Polysaccharide Interactions and Their Impact on Haze

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Protein/Polysaccharide Interactions and Their Impact on Haze Formation in White Wines Marie Dufrechou, Thierry Doco, Céline Poncet-Legrand, Francois-Xavier Sauvage, and Aude Vernhet J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02546 • Publication Date (Web): 19 Oct 2015 Downloaded from http://pubs.acs.org on October 20, 2015

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Journal of Agricultural and Food Chemistry

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Protein/polysaccharide

Interactions

and

Their

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Impact on Haze Formation in White Wines

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Marie Dufrechou1,2,3,4, Thierry Doco1, Céline Poncet-Legrand1,2,3, François-

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Xavier Sauvage1,2,3, Aude Vernhet1,2,3 1

INRA, UMR1083 SPO, F-34060 Montpellier, France

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Montpellier SupAgro, UMR1083 SPO, F-34060 Montpellier, France

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Université Montpellier I, UMR1083 SPO, F-34060 Montpellier, France

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Present address : LUNAM Université, SFR 4207 QUASAV, Groupe ESA, UPSP GRAPPE,

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55 rue Rabelais BP 30748, F-49007 Angers Cedex 01, France

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* Corresponding author:

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Marie Dufrechou

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Present address : LUNAM Université, SFR 4207 QUASAV, Groupe ESA, UPSP GRAPPE, 55

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rue Rabelais BP 30748, F-49007 Angers Cedex 01, France

[email protected]

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ACS Paragon Plus Environment

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Abstract

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Proteins in white wines may aggregate and form hazes at room temperature. This was

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previously shown to be related to pH-induced conformational changes and to occur for pH

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lower than 3.5. The aim of the present work was to study the impact of wine polysaccharides

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on pH-induced haze formation by proteins but also the consequences of their interactions with

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these proteins on the colloidal stability of white wines. To this end, model systems and

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purified global pools of wine proteins and polysaccharides were used first. Kinetics of

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aggregation, proteins involved and turbidities related to final hazes were monitored. To

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further identify the impact of each polysaccharide, fractions purified to homogeneity were

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used in a second phase. These were: 2 neutral (mannoprotein and arabinogalactan

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polysaccharides) and 2 negatively charged (rhamnogalacturonan II dimer (RG-II) and

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arabinogalactan polysaccharides). We highlighted that the impact of major wine

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polysaccharides on wine protein aggregation at room temperature was clearly less marked

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than those of the pH and the ionic strength. Polysaccharides modulated the aggregation

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kinetics and final haziness, indicating that they interfere with the aggregation process, but

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could not prevent it.

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arabinogalactan-proteins,

haze,

mannoproteins,

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Keywords:

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interactions, rhamnogalacturonan II dimer, wine proteins

protein/polysaccharide

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Introduction

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One of the major defects in bottled white wines is the formation of haze or deposits that can

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appear during transport or storage. It is often related to exposure at high temperatures but can also

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develop in properly stored wines.1-3 Haze or deposit formation is due to the aggregation of

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proteins that resist winemaking conditions (concentration usually found between the range 15-

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330 mg.L-1).4-6 These proteins mostly originate from grapes and belong to four different families:

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invertase, β-Glucanases, chitinases and thaumatin-like proteins (TLPs).7, 8 TLPs and chitinases

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are the most abundant and different isoforms can be found within each family. Previous works

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indicated their different sensitivity to heat induced unfolding and aggregation.8, 9 β-Glucanases

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and chitinases were found to be the most sensitive proteins, whereas invertase and TLPs were

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more resistant.8-10

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strongly affect the stability of wine proteins and the final haze induced by their aggregation are

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the pH and the ionic strength.11-13 The impact of the pH, studied for a range 2.5 - 4.0, was

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shown to be temperature-dependent. At ambient or at lower temperatures (below 25 °C),

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protein conformational changes induced by low pHs (≤ 3.2) were shown to be involved in the

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aggregation of some of the wine proteins.11

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The impact of non-protein compounds such as polysaccharides, polyphenols and sulfate in the

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development of heat-induced protein hazes has also been demonstrated. Like the ionic strength,

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phenolic compounds and sulfate enhance aggregate growth and significantly increase final haze.1,

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11, 13-16

Besides the temperature,4,

8

the two other physico-chemical factors that

By contrast, some specific and quantitatively minor mannoprotein or arabinogalactan

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protein (AGP) fractions, purified from wine, were shown to decrease heat-induced protein hazes

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and were thus considered as protective factors.17,

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specific mannoprotein fractions purified from yeast cell walls.19-22 However, the exact structural

18

Similar results were obtained with other


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features and mechanisms involved have not been clearly elucidated yet. In addition, a protective

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effect was only observed when these purified polysaccharides were added to the wine at

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relatively high polysaccharide to protein ratios (1/1 in mass or higher). Thus, even if naturally

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present in wines, it is at concentrations such that this protective effect is to be confirmed. Other

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studies on protein stability at room temperature also indicated that non-protein compounds in

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wine modulate the haze resulting from protein aggregation,11 and the presence of polyphenols

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and polysaccharides has been shown in a natural precipitate.1 If polyphenols, and especially

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tannins, are considered as having a triggering effect on protein aggregation in wine, 16, 23 the role

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of wine polysaccharides is not clear and has not been fully investigated yet. Their interactions

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with wine proteins deserve to be further explored.

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Polysaccharides are present in white wine at concentrations ranging from 150 to 500 mg.L-1.24

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They originate from the grape berry (pectic polysaccharides) or from yeast cell walls

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(mannoproteins). Mannoproteins have molecular weights ranging from 50 to 500 kDa.25 Most of

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them are almost neutral however fractions with different charge density have been separated from

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a mannoprotein pool purified from a red wine.26 Pectic polysaccharides are mainly

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rhamnogalacturonan II (RG-II) and polysaccharides rich in arabinose and in galactose (PRAGs).

