Current Opinion in Colloid & Interface Science 9 (2004) 305 313

www.elsevier.com/locate/cocis

Protein-stabilized emulsions
David Julian McClements*
Biopolymers and Colloids Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA
Available online 19 October 2004

Abstract
Proteins are widely used as emulsifiers to facilitate the formation, improve the stability and provide specific physicochemical properties to
oil-in-water emulsions. There have been a number of recent advances in the understanding of the ability of various types of proteins to provide
these functional properties. This article focuses on the influence of solution composition (pH, ionic strength, sugars, polyols, surfactants,
biopolymers) and environmental stresses (heating, chilling, freezing, drying) on the stability of globular protein stabilized emulsions.
D 2004 Elsevier Ltd. All rights reserved.
Keywords: Emulsions; Proteins; Surface denaturation; Thermal denaturation

1. Introduction
Many proteins are surface-active molecules that can be
used as emulsifiers because of their ability to facilitate the
formation, improve the stability and produce desirable
physicochemical properties in oil-in-water emulsions [1 ].
Proteins adsorb to the surfaces of freshly formed oil
droplets created by homogenization of oilwaterprotein
mixtures, where they facilitate further droplet disruption by
lowering the interfacial tension and retard droplet coalescence by forming protective membranes around the
droplets [2]. The ability of proteins to generate repulsive
interactions (e.g., steric and electrostatic) between oil
droplets and to form an interfacial membrane that is
resistant to rupture also plays an important role in
stabilizing the droplets against flocculation and coalescence during long-term storage [3,4 ,5 ].
The development of protein-stabilized emulsions with
improved or novel physicochemical properties relies on
understanding the interfacial behavior of adsorbed proteins, and on elucidating the relationship between
interfacial characteristics and bulk physicochemical properties of emulsions. A great deal of research has been
carried out to develop a better fundamental understanding

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* Tel.: +1 413 545 1019; fax: +1 413 545 1262.

E-mail address: mcclements@foodsci.umass.edu.
1359-0294/$ - see front matter D 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cocis.2004.09.003

of the interfacial and functional properties of proteins in

emulsions. Nevertheless, the majority of this research has
traditionally been carried out using relatively simple but
well-defined model systems, e.g., a fixed pH, ionic
strength and temperature. In practice, the functional
proteins used in industrial emulsion-based products tend
to experience a variety of different solution conditions
(pH, ionic strength, surfactants, biopolymers) and environmental stresses (thermal processing, chilling, freezing,
drying, homogenization and mechanical agitation) during
their production, storage and utilization. There has
therefore been an increasing emphasis on developing a
more fundamental understanding of the influence of
solution conditions, ingredient interactions and environmental stresses on protein functionality. This article will
therefore focus primarily on recent research carried out in
these areas.

2. Influence of environmental stresses

2.1. Aging
After adsorption to an oilwater interface a protein may
undergo substantial changes in its conformation and
interactions due to the change in its molecular environment
[1 ,6]. In an aqueous phase, a protein is surrounded

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D.J. McClements / Current Opinion in Colloid & Interface Science 9 (2004) 305313

primarily by water molecules, but at an oilwater interface,

it is surrounded by water molecules on one side and oil
molecules on the other side. Proteins undergo conformational changes after adsorption in order to maximize the
number of favorable interactions and minimize the number
of unfavorable interactions in their new environment. The
time taken for these conformational changes to occur
depends on the molecular flexibility and packing of the
adsorbed molecules [7]. Relatively flexible proteins (such as
casein) undergo relatively rapid conformational changes,
whereas more rigid globular proteins (such as lysozyme, hlactoglobulin [h-Lg] or BSA) take much longer (e.g., hours
or days). Protein conformational changes resulting from
adsorption of the protein molecules to an interface are
usually referred to as bsurface denaturationQ. The extent of
the conformational changes that occur in globular proteins
after adsorption to oil droplet surfaces in oil-in-water
emulsions depend on the nature of the oil phase [8]. The
extent of conformational changes was larger for more nonpolar oils, which may be because the hydrophobic driving
force for protein unfolding is greater.
For many globular proteins, surface denaturation leads to
an increased exposure of non-polar and sulfhydryl containing amino acids to the aqueous phase [9,10 ]. When the
droplets in globular protein-stabilized oil-in-water emulsions are in close proximity (e.g., pH close to isoelectric
point or high ionic strength) surface denaturation can
promote droplet flocculation through increased hydrophobic
attraction and disulfide bond formation between proteins
adsorbed onto different droplets. On the other hand, when
the droplets are prevented from coming into close proximity
(e.g., pH far from isoelectric point or low ionic strength)
extensive proteinprotein interactions at an interface may
lead to the formation of an interfacial membrane that is more
difficult to disrupt, which may therefore provide better
protection against droplet coalescence [3].

