# The colloidal stabilization of young red wine by Acacia senegal gum: The involvement of the protein backbone from the protein-rich arabinogalactan-proteins

Michael Nigen, Rafael Apolinar Valiente, Nerea Iturmendi, Pascale Williams, Thierry Doco, Virginie Moine, Arnaud Massot, Isabelle Jaouen, Christian Sanchez

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Michael Nigen, Rafael Apolinar Valiente, Nerea Iturmendi, Pascale Williams, Thierry Doco, et al.. The colloidal stabilization of young red wine by Acacia senegal gum: The involvement of the protein backbone from the protein-rich arabinogalactan-proteins. Food Hydrocolloids, 2019, 97, 10.1016/j.foodhyd.2019.105176. hal-02164154

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The colloidal stabilization of young red wine by *Acacia senegal* gum: The involvement of the protein backbone from the protein-rich arabinogalactan-proteins

Michaël Nigen, Rafael Apolinar Valiente, Nerea Iturmendi, Pascale Williams, Thierry Doco, Virginie Moine, Arnaud Massot, Isabelle Jaouen, Christian Sanchez

PII: S0268-005X(19)30393-5

DOI: https://doi.org/10.1016/j.foodhyd.2019.105176

Article Number: 105176

Reference: FOOHYD 105176

To appear in: *Food Hydrocolloids*

Received Date: 18 February 2019

Revised Date: 21 May 2019

Accepted Date: 18 June 2019

Please cite this article as: Nigen, Michaë., Valiente, R.A., Iturmendi, N., Williams, P., Doco, T., Moine, V., Massot, A., Jaouen, I., Sanchez, C., The colloidal stabilization of young red wine by *Acacia senegal* gum: The involvement of the protein backbone from the protein-rich arabinogalactan-proteins, *Food Hydrocolloids* (2019), doi: https://doi.org/10.1016/j.foodhyd.2019.105176.

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Graphical Abstract.
1 The colloidal stabilization of young red wine by Acacia

2 *senegal* gum: The involvement of the protein backbone from

3 the protein-rich arabinogalactan-proteins

4

5

6 Michaël Nigen<sup>a*</sup>, Rafael Apolinar Valiente<sup>a</sup>, Nerea Iturmendi<sup>c</sup>, Pascale Williams<sup>b</sup>, Thierry

7 Doco<sup>b</sup>, Virginie Moine<sup>c</sup>, Arnaud Massot<sup>c</sup>, Isabelle Jaouen<sup>d</sup> and Christian Sanchez<sup>a</sup>

8

9 <sup>a</sup> UMR1208 Ingénierie des Agropolymères et Technologies Emergentes, INRA-Montpellier

10 SupAgro-CIRAD-Université Montpellier, 2 Place Pierre Viala F-34060 Montpellier, France

11

12 <sup>b</sup> UMR 1083 Science Pour l’Œnologie, INRA-Montpellier SupAgro-Université Montpellier, 2

13 Place Pierre Viala F-34060 Montpellier, France

14

15 <sup>c</sup> BIOLAFFORT, 126 quai de la Souys, 33100 Bordeaux, France

16

17 <sup>d</sup> ALLAND & ROBERT, 9 rue Saintonge, 75003 Paris, France

18

19

20

21

22 * corresponding author. Tel.: 33 4 99 61 26 15. Fax: 33 4 99 61 30 76. E-mail:

23 michael.nigen@umontpellier.fr

24
25 **Abstract**

The arabinogalactan-proteins (AGPs) *type-biopolymers* from *Acacia senegal* gum are used as protective colloid in young red wines to prevent the precipitation of the coloring matter.

Usually, *Acacia senegal* gum is chosen based on its efficiency to stabilize iron hexacyanoferrate salts in hydro-alcoholic solution. In this study, the protective activity of *Acacia senegal* gum and its three macromolecular fractions (HIC-F1, HIC-F2 and HIC-F3), separated according to their hydrophobicity, towards the precipitation of iron hexacyanoferrate salts and polyphenols in hydro-alcoholic solutions are reported. The AGPs prevent the colloidal instability of both iron hexacyanoferrate salts and polyphenols in “model” hydro-alcoholic solutions and young red wine with a good correlation between results obtained on both systems. This result then strengthens the use of iron hexacyanoferrate salts (hydro-alcoholic – mineral test) for the evaluation of the protective activity of *A. senegal* gum in young red wine. The precipitation of iron hexacyanoferrate salts is avoided by the electrostatic binding of Ca²⁺, the driver of the instability, with the negatively charged carried by amino-acids from the protein backbone or both by amino acids and uronic acid monosaccharides localized close to the protein backbone of AGPs. The protective activity closely depends on the protein content of AGPs in both iron hexacyanoferrate salts and polyphenols hydro-alcoholic solutions: the more they are rich in proteins, the more their colloidal stabilizing efficiency is (HIC-F3>HIC-F2>HIC-F1). The differences observed in the protective activity between AGPs from the three HIC fractions are relied not only to their protein content but also to their related rate of glycosylation that modulates the protein accessibility to its environment, then their physicochemical properties as their hydrophobic behavior.

48
Keywords: Acacia gum, Arabinogalactan proteins, colloidal stability, young red wine, coloring matter

1. Introduction

Red wine is a mixture of water, alcohols, organic acid and of complex molecules at the origin of its organoleptic properties and in constant evolution during ageing. The color as well as the clarity of red wines are ones of the qualities required by the consumers. Red wines must present a colloidal stability not only at the time of bottling but also during ageing and storage until its consumption (Ribéreau-Gayon, Glories, Maujean & Dubourbieu, 2006). The phenolic compounds, responsible of the organoleptic perceptions, are also unstable molecules able to aggregate and precipitate both in young and aged red wines (Ribéreau-Gayon et al., 2006; Alcade-Eon, Garcia-Estévez, Puente, Rivas-Gonzalo & Escribano-Bailon, 2014). Among others, the phenolic compounds can react with ferric iron to form soluble complexes that may then flocculate and precipitate during ageing (Ribéreau-Gayon et al., 2006). The colloidal stability in young red wines may be ensured by several techniques as a cold treatment, fining or addition of protective colloids such as carboxymethyl cellulose, metatartaric acid, mannoproteins and Acacia gum (Waters, Pellerin & Brillouet, 1994; Waters, Pellerin & Brillouet, 1994a; Dupin, Stockdale, Williams, Jones, Markides & Waters, 2000; Escot, Feuillat, Dulau & Charpentier, 2001; Riou, Vernhet, Doco & Moutounet, 2002; Riberau-Gayon et al., 2006; Poncet-Legrand, Doco, Williams & Vernhet, 2007; Pérez□ Lamela, García□ Falcón, Simal□ Gándara & Orriols□ Fernández, 2007; Gerbaud, Gabas, Blouin & Crachereau, 2010; Teissedre, 2012; Gordillo, Cejudo-Bastante, Rodríguez-Pulido, Jara-Palacios, Ramírez-Pérez, González-Miret & Heredia, 2014). The cold and fining treatments cause the precipitation of existing particles responsible of the turbidity as well as those potentially formed during ageing. Therefore, both treatments have a clarifying and a stabilizing action (Riberau-Gayon et al., 2006). In young red wine, Acacia gum is used as a
protective colloid in order to prevent or limit the aggregation and precipitation of unstable colloids as the coloring matter (Ribereau-Gayon et al., 2006; Teissedre, 2012). In particular, Acacia gum macromolecules bind to polyphenols by the involvement of both hydrogen bonding and hydrophobic interactions that prevents their interaction and aggregation with proteins (de Freitas, Carvalho & Mateus, 2003; Mateus, Carvalho, Luis & de Freitas, 2004; Soares, Gonçalves, Fernandes, Mateus & de Freitas, 2009; Soares, Mateus & de Freitas, 2012; Chung, Rojanasasithara, Mutilangi & McClements, 2016). Hence, Acacia gum is mainly used in young red wine for its colloidal stabilizing properties. It stabilizes young red wine but does not clarify it.

