Structural Properties of Colloidal Complexes between Condensed

Article pubs.acs.org/Biomac

Structural Properties of Colloidal Complexes between Condensed Tannins and Polysaccharide Hyaluronan Florent Carn,† Sylvain Guyot,‡ Alain Baron,‡ Javier Pérez,§ Eric Buhler,† and Dražen Zanchi*,∥ †

Laboratoire Matière et Systèmes Complexes (MSC), UMR CNRS 7057, Université Paris Diderot - Paris 7, Bâtiment Condorcet, CC 7056, 75205 Paris Cedex 13, France ‡ INRA, UR117 Cidricoles et Biotransformation des Fruits et Légumes, F35653 Le Rheu, France § Synchrotron SOLEIL, L’Orme des Merisiers, Saint-Aubin BP 48, 91192 Gif-sur-Yvette, France ∥ Ecole Normale Supérieure, Département de Chimie, UMR CNRS-ENS-UPMC 8640 PASTEUR, 24 rue Lhomond, F-75231 Paris Cedex 05, France S Supporting Information *

ABSTRACT: Interactions of plant tannins with polysaccharide hyaluronan are studied by means of light scattering and small-angle X-ray scattering (SAXS). In this paper, we show that (1) the tannin−polysaccharide complexes remain stable in colloidal suspension; (2) the masses and structures of colloidal tannin−polysaccharide objects depend on the tannin degree of polymerization; and (3) the densities of tannin−polysaccharide aggregates are about 7 times lower than the density of a single solvated polysaccharide molecule. Short tannins and polysaccharides are aggregated in loose oligomeric structures whose sizes are comparable to a single polysaccharide molecule. Tannins longer than 10 nm and polysaccharides are aggregated in larger microgel-like particles whose sizes exceed 200 nm.

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INTRODUCTION The tannins protect plants from viruses, fungi, bacteria, and higher herbivores by mechanisms based on physical interactions of native or oxidized tannins with polysaccharides and proteins.1 The high affinity of tannins for other biomolecules and their strong antioxidant activity make these compounds important in food technology, pharmaceutics, and cosmetics.2,3 Nevertheless, the potential use of polyphenols in biotechnology or in therapeutics has been hindered by poorly explored interactions of these molecules with other biomacromolecules. Interactions between plant tannins and polysaccharides have been studied in the context of astringency control4 and of the clarification of beverages.5 Macroscopic gelation of polysaccharide xyloglucan with small tannin EGCG has also been reported.6 Nevertheless, the mechanisms of tannin−polysaccharide assembling remain poorly known. To understand the molecular and colloidal mechanisms that govern tannin−polysaccharide complexation, we focused on tannin−polysaccharide physical associations of a set of tannin oligomers (Figure 1) and hyaluronan (HA; Figure 2), a well-known polysaccharide that is widely represented in human and animal epithelial tissues.7,8 Condensed tannins in fruits are oligomeric and polymeric catechins. Tannins homopolymers of (−)-epicatechin have about 6% of triple bounded monomers9−11 and behave as ramified worm-like polymers with the branch persistence length of about 7 nm and the intermediate-Q scaling dimension, depending on solvent composition.9 In some fruits, tannins are more complex. © 2012 American Chemical Society

For example, grape tannins have different 3D structure because of complexity and diversity of their constitutive catechin units.12 Hyaluronan is a linear polysaccharide synthesized by bacteria that is also found in large quantities in animals and the human body, where it is mainly produced by fibroblasts and other specialized connective tissues cells.7,13 It is a semiflexible polyelectrolyte of 9 nm intrinsic persistence length8,14,15 with a repeating disaccharide structure poly((1→3)-β-D-GlcNAc-(1→4)β-D-GlcA) and a global formula C14H20NO11Na for the sodium salt form. The monomer has a molar mass equal to 401.3 g/mol and a monomer length of 1.02 nm. Polyelectrolyte HA can be described as a ″wormlike″ chain with a total persistence length LT = L0 + Le, where L0 ∼ 9 nm is the intrinsic persistence length of the uncharged polymer. Le is the electrostatic persistence length that varies, like Le ∼ κ−2, where κ−1 is the Debye screening length.8,14,15 In the analysis of tannin−HA complexation, we used 50 kDa HA fraction.

