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Effects of Epigallocatechin Gallate on β-Casein Adsorption at the Air/Water Interface Pascal Sausse, Ve´ronique Aguie´-Be´ghin, and Roger Douillard* Equipe Paroi et Mate´ riaux Fibreux, UMR FARE INRA/URCA, Centre de Recherche Agronomique, 2 Esplanade Roland Garros, BP 224, 51686 Reims Cedex 2, France Received July 26, 2002. In Final Form: October 25, 2002 Protein adsorption at the air/water interface is of first interest in foam stabilization and has been much studied. Though interactions between polyphenols and proteins in solution have been demonstrated, their effects on protein adsorption properties are totally unknown. The aim of this work is to study the effect of epigallocatechin gallate on β-casein adsorption. Protein adsorption from a polyphenol-protein solution was monitored by tensiometry and ellipsometry. The adsorption layer structure was measured by spectroscopic ellipsometry, and viscoelastic properties were monitored by an oscillating bubble method. Adsorption kinetics shows that protein adsorption is slowed by the polyphenol. While polyphenol adsorption from pure polyphenol solution is not observed, polyphenol adsorption from the mixture is observed. It seems that protein surface concentration is increased when polyphenol bulk concentration is smaller than 60 mg/L and that protein surface concentration is decreased when polyphenol bulk concentration is larger than 60 mg/L. Moreover, the elastic modulus of the layer increases after the formation of the layer. From these results, it can be concluded that β-casein adsorption properties are greatly modified by the polyphenol. However, the mechanisms involved remain to be understood.
Introduction Proteins and polyphenolic compounds occur in foodstuffs. Proteins are widely used as functional ingredients in food processing to provide properties such as foam and emulsion stabilization.1,2 Polyphenols are secondary leaf, bark, and fruit metabolites of plants. They are of great importance in the food industry since they are responsible for food organoleptic properties such as astringency, bitter taste,3-5 or formation of protein precipitate in beverages.6-8 Protein precipitation is particularly important in the case of sparkling alcoholic beverages, which contain polyphenols and where foam is stabilized by macromolecule adsorption layers.9 The precipitating properties of polyphenols are considered to be based on their protein binding ability. While polyphenol protein binding has been known and studied for many years, it is not well understood. Associations could be soluble or not and reversible or not.10 These associations depend on both polyphenol and protein chemical structures.11,12 Nevertheless, general features have been demonstrated. Polyphenol-protein affinity * Corresponding author. Tel: 33 3 26 77 35 94. Fax: 33 3 26 77 35 99. E-mail:
[email protected]. (1) Dickinson, E. Int. Dairy J. 1999, 9, 305. (2) Casanova, H.; Dickinson, E. J. Colloid Interface Sci. 1998, 207, 82. (3) Bate-Smith, E. C. Food 1954, 23, 124. (4) Luck, G.; Liao, H.; Murray, N. J.; Grimmer, H. R.; Warminski, E. E.; Williamson, M. P.; Lilley, T. H.; Haslam, E. Phytochemistry 1994, 37, 357. (5) Kielhorn, S.; Thorngate, J. H. Food Qual. Preference 1999, 10, 109. (6) Eastmond, R.; Gardner, R. J. J. Inst. Brew. 1974, 80, 192. (7) Asano, K.; Shinagawa, K.; Hashimoto, N. Am. Soc. Brew. Chem. 1982, 40, 147. (8) Kawamoto, H.; Mizutami, K.; Nakatsubo, F. Phytochemistry 1997, 46, 473. (9) Pe´ron, N.; Cagna, A.; Valade, M.; Bliard, C.; Aguie´-Be´ghin, V.; Douillard, R. Langmuir 2001, 17, 791. (10) Haslam, E. Polyphenol complexation. In Polyphenolic phenomena; Scalbert, A., Ed.; INRA Editions: Paris, 1993. (11) Zhu, M.; Phillipson, J. D.; Greengrass, P. M.; Bowery, N. E.; Cai, Y. Phytochemistry 1997, 44, 441. (12) Hagerman, A. E.; Rice, M. E.; Ritchard, N. T. J. Agric. Food Chem. 1998, 46, 2590.