Acacia gum is a natural exudate obtained from the trunk and branches of *Acacia senegal* and *Acacia seyal* trees (Williams & Phillips, 2000). Acacia gum macromolecules are highly glycosylated hydroxyproline-rich arabinogalactan-proteins (AGPs) belonging to the glycoprotein superfamily (Akiyama, Eda & Kato, 1984). AGPs from Acacia gum are mainly composed by sugars (D-galactose, L-arabinose, L-rhamnose, D-glucuronic acid, and 4-O-methyl-D-glucuronic acid) with a small fraction of proteins and minerals (Idris, Williams & Phillips, 1998; Lopez-Torrez, Nigen, Williams, Doco & Sanchez, 2015). The sugars are organized into hyperbranched polysaccharide blocks covalently linked to the polypeptide backbone in serine- and hydroxyproline-rich domains (Lopez-Torrez et al., 2015). The highly branched polysaccharide structure is formed by main chains of 1,3-linked β-D-galactopyranose substituted by side chains in O-6 position. Units of α-L-arabinofuranosyl and α-L-rhamnopyranosyl are distributed in the main and side chains while β-D-glucuronopyranosyl and 4-O-methyl-β-D-glucuronopyranosyl are mostly end-units (Anderson, Hirst & Stoddart, 1966; Anderson & Stobbart, 1966; Lopez-Torrez et al., 2015).

A. senegal gum can be defined as a continuum of AGPs differing by their sugar, amino acid and mineral content and composition, sugar to amino acid ratio, polarity, number of charges,
molar mass, size and shape (Randall, Phillips & Williams, 1989; Islam, Phillips, Sljivo, Snowden & Williams, 1997; Renard, Lavenant-Gourgeon, Ralet & Sanchez, 2006; Mejia Tamayo, Nigen, Apolinar-Valiente, Doco, Williams, Renard & Sanchez, 2018). These AGPs can be separated according to their polarity using hydrophobic interaction chromatography (HIC) with the recovery of three fractions (Randall et al., 1989; Renard et al., 2006). These three fractions were historically named arabinogalactan (AG), arabinogalactan-protein (AGP) and glycoprotein (GP) according to their protein content and elution order. However, all the ability of these three fractions to bind the β-glucosyl Yariv's reagent suggests that they all belong to the AGP family because of their reactivity to the Yariv's reactant (Osman et al. 1993). Hence, to avoid any confusion, we named them HIC-F1 (arabinogalactan, AG), HIC-F2 (arabinogalactan-protein, AGP) and HIC-F3 (glycoprotein, GP) in their order of elution, reflecting also an increasing hydrophobic index. HIC-F1 is the most abundant fraction (85-92% of the whole gum) as compared to HIC-F2 (6-16% of the whole gum) and HIC-F3 (1-3% of the whole gum). The sugar composition was similar between the three fractions, with however a larger content of arabinose in HIC-F2 and HIC-F3 and a larger content of charged sugars in HIC-F1. HIC-F3 was the richest fraction in proteins with values around 25-40%, while the amount of proteins was around 8-10% and 0.5-1% for HIC-F2 and HIC-F1, respectively. These three HIC fractions differed also by their mean molar mass ($M_w$) and high $M_w$ AGPs content (AGPs with $M_w$ upper than $10^6$ g.mol$^{-1}$). HIC-F1 was mainly composed of low $M_w$ AGPs, while HIC-F2 and HIC-F3 were richest in high $M_w$ AGPs.

In young red wines, it is advised to use *Acacia senegal* gum at a maximal concentration of 300 mg/L (Code International des Pratiques Œnologiques, Fiche Code OIV (12/72) – Edition 01/2015 II.3.3-7) even if no legal limit exists in the EC 606-2009. Before its addition in young red wines, the efficiency of *A. senegal* gum towards wine colloidal instability is evaluated according to an “efficacy test” of the International Œnological Codex (International
Oenological Codex: COEI-1-GOMARA, 2000). This "efficacy test" consists in determining the quantity of *A. senegal* gum required to prevent the flocculation of a colloidal iron hexacyanoferrate solution in hydro-alcoholic medium by calcium salt. Hence, the *A. senegal* gum efficiency towards wine colloidal instability is usually evaluated using a hydro-alcoholic – mineral solution which seems in appearance far enough from wine matrices on their biochemical and physicochemical properties. The colloidal stabilization of two so different matrices by *A. senegal* gum raises some questions on the colloidal stability mechanism and especially on the AGPs at the origin of this colloidal stabilization.

The aim of this study was to investigate the colloidal stabilizing properties of AGPs from *A. senegal* gum in three hydro-alcoholic solutions: a hydro-alcoholic – mineral solution, “wine-like” solution and young red wine. The AGPs responsible of the colloidal stability in the three hydro-alcoholic solutions were identified. Furthermore, the colloidal stabilization mechanism involving AGPs from *A. senegal* gum in hydro-alcoholic – mineral solution was characterized.

## 2. Materials and Methods

### 2.1. Materials

Instant spray dried *Acacia senegal* gum was provided by ALLAND & ROBERT Company - Natural and organic gums (Port Mort, France). *A. senegal* gum was fractionated by hydrophobic interaction chromatography (HIC) according to Renard et al. (Renard et al., 2006). Three HIC fractions were isolated that were named HIC-F1, HIC-F2 and HIC-F3 according to their elution order, and then their hydrophobic behaviour. The biochemical composition and structural properties (mean molar mass) of *A. senegal* gum and HIC fractions were described by Mejia-Tamayo (Mejia-Tamayo et al., 2018) and presented in supplementary data (Table S1).
149 The pronase was from *streptomyces griseus* (lot 10165921001, Roche). The grape marc 150 powder rich in polyphenols (64%) was provided by Grap'Sud Company (Cruviers-Lascours, 151 France). The unstable young red wine (Cabernet sauvignon/Merlot, 2015) was provided by 152 BioLaffort Company (Bordeaux, France). All other reagents were of analytical grade from 153 Sigma-Aldrich (USA). 154

155 **2.2. Hydrolysis of Acacia senegal gum and HIC-F1 fraction by pronase**

156 A. senegal gum and HIC-F1 fraction were dissolved at a concentration of 10 mg.ml⁻¹ in ultra 157 pure deionised water (18.2 mΩ resistivity) containing 0.02% NaN₃ and stirred overnight at 158 room temperature. The pH was adjusted at 6.5 using a small aliquot of NaOH solution before 159 to add pronase at a final concentration of 0.14 mg.ml⁻¹. The enzymatic hydrolysis occurred 160 during 48h at 35°C. The enzymatic reaction was stopped by removing the pronase from the 161 samples by centrifugation at 3000 rpm using a centricon Vivaspin 20 (cut-off of 50 000 Da). 162 The hydrolysis products were also removed by the centrifugation step. The samples were then 163 washed four times with ultra pure deionised water (18.2 mΩ resistivity) by centrifugation at 164 3000 rpm using a centricon Vivaspin 20 (cut-off of 50 000 Da), before their freeze-drying. 165

166 **2.3. Preparation of Acacia senegal gum and its HIC fractions**

167 Stock solution of A. senegal gum and HIC fractions were dissolved in ultra-pure 168 deionised water (18.2 mΩ resistivity) and stirred overnight at room temperature. The 169 solutions were centrifuged at 12 500 rpm for 30 min at 20°C to remove insoluble materials 170 and air bubbles. 171

172 **2.4. Electrophoretic mobility measurements**
173 The electrophoretic mobility of A. senegal gum prepared in hydro-alcoholic solutions (tartaric acid: 3 g.L⁻¹; potassium sulfate: 1 g.L⁻¹; ethanol: 12% v/v) at pH 3.1, 3.5 and 4 was determined using a Zetasizer 1000 (Malvern, United Kingdom). The measurements were performed at 25°C. The data are the average of three measurements.

