proteins and emulsifiers at liquid interfaces

Advances in Colloid and Interface Science 108–109(2004) 63–71

0001-8686/04/$ - see front matter� 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.cis.2003.10.011

Proteins and emulsifiers at liquid interfaces

Peter Wilde*, Alan Mackie, Fiona Husband, Patrick Gunning, Victor Morris

Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK

Abstract

The interfacial properties of proteins and emulsifiers have been studied extensively in the field of food colloid research.Emulsions form the basis of a huge range of food products and are generally stabilised by either protein andyor emulsifiers.Proteins have been shown to stabilise emulsions by forming a viscoelastic, adsorbed layer on the oil droplets, which form aphysical barrier to coalescence. Emulsifiers can be oil or water soluble, forming a fluid, close-packed layer at the interface witha low interfacial tension. This results in an emulsion with a small droplet size distribution, stabilised by the fluid Gibbs–Marangoni mechanism or weak electrostatic repulsion. In real food emulsions, there is usually a mixture of proteins and emulsifierscompeting for the interfacial area. This can produce a finer emulsion, however, the emulsifiers break down the viscoelasticprotein-adsorbed layer, resulting in an emulsion with reduced stability. We present a review recent work that aims to characterisethe composition, structure and physical properties of mixed protein–emulsifier interfaces, in an effort to understand the mechanismsbehind the stability behaviour of food emulsion systems.� 2004 Elsevier B.V. All rights reserved.

Keywords: Gibbs–Marangoni mechanism; Langmuir–Blodgett method; Protein–emulsifier interface; Surface rheology

1. Introduction

Proteins are the single most commonly used class offoaming and emulsifying agent used in the food industry.They are natural, non-toxic, cheap and widely available,thus making them ideal ingredients. Their interfacialproperties have made them the subject of much studyover the yearsw1,2x. They stabilise emulsions by forminga viscoelastic adsorbed layerw3x, the mechanical prop-erties of which are thought to influence the stability ofemulsions and foamsw4x. Proteins have a complexstructural morphology. It is known that the extent ofprotein adsorption is influenced by surface hydrophobic-ity w5,6x and chargew2,7x. Once adsorbed, they unfoldand rearrange their secondary and tertiary structurew8–10x to expose hydrophobic residues to the hydrophobicphase. The high concentration of protein at the surfaceleads to aggregation and the formation of interactions.Hence, the mechanical properties of the adsorbed layerdepend on the structure of the adsorbed protein, and thestrength of the interactions between themw11–13x,

*Corresponding author. Tel.:q44-1603-255-258; fax:q44-1603-507-723.

E-mail address: [email protected](P. Wilde).

which can in turn influence the stability of emulsionsformed from proteins with different secondary structuresw14,15x. In contrast, emulsifiers do not form a viscoe-lastic surface. They are more surface-active than proteinsand form a compact adsorbed layer. This layer relies oncharge repulsion or the Gibbs–Marangoni mechanismto stabilise foams and emulsions. The Gibbs–Marangonimechanism requires rapid diffusion or migration ofemulsifiers at the interface to reduce surface concentra-tion gradients that may arise. The rapid movement ofadsorbed emulsifiers drags associated continuous phase(water in foams), which restores the presence of fluidbetween bubbles or droplets and hence prevents coales-cence. Therefore, the structural properties of emulsifiersare extremely important for their surface and stabilisa-tion propertiesw16x. One of the most interesting areasin this field has been the interaction between proteinsand emulsifiers(including surfactants and lipids). Thereason for this is that although both proteins and emul-sifiers can stabilise foams and emulsions alone, theirindividual mechanisms of stabilisation are incompatible,often resulting in dramatic destabilisation when bothspecies are present at the interfacew1x. This process iscommonly known as competitive destabilisation. There-

64 P. Wilde et al. / Advances in Colloid and Interface Science 108 –109 (2004) 63–71

Fig. 1. Competitive destabilisation of protein-stabilised films by emul-sifiers. Proteins stabilise films by forming strong interactions, thepresence of emulsifiers hinders these interactions. When the film isdeformed or disturbed, the interactions are no longer strong enoughto maintain stability and the probability of rupture is increased.

