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

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Usually, Acacia senegal gum is chosen based on its efficiency to stabilize iron 28 hexacyanoferrate salts in hydro-alcoholic solution. In this study, the protective activity of 29 Acacia senegal gum and its three macromolecular fractions (HIC-F1, HIC-F2 and HIC-F3), Oenological Codex: COEI-1- GOMARA, 2000). This "efficacy test" consists in determining 124 the quantity of A. senegal gum required to prevent the flocculation of a colloidal iron 125 hexacyanoferrate solution in hydro-alcoholic medium by calcium salt. Hence, the A. senegal 126 gum efficiency towards wine colloidal instability is usually evaluated using a hydro-alcoholic 127 -mineral solution which seems in appearance far enough from wine matrices on their 128 biochemical and physicochemical properties. The colloidal stabilization of two so different 129 matrices by A. senegal gum raises some questions on the colloidal stability mechanism and 130 especially on the AGPs at the origin of this colloidal stabilization. 131 The aim of this study was to investigate the colloidal stabilizing properties of AGPs from A.  (Table S1).  A. senegal gum and HIC-F1 fraction were dissolved at a concentration of 10 mg.ml -1 in ultra 156 pure deionised water (18.2 mΩ resistivity) containing 0.02% NaN 3 and stirred overnight at 157 room temperature. The pH was adjusted at 6.5 using a small aliquot of NaOH solution before 158 to add pronase at a final concentration of 0.14 mg.ml -1 . The enzymatic hydrolysis occurred 159 during 48h at 35°C. The enzymatic reaction was stopped by removing the pronase from the 160 samples by centrifugation at 3000 rpm using a centricon Vivaspin 20 (cut-off of 50 000 Da). 161 The hydrolysis products were also removed by the centrifugation step. The samples were then 162 washed four times with ultra pure deionised water (18.2 mΩ resistivity) by centrifugation at 163 3000 rpm using a centricon Vivaspin 20 (cut-off of 50 000 Da), before their freeze-drying. 164 165  The hydro-alcoholic -grape marc solution (wine-like medium) was prepared with ultra-pure 219 deionised water (18.2 mΩ resistivity). It contained tartaric acid (2.7 g.L -1 ), potassium sulfate 220 (0.9 g.L -1 ), grape marc powder (3 g.L -1 ) and ethanol (12% v/v). The solution was gently 221 stirred during 2 h at room temperature before to adjust the pH at 3.5 using NaOH solution (1 The colloidal destabilization of the hydro-alcoholic -grape marc solution and the young red 225 wine was induced by cooling the solutions to 10°C. The efficiency of A. senegal gum and its 226 HIC fraction to prevent the destabilization of the hydro-alcoholic -grape marc solution and 227 the young red wine was studied at 10°C by varying their concentration between 0 and 2 g.L -1 . 228