177

2.5. *Multi-detector high performance size exclusion chromatography*

179 HPSEC experiments were performed using a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) coupled to one Shodex OHpak SB-G pre-column followed by four Shodex OHpak SB columns in series (SB 806 HQ, SB 805 HQ, SB 804 HQ and SB 803 HQ). 50 µl of A. senegal gum and HIC fractions solutions (1 mg.ml⁻¹) were injected. The elution was performed with 0.1 mol.L⁻¹ LiNO₃ solution containing 0.02% NaN₃ at a flow rate of 1 ml.min⁻¹ and 30°C.

184 The HPSEC system was fitted in series to a Dawn Heleos II multi-angle laser light scattering (Wyatt Technology Corp., Santa Barbara, Ca, USA) and an Optilab T-rEX refractometer (Wyatt Technology Corp., Santa Barbara, Ca, USA). The molar mass distribution and the weight-average molar mass (Mw) were calculated using ASTRA software 6.1.1.17 (Wyatt Technologies, Santa Barbara, CA). The data were analyzed using the Zimm's model (1st order) and a refractive index increment (dn/dc) of 0.155, 0.162, 0.160 and 0.145 ml.g⁻¹ for A. senegal gum, HIC-F1, HIC-F2 and HIC-F3, respectively, as determined experimentally.

191

2.6. *Colloidal stability of hydro-alcoholic - mineral solution*

193 The hydro-alcoholic – mineral solution was prepared with ultra-pure ultra-pure deionised water (18.2 mΩ resistivity) according to the International Oenological International Codex (O.I.V.). This solution was composed of tartaric acid (3 g.L⁻¹), potassium sulfate (1 g.L⁻¹), ethanol (12% v/v), potassium ferrocyanide (60 mg.L⁻¹) and iron solution (5 mg.L⁻¹).
195
196
197 When the addition of iron (Fe³⁺) was added to the hydro-alcoholic – mineral solution
containing potassium ferrocyanide (K[Fe(CN)₆]⁴⁻), induced the formation of a soluble “Prussian blue” colloidal solution of K[Fe³⁺FeII(CN)₆]⁻ is obtained. The hydro-alcoholic – mineral solution was stirred during 2 h at room temperature before to adjust its pH at 3.1 using NaOH solution (1 mol.L⁻¹). The hydro-alcoholic – mineral solution was then filtered through a 0.45 µm regenerated cellulose membrane (GE Healthcare Life Sciences, Germany). For the studies according to pH, the hydro-alcoholic – mineral solution was prepared at pH 3.1, 3.5 and 4.0.

The hydro-alcoholic – mineral solution was destabilized by the addition of CaCl₂ (22 g.L⁻¹) prepared in ultra-pure deionised water (18.2 mΩ resistivity). The added Ca²⁺ bound to K[Fe³⁺FeII(CN)₆]⁻ inducing the formation of “Prussian blue” precipitates (Ca[Fe³⁺FeII(CN)₆]₂). Most of experiments were performed at a final CaCl₂ concentration of 0.67 g.L⁻¹. Some experiments were performed at a final concentration of 0.33, 0.5, 1 and 1.33 g.L⁻¹ to investigate the influence of calcium. Each measurement was triplicated.

The efficiency of A. *senegal* gum and its HIC fraction to prevent the colloidal mineral destabilization was studied by varying their concentration in the hydro-alcoholic – mineral solution. The final concentration of A. *senegal* gum or its HIC fractions in the hydro-alcoholic – mineral solution ranged between 0 and 1 g.L⁻¹. A. *senegal* gum or HIC fraction solutions were added to the hydro-alcoholic – mineral solution before the addition of CaCl₂ solution. Each measurement was triplicated.

## 2.7. Colloidal stability of hydro-alcoholic – grape marc solution and young red wine

The hydro-alcoholic – grape marc solution (wine-like medium) was prepared with ultra-pure deionised water (18.2 mΩ resistivity). It contained tartaric acid (2.7 g.L⁻¹), potassium sulfate (0.9 g.L⁻¹), grape marc powder (3 g.L⁻¹) and ethanol (12% v/v). The solution was gently stirred during 2 h at room temperature before to adjust the pH at 3.5 using NaOH solution (1
223 mol.L⁻¹). The solution was then filtered through a 0.45 µm regenerated cellulose membrane
224 (GE Healthcare Life Sciences, Germany).

225 The colloidal destabilization of the hydro-alcoholic – grape marc solution and the young red
226 wine was induced by cooling the solutions to 10°C. The efficiency of A. senegal gum and its
227 HIC fraction to prevent the destabilization of the hydro-alcoholic – grape marc solution and
228 the young red wine was studied at 10°C by varying their concentration between 0 and 2 g.L⁻¹.
229 Each measurement was triplicated.

230
231 2.8. Characterization of the colloidal stability of hydro-alcoholic model solutions and young
232 red wine

233 The colloidal stability of the hydro-alcoholic – mineral and hydro-alcoholic – grape marc
234 solutions, and the young red wine was investigated using a Turbiscan Tower equipped with a
235 pulsed near infrared light source (λ = 880 nm) and two synchronous detectors, a transmission
236 (T) and a backscattering (BS) detectors (Formulaction, France). The samples were loaded into
237 cylindrical glass tubes and scanned throughout its entire height. The transmittance (T, in %)
238 was measured every 1 min and 50 sec during 24 h at 25°C for the hydro-alcoholic – mineral
239 solutions and during 48 h at 10°C for the hydro-alcoholic – grape marc solutions and young
240 red wine.

241 The transmittance (T) profiles were analyzed using the TowerSoft software, version 1.1.0.36
242 (Formulaction, France). ΔT (%) corresponded to the difference in transmittance between the
243 scanᵢ and the first scan. The Turbiscan Stability Index (TSI) was also determined as the
244 following:

245
$$TSI = \sum_i \frac{\sum_h |scan_i(h) - scan_{i-1}(h)|}{H}$$
246 The TSI corresponded to the transmittance variation at all measured position (h) throughout
247 the entire height (H) of the sample between the scanᵢ and the scanᵢ₋₁. The plot of TSI
248 according to time corresponds to the kinetic behavior of the samples that takes into account all
249 the physical phenomena (especially destabilization mechanism) occurring during the
250 measurement.

251 The TSI kinetics were used to plot the TSI values at 24h in hydro alcoholic – mineral solution
252 and 48h in hydro alcoholic – grape marc solution and young red wine according to AGPs
253 concentration. This representation assimilated to a “stability curve” was used to
254 experimentally determine the AGPs concentration, named critical concentration, necessary to
255 obtain the colloidal stability of the solutions.

