Adsorption of HydrophobinâProtein Mixtures at the AirâWater Interface

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Adsorption of Hydrophobin−Protein Mixtures at the Air−Water Interface: The Impact of pH and Electrolyte Ian M. Tucker,† Jordan T. Petkov,†,⊥ Jeffrey Penfold,*,‡,§ Robert K. Thomas,§ Andrew R. Cox,∥ and Nick Hedges∥ †

Unilever Research and Development Laboratory, Port Sunlight, Quarry Road East, Bebington, Wirral,CH62 4ZD, United Kingdom ISIS, STFC, Rutherford Appleton Laboratory, Chilton, Didcot, OXON OX1 0QX, United Kingdom § Physical and Theoretical Chemistry Laboratory, Oxford University, South Parks Road, Oxford OX1 3QZ, United Kingdom ∥ Unilever Research Laboratories, Sharnbrook, Beds MK44 1LQ, United Kingdom ‡

Downloaded by UNIV OF CAMBRIDGE on September 1, 2015 | http://pubs.acs.org Publication Date (Web): August 31, 2015 | doi: 10.1021/acs.langmuir.5b02403

S Supporting Information *

ABSTRACT: The adsorption of the proteins β-casein, βlactoglobulin, and hydrophobin, and the protein mixtures of βcasein/hydrophobin and β-lactoglobulin/hydrophobin have been studied at the air−water interface by neutron reflectivity, NR. Changing the solution pH from 7 to 2.6 has relatively little impact on the adsorption of hydrophobin or βlactoglobulin, but results in a substantial change in the structure of the adsorbed layer of β-casein. In β-lactoglobulin/ hydrophobin mixtures, the adsorption is dominated by the hydrophobin adsorption, and is independent of the hydrophobin or β-lactoglobulin concentration and solution pH. At pH 2.6, the adsorption of the β-casein/hydrophobin mixtures is dominated by the hydrophobin adsorption over the range of β-casein concentrations studied. At pH 4 and 7, the adsorption of β-casein/hydrophobin mixtures is dominated by the hydrophobin adsorption at low β-casein concentrations. At higher β-casein concentrations, β-casein is adsorbed onto the surface monolayer of hydrophobin, and some interpenetration between the two proteins occurs. These results illustrate the importance of pH on the intermolecular interactions between the two proteins at the interface. This is further confirmed by the impact of PBS, phosphate buffered saline, buffer and CaCl2 on the coadsorption and surface structure. The results provide an important insight into the adsorption properties of protein mixtures and their application in foam and emulsion stabilization.

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been likened to Janus particles.18 The adsorption properties of hydrophobin at the air−water interface and other interfaces have been extensively studied.18−21 At the air−water interface, it forms a dense monolayer20 which has exceptionally high surface shear elastic and viscous moduli.14,15 This gives rise to bubbles, foams, and emulsions which are especially stable.22 βCasein is a more disordered protein that lacks a well-defined tertiary structure, and this results in a greater flexibility in adopting different conformations at interfaces.23 The distribution of polar and nonpolar residues has prompted comparison of the adsorption of β-casein in the form of loops and trains with that originally envisaged for polymer chains.3 The adsorption of β-casein has been extensively studied under a wide variety of conditions.24−28 B-lactoglobulin has a more compact structure due to the presence of disulfide bonds. Hence, β-lactoglobulin has a more well-defined secondary and tertiary structure, and a globular conformation in solution.23 Studies of the adsorption of β-lactoglobulin at the air−water

INTRODUCTION Most proteins have a degree of surface activity, and can be considered as surface active polymers. They can have both polyelectrolyte and polymeric properties, and so electrostatic and hydrophobic interactions can be important in adsorbed protein layers. Protein adsorption has been extensively exploited in stabilizing foams, emulsions, dispersions, and membranes. The mechanical properties of surface layers incorporating proteins are an important aspect of their functionality. Hence, the interfacial rheology of protein containing films has been extensively studied.1−3 Proteins are frequently used in combination with surfactants or other proteins, and protein−surfactant4−11 and mixed protein12−15 adsorption has been widely studied. The focus of this paper is the study of the coadsorption of the proteins β-casein, β-lactoglobulin and hydrophobin at the air−water interface. Hydrophobin is a relatively small (∼7−10 kDa) protein which is produced by filamentous fungi.16,17 The protein is compact and robust due to the eight cysteine residues which make four intramolecular disulfide bridges. It has a globular structure with a well-defined hydrophobic patch which arises from side chain residues of leucine, valine, and analine. This results in the protein being highly surface active, and has © XXXX American Chemical Society

Received: June 30, 2015 Revised: August 18, 2015

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DOI: 10.1021/acs.langmuir.5b02403 Langmuir XXXX, XXX, XXX−XXX

