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Interfaces: their role in foam and emulsion behaviour
ELS EVI E R
Current Opinion in Colloid & Interface Science 5 (2000) 176-181 www.elsevier.nl/locate/cocis
Interfaces: their role in foam and emulsion behaviour P.J. Wilde" Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK
Abstract
In this paper, recent progress in our knowledge of how the interface influences the formation and stability of emulsions has been reviewed. Attention was focused on the effect of molecular structure, interfacial rheology, competitive adsorption and interfacial structure and composition.0 2000 Elsevier Science Ltd. All rights reserved. Keywords: Interface; Protein; Surfactant; Competitive adsorption
1. Introduction
With the exception of microemulsions, liquid foams and emulsions are in general, thermodynamically unstable. The immiscible dispersed phase will only remain as a fine dispersion for a finite period, as the dispersed droplets or bubbles will eventually coalesce and separate out into the component phases. The period of stability strongly depends on the characteristics of the interface separating the dispersed and continuous phases. The interface is composed of surface-active molecules, which can adsorb from the dispersed or continuous phase. These molecules need to be amphiphilic, and thus are attracted to the interfacial region so that their component hydrophilic and hydrophobic moieties may associate with the respective polar and non-polar phases. There are two classes of surface-active molecules, which can stabilise foams and emulsions:
1. Surfactants - these include detergents, emulsifiers and lipids. They may be water or oil soluble,
* Tel.: + 44-1603-255258; fax: + 44-1603-507723. E-mail address: [email protected] (P.J. Wilde).
and usually form a compact adsorbed layer with a low interfacial tension. 2. Polymers - amphiphilic macromolecules, the most commonly used are proteins. They typically form a visco-elastic, irreversibly adsorbed layer. The creation of foams and emulsions essentially requires the formation of fine bubbles or droplets. Apart from the energy input and the physical properties of the component phases (e.g. viscosity), the main factor controlling droplet size is the interfacial tension. Droplets become more deformable as the interfacial tension is lowered, making them easier to disperse. The ability to create foams and emulsions has been correlated with the rate of change of the interfacial tension [l-31. The important timescales for foam and emulsion formation can be sub-millisecond; therefore, the ability of a surfactant or polymer to adsorb rapidly is critical for foam and emulsion formation. The stabilisation of foams and emulsions against coalescence require different surface properties. Long-range repulsive forces can be in the form of electrostatic or steric repulsion, which prevent the close approach of dispersed phase particles. However, in concentrated foams and emulsions, the long-range
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repulsion is overcome, and other stability mechanisms become dominant. These mechanisms depend on the class of molecule used to stabilise the adsorbed layer. Surfactants stabilise foams and emulsions most effectively if they form a fluid adsorbed layer. This allows them to migrate to regions with a reduced surfactant concentration, due to perturbation during creation, mixing or transport processes. This is known as the Marangoni mechanism. In contrast, polymers are most effective when they form a solid visco-elastic adsorbed layer. This is most commonly observed in proteins, which adsorb, partially unfold and form strong interactions. This results in a visco-elastic adsorbed layer, the strength of which has been correlated with foam and emulsion stability [4,5]. The unfolding of proteins at interfaces is influenced by the structure in solution, such that flexible proteins will unfold quickly and lower the interfacial tension rapidly [5], whereas globular proteins, which have more intramolecular bonds stabilising their structure, unfold more slowly. However, they tend to form stronger intermolecular interactions and stabilise against coalescence very effectively [51. Changing the solution structure by various means has been used as a tool for improving protein functionality, probably by inducing a change in adsorbed conformation. A problem that occurs particularly in food foams and emulsions, is that there is often a mixture of polymers and surfactants competing for the interface. In concentration regimes where a mixed polymersurfactant interface exists, a reduction in stability is often observed. The underlying mechanism is thought to be a competition between the two stability mechanisms. The surfactants weaken the visco-elasticity of the adsorbed protein layer [4,6-81, and the polymers retard the fluidity of the surfactants. The result is a foam or emulsion that is less stable than those formed by the individual surfactants or polymers [9,10]. It is, therefore, not surprising that current progress in this field has tended to concentrate on: (i) adsorbed protein structure; (ii) the rheology; and (iii) the composition of interfaces. Therefore, I have reviewed the current literature in this field in these specific areas.
and Norde [ l l ] used an array of quartz plates to increase the number of interfaces that could be interrogated by circular dichroism. They showed that the adsorbed immunoglobulin possessed a different spectrum depending on the nature of the surface. Moulin et al. used a microfabricated cantilever to measure the surface stress induced by proteins adsorbed onto a gold substrate [12]. They could apparently follow long-term unfolding and interaction processes, which were found to be very different between immunoglobulin and BSA, although no firm conclusions about the source of these differences could be drawn. The limitation of these techniques in foams and emulsions was the correlation of behaviour at a solid surface with that at a fluid one. If this could be done, then it would be possible to explain the effects seen by Murray and Liang [13]. They found that spray-drying protein solutions had a detrimental effect on foam stability. This was supported by changes in their adsorption behaviour suggesting a conformational change induced by the drying process. The presence of the sugar trehalose during drying seemed to have a positive effect on foaming properties, especially for P-lactoglobulin. The corresponding adsorption behaviour suggested that the trehalose protected the proteins against denaturation. This has also been confirmed by Clarkson et al. where the presence of trehalose was seen to protect against surface denaturation induced by foaming [141, although they found that this was a minor effect compared to the effects of ionic strength and pH. Small differences in protein structure can infer major changes in functionality. The genetic variants A, B and C of P-lactoglobulin differ by only two amino acid residues, yet this was found to cause significant changes in adsorption behaviour [ 151 and emulsification properties [16]. The substitution of an aspartic acid residue in variant A to a glycine in B at residue 64 appears to increase the rate of adsorption, and the development of an elastic interface of variant B [151. Although variant B had poorer emulsification properties, its long-term stability to coalescence was higher [16].
