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Influence of an anionic surfactant on the rheology of heat-set β-lactoglobulin-stabilized emulsion gels
COLLOIDS
AND Colloids and Surfaces A: Physicochemicaland Engineering Aspects 127 (1997) 1-10
ELSEVIER
A
SURFACES
Influence of an anionic surfactant on the rheology of heat-set 13-1actoglobulin-stabilized emulsion gels Eric Dickinson *, Soon-Taek Hong Procter Department of Food Science, University of Leeds, Leeds, LS2 9JT, UK Received 14 April 1996; accepted 30 September 1996
Abstract
The influence of the anionic surfactant sodium dodecyl sulphate (SDS) on the small-deformation shear rheological behaviour of heat-set 13-1actoglobulin emulsion gels was investigated. Emulsion gels containing surfactant added after homogenization (5-8 wt% 13-1actoglobulin, 38 wt% n-tetradecane, pH 7) were prepared by in situ heat treatment (30 min at 90°C). Storage and loss moduli (frequency 1 Hz) were determined at 30°C. Emulsion gel strength was found to be sensitively dependent on the total protein content and, especially, the surfactant/protein molar ratio R. For low surfactant additions (08) inhibit gel formation. Qualitatively similar behaviour is found in 13-1actoglobulin systems containing no emulsion droplets, albeit at a substantially higher protein content. Taken together with results from complementary electrophoretic mobility and surface shear viscosity measurements, the emulsion gel small-deformation rheological behaviour can be explained in terms of various combinations of interfacial and bulk protein-surfactant interactions. These interactions may produce electrostatic protein-protein repulsion at low R, attractive SDS-mediated protein unfolding and cross-linking at intermediate R, and solubilization of protein into discrete non-associating mixed micelles at high R. Comparison with previous results indicates that surfactant effects on the rheology of heatset protein emulsion gels cannot readily be generalized. The behaviour appears to be sensitive to specific aspects of the surfactant association structure and the protein-surfactant interactions. © 1997 Elsevier Science B.V. Keywords: Emulsion gel; 13-Lactoglobulin; Protein-surfactant interactions; Sodium dodecyl sulphate; Surface viscosity; Thermal denaturation; Viscoelasticity
1. Introduction
In most food oil-in-water emulsions, both proteins and small-molecule surfactants are present during the emulsification process, and both contribute to emulsion stability and rheological properties [ 1]. As well as competing for space at the oil-water
* Corresponding author. 0927-7757/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0927-7757(96)03891-5
interface, the two kinds of surface-active species will tend to interact in the bulk phase and at the interface, possibly leading to the formation of a distinct complex [2-4]. Recent experimental studies have shown [5-8] that the nature of the protein-surfactant interaction in a concentrated emulsion containing adsorbed globular protein molecules can greatly affect the viscoelasticity of the emulsion gel formed by subsequent heat treatment. Interactions between proteins or polymers and
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low-molecular-weight surfactants have been extensively studied over the past several decades and the results are well documented in the literature [9-12]. The major thermodynamic driving force for interaction is the hydrophobic effect, i.e. the association of the alkyl chain of the surfactant molecules with the hydrophobic regions of dissolved polymer molecules. At the same time, the headgroup of ionic surfactants may also be involved in attractive interactions with oppositely charged groups along the polymer chain [2]. It is clear that the mechanism of protein-surfactant association at surfactant concentrations above the critical micelle concentration (CMC) may be substantially different from that at low concentrations (below the CMC). A consequence of protein surfactant interactions is that an aqueous solution containing a mixture of protein and surfactant may be of variable rheological character depending on the type of added surfactant (non-ionic, anionic or cationic), the surfactant concentration (with respect to the CMC) and the solution conditions (temperature, pH, ionic strength, etc.). At surfactant concentrations above the CMC, there is the possibility of separate polymer molecules becoming crosslinked via surfactant micelles to form a gel-like network. For instance, with mixed solutions of gelatin and anionic sodium dodecyl sulphate (SDS), Greener et al. [13] reported a considerable enhancement in viscosity, at surfactant concentrations above the CMC, due to cross-linking between the surfactant micelles and protein molecules. In mixtures of SDS and lysozyme, it has recently been reported [14] that the system turns into a gel at high values of the surfactant/protein molar ratio. For the case of viscous and gelled systems containing hydrophobically modified polymer and surfactant, Lindman and co-workers [15] suggested the formation of mixed aggregates involving the hydrophobic groups of the polymer molecules and surfactant micelles acting as crosslinkers between the polymer chains. A similar mechanism for the gelation of lysozyme-SDS mixtures was proposed by Mor6n and Khan [14]. Whey proteins are now becoming widely used in foods as functional ingredients for emulsification, foaming and gelation [16,17]. The gelling
properties of commercial whey protein mixtures are predominantly determined by the gelling properties the major protein component, [3lactoglobulin. It is generally accepted [ 18 21] that the theological and structural properties of heatset globular protein gels are dependent on the delicate balance of attractive and repulsive forces between the aggregating protein molecules during and after gel formation. Introducing emulsion droplets into the heat-set globular protein network is equivalent to the formation of a "filled" gel. The viscoelasticity of such a filled polymer gel is determined in large part by the nature of the interactions between the filleT particles and the polymer matrix [22]. It has been established [22,23] that the incorporation of protein-coated oil droplets produces a reinforcement of the heatset protein network and hence a larger elastic modulus. On the other hand, when the stabilizing layer around the droplets is replaced by an adsorbed layer of small-molecule surfactant (polysorbate or phospholipid), the gel strength is found to be reduced to an extent which is dependent on the protein/oil ratio and the nature of the emulsifier [6,24,25]. These results indicate that the shear rheological properties of an emulsion gel can be dependent on the structure and composition of the adsorbed layer at the oil water interface, and that the properties of the layer are influenced by the presence of surfactant. We recently demonstrated [6,26,27]that the influence of a small-molecule surfactant on the theology of a heat-set emulsion gel is dependent on whether the surfactant is added before or after emulsification. With non-ionic (non-interacting) Tween 20 added after emulsion formation, we tbund [6] that the value of the storage modulus of heat-set [3-1actoglobulin systems at neutral pH could be substantially enhanced or reduced depending on the amount of emulsifier added, as expressed in terms of the surfactant/protein molar ratio. On the other hand, addition of a surfactant that interacts more strongly with the protein, anionic DATEM (diacetyltartaric acid ester of monoglyceride) or zwitterionic lecithin (phosphatidylcholine), was found [7,8,27] to give a steady but substantial increase in the theological parameters with increasing emulsifier concentration. This
E. Dickinson, S.-T. Hong / Colloids Surfaces A: Physicochem. Eng. Aspects 127 (1997) 1-10
difference in rheological behaviour is consistent with the differing abilities of the added surfactants to displace the adsorbed protein competitively from the oil-water interface, as opposed to formation of a protein-surfactant complex at the interface or in the bulk aqueous phase [28]. In this work, we extended our investigation of the effect of surfactant-protein interactions on the rheolog~ of heat-set milk protein emulsion gels to systea4is Icontaining the more thoroughly studied anionic SDS. As well as describing the smalldeformation bulk rheological behaviour of the emulsion gels, we report here measurements of surface shear viscosity of adsorbed [3-1actoglobulin films in the presence of SDS and electrophoretic mobility data for dilute 13-1actoglobulin-stabilized emulsions containing SDS. The objective was to establish whether there is a link between the surface and bulk properties for heat-treated protein samples along the same lines as that recently established for non-heat-treated oil-in-water emulsions containing 13-1actoglobulin and sodium lauryl ether sulphate [29, 30].