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Langmuir interface have been reported,5,25 and there is some evidence for partial denaturation on adsorption.23,29 Lu et al.30 have shown how neutron reflectivity, NR, can be used to obtain important information about adsorbed amounts of proteins and the structure of the adsorbed layer at different interfaces. Subsequently the technique of NR has been applied to a variety of different systems.24−29 Building on the application of NR to the study of the adsorption of mixed surfactants31 and polymer−surfactant mixtures,32 it has been applied to the study of the adsorption of protein−surfactant mixtures.5,7,21 From these NR measurements and a range of other studies,6,8−11 a relatively clear pattern of behavior has emerged in protein−surfactant mixtures. This involves the possibility of enhanced adsorption due to surface complex formation and competitive adsorption which results in displacement of the protein from the surface at higher surfactant concentrations. Of particular relevance to this paper are the recent studies on the adsorption of protein mixtures, where it is often difficult to predict how proteins with quite different structures will compete for the interface and interact at the surface. Using fluorescence microscopy, Sengupta and Damodaran34 showed how the incompatibility between β-casein and bovine serum albumin, BSA, resulted in inhomogeneous phase separated regions. Marinova et al.13 used surface tension and surface rheology to probe the coadsorption of β-casein and BSA. In coadsorption, mixed films were formed, but for sequential adsorption the properties depended upon the order of addition. The results obtained were related to the stability of thin liquid films and foams. Mackie et al.12 used fluorescence microscopy and atomic force microscopy, AFM, to investigate the coadsorption of β-lactoglobulin and β-casein at the air−water interface, and concluded that homogeneous mixed films were formed. Radulova et al.,14 Daniov et al.,33 and Burke et al.15 have investigated the effect of β-casein on the surface properties of adsorbed hydrophobin layers, using predominantly surface rheological measurements. The rheological response was interpreted as surface layers dominated by the hydrophobin adsorption, but modified in a way consistent with the incorporation of β-casein into the surface layer. In this paper, we have used neutron reflectivity, NR, to study the adsorption and the structure of the adsorbed layers of hydrophobin with the proteins β-casein and β-lactoglobulin at the air−water interface. The more direct structural information obtained from the NR measurements provides a potentially greater insight into the way the mixed protein layers are organized, and which support and extend related studies.14,15,33 The role of pH, buffer, and CaCl2 on the mixed protein adsorption is also investigated.

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Å. On both reflectometers, the absolute reflectivity was calibrated with respect to the direct incident neutron bean intensity and the reflectivity from a D2O surface. The background scattering was subtracted using an area detector on FIGARO, and using the scattered intensity at high Q values from a single detector on INTER. The background scattering arises from the incoherent bulk scattering from the solution and there was no significant off-specular scattering evident. The measurements were made in null reflecting water, nrw (92/8 mol ratio mixture of H2O and D2O), and the solutions were contained in sealed Teflon troughs. The 25 mL solutions, prepared ex situ, were maintained at 25 °C, and each individual reflectivity profile was measured for ∼30 min. Repeated measurements, made over a time scale ∼3−4 h, showed no significant time dependence; and duplicate measurements made on separate occasions showed good reproducibility. In the kinematic approximation31 the specular reflectivity, R(Q), is related to the square of the Fourier transform of the scattering length density profile, ρ(z), where ρ(z) = Σini(z)bi, ni(z) is the number density of the ith nucleus at a distance z from the surface, and bi its neutron scattering length. ρ(z) can be manipulated through H/D isotopic substitution as D and H have different scattering lengths (−3.7 × 10−6 Å for H and 6.67 × 10−5 for D). Hence nrw, described above, will have a scattering length density of 0.0 and a neutron refractive index of 1.0, the same as air. Any reflectivity from such a surface arises from adsorption at the surface for material with a scattering length density different to unity. In the case of a single monolayer adsorbed at the interface, the adsorbed amount or area/ molecule, A, is given by A = Σb/dρ, where Σb is the scattering length of the adsorbed species (see table S1 in the Supporting Information for the Σb values of the different components in this study), and d and ρ are the thickness and scattering length density, respectively, of the adsorbed layer described as a single layer of uniform composition. In such circumstances, the reflectivity is normally evaluated using the familiar optical matrix method.37 Here the simplest model consistent with the data, which consists of 1−3 layers, is adopted, and evaluated by least-squares. Each layer is characterized by a thickness d and a scattering length density ρ. Although the discrimination between different components obtained through deuterium labeling in some mixed systems31,32 is not possible here, some discrimination is possible on the basis of the thickness and scattering length density obtained in the modeling, and this is the basis of the subsequent interpretation and discussion of the mixed protein adsorption. b. Materials. The hydrophobin, class II HFBII, was produced by a fermentation company (BAC) using yeast fermentation. It was subsequently purified by a two phase extraction at Unilever Research, Vlaardigen, using a procedure described in detail elsewhere.20,38 The extracted and purified material was freeze-dried before the preparation of the aqueous solutions. The β-casein and β-lactoglobulin were obtained from Sigma-Aldrich, as 99.9% pure, and used as supplied. High purity water (Elga Ultrapure) was used throughout ,and the D2O was obtained from Sigma-Aldrich. All the NR measurements were made in nrw. All glassware and the Teflon troughs used for the NR measurements were cleaned using alkali detergent (Decon90) and rinsed thoroughly in high purity water. The pH of the solutions was adjusted by the addition of HCl or NaOH; or with the use of PBS, phosphate buffer saline, buffer. The PBS buffer used in this study was a mixture of sodium phosphate, potassium chloride, and sodium chloride at a concentration of 0.01, 0.0027, and 0.14 M. c. Measurements Made. The adsorption at the air−water interface of β-casein was measured at β-casein concentrations from 0.05 to 0.5 wt %, at pH 2.6, 4, and 7. The hydrophobin adsorption was measured at pH 2.6, 4, and 7 at concentrations of 0.005−0.02 wt % (0.05−0.2 mg/mL). The β-lactobglobulin adsorption was measured at pH 2.6 and 7, in the concentration range 0.05−0.5 wt %. The mixed βcasein/hydrophobin measurements were made at two hydrophobin concentrations, 0.005 and 0.02 wt % (0.05 and 0.2 mg/mL), at pH 2.6, 4, and 7, and β-casein concentrations from 0.0 to 0.5 wt %. The βlactoglobulin/hydrophobin measurements were made for protein and hydrophobin concentrations similar to β-casein/hydrophobin, at pH 2.6 and 7. The effect of PBS buffer on β-casein and β-casein/

