Article pubs.acs.org/JAFC
Condensed Tannin Changes Induced by Autoxidation: Effect of the Initial Degree of Polymerization and Concentration Aude Vernhet, Stéphanie Carrillo, and Céline Poncet-Legrand* INRA, Montpellier SupAgro, and Université Montpellier 1, UMR1083 SPO, F-34060 Montpellier, France S Supporting Information *
ABSTRACT: Condensed tannins are a major class of polyphenols and play an important part in organoleptic properties of beverages. Because of their structure, they are chemically reactive. During food processing, reactions take place, leading to structural changes of the native structures to give modified tannins and pigments. Average degrees of polymerization (DPs) determined by standard depolymerization methods become irrelevant, because bonds created from oxidation are uncleavable. Small-angle X-ray scattering was used to determine the conformation of native and autoxidized tannins and assess the impact of tannins initial DP and concentration on changes induced by autoxidation. Different behaviors were observed: (i) slight increase of the DP when tannins were oxidized in dilute solutions; (ii) increase of the DP with tannins in concentrated solutions, leading to the formation of longer linear chains or branched macromolecules depending on the initial DP. KEYWORDS: tannins, oxidation, small-angle X-ray scattering, depolymerization, conformational changes
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INTRODUCTION Phenolic compounds are usually found at concentrations ranging from 0.05 to 0.35 g/L in white wines and from 0.8 to 4 g/L in red wines.1 Among them, flavonoids, which are also the most abundant class of plant polyphenols, show a large diversity of structures, from rather simple compounds to highly complex species (condensed tannins).2 The latter play a part in the development of the color and taste (astringency, bitterness) in wines. In addition, they are involved in the formation of physical instabilities, which are detrimental to product quality. Flavanoid-based tannins differ by the nature of their constitutive units (catechin, epicatechin, epigallocatechin, and epicatechin-gallate) as well as their degree of polymerization (DP). Thus, they constitute a complex mixture of (macro)molecules with different structures. This complexity is further increased during wine making and aging: once extracted, flavonoids undergo several biochemical/chemical changes, leading to the formation of modified pigments and tannins.3−5 These structural modifications are of importance in oenology as these new compounds are expected to exhibit properties that are different from those of their precursors. Identification of the main reaction pathways and of the resulting structural changes is needed to establish the relationships between wine polyphenol composition and quality. However, this identification is still a challenge due to the difficulties encountered in analyzing condensed tannins, and especially neoformed species, which represent the major part of wine tannins. The analysis of condensed tannins is often achieved by acidcatalyzed cleavage of the interflavonoid bond in the presence of a nucleophilic reagent (e.g., benzylthioether or thioglycolic acid), followed by high-performance liquid chromatography (HPLC) analysis: flavan-3-ol extender units are converted into the corresponding adducts, whereas the terminal units are released as monomeric flavan-3-ols.6−8 This gives the number average degree of polymerization (DPn) of the fraction and its composition in flavanol units but does not provide information © 2014 American Chemical Society
on its polydispersity. Furthermore, the estimation of tannin DP becomes inaccurate during wine aging due to the formation interflavonoid bonds that are no longer acid-labile under the conditions of analysis. Size distribution in native tannin fractions are reported to be obtained by size-exclusion chromatography (SEC), provided calibration has been previously done with suitable proanthocyanidin standards.9 When dealing with oxidized structures, no standards are available. Native procyanidins may be no more suitable, and interactions with chromatographic supports may be modified (i.e., some oxidized structures may be adsorbed, which is not wanted when SEC is performed). Flavanol autoxidation has mainly been studied with monomers and dimers and leads to both intermolecular and intramolecular reactions.10−12 These reactions result in the formation of new interflavanoid bonds, some of them being resistant to acid-catalyzed cleavage and thus becoming oxidation markers.13 When dealing with polymers (Figure 1), competitions between intra- and intermolecular reactions, as well as between extension and terminal units, are expected. These competitions determine changes in tannin DP, in their flexibility, and in the type of polymers produced (linear versus branched polymers). Small-angle X-ray scattering (SAXS) gives access to the weight average molecular weight and conformation of macromolecules in solutions.14 It has been used in previous works to study (i) the conformation in ethanol of tannins purified from apple parenchyma and grape seeds and (ii) the changes induced by oxidation reactions.15,16 In these experiments, we worked at high tannin concentrations (5 g/L) for DP40 apple and DP9 grape tannins and at high and low tannin concentrations (0.1 Received: Revised: Accepted: Published: 7833
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Figure 1. Examples of chemical structures of oxidized tannins. According to current knowledge on monomers and dimers, the creation of new bonds may occur on the same macromolecule (intramolecular bonding) leading to the formation of an A-type tannin or between two macromolecules (intermolecular bonding). In the case of intermolecular reactions, the new bond is formed between A and B rings. Further oxidation may lead to additional cyclization between rings A and B.
referred to as DPX, with X being the number average molecular weight determined by depolymerization methods, coupled with HPLC analysis.15 These native fractions were used to get the relationship between the average DP and the scattering intensity at null Q obtained from SAXS experiments. Apple tannin fractions were oxidized at pH 3.5 (which is considered as a relevant pH in enology) to study the effect of concentration on oxidation mechanisms (intra- vs intermolecular reactions), according to the procedure described before,16 either in dilute (0.1 g/L) or concentrated (5 g/L) solutions. Typically, native tannins were dissolved in deionized water, the pH of which was adjusted to 3.5 with trifluoroacetic acid. The solutions were centrifuged (100 000g, 90 min, 20 °C) to remove insoluble species. The supernatants were stirred at 25 °C in sealed vials to prevent any additional oxygen intake. At low concentrations, typically 10 mg of tannins (34.5 μmoles of flavanol units) was dissolved in 100 mL of buffer containing 26 μmoles of oxygen and put in a 125 mL flask (headspace of 25 mL corresponding to ∼215 μmoles of O2): in these conditions, O 2 was in large excess (almost 7 equiv). At higher concentrations, typically 15 mg of tannins (52 μmoles) was dissolved in 3 mL of buffer containing 0.78 μmoles of O2. The volume of the vials was 10 mL; thus, the headspace was 7 mL (60 μmoles of O2), resulting in a slight excess of oxygen (1.2 equiv). Oxidation was followed by UV−visible spectroscopy and depolymerization reactions after 7, 14, 26, and 40 days. Oxidations were stopped after 7, 14, 26, and 40 days; samples were pulled, freeze-dried, and kept under argon, in sealed vials, protected from light to avoid further oxidation and at −80 °C. Samples will be referred to as DPXox dil or conc (for dilute or concentrated oxidation) yd, with y being the duration of oxidation in days. For example, DP6ox conc 40d stands for an initially DP6 tannin fraction oxidized for 40 days at a concentration of 5 g/L.
and 10 g/L) for DP14 tannins. At high concentrations, we observed an increase of the weight average molecular weight of tannins upon autoxidation, attributed to intermolecular reactions.16 Our results also suggested that the concentration of tannins during autoxidation could play a key role in the competition intermolecular reaction versus intramolecular reaction.15 Working with oxidized tannins, Zanchi et al.17 observed only the formation of intramolecular bonds, but they worked in more dilute conditions and separated the fractions on chromatographic supports prior to SAXS experiments. Our present objectives were as follows: (i) to confirm that tannins are semiflexible polymers, working with a broader range of tannin fractions, from DP6 to DP80; (ii) to assess the impact of oxidation on the conformation of tannins; and (iii) to determine the key parameters that rule the formation of intermolecular or intramolecular bonds upon autoxidation reactions, especially the impact of the initial DP and concentration of tannins. To answer these questions, six different fractions of native apple tannins were studied, and kinetics of oxidation were monitored with two different DP for forty days and at two different concentrations. These concentrations, 0.1 and 5 g/L, were chosen as representative of white and red wine conditions, respectively. Apple condensed tannins were first studied instead of grape condensed tannins due to their simpler structure.
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MATERIALS
Deionized water was obtained with a Milli-Q system (Millipore, Billerica, MA, USA). Chemicals (solvents, organic acids, and reagents) were of analytical grade and purchased from VWR and Merck (Hohenbrunn, Germany). Freeze-dried powder of apple (Malus domestica) cortex tissues were kindly prepared and provided by S. Guyot (Unité de Recherches Cidricoles, INRA Rennes, France), and different apple tannin fractions were purified as described before.18,16 These fractions are 7834
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Figure 2. SAXS intensity (normalized by concentration) of native tannins in the log I−log Q representation. DP40 results16 were included in the graph. In the insert: the persistence length L obtained for the native tannin fractions by fitting SAXS spectra with the WLC model21 increases linearly with DPn up to DP40.
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positions of all particles. If the sample is a dilute solution, where the relative positions of the particles are not correlated, then S(Q) = 1 at all Q values. If the sample is a dilute solution of polydisperse macromolecules, the intensity scattered in the Q → 0 limit is proportional to a weight average of the molecular weight Mw, or the DP, and the polymer concentration C:
METHODS
UV−Visible Spectroscopy. UV−visible experiments were carried out on a UV−visible UV Safas mc2 spectrophotometer at 25 °C. Spectra of the native and oxidized fractions were recorded in ethanol, from 250 to 700 nm. Depolymerization of Tannins and HPLC-DAD Analysis. Depolymerization was carried out with 100 μL of tannin solution (at a concentration of 1g/L in methanol) combined with 100 μL of a solution of nucleophile (0.8% v/v thioglycolic acid solution in 0.2 M HCl in methanol). The mixture was heated at 90 °C for 6 min and then cooled in cold ice. HPLC analysis followed the conditions described by Vernhet et al.15 The DPn was calculated as the ratio between the summed molar concentrations of all released monomer constitutive units and the summed molar concentrations of terminal constitutive units. Small-Angle X-ray Scattering. SAXS experiments were performed on the beamline SWING, at Synchrotron Soleil (Saint-Aubin, France). The incident beam energy was 10.5 keV (λ = 1.18 Å), and the distance from the sample to the Aviex CCD detector was 1500 mm. The corresponding scattering vector Q = 4π sinθ/λ ranged from 0.007 to 0.575 Å−1, where 2θ is the scattering angle and λ is the incident wavelength. Experiments were performed at 25 °C. Several successive frames (typically 10) of 1 s each were recorded for both the sample and the solvent (EtOH). It was checked that X-rays did not cause any damage to the polyphenol molecules by comparing successive frames. The average intensity and experimental error of each set of frames were subsequently computed. Scattering from the solvent was measured and subtracted from the corresponding intensity the of tannin solution. The SAXS data were analyzed according to classical formulas for scattering from dispersions of particles or macromolecules in a homogeneous solvent.14 For such dispersions, the intensity can be decomposed as a product of the intensity scattered by a single particle and a structure factor that describes interferences arising from different particles:
I(Q → 0) ∝ M w C(ρp − ρs )2
The form factor P(Q) represents the shape of the particle and its internal electron density distribution. For macromolecules that behave like freely jointed chains, the form factor is given by the Debye scattering function19 as a function of the reduced parameter x = (QRg)2 where Rg is the radius of gyration of the macromolecules:
P(x) = 2(e−x + x − 1)2 /x 2
(3)
Different models exist in the literature for particles or polymers. Scattering curves were fitted with the Sasfit software,20 using different form factors: flexible chains fitted with eq 3; wormlike chain (WLC),21 defined by three parameters: the contour length L of the semiflexible wormlike structure, its Kuhn length b, which is the length of the statistical segment or twice the persistence length, and the radius of gyration of the cross section of the chain, Rcs; self-similar branched polymers fitted with the Fisher−Burford (FB) approximation,22 with two parameters that are the radius of gyration Rg and the self-similarity exponent Df, also called the fractal dimension. The fractal dimension is a value that gives information on the polymer structure: for instance, Df = 1.67 for a linear swollen polymer, 2 for a polymer in θ solvent and a swollen branched polymer, and 2.29 for a randomly branched ideal polymer.23 In addition to the latter model, the impact of the chain thickness was accounted for in the Fisher−Burford model, using the following equation: 2
I(Q ) = Np(ρp − ρs )2 Vp2P(Q )S(Q )
(2)
PFB,R cs(Q ) = PFB(Q )e−Q R cs
(1)
2
/2
(4)
where PFB(Q) = (1 + (2/3Df)x2)−Df/2 is the form factor calculated with the Fisher−Burford equation, whereas PFB,Rcs(Q) is the form factor obtained when the chain cross-section radius of gyration Rcs is taken into account.
