Flavan-3-ol Aggregation in Model Ethanolic Solutions: Incidence of

Riou, V.; Vernhet, A.; Doco, T.; Moutounet, M. Food Hydrocolloids 2002, 16, ..... Siboni, S. In Contact Angle, Wettability and Adhesion; Mittal, K. L...
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Langmuir 2003, 19, 10563-10572

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Flavan-3-ol Aggregation in Model Ethanolic Solutions: Incidence of Polyphenol Structure, Concentration, Ethanol Content, and Ionic Strength Ce´line Poncet-Legrand,*,† Damien Cartalade,† Jean-Luc Putaux,‡ Ve´ronique Cheynier,† and Aude Vernhet† UMR Sciences pour l’Oenologie (INRA-ENSAM-UM1) 2 Place Viala, 34060 Montpellier Cedex 1, France, and Centre de Recherches sur les Macromole´ cules Ve´ ge´ tales, CNRS BP53, 38041 Grenoble Cedex 9, France Received May 28, 2003. In Final Form: August 1, 2003 Colloidal interactions involving polyphenols play a crucial part in wine stability, clarification, and taste. Though there is some evidence that polyphenolic compounds form stacks and aggregates in hydroalcoholic solutions, only little is known about their colloidal behavior. The aim of this study was, thus, to investigate in model ethanolic solutions the colloidal aggregation of flavan-3-ol monomers and polymer fractions extracted from grape seeds as well as apple and pear parenchyma. Aggregation was studied by means of phase diagrams, and aggregates were characterized by dynamic light scattering and cryo-transmission electron microscopy. Several parameters were studied: (i) the incidence of the tannin structure (mean degree of polymerization, mDP, and percentage of galloylation) and concentration (between 10-2 and 5 g L-1) and (ii) the incidence of the ethanol content (from 2 to 20%) and ionic strength (from 10-3 to 10-1 M) of the solvent. Regarding the tannin structure, galloylation enhanced the formation of aggregates as far as monomers were concerned, but this could not be confirmed with polymers. The mDP had a complex effect: aggregation increased first with mDP for relatively low molecular weight polymers but decreased again for higher molecular weight fractions. This suggests that the higher molecular weight polymers can adopt a conformation in solution that enhances their solubility. Increasing the ionic strength resulted in a much lower tannin solubility and, when soluble, in the formation of bigger and much more polydisperse particles (salting-out effect). The ethanol content of the solvent also had a strong incidence on self-aggregation: increasing the ethanol content resulted in higher tannin solubilities and smaller and less polydisperse colloidal aggregates. This could be linked to the superficial tension properties of solvents containing various amounts of ethanol and confirmed the determinant part played by lyophobic interactions in flavan-3-ol aggregation.

Introduction Polyphenols in wine are involved in several colloidal phenomena, which are of primary importance for wine stability, clarification, and taste. For instance, they can be responsible for the formation of hazes and precipitates. Procyanidins are likely involved in the formation of protein hazes in white wines,1,2 while in red ones, polyphenol evolution during wine making and aging leads to the formation of unstable colloidal coloring matter.3 Hazes and sediments, even when they do not affect taste, are detrimental for wine quality: most of the consumers will reject the product. Furthermore, colloids may affect the efficiency of the clarification and stabilization treatments applied to ensure wine limpidity and stability.4-6 Another important property of polyphenols in enology is their well-known ability to complex with and precipitate proteins. First, this property is exploited for wine clarification by means of fining treatments. Fining consists of * Corresponding author. E-mail: [email protected]. Phone: +33499612758. Fax: +33499612683. † UMR Sciences pour l’Oenologie. ‡ Centre de Recherches sur les Macromole ´ cules Ve´ge´tales. (1) Dawes, H.; Boyes, S.; Keene, J.; Heatherbell, D. Am. J. Enol. Vitic. 1994, 45, 319-326. (2) Waters, E. J.; Peng, Z.; Pocock, K. F.; Williams, P. J. Aust. J. Grape Wine Res. 1995, I, 86-93. (3) Waters, E. J.; Peng, Z.; Pocock, K. F.; Jones, G. P.; Clarke, P.; Williams, P. J. J. Agric. Food Chem. 1994, 42, 1761-1766. (4) Escudier, J. L.; Moutounet, M.; Bernard, P. Rev. Fr. Oenol. 1987, 108, 52-57. (5) Escudier, J. L.; Moutounet, M. Rev. Fr. Oenol. 1987, 109, 44-50. (6) Vernhet, A.; Cartalade, D.; Moutounet, M. J. Membr. Sci. 2003, 211, 357-370.

introducing an exogenous protein in wine, which flocculates and precipitates, clarifying wine by removing suspended particles. This phenomenon implies interactions between the fining proteins and polyphenols, especially condensed tannins.7,8 Interactions between salivary proteins and tannins are also reported to be responsible for the astringency perception, which is an important attribute for wine quality.8-10 All these technological and organoleptic properties are usually attributed to intermolecular interactions that take place between polyphenols themselves or between polyphenols and proteins. Other wine constituents, such as polysaccharides, likely modify these interactions.11,12 A good understanding of colloidal phenomena involving polyphenols in wines is, thus, uppermost in enology (i) to be in the position to control stabilization and clarification treatments and (ii) to improve wine quality and taste. This requires an improved knowledge of the chemical structure of the involved components, along with their interactions with other wine constituents, exogenous macromolecules, and filtration media. (7) Sarni-Manchado, P.; Deleris, A.; Avallone, S.; Cheynier, V.; Moutounet, M. Am. J. Enol. Vitic. 1999, 50, 81-86. (8) Maury, C.; Sarni-Manchado, P.; Lefebvre, S.; Cheynier, V.; Moutounet, M. Am. J. Enol. Vitic. 2001, 52, 140-145. (9) Bate-Smith, E. Food 1954, 23, 124-135. (10) Sarni-Manchado, P.; Cheynier, V.; Moutounet, M. J. Agric. Food Chem. 1999, 47, 42-47. (11) Luck, G.; Liao, H.; Murray, N. J.; Grimmer, H. R.; Warminski, E. E.; Williamson, M. P.; Lilley, T. H.; Haslam, E. Phytochemistry 1994, 37, 357-371. (12) McManus, J. P.; Davis, K. G.; Beart, J. E.; Gaffney, S. H.; Lilley, T. H.; Haslam, E. J. Chem. Soc., Perkin Trans. I 1985, 1429-1438.

