Protein‐stabilized foams and emulsions

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Protein‐stabilized foams and emulsionsPeter J. Hailing a & Pieter Walstra ba Scientist in Food Research, Unilever Colworth Laboratory, Sharnbrook, Bedford, U.K.b Professor, Department of Food Science, Agricultural University, Wageningen, NetherlandsVersion of record first published: 29 Sep 2009.

To cite this article: Peter J. Hailing & Pieter Walstra (1981): Protein‐stabilized foams and emulsions, C R C Critical Reviews inFood Science and Nutrition, 15:2, 155-203

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October 1981 155

PROTEIN-STABILIZED FOAMS AND EMULSIONS

Author: Peter J. HailingUnilever Research LaboratorySharnbrook, Bedford, U.K.

Referee: Pieter WalstraDepartment of Food ScienceAgricultural UniversityWageningen, Netherlands

I. INTRODUCTION

Foams and emulsions are disperse systems; they contain two distinct phases. A liquidcontinuous phase surrounds a disperse phase, bubbles of gas, or droplets of a secondliquid immiscible with the first. The total interfacial area between the phases becomesvery large, so the characteristics of this interface have important effects on the wholesystem. Proteins tend to accumulate at both air-water and oil-water interfaces,constituting an interfacial layer and thereby altering surface properties. They are oftenhighly effective at stabilizing foams and emulsions against their tendency to revert to twobulk phases separated by a plane interface. Many food products are foams or emulsions(or both), and proteins often play a role in stabilizing these systems. The properties of thedispersion are usually crucial in determining the acceptability of the product.

This review covers studies of protein-stabilized emulsions and foams in relativelysimple systems, ranging from fundamental investigations to the more empirical"functional tests" of food proteins. The aim throughout is to identify underlyingphysicochemical principles that apply to these systems. Interpretation of most publisheddata is hindered by the failure to determine crucial parameters, particularly droplet orbubble size. Often neither authors nor their results make clear which of the severalprocesses in foam or emulsion breakdown was being followed by "stability"measurements. Hence, Sections III. and IV. start with discussions of the experimentalapproaches used and the meaning of the results obtained. It should become apparent thatno absolute values can be ascribed to proteins as foam or emulsion stabilizers, despite thehopes of some workers. The only useful comparisons are those made by the sameinvestigators with the same equipment, and the conclusions that can be drawn from thesemake up the bulk of the review.

This area has been reviewed previously by Kitchener and Mussellwhite1 (emulsionsonly), Kinsella,2 and Graham and Phillips.3"5

Studies on the more complex food foams and emulsions are not covered. The conclu-sions drawn from this review should be useful in understanding the role of protein ingredi-ents in such products, particularly in terms of their surface properties. The attempts bysome workers to look for direct comparisons between the behavior of food foams andemulsions and the model systems should, however, be treated with caution; not only arethe former more complex, but they are often rather different in nature. A special mentionis appropriate for the extensive studies on dairy emulsions (milk, cream, and similar),which have always included some very fundamental colloid science. Proteins play animportant role in these emulsions, but the situation is complicated by the presence ofother surfactants (polar lipids) and natural biological structure. Consequently, this areawill not be covered here, but has been well reviewed recently by Graf and Bauer6 andMulder and Walstra.7

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156 CRC Critical Reviews in Food Science and Nutrition

Many reviews of foams or emulsions devote much space to the extensive studies of theproperties of plane air-water or oil-water interfaces in "surface trough" experiments.However, though the behavior of proteins at such interfaces has been analyzed in somedetail,3'8"14 much less carefully controlled work has been done on the disperse systemsthemselves. Furthermore, in foams and emulsions dynamic surface properties at veryshort time scales may often be important, and these cannot be imitated in surface troughand similar experiments. In this review such studies of surface properties are only notedwhere they can be clearly related to the behavior of foams or emulsions.

Before dealing with protein-stabilized systems, a short section deals with some of thebasic theory that underlies much of their behavior. Subsequent references to theory are tothis section (Section II.) and the sources cited in it.

Frequently used symbols in this review are

1. C — concentration of protein in the aqueous phase2. d — droplet/bubble diameter3. F — surface concentration4. y — surface tension5. £ — zeta potential6. 77O — viscosity of the continuous phase7. 7r — 3.14159; surface pressure8. <f> — disperse phase volume fraction9. % — is used conventionally to give protein concentrations (10 kg.m"3)

II. BASIC SURFACE AND COLLOID SCIENCE

A. Surface PropertiesThe following terms and symbols will be used. If required, details may be found in text-

books such as Adamson.15

Interfacial or surface tension — ySurface active agents, surfactantsSurface pressure — -nSurface concentration — YAdsorbed films, contrasted with spread films

The surface pressure due to a surfactant is the reduction in y from that of a cleaninterface. The surface concentration of a surfactant is the excess in the surface layers overthat expected if the bulk concentrations persisted right up to the interface; it is a quantityper unit area.

Mention will also be made of surface rheology. Surface films of surfactant may resistdeformation, and the resistance may be characterized by viscous and elastic parameters,in a two-dimensional analogy of the rheology of bulk fluids. Both dilational and shearproperties may be determined, and these may often be very different from each other. Thesurface rheology of adsorbed protein films is often highly complex, and many differentparameters may be derived from it. However, most of these parameters tend to vary inconcert, and relationships with the behavior of foams and emulsions are at bestsemiquantitative. Hence, a loosely defined aggregate property is useful, and the term"surface rigidity" will be used as such in this review.

B. Foams and EmulsionsAgain only an outline can be given here, but the following reviews and texts may be

consulted for further details: foams,16"18 emulsions.7'18"23

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October 1981 157

1. Fundamental ParametersTwo are of crucial importance in determining the structure and behavior of any

disperse system. The volume fraction of gas or dispersed liquid is known as the (disperse)phase volume (<£). Much of the theory of disperse systems is based on all droplets orbubbles being of equal diameter (d). In practical dispersions, a range of sizes isencountered and a variety of methods have been used to arrive at an average and ameasure of spread. Since most of the relationships noted in this review aresemiquantitative, d will be used rather loosely to denote droplet or bubble size, with thetype of average noted where known.

2. StructureAt moderate phase volumes this is fairly simple, with spherical droplets or bubbles

dispersed in the continuous matrix. However, at high # it is no longer possible to pack thedisperse phase as spheres, so some deformation is required. For uniform d, it can beshown that these geometrical considerations apply above a critical <p of 0.74. At high <pthe droplets or bubbles are distorted into polyhedra with partly plane faces: the thinlayers of continuous phase liquid separating the faces of two adjacent polyhedra areknown as "lamellae", while the thicker channels where three lamellae meet are known as"plateau borders" (Figure 1A).

For a given mix of liquids either may form the continuous phase, so two emulsions canbe made, usually with very different properties. For example, the emulsions may bedescribed as oil-in-water (<j> = x) and water-in-oil (<f> = 1 — x), while the process thatinterconverts them is known as inversion.

3. StabilitySeveral different processes can be identified in the breakdown of foams or emulsions,

as illustrated in Figure IB. These processes can combine in various ways to give manypossible intermediate stages between a uniform dispersion and two completely separatedphases. Hence, the term "stability" applied to a foam or emulsion can only have meaningif the process of instability is defined.

Two of the processes in Figure 1B involve flow. Because of the usual density differencebetween the phases, gravitational (buoyancy) forces will tend to cause flow of thecontinuous phase around the disperse droplets or bubbles. At low 4> the process isnormally viewed as "creaming" or sedimentation of droplets or rise of bubbles throughthe matrix; at higher <f> it is seen as "drainage" of the latter past the disperse phase.Droplets can also be brought together in the bulk of an emulsion by flow. Such"encounters" may result from either Brownian motion or agitation of the wholeemulsion. For many systems, theoretical predictions give a reasonable guide to the ratesof these flow processes, and the expected dependence on some key parameters is given inTable 1.

The process of "disproportionation" depends on diffusion. There is an increasedpressure inside droplets or bubbles given by the Laplace equation:

4-yAP = — (for spheres)

d

so the pressure and, hence, the solubility of disperse phase material is greater for smallerbubbles; this constitutes a driving force for diffusion from small droplets or bubbles tolarger ones or to a bulk phase ("isothermal distillation"). The rate usually depends onbulk diffusion and this, in turn, depends on the solubility of the disperse phase material incontinuous liquid. As a diffusive process, some reasonable predictions of its ratedependence can again be made (Table 1).

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disperse phase

continuous f Plateauphase j border

Lamella

FIGURE 1A. Diagrammatic structure of a highphase volume foam or emulsion.

b• •P

• o•o;• •

8 o ° 0 ° O

O ° ° ° o

\ Gravitational f\separalion A. ... .

NT /Collision

*

•b;^

Disproportionati

rt O v̂ O0 ° ° ° o ° oJP ° o »O«

00 11Coalesce

Oo O oO

o o

nee

^ 8 <;

FIGURE IB. Basic processes in the breakdown of emulsions: a diagrammatic representation. Formost of these, analagous processes occur in foams. Encounters (collision) do not by themselvesconstitute breakdown, but can lead to flocculation or coalescence.

Two processes are strongly influenced by surface properties. Emulsion droplets thathave been brought together by creaming or encounters can either stick together whileremaining separated by a thin layer of continuous phase ("flocculation"), or joincompletely into one ("coalescence"). In foams <p is normally very high and the bubbles areforced together anyway, so only the latter process is important; it is often called"bursting". Though theoretical understanding of these processes remains ratherincomplete, some factors of relevance to protein-stabilized systems are noted in the nexttwo sections.

4. Colloid Forces and the DLVO TheoryWhen surfaces become very close, a number of attractive and repulsive forces between

them start to affect the further thinning of lamellae and hence the approach of droplets orbubbles, van der Waals attractions between them can become significant; repulsion canbe due to electrostatic effects or to steric interactions between adsorbed surfactants.

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October 1981 159

Table 1SOME THEORETICALLY PREDICTED

INFLUENCES ON BREAKDOWN RATES OFDISPERSE SYSTEMS

Gravitational Separation

gApd2

Creaming/bubble rise Velocity =ISJJo

(Falls for <t> ^ 0.05)

gApd2 (l-d>yDrainage (sphere structure) Velocity «•

18f7 10$

Encounter

Brownian motion Rate per droplet = 5—

4<£SAdditional in laminar shear Rate per droplet =

IT

Additional in isotropic Rate per droplet **5<t>l ——turbulence r>°

Disproportionate

Rate depends on:Driving force, proportional to yA(I/d)Diffusion rates, proportional to solubility of the disperse phase

material; and its diffusion coefficientGeometric factors determining the distance and area for

diffusion. Rate increases with <j>, (1/d)

Note: The theoretical bases of these predictions vary somewhat inreliability, but all are likely to be approximately valid. Sym-bols: g, acceleration due to gravity; Ap, phase density differ-ence; rjo, continuous phase viscosity; k, Boltzmann's constant;7r, 3.14159; S, shear rate; eo, power per unit volume.

Electrostatic repulsion depends on a surface potential, which can be related to the zetapotential (£) obtained from measurements of electrophoretic mobility. The effects ofthese colloid forces on the interaction energy between approaching surfaces were firstanalyzed in the Deryagin and Landau, Verwey and Overbeek (DLVO) theory, now muchextended and modified. For this review two conclusions are relevant:

1. Electrostatic or steric repulsions can give rise to a substantial energy barrier, oppos-ing close approach of droplets or lamellae surfaces, at separations of a few nm.

2. At larger droplet separations (of the order of 10 nm), a shallow secondary energyminimum can give rise to reversible flocculation.

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These colloid forces also affect coalescence rates. Though there is no generallyaccepted mechanism for this process, it is agreed that the rate usually increases as thelamallae become thinner, that is, as drainage proceeds or <j> becomes larger. The closeapproach of droplets past the DLVO energy barrier may also be slowed considerably byviscosity effects.167

5. Solid Particle StabilizationMicroscopic solid particles with some affinity for both phases (partially wetted by

each) will become attached to the surfaces of droplets or bubbles. As a result, these will bestabilized towards coalescence provided the particles are well wetted by the continuousphase. A typical phenomenon with solid particle stabilized dispersions is "limitedcoalescence", involving some fall in the total interfacial area until the particles reach acritical degree of packing that prevents further coalescence.

III. PROTEIN-STABILIZED EMULSIONS

A. Experimental Approaches1. Fundamental Studies

There is really no substitute for microscopic observation of the processes occurring inan emulsion, and assessment of changes in the individual droplets. King and Mukherjee24

were the first to follow the variation in droplet size with time due to coalescence in aprotein-stabilized emulsion. Kerosene or olive oil was homogenized in 1% gelatin ($0.4)and the emulsion produced allowed to stand (presumably with occasional mixing toreverse creaming). The droplet size distribution broadened, with the median d rising fromunder 1 fim to about 5 fim over 60 days. Only a few workers since have used microscopyto follow directly coalescence or flocculation in protein-stabilized emulsions.25"29 Mitaetal.28 showed the broadening of the droplet size distribution towards higher values as aBSA-stabilized benzene emulsion ages (Figure 2); disproportionation is probably partlyresponsible here. Other methods are available for following flocculation or coalescencequantitatively (though they do not tell directly which process is occurring).

Both light-scattering methods7'30'61'169'170 and the Coulter Counter7'171 have been usedwith simple protein-stabilized emulsions. Though these methods are relatively simpleand convenient, they have not received the wide use they deserve among workers withprotein systems, most of whom have not attempted to measure droplet size. Thecoalescence of single drops of oil with plane protein-stabilized oil-water interfaces hasbeen quite popular as a fundamental model for emulsions.31"38 The lifetime of the drop atthe interface depends on lamella drainage and rupture, but separate information on theseprocesses is not normally obtained. The results must be interpreted with care because thesystem differs in several important respects from a real emulsion.

1. The surface to bulk ratio is much less than in emulsions, so minor contaminants canhave large effects, as in Langmuir trough studies of surface properties. Manyanomalous results can be explained by lack of proper attention to cleanliness.

2. The drops are orders of magnitude larger than those normally found in emulsions.Biswas and Haydon33 note that with the drop size they use no electrostatic barrier tocoalescence would be expected, whatever the value of £. There is a major change inthe pattern of drainage, with "dimpling" of the drop surface, and theory suggests thatthe larger size of the lamella may affect its rupture (e.g., Vri168)

3. The lifetime observed varies widely depending on the time for which the drop and theinterface are aged (up to several hours), no doubt because of the slow formation ofequilibrium protein surface films. In real emulsions, however, the surface film isformed quickly and under very different conditions.