EXPERIMENTAL DETAILS

a. Neutron Reflectivity. The neutron reflection measurements were made at the air−water interface on the INTER reflectometer35 at the ISIS pulsed neutron source, U.K. and on the FIGARO reflectometer36 at the ILL neutron facility in France. The reflectivity, R(Q), is measured as a function of the wave vector transfer, Q, perpendicular to the surface, where Q = 4π sin θ/λ, θ is the grazing angle of incidence, and λ the neutron wavelength. On both reflectometers the measurements were made at a fixed angle of incidence, θ, and a range of wavelengths, λ (separated by time-offlight), to cover a wide wave vector transfer, Q, range. On INTER, an angle of incidence of 2.3° and a wavelength range of 0.5 to 15 Å were used to cover a Q range of 0.03−0.5 Å−1. On FIGARO, a Q range of 0.035−0.3 Å−1 was accessible using a θ of 3.8° and a λ range of 2.4−24 B


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10−6 Å−2, respectively. This gives an area/molecule ∼385 ± 25 Å2, and an adsorbed amount, Γ, of 0.43 ± 0.02 × 10−10 mol cm−2. However, in detail (see parameters in Table S2 in the Supporting Information) ,the adsorption increases slightly as the pH decreases, from 0.39 × 10−10 mol cm−2 at pH 7 to 0.47 × 10−10 mol cm−2 at pH 2.6. The results for hydrophobin are consistent with those previously reported by Zhang et al.,21 who reported an adsorbed layer thickness of 31 ± 2 Å and an adsorbed amount of 0.39 ± 0.02 × 10−10 mol cm−2. As previously observed,21 the adsorption kinetics was short compared to the measurement time at the hydrophobin concentrations studied here, and no variations that could be attributed to variations in protein MW and structure were observed. The concentration and pH dependence of the adsorption of β-casein at the air−water interface is more complex. This is illustrated in Figure 1b for 0.3 wt % β-casein at pH 2.6 and 4.0, and more fully summarized in Table 1. At pH 4 and 7, the data are best described by two layers, with a mean thickness of 45 and 48 Å, and 44 and 42 Å respectively. At pH 2.6, apart from the data at the highest concentration measured of 0.5 wt %, the data are consistent with a single layer ∼36 Å in thickness. The total amount adsorbed decreases with decreasing pH; such that at pH 4 it is 15% lower than at pH 7, and at pH 2.6 it is ∼50% lower. In the concentration range measured, the adsorption increases slightly with increasing concentration. The composition of the two layers is such that the layer adjacent to the air phase is more dense, with a volume fraction of protein ∼75%. The layer adjacent to the solvent phase contains ∼25% protein. Atkinson et al.25 also used NR to measure the adsorption of β-casein at the air−water interface. At a concentration of 0.05 wt %, from an analysis of measurements over a more limited Q range, they obtained data consistent with a two layer model with thicknesses ∼13 and ∼47 Å, and protein volume fraction ∼0.98 and ∼0.18. Although broadly qualitatively similar to the data presented here, the results are quite different quantitatively. The limited Q range of those measurements makes it more difficult to obtain a unique structural determination compared to the data presented here. The results from Fragneto et al.39 for the adsorption of β-casein at the OTS solid surface are in closer agreement with the data presented here, with a two layer model with thicknesses ∼23 and 35 Å, and protein volume fractions ∼0.61 and 0.21. The quantitative differences in this case can be more readily rationalized in terms of the different nature of the hydrophobic interfaces. Tiberg et al.27 described the adsorption of β-casein at the hydrophilic silica surface in terms of three layers, with a total thickness ∼100 Å after ∼5 h exposure. Taking into account the relative dilution of the outer layer in their analysis, the structures are again qualitatively similar to the data presented here, but again in detail different. This further highlights the importance of the nature of the surface on the partially unfolded structure at the interface. It is well established that the more disordered protein partially denature at interfaces23 and can adopt different configurations. The nature of the interface can have a significant influence, and this is highlighted in the comparison here for β-casein. Even the robust hydrophobin adopts a slightly different packing at the air−liquid and solid−liquid interfaces.44 Moitzi et al.40 determined a critical micelle concentration, cmc, for β-casein at pH 2.6 at ∼0.1 wt %, and slightly lower at higher pH. The concentration dependence of the β-casein adsorption presented here shows no obvious change as the

hydrophobin adsorption was determined at β-casein concentrations from 0.01 to 0.4 wt % and for β-casein/hydrophobin mixtures over the same β-casein concentration range and for a hydrophobin concentration of 0.005 wt % (0.05 mg/mL). The effect of CaCl2 on the adsorption was evaluated for 0.025 and 0.4 wt % β-casein at pH 2.6, 4, and 7 with 0.5 and 1.0 mM CaCl2, and for hydrophobin at 0.02 wt % (0.2 mg/mL) at pH 7 and CaCl2 concentrations 0.5 and 1.0 mM. The measurements on β-casein/hydrophobin mixtures were made for 0.02 wt % (0.2 mg/mL) hydrophobin and 0.025 and 0.4 wt % β-casein at pH 2.6, 4 and 7, for 0.5 and 1.0 mM CaCl2.

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RESULTS AND DISCUSSION a. Adsorption of β-Casein, Hydrophobin, and βLactoglobulin. The adsorption of hydrophobin at the air− water interface was measured for hydrophobin concentrations of 0.005 and 0.02 wt % (0.05 and 0.2 mg/mL) at pH 2.6, 4, and 7. The NR data for 0.005 wt % (0.05 mg/mL) hydrophobin at pH 7 are shown in Figure 1a.