high level of salt (150 mM) is added to the continuous

phase because this screens the electrostatic repulsion
between the droplets (Fig. 1). At relatively low temperatures (b65 8C), droplet flocculation is due to surface
denaturation of the globular proteins after adsorption (see
above). When h-lactoglobulin stabilized emulsions are
heated above the thermal denaturation temperature of the
adsorbed globular proteins (T m~70 8C) in the presence of
salt (150 mM NaCl), protein unfolding becomes much
more extensive, which leads to an increase in the extent
of droplet flocculation (Fig. 1). Nevertheless, it is
interesting to note that very little droplet flocculation is
observed when a h-lactoglobulin-stabilized emulsion is
heated above the thermal denaturation temperature in the
absence of salt, and then salt is added after the emulsion
has been cooled to room temperature (Fig. 1). These
results suggest that interactions between proteins adsorbed
onto different droplets are favored when the droplets are
in close proximity during heating (i.e., high salt), but that
interactions between proteins adsorbed onto the same
droplets are favored when droplets are not in close
proximity during heating (i.e., low salt). Presumably, in
the latter case, the extensive molecular rearrangements
and intra-droplet proteinprotein interactions that occur
above T m reduce the surface hydrophobicity and/or the
number of exposed sulfhydryl groups at the droplet
surfaces. This knowledge may provide a useful practical
method of reducing the susceptibility of globular protein
stabilized emulsions to droplet flocculation during heat
processing.

2.2. Thermal processing

In many practical applications, it is important to subject
protein-stabilized emulsions to thermal processing, e.g.,
cooking, pasteurization or sterilization [11,12]. Emulsions
stabilized by globular proteins are particularly sensitive to
thermal treatments, because these proteins unfold when the
temperature exceeds a critical value exposing reactive
groups originally located in their interiors, e.g., non-polar
or sulfhydryl groups [10 ]. These reactive groups increase
the attractive interactions between proteins that are adsorbed
either on the same or on different droplets, thereby altering
the susceptible of emulsions to droplet flocculation and
coalescence.
At room temperature, h-lactoglobulin stabilized emulsions (pH 7) are stable to flocculation in the absence of
added salt because of the relatively strong electrostatic
repulsion between the droplets [9,10 ]. On the other hand,
they become unstable to flocculation when a sufficiently

Fig. 1. Influence of isothermal heat treatment (3095 8C, 20 min), salt

concentration (0 or 150 mM NaCl) and order of salt addition (before or after
heating) on the mean particle diameter (d 43) of 5 wt.% n-hexadecane oil-inwater emulsions (0.5 wt.% h-lactoglobulin, pH 7.0).

D.J. McClements / Current Opinion in Colloid & Interface Science 9 (2004) 305313

The extent of droplet flocculation and the structure of the

flocs formed when whey protein stabilized emulsions (pH 7)
were subjected to thermal processing (5595 8C, 02 h)
were found to depend on the holding temperature and time,
as well as the non-adsorbed protein concentration [13 ].
These researchers found that flocs formed at relatively short
holding times were progressively disrupted when the
emulsions were held at elevated temperatures. The stability
and rheology of protein stabilized emulsions may therefore
be manipulated by controlling the protein concentration and
the heating conditions.
Emulsions stabilized by proteins that do not undergo
extensive heat-induced conformational changes (such as
caseins) are usually less susceptible to droplet aggregation
during heating [12,14].

