Michael Nigen
email: michael.nigen@umontpellier.fr

Rafael Apolinar Valiente

Nerea Iturmendi

Pascale Williams

Thierry Doco

Virginie Moine

Arnaud Massot

Isabelle Jaouen

Christian Sanchez

Michaël Nigen

Michaël Nigen

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

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

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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

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

M A N U S C R I P T A C C E P T E D

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

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

4 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 [START_REF] Akiyama | Gum Arabic is a kind of arabinogalactan protein[END_REF]. AGPs from Acacia gum are mainly composed by sugars (D-galactose, L-arabinose, L-rhamnose, D-glucuronic acid, and 4-Omethyl-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-Dgalactopyranose 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-Dglucuronopyranosyl and 4-O-methyl-β-D-glucuronopyranosyl are mostly end-units [START_REF] Anderson | Studies on uronic acid materials Part XVII some structural features of Acacia senegal gum (gum Arabic)[END_REF]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,

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

M A N U S C R I P T A C C E P T E D

ACCEPTED MANUSCRIPT 5 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 tThe 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. , 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.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6 Oenological Codex: COEI-1-GOMARA
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, "winelike" 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.

Materials and Methods

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 wereand 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).

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

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The pronase was from streptomyces griseus (lot 10165921001, Roche). The grape marc powder rich in polyphenols (64%) was provided by Grap'Sud Company (Cruviers-Lascours, France). The unstable young red wine (Cabernet sauvignon/Merlot, 2015) was provided by BioLaffort Company (Bordeaux, France). All other reagents were of analytical grade from Sigma-Aldrich (USA).

Hydrolysis of Acacia senegal gum and HIC-F1 fraction by pronase

A. senegal gum and HIC-F1 fraction were dissolved at a concentration of 10 mg.ml -1 in ultra pure deionised water (18.2 mΩ resistivity) containing 0.02% NaN 3 and stirred overnight at room temperature. The pH was adjusted at 6.5 using a small aliquot of NaOH solution before

to add pronase at a final concentration of 0.14 mg.ml -1 . The enzymatic hydrolysis occurred during 48h at 35°C. The enzymatic reaction was stopped by removing the pronase from the samples by centrifugation at 3000 rpm using a centricon Vivaspin 20 (cut-off of 50 000 Da).

The hydrolysis products were also removed by the centrifugation step. The samples were then washed four times with ultra pure deionised water (18.2 mΩ resistivity) by centrifugation at 3000 rpm using a centricon Vivaspin 20 (cut-off of 50 000 Da), before their freeze-drying.

Preparation of Acacia senegal gum and its HIC fractions

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

Electrophoretic mobility measurements

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

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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.

Multi-detector high performance size exclusion chromatography

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 LiNO 3 solution containing 0.02% NaN 3 at a flow rate of 1 ml.min -1 and 30°C.

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 (M w ) were calculated using ASTRA software 6.1.1.17 (Wyatt Technologies, Santa Barbara, CA). The data were analyzed using the Zimm's model (1 st 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.

Colloidal stability of hydro-alcoholic -mineral solution

The hydro-alcoholic -mineral solution was prepared with ultra pureultra-pure deionised water (18.2 mΩ resistivity) according to the International Oenological International Codex (O.I.V.). This solution It 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 ‰).

WhenThe addition of iron (Fe 3+ ) was added to the hydro-alcoholic -mineral solution ] is obtained. The hydro-alcoholicmineral solution was stirred during 2 h at room temperature before to adjust theits 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 CaCl 2 (22 g.L -1 ) prepared in ultra-pure deionised water (18.2 mΩ resistivity). The added Ca 2+ bound to K[Fe III Fe II (CN) 6 ] inducing the formation of "Prussian blue" precipitates (Ca[Fe III Fe II (CN) 6 ] 2 ).

Most of experiments were performed at a final CaCl 2 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 CaCl 2 solution.

Each measurement was triplicated.

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 ( 1Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 10 mol.L -1
). The solution was then filtered through a 0.45 µm regenerated cellulose membrane (GE Healthcare Life Sciences, Germany).

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

Each measurement was triplicated.