where ρp is the electron density of the particles and ρs is that of the solvent, Vp is the volume of a particle, Np is the number of particles per unit volume, P(Q) is the form factor of particle, and S(Q) is the structure factor that describes the pair correlations between the 7835
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Table 1. Scattered Intensities Extrapolated to Q → 0 I0/C and Structural Parameters Derived from the Scattering Curves Using the Gaussian, Wormlike Chain, and Fischer−Burford Modelsa Gaussian fraction DP6 DP13 DP16 DP27 DP40b DP69 DP80
I0/C 1.20 2.70 3.07 3.90 7.20 1.30 1.60
× × × × × × ×
Rg (Å) −4
10 10−4 10−4 10−4 10−4 10−3 10−3
15.6 22.3 24.6 26.7 42.2 51.9 53.7
WLC χ2 36 56 103 99 180 254 271
I0/C 1.08 2.71 3.03 3.93 7.75 1.28 1.64
× × × × × × ×
L −4
10 10−4 10−3 10−4 10−4 10−3 10−3
Fisher−Burford b
97 158.3 179.5 203 585 1250 1286
Rcs
14.4 17.9 19.8 20 16.9 7.5 8
4.1 4.9 5.1 5.3 4.5 6.3 6.7
χ2 10 12 33 46.6 76 400 400
I0/C 1.22 2.79 3.12 4.00 8.50 1.30 1.67
× × × × × × ×
−4
10 10−4 10−4 10−4 10−4 10−3 10−3
Rg
Df
χ2
15.8 23.44 27.15 28.7 57 57.2 58.2
2.63 2.43 2.31 2.35 1.91 2.25 2.28
11 11.0 26.4 19.7 44.9 17.6 11.5
a Rg: radius of gyration; L: contour length; b: Kuhn length; Rcs: radius of cross section; Df: fractal dimension; χ2 values are an indication of the quality of the fit. bData taken from ref 16.
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RESULTS AND DISCUSSION As already described in previous papers,14,15 we expected SAXS to provide information on the conformation of tannins and on the structural changes induced by autoxidation.7,8,24 The conformation of native apple tannins was studied first. Then, changes induced by autoxidation were studied using two different techniques. Conformation of Native Tannins. The conformation of native tannins was previously studied using mainly fractions of DP9 (from grape seeds) and DP40 (from apple parenchyma). In this paper, we studied six additional apple tannin fractions including higher molecular weight tannins (DP69 and DP80). The main constitutive unit in apple condensed tannins is (−)-epicatechin (95%), (+)-catechin being only present as the terminal unit.25 The average DPs of the fractions, determined using acid-catalyzed depolymerization of the fractions in the presence of a nucleophilic reagent, were 6, 13, 16, 27, 69, and 80. The yields of the depolymerization reaction were in the range 65−80%. SAXS measurements were done in ethanol at 25 °C because it is considered as a good or θ solvent for native and oxidized tannins. SAXS intensities of all the native fractions are plotted on the Figure 2. According to eq 2, I0 is proportional to the weight average molecular weight Mw, and therefore, a relation with the number average molecular weight Mn calculated from the DPn obtained through the depolymerization method was expected. We obtained a linear relation between intensities from SAXS and molecular weights from depolymerization methods: I0 = 3 × 10−5 × M n c
the cross-section radius, as explained in the next section. Results obtained previously with a DP40 tannin fraction16 have been included in the results. Also, for this fraction, the best fits were obtained with the Fisher−Burford model. The radius of gyration Rg of a linear macromolecular chain is expected to increase with the DP according to the following equation: R g = aDP ν
(6)
where ν is the Flory coefficient and a is the length of the monomer unit. Plotting ln Rg as a function of ln DP yielded ln Rg = 1.816 + 0.517 ln DP, with a correlation coefficient of 0.97. In our conditions, a equals ∼6.15 Å, which is in the same order of magnitude as results obtained by Martinez et al. using molecular modeling.26 For polymers in θ conditions, ν equals 0.5, whereas it is 0.6 for a polymer in good solvent. With ν equals 0.517, we are somewhere in between. This can be partly explained by the fact that we do not work with monodisperse fractions and/or the fact that we work with macromolecules that are linear within the range 6−40 (less than one decade) and/or the fact that the polymer adopts an intermediate behavior. Using the WLC model to fit SAXS spectra (Table 1), we obtained a value of the persistence length in the range 7.5−10 Å for DPs up to 40, whereas the radius of cross section was nearly 4.5−5 Å. These values were consistent with what we observed previously for a DP40,15,16 and a good linear relationship was found between the contour length L and the DPn (insert in the Figure 2). The length per monomer L/DPn obtained from these values (12 Å) is consistent with the dimensions of the monomeric units.26 However, for DP69 and DP80, this relationship was no longer observed: the persistence length dropped suddenly, along with an increase in Rcs, and the quality of the fit also dropped: the WLC model was no longer suitable for these two fractions. Fisher−Burford fits were improved after considering that macromolecular chains have a thickness (Table 2). Up to DP40, the fractal dimensions and the cross-section radius remained constant. For DP69 and DP80, the fractal dimension increased to 2.20 and 2.25, suggesting denser objects: in the case of polymers, it could indicate the presence of ramifications.23 In addition, the Rg values found for these two molecular weight fractions did not increase compared to the DP40 values (Table 1). The presence of ramifications was confirmed by the Kratky plots of the scattering curve (Figure 3): a point of inflection was observed with the DP69 and DP80,
(5)
The correlation coefficient was 0.97. This is not excellent, probably because of discrepancies between the number average and the weight average molecular weights brought by a slight polydispersity of the fractions with the highest degree of polymerization. In the next section, the SAXS intensities from oxidized tannins will be compared with those of native tannins, and this relation will be used to evaluate their average molecular weights. Geometrical parameters and scattered intensities at Q → 0 were determined by fitting the curves with the Gaussian, WLC, and Fisher−Burford models (Table 1). The quality of the fit was evaluated with the χ2 values: the best fits were the ones with the lowest χ2 values. Concerning the structural parameters, best fits were obtained with the WLC model up to DP27, whereas the best results were obtained with the Fisher−Burford model for DP69 and DP80, which can be implemented using 7836
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Table 2. Scattered Intensities Extrapolated to Q → 0 I0/C, Radius of Gyration Rg, Fractal Dimension Df, and CrossSection Radius Rcs of Apple Tannin Fractions, χ2 Values Are an Indication of the Quality of the Fit I0/C
fraction DP6 DP13 DP16 DP27 DP40a DP69 DP80 a
1.20 2.79 3.20 4.20 8.60 1.30 1.59
× × × × × × ×
−4
10 10−4 10−4 10−4 10‑4 10−3 10−3
Rg
Df
Rcs
χ2
16.0 24.6 27.9 31.1 60.5 56.5 59
2.0 2.0 2.0 2.0 1.7 2.25 2.2
2.6 2.5 2.5 2.5 2.8 1.5 1.0
14.9 13.1 9.8 16.1 13.5 43.5 58.1
DP and concentration. The two tannin fractions used were the DP6 and DP16, autoxidized either at 0.1 or 5 g/L. These concentrations were chosen to emphasize the concentration effect: intramolecular reactions were expected to be favored at low concentration, because in these conditions, a macromolecular chain has more chances to react with itself rather than with its neighbors. On the contrary at higher concentrations, the formations of both inter- and intramolecular bonds are expected. UV−visible spectra (Figure S1, Supporting Information) exhibited typical changes usually observed upon oxidation of tannins, that is, the appearance of humps at 320 and 420 nm. In parallel, the yields of the acid-catalyzed depolymerization reaction dropped (Table 3). This was attributed to the absence of cleavage of new covalent bonds created by oxidation12 and was used to evaluate the extent of tannin oxidation, as described later. Oxidation in “Concentrated” Systems. During oxidation, the visual aspect of solutions evolved: apparition of color changes and of a slight haze after 40 days. After centrifugation, tannins in the pellet were freeze-dried and weighed; they represented about 5%, except for DP6 after 40 days where they represented nearly 20%. They were disregarded for further experiments. The proportion of decrease of depolymerization yield was calculated as
Data taken from ref 16.