2.3. Mechanical stress

Droplet coalescence occurs in oil-in-water emulsions
when the thin film of material (the continuous phase and/or
interfacial membranes) separating the droplets is ruptured
and the fluids within the droplets (the dispersed phase)
merge together [1517 ]. Under quiescent conditions,
protein-stabilized emulsions are highly stable to droplet
coalescence because the interfacial membrane formed by the
proteins generates strong short-range repulsive forces and is
resistant to rupture [17 ]. Nevertheless, there are a number
of situations where droplet coalescence may be promoted
due to the application of mechanical stresses, such as
shearing, centrifugation or homogenization.

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2.3.1. Insufficient emulsifier

If there is insufficient emulsifier present in a system to
completely cover all of the oilwater interfaces present,
then there will be gaps in the interfacial membranes
surrounding the droplets [5 ]. Coalescence could then
occur if two gaps on different droplets came into close
proximity. This type of coalescence is likely to be most
important during homogenization where new surfaces are
continually being created by the intense forces generated
within a homogenizer [18 ].

2.3.2. Film stretching

If a sufficiently large stress is applied parallel to an
interface that is covered with emulsifier, then some of the
emulsifier molecules may be dragged along the interface,
leaving some regions where there is an excess of
emulsifier and other regions where there is a depletion
of emulsifier [17 ]. Coalescence could then occur if two
emulsifier-depleted regions on different droplets came into
close proximity during a dropletdroplet encounter. This
process is only likely to be important if the adsorption of
the emulsifier is relatively slow compared to the duration
of the applied stresses and droplet encounter frequency,
otherwise, emulsifier would have sufficient time to adsorb
to the droplet surfaces and cover the gaps. Film stretching

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307

is likely to be important in emulsions that are subjected to

intense mechanical stresses, especially if the droplets are in
close proximity, e.g., in flocculated or concentrated
emulsions.
2.3.3. Film tearing
If a sufficiently large stress is applied parallel to an
interface that is comprised of a highly cohesive layer of
emulsifier molecules, then it may cause the interfacial
membrane to buckle and tear, leaving exposed emulsifierdepleted patches that promote coalescence [17 ]. This
mechanism is likely to be important in systems where the
interfacial membranes are highly cohesive (e.g., protein
membranes with extensive cross-linking), particularly
under high applied mechanical stresses, e.g., shearing,
homogenization, centrifugation [5 ,17 ,18 ]. It is likely
to be less important in dilute, non-flocculated and
quiescent emulsions because the forces generated in these
systems are not strong enough to tear the interfacial
membranes.

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2.4. Homogenization
One of the most important roles of emulsifiers is to
facilitate the formation of small emulsion droplets during
homogenization of the oil and aqueous phases. An effective
emulsifier should rapidly adsorb to the freshly formed
droplet surfaces, reduce the interfacial tension by an
appreciable amount to facilitate droplet disruption and
provide a protective coating that prevents the droplets from
aggregating with their neighbors [2]. Protein emulsifiers
differ in the rate at which they adsorb to droplet surfaces
during homogenization, in the minimum amount that is
required to saturate the droplet surfaces, and in their ability to
protect droplets against coalescence under different environmental conditions [19]. For example, casein micelles adsorb
more rapidly to droplet surfaces than individual casein
molecules during high pressure valve homogenization, but
more protein is required to saturate the droplet surfaces for
casein micelles than individual casein molecules. The
amount of total protein present also plays a major role in
determining the stability of the droplets to aggregation
during homogenization. It is convenient to divide the
influence of emulsifier concentration on droplet size into
two regions [18 ]:

I. Insufficient Emulsifier. When the emulsifier concentration

is limiting (i.e., there is insufficient emulsifier present to
cover all of the droplet surface area created by the
homogenizer), then the droplet size is governed primarily
by the emulsifier concentration, rather than by the energy
input of the homogenizer. The surface load of the
emulsifier remains relatively constant in this regime and
is close to the value of the excess surface concentration of
the emulsifier at saturation (C sat). Under these conditions,
the mean droplet size produced by homogenization is