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October 1981 161

1 day old

3 days old•7days oldU days old

0 20 L0 60 80 100 120Globule diameter

FIGURE 2. Time dependence of dropletsize in a benzene in water emulsion stabilizedby bovine serum albumin. (From Mita, T.,Yamada, K., Matsumoto, S., and Yonezawa,D., J. Texture Stud, 4, 41, 1973. Withpermission.)

Despite all the preceding differences, there do seem to be correlations between theresults obtained with single drops and with real emulsions, as for example in pH effects(see following).

A few studies have made use of "accelerated ageing" procedures such as ultra-centrifugation, heating or solvent extraction. Sherman20 has cautioned against relatingstorage stability to these treatments, all of which can alter the mechanism as well as therate of breakdown.

2. Emulsifying CapacityAmong the "functional tests" of emulsification by proteins, this has been the most

popular since its introduction by Swift et al. An aqueous solution or suspensioncontaining the protein is vigorously stirred while oil or melted fat is run in steadily. Aftera certain volume has been added, the emulsion in the mixer undergoes a sudden changereferred to as either inversion or breaking. This is viewed as the endpoint of a titration,and the volume of oil added is taken as a measure of the emulsifying capacity (EC) of theproteins involved. The endpoint is accompanied by, and can be detected by:

1. A sudden drop in emulsion viscosity, which causes a change in stirrer motor soundand a decrease in amperage drawn40'41

2. A change in the visual appearance of the emulsion, especially if an oil-soluble dye ispresent42

3. A sudden increase in the electrical resistance of the emulsion43

The EC is normally expressed as the amount of oil emulsified by unit weight of protein(or the extract of a unit amount of tissue). The strange effects on such values of changes inconditions, particularly protein concentration, have been much discussed.

Consideration of the basic processes occurring during the EC test helps to rationalize

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the results. Micrographs of the emulsion immediately before the endpoint show that it isa normal oil-in-water system.39'44'45 A limited electron microscope study after theendpoint showed a definite change in structure, though not a simple water-in-oilemulsion.44 From the changes at the endpoint listed above it seems that at least part of thesystem becomes oil continuous, though it may correspond most closely to anoil/water/oil double emulsion. Thus, the EC test can be considered in the light offundamental work on the inversion of emulsions.

The most important factor affecting the inversion of an emulsion is the phase volume.As this is increased, inversion will tend to occur at a critical value, 4>\ equivalent to acritical oil volume per unit volume of aqueous phase. The driving force for inversionincreases particularly when <j> passes the value at which droplet deformation is necessary(0.74 for uniform diameters). Since proteins are relatively good stabilizers of oil-in-wateremulsions, <j>, might be expected to be in this region. This is exactly what is normallyobserved, as first pointed out by Acton and Saffle.46 Most results from the literature canbe recalculated to show <}>, in the range 0.65 to 0.85, so that the wide variations in ECvalues reported are due to the arbitrarily selected protein concentrations. Usually a smallamount of protein will be sufficient to prevent inversion until <f> 0.65, so much larger ECvalues can be obtained at low protein concentrations in the aqueous phase (C). Indeed, a<t>i of around 0.5 might be expected in a control without protein, giving an infinite ECvalue!

Several aspects of the performance of the test affect 4>, (or EC values). It may beincreased if the oil is added faster or if some is present initially; but this can be completelyaccounted for by a lower temperature rise (due to stirring) if the titration is completedmore quickly.44'47 Considerably reduced <frs are observed as the temperature is increased.The observed fall in surface rigidity with temperature12 may lead to increased coales-cence and inversion (see following). An increase in stirrer speed leads to a decrease in ECvalues and </>i.39'41'44'47 As expected, higher stirrer speeds tend to give smaller oil droplets,and Ivey et al.45 showed that the critical interfacial area per unit volume of emulsionremains roughly constant over a range of speeds. This may not remain valid for the veryhigh <f>,s (>0.85) that can be observed at low speeds,4 •43'48"50 where the oil may beincompletely dispersed with macroscopic oil-continuous regions present well before totalinversion.41 Agitation-induced encounters and coalescence may be partly responsible forthe effects of stirrer speed. In studies with small molecule surfactants, the materials ofconstruction of the emulsification equipment affect tja (e.g., Davies51), but this has notbeen investigated for the EC test.

In view of the previous information, the influence of proteins on $i can only betentatively and qualitatively related to physicochemical properties. An initial steepincrease in 0i is observed with increasing concentration, up to about 0.7 with a fewmilligrams of soluble protein per 10"6 m\ After this, <f>i rises more slowly with furtherincreases in protein concentration. The #i and d values observed in the EC test indicateinterfacial areas of the order of a m2 per 10~6m3 of aqueous phase. So the limiting amountof protein required is about that necessary to form a reasonably coherent film (of a fewmg. m"2). Hence the surface properties of the protein are probably the maj or source of influ-ence on the EC test. This surface role is supported by the observation that a substantialproportion of the protein becomes associated with the oil droplets during the test.39'48"52

However, the identity of the critical surface property(ies) remains unknown.

3. Functional Tests of StabilityAll the other functional tests used with proteins are essentially of stability after the

emulsion has been formed. Only rarely do the authors make clear what form of instabilitytheir test is revealing, but it is often possible to make some assessment of what may behappening.

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October 1981 163

In the "emulsifying activity" test introduced by Yasumatsu et al.,53 the emulsion iscentrifuged at low speed and the height of the unseparated layer expressed as apercentage of the total. The meaning of this test is crucially dependent on whether oil,aqueous phase, or both separate, but unfortunately this information is rarely reported. Ifno separated oil layer is formed, then only creaming and drainage will have occurred. Aspointed out by Pearce and Kinsella,30 the centrifugal field applied will cause rapidcreaming and drainage even if the droplets are completely stable to coalescence. Itappears that coalescence is usually slow under the test conditions with protein-stabilizedemulsions, while drainage is well advanced, and the droplets are closely packed and $ asthe cream layer approaches one. As a result, the fractional height observed is slightlygreater than the arbitrarily chosen <j> of the initial emulsion. This is particularly evidentfrom the results with variable 0 reported by Chen et al.,54 but it is also apparent in otherstudies.53'55 Under these conditions, the test is essentially a measure of drainage rates withrather poor sensitivity. With a few emulsions of low stability, oil separation does occurand reduces the measured height further. Similar considerations apply to analogous testsin which the emulsion is heated before centrifugation.

Another test that mainly follows creaming and drainage is the "stability rating" ofActon and Saffle.56'57 This involves standing for 24 hr, but the product of gravitationalfield and time is of the same order as in the emulsifying activity test. The water content ofthe lower half of the emulsion is determined, indicating the extent to which it has becomedepleted of oil droplets. Only in the experiments at <f> of 0.5 would any coalescence berequired to reduce the stability rating to zero. The pronounced increase in stabilityratings at higher initial <£ values are just as would be expected for drainage (Table 1).

The interpretation of tests in which an oil layer separates is hindered by the lack ofinformation on the mechanisms involved. Worthy of mention are the data of Ivey et al.45

for total decline in emulsion volume on heating, which shows an optimum range of <£ forstability (0.77 to 0.88). Though it is not clearly stated, they imply that the gradual fall instability below <j> 0.77 represented loss of aqueous phase, while oil was released above0.88. This is exactly as would be expected from the opposite effects of 4> on drainage andcoalescence (Section II).

B. Initial Breakdown Processes/. Creaming and Drainage

Some reported "stabilities" that are essentially a measure of creaming and/or drainagemay be shown to have the expected correlations with d and r)0 (Table 1). Cavoski et al.58

found that the rate of water separation from protein-stabilized tallow emulsionsincreased with d. van Eerd59 showed that actomyosin was considerably more effectivethan myosin at preventing the separation of continuous aqueous phase from emulsions ofinitial <f> 0.5; this was probably due to the higher viscosity of actomyosin solutions. Theresults of Chen et al.54 with the emulsifying activity test show that drainage became moreincomplete at high protein concentrations or with increasing levels of carboxymethylcel-lulose. Addition of 50% sucrose to the aqueous phase has a similar effect on the testresults.55 In each case a substantial increase in 77,, is probably responsible. Severalauthors have observed that increased power input during emulsion formation leads toimproved stability to creaming. This probably reflects reduction in average droplet size,but this has only been confirmed experimentally in one simple protein-stabilizedemulsion case (compare the results in two papers by Tornberg60'61). This effect is, ofcourse, well known in dairy and other emulsions.7

2. FlocculationSrivastava and Haydon25 made microscopic observations of adilute(<£0.01) emulsion

of petroleum ether in 0.01% BSA solution. Near the isoelectric point (pi) the droplets (1

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to 5 /im) flocculated extensively over a few hours. Away from the pi, where electro-phoretic mobilities showed that the droplets had a significant zeta potential (£), there was alimited equilibrium formation of dimers that was reversible on diluting the emulsion. Theresults were in reasonable agreement with the authors' suggestion that the reversible floc-culation was in the DLVO secondary energy minimum. Srivastava26 studied the details offlocculation at the pi in this system. The rate was only about one tenth of that expected forencounters, but could be increased to the theoretical value by the addition of smallmolecule surfactants. These were effective at concentrations that had been shown todestroy the rigidity of the protein film, and the flocculation rates were in reasonableagreement with his suggestion of an energy barrier proportional to the film elasticity. Themechanism seems likely to be displacement of adsorbed proteins by the other surfactants,with consequent reduction in steric repulsion between the droplets, as suggested byWalstra.85

Lata et al.62 made microscopic observations of droplets (average 1 nm) in hemoglobin-stabilized toluene emulsions (again dilute, with <f> 0.05). The pH was well away from pi,and electrophoresis showed a £ of —90 mV. Addition of various salts reduced £ andincreased the flocculation rate until it became faster than they could measure at £ less than10 m V. Calculations showed that the DLVO secondary minimum became deeper withdecreasing £.

Proteins can cause flocculation of emulsions by acting as "polymer bridges" betweendroplets. Proteins adsorbed to one droplet may also become attached to a region of bareinterface on a second, an effect that is known with other polymers. Keulemans and deBruin63 attributed the flocculation of an emulsion of corn oil in skim milk to theformation of polymer bridges. The consistency of the emulsion, which reflected thedegree of flocculation, passed through a maximum with increasing concentrations ofprotein during homogenization. At high C values, a complete surface film would beformed around most droplets before proteins could become adsorbed to two of them.Emulsions prepared at <f> of 0.6 were more viscous than those concentrated to this valueafter formation, probably reflecting the easier formation of bridges at higher <j>. Ogden etal.64 found flocculation due to polymer bridging in emulsions of paraffin oil in skim milk(glutaraldehyde-treated to fix the casein micelles). The expected effects of protein and oillevels were again observed. Flocculation of this type is, in fact, well known in dairyemulsions,7'64 and the bridging species has been shown to be the casein micelle; otherprotein species seem to be unable to cause bridging. Hence the position may be ratherdifferent from that with some other polymers, where a single molecule may be sharedbetween two interfaces. Instead, the flocculation depends on interactions betweendifferent casein molecules (in the micelle), each adsorbed to one droplet.

Such a link might also be formed by another route.7 Protein molecules often interactstrongly with each other leading, under appropriate conditions, to aggregation orprecipitation in bulk aqueous systems. Protein molecules already adsorbed to differentsurfaces may interact under similar conditions, giving rise to flocculation; this mayparticularly be responsible for the flocculation of some protein-stabilized emulsions nearthe pi.

C. Coalescence and Surface Properties1. Effect of Protein Solubility

Protein solubility appears to be the most important factor affecting performance in theemulsifying capacity test. Table 2 lists the studies in which this correlation has beenevident. It is particularly clear from the pH profiles for solubility and EC presented byCrenwelge et al.41 Several other observations are probably explained by effects onprotein solubility, particularly the decline in EC as the pH of muscle extracts approachespi, and dependence on salt concentration. Indeed it may be that solubility is the only

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Table 2CORRELATION OF EMULSIFYING CAPACITY WITH PROTEIN

SOLUBILITY

Protein(s)

Poultry muscle extractsBovine muscle extractsMiscellaneous animal tissue

extractsFish muscle proteinSoy concentrate, cottonseed flour,

globin, nonfat dried milkCod muscle extractsBeef and pork extracts

Solubility varied by

Pre/post rigor; extraction pHProteolysisDifferent extracts

SuccinylationPH

pH, storage, freeze-thawDifferent cuts, extraction conditions

Rel

656667

6841

6970

Soy and peanut flowers pH, salt concentration, heat treatment 71—73

protein property with a major effect. Using a wide variety of beef and pork meat extracts,Gillett et al.70 showed that $, in the EC test depended almost exclusively on the solubleprotein concentration. Volkert and Klein74 performed EC tests at equal soluble proteinconcentrations with four different soy products under various conditions, finding all<f>,s between 0.59 and 0.63 except for one point at the pi. However, some authors point toevidence that EC and solubility may not always vary exactly in parallel.72'73 Whatever theexact position, it would seem sensible that if EC tests are to be performed, then at leastprotein solutions of equal concentration should be used.

A correlation with protein solubility is also observed in the functional tests of stabil-ity.53'55'74"76 This may, in some cases, again reflect a need for solubility if a protein is to besurface active. Surface properties may often not be crucial during the stability testingprocedures. However, increased soluble protein concentrations may lead to smallerdroplets during the formation of the emulsion and, hence, enhanced stability to creamingand drainage.

Overall, it would appear that undissolved protein makes little or no contribution toemulsification, so that the effects of solubility are really effects of protein concentration(see following).

2. Effects ofpHThe effect of pH on coalescence has been one of the most fruitful sources of

information about the role of proteins in stabilizing emulsions. The pH will exert itseffect primarily by altering the charge of the protein molecule. Four main routes havebeen suggested by which this may in turn affect the emulsion:

1. Some proteins are incompletely soluble at the pi (see Section III.C.l.).2. The cohesiveness and, hence, rigidity of protein surface films is usually maximal at

the pi, where electrostatic repulsions between molecules are minimized. Such rigiditymay help to stabilize against droplet coalescence, especially by opposing defor-mation of the surface required in lamella rupture.

3. The surface potential of the protein film will pass through zero as its sign is reversedat the pi. Electrostatic repulsion between the surface films is the force opposinglamella thinning and, hence, rupture in the original DLVO theory.

4. Electrostatic repulsion between different parts of a single adsorbed protein moleculewill be minimized at the pi, so it will adopt its most compact conformation. Hence, aminimum in repulsion of approaching surfaces due to steric interference between theadsorbed proteins might be expected.