256

257 **3. Results**

258 **3.1. Colloidal stabilization mechanism of the hydro-alcoholic – mineral solution by A. senegal gum**
259

260 The colloidal stability of the hydro-alcoholic – mineral solution (pH 3.1) containing A. senegal gum was followed by measuring at 25°C the transmittance (T) of the solutions during 261 24h using a light scattering equipment. The figure 1 showed the temporal change in 262 transmittance (ΔT) of three hydro-alcoholic – mineral solutions without and with 0.09 and 263 0.14 g.L⁻¹ of A. senegal gum (Figure 1A, 1B and 1C). After 24h, the transmittance (T) of the 264 hydro-alcoholic – mineral solutions without and with 0.09 g.L⁻¹ of A. senegal gum increased 265 respectively by 60% and 35% in the middle of the sample (cell height: 2 – 36 mm) reflecting 266 a clarification of the solution. This phenomenon was confirmed by the loss of the blue color 267 of the solutions after 24h (Figure 1A and 1B). The clarification resulted from the formation of 268 a salt precipitate (Ca[Fe<sup>III</sup>Fe<sup>II</sup>(CN)₆]<sub>2</sub>) between K[Fe<sup>III</sup>Fe<sup>II</sup>(CN)₆] and Ca²⁺ that pelleted, as 269 evidenced by the decrease of T by 5% and 2% after 24h in the bottom of the cell (cell height: 270
1 – 1.5 mm) for the solutions without and with 0.09 g.L⁻¹ of *A. senegal* gum, respectively (Figure 1A and 1B). On the other hand, the transmittance of the hydro-alcoholic – mineral solution containing 0.14 g.L⁻¹ of *A. senegal* gum was constant during the 24h of measurement, reflecting the colloidal stability of the solution. The influence of *A. senegal* gum concentration (0 – 0.14 g.L⁻¹) on the colloidal stability kinetics of the hydro-alcoholic – mineral solution was analyzed by plotting the TSI (Turbiscan Stability Index) according to time (Figure 2A). The increase of *A. senegal* gum concentration reduced and delayed the instability mechanism occurring in the hydro-alcoholic – mineral solution. The colloidal instability was totally prevented for an *A. senegal* gum concentration above around 0.11 g.L⁻¹.

The minimal *A. senegal* gum concentration ([AG]$_{critical}$) necessary to obtain the hydro-alcoholic – mineral colloidal stability was determined by plotting the TSI values after 24h according to *A. senegal* gum concentration (stability curve) (Figure 2B). Below an *A. senegal* gum concentration of 0.05 g.L⁻¹, the TSI values at 24h was constant and similar to the control sample without *A. senegal* gum. In these experimental conditions, the concentration of *A. senegal* gum was too low. Above 0.05 g.L⁻¹ of *A. senegal* gum, the TSI values decreased progressively before to reach a constant TSI value (≤ 2) similar to the control without Ca²⁺ for *A. senegal* gum concentration upper than 0.11 g.L⁻¹. Based on the stability curve, the [AG]$_{critical}$ was 0.114 ± 0.002 g.L⁻¹ (Figure 2B).

The colloidal stabilization mechanism was further investigated by modifying the physicochemical condition as the pH of the solutions and the amount of Ca²⁺ added to induce the colloidal instability mechanism. The stability curves of hydro-alcoholic – mineral solutions prepared at pH 3.1, 3.5 and 4.0 are shown in Figure 3A. As the pH increased from 3.1 to 4.0, the stability curves were shifted towards lower *A. senegal* gum concentrations necessary to prevent the colloidal instability. The [AG]$_{critical}$ were 0.114 ± 0.002, 0.094 ± 0.003 and 0.064 ± 0.002 g.L⁻¹ at pH 3.1, 3.5 and 4.0, respectively. Hence, the colloidal
296 stabilizing properties of A. senegal gum were greatly influenced by pH and enhanced with its
297 increase in this pH range. AGPs from A. senegal gum are weakly charged glycoproteins
298 characterized by a global negative charge in aqueous solution for pH ranging from 2.0 to 10.0
299 (Burgess & Carless, 1984). Moreover, the negative AGPs µE increased from pH 2.0 to ~ 5.0
300 in aqueous solutions reflecting the increase of the global negative charge (Burgess & Carless,
301 1984; Schmitt, Sanchez, Thomas & Hardy, 1999). Thus, the electrophoretic mobility (µE) of
302 A. senegal gum in hydro-alcoholic solutions prepared at pH 3.1, 3.5 and 4.0 was measured
303 and plotted according to [AG]critical (Figure 3B). A good correlation was evidenced between
304 A. senegal gum µE and its concentration necessary to stabilize the hydro-alcoholic – mineral
305 solutions. The more the negative A. senegal gum µE was, the less the A. senegal gum quantity
306 was required to prevent the colloidal instability. Hence, negative charges carried by AGPs
307 were certainly involved in this colloidal stabilization mechanism.

308 Since the colloidal instability of the hydro-alcoholic – mineral solution is induced by the
309 addition of Ca²⁺, it was hypothesized that electrostatic interactions between Ca²⁺ and negative
310 charges of AGPs could play an important role in the stabilization mechanism. Therefore, we
311 determined the [AG]critical in hydro-alcoholic – mineral solutions (pH 3.1) containing different
312 final Ca²⁺ concentrations (0.33 to 1.33 g.L⁻¹). A close linear relationship between the
313 [AG]critical and the amount of Ca²⁺ added in the hydro-alcoholic – mineral solutions was
314 obtained (Figure 4). The increase of Ca²⁺ concentration induced also the increase of
315 [AG]critical. Based on its structural and physicochemical properties, hyperbranched and
316 negatively charged, AGPs from A. senegal gum appear as a natural mineral carrier that
317 contains ~2-3.4 wt% of ash for this Acacia gum sample, and especially Ca²⁺ (Anderson,
318 Douglas, Morrison & Weiping, 1990; Debon & Tester, 2001; Mhinzi, 2003). Lamport and
319 Varnai evidenced that Ca²⁺ bound to AGPs with a fairly strong binding constant of 6.5×10⁻⁶
320 mol.L⁻¹ (Lamport & Varnai, 2013). They also showed that the Ca²⁺ binding sites of AGPs
were not naturally fully saturated by Ca²⁺. Hence, as expected, we can easily suggest that AGPs stabilized the hydro-alcoholic – mineral solutions by its electrostatic binding with Ca²⁺, the driver of the instability mechanism.

324

325 **3.2. Identification AGPs from A. senegal gum involved in the colloidal stability mechanism of**
326 the hydro-alcoholic – mineral solution

As previously mentioned, *A. senegal* gum is a continuum of AGPs differing by their biochemical, structural and physicochemical properties (Randall et al., 1989; Renard et al., 2006). To better identify the macromolecules involved in this colloidal stabilization mechanism, *A. senegal* gum was fractionated using Hydrophobic Interaction Chromatography (HIC). The three fractions obtained were named HIC-F1, HIC-F2 and HIC-F3 according to their elution order and consequently to their increasing hydrophobic index. The biochemical composition and some structural properties of *A. senegal* gum and its HIC fractions were previously described (Mejia-Tamayo et al., 2018) and presented in supplementary data (table 1). The three HIC fractions are composed of the same sugars (D-galactose, L-arabinose, D-glucuronic acid and 4-O-methyl-D-glucuronic acid) with some differences in their proportions. The arabinose to galactose (Ara/Gal) molar ratio was 0.69, 1.03 and 1.29 for HIC-F1, HIC-F2 and HIC-F3, respectively. HIC-F1 was also richer in uronic acid (glucuronic and 4-O-methyl-glucuronic acids) than HIC-F2 and HIC-F3 fractions (21.7 wt% vs. 16.2 wt% and 14.4 wt%, respectively). Hence, the carbohydrate blocks of HIC-F1 were supposed to carry more negative charges than HIC-F2 and HIC-F3 ones. Biochemically, the main difference between HIC fractions was the protein content and consequently the protein/sugar ratio. HIC-F3 showed a higher amount of proteins (13.8 wt%) and then protein/sugar ratio (0.169) as compared to HIC-F2 (6.3 wt% and 0.069) and HIC-F1 (0.5 wt% and 0.005). Structurally, the three HIC fractions differed mainly by their mean molar mass (Mw) and high
molar mass (HM<sub>w</sub>) macromolecules content (M<sub>w</sub> > 10<sup>6</sup> g.mol<sup>-1</sup>). HIC-F2 and HIC-F3 were richer in protein-rich high molar mass (HM<sub>w</sub>) AGPs than HIC-F1 (73, 56 and 3%, respectively) (supplementary table 1).