2. Materials and methods
2.1. Materials Bovine [Mactoglobulin (lot 91H7005, purity> 99 wt%), sodium dodecyl sulphate (SDS) and ntetradecane (purity >99 wt%) were obtained from Sigma Chemicals (St. Louis, MO, USA). The molecular masses of 13-1actoglobulin and SDS assumed in calculating the surfactant protein molar ratio R were 1.84x104 and 2.88x10Zgmo1-1, respectively. Buffer salts were AnalaR-grade reagents. Water was doubly distilled. 2.2. Emulsion preparation Oil-in-water emulsions (5-8 wt% protein, 38 wt% oil, 20 mM bis-tris, pH 7) were prepared using a single-pass laboratory-scale jet homogenizer [31] operating at a constant pressure of 300 bar. The emulsion samples were degassed with a water pump; this step was necessary for the generation of reproducible rheological data. The
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emulsion droplet-size distribution and the volumesurface average diameter d32 were determined using a Malvern Mastersizer $2.01. Known amounts of the water-soluble surfactant SDS were mixed into the freshly made emulsion samples to give the required surfactant/protein molar ratio R. The emulsion samples containing surfactant were immediately transferred to the cup of the rheometer for the gelation/rheology experiments. 2.3. Heat-induced gelation and rheological measurement Small-deformation controlled-stress measurements were carried out during and after gelation as described previously [6]. Gelation was induced by heating the sample in situ in the concentric cylinder cell (i.d. 25 mm, o.d. 27.5 mm) of the Bohlin CS rheometer. A sample (2 ml) of emulsion (or protein solution) was poured carefully into the cell and covered with a thin layer of silicone oil to prevent evaporation during gelation. The gelation protocol involved heating from 30 to 90°C at 3°C min -1, maintaining the temperature at 90°C for 30 min, cooling to 30°C at I°C min -1, and then maintaining the temperature at 30°C for 20 rain. Rheological properties of the heat-treated samples were investigated by dynamic oscillatory rheometry at 30°C. Storage and loss moduli, G' and G", as a function of added emulsifier, were typically measured within the linear viscoelastic regime at a strain of 0.5% and a frequency of 1 Hz. 2.4. Surface shear viscometry The apparent surface shear viscosity at the planar interface between n-tetradecane and the dilute aqueous protein solution (0.002 wt% [3-1actoglobulin in 2 mM bis-tris buffer) was determined using the Couette-type surface rheometer described previously [32,33]. The stainless-steel biconical disk (diameter 30 mm) was suspended by a torsion wire with its edge in the plane of the fluid interface between the protein solution (370 ml) and the oil (70 ml) which were contained in a glass dish (diameter 145mm) which was thermostatically controlled at 25 __+1°C. The apparent surface shear viscosity was determined at fixed
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time intervals over a period of 2 days at a dish rotation speed of 1.27x 10 3 r a d s - 1 . When the adsorbed protein film had been aged for 24 h, the water-soluble surfactant was carefully added to the aqueous phase using a syringe without causing any significant disruption to the interfacial film.
2.5. Electrophoretic mobility Electrophoretic mobilities of droplets were determined for emulsion containing 1.6 wt% protein and 38 wt% oil (20 mM bis tris, pH 7) using a Malvern Zetasizer 4 fitted with a ZET5104 sample cell. The mobilities at room temperature were measured at a certain constant count rate of ca. 1500 kcps; this was achieved by diluting the concentrated emulsion samples extensively with buffer solutions containing the required amounts of surfactant. A small amount of diluted emulsion sample (ca. 10 ml) was then injected into the sample cell. The quoted average result for each surfactant concentration was expressed as the zeta potential ( calculated in the standard way [1] assuming a double-layer thickness small compared with the particle radius. Averages were taken over three independent sets of measurements.
protein and SDS added after emulsification at a surfactant/protein molar ratio R = 6 is shown in Fig. 1. The storage and loss moduli are plotted against time during the heating/cooling cycle. At time t=() ( T = 3 0 ° C ) the system is viscous with G'~0.6 Pa and G"~0.9 Pa. The complex modulus, defined as G*=(G'2+G"2) 1/2, tends to decrease slightly with time up to t ~ 20 min, followed by a small increase to t ~ 2 5 rain ( T ~ 9 0 ° C ) at which point the system is still more viscous than elastic at 1 Hz (G"~ 22 Pa, G'~ 17 Pa). Just beyond t = 25 min, there is a "crossover" of G' and G", which we take as indicating the formation of a mechanically significant gel network [34]. There is a considerable rate of increase of G' over the period that the temperature is maintained at 90~C, followed by an even more pronounced rate of increase during the cooling period. The moduli reach values of G' ~ 1580 Pa and G" ~ 320 Pa at the end of the temperature cycle (t = 130 min, T = 30'~C). The first general comment to be made about the plot in Fig. 1 is that the overall shape of the development pattern for both storage and loss moduli over the heating/cooling cycle is qualitatively similar to that for the previous study with a
3. Results and discussion
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We studied the rheology at 30°C of sets of concentrated heat-set [3-1actoglobulin-stabilized emulsion gels containing 5, 6 and 8 wt% protein (38 wt% oil, 20 mM bis-tris buffer, pH 7). Under the conditions of emulsification used here, the droplet-size distribution was found to be only very weakly dependent on protein concentration: the values of the average volume-surface diameter were d32=0.55_+0.01, 0.54_+0.01 and 0.52_+ 0.01 ~tm for emulsions made with 5, 6 and 8 wt% protein, respectively. All rheological measurements reported here were made at a constant strain of 0.5% and a constant frequency of 1 Hz. The dependence on strain and frequency for this type of heat-set protein-stabilized emulsion gel has been reported previously [6,7]. The development of the storage and loss moduli, G' and G", for an emulsion containing 8 wt%
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Fig. I. Development of small-deformation viscoelastic parameters during thermal processing of an oil-in-water emulsion containing SDS added at surfactant/protein molar ratio R = 6 (8 wt% !3-1actoglobulin, 38 wt% n-tetradecane, 20 mM bis tris buffer, pH 7). The storage modulus G' ( [] ) and the loss modulus G" (m) at 1 Hz are plotted against time. The different regions of the processing cycle are (a) heating from 30 to 90°C at 3°C min- 1, (b) holding at 90°C for 30 min, (c) cooling from 90 to 30°C at 1 °C min -1 and (d) holding at 30°C for 20 min.
E. Dickinson, S.-T. Hong / Colloids Surfaces A." Physicochem. Eng. Aspects 127 (1997) 1-10
emulsions containing various amounts of nonionic surfactant Tween 20 [6]. What we would like to point out specifically here, however, is that gelation was found to occur at a rather higher temperature (i.e. T=90°C) compared with that (T,.~80°C) of the corresponding system in the absence of added anionic surfactant. Several workers have commented [35-37] upon the protective effect of SDS with respect to thermal stability of globular proteins. Hegg [36] demonstrated that the binding of two molecules of SDS per 13-1actoglobulin molecule increases the thermal denaturation temperature by up to 7°C. Binding of SDS apparently induces a conformational change in the [3-1actoglobulin molecule, and the resulting protein-surfactant complex exhibits increased thermal stability [37]. This is consistent with the higher gelation temperature observed here for 13-1actoglobulin emulsions in the presence of SDS. It is generally assumed [19, 38] that all the types of molecular interactions that are involved in determining the native state of a protein are also involved at various stages during the process of thermal aggregation, albeit to different extents. The large increases in gel strength during both the heating and cooling parts of the cycle in Fig. 1 are indicative of the involvement of different kinds of protein-protein interactions during thermal processing. Electrostatic and hydrophobic interactions between adjacent polypeptide regions are mainly involved in the initial (high-temperature) stages of gelation, whereas hydrogen bonding and disulphide cross-links are probably involved to greater extent in stabilizing the final gel structure after cooling [6, 38]. Fig. 2 shows the effect of SDS added after emulsion weparation on the elastic shear modulus of heat-set emulsion gels containing 5, 6 and 8 wt% [3-1actoglobulin. The storage modulus G' at 1 Hz is plotted against the surfactant/protein molar ratio R. It is clear that the gel strength is strongly dependent on both protein content and R. For a constant protein content, a maximum gel strength occurs at R ~ 4 , and at high SDS concentrations values (R>~R~) gel formation is inhibited (i.e. G' < G"). In fact, we see that inhibition of gelation by SDS occurs at slightly different surfactant
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Fig. 2. Influence of SDS concentration on the strength of heatset emulsion gels (5-8 wt% fl-lactoglobulin, 38 wt% n-tetradecane, 20 mM bis-tris buffer, pH 7, 30°C). The storage modulus G' at 1 Hz is plotted against surfactant/protein molar ratio R for three different protein concentrations: II, 5 wt%; [3, 6 wt%; A, 8 wt%.
concentrations depending on the total protein content: R I ~ 8 for the 5 wt% protein system, compared with RI~ l0 for the 8 wt% protein system. The existence of a well-defined SDS/~-lactoglobulin ratio giving optimum emulsion gel strength in Fig. 2 is in stark contrast to the behaviour reported previously [7] for equivalent systems containing the anionic water-dispersable emulsifier DATEM, for which it was found that G' increases continuously with increasing R. One possible reason for this is that, at high surfactant concentrations, SDS displaces the protein completely from the emulsion droplet surface in a manner analogous to nonionic Tween 20, whereas DATEM does not induce such displacement [28]. Another possible reason is that, at high values of R, SDS forms nonassociating mixed micelles into which the protein molecules are separately dispersed and hence unavailable for cross-linking, whereas DATEM does not form such micelles. SDS has long been known by biochemists [3941] to bind strongly to ~-lactoglobulin (and other proteins) and to cause their "denaturation". It has been suggested [39] that the binding of SDS to a globular protein such as ~-lactoglobulin takes place in three stages. At low SDS concentrations, two or three molecules of the surfactant become tightly bound to high-affinity sites of the protein.
