) and/or hydrophobic interaction between polypeptide backbones as suggested by .

Differences between AGp, AGP and GP fractions towards aggregation in aqueous solution
could be explained by their differences in chemical composition and particularly their protein content and amino-acids composition. The protein content in AGp, AGP and GP fractions is therefore around 1.1%, 9% and 24.6%, respectively (Renard, et al., 2006) and GP fraction contains more hydrophobic amino-acids such as glycine, valine, isoleucine, leucine, phenylalanine than AGp and AGP fractions (Renard, et al., 2006). Hence, both the high protein content and the presence of hydrophobic amino-acids in higher proportion in GP fractions could explain the highest sensitivity of GP macromolecules towards self-assembly and aggregation in aqueous solution.
After harvesting, AG can be submitted to several physical treatments such as heating, spraydrying or irradiation before to be used. These treatments can also influence the extent of the natural aggregation process of AG. Aggregation behavior is enhanced when AG is heated or Interestingly, aggregating AG through controlled Maillard reaction can improve its functional properties . Consequently, the natural functional properties of AG could be improved by its self-assembly properties.

Coacervation of Acacia gum
Coacervation has generally been defined as a liquid-liquid phase separation occurring in (bio)-polymers solutions under suitable conditions (Bungenberg de Jong, 1949b). It is well accepted that coacervation corresponds to a dehydration process of (bio)-polymers. The phase separation gives rise to two incompatible and immiscible liquid phases: a (bio)polymer dense phase, called the "coacervate" phase, coexisting with a very dilute colloidal phase, the supernatant (Bamford, et al., 1950;Bungenberg de Jong, 1949a;Menger & Sykes, 1998). The dilute liquid phase remains in equilibrium with the coacervate phase. Depending on the number of (bio)-polymers involved in the phase separation, coacervation can be classified as simple or complex (Bungenberg de Jong, 1949a). In the following sections, we will discuss about the assembly of AG according to simple and complex coacervation mechanism.

Simple coacervation
Simple coacervation is a liquid-liquid phase separation where only one (bio)-polymer is involved (H.B. Bohidar, 2008;Bungenberg de Jong, 1949a). It occurs as a result of a decrease in the solubility of (bio)-polymers through water competition caused by modifying the physical chemical properties of the solvent. In aqueous solutions, simple coacervation can be promoted by the action of salts (sodium sulphate, sodium chloride), the addition of a watermiscible non-solvent (ethanol, methanol, acetone, etc…), by modifying pH or increasing/decreasing temperature, thus turning the aqueous solvent medium, good for the (bio)-polymer, into a marginal one (H.B. Bohidar, 2008;Bungenberg de Jong, 1949a;Ezpeleta, et al., 1996;Mauguet, et al., 2002;Mohanty, et al., 2003).
The addition of ethanol to AG/water dispersion led to the formation of a new dense phase containing liquid droplets, called coacervates, dispersed in a continuous phase. AG macromolecules will tend to spontaneously self-associate according to a coacervation mechanism once a critical ethanol percentage has been reached. Whatever the AG In this ternary water/ethanol/AG system, ethanol acts as a suitable dehydrating agent which shifts the energetic balance in favor of the attraction between AG macromolecules (Koets, 1944). When ethanol is added, the quality of the solvent decreases, becoming a poor solvent for the solubility of AG macromolecules, by both modifying the hydrogen bond network and the polarity of the medium (H. B. Bohidar & Mohanty, 2004;Mohanty, et al., 2003). In addition to the role of ethanol in the disturbance of hydrogen bonds network, ethanol also decreases the dielectric constant and consequently the polarity of the solvent that could favor the self-association of macromolecules (Mohanty, et al., 2003). Consequently, induced coacervation of AG macromolecules by the addition of a non-solvent such as ethanol corresponds to a dehydration process due to the modifications of solvent physical chemical properties.