Fig. 2. Contact time between oil droplets and interface before coales-cence occurs(Coalescence time) for a protein solution(0.2 mgymlb-lactoglobulin) in the presence of increasing concentrations of thewater-soluble emulsifier Tween 20(----) and the oil-soluble emulsifierSpan 80(—).

fore, we present a review of how our knowledge ofcompetitive destabilisation has progressed over theyears, focusing on recent work that has revealed the truemechanism of protein displacement by emulsifiers.

2. Influence of emulsifiers

2.1. Functionality

Low-molecular weight emulsifiers and surfactants areoften more surface-active than proteins, and will there-fore compete for interfacial area(competitive adsorp-tion). It is known that adding surfactants to protein-stabilised foams and emulsions will have a detrimentaleffect on stability w1,17–20x. Fig. 1 shows how thecompetitive adsorption is thought to destabilise lamellaethat separate bubbles and droplets in foams and emul-sions. In fact, surfactants are sometimes utilised in thisway as antifoaming agentsw17x. Surfactants are knownto displace proteins from emulsion dropletsw21–24x,but at much higher concentrations than that required fordestabilisationw19,20x. The concentration required fordestabilisation is also dependent on the structure of theemulsifier. Thus, Fig. 2 shows the coalescence stabilityof emulsion droplets stabilised byb-lactoglobulin, inthe presence of increasing concentrations of two emul-sifiers. Tween 20(polyoxyethylene sorbitan monolaur-

ate) is water soluble and Span 80(sorbitan monooleate)is oil soluble. It was found that the water-solublesurfactant was more effective at destabilising the emul-sion than the oil-soluble emulsifierw25x. Surfactantsgenerally do not interact as strongly as proteins at aninterface. In fact, above their main melting temperaturethey are freely diffusing at the interfacew26,27x, andhence their surface mechanical properties are very weakw11x. Because they are able to reduce the surface tensionto lower values than proteins, relatively small concen-trations of surfactant can affect the surface tension ofprotein solutionsw28,29x.Therefore, it was well established that emulsifiers had

a detrimental effect on protein-stabilised foams andemulsions. To be able to control and predict theseeffects, a deeper understanding of the underlying, inter-facial mechanisms was required. Therefore, several inter-facial techniques and approaches have been used overrecent years in an attempt to address these issues.

2.2. Interfacial tension

One of the simplest interfacial techniques is interfacialtension. Surface tension measurements on simple modelprotein:surfactant systems suggested that the interactionsbetween proteins and surfactants at interfaces were notsimple w30,31x. A crossover in the surface tensionbetween the surfactant and the protein–surfactant mix-ture was observed, as shown in Fig. 3. From conven-tional surface chemistry, this indicated a binding processin solution, preventing adsorption of the ligand. Indeed,strong binding was found between theb-lactoglobulinand the surfactants studiedw31x. By using an oil-solubleemulsifier, no binding could take place, and hence nocrossover in the interfacial tension was foundw25x. This

65P. Wilde et al. / Advances in Colloid and Interface Science 108 –109 (2004) 63–71

Fig. 3. Surface tension of the water-soluble emulsifier Tween 20 inthe presence(j) and absence(s) of 0.2 mgyml b-lactoglobulin. Thehigher values recorded in the presence ofb-lactoglobulin suggestbinding of the Tween 20 by the protein, preventing adsorption of theemulsifier w31x.

Fig. 4. Interference micrographs of model lamellae stabilised by(a)surfactant and(b) protein. The thickness profile gives an indicationof the relative cross-sectional thickness of the films. The elastic pro-tein interface resists collapse of the curved lamellar surface, thus slow-ing down drainage of liquid from within the lamella.

Fig. 5. Interference micrographs of mixedb-lactoglobulin:Tween 20-model lamella after(a) 5 s and(b) 15-min drainage. The initial drain-age shows characteristics of both protein and surfactant films.(b)Shows coexistence phenomena between thick protein-rich(light) andthin surfactant-rich(dark) regions.

suggested that molecular interactions in the bulk couldindeed affect the surface properties. Although the surfacetension measurements provided valuable informationregarding the interactions between the protein and emul-sifiers, the measurements did not suggest any mechanismfor the destabilisation process.