Characterization of the colloidal stability of hydro-alcoholic model solutions and young red wine

The colloidal stability of the hydro-alcoholic -mineral and hydro-alcoholic -grape marc solutions, and the young red wine was investigated using a Turbiscan Tower equipped with a pulsed near infrared light source (λ = 880 nm) and two synchronous detectors, a transmission (T) and a backscattering (BS) detectors (Formulaction, France). The samples were loaded into cylindrical glass tubes and scanned throughout its entire height. The transmittance (T, in %)

was measured every 1 min and 50 sec during 24 h at 25°C for the hydro-alcoholic -mineral solutions and during 48 h at 10°C for the hydro-alcoholic -grape marc solutions and young red wine.

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

∑ ∑ - - = i i i H h scan h scan h TSI ) ( ) (
Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

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The TSI corresponded to the transmittance variation at all measured position (h) throughout the entire height (H) of the sample between the scan i and the scan i-1 . The plot of TSI according to time corresponds to the kinetic behavior of the samples that takes into account all the physical phenomena (especially destabilization mechanism) occurring during the measurement.

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

Results

Colloidal stabilization mechanism of the hydro-alcoholic -mineral solution by A. senegal gum

The colloidal stability of the hydro-alcoholic -mineral solution (pH 3.1) containing A. characterized by a global negative charge in aqueous solution for pH ranging from 2.0 to 10.0 [START_REF] Burgess | Microelectrophoretic studies of gelatin and Acacia for the prediction of complex coacervation[END_REF]. Moreover, the negative AGPs µ E increased from pH 2.0 to ~ 5.0 in aqueous solutions reflecting the increase of the global negative charge [START_REF] Burgess | Microelectrophoretic studies of gelatin and Acacia for the prediction of complex coacervation[END_REF]Schmitt, Sanchez, Thomas & Hardy, 1999). Thus, the electrophoretic mobility (µ E ) of A. senegal gum in hydro-alcoholic solutions prepared at pH 3.1, 3.5 and 4.0 was measured and plotted according to [AG] critical (Figure 3B). A good correlation was evidenced between A. senegal gum µ E and its concentration necessary to stabilize the hydro-alcoholic -mineral solutions. The more the negative A. senegal gum µ E was, the less the A. senegal gum quantity was required to prevent the colloidal instability. Hence, negative charges carried by AGPs were certainly involved in this colloidal stabilization mechanism.

Since the colloidal instability of the hydro-alcoholic -mineral solution is induced by the addition of Ca 2+ , it was hypothesized that electrostatic interactions between Ca 2+ and negative charges of AGPs could play an important role in the stabilization mechanism. Therefore, we determined the [AG] critical in hydro-alcoholic -mineral solutions (pH 3.1) containing different final Ca 2+ concentrations (0.33 to 1.33 g.L -1 ). A close linear relationship between the

[AG] critical and the amount of Ca 2+ added in the hydro-alcoholic -mineral solutions was obtained (Figure 4). The increase of Ca 2+ concentration induced also the increase of

[AG] critical . Based on its structural and physicochemical properties, hyperbranched and negatively charged, AGPs from A. senegal gum appear as a natural mineral carrier that contains ~ 2-3.4 wt% of ash for this Acacia gum sample, and especially Ca 2+ [START_REF] Anderson | Specifications for gum Arabic (Acacia senegal); analytical data for samples collected between 1904 and 1989[END_REF]Debon & Tester, 2001;Mhinzi, 2003). Lamport and Varnai evidenced that Ca 2+ bound to AGPs with a fairly strong binding constant of 6.5×10 -6 mol.L -1 (Lamport & Varnai, 2013). They also showed that the Ca 2+ binding sites of AGPs

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

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were not naturally fully saturated by Ca 2+ . Hence, as expected, we can easily suggest that AGPs stabilized the hydro-alcoholic -mineral solutions by its electrostatic binding with Ca 2+ , the driver of the instability mechanism.

