Rafael Apolinar Valiente

Pascale Williams
email: pascale.williams@inra.fr

Michael Nigen
email: michael.nigen@umontpellier.fr

Mejia Véronica

Thierry Tamayo

Christian Doco

Sanchez

Dr Rafael Apolinar-Valiente
email: rafael.apolinar.valiente@gmail.com

Michaël Nigen

Veronica Mejia Tamayo

Thierry Doco
email: thierry.doco@inra.fr

Christian Sanchez
email: christian.sanchez@supagro.fr

Fractionation of Acacia seyal gum by ion exchange chromatography

Keywords: Acacia seyal gum, fractionation, ion exchange chromatography, polysaccharides, proteins, SEC-MALLS

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Introduction

Acacia gum is a complex polysaccharide-based plant gum exudate, obtained from trees of selected Acacia species (i.e. Acacia senegal and Acacia seyal), whom exude this substance to protect trees against external attacks. Much work can be found in literature about several aspects of Acacia senegal gum (Asen), such as its composition [START_REF] Islam | A review of recent developments on the regulatory, structural and functional aspects of gum arabic[END_REF][START_REF] Idris | Characterisation of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF]Verbeken, Dierckx, & Dewettink, 2003), polydispersity, structure [START_REF] Fincher | Arabinogalactan-proteinsstructure, biosynthesis and function[END_REF][START_REF] Idris | Characterisation of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF][START_REF] Renard | Acacia senegal gum: continuum of molecular species differing by their protein to sugar ratio, molecular weight, and charges[END_REF][START_REF] Sanchez | The Acacia gum arabinogalactan fraction is a thin oblate ellipsoid: a new model based on small-angle neutron scattering and ab initio calculation[END_REF], physicochemical properties [START_REF] Phillips | Hydration characteristics of the gum exudate from Acacia senegal[END_REF][START_REF] Renard | Acacia senegal gum: continuum of molecular species differing by their protein to sugar ratio, molecular weight, and charges[END_REF][START_REF] Mejia Tamayo | Flexibility and Hydration of Amphiphilic Hyperbranched Arabinogalactan-Protein from Plant Exudate : A Volumetric Perspective[END_REF][START_REF] Buffo | Factors affecting the emulsifying and rheological properties of gum acacia in beverage emulsions[END_REF][START_REF] Al-Assaf | Characterization and properties of Acacia senegal (L.) willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUM (TM)): Part 1-controlled maturation of Acacia senegal var. senegal to increase viscoelasticity, produce a hydrogel form and convert a poor into a good emulsifier[END_REF][START_REF] Dickinson | Interfacial structure and stability of food emulsions as affected by protein-polysaccharide interactions[END_REF]. Asen has been much more studied over years since it largely dominated the Acacia gum commerce with a market share of about 70%. However Acacia seyal gum (Asey) has been gaining interest and importance in recent years in the Acacia gum trade [START_REF] Rahim | Competition in the gum arabic market: a game theoretic modelling approach[END_REF]. Acacia gums have been largely used for different purposes in all human history [START_REF] Sanchez | Acacia gum: History of the future[END_REF]. Currently, this natural substance is employed in a variety of applications such as food, pharmaceutical and other industrial applications [START_REF] Verbeken | Exudate gums: Occurrence, production, and applications[END_REF]. Asey is constituted by about 37-44% of galactose, 33-48% of arabinose, 2-3% of rhamnose, 7-13% of glucuronic acid, 6% of 4-O methyl glucuronic acid, 1% of protein, and 4% of minerals [START_REF] Gashua | Characterization and molecular association of Nigerian and Sudanese Acacia gum exudates. Food Hydrocolloids, 51, 405-413. and Acacia seyal[END_REF][START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]. The arabinogalactan moiety of Asey is formed by a core of β-1,3 linked galactose units with branches linked through the 6 position consisting of galactose and arabinose terminated by rhamnose and glucuronic acids (Al-Assaf, Phillips, & William, 2005;[START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF][START_REF] Street | Refinement of structures previously proposed for gum Arabic and other Acacia gum exudates[END_REF]. The minor proteinaceous material is covalently attached to this arabinogalactan moiety [START_REF] Mahendran | New insights into the structural characteristics of the arabinogalactan -Protein (AGP) fraction of gum Arabic[END_REF][START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF]. Besides, Asey macromolecules present a lower hydrodynamic volume than those from Asen, that can be interpreted as a more compact conformation [START_REF] Al-Assaf | Studies on Acacia exudate gums: Part II. Molecular weight comparison of the Vulgares and Gummiferae series of Acacia gums[END_REF][START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF][START_REF] Elmanan | Studies on Acacia exudate gums: Part VI. Interfacial rheology of Acacia senegal and Acacia seyal[END_REF][START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF].

