Ethanol Concentration Influences the Mechanisms of Wine Tannin

Subscriber access provided by EMORY UNIV

Article

Ethanol concentration influences the mechanisms of wine tannin interactions with poly(L-proline) in model wine Jacqui M. McRae, Zyta M Ziora, Stella Kassara, Matthew A. Cooper, and Paul A. Smith J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00758 • Publication Date (Web): 15 Apr 2015 Downloaded from http://pubs.acs.org on April 20, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

Ethanol concentration influences the mechanisms of wine tannin interactions with poly(L-proline) in model wine Jacqui M. McRae1,3*, Zyta M. Ziora2,3, Stella Kassara1, Matthew A. Cooper2, Paul A. Smith1 1

The Australian Wine Research Institute, PO Box 197, Glen Osmond SA 5064, Australia.

2

Institute for Molecular Bioscience, University of Queensland, 306 Carmody Rd

St Lucia, QLD 4072, Australia 3

Both authors contributed equally to this paper

*Corresponding author: Tel: +61 8313 6600; Fax: +61 8313 6601

ACS Paragon Plus Environment


1

Abstract

2

Changes in ethanol concentration influence red wine astringency and yet the effect of ethanol

3

on wine tannin-salivary protein interactions is not well understood. Isothermal titration

4

calorimetry (ITC) was used to measure the binding strength between the model salivary

5

protein, poly(L-proline), PLP, and a range of wine tannins (tannin fractions from a three- and

6

a seven-year old Cabernet Sauvignon wine) across different ethanol concentrations (5, 10, 15

7

and 40% v/v). Tannin-PLP interactions were stronger at 5% ethanol than at 40% ethanol. The

8

mechanism of interaction changed for most tannin samples across the wine-like ethanol range

9

(10-15%) from a combination of hydrophobic and hydrogen-binding at 10% ethanol to only

10

hydrogen binding at 15% ethanol. These results indicate that ethanol concentration can

11

influence the mechanisms of wine tannin-protein interactions and that the previously reported

12

decrease in wine astringency with increasing alcohol may, in part, relate to a decrease tannin-

13

protein interaction strength.

14

15

Keywords: Hydrogen-bonding, hydrophobic interactions, ITC, polyproline, wine tannin


Page 2 of 30

Page 3 of 30


16

Introduction

17

Tannins, by their functional definition, interact with proteins.1 Wine tannins consist largely of

18

condensed tannins that are extracted from grapes as well as some hydrolysable tannins

19

extracted from oak such as ellagitannins.2-4 Condensed tannins are extracted from grapes

20

during winemaking and are subsequently structurally-altered due to oxidation and structural

21

rearrangement reactions including the polymerization of smaller polyphenols (Figure 1).5,6

22

They are the major contributors to red wine texture, including astringency and mouth-coating

23

characteristics and have been attributed to red wine quality.7,8 Tannin concentration is

24

directly related to the intensity of astringency and tannin composition can impact more subtle

25

sensory characteristics.9 Astringency relates to the drying or puckering sensations of wine 10

26

and is associated with the interactions between wine tannins and salivary proteins 11,12 or oral

27

epithelial cells,13 although the exact mechanism of astringency perception is not well

28

understood.10 The extent of the interaction of tannins with proteins has been implicated as a

29

measure for astringency.14 Tannin interacts with salivary proteins in three stages. In the first

30

stage, tannins bind to the protein and this interaction changes the shape of a randomly-coiled

31

protein to a more compact structure.14-16 The second stage of interaction involves hydrogen

32

bonding as the tannin-protein complexes aggregate, and in the third and final stage, further

33

hydrogen-bonding leads to the aggregates coalescing and precipitating.14,16,17

34

35

The mechanism of the first stage of tannin-protein interactions has been reported to be driven

36

by hydrophobic interactions and strengthened by hydrogen bonding;1,18,19 yet more recent

37

studies have indicated that this first stage of interaction is more likely to be driven by

38

hydrogen bonding with little or no hydrophobic interaction.20 The involvement of either

39

mechanism can depend on a number of factors, including the structure of the tannins, the

40

structure of the protein and the solvent composition in which the association occurs.



41

Interactions between flavan-3-ol dimers (Figure 1) and protein depend on the stereochemistry

42

of the dimer as well as the solvent. Epicatechin (2) dimer, procyanidin B2 (6), interactions

43

with a synthetic proline-rich protein (PRP) in 10% DMSO were shown to be dominated by

44

hydrophobic interactions,15 while interactions between the catechin (1) dimer, procyanin B3

45

(7) with the proline-rich peptide, IB7, in water were dominated by hydrogen bonding.20 The

46

structure of tannins also influences the extent of protein interaction, and larger tannins with

47

more binding sites and flexible structures have been shown to interact more readily than

48

smaller tannins.18,21-25 Sensory studies have indicated that smaller wine tannins are less

49

astringent than larger wine tannins,26,27 which may relate to the extent of interaction between

50

wine tannins and salivary proteins. Larger tannins bind proteins with a combination of

51

hydrophobic interaction and hydrogen bonding while flavan-3-ol monomer-protein

52

interactions are driven by hydrogen bonding alone.28 The strength of monomer interactions

53

with poly-(L-proline) (PLP) (5) are influenced by the relative hydrophobicity of the

54

monomer, with the more hydrophobic epicatechin gallate (3) binding more strongly than the

55

less hydrophobic, epigallocatechin (4) gallate (Figure 1).28 Conversely, comparatively

56

hydrophilic grape tannins (8) have been shown to bind more strongly to PLP than the more

57

hydrophobic wine tannins (9) (Figure 2).29 The oxidation of grape tannins during crushing