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E. Dickinson, S.-72 Hong / Colloids Surfaces A." Physicochem. Eng. Aspects 127 (1997) 1-10
As a consequence of this binding, the protein molecule undergoes unfolding, exposing a large number ( ~ 2 0 ) of weaker potential binding sites. The second stage involves cooperative surfactant binding of a predominantly ionic nature [41 ]. This may be accompanied by a third stage of very weak surfactant binding, which is presumably highly micellar in nature [42], involving complete solubilization of protein into isolated surfactant association structures. Looking at the results in Fig. 2, we can speculate that specific binding occurs between SDS and J3-1actoglobulin in the emulsions at SDS concentrations up to R ~ 2. Then, for R > 2, there is the onset of co-operative SDS-]3-1actoglobulin binding, both at the oil-water interface and in the bulk phase; this induces extensive unfolding of the protein molecules, corresponding to the first step in the thermal gelation of the protein. As further protein unfolding undoubtedly occurs when the emulsion is heated, this means that the heat-treated emulsion containing SDS (R>2) contains more extensively unfolded 13-1actoglobulin molecules than the equivalent one without added surfactant (R=0) or with surfactant added at low concentration (R ~<2). Hence, when the protein molecules aggregate to form a three-dimensional gel network at R > 2, it seems reasonable to expect an enhanced number of mechanically important cross-links between denatured protein molecules, possibly involving increased numbers of hydrophobic interactions, disulphide linkages and intermolecular hydrogen bonds. In support of this hypothesis, separate experiments in this laboratory (data not reported here) have demonstrated that the elastic modulus of a heat-set [3-1actoglobulin gels becomes increased if the protein is partially unfolded first with urea prior to thermal treatment. In the highvolume-fraction emulsions studied here, the concentrations of protein and surfactant are both high, and so SDS-mediated cross-links between unfolded protein molecules are likely to be strongly favoured. Various kinds of interactions are possible, including protein cross-linking by (i) individual surfactant molecules, with the anionic headgroup binding to a cationic group on one protein molecule and the alkyl tail interacting with the non-polar region of another; and (ii) mixed
micelles reminiscent of those found in hydrophobically modified polymer surfactant gels [15]. The dependence of the gel strength on protein content in these systems is consistent with our previously reported results for [3-1actoglobulin emulsion gels [6-8]. Increasing the overall protein content necessarily increases the local number density of contact formed between denatured protein molecules, thereby leading to an increase in gel strength. Mechanistically, the observed increase in G' with increasing protein content for 2~8), protein-protein cross-linking is inhibited again because all of the potentially available sites on the protein are surrounding by interacting SDS molecules. The binding of the anionic surfactant to ]3-1actoglobulin would be expected to lead to an increase in the net negative charge on the surfacrant protein complex. Accordingly. with the protein-stabilized emulsion, it might be expected that the net negative charge at the protein-coated droplet surface would increase in magnitude as a consequence of surfactant binding. This expectation is confirmed by the data in Fig. 3, which shows a plot of the zeta potential of the emulsion droplets as a function of surfactant concentration.
-7 5
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concentration/10-2w t
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Fig. 3. Zeta potential, ~, of protein-stabilizedemulsiondroplets ( 1.6 wt% f3-1actoglobulin,38 wt% n-tetradecane,20 mM bis-tris buffer, pH 7) in solutionsof differentsurfactantconcentration.
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Oil droplets coated solely with adsorbed 13-1actoglobulin have a zeta potential of ~= -58.4-t-1.0mV. A relatively small addition of SDS induces a sharp increase in zeta potential, which reaches a maximum (negative) value of ~ - 70 mV at surfactant concentration of about 0.06 wt%. With further surfactant addition, there is a gradual reduction in if, presumably caused by the displacement of adsorbed protein (and surfactant-protein complex) from the emulsion droplet surfactant by the excess of SDS molecules. At even higher surfactant concentrations, the measured zeta potential appears to increase again very slightly, possibly owing to the formation of a surfactant micellar structure at the oil droplet surface. Note that in these experiments we do not refer to the surfactant/protein ratio R. This is because the emulsion samples prepared for the electrophoretic mobility measurement were extremely dilute compared with those studied rheologically. Hence, in the mobility experiments, the actual values of surfactant/protein molar ratio R in Fig. 3 are hundreds of times larger than those in Fig. 2. We can reasonably infer from the results in Fig. 3 that, for the case of the concentrated emulsion systems, the addition of SDS at low levels leads to an increase in electrostatic repulsion between the droplets. This would be unfavourable for optimizing of the number of interdroplet crosslinks, because interactions between adsorbed protein molecules and between gelling protein molecules in the bulk phase would be significantly weakened. The importance of such interactions for the rheology of emulsion gels has been confirmed elsewhere [6,43]. The slight lowering of the gel strength in Fig. 2 due to small additions of SDS (i.e. R ~ 2 ) may be attributable to increased electrostatic repulsion between protein molecules caused by surfactant binding. In addition to binding to the protein, the SDS can displace adsorbed 13-1actoglobulin from the oil-water interface [44]. However, it is not obvious that this should have a serious negative effect on the rheological character of heat-set emulsion gels, since displacement of protein would be expected to form a more concentrated, and hence stronger, gel network between the droplets. A key issue here
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is whether the droplets have a reinforcing or disrupting influence on the protein gel matrix. This is in part determined by the mechanical strength of the adsorbed layer itself, and in part by the extent to which the layer is mechanically connected to the matrix. Complexation between SDS and protein would be expected to have an influence on (i) interactions between protein molecules lying wholly within the surface layer and (ii) interactions between protein molecules within the layer and those outside. Interactions of the former type are related to the surface shear rheology of the adsorbed protein film. Fig. 4 shows the effect of SDS addition on the apparent surface shear viscosity of a IMactoglobulin film adsorbed from bulk solution (0.002 wt% protein, pH 7, 25°C) at the planar ntetradecane-water interface. The SDS was added at a surfactant/protein molar ratio R = 4 to the aqueous subphase in contact with the 1-day-old 13-1actoglobulin film. It was found that the apparent surface shear viscosity after the addition of surfactant decreases slightly under the influence of continuous shearing, but it later recovers almost to the same value as that for the original 1-day-old pure ~-lactoglobulin film when subsequently kept undisturbed for several hours. Shear-thinning behaviour is commonly observed with adsorbed protein films [32,33] or protein solutions containing anionic surfactant [13]. This may be attributed [33] to the slow breaking down of protein-surfac600
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Fig. 4. Influence of SDS on surface shear viscosity of 13-1actoglobulin adsorbed at the n-tetradecane-water interface at pH 7. Apparent surface viscosity, t/s, is plotted against time. The arrow denotes the point (after 24 h) at which the surfactant (/{=4) is introduced into the aqueous subphase.