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D.J. McClements / Current Opinion in Colloid & Interface Science 9 (2004) 305313

determined by the maximum amount of surface area that

can be covered by the available emulsifier:
rmin

3Csat /
3Csat /

cS
c S V 1  /

where C sat is the excess surface concentration of the

emulsifier at saturation (in kg m2), / is the disperse
phase volume fraction, c S is the concentration of
emulsifier in the emulsion (in kg m3) and c SV is the
concentration of emulsifier in the continuous phase (in kg
m3). Different proteins have different C sat values, which
means that the size of the droplets produced using a given
amount of protein depends on protein type in this regime.
II. Excess Emulsifier. When the emulsifier concentration is
in excess (i.e., there is more emulsifier present than is
required to completely cover all of the droplet surface
area created by the homogenizer), then the droplet size
is relatively independent of emulsifier concentration and
depends primarily on the energy input of the homogenizer. Under these circumstances, the mean droplet
diameter that can be produced depends on the flow
conditions prevalent in the homogenizer (e.g., laminar,
turbulent and/or cavitational) and the physicochemical
properties of the component phases (e.g., interfacial
tension and viscosities). If the emulsifier does not form
multiple layers at the interface then the surface load of
the emulsifier will remain relatively constant in this
regime (C~C sat). Alternatively, if the emulsifier is
capable of forming multiple layers at the interface then
the surface load may increase as the overall concentration of emulsifier in the system is increased (CNC sat).
This latter affect has been observed for globular
proteins, such as whey proteins, where the surface load
increases up to a certain level as the overall emulsifier
concentration is increased [18 ].

Globular proteins may undergo appreciable denaturation

during the homogenization process, which has been
attributed to surface denaturation of the proteins after
adsorption to the droplet surfaces rather than due to the
high pressure gradients generated within the homogenizer
[20]. This kind of protein denaturation may have an
appreciable influence on the subsequent stability and
properties of emulsions, although little work has been done
in this area.
2.5. Chilling and freezing
There are many potential applications for oil-in-water
emulsions that can be chilled or frozen during storage and
then warmed or thawed prior to use, e.g., refrigerated and
frozen foods or pharmaceuticals. Storage at reduced
temperatures can be used to protect product quality against
microbial growth, enzymatic reactions and chemical
degradation. Nevertheless, many oil-in-water emulsions

are highly unstable when they are chilled and frozen,

and rapidly breakdown after reheating. The design and
manufacture of products with improved stability to cold
storage therefore depends on understanding the basic
physicochemical mechanisms that occur during the chilling
and freezing of oil-in-water emulsions. A variety of
different physicochemical processes may occur when a
food emulsion is cooled, including fat crystallization, ice
formation, freeze-concentration, interfacial phase transitions and biopolymer conformational changes [2].When
oil-in-water emulsions are cooled to temperatures where
the fat phase becomes partially crystallized (but the
aqueous phase remains completely liquid), they become
susceptible to a phenomenon known as partial coalescence.
Partial coalescence is the process whereby a fat crystal
from one partially crystalline droplet penetrates into a
liquid region of another partially crystalline droplet [2].
This process results in the formation of irregularly shaped
aggregates that usually decrease the creaming stability and
increase the shear viscosity (i.e., bthickeningQ) of the
emulsion. A number of studies have been carried out to
elucidate the factors that influence the susceptibility of oilin-water emulsions to partial coalescence, with the major
factors being the solid fat content, the thickness of the
interfacial membrane surrounding the droplets, the droplet
size and the application of mechanical agitation [2]. Partial
coalescence is a critical step in the production of some
types of food emulsions, including ice cream, whipped
cream, margarine and butter. On the other hand, it is
undesirable in other food emulsions because it leads to
deterioration of product quality, e.g., thickening of creams.
Partial coalescence may occur in the aqueous phase when
two partially crystalline fat droplets collide [21,22] or it
may occur at a gaswater interface when partially
crystalline fat droplets adsorb to the surface of a gas
bubble [23].When oil-in-water emulsions are cooled to a
temperature where the water crystallizes, there are a
number of additional physicochemical processes that occur
that can also promote emulsion instability [24]. First, when
ice crystals form in the aqueous phase the oil droplets are
forced closer together. Second, there may be insufficient
free water present to fully hydrate the emulsifier molecules
adsorbed to the droplet surfaces, which can promote
increased dropletdroplet interactions. Third, ice crystallization leads to an increase in the ionic strength of any
freeze-concentrated non-frozen aqueous phase surrounding
the emulsion droplets, which may screen electrostatic
repulsion between the droplets. Fourth, it is possible that
ice crystals formed during freezing may penetrate into the
oil droplets and disrupt their interfacial membranes, thus
making them more prone to coalescence once they are
thawed. Fifth, emulsifiers may adsorb to the surface of ice
crystals, which would reduce the amount left to cover the
emulsion droplets. Finally, emulsifiers may lose their
functionality when the temperature is decreased below a
certain level, e.g., globular proteins may be denatured. At