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Experimentally, maxima in the surface Theological parameters near the pi of theprotein do seem to be the rule.12 There are a few apparent exceptions,38 but no evidencethat these correlate with behavior in emulsions. Similarly, determinations ofelectrophoretic mobility confirm that the pi of adsorbed films is close to that of theprotein in solution, while £ tends to approach maximum levels for |pH — pi | > 2.77'109

Table 3 lists the effects of pH that have been observed in emulsions when the proteinshould remain completely soluble throughout. (Some effects of pH via solubility werenoted in the Section III.C.l.). Several different experimental approaches are included inthe table, but all should be related to the drainage and/ or rupture of protein-stabilizedlamellae. Many of the authors made parallel determinations of surface rheology, and theresults are summarized in the table. In general, stability seems to be maximal near the pi,suggesting that surface rigidity is important in resisting coalescence. Three studies3*5"25'170

indicate the opposite effect and, hence, a role for electrostatic or steric repulsion betweenthe surface films. In two of them, a comparatively low protein concentration (C) wasused. Graham et al.3'5 used 0.003% protein solutions to investigate the effects of pHbecause only at these low concentrations could instability be detected in theirultracentrifugation test. In parallel experiments with an initial C of 0.01 %, they estimatedthe surface concentration in the emulsion as only 0.1 mg.m"2. Their own studies onsurface films showed that no viscosity or elasticity could be detected at this low T. So it ishardly surprising that the repulsion force (electrostatic or steric) seems to be dominant atthese low protein concentrations. In the experiments of Srivastava and Haydon,25 thevery low <j> (0.01) and the occurrence of flocculation were also important. Other factorswere important as well. Though the overall rate of coalescence was higher at the pi, therewere many more lamellae susceptible to rupture because of extensive flocculation; atother pH values flocculation was limited and transient encounters relatively infrequentbecause of the low <f> (0.01). The authors calculated a corrected first order rate constantfor coalescence that was larger at pH 3.5, away from the pi.

None of the above factors seem likely to be responsible for the observations of de Wit etal.170 on whey protein stabilized milk-fat emulsions. At pH 4.5 and 5 (near the pi),coalescence and oil separation occurred rapidly, while at pH 4 or 5.5 the droplets werelarger than under optimal conditions. Similar instability was found if a preformedemulsion was adjusted to a pH in this region.

Since all three mechanisms for pH effects depend on electrostatic interactions, theseeffects should become less pronounced with increase in ionic strength. In the limit, thestability at all pH values should approach that found at the pi. Precisely this behaviorwas found by Mita et al.29 following the rate of coalescence of a BSA-stabilized benzeneemulsion. Increase in NaC 1 concentration reduced the rate at pH 2.9 or 7.0 to that found atpH 4.9 (near the pi); the latter was itself unaffected. Similar observations have been madein studies of emulsification with incompletely soluble proteins,72'81 for which suppressionof isoelectric precipitation by salt is well known.

3. Effects of Small Molecule SurfactantsThe addition of small molecule surfactants has been used on several occasions to

modify the surface rheology of proteins in emulsions. These agents are, in general, moresurface active than proteins, so they will tend to penetrate or even displace the proteinfilm. This usually reduces the rigidity of the surface film, though it should be noted thatother relevant properties may also be affected (e.g., £, especially with an ionic surfactant).Cumper and Alexander79 observed a substantial fall in the stability to coalescence of a y-globulin-stabilized emulsion on addition of 5% oleyl alcohol to the oil phase. This wascorrelated with a large decline in surface shear viscosity (171), and a complete loss ofelasticity (Gs). Biswas and Haydon34 found that 50 \iM tetradecyltrimethylammoniumbromide was sufficient to abolish the elasticity of a BSA film at the oil-water interface,

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Table 3EFFECT OF pH ON STABILIZATION OF EMULSIONS BY SOLUBLE PROTEINS

Protein Experimental approach Behavior Surface rheology (oil-water) Ref.

Gelatin0.01% Insulin0.25% Gelatin0.05—0.1% BSA0.1% PepsinWater soluble

muscle proteins0.01% BSA

0.5% Gelatin

Diluted egg white(ca. 0.4%)

1%BSA

0.004% BSA

0.004% Lysozyme

0.4% Wheyprotein

Emulsion stability (no details)Emulsion stability (visual)Lifetime of single oil dropsLifetime of single oil dropsLifetime of single oil dropsEmulsifying capacity

Coalescence rate in emulsion(droplet counting)

Lifetime of single oil drops

Emulsifying capacity

Coalescence rate in emulsion(droplet size determination)

Ultracentrifugal emulsionstability

Ultracentrifugal emulsionstability

Coalescence (droplet sizedetermination)

Maximum stability near piStable at pH 5 (near pi), not at pH 7Maximum near piMaximum near piMaximum near piMaximum at pH 5 (probably near pi)

Stability lower at pH 5 (near pi) thanpH3.5

Maximum near pi

Higher at pH 6 (near pi) than at pH 4or 8

Maximum stability near pi

Minimum stability near pi



TJ, only detectable at pH 5TJ, maximal near pi, G, zerorj, and G, maximal near pi77, maximal near pi—

17. and G, maximal near pi

Surface shear yield valuemaximal near pi

rj, and G, maximal near pi(but at higher C)

78793233,343480

25

35,36

49

28

5

3

170

oaoorn

0 0

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and that it also cut the half-life of single petroleum ether drops to about a quarter.Srivastava and Haydon25 extended this work, using phenol and tetradecyltri-methylammonium/tetradecyl sulphate mixtures as surfactants. Both 17, and Gscorrelated well with coalescence rates in an emulsion as well as with single drops; valuesof about 1 kg.s"1 and 10"2 Mm"1, respectively, were needed for maximal stability. (Thiscorrelation also held for changes in surface rheology induced by pH.) Similar effects oncoalescence were reported by Srivastava,26 with surfactants including decanol. Pearson82

studied emulsions of chloroform, liquid paraffin, or benzene stabilized by /?-lactoglobulin or BS A. No oil separated from the control emulsion in 24 hr, but withO.05 Mdecyltrimethylammonium bromide added it was broken completely in 10 min. (Thesurfactant alone gave emulsions of reasonable stability.) The author attempted tocorrelate his results with extensive measurements of surface rheology at the air-waterinterface.

In the light of these effects, it should be noted that most natural oils and fats containsmall molecule surfactants such as monoglycerides and free fatty acids. Lipid emulsifiersare deliberately added to many food emulsion products that also contain proteins; theirinteractions can be cooperative or at least not mutually destructive (in terms of emulsionstability). It seems likely that specific protein/emulsifier combinations and conditionsare needed for such effects.

Oortwijn and Walstra172 have reported some direct measurements of the effect of smallmolecule surfactants on protein surface concentration in a milk fat emulsion. With eitherwhey or skim milk, T reached during emulsification was substantially reduced in thepresence of more than 0.1% of either a hydrophilic (Tween® 20) or a hydrophobic(monoglyceride) emulsifier. Addition of Tween® 20 to a preformed emulsion caused thedesorption of 70% of the previously adsorbed protein.

4. Different Proteins and Protein ConcentrationsThe relative stabilizing abilities of different proteins have not often been compared in

well-defined systems. In any case this is not a particularly fruitful source of information,as differences between soluble proteins seem to be relatively small. Since the effects of pHand other conditions on a single protein can be larger, care is needed in drawingconclusions from such comparisons. Biswas and Haydon34 showed that both the half-lifeof single drops and the surface elasticity were slightly larger with 0.1% pepsin than with0.1% BSA, each at their optimal pH values. A comparison of the behavior of threedifferent proteins was an important part of the studies of Graham et al.3'5 The stabilityof their emulsions to ultracentrifugation declined slightly from BSA to lysozyme to/J-casein; this could not be readily correlated with their measurements of surfacerheological parameters. However, this is what might be expected from the very lowprotein concentrations used (discussed previously).

In the very full measurements made by Graham et al.,3'5 surface rheology was stronglydependent on protein concentration (either C or T, which were related since adsorptionwas allowed to proceed to equilibrium). In every case the rheological parameters showeda maximum, and declined to lower (often zero) values at the extreme C values of 10"5 and10"'%. There is some other evidence for the existence of such maxima.12'38 Optimumprotein concentrations for the stabilization of emulsions have not been observed, thoughIzmailova38 reported one in the stabilization of single benzene drops by a-casein. Thedrop half-life and the "interfacial strength", which appears to be a measure of surfaceshear yield stress, both showed maxima at C of 2% (in the range 0.05 to 3.5%). However,recent studies by de Feijter83 suggest that the maxima in surface rheological parametersmay be artifacts of the measuring procedure.

Concentration effects illustrate just how difficult it is to relate measured surface

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October 1981 169

properties to emulsion behavior. The surface/ volume ratio in an emulsion is many ordersof magnitude greater than in a Langmuir trough, so adsorption of protein to the dropletsoften significantly depletes the bulk. Therefore, comparisons are best made on the basisof surface concentrations, though these are not easy to determine. Of several studies ofthe effects of C, only in the low <j> emulsions of Srivastava26 it is reasonably certain thathigh F values were reached. The 0.2% BSA supplied is in excess (sufficient for F around200 mg.m2), so a significant reduction from maximal Theological parameters might beexpected; there was no change in coalescence rate. Shotton et al.27 found that a liquidparaffin emulsion was stabilized by thick surface films of gelatin. Washing with watergradually decreased F to about 3 mg.m"2, at which point the oil separated. Oortwijn andWalstra173 have recently also demonstrated that coalescence stability of protein stabilizedemulsion droplets falls markedly at F is reduced (in the region of 1 mg.m"2).

5. The Role of Surface PropertiesThe previous results clearly suggest a link between the rigidity of protein surface films

and their stabilization of emulsion droplets to coalescence. This leads to the commonlyaccepted model that the high viscoelasticity of the protein film opposes the surfacedeformations (either in shear or dilation) that are required for the later stages of drainageand for rupture of the lamellae. This model is essentially a development of early ideasabout the formation of a tough "skin" around the emulsion droplets. However,quantitation is impossible because the precise mechanism of lamella rupture remainsunknown and, hence, the identity of the crucial parameter is uncertain.

This model does not seem to be completely adequate. It is well known that mostproteins are very poor stabilizers of water-in-oil emulsions. Cockbain and McRoberts31

showed that the same applied to single drops of aqueous solution in a bulk oil phase. Asthey argued, the surface rheology is identical for oil-in-water or water-in-oil drops, so itcannot be the only determinant of lifetime. Biswas and Haydon33'34 made similarobservations on single drops and discussed the implications for the role of surfacerigidity. They pointed out that since proteins are relatively hydrophilic, the bulk of anadsorbed molecule is probably located on the water side of the interface. This must bewhat makes the behavior of aqueous lamellae so different from oil lamellae, though theywere less specific about the mechanisms involved.

Kitchener and Mussellwhite' first suggested how this effect might be mediated. Pro-teins are considered to form a thick, nondisplaceable film containing water (almost aprotein gel) on the aqueous side of the interface. When an aqueous lamella thins two ofthese films approach and further drainage is opposed by osmotic forces. This sort of be-havior has now been explored much further in the theory of "steric stabilization",18"84

though no attempt has yet been made to account for the detailed effects in a protein-stablized emulsion. The theory, as applied to synthetic polymers, would predict that thesteric or entropic force of repulsion between the surface films would be a minimum at thepi, where the protein molecules are most compact. However, the results above show aclear maximum in stability at this point.

Walstra85 has suggested that it may be instructive to consider the energy needed todesorb a protein molecule, which may be related to an activation energy for coalescence,by analogy with solid particle stabilized emulsions. This analogy is supported by theobservation of "limited coalescence" in protein-stabilized emulsions.1 The behavior ofprotein-stabilized emulsions does seen to be consistent with a role for desorption of partof the film as a prerequisite for lamella rupture. Because of the maximum inintermolecular attraction, the activation energy involved should be maximal at the pi.Small molecule surfactants aid desorption by disrupting the structure of the protein film.In pursuing this idea, the activation energy for desorption may well prove to be

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experimentally accessible. It should also be noted that a correlation might be expectedbetween the difficulty of protein desorption and the "rigidity" of the surface.

From this discussion it should be apparent that there is no quantitative model for thestabilization of aqueous lamellae by proteins, and that the qualitative mechanismsinvolved are still in dispute. It seems likely that more than one property of the proteinsurface film will be important in resisting coalescence. However, the details should not beallowed to obscure a useful general conclusion. Almost any soluble protein, at quite a lowconcentration, will adsorb to give a fairly high resistance to coalescence of emulsiondroplets under normal conditions.

6. Miscellaneous FactorsShotton et al.27 made a limited microscopic study of the increase in droplet size on

storage of gelatin-stabilized liquid paraffin emulsions. Intriguingly, an emulsion withsurface mean d of 9 nm showed noticeable coalescence after 4 days, while virtually noneoccurred if it was initially homogenized to d of 4.5 /im. Such an effect of d has beenobserved in other types of emulsion and is predicted by theories of coalescence.

Skoda and van den Tempel86 examined the effect of freezing the disperse phase(tristearin), and found that emulsions stabilized by a variety of small moleculesurfactants were completely broken on remelting while, with sodium caseinate, d was noteven changed. Addition of small molecule surfactants has been observed to reduce thefreeze-thaw stability of emulsions in sodium caseinate solutions.174 van Boekel andWalstra175'176 have made a comprehensive study of the effect of oil droplet crystallizationon the stability of emulsions with a wide range of surfactants, including caseinate. Atleast a major part of the effect of surfactant type seemed to be due to an influence on thesites at which crystals formed in the droplets.