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E. Dickinson, S.-72 Hong/Colloids Surfaces A: Physicochem. Eng. Aspects 127 (1997) 1° 10
tant complexes under shear. Based on the emulsion results of Feijter et al. [44], it might be supposed that, even at rather low SDS concentrations, there should be a limited displacement of adsorbed 13-1actoglobulin from the planar oil-water interface (e.g.~30% displacement at R ~ 2 ) . Nevertheless, in contrast to Tween 20, which induces a rapid fall in the surface shear viscosity [33], what we see in Fig. 4 is that the apparent shear viscosity of the adsorbed protein film following SDS addition is almost the same as that for the original 1-day-old pure 13-1actoglobulin film. Moreover, this behaviour extends to higher SDS concentrations (R ~ 16), as indicated in Table 1. Since the steady-state interfacial shear rheology stays relatively constant within experimental error ( _ + 1 0 m N m -1 s) over this wide range of R values, it would appear that the large effect of SDS on the rheology of the heat-set emulsion gels cannot simply be attributed to the effect of the surfactant on the viscoelasticity of the adsorbed protein layer. A more likely explanation, then, is that the large reduction in G' at high R values in the concentrated emulsions (Fig. 2) is due to a disruption by the surfactant of interactions between the adsorbed layer and the unadsorbed protein, and also, perhaps more importantly, to the disruption of the heat-set protein gel network between the droplets due to solubilization of protein into separate unconnected mixed micelles in the presence of the excess surfactant. In work on emulsions, it has been separately shown that SDS [44] or sodium lauryl ether sulTable 1 Steady-state apparent surface shear viscosity, ~/8, of [3-1actoglobulin adsorbed at the planar n-tetradecane-water interface (0.002 wt% protein, 2 m M bis-tris buffer, pH 7, 25°C) containing various amounts of added SDS (expressed as surfactant/protein molar ratio, R) R
r/~ (mN m -1 s)
0 1 4 16 64 520 1800
560 540 550 540 450 210 80
phate [30] produce almost complete displacement of interfacial protein at a surfactant concentration of ca. 0.3wt% ( R ~ 6 0 for SDS). However, the surface theology data in Table l indicate that 13-1actoglobulin is still making a major contribution to the interfacial shear viscosity at R values at least an order of magnitude larger than this. This may be because of the nature of the surface theology experiment which probes interfacial regions further away from the surface [45]. That is, even though the interface may be fully covered with a primary SDS monolayer, it is likely that considerable amounts of protein-surfactant complex still remain in the interfacial region, interacting weakly with the primary adsorbed surfactant layer, and that this in turn affects the measured interracial shear rheology. Based on the information discussed so far, it may be concluded that the small-deformation bulk rheology of a heat-set protein-stabilized emulsion gel containing SDS depends on the balance between positive contributions to G', arising from enhanced surfactant-mediated cross-links between the protein molecules, and negative contributions to G', arising from protein-protein and droplet ..... droplet electrostatic interactions (low R) and disruption of the denatured protein gel network by protein solubilization into surfactant micelles (high R). The relative contribution of these different factors would be expected to be sensitively influenced by the surfactant concentration. Taken as a whole, the results seem to suggest that the general trend of behaviour due to addition of SDS is mostly dependent on the relative amounts of [3-1actoglobulin and anionic surfactant in the system, and much less upon any special effect involving the surface of the dispersed droplets. This statement is reinforced by the rheological data in Fig. 5 for the effect of SDS on the viscoelasticity of 14 wt% heat-set 13-1actoglobulin gels (no dispersed oil droplets). The storage and loss moduli are plotted against R over the range 0---14. It can be seen that the general trend of changing gel strength is actually very similar to that exhibited by the heat-set emulsion gels in Fig. 2. That is, increasing the SDS concentration to R ~ 2 leads to a small decrease in the elastic modulus of the gel. For R >2, G' increases sharply, reaching a maxi-
E. Dickinson, S.-T. Hong / Colloids Surfaces A." Physicochem. Eng. Aspects 127 (1997) 1 10
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4. Conclusions
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We have shown that the viscoelastic properties of heat-set 13-1actoglobulin emulsion gels containing SDS are greatly dependent on protein concentration and the surfactant/protein ratio. At very low R values, the gel strength tends to decline as SDS binds strongly to specific binding sites. With increasing SDS concentration above R~2, the elastic modulus starts to increase corresponding to the SDS concentration at which cooperative surfactant binding and associated globular protein unfolding take place. Further larger additions of SDS result in a reduction in gel strength, and eventually gelation is prevented altogether (R > 10). This behaviour contrasts sharply with the previously known behaviour of systems containing the anionic water-dispersible emulsifier DATEM, in which the emulsion gel strength was found to increase continuously with R, and with systems containing the non-ionic emulsifier Tween 20, in which the emulsion gel strength was found to fall dramatically for R > 1. as the protein begins to become competitively displaced from the emulsion droplet surface. We speculate here that the increased gel strength at intermediate surfactant concentrations (from R ~ 3 to R ~ 8) is attributable to increased surfactant-induced protein unfolding and additional surfactant-mediated cross-links via surfactant molecule or mixed micellar aggregates involving assembled surfactant molecules and unfolded protein molecules. On the other hand. the increased electrostatic repulsion due to surfactant binding may account for the observed decrease in the gel strength at low R. and complete solubilization of protein into separate mixed micelles may be the reason for the low gel strength (and eventual lack of gelation) at high R. The surface shear rheology results indicate that protein-surfactant interactions within the adsorbed layer at the oil water interface have no significant correlation to the bulk rheology of heat-set protein-stabilized emulsion gels containing added SDS. Overall, it is concluded that the rheology of a heat-set protein-stabilized emulsion gel containing SDS depends on the balance between positive and negative contributions to cross-linking arising from different kinds of
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R
Fig. 5. Influenceof SDS concentration on the viscoelasticityof heat-set protein gel (14 wt% [3-1actoglobulin,pH 7, 3WC) in the absence of oil droplets. The storage modulus G' (D) and the loss modulus G" (Ill) at l Hz are plotted against the surfactant/protein molar ratio R.
mum high value of G ' ~ 4 0 0 Pa at R ~ 6 . This is then followed by a sudden drop in the elastic modulus with further increase in surfactant concentration up to R ~ 1 2 . For high surfactant contents (R> 12), a cross-over of G' and G" is observed, indicating the loss of gelation. It may be noteworthy that, whilst the 8 wt% emulsion system becomes a non-gelled viscous solution at R>~ 10, the 14 wt% protein system becomes nongelling at a slightly higher R value (i.e. R~> 12). As with the emulsions, the binding of surfactant molecules to 13-1actoglobulin could increase the protein-protein repulsion, and so alter the aggregation behaviour of the protein when heated and hence its rheology [18-21]. Therefore, the increased electrostatic repulsion between the protein molecules could account for the slight drop in gel strength observed at low surfactant concentrations (0 < R ~<2). In the vicinity of the maximum gel strength (4 ~~8 can be attributed to the increasing replacement of protein-protein contacts by protein-SDS interactions in the form of nonassociated isolated mixed micelles in the presence of excess weakly binding surfactant.
10
E. Dickinson, S.-T. Hong / Colloids Surfaces A: Physicochem. Eng. Aspects 127 (1997) 1-10
protein-surfactant interactions, essentially irrespective of the presence of dispersed emulsion droplets.
[19] [20] [21]
References [1] E. Dickinson, G. Stainsby, Colloids in Foods, Applied Science, London, 1982. [2] E. Dickinson, C.M. Woskett, in: R.D. Bee, P. Richmond, J. Mingins (Eds.), Food Colloids, Royal Society of Chemistry, Cambridge, 1989, p. 74. [3] E. Dickinson, ACS Symp. Ser. 448 (1991) 114. [4] E. Dickinson, in: E.D. Goddard, K.P. Ananthapadmanabhan (Eds.), Interactions of Surfactants with Polymers and Proteins, CRC Press, Boca Raton, FL, 1993, p. 295. [5] D.J. McClements, F.J. Monahan, J.E. Kinsella, J. Texture Stud. 24 (1993) 411. [6] E. Dickinson, S.-T. Hong, J. Agric. Food Chem. 43 (1995) 2560. [7] S.-T. Hong, E. Dickinson, in: G.O. Phillips, P.A. Williams, D.J. Wedlock (Eds.), Gums and Stabilisers for the Food Industry, Oxford University Press, Oxford, 1996, Vol. 8, p. 319. [8] E. Dickinson, Y. Yamamoto, Food Hydrocolloids, 10 (1996) 301. [9] J. Steinhardt, J.A. Reynolds, Multiple Equilibria in Proteins, Academic Press, New York, 1969. [10] E.D. Goddard, Colloids Surfaces 19 (1986) 255. [11] M.N. Jones, Chem. Soc. Rev. 21 (1992) 127. [12]E.D. Goddard, K.P. Ananthapadmanabhan (Eds.), Interactions of Surfactants with Polymers and Proteins, CRC Press, Boca Raton, FL, 1993. [13] J. Greener, B.A. Contestable, M.D. Bale, Macromolecules 20 (1987) 2490. [14] A.K. Mor6n, A. Khan, Langmuir 11 (1995) 3636. [15] B. Magny, I. Iliopoulos, R. Audebert, L. Piculell, B. Lindman, Prog. Colloid Polym. Sci. 89 (1992) 118. [16] C.V. Morr, E.Y.W. Ha, Crit. Rev. Food Sci. Nutr. 33 (1993) 431. [17] A. Kilara, in: N.S. Hettiarachchy, G.R. Ziegler (Eds.), Protein Functionality in Food Systems, Marcel Dekker, New York, 1994, p. 325. [18] A.H. Clark, C.D. Lee-Tuffnell, in: J.R. Mitchell, D.A. Ledward (Eds.), Functional Properties of Food
[22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]
[35] [36] [37] [38] [39]
[40] [41] [42] [43] [44] [45]
Macromolecules, Elsevier Applied Science, London, 1986, p. 203. A.H. Clark, S.B. Ross-Murphy, Adv. Polym. Sci. 83 (1987) 57. M. Stading, M. Langton, A.-M. Hermansson, Food Hydrocolloids 6 (1992) 455. E. Dickinson, J. Chem. Soc., Faraday Trans. 90 (1994) 173. T. van Vliet, Colloid Polym. Sci. 266 (1988) 518. J.M. Aguilera, H.G. Kessler, J. Food Sci. 54 (1989) 1213. R. Jost, F. Dannenberg, J. Rosset, Food Microstruct. 8 (1989) 23. Y.L. Xiong, J.E. Kinsella, Milchwissenschaft 46 (1991) 207. E. Dickinson, S.-T. Hong, Y. Yamamoto, Neth. Milk Dairy J., 50 (1996) 199. E. Dickinson, Y. Yamamoto, J. Food Sci., 61 (1996) 811. E. Dickinson, S.-T. Hong, J. Agric. Food Chem. 42 (1994) 1602. J. Chen, E. Dickinson, Colloids Surfaces A 100 (1995) 255, 267. J. Chen, E. Dickinson, Colloids Surfaces A 101 (1995) 77. I. Burgaud, E. Dickinson, P.V. Nelson, Int. J. Food Sci. Technol. 25 (1990) 39. E. Dickinson, B.S. Murray, G. Stainsby, J. Colloid Interface Sci. 106 (1985) 259. J. Chen, E. Dickinson, Food Hydrocolloids 9 (1995) 35. A.H. Clark, in: E. Dickinson (Ed.), Food Polymers, Gels and Colloids, Royal Society of Chemistry, Cambridge, 1991, p. 323. S.-A. Gumpen, P.O. Hegg, H. Martens, Biochim. Biophys. Acta 574 (1979) 189. P.-O. Hegg, Acta Agric. Scand. 30 (1980) 401. M. Donovan, D.M. Mulvihill, Irish J. Food Sci. Technol. 11 (1987) 77. E. Dickinson, D.J. McClements, Advances in Food Colloids, Blackie, Glasgow, 1995, ch. 3. A. Ray, R. Chatterjee, in: G.N. Ramachandran (Ed.), Conformation of Biopolymers, Academic Press, London, 1967, p. 235. R. Pitt-Rivers, F.S.O. Impiombato, Biochem. J. 109 (1968) 825. M.N. Jones, A. Wilkinson, Biochem. J. 153 (1976) 713. R.M. Hill, D.R. Briggs, J. Am. Chem. Soc. 78 (1956) 1590. R.A. Yost, J.E. Kinsella, J. Food Sci. 57 (1992) 892. J.A. de Feijter, J. Benjamins, M. Tamboer, Colloids Surfaces 27 (1987) 243. E. Dickinson, An Introduction to Food Colloids, Oxford University Press, Oxford, 1992, p. 75.