D.J. McClements / Current Opinion in Colloid & Interface Science 9 (2004) 305313

present, there is still a relatively poor understanding of the

relative importance of these various mechanisms of
emulsion instability for particular systems.The influence
of emulsifier type (whey protein isolate, casein and Tween
20) on the stability of oil-in-water emulsions to fat
crystallization and ice formation have recently been carried
out [21,22,24]. These studies have shown that dairy
proteins (WPI and casein) provide better protection against
droplet coalescence than small molecule surfactants when
the oil phase is partially crystalline, but that they can only
provide a limited degree of protection against droplet
coalescence when both water and oil crystallization occurs.
This has been attributed to the ability of the proteins to
form relatively thick interfacial membranes that are
difficult to penetrate by fat crystals. The stability of
emulsions to water crystallization can be improved by
incorporating cryoprotectants (e.g., sucrose) or by creating
multiple layers of emulsifiers around the oil droplets [25].
2.6. Drying
Protein-stabilized oil-in-water emulsions are often dried
to form powders that can be used as food ingredients in
other products (e.g., flavorings) or that can be hydrated by
consumers prior to use (e.g., dairy creamers, soups). The
emulsion should maintain its desirable physiochemical and
stability characteristics after the drying and hydration
processes [14,2628]. A variety of different unit operations
are utilized to dry food emulsion products, such as spraydrying and freeze-drying.Spray-drying may cause denaturation of globular proteins because of the relatively high
temperatures involved, or because of the generation of air
liquid interfaces in the system where proteins can adsorb
and undergo surface denaturation. Recently, it has been
shown that oil-in-water emulsions stabilized predominantly
by caseins are more stable to spray-drying than those
stabilized predominantly by whey proteins, which was
attributed to denaturation of the globular whey proteins
[14]. Freeze-drying is often used in the preparation of
specialized protein ingredients. During the freezing step the
aqueous protein solution separates into ice crystals dispersed
in a freeze concentrated solution that contains free proteins
and emulsion droplets [29]. During the drying step, the
water is removed in two stages: (i) the water in the ice
crystals is removed by sublimation, (ii) the water in the free
concentrated solution is removed by evaporation. In the
absence of cosolvents (such as sugars or polyols), proteins
usually lose their functionality during drying processes, but
in the presence of certain cosolvents, protein functionality
can be retained. At least three different physiochemical
mechanisms have been proposed to account for the ability of
cosolvents to enhance protein stability during dehydration
processes. First, cosolvents that are preferentially excluded
from protein surfaces tend to favor folded over unfolded
states of protein molecules, thereby retarding cold-, heat-,
surface- and pressure-denaturation processes. Second, some

309

cosolvents are capable of forming hydrogen bonds with the

surface of dried proteins, thereby inhibiting protein unfolding and aggregation by taking the place of water molecules.
Third, some cosolvents are capable of forming a highly
viscous glass phase around the protein molecules that
retards protein degradation by decreasing the molecular
mobility of the system. It is likely that all of these
mechanisms play some role in enhancing protein stability
to dehydration, but the relative importance of each
mechanism still needs to be established for particular
systems.