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RG-II is mostly found in its dimer form (9.5-10.5 kDa),27 and its negative charge is strongly

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influenced by the pH within wine pH-range.28 PRAGs include arabinans, arabinogalactans and

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arabinogalactan proteins (AGP). Arabinogalactans and AGP have molecular weights between 50

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and 250 kDa. As for mannoproteins, fractions with different charge densities were evidenced.25, 28

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The aim of the present work was to determine the impact of wine polysaccharides on protein

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aggregation, considering first interactions at room temperature with the impact of the pH and of

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the ionic strength. In addition to its influence on the charge and stability of wine proteins, the pH

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strongly affects the charge of some polysaccharide fractions.28 Wine proteins and acidic pectic


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polysaccharides carry opposite charges within white wine pH range (2.8 -3.5)8,

28, 29

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attractive electrostatic interactions between these macromolecules can be expected. These

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electrostatic interactions are modulated by the pH value but also by the ionic strength.30

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Interactions were first studied in model solutions with proteins and polysaccharides purified from

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a Sauvignon blanc wine. In a second phase, experiments were focused on four specific

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polysaccharides: a neutral mannoprotein (MP0), a neutral arabinogalactan-protein (AGP0), an

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acidic arabinogalactan (AGP4) and a rhamnogalacturonan II dimer (RG-II). In both experiments,

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protein and polysaccharide concentrations were increased by comparison to that found in wines

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to accelerate aggregation rates and to observe the impact of polysaccharides on aggregation

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within 15 days.

so that

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Materials and Methods

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Wines. The two Sauvignon blanc wines used for this study were elaborated at the Pech

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Rouge Experimental Unit (INRA, Gruissan, France) in 2009 (Sa1) and 2011 (Sa2). The wine

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was elaborated using classic winemaking steps. Following the fermentation, the wine was

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cold stabilized (to prevent the crystallization of tartaric salts) and clarified by filtration on a

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1 µm filtration cartridge. No enzymatic treatment and no bentonite fining were performed to

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preserve protein and polysaccharide content. Conventional enological parameters were

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analyzed according to the Vine and Wine International Organisation methods and are given

111

in Table 1. A heat test (80 °C, 30 min)4,

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protein instability. A turbidity of 10 and 33 NTU (HI88703 turbidimeter, Hanna Instrument

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Inc.) was obtained for the Sa1 and the Sa2 wines, respectively.

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was performed on both wines to assess their

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Purification of proteins and polysaccharides. Wines (3 L) were first treated with

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polyvinylpolypyrrolidone (150 mg.L-1, Sigma) during 24 h and under constant stirring to

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remove polyphenols. Wine proteins were then isolated and purified by ion exchange

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chromatography, using a cation exchange Streamline SP gel (74 mL, GE Healthcare) and a

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350 mm length*25 mm diameter column (GE healthcare), according to the method described

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previously. 11 The wine flow rate was set to 10 mL.min-1. Elution was performed at a flow

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rate of 5 mL.min-1, using 2 solutions (solution A: 13 mM tartrate buffer at pH 4.0 and

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solution B : 0.5 M NaCl in 13mM tartrate buffer pH 4.0) with the following gradient: 0-40

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min, 100% of solution A; 40-70 min, from 100% of solution A to 100% of solution B; 70-80

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min, 100% of solution B. The protein-containing fractions were identified using a UV

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detector at a wavelength of 280 nm and were pooled. Salt removal was achieved by

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extensive diafiltration (5 kDa membrane, Amicon, Millipore) using a 13 mM tartrate buffer

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at pH 4.0. The protein pools were then stored at -20 °C before use. Freeze-drying of 1 mL

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aliquots and weighing allowed us to determine the protein concentration in these stock

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solutions and to estimate that of the initial wines. The protein concentration of Sa1 and Sa2

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were respectively estimated of being 160 mg.L-1 and 150 mg.L-1.

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The total polysaccharide pool was obtained by ultrafiltration of the protein-free Sa1 wine (wine

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recovered after the column separation). To purify sample, by removing small solutes, diafiltration

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was performed using a 200 mL stirred cell equipped with a 5 kDa membrane (Amicon,

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Millipore). 200 mL of the protein-free wine were diafiltrated with pure water, up to a final

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dilution factor of small solutes (< 5 kDa) of 1600. The final polysaccharide pool was lyophilized

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and stored in a dry place.

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Protein analyses in the wines and in the protein pools. Protein analyses in wines and in

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the corresponding pools were performed by 1D SDS-PAGE. The protein concentration was

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adjusted to 0.8 g.L-1 before analysis, either by concentration using 3.0 kDa centrifugal filter

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units (Millipore) for the wines or by simple dilution (pools). Proteins (30 µg) in Laemmli

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buffer were separated on a 14% acrylamide resolving gel (gel length, 60 mm). A low

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molecular weight calibration kit (14.4 to 97 kDa, Pharmacia, Biotech) was included in each

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electrophoretic run. Gels were stained with 0.1% Coomassie Brilliant Blue R-250 (Biorad)

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in 40% of ethanol, 10% acetic acid and destained overnight in 10% acetic acid. Gels were

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then scanned at 300 dpi with an image scanner (GE Biosciences). Image analysis was carried

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out with the Totallab software (Nonlinear Dynamics Ltd) and was used to calculate the

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proportion of proteins in each stained band.8

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Carbohydrate composition of wine polysaccharides and of the purified polysaccharide

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pool. The polysaccharide compositions in the Sa1 wine and in the corresponding total

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polysaccharide pool were determined according to the method described by Doco et al.31 The

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neutral glycosyl-residue composition was determined by gas chromatography after hydrolysis

154

and conversion of monosaccharides into their alditol-acetate derivatives. The different alditol

155

acetates were identified on the basis of their retention time by comparison with standard

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monosaccharides. Neutral sugar amounts were calculated relative to two internal standards (myo-

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inositol and β-D-allose). Results represent an average of 2 experiments. Concentrations of

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mannoproteins (MP), rhamnogalacturonans II (RG-II) and polysaccharides rich in arabinose and

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galactose (PRAGs) were calculated on the basis of the neutral sugar composition using the

160

formula established previously.32


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Specific polysaccharide fractions. Four polysaccharide fractions purified to homogeneity

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and characterized at the UMR Sciences for Enology (INRA, Montpellier)25 were also used.

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These fractions were: a rhamnogalacturonan II (RG-II, acidic), a mannoprotein fraction 0

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(MP0, neutral), a type II arabinogalactan 0 (AGP0, neutral) and a type II arabinogalactan 4

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(AGP4, acidic). Their glycosyl-residue composition and their charge density are given in

167

Table 2.

168 169

Protein stability in model systems. Model solutions. Model solutions were used to study

170

the impact of polysaccharides on protein stability. Composition of the model solutions was

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as follows: 12% ethanol, 7 g.L-1 glycerol and 2 g.L-1 tartaric acid. Their pHs were adjusted at

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2.5, 3.0, 3.2, 3.5 and 4.0 with HCl 1 M or NaOH 1 M and their ionic strength at 0.02 M or

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0.15 M with NaCl. To obtain these final values, buffers at given pH and ionic strength were

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prepared with a concentration factor of 1.5 to get the required composition following the

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addition of the protein solution in 13 mM tartaric buffer at pH 4.0. Buffers were stored at 4

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°C. Lyophilized polysaccharides were dissolved in the buffer solutions (1.5 mL) and filtered

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on 0.2 µm membranes before addition of the protein solution (0.75 mL, concentration factor

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3 to get the final required protein content).