AND Colloids and Surfaces A: Physicochemicaland Engineering Aspects 127 (1997) 1-10
ELSEVIER
A
SURFACES
Influence of an anionic surfactant on the rheology of heat-set 13-1actoglobulin-stabilized emulsion gels Eric Dickinson *, Soon-Taek Hong Procter Department of Food Science, University of Leeds, Leeds, LS2 9JT, UK Received 14 April 1996; accepted 30 September 1996
Abstract
The influence of the anionic surfactant sodium dodecyl sulphate (SDS) on the small-deformation shear rheological behaviour of heat-set 13-1actoglobulin emulsion gels was investigated. Emulsion gels containing surfactant added after homogenization (5-8 wt% 13-1actoglobulin, 38 wt% n-tetradecane, pH 7) were prepared by in situ heat treatment (30 min at 90°C). Storage and loss moduli (frequency 1 Hz) were determined at 30°C. Emulsion gel strength was found to be sensitively dependent on the total protein content and, especially, the surfactant/protein molar ratio R. For low surfactant additions (0
1. Introduction
In most food oil-in-water emulsions, both proteins and small-molecule surfactants are present during the emulsification process, and both contribute to emulsion stability and rheological properties [ 1]. As well as competing for space at the oil-water
* Corresponding author. 0927-7757/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0927-7757(96)03891-5
interface, the two kinds of surface-active species will tend to interact in the bulk phase and at the interface, possibly leading to the formation of a distinct complex [2-4]. Recent experimental studies have shown [5-8] that the nature of the protein-surfactant interaction in a concentrated emulsion containing adsorbed globular protein molecules can greatly affect the viscoelasticity of the emulsion gel formed by subsequent heat treatment. Interactions between proteins or polymers and
2
E. Dickinson, S.-T. Hong / Colloids Surjitces A: Physicochem. Eng. Aspects 127 (1997) 1 10
low-molecular-weight surfactants have been extensively studied over the past several decades and the results are well documented in the literature [9-12]. The major thermodynamic driving force for interaction is the hydrophobic effect, i.e. the association of the alkyl chain of the surfactant molecules with the hydrophobic regions of dissolved polymer molecules. At the same time, the headgroup of ionic surfactants may also be involved in attractive interactions with oppositely charged groups along the polymer chain [2]. It is clear that the mechanism of protein-surfactant association at surfactant concentrations above the critical micelle concentration (CMC) may be substantially different from that at low concentrations (below the CMC). A consequence of protein surfactant interactions is that an aqueous solution containing a mixture of protein and surfactant may be of variable rheological character depending on the type of added surfactant (non-ionic, anionic or cationic), the surfactant concentration (with respect to the CMC) and the solution conditions (temperature, pH, ionic strength, etc.). At surfactant concentrations above the CMC, there is the possibility of separate polymer molecules becoming crosslinked via surfactant micelles to form a gel-like network. For instance, with mixed solutions of gelatin and anionic sodium dodecyl sulphate (SDS), Greener et al. [13] reported a considerable enhancement in viscosity, at surfactant concentrations above the CMC, due to cross-linking between the surfactant micelles and protein molecules. In mixtures of SDS and lysozyme, it has recently been reported [14] that the system turns into a gel at high values of the surfactant/protein molar ratio. For the case of viscous and gelled systems containing hydrophobically modified polymer and surfactant, Lindman and co-workers [15] suggested the formation of mixed aggregates involving the hydrophobic groups of the polymer molecules and surfactant micelles acting as crosslinkers between the polymer chains. A similar mechanism for the gelation of lysozyme-SDS mixtures was proposed by Mor6n and Khan [14]. Whey proteins are now becoming widely used in foods as functional ingredients for emulsification, foaming and gelation [16,17]. The gelling
properties of commercial whey protein mixtures are predominantly determined by the gelling properties the major protein component, [3lactoglobulin. It is generally accepted [ 18 21] that the theological and structural properties of heatset globular protein gels are dependent on the delicate balance of attractive and repulsive forces between the aggregating protein molecules during and after gel formation. Introducing emulsion droplets into the heat-set globular protein network is equivalent to the formation of a "filled" gel. The viscoelasticity of such a filled polymer gel is determined in large part by the nature of the interactions between the filleT particles and the polymer matrix [22]. It has been established [22,23] that the incorporation of protein-coated oil droplets produces a reinforcement of the heatset protein network and hence a larger elastic modulus. On the other hand, when the stabilizing layer around the droplets is replaced by an adsorbed layer of small-molecule surfactant (polysorbate or phospholipid), the gel strength is found to be reduced to an extent which is dependent on the protein/oil ratio and the nature of the emulsifier [6,24,25]. These results indicate that the shear rheological properties of an emulsion gel can be dependent on the structure and composition of the adsorbed layer at the oil water interface, and that the properties of the layer are influenced by the presence of surfactant. We recently demonstrated [6,26,27]that the influence of a small-molecule surfactant on the theology of a heat-set emulsion gel is dependent on whether the surfactant is added before or after emulsification. With non-ionic (non-interacting) Tween 20 added after emulsion formation, we tbund [6] that the value of the storage modulus of heat-set [3-1actoglobulin systems at neutral pH could be substantially enhanced or reduced depending on the amount of emulsifier added, as expressed in terms of the surfactant/protein molar ratio. On the other hand, addition of a surfactant that interacts more strongly with the protein, anionic DATEM (diacetyltartaric acid ester of monoglyceride) or zwitterionic lecithin (phosphatidylcholine), was found [7,8,27] to give a steady but substantial increase in the theological parameters with increasing emulsifier concentration. This
E. Dickinson, S.-T. Hong / Colloids Surfaces A: Physicochem. Eng. Aspects 127 (1997) 1-10
difference in rheological behaviour is consistent with the differing abilities of the added surfactants to displace the adsorbed protein competitively from the oil-water interface, as opposed to formation of a protein-surfactant complex at the interface or in the bulk aqueous phase [28]. In this work, we extended our investigation of the effect of surfactant-protein interactions on the rheolog~ of heat-set milk protein emulsion gels to systea4is Icontaining the more thoroughly studied anionic SDS. As well as describing the smalldeformation bulk rheological behaviour of the emulsion gels, we report here measurements of surface shear viscosity of adsorbed [3-1actoglobulin films in the presence of SDS and electrophoretic mobility data for dilute 13-1actoglobulin-stabilized emulsions containing SDS. The objective was to establish whether there is a link between the surface and bulk properties for heat-treated protein samples along the same lines as that recently established for non-heat-treated oil-in-water emulsions containing 13-1actoglobulin and sodium lauryl ether sulphate [29, 30].