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EXPERIMENTAL SECTION

Materials. Hyaluronan Fractions and Buffer. Fractions of hyaluronans were provided by Soliance, France. The average molecular weights used in this work were 30 kDa, (TBPM), 50 kDa (Renovhyal), 60 kDa (Renovhyal), and 100 kDa (Bashyal). Molecular weight were determined using static light scattering experiments and a classical Zimm analysis. Received: November 25, 2011 Revised: February 1, 2012 Published: February 3, 2012 751

dx.doi.org/10.1021/bm201674n | Biomacromolecules 2012, 13, 751−759

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Figure 1. Structure of tannins studied in this work. The monomer is (−)-epicatechin, made of two aromatic rings (A) and (B) and a pyrane ring (C). Monomers are linked through C4−C8 and C4−C6 interflavanol bonds. Combinations of two bonds include ramifications in the structure of polymer. monodispersity in each fraction. Most of the peaks observed on the MS base-peak chromatograms (i.e., chromatograms corresponding to the selective detection of the most intense ion versus retention time) revealed mass spectra in accordance with the expected procyanidin oligomers in the considered fraction. For example, chromatographic peaks of DP3 fraction corresponded to a mass per charge ratio m/z = 865 as the most intense ions on the mass spectra, matching the molecular weight of DP3 molecules (M = 866 Da). Methods. Ultracentrifugation. After dissolving the tannin powder in buffer and applying sonication to the solution in an ultrasound bath for 15 min, 1 mL vials of stock solutions of tannin powders in buffer (5 g/L) were centrifuged using a TLA-120.2 rotor in a Beckman Optima Max centrifuge (at 50 000 rpm, over 45 min, at 25 °C) in order to remove large aggregates and particles. Sedimentation of the tannin-hyaluronan system was achieved by ultracentrifugation of 1 mL vials (TLA 120.2 rotor, 25 °C, 120000 rpm). After centrifugation, the supernatant (100 μL) was collected from the top of the tube and analyzed by UV absorbance at 280 nm. Static and Dynamic Light Scattering (SLS and DLS). Lightscattering experiments were performed on a BI-200SM goniometer (Brookhaven instruments) equipped with two photomultipliers and cross-correlator BI-9000AT, a Mini-L30 (30 mW, 637 nm) compact diode laser, and a circulating temperature-controlled water bath (PolyScience, U.S.A.). The temperature was set at 25 °C. For DLS analysis, the homodyne intensity−intensity correlation function G(q,t) was related to the autocorrelation function of the scattered field, g(t). Inversion of the function g(t) was performed using the CONTIN procedure, providing a distribution of apparent Stokes diameters, DH. Static light scattering profiles were analyzed using BI-SLSW software and by fitting to calculated form factor as described in next subsection. The DLS and SLS experiments were performed on the samples prepared by filtered (200 nm PVDF filter) stock solutions of 50 kDa HA polysaccharide (6 g/L) and a series of filtered tannin stock solutions (DP2, DP10, DP35). Small Angle X-ray Scattering (SAXS) Experiments. The synchrotron radiation X-ray scattering data were collected on the SWING beamline of the Soleil synchrotron facility in Saclay, France. The incident beam energy was 12 keV. In most experiments the sample to detector (Aviex CCD) distance was set to 1817 mm, covering the Q range from 0.06 to 7 nm−1. All experiments were temperature-controlled at 25 °C. Typically 40 successive frames of 0.5 s each were recorded for both sample and pure solvent. It was verified that X-rays did not cause irradiation damage by comparing the successive spectra. Each frame was first angularly averaged and the final spectrum and experimental error were obtained by averaging over all frames and subtracting the pure solvent spectrum from the sample spectrum. Intensities were scaled using the scattering of water.

Figure 2. Chemical structure of hyaluronan. Monomer weight and length of the segment are, respectively, equal to m = 401.3 g/mol and b = 1.02 nm. Chemicals for buffer (glacial acetic acid, sodium acetate, sodium hydroxide) were purchased from Sigma. Experiments were done in sodium-acetate buffer at ionic strength I = 0.08 and pH = 5. At pH 5, HA was completely charged and the tannins’ oxidative degradation was less than 3% within 2 days. Stock solutions of hyaluronnans in buffer were dissolved by stirring for 24 h at 25 °C. For all experiments, the HA concentration was 3 g/L, which is below the overlap concentration for all fractions used.8 Preparation of Native Oligomeric and Polymeric Tannin Fractions. Two monodispersed procyanidin fractions DP2 and DP4 and two polydispersed DP10 and DP35 were obtained from cider apple (Kermerrien variety) using successive solvent extractions followed by a fractionation on semipreparative normal phase HPLC as described by Guyot.16 The number that follows DP for monodispersed fractions denotes the exact degree of polymerization, while for the polydispersed polymers, the value denotes the number average degree of polymerization. Epigallocatachin-gallate EGCG (purchased from Sigma), which has been extracted from green tea, was also used in this study. Characterization of Tannin Fractions. To determine the percentage of procyanidin structures, the nature of constitutive catechin units and the average degree of polymerization, all fractions were analyzed by thiolysis with subsequent reverse phase HPLC.16 All fractions contained close to 90% of native procyanidins, which were mainly constituted of (−)-epicatechin units (more than 95% of total units). Oligomers DP2 and DP4 were almost monodispersed in molecular weight, whereas DP10 and DP35 were somewhat polydispersed, with a polydispersity index (weight average over number average) IP = 1.8, where weight average was determined from the forward SAXS intensities and the number average from the thiolysis-HPLC data. Small oligomeric fractions (DP < 7) were also analyzed using liquid chromatography coupled with electrospray-ion trap mass spectrometry in the negative mode (LC-MS) to characterize the main molecular species. Conditions were similar to those previously used for the characterization of some phenolics in cider samples,17 except for the electrospray parameters, which were optimized on the pseudomolecular ions [M − H]− of tetrameric procyanidins (m/z = 1153). The LC-MS data of native oligomers fractions were in accordance with a high 752