Whiting and Richards87 showed that certain divalent cations could increase EC valuesand the stability of the emulsions produced. This may reflect an increase in rigidity of theadsorbed protein film, as appears to occur in foams (see below). Du Bois et al.,66 studyingthe papain hydrolysis of muscle homogenates, found that after there was no furthersolubilization, the EC value declined with continued proteolysis. This also seems toparallel an observation on foams, that smaller protein fragments become less effective instabilizing against coalescence. A similar observation was made by Adler-Nissen and SejrOlsen88 on proteolysis of a soy protein. Most of the more empirical studies onemulsification have used mixtures such as "muscle proteins", but little is known about thecomposition of the surface films formed under such conditions. Galluzzo andRegenstein52 observed that the EC value of chicken actomyosin was increased in thepresence of ATP to that found with myosin. They showed that when the actomyosin wasdissociated by ATP to actin and myosin, essentially only the latter was adsorbed to the oildroplets. Studies in this laboratory have shown that under some circumstances mixedprotein films can be formed, sometimes with characteristic properties.89

D. Other Properties/. Heat Stability

Because of the importance of heat treatment with many food products, it has oftenbeen applied to protein-stabilized emulsions. Increase in temperature tends to accelerateall breakdown processes in emulsions, both directly and via a reduction in r]0 andsurface rigidity. However, with many proteins thermal denaturation will cause thecontinuous aqueous phase to become more viscous again above a certain temperature, oreven to set to a gel. Thus the rate of breakdown can fall again as flow processes arestrongly inhibited. In fact, the stability of many protein-stabilized emulsions to heating ismarked compared to most other surfactants. It is possible, however, for adsorbed

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October 1981 171

proteins to cause flocculation of an emulsion on heating, under conditions when theywould normally precipitate from solution.7

Tsai et al.90 studied high oil emulsions (0 0.84) stabilized by myosin, actin, ortropomyosin-troponin. The heat stability of the emulsions correlated with the strength ofgel formed on heating the muscle protein solutions alone. However, their findings are notconclusive, as different protein concentrations were used in the gelation test, and the stablemyosin emulsion had much smaller oil droplets than with actin. The importance ofincreased viscosity in heat stability is also suggested by the results of Wang andKinsella," if reasonable assumptions are made about the relative importance of waterand oil separations in the total they reported. (Their method, "emulsifying activity", wasdiscussed previously.) Low concentrations of alfalfa protein isolate were sufficient toprevent coalescence in the unheated samples, but further increase in C caused littlereduction in creaming and drainage. Low C values could not prevent oil separation onheating, and 2% protein was needed to give the same stability as the unheated samples.However, at higher Cs, creaming and drainage were less complete than in the unheatedtest, probably because of a heat-induced viscosity increase. Adding 50% sucrose to theaqueous phase reduced creaming and drainage only in the unheated test; after heating theprotein is probably dominant in determining the matrix rheology. A similar effect can beseen from the results of Chen et al.54 with carboxymethylcellulose as viscosifier.

2. Emulsion RheologySeveral theoretical and semiempirical relationships give reasonable predictions for the

viscous properties of emulsions up to </> of around 0.6, provided there are onlyhydrodynamic interactions between droplets (see especially Reference 19). A usefulequation is that due to Eilers

/ . , l-25<fr V' = V° 1 + 1 TT7—

where <f>mil. is the phase volume for close packing of the droplets. It can be seen that 0 has amajor influence on the emulsion viscosity; van Vliet and Walstra177 have shown thatwith milk a better agreement with measured viscosity is obtained if <f> is taken to includethe volume occupied by protein and even sugar molecules.

Unfortunately, studies on simple protein-stabilized emulsions seem to have been donewithout reference to this basic theory.

Acton and Saffle56'57 reported viscosities identical to water for emulsions of <f> 0.5;perhaps the emulsions broke in the viscometer. Similarly McWatters and Holmes71'72

discuss viscosity measurements in terms of salt, pH, and concentration effects, while theemulsions had substantially varying #.

It has been shown that the viscosity of skim milk stabilized emulsions of moderate <£increased when flocculation by polymer bridging was promoted. Izmailova et al.,"measuring the yield stresses of gelatin-stabilized emulsions (<f> 0.7), found an increaseover the first 3 days of storage, which they ascribed to the slow formation of protein cross-links between the droplets. After vigorous stirring the yield stress was reduced to zero,though its failure to recover under some conditions probably indicated that the emulsionitself had been broken, ratherthanjustthecross-links.Theyieldstress was greaterwhen theoil used had a larger density difference from the aqueous phase; this may reflect slightdrainage of the emulsion prior to measurement giving a region of higher 0, close-packeddroplets, and much larger yield stress. An emulsion stabilized by 0.5% human serumalbumin had more than five times the yield stress of the gelatin system, perhaps becauseits droplets were half the size. Ramanathan et al.92 also observed an effect probably due to

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d. Emulsions stabilized by groundnut proteins were more viscous when prepared athigher mixer speed, even though <t> was slightly reduced. Such effects of d are oftenobserved in emulsions, though they may, in fact, reflect changes in the spread of sizesand, hence, 4>mn-

E. Emulsion FormationFor a fundamental study of the formation of emulsions, the key measurement is again

droplet size. This has rarely been determined in work on protein-stabilized systems.There is rather more data on the creaming rates of freshly prepared emulsions, butcaution must be exercised in relating these to initial droplet size variations; changes inT;O, or flocculation and coalescence during creaming may be responsible. Of a variety ofemulsification equipment used with proteins, the most popular have been high powermixers that give strong turbulence in low viscosity media, which will be referred to here as"blenders". The term "homogenizer" will be restricted to devices that force the processstream through a narrow valve under a high pressure drop.

/. Mechanical Effects on Droplet SizeEmulsions are normally formed from bulk liquid phases and the agitation required

obviously has an important influence on the process. Several effects have been predictedfrom theory and observed in emulsions of various types.93'94'178 This section notes howsome of these effects have been confirmed in simple protein-stabilized systems.

The droplet size is reduced as the power input rises, either in a blender45 or anhomogenizer.61 Similar behavior is suggested by measurements of creaming stability,60'95

including a study of an ultrasonic device. (A high power input is not essential to producesmall droplets, though; Cecil and Louis96 obtained droplets of 0.1 to 2 jum by gentlyinverting the tubes of emulsion over 24 hr.)

As the time of exposure to the emulsifying equipment increases, the droplet size falls atfirst, but then approaches a limiting value. This has been observed from d measurementswith a blender30 and homogenizers,30'61 and from creaming stabilities with blenders, ahomogenizer, and a sonicator.60'95

Tornberg et ai.60-61*95-97 have made a very detailed study of mechanical and equipmenteffects with several proteins. The equipment was characterized in terms of its power inputto the fluid, and of the energy density (power per unit volume) in the emulsificationchamber; theory predicts that this is the crucial variable determining the limiting dropletsize. Smaller droplets were obtained when the same total energy was supplied at highpower as compared with multiple passes at lower power, supporting a key role for energydensity. Measured creaming rates were also dependent on equipment type; it wassuggested that the relatively poor performance of the blenders (at a given power) was dueto the low energy density in this apparatus.

2. Protein Effects on Droplet SizeBefore considering the effects of proteins, it must be noted that three processes are

actually taking place simultaneously during emulsion formation. As well as dropletdisruption, surfactant molecules will adsorb to the newly created surface, and dropletswill be exposed to the threat of recoalescence; this last process is potentially much fasterthan during subsequent storage, as the vigorous agitation leads to much more frequentencounters between droplets. Recoalescence is often very important with proteins,because the development of a stabilizing surface film around new droplets is relativelyslow. Clear evidence of the importance of recoalescence comes from the studies of Pearceand Kinsella30 with a valve homogenizer. If the protein concentration in an emulsion wasreduced by dilution, further homogenization would promote net coalescence andincrease droplet size (Figure 3).

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October 1981 173

025

5 10 15 20 5 10Number of passages throughhomogenizer

FIGURE 3. Effect of the dilution of anemulsion on the process of homogenization.Droplet size was assessed by measurement ofabsorbance at 500 nm, which is proportionalto total interfacial area (and hence the in-verse square of droplet size). Emulsion sta-bilized by 2% whey protein solution, <f> = 0.4,samples diluted 1/5000 before measurement(O). Same emulsion diluted with an equalvolume of water at point shown by arrow,samples diluted 1/2500 (D). Emulsion sta-bilized by 1% whey protein solution, <p = 0.2,samples diluted 1 /2500 (A). (Reprinted fromPearce, K. N. and Kinsella, J. E., / . Agric.Food Chem., 26,716,1978. With permission.)

Most effects of proteins can be explained by likely or possible influences onrecoalescence. One study that indicates a direct effect on droplet disruption is that ofMita et al.28'29 on the emulsification of benzene in a blender. With five different proteins,the largest droplets tended to be formed for pH values near the pi. Increasing the saltconcentration raised the droplet size at a range of pH values to that found near the pi.29

Their own results with BSA confirmed that coalescence on storage was slowest underconditions where the largest droplets were obtained. Surface tension effects did not seemto be responsible either, as y was not maximal under conditions giving the largestdroplets. It is possible that these effects are due to the surface rheology of the protein filmopposing the deformation necessary for droplet breakup, so the maximum rigidity nearthe pi leads to the largest droplets. Mita et al.28 pointed out that the droplets in theseemulsions were relatively coarse compared to those obtained with small moleculesurfactants, and ascribed this to the higher y values found with proteins. More importantis probably the very high effective y values that apply to the rapidly extending surfacesduring droplet disruption; this is, in effect, another way of looking at the rigidity of theprotein surface film.

Different proteins have been shown to give emulsions of different dropletsizes,28'30'61'67'90'172 but the effects usually seem to be fairly small. An exception is the studyof Pearce and Kinsella30 where droplet sizes ranged by 2.5-fold between different solubleproteins. Yeast proteins modified by extensive succinylation gave the smallest droplets.With ovalbumin, d reached a minimum after only three homogenizer passes, and thesubsequent increase could be correlated with surface coagulation and protein depletion

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from solution; however, this was probably due to aeration. Tornberg61 found,intriguingly, that the existence and direction of protein effects on d depended on themechanical conditions applied in an homogenizer, particularly the number of passes.

Pearce and Kinsella30 found with an homogenizer that the droplet size decreased withincreasing concentrations of whey protein from 0.5 to 5%. The advantage of this highconcentration may be in the rate of adsorption to freshly formed interface. In a blender,where the lower energy density may allow longer for adsorption before collision, 0.5%ovalbumin seemed to be sufficient to give the minimum droplet size.28

Oortwijn and Walstra172 investigated the influence of <j> (0.05 to 0.50) and C (0.01 to2%) on the droplet size produced on emulsification in whey with an homogenizer. Inmilk-fat emulsions, d was unaffected by C and rose slightly only for <f> above 0.35; whilewith paraffin oil, d fell slightly with increasing protein — fat ratio (or with C/ <f>). de Wit etal.170 found that d did fall with increasing protein — fat ratio when milk fat washomogenized in a whey protein concentrate solution — while significantly larger dropletswere produced if <j> was raised from 0.04 to 0.35.

3. Effect on the Nature of the Surface FilmThree studies have recently shown how the emulsification conditions can affect the

nature of the protein surface film formed, assessed by the measurement of surfaceconcentration (F). Oortwijn and Walstra172 found that F in a whey-stabilized emulsionmade in an homogenizer increased as C was raised (in the range 0.01 to 2%) or as <p wasreduced (in the range 0.04 to 0.5). The interaction between these variables was bestdescribed by a linear dependence of F on the logarithm of the protein — fat ratio; thisheld quite well except at very high protein — fat ratios. The final value of F (usually 0.5 to2.5 mg.nf2) was established either during homogenization or shortly afterwards, nofurther increase occurring between 30 min and 24 hr after formation, even thoughunadsorbed protein was always present in the aqueous phase. There was some selectivityin the adsorption of different protein species from the whey, BSA being notably lessadsorbed than the others. Oortwijn and Walstra also showed that much higher F values(10 to 20 mg.nf2) were found with casein micelles, either isolated or in skim milk.Electron micrographs179 demonstrated that this was because the micelles remained partlyintact at the interface, though some spreading of the casein did occur. With skim milk, Fdid not fall as the protein — fat ratio was reduced; instead, when the protein becameinsufficient to cover the droplet surface, flocculation occurred, presumably by caseinmicelle bridging (mentioned previously). Sodium caseinate, in which the micelles aredestroyed, gave similar F values to whey.

Todt'80 investigated the adsorption of protein from skim milk, whey, and casein duringthe emulsification of sunflower oil ($ 0.5) in either an homogenizer or a blender. Hefound much smaller differences in F, with most values in the range 5 to 15 mg.nf2. Thedroplets obtained were several times larger than in the work of Oortwijn and Walstra.Tornberg61 found that emulsions of soybean oil (<£ 0.4) prepared in an homogenizer hadF in the range 1.5 to 3 mg.nf2 when the droplets were small, with either caseinate, whey,or soy protein. When bigger droplets were produced at lower power inputs though, muchhigher F values were found with soy isolate or whey (in the absence of salt). Hence itappears that F may generally be higher when larger droplets are produced, though it mustbe remembered that F determination by difference is at its most inaccurate here.

Protein adsorption can also be affected by mechanical factors. Todt180 found that Freached during emulsification in skim milk was substantially higher with an homogenizer(around 12 mg.nf2) than with a blender (around 5 mg.nf2). The increase was due to moreadsorption of casein, rather than whey proteins, and may reflect the facilitation oftransport of larger particles (casein micelles) by high energy turbulence.178 Tornberg61

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October 1981 175

found some evidence for a fall in T values if the power input (pressure drop) of anhomogenizer was reduced to very low values, with either whey or caseinate. Again,promotion of protein adsorption by increased turbulence may be responsible.

IV. PROTEIN-STABILIZED FOAMS

A. Experimental ApproachesThe basis of foam formation must be the creation of gas bubbles in the liquid. The three

main methods that have been used in studies on proteins (bubbling, whipping/ beating,and shaking) are discussed in the next three sections. Gas injection into a rapidly flowingliquid stream is the preferred method of generating fire-fighting foams, and has been usedin several studies on protein agents for this application.98"100 The equipment used isnormally referred to as a "branchpipe".

Two practical differences distinguish the formation of foams from that of emulsions.First, an excess of disperse phase (gas) is often available, so the volume fractionincorporated is variable rather than arbitrarily fixed. Second, the "formation" process isusually taken to include some stages which correspond to breakdown processes inemulsions. The initial uniform dispersion of moderate phase volume ("a gas emulsion")rapidly breaks down by bubble rise and. drainage. Normally measurements are not madeuntil the "true foam" has separated as a layer of very high (f>, with bubbles distorted intopolyhedral shapes. Figure 4 shows the three states involved, the volume measurementsmade, and definitions of the parameters derived from them. (Definitions of overrun andfoam volume vary, but the most common ones, as given, will be used in this review.) Thephase volume in the foam layer (#f) is usually approaching one, rarely determined assuch, but sometimes available from measurements of foam density or foam expansion,when defined as 100%X E/D= 100/(1 -<f>t). If very large amounts of gas are introduced,especially when whipping relatively concentrated protein dispersions, the liquid maybe completely converted to foam (i.e., no liquid drained when foam is judged "formed";E = C). Under these conditions the extent of gas incorporation is usually expressedas overrun which now equals </>/(l — </>).