⎛ depolymerization yield after n days ⎞ ⎟ × 100 ⎜1 − initial depolymerization yield ⎠ ⎝
(7)
As observed in previous papers,15,16 the DPn calculated from depolymerization experiments (Table 3) slightly decreased during autoxidation whereas the yield of the reaction drastically dropped (roughly −30% and −50%, respectively, for DP6 and DP16 after 26 and 40 days). As previously stated, this is attributed to the formation of new bonds, resistant to depolymerization. This means that one-third to one-half of the interflavonoid bonds underwent chemical reactions. Dimers and trimers released from the nondepolymerization of these bonds, which can be considered as oxidation markers, have been identified using UPLC/ESI-MSn.13 However their separation and quantification remains a challenge. SAXS curves are shown in Figure 4 (DP6) and in Figure 5 (DP16). Intensities normalized by the concentration increased with the time of oxidation, indicating that the weight average molecular weight of tannins increased. The scattering curves were fitted with the same models as previously (Table 4). For the DP6 tannins, the best results were obtained with the WLC and Fisher−Burford models. The contour length increased with the ratio I0/C, which is proportional to the actual DPw. The persistence length b/2 was 7.5 ± 0.4 Å, and the cross section
Figure 3. Kratky plots of native tannins fractions. The inflection points observed with DP62 and DP80 are consistent with branched and/or star polymers.
which is consistent with the appearance of branched macromolecules. This is in agreement with the results obtained by Zanchi et al.17 using ab initio modeling and Gasbor on apple parenchyma tannins having DPs of 75 and 150. To summarize, results obtained here and in previous experiments are consistent with the fact that tannins extracted from apple parenchyma (i.e., epicatechin polymers) were linear macromolecules up to DP∼40 and became more or less branched at higher DPs. However, it was previously shown that oxidation can induce branching,16 and thus, the fact that branching is due to oxidation cannot be ruled out, whether it takes place in planta or during extraction. Oxidized Tannins. SAXS experiments were performed on oxidized fractions in order to get information on (i) the evolution of the molecular weight of tannins upon oxidation and (ii) the branching versus elongation of macromolecules. Especially, we wanted to check the impact of the initial tannin
Table 3. Number Average Degree of Polymerization (DPn), Yield of Depolymerization Reaction during Oxidation at High Concentrations (5 g/L−1), and Decrease of Yield Compared to Day 0 DP6 time of oxidation 0 days 7 days 14 days 26 days 40 days
DPn 6.0 5.9 5.9 5.8 5.6
± ± ± ± ±
0.1 0.1 0.1 0.1 0.2
depolymerization yield (%) 78.0 56.0 57.0 51.5 52.0
± ± ± ± ±
1 4 4 5 4
DP16 decrease of yield (%) 28 27 34 33 7837
DPn
depolymerization yield (%)
15.4 ± 0.8
67 ± 3
16.5 ± 0.1 16.5 ± 0.2 15.0 ± 0.7
41 ± 1 32 ± 1 33 ± 3
decrease of yield (%)
39 53 51
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Scattered intensities extrapolated to Q → 0 I0/C, and structural parameters derived from the scattering curves using the WLC, Fisher−Burford, and Fisher−Burford with cross section models. Rg: radius of gyration; L: contour length; b: Kuhn length; Rcs: radius of cross section are given in angströms; Df: fractal dimension. χ2 values are an indication of the quality of the fit. a
9.8 181.2 101.3 104.0 2.5 1 1.5 1 2.0 2.2 2.2 2.35 27.9 98.0 106.6 161.0 1.20 × 10 2.20 × 10−4 2.67 × 10−4 4.0 × 10−4 10 10−4 10−4 10−4
× × × × 1.08 2.59 2.60 3.84 0 14 26 40
−4
97 128.6 144 181
14.4 14.7 15.8 19.0
4.1 4.1 4.2 4.2
10 49 36.4 49.4
−4
16.0 19 21.5 27.2
2.0 2.0 2.0 1.92
2.6 2.5 2.5 2.5
14.9 55.1 32.5 30.4
3.12 2.34 6.01 1.09
× × × ×
10 10−3 10−3 10−2
26.4 99.2 106.5 150.0
2.36 2.2 2.2 2.39
26.4 16.1 92 31
3.20 2.30 6.00 1.22
× × × ×
10 10−3 10−3 10−2
Rcs Df Rg
−4
Rcs Df Rg
Fisher−Burford with cross section
I0/C χ2 Rcs b
WLC L I0/C
was roughly 4 Å, like with linear native tannins. Parameters determined from the Fisher−Burford model with cross section were consistent with a linear polymer (Df = 2) with a constant cross section. Kratky plots (insert in Figure 4) confirmed that polymers “grew” in a linear way, probably due to mainly end to end reaction (extension). After 40 days of oxidation, I0/C increased by a factor 3.5, so was the molecular weight according to eq 2, resulting in a DP roughly equals to 21. For the DP16, poor results were obtained with the WLC models: this can be explained by the fact that polymers are no longer linear, as confirmed by the Kratky plots (insert in the Figure 5), typical of branched macromolecules. Parameters determined from the Fisher−Burford model with or without cross section (Table 4) were consistent with branched polymers (Df > 2.223). This suggests that, in this case, intermolecular reactions took place in an end-to-middle way, as already observed with DP40.16 After 40 days of oxidation, I0/ C increased by a factor 35, so was Mw. The main differences observed between DP6 and DP16 concerned (i) end-to-end reactions with the shorter native tannins, leading to slightly longer linear macromolecules, versus
oxidation time (days)
Figure 5. SAXS intensity (normalized by concentration) of native and oxidized DP16 in concentrated solutions (5 g/L) at different times of oxidation in the log I−log Q representation. Kratky plots are displayed in an insert.
DP6
Table 4. Fitting Parameters for DP6 and DP16 Tannins Oxidized at 5 g/La
χ2
I0/C
−4
Rg
Fisher−Burford
Df
χ2
Figure 4. SAXS intensity (normalized by concentration) of native and oxidized DP6 in concentrated solutions (5 g/L) at different times of oxidation in the log I−log Q representation. Kratky plots are displayed in an insert.
I0/C
DP16
Fisher−Burford with cross section
χ2
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Figure 6. Illustration of the evolution of the thiolysis yields when non-cleavable oxidation bonds are created. In this example, the thiolysis yield decreases by 33%, with 22% attributed to the formation of intermolecular bonds and 11% attributed to the formation of intramolecular bonds.
Table 5. Number Average Degree of Polymerization (DPn), Yield of Depolymerization Reaction during Oxidation at Low Concentration (0.1 g/L), and Decrease of Yield Compared to Day 0 DP6 time of oxidation 0 days 7 days 14 days 26 days 40 days a
DPn 6.0 5.8 5.8 4.9 4.6
± ± ± ± ±
0.1 0.1 0.1 0.1 0.1
depolymerization yield (%) 78.0 38.0 35.0 33.0 29.0
± ± ± ± ±
DP16 decrease of yield (%)
DPn
depolymerization yield (%)
decrease of yield (%)
51 55 58 63
15.4 ± 0.8 a 14.0 ± 0.1 b 12.3 ± 0.7
67 ± 3 a 47 ± 5 b 11 ± 3
a 30 b 84
1 1 1 2 4
No data available because of bacterial growth. bNo data available because samples could not be dissolved in ethanol.
molecular weight increased by a factor 3.5, whereas it increased by a factor 35 with DP16. These results can be used to evaluate the proportions of inter- and intramolecular bonds created during oxidation. Indeed, if during oxidation the average degree of polymerization DPn increased by a factor x, it means that (x − 1) noncleavable interflavonoid bonds were created in average, also meaning that 2(x − 1) flavanol units became non-quantitatively analyzed (Figure 6). Thus, the percentage of monomers involved in intermolecular bonds, is expected to be
end-to-middle connections with longer native tannins leading to branched macromolecules; this can be explained by the ratio between terminal and extension units (when the chain is longer, it has more chances to react with an extension unit than with a terminal unit); and (ii) the competition between intermolecular and intramolecular reactions. Indeed, the yields of the depolymerization reaction indicated that 30% of the units had reacted for DP6 versus 50% for DP16 (Table 3). Oxidation reactions can be related to the formation of either intramolecular or intermolecular bonds. Coupled with SAXS results, the different yields indicated that the ratio of intermolecular versus intramolecular reactions was totally different for the two tannins: for DP6, the weight average
ρ= 7839
2(x − 1) × 100 nx
(8)
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With our values (x = 3.5 for DP6 and x = 35 for DP16), we would have expected a loss of yield of 24% and 12%, respectively, (actual values: 30% and 50%). This means that, for DP6, intermolecular bonds represent about 80% of the newly formed bonds, whereas they represent 24% for DP16. This is probably due to the fact that longer chains have more possibilities to have adjacent monomers and thus have more possibilities to form intramolecular bonds. Oxidation in “Dilute” Systems. During oxidation, the color of solutions changes (Figure S1, Supporting Information), and as observed in concentrated conditions, the DPn obtained from depolymerization decreased (Table 5). This decrease was more pronounced than in concentrated conditions and associated with a dramatic drop of the yield of the depolymerization reaction: −63% and −84%, respectively, for DP6 and DP16 after 40 days, meaning that only 37% and 16% of the initial interflavonoid bonds remained unreacted. It was much less than what was observed at higher concentrations: 70% of unreacted interflavonoid bonds for DP6, 50% for DP16. This is likely due to the ratio between oxygen and tannins, which is much higher in the case of dilute solutions (7 equiv of O2 vs 1.2) and thus favors more oxidation reactions. SAXS curves are shown in Figures 7 (DP6) and 8 (DP16). Intensities normalized by the concentration increased with the
Figure 8. SAXS intensity (normalized by concentration) of native and oxidized DP16 in concentrated solutions (5 g/L) at different times of oxidation in the log I−log Q representation. Kratky plots are displayed in an insert.
when tannins were more concentrated. It is also worth noting that the depolymerization yield decreased by 63% versus 33% in concentrated solutions: more interflavonoid bonds were modified, but they led to the formation of more intramolecular bonds. According to eq 8, if only intermolecular reactions had occurred, we would have observed a decrease of the depolymerization yield of 16%. Thus, roughly 47% of the uncleavable bonds are due to intramolecular reactions (1/4 intermolecular, 3/4 intramolecular). For the DP16 tannins, the best results were obtained with the Fisher−Burford models, with or without cross section, depending on the oxidation time. However, χ2 values are higher than with DP6. This may be due to the presence of aggregates (even though samples were filtered) or to a high heterogeneity of the sample, making curve fitting more difficult. Parameters determined from the Fisher−Burford model with cross section were consistent with a linear polymer (Df ∼ 2) but with a slightly decreasing fractal dimension. This could reflect the local stiffening of (some of) the macromolecules: indeed the fractal dimension for rods is 1.23 The weight average molecular weight did not evolve regularly as already observed with DP6 and was higher after 14 days than after 40 days. However, with the sample oxidized 14 days, nearly 30% of the tannins were lost upon filtration prior to SAXS experiments, and thus, its composition may not be representative of the total fraction. It is also worth noting that problems of redissolution in ethanol were observed with the fraction oxidized during 26 days and that no measurement could be performed. This might be an indication of the formation of intermediate species, having different solubilities in ethanol. After 40 days, no loss upon filtration was observed, and the Mw increased by a factor 1.6, instead of 35 when tannins were oxidized at a higher concentration. The depolymerization yield decreased by 84% instead of 51% in concentrated solutions (Table 6): more interflavonoid bonds were modified, but they mainly led to the formation of intramolecular bonds. According to eq 8, if only intermolecular reactions had occurred, we would have observed a decrease of the depolymerization yield of less than 5% instead of 84%. Thus 6% of the uncleavable bonds can be attributed to intermolecular reactions and 94% to intramolecular reactions.