10.1021/la034927z CCC: $25.00 © 2003 American Chemical Society Published on Web 11/06/2003

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Figure 1. Structures of the studied monomers. Apple condensed tannins are made of epicatechin units (R1dR3dH, R2dOH) linked by C4-C6 or C4-C8 interflavanol bonds, whereas grape seed tannins are made of catechin (R1dOH, R2dR3dH), epicatechin, and epicatechin gallate (R1dR3dH, R2dG). Epigallocatechin (R1dH, R2dR3dOH) is present in wine (brought by grape skins) and green tea, epigallocatechin gallate (R1dH, R2dG, R3dOH) is also present in green tea. Table 1. Characteristics of the Different Tannin Fractions Determined by Thiolysis grape seed tannin fractions fraction number mDP percentage of galloylation mean molecular eight (Da)

F1-2 3 18.5 960

F1-3 5 22.4 1625

F2 8 26.4 2640

It was recently demonstrated, by means of dynamic light scattering (DLS) experiments, that both polymers formed by catechin-acetaldehyde reactions and procyanidins extracted from grape seeds may aggregate into colloidal particles in model ethanolic solutions.13,14 This raises the questions of tannin colloidal behavior in wine and of its incidence on interactions. Condensed tannins in wines are polymeric flavan-3-ols composed of (+)-catechin, (-)-epicatechin, (-)-epicatechin3-gallate and (-)-epigallocatechin units (Figure 1), forming procyanidins or prodelphinidin polymers. The aim of the present work was to investigate the solubility and the colloidal behavior of flavan-3-ol monomers and condensed tannins in model hydroalcoholic solutions. Several parameters were studied: first, the incidence of polyphenol structure and concentration and, second, the incidence of ethanol content and ionic strength of the model solution. Concerning the structure, the galloylation, the degree of hydroxylation, and the degree of polymerization were studied, with concentrations ranging from 10 mg L-1 to 5 g L-1. The ethanol content was varied between 2 and 20%, while the ionic strength was varied between 10-3 and 10-1 M. Experimental Section Chemicals. Deionized water was obtained with a Milli-Q system (Millipore). Absolute ethanol, acetone, and glycerol (analytical grade) were purchased from Prolabo, tartaric acid and sodium chloride were purchased from Labosi, and sodium hydroxide and formamide (analytical grade) were purchased from Carlo Erba Reagents. Flavan-3-ol monomers (catechin, epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate), hexane, and di-iodomethane (analytical grade) were obtained from Sigma. TDF4 was purchased from Franklab. Poly(methyl methacrylate) (PMMA) and poly(amide 6-6) were provided by Goodfellow. Purification and Characterization of Tannin Fractions. Grape seed tannins were purified from freeze-dried seeds of Vitis vinifera. Seeds were ground in liquid nitrogen with a Bu¨chi mixer B-400 (Bu¨chi, Switzerland) and extracted with acetone/water (60:40 v/v) under agitation at 4 °C during 1 night. The mixture was centrifuged for 10 min at 7000 g, using a Sorvall Model RC5B centrifuge. The supernatant was then filtered on a paper filter and concentrated under a vacuum at 30 °C to give the

apple (A) or pear (P) parenchyma tannins F3 15 27 4980

A1 2 0 578

A2 10 0 2882

P 28 0 8066

extract. Lipids were removed by three extractions with hexane, and the extract further concentrated to 30 mL was chromatographed on a 100 mL bed volume column (diameter, 2.7 cm; 18-cm high) of Toyopearl TSK HW-50 (F) gel (Tosoh Corporation, Tokyo, Japan). After loading the tannin extract, elution was performed with 14 bed volumes (1450 mL) of ethanol/water/ trifluoroacetyl (55:45:0.05 v/v/v) with a flow rate of 3 mL min-1 to obtain three tannin fractions: F1-1 (250 mL, discarded), F1-2 (200 mL), and F1-3 (1000 mL). This allowed the retrieval of monomers, dimers, and oligomers. The elution of five bed volumes of acetone/water (30:70 v/v) gave the fraction F2. Then, two bed volumes of acetone/water (60:40 v/v) gave the fraction F3. The organic solvents were evaporated from the different fractions so that the samples could be freeze-dried. This way, four tannin powders were obtained. They were kept under argon in sealed vials and protected from light to avoid oxidation. Those four tannin fractions were analyzed by HPLC after thiolysis, as described previously,15,16 to estimate their mean degree of polymerization (mDP) and their percentage of galloylation (Table 1). Tannin fractions extracted and purified from apple and pear parenchymas were kindly provided by Dr. C. M. G. C. Renard (from the Unite´ de Recherche Cidricole, INRA, Le Rheu, France). Their characteristics are given in Table 1. Sample Preparation. Stock solutions of tannins in absolute ethanol were prepared and stored at -40 °C. Ethanol was chosen to ensure a complete dissolution of tannins. The alcoholic stock solutions were diluted in the buffer solution (tartaric acid, 2 g L-1 unless stated otherwise; the pH was adjusted to 3.4 with 5 M sodium hydroxide), and then, the kinetics of aggregation was monitored by means of light scattering. The ethanol contents and ionic strengths of the model solutions were varied. The effect of the ethanol content (2, 5, 12, 20%) on the aggregation was studied at a constant ionic strength of 10-2 M, while the incidence of the ionic strength (10-3, 10-2, 10-1 M) was investigated for a given ethanol content (12%). The compositions of the buffers are given in Table 2. (13) Saucier, C.; Bourgeois, G.; Vitry, C.; Roux, D.; Glories, Y. J. Agric. Food Chem. 1997, 45, 1045-1049. (14) Riou, V.; Vernhet, A.; Doco, T.; Moutounet, M. Food Hydrocolloids 2002, 16, 17-23. (15) Prieur, C.; Rigaud, J.; Cheynier, V.; Moutounet, M. Phytochemistry 1994, 36, 781-784. (16) Souquet, J.-M.; Cheynier, V.; Brossaud, F.; Moutounet, M. Phytochemistry 1996, 43, 509-512.