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Aggregation kinetics. DLS experiments were carried out with a Malvern Autosizer 4700 (40

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mW He-Ne laser, λ = 633 nm, APD detection, Malvern Instruments, Malvern, UK) at an

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angle of 90° from the incident beam. Aggregation kinetics were followed by measurements

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of the scattering intensity (Is) and of the hydrodynamic diameter (Dh) of particles. Each

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measurement represented the average of 10 sub-runs and each kinetic was done in duplicate.


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The autocorrelation function of the scattered light was analyzed using the cumulant method,

185

which gives an average value of the aggregate hydrodynamic diameter (Dh) and the

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polydispersity index PI of the dispersion (0 < PI < 1). Studies were performed at 25 °C

187

during 24 hours to observe aggregation and follow their kinetics for unstable samples.

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Turbidity measurements and proteins involved in aggregation. Other samples were

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prepared in the same conditions to measure the turbidity and to determine protein

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precipitation after 15 days storage at room temperature (20 °C). Turbidity measurements

191

were performed by measuring the absorbance at a wavelength of 720 nm (Safas UVmc²

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spectrophotometer, Monaco, France). Samples were then centrifuged to remove aggregates

193

(if present) and 1D SDS-PAGE analyses were performed on the supernatants using the same

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method as detailed above. Centrifugal filter units (3.0 kDa, Millipore) were used to decrease

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the ionic strength to a value about 0.05 M. Results were compared with the protein profiles

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of the initial purified pool.

197

198

Results and discussion

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Wine protein and polysaccharide composition. The composition of the two purified protein

200

pools is compared to those of the corresponding wines in Figures 1A and 1B. Analyses and

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identification of the proteins in the Sa1 wine and in the protein pool were performed in a previous

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work.11 Four protein species were observed: thaumatin-like proteins and chitinases (band 2 to 7,

203

being the main ones), β-Glucanases (bands a and b) and invertase (band 1). Bands a and b were

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lost during the purification steps.11 A similar result was obtained for Sa2: main proteins were

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found within the range 19-28 kDa (TLPs and chitinases, bands 2 to 7).


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The polysaccharide composition of the Sa1 wine is detailed in Table 3. The monosaccharide

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composition corresponded to that of a white wine, with a total calculated concentration of 228

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mg.L-1 including rhamnogalacturonans II (38 mg.L-1), polysaccharides rich in arabinose and

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galactose (PRAGs, 41 mg.L-1) and mannoproteins (149 mg.L-1). The arabinose/galactose ratio

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was calculated at 0.48, which is usual in white wines (common ratios ranging from 0.3 to 0.8). 33

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After purification, 84.2 % of polysaccharides were recovered. Loss mainly concerned PRAGs

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(31% losses against 8% for RG-II and 11% for mannoproteins). The arabinose/galactose ratio

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was not strongly modified and mannoproteins were the major polysaccharides in both the initial

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wine (65.3%) and the purified pool (69.3%).

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Impact of wine polysaccharides on protein stability. Proteins purified from the Sa1 wine

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were used to study the overall impact of wine polysaccharides on their stability at room

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temperature. The pH was varied between 2.5 and 4.0 and two ionic strengths were studied:

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0.02 and 0.15 M. Previous results indicated a strong impact of these two parameters on wine

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protein stability at 25°C and below.11 Briefly, we showed that wine proteins remain stable at

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pH 4.0 and aggregation occurs from pH values lower than 3.5. We observed that aggregation

222

is enhanced when the pH is decreased and reaches a maximum at pH 2.5, far from protein

223

isoelectric points. Aggregation kinetics are strongly influenced by the ionic strength.

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Unstable proteins are found within the range 22-28 kDa and are mainly chitinases and some

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TLP isoforms. Later experiments12 with purified stable and unstable isoforms (two unstable

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chitinases, one stable TLP and invertase) evidence pH-induced conformational changes of the

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unstable proteins at room temperature. These modifications affect the global shape (tertiary


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structure) of the unstable chitinases but not the protein secondary structure. Though only local,

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they lead to the exposure of hydrophobic sites, which likely favor their aggregation.12

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The same conditions were selected in the present work to check the possible involvement of

231

electrostatic interactions between proteins and polysaccharides on the colloidal equilibrium of

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white wines. Some polysaccharides carry a high negative charge at wine pH (RG-II and some

233

minor PRAG structures), whereas others are mostly neutral or only carry small negative charge

234

(MP and most of the PRAGs)28. For those negatively charged, the occurrence of attractive

235

electrostatic interactions with positively charged proteins can lead to aggregation and

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precipitation (colloidal instability) irrespective to pH-induced protein aggregation.34 Considering

237

the isoelectric point of wine proteins and the impact of the pH on the charge carried by negatively

238

charged polysaccharides,28 such electrostatic attractions may occur within the whole tested pH

239

range and are expected to be the largest around pH 3.2 to 3.5. Besides their co-aggregation,

240

interactions between proteins and polysaccharides may have quite different consequences in the

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considered system: (i) formation of stable protein/polysaccharide complexes reducing the

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aggregation rate of pH unstable proteins and (ii) interactions between polysaccharides and protein

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aggregates preventing (formation of a protective layer) or enhancing (cross-linking between

244

several aggregates) their growth.34-36

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The behavior of proteins/polysaccharides mixtures were studied at a protein/polysaccharide ratio

246

of 1:1, and compared to that of the proteins alone. To accelerate aggregation kinetics, protein and

247

polysaccharide concentrations in model systems were set to 0.8 g.L-1. The stability of the

248

different systems was followed by DLS during 24 hours and haze was evaluated by visual

249

observation and turbidity measurement after 15 days. First, the stability of wine polysaccharides

250

in model systems was checked. Whatever the pH and the ionic strength, no aggregation was


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detected and polysaccharides, as expected, were stable in the tested conditions. The intensity

252

scattered by polysaccharides remained constant in the order of 200 kcounts.s-1. DLS results

253

obtained at 0.02 and 0.15 M with proteins and protein/polysaccharide mixtures (at pH 2.5, 3.0

254

and 3.2) are given in Figures 2 and 3, respectively. The whole results, including also those

255

obtained at pH 3.5 and 4.0, are summarized in Figure 4.