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Figure 3. Normalized autocorrelation function g1(t) for polysaccharide alone (3 g/L) and in presence of tannins (1 g/L). Insets show (i) scattering intensity distribution over scatterers’ hydrodynamic diameters (dashed profile is from filtered HA-DP35 sample) and (ii) Total scattered intensities and hydrodynamic diameters of dominant population (in intensity) as functions of tannin degree of polymerization DP. DH0 and I0 are values of hydrodynamic radius and of scattered intensity for the polysaccharide sample (50 kDa, 3 g/L) without added tannin. The forward scattering I(0) and the radius of gyration (Rg) were evaluated using the Guinier approximation assuming that at very small angles (Q ≪ 1/Rg) the intensity is represented as I(Q) = I(0) exp(−1/3(QRg)2). SAXS spectra of tannin solutions and of tannin-polysaccharide aggregates were also fitted to the scattering curves Fσd(RgQ) that were calculated for bushy particles with a gyration radius Rg and a self-similar structure with fractal dimension d. The function Fσd(RgQ) writes

Fσd(R gQ ) =

∫0

∞

ξd(R gQx)fσ (x)dx

Burford model for both the polymers (tannin or HA) and the aggregates. Standard errors of all fitting parameters were within less than 5%, except for the cross section radius Rc, which was extracted from high Q part of the spectra and therefore less precise.

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RESULTS DLS Experiments. In all samples, HA concentration was 3 g/L and tannin concentration was 1 g/L. After preparation, all samples remained stable over several days. Reliability of the DLS results was verified by repeating each run 3 times. A single run duration was typically 60 s. At 1 g/L of tannin the light scattering is by far dominated by HA or HA−tannin aggregates, which means that the tannin part does not adversely affect the results. Figure 3 shows the resulting correlation functions and the intensity distributions of DH obtained by the CONTIN algorithm. The corresponding dominant hydrodynamic diameters and scattering intensities are also shown. It is immediately visible that all samples contain two well-distinguished populations. The first population has DH centered between 25 and 45 nm and the second population has DH centered between 200 and 300 nm. The peak widths are approximately 40 and 400 nm, respectively. The pure HA sample has a small population that corresponds to single HA molecules. HA molecules are rather rigid linear objects and the value of DH ≈ 25 nm obtained is in agreement with data in the literature.8 The large objects correspond to a very small mass fraction of aggregates. The self-aggregation of HA at this concentration has already been reported.7 When 1 g/L of tannins were added, more aggregates were formed, as indicated by the increase of the scattering intensity by a factor of 30. Both small and large populations were affected. For small tannins, however, the large aggregate population still remained negligible in weight compared to the weight of the small particles. For that reason the weight averaged DH is largely dominated by the small particle population. Figure 3 shows that the addition of tannins shifts the mean volume of the small population by a factor of 2 and that the contribution of this species to the scattering intensity increases. However, it

(1)

with −d /2 ⎛ t2 ⎞ ⎟⎟ ξd(t ) = ⎜⎜1 + 3d /2 ⎠ ⎝

(2)

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being the Fisher-Burford form factor and

fσ (x) =

⎡ 1 ⎛ ln x ⎞2⎤ 1 ⎟ ⎥ exp⎢− ⎜ 2π σ ⎣⎢ 2 ⎝ σ ⎠ ⎥⎦

(3)

being the log-normal distribution with the width σ. From the fitting curves, the average gyration radius Rg of the particles, the width σ of their size distribution, and the dimension d that characterized their internal structure and the surface roughness were obtained. Note that for monodisperse case (σ = 0) the expression eq 1 reduces to just the Fisher−Burford function eq 1, with t = RgQ. The scattering profile from HA chains was fitted using the Fisher− Burford form factor, multiplied by the cross-section scattering formula:

⎛

F d , R g , R c(Q ) = ⎜1 + ⎜ ⎝

−d /2

R g2Q 2 ⎞ ⎟ 3d /2 ⎟⎠

exp − (R cQ )2 /2 (4)

where d, Rg, and Rc are the scaling dimension of HA, its gyration radius, and its cross-section radius, respectively. The use of the polydispersed Fisher−Burford model for fitting tannin oligomers and polymers has been justified by detailed comparison with ab initio methods.9 On the other hand, best fits of HA molecules by the same procedure also yields results that are in agreement with the literature.8,14,15 Therefore, in this work we based our analysis on the Fisher− 753


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with values higher than 2 of the dimension d, indicate that polymers are not just simple chains and that some branching takes place. Forward intensities correspond to the weight averaged degree of polymerization. The spectra correspond to noninteracting individually solvated tannin molecules, in agreement with the previously published data.9 Polysaccharide Fractions. SAXS spectra of three hyaluronan fractions: TBPM (M ≈ 30 kDa), Renovhyal (M ≈ 50 kDa), and Bashyal (M ≈ 100 kDa) are shown in Figure 5. At low Q , the SLS profiles for the two latter fractions are also shown.

is important to note that intensity due to the larger aggregates also increases, indicating that large scale aggregation is induced by tannins and that it increases with DP. The behavior observed with tannin DP35 was different. The correlation function becomes dominated by a single population of large objects, with a DH ≈ 200 nm. The intensity increases accordingly by a factor of approximately 30, indicating that the HA−tannin complex organizes into large colloidal particles. To detect the population of small species that is hidden behind the dominant 200 nm aggregates, the sample was filtered using a 200 nm PVDF filter and remeasured by DLS. From the analysis of the intensity distribution, the small population was found to account for 5% of total scattering intensity. It was also found that particles as large as 500 nm were still present after filtration. This indicates that a strong shearing breaks the most fragile large aggregates, which are recomposed after filtration into even larger structures. At this point it can be concluded that there are two populations in the HA−tannin system: small objects that measure 25 nm in size and objects that are approximately 10 times larger in size. Both populations’ intensity increase with more tannin added to the mixture. SAXS and SLS Experiments. In this section we report SAXS experiments for all tannin fractions used in this study, three HA fractions, and the mixtures of 50 kDa HA with different tannins. Only a few characteristic scattering curves were completed by SLS experiment because very limited quantities of tannins were recovered from the same batch. Tannin Fractions. Using SAXS, we first explored the tannin EGCG from tea, the monodispersed tannin oligomers from apples with DP equal to 2 and 4 and the fractions from apples with an average DP of 10 and 35 and a polydispersity index Ip of about 1.8. The SAXS intensities from solutions of EGCG, tannin oligomers and DP35 polymer in buffer are shown in Figure 4. Fisher−

Figure 5. SAXS and SLS profiles from polysaccharide hyaluronan (HA) fractions at 3 g/L (30 and 50 kDa) and 1 g/L (100 kDa). We determine the gyration radius Rg, scaling exponent d for I ∼ Q−d, and cross-section radius Rc from fits of SAXS data to monodispersed Fisher−Burford scattering function multiplied by cross section form factor (see eq 4). The forward intensities for shown spectra are 0.035, 0.055, and 0.038 cm−1 for 30, 50, and 100 kDa HA, respectively, which correspond to ratios ≈30:50:100. SLS data reveal a small ( = 100 nm.

the same procedure as the spectra for HA + DP2 tannin: the sum of HA alone and tannin alone plus the scattering contribution from aggregates. The Guinier regime for aggregates was out of 755