1. BubblingBubbling of gas through a porous sparger has been the most popular formation

method in basic studies on foams. It tends to be more reproducible and give moreuniform bubble sizes, and it allows easy monitoring of the progress of formation. Thehistory of each bubble is believed to be reasonably well defined. With small moleculesurfactants, the foam volume often reaches a steady state level, which bears a constantratio to the continuing gas flow rate. This ratio has been advanced as a fundamentalcharacterization of foaming solutions, the dynamic foam lifetime.17

A number of workers have attempted to obtain steady state foam volumes fromprotein solutions.101"107 On continued bubbling, the foam volumes of most proteinsolutions increase to levels too large to handle experimentally, and low concentrations(0.01 to 0.2%) have been used to obtain maxima. At these concentrations most of theprotein seems to be adsorbed by the bubbles passing through the solution and carried intothe foam column. Buckingham108 noted that the top of the foam column was relatively firm(assessed by penetration of a perforated weight) and composed of fine bubbles, whilecontinued bubbling produced a very different, weak, open foam below this; similarobservations have been made in this laboratory. This view is supported by theobservation of Cumper105 that the volume of foam produced was dependent on the totalamount (and not the concentration) of protein in the solution being bubbled. So theobservation of maxima under these conditions probably reflects irregular collapse of the

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o o© oo o

°o °0 o9o o

Qoo

*"6o

oo

i

Liquid Gas emulsion Foam

A Volume of liquidB Volume of gas incorporatedC Total volume of dispersionD Volume of liquid in foam I=E - B )E Volume of foam

Overrun defined a s £ l £ * 100% I =£.» 100%)A A

Foam volume defined as JL «100%A

Foam phase volume (j6f J = £ l £ = _

Gas emulsion phase volume = - 2 -

FIGURE 4. Stages and definitions in foam formation. As usual, heights in a flat-bottomed vertical-sided vessel are taken to represent volumes. It is assumed that all bubbles in the gas emulsion remainstabilized in the foam at first.

lower protein-poor foam; the maxima are found to be poorly reproducible. Because theadsorption of proteins is essentially irreversible, a true steady state is certainly notpossible. In many cases the foam volume declines beyond the maximum, rather thanmaintaining a steady level, probably due to surface coagulation (see following). From thepreceding it should be clear that a "dynamic foam lifetime" has very limited meaning inprotein systems.

Bubbling can also be used to make a foam whose stability is assessed after stopping thegas flow. The rate of increase in foam volume and the final level reached are often quotedas well. The gas supplied can only be lost by collapse at the top of the foam column, so thefoam volume at any time is essentially determined by the amount of gas passed and thestability to collapse (albeit in a foam column suffering additional mechanical stresses as itcontinually rises in its container). Often collapse is relatively insignificant and all the gassupplied remains in the foam. At sufficiently high gas flow rates very large foam volumescan be obtained, even from dilute protein solutions (values greater than 2000% werereported by Thuman et al.,103 while 1000% is normal). Foam expansions up to 160-fold(16,000%) have been measured;109 this is equivalent to <£f 0.994, while the smallest valuesobserved are around 0.9.

2. WhippingWhipping (or beating) can be carried out in a variety of devices that vigorously agitate

a liquid and its interface with a bulk gas phase. This method has been preferred for mostof the "functional tests" of proteins, as it is the standard means of gas introduction inmost aerated products. The process of bubble formation and the history of a singlebubble are not well defined, so the method has been unpopular for basic studies.However, it is not properly appreciated that bubbling is not just a simplified model of the

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October 1981 177

whipping process, but that there are some fundamental differences between them. Thewhipped foam is well mixed throughout its formation, so the stratifications often foundin bubbled protein foam columns are not observed. Every bubble remains subject tosevere mechanical stresses throughout whipping, and these will promote much morerapid coalescence than in the standing foam. The volume of air included usually goesthrough a maximum with increasing intensity of beating (severe mechanical beating is astandard method of foam breaking). Hence, the observation of maximum levels of gasincorporation during whipping reflects a much more real dynamic equilibrium betweenmechanical formation and destruction of bubbles, even in protein-stabilized systems. Inaddition, mechanical stresses can break up bubbles formed earlier into several smallerones. Finally, the greater agitation of the air-water interface of the bubbles may affect theformation of the adsorbed protein film (see following).

Overruns observed on whipping protein solutions or dispersions vary widely, fromaround 300 to over 2000%. The beating equipment used has the major influence; thewhole range of overruns can be observed with egg white (e.g., compare Nakamura andSato110 and Goodall1"). Unlike bubbling, at the moment that whipping is stopped the gasis more or less uniformly dispersed in the liquid. With the overruns noted the bubbles willrange from hardly distorted spheres (</> 0.75) to polyhedrons with very thin lamellae.

3. ShakingThis method has been used only rarely.3'4'53'76'"2"113 In terms of the fundamental

characteristics discussed in the Section IV.A.2., it resembles whipping rather thanbubbling. The rate at which gas bubbles are introduced into a solution seems to dependon the frequency and amplitude of shaking, the volume and shape of the container, andthe volume and flow properties of the liquid. It is hardly surprising that the method hasproved difficult to standardize. Foam formation by shaking tends to be slower than bybubbling or whipping under similar conditions, but this seems to be largely due to therelative inefficiency of the process in producing gas bubbles. The maximum foamvolumes obtained are also rather lower, the highest reported values being less than400%.4 Again this may be due largely to a hydrodynamic and rather trivial cause; thecontainer becomes nearly full of foam and incorporation of the small remaining volumeof bulk gas is very difficult.

4. Comparison of Foams ProducedThe first impression from the literature, which has misled many authors, is that the

type of method used to make a foam has a major effect on its properties. However, therehave been no systematic studies of foams produced from the same protein solution bydifferent methods. Foams produced by bubbling usually seem to have much lowerstability, but this may well be due to the much lower protein concentrations normal inbubbling studies. Because of this use of widely different concentrations, few results arecomparable, but Table 4 shows some observations made under roughly similarconditions. It is clear that the stability of a foam made from a given protein solution can bemarkedly affected by the method of formation — hardly surprising since key parameterslike <f> and d may vary. However, there is no evidence that the type of foaming method(bubbling, whipping, or shaking) has any systematic effect, though this cannot be ruled outeither.

5. Stability MeasurementsTwo fairly obvious measurements characterizing foam stability were introduced in

Table 4: drainage of liquid (sometimes called leakage or syneresis) and collapse of thefoam column (decrease in volume, essentially through loss of gas). However, these

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Table 4COMPARISON OF STABILITY OF FOAMS PRODUCED BY DIFFERENT

METHODS UNDER SIMILAR CONDITIONS

Protein

a-casein

/3-casein

Crude casein

BSA

Egg white

Concentration(%)

0.050.10.050.10.10.50.5

122

0.030.1

10.10.051—21—2

PH

7.27.07.27.07.07.07.0Natural6.56.5—7.05.55.55.57.07.2NaturalNatural

Method

BSBSSBSBWWBSBSBBW

Drainage(min)

!„ 4.1

t1/59.8

—lx 9

—t% 4.7t,, 3.2—————No foamt 2t 1.5-3.5

Collapse(min)

tM 24Complete in 35t1/S55None in 35UA 16t 90

V. 4 5

V. 70

t,, 80

t, 11

«H 3 2

No foam——

Ref.

114

114a

115116

: The methods are indicated by B (bubbling), W (whipping), and S (shaking). Subscripted t's in-dicate the time for the specified fraction of total drainage or collapse to occur. A first-order pro-file is not necessarily implied.

* These unpublished studies have been carried out in this laboratory by several different groups ofworkers.

macroscopic processes do not correspond directly to the microscopic events of drainage(from lamellae and plateau borders) and lamella rupture. The latter may lead just to anincrease in bubble size, without collapse, while rupture of the lamellae can be animportant source of draining liquid. When it does occur, collapse is often ragged anddifficult to measure, with large voids continuous to atmosphere appearing deep in thefoam while some bubbles remain intact at the top of the original column.

Only two studies have been made of the change in bubble sizes within a foam column asit ages. Clark and Blackman"7 photographed the side of a foam column to determinebubble size and, hence, specific surface area (m\m~3), and followed the changes in thisduring disproportionation. They showed that the specific surface area could also bemeasured by a simple light scattering technique."8 Mita et al."9 photographed the sideof wheat protein foams and calculated mean bubble volumes. The change with time of thereciprocal of this parameter, the number of bubbles per unit volume, was used as ameasure of coalescence rate. Both specific surface area and mean volumes can be relatedto a mean bubble size that refers directly to spheres with some dimensional relationshipto the polyhedral bubbles (denoted here as d'). Criticism can be made of the practicaldetails of the above methods and in particular the side and the bulk of the foam may berather different. Nevertheless, they do provide much more fundamental information onthe processes occurring in the foams, which is correspondingly easier to relate tophysicochemical properties. Since these methods would appear to be fairly simple inpractice, they are to be recommended.

The lifetime of single bubbles below a gas-water water interface has been investigatedas a model for protein-stabilized foams.103'105'"9"121 Much the same reservations apply as

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October 1981 179

for the use of single drops as a model for emulsions (see Section III. A. 1.), though the sizedisparity is less dramatic between single bubbles and their counterparts in typical foams.Although the plane surface is usually aged sufficiently, the bubbles themselves havealways been released within a minute, probably accounting for the low lifetimes observed(at most a few minutes). Despite these differences, the stabilities under differentconditions do seem to vary as for bulk foams. Nakamura120 did report some disagreementon the ranking of egg white proteins, but the foaming method used is suspect and thesingle bubbles were observed under a spread film, not an adsorbed one. An interestingcomparison of single bubbles with bulk foams is possible from the parallel studies of Mitaet al.,"9 using wheat proteins. The two methods were in agreement over the relativestabilizing abilities of different proteins and the effects of pH, but the half-life forcoalescence of a bubble in the foam was some 40 times that for one at the plane interface.A bubble in the bulk of the foam may be protected by continuous drainage of liquid intoits walls from above, as well as by the factors noted.

The drainage and rupture of single lamellae of protein solutions in air have also beenfollowed as a model for foams. Again, the model differs from the real system in much thesame ways as for single bubbles. Considerable difficulties have been experienced inobtaining reasonable stability and reproducibility, because of the tendency of the proteinto coagulate at the surfaces, giving irregular lamellae.122'123 The general impression is oneof rather variable and unpredictable results with proteins. For example, whileMussellwhite and Kitchener122 found that lamellae from BSA solutions were very thinand stable in the presence of 0.1 M salt, Yampol'skaya et al.'24 reported that they wouldrupture as soon as thin (black) areas appeared, under very similar conditions. Theproperties of the lamellae are dependent on the aging of the surfaces from which they areproduced; under otherwise similar conditions BSA solutions gave lamellae of quitedifferent stabilities when their surfaces were aged for 15 min122'123 or overnight.5 In viewof the aforementioned, great caution has been exercised here in interpreting resultsobtained with this method.

B. Stability1. Drainage

The absolute rate of drainage from protein-stabilized foams (like those of othersurfactants) usually declines with time. Many authors quote an initial rate, but this haslittle meaning in experiments where an arbitrary amount of drainage occurs during"formation" (mentioned previously). Indeed, the proportion of liquid remaining in thesefoams (1 — <t>t), at the end of a fixed formation time, often reflects drainage stability. So inthese experiments a high foam expansion (or low density) is, in fact, usually an indicationof low stability; hence, the observations that foam expansion (and so cf>i) is often higher atlower protein concentration109'115 or for more rapidly draining foams.109 A relatedobservation is that </>t decreases when bubbling at higher gas flow rates109'125'126 becausethe foam column has less time to drain during its formation. Since it contains more liquidat the end of "formation", it is observed to drain faster when the test of "stability" starts.

The drainage of polyhedral foams is usually viewed as a combination of two processes.Liquid drains from the lamellae into the Plateau Borders because of a difference inLaplace pressure; the radius of curvature of the interface at the Borders is much smaller(See Section II). At the same time, liquid drains through the Plateau Borders under theinfluence of gravity. Significant stability to both processes requires that the surfacesbehave as nearly rigid walls, due to y gradients or other surface properties; if these alloweven slight movement of the interface, major increases in drainage rates can result. Sometheoretical analyses have been made for each drainage process; for example, the drainageof a rigid disk-shaped lamella of thickness h and radius R is given by

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dh 2 APh3

^T~3 »;0R2

where AP is the Laplace pressure difference. Clearly the rate declines rapidly as drainageproceeds and the lamella becomes thin. A similar prediction can be made for drainagethrough the Plateau Borders, though it is not yet possible to combine the analyses to yielda mathematical relationship for the decline in drainage rates as 0r increases.

Several empirical equations have been fitted to experimental drainage profiles deter-mined on protein-stabilized foams. These can all be rearranged to show proportionality toa power of the volume of liquid left in the foam (i.e., 1 — 4>(). First-order behavior was ob-served by Mita et al.127 for foams of unknown <f>t obtained by bubbling from gluten solu-tions in 3 M urea, and by CooneyI28 for whey-protein foams. A second-order equation wasfitted by Mitchell et al.I09 to the drainage from foams (<f>t 0.9 to 1) formed by bubbling andstabilized by a variety of caseins, other proteins, and their acylated derivatives (Figure 5).A third-order equation often used with small molecule surfactant foams, described thedrainage of protein hydrolysate fire-fighting foams (<f>t 0.9).129 This equation has atheoretical basis in an unlikely model that suggests the major route of drainage down thefoam to be through the lamellae rather than the Plateau Borders. None of these empiricalrelationships are obeyed perfectly. With no simple method of allowing for the effects of</>f, it must be remembered that changes in this parameter may be responsible forobserved differences in drainage behavior under different conditions. The commonpractice of quoting drainage half-times is an improvement on absolute rates, but since thekinetics are rarely first order, comparisons must be made with care.

With foams prepared by whipping, there often appears to be a small delay (at most afew minutes) before drainage commences, but this may just be an artifact of themeasuring procedure. Alternatively, it could reflect some rearrangement in the foamstructure needed for drainage to commence. As well as this initial delay, Corrie100

reported an increasing rate over up to 10 min drainage from branchpipe-generatedprotein hydrolysate foams (<j>{ 0.9). The position of the maximum in rate depended uponthe height of the foam sample, though it always came before 25% of the initial liquidpresent had drained. Corrie suggested that disproportionation was important in thiseffect, reducing the total surface area and releasing liquid for drainage. Acceleratingdrainage rates have also occasionally been observed in this laboratory.