256 257

For proteins alone and for protein/polysaccharide mixture at pH 4, no aggregation was observed

258

whatever the ionic strength. At pH 3.5 at 0.15 M: the intensity scattered remained low (Figure 4)

259

and did not evolve during the experiment (not shown). The intensity scattered by the

260

protein/polysaccharide mixtures (IP+PS) was closed to the sum of the intensities scattered by the

261

macromolecule solutions considered separately (IP + IPS). This is typical of mixtures where

262

biopolymers behave independently. Despite the presence of species carrying opposite charges,

263

the Sa1 proteins and polysaccharides formed stable colloidal systems, even at the lowest ionic

264

strength, were electrostatic interactions are not expected to be screened. It indicated that if there

265

were attractive interactions between some polysaccharides and proteins, these latter neither

266

induced the formation of complexes much larger than the free species nor aggregation. This was

267

confirmed by long-term (15 days) measurements of the turbidity (Table 4). Protein aggregation

268

was observed from a pH ≤ 3.2 at 0.02 M and from a pH ≤ 3.0 at 0.15 M. At 0.02 M, aggregation

269

occurred immediately (Figure 2): lowering the pH induced an immediate increase in scattering

270

intensity, related to the formation of “polydisperse” colloidal particles with mean hydrodynamic

271

diameters Dh between 200 and 300 nm. After that, IS did not evolve strongly whereas aggregate

272

size kept increasing regularly. This behavior indicates a very quick aggregation, followed by

273

interactions between the aggregates and particle growth. As the pH decreased, aggregation was

274

strongly enhanced.12 It is important to note that except at pH 3.2, the intensities scattered by the


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protein/polysaccharide mixtures IP+PS were higher than the sum IP + IPS (Figure 4A and B). In

276

parallel, lowest values of the aggregate Dh were observed. The higher intensity and lower

277

aggregate size observed at pH 3.0 and 2.5 in presence of polysaccharides could indicate the

278

formation of higher amounts of aggregates with smaller mean sizes or the formation of more

279

dense structures related to the involvement of some polysaccharides in the aggregation. The

280

impact of wine polysaccharides on initial protein aggregation is different and less marked at pH

281

3.2, where protein aggregation develops according to much slower kinetics, than at pH 3.0 and

282

2.5. It can then be concluded from the presented results that the initial pH-induced aggregation of

283

wine proteins is modified in the presence of wine polysaccharides. However, this effect had no

284

impact on final haze: after 15 days similar turbidities were observed between model systems with

285

proteins alone and those with proteins and polysaccharides (Table 4).

286

Increasing the ionic strength allows screening potential electrostatic attractions between charged

287

polysaccharides and proteins or protein aggregates. At 0.15 M, protein aggregation develops

288

progressively (Figure 3), contrary to that observed at 0.02 M. These differences were attributed to

289

a stabilizing effect of the ionic strength on the conformation changes induced by the pH. 11 As at

290

low ionic strength, a significant impact of wine polysaccharides on protein aggregation was only

291

evidenced at low pH, i.e. pH 3.0 and 2.5 (Figure 3, Figure 4C and 4D). This impact is shown by

292

the different changes in scattering intensity and aggregate Dh observed during the first hours of

293

the aggregation kinetics. After 15 days at room temperature, the turbidities of the model systems

294

were similar at pH 2.5 whereas they were clearly smaller in the presence of polysaccharides at pH

295

3.0 (Table 4).

296

1D SDS-PAGE analyses of the non-precipitated proteins in model systems with proteins alone

297

indicated the involvement of proteins within the range 22-28 kDa in aggregation (Figure 5, A and

298

C). The same proteins were involved in the presence of polysaccharides (Figure 5B and D).


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Comparison of the band intensities and quantification by image analysis indicated that these

300

proteins were less affected in the presence of polysaccharides (the percentage of precipitated

301

proteins was calculated at pH 2.5 and 3.0) (Figure 5). The effect of polysaccharides on protein

302

amounts involved in aggregation was especially noticeable at the highest ionic strength of 0.15

303

M. It suggests the formation of stable protein/polysaccharide complexes less prone to aggregate

304

than proteins alone. However, lower protein precipitation did not necessarily correspond to lower

305

turbidity values (Table 4): only the turbidity at pH 3.0 and 0.15 M was significantly decreased in

306

the presence of polysaccharides. It is important to note that if the turbidity is related to the level

307

of protein aggregation, its value is also strongly dependent on aggregate size distribution,

308

refractive index and shape. These results thus suggest that polysaccharides interfere with protein

309

aggregation, as indicated by the initial kinetics (Figure 2 and 3) and modulate final size

310

distribution and/or structure of the aggregates.

311

For the two ionic strengths, the presence of polysaccharides thus modulated aggregation and the

312

amount of proteins involved in aggregation, indicating some effect of these macromolecules.

313

However, they did not prevent it. The impact of the ionic strength, which screens electrostatic

314

interactions between charged compounds, appeared as being more related to protein aggregation

315

than to possible electrostatic interactions between proteins and negatively charged

316

polysaccharides. Furthermore, no colloidal aggregation attributable to direct and enlarged

317

interactions between proteins and polysaccharides were observed within the tested pH range and

318

the purified protein and polysaccharide pools studied.

319 320

Polysaccharide structure and protein stability.

321

Experiments with polysaccharide fractions purified to homogeneity were performed with a new

322

protein pool, obtained from another Sauvignon blanc wine (2011, Sa2). The behavior of the


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323

proteins from the Sa2 wine (protein concentration around 150 mg.L-1) as a function of the pH was

324

compared to that of those from the Sa1 wine (protein concentration around 160 mg.L-1). Similar

325

results were obtained but aggregation was much more pronounced with the Sa2 protein pool

326

(enhanced aggregation kinetics and quick precipitation at low pHs, results not shown). This

327

higher instability, which is in accordance with the results of the heat-tests performed on the two

328

wines, can be explained by the different composition of the two protein pools. For a close total

329

protein concentration, Sa1 contained a lower content of unstable proteins (band 2 to 5, 37.5 %)

330

compared to Sa2 (band 2 to 5, 49 %) (Figure 1). Due to its higher sensitivity to pH-induced

331

aggregation, the protein concentration for experiments performed with the new pool was set to

332

0.2 g.L-1 instead of 0.8 g.L-1. This permitted to obtain kinetics close to those observed with the

333

Sa1 wine.