2.3. Model foam films (lamellae)

By directly observing model foam films or lamellae,it is possible to visualise the drainage mechanisms thatare likely to occur in the real foam or emulsion systems.The drainage of continuous phase liquid from foams isstrongly influenced by the interfacial properties of thebubbles or droplets, as the liquid flows past themw32x.It was found that freely suspended model foam filmsstabilised by surfactant drained in a fluid, chaotic man-ner w26x. The behaviour was indicative of a fluidinterface, as shown in Fig. 4a, which drained freely andrapidly until electrostatic repulsion forces slowed downthe drainage and a balance was reached between thevan der Waals attraction and the electrostatic and stericrepulsion between the interfacesw33x. The whole drain-age process typically took approximately 3 min forsurfactants. In contrast, protein-stabilised thin foam filmsdrained much more slowlyw31,34x, forming concentric,stationary Newton’s rings in the film(Fig. 4b). Thebehaviour was indicative of a rigid, elastic interface,which resisted drainage, and slowed down the flow ofliquid from the film. Typically the drainage times forsurfactant films was in the order of 2–3 min, and forproteins, in the order of 1–2 h.

Studies of mixed protein:surfactant lamellae revealedsome interesting results. As the surfactant concentrationwas increased, the drainage behaviour of the proteinfilms slowly changed. The drainage became more rapid,and the Newton’s rings became distorted as shown inFig. 5a, until eventually, at higher surfactant concentra-tions, the drainage resembled that of a pure surfactantw30,31x. This was clear evidence that the presence of asurfactant made the protein-stabilised interface morefluid i.e. less elastic, and consequently less stable. Atsufficiently high surfactant concentrations the proteinappeared to play little part in the surface properties.This was probably due to the total displacement of theprotein by the emulsifierw21–24x. Further evidencecame from measurements of the thickness of the model


lamellae. The thickness of the surfactant-stabilisedlamellae is usually much less than that of the protein.At the ionic strength normally used in these experiments,typical Tween 20 films were approximately 13-nm thick,and theb-lactoglobulin approximately 25-nm thickw31x.It was expected that the transition in lamella thickness,

from the protein-like to the surfactant-like, would pro-vide additional quantitative evidence of the disruptiveeffects of surfactants on protein films. The presence ofeven small quantities of the surfactant resulted in smallbut measurable increase in film thicknessw31x. Furtheradditions of surfactant caused a further increase in filmthickness, and an apparent coexistence of thick and thinregions(Fig. 5b). Coexistence in other protein systemswas also observedw35x. This increase in thickness wasunexpected, and there was no obvious explanation forthis effect. The observed coexistence between the thickand thin regions was thought to be due to the energyminimisation of the system, where the surfactant pre-ferred thinner regions and hence concentrated in theseareas, leaving the protein to occupy the thick regions. Itwas only much later that the true effect was explained,and found to be crucial to the whole disruption anddisplacement process. These observations will be dis-cussed later.

2.4. Surface diffusion

Observing the drainage behaviour of mixed pro-tein:emulsifier stabilised lamellae, provided qualitativeevidence that the emulsifiers were disrupting the protein-stabilised surface, but there was little quantitative infor-mation regarding the mechanism. Surface diffusionmeasurements on thin films by fluorescence recoveryafter photobleaching(FRAP) produced more quantita-tive information about the competitive adsorption pro-cess. The technique had already demonstrated the factthat protein-stabilised surfaces were immobilew34x, andsurfactant surfaces had a measurable surface lateraldiffusion coefficientw26x. Studies on mixed surfactant–protein systems showed that the interface would remainimmobile at low concentrations of surfactant, eventhough the drainage rate was seen to increase. Onlywhen the thin films drained in a surfactant-like manner,was there a measurable surface diffusionw25,31,35,36x.This was a significant finding, because it showed thatalthough the integrity of the protein film had beendisrupted at low emulsifier concentrations, indicated bythe change in drainage behaviour and increase in filmthickness, the surface was still immobile and behavinglike a protein. It was only when the surface becamevery weak that surface diffusion was detected. The otherinformation that arose from this measurement was thatthere were still significant amounts of protein present atthe surface when diffusion was measured. This wasbecause the fluorescent probe used to measure the