58

and fermentation, which form wine tannins, reduce the proportion of acid-labile interflavan

59

bonds thus creating a more rigid structure with potentially fewer binding sites than grape

60

tannins due to intramolecular bonding and steric hindrances 29-31 suggesting that overall

61

tannin structure may be more important in protein interactions than hydrophobicity. Dimers

62

and trimers with extended structures bind more readily to proteins than those with compact

63

structures where intra-molecular interactions within the polyphenol dominate.23 Proteins that

64

are larger, such as gelatin, or have more extended structures, such as salivary PRPs, also bind

65

more tannin due to the presence of more available binding sites, such as proline residues and


Page 4 of 30

Page 5 of 30


66

amine groups,11,14,18,24,32,33 compared with more globular proteins such as bovine serum

67

albumin (BSA).34-36 Binding of tannins and PRPs shows preference for available proline

68

residues that are not obscured by stereochemistry or interfering structural moieties such as

69

carbohydrate side chains of the protein 37,38 and the residual structures of PRPs such as PPII

70

helices promote tannin interactions due to the conformation of these structures.39 The

71

mechanism of interaction reportedly involves hydrophobic interactions and hydrogen

72

bonding although there are differences in the reported literature about which is the driving

73

mechanism of the first stage of interaction.15,23,29,40 Hydrophobic interactions involve π-π

74

bonding between the planar surfaces of the phenolic tannin structure and the proline ring of

75

the PLP moiety,15 while hydrogen bonding occurs between the carbonyl groups of the proline

76

and the hydroxyl groups of the tannin.1,23 Conversely, interactions between tannins and BSA

77

have been found to be non-specific and are likely to be driven by hydrogen-bonding.34,41

78

79

Solvent composition, particularly ethanol concentration, can influence tannin solubility as

80

well as tannin-protein interactions and astringency.11,18,24 In the wine matrix, the ethanol

81

concentration generally ranges from 10-15%, and this has been shown to influence the wine

82

astringency 42-44 with the intensity of overall astringency decreasing with increasing ethanol

83

concentrations (from 0% to 15% ethanol).44 The concentration of ethanol in wine can also

84

alter the astringency sub-qualities, including ‘velvety’ and ‘silkiness’,44 potentially by

85

changing the extent of tannin-salivary protein interactions or via direct impact on the oral

86

surfaces.42,43 More research is also underway to decrease the concentration of ethanol in

87

wine, such as the development of low alcohol yeast strains,45 which may also impact wine

88

astringency. Wine tannin size and shape are not directly affected by ethanol concentration 46

89

and therefore the changes in astringency with ethanol are more likely to be associated with

90

the physicochemical properties of the different tannins in solvents with different ethanol



91

concentrations.16 Ethanol can disrupt tannin-cell wall binding of apple pulp at concentrations

92

between 20-40% 22 demonstrating the influence of ethanol on non-covalent tannin-protein

93

binding. Storage of red wine can decrease the astringency of a wine as a result of decreased

94

tannin concentrations as well as structural changes in wine tannins.6,47 Tannin structures

95

change over time as a result of gradual oxidation and structural rearrangement reactions that

96

occur under the acidic conditions of wine (pH generally 3.0 to 4.0). Isothermal titration

97

calorimetry (ITC) experiments have indicated that this can reduce the strength of the tannin-

98

protein binding, potentially contributing to the decrease in astringency intensity.29

99

100

In this study, the binding strengths between PLP and a range of different wine tannins and

101

tannin fractions were measured using isothermal titration calorimetry (ITC) across a range of

102

ethanol concentrations (5, 10, 15 and 40% ethanol) to assess the impact of ethanol on tannin-

103

peptide interactions for tannins with different structures.

104 105

Materials and methods

106

Chemicals. All solvents used were high-performance liquid chromatography (HPLC) grade,

107

all chemicals were analytical reagent grade, and water was sourced from a Milli-Q

108

purification system. Ethanol was purchased from Merck Australia (Kilsyth, VIC, Australia).

109

Ammonium formate and poly-(L-proline) (5, DPn 58, molecular weight 5 600 Da) were

110

purchased from Sigma-Aldrich (Castle Hill, NSW, Australia), and formic acid (99% w/w AR

111

grade) was purchased from Chem-Supply.

112 113

Tannin isolation and composition. Wine tannin was isolated from a three- and a seven-year

114

old Cabernet Sauvignon wine and fractionated as described previously 26 to give six tannin


Page 6 of 30

Page 7 of 30


115

samples: total tannin (TT), aqueous (Aq) tannin and butanol-soluble (Bu) tannin for each

116

vintage wine. Fractionation of the TT tannins produced greater proportions of Aq tannin than

117

Bu tannin for both wines with Aq/Bu mass ratios of 3.6:1 and 2.9:1 for the younger and older

118

wines, respectively. The molar extinction coefficients (ɛ) of each tannin sample were

119

determined as described previously 26,29 and used in calculating the concentration of tannin in

120

the stock solutions for ITC analysis. Tannin analysis was performed in a previous study 26

121

using gel permeation chromatography for molecular size, phloroglucinolysis for percent yield

122

(proportions of acid-labile inter-flavan bonds) and subunit composition and octanol-water

123

coefficients (Log P). The percent yield of the young wine tannin samples was 30.2 ± 4.2%

124

and for the aged wine tannin samples was 13.2 ± 2.3%, demonstrating the formation of non-

125

acid labile bonds in tannins as the wine ages. The percent of epicatechin gallate subunits (3)

126

was 3.4 ± 0.5% and 2.0 ± 0.2% for young and aged wines, respectively. There was no

127

significant difference in the percent epigallocatechin between tannin samples of different

128

ages.