The stability curves of the hydro-alcoholic – mineral solution in the presence of the three HIC fractions are shown in the Figure 5. They can be classified from the most effective to the least in terms of stabilization as: HIC-F3 > HIC-F2 > HIC-F1. Thus, the critical concentrations of HIC-F1, HIC-F2 and HIC-F3 fractions were 0.570, 0.090 and 0.012 g.L<sup>-1</sup>, respectively.

Though HIC-F3 was the minor fraction in weight (around 1 wt% of whole A. *senegal* gum), its efficiency for the colloidal stabilization of the hydro-alcoholic – mineral solution was 48, 10 and 7.5 times larger than those of HIC-F1, A. *senegal* gum and HIC-F2, respectively.

Hence, the colloidal stability properties of A. *senegal* gum towards the hydro-alcoholic – mineral solution were mainly due to its two minor fractions in weight, HIC-F2 and HIC-F3 (around 11 wt% of whole A. *senegal* gum), that were especially richer in protein-rich HM<sub>w</sub> AGPs than HIC-F1 (Table 1).

HIC-F1 (89 wt% of whole A. *senegal* gum) that was characterized by poor colloidal stabilizing property contained also a low amount (3%) of protein-rich HM<sub>w</sub> AGPs. To precise whether the macromolecular origin of the colloidal stabilizing properties is due to this fraction, its HM<sub>w</sub> AGPs were removed by an enzymatic hydrolysis treatment using the pronase, a protease mixture. Several studies showed the specific hydrolysis activity of pronase for protein-rich HM<sub>w</sub> AGPs from A. *senegal* gum without degrading and affecting the structural properties of low M<sub>w</sub> (LM<sub>w</sub>) AGPs (Connolly, Fenyo & Vandevelde, 1988; Randall et al., 1989; Flindt, Al-Assaf, Phillips & Williams, 2005; Mahendran, Williams, Phillips, Al-Assaf & Baldwin, 2008; Renard, Lavenant-Gourgeon, Lapp, Nigen & Sanchez, 2014). The refractive index (RI) signal and the M<sub>w</sub> distribution of HIC-F1 before and after the enzymatic treatment are shown in Figure 6A. The pronase hydrolyzed the protein-rich AGPs decreasing
the HM<sub>w</sub> AGPs content from 3 to 1.7%, without affecting the quantity and the structural properties of LM<sub>w</sub> AGPs (Figure 6A). The colloidal stabilizing properties of the hydrolyzed HIC-F1 towards the hydro-alcoholic – mineral solution were characterized and compared to HIC-F1 (Figure 6C). The hydro-alcoholic – mineral solutions was totally unstable until 1.2 g.L<sup>-1</sup> of hydrolyzed HIC-F1, a concentration twice more than the minimal HIC-F1 concentration necessary to totally prevent the colloidal instability ([HIC-F1]<sub>critical</sub> = 0.570 g.L<sup>-1</sup>). Hence, the removal of less than 1.5% of AGPs from HIC-F1, corresponding to HM<sub>w</sub> AGPs, induced the loss of its colloidal stabilizing properties. The main contribution of protein-rich HM<sub>w</sub> AGPs was also confirmed by studying the colloidal stabilizing properties of hydrolyzed A. senegal gum by pronase. After the enzymatic treatment, the content of protein-rich HM<sub>w</sub> (M<sub>w</sub> > 10<sup>6</sup> g.mol<sup>-1</sup>) AGPs decreased from 14 to 6% (Figure 6B). As observed for HIC-F1, A. senegal gum also lost its stabilizing properties after the pronase treatment: the hydro-alcoholic – mineral solutions containing 0.12 g.L<sup>-1</sup> of hydrolyzed A. senegal gum was totally unstable while [AG]<sub>critical</sub> was found to be 0.114 g.L<sup>-1</sup>. These results highlighted the major role of the protein-rich AGPs in the colloidal stabilizing mechanism of the hydro-alcoholic – mineral solution. Hence, HIC-F2 and HIC-F3 fractions prevented the colloidal instability of the hydro-alcoholic – mineral solution (i.e. formation of salt precipitates) by their electrostatic binding with Ca<sup>2+</sup>. The key role of the protein-rich AGPs on the functional properties of A. senegal gum was also evidenced by studying its interfacial properties (Elmaman, Al-Assaf, Phillips & Williams, 2008). In this study, the removal of the protein-rich AGPs by a pronase treatment resulted in the loss of the interfacial properties of A. senegal gum.

393

394 3.3. Colloidal stabilization of a hydro-alcoholic – grape marc solution and an unstable young

395 red wine by AGPs from A. senegal gum
In order to correlate the results obtained on the hydro-alcoholic – mineral solution to the wine matrix, the stabilizing properties of *A. senegal* gum and its HIC fractions were characterized on an unstable synthetic hydro-alcoholic – grape marc solution (wine-like medium) and an unstable young red wine.

The hydro-alcoholic – grape marc solution and the young red wine that were stable at 25°C (control sample) were destabilized upon cooling to 10°C that induced the precipitation of the unstable colloidal substances as the coloring matter (Ribereau-Gayon et al., 2006; Alcade-Eon et al., 2014). The colloidal stability kinetics of the hydro-alcoholic – grape marc solution and the young red wine supplemented with *A. senegal* gum and HIC fractions were followed by measuring the transmittance of the solution during 48 h at 10°C. The increase of *A. senegal* gum concentration from 0 to 0.5 g.L⁻¹ led to the colloidal stabilization of the hydro-alcoholic – grape marc solution (Figure 7A). The solution supplemented with 0.5 g.L⁻¹ of *A. senegal* gum presented a similar kinetic behavior as the control sample at 25°C suggesting that AGPs from *A. senegal* gum totally prevented the colloidal instability. This result was in agreement with the use of *A. senegal* gum, and more globally the AGPs, as protective colloids in wines and model beverages to prevent the aggregation and precipitation of polyphenols and proteins (Ribereau-Gayon et al., 2006; Waters et al., 1994; Riou et al., 2002; Mateus et al., 2004; Luck, Liao, Murray, Grimmer, Warminski, Williamson, Lilley & Haslam, 1994; Liang, Liu, Qi, Su, Yu, Wang & He, 2013; Soares et al., 2009; de Freitas et al., 2003), and the chemical modifications of anthocyanins (Chung et al., 2016). The efficiency of *A. senegal* gum and its HIC fractions towards the colloidal instability of this hydro-alcoholic – grape marc solution were characterized by plotting the stability curves after 48h of kinetics (Figure 7B). The critical concentrations of *A. senegal* gum, HIC-F1, HIC-F2 and HIC-F3 fractions were found to be 0.245 ± 0.010, 0.996 ± 0.090, 0.118 ± 0.014 and 0.027 ± 0.003 g.L⁻¹, respectively. The colloidal stabilizing properties of *A. senegal* gum, HIC-F2 and HIC-F3 fractions were also
421 confirmed on an unstable young red wine (Figure 7C). They totally prevented the young red
422 wine colloidal instability at 10°C for concentrations of 0.086 ± 0.007, 0.045 ± 0.006 and
423 0.015 ± 0.002 g.L⁻¹, respectively. Unlike the other two HIC fractions, HIC-F1 was not
424 efficient for the colloidal stabilization of the young red wine even at a concentration of 2 g.L⁻¹
425 (data not shown).