depends on polyphenol molecular size: the larger the molecular size, the higher the affinity.13,14 Oxidation occurs spontaneously at room temperature in the presence of oxygen. Among the oxidation products, polyphenol oligomers are found as well as other compounds with molecular sizes larger than that of the original polyphenol.15-17 Due to their molecular sizes, these oxidation products bind more easily to proteins and mixture properties evolve with time.18 Protein structure also has an important role on these interactions, especially the amino acid composition, since it has been shown that proline-rich proteins have a greater affinity for polyphenols than other proteins.19-22 Although protein adsorption properties are of first interest and although polyphenol-protein interactions in solution have been much studied, the effect of polyphenols on protein adsorption properties has practically not been studied. Only one study reports the effects of catechin on the adsorption and foam properties of a proteinsurfactant mixture (β-lactoglobulin-Tween 20). The data may indicate that catechin increases the foamability and the foam stability of the mixture.23 The simpler case of a (13) Artz, W. E.; Bishop, P. D.; Keith Dunker, A.; Shanus, E. G.; Swanson, B. G. J. Agric. Food Chem. 1987, 35, 417. (14) De Freitas, V.; Mateus, N. J. Agric. Food Chem. 2001, 49, 940. (15) Berkowitz, J. E.; Coggon, P.; Sanderson, G. W. Phytochemistry 1971, 10, 2271. (16) Hashimoto, F.; Nonaka, G.-I.; Nishioka, I. Chem. Pharm. Bull. 1992, 40, 1383. (17) Haslam, E. Polyphenol complexation. A study in molecular recognition. In Phenolic compounds in food and their effects on health I; American Chemical Society: Washington, DC, 1992. (18) Asano, K.; Ohtsu, K.; Shinagawa, K.; Hashimoto, N. Agric. Biol. Chem. 1984, 48, 1139. (19) Baxter, N. J.; Lilley, T. H.; Haslam, E.; Williamson, M. P. Biochemistry 1997, 36, 5566. (20) Murray, N. J.; Williamson, M. P.; Lilley, T. H.; Haslam, E. Eur. J. Biochem. 1994, 219, 923. (21) Charlton, A. J.; Baxter, N. J.; Lilley, T. H.; Haslam, E.; McDonald, C. J.; Williamson, M. P. FEBS Lett. 1996, 382, 289. (22) Lu, Y.; Bennick, A. Arch. Oral Biol. 1998, 43, 717. (23) Sarker, D. K.; Wilde, P. J.; Clark, D. C. J. Agric. Food Chem. 1995, 43, 295.
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polyphenol-protein mixture without competitive adsorption of surfactants is yet unknown. To afford experimental data in this field, a study has been initiated with the model system epigallocatechin gallate-β-casein at the air/water interface. The properties of the model compounds are well documented. Epigallocatechin gallate (Egcg) is a small phenolic compound with regard to its molecular mass (458 g/mol). Its affinity for several proteins has been demonstrated.24-26 β-Casein adsorbs strongly at a fluid interface, and its adsorption layer is relatively well characterized. Its flexible structure in solution is reminiscent of that of a random coil polymer because of a low level of secondary structure.27,28 It is a relatively proline-rich protein (16%). Moreover, its polyphenol affinity has been demonstrated with pentagalloyl glucose.29
time was set as the time needed for surface pressure to reach 1 mN/m. This value is 10 times higher than the background noise but always smaller than the usual induction time. Thus the induction time is measured in the region where the dynamics is dominated by diffusion in the bulk. Ellipsometry. Measurements were performed using a spectroscopic phase modulated ellipsometer (UVISEL, Jobin Yvon, Longjumeau, France). It was equipped with a xenon arc source. Both the polarizer and the analyzer were set to the 45° configuration angle. The photoelastic modulator, activated at a 50 kHz frequency, was set to the 0° configuration orientation. The spectroscopic measurements were monitored between 250 and 810 nm. The incident angle was set to 53.4°. All measurements were done at the air/water interface in an air-conditioned room at 20 ( 1 °C. The two ellipsometric angles Ψ and ∆ are linked to the two reflectivity coefficients rp and rs, in the directions parallel and perpendicular to the incident plane, respectively, by