129

130

Isothermal titration calorimetry. An autoITC 200 (GE life sciences) was used to measure

131

the change in heat induced by tannin-PLP interactions and data were analysed using the

132

MicroCal Origin version 7.0 software package adapted for auto-ITC data analysis. Tannin

133

fractions were prepared for ITC and titrated using the method previously described.29 Briefly,

134

tannin samples (2 mM) in buffer solution (10 mM ammonium formate) were titrated into a

135

cell containing 0.075, 0.050 or 0.033 mM PLP in the same buffer solution. The titration

136

consisted of 17 x 2 µL injections at 25°C. The concentration of ethanol was varied in the

137

buffer solution (5, 10, 15 and 40% v/v ethanol) and this concentration was the same in both

138

the titrant and PLP solutions for each experiment. Wine tannins were not soluble without

139

ethanol, hence 5% ethanol was considered the low alcohol control sample. Solutions of 5-



140

15% ethanol were at pH 3.5 and the 40% ethanol solution was pH 4.0 due to the reduced

141

proportion of the buffer solution. The influence of the different pH was considered to have a

142

lesser impact than ethanol concentration based on previous reports.18,44,48 Experiments were

143

replicated six times and results were averaged. Thermograms and binding isotherms were

144

used to determine the enthalpy (∆H), binding constants (K), stoichiometry (N), and entropy

145

(T∆S) using a single-site binding model. The change in standard Gibbs free energy (∆G) was

146

calculated using the Gibbs-Helmholz thermodynamic equation: ∆G = -RTlnK, where R is the

147

ideal gas constant (1.985 cal mol-1K-1) and T is the temperature (298 K). Examples of the

148

thermograms and binding isotherms for the different wine tannins are given in Figure 3.

149

150

Statistical analysis. Differences in thermodynamic parameters between different tannins in

151

each matrix and between the same tannin in different matrices were determined using

152

ANOVAs and Tukey analyses with GraphPad Prism statistics software.

153 154

Results and Discussion

155

Wine tannin-peptide interactions. Tannins from a three- and a seven-year old Cabernet

156

Sauvignon wine were isolated and fractionated in previous experiments 26 to produce a range

157

of wine tannins with different structural properties: total (TT) tannin, aqueous (Aq) tannin

158

and butanol-soluble (Bu) tannin. The Bu tannins were consistently smaller with molecular

159

weights of 1 865 ± 92 g mol-1 compared with 3 020 ± 182 g mol-1 for the TT and Aq tannins

160

(Table 1).26 The most notable difference between young and aged wine tannins was the

161

proportion of acid-labile interflavan bonds at around 30% and 13%, respectively.26 Gradual

162

oxidation and structural rearrangement of tannin subunits over time have been shown to

163

cause this reduction in acid-labile bonds.30,47,49


Page 8 of 30

Page 9 of 30


164

165

The strength of the interactions between wine tannins and the peptide, poly-L-proline (PLP)

166

(5), was measured across a range of ethanol concentrations, 5% to 40% v/v, using isothermal

167

titration calorimetry (ITC). Comparisons of the strength of tannin-peptide interactions for the

168

different tannin samples were made at 10% ethanol (Table 2). The mechanisms of binding

169

were assessed by comparing the change in enthalpy (∆H), where a favourable change is

170

indicated by a negative ∆H value and has been associated with hydrogen bonding, and the

171

change in entropy (T∆S), where a favourable change is indicated by a positive T∆S value and

172

has been associated with hydrophobic interactions. Overall binding strength was assessed as a

173

change in Gibbs free energy (∆G), which is the sum of the changes in enthalpy and in

174

entropy. The negative value of the change in Gibbs free energy indicates a spontaneous

175

process. The binding constants (K) and stoichiometry (N) for each tannin sample were

176

calculated based on a single-site binding model. Greater K values indicated stronger PLP-

177

tannin interactions and greater N values indicated that more tannin molecules bound to each

178

PLP molecule. For all tannin samples, binding with PLP was spontaneous and exothermic. At

179

10% ethanol, the changes in enthalpy and in entropy were favourable (Table 2), suggesting

180

that PLP-wine tannin interactions involved both hydrophobic interactions and hydrogen

181

bonding as previously reported.28,29

182

183

Hydrogen bonding contribution (∆H)was greater in the young wine tannins when compared

184

with the same type of tannin from the aged wine at 10% ethanol (Table 2), which is

185

consistent with previous reports.29 The contribution of hydrophobic interactions were greater

186

in the Bu tannins and in the aged wine tannins compared with young wine tannins, as

187

suggested by the greater T∆S values. The combination of changes in enthalpy and entropy



188

influenced the overall strength, ∆G, of the tannins. For the younger wine tannins, ∆G was

189

greater for Aq3 and similar for TT3 and Bu3, while the ∆G for the tannins from aged wines

190

was similar for Aq7 and Bu7 and less for TT7. The binding association constant (K) for Aq3

191

tannin-peptide interactions at 10% ethanol was around double that of the other tannins. This

192

may reflect a three-dimensional conformation of this tannin in this solution that was more

193

favourable for peptide binding. Tannins that have more extended structures have been shown

194

to interact more readily with proteins 23,34 and Aq3 may have a more extended structure with

195

more available binding sites. The molecular weight of the Aq tannin from the younger wine is

196

slightly lower than that of the Aq tannin from the aged wine, however changes in the tannin

197

structure that are known to occur with wine aging results in tannins that have potentially