Figure 1. NR data for (a) 0.005 wt % (0.05 mg/mL) hydrophobin in nrw at pH 7 and (b) 0.3 wt % β-casein in nrw at (red) pH 2.6 and (blue) pH 4.0. The solid lines are model calculations as described in the main text, and for the key model parameters in Table 1.

The data are well described by a single layer of uniform composition. At the concentrations measured (0.005 and 0.02 wt % (0.05 and 0.2 mg/mL)), the adsorption is broadly independent of hydrophobin concentration and solution pH. The mean values for the thickness of the adsorbed layer, d, and the scattering length density, ρ, are 30 ± 2 Å and 1.4 ± 0.2 × C


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Table 1. Key Model Parameters from Analysis of NR Data for β-Casein Adsorbed at the Air−Water Interface β-casein concn (wt %)

pH

d1 (±2 Å)

ρ1 (±0.2 × 10‑6 Å−2)

d2 (±2 Å)

ρ2 (±0.1 × 10‑6 Å−2)

0.05 0.15 0.3 0.5 0.05 0.15 0.3 0.5 0.05 0.15 0.3 0.5

7 7 7 7 4 4 4 4 2.6 2.6 2.6 2.6

42 44 48 43 40 44 43 54 33 34 40 36

1.0 1.0 1.0 1.1 1.0 0.9 0.9 0.9 0.8 0.7 0.6 1.0

43 41 43 40 44 46 48 54

0.4 0.4 0.4 0.5 0.3 0.3 0.3 0.3

43

0.3

Over a more limited pH range, they observe a transition from a double layer to single layer adsorption consistent with an increase in adsorption. The structure rearranged from two layers into a single uniform layer with time. The rearrangement was faster as the pH is reduced, such that at lower pH the structure is always in the form of a single layer. Vogtt et al.,41 in solution studies, report the structure of β-lactoglobulin as composed of a dimer of two 17 Å spheres which dissociate with increasing temperature, and which is close to the globular dimension from the crystal structure of 18 Å. The results of Horne et al.5 and Vogtt et al.41 are consistent with the results reported here, whereas the interpretation of results of Atkinson et al.25 are compromised by the limited Q range over which the measurements were made. b. Adsorption of β-Casein/Hydrophobin. The coadsorption of β-casein and hydrophobin at the air−water interface was measured by NR for two different hydrophobin concentrations (0.005 and 0.02 wt % (0.05 and 0.2 mg/ mL)), at a range of β-casein concentrations (from 0.0 to 0.5 wt %), and at pH 2.6, 4, and 7. At pH 2.6, the NR data for the mixed adsorption are consistent with a single layer of uniform composition, and the key model parameters are summarized in Table 3 (for 0.005 wt % (0.05 mg/mL) hydrophobin) and in Table S3 (for 0.02 wt % (0.2 mg/mL hydrophobin)). The values of d and ρ at pH 2.6 with the addition of β-casein (see Tables 3a and S3a) are similar to those obtained in the absence of β-casein, and show no systematic variation as the βcasein concentration increases. This implies that at pH 2.6 the adsorption is totally dominated by the hydrophobin, and that there is little or no β-casein at the surface. At pH 4 and 7 the variation in the NR data with the addition of β-casein is different (see Tables 3b,c and S3b,c). At the lowest β-casein concentration measured (0.025 wt %), the data are similar to that observed in the absence of β-casein. That is, they are consistent with a single layer in which the surface adsorption is dominated by the hydrophobin. The exception to this is the data for 0.2 mg/mL hydrophobin at pH 4 (see Table S3b), where at all β-casein concentrations the data are consistent with a two layer model. In general, at pH 4 and 7 and for β-casein concentrations >0.025 wt % the data are consistent with a two layer model. Figure 2 shows the difference in the NR data as the β-casein concentration increases. As the β-casein concentration increases, the thickness and density of the initial layer (adjacent to the air phase) remain within error constant, and the more dilute/diffuse layer (adjacent to the solvent phase) increases in thickness, as shown in Figure 2 and Table 3. These initial values for the

concentration changes through the apparent cmc. Hence it can be assumed that competition between adsorption and selfassembly does not contribute to the pattern of adsorption. Moitzi et al.40 showed that the aggregated state in solution is different at the extremes of pH. When cationic at low pH, the aggregation number is relatively low, and higher at high pH when the protein is anionic. Furthermore, they argue that the protein in its cationic state loses the distinct separation between its hydrophobic and hydrophilic regions, which gives rise to the lower aggregation number. This is consistent with the lower adsorption observed here at low pH, and the change in the surface structure to a single more compact layer at low pH. The adsorption of β-lactoglobulin was measured at the air− water interface in nrw at pH 2.6 and 7, in the protein concentration range of 0.05−0.5 wt %. Independent of the protein concentration and solution pH, the adsorption is consistent with a single layer of uniform composition. At pH 7, the layer is ∼22 Å thick, and at pH 2.6 it is slightly thicker, ∼25 Å. The key model parameters (d, ρ) are summarized in Table 2. Table 2. Key Model Parameters from Analysis of NR Data for β-Lactoglobulin at the Air−Water Interface at (a) pH 7 and (b) pH 2.6 (a) β-lactoglobulin concn (wt %)

d (±2 Å)

ρ (±0.2 × 10‑6 Å‑2)

0.05 0.1 0.2 0.3 0.5

22 21 19 25 22

0.9 0.9 1.2 0.9 1.0

β-lactogobulin concn (wt %)

d (±2 Å)

ρ (±0.2 × 10‑6 Å‑2)