Complex coacervation between AG and proteins occurs by charge neutralization with the involvement of non-specific electrostatic interactions between de-protonated carboxyl groups of AG macromolecules and the protonated amino groups of proteins. As complex coacervation mechanism mainly occurs by the involvement of weak electrostatic interactions, this assembly mechanism is substantially influenced by the physical chemical properties of the solvent (pH, ionic strength, nature of salts and temperature) and the structural and physical chemical properties (global charge, charge distribution, flexibility and concentration) of biopolymers (polysaccharides and proteins) (Schmitt, et al., 1998;Ye, 2008). The identification of the specific conditions resulting in a two phase system, named phase diagram and where binodal curve is determined, is a tedious work that requires time and large quantities of raw material. Trying to overcome these major drawbacks, phase diagram of a β-lactoglobulin -AG mixture has been recently determined through an innovative miniaturized approach based on millifluidic (Amine et al., submitted). In this work authors proved that by using turbidity measurements based on image analysis within only 2 µl biopolymers droplets, binodal curve was able to be determined with a good agreement with those obtained in bulk. This method should find applications for the screening of numerous protein-polysaccharide mixtures for industrial issues.
Indeed, the formation of complex coacervates between AG and proteins is of industrial interest to value them, by enhancing their functional properties and developing novel biopolymer assemblies. One of the first and the most important industrial applications of complex coacervation is microencapsulation. Polysaccharide/protein microcapsules are used in many industries (food, pharmaceutical, cosmetics, agricultural, etc…) to entrap and protect sensitive molecules (aroma compounds, bioactives, drugs, enzymes) against processing (heat, shear, redox potential, etc…) and storage conditions (oxygen, temperature and moisture). The use of microcapsules is also a mean to deliver the encapsulated interest

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30 molecules to the specific target with the optimal kinetic by changing the physical chemical conditions (pH), mechanical process (chewing) or enzymatic action (Schmitt, et al., 2011).
Several AG/protein (gelatin, whey proteins, gliadins, pea globulin) microcapsules were developed and elaborated to encapsulate different molecules such as lemon and orange flavors, oil, pesticides and flavonoid compounds (Aberkane, et al., 2012 ). The preparation of microparticles by complex coacervation was similarly performed on AG/polysaccharide mixtures such as AG and chitosan (Butstraen & Salaün, 2014).
The rheological properties of solutions could also be modified by the formation of complex coacervates between AG and proteins. The rheological properties of protein and polysaccharide in associative conditions result in different behaviors compare to each individual biopolymer. It is expected that the bulk viscosity of the system is improved with the formation of microstructures as complex coacervates (Schmitt, et al., 2011). The characterization of the rheological properties of AG/whey protein coacervates evidenced that these assemblies displayed a viscous character . As electrostatic interactions mainly stabilize complex coacervates, protein/polysaccharide molar ratio, pH and ionic strength are key parameters for the rheological properties of complex coacervates.
The maximum of viscosity is obtained for mixing conditions leading to the complete charge neutralization of the two biopolymers. This viscous behavior was similarly evidenced on AG/Chitosan complex coacervates (Espinosa-Andrews, et al., 2013). In their work, they highlighted an interrelationship between the biopolymers mass ratio, the charge density and the viscoelastic properties of the coacervated phase.
The complex coacervates formed between AG and proteins gather the surface properties of each biopolymer. Hence, complex coacervates can also be used to stabilize air/water or oil/water interfaces in a variety of foamed and emulsified products (Dickinson, 2008). ACCEPTED MANUSCRIPT 31 charge neutralization ratio (pH 4.2) was as high as the pure adsorbed protein in the same condition . However, the viscoelastic properties of the film formed by AG/-lactoglobulin complexes were stronger than those of the pure protein and the gas permeability of the film was significantly reduced compare to pure -lactoglobulin. Similar results were obtained in chilled dairy foams using whey protein isolate/AG complexes (Schmitt & Kolodziejczyk, 2010). Consequently, the stability of the foam can be improved by the adsorption of AG/protein complexes at the air bubble interfaces. Emulsions can also be stabilized by AG/protein complexes or coacervates. Ducel et al. evidenced that AG/pea globulin and AG/α-gliadin complexes or coacervates tend to decrease more strongly the oil/water interfacial tension than the pure protein (Ducel, Saulnier, Richard, & Boury, 2005).
