Protein/Polysaccharide Interactions and Their Impact On Haze Formation in White Wines

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Article
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
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Page 1 of 35 Journal of Agricultural and Food Chemistry
1 Protein/polysaccharide Interactions and Their
2 Impact on Haze Formation in White Wines

3 Marie Dufrechou1,2,3,4, Thierry Doco1, Céline Poncet-Legrand1,2,3, François-
4 Xavier Sauvage1,2,3, Aude Vernhet1,2,3
1
5 INRA, UMR1083 SPO, F-34060 Montpellier, France
2
6 Montpellier SupAgro, UMR1083 SPO, F-34060 Montpellier, France
3
7 Université Montpellier I, UMR1083 SPO, F-34060 Montpellier, France
4
8 Present address : LUNAM Université, SFR 4207 QUASAV, Groupe ESA, UPSP GRAPPE,
9 55 rue Rabelais BP 30748, F-49007 Angers Cedex 01, France
10
11
12
13
14
15 * Corresponding author:
16 Marie Dufrechou m.dufrechou@groupe-esa.com
17 Present address : LUNAM Université, SFR 4207 QUASAV, Groupe ESA, UPSP GRAPPE, 55
18 rue Rabelais BP 30748, F-49007 Angers Cedex 01, France
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Journal of Agricultural and Food Chemistry Page 2 of 35
22 Abstract
23 Proteins in white wines may aggregate and form hazes at room temperature. This was
24 previously shown to be related to pH-induced conformational changes and to occur for pH
25 lower than 3.5. The aim of the present work was to study the impact of wine polysaccharides
26 on pH-induced haze formation by proteins but also the consequences of their interactions with
27 these proteins on the colloidal stability of white wines. To this end, model systems and
28 purified global pools of wine proteins and polysaccharides were used first. Kinetics of
29 aggregation, proteins involved and turbidities related to final hazes were monitored. To
30 further identify the impact of each polysaccharide, fractions purified to homogeneity were
31 used in a second phase. These were: 2 neutral (mannoprotein and arabinogalactan
32 polysaccharides) and 2 negatively charged (rhamnogalacturonan II dimer (RG-II) and
33 arabinogalactan polysaccharides). We highlighted that the impact of major wine
34 polysaccharides on wine protein aggregation at room temperature was clearly less marked
35 than those of the pH and the ionic strength. Polysaccharides modulated the aggregation
36 kinetics and final haziness, indicating that they interfere with the aggregation process, but
37 could not prevent it.
38
39
40 Keywords: arabinogalactan-proteins, haze, mannoproteins, protein/polysaccharide
41 interactions, rhamnogalacturonan II dimer, wine proteins
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43
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45 Introduction
46 One of the major defects in bottled white wines is the formation of haze or deposits that can
47 appear during transport or storage. It is often related to exposure at high temperatures but can also
48 develop in properly stored wines.1-3 Haze or deposit formation is due to the aggregation of
49 proteins that resist winemaking conditions (concentration usually found between the range 15-
50 330 mg.L-1).4-6 These proteins mostly originate from grapes and belong to four different families:
51 invertase, β-Glucanases, chitinases and thaumatin-like proteins (TLPs).7, 8 TLPs and chitinases
52 are the most abundant and different isoforms can be found within each family. Previous works
53 indicated their different sensitivity to heat induced unfolding and aggregation.8, 9 β-Glucanases
54 and chitinases were found to be the most sensitive proteins, whereas invertase and TLPs were
55 more resistant.8-10 Besides the temperature,4, 8

the two other physico-chemical factors that
56 strongly affect the stability of wine proteins and the final haze induced by their aggregation are
57 the pH and the ionic strength.11-13 The impact of the pH, studied for a range 2.5 - 4.0, was
58 shown to be temperature-dependent. At ambient or at lower temperatures (below 25 °C),
59 protein conformational changes induced by low pHs (≤ 3.2) were shown to be involved in the
60 aggregation of some of the wine proteins.11
61 The impact of non-protein compounds such as polysaccharides, polyphenols and sulfate in the
62 development of heat-induced protein hazes has also been demonstrated. Like the ionic strength,
63 phenolic compounds and sulfate enhance aggregate growth and significantly increase final haze.1,
11, 13-16
64 By contrast, some specific and quantitatively minor mannoprotein or arabinogalactan
65 protein (AGP) fractions, purified from wine, were shown to decrease heat-induced protein hazes
66 and were thus considered as protective factors.17, 18