3. Influence of aqueous phase composition

3.1. pH and ionic strength
In practical applications, proteins may have to exhibit
their desirable functional properties in products that have a
wide range of different pH values and ionic strengths. The
interfacial membranes formed by proteins are usually
relatively thin and electrically charged, hence, the major
mechanism preventing droplet flocculation in proteinstabilized emulsions is electrostatic repulsion, rather than
steric repulsion [30]. Protein-stabilized emulsions are therefore particularly sensitive to pH and ionic strength effects.
They tend to flocculate at pH values close to the isoelectric
point of the adsorbed proteins and when the ionic strength
exceeds a particular level, because the electrostatic repulsion
between the droplets is then no longer sufficiently strong to
overcome the various attractive interactions, e.g., van der
Waals, hydrophobic or depletion [19,31]. Multivalent
counter-ions are particularly efficient at promoting emulsion
instability because they are more effective at screening
electrostatic interactions (reducing the Debye screening
length) and because they can bind to droplet surfaces
thereby reducing the f-potential [32]. The difference in the
effectiveness of monovalent (Na+) and divalent (Ca2+)
counter-ions at promoting droplet flocculation in a whey
protein stabilized emulsion (pH 7) is highlighted in Fig. 2. A
variety of strategies have been developed to improve the
stability of protein-stabilized emulsions to droplet flocculation induced by pH or ionic strength effects:
! Multivalent counter-ions, such as Ca2+, Fe2+or Fe3+,
can be sequestered by binding to chelating agents
(such as EDTA, citrates, phosphates) or by binding to
biopolymers (such as casein macro-peptide, phosvitin,
feritin) [3336].
! Ionic surfactants that adsorb to the surface of the oil
droplets can be added to a protein-stabilized emulsion
to change the pH dependence of the f-potential of the
droplets, thereby changing the range of pH values that
the emulsion is stable to flocculation [37].
! Electrically charged biopolymers that adsorb to the
surface of oppositely charged droplets can be added to

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D.J. McClements / Current Opinion in Colloid & Interface Science 9 (2004) 305313

Fig. 2. Effect of (a) potassium chloride and (b) calcium chloride on mean
particle diameter of diluted 7 wt.% soybean oil-in-water emulsions
stabilized by WPI. The multivalent counter-ion is more effective at
destabilizing the emulsions due to ion-binding and screening effects.

a protein-stabilized emulsion to change the pH dependence of the f-potential of the droplets, thereby
changing the range of pH values that the emulsion is
stable to flocculation [38 ,39].

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3.2. Sugars and polyols

The aqueous phase surrounding the oil droplets in many
commercial emulsion-based products often contains a
variety of different kinds of small polar molecules, such
as sugars and polyols [29]. These relatively small molecules
may influence the molecular and functional properties of the
adsorbed proteins in oil-in-water emulsions in a variety of
different ways, and therefore it is important to quantify their
influence on protein functionality in emulsions.The influence of a neutral cosolvent (sucrose) on the kinetics of
droplet aggregation promoted by surface denaturation in hlactoglobulin stabilized oil-in-water emulsions has recently

been examined [40]. When sucrose was added to the

emulsions immediately after homogenization, the rate of
particle growth decreased as the sucrose concentration was
increased (040 wt.%). On the other hand, when sucrose
was added to the emulsions after a few hours of aging it
actually promoted a greater extent of droplet flocculation. A
number of physicochemical mechanisms were postulated to
account for the observed effects of sucrose on the stability of
the emulsions to droplet flocculation. First, sucrose
increases the viscosity of the continuous phase, which
should slow down the rate of dropletdroplet collisions.
Second, sucrose alters the physicochemical properties of the
aqueous solution surrounding the droplets (e.g., density,
dielectric constant, refractive index, osmotic pressure),
which may change the height of the repulsive energy barrier
and alter the fraction of collisions leading to aggregation.
Third, sucrose puts the proteins under osmotic stress, which
may slow down the kinetics of protein surface denaturation
[29]. Fourth, sucrose increases the attractive interactions
between emulsion droplets through a short-range bmolecular
depletion interactionQ, which may strengthen the bonds
between flocculated droplets. More research is required to
determine the relative importance of these and other
mechanisms in specific emulsion systems.The influence of
sucrose on the extent of droplet flocculation occurring in
oil-in-water emulsions stabilized by a globular protein after
thermal processing has also been examined [40]. Sucrose
(040 wt.%) and NaCl (150 mM NaCl) were added to nhexadecane oil-in-water emulsions stabilized by h-Lg (pH
7.0) either before or after isothermal heat treatment (3095
8C for 20 min). When salt was added to emulsions before
heat treatment, appreciable droplet flocculation was
observed below the thermal-denaturation temperature of
the adsorbed h-Lg (T m~70 8C), and more extensive
flocculation was observed above T m. On the other hand,
when salt was added to emulsions after heat treatment,
appreciable droplet flocculation still occurred below T m, but
little flocculation was observed above T m (see above).
Addition of sucrose to the emulsions increased T m, and
either promoted or suppressed droplet flocculation depending on whether it was added before or after heat treatment.
These results were interpreted in terms of the influence of
sucrose on protein conformational stability, proteinprotein
interactions and the physiochemical properties of aqueous
solutions. The presence of high levels of sucrose increased
the attraction between emulsion droplets once the protein
molecules had unfolded, but it meant that the emulsions had
to be heated to a higher temperature to promote protein
unfolding.
3.3. Surfactants
Emulsions often contain small molecule surfactants,
which may be either non-ionic or ionic, e.g., lecithin,
polysorbates, spans. These surfactants can alter the stability
of protein-stabilized emulsions either directly or indirectly.-