The coacervates films were characterized by a long relaxation time and a high surface elasticity. In addition, the authors reported that charged complex coacervates were more efficient to stabilize oil droplets.

Surface properties: adsorption at solid-liquid and liquid-liquid interfaces
Surface properties of AG, and of a number of plant gum exudates, are unique in the polysaccharide world. By surface properties, we mean both the ability of AG to decrease interfacial tension between gas-water, liquid-liquid or solid-liquid interfaces, and to stabilize these interfaces through steric and electrostatic interactions and hydration forces (Adamson & Gast, 1997). These properties can be used to form and stabilize foams (Redgwell, Schmitt, Beaulieu, & Curti, 2005), emulsions and to stabilize solid nanoparticles. It can be noticed that studies on foaming properties of AG are rare as compared to studies on stabilization of liquid or solid particles, especially nanoparticles.
AG was also used to stabilize latex nanoparticles as a model system of interface with a surface coverage of about 0.5 -5 mg/m 2 depending on solvent conditions and initial AG concentration (Gashua, Williams, & Baldwin, 2016;M. L. Snowden, Phillips, & Williams, 1987). The surface coverage was found to be similar at liquid-liquid interfaces in O/W emulsions (Randall, Phillips, & Williams, 1988) . The high M w fraction of AG, AGP, was found to be the most effective to be adsorbed at the interface after only 15 min while AGp fraction was ineffective in the stabilization of the latex dispersions (M. L. Snowden, et al., 1987).
Electronic spin resonance data indicated that AG adsorbed at the solid-liquid interface with approximately half of its segments close to the surface in trains and the other half in loops and tails extending away from the surface into solution (M. L. Snowden, et al., 1987). An alternative model of end-on or multilayer adsorption was recently proposed to explain the high layer thickness after adsorption of AG on latex particles (Gashua, et al., 2016). As noted above, AG has also been explored as coating agent of nanomaterials for biomedical applications, namely magnetic nanoparticles (Ali, Ziada, & Blunden, 2009;Banerjee & Chen, 2008Palma, et al., 2015) . AG coupled magnetic nanosystem could therefore find applications as a MRI contrast agent for cell-labeling applications. Other applications concerned the use of AG as a nontoxic material in the production of readily administrable biocompatible gold nanoparticles for diagnostic and therapeutic applications in nanomedicine (Kattumuri, et al., 2007) (Kattumuri, Katti, Bhaskaran, Boote, Casteel, Fent, Roberton, Chandrasekhar, Kannan & Katti, 2007) or to improve antioxydant properties of nanoparticles (Kong, et al., 2014).
It is today widely accepted that it is the protein-rich high-molecular weight fraction of AG, the AGP complex, which mainly provides the surface properties of gum. A fair non-linear relationship between AGP concentration and emulsion stability was recently shown with a seemingly AGP critical concentration of about 10% (Nishino, Katayama, Sakata, Al-Assaf, & Phillips, 2012). Here emulsion stability was checked following heating at 60°C for 3 weeks and we may wonder whether surface properties of AGP were highlighted or heat-induced aggregation of AGP at the interfaces, in line with the process of "Super Gum" formation described below Aoki, Al-Assaf, Katayama, & Phillips, 2007;. We remember here that "AGP" molecular fraction both encompasses the so-called AGP and the high M w GP fractions as well as minor concentrations of the AGp fraction. One direct consequence is that companies using gum in their products, for instance to make stable oil-in-water (o/w) emulsions, want to obtain gum samples with high proportions of AGP, e.g. above 12%. This explains also why modified gum with higher content in high-molecular weight fractions was developed recently through Despite its good surface properties, AG is far from being as efficient as proteins to form and stabilize oil-in-water emulsions. An oil to emulsifier ratio of about 1:1 is therefore needed for AG while a lower ratio of 1:10 is common for proteins. It is