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A Comparison of the Functional and Interfacial Properties of β-Casein and Dephosphorylated β-Casein
JOURNAL OF COLLOID AND INTERFACE SCIENCE ARTICLE NO.
195, 77–85 (1997)
CS975137
A Comparison of the Functional and Interfacial Properties of b-Casein and Dephosphorylated b-Casein Fiona A. Husband, Peter J. Wilde, 1 Alan R. Mackie, and Martin J. Garrood Food Biophysics Department, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney Lane, Norwich NR4 7UA, United Kingdom Received April 9, 1997; accepted August 19, 1997
phorylated, according to the specifications of the supplier (Sigma Chemical Co.). This results in the net negative charge of the N-terminal 50 amino acids, at pH 7.0, decreasing from approximately 011 to 03. This decrease in net negative charge is not accompanied by a decrease in solubility (5). However a decrease in emulsion and foam stability has been reported as a result of dephosphorylating total casein (5) but the underlying mechanisms remain unclear. In this paper we report results of an investigation of the foaming and emulsification properties of b-casein and 80% dephosphorylated b-casein. The properties of the adsorbed interfacial layer are important in determining functional behavior. Previous studies on emulsion stability of proteins (8) showed that b-casein formed emulsions which were more stable against coalescence than b-lactoglobulin. This was unexpected, as it has been thought that the principle mechanism by which proteins stabilize emulsions is physically preventing coalescence through the formation of a highly viscoelastic adsorbed layer (8, 17). The interfacial elasticity of b-casein is much less than that of b-lactoglobulin; therefore the emulsion stability results contradicted the interfacial behavior. Therefore the emphasis of this paper is to attempt to investigate some mechanisms underlying the formation and stability of b-casein stabilized emulsions and foams by interfacial techniques. The findings are discussed in view of the current loop-and-train model of Leaver and Dalgleish (28).
The functional and interfacial properties of b-casein and dephosphorylated b-casein (DeP b-casein) were studied at pH 7.0 in 10 mM phosphate buffer. A decrease in emulsion stability and an increase in foamability was observed. Results from a variety of interfacial techniques including electrophoretic mobility, thin film thickness, surface and interfacial tension, surface rheology, adsorbed layer thickness, and adsorption isotherms of dephosphorylated b-casein and b-casein are reported. The results demonstrate that the phosphorylated groups of the N-terminal region of bcasein are important for stabilizing emulsions. This is either as a direct result of charge repulsion between b-casein N-terminal regions or more probably as an indirect result of the reduced Nterminal charge permitting DeP b-casein to adopt a different interfacial conformation resulting in a loss or reduction of a steric barrier. q 1997 Academic Press Key Words: casein; emulsion; foam; interface.
INTRODUCTION
Casein, the principal group of proteins in milk, is widely used for its functional properties (i.e., foaming and emulsifying) within the food industry. b-Casein, one of the major casein proteins (1), is the most surface active (2) and is known to adsorb strongly to a variety of hydrophobic interfaces (3). Although widely used in the food industry, mainly for its ability to form and stabilize emulsions, the underlying mechanisms relating stability and interfacial properties of casein are not fully understood. The structure of b-casein is primarily aperiodic with charged residues, specifically phosphoserine, clustered in the N-terminal region of the molecule. The remainder of the molecule is mainly hydrophobic with very few charged residues ( 4). This confers an amphiphilic nature to the b-casein molecule which is very rare among proteins. The dephosphorylation of b-casein, in this case, produced a variety of molecules which are, on average, 80% dephos-
MATERIALS AND METHODS
b -Casein, minimum purity of 90% (C-6905, lot 12H9550), 80% dephosphorylated (DeP) b-casein (C-8157, lot 81H9620), and latex beads, average diameter 0.1 mM (LB-1), were purchased from Sigma Chemical Company. A sample of b-casein, minimum purity 95%, produced according to the method of Leaver and Law (36), was also kindly provided by the Hannah Research Institute, Scotland. Protein concentration was measured spectrophotometrically using an absorbance coefficient of 0.44 mg 01 .ml.cm01 . Surface chemically pure water (surface tension ú72.8 mN/m)
1 To whom correspondence should be addressed. Fax: ( /44) 1603507723. E-mail: [email protected].
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0021-9797/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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at 207C) obtained by steam distillation of deionized water from potassium permanganate was used throughout this study. All other chemicals were of ‘‘AnalaR’’ grade from BDH chemical company and were used without further purification. All samples were prepared using 10 mM phosphate buffer, pH 7.0, unless otherwise stated.
in surface area was approximately 5% of the total area within the trough. This was found to be within the elastic region of the response, thus ensuring that the protein interface was not ‘‘overstretched.’’ The samples (concentration 0.004 mg/ ml) were prepared immediately before use, and measurements were taken at 1-min intervals.
Emulsion Preparation and Characterization
Surface and Interfacial Tension
Oil -in-water emulsions were made using a low-volume single-pass valve homogenizer (Emulsiflex-20,000-B-3, Avestin, Ottawa) operating at 32 MPa at room temperature (7). The oil phase (10 wt.% of the total emulsion) was ntetradecane; the aqueous phase consisted of 1.5 mg/ml bcasein dissolved in 10 mM phosphate buffer, pH 7.0. All experiments were performed in triplicate at room temperature. The volume mean diameter (D4,3 ) of freshly prepared emulsions was determined by light scattering using a Coulter LS-230 particle sizer (Coulter Corporation, Miami, FL).
The surface and interfacial tensions were measured using the pendant drop technique. A pendant drop of aqueous phase was held in air by the tip of a modified syringe for the measurement of surface tension. Interfacial tension was measured by a sessile drop of n-tetradecane held in the aqueous phase. The image was digitized with a Pulnix TM500 monochrome camera, using a MuTech MV200 Frame grabber and PC. The shape of the drop image was analyzed by the selected plane method (13). Data presented are averages of at least three measurements. A sample concentration of 0.015 mg/ml was used in all cases, and the surface or interfacial tension was measured every 4 s.
Orthokinetic Stability The orthokinetic stability of the emulsions was studied first under continuous shear by stirring with a 5-mm diameter paddle in a 12-ml glass vial as described previously (8), and second under continuous turbulent flow and measured within the stirred (640 rpm) small volume module sample chamber of the LS-230. The control for these experiments was unstirred samples.
Adsorbed Layer Thickness
The foamability and foam stability were studied by a micro-conductimetric method as described previously (9, 10). A 2-ml sample of solution was sparged using prehumidifed white-spot nitrogen. The drainage of liquid and film rupture were followed conductometrically. Foamability was defined as the conductivity at time zero, immediately after the cessation of sparging. The foam stability was calculated by expressing the foam conductivity after 5 min drainage as a percentage of the initial foam conductivity.
The adsorbed layer thickness of b-casein and DeP bcasein was determined by measuring the hydrodynamic radius of latex beads coated with protein and compared with the measured radius of uncoated beads. The hydrodynamic radius was measured by photon correlation spectroscopy (PCS) using a 35-mW HeNe Laser. The scattering was measured at 907, and analyzed by a Malvern 64-channel linear correlator MDP7026. In the data reduction, the method of cumulants was followed for the departures from single exponential behavior (33), the key elements of which have been given by Pusey et al. (34). This method yields a z-averaged hydrodynamic radius and some indication of the size distibution and skewness. The incubation mixture consisted of 0.01% polystyrene latex and 4 mg/m 2 protein, in a 100 mM phosphate buffer, pH 7.0. The protein was allowed to adsorb for 3–4 h before measurement.
Film Thickness
Electrophoretic Mobility
The thickness of aqueous thin films, suspended in either air or oil, was measured using an interferometric technique described previously (11). Each sample, concentration 1.5 mg/ml, was allowed to equilibrate for at least 15 min, and the data presented are averages of at least three measurements.
The electrophoretic mobility and Zeta potential ( z ) of bcasein stabilized emulsion droplets were determined using a Malvern Instruments Zetasizer 3 (Malvern Instruments, Malvern, UK). The emulsions were diluted with 10 mM phosphate buffer at 257C. Data presented are averages of at least 12 measurements.