diffusion was covalently bound to the protein. Theflourophore was present in only small quantities so thesurface properties of the protein were not affected. Itwas found that much of the fluorescence signal was stillpresent upon the detection of mobilityw30,35,36x. Muchgreater emulsifier concentrations were required to dis-place the protein from the surface. In fact, these meas-urements were used to compare the competitivedisplacement behaviour at the oil–water and air–waterinterfacesw30,35x. Therefore, the FRAP measurements,in conjunction with the other thin film measurementsand surface tension data, gave valuable informationabout the process of competitive destabilisation. It dem-onstrated that at low concentrations the surfactant couldadsorb alongside the protein and weaken the interfaciallayer, as evidenced by the film drainage behaviour. Theprotein was eventually disrupted sufficiently to cause itto diffuse freely at the surface, perhaps as a result ofbinding the surfactant, followed by a gradual displace-ment of the protein.The FRAP technique was also used to study the

coexistence phenomenon observed in some mixed pro-tein:surfactant films. Large phase separations wereapparent inb-casein:Tween 20 thin filmsw35x. Thesewere large enough to investigate the diffusion propertiesin the different regions. Fluorescent measurements clear-ly showed that the thinner regions were deficient ofprotein, and by using a fluorescent surfactant analogue,it was possible to show that the thin regions were fluid,and the thick regions, which were rich in protein, wereimmobile. This coexistence of regions or phase separa-tion was again thought to be induced by the differentequilibrium thicknesses between the protein and thesurfactant. Indeedb-casein forms even thicker filmsthan b-lactoglobulin due to the large electrostatic andsteric repulsion forces, therefore enhancing this effect.It would not be until much later that the significance ofthese measurements would be appreciated. It was foundthat the phase separation of theb-casein and surfactantwas so effective that it was very difficult to measureany diffusion of the labelled protein. This again wouldhave implications in future investigations. This wasthought to be clear evidence of the importance ofprotein–surfactant binding on competitive adsorption.For b-lactoglobulin, which, unlikeb-casein, bound tosurfactants, it was clearly possible for the complex tobe solubilised in a surface dominated by surfactant,although some phase separation was observedw37x.

2.5. Interfacial rheology

Interfacial rheology has been shown to be a verysensitive measure of the impact of surfactants andemulsifiers on the adsorbed protein layersw3,13x. Fromobservations of the drainage behaviour of the modellamellae, it was thought that the increased drainage rates


Fig. 6. Surface dilatational moduli of 0.1 mgyml b-lactoglobulin,(a)before and(b) after treatment with activated charcoal, to remove lipidcontamination.

at low emulsifier concentrations, were due to a weak-ening of the interfacial film. This agreed with measure-ments of the interfacial shear rheologyw38x. Thisdemonstrated that this interfacial rheology was a pow-erful tool for studying the early stages of competitiveadsorption, and was complimentary to the FRAP tech-nique. Surface dilatational rheology also showed a weak-ening of the mixed interfacew39x. This indeed confirmedthe idea that the increased rates of thin film drainagewere due to a weaker but not completely disruptedprotein surface. However, the surface dilatational rheol-ogy seemed to be less sensitive to the presence of thesurfactants than the surface shear measurements. This isbecause the dilatational technique measures the responseto compression of the surface, and the surface shearmeasures directly the strain resulting from and appliedmechanical stress. The result is that the shear measure-ments are more sensitive to the molecular interactions,which make up the network, and the dilatational methodis more sensitive to the surface composition. Hence,using both methods can result in a more completeunderstanding of the composition and mechanical prop-erties of the film.Emulsifiers and surfactants were not the only surface-

active molecules to disrupt adsorbed protein films.Minute concentrations of lipids present in commercialprotein preparations were found to disrupt the surfaceshear and dilatational rheologyw40x. Fig. 6 shows howremoving the lipid contamination with activated charcoaldramatically improved the surface elasticity. This dem-onstrated that the surface rheology of adsorbed proteinfilms were extremely sensitive to very small quantitiesof lipids. Ethanol was also found, under specific condi-tions, to destabilise protein foamsw41x, by acting like a

poor surfactant, it could weaken the surface elasticity,which increased the drainage rates from foams andpromoted coalescence.So the true mechanism of competitive adsorption and