Similar results were obtained with other
67 specific mannoprotein fractions purified from yeast cell walls.19-22 However, the exact structural
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68 features and mechanisms involved have not been clearly elucidated yet. In addition, a protective
69 effect was only observed when these purified polysaccharides were added to the wine at
70 relatively high polysaccharide to protein ratios (1/1 in mass or higher). Thus, even if naturally
71 present in wines, it is at concentrations such that this protective effect is to be confirmed. Other
72 studies on protein stability at room temperature also indicated that non-protein compounds in
73 wine modulate the haze resulting from protein aggregation,11 and the presence of polyphenols
74 and polysaccharides has been shown in a natural precipitate.1 If polyphenols, and especially
75 tannins, are considered as having a triggering effect on protein aggregation in wine, 16, 23 the role
76 of wine polysaccharides is not clear and has not been fully investigated yet. Their interactions
77 with wine proteins deserve to be further explored.
78 Polysaccharides are present in white wine at concentrations ranging from 150 to 500 mg.L-1.24
79 They originate from the grape berry (pectic polysaccharides) or from yeast cell walls
80 (mannoproteins). Mannoproteins have molecular weights ranging from 50 to 500 kDa.25 Most of
81 them are almost neutral however fractions with different charge density have been separated from
82 a mannoprotein pool purified from a red wine.26 Pectic polysaccharides are mainly
83 rhamnogalacturonan II (RG-II) and polysaccharides rich in arabinose and in galactose (PRAGs).
84 RG-II is mostly found in its dimer form (9.5-10.5 kDa),27 and its negative charge is strongly
85 influenced by the pH within wine pH-range.28 PRAGs include arabinans, arabinogalactans and
86 arabinogalactan proteins (AGP). Arabinogalactans and AGP have molecular weights between 50
87 and 250 kDa. As for mannoproteins, fractions with different charge densities were evidenced.25, 28
88 The aim of the present work was to determine the impact of wine polysaccharides on protein
89 aggregation, considering first interactions at room temperature with the impact of the pH and of
90 the ionic strength. In addition to its influence on the charge and stability of wine proteins, the pH
91 strongly affects the charge of some polysaccharide fractions.28 Wine proteins and acidic pectic
4
92 polysaccharides carry opposite charges within white wine pH range (2.8 -3.5)8, 28, 29
so that
93 attractive electrostatic interactions between these macromolecules can be expected. These
94 electrostatic interactions are modulated by the pH value but also by the ionic strength.30
95 Interactions were first studied in model solutions with proteins and polysaccharides purified from
96 a Sauvignon blanc wine. In a second phase, experiments were focused on four specific
97 polysaccharides: a neutral mannoprotein (MP0), a neutral arabinogalactan-protein (AGP0), an
98 acidic arabinogalactan (AGP4) and a rhamnogalacturonan II dimer (RG-II). In both experiments,
99 protein and polysaccharide concentrations were increased by comparison to that found in wines
100 to accelerate aggregation rates and to observe the impact of polysaccharides on aggregation
101 within 15 days.
102
103 Materials and Methods
104 Wines. The two Sauvignon blanc wines used for this study were elaborated at the Pech
105 Rouge Experimental Unit (INRA, Gruissan, France) in 2009 (Sa1) and 2011 (Sa2). The wine
106 was elaborated using classic winemaking steps. Following the fermentation, the wine was
107 cold stabilized (to prevent the crystallization of tartaric salts) and clarified by filtration on a
108 1 µm filtration cartridge. No enzymatic treatment and no bentonite fining were performed to
109 preserve protein and polysaccharide content. Conventional enological parameters were
110 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, 6