Only circumstantial evidence is available for the effect of bubble size on drainage, asthis has never been systematically varied for a single protein. Foams prepared bybubbling that have smaller bubbles tend to have lower expansions (i.e., lower 4>t).Cumper105 noticed this effect on increasing protein concentration, while Mitchell etal.109

found it when comparing various natural and modified caseins. Because high <f>t values infoams prepared by bubbling reflect rapid drainage (mentioned previously), this suggeststhat the rate may increase with d'. The opposite effect is indicated by the slower drainageand larger d' of gluten-stabilized foams prepared by bubbling, as compared with glutenor gliadin.119'130

A decrease in the rate of drainage when the bulk viscosity of the liquid (TJO) is raised isevident from many reported results, listed in Table 5. As long ago as 1934, Barmorem

clearly appreciated the influence of viscosity on the drainage of his whipped egg whitefoams. The results reported by Nakamura and Sato135 are particularly clear, in that theeffects of six different viscosifiers could be quantitatively accounted for on the basis ofthe increases in r)o they caused. Nevertheless, the importance of rjo is still overlookedby many workers and a number of the correlations in Table 5 were not noted by the citedauthors. The table also includes correlations with the rate of collapse, which probablyreflect the role of drainage in facilitating this process.

Since many protein solutions or dispersions are in fact non-Newtonian, we might

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October 1981 181

30

2< • - Casein

Time (mins)10 15

FIGURE 5. Second-order drainage of casein foams. 0.05% w/v Caseinsolutions in phosphate buffer pH 7.2 (ionic strength 0.05) were foamed bybubbling air. By measuring the height of the liquid-foam boundary, the volumeof liquid in the foam was determined, when bubbling was stopped after a fullcolumn had been generated (Vo), and at various times of drainage thereafter(V). (From Mitchell, J. R., Adams, D. J., Evans, M. T. A., Irons, L., andMusselwhite, P. R., unpublished work, 1967 to 1970.)

expect a more precise relationship with apparent viscosity at an appropriate, low shearrate. If the liquid phase has a yield stress, this may completely stop drainage.

2. CollapseInvestigations of the time course of collapse of protein-stabilized foams have revealed

several different types of profile and varied relationships with drainage. No attemptshave been made to analyze the results quantitatively. Mitchell et al.109 compared thecollapse of foams made by bubbling solutions of various caseins and their acylderivatives. In most cases collapse proceeded at a fairly steady rate, if anything, slowingdown with time. With hydrophobic derivatives the rate was very slow and was notcorrelated with the much faster drainage of most of the liquid. Photomicrographs ofbutyryl /3-casein foams taken after the phase of rapid drainage showed a high 4> structurewith thin lamellae and narrow Plateau Borders; they also revealed some coalescence ofbubbles in the bulk of the foam prior to collapse. With unmodified caseins and their

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Table 5IMPROVED FOAM STABILITY DUE TO INCREASED LIQUID VISCOSITY

00

Protein

Egg white

Egg white proteinsBovine or human albuminGliadinSoy isolateEgg white proteins

Egg whiteCasein, whey and mixtures

Whey concentratesWheyModified fish proteinGlutenSunflower concentrate

Foaming method

W

WBWWW

ww

wwwB

W

Measured

Drainage

DrainageCollapseDrainageDrainageDrainage

DrainageDrainage and

collapseDrainageDrainageDrainageDrainageDrainage

Viscosity altered by

Varying whipping time,different egg whites,varying pH

Adding water or ovomucinAdding sucroseAdding sucroseAdding sucroseMixing different proteins,

adding viscosifiersAdding alginateAdding guar gum and

carrageenanAdding sucroseAdding CM-celluloseAdding sucroseAdding sucroseAdding sucrose

Viscosity measured?

Yes

YesNoNoNoYes

NoNo

NoNoNoYesNo

Ref,

131

132114133134135

111136

116137138127139

s-

8.

©

Note: The foam formation method is indicated by W (whipping) or B (bubbling).

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hydrophilic derivatives, collapse followed closely on drainage. Examination of a maleyl/3-casein foam showed that it did seem to drain to a typical high <f> structure immediatelybefore collapse. /?-casein itself was unusual in that collapse was minimal at first, butaccelerated rapidly after a period of drainage, and was then completed in a short time.Photomicrographs showed that the bubbles remained only slightly distorted spheres, nohigh <f> structure was formed, and a great deal of coalescence occurred in the bulk of thefoam before collapse. It may be that the rapid collapse phase of this foam starts after thebubbles have coarsened to a critical degree.

Subsequent studies in this laboratory109'136 have shown collapse profiles of each ofthese types with a number of other proteins. Accelerating rates seem to be fairly common.Graham and Phillips4 reported that foams prepared by shaking solutions of BSA,lysozyme, or /3-casein all collapsed smoothly, with little evidence of accelerating rates (inmarked contrast to the results of Mitchell et al.109 with very similar/?-casein solutions).Interestingly, the rate of decline in foam volume during collapse was independent of theheight of the foam column (i.e., whether it was in a wide or a narrow vessel).

3. Effects of Protein SolubilityThe relationship between the solubility of proteins and their foaming behavior is much

more complex than for emulsification. Correlations between foam formation or stabilityand the solubility of the protein involved have been observed in some cases (Table 6).However, a number of findings do not fit this simple behavior. While Watts141 observed aweak minimum in overrun and drainage stability (by whipping) near the isoelectric point(pi) of the protein in soy flour dispersions, Watts and Ulrich142 found that thesupernatant at the pi ,gave higher overruns and stabilities than the original flour. Asimilar effect was observed with cottonseed flour by Lawhon et al.,143 who presentedsome evidence that the improvement was due to the removal of residual oil (seefollowing). Both of these studies still suggest that only soluble protein contributes tofoaming, though.

Two studies have provided evidence for the stabilization of foams by proteinprecipitates, acting as solid particles. Hermansson et al.,144 on whipping 10% dispersionsof fish protein concentrate, found that overrun and drainage stability were maximal at anionic strength of 0.5M, where solubility showed a clear minimum. Removal of theinsoluble material reduced the stability though the overrun was unchanged, so theauthors attributed these effects to solid particle stabilization. It would seem that a specifictype of precipitated protein must be involved, since 97% of the concentrate remainedinsoluble under all conditions. Suspensions of the insoluble material alone did not foam.Bee et al.145 found that the stability to collapse of whipped 2%-casein dispersions wasmaximal at pH 5, near the pi. Removal of the insoluble material increased the rate ofcollapse at pH 5 and eliminated significant variation with pH (Figure 6). When the samesolutions were foamed by bubbling there appeared to be a clear minimum in stabilityaround pH 4.5, though the method used (loss of gas from the foam during formation) wasa little suspect. Casein micelle particles are also believed to be of importance in thestabilization of skim-milk foams.7 Solid particles appear to be particularly effective ingiving stability to disproportionation, and their precise action in these foams is discussedlater in connection with this process.

4. Effects ofpHThe usefulness of pH effects in examining the role of protein surface properties in the

stabilization of disperse systems was explained in the discussion of emulsions (SectionIII.). Studies of the surface rheology of protein films at the air-water interface againusually indicate a maximum in rigidity near the pi.12 A single report of the

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Table 6CORRELATIONS OBSERVED BETWEEN PROTEIN SOLUBILITY AND

IMPROVED FOAMING PERFORMANCE

Protein

GliadinSoy isolateCaseinSoy protein productsSunflower mealAlfalfa leaf proteinSunflower flour

Foaming method

WWBS

wsw

Measured

DrainageOverrun, drainageFoam formationFoam volume, collapseOverrun, drainageCollapseOverrun, drainage

Solubility altered by

Adding saltChanging pHChanging pH, adding saltDifferent productsChanging pHChanging pHChanging pH

Ref

13313410953

140112139

Note: The foams were prepared by whipping (W), bubbling (B), or shaking (S).

280

2U0-

Caseindispersion

I 03

PH ' 5 6 ?

FIGURE 6. Effect of casein precipitate particles on foam stability. 2% w/v Casein solutions wereadjusted to the pH specified and whipped. The time for the foam to collapse to half its initial volume wasrecorded. (From Bee, R. D., Breslaw, E. S., England, R. R., Graham, D. E., and Birkett, R. J.,unpublished work, 1973 to 1975.)

electrophoretic mobility of air bubbles in gelatin gels suggests that the zeta potential (£)does pass through zero at the pi as expected.77

Many investigations have been made of the effects of pH on the foaming of proteinsolutions, but their implications are often doubtful. Studies in which solubility effectsseemed to be dominant were noted in Section IV.B.3. Of results obtained with completelysoluble proteins, Table 7 lists those that can be unambiguously interpreted. The almostunanimous conclusion is that stability is at a maximum near the pi. It is difficult to assesswhether pH directly affects lamella drainage or rupture because of the interdependenceof these processes in causing macroscopic foam drainage and collapse. Mita et al. found

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October 1981 185

that drainage stability was clearly maximal near the pi,127 under conditions where lamellarupture was very much slower.1" This suggests a direct effect of pH on lamella drainage,but one on rupture seems likely as well. Only for one of the studies listed in Table 7, thatof Cumper,105 have parallel measurements of surface rheology been made.146 The surfaceelasticity was maximal near the pi for films of pepsin and /?-globulin, while the surfaceviscosity of the nonelastic insulin films was qualitatively assessed to show a maximumnear the pi.

Some more doubtful results on pH dependence are listed in Table 8, partly to providemore data for cautious interpretation and partly because of the significance that has beenattached to certain anomalous effects. Again a maximum in foaming properties is usuallyfound near the pi, but among these results there are several exceptions. Mitchell et al.109

suggested that the pronounced minimum in the "steady-state" foam volume of /3-lacto-globulin106 may be due to its rapid surface coagulation near the pi. Another general trend isan increase in foam stability with many proteins towards extremes of pH, and this issupported by two more reliable results from Table 8.

Table 9 lists four studies of the effects of pH on the whipping of egg white. No simpleresults are obtained, but maxima seem to be possible either in the natural pH range (8 to9) or near the pi of most of the proteins present (4 to 5). The former optimum may partlyreflect the maximum in viscosity observed in this region,131 though this might be expectedto have an adverse effect on overrun (see following). Perhaps a protein component thatplays an important surface role in the foaming of egg white is isoelectric in the pH 8 to 9region. Bailey149 showed that pH had a complex effect on the rate of whipping tomaximum overrun and drainage stability, and this may contribute to the variations inresults observed.

Suppression of pH effects by high salt concentrations gives an indication of an ultimateelectrostatic basis, as discussed for emulsions. Addition of salts increased foaming awayfrom the pi on bubbling solutions of soy proteins102 or egg albumin.103 Addition of NaClto gluten solutions at pH 3.5 progressively decreased the rate of drainage from theirfoams to the value found near the pi.127 Again there are anomalous effects. Cumper105

found that increasing NaCl concentrations caused a marked increase in the "steady state"foam volume or in the lifetime of single bubbles, with pepsin at its optimum pH (3.7).Smaller enhancements were found with /J-globulin (bubbling) or insulin (single bubbles) attheir pH optima. These effects seemed to be due to an inhibition of surface coagulation bysalt.

The maximum in lamella stability near the pi indicates that electrostatic repulsion ofthe protein surface films is not generally important in this pH region, not surprisinglybecause the lamella thickness is probably much greater than the range of colloid forces.The increase in stability at extremes of pH might be attributed to a role for £ under theseconditions; Cumper and Alexander105'146 found that surface rigidity remained low belowpH 2.5, though the foaming of /3-globulin, pepsin, and insulin increased again.Denaturation of the proteins is probably important at these pH extremes (see following).A dip in the middle of the maximum around the pi has been observed with gelatin foamsprepared by bubbling104 and with single bubble lifetimes.105 Possibly this might reflect anincrease in stability when £ is not precisely zero.

5. Effects of Lipids and Other SurfactantsSmall quantities of contaminating lipid can cause dramatic reductions in the foaming

abilities of proteins, and this effect is often very important in potential food ingredients.Table 10 lists reports of damage to the foaming properties that correlated with anincrease in lipid content. The amount of lipid involved is often very low (e.g., 0.1 % of soyphosphatides133). It seems likely that surface active lipids are responsible for these effects

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Table 7THE EFFECTS OF pH ON PROTEIN-STABILIZED FOAMS AND MODELS OF THEM

Method

Single bubblesFoam (B)

Single bubblesSingle lamellaeFoam (S)

Foam (B)Single bubblesFoam (B)

Measurement

LifetimeStability to collapseStability to collapseStability to collapseLifetimeLifetimeFoam volumeFoam volumeStability to collapse

Stability to collapseStability to drainageLifetimeStability to bubble

coalescenceStability to drainage

Protein(s)

SalmineHemoglobinGelatinOvalbuminInsulin, pepsin, a-globulinPepsinBSALysozymeBSA

Lysozyme'Gluten, glutenin, gliadin'Gluten, glutenin, gliadin'Gluten, glutenin, gliadin

Ovalbumin

pH dependence

Near pi

MaximumMaximumNo clear effectsMaximumMaximumMaximumLittle changeSharp increase at pH 11Higher at pH 5.5 (near pi)

than 2.5 or 8, higher stillat 12

Falls at pH 12 (pi 11.5)MaximumMaximumMaximum

Maximum

Other

_Also rising to pH 2

Little changeSharp increase at pH 11Higher at pH 5.5 (near pi)

than 2.5 or 8, higher stillat 12

Falls at pH 12 (pi 11.5)—

—

Ref.

103104104104105122,123444

4127,130119119

126

•

3"a.

o3

Note: Foams were prepared by bubbling (B) or shaking (S).

"These proteins were dissolved in 3M urea.

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Table 8MORE AMBIGUOUS EVIDENCE FOR THE EFFECTS OF pH ON PROTEIN—STABILIZED FOAMS

Foaming method

B

W

Measurement

Time to fixed foamvolume (order of100s)

"Steady state" foamvolume







"Foaminess"


Stability todrainage

Protein(s)

Gelatin

Soy

Egg albumin

Salmine

Hemoglobin

Ovalbumin,gelatin

Insulin,^-globulin

Pepsin

5 egg whiteproteins

/3-lactoglobulin

Whey

pH dependence

Near p(

Maximum formationrate

Maximum

Maximum pH 4(pi 4.8)

Maximum at pH 5and 10, low atextremes (pi 12)

Maximum

Maximum

Maximum

Maximum at pH 3.7(PI2)

Maximum

Minimum

Maximum at pH 4(near pi of majorcomponents)

Other

_

High < 2 and > 12

High < 1 and > 12

Maximum at pH 5and 10, low atextremes (pi 12)

High < 2

Rising again pH 2

High < 2 and > 12

n ^ A A H apt n tf4^Vftvl^4vHBl

ivieaning uouonuibecause of

Method used

Method used; proteininsolubility

Method used

Method used

Method used

Method used

Method used

Method used

Method unclear

Method used

Protein insolubility

Ref.