2. Adsorbed protein structure
3. Interfacial rheology
Secondary and tertiary structures of proteins in solution can be easily determined using a range of spectrometric techniques. The structure of adsorbed proteins is more difficult due to various technical complications. The signal intensity from a single interface is usually small and the resultant signal to noise ratio can make interpretation difficult. Measurements of proteins adsorbed onto particles or droplets creates light scattering problems. Vermeer
The surface rheology of adsorbed protein layers has long been known to be important for the stabilisation of foams and emulsions [4,17]. The visco-elastic properties of the surface have often been correlated with functionality [2-4,181. A recent review by Dickinson [19'1 brought together certain aspects of the structure and surface rheology of proteins, and the correlation with the formation and stability of foams and emulsions. A fascinating article by Izmailova et
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al. [20’] reviewed the contribution made by Rehbinder to this area and offered new results showing how the coalescence stability of protein foams and emulsions can be directly related to the mechanical properties of the adsorbed protein layer. Rehbinder put forward the concept that the elasticity of protein interfaces was responsible for their stabilising ability. This concept was developed further with some new results confirming the idea that it is not simply the value of the elastic modulus that is important for stability, but the stress at which the elasticity breaks down. However care must also be taken, as some proteins such as p-casein are excellent at stabilising emulsions by steric repulsion, but have very poor surface rheological properties [21]. The surface visco-elastic properties of proteins can also be important for formation of and drainage from foams. Prins [22’] showed, by using the overflowing cylinder, that proteins can slow the flow of liquid close to the surface. This can retard liquid drainage from foams, and is thought to contribute to the slower drainage rates observed in protein-stabilised foams compared to those stabilised by surfactants. In addition, Prins also postulated that this ‘stagnant surface behaviour’ enabled the bubbles and droplets to be more easily dispersed when the surface tension was taken into account. In the absence of protein, the droplet break-up was strongly dependent on the relative viscosities of the continuous and dispersed phases. In contrast, in the presence of protein, the droplets were dispersed more easily and independently of liquid viscosity. This clearly demonstrated the contribution made by the surface rheology of proteins to the functionality of foams and emulsions. The presence of surfactants will reduce the surface visco-elasticity of adsorbed protein layers [4,6,7] often resulting in a drop in coalescence stability [4,9,10,23]. Chen and Dickinson showed that by displacing proteins from emulsion droplets, the visco-elasticity of emulsion gels could be significantly reduced [24]. The interaction between the emulsion droplets and the gel matrix was thought to be influenced by the amount of adsorbed protein. Protein adsorption and the development of the visco-elastic surface can be significantly affected by protein conformation. Roth et al. [25] found that ageing and heat treating proteins at the interface could result in higher surface viscosities, depending on the temperature, and the length of heat treatment. Certainly the interfaces heated to the higher temperatures studied formed a more viscous interface. Some of the more visco-elastic interfaces were also more resistant to the effects of added surfactant, although there was no clear correlation for all the treatments studied. Petkov et al. demonstrated a new method of simultaneously measuring the surface dilational and shear parameters in protein films
[26]. Again, this technique demonstrates how sensitive surface rheology is to the disruptive effects which surfactants have on protein adsorbed layers. One way to restore stability to a surfactant destabilised foam is to restore the surface visco-elasticity. Propylene glycol alginate (PGA) has been used to improve beer foam stability. A detailed study of this improvement showed that PGA restored the surface elasticity of a mixed protein-surfactant interface [27]. This was thought to be due to an electrostatic ‘crosslinking’ mechanism, which partially restored the protein-protein interactions that had been disrupted by the presence of surfactant. The interfacial rheology of wheat proteins is of importance to the baking industry. Yet a recent review of the area [28] shows that there is distinct gap in our knowledge on effect of their surface properties on baking quality. It is likely, therefore, that the surface properties of wheat proteins will receive much attention in the near future. It is not only the surface rheology of proteins that is important to functionality. Recent studies on the surface rheology of surfactants have also shown a correlation with foam formation and stability. Kanicky et al. [291 showed that sodium laurate solutions showed maximal surface viscosity at pH values close to their pK,. This corresponded with maximal foam formation, and foam stability. They showed that the interfacial packing was highest at the pK,, as expected, as the electrostatic repulsion between the molecules was minimal. This also corresponded with improved emulsification and reduced water evaporation. Changing the chain length of the sodium laurate changed the pK, such that the foaming properties were more dependent on the pH than the molecular structure of the surfactant. Fruhner et al. studied a range of different surfactants [30’]. By using the oscillating bubble technique at high frequencies (up to 500 Hz), they were able to obtain surface dilational visco-elastic data for soluble surfactants. They found that the surface needed to develop a significant viscous component before the solutions were able to create a stable foam. At very low concentrations, below that which would support a stable foam, purely elastic behaviour was observed. At high concentrations, the surfactants which were able to support foams such as SDS, CTAB and Triton-X 100, developed a viscous component. Surfactants which did not form stable foams, such as nonanol and dodecanol, remained purely elastic. The differences are thought to be due to the improved hydration of the headgroups belonging to the foam active group of surfactants. 4. Surface composition and structure Competitive destabilisation of proteins by surfac-
P.J. Wilde /Current Opinion in Colloid & Interface Science 5 (2000) 176-181
tants has been an area of intense interest in food colloids for many years [4,6,9,10,231. Although it is known that surfactants reduce the surface visco-elasticity of protein films, as discussed in Section 3, until recently, however, the mechanisms underlying this effect remained elusive. Our understanding of the competitive adsorption mechanism has been significantly advanced recently by the ability to visualise the structure of mixed protein-surfactant interfaces. Atomic force microscopy (AFM) is a powerful method for imaging surface detail on flat substrates at molecular resolution. It has been used to study protein adsorption onto both solid [31] and liquid interfaces [32]. Recent work by Morris et al. [33",34'] have been able to image mixed protein-surfactant interfaces. In contrast to the assumption that mixed interfaces contain a homogenous distribution of protein and surfactant, these studies clearly show a demixing phenomenon, an example of which is shown in Fig. 1. The power of the technique is that it can impart interfacial thickness information with extremely high spatial resolution. This has led to the proposed 'orogenic' model of protein displacement. The observations showed that the surfactant domains exerted a lateral surface pressure and compressed protein regions. As more surfactant was adsorbed, the surface pressure increased until the protein regions collapsed and were eventually displaced. These studies have helped explain: (i) why the stability of protein foams and emulsions is often affected at very low concentrations of surfactant, before any protein is displaced [10,231; (ii) observed increases in the thickness or concentration of adsorbed protein layers observed in mixed protein-surfactant interfaces [19,35']; and (iii) why proteins with a higher viscoelastic modulus are more difficult to displace [lo]. The
Fig. 1. AFM image of a Langmuir-Blodgett film of a mixed plactoglobulin-Tween 20 film at the air-water interface. The light regions denote the surfactant domains, and the dark regions represent the thicker protein regions. Image size is 2 x 2 pm.