426

4. Discussion

428 The colloidal stabilizing properties of A. senegal gum and its HIC fractions isolated from
429 Hydrophobic Interaction Chromatography were investigated on three colloidal hydro-
430 alcoholic matrices: a hydro-alcoholic – mineral matrix, a hydro-alcoholic – grape marc
431 solution (“wine-like” matrix) and an unstable young red wine. Whatever the hydro-alcoholic
432 matrix tested, the HIC fractions can be classified in the same following order according to
433 their colloidal stabilizing efficiency: HIC-F1 << HIC-F2 < HICF3. A. senegal gum that is a
434 mixture of the HIC fractions presented some intermediate properties between those of HIC-F1
435 and HIC-F2. Before its addition in young red wines, Usually, A. senegal gum added in young
436 red wines is usually chosen based on its ability to stabilize a hydro-alcoholic – mineral matrix.
437 When we plot the critical concentrations of A. senegal gum and its HIC fractions determined
438 in hydro-alcoholic – mineral solution as a function of those determined in hydro-alcoholic –
439 grape marc solution or young red wine (Figure 8), a good correlation between both systems is
440 obtained. The more the AGPs from A. senegal gum are efficient to stabilize the hydro-
441 alcoholic – mineral solution, the better they are to stabilize the polyphenols in young red
442 wines. Hence, these results confirmed and strengthened the relevance and the use of the
443 hydro-alcoholic – mineral test for the evaluation of A. senegal gum colloidal stabilizing
444 properties before its addition in young red wine.
In the hydro-alcoholic – mineral solution, the colloidal stability was assessed by the electrostatic binding of Ca²⁺, the “positively charged” driver of the colloidal instability, with the negatively charged AGPs. According to the monosaccharide and amino acid compositions of Acacia *senegal* gum (Mejia-Tamayo et al., 2018), the negative charges of AGPs are carried out both by the carbohydrate moiety through the carboxylic groups of glucuronic and 4-O-methyl glucuronic acid monosaccharides, and the polypeptidic backbone by the carboxylic groups of aspartic and glutamic acid amino acid side chains. The content and proportion of these negatively charged uronic acids and amino acids are fraction dependent with a large excess of uronic acids in the three fractions. HIC-F1 has a higher uronic acid content (20.9 wt% of whole fraction) as compared to HIC-F2 and HIC-F3 (14.9 wt% and 11.7 wt%, respectively) (supplementary table 1). Recently, Grein-Iankovski et al. (2018) characterized an acid dissociation constant (pKₐ) between 3 and 4 for the AGPs from A. *senegal* gum that was attributed to the carboxylic group of glucuronic acids. This chemical property suggests a partial ionization of the carboxylic groups in the range of the pH studied in the hydro-alcoholic solutions, and moreover the increase of the negative charge of AGPs with the increase of pH from 3.1 to 4.0. This result is in well accordance with the decrease of the critical concentration of A. *senegal* gum when the pH is increased from 3.1 to 4.0. Recently, tThe binding of Ca²⁺ with the carboxyl group of two uronic acids of a conserved O-Hyp-linked arabinogalactan polysaccharide was demonstrated using molecular dynamic simulations (Lamport & Varnai, 2013). Hence, based on the excess of uronic acid in the three HIC fractions and the molecular dynamic simulations it could be expected as a first approximation that the mineral colloidal stability occurred mainly by the binding of Ca²⁺ to the uronic acids of carbohydrate blocks. However, our results showed that HIC-F1, the richest fraction in uronic acids, had the weakest colloidal stabilizing properties, while HIC-F3, the one with the least uronic acids content, was the most effective fraction for the colloidal
stabilization of the hydro-alcoholic – mineral solution (Figure 5). Hence, the stabilizing
properties of AGPs could not be only explained by their uronic acids content. We also showed
a significant decrease of the stabilizing properties of HIC-F1 and A. senegal gum after the
hydrolysis of their protein-rich AGPs by a pronase treatment. The pronase is a cocktail of
proteases hydrolyzing the accessible polypeptidic backbone of protein-rich AGPs without
degrading the low molar mass (LMw) AGPs poor in proteins (Renard et al. 2014). This
enzymatic treatment gave rise to macromolecules presenting similar molar masses (Mw from
1.8 to 6.0x10⁵ g.mol⁻¹) and conformational properties as low molar mass (LMw) AGPs
naturally found in A. senegal gum (Connolly et al., 1988, Randall et al., 1989, Aoki, Al-
Assaf, Katayama & Phillips, 2007, Renard et al., 2014). Since the conformational properties,
and probably the sugar organization, of the released carbohydrate blocks of AGPs was
seemingly not disturbed by the pronase treatment, this suggested that the uronic acids of the
carbohydrate blocks were certainly not directly involved in the electrostatic Ca²⁺ binding in
this colloidal stability test. Similar results were obtained on AGPs purified from a red wine by
Waters et al. These authors showed that the greatest protein haze protective activity in
Chardonnay wine was reached with AGPs presenting the highest protein content. Moreover,
this protective activity was not affected by the sugar hydrolysis of AGPs using α-
arabinofuranosidase and endo-(1→6)-β-D-galactanase, while it was totally lost when the
protein moiety was altered with the loss of 75% of initial protein after smith degradation
treatment (Waters et al., 1994). These results revealed the key role of the accessible
polypeptidic backbone of AGPs in their colloidal stabilizing properties. In the hydro-alcoholic
– mineral solution, it could be hypothesized that the electrostatic binding of Ca²⁺ occurred
with only the negatively charged amino acids (aspartic and glutamic acids) or with the
involvement of both negatively charged amino acids and uronic acid monosaccharides
localized close to the protein backbone.
As observed in the hydro-alcoholic – mineral solution, the protein-rich AGPs from *A. senegal* gum, HIC-F2 and HIC-F3 fractions, were also found to be the most effective for the colloidal stabilization of the hydro-alcoholic – grape marc solution and the unstable young red wine.

The surface and colloidal stabilizing properties of the protein-rich AGPs were previously demonstrated in other type of matrices. After *A. senegal* gum fractionation by size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC), Ray et al. showed that the protein-rich AGPs made the best emulsions in model beverage (Ray, Bird, Iacobucci & Clark, 1995). Protein-rich AGPs were also found to be the most effective in decreasing the interfacial tension of n-hexadecane-water interface (Castellani, Al-Assaf, Axelos, Phillips & Anton, 2010), to preferentially adsorb at the interface of latex dispersions (Snowden, Phillips & Williams, 1987) and oil droplets (Randall, Phillips & Williams, 1988), and to stabilize carbon nanotube dispersions (Li, Zhang, Jin & Cai, 2018).

The stabilizing properties of the protein-rich AGPs towards such a chemical diversity of molecules (minerals, polyphenols, proteins, oils, latex, etc...) could be relied to their intrinsic properties. Structurally, the three HIC fractions seems not so different. AGPs from these fractions are hyperbranched glycoproteins adopting more or less extended ellipsoidal conformations (Sanchez, Schmitt, Kolodziejczyk, Lapp, Gaillard & Renard, 2008; Renard, Garnier, Lapp, Schmitt & Sanchez, 2012; Renard, Garnier, Lapp, Schmitt & Sanchez, 2013; Renard et al., 2014; Lopez-Torrez et al., 2015). AGPs from HIC-F2 and HIC-F3 fractions also appeared as more “flexible” than those of HIC-F1 (Mejia-Tamayo et al., 2018).