198

more rigid structures with fewer acid-labile bonds.6,30 Hence, Aq3 is likely to have a greater

199

proportion of available binding sites than Aq7 despite a smaller molecular weight. The

200

stoichiometry (N) of the interaction refers to the number of tannin molecules that bind to each

201

PLP molecule. The number of tannins that can bind with each PLP molecule depends on the

202

number of available binding site, which relates to the molecular weight of the tannin as well

203

as the structure. The calculated N was inversely proportional to the molecular weight of the

204

tannin sample, indicating that the PLP molecules were able to bind more smaller tannins and

205

fewer larger tannins (Tables 1 and 2). More aged wine tannins also bound to each PLP

206

molecule compared with the younger wine counterparts, suggesting that these tannins have

207

fewer available binding sites.29 The structural changes of the tannins in wine over time form a

208

more rigid structure as indicated by a reduction in the number of acid-labile interflavan

209

bonds. Many more Bu tannins were able to bind to each PLP compared with either Aq or TT

210

tannins, further suggesting that these tannins contained fewer available sites for peptide

211

binding. The Aq tannin from the younger wine (Aq3) demonstrated the strongest peptide


Page 10 of 30

Page 11 of 30


212

interactions of the measured tannins in 10% ethanol and this is likely to relate to its 3D-

213

structure.46

214

215

Tannin-peptide interactions at different ethanol concentrations. Tannin astringency

216

reportedly decreases as ethanol concentration in model wines increases from 10% to 15% 43

217

and the reason for this may relate to changes in the mechanisms of tannin-salivary protein

218

interactions. In this study, ITC measurements were used to assess the strength of the

219

interactions between wine tannins and PLP across a range of ethanol concentrations, 5% to

220

40% v/v, as well as any differences in the changes in enthalpy and in entropy that may

221

suggest differences in binding mechanisms. The binding strengths between the wine tannins

222

and PLP as measured with changes in Gibbs free energy (∆G) and binding constants (K)

223

indicated that the wine tannin-PLP interactions were weaker at the greater ethanol

224

concentrations (Figure 4). Ethanol has been shown to disrupt tannin-apple cell wall binding at

225

higher concentrations such as 40% ethanol 22 and the weak interaction between tannin and

226

PLP at this higher ethanol concentration is also demonstrated here. The weaker tannin-

227

peptide interactions at 15% ethanol compared with 10% ethanol may, at least in part,

228

contribute to the previously observed reduction in wine astringency across this ethanol

229

concentration range.43,44

230

231

The mechanism of binding of the tannin samples with PLP also changed over the wine-like

232

ethanol range and differences were observed in tannins with different solubilities. For the TT

233

and Aq tannins, the change in entropy (T∆S) was favourable (positive) at 10% ethanol and

234

unfavourable (negative) at 15% ethanol, while the Bu tannins also demonstrated slightly

235

favourable entropy at 15% ethanol. The change in enthalpy (∆H) was favourable for all



236

tannin samples. Favourable ∆H is associated with binding due to hydrogen bonding, while

237

favourable T∆S has been associated with hydrophobic interactions and the increased disorder

238

due to the displacement of bulk water.28 The favourable T∆S at 10% ethanol and

239

unfavourable T∆S at 15% ethanol suggested that there were differences in the mechanism of

240

tannin-peptide interactions across the wine-like ethanol range. Below 15% ethanol, there is

241

evidence of hydrophobic interactions between tannins and PLP, and for solutions with

242

ethanol concentrations at and above 15%, peptide association appears to be driven only by

243

hydrogen bonding. The combination of favourable ∆H (negative) and unfavourable T∆S

244

(negative) contributed to the weaker overall binding (∆G) in solutions containing 40%

245

ethanol (Figure 4).

246

247

The different tannin samples varied in the strengths and mechanisms of peptide binding. The

248

T∆S was favourable for the Bu tannins at 15% ethanol, suggesting that the structure of these

249

tannins promoted more hydrophobic interactions than the Aq and TT tannins, which showed

250

unfavourable T∆S at the same ethanol concentration. The differences between the changes in

251

enthalpy and in entropy for the different tannin fractions were more pronounced at 40%

252

ethanol compared with 5% ethanol, and this may relate to differences in tannin structure.

253

Smaller tannins, such as Bu tannins, have fewer binding sites for peptide interactions than

254

larger tannins such as TT and Aq and therefore these interactions are more readily disrupted

255

by higher ethanol concentrations. The number of TT and Aq tannin molecules that interacted

256

with each PLP molecule (N) decreased slightly across the ethanol concentrations (Figure 5),

257

whereas the number of Bu tannins that interacted with PLP ranged from around 4.6 to 5.6,

258

independently of the ethanol concentration. This also may relate to the structure of these

259

tannins and the limited number of available binding sites.


Page 12 of 30

Page 13 of 30


260

261

Different reports on the primary mechanism for tannin-protein interactions have indicated

262

either hydrophobically-driven interactions or hydrogen-bond driven interactions 15,23,40 and

263

the reason for this discrepancy is likely to relate to the solvent system used as well as the

264

particular tannin structure used in the analysis. Similarly, reports on the impacts of ethanol on

265

sensory analysis have varied from ethanol increasing astringency to decreasing astringency

266

43,50

267

the solvents used for analysis. More hydrophilic tannins such as grape skin tannins at higher

268

ethanol concentrations are likely to interact with proteins primarily with hydrogen bonding,

269

while more hydrophobic grape seed tannins may be driven by a combination of hydrophobic

270

and hydrophilic interactions.22,28 Similarly, the astringency of tannins in model wines will

271

depend on the structure and physicochemical properties of the tannins in the study,