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Table 2. Compositions of the Buffers name of buffer EtOH 2% EtOH 5% EtOH 12% EtOH 20% I 10-3 M I 10-2 M I 10-1 M

ethanol content (% v/v) 2 5 12 20 12 12 12

salt concentration L-1

2g tartaric acid 2 g L-1 tartaric acid 2 g L-1 tartaric acid 2 g L-1 tartaric acid 0.2 g L-1 tartaric acid 2 g L-1 tartaric acid 2 g L-1 tartaric acid + 0.1 M NaCl

Phase Diagrams. Samples were prepared as previously described, with tannin concentrations ranging from 0.1 to 5 g L-1 and ethanol content ranging from 2 to 20%. After mixing the buffer and the tannin stock solutions, the samples were allowed to stand for 24 h at room temperature prior to visual examination and centrifugation. When the solid precipitates were formed, pellets were analyzed by thiolysis to determine the mDP of the “insoluble” tannins. Cryo-Transmision Electron Microscopy. According to the method described elsewhere,17,18 thin liquid films of the suspensions were prepared on NetMesh (Pelco, U.S.A.) “lacey” carbon membranes and quench-frozen in liquid ethane. Once mounted in a Gatan 626 cryo-holder cooled with liquid nitrogen, the specimens were transferred to the microscope and observed at low temperature (-180 °C). Images were recorded on Kodak SO163 films, in low dose conditions, using a Philips CM 200 “Cryo” electron microscope operated at 80 kV. DLS. DLS measurements were carried out with a Malvern Zetasizer 3000 HS (Malvern Instruments, Malvern, U.K.), equipped with a 10 mW He-Ne laser, and at a wavelength of 633 nm. The sample cell was thermostated at 25 ( 0.1 °C. Measurements were carried out at an angle of 90° from the incident beam. The time-dependence of the scattered light was monitored, and the autocorrelation function G(t) of the particles was measured. The diffusion coefficient of the particle (D) was derived from this function, and hydrodynamic radii of the particles RH were calculated using the Stokes-Einstein equation. All the calculations were made assuming that particles were spherical, and, thus, this equation can be written as

D)

kT 6πηRH

(1)

where k is the Boltzmann constant, T is the temperature, and η is the solvent viscosity. The cumulant method was used to fit the autocorrelation curves, and the results obtained with this method are referred to as Zav (average size). Time zero was considered to be the mixing of the stock solution with the buffer, and each measurement was the average of 10 subruns. Incidence of Ethanol on the Surface Tension Components of Model Solutions. Total Surface Tension. The surface tension, γTL, of water/ethanol mixtures with 2, 5, 12, and 20% (v/v) ethanol was measured at 25 °C by the Wilhelmy plate method using a K12 tensiomemeter (Kru¨ss, Germany). Lifshitz-van der Waals and Lewis Acid-Base Components of the Surface Tension. The total surface tension of a given liquid (or solid) can be divided into two additive components: AB γTL ) γLW L + γL

(2)

where γLW L represents the Lifshitz-van der Waals component of γTL, related to electrodynamic Lifshitz-van der Waals interactions, and γAB L represents the polar, Lewis acid-base component of γTL, related to electron-donor-electron-acceptor interactions.19,20 Acid-base interactions are asymmetric in nature, and the acid-base component is expressed as (17) Dubochet, J.; Adrian, M.; Chang, J.-J.; Homo, J.-C.; Lepault, J.; McDowell, A.; Schultz, P. Q. Rev. Biophys. 1988, 129-228. (18) Putaux, J.-L.; Bule´on, A.; Borsali, R.; Chanzy, H. Int. J. Biol. Macromol. 1999, 26, 145-150.

(3)

where γ+ L represents the electron-acceptor and γL represents the AB electron-donor parameters of γL . The use of contact angle (θ) measurements between solids (S) and liquids (L) leads to the determination of the work of adhesion AB WTSL (WTSL ) WLW SL + WSL ), given by

WTSL ) γTL(1 + cos θ) ) γTSL - γTS - γTL

(4)

where γTSL is the interfacial tension between the liquid and the solid. According to the acid-base theory, AB LW LW 2 + γTSL ) γLW SL + γSL ) (xγS - xγL ) + 2(xγS γS +

xγ+L γ-L - xγ+S γ-L - xγ-S γ+L )

(5)

Combining eqs 4 and 5 yields LW + - + γL(1 + cos θ) ) 2(xγLW S γL + xγS γL + xγS γL )

(6)