Materials and Methods Polyphenol and Protein. Epigallocatechin gallate (purity > 95%) was obtained from Sigma. β-Casein was purified according to the method of Mercier et al.30 Acid casein was prepared from the skimmed milk of a single cow homozygous for the three major caseins (RS1B, βB, and κB) and freeze-dried. Acid casein was then fractionated by ion exchange chromatography on DEAE 5PW using a NaCl gradient in a 20 mM imidazole buffer (pH 7), 3.3 M urea, and 1 mM DTT (dithiothreitol). The fraction corresponding to β-casein was rechromatographed in the same conditions, and its purity was checked by polyacrylamide gel electrophoresis. The extinction coefficient used to determine the β-casein concentration in solution is E1% 1cm ) 4.6. Dynamic Bubble Tensiometer. The dynamic bubble tensiometer is an I.T.Concept (Longessaigne, France) instrument, similar to the one previously described.31 For its operation, a bubble is formed at the tip of the needle of a syringe whose plunger is driven by an electric motor. The image of the bubble is taken from a CCD camera and digitized in a microcomputer. The interfacial tension γ is determined by analyzing the profile of the bubble according to the Laplace equation:
2 1 d (x sin θ) ) - cz x dx b
(1)
where x and z are the Cartesian coordinates at any point of the bubble profile, b is the radius of curvature at the apex of the bubble, θ is the angle of the tangent to the bubble profile, and c ) 2/a2, where a is the capillary constant (a ) (2γ/(Fg))1/2 where F is the difference between the densities of the two phases and g is the acceleration of gravity). The area of the bubble and the interfacial tension were calculated several times per second. Moreover, a sinusoidal fluctuation of the area, A, of the bubble made possible the calculation of the dilational modulus at a chosen amplitude and frequency (0.1 Hz in this study):
)
dγ d(ln A)
(2)
Induction Time. Usually the induction time is taken as the intersection between the time axis and the linear increase of the surface pressure increase. For the sake of precision, the induction (24) Bacon, J. R.; Rhodes, M. J. C. J. Agric. Food Chem. 1998, 46, 5083. (25) Naurato, N.; Wong, P.; Lu, Y.; Wroblewski, K.; Bennick, A. J. Agric. Food Chem. 1999, 47, 2229. (26) Wroblewski, K.; Muhandiram, R.; Chakrabartty, A.; Bennick, A. Eur. J. Biochem. 2001, 268, 4384. (27) Swaisgood, H. E. Developments in Dairy Chemistry; Applied Science Publishers: London, 1982. (28) Razumovsky, L.; Damodaran, S. Langmuir 1999, 15, 1392. (29) Spencer, C. M.; Cai, Y.; Martin, R.; Gaffney, S. H.; Goulding, P. N.; Magnolato, D.; Lilley, T. H.; Haslam, E. Phytochemistry 1988, 27, 2397. (30) Mercier, J. C.; Maubois, J. L.; Poznanski, S.; Ribadeau-Dumas, B. Bull. Soc. Chim. Biol. 1968, 50, 521. (31) Benjamins, J.; Cagna, A.; Lucassen-Reynders, E. H. Colloids Surf. 1996, 114, 245.
rp ) tan(Ψ) exp(i∆) rs
(3)
The fixed wavelength chosen for the kinetic measurements corresponds to the Brewster conditions defined by ∆ ) (π/2. The ellipticity coefficient measured in the Brewster conditions for the substrate, FB, is defined by
FB ) tan(Ψ) sin(∆)
(4)
In the case of thin layers for which the thickness h is largely lower than the wavelength λ, Brewster angle ellipticity is given by the Drude equation:32
FB )
2 2 π xn0 + n2 λ n02 - n22
∫
+∞
-∞
[n12(z) - n02][n12(z) - n22] (5) dz n12(z)
where n0, n1, and n2 are the indices of the upper phase (air), the layer, and the lower phase (buffer), respectively. Assuming that n1 is constant and n0 ) 1, one finds
[n12 - 1][n12 - n22]
FB ∝ h
n12
(6)
Thus, ellipticity is proportional to the layer thickness and increases as the square of the layer index. Therefore, as a first approximation, ellipticity increases with the total surface concentration including protein and polyphenol surface concentration. To obtain protein and polyphenol surface concentrations, thickness and optical indexes of the layers have been determined from ellipsometric spectra. The complex refractive index of the substrate, N2 ) n2 + ik2, was obtained from a pure buffer spectrum by analytical inversion of the Fresnel equations.33 The thickness of the layer was obtained as the value which permits the numerical inversion of the Fresnel equations with a practically null value of the complex refractive index in the range 350-600 nm when the protein and the polyphenol do not absorb light.34 This calculation gives the n1 and k1 spectrum between 250 and 810 nm. Since β-casein absorption at 280 nm is negligible, the polyphenol surface concentration can be deduced from the relation between the imaginary refractive index of the layer k1 and the bulk extinction coefficient of the polyphenol at 280 nm ():
4πk1 ) c1 ln 10 λ
(7)
(32) Drude, P. Ann. Phys. Chem. (Leipzig) 1891, 43, 126. (33) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and polarized light; Elsevier Science: New York, 1987. (34) Martensson, J.; Arwin, H. Langmuir 1995, 11, 963.