272

particularly given the differences in binding strength and mechanisms of interaction for wine

273

tannins over the wine-like ethanol concentration range. This demonstrates the need for

274

reporting the hydrophobicity of the tannins used in structure-function experiments.

and this again may relate to the structure and relative hydrophobicity of the tannins and

275

276

Impact of wine age on tannin-peptide interactions. Red wine astringency reportedly

277

decreases with wine age 51 and this may relate to changes in the properties of the tannins,

278

which subsequently impact the tannin binding capacity.29,47 The tannin samples selected for

279

this trial were isolated from red wine of three- and seven years of age, to compare the binding

280

strength of wine tannin after different periods of aging. For Aq and TT tannins, those from

281

the younger wine demonstrated slightly stronger binding (∆G) across the different ethanol

282

concentrations, even though the molecular weights were similar or smaller than those of the

283

tannins from the aged wine (Table 1, Figure 4). The is in agreement with previous reports.29



284

During wine aging, wine tannin structures change due to gradual oxidation and structural

285

rearrangement reactions, which can reduce the number of hydroxyl groups available for

286

hydrogen bonding and potentially increase the proportion of hydrophobic binding sites30,31

287

Differences in the ∆H and T∆S for younger and aged wine tannins suggested differences in

288

the mechanisms of interactions. Peptide interactions with aged wine tannins produced more

289

favourable changes in entropy and smaller changes in enthalpy than with the younger wine

290

tannins (Table 2), suggesting that the structures of these tannins promoted more hydrophobic

291

interactions. The stoichiometry (N) indicated that more aged wine tannin molecules bind to

292

PLP than the same fraction of young wine tannin molecules, suggesting that aged wine

293

tannins may be more compact and have fewer binding sites for peptide interaction than

294

younger wine tannins. The weaker binding of more aged wine tannins may, in part, contribute

295

to the decrease in wine astringency with aging since protein interactions are associated with

296

astringency.14,52

297

298

In summary, the strength of wine tannin-PLP binding was shown to decrease with increasing

299

ethanol concentration and the mechanisms of interaction changed between 10% and 15%

300

ethanol. For the larger tannins, peptide interactions consisted of favourable changes in both

301

entropy and in enthalpy at 10% ethanol, and at 15% ethanol, interactions involved favourable

302

changes in enthalpy and unfavourable changes in entropy. Peptide interactions with smaller

303

tannins showed slightly favourable changes in entropy at 15% ethanol. This suggests that at

304

lower ethanol concentrations, peptide interactions with wine tannin involves a combination of

305

hydrophobic interactions and hydrogen bonding, while only hydrogen bonding occurred in

306

the higher ethanol concentration. Tannins from aged wines also demonstrated more

307

hydrophobic interactions than from younger wines. These results indicate that the solvent

308

composition and wine tannin structure can influence the mechanisms for tannin-protein


Page 14 of 30

Page 15 of 30


309

interaction which, in turn, may influence wine astringency. This may contribute to some of

310

the differences observed in wine styles with different astringency qualities. Additionally, this

311

work demonstrates the need for reporting the relative physical chemistry characteristics such

312

as hydrophobicity of the tannins used in structure-function experiments.

313

314

Supporting Information Available: Tables S1-S3: Thermodynamics parameters for

315

interactions between different wine tannins with PLP at 5%, 15% and 40% ethanol. This

316

material is available free of charge via the Internet at http://pubs.acs.org.

317

318

References

319

1. Haslam, E. Natural polyphenols (vegetable tannins) as drugs: Possible modes of action. J

320 321 322 323

Nat Prod 1996, 59, 205-215. 2. Vivas, N.; Glories, Y. Role of oak wood ellagitannins in the oxidation process of red wines during aging. Am J Enol Viticult 1996, 47, 103-107. 3. Vivas, N.; Nonier, M. F.; Gaulejac, N. V. d. Structural characterization and analytical

324

differentiation of grape seeds, skins, stems and Quebracho tannins. Bulletin l'OIV

325

2004, 77, 643-659

326 327

4. Herderich, M. J.; Smith, P. A. Analysis of grape and wine tannins: Methods, applications and challenges. Australian Journal of Grape and Wine Research 2005, 11, 1-10.

328

5. Monagas, M.; Bartolome, B.; Gomez-Cordoves, C. Updated knowledge about the presence

329

of phenolic compounds in wine. Critical Reviews in Food Science and Nutrition 2005,

330

45, 85-118.



331

6. Cheynier, V.; Duenas-Paton, M.; Salas, E.; Maury, C.; Souquet, J. M.; Sarni-Manchado,

332

P.; Fulcrand, H. Structure and properties of wine pigments and tannins. Am. J. Enol.

333

Vitic. 2006, 57, 298-305.

334

7. Mercurio, M. D.; Dambergs, R. G.; Cozzolino, D.; Herderich, M. J.; Smith, P. A.

335

Relationship between red wine grades and phenolics. 1. Tannin and total phenolics

336

concentrations. J Agr Food Chem 2010, 58, 12313-12319.

337

8. Smith, P.; Mercurio, M.; Dambergs, R.; Francis, L.; Herderich, M. Red grape and wine

338

quality - the roles and relevance of tannin. Australian & New Zealand Wine Industry

339

Journal 2007, 47-52.

340

9. Vidal, S.; Courcoux, P.; Francis, L.; Kwiatkowski, M.; Gawel, R.; Williams, P.; Waters,

341

E.; Cheynier, V. Use of an experimental design approach for evaluation of key wine

342

components on mouth-feel perception. Food Qual Prefer 2004, 15, 209-217.