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the reach of our data, but the beginning of the curvature provides enough information to estimate the gyration ratio of the smallest population of aggregates. The SLS result for HA + 0.25 g/L tannin indicates that even at much lower Q the scattering still increases. By applying the Fisher-Burford fit we find that this profile corresponds to the aggregates with Rg ≈ 100 nm. On the other hand, the DLS results show that the maximal size of aggregates (hydrodynamic diameter DH) is about 300 nm. The forward intensity increases by a factor of at least 20 compared to the pure polysaccharide sample, indicating that many polysaccharide molecules aggregate together. The aggregates are larger than the aggregates found in the case of HA with DP2 tannin, with a gyration radii of aggregates between 30 and 50 nm, as shown on the inset of Figure 8. These results are in agreement with the sizes determined by DLS. We confirm that the low-Q scattering is dominated by polydispersed population of large aggregates, whose average size increases with tannin concentration. The internal structure of aggregates is related to the scaling dimension d. It can be easily measured because the scattering intensity follows a scaling law over almost two decades of intensity. Its value of d = 2.36 is independent of tannin concentration for t > 0.05 g/L. This is a typical value for a bushy fractal object. Ultracentrifugation of HA−Tannin Solutions. To explore sedimentation of HA molecules alone in solution, a 60 kDa fraction (very similar to the 50 kDa one) was ultracentrifuged at 120 krpm in TLA120.2 rotor (800000 g), 1 mL tubes, over 40 min. The SAXS spectra of the sample before centrifugation and of the 100 μL supernatant were compared. No migration of macromolecules was observed in spite of their high molecular weight. Only the magnitude of the low-Q up-turn of the scattering intensity decreases with centrifugation, because of the sedimentation of the aggregates that represent a very small fraction of polysaccharide. The remaining spectrum is nonaffected by centrifugation within less than 0.1% of scattering intensity. These SAXS data are shown in Figure S-3 (see Supporting Information). A tiny fraction of aggregates observed corresponds to the population of large particles detected by DLS and SLS on filtered HA samples (Figure 3). In order to quantify sedimentation of tannin-polysaccharide complexes, solutions of 50 kDa polysaccharide (3 g/L) in the presence of several tannin oligomers (1 g/L) were centrifuged (at 120 × 103 rpm in TLA120.2 rotor over 45 min.). After each centrifugation, a supernatant fraction (100 μL) was collected. The same experimental procedure was performed with samples containing tannins alone. The concentration of tannin in supernatant ts and in samples before centrifugation t were measured by UV absorption at 280 nm. It should be noted that polysaccharides have a negligible contribution to absorption at this wavelength. The experimental error of this procedure was within 5%, mostly attributed to the manual protocol of collecting supernatant. To determine the concentrations by UV spectrometry, very precise dilutions (error c M = > > I< c/M< of approximately 10−20 for HA-DP35 system was estimated from the SAXS data. Knowing I>/I< from the CONTIN analysis of DLS data, we get that for HA-DP35 system c> > c 1. From the SAXS experiments, we have acquired the following structural information about HA-tannin aggregates: their radii of gyration Rg, scaling dimension d, and that the conformation of tannins and of HA macromolecules within aggregates are the same as if these molecules were dissolved individually. Consequently, we can estimate the aggregation number using the formula: ⎛ R agg ⎞d g N∼⎜ 0 ⎟ ⎜ R ⎟ ⎝ g ⎠

Figure 10. Polysaccharide average aggregation number N as function of tannin concentration t for 50 kDa hyaluronan at concentration s = 3 g/L obtained (i) dividing the forward intensity by its value for HA alone, N = I/I0 (ignoring tannin contribution); (ii) from geometrical considerations, using eq 6; and (iii) taking tannin contribution into account, using Langmuir model and approximate formula for scattering intensity of a complex as described in text, using eq 10. Quantity of bound tannins as number of catechin units per polysaccharide molecule (nc) was calculated using affinities K = 0.5 for DP35 and K = 0.04 for DP2, parameter η = 0.2. Polydispersity factor calculated from DLS data is αp = 0.43 for HA + DP35 sample and αp = 1 for HA + DP2 sample.

bound into nc/DP tannin macromolecules and ξ is the number of objects (1,nc) per unit of volume. The amplitude A(1,nc) is

(6)

where Rgagg and Rg0 are the radii of gyration of the aggregate and of a single (tannin-dressed) HA macromolecule, respectively. It should be noted that here we use the radii of gyration Rgagg of the smallest aggregates, determined by fits of SAXS data for Q of the order of 0.1 nm−1, because the weight average of gyration radius is closest to these values. The resulting values of N for HA and two tannins DP35 and DP2 are shown in Figure 10. For DP35, the value of d = 2.36 was fairly well determined. One sees that DP35 tannin makes progressively larger aggregates as one increases tannin concentration. On the other hand, the size of aggregates of HA with DP2 tannins are independent of t and only twice the size of bare HA, making the concept of scaling useless. However, we used d = 1.6 for DP2 samples, keeping in mind that this is only a rough approximation. Another simple way to determine the mass of HA−tannin assemblies is based on the analysis of the forward scattering intensity. The same analysis will also give us the fraction of bound tannins contributing to HA−tannin aggregates. We will determine the aggregation number N as function of tannin concentration t for a given tannin oligomer with degree of polymerization DP. From the experiment, we have the forward scattering intensity I0(t) = A0(t)2, with A0 being the total forward scattering amplitude. Assuming that non-negligible mass fraction αp of total HA and tannin contributes to the largest aggregates (N, Nnt) and therefore dominates the scattering, the forward scattering intensity reads I0(t ) = N α pI(1, nc)

A(1, nc) = ρs + ρcnc

(8)

where ρs and ρc are the contrasts of one HA and one catechin, respectively. They can be found from the scattering plots on just HA or just tannin (Figures 4 and 5) from the relation ρs,c =