From a theoretical point of view and according to van Osset + et al.,21,22 γLW L , γ , and γ of a given liquid (or solid) can be determined by means of contact angle measurements between this liquid (or solid) and three solids (or liquids) of known surfacetension components: one totally apolar (γTS ) γLW S ), which allows the determination of the Lifshitz-van der Waals component of the surface tension, and at least two polar ones, which allows calculation the acid and base parameters of the surface tension. This approach, referred to as the vOCG theory, was used here to follow the evolution of the acid-base properties of the water/ ethanol mixtures as a function of their ethanol content. Because the electron-donor and electron-acceptor parameters of polar liquids and solids cannot be measured directly,19,20 the values determined for the acid-base parameters are only relative ones and are dependent on the acidic and basic components chosen for the reference liquid (water). van Oss and co-workers proposed the assumption that, for water, γ+ ) γ- ) 25.5 mJ m-2. This scale has been criticized by several authors23,24 because liquids or solids always appear as predominantly basic, which is often in contradiction with their acid-base properties determined by means of different methods and with their chemical composition. Different scales have then been proposed, assuming γ+ L /γL ratios for water larger than 1 (water being considered more acidic than basic).24-26 Della Volpe and Siboni24 also suggested the application of a complete calculation procedure, along with an extended set of probe solvents. Other authors suggested that some of the problems encountered in the application of the vOCG approach are related to the fact that the spreading pressure, πe, is neglected in the resolution of eq 4.25 The spreading pressure is defined as the difference between the surface free energy of a solid in equilibrium with vacuum (γS) and the surface free energy of this solid in equilibrium with the vapor of a given liquid (γSV, γS ) γSV + πe). There is still no general agreement concerning the incidence of πe on contact angle measurements.19,25-28 The most common but debated hypothesis is that this term is very low for finite contact angle values and can be neglected. The problem when this term is considered is that a reliable experimental (19) van Oss, C. J. Interfacial Forces in Aqueous Media; Marcel Dekker: New York, 1994. (20) Fowkes, F. M. J. Adhes. Sci. Technol. 1990, 4, 669-691. (21) van Oss, C. J.; Ju, L.; Chaudhury, M. K.; Good, R. J. J. Colloid Interface Sci. 1989, 128, 313-319. (22) van Oss, C. J.; Good, R. J. J. Macromol. Sci., Chem. 1989, A26, 1183-1203. (23) Morra, M. J. Colloid Interface Sci. 1996, 182, 312-314. (24) Della Volpe, C.; Siboni, S. J. Colloid Interface Sci. 1997, 195, 121-136. (25) Lee, L.-H. J. Colloid Interface Sci. 1999, 214, 64-78. (26) Della Volpe, C.; Siboni, S. J. Adhes. Sci. Technol. 2000, 14, 235272. (27) Bellon-Fontaine, M. N.; Cerf, O. J. Adhes. Sci. Technol. 1990, 4, 475-480. (28) Busscher, H. J.; Kip, G. A. M.; van Silfhout, A.; Arends, J. J. Colloid Interface Sci. 1986, 114, 307-313.

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Table 3. Superficial Tension Components and Acid-Base Parameters (mJ m-2) of the Test Liquids Used for the Characterization of Solid Surfaces19

γTL γLW L γ+ L γL γAB L

di-iodomethane

water

formamide

glycerol

50.8 50.8 0 0 0

72.8 21.8 25.5 25.5 51.0

58 39 2.28 39.6 19

64.0 34 3.92 57.4 30

procedure does not yet exist to evaluate its incidence in experimental conditions similar to those of contact angle measurements.29 Because neither an absolute acid-base scale nor widely admitted experimental and calculation procedures are defined yet, the vOCG approach was used here to evaluate the relative values of the γ+ L and γL parameters of the different water/ ethanol mixtures, with the scale and calculation procedure proposed by its authors. This does not allow a direct comparison between acidic and basic components of a given material. Only acid and base properties of solvents between themselves and with the reference liquid (water) can be compared. Solid Surfaces Used for the Characterization of Surface Tension Components of Water/Ethanol Mixtures. The solid surfaces used for the evaluation of the superficial tension parameter of the different water/ethanol mixtures were Parafilm (hydrocarbon wax, apolar) and polar PMMA and poly(amide 6-6) (Nylon). Parafilm was used as such for contact angle measurements while the PMMA and nylon surfaces were first extensively washed as follows: extensive washing with a 2% TDF solution at 80 °C, extensive rinsing with hot (80 °C) milli-Q water (10 steps), and extensive rinsing with cold milli-Q water (10 steps). The surfaces were then dried under air, and their surface tension properties were determined by means of contact angle measurements with the different test liquids (apolar di-iodomethane, polar water, glycerol, and formamide), as well as with the water/ethanol mixtures. Except with di-iodomethane, with which contact angles exhibited only small variations, each result (θ) represents a mean of at least 40 measurements. The surface tension properties of the test liquids are given in Table 3.

Results Influence of Tannin Concentration and Structure on Aggregation. Because we were interested in the colloidal behavior of flavan-3-ols in wines, we first studied their apparent solubility in a relevant solvent (i.e., water containing 2 g L-1 tartaric acid and 12% ethanol, pH adjusted to 3.4). When no visible haze was detected (i.e., at low tannin concentration, no phase separation), DLS was used to investigate the presence of colloidal aggregates and determine their size as well as their polydispersity. A phase diagram was established by varying the tannin concentration for the different grape fractions and monomers (Figure 2). We first expressed the concentration in g L-1 (Figure 2a). This representation did not allow a discrimination between mDP values of 5, 8, and 15. The concentration was then expressed in mM, keeping in mind that the given values represent a mean concentration, due to the fraction polydispersity. These mean molar concentrations were calculated from the composition of the fractions (Table 1). Figure 2b clearly shows a decrease of solubility as the mDP increased, with only slight differences between the mDP 8 and 15 fractions. Because higher molecular weight tannins precipitated as soon as their concentration reached 2 g L-1, DLS experiments were performed at concentrations of 1 g L-1 and less, unless otherwise stated. (29) Della Volpe, C.; Maniglio, D.; Siboni, S. In Contact Angle, Wettability and Adhesion; Mittal, K. L., Ed.; 2002; Vol. 2, pp 45-71.

Figure 2. Phase diagrams of flavan-3-ols extracted from grape seeds in a model solution (2 g L-1 tartaric acid, 12% EtOH, pH ) 3.4). Tannin concentrations are given in g L-1 (a) or in mM (b). Table 4. Aggregation of Flavan-3-ol Monomers and Proanthocyanidin Polymers. ++ Indicates that a Particle Size Could Be Measured, - Means that No Aggregation Was Observed, ( Indicates an Intermediate Behavior: the Scattered Intensity Was Not High Enough to Allow Accurate Measurements particle formation Flavan-3-ol Monomers catechin epicatechin epigallocatechin epicatechin gallate ++ epigallocatechin gallate +/-

Zav (nm) at 1 g L-1 concentration / / / 150 110

Procyanidin Fractions, Non-Galloylated Tannins (Epicatechin) apple mDP 2 ++ 380 apple mDP 10 ++ 560 pear mDP 28 ++ 100 Procyanidin Fractions, Partly Galloylated Tannins (Epicatechin, Catechin, Epicatechin Gallate) grape seed mDP 3 ++ 185 grape seed mDP 5 ++ 1450a grape seed mDP 8 ++ 410 grape seed mDP 15 ++ 505 a This Z , much higher than λ, cannot be considered an absolute av value. It can only be concluded that particles formed with mDP 5 are larger.