Asen has been demonstrated as a continuum of macromolecules which shows varying protein/sugar ratios, molar masses and charge density [START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF][START_REF] Renard | Acacia senegal gum: continuum of molecular species differing by their protein to sugar ratio, molecular weight, and charges[END_REF]. Consequently, the fractionation of Asen was done by different methods such as the anion-exchange chromatography [START_REF] Osman | Characterization of gum arabic fractions obtained by anionexchange chromatography[END_REF], the size exclusion chromatography (SEC) [START_REF] Ray | Functionality of gum arabic. Fractionation, characterization and evaluation of gum fractions in citrus oil emulsions and model beverages[END_REF] or the hydrophobic interaction chromatography (HIC), which was the most used fractionation procedure [START_REF] Randall | Fractionation and characterization of gum from Acacia senegal[END_REF][START_REF] Osman | The molecular characterisation from Acacia Senegal of the polysaccharide gum[END_REF][START_REF] Ray | Functionality of gum arabic. Fractionation, characterization and evaluation of gum fractions in citrus oil emulsions and model beverages[END_REF][START_REF] Renard | Acacia senegal gum: continuum of molecular species differing by their protein to sugar ratio, molecular weight, and charges[END_REF]. Using HIC, three main AGP fractions can be obtained from Asen: (i) a dominant fraction in percentage with low protein content and low mean molar mass (HIC-F1), (ii) a fraction presenting a high molar mass and rich in protein (HIC-F2) and, finally, (iii) a fraction showing high molar mass and the highest protein content (HIC-F3) [START_REF] Renard | Acacia senegal gum: continuum of molecular species differing by their protein to sugar ratio, molecular weight, and charges[END_REF]. In a previous work, we presented the successful fractionation of Asen by Ionic Exchange Chromatography (IEC) from which two fractions were obtained: one protein-poor fraction with low molar mass and one protein-rich fraction with high-molar mass showing a great tendency to aggregate [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF]. In comparison with Asen, fractionation of Asey has been notably less reported. [START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF] undertook both GPC and HIC to compare gums from Asey and Asen varieties. Using HIC fractionation, they found for Asey practically the same class of fractions than those found for Asen. On the other hand, they found a fourth fraction with low molar mass and rich in protein when Asey was fractionated by GPC, which was not observed in Asen, concluding that separation of Asey by HIC was not complete.

Trying to develop a deeper understanding about the not greatly studied characteristics and functional properties of Asey gum, we considered that it would be useful to recuperate a fraction showing higher Mw together with higher protein content than those found in starting Asey gum. Previously, we succeeded in achieving an Asen fraction showing these two characteristics, as compared to the starting Asen gum [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF]. Thus, our main objective was to obtain a high Mw and high-protein amount fraction from Asey using IEC. This technique separates macromolecules in accordance with the proportion of anionic groups (mainly carboxylate groups from acid sugar residues) interacting with the positively charged sites on the gel. The retention could also be affected by other parameters, such as the molecular size and the structure of the macromolecule [START_REF] Medved | Ion-exclusion chromatography of organic acids: Regularities in the retention of aliphatic carboxylic acids[END_REF], as well as the type and the hydrophobicity of the gel [START_REF] Ohta | Ion-exclusion chromatography of benzenecarboxylic acids on an unmodified silica-gel column[END_REF].

Asey has been therefore fractionated by IEC through the use of DEAE Sephacel gel as stationary phase. Two main fractions were recovered, one of which was rich in protein with very high molar mass. These fractions were subsequently characterized in terms of biochemical composition (amino acid and sugar residues), structure (weight-average molar masses Mw, number-average molar mass Mn, polydispersity index Mw/Mn, intrinsic viscosity [η]) and volumetric properties (partial specific volume vs° and partial specific adiabatic compressibility βs° coefficients and hydrodynamic volume from intrinsic viscosity).

Material and Methods

Material

Spray-dried Acacia gum type from Acacia seyal trees (Lot: OF110724) was provided by ALLAND & ROBERT Company -Natural and organic gums (Port Mort, France).

Preparation of Acacia seyal gum (Asey) dispersions

Asey dispersions were prepared by weight (wt %). Known amounts of Asey powder (650 g, corresponding to 629 g in dry terms) were dispersed in water (6500 mL) and gently stirred for 24 h at room temperature (20 °C). The pH of dispersions was subsequently adjusted at 3.5 using HCl 1 N, HCl 0.1 N, or NaOH 0.5 N solutions.

Ion exchange chromatography (IEC)

IEC was performed at room temperature on DEAE Sephacel (Sigma Aldrich, St. Louis, Mo) column (54 x 20 cm), and fractions IEC-F1 and IEC-F2 were obtained following the methodology described by [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF]. The use of buffer was excluded because our work is focused not only from a laboratory point of view but also from the possibility of an industrial production perspective. The experience was conducted in triplicate, and the results were reproducible. Therefore, we considered that although the use of a buffer for controlling pH is the more scientifically rigorous choice, not using buffer eases the laboratory procedure and it opens the possibility of future industrial food applications.

Yariv detection

β-glucosyl Yariv reagent has been widely used to identify AGPs [START_REF] Osman | The molecular characterisation from Acacia Senegal of the polysaccharide gum[END_REF][START_REF] Paulsen | The Yariv reagent: Behaviour in different solvents and interaction with a gum arabic arabinogalactanprotein[END_REF]. Petri dishes containing 1% agarose gel in 10 mM Tris buffer, pH 7.3, with 0.9% NaCl and 1 mM CaCl2 were used. Consequently, β-D-Glucosyl Yariv reagent (Biosupplies, Victoria, Australia; 40 μL, 1 mg•mL -1 ) was delivered to a central well, placing fractions IEC-F1 and IEC-F2 in equidistant peripheral wells. The Petri dishes were left overnight at 25°C. We used arabinogalactan from larch wood (Sigma Aldrich, St. Louis, Mo) as negative control.

Amino acid analysis

Total amino acids were analysed with a Biochrom 30 analyser (BIOCHROM 30, Cambridge, UK). Amino acid composition of samples was determined after acid hydrolysis (6 N HCl) and heating (110°C, 24 h). Norleucine was used as internal standard.

Sugar composition as trimethylsilyl derivatives

The neutral and acidic sugar composition was determined after solvolysis with anhydrous MeOH containing 0.5 M HCl (80 ºC, 16 h), by GC of their per-O-trimethylsilylated methyl glycoside derivatives [START_REF] Doco | Determination of the neutral and acidic glycosyl residue compositions of plant cell polysaccharides by GC-EI-MS analysis of the trimethylsilyl methyl glucoside derivatives[END_REF][START_REF] Apolinar-Valiente | Effect of enzyme additions on the oligosaccharide composition of Monastrell red wines from four different wine-growing origins in Spain[END_REF].