Foaming Properties
Surface Dilational Rheology Surface dilational studies were performed according to the ‘‘ring trough’’ method of Kokelaar (12), measuring the elastic modulus and surface tension of adsorbing protein as a function of time. The presence of contamination and the effect of b-casein aggregation were examined. The change
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RESULTS
Emulsion Studies Initially, the orthokinetic stability of emulsions stabilized by each protein was determined. The b-casein emulsion
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However, an increase in the D4,3 value of DeP b-casein was observed after 60 min, whereas no increase in the D4,3 value of b-casein was observed over a similar time. At further extended times (i.e., 90 min) the presence of a separate oil layer was observed, hence the increase in D4,3 was due to oil droplet coalescence and not flocculation. The emulsifying capacity of b-casein and DeP b-casein can be seen in Fig. 1 at time Å 0 min. The results show that b-casein has a D4,3 of 1.3 mm slightly smaller than that of 1.8 mm for DeP b-casein. Both emulsions displayed a monomodal distribution. Electrophoretic Mobility
FIG. 1. The D4,3 values of a 10 wt.% n-tetradecane oil-in-water emulsion stabilized by 1.5 mg ml 01 b-casein ( j ) and DeP b-casein ( h ) as a function of time under continuous turbulent flow and measurement.
showed no increase in D4,3 (i.e., little or no coalescence) after 4 h under continuous shear. However, the DeP b-casein emulsion D4,3 value increased so quickly (within 9 min) that determination of its orthokinetic stability proved difficult due to rapid formation of a separate oil layer on the surface of the emulsion. Therefore in order to follow the kinetics of coalescence of the DeP sample, a more gentle turbulence was required. The most applicable method was that of continuous circulation and measurement within the sizing apparatus (Coulter LS230) of a more dilute sample. This slowed down the dynamics of the coalescence of the DeP sample sufficiently to enable determination of D4,3 with stirring time. Figure 1 shows the D4,3 values of the two different samples under continuous, turbulent flow and measurement. There is a clear difference between the stability of b-casein and DeP b-casein. The b-casein emulsion changed very little during the time of the experiment, whereas the D4,3 values of the DeP b-casein emulsion increased significantly even after only 2 min. Values obtained after 3 min for the DeP bcasein are not shown, as the decrease in the scattering density due to coalescence meant that analysis of the droplet size distribution was no longer reliable. The particle size distribution of the native b-casein was monomodal throughout this experiment. However the size distribution of the DeP bcasein was initially monomodal, but rapidly developed a polydisperse distribution, exhibiting an additional population of larger droplets. The static control experiments, where the samples were not subjected to turbulent flow, showed no change from their initial D4,3 values during the time course of the experiment.
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Emulsion droplets stabilized by b-casein had a zeta potential of 032.47 { 0.42 mV compared to 025.0 { 0.42 mV for the DeP b-casein sample. This clearly shows that emulsion droplets stabilized by DeP b-casein possessed a net charge which was less negative than the native sample, consistent with the removal of phosphate groups during the dephosphorylation process. Foaming Properties Figure 2 shows the foamability (initial conductivity) of both samples at increasing concentrations. The experimental errors are { 1% for these samples, showing that there is a clear difference between b-casein and DeP b-casein. The DeP b-casein had a higher initial conductivity at all concentrations. The difference between the two curves was greater at lower concentrations, with DeP b-casein having a value
FIG. 2. The initial conductivity values (foamability) of foams stabilized by b-casein ( j ) and DeP b-casein ( h ) as a function of increasing protein concentration. Buffer: 10 mM phosphate, pH 7.0.
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TABLE 1 Thin Film Thickness of b-Casein and DeP b-Casein Suspended in Air or Oil Sample
Air–water thickness (nm)
Oil–water thickness (nm)
b-Casein light gray DeP b-casein light gray DeP b-casein dark gray
42 { 2 41 { 2 29 { 2
38 { 3 N.D. 28 { 2
Note. N.D. Å not determined.
Thin Film Observations and Thickness
FIG. 3. The foam stability of b-casein ( j ) and DeP b-casein ( h ) as a function of increasing protein concentration. Buffer: 10 mM phosphate, pH 7.0.
approximately 30% greater than b-casein; this decreased to approximately 11% at higher concentrations. Figure 3 shows the foam stability of both samples at increasing concentrations. There is no significant difference between b-casein and DeP b-casein at any concentration. An increase in foam stability, with increasing concentration, to a plateau value would be expected using this method.
The drainage patterns of model thin films, suspended in air or oil, were similar for both samples. Both b-casein and DeP b-casein had symmetrical (or protein-like) drainage. The drainage rate to an equilibrated light gray (thick) film was similar in both cases. The DeP b-casein then continued to slowly drain further, in some areas to a dark gray (thinner) film. These thinner areas were static and co-existed with the thicker areas, as shown in Fig. 4b. Both samples had similar thin film stability. No thinner areas were observed for bcasein (Fig. 4a). Table 1 shows the effect of dephosphorylation on equilibrated thin film thickness at both the air–water and oil– water interfaces. Both samples had similar thicknesses for the light gray region of the films. Comparing light gray and dark gray areas of the film, there was an approximately 25% decrease in film thickness. A similar decrease was observed in the oil–water films.
FIG. 4. Thin films of: (a) b-casein showing a layer of uniform thickness and (b) DeP b-casein showing two coexisting areas of differing thickness (see Table 1). 1.5 mg.ml 01 protein concentration, 10 mM phosphate buffer, pH 7.0, 15 min equilibration time.
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FIG. 5. The elastic modulus of 0.004 mg.ml 01 b-casein ( j ) and 0.004 mg.ml 01 DeP b-casein ( h ) as a function of surface tension. Buffer: 10 mM phosphate, pH 7.0.
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FIG. 6. The surface tension (air–water) of b-casein ( m ) and DeP bcasein ( n ) and interfacial tension (n-tetradecane–water) of b-casein ( j ) and DeP b-casein ( h ) as a function of time. Protein concentration 0.015 mg.ml 01 , 10 mM phosphate buffer, pH 7.0.
Surface Dilational Rheology In a protein system, the elastic modulus should increase with time and eventually reach a plateau value at extended times. In the case of b-casein, it has been observed that the modulus achieves a maximum. Extensive studies have shown that this behavior is intrinsic to the structure of bcasein, and is not due in any part to contamination or other artifacts. The surface elastic modulus is shown in Fig. 5 as a function of surface tension. Both samples have a maximum value in the elastic modulus of similar magnitude. The elastic modulus maximum occurs at different surface tensions, 66 mN/m for b-casein and 64 mN/m for DeP b-casein. The DeP b-casein has a much broader peak and decreased to lower values of approximately 11 mN/m in comparison to 15 mN/m for b-casein. The absence of contamination was verified by a variety of methods (charcoal treatment, ion exchange, and chloroform/ methanol lipid extraction), and subsequent observation confirmed identical dilation rheology of the treated samples. bCasein, from the Hannah Research Institute, showed identical behavior. As a further check on the dilation ‘‘ring trough’’ method, the elasticity of b-casein was also examined by compressing a spread monolayer on a Langmuir trough (data not shown). The effect of b-casein aggregation was also examined by studying the b-casein surface dilational rheology at 207C ( b-casein aggregated) and at 47C ( b-casein existing as a monomer (26)). The results of the above treatments (data not shown) and conditions indicate that the unusual surface rheological behavior of b-casein was due to the structural properties of the protein and not
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to contamination, experimental aberrations, sample variance, or state of aggregation. Surface and Interfacial Tension Figure 6 shows the surface tension of b-casein and DeP b-casein as a function of time. The data are represented by a line, and the markers are for identification only. The rate
FIG. 7. The effect of applied concentration on adsorbed layer thickness of b-casein ( j ) and DeP b-casein ( h ) adsorbed to latex beads. Buffer: 100 mM phosphate buffer, pH 7.0.