Effects of Epigallocatechin Gallate on Adsorption ΓEgcg ) c1h ΓEgcg )
Langmuir, Vol. 19, No. 3, 2003 739 (8)
4π kh λ ln 10 1
(9)
where ΓEgcg is the polyphenol surface concentration, h is the layer thickness, and λ is the wavelength. The β-casein surface concentration, Γcas, can be estimated from the formula35
Γcas
) h(n - n ) (dn dc ) 1
2
(10)
where n1 is the buffer refractive index, n2 is the real layer refractive index, and dn/dc is the refractive index increment of the β-casein. This formula is valid when the adsorption layer is formed of one compound. It could be generalized to the case where the adsorption layer is formed of two compounds, β-casein and Egcg:
Γcas
(dn dc )
cas
(dn dc )
+ ΓEgcg
Egcg
) h(n1 - n2)
(11)
where ΓEgcg is determined by the method previously described. With this method, even if the layer thickness, h, and the optical indexes, n1 and k1, are not precisely determined, the errors in h and n1 or k1 are covariant in a way which allows determination of surface concentrations ΓEgcg or Γcas with more accuracy than h, n1, or k1 alone.36 Refractive Index Increment. The physical quantity dn/dc was measured for solutions of Egcg using a Schmidt-Haensch DUR-W2 refractometer and found to be 0.19 cm3 g-1. β-Casein dn/dc was kept to 0.18 cm3 g-1 as previously measured.37 Sample Preparation. Protein-polyphenol mixtures were prepared in a 10 mM MOPS (morpholinopropane sulfonic acid) buffer, pH 7. The protein bulk concentration was fixed to 10 mg/L in all the mixtures. Egcg solutions were prepared just before use in order to reduce oxidation. Aged samples were aged in a dark room at 20 °C.
Results and Discussion Polyphenol Surface Properties. Measurements of surface tension and ellipticity of a pure Egcg solution (100 mg/L) show that surface tension remains that of the buffer and that the ellipticity remains slightly negative like that of the buffer. Accordingly, in the frame of this work, polyphenol adsorption can be neglected. On account of the molecular structure of Egcg, this result is not striking, because hydrophilic hydroxyl groups are distributed all around the molecule, which is not polarized with respect to hydrophilic and hydrophobic chemical groups. Protein Adsorption Kinetics. Protein adsorption generally exhibits a typical kinetics, which can be divided in three phases (Figure 1). In the first one, which is called the induction time, the surface pressure remains practically zero, while protein adsorption occurs. In the second one, surface pressure increases rapidly, and in the third one, the surface pressure stabilizes or increases slowly. Induction times of β-casein solutions vary as described for other proteins:38 they decrease with increasing protein bulk concentration (Figure 2a). Consequently, a stable induction time is expected for the samples with constant protein bulk concentrations (10 mg/L). On the contrary, mixtures of protein and polyphenol exhibit a striking increase of induction time with the polyphenol bulk concentration (Figure 2b). The rise in induction time begins (35) De Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759. (36) Bain, C. D. Curr. Opin. Colloid Surf. Sci. 1998, 3, 287. (37) Puff, N.; Marchal, R.; Aguie´-Be´ghin, V.; Douillard, R. Langmuir 2001, 17, 2206. (38) Ybert, C.; Di Meglio, J. M. Langmuir 1998, 14, 471.