Identification AGPs from A. senegal gum involved in the colloidal stability mechanism of 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-F1was 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 (M w ) and high

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
15 molar mass (HM w ) macromolecules content (M w > 10 6 g.mol -1 ). HIC-F2 and HIC-F3 were richer in protein-rich high molar mass (HM w ) 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 -1 , 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-alcoholicmineral 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 w 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 w AGPs. To precise whether the macromolecular origin of the colloidal stabilizing properties is due to this fraction, its HM w AGPs were removed by an enzymatic hydrolysis treatment using the pronase, a protease mixture. Several studies showed the specific hydrolysis activity of pronase the HM w AGPs content from 3 to 1.7%, without affecting the quantity and the structural properties of LM w 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 -1 of hydrolyzed HIC-F1, a concentration twice more than the minimal HIC-F1 concentration necessary to totally prevent the colloidal instability ([HIC-F1] critical = 0.570 g.L - 1 ). Hence, the removal of less than 1.5% of AGPs from HIC-F1, corresponding to HM w AGPs, induced the loss of its colloidal stabilizing properties. The main contribution of protein-rich HM w AGPs was also confirmed by studying the colloidal stabilizing properties of hydrolyzed A. senegal gum by pronase. After the enzymatic treatment, the content of proteinrich HM w (M w > 10 6 g.mol -1 ) 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 -1 of hydrolyzed A. senegal gum was totally unstable while [AG] critical was found to be 0.114 g.L -1 . These results highlighted the major role of the protein-rich AGPs in the colloidal stabilizing mechanism of the hydroalcoholic -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 2+ . 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 proteinrich AGPs by a pronase treatment resulted in the loss of the interfacial properties of A.

senegal gum.

Colloidal stabilization of a hydro-alcoholic -grape marc solution and an unstable young red wine by AGPs from A. senegal gum

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

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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;[START_REF] Alcade-Eon | Color stabilization of red wines. A chemical and colloidal approach[END_REF]. 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

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

18 confirmed on an unstable young red wine (Figure 7C). They totally prevented the young red wine colloidal instability at 10°C for concentrations of 0.086 ± 0.007, 0.045 ± 0.006 and 0.015 ± 0.002 g.L -1 , respectively. Unlike the other two HIC fractions, HIC-F1 was not efficient for the colloidal stabilization of the young red wine even at a concentration of 2 g.L -1 (data not shown).

Discussion

The colloidal stabilizing properties of A. senegal gum and its HIC fractions isolated from Hydrophobic Interaction Chromatography were investigated on three colloidal hydroalcoholic matrices: a hydro-alcoholic -mineral matrix, a hydro-alcoholic -grape marc solution ("wine-like" matrix) and an unstable young red wine. Whatever the hydro-alcoholic matrix tested, the HIC fractions can be classified in the same following order according to their colloidal stabilizing efficiency: HIC-F1 << HIC-F2 < HICF3. A. senegal gum that is a mixture of the HIC fractions presented some intermediate properties between those of HIC-F1

and HIC-F2. Before its addition in young red wines,Usually, A. senegal gum added in young red wines is usually chosen based on its ability to stabilize a hydro-alcoholic -mineral matrix.

When we plot the critical concentrations of A. senegal gum and its HIC fractions determined in hydro-alcoholic -mineral solution as a function of those determined in hydro-alcoholicgrape marc solution or young red wine (Figure 8), a good correlation between both systems is obtained. The more the AGPs from A. senegal gum are efficient to stabilize the hydroalcoholic -mineral solution, the better they are to stabilize the polyphenols in young red wines. Hence, these results confirmed and strengthened the relevance and the use of the hydro-alcoholic -mineral test for the evaluation of A. senegal gum colloidal stabilizing properties before its addition in young red wine.

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

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In the hydro-alcoholic -mineral solution, the colloidal stability was assessed by the electrostatic binding of Ca 2+ , 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-Omethyl 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 (pK a ) 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 hydroalcoholic 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 Ca 2+ with the carboxyl group of two uronic acids of a conserved O-Hyplinked 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 Ca 2+ 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

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

M A N U S C R I P T A C C E P T E D

ACCEPTED MANUSCRIPT 20 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 (LM w ) AGPs poor in proteins (Renard et al. 2014). This enzymatic treatment gave rise to macromolecules presenting similar molar masses (M w from 1.8 to 6.0x10 5 g.mol -1 ) and conformational properties as low molar mass (LM w ) AGPs naturally found in A. senegal gum (Connolly et al., 1988, Randall et al., 1989[START_REF] Aoki | Characterization and properties of Acacia senegal (L.) Willd. Var. senegal with enhanced properties (Acacia (sen) SUPER GUM (TM): Part 2-Mechanism of the maturation process[END_REF], 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 Ca 2+ 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 αarabinofuranosidase 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 Ca 2+ 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.