334

The impact of the four selected polysaccharide fractions was studied at 3 different pHs (2.5, 3.0

335

and 3.5) and an ionic strength of 0.02 M. The latter was chosen because most white wines have

336

ionic strengths lying within the range 0.02 – 0.04 M. The selected polysaccharides were: neutral

337

mannoproteins (MP0), which represent the major polysaccharides in white wines; RG-II dimer

338

(an acidic pectic polysaccharide) and two PRAGS, a neutral (AGP0) and an acidic one (AGP4)

339

(Table 2). The negative charge of RG-II and AGP4, related to uronic acids, strongly increases

340

within the tested pH range (Table 2). Two different polysaccharide concentrations were used. The

341

first one was chosen accounting for the protein/polysaccharide ratios found in the Sa1 white wine,

342

i.e.: proteins/MP0 1:1, proteins/AGP0 or AGP4 1:0.2 and proteins/RG-II 1:0.2. To emphasize their

343

possible impact, polysaccharide concentrations were increased to get the following ratios:

344

proteins/MP0 1:4, proteins/AGP0 or AGP4 1:1 and proteins/RG-II 1:1. At their highest

345

concentration, the intensity scattered by the polysaccharide model solutions were 70, 18, 25 and

346

12 kcounts.s-1 for MP0, AGP0, AGP4 and RG-II, respectively. They were thus negligible by


15


Page 16 of 35

347

comparison to the intensity scattered by the protein solution. At pH 3.5, protein aggregation

348

was small and did not allow us to observe an impact of polysaccharides. Besides, if

349

electrostatic interactions occurred between the negatively charged RGII or AGP4 and

350

positively charged wine proteins, they did not lead to aggregation and haze formation (Table

351

5). Results of DLS experiments at pH 3.0 and 2.5 are shown Figures 6 and 7. Turbidities

352

after 15 days are summarized in Table 5.

353

The average Dh values obtained by DLS during protein aggregation allowed distinguishing

354

between two different phases (Figure 6B and D; Figure 7B and D) : a first phase with a quick

355

aggregation and a pseudo-stabilization of the average Dh at a value around 700 nm, and then a

356

second phase where Dh continue to increase regularly till the end of the experiment. The first

357

phase last during the first 6 hours of the experiment at pH 3.0 and was shortened to 3 hours at pH

358

2.5, likely in relation with enhanced conformational changes of the involved proteins.12 Enlarged

359

protein aggregation observed in the second phase led to the formation of very polydisperse

360

suspensions. Due to the enhanced polydispersity observed during the second phase of the

361

aggregation, changes in mean Dh could only be compared without ambiguity during the first

362

phase. Though polysaccharides did not prevent aggregation, modifications in aggregate mean size

363

and growth were evidenced at the highest polysaccharide concentrations for all fractions except

364

RGII (Figure 6D and 7D). At pH 3.0, aggregate Dh in the presence of AGP0, AGP4 and MP0

365

were between 40% (AGP0 and AGP4) to 70% (MP0) smaller than that formed by proteins alone

366

whereas at pH 2.5, aggregates were 30 (MP0), 55 (AGP0) and 75 (AGP4) % smaller. At their

367

concentration in wines, only MP0 affected the initial aggregate size (Figures 6B and 7B). Either at

368

the wine ratio (1:1) or at a higher one (1:4), the intensity scattered by mixtures was closed to that

369

scattered by the proteins alone for all polysaccharides but AGP0 at pH 3.0.


16

Page 17 of 35


370

Thus, the presence of MP0, AGP0 and AGP4 at high protein:polysaccharide ratios (1:1 or 1:4) had

371

an effect on aggregate size and growth during the first hours of the pH-induced aggregation of

372

wine proteins. However, results obtained by turbidity measurements after 15 days showed that

373

the initial decrease of aggregate size and growth did not necessarily result in lower hazes (Table

374

5). At pH 2.5 and considering the standard deviation, turbidities of systems with proteins alone

375

and proteins in mixture with polysaccharides were close except for MP0 at its highest ratio, for

376

which a decrease in haziness was observed (Table 5). This impact of MP0 was not observed at pH

377

3.0. At this pH, only AGP0 and AGP4 modified final haze and in that case higher turbidities were

378

obtained

379

characterization of the aggregates formed would be needed to explain differences observed

380

between the two AGPs and MP0. Indeed, the intensity scattered by proteins and

381

polysaccharide/proteins mixtures, and thus final haze, not only depends on aggregate mean size

382

and number but also by their structure (density, refractive index) and shape. Samples were

383

centrifuged to remove precipitated proteins and supernatants were analyzed by 1D SDS-PAGE.

384

In accordance with previous results, precipitated proteins when the pH was decreased were

385

proteins within the bands 22-28 kDa and purified polysaccharides did not modify the

386

precipitation pattern (results not shown).

387

Using purified wine polysaccharide fractions and high concentrations confirmed then the results

388

obtained previously with the polysaccharide pool:

389

- Acidic wine polysaccharides formed stable colloidal systems with wine proteins despite

390

opposite charges: no enlarged aggregation was observed for pH above 3.2, where the pH-

391

induced aggregation of wine proteins is not observed. Beside, no relationship appeared in the

392

present study between this acidic character and their impact on pH-induced protein

than with protein alone (factor 2 with AGP0 and 1.4 for AGP4). An in-depth


17


Page 18 of 35

393

aggregation. RG-II, the smallest (≈ 10 kDa) and the most acidic (at pH 3.0 ≈ -32.5 C/g) did not

394

affect pH-induced aggregation and no strong differences were observed between the neutral

395

AGP0 and the acidic AGP4.

396

- As far as they may modulate initial aggregation and final haziness, indicating that they

397

interfere in the aggregation process, wine polysaccharides cannot be considered as having a

398

determinant effect on protein stability in properly stored wines, i.e. when protein aggregation

399

is not induced by their exposure to high temperatures. These results are in agreement with

400

those obtained by Gazzola et al.23 within the context of the heat-induced aggregation of

401

purified chitinases and TLPs: though heat-induced aggregation results in conformational

402

changes very different than those observed at the ambient temperature, they did not evidence a

403

strong impact of polysaccharides (pool purified from a Chardonnay wine) on protein

404

aggregation.

405 406 407

Abbreviations used

408

AGP0: neutral arabinogalactan protein, AGP4: acidic arabinogalactan protein, Dh: hydrodynamic

409

diameter, DLS: Dynamic light scattering, IS: scattered intensity, MP0: mannoproteins, RG-II:

410

rhamnogalacturonan type II, Sa1: Sauvignon blanc 2009, Sa2: Sauvignon blanc 2011,

411

412

413

Acknowledgment

414

The authors are grateful to Mrs Pascale Williams and Mrs Pilar Fernández of INRA-Sciences for

415

enology for her help with polysaccharide analysis and protein/polysaccharide experiments by


18

Page 19 of 35


416

DLS respectively. They also thank the Pech-Rouge experimental unit (Gruissan, France) which

417

provided the Sauvignon blanc wines 2009 and 2011.