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Fig. 2. Brownian dynamic simulation of the competitive displacement of protein-like (dark) particles, by surfactant-like displacer (light) particles. Attention is drawn to the similarity with Fig. 1. Reproduced from [390°] with permission.
only weakness to this approach is that the interface needs to be transferred to a solid substrate by the Langmuir-Blodgett method. The orogenic mechanism was confirmed in situ by following the displacement of a protein from a solid surface [36']. In situ measurements on fluid interfaces can be performed using Brewster angle microscopy (BAM). Studies by Patino et al. [37,38"] have shown that it is possible to image interfaces formed by proteins and surfactants. The surfactants have a high refractive index compared to the proteins, allowing discrimination between the two species. They found that mixed p-casein and monopalmitin films phase separated at the interface. They also showed an increase in interfacial film thickness. Although BAM has much lower resolution than AFM, the approaches are highly complimentary, as BAM can observe these phenomena in situ at the interface. The orogenic model was further supported in a separate computer simulation study. By using a Brownian dynamics approach Wijmans and Dickinson [39"] simulated the competitive displacement process. The results showed a remarkable similarity to the experimental observations made using AFM [33",34'] and BAM [38"], whereby the surfactants adsorbed and formed distinct domains, compressing and increasing the thickness of the protein regions. Fig. 2 shows an example of one of these simulations, and the likeness between that and Fig. 1 is quite remarkable. They also support the importance of the rheology of the protein network in the process, and also that repulsive forces between the protein and surfactant promote the phase separation.
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5. Conclusions
Understanding the role of interfaces on the formation and stability of foams and emulsions has been significantly advanced in recent years. There appears to be a consensus of opinion that the surface rheological behaviour of both proteins and surfactants is of paramount importance. However, rheology is a complex area, and the specific surface rheological parameters, which are most important for these applications, are still being defined. The main objectives for the future are to understand how factors such as molecular structure, component interactions and surface composition of mixed interfaces, influence and control the various interfacial rheological parameters which are regarded as important contributory factors to foam and emulsion behaviour.
Acknowledgements
The author acknowledges the support of the BBSRC through their core strategic grant to the Institute of Food Research. References and recommended reading
DO
of special interest of outstanding interest
[l] Graham DE, Phillips MC. The conformation of proteins at the a/w interface and their role in stabilising foams. In: Akers RJ, editor. Foams. London: Academic Press, 1976: 195-215. [2] Kim SH, Kinsella JE. Surface activity of food proteins: relationships between surface pressure development, viscoelasticity of interfacial films and foam stability of bovine serum albumin. J Food Sci 1985;501526-1530. [3] Nakai S, Li-Chan E. Recent advances in structure and function of food proteins, QSAR approach. Crit Rev Food Sci Nutr 1993;33:477-499. [4] Halling PJ. Protein-stabilised foams and emulsions. CRC Crit Rev Food Sci Nutr 1981; Oct: 155-203. [5] Mitchell JR. Foaming and emulsifying properties of proteins. In: Hudson BJF, editor. Developments in food proteins 4. London: Elsevier, 1986:291-338. [6] MacRitchie F. Proteins at interfaces. In: Anfinsen CB, Edsall JT, Richards FM, editors. Advances in protein chemistry vol. 32. New York Academic Press, 1978:283-326. [7] Courthaudon J-L, Dickinson E, Matsumura Y, Clark DC. Competitive adsorption of p-lactoglobulin Tween 20 at the oil-water interface. Colloids Surf 1991;56:293-300. [8] Wiistneck R, Kragel J, Miller R, Wilde PJ, Clark DC. Adsorption of surface-active complexes between p-casein, Plactoglobulin and ionic surfactants and their shear rheological behaviour. Colloid Surf A: Physicochem Eng Aspects 1996;114255-265. [9] Coke M, Wilde PJ, Russell EJ, Clark DC. The influence of surface composition and molecular diffusion on the stability of foams formed from protein detergent mixtures. J Colloid Interface Sci 1990;138:489-504.