2. Materials and methods
2.1. Materials Bovine [Mactoglobulin (lot 91H7005, purity> 99 wt%), sodium dodecyl sulphate (SDS) and ntetradecane (purity >99 wt%) were obtained from Sigma Chemicals (St. Louis, MO, USA). The molecular masses of 13-1actoglobulin and SDS assumed in calculating the surfactant protein molar ratio R were 1.84x104 and 2.88x10Zgmo1-1, respectively. Buffer salts were AnalaR-grade reagents. Water was doubly distilled. 2.2. Emulsion preparation Oil-in-water emulsions (5-8 wt% protein, 38 wt% oil, 20 mM bis-tris, pH 7) were prepared using a single-pass laboratory-scale jet homogenizer [31] operating at a constant pressure of 300 bar. The emulsion samples were degassed with a water pump; this step was necessary for the generation of reproducible rheological data. The
3
emulsion droplet-size distribution and the volumesurface average diameter d32 were determined using a Malvern Mastersizer $2.01. Known amounts of the water-soluble surfactant SDS were mixed into the freshly made emulsion samples to give the required surfactant/protein molar ratio R. The emulsion samples containing surfactant were immediately transferred to the cup of the rheometer for the gelation/rheology experiments. 2.3. Heat-induced gelation and rheological measurement Small-deformation controlled-stress measurements were carried out during and after gelation as described previously [6]. Gelation was induced by heating the sample in situ in the concentric cylinder cell (i.d. 25 mm, o.d. 27.5 mm) of the Bohlin CS rheometer. A sample (2 ml) of emulsion (or protein solution) was poured carefully into the cell and covered with a thin layer of silicone oil to prevent evaporation during gelation. The gelation protocol involved heating from 30 to 90°C at 3°C min -1, maintaining the temperature at 90°C for 30 min, cooling to 30°C at I°C min -1, and then maintaining the temperature at 30°C for 20 rain. Rheological properties of the heat-treated samples were investigated by dynamic oscillatory rheometry at 30°C. Storage and loss moduli, G' and G", as a function of added emulsifier, were typically measured within the linear viscoelastic regime at a strain of 0.5% and a frequency of 1 Hz. 2.4. Surface shear viscometry The apparent surface shear viscosity at the planar interface between n-tetradecane and the dilute aqueous protein solution (0.002 wt% [3-1actoglobulin in 2 mM bis-tris buffer) was determined using the Couette-type surface rheometer described previously [32,33]. The stainless-steel biconical disk (diameter 30 mm) was suspended by a torsion wire with its edge in the plane of the fluid interface between the protein solution (370 ml) and the oil (70 ml) which were contained in a glass dish (diameter 145mm) which was thermostatically controlled at 25 __+1°C. The apparent surface shear viscosity was determined at fixed
4
E. Dickinson, S.-T. Hong / Colloids Surfaces Am"Physicochem. Eng. Aspects 127 (1997) 11 0
time intervals over a period of 2 days at a dish rotation speed of 1.27x 10 3 r a d s - 1 . When the adsorbed protein film had been aged for 24 h, the water-soluble surfactant was carefully added to the aqueous phase using a syringe without causing any significant disruption to the interfacial film.
2.5. Electrophoretic mobility Electrophoretic mobilities of droplets were determined for emulsion containing 1.6 wt% protein and 38 wt% oil (20 mM bis tris, pH 7) using a Malvern Zetasizer 4 fitted with a ZET5104 sample cell. The mobilities at room temperature were measured at a certain constant count rate of ca. 1500 kcps; this was achieved by diluting the concentrated emulsion samples extensively with buffer solutions containing the required amounts of surfactant. A small amount of diluted emulsion sample (ca. 10 ml) was then injected into the sample cell. The quoted average result for each surfactant concentration was expressed as the zeta potential ( calculated in the standard way [1] assuming a double-layer thickness small compared with the particle radius. Averages were taken over three independent sets of measurements.
protein and SDS added after emulsification at a surfactant/protein molar ratio R = 6 is shown in Fig. 1. The storage and loss moduli are plotted against time during the heating/cooling cycle. At time t=() ( T = 3 0 ° C ) the system is viscous with G'~0.6 Pa and G"~0.9 Pa. The complex modulus, defined as G*=(G'2+G"2) 1/2, tends to decrease slightly with time up to t ~ 20 min, followed by a small increase to t ~ 2 5 rain ( T ~ 9 0 ° C ) at which point the system is still more viscous than elastic at 1 Hz (G"~ 22 Pa, G'~ 17 Pa). Just beyond t = 25 min, there is a "crossover" of G' and G", which we take as indicating the formation of a mechanically significant gel network [34]. There is a considerable rate of increase of G' over the period that the temperature is maintained at 90~C, followed by an even more pronounced rate of increase during the cooling period. The moduli reach values of G' ~ 1580 Pa and G" ~ 320 Pa at the end of the temperature cycle (t = 130 min, T = 30'~C). The first general comment to be made about the plot in Fig. 1 is that the overall shape of the development pattern for both storage and loss moduli over the heating/cooling cycle is qualitatively similar to that for the previous study with a
3. Results and discussion
I
b
~1
c
I
1500 °
d_
/ J /
We studied the rheology at 30°C of sets of concentrated heat-set [3-1actoglobulin-stabilized emulsion gels containing 5, 6 and 8 wt% protein (38 wt% oil, 20 mM bis-tris buffer, pH 7). Under the conditions of emulsification used here, the droplet-size distribution was found to be only very weakly dependent on protein concentration: the values of the average volume-surface diameter were d32=0.55_+0.01, 0.54_+0.01 and 0.52_+ 0.01 ~tm for emulsions made with 5, 6 and 8 wt% protein, respectively. All rheological measurements reported here were made at a constant strain of 0.5% and a constant frequency of 1 Hz. The dependence on strain and frequency for this type of heat-set protein-stabilized emulsion gel has been reported previously [6,7]. The development of the storage and loss moduli, G' and G", for an emulsion containing 8 wt%
~ooo-
500 -
0
m : : - T
0
20
I 40
I 60
I 80
I 100
I 120
time/rain
Fig. I. Development of small-deformation viscoelastic parameters during thermal processing of an oil-in-water emulsion containing SDS added at surfactant/protein molar ratio R = 6 (8 wt% !3-1actoglobulin, 38 wt% n-tetradecane, 20 mM bis tris buffer, pH 7). The storage modulus G' ( [] ) and the loss modulus G" (m) at 1 Hz are plotted against time. The different regions of the processing cycle are (a) heating from 30 to 90°C at 3°C min- 1, (b) holding at 90°C for 30 min, (c) cooling from 90 to 30°C at 1 °C min -1 and (d) holding at 30°C for 20 min.
E. Dickinson, S.-T. Hong / Colloids Surfaces A." Physicochem. Eng. Aspects 127 (1997) 1-10
emulsions containing various amounts of nonionic surfactant Tween 20 [6]. What we would like to point out specifically here, however, is that gelation was found to occur at a rather higher temperature (i.e. T=90°C) compared with that (T,.~80°C) of the corresponding system in the absence of added anionic surfactant. Several workers have commented [35-37] upon the protective effect of SDS with respect to thermal stability of globular proteins. Hegg [36] demonstrated that the binding of two molecules of SDS per 13-1actoglobulin molecule increases the thermal denaturation temperature by up to 7°C. Binding of SDS apparently induces a conformational change in the [3-1actoglobulin molecule, and the resulting protein-surfactant complex exhibits increased thermal stability [37]. This is consistent with the higher gelation temperature observed here for 13-1actoglobulin emulsions in the presence of SDS. It is generally assumed [19, 38] that all the types of molecular interactions that are involved in determining the native state of a protein are also involved at various stages during the process of thermal aggregation, albeit to different extents. The large increases in gel strength during both the heating and cooling parts of the cycle in Fig. 1 are indicative of the involvement of different kinds of protein-protein interactions during thermal processing. Electrostatic and hydrophobic interactions between adjacent polypeptide regions are mainly involved in the initial (high-temperature) stages of gelation, whereas hydrogen bonding and disulphide cross-links are probably involved to greater extent in stabilizing the final gel structure after cooling [6, 38]. Fig. 2 shows the effect of SDS added after emulsion weparation on the elastic shear modulus of heat-set emulsion gels containing 5, 6 and 8 wt% [3-1actoglobulin. The storage modulus G' at 1 Hz is plotted against the surfactant/protein molar ratio R. It is clear that the gel strength is strongly dependent on both protein content and R. For a constant protein content, a maximum gel strength occurs at R ~ 4 , and at high SDS concentrations values (R>~R~) gel formation is inhibited (i.e. G' < G"). In fact, we see that inhibition of gelation by SDS occurs at slightly different surfactant
5
2000
1600
A
1200
b
800
400
0 2
4
6
8
10
12
R
Fig. 2. Influence of SDS concentration on the strength of heatset emulsion gels (5-8 wt% fl-lactoglobulin, 38 wt% n-tetradecane, 20 mM bis-tris buffer, pH 7, 30°C). The storage modulus G' at 1 Hz is plotted against surfactant/protein molar ratio R for three different protein concentrations: II, 5 wt%; [3, 6 wt%; A, 8 wt%.
concentrations depending on the total protein content: R I ~ 8 for the 5 wt% protein system, compared with RI~ l0 for the 8 wt% protein system. The existence of a well-defined SDS/~-lactoglobulin ratio giving optimum emulsion gel strength in Fig. 2 is in stark contrast to the behaviour reported previously [7] for equivalent systems containing the anionic water-dispersable emulsifier DATEM, for which it was found that G' increases continuously with increasing R. One possible reason for this is that, at high surfactant concentrations, SDS displaces the protein completely from the emulsion droplet surface in a manner analogous to nonionic Tween 20, whereas DATEM does not induce such displacement [28]. Another possible reason is that, at high values of R, SDS forms nonassociating mixed micelles into which the protein molecules are separately dispersed and hence unavailable for cross-linking, whereas DATEM does not form such micelles. SDS has long been known by biochemists [3941] to bind strongly to ~-lactoglobulin (and other proteins) and to cause their "denaturation". It has been suggested [39] that the binding of SDS to a globular protein such as ~-lactoglobulin takes place in three stages. At low SDS concentrations, two or three molecules of the surfactant become tightly bound to high-affinity sites of the protein.