These experiments allowed us to prove aggregation and to determine the size of the aggregates formed in the case of monomers (DP ) 1 for catechin, epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and procyanidin fractions. Procyanidin fractions here were grape seed tannins (partly galloylated) but also tannins from apple and pear parenchyma that are only composed of epicatechin units (non-galloylated). The results are summarized in Table 4. No aggregation was observed (i.e., no correlation function was measurable) with non-galloylated monomers. By contrast, the formation of colloidal aggregates could be clearly demonstrated for epicatechin gallate, whereas a weak correlation was

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Figure 3. Time-dependence of the aggregation of grape seed tannins: evolution of the aggregate size monitored by DLS. (Tannin concentration: 1 g L-1 in (v/v) water/ethanol 12%, 2 g L-1 tartaric acid).

Figure 5. Evolution of the particle size monitored by DLS as a function of the tannin concentration. Polydispersity indexes after 900 min are indicated on the plot for each concentration.

Figure 4. Aggregation kinetics of the mDP 5 grape seed fraction in the model wine solution as a function of the tannin concentration (DLS measurements). Polydispersity indexes after 900 min are indicated on the plot for each concentration.

Figure 6. Aggregation kinetics of the mDP 10 apple fraction in the model wine solutions as a function of the ionic strength, monitored by DLS.

observed with epigallocatechin gallate. In contrast to monomers, all tannin fractions (mDP 2-28) exhibited a clear self-aggregation, underlying the incidence of the mDP on this phenomenon (Table 4). Aggregation occurred as soon as the tannins were introduced in the model solutions and was time-dependent (Figure 3): the mean apparent diameter (Zav) of the aggregates increased more or less quickly (depending on the fraction) before stabilizing and reaching a (pseudo)plateau. The polydispersity index of the particles also increased very quickly and finally reached 1 for each fraction, indicating that the aggregates were very polydisperse and that the size given by the cumulant method was overestimated. The average particle sizes given in Table 4 are those measured at the plateau. No phase separation occurred during several days; the aggregates were metastable. The self-aggregation of molecules is known to occur above a given concentration, often called the critical aggregation concentration. Decreasing the tannin concentration decreased aggregation, in terms of scattered intensity, aggregate size, and polydispersity index. The aggregation kinetics of the mDP 5 grape seed fraction at different concentrations is shown in Figure 4, while Figure 5 summarizes the plateau results of all the procyanidins. The general trend was an increase of the average size of the particles and of the polydispersity index with the concentration. It is, however, worth noting that at the lowest concentrations (i.e., 0.1 and 0.01 g L-1), no detectable aggregation occurred for the mDP 3 and 15 grape seed fractions and the mDP 28 pear fraction. Considering all the results, there was no simple relationship between the mDP and the tannin tendency to aggregate. Aggregation increased first with the mDP of tannins and seemed to reach a maximum for quite low molecular weight procyanidins (mDP 5 for grape seed tannins, mDP 10 for apple tannins) before decreasing for higher molecular weight polyphenols.

Figure 7. Average particle sizes (DLS measurements) obtained with different tannin fractions in 12% ethanol, at different ionic strengths, pH ) 3.4. Asterisks indicate that precipitation occurred.

Influence of the Medium. Among the parameters that play a crucial role in colloidal phenomena are the ionic strength and the solvent properties. The two parameters may greatly vary, depending upon the grape variety and cultural conditions, as well as upon the processes used for wine elaboration.30 The effects of ionic strength and ethanol content were, thus, studied. Ionic Strength. The incidence of ionic strength on tannin aggregation was investigated within the range 10-3-10-1 M for a tannin concentration of 1 g L-1. Increasing the ionic strength strongly enhanced the tannin self-aggregation (Figure 6) rate, average particle size, and polydispersity. The results obtained with all the fractions are given in Figure 7. In all cases, except for pear parenchyma tannins, mDP 28, the size and the polydispersity index of the aggregates increased with the ionic strength, sometimes leading to precipitation (asterisks above the bars). Ethanol Content. We first made a phase diagram at different concentrations with grape seed tannins (Figure (30) Flanzy, C. Oenologie. Fondements scientifiques et technologiques., Technique et Documentation ed.; Lavoisier: Paris, 1998.

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Figure 9. Influence of the ethanol concentration on the kinetics of aggregation, grape seed tannins mDP 8 at 1 g L-1, in 2 g L-1 tartaric acid, pH ) 3.4, studied by DLS. Values given above each curve are the polydispersity indexes for a given ethanol concentration.

Figure 10. Average sizes (DLS) of the particles obtained with different tannin fractions at different ethanol contents, 1 × 10-2 M, pH ) 3.4, tannin concentration of 1 g L-1. Asterisks indicate a phase separation after a few hours.

Figure 8. Phase diagrams of flavan-3-ols in a model solution: (a) mDP 3, (b) mDP 5, (c) mDP 8, and (d) mDP 15. Open symbols indicate that the tannins are soluble; closed symbols indicate a phase separation.

8a-d). For a given mDP, the general trend was a clear increase of tannin solubility as the ethanol content increased. When the ethanol concentration reached 20%, all fractions were soluble up to 5 g L-1. We did not try higher concentrations because they are not relevant to wine. In contrast to what was observed at 12% ethanol (decrease of solubility as the mDP increased), there was no direct relationship between the mDP and the solubility for ethanol contents below 5%. In this case, the solubility decreased from 1.8 to 0.18 mM when the mDP increased from 3 to 8, before increasing again to 0.4 mM for the mDP 15 fraction. The results of the DLS measurements (aggregation kinetics), performed at a concentration of 1 g L-1, are shown in Figure 9 for the case of mDP 8. The trend observed for this fraction was quite general. The plateau values obtained for all the procyanidin fractions are shown in Figure 10. An increase of the particle size when the ethanol content decreased was observed, even leading to haze formation in some cases (asterisks above the bars): grape seed tannins, mDP 5 and 8 at 2 and 5% ethanol, mDP 3 at 2% ethanol (in accordance with the phase diagram). Note also that the polydispersity index evolved in the same way: smaller particles corresponded to less polydisperse aggregates.