147

102

103

103

104

104

105

105

148

106

116

Note: Foams were prepared by bubbling (B) or whipping (W). The problems in interpreting results obtained by certain experimental approaches were discussedin the text.

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Table 9EFFECTS OF pH ON THE WHIPPING OF EGG WHITE

Type of egg white

FreshFrozen

Homogenized

Reconstituted

Measured

Drainage stabilityOverrunDrainage stabilityOverrunDrainage stabilityOverrunDrainage stability

pH dependence

Maximum at 8Maximum near 8.7Generally highest at 5,7Maximum at 4.7Maximum at 8.6Weak maximum 3—4No clear trend

Ref

131149

110

111

in the same way that small molecule surfactants affect emulsions. The most effectivelipids seem to be those bound tightly to proteins and, hence, likely to be polar.109'134 Incontrast, relatively pure nonpolar fats and oils can often be added in much largerquantities without serious effect (e.g., vegetable oil116 or butterfat109).

Much information on the way in which moderate or high quantities of fat affectfoaming is available for dairy emulsions and related products; in these systems fatdroplets may sometimes enhance foaming by a specific mechanism, as in whippingcream.7 One interesting observation is that oil droplets containing solid crystals areparticularily deleterious to foaming (at low oil contents), and this can be explained bytheories of antifoam action.

6. Effects of Protein ConcentrationSome conclusions and rather more speculation can be based on the observed effects on

foam stability of the protein concentration in the aqueous phase (C). Table 11 lists resultsobtained with the reasonably unambiguous experimental approaches, and includeseffects on foam formation that will be discussed later. The four investigations made in thelow concentration range seem to agree on a rapid increase in stability up to C of the orderof 0.1%. This effect is probably dependent on surface properties. For a typical foam ofd' 0.15 mm and 4> 0.95,0.1 % proteinin the liquid phase before foaming would give a surfaceconcentration (F) of 1 mg.m2 if completely adsorbed. This T represents a fairly coherentfilm and is the level at which surface rigidity generally becomes substantial.3"5 Because ofserious practical difficulties, no experimental estimates are available for T in protein-stabilized foams, and the problems in dealing with this parameter were discussed byGraham and Phillips.4

There is less agreement about effects in the high concentration range (Table 11). Whilein two studies there is little change in stability between 0.1% and 0.5 or 2%, the moregeneral behavior seems to be a steady increase up to very high C. One obvious mechanismfor this effect is the increase in liquid viscosity brought about by the protein. This wouldbe particularly important with the more denatured food ingredient proteins, whoseeffects on r)o would be greater. Gray and Stone115 commented on the link between theincreasing TJO of more concentrated gelatin solutions and the decreasing drainage ratefrom their foams. Nakamura and Sato135 investigated the dilution of egg white so as tomaintain 770 while reducing C from 10 to 2%. A significant part of the effect of C,though not all of it, could evidently be accounted for by changes in -q0. In systems likethis that contain more than one protein, an important surface role of a minor componentmay account for the continued increase of foam stability at high total C. Indeed, there isevidence that minor components may be important in the surface films in egg whitefoams.110'132'135'132 Some effect on surface properties at high concentrations of a singleprotein cannot be ruled out, though, and seems to be the only possibility in the

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Table 10REPORTED REDUCTIONS IN FOAM FORMATION OR STABILITY WITH INCREASE IN

LIPID CONTENT

Foaming method

WW

ww

wBSW

wsB

WW

Protein(s)

Whey, egg albuminEgg whiteSoy flourGliadin

Soy isolatesCaseinSoy protein productsCottonseed flourWhey concentratesAlfalfa leaf proteinsGluten

Whey protein concentratesSkim milk

Lipid varied by

Adding fatAdding olive oilSolvent extractionAdding cottonseed oil or soy

phospholipidsAlcohol washingDifferent de-fatting proceduresDifferent productsSolvent extractionDifferent productsDifferent extracts or acetone washingDifferent de-fatting procedures

or adding oilsWhey clarified or notExtent of fat removal

Affected

Collapse (qualitative)Overrun, drainageAbility to whipOverrun, drainage

DrainageAll foaming propertiesFoam volumeOverrun, foam viscosityOverrunFoam volume, collapseDrainage

Overrun, drainageOverrun, drainage

Ref.

150149141133

13410953

143116112127

181Well known, e.g., 7

Note: Foams were produced by whipping (W), bubbling (B), or shaking (S).

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Table 11EFFECT OF INCREASING PROTEIN CONCENTRATION ON FOAM FORMATION AND STABILITY

Protein C range (%) Foaming method

Soy flourSoy extract

Egg albumin

Gelatin

Peptone

BSAGliadinSoy isolateCasein

Fish proteinconcentrate

Whey concentrate

Egg whiteProtein hydrolysate

(fire-fighting)Sunflower concentrateSkim milk powderOvalbuminWhey concentrates

2—201.5—5

0.05—2

0.05—2

0.05—2

0.005—41.5—90.5 -50.01—0.5

2—40

0.1—13

0.1—133—8

0.5—1210—300.01—11-14

Effect of increasing C on

Foam formation

ww

B

B

B

BWWB

W

W

WBP

WWBW

Overrun maximal about 8%Overrun increases slowly

—

—

—

Overrun maximal at 3%Overrun maximal at 3%

Overrun maximal at 10%

—

Overrun shows little change

Overrun plateau's above 2%Overrun maximal at 15%

Overrun maximal at 6-11%

Foam stability

Drainage stability increasesmore quickly

Drainage stability nearly constantabove 0.1%

Drainage stability increasessteadily

Drainage stability increasesslightly

Collapse stability increases steadilyDrainage stability increases steadilyDrainage stability plateau's above 3%Drainage and collapse stabilities both

plateau above 0.1%Drainage stability increases steadily

Drainage stability increases rapidly upto 0.3%, then increases steadily

Drainage stability increases steadilyDrainage stability increases steadily

Drainage stability increases steadilyDrainage stability increases steadilyDrainage stability increases slowlyDrainage stability increases steadily

Rcf.

141

142

115

115

115

114133134109

151

116

116100

139182126181

So

2

Soa.

3

a.

o

Note: Foams were prepared by whipping (W), bubbling (B), or in a branchpipe (BP).

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October 1981 191

experiments with up to 4% BSA."4 Perhaps an effect on adsorption rates is responsible.There have been no reports of a decrease in stability as C is raised. In contrast, Graham

et al.3'5 reported that the air-water surface rheological parameters were maximal atintermediate V values, though these measurements have been criticized (see discussionunder emulsions, Section III.).

7. Comparison of Different ProteinsIt is clear that soluble proteins can differ widely in their ability to stabilize foams in

contrast to the apparent position with emulsions. This section will discuss those of theobserved differences that seem to shed light on the critical physicochemical propertiesinvolved.

Mitchell et al.109 showed that hydrophobic derivatives of caseins made by acylationhad a much enhanced ability to stabilize thin lamellae in foams and retard collapse. Theysuggested that increased stability was due to higher surface rigidity, itself resulting fromgreater cohesive interactions between the modified proteins. Modification had littleeffect on drainage rates. Hydrophilic derivatives of casein with increased charge and,hence, probably less cohesion, showed reduced stabilities to drainage and collapse.Similar effects were found on acylation of several other proteins. A particularly largeenhancement was obtained on making a hydrophobic derivative of cytochromec, whosepi would be shifted much nearer to the pH used. Graham and Phillips4 found that thestability to collapse of foams prepared by shaking decreased in the order lysozyme >BSA > /?-casein. This correlated with the relative magnitudes of various surfacerheological parameters determined on adsorbed films of these proteins. In turn, theseseemed to be related to the high degree of protein structure remaining in lysozyme films,while ^-casein had a flexible, random structure even in solution.4'14 Heat denaturedlysozyme gave reduced stability, though it foamed more readily. Mita et al. '" found thatglutenin was more effective at preventing bubble coalescence in bulk foams than eithergluten or gliadin. Glutenin is composed of very large aggregates (MW> 106), and if thesewere dissociated by breaking intermolecular disulfide bonds, then its superior stabilizingproperties were lost. The aggregates would be expected to give a more rigid surface filmand to be more difficult to desorb. However, the increased coalescence stability mightequally be attributed to some effect of the larger bubble sizes in these foams.

There is evidence from some less fundamental studies that an appreciable degree oftertiary structure in the dissolved protein (and hence presumably in the surface film) isneeded for maximal foam stability. Partly hydrolyzed proteins, especially from soy, havebeen widely used as whipping/foaming agents in foods.134 The evidence from very limitedstudies in simple systems suggests that they give lower stability than the unhydrolyzed ma-terial, though the foam may be formed more readily.109>I34 A decline in stability to foam col-lapse has been observed with increasing alkaline125 or enzymic88'153 hydrolysis of soy pro-tein. A similar decline in drainage stability has been observed during enzymic hydrolysis ofegg white,154 whey protein,137 and fish protein.138 A minimum molecular weight for goodfoaming was observed on fractionation of the proteins and peptides of beer.155

Partial denaturation without loss of solubility may sometimes increase drainagestability, though. This effect is particularly marked if whey proteins are moderatelyheated prior to whipping.116'137'150'156 Peter and Bell150 found that acid, alkali, or sulfitetreatment could also be beneficial. Richert et al.156 reported that one set of heatingconditions gave a solution of high viscosity that whipped to a stiff foam of very highdrainage stability. In this case the foam properties are probably caused by the increasedT/O, but other factors would seem to be important under different conditions, de Wit181

found that whey proteins from which fat had been carefully removed no longer required aheat treatment to give go od foaming performance. This suggested that the role of heating

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may be to lessen interference by the fat (see preceding). Heating of soy protein solutionsalso improved the drainage stability of foams whipped from them.134 Mussellwhite123

noted that denaturation of BSA with 8M urea and sulfite led to a more stable foam thanwith the native protein.

A recent study by Horiuchi et al."3 has compared the foaming of polypeptidesobtained by enzymic hydrolysis. The stability to collapse of foams prepared by shakingdeclined with this order of original proteins: gluten, egg albumen, soy, casein, gelatin.The same order was found for "surface hydrophobicity" (determined by a dye bindingtechnique) and for surface activity (estimated from adsorption isotherms). Since all thesematerials probably contain little rigid structure, their hydrophilic/hydrophobicproperties may have enhanced importance, as with small molecule surfactants.

8. Effect of Multivalent Metal IonsSeveral observations have indicated an increase in stability of foams on addition of

multivalent metal ions to protein solutions.102'103'128'150 Such ions are routinely included inthe formulation of hydrolyzed protein solutions for fire-fighting foams, and have beenthe subject of a large number of patents.100 This effect may be related to an observationthat multivalent cations can increase the dilational modulus of protein films,157 probablyby bridging between carboxyl groups.

9. The Role of Surf ace PropertiesMuch of the discussion of the ways in which protein surface properties can affect

emulsion stability is also relevant to foams. Similarly, any conclusions drawn arenecessarily as tentative. The most common view is again that the high rigidity of proteinsurface films opposes the deformations necessary to initiate rupture of foam lamellae. Thisis consistent with most of the results presented in the last five headings. Nevertheless, theexperimental evidence is equally consistent with desorption from the protein film beingthe key event in activation of lamella rupture, another view discussed for emulsions.Besides the effects of pH and added surfactants, it also predicts that rupture would beslower at high T (because of the need to desorb more protein) and with natural ormodified proteins having greater cohesive interactions. The glutenin aggregates in thestudies of Mita et al."9 might act even more like solid particles, with large desorptionenergies giving the observed high stabilities.

In polyhedron foams the drainage of the thin lamellae has an important effect onrupture; all theories agree that bursting is more likely the thinner the lamella. Such aneffect explains the common correlation between drainage and collapse rates. Surfaceproperties can have a considerable effect on lamella drainage. If the surfaces remain rigid,the viscous drainage of liquid from between them is very slow, but a process involvingrelative motion of whole areas of the lamella ("marginal regeneration") can increase ratessubstantially.17 Surface rheology is clearly important here. Studies on single lamellae of/?-casein solutions established that they drained rapidly by marginal regeneration, whilewith BSA, slow draining rigid lamellae were formed.5

In theory, the drainage of foam lamellae can also be slowed by repulsive forces (DLVOtype or similar) between their surfaces. If these become large enough to balance thedriving force for drainage, then metastable lamellae of characteristic thickness may beobtained. Such lamellae (in the isolated form) can sometimes be prepared from proteinsolutions,3'5'122'123 but the repulsive force between their surfaces is something of a mystery.Their thickness seems to be maximal near the pi,122'123 where both electrostatic andentropic repulsion are expected to be at a minimum. Whether such metastable lamellaecan be formed in foams is less clear. The lack of correlation between drainage andcollapse in some foams has been ascribed to the formation of metastable lamellae.

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October 1981 193

However, it is possible that lamella rupture follows a very slow final phase of drainagethat occurs well after the "half-life" has been completed and that has not beenexperimentally investigated. Graham3 observed microscopically that the rupturinglamellae in lysozyme foams were of the order of 100 nm thick, so that colloid forcesbetween their surfaces would not be significant; they must have been still draining, even ifvery slowly.

If lamellae remain this thick in all protein stabilized foams, then neither electrostatic orsteric repulsion can be of importance in stabilizing them. Lamellae in foams might beexpected to rupture at greater thicknesses than for emulsions because they are typicallymuch larger in area (bubbles are bigger than emulsion droplets). Another important,factor in foams, whose role has not been investigated with proteins, is water evaporation;among other things, this can be a route to very thin lamellae. It has been suggested thatthe rupture of lamellae in polyhedron foams may be initiated by mechanical stresses in thestructure.158 Two endogenous mechanisms can be involved in setting up such stresses,disproportionation (discussed below), and the rupture of other lamellae. Mita et al."9

found that lamella rupture in foams stabilized by wheat proteins followed a first-orderprogress curve. Hence, in this system any stresses set up by one rupture seem not to affectany other lamellae.