+
[lo] Cornec M, Wilde PJ, Gunning PA et al. Emulsion stability as affected by competitive adsorption between an oil-soluble emulsifier and milk proteins at the interface. J Food Sci 1998;63:39-43. [ l l ] Vermeer AWP, Norde W. CD spectroscopy of proteins adsorbed at flat hydrophilic quartz and hydrophobic Teflon surfaces. J Colloid Interface Sci 2000;225:394-397. [12] Moulin AM, Oshea SJ, Badley RA, Doyle P, Welland ME. Measuring surface-induced conformational changes in proteins. Langmuir 1999;15:8776-8779. [13] Murray BS, Liang H-J. Enhancement of the foaming properties of protein dried in the presence of trehalose. J Agric Food Chem 1999;47:4984-4991. [14] Clarkson JR, Cui ZF, Darton RC. Effect of solution conditions on protein damage in foam. Biochem Eng J 2000;4: 107-114. [15] Mackie AR, Husband FA, Holt C, Wilde PJ. Adsorption of p-lactoglobulin variants A and B to the air-water interface. Int J Food Sci Technol 1999;34:509-516. [16] Euston SR, Hirst RL, Hill JP. The emulsifying properties of P-lactoglobulin genetic variants A, B and C. Colloid Surf B - Biointerfaces 1999;12:193-202. [17] Neurath H, Bull HB. The surface activity of proteins. Chem Rev 1938;23:391-435. [18] Phillips MC. Protein conformation at liquid interfaces and its role in stabilising emulsions and foams. Food Technol 1981;35:50-57. [19] Dickinson E. Adsorbed protein layers at fluid interfaces: interactions, structure and surface rheology. Colloid Surf B - Biointerfaces 1999;15:161-176. A useful review of the properties of adsorbed protein layers. Particular attention is paid to the adsorption and surface rheology of P-lactoglobulin in the presence and absence of surfactants. A simulation model of protein adsorption, and neutron reflectivity data of competitive displacement of P-lactoglobulin is presented and discussed. [20] Izmailova VN, Yampolskaya GP, Tulovskaya ZD. Development of the Rehbinder’s concept on structure-mechanical barrier in stability of dispersions stabilised with proteins. Colloid Surf A - Physicochem Eng Aspects 1999;160:89-106. Rehbinder’s concept that proteins stabilise foams and emulsions by forming a mechanical barrier was proposed around 70 years ago. This manuscript reviews this concept and presents new data showing the importance of the elastic limit of the protein adsorbed layer. Beyond this limit, the surface begins to flow, and the probability of coalescence increases dramatically. [21] Husband FA, Wilde PJ, Mackie AR, Garrood MJ. A comparison of the functional and interfacial properties of p-casein and dephosphorylated p-casein. J Colloid Interface Sci 1997;195:77-85. [22] Prins A. Stagnant surface behaviour and its effect on foam and film stability. Colloids Surf A - Physicochem Eng Aspects 1999;149:467-473. Prins demonstrates how the elastic surface formed by proteins can alter the fluid flow adjacent to that surface. This is referred to as a stagnant layer. The effect is shown to not only reduce the drainage of liquid from foams, but is also shown to aid bubble and droplet break-up, by inducing large surface tension gradient across the surface of a droplet under shear. [23] Dickinson E, Owusu RK, Williams A. Orthokinetic destabilization of a protein-stabilised emulsion by a watersoluble surfactant. J Chem Soc Faraday Trans 1993;89: 865-866. [24] Chen J, Dickinson E. Effect of monoglycerides and diglycerol-esters on viscoelasticity of heat-set whey protein emulsion gels. Int J Food Sci Technol 1999;34:493-501.
P.J. Wilde /Current Opinion in Colloid & Interface Science 5 (2000) 176-181 Roth S, Murray BS, Dickinson E. Interfacial shear rheology of aged and heat-treated p-lactoglobulin films: displacement by nonionic surfactant. J Agric Food Chem 2000;48: 1491-1497. Petkov JT, Gurkov TD, Campbell BE, Bonvankar RP. Dilatational and shear elasticity of gel-like protein layers on air/water interface. Langmuir 2000;16:3703-3711. Sarker DK, Wilde PJ. Restoration of protein foam stability through electrostatic propylene glycol alginate-mediated protein-protein interactions. Colloid Surf B - Biointerfaces 1999;15:203-213. Ornebro J, Nylander T, Eliasson AC. Interfacial behaviour of wheat proteins. J Cereal Sci 2000;31:195-221. Kanicky JR, Poniatowski AF, Mehta NR, Shah DO. Cooperativity among molecules at interfaces in relation to various technological processes: Effect of chain length on the pK(a) of fatty acid salt solutions. Langmuir 2000;16:172-177. Fruhner H, Wantke KD, Lunkenheimer K. Relationship between surface dilational properties and foam stability. Colloids Surf A - Phvsicochem Eng- Aspects - 2000;162:193-202. Surface dilational parameters of surfactants were measured at high frequencies by the oscillating bubble technique. They demonstrated that for a surfactant to create stable foams, the surface modulus had to develop a significant viscous component. [31] McMaster TJ, Miles MJ, Shewry PR, Tatham AS. In situ surface adsorption of the protein C hordein using atomic force microscopy. Langmuir 2000;16:1463-1468. [32] Johnson CA, Yuan Y, Lenhoff AM. Adsorbed layers of ferritin at solid and fluid interfaces studied by atomic force microscopy. J Colloid Interface Sci 2000;223:261-272. [33] Mackie AR, Gunning AP,Wilde PJ, Morris VJ. The orogenic OD displacement of protein from the air/water interface by surfactant. J Colloid Interface Sci 1999;210:157-166. AFM was used to image Langmuir-Blodgett films of mixed protein surfactant interfaces. The adsorbed proteins and surfactants were found to be phase separated. A new physical mechanism of competitive displacement termed ‘orogenic’ displacement was proposed, whereby the surfactant domains exerted a lateral surface pressure compressing the protein layer. As the surface pressure increased, the protein regions became more compressed causing it to collapse, crumple and finally be displaced. The ease by which a protein was displaced was directly correlated with its surface rheological properties. [34] Mackie AR, Gunning AP, Wilde PJ, Morris VJ. Orogenic
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displacement of protein from the oil/water interface. Langmuir 2000;16:2243-2247. The orogenic mechanism was further investigated at the oil water interface and the generality of the mechanism was confirmed. The origins of the process were also investigated. The proteins formed a disordered assembly at the interface, and small defects in the surface packing were thought to be responsible for allowing surfactants to adsorb into the protein layer. [35] Cornec M, Narsimhan G. Adsorption and exchange of plactoglobulin onto spread monoglyceride monolayers at the air-water interface. Langmuir 2000;16:1216-1225. [36] Gunning AP,Mackie AR, Wilde PJ, Morris VJ. In-situ observation of surfactant-induced displacement of protein by atomic force microscopy. Langmuir 1999;15:4636-4640. AFM could not be used to follow competitive displacement in situ at a fluid interface. The process was therefore monitored on a solid, hydrophobic surface in real time. The same orogenic displacement process was found to occur with the solid substrate, further supporting the previous studies of Langmuir-Blodgett films. [37] Patino JMR, Sanchez CC, Nino MRR. Is Brewster angle microscopy a useful technique to distinguish between isotropic domains in p-casein-monoolein mixed monolayers at the air-water interface. Langmuir 1999;15:4777-4788. [38] Patino JMR, Sanchez CC, Nino MRR. Analysis of p-caseinOD monopalmitin mixed films at the air-water interface. J Agric Food Chem 1999;47:4998-5008. Brewster angle microscopy was used to image mixed protein-surfactant systems, at the air-water interface. This demonstrated for the first time that the orogenic mechanism of competitive displacement was a valid model, because the measurements were performed in situ on a fluid interface, and the same phase separation and thickening of the interfacial layers were observed. [39] Wijmans CM, Dickinson E. Brownian dynamics simulation of 00 the displacement of a protein monolayer by competitive absorption. Langmuir 1999;15:8344-8348. Brownian dynamic simulations of the competitive displacement process between protein-like and surfactant-like particles, independently displaced many features of the orogenic mechanism observed experimentally. The simulations demonstrated the importance of the strength of the protein network and showed that repulsive forces between the protein and surfactants encouraged phase separation. This approach should prove to be a powerful tool for investigating the role of specific inter-molecular interaction processes on the development of interfacial structure.