Cryo-transmission electron microscopy (cryo-TEM) experiments were performed to confirm the increase of aggregation and particle size when the ethanol content decreased and the ionic strength increased. The technique was preferred to more conventional sample preparation methods because it allowed visualization of the particles embedded in the vitrified medium, preventing artifacts arising from the detrimental effects of air-drying or interaction with a staining solution of heavy salts. With 12% EtOH and 0.2 g L-1 tartaric acid (Figure 11a), relatively dense spheroidal particles were observed with a size varying from 100 to 300 nm and a granular aspect. When the ethanol content was increased to 20% (Figure 11b), the aspect of the sample was rather different. We increased the tannin concentration to 10 g L-1 to allow the observation of aggregated structures. At this concentration and ethanol content, no phase separation was observed. While an aggregation was clearly detected, it led to the formation of gel-like networks of 50-nm-large aggregates with a granular aspect and a low density. When the tartaric acid concentration was raised, maintaining ethanol at 20%, compact aggregates were observed with a size ranging from 100 to 150 nm (Figure 11c) and exhibiting a granular structure similar to that of the particles seen in Figure 11a. The evolution observed by cryo-TEM was consistent with the DLS results but was only qualitative because we had to work at different tannin concentrations. While the basic “grains” observed in the three samples may correspond to isolated polyphenol molecules, it is difficult to precisely measure their size from the images. Cryo-TEM imaging makes use of the phase contrast created by the defocus of the microscope objective lense. Defocusing generates Fresnel fringes around objects.17,18 While these fringes help to detect objects that are small or have a low density, they may artificially increase their apparent size. The real size would be achieved from in-

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surface tension measurements performed on ethanol/ water mixtures are reported in Table 5. In accordance with literature data, increasing the ethanol concentration strongly decreased the total superficial tension of the solvent.31 Contact angle measurements were performed between the ethanol/water mixtures and different solid surfaces to distinguish, within the evolution of γTL, the evolution of the Lifshitz-van der Waals component, γLW L , and that of . It was assumed that no the Lewis acid-base one, γAB L preferential adsorption of one of the solution components occurred at the interfaces: this is a possible source of error when measuring the contact angles between liquid mixtures and surfaces.28 In other respects, the spreading pressure πe was neglected. These two assumptions are potential limits of the approach used to characterize the evolution of the acid-base properties of the water/ethanol mixtures. Solid surfaces were first characterized by a set of test liquids, that is, di-iodomethane, water, formamide, and glycerol. The contact angle values measured between liquids and solids and the derived surface tension properties of solids are given in Table 6. From its chemical composition, Parafilm is considered as an apolar surface (γTL ) γLW L ). Its surface free energy was, thus, directly derived from the contact angle obtained with di-iodomethane. The properties found for PMMA and nylon 6-6 were in good agreement with the literature data. Within the vOCG scale, PMMA appears as monopolar and basic. Contact angle measurements of the water/ethanol mixtures on Parafilm showed that the decrease of γTL as the ethanol content increased was mainly related to a LW decrease of γAB L (Table 5). Indeed, γL remained almost constant between 2 and 20% ethanol and close to that of pure water and ethanol. The contact angle on PMMA then allowed the estimatation, within γAB L , of the evolution of the γ+ L and γL parameters. The consistency of these parameters was verified by contact angle measurements on nylon. Because these values are only relative and dependent on the scale chosen for water, the results were expressed finally as ratios between the mixture components and water (Table 5). These ratios indicated a strong modification of the acid-base characteristics of the water/ ethanol mixtures with the ethanol content. The general trend was a decrease of the acidic parameter of the mixture with regard to pure water as the ethanol content increased, along with an increase of its basic parameter (always with regard to water). Discussion Figure 11. Cryo-TEM images of a grape seed tannin fraction in different model solutions: (a) 12% (v/v) EtOH, 0.2 g L-1 tartaric acid, 1 g L-1 tannin; (b) 20% (v/v) EtOH, 0.2 g L-1 tartaric acid, 10 g L-1 tannin; and (c) 20% (v/v) EtOH, 2 g L-1 tartaric acid, 1 g L-1 tannin (scale bars, 100 nm).

focus images, but in that case, the sample would have no contrast in the embedding ice. For this reason, the size of the grains derived from images such as those in Figure 11 only provide an upper limit that we estimated to be around 4 nm, which is of the same order of magnitude as the expected size of a mDP 8 condensed tannin molecule. The surface tension properties of suspending media play a determinant part in the solubility and colloidal behavior of biopolymers.22 It was, thus, of importance here to characterize the evolution of these properties as a function of the ethanol content of the model solutions. Results of

Tannin Structure and Concentration. The difference in the aggregation behavior exhibited by the flavan3-ol monomers indicated a clear incidence of galloylation on molecular interactions: the additional trihydroxylated aromatic ring (Figure 1) brought by the gallic acid favors self-association, as already demonstrated by NMR.32 It was also shown to favor intermolecular interactions between flavan-3-ols and proteins.12,33-36 The much lower (31) O’Neill, M.; Cass, E.; McMillan, N. D. J. Am. Soc. Brew. Chem. 2001, 59, 90-95. (32) Baxter, N. J.; Lilley, T. H.; Haslam, E.; Williamson, M. P. Biochemistry 1997, 36, 5566-5577. (33) Ezaki-Furuichi, E.; Nonaka, G. I.; Nishioka, I.; Hayashi, K. Agric. Biol. Chem. 1987, 51, 115-120. (34) Ricardo da Silva, J. M.; Cheynier, V.; Souquet, J.-M.; Moutounet, M.; Cabanis, J.-C.; Bourzeix, M. J. Sci. Food Agric. 1991, 57, 111-125. (35) Charlton, A. J.; Haslam, E.; Williamson, M. P. J. Am. Chem. Soc. 2002, 124, 9899-9905.