was performed on both wines to assess their
112 protein instability. A turbidity of 10 and 33 NTU (HI88703 turbidimeter, Hanna Instrument
113 Inc.) was obtained for the Sa1 and the Sa2 wines, respectively.
114
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115 Purification of proteins and polysaccharides. Wines (3 L) were first treated with
116 polyvinylpolypyrrolidone (150 mg.L-1, Sigma) during 24 h and under constant stirring to
117 remove polyphenols. Wine proteins were then isolated and purified by ion exchange
118 chromatography, using a cation exchange Streamline SP gel (74 mL, GE Healthcare) and a
119 350 mm length*25 mm diameter column (GE healthcare), according to the method described
120 previously. 11 The wine flow rate was set to 10 mL.min-1. Elution was performed at a flow
121 rate of 5 mL.min-1, using 2 solutions (solution A: 13 mM tartrate buffer at pH 4.0 and
122 solution B : 0.5 M NaCl in 13mM tartrate buffer pH 4.0) with the following gradient: 0-40
123 min, 100% of solution A; 40-70 min, from 100% of solution A to 100% of solution B; 70-80
124 min, 100% of solution B. The protein-containing fractions were identified using a UV
125 detector at a wavelength of 280 nm and were pooled. Salt removal was achieved by
126 extensive diafiltration (5 kDa membrane, Amicon, Millipore) using a 13 mM tartrate buffer
127 at pH 4.0. The protein pools were then stored at -20 °C before use. Freeze-drying of 1 mL
128 aliquots and weighing allowed us to determine the protein concentration in these stock
129 solutions and to estimate that of the initial wines. The protein concentration of Sa1 and Sa2
130 were respectively estimated of being 160 mg.L-1 and 150 mg.L-1.
131 The total polysaccharide pool was obtained by ultrafiltration of the protein-free Sa1 wine (wine
132 recovered after the column separation). To purify sample, by removing small solutes, diafiltration
133 was performed using a 200 mL stirred cell equipped with a 5 kDa membrane (Amicon,
134 Millipore). 200 mL of the protein-free wine were diafiltrated with pure water, up to a final
135 dilution factor of small solutes (< 5 kDa) of 1600. The final polysaccharide pool was lyophilized
136 and stored in a dry place.
137
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138 Protein analyses in the wines and in the protein pools. Protein analyses in wines and in
139 the corresponding pools were performed by 1D SDS-PAGE. The protein concentration was
140 adjusted to 0.8 g.L-1 before analysis, either by concentration using 3.0 kDa centrifugal filter
141 units (Millipore) for the wines or by simple dilution (pools). Proteins (30 µg) in Laemmli
142 buffer were separated on a 14% acrylamide resolving gel (gel length, 60 mm). A low
143 molecular weight calibration kit (14.4 to 97 kDa, Pharmacia, Biotech) was included in each
144 electrophoretic run. Gels were stained with 0.1% Coomassie Brilliant Blue R-250 (Biorad)
145 in 40% of ethanol, 10% acetic acid and destained overnight in 10% acetic acid. Gels were
146 then scanned at 300 dpi with an image scanner (GE Biosciences). Image analysis was carried
147 out with the Totallab software (Nonlinear Dynamics Ltd) and was used to calculate the
148 proportion of proteins in each stained band.8
149
150 Carbohydrate composition of wine polysaccharides and of the purified polysaccharide
151 pool. The polysaccharide compositions in the Sa1 wine and in the corresponding total
152 polysaccharide pool were determined according to the method described by Doco et al.31 The
153 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
156 monosaccharides. Neutral sugar amounts were calculated relative to two internal standards (myo-
157 inositol and β-D-allose). Results represent an average of 2 experiments. Concentrations of
158 mannoproteins (MP), rhamnogalacturonans II (RG-II) and polysaccharides rich in arabinose and
159 galactose (PRAGs) were calculated on the basis of the neutral sugar composition using the
160 formula established previously.32
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161
162 Specific polysaccharide fractions. Four polysaccharide fractions purified to homogeneity
163 and characterized at the UMR Sciences for Enology (INRA, Montpellier)25 were also used.
164 These fractions were: a rhamnogalacturonan II (RG-II, acidic), a mannoprotein fraction 0
165 (MP0, neutral), a type II arabinogalactan 0 (AGP0, neutral) and a type II arabinogalactan 4
166 (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
171 as follows: 12% ethanol, 7 g.L-1 glycerol and 2 g.L-1 tartaric acid. Their pHs were adjusted at