418

References

419

420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460

1. 2.

3. 4. 5.

6. 7.

8. 9.

10.

11.

12. 13.

14. 15.

16.

Esteruelas, M.; Poinsaut, P.; Sieczkowski, N.; Manteau, S.; Fort, M. F.; Canals, J. M.; Zamora, F., Characterization of natural haze protein in sauvignon white wine. Food Chem. 2009, 113, 1, 28-35. Pocock, K. F.; Waters, E. J., Protein haze in bottled white wines: How well do stability tests and bentonite fining trials predict haze formation during storage and transport? Aust. J. Grape Wine Res. 2006, 12, 3, 212-220. Sarmento, M. R.; Oliveira, J. C.; Slatner, M.; Boulton, R. B., Influence of intrinsic factors on conventional wine protein stability tests. Food Control 2000, 11, 6, 423-432. Bayly, Francis C.; Berg, H. W., Grape and wine proteins of white wine varietals. Am. J. Enol. Vitic. 1967, 18, 1, 18-32. Vincenzi, S.; Mosconi, S.; Zoccatelli, G.; Pellegrina, C. D.; Veneri, G.; Chignola, R.; Peruffo, A.; Curioni, A.; Rizzi, C., Development of a new procedure for protein recovery and quantification in wine. Am. J. Enol. Vitic. 2005, 56, 2, 182-187. Hsu, J. C.; Heatherbell, D. A., Isolation and characterization of soluble proteins in grapes, grape juice, and wine. Am. J. Enol. Vitic. 1987, 38, 1, 6-10. Dambrouck, T; Marchal, R; Cilindre, C; Parmentier, M; Jeandet, P, Determination of the Grape Invertase Content (Using PTA−ELISA) following Various Fining Treatments versus Changes in the Total Protein Content of Wine. Relationships with Wine Foamability. J. Agric. Food Chem. 2005, 53, 22, 8782–8789. Sauvage, F-X.; Bach, B.; Moutounet, M.; Vernhet, A., Proteins in white wines: Thermo-sensitivity and differential adsorbtion by bentonite. Food chem. 2010, 118, 1, 26-34. Falconer, R.J. ; Marangon, M.; Van Sluyter, S.C. ; Neilson, K.A.; Chan, C.; Waters, E.J., Thermal Stability of Thaumatin-like Protein, Chitinase, and invertase Isolated from Sauvignon blanc and Semillon Juice and Their Role in Haze Formation in Wine Journal of Agricultural and Food Chemistry 2010, 58, 975-980. Pocock, K. F.; Hayasaka, Y.; McCarthy, M. G.; Waters, E. J., Thaumatin-like proteins and chitinases, the haze-forming proteins of wine, accumulate during ripening of grape (Vitis vinifera) berries and drought stress does not affect the final levels per berry at maturity. Journal of Agricultural and Food Chemistry 2000, 48, 5, 1637-1643. Dufrechou, M.; Poncet-Legrand, C.; Sauvage, F-X.; Vernhet, A., Stability of white wine proteins: combined effect of pH, ionic strength, and temperature on their aggregation. J. Agric. Food Chem. 2012, 60, 5, 13081319. Dufrechou, M.; Vernhet, A.; Roblin, P.; Sauvage, F-X.; Poncet-Legrand, C., White Wine Proteins: How Does the pH Affect Their Conformation at Room Temperature? Langmuir 2013, 29, 33, 10475-10482. Marangon, M.; Sauvage, F. X.; Waters, E. J.; Vernhet, A., Effects of Ionic Strength and Sulfate upon Thermal Aggregation of Grape Chitinases and Thaumatin-like Proteins in a Model System. J. Agric. Food Chem. 2011, 59, 6, 2652-2662. Besse, C.; Clark, A.; Scollary, G., Investigation of the role of total and free copper in protein haze formation. Australian Grapegrower & Winemaker 2000No. 437, 19-20. Pocock, K. F.; Alexander, G. M.; Hayasaka, Y.; Jones, P. R.; Waters, E. J., Sulfate - a candidate for the missing essential factor that is required for the formation of protein haze in white wine. J. Agric. Food Chem. 2007, 55, 5, 1799-1807. Marangon, M.; Vincenzi, S.; Lucchetta, M.; Curioni, A., Heating and reduction affect the reaction with tannins of wine protein fractions differing in hydrophobicity. Anal. Chim. Acta 2010, 660, 1-2, 110-118.


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461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506

17. 18. 19.

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28. 29. 30. 31. 32. 33. 34. 35. 36.