Figure 1. Effects of β-casein and Egcg bulk concentrations on the kinetics of surface pressure. Adsorption kinetics of β-casein alone: (b) 10 mg/L, (9) 6 mg/L, and (2) 2 mg/L. Mixtures of β-casein 10 mg/L and Egcg: (O) 20 mg/L, (0) 50 mg/L, and (4) 70 mg/L.
when the polyphenol concentration reaches 20 mg/L and rapidly increases with Egcg concentration. For high polyphenol concentrations (>80 mg/L), mixture induction times are 100 times higher than that of β-casein alone at the same concentration. Like surface pressure, ellipticity variation during adsorption is slowed for polyphenol concentrations higher than 50 mg/L (Figure 3). Hence, Egcg slows the adsorption of the protein and its effect on adsorption kinetics is similar to that of a decrease of β-casein bulk concentration. From surface pressure kinetics at long times (>1000 s), it can be seen that for mixtures and β-casein solutions with the same induction time, the kinetics differ when time is larger than the induction time. Indeed, in mixtures, the surface pressure rise is slower than that of the pure casein, and this difference increases with the polyphenol concentration. This indicates that the adsorption layer, its components or its structure, is not the same for pure protein and for the mixture. Several studies have shown that at short times, adsorption can be modeled by a diffusion-controlled model.39-41 This is due first to the fact that protein surface concentration is low enough to neglect the energy barrier due to the presence of the protein at the interface and second to the practical irreversibility of protein adsorption. Hence, at early time the surface concentration Γ is controlled by a diffusion law, which gives the simple relation:42
xDtπ
Γ(t) ) 2c0
(12)
where c0 is the protein bulk concentration and D is the protein diffusion coefficient. Moreover, this relation can be extended to the induction time,38 since during all the (39) MacRitchie, F.; Alexander, A. E. J. Colloid Sci. 1963, 18, 453. (40) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403. (41) De Feijter, J. A.; Benjamins, J. Adsorption kinetics of proteins at the air-water interface. In Food emulsions and foams: based on the proceedings of an international symposium organised by the Food Chemistry Group of the Royal Society of Chemistry at Leeds from 24th26th March 1986; Dickinson, E., Ed.; Special Publication No. 58; Royal Society of Chemistry: London, 1987; p 72. (42) Ward, A. F. H.; Tordai, L. J. Chem. Phys. 1946, 14, 453.
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Figure 3. Effects of β-casein and Egcg bulk concentrations on the kinetics of ellipticity. Adsorption layers were formed from mixtures of β-casein 10 mg/L and Egcg: (O) Egcg 0 mg/L, (b) Egcg 9.4 mg/L, (0) Egcg 18.7 mg/L, (9) Egcg 55 mg/L, (2) Egcg 63.4 mg/L, and (4) Egcg 80 mg/L.
Figure 2. (a) Effect of the β-casein bulk concentration on the induction time τ. (b) Effect of the Egcg bulk concentration on the induction time of a 10 mg/L β-casein solution.
induction time the surface pressure is practically zero. Therefore, it can be assumed that the origin of the change of adsorption kinetics reported here is a change in parameters controlling diffusion: the protein diffusion coefficient or the protein bulk concentration. Therefore, assuming that the protein diffusion coefficient in the mixture is not much affected by the phenolics, the β-casein bulk effective concentration can be deduced from mixture induction times (Figure 2b) and from the calibration curve obtained with the induction time of β-casein at various concentrations (Figure 2a). The effective concentration of a mixture is the concentration of β-casein alone which gives the same induction time as the mixture. The effect of Egcg on the adsorption kinetics can be quantified as the ratio of the effective to the total β-casein in solution. This ratio plotted as a function of the polyphenol concentration shows that protein adsorption is significantly perturbed near 40 mg/L Egcg and quickly decreases for higher concentrations (Figure 4). For Egcg concentrations higher than 90 mg/L, the effective protein is less than 10% of the total protein. It can be seen from eq 12 that the diffusion coefficient is proportional to the reciprocal of the induction time; consequently, if the adsorption kinetics modification is due only to a decrease of the diffusion coefficient, then
Figure 4. Effect of the Egcg bulk concentration on the reduced effective β-casein concentration R. R is the ratio of effective to total β-casein (10 mg/L). The effective casein of a mixture is equal to the β-casein bulk concentration of a solution with the same induction time.