172 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
173 0.15 M with NaCl. To obtain these final values, buffers at given pH and ionic strength were
174 prepared with a concentration factor of 1.5 to get the required composition following the
175 addition of the protein solution in 13 mM tartaric buffer at pH 4.0. Buffers were stored at 4
176 °C. Lyophilized polysaccharides were dissolved in the buffer solutions (1.5 mL) and filtered
177 on 0.2 µm membranes before addition of the protein solution (0.75 mL, concentration factor
178 3 to get the final required protein content).
179 Aggregation kinetics. DLS experiments were carried out with a Malvern Autosizer 4700 (40
180 mW He-Ne laser, λ = 633 nm, APD detection, Malvern Instruments, Malvern, UK) at an
181 angle of 90° from the incident beam. Aggregation kinetics were followed by measurements
182 of the scattering intensity (Is) and of the hydrodynamic diameter (Dh) of particles. Each
183 measurement represented the average of 10 sub-runs and each kinetic was done in duplicate.
8
184 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
186 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.
188 Turbidity measurements and proteins involved in aggregation. Other samples were
189 prepared in the same conditions to measure the turbidity and to determine protein
190 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
192 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
194 method as detailed above. Centrifugal filter units (3.0 kDa, Millipore) were used to decrease
195 the ionic strength to a value about 0.05 M. Results were compared with the protein profiles
196 of the initial purified pool.
197
198 Results and discussion
199 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
201 identification of the proteins in the Sa1 wine and in the protein pool were performed in a previous
202 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
204 lost during the purification steps.11 A similar result was obtained for Sa2: main proteins were
205 found within the range 19-28 kDa (TLPs and chitinases, bands 2 to 7).
9
206
207 The polysaccharide composition of the Sa1 wine is detailed in Table 3. The monosaccharide
208 composition corresponded to that of a white wine, with a total calculated concentration of 228
209 mg.L-1 including rhamnogalacturonans II (38 mg.L-1), polysaccharides rich in arabinose and
210 galactose (PRAGs, 41 mg.L-1) and mannoproteins (149 mg.L-1). The arabinose/galactose ratio
211 was calculated at 0.48, which is usual in white wines (common ratios ranging from 0.3 to 0.8). 33
212 After purification, 84.2 % of polysaccharides were recovered. Loss mainly concerned PRAGs
213 (31% losses against 8% for RG-II and 11% for mannoproteins). The arabinose/galactose ratio
214 was not strongly modified and mannoproteins were the major polysaccharides in both the initial
215 wine (65.3%) and the purified pool (69.3%).
216 Impact of wine polysaccharides on protein stability. Proteins purified from the Sa1 wine
217 were used to study the overall impact of wine polysaccharides on their stability at room
218 temperature. The pH was varied between 2.5 and 4.0 and two ionic strengths were studied:
219 0.02 and 0.15 M. Previous results indicated a strong impact of these two parameters on wine
220 protein stability at 25°C and below.11 Briefly, we showed that wine proteins remain stable at
221 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.
224 Unstable proteins are found within the range 22-28 kDa and are mainly chitinases and some
225 TLP isoforms. Later experiments12 with purified stable and unstable isoforms (two unstable
226 chitinases, one stable TLP and invertase) evidence pH-induced conformational changes of the
227 unstable proteins at room temperature. These modifications affect the global shape (tertiary
10
228 structure) of the unstable chitinases but not the protein secondary structure. Though only local,
229 they lead to the exposure of hydrophobic sites, which likely favor their aggregation.12
230 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
232 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
236 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
241 considered system: (i) formation of stable protein/polysaccharide complexes reducing the
242 aggregation rate of pH unstable proteins and (ii) interactions between polysaccharides and protein
243 aggregates preventing (formation of a protective layer) or enhancing (cross-linking between
244 several aggregates) their growth.34-36