343 344 345

10. Bajec, M. R.; Pickering, G. Astringency: Mechanisms and perception. Critical Reviews in Food Science and Nutrition 2008, 48, 858-875. 11. Pascal, C.; Poncet-Legrand, C.; Cabane, B.; Vernhet, A. Aggregation of a proline-rich

346

protein induced by epigallocatechin gallate and condensed tannins: Effect of protein

347

glycosylation. J Agr Food Chem 2008, 56, 6724-6732.

348

12. Tarascou, I.; Barathieu, K.; Simon, C.; Ducasse, M. A.; Andre, Y.; Fouquet, E.; Dufourc,

349

E. J.; de Freitas, V.; Laguerre, M.; Pianet, I. A 3D structural and conformational study

350

of procyanidin dimers in water and hydro-alcoholic media as viewed by NMR and

351

molecular modeling. Magn Reson Chem 2006, 44, 868-880.

352 353

13. Payne, C.; Bowyer, P. K.; Herderich, M.; Bastian, S. E. P. Interaction of astringent grape seed procyanidins with oral epithelial cells. Food Chemistry 2009, 115, 551-557.


Page 16 of 30

Page 17 of 30

354


14. Jöbstl, E.; O’Connell, J.; Fairclough, J. P. A.; Williamson, M. P. Molecular Model for

355

Astringency Produced by Polyphenol/Protein Interactions. Biomacromolecules 2004,

356

5, 942-949.

357

15. Baxter, N. J.; Lilley, T. H.; Haslam, E.; Williamson, M. P. Multiple interactions between

358

polyphenols and a salivary proline-rich protein repeat result in complexation and

359

precipitation. Biochemistry 1997, 36, 5566-5577.

360

16. Zanchi, D.; Poulain, C.; Konarev, P.; Tribet, C.; Svergun, D. I. Colloidal stability of

361

tannins: astringency, wine tasting and beyond. Journal of Physics: Condensed Matter

362

2008, 20, 494224.

363

17. Dinnella, C.; Recchia, A.; Fia, G.; Bertuccioli, M.; Monteleone, E. Saliva characteristics

364

and individual sensitivity to phenolic astringent stimuli. Chemical Senses 2009, 34,

365

295-304.

366 367 368

18. Charlton, A.; Baxter, N.; Khan, M.; Moir, A.; Haslam, E.; Davies, A.; Williamson, M. Polyphenol/peptide binding and precipitation. J Agr Food Chem 2002, 50 1593-1601. 19. Le Bourvellec, C.; Renard, C. M. G. C. Interactions between polyphenols and

369

macromolecules: Quantification methods and mechanisms. Critical Reviews in Food

370

Science and Nutrition 2012, 52, 213-248.

371

20. Simon, C.; Barathieu, K.; Laguerre, M.; Schmitter, J.-M.; Fouquet, E.; Pianet, I.; Dufourc,

372

E. J. Three-dimensional structure and dynamics of wine tannin-saliva protein

373

complexes. a multitechnique approach. Biochemistry 2003, 42, 10385-10395.

374

21. Bindon, K.; Smith, P.; Kennedy, J. A. Interaction between Grape-Derived

375

Proanthocyanidins and Cell Wall Material. 1. Effect on Proanthocyanidin

376

Composition and Molecular Mass. J Agr Food Chem 2010, 58, 2520–2528.

377 378

22. Le Bourvellec, C.; Guyot, S.; Renard, C. M. G. C. Non-covalent interaction between procyanidins and apple cell wall material: Part I. Effect of some environmental



379

parameters. Biochimica et Biophysica Acta (BBA) - General Subjects 2004, 1672,

380

192-202.

381

23. Cala, O.; Pinaud, N.; Simon, C.; Fouquet, E.; Laguerre, M.; dufourc, E. J.; Pianet, I. NMR

382

and molecular modeling of wine tannins binding to saliva proteins: revisiting

383

astringency from molecular and colloidal prospects. Faseb J 2010, 24, 1-10.

384

24. Hagerman, A. E.; Rice, M. E.; Ritchard, N. T. Mechanisms of protein precipitation for

385

two tannins, pentagalloyl glucose and epicatechin16 (4f8) catechin (procyanidin). J

386

Agr Food Chem 1998, 46, 2590-2595.

387

25. Cheynier, V.; Prieur, C.; Guyot, S.; Rigaud, J.; Moutounet, M. The structures of tannins

388

in grapes and wines and their interactions with proteins. In Wine - Nutritional and

389

therapeutic benefits T. R. Watkins, Ed.; ACS: Washington DC, 1997; pp 81-93

390

26. McRae, J. M.; Schulkin, A.; Kassara, S.; Holt, H.; Smith, P. A. Sensory properties of

391

wine tannin fractions: Implications for in-mouth sensory properties. J Agr Food Chem

392

2013, 61, 719-727.

393

27. Vidal, S.; Francis, L.; Guyot, S.; Marnet, N.; Kwiatkowski, M.; Gawel, R.; Cheynier, V.;

394

Waters, E. J. The mouth-feel properties of grape and apple proanthocyanidins in a

395

wine-like medium. J Sci Food Agr 2003, 83, 564-573.

396

28. Poncet-Legrand, C.; Gautier, C.; Cheynier, V.; Imberty, A. Interactions between flavan-3-

397

ols and poly(L-proline) studied by Isothermal Titration Calorimetry: Effect of the

398

tannin structure. J Agr Food Chem 2007, 55 9235-9240.

399

29. McRae, J. M.; Falconer, R. J.; Kennedy, J. A. Thermodynamics of grape and wine tannin

400

interaction with polyproline: Implications for red wine astringency. J Agr Food Chem

401

2010, 58, 12510–12518.