Glycosyl-linkage compositions.

The glycosyl-linkage compositions of the partially methylated alditol acetates were determined by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) as previously described [START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF].

Molar mass distribution and intrinsic viscosity.

Fractions IEC-F1 and IEC-F2 from Asey were analysed by size exclusion chromatography (SEC) as described by [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF]. The SEC line was constituted, depending on the analysis, by an OHPAK SB-G guard pre-column followed by four columns (OHPAK SB 803,804,805 and 806 HQ,Shodex) for Asey and fraction IEC-F2 and by an OHPAK SB-G guard pre-column followed by one column (OHPAK SB 805 HQ, Shodex) for fraction IEC-F1. In the latter, only one Shodex column was used in order to decrease anomalous elution of high Mw hyperbranched macromolecules. The AGPs were eluted with filtered (0.1 μm filter, Millipore) solution (0.1 M LiNO3 + 0.02% NaN3) at a flow rate of 1 mL•min -1 and 30°C. The samples were dissolved in filtered Milli-Q water (1 mg•mL -1 ), stirred gently (24 h) and centrifuged (10 000 rpm, 10 min). Subsequently, 75 µL of IEC-F2 fraction and Asey and 10 µL IEC-F1 fraction were injected. Intrinsic viscosity was determined using an online viscometer (Viscostar II Wyatt, Santa Barbara, CA, USA) and a differential refractometer (Optilab T-Rex, Wyatt, Santa Barbara, CA, USA).. The refractive index increment values (dn/dc) were determined as 0.155, 0.158 and 0.155 mL•g -1 for Asey, IEC-F1 and IEC-F2, respectively.

Partial specific volume and partial specific adiabatic compressibility

Volumetric properties of biopolymers [partial specific volume (vs°) and partial specific adiabatic compressibility (βs°)] can be calculated from measurements of density and sound velocity [START_REF] Mejia Tamayo | Flexibility and Hydration of Amphiphilic Hyperbranched Arabinogalactan-Protein from Plant Exudate : A Volumetric Perspective[END_REF], which were simultaneously measured (25° C) using a DSA 5000M sonodensimeter (Anton Paar, France), as previously described [START_REF] Mejia Tamayo | Flexibility and Hydration of Amphiphilic Hyperbranched Arabinogalactan-Protein from Plant Exudate : A Volumetric Perspective[END_REF]. Dispersions were dialyzed overnight against sodium acetate buffer (10 mM, pH 5) to reach isopotential equilibrium, centrifuged (12 000 rpm, 30 min, 20°C) to remove insoluble materials and degassed 15 min to remove dissolved air (300 Ultrasonik bath, Ney, Yucaipa, CA, USA). Measurements were duplicated.

FTIR spectral acquisition.

Spectral acquisitions were performed on dry gum samples using a Vertex 70V Fouriertransform mid-spectrometer equipped with an ATR Diamond cell (SPECAC, Smyrna, GA, USA). Each recorded spectrum was the average of 128 repetitions from 900 to 1800 cm -1 with a 4 cm -1 spectral resolution. A micrometric screw applying constant pressure ensured good contact between the sample and the crystal. The spectra were acquired and analysed using OPUS Software version 7.5. A sample-less spectrum was recorded between two polysaccharide fractions to monitor the stability of the background. Each spectrum was subjected to linear standardisation at absorbance values from 0 to 1 for the respective wave numbers of 1800 and 1025 cm -1 for proper spectra exposure and interpretation.

Results and discussion

The aim of the present study was the fractionation of Asey using IEC in order mainly to recover one AGPs fraction (IEC-F1) with high molar mass and high protein content. It is necessary to mention that, in our view, it was also important to characterize our material of departure in order to show that it corresponded to a "classical" Asey. All the information about Asey will be discussed in the corresponding section.

Fractionation of Asey by IEC: yield of the recuperation

The IEC fractionation procedure allowed recovering two fractions, IEC-F1 and IEC-F2, which correspond respectively to 1.4% and 82.6% of the initial gum. The losses of material can occur during the fractionation, concentration, diafiltration and spray-drying steps; nevertheless, we consider that the total yield (84.0% of initial gum) was satisfactory. Supplementary data (Fig. 1) shows the precipitation of both fractions IEC-F1 and IEC-F2 by Yariv's reagent, corroborating that they belong to AGP family. Fractions from Acacia senegal gum obtained by different fractionation techniques, such as preparative GPC [START_REF] Qi | Gum Arabic glycoprotein in a twisted hairy rope[END_REF], HIC [START_REF] Osman | The molecular characterisation from Acacia Senegal of the polysaccharide gum[END_REF] or IEC [START_REF] Mejia Tamayo | Flexibility and Hydration of Amphiphilic Hyperbranched Arabinogalactan-Protein from Plant Exudate : A Volumetric Perspective[END_REF], were also found to interact with Yariv reagent.

Fractions IEC-F1 and IEC-F2: composition and structure

Knowing that our principal aim is to isolate an AGPs fraction presenting high molar mass and high content of protein, we will look more closely at the results of the fraction IEC-F1.

Regarding Asey and IEC-F2, their data are also exhibited in the different Tables andFigures, being similar both of them.