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Table 5. Superficial Tension (γTL) and Lifshitz-van der Waals (γLW L ) and Acid-Base ( a γAB L ) Components of the Water/Ethanol Mixtures γTL

(mJ/m2)

ΘParafilm (deg) ΘPMMA (deg) Θnylon (deg) b (mJ/m2) γLW L c (mJ/m2) γAB L

2% ethanol

5% ethanol

12% ethanol

20% ethanol

65.1 ( 0.05 105 (3 74 ( 4 51 ( 3 21.8 ( 2 43.3 ( 2

58.2 ( 0.05 101 ( 3 72 ( 3 51 ( 3 20.8 ( 2 37.4 ( 2

48.4 ( 0.05 93 ( 3 62 ( 3 39 ( 3 19.8 ( 2 28.6 ( 2

41.8 ( 0.05 83 ( 3 58 ( 3 33 ( 3 20.6 ( 2 21.2 ( 2

PMMA d 2 γ+ L (mJ/m ) -d γL (mJ/m2)

13.9 33.6

- 1/2 + 1/2 e (γ+ + (γ(mJ/m2) S γL ) S γL ) + - 1/2 - + 1/2 f (γS γL ) + (γS γL ) (mJ/m2) + γ+ w/γL - γw/γL

23.7 23.3 1.8 0.8

7.8 44.8

4.7 43.5

0.5 216.6

18.3 19.0 3.3 0.6

14.0 15.7 5.4 0.6

9.7 13.6 49.0 0.1

Nylon

a γ+, acid parameter of γAB; γ-, base parameter of γAB; subscript w for water; Θ, contact angle between the water/ethanol solutions and L L L L T LW d the solid surfaces. b From the contact angle on Parafilm. c γAB From the contact angle on PMMA. e From Θnylon: L ) γL - γL .

xγ+nylonγ-L + xγ-nylonγ+L ) f

(1 + cos θ) T LW γL - xγLW L γnylon 2

+ Calculated from γ+ L , γL , γnylon, and γnylon determined previously.

Table 6. Surface Tension Components of the Solid Surfaces Used for Characterization of the Acid-Base Properties of the Water/Ethanol Solutions

2 γLW L (mJ/m ) 2) γ+ (mJ/m L 2 γL (mJ/m ) AB γL (mJ/m2) γTL (mJ/m2)

di-iodomethane water formamide glycerol

Parafilm

nylon

PMMA

26.6

41.1 0.45 27.1 7.0 48.1

43.4 ∼0 8.3 0 43.4

0 26.6

Contact Angles (deg) 63 (3 37 ( 2 111 ( 1 51 ( 2 n.d. 41 ( 2 n.d. 48 ( 2

32 (3 76 (3 63 (3 68 (3

signal found for epigallocatechin gallate by comparison to epicatechin gallate is likely related to its higher degree of hydroxylation, which confers to this molecule a higher solubility. Unlike monomers, the condensed tannins did not allow us to come to a conclusion about the galloylation effect: non-galloylated apple tannins mDP 10 and partly galloylated grape seed tannins mDP 8 and 15 did not have a significantly different behavior. Comparison between monomers and polymerized tannins demonstrated the strong incidence of the mDP on self-aggregation. A significantly higher aggregation was evidenced for mDP values as low as 3 in the case of the partly galloylated tannins (grape seeds) and 2 in the case of the non-galloylated tannins (apple and pear parenchyma). Considering both the DLS and the phase diagram results, it appeared that in the case of tannin fractions, the solubility and the size of the particles formed were correlated. The scheme described in Figure 12 can, thus, be proposed. At very low concentrations, polyphenols do not form measurable particles. When the concentration reaches a critical value, small finite-sized aggregates are formed (small size, low polydispersity index). Increasing further the concentration enhances aggregation (higher (36) Charlton, A.; Baxter, N. J.; Khan, M. L.; Moir, A. J. G.; Haslam, E.; Davis, A. P.; Williamson, M. P. J. Agric. Food Chem. 2002, 50, 15931601.

particle sizes, high polydispersity index), finally leading to phase separation.37 The concentrations at which aggregation and phase separation occur are dependent on the tannin structure, as well as on the ionic strength and on the ethanol content of the model solution. It has been observed that flavan-3-ols’ affinity for proteins such as gelatin increases with their mDP.38 This is usually assigned to an increase of the polyphenol hydrophobicity with the mDP. The present results are in accordance with this: the aggregation increased with the mDP for values up to 8-10 (MW ) 3000). However, this relation was not verified in the case of the higher molecular weight tannins. From our results, the fractions studied could be ranked by decreasing order of solubility as follows: mDP 3 > mDP 15 > mDP 5 ≈ mDP 8 (grape seed tannins); mDP 28 > mDP 2 > mDP 10 (apple and pear tannins). The higher solubility of the mDP 15 and 28 fractions cannot be attributed to their composition because mDP 8 and 15 grape seed fractions have roughly the same compositions in terms of the relative percentage of monomeric units, while apple and pear fractions are only made of epicatechin. The polydispersity in terms of the size and composition within each fraction is not controlled, and a small amount of high molecular weight tannins in a small mDP fraction could play an important part in its solubility. However, thiolysis made on pellets obtained after centrifugation when phase separation occurred showed no difference between the composition of molecules involved in the precipitates and that of the supernatant, confirming that the polydispersity of the fraction is not of concern here. Then, two other assumptions can be made: (i) above a given DP, the molecule adopts a conformation that enhances its solubility and (ii) aggregates formed have different structures. DLS provides a mean hydrodynamic radius but gives no information on the aggregation numbers or on the structure of the aggregates; they can be more or less loose, depending on the conformation of the polymers in solution. The elucidation of this (37) Israelachvili, J. Intermolecular and Surfaces Forces, 2nd ed.; Academic Press: New York, 1991. (38) Oh, H. I.; Hoff. J. E. J. Food Sci. 1979, 44, 87-89.

