Professional Documents
Culture Documents
Eric Dickinson
Procter Department of Food Science, University of L eeds, L eeds, UK L S2 9JT
Physical properties of oil-in-water emulsions stabilized by milk proteins are determined largely by the nature of the adsorbed
layer at the surface of the dispersed droplets. There are two distinct classes of protein : the disordered caseins (especially a -casein
s1
and b-casein) and the globular whey proteins (especially b-lactoglobulin). Substantial dierences exist between these two classes in
terms of adsorbed layer structure and surface rheological properties at the oil/water interface. Theoretical modelling of adsorbed
layers of a -casein and b-casein with a simple self-consistent-eld approach is useful for understanding the excellent stabilizing
s1
properties of the caseins, and in interpreting the aggregation behaviour of casein-based emulsions as a function of ionic strength
and pH. The creaming behaviour and droplet occulation are sensitive also to the concentration of non-adsorbed casein. In
systems containing milk protein and small-molecule surfactant, competitive adsorption has a strong inuence on orthokinetic
emulsion stability, and on the viscoelasticity of heat-set b-lactoglobulin-stabilized emulsion gels. Computer simulation of model
particle gel networks shows considerable promise for providing new insight into the relationship between interparticle interactions and the structure and rheology of emulsion gels.
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Fig. 1 Suggested representations of b-casein adsorbed at a polystyrene latex surface.37 (a) The looptrain model with a boundary
between the two regions at around residue number 50. (b) The bloband-spring model in which the blob represents the cluster of negatively charged residues around residue number 20 : (i) native protein at
low ionic strength, (ii) dephosphorylated protein at low ionic strength
or native protein at high ionic strength, and (iii) native protein with
bound calcium ions.
Fig. 2 Inuence of pH on total segment density prole o(z) for bcasein-like polymer predicted from SCF theory at ionic strength 0.01
M and bulk concentration 5 ] 10~6.51
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Fig. 4 Sketches of representative adsorbed congurations of (a) bcasein and (b) a -casein
s1
understood48,51 in terms of the poorer solubility of the dephosphorylated polymer, and it conrms the importance of
the phosphoserine residues in b-casein in maintaining a thick
steric stabilizing layer whilst avoiding interfacial protein precipitation (i.e. multilayer formation).
Without attempting to optimize the choice of the model
parameters, there is generally good agreement between the
structure of the adsorbed b-casein-like polymer predicted from
the SCF theory and the information obtained experimentally
from the various experimental techniques. A powerful emerging technique for the study of interfacial structure is specular
neutron reection.38 Sets of reectivity data obtained for bcasein adsorbed at liquid39 and solid40 surfaces have been
analysed in terms of a two-layer model consisting of a thin
dense inner layer and a lower density outer layer.1 At pH
values in the range 5.57.0 at the air/water interface, the ts to
the data indicate41 a dense protein-rich inner layer (o B 95%)
and a more diuse outer layer (o B 15%) extending into the
bulk phase. Model-independent Guinier analysis shows52 an
increase in adsorbed amount and a thickening of the layer as
the pH is lowered from 7 to 5.5. Independent experimental
conrmation of this eect of pH on the protein layer coverage
and structure has been obtained in a recent surface plasmon
resonance study53 of b-casein adsorption onto a selfassembled monolayer of octadecyl mercaptan on gold.54,55
On tting the kinetic data to a simple double-exponential
function,56 the surface plasmon resonance technique has
demonstrated53 that the rate of adsorption of b-casein at the
hydrophobic solid surface at pH 5.5 is less than half that at
pH 7.0. The very dilute tail region, predicted from the SCF
theory for the adsorbed b-casein-like polymer, is consistent
with the large hydrodynamic layer thickness (1015 nm)
derived from dynamic light-scattering.25,36,37 Mobility of the
phosphoserine-rich tail region in adsorbed b-casein in emulsions has been conrmed by 31P NMR.57 Dephosphorylation
of b-casein has been found37,58 to lead to a reduction in
hydrodynamic layer thickness accompanied by a marginal
increase in adsorbed amount ; this again agrees qualitatively
with predictions from SCF theory.48
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complete protein displacement (say R [ 20) is greater in emulsions than in planar interface studies (see Fig. 9) because of the
higher surface/volume ratio. The value of R required for complete displacement has been found88,89,92 to be higher for
ionic surfactants like sodium dodecyl sulfate (SDS) or sodium
lauryl ether sulfate (SLES) which form strong interfacial complexes with the adsorbed protein. On the other hand, oilsoluble surfactants such as sorbitan monooleate (Span 80) or
monoglycerides tend to be less eective at protein displacement.88,93h95 The competitive adsorption behaviour is also
sensitive to changes in protein structure induced by physical
processing. Ageing or heating of emulsions prepared with blactoglobulin reduces the ability of the surfactant to displace
the protein from the oil/water interface.9,94,96 An additional
eect on the competitive adsorption behaviour is the nature of
the oil phase, especially its polarity (hydrocarbon oil vs. triglyceride oil)97,98 and the state of crystallization.99
Surface shear rheology is very sensitive to the structure and
dynamics of adsorbed layers.11,100h102 Hence, it can provide a
valuable probe of the competitive adsorption of proteins with
surfactants and of interfacial proteinsurfactant interactions.