̃ Ms,cIs,c NA

(9)

where M and I ̃ are the molecular weight and forward scattering intensity (per g/L) of HA (s) and catechin (c). NA is the Avogadro constant. In the first, roughest approximation, we conider that equilibrium can be represented by a sum of two populations: (i) the polysaccharide−tannin polydispersed (or multipopulation) assemblies (N, Nnt) involving b tannins in (g/L) and all polysaccharides at concentration of s (g/L) and (ii) the remaining free tannins at concentration f = t − b (g/L). In other words, we neglect HA molecules free of tannins, which is a good approximation if we know that their molecular weight is of the order of 50 kDa so that the probability of having the whole sequence (250 units) free of tannin is negligible. From eqs 7−9, we get the following expression for the aggregation number: I (t ) N= 0 s αPI0

1

(1 +

γ

b s

2

)

(10)

where is the scattering intensity of just HA at 3 g/L and γ ≡ Ic̃ Ms/(Is̃ Mc). For 50 kDa HA, one finds γ = 3.27. Scattering intensities of the HA chain and catechin were measured, as well as I0(t). The only parameter that is not obtained from our I0s

(7)

where I(1, nc) = ξA(1, nc)2 is the scattering intensity on one HA molecule dressed by nc tannin monomers (catechins), 757


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experiments is the bound tannin concentration b. It can nevertheless be estimated if we assume that the tannin adsorption to polysaccharides is described by an effective Langmuir model: b=

ηsKf 1 + Kf

used ionic strength of 80 mM the Debye length (interaction range) is ∼1 nm, which is shorter than tannins used in this study and well below the sizes of HA-tannin assemblies. Internal structure and compactness of aggregates depend on tannin length as indicated by the increase of the scaling dimension of aggregates with increasing tannin length. Moreover, aggregates’ structure evolves from extended coil (d < 2) to 3D bushy structures with (d = 2.36) for DP35. Within aggregates, HA and tannin molecules keep their conformations just as if they were individually solvated. The aggregates are therefore bushy (d < 3) structures formed of coil-like HA chains reticulated by tannins. Therefore, the resulting density within an effective hydrodynamic sphere of aggregates can be even lower than the hydrodynamic sphere density of just one HA coil. We can estimate the density of HA and tannin within aggregates (ρagg) as follows:

(11)

where K is HA-tannin effective affinity in L/g, ηs is maximal concentration of tannins that can be bound by s (g/L) of HA, and f = t − b is free tannin concentration. The affinity K and parameter η have been measured for pectins and celluloses by Le Bourvellec et al.21 According to this latter work, η = 1.5 and K is given by the following empirical relation K = a(T , I )

DP − 1 B + 1 + DP

(12)

where a(T,I) depends on temperature T (an almost linear increase by a factor of 2 from 5 to 35 °C) and on the ionic strength I (logarithmic increase by about 20% per decade of I between 10 μM and 1 M). In our experimental conditions (T = 25 °C, I = 80 mM, pH = 5), the resulting values were a = 1.1 L/g and B = 24. If we use the above values for η and K, the aggregation number deviates strongly from values estimated by geometrical considerations. Moreover, the dependency N(t) decreases above 1 g/L of tannin. These are indications that the value 1.5 of η is overestimated for HA. Indeed, the difference between the present system and the one studied previously21 is that, in our case, complexes are soluble or form mesoscopic aggregates, while in the previous study,21 the substrate for tannins was solid phase polysaccharides in suspension. In that case, along with tannin adsorption to polysaccharide substrate, subsequent tannin− tannin stacking took place on the surface, which increased drastically the effective value of η. Figure 10 shows N as function of t for DP35 and DP2 calculated by three methods. The first method is dividing the scattering intensity at a given tannin concentration by just the polysaccharide intensity, that is, ignoring the correction factor (1 + √γ(b/s))2 in eq 10. This is a good approximation if contribution of tannins to scattering on aggregates can be neglected. On the second curve, N(t) is calculated from geometrical considerations, using eq 6. On the third curve, N(t) is calculated from eq 10, using affinities K = 0.5 for DP35 and K = 0.04 for DP2, parameter η = 0.2, and polydispersity factor calculated from DLS data αp = 0.43 for HA + DP35 sample and αp = 1 for HA + DP2 sample. For the latter set of parameters, the aggregation number values are in reasonable agreement with the values found by purely geometrical means. Notice that the values of affinities K are in good agreement with the formula in eq 12, but the value of 0.2 for η used is about seven times lower than η obtained in literature for pectins and celluloses.21 The amount of bound tannins measured in catechins per HA molecule denoted by nc is also shown in Figure 10. In the present model, the polydispersity and tannin−HA partitioning over aggregates are taken into account only in a very rough approximation. A more realistic model could include tannin-driven micellization theory (e.g., shell model, used in refs 22 and 23, for modelization of tannin−protein micelles), but this is beyond the scope of the present analysis. Curves for N(t) and the dependency I0(t) clearly tend toward some saturation value, together with the quantity of bound tannins. This indicates that the maximal size of aggregates is determined by tannin−HA interactions and not by electrostatic repulsion. This fact is plausible because at the