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

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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

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

22 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,

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 23
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.

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 Ca 2+ , the driver of the instability.

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176 M w (g.mol -1 ) 6.8 × 10 5 3.5 ×10 5 1.5 ×10 6 1.6 ×10 6

High M w content (%) a 14 3 73 56

a The high M w corresponded to macromolecules with a M w upper than 10 6 g.mol -1 .

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

senegal

  gum was followed by measuring at 25°C the transmittance (T) of the solutions during 24h using a light scattering equipment. The figure1showed the temporal change in transmittance (∆T) of three hydro-alcoholic -mineral solutions without and with 0.09 and 0.14 g.L -1 of A. senegal gum (Figure1A, 1B and 1C). After 24h, the transmittance (T) of the hydro-alcoholic -mineral solutions without and with 0.09 g.L -1 of A. senegal gum increased respectively by 60% and 35% in the middle of the sample (cell height: 2 -36 mm) reflecting a clarification of the solution. This phenomenon was confirmed by the loss of the blue color of the solutions after 24h (Figure1A and 1B). The clarification resulted from the formation of a salt precipitate (Ca[Fe III Fe II (CN) 6 ] 2 ) between K[Fe III Fe II (CN) 6 ] and Ca 2+ that pelleted, as evidenced by the decrease of T by 5% and 2% after 24h in the bottom of the cell (cell height:Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. (2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019) 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-alcoholicmineral solution was analyzed by plotting the TSI (Turbiscan Stability Index) according to time (Figure2A). 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 hydroalcoholic -mineral colloidal stability was determined by plotting the TSI values after 24h according to A. senegal gum concentration (stability curve) (Figure2B). 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 Ca 2+ 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 (Figure2B). The colloidal stabilization mechanism was further investigated by modifying the physicochemical condition as the pH of the solutions and the amount of Ca 2+ 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 Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. (2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 13 stabilizing properties of A. senegal gum were greatly influenced by pH and enhanced with its increase in this pH range. AGPs from A. senegal gum are weakly charged glycoproteins

  for protein-rich HM w AGPs from A. senegal gum without degrading and affecting the structural properties of low M w (LM w ) 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 w distribution of HIC-F1 before and after the enzymatic treatment are shown in Figure 6A. The pronase hydrolyzed the protein-rich AGPs decreasing Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. (2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019