6
E. Dickinson, S.-72 Hong / Colloids Surfaces A." Physicochem. Eng. Aspects 127 (1997) 1-10
As a consequence of this binding, the protein molecule undergoes unfolding, exposing a large number ( ~ 2 0 ) of weaker potential binding sites. The second stage involves cooperative surfactant binding of a predominantly ionic nature [41 ]. This may be accompanied by a third stage of very weak surfactant binding, which is presumably highly micellar in nature [42], involving complete solubilization of protein into isolated surfactant association structures. Looking at the results in Fig. 2, we can speculate that specific binding occurs between SDS and J3-1actoglobulin in the emulsions at SDS concentrations up to R ~ 2. Then, for R > 2, there is the onset of co-operative SDS-]3-1actoglobulin binding, both at the oil-water interface and in the bulk phase; this induces extensive unfolding of the protein molecules, corresponding to the first step in the thermal gelation of the protein. As further protein unfolding undoubtedly occurs when the emulsion is heated, this means that the heat-treated emulsion containing SDS (R>2) contains more extensively unfolded 13-1actoglobulin molecules than the equivalent one without added surfactant (R=0) or with surfactant added at low concentration (R ~<2). Hence, when the protein molecules aggregate to form a three-dimensional gel network at R > 2, it seems reasonable to expect an enhanced number of mechanically important cross-links between denatured protein molecules, possibly involving increased numbers of hydrophobic interactions, disulphide linkages and intermolecular hydrogen bonds. In support of this hypothesis, separate experiments in this laboratory (data not reported here) have demonstrated that the elastic modulus of a heat-set [3-1actoglobulin gels becomes increased if the protein is partially unfolded first with urea prior to thermal treatment. In the highvolume-fraction emulsions studied here, the concentrations of protein and surfactant are both high, and so SDS-mediated cross-links between unfolded protein molecules are likely to be strongly favoured. Various kinds of interactions are possible, including protein cross-linking by (i) individual surfactant molecules, with the anionic headgroup binding to a cationic group on one protein molecule and the alkyl tail interacting with the non-polar region of another; and (ii) mixed
micelles reminiscent of those found in hydrophobically modified polymer surfactant gels [15]. The dependence of the gel strength on protein content in these systems is consistent with our previously reported results for [3-1actoglobulin emulsion gels [6-8]. Increasing the overall protein content necessarily increases the local number density of contact formed between denatured protein molecules, thereby leading to an increase in gel strength. Mechanistically, the observed increase in G' with increasing protein content for 2
-7 5
-7O
~
-85
~
o
~-
~ ~
-6 0
-55 -5 0
I
I
I
p
7
14
21
28
concentration/10-2w t
35
%
Fig. 3. Zeta potential, ~, of protein-stabilizedemulsiondroplets ( 1.6 wt% f3-1actoglobulin,38 wt% n-tetradecane,20 mM bis-tris buffer, pH 7) in solutionsof differentsurfactantconcentration.
E. Dickinson, S.-T. Hong / Colloids Surfaces A: Physicochem. Eng. Aspects 127 (1997) 1-10
Oil droplets coated solely with adsorbed 13-1actoglobulin have a zeta potential of ~= -58.4-t-1.0mV. A relatively small addition of SDS induces a sharp increase in zeta potential, which reaches a maximum (negative) value of ~ - 70 mV at surfactant concentration of about 0.06 wt%. With further surfactant addition, there is a gradual reduction in if, presumably caused by the displacement of adsorbed protein (and surfactant-protein complex) from the emulsion droplet surfactant by the excess of SDS molecules. At even higher surfactant concentrations, the measured zeta potential appears to increase again very slightly, possibly owing to the formation of a surfactant micellar structure at the oil droplet surface. Note that in these experiments we do not refer to the surfactant/protein ratio R. This is because the emulsion samples prepared for the electrophoretic mobility measurement were extremely dilute compared with those studied rheologically. Hence, in the mobility experiments, the actual values of surfactant/protein molar ratio R in Fig. 3 are hundreds of times larger than those in Fig. 2. We can reasonably infer from the results in Fig. 3 that, for the case of the concentrated emulsion systems, the addition of SDS at low levels leads to an increase in electrostatic repulsion between the droplets. This would be unfavourable for optimizing of the number of interdroplet crosslinks, because interactions between adsorbed protein molecules and between gelling protein molecules in the bulk phase would be significantly weakened. The importance of such interactions for the rheology of emulsion gels has been confirmed elsewhere [6,43]. The slight lowering of the gel strength in Fig. 2 due to small additions of SDS (i.e. R ~ 2 ) may be attributable to increased electrostatic repulsion between protein molecules caused by surfactant binding. In addition to binding to the protein, the SDS can displace adsorbed 13-1actoglobulin from the oil-water interface [44]. However, it is not obvious that this should have a serious negative effect on the rheological character of heat-set emulsion gels, since displacement of protein would be expected to form a more concentrated, and hence stronger, gel network between the droplets. A key issue here
7
is whether the droplets have a reinforcing or disrupting influence on the protein gel matrix. This is in part determined by the mechanical strength of the adsorbed layer itself, and in part by the extent to which the layer is mechanically connected to the matrix. Complexation between SDS and protein would be expected to have an influence on (i) interactions between protein molecules lying wholly within the surface layer and (ii) interactions between protein molecules within the layer and those outside. Interactions of the former type are related to the surface shear rheology of the adsorbed protein film. Fig. 4 shows the effect of SDS addition on the apparent surface shear viscosity of a IMactoglobulin film adsorbed from bulk solution (0.002 wt% protein, pH 7, 25°C) at the planar ntetradecane-water interface. The SDS was added at a surfactant/protein molar ratio R = 4 to the aqueous subphase in contact with the 1-day-old 13-1actoglobulin film. It was found that the apparent surface shear viscosity after the addition of surfactant decreases slightly under the influence of continuous shearing, but it later recovers almost to the same value as that for the original 1-day-old pure ~-lactoglobulin film when subsequently kept undisturbed for several hours. Shear-thinning behaviour is commonly observed with adsorbed protein films [32,33] or protein solutions containing anionic surfactant [13]. This may be attributed [33] to the slow breaking down of protein-surfac600
"~
%
450
T 300
150
I
I
I
I
I
10
20
30
40
50
60
time I h
Fig. 4. Influence of SDS on surface shear viscosity of 13-1actoglobulin adsorbed at the n-tetradecane-water interface at pH 7. Apparent surface viscosity, t/s, is plotted against time. The arrow denotes the point (after 24 h) at which the surfactant (/{=4) is introduced into the aqueous subphase.