Current Opinion in Colloid & Interface Science 5 (2000) 176-181 www.elsevier.nl/locate/cocis
Interfaces: their role in foam and emulsion behaviour P.J. Wilde" Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK
Abstract
In this paper, recent progress in our knowledge of how the interface influences the formation and stability of emulsions has been reviewed. Attention was focused on the effect of molecular structure, interfacial rheology, competitive adsorption and interfacial structure and composition.0 2000 Elsevier Science Ltd. All rights reserved. Keywords: Interface; Protein; Surfactant; Competitive adsorption
1. Introduction
With the exception of microemulsions, liquid foams and emulsions are in general, thermodynamically unstable. The immiscible dispersed phase will only remain as a fine dispersion for a finite period, as the dispersed droplets or bubbles will eventually coalesce and separate out into the component phases. The period of stability strongly depends on the characteristics of the interface separating the dispersed and continuous phases. The interface is composed of surface-active molecules, which can adsorb from the dispersed or continuous phase. These molecules need to be amphiphilic, and thus are attracted to the interfacial region so that their component hydrophilic and hydrophobic moieties may associate with the respective polar and non-polar phases. There are two classes of surface-active molecules, which can stabilise foams and emulsions:
1. Surfactants - these include detergents, emulsifiers and lipids. They may be water or oil soluble,
* Tel.: + 44-1603-255258; fax: + 44-1603-507723. E-mail address: [email protected] (P.J. Wilde).
and usually form a compact adsorbed layer with a low interfacial tension. 2. Polymers - amphiphilic macromolecules, the most commonly used are proteins. They typically form a visco-elastic, irreversibly adsorbed layer. The creation of foams and emulsions essentially requires the formation of fine bubbles or droplets. Apart from the energy input and the physical properties of the component phases (e.g. viscosity), the main factor controlling droplet size is the interfacial tension. Droplets become more deformable as the interfacial tension is lowered, making them easier to disperse. The ability to create foams and emulsions has been correlated with the rate of change of the interfacial tension [l-31. The important timescales for foam and emulsion formation can be sub-millisecond; therefore, the ability of a surfactant or polymer to adsorb rapidly is critical for foam and emulsion formation. The stabilisation of foams and emulsions against coalescence require different surface properties. Long-range repulsive forces can be in the form of electrostatic or steric repulsion, which prevent the close approach of dispersed phase particles. However, in concentrated foams and emulsions, the long-range
1359-0294/00/$ - see front matter 0 2000 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 9 - 0 2 9 4 ( 0 0 ) 0 0 0 5 6 - X
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repulsion is overcome, and other stability mechanisms become dominant. These mechanisms depend on the class of molecule used to stabilise the adsorbed layer. Surfactants stabilise foams and emulsions most effectively if they form a fluid adsorbed layer. This allows them to migrate to regions with a reduced surfactant concentration, due to perturbation during creation, mixing or transport processes. This is known as the Marangoni mechanism. In contrast, polymers are most effective when they form a solid visco-elastic adsorbed layer. This is most commonly observed in proteins, which adsorb, partially unfold and form strong interactions. This results in a visco-elastic adsorbed layer, the strength of which has been correlated with foam and emulsion stability [4,5]. The unfolding of proteins at interfaces is influenced by the structure in solution, such that flexible proteins will unfold quickly and lower the interfacial tension rapidly [5], whereas globular proteins, which have more intramolecular bonds stabilising their structure, unfold more slowly. However, they tend to form stronger intermolecular interactions and stabilise against coalescence very effectively [51. Changing the solution structure by various means has been used as a tool for improving protein functionality, probably by inducing a change in adsorbed conformation. A problem that occurs particularly in food foams and emulsions, is that there is often a mixture of polymers and surfactants competing for the interface. In concentration regimes where a mixed polymersurfactant interface exists, a reduction in stability is often observed. The underlying mechanism is thought to be a competition between the two stability mechanisms. The surfactants weaken the visco-elasticity of the adsorbed protein layer [4,6-81, and the polymers retard the fluidity of the surfactants. The result is a foam or emulsion that is less stable than those formed by the individual surfactants or polymers [9,10]. It is, therefore, not surprising that current progress in this field has tended to concentrate on: (i) adsorbed protein structure; (ii) the rheology; and (iii) the composition of interfaces. Therefore, I have reviewed the current literature in this field in these specific areas.
and Norde [ l l ] used an array of quartz plates to increase the number of interfaces that could be interrogated by circular dichroism. They showed that the adsorbed immunoglobulin possessed a different spectrum depending on the nature of the surface. Moulin et al. used a microfabricated cantilever to measure the surface stress induced by proteins adsorbed onto a gold substrate [12]. They could apparently follow long-term unfolding and interaction processes, which were found to be very different between immunoglobulin and BSA, although no firm conclusions about the source of these differences could be drawn. The limitation of these techniques in foams and emulsions was the correlation of behaviour at a solid surface with that at a fluid one. If this could be done, then it would be possible to explain the effects seen by Murray and Liang [13]. They found that spray-drying protein solutions had a detrimental effect on foam stability. This was supported by changes in their adsorption behaviour suggesting a conformational change induced by the drying process. The presence of the sugar trehalose during drying seemed to have a positive effect on foaming properties, especially for P-lactoglobulin. The corresponding adsorption behaviour suggested that the trehalose protected the proteins against denaturation. This has also been confirmed by Clarkson et al. where the presence of trehalose was seen to protect against surface denaturation induced by foaming [141, although they found that this was a minor effect compared to the effects of ionic strength and pH. Small differences in protein structure can infer major changes in functionality. The genetic variants A, B and C of P-lactoglobulin differ by only two amino acid residues, yet this was found to cause significant changes in adsorption behaviour [ 151 and emulsification properties [16]. The substitution of an aspartic acid residue in variant A to a glycine in B at residue 64 appears to increase the rate of adsorption, and the development of an elastic interface of variant B [151. Although variant B had poorer emulsification properties, its long-term stability to coalescence was higher [16].