Sugar composition

Table 1A presents the neutral sugar and uronic acid composition of Asey. We found high content of arabinose (48.5%) and galactose (34.2%), whereas rhamnose only represents 3.2%. Concerning the uronic acids, glucuronic acid and 4-O-methyl glucuronic acid show values of 7.7% and 6.4%, respectively. This sugar composition corresponds with data largely reported in literature [START_REF] Biswas | Evaluation of methods for measuring amino acid hydrophobicities and interactions[END_REF][START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF][START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF][START_REF] Andres-Brull | Studies on acacia gums: Part VII effect of exudates form and tree age on the characteristics of Acacia seyal var. seyal and Acacia seyal var. fistula[END_REF][START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]Gashua, Williams, & Baldwin, 2016). When characteristic ratio Ara/Gal of Asey was calculated, it presents a value of 1.4, which is in general also coherent with values found in literature [START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF][START_REF] Andres-Brull | Studies on acacia gums: Part VII effect of exudates form and tree age on the characteristics of Acacia seyal var. seyal and Acacia seyal var. fistula[END_REF][START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]. It can be noted that this ratio could notably vary depending on the origin of the Asey gum [START_REF] Biswas | Evaluation of methods for measuring amino acid hydrophobicities and interactions[END_REF][START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF].

Regarding the neutral sugar and uronic acid composition of the IEC-F1 fraction (Table 1A), arabinose appears as its major component (54%), followed by galactose (29%). This corresponds to data previously reported for fractions from Asey after separation by different separation methods [START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF][START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF]. Fraction IEC-F1 shows the greatest value (1.8) for the calculated Ara/Gal ratio. This behaviour could indicate longer side branches of arabinose than the other two studied samples. [START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF] suggested that a higher content of long arabinose side chains that may self-organize and interact between them (e.g. hydrogen bonding, steric effect, etc.) could imply a more compact structure. The calculated characteristic ratios are quite similar to those found in Asey fractions by GPC separation (1.1-2.8) [START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF] and by HIC (around 1.2) [START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF]. In both cases, the different separation technique together with the above mentioned origin influence could explain the variations observed. Regarding glucuronic acid, the highest percentage appears in fraction IEC-F1 (10%). Using IEC separation, the most negative charged macromolecules should be more retained by the positively charged column.

It would be logical to think that the first eluted fraction would present less compound showing negative charges, such as glucuronic acid. The opposite is, however, found. Our observation concerning glucuronic acid may be caused by steric hindrance of some groups to interact with the DEAE gel. In the same line, [START_REF] Osman | Characterization of gum arabic fractions obtained by anionexchange chromatography[END_REF] reported that the order of elution of the fractions separated by IEC does not follow their glucuronic acid amount, suggesting the possibility of a steric disability. Previously, we also proposed this behavior during fractionation of Asen by IEC [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF].

From the glycosidic linkages reported in Table 1B, the ratio of terminal to branched residues can be calculated. For this purpose, we have calculated the terminal units (TU) as follows: The coefficients used in these two formulas were reported by [START_REF] Sweet | Quantitative-analysis by various glc response-factor theories for partially methylated and partially ethylated alditol acetates[END_REF]. The TU/BU ratio is higher in fraction IEC-F1 (0.57) compared fraction IEC-F2 (0.47). The highest TU/BU ratio of fraction IEC-F1 could suggest a less branched structure of the carbohydrate moiety. The data given in Table 1B enables us to calculate the content of free OH in the polysaccharide portion, as shown below in section 3.2.6. 13

Amino acid composition

Table 2A shows the total amino acid content and amino acid composition of Asey and the two IEC fractions. The total content of amino acids for Asey is 7 mg•g -1 of sample, which is in agreement with those obtained by other authors [START_REF] Elmanan | Studies on Acacia exudate gums: Part VI. Interfacial rheology of Acacia senegal and Acacia seyal[END_REF][START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]Gashua et al., 2016). Regarding the amino acid composition of Asey, hydroxyproline is by far the major amino acid, followed by serine, and in less amount leucine, aspartic acid, proline and threonine, as previously presented in literature [START_REF] Osman | The molecular characterisation from Acacia Senegal of the polysaccharide gum[END_REF][START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF][START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF][START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]. Minor amino acids were isoleucine, lysine, arginine and tyrosine (Table 2A).

Fraction IEC-F1 presents much lower content of hydroxyproline (6%) and serine (8%) amino acids compared to fraction IEC-F2 (34% and 16%, respectively) and Asey (32% and 13%, respectively). [START_REF] Mahendran | New insights into the structural characteristics of the arabinogalactan -Protein (AGP) fraction of gum Arabic[END_REF] reported that the link between the protein fraction and the polysaccharide fraction are both O-serine and O-hydroxyproline residues. Therefore, these results could indicate a lower number of polysaccharide chains linked to proteins in fraction IEC-F1. On the other hand, aspartic acid appears as the most abundant amino acid of fraction IEC-F1 (15%), showing also high amounts of leucine (10%) and valine (9%). When [START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF] separated the Asey gum by GPC, they also detected that aspartic acid was the major amino acid in several fractions, whereas hydroxyproline presented low values in some of these or other fractions.

We have calculated the percentages of several amino acid families trying to observe a possible influence of the charges during the separation of fractions by IEC. In particular, we determined the percentages of negatively charged, positively charged, polar (hydrophilic) and non-polar (hydrophobic) amino acid residues (Table 2B). Negatively charged amino acids are estimated as the sum of aspartic acid and glutamic acid. The sum of histidine, lysine and arginine is considered as positively charged amino acids. Hydrophilic or polar amino acids are calculated as the sum of serine, threonine and tyrosine. Finally, hydrophobic or non-polar amino acids are calculated as the sum of alanine, glycine, isoleucine, leucine, phenylalanine, proline and valine.