⎛ 0 ⎞3 ⎛ R (t ) ⎞d ⎛ R 0 ⎞3 N (Ms + ncMc) ⎜ R g ⎟ g g ⎟ = ∼⎜ 0 ⎟ ⎜ ⎜ R ⎟ ⎜ R (t ) ⎟ ⎜ R g (t ) ⎟ ρsug Ms ⎝ g ⎠ ⎝ g ⎠ ⎝ ⎠ ρagg

(13)

where ρsug is the density of solvated HA chain. Equation 13 provides ρagg/ρsug ≈ 0.13 for the largest HA-DP35 aggregates. Consequently, the effective density of HA plus tannin in aggregates is about 7 times lower than the density of just HA chain. This result means that the polysaccharide-tannin complex forms a more stable suspension than just tannin or polysaccharide alone. Our results show that the structure and mass of tanninpolysaccharide assemblies depend essentially on tannin length and that it is not very sensitive to tannin concentration. For variation of the tannin concentration from 0.05 to 5 g/L, the aggregation number increases only by a factor of 2 or 3. On the other hand, it increases by a factor of about 25 if the tannin degree of polymerization changes from 2 to 35. The size of dominant aggregates, as indicated by our measurements of hydrodynamic and gyration radii, is of the order of a single polysaccharide molecule for short tannins (DP up to 10) and become of the order of 100 nm for longer tannins (DP=35). These findings, together with the fact that the internal structure of aggregates evolves from coil-like (scaling dimension d < 2) to microgel-like (2 < d < 3), are indications that there exists some crossover end-to-end tannin length l*t = lt(DP*). Tannins shorter than this length can form only small oligomeric aggregates containing not more than a few polysaccharide molecules dressed by tannins. Long tannins form mostly larger aggregates, which are globular and contain a large portion of water, of up to 50 polysaccharide molecules and up to 25 catechin units per HA. The crossover tannin length, l*t, can be estimated knowing that lt ≈ 0.34 nm × DP, according to a previous study,9 and that the crossover from oligomers (micelles) to microgel aggregates occurs between DP = 10 and DP = 35, i.e. for tannin lengths lt between 3.4 and 12 nm. This suggests that the relevant quantity might be the polysaccharide persistence length Lp = 9 nm = lt(DP*), corresponding to DP* = 26. Tannins with DP ≲ DP* form with polysaccharides mostly oligomers, while tannins with DP ≳ DP* aggregate with polysaccharides into microgel particles with Rg ≳ 100 nm. Although in some respects tannin−polysaccharide complexation is similar to tannin-assisted aggregation of proteins, there is a capital difference between the two phenomena. Typically, unfolded proteins form with tannins macroscopic aggregates 758


Biomacromolecules

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causing precipitation3 or, in some cases,22,23 micelle-like assemblies. In both cases, protein compactness is enhanced within the aggregate. This is the molecular manifestation of astringency (from Latin adstringere, meaning “to bind fast”), the main property of tannins. On the contrary, we show that polysaccharides do not collapse nor form closely packed aggregates in the presence of tannins, but create either loose oligomers or microgels that are even less compact than single polysaccharide coils. The difference is easily observable already by moderate centrifugation, where tanninprotein micelles are found in the pellet, while tannin−polysaccharide complexes remain stable even at ultracentrifugation of 8 × 105g. The explanation of this difference is in the physical properties of proteins and polysaccharides. Unfolded proteins are typically very flexible, have negligible charge and contain hydrophobic blocs likely to assemble together into micelles. Polysaccharides like HA are semiflexible polyelectrolytes, which remain well solvated. Tannins longer than Debye length can bridge two HA molecules, but the HA molecules remain as far as possible from one another, avoiding any contact because of Coulomb repulsion. High rigidity of the polysaccharide string enhances even more the hydrodynamic volume of aggregates.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS D.Z. is indebted to Christophe Tribet for help and advice. REFERENCES