245 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
11
251 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
12
275 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).
13
299 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
14
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
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
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 than with protein alone (factor 2 with AGP0 and 1.4 for AGP4). An in-depth
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
17
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
416 DLS respectively. They also thank the Pech-Rouge experimental unit (Gruissan, France) which
417 provided the Sauvignon blanc wines 2009 and 2011.
418
419 References
420 1. Esteruelas, M.; Poinsaut, P.; Sieczkowski, N.; Manteau, S.; Fort, M. F.; Canals, J. M.; Zamora, F.,
421 Characterization of natural haze protein in sauvignon white wine. Food Chem. 2009, 113, 1, 28-35.
422 2. Pocock, K. F.; Waters, E. J., Protein haze in bottled white wines: How well do stability tests and bentonite
423 fining trials predict haze formation during storage and transport? Aust. J. Grape Wine Res. 2006, 12, 3,
424 212-220.
425 3. Sarmento, M. R.; Oliveira, J. C.; Slatner, M.; Boulton, R. B., Influence of intrinsic factors on conventional
426 wine protein stability tests. Food Control 2000, 11, 6, 423-432.
427 4. Bayly, Francis C.; Berg, H. W., Grape and wine proteins of white wine varietals. Am. J. Enol. Vitic. 1967, 18,
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430 Rizzi, C., Development of a new procedure for protein recovery and quantification in wine. Am. J. Enol.
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432 6. Hsu, J. C.; Heatherbell, D. A., Isolation and characterization of soluble proteins in grapes, grape juice, and
433 wine. Am. J. Enol. Vitic. 1987, 38, 1, 6-10.
434 7. Dambrouck, T; Marchal, R; Cilindre, C; Parmentier, M; Jeandet, P, Determination of the Grape Invertase
435 Content (Using PTA−ELISA) following Various Fining Treatments versus Changes in the Total Protein
436 Content of Wine. Relationships with Wine Foamability. J. Agric. Food Chem. 2005, 53, 22, 8782–8789.
437 8. Sauvage, F-X.; Bach, B.; Moutounet, M.; Vernhet, A., Proteins in white wines: Thermo-sensitivity and
438 differential adsorbtion by bentonite. Food chem. 2010, 118, 1, 26-34.
439 9. Falconer, R.J. ; Marangon, M.; Van Sluyter, S.C. ; Neilson, K.A.; Chan, C.; Waters, E.J., Thermal Stability of
440 Thaumatin-like Protein, Chitinase, and invertase Isolated from Sauvignon blanc and Semillon Juice and
441 Their Role in Haze Formation in Wine Journal of Agricultural and Food Chemistry 2010, 58, 975-980.
442 10. Pocock, K. F.; Hayasaka, Y.; McCarthy, M. G.; Waters, E. J., Thaumatin-like proteins and chitinases, the
443 haze-forming proteins of wine, accumulate during ripening of grape (Vitis vinifera) berries and drought
444 stress does not affect the final levels per berry at maturity. Journal of Agricultural and Food Chemistry
445 2000, 48, 5, 1637-1643.
446 11. Dufrechou, M.; Poncet-Legrand, C.; Sauvage, F-X.; Vernhet, A., Stability of white wine proteins: combined
447 effect of pH, ionic strength, and temperature on their aggregation. J. Agric. Food Chem. 2012, 60, 5, 1308-
448 1319.
449 12. Dufrechou, M.; Vernhet, A.; Roblin, P.; Sauvage, F-X.; Poncet-Legrand, C., White Wine Proteins: How Does
450 the pH Affect Their Conformation at Room Temperature? Langmuir 2013, 29, 33, 10475-10482.
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452 Aggregation of Grape Chitinases and Thaumatin-like Proteins in a Model System. J. Agric. Food Chem.
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455 Australian Grapegrower & Winemaker 2000No. 437, 19-20.
456 15. Pocock, K. F.; Alexander, G. M.; Hayasaka, Y.; Jones, P. R.; Waters, E. J., Sulfate - a candidate for the missing
457 essential factor that is required for the formation of protein haze in white wine. J. Agric. Food Chem. 2007,
458 55, 5, 1799-1807.
459 16. Marangon, M.; Vincenzi, S.; Lucchetta, M.; Curioni, A., Heating and reduction affect the reaction with
460 tannins of wine protein fractions differing in hydrophobicity. Anal. Chim. Acta 2010, 660, 1-2, 110-118.
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461 17. Waters, E. J.; Pellerin, P.; Brillouet, J. -M., A Saccharomyces mannoprotein that protects wine from protein
462 haze. Carbohydr. Polym. 1994, 23, 3, 185-191.
463 18. Waters, E. J.; Pellerin, P.; Brillouet, J. M., A Wine Arabinogalactan-Protein That Reduces Heat-Induced