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Figure 12. Aggregation scenario.

question will need further experimentation among others by static light scattering or small-angle neutron scattering. Influence of the Medium. The results obtained both by DLS (Figures 7 and 10) and cryo-TEM (Figure 11) were consistent and indicated an increase in the aggregation process when the ethanol content was decreased or the ionic strength was raised. In the absence of specific bonds, the solubility of biopolymers in an aqueous solvent is governed by electrostatic, Lifshitz-van der Waals and polar, Lewis acid-base interactions.19,21 In aqueous media, Lewis acid-base interactions mainly refer to the formation of hydrogen bonds. Electrostatic repulsion between two similar entities may ensure their stability and, thus, their solubility. Polyphenols are amphipathic molecules, with “hydrophobic” rings and “hydrophilic”, weak-acid, hydroxyl groups (pKa between 9 and 10). Considering these pKa values, there should be no charged groups in the procyanidin fractions at the pH of the model solutions (3.4). The solubility of the aggregates was considered as being only dependent on London-van der Waals and polar forces. Lifshitz-van der Waals and polar forces occur between the solutes, between the solvent molecules (solvent cohesion), and between the solute and the solvent, depending on their respective properties. Lifshitz-van der Waals forces between two similar entities immersed in a solvent are always attractive.19,37 Thus, the solubility of uncharged compounds can only be achieved by means of repulsive acid-base interactions (“hydrophilic” repulsion in aqueous media), which have to overcome Lifshitz-van der Waals attraction. It has been demonstrated that, for most biopolymers in aqueous solvents, ∆GLW is of appreciable magnitude but remains low.19 The present results have shown that increasing the ethanol concentration in the solvent between 2 and 20% did not significantly modify γLW L . It can then be considered that the Lifshitz-van der Waals interactions were not affected by the ethanol content of the medium. Differences observed in tannin aggregation when the ethanol content varied can be, thus, attributed to modified acid-base interactions. Increasing the ethanol content in the medium led to a clear decrease of γAB L , along with a modification of the acid-base properties of the solvent. The decrease in γAB L indicates a decrease in the solvent cohesion related to acid-base interactions, which can already favor tannin solvation. However, this effect alone is insufficient: the addition of ethanol in an aqueous solution often leads to protein and polysaccharide aggregation, unlike what is observed here with polyphenols. From their chemical composition, it can be supposed that flavan-3-ols are predominantly acidic. The polar groups of these molecules are hydroxyl groups, which are known to be H donors.39,40 This hypothesis is supported by their good solubility in rather basic solvents41,42 and by their affinity for polymers (poly(vinylpyrrolidone),43 nylon,44 etc.) that are classified by the vOCG scale as much less acidic

than water. It can then be considered that the evolution + of γ+ W/γL (decreasing acid character with regard to water) - and γW/γL (increasing basic character with regard to water) as the ethanol content increases promotes acidbase interactions between tannins and the solvent. This, along with the strong decrease of solvent-solvent polar interactions, favors tannin solvation (hydrophilic repulsion). A decrease of solubility when the ionic strength is increased has been frequently observed, for instance, in the case of nonpolar organic solutes, hydrophobic interactions between nonpolar residues increase with the addition of salt, leading to the well-known salting-out effect.45 These effects can, for instance, lower the surfactant critical micellar concentrations.37,46 In our system, because polyphenol aggregates are not charged (and, thus, electrostatic interactions are not playing a decisive part), the large incidence of the ionic strength confirms that hydrophobic (lyophobic in the case of water/ethanol mixtures) attraction plays a determinant part in flavan3-ol aggregation. Conclusion This work investigated the incidence of the flavan-3ols’ structure on their aggregation and solubility. Aggregation was studied by means of DLS and cryo-TEM. These results underlined the strong incidence of their mDP on this property and evidenced that, unlike what has been commonly thought, there was no direct and simple relationship between the decrease in the solubility and the increase in the mDP. This first approach was performed with tannins extracted from grape seeds and from apple and pear parenchyma, which are relatively easy to purify. However, tannins in wine essentially originate from grape skins and differ from seed tannins by the presence of trihydroxylated units (15-30%). Comparison between monomers indicated that the third hydroxyl group on the B ring of the molecule could play a part in the solubility. The skin tannin behavior has to be further investigated and compared to that of the seed tannin. Furthermore, in red wines, tannin and antho(39) Good, R. J.; Srivatsa, N. R.; Islam, M.; Huang, H. T. L.; van Oss, C. J. J. Adhes. Sci. Technol. 1990, 4, 607-617. (40) Haslam, E. Practical polyphenolics. From structure to molecular recognition and physiological action; Cambridge University Press: Cambridge, 1998. (41) Hagerman, A. E.; Butler, L. G. J. Agric. Food Chem. 1980, 28, 947-952. (42) Nitao, J. K.; Birr, B. A.; Nair, M. G.; Herms, D. A.; Mattson, W. J. J. Agric. Food Chem. 2001, 49, 2207-2214. (43) Doner, L. W.; Be´card, G.; Irwin, P. L. J. Agric. Food Chem. 1993, 41, 753-757. (44) Oh, H. I.; Hoff, J. E.; Armstrong, G. S.; Haff, L. A. J. Agric. Food Chem. 1980, 28, 394-398. (45) Robinson, D. R.; Jencks, W. P. J. Am. Chem. Soc. 1965, 87, 24702479. (46) Miyagishi, S.; Okada, K.; Asakawa, T. J. Colloid Interface Sci. 2001, 238, 91-95.

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cyanin evolution during aging leads to the formation of derived pigments: their chemical structure is not fully known yet and their behavior toward self-aggregation will be studied along with their identification and purification. This work also evidenced the strong incidence of the ethanol content and of the ionic strength on aggregation. Cryo-microscopy observations allowed us to observe different types of aggregates: relatively dense aggregates when the ionic strength was high or the ethanol concentration was low and looser or gel-like aggregates when the ethanol content was raised. This is very important in enology considering the diversity of the products in terms of the ethanol percentage and salt composition. Depending

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on the raw material and on the wine-making process, these two parameters will be determinant in the final polyphenolic composition of the wine: they will modulate the tannin solubility as well as their ability to form metastable colloidal aggregates. This will in turn influence their implication in hazes and precipitates, and their interaction with endogenous polysaccharides, clarification media, and fining proteins. Finally, these two parameters may be of importance in the interactions between flavan-3-ols and salivary proteins, which are considered responsible for wine astringency. LA034927Z