Page 18 of 30

Page 19 of 30

402


30. Poncet-Legrand, C.; Cabane, B.; Bautista-Ortín, A.; Carrillo, S.; Fulcrand, H.; Pérez, J.;

403

Vernhet, A. Tannin oxidation: Intra- versus intermolecular reactions.

404

Biomacromolecules 2010, 11, 2376–2386.

405

31. Vernhet, A.; Dubascoux, S.; Cabane, B.; Fulcrand, H.; Dubreucq, E.; Poncet-LeGrand, C.

406

Characterization of oxidized tannins: comparison of depolymerization methods,

407

assymetric flow field-flow fractionation and small-angle X-ray scattering. Anal

408

Bioanal Chem 2011, 401, 1559-1569.

409

32. Boze, H.; Marlin, T.; Durand, D.; Pérez, J.; Vernhet, A.; Canon, F.; Sarni-Manchado, P.;

410

Cheynier, V.; Cabane, B. Proline-rich salivary proteins have extended conformations.

411

Biophysical Journal 2010, 99, 656–665.

412

33. Charlton, A. J.; Baxter, N. J.; Lilley, T. H.; Haslam, E.; McDonald, C. J.; Williamson, M.

413

P. Tannin interactions with a full-length human salivary proline-rich protein display a

414

stronger affinity than with single proline-rich repeats. FEBS letters 1996, 382, 289-

415

292.

416

34. Frazier, R. A.; Papadopoulou, A.; Mueller-Harvey, I.; Kissoon, D.; Green, R. J. Probing

417

protein-tannin interactions by isothermal titration microcalorimetry. J Agr Food Chem

418

2003, 51, 5189-5195.

419

35. Deaville, E. R.; Green, R. J.; Mueller-Harvey, I.; Willoughby, I.; Frazier, R. A.

420

Hydrolyzable Tannin Structures Influence Relative Globular and Random Coil

421

Protein Binding Strengths. J. Agric. Food Chem. 2007, 55, 4554-4561.

422

36. Frazier, R. A.; Deaville, E. R.; Green, R. J.; Stringanod, E.; Willoughbyd, I.; Plante, J.;

423

Mueller-Harvey, I. Interactions of tea tannins and condensed tannins with proteins.

424

Journal of Pharmaceutical and Biomedical Analysis 2010, 51, 490-495.

425

37. Lu, Y.; Bennick, A. Interaction of tannin with human salivary proline-rich proteins.

426

Archives of Oral Biology 1998, 43, 717-728.



427

38. Luck, G.; Liao, H.; Murray, N. J.; Grimmer, H. R.; Warminski, E. E.; Williamson, M. P.;

428

Lilley, T. H.; Haslam, E. Polyphenols, astringency and proline-rich proteins.

429

Phytochemistry 1994, 37, 357-371.

430

39. Pascal, C.; Paté, F.; Cheynier, V.; Delsuc, M. Study of the interactions between a proline-

431

rich protein and a flavan-3-ol by NMR: Residual structures in the natively unfolded

432

protein provides anchorage points for the ligands. Biopolymers 2009, 91, 745-756.

433

40. Richard, T.; Lefeuvre, D.; Descendit, A.; Quideau, S.; Monti, J. P. Recognition characters

434

in peptide-polyphenol complex formation. Biochim. Biophys. Acta-Gen. Subj. 2006,

435

1760, 951-958.

436 437 438 439 440

41. Oh, H. I.; Hoff, J. E.; Armstrong, G. S.; Haff, L. A. Hydrophobic interaction in tanninprotein complexes. J Agr Food Chem 1980, 28, 394-398. 42. Kallithraka, S.; Bakker, J.; Clifford, M. N. Effect of pH on astringency in model solutions and wines. J Agr Food Chem 1997, 45, 2211-2216. 43. Fontoin, H.; Saucier, C.; Teissedre, P.-L.; Glories, Y. Effect of pH, ethanol and acidity on

441

astringency and bitterness of grape seed tannin oligomers in model wine solution.

442

Food Qual Prefer 2008, 19, 286-291.

443

44. Demiglio, P.; Pickering, G. J. The influence of ethanol and pH on the taste and mouthfeel

444

sensations elicited by red wine. Journal of Food, Agriculture and Environment 2008,

445

6, 143-150.

446 447 448

45. Carrau, F.; Gaggero, C.; Aguilar, P. S. Yeast diversity and native vigor for flavor phenotypes. Trends in Biotechnology 2015, 33, 148-154. 46. McRae, J. M.; Kirby, N. M.; Mertens, H. D. T.; Kassara, S.; Smith, P. A. Measuring the

449

molecular dimensions of wine tannins: comparison of small-angle X-ray scattering,

450

gel-permeation chromatography and mean degree of polymerization. J Agr Food

451

Chem 2014, 62, 7216-7224.


Page 20 of 30

Page 21 of 30

452


47. McRae, J. M.; Kassara, S.; Kennedy, J. A.; Waters, E. J.; Smith, P. A. Effect of wine pH

453

and bottle closure on tannins. J Agr Food Chem 2013, 61, 11618-11627.

454

48. McManus, J. P.; Davis, K. G.; Beart, J. E.; Gaffney, S. H.; Lilley, T. H.; Haslam, E.

455

Polyphenol interactions. part 1. introduction; some observations on the reversible

456

complexation of polyphenols with proteins and polysaccharides. Journal of the

457

Chemical Society-Perkin Transactions 2 1985, 1429-1438.