Figure 7. SAXS intensity (normalized by concentration) of native and oxidized DP6 in dilute solutions (0.1 g/L) at different times of oxidation in the log I−log Q representation. Kratky plots are displayed in an insert.
time of oxidation until day 26, indicating that the weight average molecular weight of tannins increased, before dropping at day 40. The scattering curves were fitted with the same models as previously (Tables 6 and 7). For the DP6 tannins, the best results were obtained with the Fisher−Burford models, with or without cross section, depending on the oxidation time. However, results obtained with the WLC were correct and showed that the persistence length b/2 evolved when the oxidation time increased and was 10.5 Å at 26 days. The cross section was still in the range 4.1−5 Å. Parameters determined from the Fisher−Burford model with cross section are consistent with a linear polymer (Df ∼ 2) with a constant cross section. However, at longer oxidation times, the fractal dimension slightly increased. The weight average molecular weight did not increase regularly: it was surprisingly higher after 26 days than after 40 days. After 40 days, the Mw was increased by a factor of 2 in dilute solution, instead of 3.5 7840
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Table 6. Fitting Parameters for DP6 Tannins Oxidized at 0.1 g/La WLC oxidation time (days) 0 7 26 40
I0/C 1.08 2.04 3.60 2.18
× × × ×
10−4 10−4 10−4 10−4
Fisher−Burford
L
b
Rcs
χ2
97 133 174 140
14.4 16.0 21.0 16.8
4.1 4.3 5.0 4.6
10 21.6 18.3 15.6
I0/C 1.20 2.10 3.50 2.43
× × × ×
10−4 10−4 10−4 10−4
Fisher−Burford with cross section
Rg
Df
χ2
16.0 20.6 26.9 21.3
2.59 2.35 2.27 2.43
11 18.9 12.9 13.0
I0/C 1.20 2.10 3.70 2.40
× × × ×
10−4 10−4 10−4 10−4
Rg
Df
Rcs
χ2
16.0 20.6 27.6 21.3
2.00 2.00 2.05 2.10
2.6 2.5 2.3 2.5
14.9 16.0 37.9 11.9
a Scattered intensities extrapolated to Q → 0 I0/C and structural parameters derived from the scattering curves using the Gaussian, WLC, and Fischer−Burford models. Rg: radius of gyration; L: contour length; b: Kuhn length; Rcs: radius of cross section; Df: fractal dimension; χ2 values are an indication of the quality of the fit.
Table 7. Fitting Parameters for DP16 Tannins Oxidized at 0.1 g/La WLC
Fisher−Burford
Fisher−Burford with cross section
oxidation time (days)
I0/C
L
b
Rcs
χ2
I0/C
Rg
Df
χ2
I0/C
Rg
Df
Rcs
χ2
0 14 40
3.03 × 10−4 8.90 × 10−4 4.90 × 10−4
179.5 789.0 280.0
19.8 24.2 25.0
5.1 5.4 5.0
33 98 214
3.12 × 10−4 9.80 × 10−4 5.30 × 10−4
26.4 78.8 41.6
2.36 1.9 1.97
26.4 47.8 157.0
3.20 × 10−4 1.09 × 10−3 5.20 × 10−4
27.9 83.0 40.8
2.0 1.9 1.9
2.5 2.0 2.0
9.8 102.4 164.0
Scattered intensities extrapolated to Q → 0 I0/C and structural parameters derived from the scattering curves using the Gaussian, WLC, and Fischer−Burford models. Rg: radius of gyration; L: contour length; b: Kuhn length; Rcs: radius of cross section; Df: fractal dimension; χ2 values are an indication of the quality of the fit. a
To summarize our results, we confirmed that usual analytical methods (depolymerization followed by HPLC analysis) provide information that must be cautiously interpreted when dealing with autoxidized tannins. Indeed, upon oxidation, conversion yields dramatically decrease, meaning that the proportion of identified monomers can be as low as 10%. The decrease of conversion yield is by itself an indicator for tannin oxidation and should be indicated. Using SAXS allowed determination of the conformations of native tannins and evidence of conformational changes upon oxidation. Native tannins can be considered as wormlike chains (or semiflexible polymers) having a relatively short persistence length (7.5−10 Å) and a cross section in the range 4.5−5 Å. However, for chains having more than 40 monomers, results indicated some degree of branching. Upon oxidation, several scenarios were observed (also summarized in Table 8)
with DP16 tannins oxidized at high concentrations: in this case, intermolecular reactions are favored, and as the ratio extension units/terminal units is higher than with DP6, the probability for the chains to react end-tomiddle increases. (iii) In the case of dilute solutions of DP16, a relative stagnation of the molecular weight (X1.7 compared to X35) and slight decrease of the fractal dimension were observed, possibly accounting for the partial stiffening of macromolecules in connection with (mainly) intramolecular reactions The impact of the initial concentration and DP of condensed tannins on autoxidation mechanisms was evidenced: oxidation in dilute and concentrated solutions leads to the formation of different structures, the solubility of which is very different. Concentrations (0.1 and 5 g/L) were chosen to mimic those of condensed tannins in white and red wines. However, it must be kept in mind that these results apply to the simple model systems considered first: apple tannin fractions (mainly epicatechin subunits) with different DP, given oxygen/flavanol unit ratios, water acidified to pH 3.5. When dealing with complex systems such as wine, the initial DP of condensed tannins is polydispersed, and the constitutive units of grape tannins include not only epicatechin and catechin, but also epigallocatechin and epicatechin-gallate. The reactivity of the latter may be different and influence structural changes. Further studies will have to consider this complexity. Last, but not least, other modified tannins are formed, for instance ethyl-linked tannins and anthocyanins/tannins adducts.
Table 8. Proportions of Oxidation Bonds Created Either Intermolecularly or Intramolecularly tanin fraction
intermolecular bonds (%)
intramolecular bonds (%)
DP6ox conc 40d DP16ox conc 40d DP6ox dil 40d DP16ox dil 40d
80 (end to end) 24 (end to middle) 25 6
20 76 75 94
depending on the initial tannin concentration and its initial degree of polymerization: (i) Increase of the weight average molecular weight of tannins but no conformational changes (the chains are getting longer, but no polymer branching is observed) in the case of DP6 tannins. In this case, intermolecular and intramolecular reactions occur, but it is worth noting that the proportion of intramolecular reactions is higher when the concentration is lower: in dilute solutions a chain has more chances to react with itself than with a faraway neighbor. (ii) Huge increase of the weight average molecular weight (X35), with the formation of branched macromolecules
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ASSOCIATED CONTENT
S Supporting Information *
UV−visible spectra of native and oxidized tannins. This material is available free of charge via the Internet at http:// pubs.acs.org. 7841
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(17) Zanchi, D.; Konarev, P. V; Tribet, C.; Baron, A.; Svergun, D. I.; Guyot, S. Rigidity, conformation, and solvation of native and oxidized tannin macromolecules in water-ethanol solution. J. Chem. Phys. 2009, 130, 245103. (18) Poncet-Legrand, C.; Cartalade, D.; Putaux, J.-L.; Cheynier, V.; Vernhet, A. Flavan-3-ol aggregation in model ethanolic solutions: Incidence of polyphenol structure, concentration, ethanol content, and ionic strength. Langmuir 2003, 19, 10563−10572. (19) Debye, P. Molecular-weight determination by light scattering. J. Phys. Chem. 1947, 51, 18−32. (20) Kohlbrecher, J. SASfit: A program for fitting simple structural models to small angle scattering data. http://sans.web.psi.ch/ SANSSoft/sasfit.pdf, 2009. (21) Pedersen, J. S.; Schurtenberger, P. Scattering functions of semiflexible polymers with and without excluded volume effects. Macromolecules 1996, 29, 7602−7612. (22) Fisher, M. E.; Burford, R. J. Theory of critical-point scattering and correlations. I. The Ising model. Phys. Rev. 1967, 156, 583. (23) Beaucage, G. Small-angle scattering from polymeric mass fractals of arbitrary mass-fractal dimension. J. Appl. Crystallogr. 1996, 29, 134− 146. (24) Matthews, S.; Mila, I.; Scalbert, A.; Pollet, B.; Lapierre, C.; duPenhoat, C.; Rolando, C.; Donnelly, D. M. X. Method for estimation of proanthocyanidins based on their acid depolymerization in the presence of nucleophiles. J. Agric. Food Chem. 1997, 45, 1195− 1201. (25) Guyot, S.; Marnet, N.; Laraba, D.; Sanoner, P.; Drilleau, J.-F. Reversed-phase HPLC following thiolysis for quantitative estimation and characterization of the four main classes of phenolic compounds in different tissue zones of a french cider apple variety (Malus domestica Var. Kermerrien). J. Agric. Food Chem. 1998, 46, 1698−1705. (26) Martinez, S. Inhibitory mechanism of mimosa tannin using molecular modeling and substitutional adsorption isotherms. Mater. Chem. Phys. 2002, 77, 97−102.
AUTHOR INFORMATION
Corresponding Author
*Phone: 33 (0)4 99 61 20 23; fax: 33 (0)4 99 61 28 57; e-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We acknowledge the Synchrotron SOLEIL for provision of synchrotron radiation facilities, and we thank Pierre Roblin for assistance in using beamline SWING, Hélène Fulcrand, Laetitia Mouls and Virginie Hugouvieux for help in fruitful discussions.