Biochemically, HIC fractions presented some more marked differences, especially in their protein content and consequently in their protein to sugar ratio (supplementary table 1) that influenced their hydrophobic and hydration properties (Mejia-Tamayo et al., 2018). HIC-F1, HIC-F2 and HIC-F3 were eluted in that order according to their protein content and increasing hydrophobic behavior. From our results, the higher the HIC fraction is rich in
protein, the more effective it is for the colloidal stabilization of the three hydro-alcoholic solutions. In the hydro-alcoholic – grape marc solution and young red wine, these results were in accordance with the putative stabilization of polyphenols by their binding with AGPs via the establishment of hydrophobic interactions and hydrogen bonding (de Freitas et al., 2003; Mateus et al., 2004; Soares et al., 2009; Soares et al., 2012; Chung et al., 2016). Hence, the protein content and certainly the amino acids composition and distribution in the protein backbone of AGPs from *A. senegal* gum appeared as the main factor for the expression of their colloidal stabilizing properties in hydro-alcoholic matrices. This behavior agrees with the work performed by Dickinson et al. dealing with the surface properties of Acacia gums. Based on the surface activity of various Acacia gums with different protein content, these authors previously evidenced a good correlation between the Acacia gum protein content and its interfacial properties (Dickinson, Murray, Stainsby & Anderson, 1988; Dickinson, 2003). The protein content appeared also as essential for the colloidal stabilizing properties of mannoproteins, other glycoproteins considered as protective colloids in wine. The addition of mannoproteins containing a high proportion of proteins (10 to 30%) to red wine prevented the coloring matter precipitation and protein haze in white wine (Waters et al., 1994a; Escot et al., 2001; Charpentier, Escot, Gonzalez, Dulau & Feuillat, 2004; Poncet et al., 2007), while the mannoproteins with a low protein content were devoid of protective activity (Charpentier et al., 2004; Guadalupe, Palacios & Ayestaran, 2007; Poncet et al., 2007; Guadalupe & Ayestaran, 2008).

It is useful to remember that AGPs are constituted by a protein backbone covalently linked to hyperbranched carbohydrate blocks that partially hindered it from its environment. Therefore, if we consider the protein as the major component for the colloidal stabilizing properties of AGPs, it would seem appropriate to consider not only the protein content but also its accessibility to its environment that is closely link to the rate of glycosylation. HIC-F1,
presenting the weakest colloidal stabilizing properties, is highly glycosylated as compared to HIC-F2 and HIC-F3: their protein to sugar ratios (w/w) were 0.005, 0.069 and 0.169, respectively. The high glycosylation rate of HIC-F1 cause the protein less accessible to its environment as demonstrated by its resistance to the protein hydrolysis assay (Flindt et al., 2005; Renard et al., 2014). Moreover, the rate of glycosylation also influenced the structural and physicochemical properties of HIC fractions. The more the AGPs were glycosylated, then hydrated, the less their flexibility and hydrophobic behavior were marked (Mejia Tamayo et al., 2018). Therefore, we can advance that the surface properties of AGPs are mainly due to the physicochemical properties of their protein backbone which are subtly modulated and controlled by their rate of glycosylation.

## 5. Conclusion

In this research, the colloidal stabilizing properties of arabinogalactan-proteins from A. senegal gum in hydro-alcoholic – mineral and hydro-alcoholic – polyphenols solutions were investigated. The AGPs prevented the colloidal instability of both calcium iron hexacyanoferrate salts in “model” hydro-alcoholic solution and polyphenols in young red wine. A good relationship was evidenced between the stabilizing properties of AGPs determined in these two hydro-alcoholic solutions. The protein moiety of the AGPs appeared to be essential for these functional properties whatever the hydro-alcoholic solutions. The more the AGPs were rich in proteins, the more their colloidal stabilizing efficiency were. In the hydro-alcoholic – mineral solution, the AGPs avoided the precipitation of potassium ferrocyanide salts by their electrostatic binding with Ca²⁺, the driver of the instability.

## 6. Acknowledgements
The authors would like to thank BioLaffort Company (Bordeaux, France) for the financial support and Alland&Robert Company (Port-Mort, France) for the gift of Acacia *senegal* gum sample.

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682 Randall, R.C., Phillips, G.O., & Williams, P.A. (1988). The role of the proteinaceous component on the emulsifying properties of gum arabic. *Food Hydrocolloids*, **2**, 131-140.

684 Randall, R.C., Phillips, G.O., & Williams, P.A. (1989). Fractionation and characterization of gum from *Acacia senegal*. *Food Hydrocolloids*, **3**, 65-75.

686 Ray, A.K., Bird, P.B., Iacobucci, A.I., & Clark, B.C. (1995). Functionality of gum Arabic. Fractionation, characterization and evaluation of gum fractions in citrus oil emulsions and model beverages. *Food Hydrocolloids*, **9**, 123-131.
689 Renard, D., Lavenant-Gourgeon, L., Ralet, M.C., & Sanchez C. (2006). Acacia *senegal* gum: continuum of molecular species differing by their protein to sugar ratio, molecular weight, and charges. *Biomacromolecules*, 7, 2637-2649.

692 Renard, D., Garnier, C., Lapp, A., Schmitt, C., & Sanchez, C. (2012). Structure of arabinogalactan-protein from Acacia gum: From porous ellipsoids to supramolecular architectures. *Carbohydrate Polymers*, 90, 322-332.

695 Renard, D., Garnier, C., Lapp, A., Schmitt, C., & Sanchez C. (2013). Corrigendum to "Structure of arabinogalactan-protein from Acacia gum: From porous ellipsoids to supramolecular architectures" [Carbohydrate Polymers 90 (2012) 322-332]. *Carbohydrate Polymers*, 97, 864-867.

699 Renard, D., Lavenant-Gourgeon, L., Lapp, A., Nigen, M., & Sanchez, C. (2014). Enzymatic hydrolysis studies of arabinogalactan-protein structure from Acacia gum: The self-similarity hypothesis of assembly from a common building block. *Carbohydrate Polymers*, 112, 648-661.

703 Ribéreau-Gayon, P., Glories, Y., Maujean, A., & Dubourbieu, D. (2006). *Handbook of Enology. Vol. 2. The Chemistry of Wine Stabilization and Treatments*, 2nd ed.; Wiley: Chichester, UK.

706 Riou, V., Vernhet, A., Doco, T., & Moutounet, M. (2002). Aggregation of grape seed tannins in model wine – effect of wine polysaccharides. *Food Hydrocolloids*, 16, 17-23.

708 Sanchez, C., Schmitt, C., Kolodziejczyk, E., Lapp, A., Gaillard, C., & Renard, D. (2008). The Acacia gum arabinogalactan fraction is a thin oblate ellipsoid: A new model based on small-angle neuron scattering and ab initio calculation. *Biophysical Journal*, 94, 629-639.

711 Schmitt, C., Sanchez, C., Thomas, F., & Hardy, J. (1999). Complex coacervation between β-lactoglobulin and acacia gum in aqueous medium. *Food Hydrocolloids*, 13, 483-496.
713 Snowden, M.L., Phillips, G.O., & Williams, P.A. (1987). Functional characteristics of gum arabic. *Food Hydrocolloids, 1*, 291-300.