disruption of protein interfaces by surfactants was grad-ually being revealed. The protein alone(particularlyglobular) formed a viscoelastic surface, small concentra-tions of surfactants gradually adsorbed alongside theprotein, possibly assisted by binding, weakening theprotein interfacew39,42x. The weakening process contin-ued, causing thin films to drain more rapidly. No proteinhad been displaced at this time, it was thought that theco-adsorption of the surfactant was merely preventinginter-protein bonds from forming, which were vital forthe development of a viscoelastic interface. This wassupported by the fact that the surface shear propertieswere more sensitive to surfactants than the surfacedilatational rheologyw40,43x. The less elastic interfacewas thought to be the source of the instability observedin foams w31,42,44x and emulsionsw25,45x. As thesurfactant concentration increased, the surface was even-tually mobilised, and the surfactant dominated the sur-face behaviour, and stabilised the thin films by theGibbs–Marangoni mechanism. The stability of the films,foams and sometimes emulsions, then increasedw17,31,42,44,45x as expected from the increasing con-centrations of surfactant.

2.6. Interfacial imaging

Although much was known about the surface activityand the physical properties of mixed protein:emulsifierinterfaces, the true mechanism of competitive adsorptionand displacement was unknown. The main issue wasthat the reason why some proteins were more resistantto destabilisation and displacement could not beexplained. For example, hydrophobic proteins were moreresistant to the destabilising effects of surfactantsw41,46x, andb-lactoglobulin was more resistant thanb-caseinw25,35,45x. The difference between the two latterproteins is thatb-lactoglobulin forms a much moreelastic surface thanb-casein. The effect of proteinhydrophobicity could be argued along the lines ofcompetition due to the surface activity. However, it wasnot entirely clear why a globular protein, which formeda more elastic interface, should be more resistant tosurfactants. The supposition was that the interactionsbetween the proteins were stronger, therefore moredifficult to disrupt.Confocal microscopy had been used to visualise

protein displacement by a surfactantw47x, but the reso-lution was not great enough to visualise the mechanismof displacement. Brewster angle microscopy(BAM),had been used to study mixed protein:emulsifier inter-faces, and had seen interesting structures forming in theinterface w48x. However, the full displacement process


Fig. 7. AFM images of mixedb-lactoglobulin:Tween 20 interfaces. Light regions represent the protein network, and the darker regions representsurfactant domains. At low surface pressures, the emulsifier adsorbs into the protein network, forming small domains(a); as more surfactant isadded, the surface pressure in the domains increases and they expand(b); until the protein is compressed into narrow regions(c); finally theprotein network fails, and the protein begins to be displaced(d). Image sizes are(a) 1 mm; (b) 3.2mm; (c) 6 mm and(d) 8 mm.

had not been followed. The most significant break-through was the use of atomic force microscopy(AFM)which is used to image structures on the nanometrescale, on atomically flat substrates. Interfaces, beingextremely flat, are ideal subjects for AFM. Previousobservations of coexistence of protein and surfactantphases in model thin filmsw31,35x, were thought, aspreviously discussed, to be due to the film thinningprocess. However, separate experimental observationssuggested that this effect might also occur in monolay-ers. Model systems of different milk proteins and Tween20, were studied in spread and adsorbed protein layers.The interfaces were transferred onto a mica substrate bythe Langmuir–Blodgett methodw49x, and imaged usingAFM. These studies revealed that phase separationbetween the protein and the surfactant did indeed occurat a single interface. Fig. 7 shows some example imagesof the mixed protein:surfactant interfaces, showing theseparate protein and surfactant domains. The imagesshow the progressive displacement ofb-lactoglobulinby the surfactant Tween 20 as the surface pressure isincreased by additions of surfactant. The appearance ofthe domains is not unlike the model foam lamellaeshown in Fig. 5b.