this coefficient would be 50 times lower at a high polyphenol concentration than for pure protein. Such a change in the diffusion coefficient seems impossible, whereas a decrease in the β-casein bulk concentration could explain this slowed adsorption kinetics. The calculated effective concentration means the quantity of available protein, which can still adsorb in the presence of Egcg. This suggests that polyphenol prevents the adsorption of a fraction of the total casein. A first explanation for this lowered available fraction could be the formation of a precipitate or soluble aggregate as has already been observed in polyphenol-protein solution,43,44 but protein bulk concentration (10 mg/L) is too low to observe a precipitate. But the slow adsorption of the protein in the presence of polyphenol could also be explained by an energy barrier. Generally, the change in protein conformation during adsorption leads to a lower surface energy of the protein. Interactions between protein (43) Siebert, K. J.; Troukhanova, N. V.; Lynn, P. Y. J. Agric. Food Chem. 1996, 44, 80. (44) Siebert, K. J.; Carrasco, A.; Lynn, P. Y. J. Agric. Food Chem. 1996, 44, 1997.
Effects of Epigallocatechin Gallate on Adsorption
and polyphenol could reduce the flexibility of the casein and prevent the change in protein conformation. Another explanation is that the hydrophobic/hydrophilic balance of the protein is changed by the association with polyphenol. Kinetics measurements show clearly that protein adsorption is modified by the polyphenol. The main result is the slower adsorption of the mixture. However, the reasons for the low adsorption rate of the mixture remain to be elucidated. Moreover, after the induction time the surface pressure of the mixture is lower than that of β-casein alone. Above the induction time, the adsorption is even slower than that of the solution with the effective protein concentration. This fact suggests that the polyphenol is present in the layer and may cause the effects described above. Nevertheless, direct evidence of the presence of the polyphenol in the layer is needed. Adsorption Layer Structure. Whereas the induction time continuously increases with polyphenol concentration, suggesting that the protein adsorption rate decreases, the ellipticity variation rate does not adhere precisely to that trend (Figure 3). Initial ellipticity increase rates of mixtures whose polyphenol concentration is lower than 40 mg/L are slightly higher than those of protein alone at the same concentration. For polyphenol concentrations larger than 40 mg/L, the initial ellipticity rate decreases rapidly and becomes smaller than that of pure β-casein for polyphenol concentrations higher than 60 mg/L. Thus the reduction of adsorption rate is observed by ellipsometry as previously by surface tension, but only for polyphenol concentrations larger than 60 mg/L. Moreover, when the polyphenol concentration is smaller than 60 mg/L, the equilibrium value of the ellipticity is larger than that of pure β-casein. Thus, protein adsorption seems to be greater with such polyphenol concentrations (20-60 mg/L), a fact which was not noticed from surface pressure kinetics. The higher ellipticity value observed at low polyphenol concentrations could be due to the adsorption of more protein at the interface or to the occurrence of polyphenol in the adsorption layer. Optical refractive indexes of the layer calculated from ellipsometric data show that the layer structure is obviously affected by the polyphenol. First, the real index is higher in the presence of polyphenol (Figure 5a). Second, an absorption spectrum appears in interfacial layers of mixtures but not with the pure protein (Figure 5b). The maximum of absorption is around 275 nm. Bulk measurements show that the maximum of absorption of the β-casein is at 276 nm, and that of the polyphenol is at 275 nm. Ellipsometric spectra are not accurate enough to assert that absorption is due only to the polyphenol. Nevertheless, the protein extinction coefficient (0.46 L g-1 cm-1) is much smaller than that of the polyphenol (23 L g-1 cm-1) and absorption is observed only in mixtures. This allows one to conclude that because of its weaker extinction coefficient protein absorption in the layer is negligible with regard to the polyphenol absorption and that absorption is evidence for occurrence of the polyphenol in the layer. Concentrations calculated from refractive and absorption indexes of the layer 1 and 24 h after the beginning of the adsorption are shown in parts a and b of Figure 6, respectively. The first result is that the ellipticity increase with respect to the ellipticity of a layer formed from a pure β-casein solution is due not only to the occurrence of the polyphenol but also to an excess concentration of β-casein. Indeed, the surface concentration of β-casein increases between 0 and 20 mg/L Egcg, remains constant between 20 and 50 mg/L, and then decreases to reach a
Langmuir, Vol. 19, No. 3, 2003 741
Figure 5. Effects of β-casein and Egcg adsorption on the real part n (a) and complex part k (b) of the refractive index of the layer evaluated from ellipsometric data: (0) MOPS buffer; (O) β-casein 10 mg/L, alone; (4) β-casein 10 mg/L-Egcg 37 mg/L, 1 h after the beginning of adsorption; (3) β-casein 10 mg/LEgcg 37 mg/L, 24 h after the beginning of adsorption.