715 Soares, S.I., Gonçalves, R.M., Fernandes, I., Mateus, N., & de Freitas, V. (2009). Mechanistic approach by which polysaccharides inhibit α-amylase/procyanidin aggregation. *Journal of Agricultural and Food Chemistry*, 57, 4352-4358.

718 Soares, S., Mateus, N., & de Freitas, V. (2012). Carbohydrates inhibit salivary proteins precipitation by condensed tannins. *Journal of Agricultural and Food Chemistry*, 60, 3966-3972.

721 Teissedre, P.L. (2012). Arabic Gum in Oenology. In: Kennedy J.F, Phillips G.O., Williams P.A. (eds) Arabic gum. RSC Publishing, Cambridge.

723 Waters, E.J., Pellerin, P., & Brillouet, J-M. (1994). A wine arabinogalactan-protein that reduces heat-induced wine protein haze. *Bioscience Biotechnology and Biochemistry*, 58, 43-48.

726 Waters, E.J., Pellerin, P., & Brillouet, J-M. (1994a). A Saccharomyces mannoprotein that protects wine from protein haze. *Carbohydrate polymers*, 23, 185-191.

728 Williams, P.A., & Phillips, G.O. (2000). Gum Arabic. In G.O. Phillips, & P.A. Williams (Eds.), Handbook of hydrocolloids, Boca Raton: CRC Press.

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Figure 1. Transmittance changes of hydro-alcoholic – mineral solutions without (A) and with 731 0.09 (B) and 0.14 g.L⁻¹ (C) of *A. senegal* gum. The transmittance was registered at 25°C 732 during 24h. Pictures of the solutions before and after 24h of kinetic are presented on the right. 733

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>
Figure 2. (A) Colloid stability kinetics of hydro-alcoholic – mineral solutions containing various *A. senegal* gum concentrations: 0 (●), 0.05 (●), 0.08 (●), 0.09 (●), 0.10 (●), 0.11 (●) and 0.14 (●) g.L⁻¹. (B) Colloidal stability curve of hydro-alcoholic – mineral solution in presence of *A. senegal* gum after 24h of kinetic. The experiments were performed at 25°C.

The line (figure 2B) is shown to guide the eyes. The experiments were triplicated.

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>
Figure 3. (A) Colloidal stability curves of hydro-alcoholic – mineral solutions in presence of A. senegal gum (AG) at pH 3.1 (○), 3.5 (○) and 4.0 (○) after 24h of kinetic. (B) Relationship between the electrophoretic mobility of A. senegal gum (AG) and its critical concentration in hydro-alcoholic – mineral solutions. The lines (figure 3A) are shown to guide the eyes. The experiments were triplicated.

<figure><img src="image_9.png" /></figure>
Figure 4. Relationship between the concentration of Ca²⁺ in the hydro-alcoholic – mineral solution and the critical stabilizing concentration in A. senegal gum ([AG]crit). The experiments were triplicated.

<figure><img src="image_10.png" /></figure>
Figure 5. Colloidal stability curves of hydro-alcoholic – mineral solution in presence of A.

senegal gum (○), HIC-F1 (○), HIC-F2 (○) and HIC-F3 (○) fractions after 24h of kinetic. The experiments were performed at 25°C. The lines are shown to guide the eyes. The inset figure is a zoom of the left part. The experiments were triplicated.

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<figure><img src="image_11.png" /></figure>

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Figure 6. Molar mass distribution (thick line) and normalized refractive index signal (thin line) of HIC-F1 (A) and *A. senegal* gum (B) before (black) and after (red) pronase treatment. Colloidal stability curves of hydro-alcoholic – mineral solution in presence of HIC-F1 (C) and *A. senegal* gum (D) before (○) and after (●) pronase treatment after 24h of kinetic. The lines (figures 6C and 6D) are shown to guide the eyes. The colloidal stability experiments were triplicated.

<figure><img src="image_12.png" /></figure>
Figure 7. (A) Colloid stability kinetic of hydro-alcoholic – grape marc solutions according to A. senegal gum (AG) concentration at 10°C. The concentrations of A. senegal gum were 0 (●), 0.01 (●), 0.025 (●), 0.05 (●), 0.1 (●) and 0.5 (●) g.L⁻¹. The colloidal stability kinetic of the hydro-alcoholic – grape marc solution at 25°C without A. senegal gum is shown as a control (●) (B) Colloidal stability curves of hydro-alcoholic – grape marc solution in presence of A. senegal gum (○), HIC-F1 (○), HIC-F2 (○) and HIC-F3 (○) fractions after 48h of kinetic at 10°C. (C) Colloidal stability curves of a young red wine in presence of A. senegal gum (○), HIC-F2 (○) and HIC-F3 (○) fractions after 48h of kinetic at 10°C. The lines (figures 7B and 7C) are shown to guide the eyes. The inset figure is a zoom of the left part. The experiments were triplicated.

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<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>
Figure 8. Relationship between the critical concentrations of A. senegal gum (AG) and its HIC-fractions determined in hydro-alcoholic – mineral solution and those determined in hydro-alcoholic grape marc solution (A) and young red wine (B). The experiments were triplicated.

<figure><img src="image_16.png" /></figure>

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826 Supplementary table 1. Biochemical and structural properties of Acacia *senegal* gum (AG),

827 and its HIC fractions.

828

<table><thead><tr><th></th><th>AG</th><th>HIC-F1</th><th>HIC-F2</th><th>HIC-F3</th></tr></thead><tbody><tr><td>Total dry matter (mg.g<sup>-1</sup>)</td><td>893.4</td><td>921.6</td><td>926.2</td><td>921.9</td></tr><tr><td>Sugars (mg.g<sup>-1</sup>)</td><td>944.4</td><td>961.3</td><td>918.3</td><td>813.0</td></tr><tr><td>Galactose</td><td>363.6</td><td>374.9</td><td>315.9</td><td>270.7</td></tr><tr><td>Arabinose</td><td>281.4</td><td>257.6</td><td>326.9</td><td>311.4</td></tr><tr><td>Rhamnose</td><td>120.9</td><td>120.2</td><td>125.8</td><td>113.0</td></tr><tr><td>Glucuronic acid</td><td>168.1</td><td>195.1</td><td>143.3</td><td>111.4</td></tr><tr><td>4-O-Me-Glucuronic acid</td><td>9.4</td><td>13.5</td><td>5.5</td><td>5.7</td></tr><tr><td>Proteins (mg.g<sup>-1</sup>)</td><td>21.5</td><td>4.9</td><td>63.1</td><td>137.7</td></tr><tr><td>Ash (mg.g<sup>-1</sup>)</td><td>34.1</td><td>30.5</td><td>19.3</td><td>49.3</td></tr><tr><td>M<sub>w</sub> (g.mol<sup>-1</sup>)</td><td>6.8 &times; 10<sup>5</sup></td><td>3.5 &times; 10<sup>5</sup></td><td>1.5 &times; 10<sup>6</sup></td><td>1.6 &times; 10<sup>6</sup></td></tr><tr><td>High M<sub>w</sub> content (%)<sup>a</sup></td><td>14</td><td>3</td><td>73</td><td>56</td></tr></tbody></table>

829

830 <sup>a</sup> The high M<sub>w</sub> corresponded to macromolecules with a M<sub>w</sub> upper than 10<sup>6</sup> g.mol<sup>-1</sup>.

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**Highlights.**

- AGPs from Acacia *senegal* gum prevent the coloring matter precipitation

- The more AGPs are rich in proteins, the more their stabilizing efficiency are

- Stabilizing properties of AGPs are correlated in “synthetic” solutions and red wine

- In “synthetic” mineral solution, AGPs avoid the precipitation by Ca²⁺ binding