Preparation of Acacia senegal gum and its HIC fractions
Each measurement was triplicated. 229 progressively before to reach a constant TSI value (≤ 2) similar to the control without Ca 2+ for 286 A. senegal gum concentration upper than 0.11 g.L -1 . Based on the stability curve, the 287 [AG] critical was 0.114 ± 0.002 g.L -1 ( Figure 2B). 288 The colloidal stabilization mechanism was further investigated by modifying the 289 physicochemical condition as the pH of the solutions and the amount of Ca 2+ added to induce 290 the colloidal instability mechanism. The stability curves of hydro-alcoholic -mineral 291 solutions prepared at pH 3.1, 3.5 and 4.0 are shown in Figure 3A. As the pH increased from A. senegal gum µ E and its concentration necessary to stabilize the hydro-alcoholic -mineral 304 solutions. The more the negative A. senegal gum µ E was, the less the A. senegal gum quantity 305 was required to prevent the colloidal instability. Hence, negative charges carried by AGPs 306 were certainly involved in this colloidal stabilization mechanism. 307 Since the colloidal instability of the hydro-alcoholic -mineral solution is induced by the 308 addition of Ca 2+ , it was hypothesized that electrostatic interactions between Ca 2+ and negative 309 charges of AGPs could play an important role in the stabilization mechanism. Therefore, we 310 Varnai evidenced that Ca 2+ bound to AGPs with a fairly strong binding constant of 6.5×10 -6 319 mol.L -1 (Lamport & Varnai, 2013). They also showed that the Ca 2+ binding sites of AGPs Hence, the colloidal stability properties of A. senegal gum towards the hydro-alcoholic -356 mineral solution were mainly due to its two minor fractions in weight, HIC-F2 and HIC-F3 357 (around 11 wt% of whole A. senegal gum), that were especially richer in protein-rich HM w 358 AGPs than HIC-F1 (Table 1). In order to correlate the results obtained on the hydro-alcoholic -mineral solution to the wine 396 matrix, the stabilizing properties of A. senegal gum and its HIC fractions were characterized 397 on an unstable synthetic hydro-alcoholic -grape marc solution (wine-like medium) and an 398 unstable young red wine. 399 The hydro-alcoholic -grape marc solution and the young red wine that were stable at 25°C 400 As observed in the hydro-alcoholic -mineral solution, the protein-rich AGPs from A. senegal 495 gum, HIC-F2 and HIC-F3 fractions, were also found to be the most effective for the colloidal 496 stabilization of the hydro-alcoholic -grape marc solution and the unstable young red wine. 497 The surface and colloidal stabilizing properties of the protein-rich AGPs were previously 498 demonstrated in other type of matrices. After A. senegal gum fractionation by size exclusion 499 chromatography (SEC) and hydrophobic interaction chromatography (HIC), Ray et al. 500 showed that the protein-rich AGPs made the best emulsions in model beverage (Ray,Bird,501 Iacobucci & Clark, 1995). Protein-rich AGPs were also found to be the most effective in 502 decreasing the interfacial tension of n-hexadecane-water interface (Castellani, Al-Assaf, 503 Axelos, Phillips & Anton, 2010), to preferentially adsorb at the interface of latex dispersions 504 (Snowden, Phillips & Williams, 1987) and oil droplets (Randall, Phillips & Williams, 1988), 505 and to stabilize carbon nanotube dispersions (Li, Zhang, Jin & Cai, 2018). 506 The stabilizing properties of the protein-rich AGPs towards such a chemical diversity of 507 molecules (minerals, polyphenols, proteins, oils, latex, etc…) could be relied to their intrinsic 508 properties. Structurally, the three HIC fractions seems not so different. AGPs from these 509 It is useful to remember that AGPs are constituted by a protein backbone covalently linked to 540 hyperbranched carbohydrate blocks that partially hindered it from its environment. Therefore, 541 if we consider the protein as the major component for the colloidal stabilizing properties of 542 AGPs, it would seem appropriate to consider not only the protein content but also its 543 accessibility to its environment that is closely link to the rate of glycosylation. HIC-F1,

Conclusion 556
In this research, the colloidal stabilizing properties of arabinogalactan-proteins from A. 557 senegal gum in hydro-alcoholic -mineral and hydro-alcoholic -polyphenols solutions were 558 investigated. The AGPs prevented the colloidal instability of both calcium iron 559 hexacyanoferrate salts in "model" hydro-alcoholic solution and polyphenols in young red 560 wine. A good relationship was evidenced between the stabilizing properties of AGPs 561 determined in these two hydro-alcoholic solutions. The protein moiety of the AGPs appeared 562 to be essential for these functional properties whatever the hydro-alcoholic solutions. The 563 more the AGPs were rich in proteins, the more their colloidal stabilizing efficiency were. In 564 the hydro-alcoholic -mineral solution, the AGPs avoided the precipitation of potassium 565 ferrocyanide salts by their electrostatic binding with Ca 2+ , the driver of the instability.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
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 Ca 2+ binding