D.J. McClements / Current Opinion in Colloid & Interface Science 9 (2004) 305313

Surfactants may directly influence protein functionality by

binding to them, because this can lead to substantial changes
in protein conformation and interactions [41,42]. Ionic
surfactants may bind to protein molecules through a
combination of electrostatic and hydrophobic interactions,
whereas non-ionic surfactants may bind to proteins through
hydrophobic interactions. Once a surfactant has bound to a
protein, it may either stabilize or destabilize the protein
structure and/or it may either promote or oppose protein
aggregation depending on surfactant type, surfactant concentration and environmental conditions [37,42]. These
alterations in the molecular characteristics of globular
proteins may lead to changes in their ability to absorb to
interfaces and to stabilize emulsions. It is therefore
important to improve our understanding of the origin and
nature of proteinsurfactant interactions and of their
influence on protein functionality.Small molecule surfactants may also influence protein functionality in a more
indirect manner. Surfactants are surface-active molecules
that normally compete with proteins for the available oil
water interface, and thus can displace some or all of the
protein from the interface [4,43 ,44]. The composition of
an interface depends on the relative surface activity and
concentration of the protein and surfactant molecules, as
well as the time that each component is introduced into the
system. Interfacial composition alters the magnitude, range
and sign of the interactions between droplets, as well as
the rheology of the interface, and therefore it may alter the
overall physicochemical properties and stability of emulsions. The competition between protein and surfactant
molecules at interfaces may therefore play an important
role in determining protein functionality in food emulsions.
The application of new imaging techniques and computer
simulations has provided a good understanding of the
microstructure of interfaces containing a mixture of
proteins and small molecule surfactants [43 ,44,45]. When
a protein-coated interface is brought into contact with an
aqueous solution containing surfactant a sequence of
events occurs that depends on the surfactant concentration.
At relatively low surfactant concentrations, surfactant
molecules adsorb to the interface and form small islands
of surfactant located within the protein network. As the
surfactant concentration is increased the size of the
surfactant-rich regions expands, restricting the protein
network to a smaller surface area. At relatively high
surfactant concentrations, the protein region increases
appreciably in thickness and eventually the protein
molecules are completely displaced from the interface.
The two-dimensional phase separation of the interface into
protein-rich and surfactant-rich domains can clearly be
observed using microscopy techniques and computer
simulations [43 45]. The structure of the domains formed
depends on the strength of the attractive interactions
between the adsorbed proteins. Proteins such as hlactoglobulin, which have strong interfacial interactions,
tend to form irregular-shaped domains, whereas those such

311

as casein, which have weak interfacial interactions tend to

form circular-shaped domains [4 ].

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3.4. Biopolymers
Protein-stabilized oil-in-water emulsions may contain
one or more types of biopolymer in the continuous phase.
These biopolymers could be a fraction of the protein used as
an emulsifier that did not adsorb to the droplet surfaces
because they were already saturated with protein, or they
could be other functional ingredients, such as thickening
agents or gelling agents. These biopolymers may interact
with the adsorbed proteins, either directly or indirectly, and
influence the stability of the emulsion through a variety of
mechanisms [38 ].