464 Wine Protein Haze. Biosci., Biotechnol., Biochem. 1994, 58, 1, 43-48.
465 19. Dupin, I. V.; Stockdale, V. J.; Williams, P. J.; Jones, G. P.; Markides, A. J.; Waters, E. J., Saccharomyces
466 cerevisiae mannoproteins that protect wine from protein haze: evaluation of extraction methods and
467 immunolocalization. J. Agric. Food Chem. 2000, 48, 4, 1086-1095.
468 20. Dupin, I. V. S.; McKinnon, B. M.; Ryan, C.; Boulay, M.; Markides, A. J.; Jones, G. P.; Williams, P. J.; Waters, E.
469 J., Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: Their release during
470 fermentation and lees contact and a proposal for their mechanism of action. J. Agric. Food Chem. 2000, 48,
471 8, 3098-3105.
472 21. Ledoux, V.; Dulau, L.; Dubourdieu, D., Interprétation de l'amélioration de la stabilité protéique des vins au
473 cours de l'élevage sur lies. (An explanantion for the improvement of protein stability of wines during aging
474 on yeast lees). J. Int. Sci. Vigne Vin 1992, 26, 239-251.
475 22. Moine-Ledoux, V.; Dubourdieu, D., An invertase fragment responsible for improving the protein stability of
476 dry white wines. J. Sci. Food Agric. 1999, 79, 4, 537-543.
477 23. Gazzola, D.; Van Sluyter, S.C.; Curioni, A.; Waters, E.J.; Marangon, M., Roles of proteins, polysaccharides,
478 and phenolics in haze formation in white wine via reconstitution experiments. J. Agric. Food Chem. 2012,
479 60, 42, 10666-10673.
480 24. Pellerin, P.; Cabanis, J.C., Les glucides, in Oenologie - Fondements scientifiques et technologiques, C.Flanzy.
481 Lavoisier Tec&Doc: Paris, 1998, 40-93.
482 25. Vidal, S.; Williams, P.; Doco, T.; Moutounet, M.; Pellerin, P., The polysaccharides of red wine: total
483 fractionation and characterization. Carbohydr. Polym. 2003, 54, 4, 439-447.
484 26. Coimbra, M. A.; Goncalves, F.; Barros, A. S.; Delgadillo, I., Fourier transform infrared spectroscopy and
485 chemometric analysis of white wine polysaccharide extracts. J. Agric. Food Chem. 2002, 50, 12, 3405-3411.
486 27. Oneill, M. A.; Warrenfeltz, D.; Kates, K.; Pellerin, P.; Doco, T.; Darvill, A. G.; Albersheim, P.,
487 Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is
488 covalently cross-linked by a borate ester - In vitro conditions for the formation and hydrolysis of the dimer.
489 J. Biol. Chem. 1996, 271, 37, 22923-22930.
490 28. Vernhet, A.; Pellerin, P.; Prieur, C.; Osmianski, J.; Moutounet, M., Charge properties of some grape and
491 wine polysaccharide and polyphenolic fractions. Am. J. Enol. Vitic. 1996, 47, 1, 25-30.
492 29. Dawes, H.; Boyes, S.; Keene, J.; Heatherbell, D., Protein instability of wines: influence of protein isolelectric
493 point. Am. J. Enol. Vitic. 1994, 45, 3, 319-326.
494 30. Israelachvili, J., Intermolecular & surface forces. Second edition ed. 1992.
495 31. Doco, T.; Williams, P.; Cheynier, V., Effect of flash release and pectinolytic enzyme treatments on wine
496 polysaccharide composition. J. Agric. Food Chem. 2007, 55, 16, 6643-6649.
497 32. Doco, T.; Quellec, N.; Moutounet, M.; Pellerin, P., Polysaccharide patterns during the aging of Carignan
498 noir red wines Am. J. Enol. Vitic. 1999, 50, 25-32.
499 33. Doco, T.; Vuchot, P.; Cheynier, V.; Moutounet, M., Structural modification of wine arabinogalactans during
500 aging on lees. Am. J. Enol. Vitic. 2003, 54, 3, 150-157.
501 34. Doublier, J.L.; Garnier, C.; Renard, D; Sanchez, C., Protein-polysaccharide interactions. Curr. Opin. Colloid
502 Interface Sci. 2001, 5, 3-4, 202-214.
503 35. Samant, S.K.; Singhal, R.S.; HKulkarni, P.R.; Rege, D.V., Protein-polysaccharide interactions: a new
504 approach in food formulations. Int. J. Food Sci. Technol. 1993, 28, 6, 547-562.
505 36. Turgeon, S.L.; Beaulieu, M.; Schmitt, C.; Sanchez, C., Protein-polysaccharide interactions: phase-ordering
506 kinetics, thermodynamic and structural aspects. Curr. Opin. Colloid Interface Sci. 2003, 8, 4-5, 401-414.
507
508
20
509 Figure Captions
510 Figure 1: A) 1D SDS-PAGE profile of the Sa1 Sauvignon blanc wine 2009 (molecular weight
11
511 (MW) standards on left) and of its purified protein pool (from reference ) B) 1D SDS-PAGE
512 profile of the Sa2 Sauvignon blanc wine 2011 (molecular weight (MW) standards on left) and of
513 its purified protein pool.
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 for the different model systems : polysaccharides (IPS), proteins (IP),
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
21
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
Table 1: Conventional enological analyses of Sauvignon blanc 2009 (Sa1) and 2011 (Sa2)
Sa1 Sa2
Ethanol (% v/v) 11.5 9.5