2. Adsorbed protein structure
3. Interfacial rheology
Secondary and tertiary structures of proteins in solution can be easily determined using a range of spectrometric techniques. The structure of adsorbed proteins is more difficult due to various technical complications. The signal intensity from a single interface is usually small and the resultant signal to noise ratio can make interpretation difficult. Measurements of proteins adsorbed onto particles or droplets creates light scattering problems. Vermeer
The surface rheology of adsorbed protein layers has long been known to be important for the stabilisation of foams and emulsions [4,17]. The visco-elastic properties of the surface have often been correlated with functionality [2-4,181. A recent review by Dickinson [19'1 brought together certain aspects of the structure and surface rheology of proteins, and the correlation with the formation and stability of foams and emulsions. A fascinating article by Izmailova et
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al. [20’] reviewed the contribution made by Rehbinder to this area and offered new results showing how the coalescence stability of protein foams and emulsions can be directly related to the mechanical properties of the adsorbed protein layer. Rehbinder put forward the concept that the elasticity of protein interfaces was responsible for their stabilising ability. This concept was developed further with some new results confirming the idea that it is not simply the value of the elastic modulus that is important for stability, but the stress at which the elasticity breaks down. However care must also be taken, as some proteins such as p-casein are excellent at stabilising emulsions by steric repulsion, but have very poor surface rheological properties [21]. The surface visco-elastic properties of proteins can also be important for formation of and drainage from foams. Prins [22’] showed, by using the overflowing cylinder, that proteins can slow the flow of liquid close to the surface. This can retard liquid drainage from foams, and is thought to contribute to the slower drainage rates observed in protein-stabilised foams compared to those stabilised by surfactants. In addition, Prins also postulated that this ‘stagnant surface behaviour’ enabled the bubbles and droplets to be more easily dispersed when the surface tension was taken into account. In the absence of protein, the droplet break-up was strongly dependent on the relative viscosities of the continuous and dispersed phases. In contrast, in the presence of protein, the droplets were dispersed more easily and independently of liquid viscosity. This clearly demonstrated the contribution made by the surface rheology of proteins to the functionality of foams and emulsions. The presence of surfactants will reduce the surface visco-elasticity of adsorbed protein layers [4,6,7] often resulting in a drop in coalescence stability [4,9,10,23]. Chen and Dickinson showed that by displacing proteins from emulsion droplets, the visco-elasticity of emulsion gels could be significantly reduced [24]. The interaction between the emulsion droplets and the gel matrix was thought to be influenced by the amount of adsorbed protein. Protein adsorption and the development of the visco-elastic surface can be significantly affected by protein conformation. Roth et al. [25] found that ageing and heat treating proteins at the interface could result in higher surface viscosities, depending on the temperature, and the length of heat treatment. Certainly the interfaces heated to the higher temperatures studied formed a more viscous interface. Some of the more visco-elastic interfaces were also more resistant to the effects of added surfactant, although there was no clear correlation for all the treatments studied. Petkov et al. demonstrated a new method of simultaneously measuring the surface dilational and shear parameters in protein films
[26]. Again, this technique demonstrates how sensitive surface rheology is to the disruptive effects which surfactants have on protein adsorbed layers. One way to restore stability to a surfactant destabilised foam is to restore the surface visco-elasticity. Propylene glycol alginate (PGA) has been used to improve beer foam stability. A detailed study of this improvement showed that PGA restored the surface elasticity of a mixed protein-surfactant interface [27]. This was thought to be due to an electrostatic ‘crosslinking’ mechanism, which partially restored the protein-protein interactions that had been disrupted by the presence of surfactant. The interfacial rheology of wheat proteins is of importance to the baking industry. Yet a recent review of the area [28] shows that there is distinct gap in our knowledge on effect of their surface properties on baking quality. It is likely, therefore, that the surface properties of wheat proteins will receive much attention in the near future. It is not only the surface rheology of proteins that is important to functionality. Recent studies on the surface rheology of surfactants have also shown a correlation with foam formation and stability. Kanicky et al. [291 showed that sodium laurate solutions showed maximal surface viscosity at pH values close to their pK,. This corresponded with maximal foam formation, and foam stability. They showed that the interfacial packing was highest at the pK,, as expected, as the electrostatic repulsion between the molecules was minimal. This also corresponded with improved emulsification and reduced water evaporation. Changing the chain length of the sodium laurate changed the pK, such that the foaming properties were more dependent on the pH than the molecular structure of the surfactant. Fruhner et al. studied a range of different surfactants [30’]. By using the oscillating bubble technique at high frequencies (up to 500 Hz), they were able to obtain surface dilational visco-elastic data for soluble surfactants. They found that the surface needed to develop a significant viscous component before the solutions were able to create a stable foam. At very low concentrations, below that which would support a stable foam, purely elastic behaviour was observed. At high concentrations, the surfactants which were able to support foams such as SDS, CTAB and Triton-X 100, developed a viscous component. Surfactants which did not form stable foams, such as nonanol and dodecanol, remained purely elastic. The differences are thought to be due to the improved hydration of the headgroups belonging to the foam active group of surfactants. 4. Surface composition and structure Competitive destabilisation of proteins by surfac-
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tants has been an area of intense interest in food colloids for many years [4,6,9,10,231. Although it is known that surfactants reduce the surface visco-elasticity of protein films, as discussed in Section 3, until recently, however, the mechanisms underlying this effect remained elusive. Our understanding of the competitive adsorption mechanism has been significantly advanced recently by the ability to visualise the structure of mixed protein-surfactant interfaces. Atomic force microscopy (AFM) is a powerful method for imaging surface detail on flat substrates at molecular resolution. It has been used to study protein adsorption onto both solid [31] and liquid interfaces [32]. Recent work by Morris et al. [33",34'] have been able to image mixed protein-surfactant interfaces. In contrast to the assumption that mixed interfaces contain a homogenous distribution of protein and surfactant, these studies clearly show a demixing phenomenon, an example of which is shown in Fig. 1. The power of the technique is that it can impart interfacial thickness information with extremely high spatial resolution. This has led to the proposed 'orogenic' model of protein displacement. The observations showed that the surfactant domains exerted a lateral surface pressure and compressed protein regions. As more surfactant was adsorbed, the surface pressure increased until the protein regions collapsed and were eventually displaced. These studies have helped explain: (i) why the stability of protein foams and emulsions is often affected at very low concentrations of surfactant, before any protein is displaced [10,231; (ii) observed increases in the thickness or concentration of adsorbed protein layers observed in mixed protein-surfactant interfaces [19,35']; and (iii) why proteins with a higher viscoelastic modulus are more difficult to displace [lo]. The
Fig. 1. AFM image of a Langmuir-Blodgett film of a mixed plactoglobulin-Tween 20 film at the air-water interface. The light regions denote the surfactant domains, and the dark regions represent the thicker protein regions. Image size is 2 x 2 pm.