(1) Singleton, V.; Noble, A. C. In Advance in Food Research; Chichester, C. O., Ed.; American Chemical Society: Washington, DC, 1976; pp 47−70. (2) Flanzy, C. Oenologie - Fondements Scientifiques et Technologiques; Technique Lavoisier: Paris, 1998; p 1311. (3) Cheynier, V.; Duenas-Paton, M.; Salas, E.; Maury, C.; Souquet, J. M.; Sarni-Manchado, P.; Fulcrand, H. Structure and properties of wine pigments and tannins. Am. J. Enol. Vitic. 2006, 57, 298−305. (4) Dueñ as, M.; Fulcrand, H.; Cheynier, V. Formation of anthocyanin−flavanol adducts in model solutions. Anal. Chim. Acta 2006, 563, 15−25. (5) Fulcrand, H.; Duenas, M.; Salas, E.; Cheynier, V. Phenolic reactions during winemaking and aging. Am. J. Enol. Vitic. 2006, 57, 289−297. (6) Matthews, S.; Mila, I.; Scalbert, A.; Donnelly, D. M. X. Extractable and non-extractable proanthocyanidins in barks. Phytochemistry 1997, 45, 405−410. (7) Rigaud, J.; Perez-Ilzarbe, X.; Ricardo da Silva, J. M.; Cheynier, V. Micro method for the identification of proanthocyanidin using thiolysis monitored by high-performance liquid chromatography. J. Chromatogr. 1991, 540, 401−405. (8) Souquet, J.-M.; Cheynier, V.; Brossaud, F.; Moutounet, M. Polymeric proanthocyanidins from grape skins. Phytochemistry 1996, 43, 509−512. (9) Kennedy, J. A.; Taylor, A. W. Analysis of proanthocyanidins by high-performance gel permeation chromatography. J. Chromatogr. A 2003, 995, 99−107. (10) Guyot, S.; Vercauteren, J.; Cheynier, V. Structural determination of colourless and yellow dimers resulting from (+)-catechin coupling catalysed by grape polyphenoloxidase. Phytochemistry 1996, 42, 1279− 1288. (11) Kusano, R.; Tanaka, T.; Matsuo, Y.; Kouno, I. Structures of epicatechin gallate trimer and tetramer produced by enzymatic oxidation. Chem. Pharm. Bull. (Tokyo) 2007, 55, 1768−1772. (12) Tanaka, T.; Matsuo, Y.; Kouno, I. A novel black tea pigment and two new oxidation products of epigallocatechin-3-O-gallate. J. Agric. Food Chem. 2005, 53, 7571−7578. (13) Mouls, L.; Fulcrand, H. UPLC-ESI-MS study of the oxidation markers released from tannin depolymerization: Toward a better characterization of the tannin evolution over food and beverage processing. J. Mass Spectrom. 2012, 47, 1450−1457. (14) Pedersen, J. S. Analysis of small-angle scattering data from colloids and polymer solutions: modeling and least-squares fitting. Adv. Colloid Interface Sci. 1997, 70, 171−210. (15) Vernhet, A.; Dubascoux, S.; Cabane, B.; Fulcrand, H.; Dubreucq, E.; Poncet-Legrand, C. Characterization of oxidized tannins: Comparison of depolymerization methods, asymmetric flow field-flow fractionation and small-angle X-ray scattering. Anal. Bioanal. Chem. 2011, 401, 1559−1569. (16) Poncet-Legrand, C.; Cabane, B.; Bautista-Ortin, A.-B.; Carrillo, S.; Fulcrand, H.; Pérez, J.; Vernhet, A. Tannin oxidation: Intra- versus intermolecular reactions. Biomacromolecules 2010, 11, 2376−2386. 7842
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Condensed Tannin Changes Induced by Autoxidation: Effect of the Initial Degree of Polymerization and Concentration Aude Vernhet, Stéphanie Carrillo, and Céline Poncet-Legrand* INRA, Montpellier SupAgro, and Université Montpellier 1, UMR1083 SPO, F-34060 Montpellier, France S Supporting Information *
ABSTRACT: Condensed tannins are a major class of polyphenols and play an important part in organoleptic properties of beverages. Because of their structure, they are chemically reactive. During food processing, reactions take place, leading to structural changes of the native structures to give modified tannins and pigments. Average degrees of polymerization (DPs) determined by standard depolymerization methods become irrelevant, because bonds created from oxidation are uncleavable. Small-angle X-ray scattering was used to determine the conformation of native and autoxidized tannins and assess the impact of tannins initial DP and concentration on changes induced by autoxidation. Different behaviors were observed: (i) slight increase of the DP when tannins were oxidized in dilute solutions; (ii) increase of the DP with tannins in concentrated solutions, leading to the formation of longer linear chains or branched macromolecules depending on the initial DP. KEYWORDS: tannins, oxidation, small-angle X-ray scattering, depolymerization, conformational changes
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INTRODUCTION Phenolic compounds are usually found at concentrations ranging from 0.05 to 0.35 g/L in white wines and from 0.8 to 4 g/L in red wines.1 Among them, flavonoids, which are also the most abundant class of plant polyphenols, show a large diversity of structures, from rather simple compounds to highly complex species (condensed tannins).2 The latter play a part in the development of the color and taste (astringency, bitterness) in wines. In addition, they are involved in the formation of physical instabilities, which are detrimental to product quality. Flavanoid-based tannins differ by the nature of their constitutive units (catechin, epicatechin, epigallocatechin, and epicatechin-gallate) as well as their degree of polymerization (DP). Thus, they constitute a complex mixture of (macro)molecules with different structures. This complexity is further increased during wine making and aging: once extracted, flavonoids undergo several biochemical/chemical changes, leading to the formation of modified pigments and tannins.3−5 These structural modifications are of importance in oenology as these new compounds are expected to exhibit properties that are different from those of their precursors. Identification of the main reaction pathways and of the resulting structural changes is needed to establish the relationships between wine polyphenol composition and quality. However, this identification is still a challenge due to the difficulties encountered in analyzing condensed tannins, and especially neoformed species, which represent the major part of wine tannins. The analysis of condensed tannins is often achieved by acidcatalyzed cleavage of the interflavonoid bond in the presence of a nucleophilic reagent (e.g., benzylthioether or thioglycolic acid), followed by high-performance liquid chromatography (HPLC) analysis: flavan-3-ol extender units are converted into the corresponding adducts, whereas the terminal units are released as monomeric flavan-3-ols.6−8 This gives the number average degree of polymerization (DPn) of the fraction and its composition in flavanol units but does not provide information © 2014 American Chemical Society
on its polydispersity. Furthermore, the estimation of tannin DP becomes inaccurate during wine aging due to the formation interflavonoid bonds that are no longer acid-labile under the conditions of analysis. Size distribution in native tannin fractions are reported to be obtained by size-exclusion chromatography (SEC), provided calibration has been previously done with suitable proanthocyanidin standards.9 When dealing with oxidized structures, no standards are available. Native procyanidins may be no more suitable, and interactions with chromatographic supports may be modified (i.e., some oxidized structures may be adsorbed, which is not wanted when SEC is performed). Flavanol autoxidation has mainly been studied with monomers and dimers and leads to both intermolecular and intramolecular reactions.10−12 These reactions result in the formation of new interflavanoid bonds, some of them being resistant to acid-catalyzed cleavage and thus becoming oxidation markers.13 When dealing with polymers (Figure 1), competitions between intra- and intermolecular reactions, as well as between extension and terminal units, are expected. These competitions determine changes in tannin DP, in their flexibility, and in the type of polymers produced (linear versus branched polymers). Small-angle X-ray scattering (SAXS) gives access to the weight average molecular weight and conformation of macromolecules in solutions.14 It has been used in previous works to study (i) the conformation in ethanol of tannins purified from apple parenchyma and grape seeds and (ii) the changes induced by oxidation reactions.15,16 In these experiments, we worked at high tannin concentrations (5 g/L) for DP40 apple and DP9 grape tannins and at high and low tannin concentrations (0.1 Received: Revised: Accepted: Published: 7833
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Figure 1. Examples of chemical structures of oxidized tannins. According to current knowledge on monomers and dimers, the creation of new bonds may occur on the same macromolecule (intramolecular bonding) leading to the formation of an A-type tannin or between two macromolecules (intermolecular bonding). In the case of intermolecular reactions, the new bond is formed between A and B rings. Further oxidation may lead to additional cyclization between rings A and B.
referred to as DPX, with X being the number average molecular weight determined by depolymerization methods, coupled with HPLC analysis.15 These native fractions were used to get the relationship between the average DP and the scattering intensity at null Q obtained from SAXS experiments. Apple tannin fractions were oxidized at pH 3.5 (which is considered as a relevant pH in enology) to study the effect of concentration on oxidation mechanisms (intra- vs intermolecular reactions), according to the procedure described before,16 either in dilute (0.1 g/L) or concentrated (5 g/L) solutions. Typically, native tannins were dissolved in deionized water, the pH of which was adjusted to 3.5 with trifluoroacetic acid. The solutions were centrifuged (100 000g, 90 min, 20 °C) to remove insoluble species. The supernatants were stirred at 25 °C in sealed vials to prevent any additional oxygen intake. At low concentrations, typically 10 mg of tannins (34.5 μmoles of flavanol units) was dissolved in 100 mL of buffer containing 26 μmoles of oxygen and put in a 125 mL flask (headspace of 25 mL corresponding to ∼215 μmoles of O2): in these conditions, O 2 was in large excess (almost 7 equiv). At higher concentrations, typically 15 mg of tannins (52 μmoles) was dissolved in 3 mL of buffer containing 0.78 μmoles of O2. The volume of the vials was 10 mL; thus, the headspace was 7 mL (60 μmoles of O2), resulting in a slight excess of oxygen (1.2 equiv). Oxidation was followed by UV−visible spectroscopy and depolymerization reactions after 7, 14, 26, and 40 days. Oxidations were stopped after 7, 14, 26, and 40 days; samples were pulled, freeze-dried, and kept under argon, in sealed vials, protected from light to avoid further oxidation and at −80 °C. Samples will be referred to as DPXox dil or conc (for dilute or concentrated oxidation) yd, with y being the duration of oxidation in days. For example, DP6ox conc 40d stands for an initially DP6 tannin fraction oxidized for 40 days at a concentration of 5 g/L.
and 10 g/L) for DP14 tannins. At high concentrations, we observed an increase of the weight average molecular weight of tannins upon autoxidation, attributed to intermolecular reactions.16 Our results also suggested that the concentration of tannins during autoxidation could play a key role in the competition intermolecular reaction versus intramolecular reaction.15 Working with oxidized tannins, Zanchi et al.17 observed only the formation of intramolecular bonds, but they worked in more dilute conditions and separated the fractions on chromatographic supports prior to SAXS experiments. Our present objectives were as follows: (i) to confirm that tannins are semiflexible polymers, working with a broader range of tannin fractions, from DP6 to DP80; (ii) to assess the impact of oxidation on the conformation of tannins; and (iii) to determine the key parameters that rule the formation of intermolecular or intramolecular bonds upon autoxidation reactions, especially the impact of the initial DP and concentration of tannins. To answer these questions, six different fractions of native apple tannins were studied, and kinetics of oxidation were monitored with two different DP for forty days and at two different concentrations. These concentrations, 0.1 and 5 g/L, were chosen as representative of white and red wine conditions, respectively. Apple condensed tannins were first studied instead of grape condensed tannins due to their simpler structure.