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of decrease and final values of the surface tension were similar for b-casein and DeP b-casein. The interfacial tension of b-casein and DeP b-casein against n-tetradecane, as a function of time, is shown in Fig. 6. Similar surface tension values were observed for both proteins during the experiment. Adsorbed Layer Thickness The adsorbed layer thickness is shown in Fig. 7. At low applied concentrations (i.e., less than 2.0 mg/m 2 ), there is no difference between b-casein and DeP b-casein. The main difference between the two proteins is reflected in the maximal plateau level of thickness, with the DeP b-casein having a maximal thickness 35% less than that observed for bcasein. DISCUSSION
The foam and emulsion properties of b-casein and dephosphorylated b-casein are discussed in terms of their interfacial properties. Emulsion Properties Dephosphorylating b-casein has a drastic effect on emulsion orthokinetic stability (Fig. 1). The DeP b-casein stabilized emulsion droplets tend to coalesce rapidly even under low turbulence and high dilution, compared to native bcasein which is extremely stable against coalescence for several hours at higher droplet concentrations and high turbulence. This is in agreement with Cornec et al. (8) who reported a similar high orthokinetic stability of b-casein. Chen et al. (17) reported a similar decrease in orthokinetic stability of sodium caseinate in the presence of calcium. This suggests that the high net negative charge on the N-terminal region of b-casein is important in the stability mechanism. Emulsion stability may be due to a charge repulsion effect of the protruding N-terminal loop or tail region (6, 28). Leermakers et al. (18) suggested from the Scheutjens–Fleer self-consistent field theory on a b-casein look-alike molecule that the N-terminal region is more likely to be a tail rather than a loop. The effect of dephosphorylation on the adsorbed layer thickness was observed by PCS (Fig. 7). The plateau value observed for b-casein agrees well with values previously reported (24). The observed plateau value (7 nm) for dephosphorlyated b-casein is approximately 4 nm less than that of b-casein (11 nm). This suggests that the N-terminal loop or tail has a more compact structure when dephosphorylated. This agrees well with Brooksbank et al. (23), who also observed a 4-nm decrease in the thickness of the adsorbed layer. The reduction in the hydrodynamic thickness of the adsorbed DeP b-casein was predicted previously (18) by use of the Scheutjens–Fleer self-consistent field theory and
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a b-casein look-alike molecule. The more compact structure of the N-terminal region may reduce the ability of the protein to sterically stabilize against coalescence. Brooksbank (23) also shows from the adsorption isotherm that there appears to be no large conformational changes of the protein, since the surface area of the latex bead occupied by the protein was similar whether native or dephosphorylated. The decrease in adsorbed film thickness, on latex, as a result of a net charge decrease also agrees with specular neutron reflectance measurements (37) and predictions from the Scheutjens–Fleer self-consistent field theory. The nuclear reflectance measurements suggest an adsorbed layer thickness lower for DeP b-casein than for b-casein. The decrease was proposed to be mainly in the outer more diffuse region of the adsorbed layer. A decrease in adsorbed layer thickness was previously reported (25) for b-casein in the presence of calcium. Again this decrease was thought to be mainly in the outer more diffuse portion of the adsorbed layer, perhaps as a result of calcium interacting principally with the charged phosphoserine and carboxylate residues, hence causing a local net charge reduction. The film thickness data presented here clearly show that dephosphorylation not only affects the adsorbed layer thickness, but also has a direct effect upon the repulsion forces (electrostatic and/or steric) between oil droplets. b-Casein stabilized oil–water film thickness was around 40 nm, similar to previously quoted values of air–water thin films (19), which is around 10 nm thicker than other protein stabilized thin films examined (20). The majority of the DeP b-casein thin film was considerably thinner (28 nm), more similar to thicknesses obtained with other proteins. The co-existence of thicker and thinner regions suggests phase separation and is possibly due to the fact that the b-casein molecules in this sample are on average 80% dephosphorylated. The distribution of dephosphorylation is not clear, it is possible that there is a fraction of the dephosphorylated sample that is not dephosphorylated, hence the region with a thickness similar to native b-casein. Also, the ‘‘phase separation’’ phenomenon of the different film thicknesses may only result from film formation; that is, the driving force behind separating the different regions is their different equilibrium thicknesses. Therefore this effect is perhaps unlikely to occur on a planar interface or surface of an oil droplet. In addition, the reduction in film thickness cannot be as a result of contaminating surfactant since the drainage pattern and rate would be much more chaotic and faster, respectively, and a typical surfactant film would have a thickness of approximately 12 nm (20). Zeta potential measurements on the emulsions showed that dephosphorylating the b-casein makes the surface charge of the droplets less negative, which is expected since dephosphorylation involves the removal of up to 5 negatively charged groups from the b-casein molecule. This change in charge density is likely to effect the thickness of thin films,
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since the thickness is dominated by charge repulsion between the approaching interfaces (11). Therefore the observed decrease in film thickness upon dephosphorylation may be due to either the reduction in adsorbed layer thickness or the change in surface charge of the adsorbed layer, or both. The reduction in adsorbed layer thickness accounts for a large proportion of the observed decrease in equilibrium thin film thickness (Table 1). It is likely therefore that the remainder is accounted for by the change in charge density. However it is difficult, if not impossible, to separate the steric repulsion from the electrostatic repulsion effects, since it is not possible to remove the charge without affecting the thickness of the adsorbed layer. The emulsifying capacity of both proteins are similar. Considering that the Dep b-casein stabilized emulsion was observed to be far less stable, the slightly larger D4,3 value observed for DeP b-casein may be a result of droplet coalescence during turbulent conditions encountered shortly after the homogenization process. Unlike surfactants, proteins stabilize interfaces by forming a rigid, viscoelastic adsorbed layer which is capable of physically resisting deformation and coalescence (7, 8, 10, 11). The strength and integrity of the protein adsorbed layer is paramount to the stability of protein stabilized emulsions (8, 17, 31). Once adsorbed, proteins unfold and form protein– protein interactions which are mainly responsible for the viscoelastic properties. The surface elastic modulus is a good indicator of the strength of these interactions. For the majority of surface active proteins, an increase in the elastic modulus occurs with time until a plateau value is approached. This is not the case for b-casein and DeP b-casein. The maximum in elasticity (Fig. 5), also previously reported elsewhere (14, 15), appears as a result of two consecutive processes. The first step of the process involves the adsorption of b-casein from the bulk solution. The rate of the initial step (i.e., increase in elastic modulus) increases with increasing surface concentration. There was only a slight difference between the native and DeP b-casein during this step, with the b-casein increase being slightly steeper than that of the DeP b-casein. The magnitude of the elastic modulus of b-casein is very low (15 mNm01 after 40 min) compared to other proteins, for example, b-lactoglobulin may have an elastic modulus in excess of 140 mN.m01 under similar conditions (i.e., concentration, pH, ionic strength, and temperature). The highly stable emulsions produced by b-casein were therefore unexpected considering its poor interfacial rheological properties. The second step of the process, i.e., the decrease in elastic modulus, could be due to contamination (16), fracturing of the interface, or a rearrangement of b-casein at the interface. As stated under Results, the effects of contamination, surface fracturing or overstretching the interface, surface aggregation, and methodological artifacts were all investigated thoroughly and discounted. Therefore we conclude that the ob-
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served maximum in the surface elasticity is due to molecular rearrangement of the b-casein molecule at the interface. A change in b-casein conformation at the interface is implied from studies of the adsorbed layer thickness of bcasein (27) which suggest that b-casein can adopt different conformations at low and high surface coverages (i.e., 1 and 3 mg.m02 , respectively), since the adsorbed layer thickness increases from approx. 5 to 10 nm, as determined by quasielastic light scattering. Theoretical brush thickness calculations (35) presented show a similar thickness (5.4 nm) at 1.6 mg.m02 , and brush thickness increases with increasing concentration, which supports the idea of conformational change. Leaver and Dalgleish (28) suggested the loop-andtrain model of b-casein conformation at the interface, with the N-terminal 50 amino acids forming a loop or tail into the bulk solution while the remainder of the molecule is much more closely associated with the interface. This is supported by the kinetics of the trypsin treatment of high surface coverage b-casein stabilized emulsions (28) showing that the N-terminal is more susceptible to protease treatment than the C-terminal, and hence the N-terminal is more accessible. Also trypsinolysis causes a decrease in the adsorbed layer from 10 to 5 nm thick. The 5-nm thickness putatively represents the 50-209 peptide of b-casein. The surface rheological properties of proteins at the oil– water interface have previously been reported (29, 30). Benjamins et al. (30) reported differences between air–water and oil–water surface rheology of three different proteins (none of which were b-casein), using the dynamic drop tensiometer and purified sunflower oil. The main difference was a decrease in the elastic modulus observed at the oil– water interface only. Benjamins et al. (30) suggest this to be as a result of a ‘‘collapse-type’’ phenomonon. This collapse-type phenomenon could be due to low levels of surface active impurities in the triglyceride oil disrupting the protein adsorbed layer. Disruption of the adsorbed protein layer at the interface by oil–soluble surfactants has been reported previously (8). The effect of contaminating surfactant on surface rheological properties has been reported previously (16) at the air–water interface. The presence of a contaminating surfactant produced a decrease in the elastic modulus. In contrast Williams and Prins (29) reported similar surface rheological properties of b-casein at the air–water and oil– water interface, using the ring trough method and paraffin oil. The paraffin oil in that study is more closely related to the n-tetradecane used in this investigation than the sunflower oil used by Benjamins (30). Therefore the air–water surface rheology results presented here may be a good indicator of the expected behavior of both proteins at the ntetradecane oil–water interface. To summarize, despite the poor interfacial elasticity of bcasein, compared to all other proteins examined, it is capable of producing emulsions which are highly stable to coalescence. The evidence presented here strongly suggests that
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the phosphorylated loop or tail region of the molecule is, in the main, responsible for this high stability. Removal of the charge by dephosphorylation reduces the electrostatic repulsion between interfaces and b-casein adopts a more compact adsorbed layer, thus reducing any steric hindrance of the loop or tail region. The stability of emulsions stabilized by dephosphorylated b-casein are consequently very poor. Foam Properties The difference observed in foamability of the two samples is not a result of different created bubble sizes. As reported previously (21), any differences in surface tension between samples would not result in differences in created bubble size using the microconductivity apparatus. The bubble size is controlled by using a single orifice and constant gas flow for sparging. There is also no difference in the rates of adsorption to the interface of the gas bubble, as can be seen from the surface tension data (Fig. 6). Although the surface tension data reported here are at a static interface, it has been reported previously (22) that the surface tension at a dynamic interface, as measured by the overflowing cylinder, shows trends similar to the static interface results. Hence no differences in surface tension profiles of the two proteins are expected at a dynamic interface. The difference in foamability requires further investigation. The foam stability data are identical for both proteins despite the fact that the thin film thickness data showed thinner areas within the DeP b-casein films. However, this change in foam film thickness is unlikely to make much difference to the conductivity as most of the liquid is contained within the Plateau borders. Thin film drainage and surface rheology data show little difference; therefore the rate of drainage of liquid from the foam is unlikely to differ between the proteins. It has been reported previously ( 5 ) that dephosphorylated whole casein produced a smaller foam volume, and foam collapse was quicker. The difference in results reflects the difference in functional properties of b-casein and whole casein which consist mainly of four proteins:- as1 , as2 , b, and k in the ratio 40:10:35:12, all of which are phosphorylated and surface active. It should also be noted that in the single-orifice sparging method presented both the bubble size distribution and the foam volume are controlled, and foam drainage rather than volume is measured. In summary, the massive reduction in emulsion orthokinetic stability with a reduction in charge suggests that charge repulsion plays an important role in emulsion stability. However, the adsorbed layer thickness data suggest that there is also a conformational change upon dephosphorylation. This conformational change at the interface which produces a more compact structure will minimize the steric stabilization of the emulsion droplets by the N-terminal loop/tail region.