The first eluted fraction (IEC-F1) should present a lower content of negative charged macromolecules, which would interact with the positively charged gel. Instead, the IEC-F1 fraction shows the highest percentage of negatively charged amino acids (23%) compared to fraction IEC-F2 (8%) and Asey (12%). That could be explained, like previously suggested for the sugars, by the fact that some negatively charged amino acids may be sterically hindered to interact with gel.

Concerning the polarity/hydrophobicity, we have found that fraction IEC-F1 shows the highest percentages for the hydrophobic amino acids (47%) and the lowest value for the hydrophilic amino acids (14%). On the other hand, fraction IEC-F2 presents the opposite trend: the lowest percentage of hydrophobic amino acids (26%) and the highest percentage of hydrophilic amino acids (23%). Therefore, a different hydrophobicity between amino acids from the two obtained fractions separated by IEC has been found.

Regarding the total content of amino acids, fraction IEC-F1 shows clearly the highest value (74 mg amino acid•g -1 of sample), matching with our previous results about IECfractionation of Asen [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF]. Literature shows varying protein contents between fractions when Asey gum was fractionated by HIC [START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF] or by GPC [START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF]. Our results are corroborated by the infrared spectra of fractions IEC-F1 and IEC-F2 (Supplementary Figure 2). The absorbance is clearly higher at 1650 and at 1545 cm -1 for fraction IEC-F1, indicating the emergence of Amide I and II vibration bands of proteins, respectively [START_REF] Bertrand | La spectroscopie infra-rouge et ses applications analytiques[END_REF][START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]. In accord with [START_REF] Barth | Infrared spectroscopy of proteins[END_REF], they mainly depend on the secondary structure of the polypeptide backbone, being influenced by the nature of the side chains. The whole infrared spectra of our AGP samples (Supplementary Figure 2) resemble, as expected, to the Asey spectrum found by [START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF] and also to those of AGPs from wine reported by [START_REF] Boulet | Fourier transform infrared spectroscopy study of wine polysaccharides[END_REF] in the range between 1800 and 800 cm -1 .

Size exclusion chromatograms and Mw distribution

Asey chromatogram (Figure 1A) shows one main peak between 26 and 33 min elution time (RI signal). This profile, together with the obtained Mw distribution as a function of the elution time (Figure 1A), is in coherence with those previously reported by several authors [START_REF] Gashua | Characterization and molecular association of Nigerian and Sudanese Acacia gum exudates. Food Hydrocolloids, 51, 405-413. and Acacia seyal[END_REF][START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]. Figure 1B exhibits the molar mass distribution analysis of Asey. It was divided into three selected ranges in order to estimate the relative percentage in small, medium and large molar masses: range 1 (below 5 x 10 5 g•mol - 1 ), range 2 (between 5 x 10 5 and 1 x 10 6 g•mol -1 ) and range 3 (above 1 x 10 6 g•mol -1 ). Thus, we have obtained higher values for range 1 (42%) and 2 (34%) in comparison with the range 3 (24%).

The chromatogram of fraction IEC-F1 presents one major peak with elution time values (RI signal) ranging from 6.4 to 8.9 min (Figure 1A). This population presents only high Mw AGPs (upper than 10 6 g•mol -1 ). A second lower but well-defined peak can be deduced between 8.9 and 11 min, showing AGPs with low Mw (below than 10 6 g•mol -1 ).

Figure 1B shows that fraction IEC-F1 has much higher percentage of cumulative molar mass in range 3 (82%) compared to ranges 1 (12%) and 2 (6%). As expected, our procedure enables to obtain high Mw AGP from Asey, corresponding to the fraction IEC-F1. However, IEC separation seems not as good for Asey as we previously found for Asen [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF], where we reported 96% of fraction IEC-F1 in the range 3. One likely explanation could be the higher starting percentage of macromolecules from range 3 (>10 6 g•mol -1 ) in the case of Asey (24%) in comparison to Asen (15%, from [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF]. This high number of macromolecules would result in a steric hindrance which should hinder that macromolecules from ranges 1 and 2 should be fixed to the gel [START_REF] Osman | Characterization of gum arabic fractions obtained by anionexchange chromatography[END_REF]. In such a way, these macromolecules from ranges 1 and 2 would pass easily through the column and would be eluted as an integral part of fraction IEC-F1. Therefore, the hydrodynamic volume of the macromolecules contributes in the efficiency of IEC separation method. Another explanation, not in conflict with the previous account, could be the higher negative attractive potential interaction in Asen compared to Asey due to the charged residues content [START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]. This behaviour would explain the lower separation for Asey compared to our previous work [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF].

Static and dynamic molecular parameters

The static and dynamic parameters of Asey and fractions IEC-F1 and IEC-F2 are given in Table 3A. Asey shows values of 7.8 x 10 5 g•mol -1 for Mw and 4.4 x 10 5 g•mol -1 for Mn. These values are close to those found in literature [START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF][START_REF] Elmanan | Studies on Acacia exudate gums: Part VI. Interfacial rheology of Acacia senegal and Acacia seyal[END_REF][START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]Gashua et al., 2016).

Fraction IEC-F2 displays weight-average molar mass (Mw) and number-average molar mass (Mn) of 8.1 x 10 5 g•mol -1 and 4.7 x 10 5 g•mol -1 , respectively. In contrast, fraction IEC-F1 presents much higher values for Mw (3.1 x 10 6 g•mol -1 ) and Mn (1.3 x 10 6 g•mol -1 ). [START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF] and [START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF] also detected differences in Mn and Mw between fractions when Asey was separated, respectively, by GPC and by HIC. However, IEC technique allows to obtain a fraction whose Mw is largely greater than the starting Asey gum. As mentioned earlier, [START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in relation to Acacia senegal[END_REF] obtained two high protein-content fractions by HIC: the classical fractions HIC-F2 and HIC-F3. These fractions display, respectively, Mw values smaller (-3.8%) and larger (+17%) than the Mw value of starting Asey gum. On the other hand, we obtain by IEC a fraction (IEC-F1) whose Mw represents an increase of 283% with regard to the starting Asey gum Mw. Concerning the polydispersity data (Mw/Mn), Asey presents a value of 1.8, which is also close to values reported by other authors [START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]Gashua et al., 2016). Fraction IEC-F2 presents similar polydispersity (1.7) than Asey (1.8), whereas fraction IEC-F1 shows the highest value for this parameter (2.6).