then not surprising that proteinrich macromolecules play an important role in the emulsifying/stabilizing properties of AGs (Randall, et al., 1988;Ray, et al., 1995). It was then reported with samples of various AG species, having nitrogen contents in the range from 0.1% to 7.5%, a good correlation between the nitrogen content of the gum and its limiting long-time interfacial tension and between the emulsifying capacity and the initial rate of change of tension with time . It was also shown by SEC on supernatants after removing oil droplets that protein-rich fractions adsorb strongly at the oil-water interface (Randall, et al., 1988). Similar results using the same methodological approach were obtained by others (Alftren, Penarrieta, Bergenstahl, & Nilsson, 2012;Flindt, Al-Assaf, Phillips, & Williams, 2005;Padala, et al., 2009) . However, a careful examination of results reveal that while a preferential adsorption onto oil-water interfaces of protein-rich fractions occurred, all ACCEPTED MANUSCRIPT 35 molecular fractions seems to be present at the interfaces with however different adsorption kinetics. In the frame of the wattle-blossom model, it is supposed that, in analogy with block co-polymers, the more hydrophobic protein chain anchors at the interface while the protruding hydrophilic carbohydrate blocks attached to this chain provide a strong steric barrier towards flocculation and coalescence (Islam, et al., 1997;Randall, et al., 1989).
However, numerous hydrophilic hydroxyamino acid residues are present in the polypeptide chain, and no structural data today exist about the spatial position (buried or in periphery) of the polypeptide chain, casting some doubt on the model proposed by Islam et al. and Randall et al. In fact, the efficiency of AG is better related to the way it adsorbs onto interfaces and the mechanical properties provided by interfacial films rather than to a low interfacial tension (Shotton & Wibberley, 1961). Surface charged groups provide the basis for some electrostatic contribution to the colloidal stabilization, however the relative low value of the (negative) zeta potential, 10-20 mV under beverage emulsion conditions (Jayme, et al., 1999;Ray, et al., 1995) , and the pH-independent destabilization mechanism (coalescence), indicates that the primary mechanism is steric stabilization (Dickinson, 2003;Trindade, et al., 2008). It is better to say electro-steric stabilization mechanism as suggested by Jayme et al.
Electro-steric forces, probably including hydration forces, and elastic interfaces are at the origin of emulsion stabilization by AG, especially against coalescence .
Since were induced through controlled Maillard reaction incubating kibbled gum many weeks at 60°C under controlled moisture mimicking natural maturation process according to the authors (Al-Assaf, Phillips, Sasaki, & Katayama, 2003). This process resulted in an increase of M w of AG from about 4.2x10 5 g.mol -1 up to about 20x10 5 g.mol -1 .
Modified gums allow the production of oil-in-water emulsions with smaller droplets, improved interfacial viscoelasticity and emulsion stability as compared to the unmodified control gum. The effect of AG molecular weight (M w ) on emulsion stability (Dickinson, et al., 1991a) or surface rheological properties (Nakamura, 1986) was previously demonstrated. In this case, the initial size of emulsion droplets was not impacted by M w but emulsion stability was improved with high M w . Similar results using matured gums were obtained in other studies Castellani, Gaillard, et al., 2010;X. L. Yao, et al., 2013). In addition, it was shown that modified gums have the ability to decrease interfacial tension faster than unmodified gums, which is an important parameter in determining emulsifying capacity as noted above Castellani, Guibert, et al., 2010;X. L. Yao, et al., 2013). The improved stability of produced emulsions could be due partly to the higher viscoelasticity of interfaces formed from matured gums Castellani, Guibert, et al., 2010). It is interesting to note that surface properties of matured gums are very close to that of the AGP fraction as obtained by HIC chromatography (Castellani, Gaillard, et al., 2010).