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Waters, E. J.; Pellerin, P.; Brillouet, J. -M., A Saccharomyces mannoprotein that protects wine from protein haze. Carbohydr. Polym. 1994, 23, 3, 185-191. Waters, E. J.; Pellerin, P.; Brillouet, J. M., A Wine Arabinogalactan-Protein That Reduces Heat-Induced Wine Protein Haze. Biosci., Biotechnol., Biochem. 1994, 58, 1, 43-48. Dupin, I. V.; Stockdale, V. J.; Williams, P. J.; Jones, G. P.; Markides, A. J.; Waters, E. J., Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: evaluation of extraction methods and immunolocalization. J. Agric. Food Chem. 2000, 48, 4, 1086-1095. Dupin, I. V. S.; McKinnon, B. M.; Ryan, C.; Boulay, M.; Markides, A. J.; Jones, G. P.; Williams, P. J.; Waters, E. J., Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: Their release during fermentation and lees contact and a proposal for their mechanism of action. J. Agric. Food Chem. 2000, 48, 8, 3098-3105. Ledoux, V.; Dulau, L.; Dubourdieu, D., Interprétation de l'amélioration de la stabilité protéique des vins au cours de l'élevage sur lies. (An explanantion for the improvement of protein stability of wines during aging on yeast lees). J. Int. Sci. Vigne Vin 1992, 26, 239-251. Moine-Ledoux, V.; Dubourdieu, D., An invertase fragment responsible for improving the protein stability of dry white wines. J. Sci. Food Agric. 1999, 79, 4, 537-543. Gazzola, D.; Van Sluyter, S.C.; Curioni, A.; Waters, E.J.; Marangon, M., Roles of proteins, polysaccharides, and phenolics in haze formation in white wine via reconstitution experiments. J. Agric. Food Chem. 2012, 60, 42, 10666-10673. Pellerin, P.; Cabanis, J.C., Les glucides, in Oenologie - Fondements scientifiques et technologiques, C.Flanzy. Lavoisier Tec&Doc: Paris, 1998, 40-93. Vidal, S.; Williams, P.; Doco, T.; Moutounet, M.; Pellerin, P., The polysaccharides of red wine: total fractionation and characterization. Carbohydr. Polym. 2003, 54, 4, 439-447. Coimbra, M. A.; Goncalves, F.; Barros, A. S.; Delgadillo, I., Fourier transform infrared spectroscopy and chemometric analysis of white wine polysaccharide extracts. J. Agric. Food Chem. 2002, 50, 12, 3405-3411. Oneill, M. A.; Warrenfeltz, D.; Kates, K.; Pellerin, P.; Doco, T.; Darvill, A. G.; Albersheim, P., Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester - In vitro conditions for the formation and hydrolysis of the dimer. J. Biol. Chem. 1996, 271, 37, 22923-22930. Vernhet, A.; Pellerin, P.; Prieur, C.; Osmianski, J.; Moutounet, M., Charge properties of some grape and wine polysaccharide and polyphenolic fractions. Am. J. Enol. Vitic. 1996, 47, 1, 25-30. Dawes, H.; Boyes, S.; Keene, J.; Heatherbell, D., Protein instability of wines: influence of protein isolelectric point. Am. J. Enol. Vitic. 1994, 45, 3, 319-326. Israelachvili, J., Intermolecular & surface forces. Second edition ed. 1992. Doco, T.; Williams, P.; Cheynier, V., Effect of flash release and pectinolytic enzyme treatments on wine polysaccharide composition. J. Agric. Food Chem. 2007, 55, 16, 6643-6649. Doco, T.; Quellec, N.; Moutounet, M.; Pellerin, P., Polysaccharide patterns during the aging of Carignan noir red wines Am. J. Enol. Vitic. 1999, 50, 25-32. Doco, T.; Vuchot, P.; Cheynier, V.; Moutounet, M., Structural modification of wine arabinogalactans during aging on lees. Am. J. Enol. Vitic. 2003, 54, 3, 150-157. Doublier, J.L.; Garnier, C.; Renard, D; Sanchez, C., Protein-polysaccharide interactions. Curr. Opin. Colloid Interface Sci. 2001, 5, 3-4, 202-214. Samant, S.K.; Singhal, R.S.; HKulkarni, P.R.; Rege, D.V., Protein-polysaccharide interactions: a new approach in food formulations. Int. J. Food Sci. Technol. 1993, 28, 6, 547-562. Turgeon, S.L.; Beaulieu, M.; Schmitt, C.; Sanchez, C., Protein-polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects. Curr. Opin. Colloid Interface Sci. 2003, 8, 4-5, 401-414.

507 508


20

Page 21 of 35


509

Figure Captions

510

Figure 1: A) 1D SDS-PAGE profile of the Sa1 Sauvignon blanc wine 2009 (molecular weight

511

(MW) standards on left) and of its purified protein pool (from reference

512

profile of the Sa2 Sauvignon blanc wine 2011 (molecular weight (MW) standards on left) and of

513

its purified protein pool.

11

) B) 1D SDS-PAGE

514 515

Figure 2: Dynamic light scattering experiments performed with the Sa1 wine proteins (protein

516

concentration, 0.8 g.L-1) without and with the polysaccharides purified from the same wine

517

(polysaccharide concentration, 0.8 g.L-1) and at a ionic strength of 0.02 M. Is: light scattering

518

intensity (kcounts.s-1) and Dh: average hydrodynamic diameter (nm). A) and B): pH 2.5; C) and

519

D): pH 3.0; E) and F): pH 3.2.

520 521

Figure 3: Dynamic light scattering experiments performed with the Sa1 wine proteins (protein

522

concentration, 0.8 g.L-1) without and with the polysaccharides purified from the same wine

523

(polysaccharide concentration, 0.8 g.L-1) and at a ionic strength of 0.15 M. Is: light scattering

524

intensity (kcounts.s-1) and Dh: average hydrodynamic diameter (nm). A) and B): pH 2.5; C) and

525

D): pH 3.0; E) and F): pH 3.2.

526 527

Figure 4: Scattered intensity and hydrodynamic diameter determined by DLS after 6 hours at

528

25°C

529

proteins/polysaccharides (IP+PS). Different pHs (2.5 to 4.0) and ionic strengths (0.02 M and

530

0.15M) were tested. A) Scattered intensity (IS) at 0.02 M, B) hydrodynamic diameter (Dh) at

531

0.02M, C) Is at 0.15 M, D) Dh at 0.15 M. The sum of the intensity scattered by model systems

for

the

different

model

systems

:

polysaccharides


(IPS),

proteins

(IP),

21


Page 22 of 35

532

with only polysaccharides and proteins IP + IPS was calculated and compared to the scattered

533

intensity of model systems with proteins/polysaccharides. These values are expected to be the

534

same if there are no interactions between polysaccharides and proteins.

535 536

Figure 5: 1D SDS-PAGE of non-precipitated proteins in model systems with wine proteins and

537

wine proteins/polysaccharides after 15 days at 20 °C at different pHs (2.5 to 4.0) and ionic

538

strength (0.02 M and 0.15 M).

539 540

Figure 6: Impact of polysaccharides (RG-II, MP0, AGP0 and AGP4) on aggregation. Kinetics

541

were followed by DLS at 25 °C. Model systems at pH 3.0 and 0.02 M were used and two

542

different ratios proteins/polysaccharides were used: A) Is and B) Dh at a wine ratio; C) Is and D)

543

Dh at a concentrated ratio.

544 545

Figure 7: Impact of polysaccharides (RG-II, MP0, AGP0 and AGP4) on aggregation. Kinetics

546

were followed by DLS at 25 °C. Model systems at pH 2.5 and 0.02 M were used and two

547

different proteins/polysaccharide ratios were used: A) Is and B) Dh at a wine ratio; C) Is and D)

548

Dh at a concentrated ratio.