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Fig. 2. Brownian dynamic simulation of the competitive displacement of protein-like (dark) particles, by surfactant-like displacer (light) particles. Attention is drawn to the similarity with Fig. 1. Reproduced from [390°] with permission.
only weakness to this approach is that the interface needs to be transferred to a solid substrate by the Langmuir-Blodgett method. The orogenic mechanism was confirmed in situ by following the displacement of a protein from a solid surface [36']. In situ measurements on fluid interfaces can be performed using Brewster angle microscopy (BAM). Studies by Patino et al. [37,38"] have shown that it is possible to image interfaces formed by proteins and surfactants. The surfactants have a high refractive index compared to the proteins, allowing discrimination between the two species. They found that mixed p-casein and monopalmitin films phase separated at the interface. They also showed an increase in interfacial film thickness. Although BAM has much lower resolution than AFM, the approaches are highly complimentary, as BAM can observe these phenomena in situ at the interface. The orogenic model was further supported in a separate computer simulation study. By using a Brownian dynamics approach Wijmans and Dickinson [39"] simulated the competitive displacement process. The results showed a remarkable similarity to the experimental observations made using AFM [33",34'] and BAM [38"], whereby the surfactants adsorbed and formed distinct domains, compressing and increasing the thickness of the protein regions. Fig. 2 shows an example of one of these simulations, and the likeness between that and Fig. 1 is quite remarkable. They also support the importance of the rheology of the protein network in the process, and also that repulsive forces between the protein and surfactant promote the phase separation.
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5. Conclusions
Understanding the role of interfaces on the formation and stability of foams and emulsions has been significantly advanced in recent years. There appears to be a consensus of opinion that the surface rheological behaviour of both proteins and surfactants is of paramount importance. However, rheology is a complex area, and the specific surface rheological parameters, which are most important for these applications, are still being defined. The main objectives for the future are to understand how factors such as molecular structure, component interactions and surface composition of mixed interfaces, influence and control the various interfacial rheological parameters which are regarded as important contributory factors to foam and emulsion behaviour.
Acknowledgements
The author acknowledges the support of the BBSRC through their core strategic grant to the Institute of Food Research. References and recommended reading
DO
of special interest of outstanding interest
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[lo] Cornec M, Wilde PJ, Gunning PA et al. Emulsion stability as affected by competitive adsorption between an oil-soluble emulsifier and milk proteins at the interface. J Food Sci 1998;63:39-43. [ l l ] Vermeer AWP, Norde W. CD spectroscopy of proteins adsorbed at flat hydrophilic quartz and hydrophobic Teflon surfaces. J Colloid Interface Sci 2000;225:394-397. [12] Moulin AM, Oshea SJ, Badley RA, Doyle P, Welland ME. Measuring surface-induced conformational changes in proteins. Langmuir 1999;15:8776-8779. [13] Murray BS, Liang H-J. Enhancement of the foaming properties of protein dried in the presence of trehalose. J Agric Food Chem 1999;47:4984-4991. [14] Clarkson JR, Cui ZF, Darton RC. Effect of solution conditions on protein damage in foam. Biochem Eng J 2000;4: 107-114. [15] Mackie AR, Husband FA, Holt C, Wilde PJ. Adsorption of p-lactoglobulin variants A and B to the air-water interface. Int J Food Sci Technol 1999;34:509-516. [16] Euston SR, Hirst RL, Hill JP. The emulsifying properties of P-lactoglobulin genetic variants A, B and C. Colloid Surf B - Biointerfaces 1999;12:193-202. [17] Neurath H, Bull HB. The surface activity of proteins. Chem Rev 1938;23:391-435. [18] Phillips MC. Protein conformation at liquid interfaces and its role in stabilising emulsions and foams. Food Technol 1981;35:50-57. [19] Dickinson E. Adsorbed protein layers at fluid interfaces: interactions, structure and surface rheology. Colloid Surf B - Biointerfaces 1999;15:161-176. A useful review of the properties of adsorbed protein layers. Particular attention is paid to the adsorption and surface rheology of P-lactoglobulin in the presence and absence of surfactants. A simulation model of protein adsorption, and neutron reflectivity data of competitive displacement of P-lactoglobulin is presented and discussed. [20] Izmailova VN, Yampolskaya GP, Tulovskaya ZD. Development of the Rehbinder’s concept on structure-mechanical barrier in stability of dispersions stabilised with proteins. Colloid Surf A - Physicochem Eng Aspects 1999;160:89-106. Rehbinder’s concept that proteins stabilise foams and emulsions by forming a mechanical barrier was proposed around 70 years ago. This manuscript reviews this concept and presents new data showing the importance of the elastic limit of the protein adsorbed layer. Beyond this limit, the surface begins to flow, and the probability of coalescence increases dramatically. [21] Husband FA, Wilde PJ, Mackie AR, Garrood MJ. A comparison of the functional and interfacial properties of p-casein and dephosphorylated p-casein. J Colloid Interface Sci 1997;195:77-85. [22] Prins A. Stagnant surface behaviour and its effect on foam and film stability. Colloids Surf A - Physicochem Eng Aspects 1999;149:467-473. Prins demonstrates how the elastic surface formed by proteins can alter the fluid flow adjacent to that surface. This is referred to as a stagnant layer. The effect is shown to not only reduce the drainage of liquid from foams, but is also shown to aid bubble and droplet break-up, by inducing large surface tension gradient across the surface of a droplet under shear. [23] Dickinson E, Owusu RK, Williams A. Orthokinetic destabilization of a protein-stabilised emulsion by a watersoluble surfactant. J Chem Soc Faraday Trans 1993;89: 865-866. [24] Chen J, Dickinson E. Effect of monoglycerides and diglycerol-esters on viscoelasticity of heat-set whey protein emulsion gels. Int J Food Sci Technol 1999;34:493-501.