Asey gum presents a larger intrinsic viscosity ([η]

) value (23 mL•g -1 ) than those reported previously (12-18 mL•g -1 ) [START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF][START_REF] Elmanan | Studies on Acacia exudate gums: Part VI. Interfacial rheology of Acacia senegal and Acacia seyal[END_REF][START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]. These variations may be due to differences in the age of trees [START_REF] Idris | Characterisation of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF] and/or the origin of the gum [START_REF] Gashua | Characterization and molecular association of Nigerian and Sudanese Acacia gum exudates. Food Hydrocolloids, 51, 405-413. and Acacia seyal[END_REF]. Fraction IEC-F2 shows similar values (22 mL•g -1 ) to Asey gum, whereas, as expected, the highest intrinsic viscosity ([η]) is found for fraction IEC-F1 (36 mL•g -1 ). This parameter in Acacia gum has been suggested as strongly linked to the molar mass as well as to the protein content (Chikamai, Banks, Anderson, & Weiping, 1996). In our case, the [η] value (Table 3A) and the amino acid total content (Table 2A) of fraction IEC-F1 are in perfect coherence with these two remarks, which can be explained by the fact that protein-rich macromolecules in Acacia gum display generally high Mw.

The average sphere-equivalent hydrodynamic radius (RH) of Asey gum (13.6 nm, Table 3A) was calculated from the intrinsic viscosity following RH = (([η]•Mv•3)/(10•π•NA)) 1/3 . In this formula, [η] is the intrinsic viscosity and Mv the viscosity-weighted molar mass. This RH value was close to the 9.3 nm value previously determined on another Asey batch [START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]. Values in the range 8.4-9.0 nm were also found by [START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF]. These values are logically close to the hydrodynamic radius determined on IEC-F2 (≅ 13.4 nm) and smaller than the RH of . Regarding the radius of gyration (Rg), values of about 32 nm were found for IEC-F1, which according to our experience is quite common with high Mw AGP. Only Rg values above 10 nm were considered to calculate Rg, corresponding to about 53% of macromolecules from Asey, 61% of macromolecules from IEC-F1 and 53% of macromolecules from IEC-F2.

Figure 2A shows the results of Mark-Houwink-Sakurada plot for IEC fractions, which relates the intrinsic viscosity [η] as a function of Mw. These two parameters are linked by the equation [η] = KαMw α , where Kα is a constant and α an exponent value called hydrodynamic coefficient [START_REF] Burchard | Solution Properties of Branched Macromolecules[END_REF]. The theoretical values for α vary from 0 (sphere) to 1.8 (rod) with 0.5-0.8 intermediate values for flexible polymers depending on the solvent quality [START_REF] Ross-Murphy | Physical Techniques for the Study of Food Biopolymers[END_REF]. Mark-Houwink-Sakurada log-log plots from IEC-F1 and IEC-F2 can be described by one single slope (exponent of the power law), indicating one single conformation for these AGP in solution. Slope values were 0.31 for IEC-F2 and 0.39 for IEC-F1. Generally, slope values within 0.3-0.5 are classically found for hyperbranched structures [START_REF] Callaghan | The size and shape of amylopectin: a study using pulsed field gradient nuclear magnetic resonance[END_REF][START_REF] Lelievre | The size and shape of amylopectin: a study using analytical ultracentrifugation[END_REF][START_REF] Millard | Light-scattering molecular weights and intrinsic viscosities of processed waxy maize starches in 90% dimethyl sulfoxide and H2O[END_REF][START_REF] Rolland-Sabaté | Online determination of structural properties and observation of deviations from power law behavior[END_REF][START_REF] Li | Experimental and theoretical studies of scaling of sizes and intrinsic viscosity of hyperbranched chains in good solvents[END_REF]. The higher slope value for fraction IEC-F1 (0.39) would indicate a more anisotropic shape, a different density or a different affinity for solvent.

The ratio of geometric to hydrodynamic radius (ρ = Rg•RH -1 ), also called asymmetry [START_REF] Adolphi | Coil dimensions and conformation of macromolecules in qaueous media from flow field-flow fractionation/multi-angle laser light scattering illustrated by studies on pullulan[END_REF] or anisotropy parameter [START_REF] Mansfield | Shape characteristics of equilibrium and nonequilibrium fractal clusters[END_REF], is a structural parameter. It can be affected by macromolecular flexibility and polydispersity [START_REF] Adolphi | Coil dimensions and conformation of macromolecules in qaueous media from flow field-flow fractionation/multi-angle laser light scattering illustrated by studies on pullulan[END_REF] but also by particle homogeneity and density. Only Rg values above 10 nm were considered to calculate ρ ratio, corresponding to about 53% of macromolecules from Asey, 61% of macromolecules from IEC-F1 and 53% of macromolecules from IEC-F2. In the case of fraction IEC-F1, this ratio remains constant around 0.9-1.1 (Figure 2B), which is close to the values found with dendrimers or hyperbranched polymers [START_REF] Lopez-Torrez | Food Hydrocolloids Acacia senegal vs . Acacia seyal gums -Part 1 : Composition and structure of hyperbranched plant exudates[END_REF]. On the other hand, the ρ ratio for IEC-F2 increases with Mw from 0.65 to 0.90, which would indicate that AGP with high Mw are more anisotropic than low Mw AGP since the theoretical ratio for hard homogeneous sphere is around 0.8.