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MATERIALS
Deionized water was obtained with a Milli-Q system (Millipore, Billerica, MA, USA). Chemicals (solvents, organic acids, and reagents) were of analytical grade and purchased from VWR and Merck (Hohenbrunn, Germany). Freeze-dried powder of apple (Malus domestica) cortex tissues were kindly prepared and provided by S. Guyot (Unité de Recherches Cidricoles, INRA Rennes, France), and different apple tannin fractions were purified as described before.18,16 These fractions are 7834
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Figure 2. SAXS intensity (normalized by concentration) of native tannins in the log I−log Q representation. DP40 results16 were included in the graph. In the insert: the persistence length L obtained for the native tannin fractions by fitting SAXS spectra with the WLC model21 increases linearly with DPn up to DP40.
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positions of all particles. If the sample is a dilute solution, where the relative positions of the particles are not correlated, then S(Q) = 1 at all Q values. If the sample is a dilute solution of polydisperse macromolecules, the intensity scattered in the Q → 0 limit is proportional to a weight average of the molecular weight Mw, or the DP, and the polymer concentration C:
METHODS
UV−Visible Spectroscopy. UV−visible experiments were carried out on a UV−visible UV Safas mc2 spectrophotometer at 25 °C. Spectra of the native and oxidized fractions were recorded in ethanol, from 250 to 700 nm. Depolymerization of Tannins and HPLC-DAD Analysis. Depolymerization was carried out with 100 μL of tannin solution (at a concentration of 1g/L in methanol) combined with 100 μL of a solution of nucleophile (0.8% v/v thioglycolic acid solution in 0.2 M HCl in methanol). The mixture was heated at 90 °C for 6 min and then cooled in cold ice. HPLC analysis followed the conditions described by Vernhet et al.15 The DPn was calculated as the ratio between the summed molar concentrations of all released monomer constitutive units and the summed molar concentrations of terminal constitutive units. Small-Angle X-ray Scattering. SAXS experiments were performed on the beamline SWING, at Synchrotron Soleil (Saint-Aubin, France). The incident beam energy was 10.5 keV (λ = 1.18 Å), and the distance from the sample to the Aviex CCD detector was 1500 mm. The corresponding scattering vector Q = 4π sinθ/λ ranged from 0.007 to 0.575 Å−1, where 2θ is the scattering angle and λ is the incident wavelength. Experiments were performed at 25 °C. Several successive frames (typically 10) of 1 s each were recorded for both the sample and the solvent (EtOH). It was checked that X-rays did not cause any damage to the polyphenol molecules by comparing successive frames. The average intensity and experimental error of each set of frames were subsequently computed. Scattering from the solvent was measured and subtracted from the corresponding intensity the of tannin solution. The SAXS data were analyzed according to classical formulas for scattering from dispersions of particles or macromolecules in a homogeneous solvent.14 For such dispersions, the intensity can be decomposed as a product of the intensity scattered by a single particle and a structure factor that describes interferences arising from different particles:
I(Q → 0) ∝ M w C(ρp − ρs )2
The form factor P(Q) represents the shape of the particle and its internal electron density distribution. For macromolecules that behave like freely jointed chains, the form factor is given by the Debye scattering function19 as a function of the reduced parameter x = (QRg)2 where Rg is the radius of gyration of the macromolecules:
P(x) = 2(e−x + x − 1)2 /x 2
(3)
Different models exist in the literature for particles or polymers. Scattering curves were fitted with the Sasfit software,20 using different form factors: flexible chains fitted with eq 3; wormlike chain (WLC),21 defined by three parameters: the contour length L of the semiflexible wormlike structure, its Kuhn length b, which is the length of the statistical segment or twice the persistence length, and the radius of gyration of the cross section of the chain, Rcs; self-similar branched polymers fitted with the Fisher−Burford (FB) approximation,22 with two parameters that are the radius of gyration Rg and the self-similarity exponent Df, also called the fractal dimension. The fractal dimension is a value that gives information on the polymer structure: for instance, Df = 1.67 for a linear swollen polymer, 2 for a polymer in θ solvent and a swollen branched polymer, and 2.29 for a randomly branched ideal polymer.23 In addition to the latter model, the impact of the chain thickness was accounted for in the Fisher−Burford model, using the following equation: 2
I(Q ) = Np(ρp − ρs )2 Vp2P(Q )S(Q )
(2)
PFB,R cs(Q ) = PFB(Q )e−Q R cs
(1)
2
/2
(4)
where PFB(Q) = (1 + (2/3Df)x2)−Df/2 is the form factor calculated with the Fisher−Burford equation, whereas PFB,Rcs(Q) is the form factor obtained when the chain cross-section radius of gyration Rcs is taken into account.
where ρp is the electron density of the particles and ρs is that of the solvent, Vp is the volume of a particle, Np is the number of particles per unit volume, P(Q) is the form factor of particle, and S(Q) is the structure factor that describes the pair correlations between the 7835
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Table 1. Scattered Intensities Extrapolated to Q → 0 I0/C and Structural Parameters Derived from the Scattering Curves Using the Gaussian, Wormlike Chain, and Fischer−Burford Modelsa Gaussian fraction DP6 DP13 DP16 DP27 DP40b DP69 DP80
I0/C 1.20 2.70 3.07 3.90 7.20 1.30 1.60
× × × × × × ×
Rg (Å) −4
10 10−4 10−4 10−4 10−4 10−3 10−3
15.6 22.3 24.6 26.7 42.2 51.9 53.7
WLC χ2 36 56 103 99 180 254 271
I0/C 1.08 2.71 3.03 3.93 7.75 1.28 1.64
× × × × × × ×
L −4
10 10−4 10−3 10−4 10−4 10−3 10−3
Fisher−Burford b
97 158.3 179.5 203 585 1250 1286
Rcs
14.4 17.9 19.8 20 16.9 7.5 8
4.1 4.9 5.1 5.3 4.5 6.3 6.7
χ2 10 12 33 46.6 76 400 400
I0/C 1.22 2.79 3.12 4.00 8.50 1.30 1.67
× × × × × × ×
−4
10 10−4 10−4 10−4 10−4 10−3 10−3
Rg
Df
χ2
15.8 23.44 27.15 28.7 57 57.2 58.2
2.63 2.43 2.31 2.35 1.91 2.25 2.28
11 11.0 26.4 19.7 44.9 17.6 11.5
a Rg: radius of gyration; L: contour length; b: Kuhn length; Rcs: radius of cross section; Df: fractal dimension; χ2 values are an indication of the quality of the fit. bData taken from ref 16.
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RESULTS AND DISCUSSION As already described in previous papers,14,15 we expected SAXS to provide information on the conformation of tannins and on the structural changes induced by autoxidation.7,8,24 The conformation of native apple tannins was studied first. Then, changes induced by autoxidation were studied using two different techniques. Conformation of Native Tannins. The conformation of native tannins was previously studied using mainly fractions of DP9 (from grape seeds) and DP40 (from apple parenchyma). In this paper, we studied six additional apple tannin fractions including higher molecular weight tannins (DP69 and DP80). The main constitutive unit in apple condensed tannins is (−)-epicatechin (95%), (+)-catechin being only present as the terminal unit.25 The average DPs of the fractions, determined using acid-catalyzed depolymerization of the fractions in the presence of a nucleophilic reagent, were 6, 13, 16, 27, 69, and 80. The yields of the depolymerization reaction were in the range 65−80%. SAXS measurements were done in ethanol at 25 °C because it is considered as a good or θ solvent for native and oxidized tannins. SAXS intensities of all the native fractions are plotted on the Figure 2. According to eq 2, I0 is proportional to the weight average molecular weight Mw, and therefore, a relation with the number average molecular weight Mn calculated from the DPn obtained through the depolymerization method was expected. We obtained a linear relation between intensities from SAXS and molecular weights from depolymerization methods: I0 = 3 × 10−5 × M n c
the cross-section radius, as explained in the next section. Results obtained previously with a DP40 tannin fraction16 have been included in the results. Also, for this fraction, the best fits were obtained with the Fisher−Burford model. The radius of gyration Rg of a linear macromolecular chain is expected to increase with the DP according to the following equation: R g = aDP ν
(6)
where ν is the Flory coefficient and a is the length of the monomer unit. Plotting ln Rg as a function of ln DP yielded ln Rg = 1.816 + 0.517 ln DP, with a correlation coefficient of 0.97. In our conditions, a equals ∼6.15 Å, which is in the same order of magnitude as results obtained by Martinez et al. using molecular modeling.26 For polymers in θ conditions, ν equals 0.5, whereas it is 0.6 for a polymer in good solvent. With ν equals 0.517, we are somewhere in between. This can be partly explained by the fact that we do not work with monodisperse fractions and/or the fact that we work with macromolecules that are linear within the range 6−40 (less than one decade) and/or the fact that the polymer adopts an intermediate behavior. Using the WLC model to fit SAXS spectra (Table 1), we obtained a value of the persistence length in the range 7.5−10 Å for DPs up to 40, whereas the radius of cross section was nearly 4.5−5 Å. These values were consistent with what we observed previously for a DP40,15,16 and a good linear relationship was found between the contour length L and the DPn (insert in the Figure 2). The length per monomer L/DPn obtained from these values (12 Å) is consistent with the dimensions of the monomeric units.26 However, for DP69 and DP80, this relationship was no longer observed: the persistence length dropped suddenly, along with an increase in Rcs, and the quality of the fit also dropped: the WLC model was no longer suitable for these two fractions. Fisher−Burford fits were improved after considering that macromolecular chains have a thickness (Table 2). Up to DP40, the fractal dimensions and the cross-section radius remained constant. For DP69 and DP80, the fractal dimension increased to 2.20 and 2.25, suggesting denser objects: in the case of polymers, it could indicate the presence of ramifications.23 In addition, the Rg values found for these two molecular weight fractions did not increase compared to the DP40 values (Table 1). The presence of ramifications was confirmed by the Kratky plots of the scattering curve (Figure 3): a point of inflection was observed with the DP69 and DP80,
(5)
The correlation coefficient was 0.97. This is not excellent, probably because of discrepancies between the number average and the weight average molecular weights brought by a slight polydispersity of the fractions with the highest degree of polymerization. In the next section, the SAXS intensities from oxidized tannins will be compared with those of native tannins, and this relation will be used to evaluate their average molecular weights. Geometrical parameters and scattered intensities at Q → 0 were determined by fitting the curves with the Gaussian, WLC, and Fisher−Burford models (Table 1). The quality of the fit was evaluated with the χ2 values: the best fits were the ones with the lowest χ2 values. Concerning the structural parameters, best fits were obtained with the WLC model up to DP27, whereas the best results were obtained with the Fisher−Burford model for DP69 and DP80, which can be implemented using 7836
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Table 2. Scattered Intensities Extrapolated to Q → 0 I0/C, Radius of Gyration Rg, Fractal Dimension Df, and CrossSection Radius Rcs of Apple Tannin Fractions, χ2 Values Are an Indication of the Quality of the Fit I0/C
fraction DP6 DP13 DP16 DP27 DP40a DP69 DP80 a
1.20 2.79 3.20 4.20 8.60 1.30 1.59
× × × × × × ×
−4
10 10−4 10−4 10−4 10‑4 10−3 10−3
Rg
Df
Rcs
χ2
16.0 24.6 27.9 31.1 60.5 56.5 59
2.0 2.0 2.0 2.0 1.7 2.25 2.2
2.6 2.5 2.5 2.5 2.8 1.5 1.0
14.9 13.1 9.8 16.1 13.5 43.5 58.1
DP and concentration. The two tannin fractions used were the DP6 and DP16, autoxidized either at 0.1 or 5 g/L. These concentrations were chosen to emphasize the concentration effect: intramolecular reactions were expected to be favored at low concentration, because in these conditions, a macromolecular chain has more chances to react with itself rather than with its neighbors. On the contrary at higher concentrations, the formations of both inter- and intramolecular bonds are expected. UV−visible spectra (Figure S1, Supporting Information) exhibited typical changes usually observed upon oxidation of tannins, that is, the appearance of humps at 320 and 420 nm. In parallel, the yields of the acid-catalyzed depolymerization reaction dropped (Table 3). This was attributed to the absence of cleavage of new covalent bonds created by oxidation12 and was used to evaluate the extent of tannin oxidation, as described later. Oxidation in “Concentrated” Systems. During oxidation, the visual aspect of solutions evolved: apparition of color changes and of a slight haze after 40 days. After centrifugation, tannins in the pellet were freeze-dried and weighed; they represented about 5%, except for DP6 after 40 days where they represented nearly 20%. They were disregarded for further experiments. The proportion of decrease of depolymerization yield was calculated as
Data taken from ref 16.