pH 3.2 3.2
Total acidity (g.L−1 H2SO4) 4.9 5.0
Total SO2 (mg.L−1) 87 91
Free SO2 (mg.L−1) 25 19
Total polyphenol Index 4.6 6.3
K+ (mg.L−1) 566 886
Na+ (mg.L−1) 12 16
Ca2+ (mg.L−1) 80 69
Mg2+ (mg.L−1) 70 64
Conductivity (mS) 1.36 1.92
Turbidity (NTU) 10 33
Protein content (mg.L-1) 160 150
Dufrechou et al : table 1

Table 2: Glycosyl residue composition, molecular mass, protein content and charge
density of the four purified polysaccharides 23
RG-II AGP0 AGP4 MP0
proteins a 3.6 0.8 1.6

b
Neutral
Arabinose 8.4 ± 0.4 40.3 ± 2.8 24.3 ± 0.8 5.2 ± 1.4
Rhamnose 15.5 ± 0.4 1.0 ± 0.6 14.1 ± 0.2 0.4 ± 0.1
Fucose 4.1 ± 0.2 Nd 0.3 ± 0.2
Xylose Nd 0.6 ± 0.0 2.6 ± 0.1
Mannose 0.15 ± 0.1 0.4 ± 0.1 5.0 ± 0.5 88.8 ± 2.0
Galactose 5.3 ± 0.2 51.8 ± 1.4 28.8 ± 0.3 2.9 ± 0.3
Apiose 5.8 ± 0.3
2-O-Me-Xylose 5.7 ± 0.4
2-O-Me-Fucose 5.0 ± 0.3
Glucose 1.6 ± 0.5 0.8 ± 0.1 2.6 ± 0.6
b
Acidic
Galacturonic acid 33.6 ± 1.8 Nd 9.6 ± 0.3
Glucuronic acid 3.3 ± 0.1 4.2 ± 0.4 14.7 ± 0.5
Aceric acid 8.5 ± 1.1
DHA 2.5 ± 0.1
KDO 3.0 ± 0.1
Apparent MW (kDa) 10.5 75 177 62
Charge density (C.g-1)