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Minimizing these electrostatic and steric effects means that the emulsion orthokinetic stability will rely more upon the interfacial rheology (8, 31). Therefore as the interfacial rheology of b-casein is very low compared to other proteins (32), it is therefore not surprising that the orthokinetic stability of the DeP b-casein is so poor. The foaming properties are very similar for both proteins. The difference in foamability of the proteins cannot be readily explained by a charge and/or steric effect; therefore alternative explanations (e.g., isoelectric point, local conductivity effects) require further investigation. The lack of any effect of dephosphorylation on the foam stability is suprising considering the dramatic effect on emulsion stability. Further work is necessary to identify the primary stabilization mechanisms responsible for the different behavior of foams and emulsions. CONCLUSIONS
The results presented suggest that the mechanism of emulsion orthokinetic stability by b-casein depends on the charged phosphate groups in the N-terminal domain. Removal of the phosphate groups decreased the film thickness due to a reduction of the electrostatic and/or steric repulsion. The poor elastic modulus of b-casein meant that without the charge/steric stabilizing effect of the phosphate groups in the N-terminal region, there was a drastic reduction in emulsion orthokinetic stability. Dephosphorylation of b-casein had no effect on foam stability as measured by the microconductivity technique, but an increase in foamability was measured. None of the interfacial measurements presented appear to elucidate an underlying mechanism for the foaming behavior. ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from BBSRC, and to Jeff Leaver of the Hannah Research Institute for supplying pure b-casein.
REFERENCES 1. Davies, D. T., and Law, A. J. R., J. Dairy Res. 44, 447 (1977). 2. Mitchell, J., Irons, L., and Palmer, G. J., Biochim. Biophys. Acta 200, 138 (1970). 3. Dickinson, E., J. Chem. Soc. Faraday Trans. 88, 2973 (1992). 4. Swasigood, H. E., in ‘‘Developments in Dairy Chemistry-1’’ (P. F. Fox, Ed.), p. 1. Applied Science, London, 1982. 5. Van Hekken, D. L., and Strange, E. D., J. Dairy Sci. 76(11), 3384 (1993). 6. Horne, D. S., and Leaver, J., Food Hydrocolloids 9(2), 91 (1995). 7. Mackie, A. R., Wilde, P. J., Wilson, D. R., and Clark, D. C., J. Chem. Soc. Faraday Trans. 89(15), 2755 (1993). 8. Cornec, M., Mackie, A. R., Wilde, P. J., and Clark, D. C., Colloids Surf. A: Physicochem. Eng. Aspects 114, 237 (1996). 9. Clark, D. C., Wilde, P. J., and Wilson, D. R., Colloids Surf. 59, 209 (1991).
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10. Coke, M., Wilde, P. J., Russell E. J., and Clark, D. C., J. Colloid Interface Sci. 138, 489 (1990). 11. Clark, D. C., Coke, M., Mackie, A. R., Pinder, A. C., and Wilson, D. R. J. Colloid Interface Sci. 138, 207 (1990). 12. Kokelaar, A. J. J., Prins, A., and de Gee, M., J. Colloid Interface Sci. 146, 507 (1991). 13. Ambwani, D. S., and Fort, T., Jr., in ‘‘Surface and Colloid Science, Vol. II’’ (R. J. Good and R. R. Stromberg, Eds.), p. 93. Plenum, New York, 1979. 14. Benjamins, J., de Feijter, J. A., Evans, M. T. A., Graham, D. E., and Phillips, M. C., Faraday Discuss. Chem. Soc. 159, 218 (1975). 15. Graham, D. E., and Philips, M. C., J. Colloid Interface Sci. 76, 227 (1980). 16. Clark, D. C., Husband, F. A., Wilde, P. J., Cornec, M., Miller, R., Kragel, J., and Wustneck, R., J. Chem. Soc. Faraday Trans. 91(13), 1991 (1995). 17. Chen, J., Dickinson, E., and Iveson, G., Food Structure 12, 135 (1993). 18. Leermakers, F. A. M., Atkinson, P. J., Dickinson, E., and Horne, D. S., J. Colloid Interface Sci. 178, 681 (1996). 19. Pittia, P., Wilde, P. J., and Clark, D. C., Food Hydrocolloids 10(3), 335 (1996). 20. Wilde, P. J., and Clark, D. C., J. Colloid Interface Sci. 155, 48 (1993). 21. Wilde, P. J., J. Colloid Interface Sci. 178, 733 (1996). 22. Bergink-Martens, D. J. M., ‘‘Interface Dilation: The Overflowing Cylinder Technique.’’ Ph.D. thesis, Agricultural University, Wageningen (1993).
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23. Brooksbank, D. V., Davidson, C. M., Horne, D. S., and Leaver, J., J. Chem. Soc. Faraday Trans. 89(18), 3419 (1993). 24. Dalgleish, D. G., Colloids Surf. 65, 9 (1990). 25. Atkinson, P. J., Dickinson, E., Horne, D. S., and Richardson, R. M., J. Chem. Soc. Faraday Trans. 91(17), 2847 (1995). 26. Sullivan, R. A., Fitzpatrick, M. M., Stanton, E. K., Annino, R., Kissel, G., and Palermiti, F., Arch. Biochem. Biophys. 55, 455 (1955). 27. Fang, Y., and Dalgleish, D. G., J. Colloid Interf. Sci. 156, 329 (1993). 28. Leaver, J., and Dalgleish, D. G., Biochim. Biophys. Acta 1041, 217 (1990). 29. Williams, A., and Prins, A., Colloids Surf. A: Physicochem. Eng. Aspects 114, 267 (1996). 30. Benjamins, J., Cagna, A., and Lucassen-Reynders, E. H., Colloids Surf. A: Physicochem. Eng. Aspects 114, 245 (1996). 31. Dickinson, E., Owuzu, R. K., and Williams, A., J. Chem. Soc. Faraday Trans. 89(5), 865 (1993). 32. Murray, B., and Dickinson, E., Food Sci. Technol. Int. 2(3), 131 (1996). 33. Koppel, D. E., J. Chem. Phys. 57, 4814 (1972). 34. Pusey, P. N., Koppel, D. E., Schaefer, D. W., Camerini-Otero, R. D., and Koenih, S. H., Biochemistry 13, 952 (1972). 35. Mackie, A. R., Mingins, J., and North, A. N., J. Chem. Soc. Faraday Trans. 87(13), 3043 (1991). 36. Leaver, J., and Law, A. J. R., J. Dairy Res. 59, 557 (1992). 37. Atkinson, P. J., Dickinson, E., Horne, D. S., Leaver, J., Leermakers, F. A. M., and Richardson, R. M., in ‘‘Food Colloids: Proteins, Lipids and Polysaccharides’’ (E. Dickinson and B. Bergenstahl, Eds.), p. 217. Royal Society of Chemistry, Cambridge, 1997.