surface concentration lower than that of β-casein alone. This general trend is the same 1 and 24 h after the beginning of the adsorption. However, the values are not the same. In the medium range, the total surface concentration is 3.2 mg/m2 1 h after the beginning of the adsorption and 4.2 mg/m2 after 24 h. These values have to be compared to those of β-casein alone: 2.1 mg/m2 after 1 h and 2.2 mg/m2 after 24 h. The second point to mention is the proportion of polyphenol in the layer which increases until the Egcg concentration reaches 20 mg/L and remains constant for higher concentrations: approximately 6 polyphenol molecules for 1 protein 1 h after the beginning of the adsorption and 15 polyphenol molecules for 1 protein 24 h after. The constant molar ratio implies that β-casein and Egcg surface concentrations evolve in the same way with Egcg bulk concentration. Therefore, the polyphenol fraction and the total surface concentration largely increase with time when the polyphenol occurs in the bulk. Two points can be noticed from the comparison of surface pressure and surface concentration. First, the low β-casein surface concentration at high polyphenol concentration is consistent with the low effective bulk concentration of protein calculated from the induction time in the previous section. Second, at medium concentrations (20-60 mg/L) the protein surface concentration is increased while the
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Figure 7. Effects of Egcg on the relation between the dilational modulus and the surface pressure π during the first 30 min of adsorption: (O) β-casein 10 mg/L alone, (0) Egcg 15.6 mg/L, (4) Egcg 30.5 mg/L, and (3) Egcg 44.9 mg/L.
Figure 6. Effects of the Egcg bulk concentration on the surface concentration of β-casein and Egcg and on their molar ratio. Surface concentrations 1 h (a) and 24 h (b) after the beginning of adsorption: (O) β-casein surface concentration, (4) total surface concentration (β-casein + Egcg), and (9) molar ratio Egcg/β-casein in the layer.
surface pressure is decreased. Hence, it seems that the adsorption for polyphenol-protein association leads to a lower surface tension decrease than that for β-casein alone. The description of the adsorption layer structure is rough. Like the nature of the compounds adsorbed, the organization of the compounds in the layer could change with polyphenol concentration. Such a change in layer organization could explain the mixture surface properties. But now, the question of why the surface concentration of the mixture increases while the surface pressure decreases remains to be asked. The behavior of mixture layers is rather complex; the enhanced surface concentrations and the increase in polyphenol ratio with time were unexpected. However, an increase in polyphenol and protein surface concentrations with time could be related to polyphenol oxidation. Oxidation Effects. During the first 30 min of adsorption, no difference of the dilational modulus is observed between a β-casein layer and a β-casein-Egcg one (Figure 7). Moreover, no difference is noticed in these conditions in the relation between the dilational modulus and the surface pressure. In fact, a scaling law approach of polymers shows that the slope of the /π curve is linked to the Flory exponent of the β-casein polymer chain at the interface.47,48 Although structural differences have been observed by ellipsometry, it seems that the molecular
Figure 8. Effects of Egcg and DTT on the kinetics of the dilational modulus of an adsorption layer of β-casein: (b) β-casein 10 mg/L alone, (9) β-casein 10 mg/L + Egcg 10 mg/L, (2) β-casein 10 mg/L + Egcg 40 mg/L, (1) β-casein 10 mg/L + Egcg 80 mg/L, (O) β-casein 10 mg/L + DTT 10 mM, and (4) β-casein 10 mg/L + Egcg 40 mg/L + DTT 10 mM.
organization of β-casein at the interface is not largely affected by the presence of the polyphenol in the first 30 min of adsorption. After 1 h, the dilational modulus begins to increase (Figure 8). The rate of increase of the modulus is related to the polyphenol concentration. This rate is higher for 40 mg/L than for 10 and 80 mg/L. The lower rate of increase of the modulus with 10 mg/L Egcg bulk concentration could be explained by the lower polyphenol proportion in the layer, and that at 80 mg/L of Egcg by the longer time necessary to form the layer and the lower surface concentration. The time needed for a significant increase of the dilational modulus contrasts with the effect on adsorption kinetics. The effect on adsorption kinetics (45) Sengupta, T.; Damodaran, S. J. Colloid Interface Sci. 1998, 206, 407. (46) Sengupta, T.; Razumovsky, L.; Damodaran, S. Langmuir 1999, 15, 6991. (47) Puff, N.; Cagna, A.; Aguie´-Be´ghin, V.; Douillard, R. J. Colloid Interface Sci. 1998, 208, 405. (48) Aguie´-Be´ghin, V.; Leclerc, E.; Daoud, M.; Douillard, R. J. Colloid Interface Sci. 1999, 214, 143.