0.05 0.1 0.2 0.3 0.5

25 20 23 31 27

0.8 0.9 0.8 0.7 0.8

(b)

Although there is a slight change in the surface structure, as indicated by the change in the adsorbed layer thickness, the amount adsorbed is independent of protein concentration and pH. Horne et al.5 report an adsorbed layer thickness ∼20 Å for β-lactoglobulin at the air−water interface. Atkinson et al.25 in an earlier NR study interpret the adsorption of β-lactoglobulin at the air−water interface initially in terms of two layers, with a dense initial layer ∼10 Å and a more diffuse dilute layer ∼20 Å. D


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Table 3. Key Model Parameters from Analysis of NR Data for β-Casein/0.005 wt % (0.05 mg/mL) Hydrophobin at (a) pH 2.6, (b) pH 4, and (c) pH 7 (a) β-casein concn (wt %) 0.0 0.05 0.15 0.3 0.5


ρ (±0.2 × 10‑6 Å‑2)

d (±2 Å) 33 34 32 34 31 (b)

1.5 1.0 1.0 1.2 1.0

β-casein concn (wt %)

d1 (±2 Å)

ρ1 (±0.2 × 10‑6 Å‑2)

d2 (±2 Å)

ρ2 (±0.1 × 10‑6 Å‑2)

0.0 0.025 0.05 0.15 0.3 0.5

28 30 36 33 30 28

1.4 1.2 1.1 1.2 1.2 1.2 (c)

52 38 97 93

0.2 0.3 0.2 0.1


d1 (±2 Å)

ρ1 (±0.2 × 10‑6 Å‑2)

d2 (±2 Å)

ρ2 (±0.1 × 10‑6 Å‑2)

0.0 0.025 0.05 0.15 0.2 0.3 0.5

29 33 42 41 32 28 28

1.3 1.2 0.8 0.9 1.2 1.2 1.2

40 39 46 73 83

0.2 0.3 0.2 0.1 0.1

marked contrast to what is observed for β-lactoglobulin/ surfactant mixtures,5 hydrophobin/surfactant mixtures,21 and βlactoglobulin/β-casein mixtures,12 where competitive adsorption occurs. It is also different from the results for β-casein/ hydrophobin mixtures presented here and reported elsewhere,14 where coadsorption occurs. Wang et al.42 showed that the disproportionation of bubbles stabilized by hydrophobin/milk protein mixtures (sodium caseinate, β-lactoglobulin) are improved by the presence of hydrophobin. They assumed that sodium caseinate can compete more effectively than the less surface active β-lactoglobulin with hydrophobin. As such, the β-lactoglobulin that only initially adsorbs is progressively displaced by the hydrophobin at the interface as the bubbles shrink. These observations are consistent with the more direct observations reported here. d. Effects of PBS Buffer. Predominantly in this study HCl and NaOH have been used to adjust the solution pH, and in particular pH has been shown to have an impact upon the adsorption and the structure of the adsorbed layer of β-casein. However, it is also well-established that the use of buffer, through its finite electrolyte concentration, can have a profound effect on protein self-assembly.40,43 NR measurements of βcasein and β-casein/hydrophobin adsorption at the air−water interface have been made in the presence of PBS buffer, in order to establish the impact of buffer on the adsorption. The measurements were made at pH 7.4 with PBS buffer at a salt concentration ∼0.14 M. The NR data for β-casein in the concentration range 0.01−0.4 wt % in the presence of PBS buffer are best described as a single layer with a mean thickness of ∼42 Å and scattering length density of ∼0.8 × 10−6 Å−2. There is little systematic variation in the adsorption with βcasein concentration, as shown in the summary of the key model parameters in Table S5 in the Supporting Information. Although the surface structure is broadly similar to that

thickness of the second layer are similar to those encountered for β-casein alone. The much thicker layers at higher β-casein concentrations may be associated with the onset of a more complex surface structure involving hydrophobin, such as was observed in some hydrophobin/surfactant mixtures.49 However, the layers are too dilute to warrant a more detailed analysis. The evolution in the structure is broadly similar at pH 4 and 7. The results are consistent with an adsorbed layer of hydrophobin onto which β-casein in a disordered structural arrangement is adsorbed. Some penetration of the β-casein into the initial hydrophobin layer could be expected; but it is difficult to determine this from the data. The surface structure is consistent with that proposed by Radulova et al.14 on the basis of surface rheological measurements. An approximate schematic representation of the mixed surface structure is illustrated in Figure 3. c. Adsorption of β-Lactoglobulin/Hydrophobin. The adsorption of β-lactoglobulin/hydrophobin mixtures was measured by NR at the air−water (nrw) interface for hydrophobin concentrations of 0.005 and 0.02 wt % (0.05 and 0.2 mg/mL), β-lactoglobulin concentrations in the range 0.0 to 0.5 wt % and at pH 2.6 and 7. The key model parameters from the analysis of the NR data are summarized in Table S4 in the Supporting Information. The data were all consistent with a single layer of uniform density at the interface. The mean thickness and scattering length density of the adsorbed layer was independent of pH and β-lactoglobulin concentrations; and the mean values of d and ρ were 28 Å and 1.3 × 10−6 Å−2. These values are systematically and significantly larger than those obtained for β-lactoglobulin adsorption (see Table 2), but are similar to those obtained for hydrophobin. Hence, it is assumed that over the range of pH and concentrations studied the hydrophobin dominates the mixed adsorption, and that there is little or no β-lactoglobulin at the interface. This is in E