458

49. Vidal, S.; Cartalade, D.; Souquet, J.-M.; Fulcrand, H.; Cheynier, V. Changes in

459

proanthocyanidin chain length in winelike model solutions. J Agr Food Chem 2002,

460

50, 2261-2266.

461

50. Obreque-Slíer, E.; Peña-Neira, A.; López-Solís, R. Enhancement of both salivary protein-

462

enological tannin interactions and astringency perception by ethanol. J Agr Food

463

Chem 2010, 58, 3729-3735.

464

51. Chira, K.; Pacella, N.; Jourdes, M.; Teissedre, P.-L. Chemical and sensory evaluation of

465

Bordeaux wines (Cabernet-Sauvignon and Merlot) and correlation with wine age.

466

Food Chemistry 2011, 126, 1971-1977.

467

52. Hofmann, T.; Glabasnia, A.; Schwarz, B.; Wisman, K. N.; Gangwer, K. A.; Hagerman,

468

A. E. Protein binding and astringent taste of a polymeric procyanidin, 1,2,3,4,6-penta-

469

O-galloyl-beta-D-glucopyranose, castalagin, and grandinin. J. Agric. Food Chem.

470

2006, 54, 9503-9509.

471 472

Financial support:

473

JMM, SK and PAS from The Australian Wine Research Institute, a member of the Wine

474

Innovation Cluster at the Waite Precinct in Adelaide, acknowledge the support of Australian

475

grape growers and winemakers through their investment body, the Australian Grape and

476

Wine Authority, with matching funds from the Australian Government.



477

Figure Captions

478

Figure 1. The structures of flavan-3-ol monomers (wine and grape tannin subunits) and

479

dimers including catechin (1), epicatechin (2), epicatechin gallate (3), and epigallocatechin

480

(4); flavan-3-ol dimers, B2 (6) and B3 (7), as well as the structure of poly-(L-proline) (5)

481

Figure 2. Examples of grape tannin (8) and wine tannin (9) structures showing B ring ether-

482

linkages, A-type linkages and ethyl linkages.

483

Figure 3. Thermograms and binding isotherms of wine tannin samples interacting with PLP

484

(0.075 mM) in 10% ethanol. a) TT3; b) Bu3; and c) Aq3.

485

Figure 4. Overall binding strength of wine tannins samples and PLP across the range of

486

ethanol concentrations as measured using a) changes in Gibbs free energy (∆G) and b)

487

binding association constants (K).

488

Figure 5. The impact of ethanol concentration on the interaction between wine tannin

489

samples and PLP as measured with a) change in enthalpy (∆H); b) change in entropy (T∆S);

490

and the c) the stoichiometry (N) of interaction (number of tannin molecules binding per PLP

491

molecule).


Page 22 of 30

Page 23 of 30


Tables Table 1. Characteristics of Wine Tannins used in This Study including the Molecular Weight (MW), Octanol/Water Partition Coefficient (Log P), and Extinction Coefficient (ɛ). Tannin

MW

Sample

(g mol-1) a

Log P a

ɛ (x103) (M-1cm-1)

TT3

2 930

-0.61

44.3

Aq3

3 030

-1.31

37.4

Bu3

1 800

-0.7

27.1

TT7

2 850

-0.55

36.3

Aq7

3 270

-1.34

45.6

Bu7

1 930

-0.72

29.2

a

Results from McRae et al. 26



Page 24 of 30

Table 2. Thermodynamics Parameters for Interactions between Different Wine Tannins and PLP at 10% Ethanol including the Change in Enthalpy (∆H), Change in Entropy (T∆S), Change in Gibbs Free Energy (∆G), Stoichiometry (N), and Binding Association Constant (K). Values are shown as the Average of Six Results ± One Standard Deviation and Letters Indicate Values in Columns that are Significantly Different (p < 0.05).

Tannin

∆H

T∆S

∆G

K

Fraction

(kcal mol-1)

(kcal mol-1)

(kcal mol-1)

(106 M-1)

TT3

-6.93 ± 0.09a

1.26 ± 0.20d

-8.19 ± 0.11bc

1.03 ± 0.20bc

3.06 ± 0.09d

Aq3

-7.09 ± 0.38a

1.59 ± 0.29d

-8.68 ± 0.09a

2.34 ± 0.35a

2.71 ± 0.11e

Bu3

-4.83 ± 0.08c

3.31 ± 0.07b

-8.14 ± 0.00c

0.93 ± 0.01c

5.50 ± 0.15b

TT7

-5.90 ± 0.18b 2.21 ± 0.16c

-8.11 ± 0.01d

0.89 ± 0.02d

3.41 ± 0.08c

Aq7

-6.04 ± 0.10b 2.22 ± 0.10c

-8.27 ± 0.02b

1.16 ± 0.03b

3.32 ± 0.10c

Bu7

-4.44 ± 0.13c

-8.28 ± 0.04b

1.18 ± 0.08b

5.68 ± 0.01a

3.84 ± 0.10a


N

Page 25 of 30


Figure 1



Page 26 of 30

Figure 2

OCH3 HO OH O

HO

OH

Glu-O

OH OH

9

OH HO

OH OH HO

O+

H 3CO

OH

OH HO

OH

OH

OH O

OH OH OH

HO

O O

OH

O OH

OH HO

O

8

OH OH

OH OH HO

O

O

HO OH HO

OH OH

O O

OH

OH

HO OH OH

OH


OH

OH

Page 27 of 30


Figure 3



Figure 4


Page 28 of 30

Page 29 of 30


Figure 5



Table of Contents Graphic


Page 30 of 30

Ethanol Concentration Influences the Mechanisms of Wine Tannin

Recommend Documents