  .,Al-Assaf, S., Axelos, M., Phillips, G.O., & Anton, M. (2010). Hydrocolloids with emulsifying capacity. Part-2 Adsorption properties at the n-hexadecane-water interface.FoodHydrocolloids, 24, 121-130. Charpentier, C., Escot, S., Gonzalez, E., Dulau, L., & Feuillat, M. (2004). Influence of yeast glycosylated proteins on tannins aggregation in model solution. Journal International des Sciences de la Vigne et duVin, 38, 209-218. Chung, C., Rojanasasithara, T., Mutilangi, W., & McClements, D.J. (2016). Enhancement of colour stability of anthocyanins in model beverages by gum Arabic addition. FoodChemistry, 201, 14-22. Connolly, S., Fenyo, J.C., & Vandevelde, M.C. (1988). Effect of a proteinase on the macromolecular distribution of Acacia senegal gum. Carbohydrate Polymers, 8, 23-32. Debon, S.J.J., & Tester, R.F.(2001). In vitro binding of calcium, iron and zinc by non-starch polysaccharides. FoodChemistry, 73, 401-410. Dickinson, E., Murray, B.S., Stainsby, G., & Anderson, D.M.W. (1988). Surface activity and emulsifying behavior of some Acacia gums. FoodHydrocolloids, 2, 477-490. Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. FoodHydrocolloids, 17, 25-39. de Freitas, V., Carvalho, E., & Mateus, N. (2003). Study of carbohydrate influence on proteintannin aggregation by nephelometry. Food Chemistry, 81, 503-509. Dupin, I.V.S., Stockdale, V.J., Williams, P.J., Jones, G.P., Markides, A.J., & Waters, E.J. (2000). Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: evaluation of extraction methods and immunolocalization. Journal of Agricultural and Food Chemistry, 48, 1086-1095. Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. (2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019., Al-Assaf, S., Phillips, G.O., & Williams, P.A. (2008). Studies on Acacia exudate gums: Part VI. Interfacial rheology of Acacia senegal and Acacia seyal. Food Hydrocolloids, 22, 682-689. Escot, S., Feuillat, M., Dulau, L., & Charpentier, C. (2001). Release of polysaccharides by yeasts and the influence of released polysaccharides on colour stability and wine astringency. Australian Journal of Grape and Wine research, 7, 153-159. Flindt, C., Al-Assaf, S., Phillips, G.O., & Williams, P.A. (2005). Studies on Acacia exudate gums: Part V. Structural features of Acacia seyal. Food Hydrocolloids, 19, 687-701. Gerbaud, V., Gabas, N., Blouin, J., & Crachereau, J.C. (2010). Stuy of wine acid salt stabilization by addition of carboxymethylcellulose (CMC) : comparison with the « protective colloids » effect. Journal International des Sciences de la Vigne et du Vin, 44, 231-242. Gordillo, B., Cejudo-Bastante, M.J., Rodríguez-Pulido, F.J., Jara-Palacios, M.J., Ramírez-Pérez, P., González-Miret, M.L., & Heredia, F.J. (2014) Impact of adding white pomace to red grapes on the phenolic composition and color stability of syrah wines from a warm climate. Journal of Agricultural and Food Chemistry, 62, 2663-2671. Grein-Iankovski, A., Ferreira, J.G.L., Orth, E.S., Sierakowski, M.R., Cardoso, M.B., Simas, F.F., & Riegel-Vidotti, I.C. (2018). A comprehensive study of the relation between structural and physical chemical properties of acacia gums. Food Hydrocolloids, 85, 167-175. Guadalupe, Z., Palacios, A., & Ayestaran, B. (2007). Maceration enzymes and mannoproteins: A possible strategy to increase colloidal stability and color extraction in red wines. The Journal of Agricultural and Food Chemistry, 55, 4854-4862. Guadalupe, Z., & Ayestaran, B. (2008). Effect of commercial mannoprotein addition on polysaccharide, polyphenolic, and color composition in red wines. The Journal of Agricultural and Food Chemistry, 56, 9022-9029.Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. (2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019

Figure 1 .Figure 4 .Figure 5 .Figure 6 .Figure 8 .

 14568 Figure 1. Transmittance changes of hydro-alcoholic -mineral solutions without (A) and with 0.09 (B) and 0.14 g.L -1 (C) of A. senegal gum. The transmittance was registered at 25°C

-

  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 Ca 2+ binding

  Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. (2019). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

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	senegal trees of different age and location using multidetection gel permeation continuum of molecular species differing by their protein to sugar ratio, molecular weight, arabic. Food Hydrocolloids, 1, 291-300.
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Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. (

2019

). 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, 97. , DOI : 10.1016/j.foodhyd.2019.105176

.

  Biochemical and structural properties of Acacia senegal gum (AG),

		ACCEPTED MANUSCRIPT
	Supplementary table 1and its HIC fractions.
		AG	HIC-F1	HIC-F2	HIC-F3
	Total dry matter (mg.g -1 )	893.4	921.6	926.2	921.9
	Sugars (mg.g -1 )	944.4	961.3	918.3	813.0
	Galactose	363.6	374.9	315.9	270.7
	Arabinose	281.4	257.6	326.9	311.4
	Rhamnose	120.9	120.2	125.8	113.0
	Glucuronic acid	168.1	195.1	143.3	111.4
	4-O-Me-Glucuronic acid	9.4	13.5	5.5	5.7
	Proteins (mg.g -1 )	21.5	4.9	63.1	137.7
	Ash (mg.g -1 )	34.1	30.5	19.3	49.3
					40

Acknowledgements

Version postprint

Comment citer ce document : Nigen, M., Apolinar Valiente, R., Iturmendi, N., Williams, P., Doco, T., Moine, Massot, Jaouen, I., Sanchez, C. ( 2019). The colloidal stabilization of young red wine by Acacia senegal gum:

The involvement of the protein backbone from the protein-rich arabinogalactan-proteins.