Although these results using matured AGs seem conclusive on the interfacial properties of high M w protein-rich macromolecules, some comments may be of interest. The first remark is that few studies have been done to definitely conclude on the benefit of matured gums regarding initial emulsion droplet size and emulsion stability. In addition, emulsions have never been prepared under the same experimental conditions (homogenizing conditions, gum concentration, oil to emulsifier ratio, dispersion pH, etc…), rendering the comparison of results difficult. For instance, it was shown that initial emulsion droplet size was significantly lower for oil to emulsifier ratios above 1 but was not different for ratios below 1 (Kateyama, et al., 2006). Another concern deals with the nature of supramolecular structures produced  . Obviously it can be anticipated that the global architecture of supramolecular structures is also modified. In these conditions, how to compare matured high M w macromolecules with a smaller AGP fraction of control gum?
Another approach to demonstrate the role of protein-rich high M w macromolecules in AG surface properties was to hydrolyze it with protease-type enzymes. In this case, loss of emulsification properties was observed (Chikamai, Banks, Anderson, & Weiping, 1996).
However, by hydrolyzing AG while measuring interfacial viscoelasticity showed that, after 510 min hydrolysis, interfacial viscoelasticity decreased but remained high. It is possible that hydrolyzing the gum in the bulk or at an interface did not produce the same results. One can note that high M w macromolecules present in Acacia seyal gum are also hydrolyzed by pronase but to a lesser extent, which can be partly due to its more compact conformation (Elmanan, et al., 2008;Flindt, et al., 2005). As AGP architecture is modified after hydrolysis, one may wonder whether the loss in surface properties is due to a decrease in M w or to a change in macromolecular conformation. In fact, both chemical structure of the polysaccharide component (Connolly, et al., 1987;Mahendran, et al., 2008;Randall, et al., 1988) and AGP conformation remain largely unaffected by proteases, suggesting a selfsimilar structure for the AGP component (Renard, Lavenant-Gourgeon, et al., 2014).
Owing to the expected surface properties of protein-rich molecular fractions of AG, some scarce studies tried to unravel the specific properties of individual macromolecular components. Unlike one study where four fractions were obtained by SEC (Ray, et al., 1995), the remaining studies were classically concerned by HIC fractions, i.e. AGp, AGP and GP Castellani, Gaillard, et al., 2010;Fauconnier, et al., 2000;Lopez-Franco, et al., 2004;Ray, et al., 1995). Among these studies, one of them studied the effect of fractions on oil-in-water emulsions characteristics (Ray, et al., 1995) and three studies focused on interfacial properties of fractions as determined by interfacial tension measurements and Langmuir-Blodgett films Castellani, Gaillard, et al., 2010;Fauconnier, et al., 2000;Lopez-Franco, et al., 2004). It was clear from

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Comment citer ce document : Sanchez, C., Nigen, M., Mejia Tamayo, V., Doco, T., Williams, P., Amine, C.,   . It is important to note that experiments were done at 0.05wt% gum concentration and that similar experiments at higher gum concentration should be instructive. In terms of surface pressure and limiting area, GP fraction was again the more efficient and AGp fraction the less efficient (Castellani, Gaillard, et al., 2010;Fauconnier, et al., 2000;Lopez-Franco, et al., 2004). Results were unclear in terms of surface elasticity as determined from Langmuir-Blodgett film experiments. One study showed that the whole gum produced more elastic films than fractions which formed films with similar elasticity (Lopez-Franco, et al., 2004). Another study showed that films from AGp fractions were more elastic than films made from AGP or GP fractions, the latter being the less elastic (Fauconnier, et al., 2000). These results were obtained using the same Acacia senegal gum concentration (10wt%) but different subphase composition, which may explain in part the observed differences.
Although there is no doubt about the important surface activity of AG protein-rich molecular fractions, and of the important role of high M w fractions in the stabilization of emulsions, nitrogen content alone cannot be used to predict performance of AGs for emulsification.