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A Comparison of the Functional and Interfacial Properties of b-Casein and Dephosphorylated b-Casein Fiona A. Husband, Peter J. Wilde, 1 Alan R. Mackie, and Martin J. Garrood Food Biophysics Department, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney Lane, Norwich NR4 7UA, United Kingdom Received April 9, 1997; accepted August 19, 1997
phorylated, according to the specifications of the supplier (Sigma Chemical Co.). This results in the net negative charge of the N-terminal 50 amino acids, at pH 7.0, decreasing from approximately 011 to 03. This decrease in net negative charge is not accompanied by a decrease in solubility (5). However a decrease in emulsion and foam stability has been reported as a result of dephosphorylating total casein (5) but the underlying mechanisms remain unclear. In this paper we report results of an investigation of the foaming and emulsification properties of b-casein and 80% dephosphorylated b-casein. The properties of the adsorbed interfacial layer are important in determining functional behavior. Previous studies on emulsion stability of proteins (8) showed that b-casein formed emulsions which were more stable against coalescence than b-lactoglobulin. This was unexpected, as it has been thought that the principle mechanism by which proteins stabilize emulsions is physically preventing coalescence through the formation of a highly viscoelastic adsorbed layer (8, 17). The interfacial elasticity of b-casein is much less than that of b-lactoglobulin; therefore the emulsion stability results contradicted the interfacial behavior. Therefore the emphasis of this paper is to attempt to investigate some mechanisms underlying the formation and stability of b-casein stabilized emulsions and foams by interfacial techniques. The findings are discussed in view of the current loop-and-train model of Leaver and Dalgleish (28).
The functional and interfacial properties of b-casein and dephosphorylated b-casein (DeP b-casein) were studied at pH 7.0 in 10 mM phosphate buffer. A decrease in emulsion stability and an increase in foamability was observed. Results from a variety of interfacial techniques including electrophoretic mobility, thin film thickness, surface and interfacial tension, surface rheology, adsorbed layer thickness, and adsorption isotherms of dephosphorylated b-casein and b-casein are reported. The results demonstrate that the phosphorylated groups of the N-terminal region of bcasein are important for stabilizing emulsions. This is either as a direct result of charge repulsion between b-casein N-terminal regions or more probably as an indirect result of the reduced Nterminal charge permitting DeP b-casein to adopt a different interfacial conformation resulting in a loss or reduction of a steric barrier. q 1997 Academic Press Key Words: casein; emulsion; foam; interface.
INTRODUCTION
Casein, the principal group of proteins in milk, is widely used for its functional properties (i.e., foaming and emulsifying) within the food industry. b-Casein, one of the major casein proteins (1), is the most surface active (2) and is known to adsorb strongly to a variety of hydrophobic interfaces (3). Although widely used in the food industry, mainly for its ability to form and stabilize emulsions, the underlying mechanisms relating stability and interfacial properties of casein are not fully understood. The structure of b-casein is primarily aperiodic with charged residues, specifically phosphoserine, clustered in the N-terminal region of the molecule. The remainder of the molecule is mainly hydrophobic with very few charged residues ( 4). This confers an amphiphilic nature to the b-casein molecule which is very rare among proteins. The dephosphorylation of b-casein, in this case, produced a variety of molecules which are, on average, 80% dephos-
MATERIALS AND METHODS
b -Casein, minimum purity of 90% (C-6905, lot 12H9550), 80% dephosphorylated (DeP) b-casein (C-8157, lot 81H9620), and latex beads, average diameter 0.1 mM (LB-1), were purchased from Sigma Chemical Company. A sample of b-casein, minimum purity 95%, produced according to the method of Leaver and Law (36), was also kindly provided by the Hannah Research Institute, Scotland. Protein concentration was measured spectrophotometrically using an absorbance coefficient of 0.44 mg 01 .ml.cm01 . Surface chemically pure water (surface tension ú72.8 mN/m)
1 To whom correspondence should be addressed. Fax: ( /44) 1603507723. E-mail: [email protected].
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at 207C) obtained by steam distillation of deionized water from potassium permanganate was used throughout this study. All other chemicals were of ‘‘AnalaR’’ grade from BDH chemical company and were used without further purification. All samples were prepared using 10 mM phosphate buffer, pH 7.0, unless otherwise stated.
in surface area was approximately 5% of the total area within the trough. This was found to be within the elastic region of the response, thus ensuring that the protein interface was not ‘‘overstretched.’’ The samples (concentration 0.004 mg/ ml) were prepared immediately before use, and measurements were taken at 1-min intervals.
Emulsion Preparation and Characterization
Surface and Interfacial Tension
Oil -in-water emulsions were made using a low-volume single-pass valve homogenizer (Emulsiflex-20,000-B-3, Avestin, Ottawa) operating at 32 MPa at room temperature (7). The oil phase (10 wt.% of the total emulsion) was ntetradecane; the aqueous phase consisted of 1.5 mg/ml bcasein dissolved in 10 mM phosphate buffer, pH 7.0. All experiments were performed in triplicate at room temperature. The volume mean diameter (D4,3 ) of freshly prepared emulsions was determined by light scattering using a Coulter LS-230 particle sizer (Coulter Corporation, Miami, FL).
The surface and interfacial tensions were measured using the pendant drop technique. A pendant drop of aqueous phase was held in air by the tip of a modified syringe for the measurement of surface tension. Interfacial tension was measured by a sessile drop of n-tetradecane held in the aqueous phase. The image was digitized with a Pulnix TM500 monochrome camera, using a MuTech MV200 Frame grabber and PC. The shape of the drop image was analyzed by the selected plane method (13). Data presented are averages of at least three measurements. A sample concentration of 0.015 mg/ml was used in all cases, and the surface or interfacial tension was measured every 4 s.
Orthokinetic Stability The orthokinetic stability of the emulsions was studied first under continuous shear by stirring with a 5-mm diameter paddle in a 12-ml glass vial as described previously (8), and second under continuous turbulent flow and measured within the stirred (640 rpm) small volume module sample chamber of the LS-230. The control for these experiments was unstirred samples.
Adsorbed Layer Thickness
The foamability and foam stability were studied by a micro-conductimetric method as described previously (9, 10). A 2-ml sample of solution was sparged using prehumidifed white-spot nitrogen. The drainage of liquid and film rupture were followed conductometrically. Foamability was defined as the conductivity at time zero, immediately after the cessation of sparging. The foam stability was calculated by expressing the foam conductivity after 5 min drainage as a percentage of the initial foam conductivity.
The adsorbed layer thickness of b-casein and DeP bcasein was determined by measuring the hydrodynamic radius of latex beads coated with protein and compared with the measured radius of uncoated beads. The hydrodynamic radius was measured by photon correlation spectroscopy (PCS) using a 35-mW HeNe Laser. The scattering was measured at 907, and analyzed by a Malvern 64-channel linear correlator MDP7026. In the data reduction, the method of cumulants was followed for the departures from single exponential behavior (33), the key elements of which have been given by Pusey et al. (34). This method yields a z-averaged hydrodynamic radius and some indication of the size distibution and skewness. The incubation mixture consisted of 0.01% polystyrene latex and 4 mg/m 2 protein, in a 100 mM phosphate buffer, pH 7.0. The protein was allowed to adsorb for 3–4 h before measurement.
Film Thickness
Electrophoretic Mobility
The thickness of aqueous thin films, suspended in either air or oil, was measured using an interferometric technique described previously (11). Each sample, concentration 1.5 mg/ml, was allowed to equilibrate for at least 15 min, and the data presented are averages of at least three measurements.
The electrophoretic mobility and Zeta potential ( z ) of bcasein stabilized emulsion droplets were determined using a Malvern Instruments Zetasizer 3 (Malvern Instruments, Malvern, UK). The emulsions were diluted with 10 mM phosphate buffer at 257C. Data presented are averages of at least 12 measurements.