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The effect of the addition of PBS buffer on β-casein/ hydrophobin mixtures was measured at a fixed hydrophobin concentration of 0.005 wt % (0.05 mg/mL) and β-casein concentrations from 0.01 to 0.4 wt %. The NR data, over the entire β-casein concentration range measured, are consistent with the adsorption of a monolayer of uniform composition: as summarized in Table S6 in the Supporting Information. This is similar to what was observed for β-casein/hydrophobin mixtures at pH 2.6. The values of d and ρ in Table S6 in the Supporting Information suggest that for β-casein concentrations of 0.2 wt % and lower the surface is dominated by the hydrophobin adsorption. However, the mean thickness of the layer, 26 Å, is thinner than that for hydrophobin in the absence of buffer. Penfold et al.44 discussed the thickness of adsorbed hydrophobin layers at the air−water and liquid−solid interfaces in comparison to the molecular dimensions of hydrophobin. At the hydrophilic and hydrophobic solid surfaces, the monolayer and bilayer dimensions of 20 and 41 Å obtained from NR studies closely match the molecular dimensions for the orientation expected at those interfaces. At the air−water interface, the thicker monolayer, ∼30 Å, was associated with some orientational and vertical disorder at the interface. The presence of PBS buffer seems to have the effect of reducing that disorder, and the thickness of the layer more closely match that expected from the molecular dimensions, in a way that just changing pH does not. This implies that the disorder arises from electrostatic repulsion between hydrophobin molecules, and that the electrolyte of the PBS buffer partially screens those interprotein interactions. At the β-casein concentration of 0.4 wt %, the scattering length density of the adsorbed layer has decreased significantly. This could be interpreted that the βcasein has replaced the hydrophobin at the interface, but the thickness of the adsorbed layer is small compared to that encountered for β-casein in PBS buffer or at lower pH. Hence it is more likely that this change is associated with a partial decrease in the adsorption of the hydrophobin due to β-casein/ hydrophobin complex formation in solution, as was encountered in hydrophobin/nonionic surfactant mixtures.21 e. Impact of CaCl2. The role of calcium on the structure and functionality of milk related proteins is well established,45 and the impact of calcium on milk protein adsorption,28 and especially on competitive adsorption in mixed proteins,46,47 has been studied. Here the impact of the addition of CaCl2 on the adsorption of hydrophobin, β-casein, and hydrophobin/βcasein mixtures at the air−water interface has been studied. The effect of CaCl2 on the hydrophobin adsorption was measured at two CaCl2 concentrations, 0.5 and 1.0 mM, for hydrophobin concentrations of 0.005 and 0.02 wt % (0.05 and 0.2 mg/mL), and at pH 2.6, 4.0, and 7.0. Within error, the addition of CaCl2 has no impact on the adsorption or the structure of the adsorbed layer of hydrophobin, at either hydrophobin concentration or at any pH. This is consistent with the previously reported weak variation in the adsorption with pH and added electrolyte,21 and the dominance of the hydrophobic interaction between the hydrophobin molecules at the interface. The adsorption of β-casein in the presence of CaCl2 was studied at two CaCl2 concentrations (0.5 and 1.0 mM) and two β-casein concentrations (0.025 and 0.4 wt %) at pH 2.6, 4, and 7. The results at pH 4 and 7 are broadly similar, and the addition of 0.5 mM CaCl2 has little impact upon the adsorption. At the higher CaCl2 concentration, 1 mM, there is a slight change in the adsorption which is independent of the

Figure 2. NR data at the air−water (nrw) interface for (a) 0.005 wt % (0.05 mg/mL) hydrophobin/0.05 wt % β-casein at pH 7, and (b) 0.005 wt % (0.05 mg/mL) hydrophobin/0.3 wt % β-casein at pH 4. The solid lines are model fits as described in the text and for the parameters summarized in Table 3.

Figure 3. Schematic representation of the mixed hydrophobin/βcasein surface layer at the air−water interface.

obtained at pH 2.6, the adsorbed layer is systematically thicker, ∼45 Å compared to a mean thickness ∼36 Å at pH 2.6 in the absence of buffer. Hence, in the presence of PBS buffer, the βcasein adopts a different conformation at the surface compared to the structure at pH 2.6. The addition of electrolyte (0.14 M in PBS buffer) has the effect of reducing or screening the electrostatic interactions within the protein, and between proteins, resulting in a different layer structure. The more disordered β-casein has a greater ability to adopt different conformations at the interface compared to the globular proteins such as Lysozyme and β-lactoglobulin, which only partially disorder.43 F


Article

Langmuir

Table 4. Key Model Parameters from the Analysis of NR Data in the Presence of CaCl2 for (a) β-Casein and (b) β-Casein/ Hydrophobin Mixtures


(a) β-casein concn (wt %)

pH

CaCl2 concn (mM)

0.4 0.4 0.025 0.4 0.025 0.4

7 7 2.6 2.6 2.6 2.6

0.0 1.0 0.5 0.5 1.0 1.0


hydrophobin concn, wt % (mg/mL)

pH

CaCl2 concn (mM)

0.2 0.4 0.4

0.02 (0.2) 0.02 (0.2) 0.02 (0.2)

4 7 7

1.0 0.5 1.0

d1 (±2 Å)

ρ1 (±0.2 × 10‑6 Å‑2)

d2 (±2 Å)

ρ2 (±0.2 × 10‑6 Å‑2)

