Trends in Food Science & Technology 9 (1998) 143±151

Review
Molecular basis of
protein functionality importance of understanding the molecular basis for
protein functionality [1]. As a direct consequence of its

with special support for basic research in this area, the dairy indus-
try has been able to convert bovine whey proteins, tra-
ditionally a waste product of cheese production, into
consideration of value-added food ingredients [2]. Whey proteins are
now widely used in foods as gelling agents, emulsi®ers,

cold-set gels
texture modi®ers, thickening agents and foaming agents
[2]. In addition to their functional characteristics, they
also have the advantages of high nutritive value and

derived from heat- generally recognized as safe (GRAS) status, making them
`label-friendly' ingredients. Whey protein ingredients
may be purchased in several di€erent forms, the most
denatured whey common of which are whey protein isolate (WPI) and
whey protein concentrate (WPC). WPI ingredients have
higher protein concentrations (>90%) and less impuri-
ties than WPC ingredients (50±75%) and, consequently,
are more expensive to purchase because they require
Cory M. Bryant* and more processing [3]. For this reason, WPI tends to be
used in more specialized applications than WPC. The
D. Julian McClements
Department of Food Science, University of
Massachusetts, Amherst, MA 01003, USA (fax: +1-
413-545-1262; e-mail: cbryant@foodsci.umass.edu)
Whey proteins are widely used as ingredients in foods
because of their unique functional properties, i.e. emulsi®-
cation, gelation, thickening, foaming and water-binding
capacity. This review describes the recent development of
cold-setting whey protein ingredients and their potential
application in foods. We emphasize the importance of an
understanding of the molecular basis of protein function-
ality to the development of ingredients. # 1998 Elsevier
Science Ltd. All rights reserved
Increasing preferences of consumers for more tasty,
healthy, convenient, and natural food products has
provided the dairy industry with a unique opportunity
to develop and supply milk protein ingredients for
improving the functional properties of food products.
The development of these ingredients has largely
depended on recognition by food scientists of the
*Corresponding author.
0924-2244/98/$19.00. Copyright # 1998 Elsevier Science Ltd. All rights reserved
PII: S 0 92 4 - 2 24 4 ( 9 8 ) 0 0 03 1 - 4
principal components of whey protein are -lactoglo-
bulin, -lactalbumin, bovine serum albumin (BSA) and
the immunoglobulins [4]. Typical relative abundancies,
isoelectric points, molecular weights and denaturation
temperatures of these proteins are listed in Table 1.
Whey protein ingredients also contain various impuri-
ties, such as moisture, lipid, lactose and minerals. The
functional properties of whey protein ingredients
depend not only on their composition but also on their
degree of denaturation. Proteins may be denatured to
some extent by the various steps used in their produc-
tion [3, 6], and this usually has an adverse e€ect on their
functional properties. As a result, two protein ingre-
dients with exactly the same composition can exhibit
very di€erent functional properties due to di€erences in
their degree of denaturation.
In this article, we are primarily concerned with the
use of whey proteins as thickening or gelling agents in
foods. This application of whey proteins relies on the
unfolding and aggregation of the protein molecules in
aqueous solution. Traditionally, foods containing whey
protein ingredients had to be heated above 65 C before
the proteins would form gels or thicken solutions, which
limited their application in many types of food products
[2, 7]. We describe a technique which has recently been
developed to produce whey protein ingredients that are
capable of thickening solutions or creating gels at
144 C.M. Bryant and D.J. McClements/Trends in Food Science & Technology 9 (1998) 143±151