Foaming Properties
Surface Dilational Rheology Surface dilational studies were performed according to the ‘‘ring trough’’ method of Kokelaar (12), measuring the elastic modulus and surface tension of adsorbing protein as a function of time. The presence of contamination and the effect of b-casein aggregation were examined. The change
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RESULTS
Emulsion Studies Initially, the orthokinetic stability of emulsions stabilized by each protein was determined. The b-casein emulsion
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However, an increase in the D4,3 value of DeP b-casein was observed after 60 min, whereas no increase in the D4,3 value of b-casein was observed over a similar time. At further extended times (i.e., 90 min) the presence of a separate oil layer was observed, hence the increase in D4,3 was due to oil droplet coalescence and not flocculation. The emulsifying capacity of b-casein and DeP b-casein can be seen in Fig. 1 at time Å 0 min. The results show that b-casein has a D4,3 of 1.3 mm slightly smaller than that of 1.8 mm for DeP b-casein. Both emulsions displayed a monomodal distribution. Electrophoretic Mobility
FIG. 1. The D4,3 values of a 10 wt.% n-tetradecane oil-in-water emulsion stabilized by 1.5 mg ml 01 b-casein ( j ) and DeP b-casein ( h ) as a function of time under continuous turbulent flow and measurement.
showed no increase in D4,3 (i.e., little or no coalescence) after 4 h under continuous shear. However, the DeP b-casein emulsion D4,3 value increased so quickly (within 9 min) that determination of its orthokinetic stability proved difficult due to rapid formation of a separate oil layer on the surface of the emulsion. Therefore in order to follow the kinetics of coalescence of the DeP sample, a more gentle turbulence was required. The most applicable method was that of continuous circulation and measurement within the sizing apparatus (Coulter LS230) of a more dilute sample. This slowed down the dynamics of the coalescence of the DeP sample sufficiently to enable determination of D4,3 with stirring time. Figure 1 shows the D4,3 values of the two different samples under continuous, turbulent flow and measurement. There is a clear difference between the stability of b-casein and DeP b-casein. The b-casein emulsion changed very little during the time of the experiment, whereas the D4,3 values of the DeP b-casein emulsion increased significantly even after only 2 min. Values obtained after 3 min for the DeP bcasein are not shown, as the decrease in the scattering density due to coalescence meant that analysis of the droplet size distribution was no longer reliable. The particle size distribution of the native b-casein was monomodal throughout this experiment. However the size distribution of the DeP bcasein was initially monomodal, but rapidly developed a polydisperse distribution, exhibiting an additional population of larger droplets. The static control experiments, where the samples were not subjected to turbulent flow, showed no change from their initial D4,3 values during the time course of the experiment.
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Emulsion droplets stabilized by b-casein had a zeta potential of 032.47 { 0.42 mV compared to 025.0 { 0.42 mV for the DeP b-casein sample. This clearly shows that emulsion droplets stabilized by DeP b-casein possessed a net charge which was less negative than the native sample, consistent with the removal of phosphate groups during the dephosphorylation process. Foaming Properties Figure 2 shows the foamability (initial conductivity) of both samples at increasing concentrations. The experimental errors are { 1% for these samples, showing that there is a clear difference between b-casein and DeP b-casein. The DeP b-casein had a higher initial conductivity at all concentrations. The difference between the two curves was greater at lower concentrations, with DeP b-casein having a value
FIG. 2. The initial conductivity values (foamability) of foams stabilized by b-casein ( j ) and DeP b-casein ( h ) as a function of increasing protein concentration. Buffer: 10 mM phosphate, pH 7.0.
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TABLE 1 Thin Film Thickness of b-Casein and DeP b-Casein Suspended in Air or Oil Sample
Air–water thickness (nm)
Oil–water thickness (nm)
b-Casein light gray DeP b-casein light gray DeP b-casein dark gray
42 { 2 41 { 2 29 { 2
38 { 3 N.D. 28 { 2
Note. N.D. Å not determined.
Thin Film Observations and Thickness
FIG. 3. The foam stability of b-casein ( j ) and DeP b-casein ( h ) as a function of increasing protein concentration. Buffer: 10 mM phosphate, pH 7.0.
approximately 30% greater than b-casein; this decreased to approximately 11% at higher concentrations. Figure 3 shows the foam stability of both samples at increasing concentrations. There is no significant difference between b-casein and DeP b-casein at any concentration. An increase in foam stability, with increasing concentration, to a plateau value would be expected using this method.
The drainage patterns of model thin films, suspended in air or oil, were similar for both samples. Both b-casein and DeP b-casein had symmetrical (or protein-like) drainage. The drainage rate to an equilibrated light gray (thick) film was similar in both cases. The DeP b-casein then continued to slowly drain further, in some areas to a dark gray (thinner) film. These thinner areas were static and co-existed with the thicker areas, as shown in Fig. 4b. Both samples had similar thin film stability. No thinner areas were observed for bcasein (Fig. 4a). Table 1 shows the effect of dephosphorylation on equilibrated thin film thickness at both the air–water and oil– water interfaces. Both samples had similar thicknesses for the light gray region of the films. Comparing light gray and dark gray areas of the film, there was an approximately 25% decrease in film thickness. A similar decrease was observed in the oil–water films.
FIG. 4. Thin films of: (a) b-casein showing a layer of uniform thickness and (b) DeP b-casein showing two coexisting areas of differing thickness (see Table 1). 1.5 mg.ml 01 protein concentration, 10 mM phosphate buffer, pH 7.0, 15 min equilibration time.
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FIG. 5. The elastic modulus of 0.004 mg.ml 01 b-casein ( j ) and 0.004 mg.ml 01 DeP b-casein ( h ) as a function of surface tension. Buffer: 10 mM phosphate, pH 7.0.
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FIG. 6. The surface tension (air–water) of b-casein ( m ) and DeP bcasein ( n ) and interfacial tension (n-tetradecane–water) of b-casein ( j ) and DeP b-casein ( h ) as a function of time. Protein concentration 0.015 mg.ml 01 , 10 mM phosphate buffer, pH 7.0.
Surface Dilational Rheology In a protein system, the elastic modulus should increase with time and eventually reach a plateau value at extended times. In the case of b-casein, it has been observed that the modulus achieves a maximum. Extensive studies have shown that this behavior is intrinsic to the structure of bcasein, and is not due in any part to contamination or other artifacts. The surface elastic modulus is shown in Fig. 5 as a function of surface tension. Both samples have a maximum value in the elastic modulus of similar magnitude. The elastic modulus maximum occurs at different surface tensions, 66 mN/m for b-casein and 64 mN/m for DeP b-casein. The DeP b-casein has a much broader peak and decreased to lower values of approximately 11 mN/m in comparison to 15 mN/m for b-casein. The absence of contamination was verified by a variety of methods (charcoal treatment, ion exchange, and chloroform/ methanol lipid extraction), and subsequent observation confirmed identical dilation rheology of the treated samples. bCasein, from the Hannah Research Institute, showed identical behavior. As a further check on the dilation ‘‘ring trough’’ method, the elasticity of b-casein was also examined by compressing a spread monolayer on a Langmuir trough (data not shown). The effect of b-casein aggregation was also examined by studying the b-casein surface dilational rheology at 207C ( b-casein aggregated) and at 47C ( b-casein existing as a monomer (26)). The results of the above treatments (data not shown) and conditions indicate that the unusual surface rheological behavior of b-casein was due to the structural properties of the protein and not
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to contamination, experimental aberrations, sample variance, or state of aggregation. Surface and Interfacial Tension Figure 6 shows the surface tension of b-casein and DeP b-casein as a function of time. The data are represented by a line, and the markers are for identification only. The rate
FIG. 7. The effect of applied concentration on adsorbed layer thickness of b-casein ( j ) and DeP b-casein ( h ) adsorbed to latex beads. Buffer: 100 mM phosphate buffer, pH 7.0.