549

550

551


22

Page 23 of 35


Table 1: Conventional enological analyses of Sauvignon blanc 2009 (Sa1) and 2011 (Sa2)

Sa1 Ethanol (% v/v) pH Total acidity (g.L−1 H2SO4) Total SO2 (mg.L−1) Free SO2 (mg.L−1) Total polyphenol Index K+ (mg.L−1) Na+ (mg.L−1) Ca2+ (mg.L−1) Mg2+ (mg.L−1) Conductivity (mS) Turbidity (NTU) Protein content (mg.L-1)

11.5 3.2 4.9 87 25 4.6 566 12 80 70 1.36 10 160

Dufrechou et al : table 1


Sa2 9.5 3.2 5.0 91 19 6.3 886 16 69 64 1.92 33 150


Page 24 of 35

Table 2: Glycosyl residue composition, molecular mass, protein content and charge density of the four purified polysaccharides 23

RG-II

AGP0

AGP4

MP0

3.6

0.8

1.6

40.3 ± 2.8 1.0 ± 0.6 Nd 0.6 ± 0.0 0.4 ± 0.1 51.8 ± 1.4

24.3 ± 0.8 14.1 ± 0.2 0.3 ± 0.2 2.6 ± 0.1 5.0 ± 0.5 28.8 ± 0.3

5.2 ± 1.4 0.4 ± 0.1

88.8 ± 2.0 2.9 ± 0.3

1.6 ± 0.5

0.8 ± 0.1

2.6 ± 0.6

33.6 ± 1.8 3.3 ± 0.1 8.5 ± 1.1 2.5 ± 0.1 3.0 ± 0.1

Nd 4.2 ± 0.4

9.6 ± 0.3 14.7 ± 0.5

10.5

75

177

proteins a Neutral

b

Arabinose Rhamnose Fucose Xylose Mannose Galactose Apiose 2-O-Me-Xylose 2-O-Me-Fucose Glucose Acidic

8.4 ± 0.4 15.5 ± 0.4 4.1 ± 0.2 Nd 0.15 ± 0.1 5.3 ± 0.2 5.8 ± 0.3 5.7 ± 0.4 5.0 ± 0.3

b

Galacturonic acid Glucuronic acid Aceric acid DHA KDO Apparent MW (kDa)

62

Charge density (C.g-1) pH 3.0 -31.5 -3.6 -112.5 pH 4.0 -163.3 -5.2 -163.7 a: percent of dry matter, b: Molar ratios, Nd: non-detected

Dufrechou et al : Table 2


-6.6 -10.2

Page 25 of 35


Table 3: Polysaccharide analyses of the Sa1 wine and of the corresponding purified polysaccharide pool. A) Neutral sugar composition B) Polysaccharide contents calculated from the neutral sugar composition 29

A)

2-OMeFuc*

Rha*

Fuc*

2-OMeXyl*

Ara*

Api*

Xyl*

Man*

Gal*

Glu*

0.7±0.1

3.3±0.1

0.6±0.0

0.5±0.1

10.9±0.7

0.5±0.1

0.9±0.1

119.6±7.9

22.5±4.7

6.7±6.4

0.7±0.2

2.7±0.3

0.7±0.2

0.3±0.0

8.1±1.4

0.6±0.4

0.6±0.4

106.3±41.1

14.6±5.5

5.5±1.5

Wine polysaccharides (mg.L-1) Purified polysaccharide fraction (mg.L-1)

* Respectively: 2-OMeFucose, Rhamnose, Fucose, 2-OMeXylose, Arabinose, Apiose, Xylose, Mannose, Galactose, Glucose B) RG-II

PRAG

MP

Total

Wine (mg.L-1)

38

41

149

228

Purified polysaccharide fraction (mg.L-1)

31

28

133

192




Page 26 of 35

Table 4: Impact of polysaccharides on turbidity estimated by measurements of the absorbance at 720 nm after 15 days of storage at 20°C. Model systems containing protein and protein/polysaccharide mixtures were compared at different ionic strength (0.02 and 0.15 M) and pHs (2.5 to 4.0). A visual haze is observed for value higher than 0.01 a.u.

Ionic strength 0.02 M 0.15 M

pH Model system

2.5

3.0

3.2

3.5

4.0

Proteins

0.031 ± 0.002

0.029 ± 0.001

0.021 ± 0.001

0.006 ± 0.002

0.003 ± 0.000

Prot/Polysacc.

0.031 ± 0.003

0.028 ± 0.001

0.021 ± 0.002

0.006 ± 0.001

0.008 ± 0.001

Proteins

0.037 ± 0.005

0.027 ± 0.001

0.002 ± 0.003

0.002 ± 0.000

0.002 ± 0.001

Prot/Polysacc.

0.044 ± 0.003

0.016 ± 0.000

0.006 ± 0.001

0.006 ± 0.001

0.003 ± 0.001



Page 27 of 35


Table 5: Impact of polysaccharides on turbidity was estimated by spectrophotometry at 720 nm. Model systems with wine proteins/RG-II, MP0, AGP0 or AGP4 were compared at different pHs (2.5, 3.0 and 3.5) and concentration after 15 days at room temperature A visible haze was observed from a value of 0.01 a.u.

pH Model system

2.5

3.0

3.5

Proteins

0.021 ± 0.002

0.011 ± 0.002

0.001 ± 0.001

Proteins/RGII

1:1

0.026 ± 0.001

0.013 ± 0.003

0.001 ± 0.001

1:0.2

0.021 ± 0.000

0.013 ± 0.003

0.000 ± 0.000

1:4

0.013 ± 0.001

0.009 ± 0.002

0.000 ± 0.000

1:1

0.016 ± 0.004

0.014 ± 0.000

0.000 ± 0.000

1:1

0.027 ± 0.004

0.018 ± 0.001

0.000 ± 0.000

1:0.2

0.022 ± 0.001

0.023 ± 0.003

0.000 ± 0.000

1:1

0.022 ± 0.000

0.017 ± 0.000

0.000 ± 0.000

1:0.2

0.026 ± 0.004

0.015 ± 0.002

0.001 ± 0.001

Proteins/MP0 Proteins/AGP0 Proteins/AGP4

Ratios




Page 28 of 35

Dufrechou et al., figure 1 A)

B)

Band number Purified pool Sa1 (%) Purified pool Sa2 (%)

1

2

3

4

5

6

9

3

5

12

17.5

15.5

38

5.3

8.5

7.7

9.6

23.2

14.2

31.5


7

Page 29 of 35


Dufrechou et al., Figure 2

A)

B)

C)

D)

E)

F)



Dufrechou et al.,Figure 3 A)

B)

C)

D)

E)

F)


Page 30 of 35

Page 31 of 35


Dufrechou et al., Figure 4 A)

B) Ionic strength - 0.02 M

Ionic strength - 0.02 M

C)

D) Ionic strength - 0.15 M


Ionic strength - 0.15 M


Page 32 of 35


Percentage of precipitated proteins (%) 0.02 M 0.15 M pH 2.5 pH 3.0

Proteins Proteins/Polysaccharides Proteins Proteins/Polysaccharides

25 17 18 9


41 26 36 22

Page 33 of 35


Dufrechou et al., Figure 6 A)

B)

C)

D)




A)

B)

C)

D)


Page 34 of 35

Page 35 of 35


85x47mm (300 x 300 DPI)


Polysaccharide Interactions and Their Impact on Haze

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