(1) Pourcel, L.; Routaboul, J.-M.; Cheynier, V.; Lepiniec, L.; Debeaujon, I. Trends Plant Sci. 2007, 12, 29−36. (2) Plant Polyphenols: Synthesis, Properties, Significance, Hemingway, R. W., Laks, P. E., Eds.; Plenum Press: New York, 1992. (3) Haslam, E. Practical Polyphenolics: From Structure to Molecular Recognition and Physiological Action; Cambridge University Press: Cambridge, 1998. (4) Le Bourvellec, C.; Le Quéré, J.-M.; Renard, C. M. G. C. J. Agric. Food Chem. 2007, 55, 7896−7904. (5) Poncet-Legrand, C.; Doco, T.; Williams, P; Vernhet, A. Am. J. Enol. Vitic. 2007, 58, 87−91. (6) Nitta, Y.; Fang, Y.; Takemasa, M.; Nishinari, K. Biomacromolecules 2004, 5, 1206−1213. (7) Esquenet, C.; Buhler, E. Macromolecules 2002, 35, 3708−3716. (8) Buhler, E.; Boué, F. Macromolecules 2004, 37, 1600−1610. (9) Zanchi, D.; Svergun, D.; Konarev, P.; Baron, A.; Guyot, S. J. Chem. Phys. 2009, 130, 245103. (10) Porter, L. J. In Plant Polyphenols: Synthesis, Properties, Significance; Hemingway, R. W., Laks, P. E., Eds.; Plenum Press: New York, 1992; p 245. (11) Newman, R. H.; Porter, L. J.; Foo, L. Y.; Johns, S. R.; Willing, R. I. Magn. Reson. Chem. 1987, 25, 118−124. (12) Poncet-Legrand, C.; Cabane, B.; Bautista-Ortin, A.-B.; Carrillo, S.; Fulcrand, H.; Pérez, J.; Vernhet, A. Biomacromolecules 2010, 11, 2376−2386. (13) Morfin, I.; Buhler, E.; Cousin, F.; Grillo, I.; Boué, F. Biomacromolecules 2011, 12, 859−870. (14) Buhler, E.; Boué, F. Eur. Phys. J. E: Soft Matter Biol. Phys. 2003, 10, 89−92. (15) Bonnet, F.; Schweins, R.; Boué, F.; Buhler, E. Europhys. Lett. 2008, 83, 48002. (16) Guyot, S.; Marnet, N.; Drilleau, J. F. J. Agric. Food Chem. 2001, 49, 14−20. (17) Bernillon, S.; Guyot, S.; Renard, C. M. G. C. Rapid Commun. Mass Spectrom. 2004, 18, 939−943. (18) Fisher, M. E.; Burford, R. J. Phys. Rev. 1967, 156, 583−622. (19) Beaucage, G. Phys. Rev. E 2004, 70, 031401. (20) Zanchi, D.; Poncet-Legrand, C.; Tribet, C.; Schweins, R.; Cabane, B.; Cartalade, D.; Vernhet, A. Langmuir 2007, 23, 9949−9959. (21) Le Bourvellec, C.; Le Quéré, J.-M.; Sanoner, P.; Drilleau, J.-F.; Guyot, S. J. Agric. Food Chem. 2004, 52, 122−130. (22) Zanchi, D.; Narayanan, T.; Hagenmuller, D.; Baron, A.; Guyot, S.; Cabane, B.; Bouhallab, S. Europhys. Lett. 2008, 82, 58001. (23) Zanchi, D.; Poulain, C.; Konarev, P.; Tribet, C.; Svergun, D. I. J. Phys.: Condens. Matter 2008, 20, 494224.

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CONCLUSIONS Interactions between polysaccharide hyaluronan (HA) and plant tannins were investigated by means of light scattering and small-angle X-ray scattering. Structural study of the system revealed that tannins and polysccharides form finitesize complexes, typically partitioned in two populations: (i) oligomeric (N ≲ 5) coil-like aggregates and (ii) large reticulated bushy objects (or microgels) with a fractal dimension of about 2.4 and a hydrodynamic diameter going up to ten times the size of a single HA coil. The sizes and structures of these objects are determined by tannin length and are stable at least for concentrations from 0.05 to 5 g/L of tannin with 3 g/L of polysaccharide. Short tannins (lt ≲ 10 nm) are aggregated with polysaccharides in small oligomeric (N < 5) coil-like aggregates with a size of up to 2 times single HA molecule. Tannins with lt ≳ 10 nm are aggregated with polysaccharides in large bushy objects (or microgels) with a fractal dimension of about 2.4 and a hydrodynamic diameter up to ten times the size of a single HA coil. This suggests that the characteristic tannin length of 10 nm is determined by the persistence length of polysaccharide. However, more detailed experiments are needed to confirm this conjecture. Within aggregates, polysaccharide and tannin molecules keep their native conformations. Consequently, tannin−polysaccharide aggregates have very low compactness. (Tannin−HA microgel density is about 7 times lower than the density of solvated polysaccharide alone!) Moreover, the aggregates remain charged. For both reasons, they remain stable in suspension up to a very high centrifugation speed.

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Article

ASSOCIATED CONTENT

S Supporting Information *

SAXS profiles for HA at 3 g/L with a range of concentration of DP2 tannin, including the spectra of tannin only and HA only with corresponding fits; SAXS data for HA with tannin polymer DP35; and SAXS profiles of simply dissolved, filtered, and ultracentrifuged sample HA 60 kDa. This material is available free of charge via the Internet at http://pubs.acs.org. 759


Structural Properties of Colloidal Complexes between Condensed

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