pH 3.0 -31.5 -3.6 -112.5 -6.6
pH 4.0 -163.3 -5.2 -163.7 -10.2
a: percent of dry matter, b: Molar ratios, Nd: non-detected
Dufrechou et al : Table 2

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*
Wine polysaccharides
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
(mg.L-1)
Purified polysaccharide
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
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 31 28 133 192

fraction (mg.L-1)

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 pH
strength 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
0.02 M 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
0.15 M Prot/Polysacc. 0.044 ± 0.003 0.016 ± 0.000 0.006 ± 0.001 0.006 ± 0.001 0.003 ± 0.001

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 Ratios 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
Proteins/MP0 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
Proteins/AGP0 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
Proteins/AGP4 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

Dufrechou et al., figure 1
A) B)
1 2 3 4 5 6 7
Band number
9 3 5 12 17.5 15.5 38
Purified pool Sa1 (%)
5.3 8.5 7.7 9.6 23.2 14.2 31.5

Purified pool Sa2 (%)

Dufrechou et al., Figure 2
A) B)
C) D)
E) F)

Dufrechou et al.,Figure 3
A) B)
C) D)
E) F)

A) B)
Ionic strength - 0.02 M Ionic strength - 0.02 M
C) D)
Ionic strength - 0.15 M Ionic strength - 0.15 M

Percentage of precipitated proteins

(%)
0.02 M 0.15 M
pH 2.5 Proteins 25 41
Proteins/Polysaccharides 17 26
pH 3.0 Proteins 18 36
Proteins/Polysaccharides 9 22

A) B)
C) D)

A) B)
C) D)

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Journal of Agricultural and Food Chemistry is published by the American Chemical

1 Protein/polysaccharide Interactions and Their

2 Impact on Haze Formation in White Wines

4 Xavier Sauvage1,2,3, Aude Vernhet1,2,3

9 55 rue Rabelais BP 30748, F-49007 Angers Cedex 01, France

16 Marie Dufrechou m.dufrechou@groupe-esa.com

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

ACS Paragon Plus Environment 1

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

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

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

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

37 could not prevent it.

40 Keywords: arabinogalactan-proteins, haze, mannoproteins, protein/polysaccharide

41 interactions, rhamnogalacturonan II dimer, wine proteins

ACS Paragon Plus Environment 2

55 more resistant.8-10 Besides the temperature,4, 8

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

60 aggregation of some of the wine proteins.11

66 and were thus considered as protective factors.17, 18

77 with wine proteins deserve to be further explored.

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

93 attractive electrostatic interactions between these macromolecules can be expected. These

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

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

101 within 15 days.

103 Materials and Methods

111 in Table 1. A heat test (80 °C, 30 min)4, 6

136 and stored in a dry place.

148 proportion of proteins in each stained band.8

150 Carbohydrate composition of wine polysaccharides and of the purified polysaccharide

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

160 formula established previously.32

162 Specific polysaccharide fractions. Four polysaccharide fractions purified to homogeneity

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

178 3 to get the final required protein content).

196 of the initial purified pool.

198 Results and discussion

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

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

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

244 several aggregates) their growth.34-36

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

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

295 3.0 (Table 4).

310 distribution and/or structure of the aggregates.

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

318 the purified protein and polysaccharide pools studied.

320 Polysaccharide structure and protein stability.

333 Sa1 wine.

352 after 15 days are summarized in Table 5.

386 precipitation pattern (results not shown).

388 obtained previously with the polysaccharide pool:

395 AGP0 and the acidic AGP4.

407 Abbreviations used

417 provided the Sauvignon blanc wines 2009 and 2011.

509 Figure Captions