10. DisproportionationMicroscopic observation has shown that disproportionation occurs in protein-

stabilized foams.3'117>14S The effects of changing the gas phase are likely to reflectdisproportionation as well; since this process depends on diffusion of the gas, itssolubility in the aqueous phase is a major influence. (A few observations comparing thebubbling of air and CO2 through dilute protein solutions must be discarded in view of thepronounced effect of CO2 on pH even with some buffer present.3)

Clark and Blackman117 showed that the decline in surface area of protein hydrolysatefire-fighting foams over 100 min or so was largely due to disproportionation. The bubbleswere only slightly distorted from spherical, with an average diameter of about 0.15 mm.The authors correlated the progress curve with a model that assumed the major resistanceto diffusion lay in the surface films. This seems unlikely to be correct in view of the highpermeability of protein films159 and the relatively thick aqueous layers between thebubbles. Substituting for air as the disperse phase caused an increase in rate, small withO2 and much larger with CO2, in line with the relative solubilities of these gases.

Disproportionation usually leads to the rapid disappearance of small bubbles fromfoams. However, they can be protected from complete disappearance by dispro-portionation if the compression of their surface film leads to a permanent decrease insurface tension, which is particularly likely with difficult to desorb protein surface films.(The critical condition has been shown to be a surface dilational modulus [e = dy/ d2nA]greater than half 7.) Bee et al.145 made detailed investigations of this mechanism ofstabilization by microscopic observation of very small single bubbles disproportionatingto the atmosphere. Accelerating rates of shrinkage were found with bubbles in 0.1 %solutions of lysozyme or /J-casein; parallel determination of surface properties showedthat the critical condition for stability, 2e>y, could never be satisfied with thesesolutions. In a mixed protein system containing suspended particles, however, after aninitial shrinkage a limiting bubble size was reached that remained stable over 70 hr. Atrue e value for this material proved difficult to measure, but was clearly very large.Filtering the suspension showed that the particles were responsible for both the stabilityto complete disproportionation and the high e. A similar effect probably accounts for theobservation by Clark and Blackman117 that the small bubbles in their protein-stabilized

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foams tended to shrink only to a limiting size (at least over tens of minutes), in contrast toaccelerating shrinkage with small molecule surfactants.

It has been suggested that changes in bubble size resulting from disproportionationmight set up considerable stresses in polyhedron foams, and it has been proposed thatthese might initiate lamella rupture. Bee et al.l4> suggested that the prevention ofdisproportionation by solid particle effects was behind their observation that caseinprecipitates stabilized foams to collapse. Interestingly, the protein precipitate did notappear to stabilize foams prepared by bubbling in which more uniform bubble sizesmight render disproportionation less important. Graham3 made a microscope analysis ofBSA-stabilized foams and suggested that the disproportionation of small sphericalbubbles suspended in the lamellae between the main bubbles set up stresses leading torupture of these main lamellae. This was supported by the observation that collapse ratesincreased as the gas phase was changed in the order N2, air, O2, CO2 (i.e., increasingsolubility).

C. Foam Formation/. Amount of Foam: Mechanical Effects

Though the equipment and conditions used in foam formation are well known to affectthe amount formed, no systematic studies have been made in protein-stabilized systems.Bailey149 found that two types of beater performed very differently on the same batch ofegg white. In whipping (and in shaking) the hydrodynamics of the system are veryimportant in determining the rate of air bubble formation. This probably explains theobservation that overrun decreases in whipping protein solutions of higher vis-cosity,"1'133"135 even though subsequent foam stability is increased. High viscosity cansometimes completely prevent the incorporation of air into the liquid.'50 Viscosity effectsare probably also responsible for the fall in overrun often observed at high proteinconcentrations (Table 11).

2. Amount of Foam: Surface PropertiesThe newly formed bubbles will rapidly be exposed to the threat of coalescence with

each other or with atmosphere: in bubbling methods principally because of the rapidityof the initial breakdown processes; in whipping or shaking largely because of continuedvigorous agitation. Hence, the bubbles must quickly gain a stabilizing surface film if theyare to survive. However, development of surface pressure (TT) and surface rigidity insurface trough experiments is so slow with most proteins that foam formation might beexpected to be impossible. To resolve this paradox, it is likely that agitation of theaqueous phase will accelerate adsorption (via convective effects on transport), whilecompressional oscillations of the interface might speed up surface denaturation. Gilesand Lucassen160 have demonstrated that development of the surface pressure of bovineplasma albumin solutions is accelerated by taking the interface through compression/ ex-pansion cycles.

By developing a radio-tracer method for direct measurement of F in adsorbed films,Graham and Phillips3'4 have demonstrated that the slow step in development of n andsurface rigidity is, in fact, rearrangement of protein structure in the interface rather thanadsorption.

Mac Ritchie and Alexander114 observed that solutions of human albumin, pepsin, andlysozyme could not be foamed by bubbling, while that of BSA could. The first threesolutions were shown to give a much slower buildup of surface rigidity on adsorption.They could be foamed if sucrose was added and this also caused an increased rate ofsurface film formation.

Lauwers and Ruyssen106 observed a correlation between the maximum foam volumes

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October 1981 195

obtained by bubbling /3-lactoglobulin solutions of different concentrations and the ratesof n development on adsorption at the same C value. Mitchell et al.109 showed that as-casein foamed poorly on bubbling compared with other caseins and gave a slower TTincrease on adsorption. It foamed as well as the rapidly surface denatured /J-casein onshaking; this interesting result may reflect the greater agitation of the surfaces and thelonger period of foam formation. There was also a general correlation between the ratesof v development and of foam volume build-up on bubbling, with solutions of caseinsand their acyl derivatives. Graham and Phillips4'161 demonstrated that the very differentrates of foam formation on shaking protein solutions could be related to the rates of vincrease on adsorption; in each case /J-casein > BSA > lysozyme. Previous partial heatdenaturation of BSA or lysozyme led to parallel increases in both rates.

Graham and Phillips4'14 argue that the rate of surface denaturation and TT developmentcan in turn be related to the structural characteristics of the protein. The rate is faster formore flexible random-structured proteins than for those with a tightly held tertiarystructure. There is further evidence in support of this effect of protein structure frommore empirical studies. Partially hydrolyzed soy proteins whip to larger foam volumesthan the original materials,134'153 and progressive increases in overrun are found onenzymic hydrolysis of egg white,154 whey protein,137 or succinylated solubilized fishprotein.138

Increases in the amount of foam formed have been observed on partial heatdenaturation of soy proteins134'141 or ovalbumin.121 Interestingly, Watts141 showed thatthe overrun obtained with the heated soy protein could be equalled by longer whipping ofthe untreated material, as might be expected for an effect on surface denaturation rate.Mussellwhite123 found that foam formation on shaking solutions of lysozyme or pepsincould be enhanced by denaturation with 8 M urea and sulfite. Increased foam formationfrom protein solutions at extremes of pH was noted in Tables 7 and 8.

3. Surface CoagulationThe foam volume produced from protein solutions is quite often observed to reach a

maximum level and then decline. This effect, which is not found with small moleculesurfactants, is usually attributed to the coagulation of proteins at an agitated air-waterinterface. The protein is converted to insoluble particles that have lost foamingproperties, so the foam collapses and they are desorbed into the bulk liquid. Surfacecoagulation is particularly important in the foaming of dilute solutions (e.g., Reference105), as would be predicted since the process in zero order in C.162 The rate of coagulation issometimes increased near the pi of the protein.109'162 Cumper105 reported that high saltconcentrations reduced the rate of coagulation and hence raised the maximumfoam volume, though concentrations up to 0.1 M had little effect in the studiesof Mitchell et al.109 Henson et al.163 compared the relative tendency to surfacecoagulation of a series of proteins with their foaming behavior in dilute solution.109

Proteins that were more susceptible to surface coagulation showed the expected declinein foam volume as bubbling was continued. In Langmuir trough experimentscoagulation can be induced by compression of the surface film to a critical pressure, andMacRitchie and Owens164 calculated the ratio of IT reached by adsorption to this criticalvalue for BSA and ovalbumin. They related the higher ratio for the latter to its greatersusceptibility to coagulation during foaming.

Though the zero-order kinetics of surface coagulation tend to reduce its impor-tance in more concentrated protein solutions, this may be partly compensatedfor by the more intense agitation in the whipping method normally used withthese systems. The phenomenon of "overheating", a decrease in overrun or stabilityfrom the maximum level as whipping is prolonged, is well known with egg white.

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Ovalbumin is known to be exceptionally sensitive to surface coagulation,109 andprecipitated protein can be demonstrated in whipped egg white, though it is only arelatively small part of the total."6'132'135 In the overheating of egg white, the stability tosubsequent drainage starts to fall if whipping is prolonged beyond very short times,before the overrun has reached its maximum value.131'134'149 In discussions of overheatingit is not always remembered that fresh egg white is not a simple solution of proteins, butcontains natural biological structures. Barmore131 showed that the decline in drainagestability on overheating correlated with a fall in viscosity of the liquid draining from thefoam. Forsythe and Bergqvist165 showed that blending (in the absence of aeration)reduced the viscosity of egg white by breaking down microscopically visible fibrousstructures. These viscosity changes seem to be an important factor in overheating and,indeed, Barmore131 found no evidence for the effect with reconstituted dried egg white,over relatively short whipping times. However, some evidence of overheating wasobserved with prehomogenized egg white by Nakamura and Sato.110 Goodall111 showedclearly that reconstituted egg whites from various sources could be overbeaten, thoughthey gave maxima in overrun and stability at about the same whipping times. Carefulexamination of the results of Barmore131 reveals that the fall in viscosity is not sufficientto explain all the increases in drainage rates at longer whipping times. Thus, it seemslikely that surface coagulation, perhaps of a specific protein component,110'135 is alsoimportant in the overheating of egg white.

Overheating effects seem to be much less severe with other proteins. A soy isolate gavea maximum in overrun, but no decline in drainage stability, with whipping times up to tentimes that optimal for egg white.134 McDonald and Pence133 found only slight falls inoverrun and stability at long whipping times with 3% solution of gliadin. Both overrunand drainage stability rose to a level plateau with dispersions of fish proteinconcentrate151 or whey protein.U6 Precipitation*of protein following surface coagulationseems also to be less than with egg white. DeVilbiss et al.116 could only detect it inwhipped whey-protein solutions for C less than 1%.

4. Bubble SizeThis important parameter can vary enormously in protein-stabilized foams, from 1 /um

to 10 mm, and is clearly affected by the conditions of formation. However, there is littleexperimental evidence as to how it is determined. The results of Cumper105 on foamsproduced by bubbling indicate that d' was smaller under conditions giving increasedmaximum foam volumes: higher protein concentration (C); insulin compared to otherproteins; high salt concentrations; and prior partial heat denaturation. These conditionsalso minimized surface coagulation, which might normally lead to coalescence in thefoam column and hence larger d'. Chang et al.99 prepared foams from a proteinhydrolysate solution by injecting air and then passing through a packed column. As moreair was introduced d' was decreased, while it was nearly independent of C (1.5 to 6%).Graham and Phillips4 observed that smaller bubbles were obtained on shaking solutionsof lysozyme than with /3-casein. They suggested that the slow surface denaturation oflysozyme meant that only small bubbles were likely to gain a stabilizing protein filmbefore being lost. However, Graham3 presents photographs showing even smallerbubbles with BSA, which surface denatures at an intermediate rate. Mitchell et al.109 alsoobserved that acylation of /?-casein, which reduces the rate of w development onadsorption, increased the d' values in foams made by bubbling.

Mita et al.119 have made an extensive study of bubble sizes in foams prepared bybubbling solutions of wheat proteins in 3 M urea. The bubbles tended to be larger nearthe pi, an effect that was particularly marked with glutenin. Surface tension, however,was at a minimum near the pi, which would be expected to lead to release of smaller

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bubbles from the sparger; the size of single bubbles released from a nozzle into the proteinsolutions was indeed found to have this dependence on pH. Mita et al. suggested that themaximal surface rigidity hear the pi would oppose the formation of small bubbles andthat changes in surface rheology were also responsible for the decrease in d' on increasingthe temperature or on breaking the intermolecular disulfide bonds in glutenin. However,it seems unlikely that a protein surface film could be formed rapidly enough to influencethe process of bubble detachment from the sparger. A possible explanation for the muchlarger d' in glutenin-stabilized foams comes from the view of the aggregates in this proteinas approximating to solid particles; "limited coalescence" of bubbles may take place inthe foam column during formation.

D. Foam RheologyA few studies have been made of the response of protein-stabilized foams to applied

stresses. Grove et al.98 studied the viscosity of protein hydrolysate fire-fighting foams.Though the foams were of high overrun when released from the equipment, themeasurements were made on a stream under pressure to simulate the resistance to flow indelivery systems. As the pressure was increased, the air bubbles were compressed and <f>was reduced. In the range of # investigated (which corresponded to gas emulsions orsphere foams) the combined effects of pressure and the weight of gas present on foamviscosity (tjf) was determined and could be accounted for solely by the resulting <f> value.(Log ?jf was roughly proportional to 0.) Viscosity was independent of flow rate in theregion studied, but there was evidence that it became much larger at low shear rates. Notethat at these <j> values the rheology would be expected to be similar to that of typicalemulsions (see preceding).

In polyhedron foams rheology is expected to be very different, with lamella propertiesbeing of special importance and elastic behavior resulting from Laplace pressurechanges. Lawhon et al.'43>'66 measured the apparent viscosity at fixed shear rate for foamswhipped from dispersions of various oilseed proteins. Addition of sucrose alwaysincreased tjf, suggesting that rj0 may contribute to this. The presence of small amountsof lipid caused a substantial drop in rjt, suggesting a contribution from surface rigidity(see preceding). There is some support for this from the studies of Corrie100 on fullyexpanded protein hydrolysate foams, which had a yield stress that was maximal near thelikely pi. It may also account for the decline in foam strengths (assessed by the rate of fallof a perforated weight) above an optimum temperature.108'126

V. CONCLUSIONS

Despite a considerable volume of work, the definite conclusions that can be drawn arerather limited. A number of effects of protein physicochemical properties on foam andemulsion behavior are reasonably well established. In some cases these effects seem todepend on expected influences on flow processes; but the mechanisms of those processesdominated by surface properties remain in dispute.

ACKNOWLEDGMENTS

The author is very grateful to Unilever Limited for permission to publish this review,and to the following members of Unilever Research for most helpful discussions: Dr. R.D. Bee, Dr. D. F. Darling, Dr. J. A. de Feijter, Dr. R. B. Leslie, Dr. P. J. Lillford, Dr. J.Lucassen, Dr. M. G. Jones, Dr. M. van den Tempel, and Dr. N. Thomas. He would alsolike particularly to thank the referee, Dr. P. Walstra, for his detailed and usefulcomments and helpful discussion.

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Documents

Protein‐stabilized foams and emulsions