Gums with similar protein content may exhibit significant differences in emulsifying capacity and emulsion stability (Dickinson, et al., 1991b), confirming the view that it is the nature and distribution of the proteinaceous component of the gum which is important, not just its overall amount. Gums with higher protein content do not also necessarily produce more stable emulsions and some Acacia seyal gum samples with a much lower protein content (0.8%) have been found to give better emulsion stability than Acacia senegal gum (R. A. Buffo, et al., 2001). More surprisingly, it was shown recently that high M w arabinogalactan macromolecules extracted from Peach exudate and not containing protein displayed better emulsifying and emulsion stabilizing properties than Acacia senegal gum (Qian, Cui, Wang, Wang, & Zhou, 2011). It has been suggested that AGP surface properties depend considerably on the polysaccharide component (Goodrum, et al., 2000). Anderson (1978) suggested that the superior emulsifying power of gum Arabic may be related to the significant proportions (< 10 mol%) of terminal Rhap groups, which possess hydrophobic centers (D. M. W. Anderson, 1978). In addition, the -1,3 galactan backbone should form an

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Comment citer ce document : Sanchez, C., Nigen, M., Mejia Tamayo, V., Doco, T., Williams, P., Amine, C.,   Kitazawa et al. (Kitazawa, et al., 2013), the inside of the helix comprising a hydrophobic surface with the bulk of the galactan hydroxyl groups oriented toward the outer surface of the helix (Goodrum, et al., 2000). The "hydrophilic" carbohydrate blocks attached to the polypeptide chain could therefore have a substantial impact on the adsorption of AG onto hydrophobic surfaces.
The presence of surface active components in AG implies that the nature of dispersed phase is important to determine emulsion formation and stability. Based on the varying degree of stability against washing of emulsions made with different oils, it was suggested that the efficiency with which AG is adsorbed depends at least in part on the nature and polarity of oil (Dickinson, et al., 1991b;Shotton, et al., 1960). A non polar dispersed phase will therefore display a higher interfacial tension with water and will favor adsorption of the more tensioactive components. This is the basis of the extensive use of AG to stabilize orange oil emulsions, limonene, the main orange oil component, being a non polar molecule. This also may explain why the hydrophobic GP fraction adsorbs faster at interfaces than the AGP fraction when the non polar n-hexadecane is used (Castellani, Gaillard, et al., 2010). For instance, hexadecane-in-water emulsions made with AG are more stable than decanol-inwater emulsions (Chanamai & McClements, 2002). In addition, both polarity and water solubility of the dispersed phase influence the destabilization mechanism. With a high polarity and high water-soluble oil (e.g. decanol), oil-in-water emulsions are unstable to both Ostwald ripening and coalescence when stabilized by a weakly adsorbing biopolymer such as AG (Chanamai, et al., 2002). When a low polarity and high water-soluble oil is used (e.g. decane), emulsions are stable to coalescence, but unstable to Ostwald ripening. Finally, with low polarity and low water-soluble oil (e.g. hexadecane), emulsions are stable to both Ostwald ripening and coalescence (Chanamai, et al., 2002).
Finally, it is important to mention that trace levels of lipids, probably attached to the "AGP" macromolecules, would improve the surface properties of AG (M. P. Yadav, et al., 2007;M.P. Yadav, et al., 2012). This points out the potential role of minor components present in AGs on their surface properties, e.g. free proteins, peptides, oleoresin, feruclic acids. These low M w components could explain that some gums with low AGP concentrations, for instance lower than 10%, still display excellent ability to stabilize oil-in-water emulsions or that gums

Enzymatic modifications of Acacia gum
Modifications of AGs with enzymes first occurred during its maturation process. The gum from the earliest exudation, called "green gum", is not entirely soluble yielding a glairy mucus-like fluid from which a perfect solution separates after a certain time. After storage during several months, a change is observed probably due to enzymes, so that gum becomes fluid and entirely soluble (Reinitzer, 1909). Neither the nature of these enzymes nor their origin is known. A recent Ph.D. student has studied these changes during gum maturation and it appeared that green gum displayed a peculiar rheological behaviour and was highly heterogeneous with part of kibbles containing very high M w AGP (Cozic, 2007). Upon storage, the gum recovered a classical distribution of molecular fractions.