Thermodynamic volumetric parameters

Partial specific volume and partial specific adiabatic compressibility coefficient are thermodynamic parameters which can be related to solvent-solute and solute-solute interactions. These two volumetric properties have been used to describe the structure and flexibility changes of macromolecules when submitted to changes in their environment [START_REF] Gekko | Compressibility-structure relationship of globular proteins[END_REF][START_REF] Hoiland | Partial Molar Volumes of Biochemical Model Compounds in Aqueous Solution[END_REF][START_REF] Gekko | Flexibility of food proteins as revealed by compressibility[END_REF]. The partial specific volume (vs°) can be defined as the change of the system volume due to the addition of an infinitesimal amount of the solute, whereas the partial adiabatic compressibility coefficient (βs°) is defined as the change of the system pressure caused by the addition of an infinitesimal amount of the solute [START_REF] Gekko | Compressibility-structure relationship of globular proteins[END_REF][START_REF] Hoiland | Partial Molar Volumes of Biochemical Model Compounds in Aqueous Solution[END_REF][START_REF] Gekko | Flexibility of food proteins as revealed by compressibility[END_REF][START_REF] Mejia Tamayo | Flexibility and Hydration of Amphiphilic Hyperbranched Arabinogalactan-Protein from Plant Exudate : A Volumetric Perspective[END_REF]. Both of them depend mainly on the intrinsic contribution of the solute and its hydration. The first one concerns the constitutive volume of the molecule itself, that is to say, the atoms forming the molecule (van der Waals volume) and the cavities (voids) within the molecule. On the other hand, the hydration contribution concerns the solute-solvent interactions, and specially the changes in physical properties of water upon interaction with AGP surface residues. Changes in vs° and βs° are mainly attributed to changes in the cavities or hydration. The first one produces a positive effect on vs°. On the other hand, hydration causes a negative effect on vs°, which indicates the occurrence of a more compressible (more flexible) structure [START_REF] Gekko | Compressibility of globular proteins in water at 25° C[END_REF]Chalikian & Breslauer, 1996). These two properties can be determined using ultrasound measurements as previously described by Mejia Tamayo et al. (2018).

Table 3B gives vs° and βs° of the fractions IEC-F1 and IEC-F2. Greater values of vs° and βs° are observed in IEC-F1 (0.607 cm 3 •g -1 and -7.4 x 10 -11 Pa -1 , respectively) compared to those of fraction IEC-F2 (0.582 cm 3 •g -1 and -12.4 x 10 -11 Pa -1 , respectively) and those of Asey (0.577 cm 3 •g -1 and -13.2 x 10 -11 cm 3 •g -1 , respectively). Therefore, it can be concluded that the fraction IEC-F1 displays a more compressible and less hydrated structure than the IEC-F2 fraction. Moreover, fraction IEC-F1 from Asey presents less negative βs° value than the similar fraction obtained from Asen (-7.4 x 10 -11 vs -9.4 x 10 -11 Pa -1 ) [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF], indicating less hydration of the former. On the other hand, the same compressibility values were found for IEC-F2 fraction coming from the two gums. It can be noted that a link between protein interfacial properties and their molecular compressibility was clearly demonstrated with globular proteins [START_REF] Gekko | Flexibility of food proteins as revealed by compressibility[END_REF][START_REF] Damodaran | Amino acids, peptides and proteins[END_REF]. It can then be assumed that fraction IEC-F1 from Asey would present better interfacial properties as compared to unfractionated Asey. All these results are in agreement with the data of IEC fractions from Asen previously reported by [START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF].

well as the greatly highest value of total amino acid content. The molar mass distribution of fraction IEC-F1 shows difference compared to fraction IEC-F2 and also to Asey gum, giving great percentages (82%) in the range 3 (molar masses above 1 x 10 6 g•mol -1 ). Fraction IEC-F1 also shows the largely highest values of Mw, Mn and intrinsic viscosity. The higher hydrodynamic coefficient from the Mark-Houwink-Sakurada analysis corresponds to fraction IEC-F1, which would implicate more anisotropic conformation compared to fraction IEC-F2.

The anisotropy parameter (ρ) remains constant around 0.9-1.1 in fraction IEC-F1, whereas it enhances from 0.65 to 0.90 in fraction IEC-F2. Besides, from the partial specific volume (vs°) and the partial specific adiabatic compressibility (βs°) coefficients, a more flexible and less hydrated structure in the fraction IEC-F1 compared to Asey gum can be concluded.

Finally, from a large part of the obtained data, we could conclude a steric hindrance of some groups to interact with the DEAE gel during IEC fractionation. Based on the results shown in this work, further studies should be carried out in order to improve the knowledge about the potential physico-chemical characteristics of obtained Asey fractions and, hence, to raise their potential applications. a The acid hydrolysis used in amino acid composition analysis converts asparagine to aspartic acid and glutamine to glutamic acid, or destroys some amino acids such as tryptophan, so these residues are not measured and not reported in the table.

b AAs: amino acids; asp: aspartic acid; glu: glutamic acid; arg: arginine; his: histidine; lys: lysine; ser: serine; thr: threonine; tyr: tyrosine; ala: alanine; gly: glycine; ile: isoleucine; leu: leucine; phe: phenylalanine; pro: proline; val: valine. Cumulative percentage ranges (%) for molar masses of Acacia seyal gum (Asey) and fractions IEC-F1 and IEC-F2; range 1 (■): molar mass below 5 x 10 5 g•mol -1 , range 2 (■): molar mass between 5 x 10 5 and 1 x 10 6 g•mol -1 and range 3 (■): molar mass above 1 x 10 6 g•mol -1 (Fig. B).