0.9 0.8 0.8 0.8 0.7 0.7

42 51

0.3 0.3

55

0.2

65

0.3

45 46 42 46 36 46 (b)

d1 (±2 Å) ρ1 (±0.2 × 10‑6 Å‑2) 32 21 22

0.9 1.3 1.3

β-casein concentration, resulting in a slightly thicker second layer in the two layer structure. The key model parameters for the resulting one and two layer structures are summarized in Table 4a. At pH 2.6, the most notable impact of the addition of CaCl2 is at the higher β-casein concentration, where there is an increase in the thickness of the second layer, compared to that observed in the absence of CaCl2. At the lower β-casein concentration, the changes are less significant. Clearly, at sufficiently high β-casein and CaCl2 concentrations, the addition of CaCl2 does have a slight impact on the structure of the adsorbed layer of β-casein. This occurs at mM concentrations of CaCl2, compared to the effects of electrolyte present in the PBS buffer at ∼100 times that concentration. Hence, the specific binding of Ca2+ ions has only a slight effect upon the conformation of the outer disordered and a slightly more dilute component, resulting in a more extended conformation. Atkinson et al.25 reported a decrease in the adsorption with the addition of Ca2+ at the air−water interface, and a decrease in the outer layer thickness in a two layer model. Follows et al.28 reported a densification of the inner layer and a more diffuse outer layer in the adsorption of β-casein at the hydrophilic silica surface, in the presence of a calcium containing buffer at relatively high concentrations compared to the CaCl2 concentrations used in this study. Velev et al.48 reported a decrease in film thickness for β-casein adsorption in foam and emulsion studies, but at relatively high calcium concentrations compared to those used here. The measurements they reported were also made in the presence of 150 mM NaCl. At the relatively low calcium concentrations used in this study, Velev et al. reported changes in the surface layer that were relatively small and insignificant. The results of Atkinson et al.25 are different in comparison to the data presented here. However, as their data were measured over a limited Q range there is much greater uncertainty in the interpretation of that data. The results of Follows et al.28 are qualitatively similar to the data presented here. However, a more detailed comparison between the data presented here and the results of Follows et al.28 and Velev et al.48 is hampered by the role of the different interfaces encountered and different solution conditions. For the β-casein/hydrophobin mixtures, NR measurements were made for a hydrophobin concentration of 0.02 wt % (0.2 mg/mL) and three β-casein concentrations, 0.025, 0.2, and 0.4

d2 (±2 Å) ρ2 (±0.2 × 10‑6 Å‑2) 87 20 20

0.1 0.2 0.2

d3 (±2 Å) ρ3 (±0.2 × 10‑6 Å‑2) 87 100

0.1 0.1

wt %, at pH 7, 4 and 2.6, and for 0.5 and 1.0 mM added CaCl2. The key results, where significant changes due to the addition of CaCl2 were observed, are summarized in Table 4b. At pH 2.6, the NR data for 0.025, 0.2, and 0.4 wt % β-casein with 0.02 wt % (0.2 mg/mL) hydrophobin does not significantly alter with the addition of either 0.5 or 1.0 mM CaCl2, and the data are consistent with a single later similar to that observed in the absence of CaCl2. At pH 4 and 7, the effects of the addition of CaCl2 are similar. At the lower β-casein concentration, there is little effect. At the higher β-casein concentration (see Table 4b for 0.2 and 0.4 wt % β-casein with 0.02 wt % (0.2 mg/mL hydrophobin)), a more extended and diffuse structure is obtained, and at the highest β-casein concentration three layers are required to describe the surface structure. As was reported in the data for hydrophobin/β-casein in the absence of Ca2+, this more extended diffuse structure may be associated with the onset of a more complex surface structure as was observed in ref 49. As the effect of CaCl2 on hydrophobin adsorption is small, it is assumed that the effects obtained for the β-casein/ hydrophobin mixtures are due to the impact of CaCl2 on the βcasein and the β-casein/hydrophobin interaction at the interface.

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SUMMARY The compact and robust protein hydrophobin is highly surface active, as previously reported.16,21 It forms a dense well-defined monolayer at the air−water interface which is largely independent of solution pH, as described by Zhang et al.21 The globular protein, β-lactoglobulin, adsorbed at the air− water interface to form a monolayer with dimensions similar to its solution and crystal structure dimensions. The thickness of the adsorbed layer and the pH dependence are such that partial disordering or denaturation at the interface occurs and is more pronounced at low pH; and the results are broadly consistent with solution studies41 and previous adsorption studies.5,25 The more disordered protein, β-casein, adsorbs at the air−water interface with a less compact structure, which can be described as a dense initial layer and an outer layer that is more dilute and diffuse at pH 4 and 7. At pH 2.6, the adsorption is in the form of a single more compact surface layer. The results are broadly consistent with related previous studies but which were all made in slightly different circumstances,25,27,39 and this in part accounts for the differences encountered. The pH dependence, especially at low pH, arises from the loss of distinct separation G