⎛ depolymerization yield after n days ⎞ ⎟ × 100 ⎜1 − initial depolymerization yield ⎠ ⎝
(7)
As observed in previous papers,15,16 the DPn calculated from depolymerization experiments (Table 3) slightly decreased during autoxidation whereas the yield of the reaction drastically dropped (roughly −30% and −50%, respectively, for DP6 and DP16 after 26 and 40 days). As previously stated, this is attributed to the formation of new bonds, resistant to depolymerization. This means that one-third to one-half of the interflavonoid bonds underwent chemical reactions. Dimers and trimers released from the nondepolymerization of these bonds, which can be considered as oxidation markers, have been identified using UPLC/ESI-MSn.13 However their separation and quantification remains a challenge. SAXS curves are shown in Figure 4 (DP6) and in Figure 5 (DP16). Intensities normalized by the concentration increased with the time of oxidation, indicating that the weight average molecular weight of tannins increased. The scattering curves were fitted with the same models as previously (Table 4). For the DP6 tannins, the best results were obtained with the WLC and Fisher−Burford models. The contour length increased with the ratio I0/C, which is proportional to the actual DPw. The persistence length b/2 was 7.5 ± 0.4 Å, and the cross section
Figure 3. Kratky plots of native tannins fractions. The inflection points observed with DP62 and DP80 are consistent with branched and/or star polymers.
which is consistent with the appearance of branched macromolecules. This is in agreement with the results obtained by Zanchi et al.17 using ab initio modeling and Gasbor on apple parenchyma tannins having DPs of 75 and 150. To summarize, results obtained here and in previous experiments are consistent with the fact that tannins extracted from apple parenchyma (i.e., epicatechin polymers) were linear macromolecules up to DP∼40 and became more or less branched at higher DPs. However, it was previously shown that oxidation can induce branching,16 and thus, the fact that branching is due to oxidation cannot be ruled out, whether it takes place in planta or during extraction. Oxidized Tannins. SAXS experiments were performed on oxidized fractions in order to get information on (i) the evolution of the molecular weight of tannins upon oxidation and (ii) the branching versus elongation of macromolecules. Especially, we wanted to check the impact of the initial tannin
Table 3. Number Average Degree of Polymerization (DPn), Yield of Depolymerization Reaction during Oxidation at High Concentrations (5 g/L−1), and Decrease of Yield Compared to Day 0 DP6 time of oxidation 0 days 7 days 14 days 26 days 40 days
DPn 6.0 5.9 5.9 5.8 5.6
± ± ± ± ±
0.1 0.1 0.1 0.1 0.2
depolymerization yield (%) 78.0 56.0 57.0 51.5 52.0
± ± ± ± ±
1 4 4 5 4
DP16 decrease of yield (%) 28 27 34 33 7837
DPn
depolymerization yield (%)
15.4 ± 0.8
67 ± 3
16.5 ± 0.1 16.5 ± 0.2 15.0 ± 0.7
41 ± 1 32 ± 1 33 ± 3
decrease of yield (%)
39 53 51
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Scattered intensities extrapolated to Q → 0 I0/C, and structural parameters derived from the scattering curves using the WLC, Fisher−Burford, and Fisher−Burford with cross section models. Rg: radius of gyration; L: contour length; b: Kuhn length; Rcs: radius of cross section are given in angströms; Df: fractal dimension. χ2 values are an indication of the quality of the fit. a
9.8 181.2 101.3 104.0 2.5 1 1.5 1 2.0 2.2 2.2 2.35 27.9 98.0 106.6 161.0 1.20 × 10 2.20 × 10−4 2.67 × 10−4 4.0 × 10−4 10 10−4 10−4 10−4
× × × × 1.08 2.59 2.60 3.84 0 14 26 40
−4
97 128.6 144 181
14.4 14.7 15.8 19.0
4.1 4.1 4.2 4.2
10 49 36.4 49.4
−4
16.0 19 21.5 27.2
2.0 2.0 2.0 1.92
2.6 2.5 2.5 2.5
14.9 55.1 32.5 30.4
3.12 2.34 6.01 1.09
× × × ×
10 10−3 10−3 10−2
26.4 99.2 106.5 150.0
2.36 2.2 2.2 2.39
26.4 16.1 92 31
3.20 2.30 6.00 1.22
× × × ×
10 10−3 10−3 10−2
Rcs Df Rg
−4
Rcs Df Rg
Fisher−Burford with cross section
I0/C χ2 Rcs b
WLC L I0/C
was roughly 4 Å, like with linear native tannins. Parameters determined from the Fisher−Burford model with cross section were consistent with a linear polymer (Df = 2) with a constant cross section. Kratky plots (insert in Figure 4) confirmed that polymers “grew” in a linear way, probably due to mainly end to end reaction (extension). After 40 days of oxidation, I0/C increased by a factor 3.5, so was the molecular weight according to eq 2, resulting in a DP roughly equals to 21. For the DP16, poor results were obtained with the WLC models: this can be explained by the fact that polymers are no longer linear, as confirmed by the Kratky plots (insert in the Figure 5), typical of branched macromolecules. Parameters determined from the Fisher−Burford model with or without cross section (Table 4) were consistent with branched polymers (Df > 2.223). This suggests that, in this case, intermolecular reactions took place in an end-to-middle way, as already observed with DP40.16 After 40 days of oxidation, I0/ C increased by a factor 35, so was Mw. The main differences observed between DP6 and DP16 concerned (i) end-to-end reactions with the shorter native tannins, leading to slightly longer linear macromolecules, versus
oxidation time (days)
Figure 5. SAXS intensity (normalized by concentration) of native and oxidized DP16 in concentrated solutions (5 g/L) at different times of oxidation in the log I−log Q representation. Kratky plots are displayed in an insert.
DP6
Table 4. Fitting Parameters for DP6 and DP16 Tannins Oxidized at 5 g/La
χ2
I0/C
−4
Rg
Fisher−Burford
Df
χ2
Figure 4. SAXS intensity (normalized by concentration) of native and oxidized DP6 in concentrated solutions (5 g/L) at different times of oxidation in the log I−log Q representation. Kratky plots are displayed in an insert.
I0/C
DP16
Fisher−Burford with cross section
χ2
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Figure 6. Illustration of the evolution of the thiolysis yields when non-cleavable oxidation bonds are created. In this example, the thiolysis yield decreases by 33%, with 22% attributed to the formation of intermolecular bonds and 11% attributed to the formation of intramolecular bonds.
Table 5. Number Average Degree of Polymerization (DPn), Yield of Depolymerization Reaction during Oxidation at Low Concentration (0.1 g/L), and Decrease of Yield Compared to Day 0 DP6 time of oxidation 0 days 7 days 14 days 26 days 40 days a
DPn 6.0 5.8 5.8 4.9 4.6
± ± ± ± ±
0.1 0.1 0.1 0.1 0.1
depolymerization yield (%) 78.0 38.0 35.0 33.0 29.0
± ± ± ± ±
DP16 decrease of yield (%)
DPn
depolymerization yield (%)
decrease of yield (%)
51 55 58 63
15.4 ± 0.8 a 14.0 ± 0.1 b 12.3 ± 0.7
67 ± 3 a 47 ± 5 b 11 ± 3
a 30 b 84
1 1 1 2 4
No data available because of bacterial growth. bNo data available because samples could not be dissolved in ethanol.