8
E. Dickinson, S.-72 Hong/Colloids Surfaces A: Physicochem. Eng. Aspects 127 (1997) 1° 10
tant complexes under shear. Based on the emulsion results of Feijter et al. [44], it might be supposed that, even at rather low SDS concentrations, there should be a limited displacement of adsorbed 13-1actoglobulin from the planar oil-water interface (e.g.~30% displacement at R ~ 2 ) . Nevertheless, in contrast to Tween 20, which induces a rapid fall in the surface shear viscosity [33], what we see in Fig. 4 is that the apparent shear viscosity of the adsorbed protein film following SDS addition is almost the same as that for the original 1-day-old pure 13-1actoglobulin film. Moreover, this behaviour extends to higher SDS concentrations (R ~ 16), as indicated in Table 1. Since the steady-state interfacial shear rheology stays relatively constant within experimental error ( _ + 1 0 m N m -1 s) over this wide range of R values, it would appear that the large effect of SDS on the rheology of the heat-set emulsion gels cannot simply be attributed to the effect of the surfactant on the viscoelasticity of the adsorbed protein layer. A more likely explanation, then, is that the large reduction in G' at high R values in the concentrated emulsions (Fig. 2) is due to a disruption by the surfactant of interactions between the adsorbed layer and the unadsorbed protein, and also, perhaps more importantly, to the disruption of the heat-set protein gel network between the droplets due to solubilization of protein into separate unconnected mixed micelles in the presence of the excess surfactant. In work on emulsions, it has been separately shown that SDS [44] or sodium lauryl ether sulTable 1 Steady-state apparent surface shear viscosity, ~/8, of [3-1actoglobulin adsorbed at the planar n-tetradecane-water interface (0.002 wt% protein, 2 m M bis-tris buffer, pH 7, 25°C) containing various amounts of added SDS (expressed as surfactant/protein molar ratio, R) R
r/~ (mN m -1 s)
0 1 4 16 64 520 1800
560 540 550 540 450 210 80
phate [30] produce almost complete displacement of interfacial protein at a surfactant concentration of ca. 0.3wt% ( R ~ 6 0 for SDS). However, the surface theology data in Table l indicate that 13-1actoglobulin is still making a major contribution to the interfacial shear viscosity at R values at least an order of magnitude larger than this. This may be because of the nature of the surface theology experiment which probes interfacial regions further away from the surface [45]. That is, even though the interface may be fully covered with a primary SDS monolayer, it is likely that considerable amounts of protein-surfactant complex still remain in the interfacial region, interacting weakly with the primary adsorbed surfactant layer, and that this in turn affects the measured interracial shear rheology. Based on the information discussed so far, it may be concluded that the small-deformation bulk rheology of a heat-set protein-stabilized emulsion gel containing SDS depends on the balance between positive contributions to G', arising from enhanced surfactant-mediated cross-links between the protein molecules, and negative contributions to G', arising from protein-protein and droplet ..... droplet electrostatic interactions (low R) and disruption of the denatured protein gel network by protein solubilization into surfactant micelles (high R). The relative contribution of these different factors would be expected to be sensitively influenced by the surfactant concentration. Taken as a whole, the results seem to suggest that the general trend of behaviour due to addition of SDS is mostly dependent on the relative amounts of [3-1actoglobulin and anionic surfactant in the system, and much less upon any special effect involving the surface of the dispersed droplets. This statement is reinforced by the rheological data in Fig. 5 for the effect of SDS on the viscoelasticity of 14 wt% heat-set 13-1actoglobulin gels (no dispersed oil droplets). The storage and loss moduli are plotted against R over the range 0---14. It can be seen that the general trend of changing gel strength is actually very similar to that exhibited by the heat-set emulsion gels in Fig. 2. That is, increasing the SDS concentration to R ~ 2 leads to a small decrease in the elastic modulus of the gel. For R >2, G' increases sharply, reaching a maxi-
E. Dickinson, S.-T. Hong / Colloids Surfaces A." Physicochem. Eng. Aspects 127 (1997) 1 10
9
400
4. Conclusions
3O0
We have shown that the viscoelastic properties of heat-set 13-1actoglobulin emulsion gels containing SDS are greatly dependent on protein concentration and the surfactant/protein ratio. At very low R values, the gel strength tends to decline as SDS binds strongly to specific binding sites. With increasing SDS concentration above R~2, the elastic modulus starts to increase corresponding to the SDS concentration at which cooperative surfactant binding and associated globular protein unfolding take place. Further larger additions of SDS result in a reduction in gel strength, and eventually gelation is prevented altogether (R > 10). This behaviour contrasts sharply with the previously known behaviour of systems containing the anionic water-dispersible emulsifier DATEM, in which the emulsion gel strength was found to increase continuously with R, and with systems containing the non-ionic emulsifier Tween 20, in which the emulsion gel strength was found to fall dramatically for R > 1. as the protein begins to become competitively displaced from the emulsion droplet surface. We speculate here that the increased gel strength at intermediate surfactant concentrations (from R ~ 3 to R ~ 8) is attributable to increased surfactant-induced protein unfolding and additional surfactant-mediated cross-links via surfactant molecule or mixed micellar aggregates involving assembled surfactant molecules and unfolded protein molecules. On the other hand. the increased electrostatic repulsion due to surfactant binding may account for the observed decrease in the gel strength at low R. and complete solubilization of protein into separate mixed micelles may be the reason for the low gel strength (and eventual lack of gelation) at high R. The surface shear rheology results indicate that protein-surfactant interactions within the adsorbed layer at the oil water interface have no significant correlation to the bulk rheology of heat-set protein-stabilized emulsion gels containing added SDS. Overall, it is concluded that the rheology of a heat-set protein-stabilized emulsion gel containing SDS depends on the balance between positive and negative contributions to cross-linking arising from different kinds of
200
6 100
0
, 2
4
,
6
8
10
12
14
R
Fig. 5. Influenceof SDS concentration on the viscoelasticityof heat-set protein gel (14 wt% [3-1actoglobulin,pH 7, 3WC) in the absence of oil droplets. The storage modulus G' (D) and the loss modulus G" (Ill) at l Hz are plotted against the surfactant/protein molar ratio R.
mum high value of G ' ~ 4 0 0 Pa at R ~ 6 . This is then followed by a sudden drop in the elastic modulus with further increase in surfactant concentration up to R ~ 1 2 . For high surfactant contents (R> 12), a cross-over of G' and G" is observed, indicating the loss of gelation. It may be noteworthy that, whilst the 8 wt% emulsion system becomes a non-gelled viscous solution at R>~ 10, the 14 wt% protein system becomes nongelling at a slightly higher R value (i.e. R~> 12). As with the emulsions, the binding of surfactant molecules to 13-1actoglobulin could increase the protein-protein repulsion, and so alter the aggregation behaviour of the protein when heated and hence its rheology [18-21]. Therefore, the increased electrostatic repulsion between the protein molecules could account for the slight drop in gel strength observed at low surfactant concentrations (0 < R ~<2). In the vicinity of the maximum gel strength (4 ~
10
E. Dickinson, S.-T. Hong / Colloids Surfaces A: Physicochem. Eng. Aspects 127 (1997) 1-10
protein-surfactant interactions, essentially irrespective of the presence of dispersed emulsion droplets.
[19] [20] [21]
References [1] E. Dickinson, G. Stainsby, Colloids in Foods, Applied Science, London, 1982. [2] E. Dickinson, C.M. Woskett, in: R.D. Bee, P. Richmond, J. Mingins (Eds.), Food Colloids, Royal Society of Chemistry, Cambridge, 1989, p. 74. [3] E. Dickinson, ACS Symp. Ser. 448 (1991) 114. [4] E. Dickinson, in: E.D. Goddard, K.P. Ananthapadmanabhan (Eds.), Interactions of Surfactants with Polymers and Proteins, CRC Press, Boca Raton, FL, 1993, p. 295. [5] D.J. McClements, F.J. Monahan, J.E. Kinsella, J. Texture Stud. 24 (1993) 411. [6] E. Dickinson, S.-T. Hong, J. Agric. Food Chem. 43 (1995) 2560. [7] S.-T. Hong, E. Dickinson, in: G.O. Phillips, P.A. Williams, D.J. Wedlock (Eds.), Gums and Stabilisers for the Food Industry, Oxford University Press, Oxford, 1996, Vol. 8, p. 319. [8] E. Dickinson, Y. Yamamoto, Food Hydrocolloids, 10 (1996) 301. [9] J. Steinhardt, J.A. Reynolds, Multiple Equilibria in Proteins, Academic Press, New York, 1969. [10] E.D. Goddard, Colloids Surfaces 19 (1986) 255. [11] M.N. Jones, Chem. Soc. Rev. 21 (1992) 127. [12]E.D. Goddard, K.P. Ananthapadmanabhan (Eds.), Interactions of Surfactants with Polymers and Proteins, CRC Press, Boca Raton, FL, 1993. [13] J. Greener, B.A. Contestable, M.D. Bale, Macromolecules 20 (1987) 2490. [14] A.K. Mor6n, A. Khan, Langmuir 11 (1995) 3636. [15] B. Magny, I. Iliopoulos, R. Audebert, L. Piculell, B. Lindman, Prog. Colloid Polym. Sci. 89 (1992) 118. [16] C.V. Morr, E.Y.W. Ha, Crit. Rev. Food Sci. Nutr. 33 (1993) 431. [17] A. Kilara, in: N.S. Hettiarachchy, G.R. Ziegler (Eds.), Protein Functionality in Food Systems, Marcel Dekker, New York, 1994, p. 325. [18] A.H. Clark, C.D. Lee-Tuffnell, in: J.R. Mitchell, D.A. Ledward (Eds.), Functional Properties of Food
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