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of decrease and final values of the surface tension were similar for b-casein and DeP b-casein. The interfacial tension of b-casein and DeP b-casein against n-tetradecane, as a function of time, is shown in Fig. 6. Similar surface tension values were observed for both proteins during the experiment. Adsorbed Layer Thickness The adsorbed layer thickness is shown in Fig. 7. At low applied concentrations (i.e., less than 2.0 mg/m 2 ), there is no difference between b-casein and DeP b-casein. The main difference between the two proteins is reflected in the maximal plateau level of thickness, with the DeP b-casein having a maximal thickness 35% less than that observed for bcasein. DISCUSSION
The foam and emulsion properties of b-casein and dephosphorylated b-casein are discussed in terms of their interfacial properties. Emulsion Properties Dephosphorylating b-casein has a drastic effect on emulsion orthokinetic stability (Fig. 1). The DeP b-casein stabilized emulsion droplets tend to coalesce rapidly even under low turbulence and high dilution, compared to native bcasein which is extremely stable against coalescence for several hours at higher droplet concentrations and high turbulence. This is in agreement with Cornec et al. (8) who reported a similar high orthokinetic stability of b-casein. Chen et al. (17) reported a similar decrease in orthokinetic stability of sodium caseinate in the presence of calcium. This suggests that the high net negative charge on the N-terminal region of b-casein is important in the stability mechanism. Emulsion stability may be due to a charge repulsion effect of the protruding N-terminal loop or tail region (6, 28). Leermakers et al. (18) suggested from the Scheutjens–Fleer self-consistent field theory on a b-casein look-alike molecule that the N-terminal region is more likely to be a tail rather than a loop. The effect of dephosphorylation on the adsorbed layer thickness was observed by PCS (Fig. 7). The plateau value observed for b-casein agrees well with values previously reported (24). The observed plateau value (7 nm) for dephosphorlyated b-casein is approximately 4 nm less than that of b-casein (11 nm). This suggests that the N-terminal loop or tail has a more compact structure when dephosphorylated. This agrees well with Brooksbank et al. (23), who also observed a 4-nm decrease in the thickness of the adsorbed layer. The reduction in the hydrodynamic thickness of the adsorbed DeP b-casein was predicted previously (18) by use of the Scheutjens–Fleer self-consistent field theory and
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a b-casein look-alike molecule. The more compact structure of the N-terminal region may reduce the ability of the protein to sterically stabilize against coalescence. Brooksbank (23) also shows from the adsorption isotherm that there appears to be no large conformational changes of the protein, since the surface area of the latex bead occupied by the protein was similar whether native or dephosphorylated. The decrease in adsorbed film thickness, on latex, as a result of a net charge decrease also agrees with specular neutron reflectance measurements (37) and predictions from the Scheutjens–Fleer self-consistent field theory. The nuclear reflectance measurements suggest an adsorbed layer thickness lower for DeP b-casein than for b-casein. The decrease was proposed to be mainly in the outer more diffuse region of the adsorbed layer. A decrease in adsorbed layer thickness was previously reported (25) for b-casein in the presence of calcium. Again this decrease was thought to be mainly in the outer more diffuse portion of the adsorbed layer, perhaps as a result of calcium interacting principally with the charged phosphoserine and carboxylate residues, hence causing a local net charge reduction. The film thickness data presented here clearly show that dephosphorylation not only affects the adsorbed layer thickness, but also has a direct effect upon the repulsion forces (electrostatic and/or steric) between oil droplets. b-Casein stabilized oil–water film thickness was around 40 nm, similar to previously quoted values of air–water thin films (19), which is around 10 nm thicker than other protein stabilized thin films examined (20). The majority of the DeP b-casein thin film was considerably thinner (28 nm), more similar to thicknesses obtained with other proteins. The co-existence of thicker and thinner regions suggests phase separation and is possibly due to the fact that the b-casein molecules in this sample are on average 80% dephosphorylated. The distribution of dephosphorylation is not clear, it is possible that there is a fraction of the dephosphorylated sample that is not dephosphorylated, hence the region with a thickness similar to native b-casein. Also, the ‘‘phase separation’’ phenomenon of the different film thicknesses may only result from film formation; that is, the driving force behind separating the different regions is their different equilibrium thicknesses. Therefore this effect is perhaps unlikely to occur on a planar interface or surface of an oil droplet. In addition, the reduction in film thickness cannot be as a result of contaminating surfactant since the drainage pattern and rate would be much more chaotic and faster, respectively, and a typical surfactant film would have a thickness of approximately 12 nm (20). Zeta potential measurements on the emulsions showed that dephosphorylating the b-casein makes the surface charge of the droplets less negative, which is expected since dephosphorylation involves the removal of up to 5 negatively charged groups from the b-casein molecule. This change in charge density is likely to effect the thickness of thin films,
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since the thickness is dominated by charge repulsion between the approaching interfaces (11). Therefore the observed decrease in film thickness upon dephosphorylation may be due to either the reduction in adsorbed layer thickness or the change in surface charge of the adsorbed layer, or both. The reduction in adsorbed layer thickness accounts for a large proportion of the observed decrease in equilibrium thin film thickness (Table 1). It is likely therefore that the remainder is accounted for by the change in charge density. However it is difficult, if not impossible, to separate the steric repulsion from the electrostatic repulsion effects, since it is not possible to remove the charge without affecting the thickness of the adsorbed layer. The emulsifying capacity of both proteins are similar. Considering that the Dep b-casein stabilized emulsion was observed to be far less stable, the slightly larger D4,3 value observed for DeP b-casein may be a result of droplet coalescence during turbulent conditions encountered shortly after the homogenization process. Unlike surfactants, proteins stabilize interfaces by forming a rigid, viscoelastic adsorbed layer which is capable of physically resisting deformation and coalescence (7, 8, 10, 11). The strength and integrity of the protein adsorbed layer is paramount to the stability of protein stabilized emulsions (8, 17, 31). Once adsorbed, proteins unfold and form protein– protein interactions which are mainly responsible for the viscoelastic properties. The surface elastic modulus is a good indicator of the strength of these interactions. For the majority of surface active proteins, an increase in the elastic modulus occurs with time until a plateau value is approached. This is not the case for b-casein and DeP b-casein. The maximum in elasticity (Fig. 5), also previously reported elsewhere (14, 15), appears as a result of two consecutive processes. The first step of the process involves the adsorption of b-casein from the bulk solution. The rate of the initial step (i.e., increase in elastic modulus) increases with increasing surface concentration. There was only a slight difference between the native and DeP b-casein during this step, with the b-casein increase being slightly steeper than that of the DeP b-casein. The magnitude of the elastic modulus of b-casein is very low (15 mNm01 after 40 min) compared to other proteins, for example, b-lactoglobulin may have an elastic modulus in excess of 140 mN.m01 under similar conditions (i.e., concentration, pH, ionic strength, and temperature). The highly stable emulsions produced by b-casein were therefore unexpected considering its poor interfacial rheological properties. The second step of the process, i.e., the decrease in elastic modulus, could be due to contamination (16), fracturing of the interface, or a rearrangement of b-casein at the interface. As stated under Results, the effects of contamination, surface fracturing or overstretching the interface, surface aggregation, and methodological artifacts were all investigated thoroughly and discounted. Therefore we conclude that the ob-
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served maximum in the surface elasticity is due to molecular rearrangement of the b-casein molecule at the interface. A change in b-casein conformation at the interface is implied from studies of the adsorbed layer thickness of bcasein (27) which suggest that b-casein can adopt different conformations at low and high surface coverages (i.e., 1 and 3 mg.m02 , respectively), since the adsorbed layer thickness increases from approx. 5 to 10 nm, as determined by quasielastic light scattering. Theoretical brush thickness calculations (35) presented show a similar thickness (5.4 nm) at 1.6 mg.m02 , and brush thickness increases with increasing concentration, which supports the idea of conformational change. Leaver and Dalgleish (28) suggested the loop-andtrain model of b-casein conformation at the interface, with the N-terminal 50 amino acids forming a loop or tail into the bulk solution while the remainder of the molecule is much more closely associated with the interface. This is supported by the kinetics of the trypsin treatment of high surface coverage b-casein stabilized emulsions (28) showing that the N-terminal is more susceptible to protease treatment than the C-terminal, and hence the N-terminal is more accessible. Also trypsinolysis causes a decrease in the adsorbed layer from 10 to 5 nm thick. The 5-nm thickness putatively represents the 50-209 peptide of b-casein. The surface rheological properties of proteins at the oil– water interface have previously been reported (29, 30). Benjamins et al. (30) reported differences between air–water and oil–water surface rheology of three different proteins (none of which were b-casein), using the dynamic drop tensiometer and purified sunflower oil. The main difference was a decrease in the elastic modulus observed at the oil– water interface only. Benjamins et al. (30) suggest this to be as a result of a ‘‘collapse-type’’ phenomonon. This collapse-type phenomenon could be due to low levels of surface active impurities in the triglyceride oil disrupting the protein adsorbed layer. Disruption of the adsorbed protein layer at the interface by oil–soluble surfactants has been reported previously (8). The effect of contaminating surfactant on surface rheological properties has been reported previously (16) at the air–water interface. The presence of a contaminating surfactant produced a decrease in the elastic modulus. In contrast Williams and Prins (29) reported similar surface rheological properties of b-casein at the air–water and oil– water interface, using the ring trough method and paraffin oil. The paraffin oil in that study is more closely related to the n-tetradecane used in this investigation than the sunflower oil used by Benjamins (30). Therefore the air–water surface rheology results presented here may be a good indicator of the expected behavior of both proteins at the ntetradecane oil–water interface. To summarize, despite the poor interfacial elasticity of bcasein, compared to all other proteins examined, it is capable of producing emulsions which are highly stable to coalescence. The evidence presented here strongly suggests that
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the phosphorylated loop or tail region of the molecule is, in the main, responsible for this high stability. Removal of the charge by dephosphorylation reduces the electrostatic repulsion between interfaces and b-casein adopts a more compact adsorbed layer, thus reducing any steric hindrance of the loop or tail region. The stability of emulsions stabilized by dephosphorylated b-casein are consequently very poor. Foam Properties The difference observed in foamability of the two samples is not a result of different created bubble sizes. As reported previously (21), any differences in surface tension between samples would not result in differences in created bubble size using the microconductivity apparatus. The bubble size is controlled by using a single orifice and constant gas flow for sparging. There is also no difference in the rates of adsorption to the interface of the gas bubble, as can be seen from the surface tension data (Fig. 6). Although the surface tension data reported here are at a static interface, it has been reported previously (22) that the surface tension at a dynamic interface, as measured by the overflowing cylinder, shows trends similar to the static interface results. Hence no differences in surface tension profiles of the two proteins are expected at a dynamic interface. The difference in foamability requires further investigation. The foam stability data are identical for both proteins despite the fact that the thin film thickness data showed thinner areas within the DeP b-casein films. However, this change in foam film thickness is unlikely to make much difference to the conductivity as most of the liquid is contained within the Plateau borders. Thin film drainage and surface rheology data show little difference; therefore the rate of drainage of liquid from the foam is unlikely to differ between the proteins. It has been reported previously ( 5 ) that dephosphorylated whole casein produced a smaller foam volume, and foam collapse was quicker. The difference in results reflects the difference in functional properties of b-casein and whole casein which consist mainly of four proteins:- as1 , as2 , b, and k in the ratio 40:10:35:12, all of which are phosphorylated and surface active. It should also be noted that in the single-orifice sparging method presented both the bubble size distribution and the foam volume are controlled, and foam drainage rather than volume is measured. In summary, the massive reduction in emulsion orthokinetic stability with a reduction in charge suggests that charge repulsion plays an important role in emulsion stability. However, the adsorbed layer thickness data suggest that there is also a conformational change upon dephosphorylation. This conformational change at the interface which produces a more compact structure will minimize the steric stabilization of the emulsion droplets by the N-terminal loop/tail region.