molecular weight increased by a factor 3.5, whereas it increased by a factor 35 with DP16. These results can be used to evaluate the proportions of inter- and intramolecular bonds created during oxidation. Indeed, if during oxidation the average degree of polymerization DPn increased by a factor x, it means that (x − 1) noncleavable interflavonoid bonds were created in average, also meaning that 2(x − 1) flavanol units became non-quantitatively analyzed (Figure 6). Thus, the percentage of monomers involved in intermolecular bonds, is expected to be
end-to-middle connections with longer native tannins leading to branched macromolecules; this can be explained by the ratio between terminal and extension units (when the chain is longer, it has more chances to react with an extension unit than with a terminal unit); and (ii) the competition between intermolecular and intramolecular reactions. Indeed, the yields of the depolymerization reaction indicated that 30% of the units had reacted for DP6 versus 50% for DP16 (Table 3). Oxidation reactions can be related to the formation of either intramolecular or intermolecular bonds. Coupled with SAXS results, the different yields indicated that the ratio of intermolecular versus intramolecular reactions was totally different for the two tannins: for DP6, the weight average
ρ= 7839
2(x − 1) × 100 nx
(8)
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With our values (x = 3.5 for DP6 and x = 35 for DP16), we would have expected a loss of yield of 24% and 12%, respectively, (actual values: 30% and 50%). This means that, for DP6, intermolecular bonds represent about 80% of the newly formed bonds, whereas they represent 24% for DP16. This is probably due to the fact that longer chains have more possibilities to have adjacent monomers and thus have more possibilities to form intramolecular bonds. Oxidation in “Dilute” Systems. During oxidation, the color of solutions changes (Figure S1, Supporting Information), and as observed in concentrated conditions, the DPn obtained from depolymerization decreased (Table 5). This decrease was more pronounced than in concentrated conditions and associated with a dramatic drop of the yield of the depolymerization reaction: −63% and −84%, respectively, for DP6 and DP16 after 40 days, meaning that only 37% and 16% of the initial interflavonoid bonds remained unreacted. It was much less than what was observed at higher concentrations: 70% of unreacted interflavonoid bonds for DP6, 50% for DP16. This is likely due to the ratio between oxygen and tannins, which is much higher in the case of dilute solutions (7 equiv of O2 vs 1.2) and thus favors more oxidation reactions. SAXS curves are shown in Figures 7 (DP6) and 8 (DP16). Intensities normalized by the concentration increased with the
Figure 8. SAXS intensity (normalized by concentration) of native and oxidized DP16 in concentrated solutions (5 g/L) at different times of oxidation in the log I−log Q representation. Kratky plots are displayed in an insert.
when tannins were more concentrated. It is also worth noting that the depolymerization yield decreased by 63% versus 33% in concentrated solutions: more interflavonoid bonds were modified, but they led to the formation of more intramolecular bonds. According to eq 8, if only intermolecular reactions had occurred, we would have observed a decrease of the depolymerization yield of 16%. Thus, roughly 47% of the uncleavable bonds are due to intramolecular reactions (1/4 intermolecular, 3/4 intramolecular). For the DP16 tannins, the best results were obtained with the Fisher−Burford models, with or without cross section, depending on the oxidation time. However, χ2 values are higher than with DP6. This may be due to the presence of aggregates (even though samples were filtered) or to a high heterogeneity of the sample, making curve fitting more difficult. Parameters determined from the Fisher−Burford model with cross section were consistent with a linear polymer (Df ∼ 2) but with a slightly decreasing fractal dimension. This could reflect the local stiffening of (some of) the macromolecules: indeed the fractal dimension for rods is 1.23 The weight average molecular weight did not evolve regularly as already observed with DP6 and was higher after 14 days than after 40 days. However, with the sample oxidized 14 days, nearly 30% of the tannins were lost upon filtration prior to SAXS experiments, and thus, its composition may not be representative of the total fraction. It is also worth noting that problems of redissolution in ethanol were observed with the fraction oxidized during 26 days and that no measurement could be performed. This might be an indication of the formation of intermediate species, having different solubilities in ethanol. After 40 days, no loss upon filtration was observed, and the Mw increased by a factor 1.6, instead of 35 when tannins were oxidized at a higher concentration. The depolymerization yield decreased by 84% instead of 51% in concentrated solutions (Table 6): more interflavonoid bonds were modified, but they mainly led to the formation of intramolecular bonds. According to eq 8, if only intermolecular reactions had occurred, we would have observed a decrease of the depolymerization yield of less than 5% instead of 84%. Thus 6% of the uncleavable bonds can be attributed to intermolecular reactions and 94% to intramolecular reactions.
Figure 7. SAXS intensity (normalized by concentration) of native and oxidized DP6 in dilute solutions (0.1 g/L) at different times of oxidation in the log I−log Q representation. Kratky plots are displayed in an insert.
time of oxidation until day 26, indicating that the weight average molecular weight of tannins increased, before dropping at day 40. The scattering curves were fitted with the same models as previously (Tables 6 and 7). For the DP6 tannins, the best results were obtained with the Fisher−Burford models, with or without cross section, depending on the oxidation time. However, results obtained with the WLC were correct and showed that the persistence length b/2 evolved when the oxidation time increased and was 10.5 Å at 26 days. The cross section was still in the range 4.1−5 Å. Parameters determined from the Fisher−Burford model with cross section are consistent with a linear polymer (Df ∼ 2) with a constant cross section. However, at longer oxidation times, the fractal dimension slightly increased. The weight average molecular weight did not increase regularly: it was surprisingly higher after 26 days than after 40 days. After 40 days, the Mw was increased by a factor of 2 in dilute solution, instead of 3.5 7840
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Table 6. Fitting Parameters for DP6 Tannins Oxidized at 0.1 g/La WLC oxidation time (days) 0 7 26 40
I0/C 1.08 2.04 3.60 2.18
× × × ×
10−4 10−4 10−4 10−4
Fisher−Burford
L
b
Rcs
χ2
97 133 174 140
14.4 16.0 21.0 16.8
4.1 4.3 5.0 4.6
10 21.6 18.3 15.6
I0/C 1.20 2.10 3.50 2.43
× × × ×
10−4 10−4 10−4 10−4
Fisher−Burford with cross section
Rg
Df
χ2
16.0 20.6 26.9 21.3
2.59 2.35 2.27 2.43
11 18.9 12.9 13.0
I0/C 1.20 2.10 3.70 2.40
× × × ×
10−4 10−4 10−4 10−4
Rg
Df
Rcs
χ2
16.0 20.6 27.6 21.3
2.00 2.00 2.05 2.10
2.6 2.5 2.3 2.5
14.9 16.0 37.9 11.9
a Scattered intensities extrapolated to Q → 0 I0/C and structural parameters derived from the scattering curves using the Gaussian, WLC, and Fischer−Burford models. Rg: radius of gyration; L: contour length; b: Kuhn length; Rcs: radius of cross section; Df: fractal dimension; χ2 values are an indication of the quality of the fit.
Table 7. Fitting Parameters for DP16 Tannins Oxidized at 0.1 g/La WLC
Fisher−Burford
Fisher−Burford with cross section
oxidation time (days)
I0/C
L
b
Rcs
χ2
I0/C
Rg
Df
χ2
I0/C
Rg
Df
Rcs
χ2
0 14 40
3.03 × 10−4 8.90 × 10−4 4.90 × 10−4
179.5 789.0 280.0
19.8 24.2 25.0
5.1 5.4 5.0
33 98 214
3.12 × 10−4 9.80 × 10−4 5.30 × 10−4
26.4 78.8 41.6
2.36 1.9 1.97
26.4 47.8 157.0
3.20 × 10−4 1.09 × 10−3 5.20 × 10−4
27.9 83.0 40.8
2.0 1.9 1.9
2.5 2.0 2.0
9.8 102.4 164.0
Scattered intensities extrapolated to Q → 0 I0/C and structural parameters derived from the scattering curves using the Gaussian, WLC, and Fischer−Burford models. Rg: radius of gyration; L: contour length; b: Kuhn length; Rcs: radius of cross section; Df: fractal dimension; χ2 values are an indication of the quality of the fit. a
To summarize our results, we confirmed that usual analytical methods (depolymerization followed by HPLC analysis) provide information that must be cautiously interpreted when dealing with autoxidized tannins. Indeed, upon oxidation, conversion yields dramatically decrease, meaning that the proportion of identified monomers can be as low as 10%. The decrease of conversion yield is by itself an indicator for tannin oxidation and should be indicated. Using SAXS allowed determination of the conformations of native tannins and evidence of conformational changes upon oxidation. Native tannins can be considered as wormlike chains (or semiflexible polymers) having a relatively short persistence length (7.5−10 Å) and a cross section in the range 4.5−5 Å. However, for chains having more than 40 monomers, results indicated some degree of branching. Upon oxidation, several scenarios were observed (also summarized in Table 8)
with DP16 tannins oxidized at high concentrations: in this case, intermolecular reactions are favored, and as the ratio extension units/terminal units is higher than with DP6, the probability for the chains to react end-tomiddle increases. (iii) In the case of dilute solutions of DP16, a relative stagnation of the molecular weight (X1.7 compared to X35) and slight decrease of the fractal dimension were observed, possibly accounting for the partial stiffening of macromolecules in connection with (mainly) intramolecular reactions The impact of the initial concentration and DP of condensed tannins on autoxidation mechanisms was evidenced: oxidation in dilute and concentrated solutions leads to the formation of different structures, the solubility of which is very different. Concentrations (0.1 and 5 g/L) were chosen to mimic those of condensed tannins in white and red wines. However, it must be kept in mind that these results apply to the simple model systems considered first: apple tannin fractions (mainly epicatechin subunits) with different DP, given oxygen/flavanol unit ratios, water acidified to pH 3.5. When dealing with complex systems such as wine, the initial DP of condensed tannins is polydispersed, and the constitutive units of grape tannins include not only epicatechin and catechin, but also epigallocatechin and epicatechin-gallate. The reactivity of the latter may be different and influence structural changes. Further studies will have to consider this complexity. Last, but not least, other modified tannins are formed, for instance ethyl-linked tannins and anthocyanins/tannins adducts.
Table 8. Proportions of Oxidation Bonds Created Either Intermolecularly or Intramolecularly tanin fraction
intermolecular bonds (%)
intramolecular bonds (%)
DP6ox conc 40d DP16ox conc 40d DP6ox dil 40d DP16ox dil 40d
80 (end to end) 24 (end to middle) 25 6
20 76 75 94
depending on the initial tannin concentration and its initial degree of polymerization: (i) Increase of the weight average molecular weight of tannins but no conformational changes (the chains are getting longer, but no polymer branching is observed) in the case of DP6 tannins. In this case, intermolecular and intramolecular reactions occur, but it is worth noting that the proportion of intramolecular reactions is higher when the concentration is lower: in dilute solutions a chain has more chances to react with itself than with a faraway neighbor. (ii) Huge increase of the weight average molecular weight (X35), with the formation of branched macromolecules
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ASSOCIATED CONTENT
S Supporting Information *
UV−visible spectra of native and oxidized tannins. This material is available free of charge via the Internet at http:// pubs.acs.org. 7841
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Article
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AUTHOR INFORMATION
Corresponding Author
*Phone: 33 (0)4 99 61 20 23; fax: 33 (0)4 99 61 28 57; e-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We acknowledge the Synchrotron SOLEIL for provision of synchrotron radiation facilities, and we thank Pierre Roblin for assistance in using beamline SWING, Hélène Fulcrand, Laetitia Mouls and Virginie Hugouvieux for help in fruitful discussions.
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dx.doi.org/10.1021/jf501441j | J. Agric. Food Chem. 2014, 62, 7833−7842