Surfactants were added to the Langmuir trough toincrease the surface pressure and displace the protein.Langmuir–Blodgett films were sampled at differentsurface pressures. Further analysis of the imagesrevealed the true nature of the competitive adsorptionprocess. As the surfactant adsorbed into the proteinlayer, the surface pressure increased. In the early stages,the surfactant was observed forming small domains inthe protein layer. As the surface pressure increased, thedomains grew in size as shown in Fig. 8, and eventuallymerged, finally displacing the protein from the surface.This was a whole new concept of the process ofsurfactant displacement of proteins. This was a purelyphysical process. The surfactants probably adsorbed intodefects in the protein filmw50x the surfactant wouldform domains, and apply a surface pressure against thesurrounding protein effectively squeezing the proteinfrom the surface.The process was found to be repeatable, and the

extent of displacement was found to be highly dependenton surface pressurew49–51x. The AFM also showedthat the protein layer increased in thickness(Fig. 8) asit was squeezed out. Estimates of protein volume showedthat even when the surfactant domains occupied signif-


Fig. 8. Interfacial area occupied by protein(hn) and thickness of the protein regions(jm) as a function of surface pressure for the proteinsb-casein(nm) andb-lactoglobulin(hj).

icant areas of the surface, little or no protein had beendisplaced into solution, but simply crumpled intoincreasingly thicker areas. This process was termedorogenic displacement, after the plate tectonic processthat created mountain ranges.The effects of surfactant binding was tested, by

studying proteins that did or did not bind surfactants,and little difference was found between the two. Thedirect interaction between the surfactant and the proteinwas only at the boundary between the protein andsurfactant domains. The main effect was the compres-sion of the protein layers by the surfactant domains thatpossessed a greater surface pressure. This also explainedwhy different proteins were more easily displaced thanothers. For example,b-casein was disrupted and dis-placed at lower surfactant concentrations thanb-lacto-globulin w25x, we showed(Fig. 8) that the surfaceelasticity and collapse pressure ofb-casein was lowerthan that ofb-lactoglobulin w49x. This meant thatb-casein had a lower elastic resistance to the expandingsurface pressure applied by the surfactant domains,henceb-casein was displaced at lower surface pressures.The evidence was also visible from the images of theinterface. Surfactants displacingb-casein formed morerounded, circle-like domains, because the protein filmoffered little resistance to the expansion of the domains.In contrast, bothb-lactoglobulin anda-lactalbumin,which both form more elastic interfaces, offered moreresistance to the expanding domains, and consequentlythey appeared irregular and jagged in shape, as presum-ably the expanding surfactant domains had to spread inthe direction of least resistance.

The limitation of this technique was that the surfacehad to be transferred onto a mica substrate before theAFM could image the film. To substantiate these find-ings, the displacement of a protein film was observedfrom a graphite surface in situw52x. This showed thesame orogenic displacement behaviour that had beenobserved from the air–water surface, thus validating ouroriginal observations. Further BAM measurements wereundertakenw53x, observing the same displacement pro-cess in situ at the air–water interface, albeit at a lowerresolution. In addition, Brownian dynamic simulationsof mixed interfaces showing the similar phase separationand orogenic mechanismsw54x.We are now confident that the basic principles of

competitive adsorption and displacement of proteins byemulsifiers and surfactants are understood, and can besummarised as follows: proteins stabilise foams andemulsions by forming a viscoelastic, immobile adsorbedlayer. Emulsifiers are more surface-active and can lowerthe interfacial tension to lower values than proteins.Therefore, emulsifiers can adsorb into the protein-adsorbed layer, probably into packing defects at first.As more surfactants adsorb into these defects, they formsurfactant-rich domains. Consequently the interfacebecomes weaker, and resultant foams and emulsionsbecome less stable, due to increased drainage andcoalescence rates. For the displacement process to pro-ceed, the surface pressure in the surfactant domains mustbe greater than the combined surface pressure in theprotein-rich region plus the elastic resistance of theprotein network. When the surface pressure in theprotein areas eventually reaches the collapse point, the


protein layer will ‘crumple’ and thicken. Then, eventu-ally the protein network fails and is displaced from thesurface.

3. Summary

The interfacial properties of mixed proteins and emul-sifiers are very important for understanding the func-tionality of food emulsion systems. Protein structure isvery important for the development of an elastic inter-face, and hence the coalescence stability of emulsions.The presence of low levels of emulsifier is detrimentalto emulsion stability, which has been explained throughthe orogenic displacement model. This model explainsthe importance of the interfacial rheology of the protein-adsorbed layer in resisting disruption and destabilisationby surfactants and emulsifiers. This knowledge shouldhelp us to devise strategies to control the stability of awhole range of emulsion applications.

Acknowledgments

The authors gratefully acknowledge the Biotechnolo-gy and Biological Sciences Research Council for fund-ing through the core strategic grant to the Institute.

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