The displacement of b-lactoglobulin from the oil/water interface by non-ionic surfactant is associated with a drop in
apparent surface shear viscosity by several orders of magnitude.91,94,103 Although non-ionic surfactants generally interact weakly with proteins in solution, the existence of a
strongly hydrophobic binding site on b-lactoglobulin leads to
a specic 1 : 1 complex with Tween 20 which has implications
for the composition and properties of the adsorbed
layer.104h106 Eects of proteinsurfactant interactions are
even more important for anionic or cationic surfactants, to
such an extent that the surfactantprotein complexes may
have completely dierent surface rheological properties from
those of the pure proteins.103,107,108
The orthokinetic stability of an oil-in-water emulsion containing fully liquid droplets is predominantly determined by
the structure and interactions of the adsorbed protein layer.
The disruption of the adsorbed protein layer on addition of a
water-soluble surfactant (e.g. Tween 20) has been shown109 to
lead to a substantial reduction in coalescence stability of
model b-lactoglobulin-stabilized emulsions under turbulent
ow conditions. Oil droplets containing fat crystals are especially susceptible to partial coalescence when stirred, and in
dairy-type emulsions the nature of the fat crystal morphology
is generally the most important factor controlling the orthokinetic destabilization rate.110h112 Combined aeration and
freezing during ice-cream making causes the emulsion to
undergo partial coalescence, with clumps and clusters of fat
droplets forming an internal network in the frozen
product.81,82 The rate of fat destabilization is enhanced by the
presence of surfactant. Fig. 10 shows the eect of Tween 80
addition on the percentage of fat destabilized during whipping
of ice-cream mix in a barrel freezer.78 It has been shown113
that air incorporation or shearing alone (independent of ice
crystallization) does not lead to the same high rate of fat
destabilization as when whipping and freezing occur simultaneously.
In agreement with theories of depletion occulation by nonadsorbing polymers,114 it has been found6,115 that ne
casein-stabilized oil-in-water emulsions containing low concentrations of hydrocolloids such as xanthan gum or guar
gum exhibit poor creaming stability. Recently, it has also been
found116,117 that, in casein-stabilized emulsions containing
no added hydrocolloid, the presence of excess casein itself
induces a substantial reduction in creaming stability, presumably also as a result of a depletion occulation mechanism.
J. Chem. Soc., Faraday T rans., 1998, V ol. 94
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Fig. 12 A network of aggregated particles in two dimensions produced by computer simulation. The particle areal packing fraction is
ca. 50%. (Assuming each particle represents a single protein molecule,
this cross-linked planar structure may be a useful model of a gel-like
adsorbed globular protein layer.)
Fig. 13 Inuence of anionic surfactant SDS on the elasticity of heatset b-lactoglobulin-stabilized emulsion gels (38 wt.% n-tetradecane,
58 wt.% protein, pH 7.0). Storage shear modulus G@ at 1 Hz and
30 C is plotted against surfactant/protein molar ratio R for three different b-lactoglobulin concentrations : (=) 5, (K) 6 and (>) 8 wt.%.
(Reproduced with permission from ref. 108.)
drastic lowering of the modulus again. This can be attributed,140 at least in part, to protein displacement from the oil/
water interface (cf. the behaviour of Tween 20). But another
contributing factor is the incorporation of denatured protein
into non-associating mixed proteinsurfactant micelles, so
that the resulting b-lactoglobulin aggregates become highly
compartmentalized and hence unavailable for network formation.