Fig. 1 .

 1 Fig. 1. Size exclusion chromatograms showing the elution profiles monitored by refractometer: relative refractive index (thick line) and molar mass (Mw: g•mol -1 ; thin line) of

Fig. 2 .

 2 Fig. 2. A: Mark-Houwink-Sakurada plot showing the intrinsic viscosity ([η]; mL•g -1 ) as a function of molar mass (Mw; g•mol -1 ) for Acacia seyal gum (Asey, ▬) as well as of fractions

Table 1 .

 1 Neutral sugars and uronic acids composition (% molar) (1A) and glycosidiclinkages composition (% molar) (1B) of Acacia seyal gum (Asey) and fractions IEC-F1 and IEC-F2 obtained using Ion Exchange Chromatography (IEC).

	Asey	Fraction IEC-F1	Fraction IEC-F2

Table 2 .

 2 Amino acid composition (% by mass of each amino acid relative to total mass of all amino acids), total amino acid content (mg amino acid•g -1 of sample) (2A) and amino acid

	families (%) (2B) of Acacia seyal gum (Asey) and fractions IEC-F1 and IEC-F2 obtained
	using Ion Exchange Chromatography (IEC).
	2A Amino Acid (%)	Asey	Fraction IEC-F1	Fraction IEC-F2
	Alanine	3.0	4.0	1.9
	Arginine	1.8	2.8	1.4
	Aspartic acid a	6.7	15.3	4.8
	Glutamic Acid a	5.2	7.5	3.4
	Glycine	3.3	5.3	2.6
	Histidine	3.6	2.5	5.2
	Hydroxyproline	31.6	5.6	34.3
	Isoleucine	1.6	4.2	0.5
	Leucine	7.8	9.8	7.7
	Lysine	2.0	5.0	1.7
	Phenylalanine	2.5	8.5	2.4
	Proline	6.8	6.6	7.4
	Serine	13.2	7.6	16.3
	Threonine	5.0	4.7	5.3
	Tyrosine	1.8	1.4	1.2
	Valine	4.6	9.0	3.9
	Total amino acid content (mg amino acid•g -1 of sample)	7.3	74.5	6.2
	2B Amino Acid Families (%)
	Negatively charged AAs (asp+glu) b	11.8	22.8	8.2
	Positively charged AAs (arg+his+lys) b	7.3	10.3	8.3
	Hydrophilic or polar AAs (ser+thr+tyr) b	20.0	13.7	22.8
	Hydrophobic or non-polar AAs (ala+gly+ile+leu+phe+pro+val) b	29.6	47.4	26.4

Table 4 .

 4 Basic molecular characteristics of Acacia seyal gum (Asey), as well as fractions IEC-F1 and IEC-F2 obtained using Ion Exchange Chromatography (IEC).

	Asey	Fraction IEC-F1	Fraction IEC-F2

a Sugars percentage was determined by the difference of proteins and minerals from 100% of sample.

Acknowledgements

Author R. Apolinar-Valiente is the holder of a postdoctoral fellowship from ALLAND & ROBERT Company -Natural and organic gums (Port Mort, France). This study was made possible thanks to its financial assistance, being included within the DIVA research programme.

Compliance with ethical standards

Basic molecular characteristics

Using several biochemical and structural properties, we have estimated the contribution of the polysaccharide and protein moiety of IEC fractions to basic molecular properties (Table 4). By means of the sugar (Table 1) and amino acid (Table 2) composition, we have calculated the average Mw of the polysaccharide and protein part. The content of free OH in the polysaccharide portion was estimated by taking into account the linkage of the neutral sugars (Table 1B) and the number of possible interacting sites of the protein portion Observing the protein moiety, the fraction IEC-F1 shows a much greater number of potential charged and polar interacting sites (~1 200) as compared to fraction IEC-F2 (35). Their corresponding content of amino acid residues (~1 800 and 40, respectively) explains this behavior. Concerning the polysaccharide part, the number of charges contributed by fraction IEC-F1 also presents much higher values (~47 000) than those from fraction IEC-F2 (~12 000), which is explained by their corresponding content of sugar residues (~17 000 and ~5 000, respectively).

Conclusions

We have succeeded to recover a fraction rich in protein and with high molar mass from Asey gum by ion exchange chromatography. This fraction, called IEC-F1, has been deeply characterized, together with the other obtained fraction, named IEC-F2. Significant differences between the characteristic ratio arabinose/galactose, the glucuronic acid content and the ratio of terminal to branched glycosidic residues of IEC-F1 and IEC-F2 fractions were observed. Fraction IEC-F1 shows the lowest content of hydroxyproline and serine, as

Conflict of interest

The authors declare that they have no conflict of interest.

Compliance with ethics requirements

This article does not contain any studies with human or animal subjects.

Table 3. Molecular parameters determined by on line SEC-MALLS, differential refractometer and viscometer (3A) and thermodynamic parameters (partial specific volume (vs°) and partial specific adiabatic compressibility (βs°)) (3B) of Acacia seyal gum (Asey) and fractions IEC-F1 and IEC-F2 obtained using Ion Exchange Chromatography (IEC).

3A Molecular parameters

Asey

Fraction IEC-F1

Fraction IEC-F2

Mw (g•mol -1 ) 7.8 x 10 5 3.1 x 10 6 8.1 x 10 5 Mn (g•mol -1 ) 4.4 x 10 5 1.