Article


Langmuir Author Contributions

between the hydrophobic and hydrophilic regions of the protein, and the differences in charge distribution within the protein, as described in complementary solution studies.40 In the protein/hydrophobin mixed adsorption the pattern of adsorption depends on the protein structures and solution pH. For hydrophobin/β-lactoglobulin mixtures the surface is dominated by the hydrophobin adsorption and the βlactoglobulin does not compete for the surface. This is consistent with the observations of Wang et al.,42 but different to that observed in some other protein mixtures12 and protein/ surfactant mixtures5,14,21 where competitive adsorption is observed. For the β-casein/hydrophobin mixture the surface properties depend upon the relative concentrations of the two proteins and the solution pH. At pH 2.6 the adsorption is dominated by the hydrophobin adsorption; with little or no βcasein at the interface, as was encountered with the hydrophobin/β-lactoglobulin mixture. At higher pH β-casein adsorbed onto and partially into the hydrophobin layer at the interface, to form a partially mixed layer at the interface, as observed in other hydrophobin/protein studies.12,42 The structure at the surface is similar to that inferred indirectly by Radulova et al.14 from surface rheology measurements. The effects of the addition of PBS buffer and CaCl2 on the adsorption and structure of the adsorbed layers of hydrophobin, β-casein, β-lactoglobulin and β-casein/hydrophobin mixtures highlight the relative role of intra and interprotein interactions at the surface. PBS buffer and CaCl2 have little effect on the hydrophobin adsorption, similar to that reported for the variations with pH here and previously.21 This reinforces the assumption that the hydrophobic interactions dominate the surface behavior of the hydrophobin. The addition of PBS buffer to β-casein results in a more compact structure at the interface, but different to that observed at low pH. The effect of screening the electrostatic interactions results in a different conformation at the interface, and one more closely resembling the more compact globular proteins.43 In the β-casein/ hydrophobin mixtures the addition of PBS buffer resulted in a surface dominated by the hydrophobin adsorption. This is similar to that observed at low pH, except that a more ordered thinner layer results. The addition of CaCl2 results in a different impact upon the charge distribution within the adsorbed βcasein. At all pH this results in a more extended and disordered structure at the interface; consistent with other related observations.28 Similar observations were made for the hydrophobin/β-casein mixed adsorption.

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All the authors have given their approval for the submitted manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the provision of neutron beam time on the INTER (ISIS) and FIGARO (ILL) reflectometers, and the scientific and technical support of the Instrument Scientists and technical staff.

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(1) Murray, B. S. Rheological properties of protein films. Curr. Opin. Colloid Interface Sci. 2011, 16, 27−35. (2) Maldonado-Valderrama, J.; Rodriquez Patino, J. M. Interfacial rheology of protein-surfactant mixtures. Curr. Opin. Colloid Interface Sci. 2010, 15, 271−282. (3) Wierenga, P. D.; Gruppen, H. New views on foams from protein solutions. Curr. Opin. Colloid Interface Sci. 2010, 15, 365−373. (4) Interaction of surfactants with polymers and proteins; Goddard, E. D., Anathapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993. (5) Horne, D. S.; Atkinson, P. J.; Dickinson, E.; Pinfield, V. J.; Richardson, R. M. Neutron reflectivity of competitive adsorption of βlactoglobulin and non-ionic surfactant at the air-water interface. Int. Dairy J. 1998, 8, 73−77. (6) Kragel, J.; Wüstneck, R.; Husband, F.; Wilde, P. J.; Makievski, A. V.; Grigoriev, D. O.; Li, J. B. Properties of mixed protein/surfactant adsorption layers. Colloids Surf., B 1999, 12, 399−407. (7) Green, R. J.; Su, T. J.; Joy, H.; Lu, J. R. Interaction of lysozyme and sodium dodecyl sulphate at the air-liquid interface. Langmuir 2000, 16, 5797−5805. (8) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Wilde, P. J.; Rodriquez Patino, J. In-situ measurements of the displacement of protein films from the air/water interface by surfactants. Biomacromolecules 2001, 2, 1001−1006. (9) Kotsmar, Cs.; Grigoriev, D. O.; Xu, F.; Aksenenko, E. V.; Fainerman, V. B.; Leser, M. E.; Miller, R. Equilibrium of adsorption of mixed milk protein/surfactant solutions at the air/water interface. Langmuir 2008, 24, 13977−13984. (10) Woodward, N. C.; Gunning, A. P.; Mackie, A. R.; Wilde, P. J.; Morris, V. J. Comparison of the orogenic displacement of Sodium Caseinate with the Caseins from the air-water interface by non-ionic surfactants. Langmuir 2009, 25, 6739−6744. (11) Kotsmar, Cs.; Kragel, J.; Kovalchuk, V. I.; Aksenenko, E. V.; Fainerman, V. B.; Miller, R. Dilation and shear rheology of mixed βcasein/surfactant adsorption layers. J. Phys. Chem. B 2009, 113, 103− 113. (12) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Wilde, P. J.; Morris, V. J. Orogenic displacement in mixed β-lactoglobulin/β-casein films at the air-water interface. Langmuir 2001, 17, 6593−6598. (13) Marinova, K. G.; Stanimirova, R. D.; Georgiev, M. T.; Alexandrov, N. A.; Basheva, E. S.; Kralchevsky, P. A. Co-adsorption of proteins β-casein and BSA in relation to the stability of thin liquid films and foams. In Colloid and Interface Chemistry for Nanotechnology; Kralchevsky, P., Miller, R., Ravera, F., Eds.; CRC Press, Boca Raton, FL, 2013; Chapter 18. (14) Radulova, G. M.; Golemanov, K.; Danov, K. D.; Kralchevsky, P. A.; Stoyanov, S. D.; Arnaudov, L. N.; Blijdenstein, B. J.; Pelan, E. G.; Lips, A. Surface shear rheology of adsorption layers from the protein HFBII hydrophobin: effect of added β-casein. Langmuir 2012, 28, 4168−4177. (15) Burke, J.; Cox, A.; Petkov, J.; Murray, B. S. Interfacial rheology and stability of air bubbles stabilised by mixtures of hydrophobin and β-casein. Food Hydrocolloids 2014, 34, 119−127.

ASSOCIATED CONTENT

S Supporting Information *

The material is available free of charge via the Internet at http://pubs.acs.org/. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02403. Tables of key model parameters from the analysis of some of the NR data and of the Σb values for the different components (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: jeff[email protected]. Present Address

⊥ J.T.P.: KLK Oleo, SDN BHD, Menara KLK, Mutiara Damansara, 47810 Petaling, Jaya Selanger, Malaysia.

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