Gel-like characteristics may be induced in a concentrated
globular protein-stabilized emulsion without heating through
the addition of a second polymer that has an attractive electrostatic interaction with the adsorbed protein surface and so
can form polymer bridges between adjacent droplets.141
Bridging occulation is strong when the added polymer is of
opposite charge to the adsorbed protein (e.g. cationic gelatin
added to b-lactoglobulin-coated droplets at neutral pH)140
and relatively weak when the added polymer is of the same
charge as the adsorbed protein (e.g. anionic dextran sulfate or
i-carrageenan added to BSA-coated droplets at neutral
pH).142,143 An emulsion gel with covalent cross-links between
protein-coated droplets may be induced by treating the emulsion with the enzyme transglutaminase.144h146 At large deformations, the modulus of the enzyme-set emulsion gel
(containing chemical cross-links) increases with strain,
whereas that for the equivalent heat-set emulsion gel (with
predominantly physical bonds) has qualitatively the opposite
behaviour.140,144
Improved understanding of the quantitative relationship
between interparticle interactions and the rheology of emulsion gels can be derived from simple models of network structures formed from idealized systems of aggregating
particles.147 Recent progress has been made by a consideration of the properties of idealized model particle gels generated by computer simulation.148h151 An important aspect of
the data treatment is the use of fractal scaling analysis150 to
describe the intermediate-range microstructure. While it is the
(semi-)permanent cross-links formed during aggregation that
are mainly responsible for holding together the network structure in a strong elastic particle gel, the structure also depends
on the nature of weak reversible forces that act between the
particles before and during the formation of the cross-links.
These combined features can be taken account of in a Brownian dynamics simulation model152 which incorporates exible
irreversible cross-links, as well as non-bonded particleparticle
interactions. Such a model was used to generate the twoJ. Chem. Soc., Faraday T rans., 1998, V ol. 94
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Conclusions
This article has described recent progress in understanding the
relationship between the properties of adsorbed protein layers
and the stability and rheology of protein-based oil-in-water
emulsion systems. Emphasis here on the surface properties of
the major milk proteins (a -casein, b-casein and bs1
lactoglobulin) seems justied for a number of reasons : (i) the
interesting molecular contrast between the disordered caseins
and the globular whey proteins, (ii) the emergence of new
experimental data on adsorbed layer structure and surface
rheology for these pure proteins, (iii) the challenge posed by
the dierent theoretical predictions of interlayer interaction
potentials for the two dierent caseins, and (iv) the importance
of these proteins in relation to the processing of dairy-based
food colloids.
Based on recent progress, there is good reason to expect
that research in this eld will continue to benet from
advances in instrumental techniques for the study of macromolecular structure and dynamics at wet surfaces, and from
developments in the numerical modelling of complex
assemblies of surfactants and biopolymers. Further steps
towards the much greater complexity of real food systems can
be explored by extending these powerful experimental and
computational techniques to mixed proteinsurfactant
systems.
Milk protein-stabilized emulsions are susceptible to changes
in physical properties on quiescent storage or when subjected
to stirring. They can be converted into emulsion gels by
various processing methods such as heating, high-pressure
treatment, addition of polymers, or enzymic cross-linking. An
important challenge for us now is to be able to relate the
structural and rheological characteristics of these colloidal
systems to the nature of the interdroplet interactions
occurring during and after gelation. One powerful approach
towards establishing the complex link between interactions
and rheology is through computer simulation of particle gel
networks. The success of recent comparisons between simulation and experiment indicates that some encouraging progress
is beginning to be made.
Nevertheless, important issues do remain unresolved. First,
despite some exciting new advances, we still know little about
the molecular congurations of adsorbed proteins. Much
more detailed work is required using a wide range of spectroscopic and scattering techniques in order to establish the
structure of adsorbed proteins at oil/water and air/water interfaces to the same level of detail as has been established for the
structure of native proteins in solution. Secondly, we need to
know a lot more about the congurational structure of individual proteins in mixed lms containing surfactants and
other proteins. Thirdly, there is still disappointingly scant
understanding of the relationship (if any) between surface
rheological properties of adsorbed protein layers and stability
of protein-stabilized emulsions. This is not because of any
shortage of data in the literature on the static and dynamic
properties of pure protein layers or mixed proteinlipid lms,
far from it. What is lacking in most cases is any clear indication of useful quantitative links between the surface lm measurements and the colloidal stability properties. Without the
establishment of such links, a sceptical food scientist would be
justied in questioning the practical value of such data.
Fourthly, there is the question of the structural nature of the
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