Enzymatic modifications of AG, especially protein hydrolysis, were used primarily to demonstrate that most of proteins were associated with the higher molecular weight component of the gum and contribute to the elaboration of the wattle-blossom model (Connolly, et al., 1987(Connolly, et al., , 1988Randall, et al., 1988). It was then demonstrated that protease treatment of Acacia senegal gum mostly degraded the protein-rich high M w component of the gum, i.e. both AGP and high M w GP fractions, but did not degrade or marginally AGp and low M w GP components Connolly, et al., 1987Connolly, et al., , 1988Flindt, et al., 2005;Mahendran, et al., 2008;Osman, et al., 1993;Randall, et al., 1988Randall, et al., , 1989Renard, Lavenant-Gourgeon, et al., 2014). The lower M w obtained after hydrolysis was in the range 1.7-2x10 5 g.mol -1 , depending on the enzymes and conditions used with the lower values obtained with papain . This can be considered as the nominal building block of the gum as obtained by protein enzymatic hydrolysis. The limiting intrinsic viscosity values obtained were in the range 12-15 mL.g -1 (Chikamai, et al., 1996;Connolly, et al., 1987Connolly, et al., , 1988Renard, Lavenant-Gourgeon, et al., 2014). The decrease of gum viscosity after enzyme hydrolysis has been suggested as a way to improve their processing (Chikamai, et al., 1996).
When using specific enzymes of polysaccharide such a -galactosidase, a limited 8% decrease in galactose content was observed in parallel to a 27% increase in protein content (Chikamai, et al., 1996). Analysis by SEC showed a broadening of the AGP fraction and a properties (Heidebach, Sass, & de With, 2013). Very recently, a glucuronosyltransferase was isolated from Arabidopsis thaliana and used with AG. It was found that glucuronic acids were incorporated up to 1/3 of AG total weight. Oil-in-water emulsions made by the enzymemodified gum arabic were slightly smaller in droplets size and remarkably more stable compare to emulsions made with native AG (Dilokpimol & Geshi, 2014).

Conclusions and future prospects
Acacia gum is a plant exudate mainly produced in sub-sahalian regions of Africa. It is a natural ingredient of geo-political and economic importance. Two gums are authorized for uses, Acacia senegal gum and Acacia seyal gum. The first one was in the past the most used and studied whereas a growing demand of low cost natural gum can explain the growing part of Acacia seyal sales in recent years. Acacia gums are used by humans since prehistoric times and continue to be widely used today, the World demand having risen by 25% since the last ten years. Gums are mainly used in Food industry (confectionary, drinking industry) but also in non-food applications (pharmacy, cosmetics, materials).
Acacia senegal gums are composed of arabinogalactan-proteins (AGP) type biopolymers. It contains a continuum of hyperbranched amphiphilic charged polysaccharide-protein complexes differing by the amount of protein, type of sugars, sugar to amino-acid ratios, degree of branching, conformation and physicochemical properties. It also contains minor components such as minerals, polyphenols and traces of lipids. Arabinogalactan-proteins in Acacia senegal gums have generally anisotropic shapes and can be described as highly porous ellipsoidal objects. In fact, these macromolecules are kinds of sponges, which can explain their ability to interact with different kinds of entities (e.g. proteins, minerals, polyphenols, etc...). Besides their known biological properties, Acacia gum biopolymers display interesting functional properties such as high affinity for water, low newtonian viscosity even at quite high gum concentrations and the ability to adsorb and stabilize gazliquid, liquid-liquid and solid-liquid interfaces. Surface properties of gums are strongly related to the presence of protein-rich high molecular weight species.

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Comment citer ce document : Sanchez, C., Nigen, M., Mejia Tamayo, V., Doco, T., Williams, P., Amine, C., Renard Maturation mechanisms of Acacia gums: once exuded, gums evolve with time through sun drying, oxidation, (enzymatic) hydrolysis and interactions between AGP and between AGP and minor components upon storage and processing. The consequences are a change in biopolymer composition and distribution, structure and functional properties. Probably one of the most challenging questions is the mechanism leading to the known gum from the "green gum". Initial glairy gum is composed by very high M w AGP that imparts specific rheological behaviour. With time, even in the dry state, the M w of AGP decreases, which is may be caused by the action of some glycosidases and/or proteases. The presence of these enzymes has never been reported. Apart from hydrolysis, gum can also be oxidized which can have significant functional consequences, especially when it is rich in polyphenols.
