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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

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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

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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-

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protein interaction strength.

14

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


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Introduction

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

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

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

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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

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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

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

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

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

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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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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mechanism of interaction reportedly involves hydrophobic interactions and hydrogen

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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peptide interactions for tannins with different structures.

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Materials and methods

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

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

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

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

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

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grade) was purchased from Chem-Supply.

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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significant difference in the percent epigallocatechin between tannin samples of different

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ages.

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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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

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

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Statistical analysis. Differences in thermodynamic parameters between different tannins in

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

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ANOVAs and Tukey analyses with GraphPad Prism statistics software.

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Results and Discussion

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

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

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

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

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

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

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

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

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


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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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bonding as previously reported.28,29

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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structure.46

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Tannin-peptide interactions at different ethanol concentrations. Tannin astringency

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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

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

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

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

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

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

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

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

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

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

229

concentration range.43,44

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

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

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

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

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



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

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

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

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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

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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

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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).

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

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

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

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

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

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

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

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

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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,

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

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tannins and the limited number of available binding sites.


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

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

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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

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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,

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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

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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


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

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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.

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References

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

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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

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

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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 ... - ACS Publications

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