P.J. Wilde /Current Opinion in Colloid & Interface Science 5 (2000) 176-181 Roth S, Murray BS, Dickinson E. Interfacial shear rheology of aged and heat-treated p-lactoglobulin films: displacement by nonionic surfactant. J Agric Food Chem 2000;48: 1491-1497. Petkov JT, Gurkov TD, Campbell BE, Bonvankar RP. Dilatational and shear elasticity of gel-like protein layers on air/water interface. Langmuir 2000;16:3703-3711. Sarker DK, Wilde PJ. Restoration of protein foam stability through electrostatic propylene glycol alginate-mediated protein-protein interactions. Colloid Surf B - Biointerfaces 1999;15:203-213. Ornebro J, Nylander T, Eliasson AC. Interfacial behaviour of wheat proteins. J Cereal Sci 2000;31:195-221. Kanicky JR, Poniatowski AF, Mehta NR, Shah DO. Cooperativity among molecules at interfaces in relation to various technological processes: Effect of chain length on the pK(a) of fatty acid salt solutions. Langmuir 2000;16:172-177. Fruhner H, Wantke KD, Lunkenheimer K. Relationship between surface dilational properties and foam stability. Colloids Surf A - Phvsicochem Eng- Aspects - 2000;162:193-202. Surface dilational parameters of surfactants were measured at high frequencies by the oscillating bubble technique. They demonstrated that for a surfactant to create stable foams, the surface modulus had to develop a significant viscous component. [31] McMaster TJ, Miles MJ, Shewry PR, Tatham AS. In situ surface adsorption of the protein C hordein using atomic force microscopy. Langmuir 2000;16:1463-1468. [32] Johnson CA, Yuan Y, Lenhoff AM. Adsorbed layers of ferritin at solid and fluid interfaces studied by atomic force microscopy. J Colloid Interface Sci 2000;223:261-272. [33] Mackie AR, Gunning AP,Wilde PJ, Morris VJ. The orogenic OD displacement of protein from the air/water interface by surfactant. J Colloid Interface Sci 1999;210:157-166. AFM was used to image Langmuir-Blodgett films of mixed protein surfactant interfaces. The adsorbed proteins and surfactants were found to be phase separated. A new physical mechanism of competitive displacement termed ‘orogenic’ displacement was proposed, whereby the surfactant domains exerted a lateral surface pressure compressing the protein layer. As the surface pressure increased, the protein regions became more compressed causing it to collapse, crumple and finally be displaced. The ease by which a protein was displaced was directly correlated with its surface rheological properties. [34] Mackie AR, Gunning AP, Wilde PJ, Morris VJ. Orogenic
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displacement of protein from the oil/water interface. Langmuir 2000;16:2243-2247. The orogenic mechanism was further investigated at the oil water interface and the generality of the mechanism was confirmed. The origins of the process were also investigated. The proteins formed a disordered assembly at the interface, and small defects in the surface packing were thought to be responsible for allowing surfactants to adsorb into the protein layer. [35] Cornec M, Narsimhan G. Adsorption and exchange of plactoglobulin onto spread monoglyceride monolayers at the air-water interface. Langmuir 2000;16:1216-1225. [36] Gunning AP,Mackie AR, Wilde PJ, Morris VJ. In-situ observation of surfactant-induced displacement of protein by atomic force microscopy. Langmuir 1999;15:4636-4640. AFM could not be used to follow competitive displacement in situ at a fluid interface. The process was therefore monitored on a solid, hydrophobic surface in real time. The same orogenic displacement process was found to occur with the solid substrate, further supporting the previous studies of Langmuir-Blodgett films. [37] Patino JMR, Sanchez CC, Nino MRR. Is Brewster angle microscopy a useful technique to distinguish between isotropic domains in p-casein-monoolein mixed monolayers at the air-water interface. Langmuir 1999;15:4777-4788. [38] Patino JMR, Sanchez CC, Nino MRR. Analysis of p-caseinOD monopalmitin mixed films at the air-water interface. J Agric Food Chem 1999;47:4998-5008. Brewster angle microscopy was used to image mixed protein-surfactant systems, at the air-water interface. This demonstrated for the first time that the orogenic mechanism of competitive displacement was a valid model, because the measurements were performed in situ on a fluid interface, and the same phase separation and thickening of the interfacial layers were observed. [39] Wijmans CM, Dickinson E. Brownian dynamics simulation of 00 the displacement of a protein monolayer by competitive absorption. Langmuir 1999;15:8344-8348. Brownian dynamic simulations of the competitive displacement process between protein-like and surfactant-like particles, independently displaced many features of the orogenic mechanism observed experimentally. The simulations demonstrated the importance of the strength of the protein network and showed that repulsive forces between the protein and surfactants encouraged phase separation. This approach should prove to be a powerful tool for investigating the role of specific inter-molecular interaction processes on the development of interfacial structure.