Composition, structure and functional properties of Acacia seyal gum: for economic reasons, Acacia seyal gum represents actually 50-75% of sales. However, it has been much less studied than Acacia senegal gum. The differences between both gums in terms of sugars and amino-acid compositions are mostly known, as well as the major differences in M w distribution. However, Acacia seyal gum is richer in minerals and polyphenols, less rich in proteins, more compact, more unstable in solution, less charged, less surface active, less hydrolysable by enzymes. The structure and conformation of AGP from Acacia seyal are unknown and their functional properties badly known. We do not know the reasons for the molecule compactness and whether such a compactness can be modified changing the solvent polarity or temperature. We know that assemblies with proteins are possible but stabilization of assemblies appear difficult. We do not know the structure of oil-in-water interfaces covered by this gum. However, we know that stabilization of emulsions or suspensions can be achieved using high gum concentrations. Determine the amino-acid sequences is challenging because sugar blocks have to be degraded either by chemical or enzymatic treatments or both. Please note that the charge density of AGP should be determined as well, both to better understand the structure and the macromolecular functional properties. Nano/microparticles-based on AGP assembly with other biopolymers: formation, stabilization, industrial scaling: the formation of nano/microparticles based on electrostatic interactions between Acacia senegal gum and proteins is not a difficult task at the lab scale.
On the other hand, the stabilization of these assemblies against changes in pH or ionic strength is a bottleneck that has limited up to now their industrial applications. Since

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46 assemblies are obtained through weak interactions, cross-linking stabilization without chemical treatments imply either the use of enzymes or physical treatments. Using enzymes to stabilize Acacia gum-protein microparticles has been reported, this is a possible but expensive way. Another way is protein denaturation by heating, which has been proven to be efficient at the lab scale. An important challenge is the formation and stabilization of Acacia gum-protein nano/microparticles at the industrial scale. Issues about mixing conditions and heating in large volumes need to be considered.
Enzymatic modification of Acacia gum: the possibility to modify the structure and functional properties of Acacia gums clearly represents the best future way of innovation. Actually, the only way to propose new ingredients based on Acacia gums is i) to enrich them with AGP, ii) to purify AGP, iii) to assemble them with proteins, iv) to graft lipids by chemical reaction.
Enzymes can potentially expand the modification possibilities of gums. Two ways should be explored. The first one concerns hydrolysis of gums. This can be achieved either by the use of proteases or/and glycosidases. Proteases have demonstrated their ability to degrade Acacia gums and to significantly decrease their viscosity, which is of particular use in industry. However, protein-rich fractions are degraded, which impairs their surface properties. A major bottleneck is then to decrease the gum viscosity while maintaining high surface properties. The use of glycosidases may be a solution, however their efficiency is impaired by the hyperbranched characteristic of gums. Fortunately, a number of enzymes or enzymatic cocktails are actually available and must be screened. Enzymes with more specific activities could be found but the questions of their availability and costs should be questioned. In particular, the possibility to increase the concentration of charged carboxylate groups removing methyl groups appears interesting. The second way, probably the most promising, is to graft onto the gum specific molecules or chemical groups. Then one can imagine to graft lipids or oligopeptides to improve amphipathic properties of gums or to graft carboxylate groups to increase the gum charge density. It remains to find efficient enzymes compatible with an industrial use then to optimize grafting processes. Finally, one major bottleneck is to crosslink gum molecules by enzymes to form 100% gum nano/microparticles. This should open a lot of new applications as texturing agents and stabilized microcapsules for aroma protection.

Figure captions
Figure 1: R h conformation plots of spray-dried (black) and raw (red) Acacia senegal (A) and seyal (B) gums. 202 spray-dried and 100 raw A. senegal gums, 28 spray-dried and 6 raw A.