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3.4.1. Depletion flocculation

The presence of non-adsorbing biopolymers in the
aqueous phase of a protein-stabilized emulsion causes an
increase in the attractive force between the droplets due to
an osmotic effect associated with the exclusion of the
biopolymers from a narrow region surrounding each droplet
[2,46]. This attractive force increases as the concentration of
biopolymers increases, until eventually it may become large
enough to overcome the repulsive interactions between the
droplets and cause them to flocculate. This type of droplet
aggregation is usually referred to as depletion flocculation.
A wide variety of different biopolymers have previously
been shown to be capable of inducing depletion flocculation
when added in sufficiently high concentrations, including
polysaccharides (xanthan gum, gum arabic, modified starch,
maltodextrin, pectin, carrageenan) and proteins (whey and
caseinate). The lowest concentration required to cause
depletion flocculation is referred to as the critical flocculation concentration (CFC). The CFC decreases as the size
of the emulsion droplets increases and the effective volume
fraction of the biopolymers increases [46]. The flocculation
rate initially increases as the concentration of non-adsorbing
biopolymers is increased because of the enhanced attraction
between the droplets, i.e., a higher collision efficiency.
However, once the concentration of biopolymers exceeds a
certain concentration, the flocculation rate often decreases
because the viscosity of the continuous phase increases so
much that the movement of the droplets is severely retarded,
i.e., a lower collision frequency.In general, any change in
solution conditions that alters the effective volume of the
non-adsorbed biopolymers will influence the CFC, e.g.,
temperature, ionic strength, solvent quality [38 ].

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3.4.2. Bridging flocculation

Electrically charged biopolymers are capable of adsorbing to the surfaces of oppositely charged emulsion droplets
through electrostatic interactions. At certain biopolymer
and droplet concentrations, the biopolymers promote
droplet flocculation due to charge neutralization and
bridging effects [38 ]. Since the driving force for this

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D.J. McClements / Current Opinion in Colloid & Interface Science 9 (2004) 305313

type of bridging flocculation is electrostatic in origin, the

stability of the emulsions is strongly dependent on pH and
ionic strength, as well as the electrical characteristics of the
adsorbed proteins and the biopolymers. A large number of
studies have examined the factors that influence bridging
flocculation in protein stabilized emulsions. Emulsions
stabilized by h-lactoglobulin undergo bridging flocculation
by charged polysaccharides (e.g., pectin, carrageenan,
dextran sulfate) at pH values where the polysaccharide and
protein have opposite charges [38 ,39,4749]. Bridging
flocculation is most pronounced at polysaccharide concentrations where the droplet surfaces are only partially covered.
If there is a high concentration of polysaccharide present in
the continuous phase, then the propensity for bridging
flocculation to occur is reduced because there is sufficient
biopolymer to completely cover the droplet surfaces. Nevertheless, if the free biopolymer concentration gets sufficiently
high then depletion flocculation may occur.

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3.4.3. Multiple-layer formation

Electrically charged biopolymers are capable of adsorbing to the surfaces of oppositely charged emulsion
droplets. At sufficiently high biopolymer concentrations
and low droplet concentrations, biopolymers tend to adsorb
to the surface of protein-coated droplets to form an
interfacial layer consisting of protein+biopolymer [39
42,43 ,4449]. Recent experiments in our laboratory have
shown that droplets coated by multilayered interfacial
membranes often have improved stability to environmental
stresses (such as thermal treatments, drying, freezing and
mechanical agitation) than those stabilized by singlelayered membranes because of the increase in interfacial
thickness and rheology.

4. Conclusions
There have been significant advances in our understanding of the factors that influence the functionality of
proteins in oil-in-water emulsions. This article has mainly
focused on advances in the understanding of the influence of
solution composition and environmental stresses on protein
functionality. Improved knowledge of these effects should
facilitate the rational and systematic design and production
of emulsion-based products.
References and recommended reading
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An excellent up-to-date introduction and overview of the molecular and
physicochemical basis of protein functionality in biological systems.

! of special interest
!! of outstanding interest

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