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Minimizing these electrostatic and steric effects means that the emulsion orthokinetic stability will rely more upon the interfacial rheology (8, 31). Therefore as the interfacial rheology of b-casein is very low compared to other proteins (32), it is therefore not surprising that the orthokinetic stability of the DeP b-casein is so poor. The foaming properties are very similar for both proteins. The difference in foamability of the proteins cannot be readily explained by a charge and/or steric effect; therefore alternative explanations (e.g., isoelectric point, local conductivity effects) require further investigation. The lack of any effect of dephosphorylation on the foam stability is suprising considering the dramatic effect on emulsion stability. Further work is necessary to identify the primary stabilization mechanisms responsible for the different behavior of foams and emulsions. CONCLUSIONS
The results presented suggest that the mechanism of emulsion orthokinetic stability by b-casein depends on the charged phosphate groups in the N-terminal domain. Removal of the phosphate groups decreased the film thickness due to a reduction of the electrostatic and/or steric repulsion. The poor elastic modulus of b-casein meant that without the charge/steric stabilizing effect of the phosphate groups in the N-terminal region, there was a drastic reduction in emulsion orthokinetic stability. Dephosphorylation of b-casein had no effect on foam stability as measured by the microconductivity technique, but an increase in foamability was measured. None of the interfacial measurements presented appear to elucidate an underlying mechanism for the foaming behavior. ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from BBSRC, and to Jeff Leaver of the Hannah Research Institute for supplying pure b-casein.
REFERENCES 1. Davies, D. T., and Law, A. J. R., J. Dairy Res. 44, 447 (1977). 2. Mitchell, J., Irons, L., and Palmer, G. J., Biochim. Biophys. Acta 200, 138 (1970). 3. Dickinson, E., J. Chem. Soc. Faraday Trans. 88, 2973 (1992). 4. Swasigood, H. E., in ‘‘Developments in Dairy Chemistry-1’’ (P. F. Fox, Ed.), p. 1. Applied Science, London, 1982. 5. Van Hekken, D. L., and Strange, E. D., J. Dairy Sci. 76(11), 3384 (1993). 6. Horne, D. S., and Leaver, J., Food Hydrocolloids 9(2), 91 (1995). 7. Mackie, A. R., Wilde, P. J., Wilson, D. R., and Clark, D. C., J. Chem. Soc. Faraday Trans. 89(15), 2755 (1993). 8. Cornec, M., Mackie, A. R., Wilde, P. J., and Clark, D. C., Colloids Surf. A: Physicochem. Eng. Aspects 114, 237 (1996). 9. Clark, D. C., Wilde, P. J., and Wilson, D. R., Colloids Surf. 59, 209 (1991).
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10. Coke, M., Wilde, P. J., Russell E. J., and Clark, D. C., J. Colloid Interface Sci. 138, 489 (1990). 11. Clark, D. C., Coke, M., Mackie, A. R., Pinder, A. C., and Wilson, D. R. J. Colloid Interface Sci. 138, 207 (1990). 12. Kokelaar, A. J. J., Prins, A., and de Gee, M., J. Colloid Interface Sci. 146, 507 (1991). 13. Ambwani, D. S., and Fort, T., Jr., in ‘‘Surface and Colloid Science, Vol. II’’ (R. J. Good and R. R. Stromberg, Eds.), p. 93. Plenum, New York, 1979. 14. Benjamins, J., de Feijter, J. A., Evans, M. T. A., Graham, D. E., and Phillips, M. C., Faraday Discuss. Chem. Soc. 159, 218 (1975). 15. Graham, D. E., and Philips, M. C., J. Colloid Interface Sci. 76, 227 (1980). 16. Clark, D. C., Husband, F. A., Wilde, P. J., Cornec, M., Miller, R., Kragel, J., and Wustneck, R., J. Chem. Soc. Faraday Trans. 91(13), 1991 (1995). 17. Chen, J., Dickinson, E., and Iveson, G., Food Structure 12, 135 (1993). 18. Leermakers, F. A. M., Atkinson, P. J., Dickinson, E., and Horne, D. S., J. Colloid Interface Sci. 178, 681 (1996). 19. Pittia, P., Wilde, P. J., and Clark, D. C., Food Hydrocolloids 10(3), 335 (1996). 20. Wilde, P. J., and Clark, D. C., J. Colloid Interface Sci. 155, 48 (1993). 21. Wilde, P. J., J. Colloid Interface Sci. 178, 733 (1996). 22. Bergink-Martens, D. J. M., ‘‘Interface Dilation: The Overflowing Cylinder Technique.’’ Ph.D. thesis, Agricultural University, Wageningen (1993).
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23. Brooksbank, D. V., Davidson, C. M., Horne, D. S., and Leaver, J., J. Chem. Soc. Faraday Trans. 89(18), 3419 (1993). 24. Dalgleish, D. G., Colloids Surf. 65, 9 (1990). 25. Atkinson, P. J., Dickinson, E., Horne, D. S., and Richardson, R. M., J. Chem. Soc. Faraday Trans. 91(17), 2847 (1995). 26. Sullivan, R. A., Fitzpatrick, M. M., Stanton, E. K., Annino, R., Kissel, G., and Palermiti, F., Arch. Biochem. Biophys. 55, 455 (1955). 27. Fang, Y., and Dalgleish, D. G., J. Colloid Interf. Sci. 156, 329 (1993). 28. Leaver, J., and Dalgleish, D. G., Biochim. Biophys. Acta 1041, 217 (1990). 29. Williams, A., and Prins, A., Colloids Surf. A: Physicochem. Eng. Aspects 114, 267 (1996). 30. Benjamins, J., Cagna, A., and Lucassen-Reynders, E. H., Colloids Surf. A: Physicochem. Eng. Aspects 114, 245 (1996). 31. Dickinson, E., Owuzu, R. K., and Williams, A., J. Chem. Soc. Faraday Trans. 89(5), 865 (1993). 32. Murray, B., and Dickinson, E., Food Sci. Technol. Int. 2(3), 131 (1996). 33. Koppel, D. E., J. Chem. Phys. 57, 4814 (1972). 34. Pusey, P. N., Koppel, D. E., Schaefer, D. W., Camerini-Otero, R. D., and Koenih, S. H., Biochemistry 13, 952 (1972). 35. Mackie, A. R., Mingins, J., and North, A. N., J. Chem. Soc. Faraday Trans. 87(13), 3043 (1991). 36. Leaver, J., and Law, A. J. R., J. Dairy Res. 59, 557 (1992). 37. Atkinson, P. J., Dickinson, E., Horne, D. S., Leaver, J., Leermakers, F. A. M., and Richardson, R. M., in ‘‘Food Colloids: Proteins, Lipids and Polysaccharides’’ (E. Dickinson and B. Bergenstahl, Eds.), p. 217. Royal Society of Chemistry, Cambridge, 1997.
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