Effects of Epigallocatechin Gallate on Adsorption
Figure 9. Effect of Egcg and DTT on the reduced effective β-casein concentration during aging: (O) β-casein 10 mg/L + Egcg 20 mg/L and (0) β-casein 10 mg/L + Egcg 20 mg/L + DTT 10 mM.
appears as soon as the adsorption begins, whereas the modulus effect is not visible at the beginning of the adsorption. Thus, interactions involved in adsorption kinetics and in modulus effects must not be the same. Furthermore, the addition of an antioxidant (DTT) avoids the modulus increase. These observations imply that the origin of the modulus effect could be linked with oxidation, which is a common process with Egcg. It has been reported that polyphenol oxidation products could react with protein10,49 and bring about cross-linking. These covalent bonds could produce a reticulation in the adsorption layer, which would explain the modulus increase. Like the modulus, the induction time increases with aging of the solution: aged solutions have a longer induction time than fresh solutions. Consequently, the effective β-casein concentration decreases with aging time (Figure 9). Thus, the β-casein of the 20 mg/L Egcg-protein mixture is totally available prior to aging, while the available concentration is divided by 2 after 5 h of aging. For the same mixture with addition of DTT, the available concentration is divided by 1.2 after 5 h of aging. Thus, the available fraction of β-casein decreases slowly when DTT is added. As in the case of the modulus, the effect of aging on the induction time becomes significant after several hours and is prevented by the addition of an antioxidant. Thus, oxidation seems to occur in the bulk as at the interface. The change in the nature of the phenolic compound involves a change in protein affinity or reactiv(49) Haslam, E. Complexation and oxidative transformation of polyphenols. In Polyphenol 94; INRA Editions: Paris, 1994; p 45.
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ity. Consequently, the surface properties of these new oxidized polyphenol-protein associations must not be the same as those of the Egcg-protein association. The importance of the aging effect shows that after several hours the system cannot be considered as being built up with the same molecules. Accordingly, the optical properties such as the bulk extinction coefficient used in the ellipsometric model may change with the oxidation of the phenolic compound. These observations point to the fact that oxidation could also be at the origin of the increase of the calculated molar ratio in the layer or of the differences of adsorption kinetics observed for β-casein and mixtures by ellipsometry. Nevertheless, the refractive index increments of organic compounds are close, as observed for protein and polyphenol. The refractive index increments of the polyphenol and of the oxidation products may also be very close. On the contrary, absorption spectra are sensitive to the nature of the compound. But as far as our data can be interpreted, the spectra are qualitatively the same 1 and 24 h after the beginning of adsorption. Therefore, even if a part of the properties observed could be attributed to oxidation products, the concentration calculated may be close to the actual one. Conclusion From this simple experimental model, it can be concluded that polyphenol can largely change β-casein surface properties. Adsorption of β-casein is slowed by the polyphenol. Moreover, adsorption of polyphenol-protein associations occurs. The higher surface concentration observed at medium polyphenol concentrations indicates that the polyphenol effect is not limited to a reduction of protein adsorption. Further work is needed to determine the mechanisms involved in the change of β-casein surface properties caused by the polyphenol. The enhanced surface concentration at medium polyphenol concentrations is not explained yet. The reason for the low rate of adsorption of the mixture could be explained by the protein bulk state, for instance, by the formation of aggregates. The fact that oxidation enhances the effects of the original polyphenol indicates that polyphenol structure could play an important role in these interactions at the interface. To determine the effect of the polyphenol molecular structure on β-casein surface properties, the effects of other polyphenols on β-casein adsorption have to be described. Acknowledgment. This work was supported by the Champagne-Ardenne CPER participants through Europol’Agro (Reims) program management and Moe¨t et Chandon. Thanks go to V. Cheynier, B. Robillard, M. Valade, J. Meunier, and B. Monties for related discussions. LA026304B