Table 1. Physical characteristics of the major whey proteins biopolymers in solution, since there is little change in
a the van der Waals interactions in the folded and unfol-
Protein pI M.W. % Td
ded states. Nevertheless, if a biopolymer molecule is
-lactoglobulin 5.2 18 400
-lactalbumin 4.8±5.1 14 200
bovine serum albumin 4.8±5.1 66 000
immunoglobulins 5.5±6.8 15±96104
a
Td represents the temperature of denaturation.
After references [2, 4, 5].
ambient and even refrigeration temperatures. These
cold-set ingredients can be used to produce foods with
di€erent textures, appearances and water-holding capaci-
ties than traditional whey protein ingredients.
Molecular basis of protein functionality
The functional properties of protein ingredients are
ultimately determined by the unique structure and
interactions of the protein molecule [8]. An under-
standing of the molecular basis of protein functionality
is therefore essential to the development and application
of new protein ingredients. In this section, we highlight
some of the most important types of molecular interac-
tions that govern the conformation and aggregation of
proteins in foods. The general characteristics of the
molecular interactions between protein molecules are
summarized in Table 2.
Steric and van der Waals interactions
There is an extremely strong repulsive interaction
between atoms or molecules at close separations
because of the overlap of their electron clouds [9]. This
steric overlap repulsion de®nes the size and shape of
atoms and molecules, and determines how closely they
can pack together. Steric overlap interactions play a
particularly important role in determining the possible
conformations of proteins in solution because the
molecule cannot adopt any spatial arrangements in
which two or more segments occupy the same space.
At the molecular level, van der Waals interactions
operate between all types of molecules and tend to have
fairly similar magnitudes [9]. Consequently, they only
play a minor role in determining the conformation of
60 78 large enough to act like a colloidal particle, then there
22 62 may be a signi®cantly strong van der Waals attraction,
5.5 64
9.1 72
which favors its aggregation with other biopolymer
molecules [10].
Hydrophobic interactions
Hydrophobic interactions play a major role in deter-
mining the conformation and interactions of protein
molecules in solution [9, 11]. They manifest themselves
as strong attractive forces between non-polar groups
separated by water [9, 12]. However, they actually ori-
ginate due to the ability of water molecules to form
relatively strong hydrogen bonds with other water
molecules, whereas non-polar molecules can only form
relatively weak van der Waals bonds with their neigh-
bors [9]. When a non-polar molecule is introduced into
water it causes the water molecules in its immediate
vicinity to rearrange themselves, which changes both the
interaction energy and entropy of the system. These
changes are thermodynamically unfavorable and so the
system attempts to minimize the contact area between
water and non-polar groups, which produces an attrac-
tive force between the non-polar groups [12±14]. Direct
force measurements between macroscopic non-polar
surfaces have shown that hydrophobic interactions are
relatively strong and long range (0 to 10 nm), however
such measurements for individual molecules are unclear
[9]. One of the characteristic features of hydrophobic
interactions is their tendency to increase in strength as
the temperature is raised [15, 16] up to 60±70 C at
which point they begin slowly to lose strength as tem-
perature is further increased [6, 17].
Electrostatic interactions
Electrostatic interactions act between species which
have a permanent electrical charge, for example, dipoles
or ions. They may be either attractive or repulsive
depending on the relative magnitude of the charges car-
ried [18]. When the charges have the same sign, the
interaction is repulsive, but when they have di€erent

Table 2. General characteristics of molecular interactions between two similar protein molecules in aqueous solution
Type Sign Strength Range pH I.S. Temperature
Hydrophobic Attractive Strong Long No No Increases
Electrostatic Repulsive Weak!Stronga Short!Longa Yes Decreases Increases
Hydrogen bonding Attractive Weak Short No No Decreases
Hydration Repulsive Strong Short Nob Nob Decreases
Van der Waals Attractive Weak Short No No ±
Steric repulsion Repulsive Strong Short No No ±
Disul®de bonds Attractive Very Strong Short Yes No
a
Depends on pH and ionic strength (I.S.).
b
Indirectly depends on pH and I.S. because these factors in¯uence the degree of protein hydration.
 C.M. Bryant and D.J. McClements/Trends in Food Science & Technology 9 (1998) 143±151
signs it is attractive [9]. Proteins may be involved in a
variety of di€erent types of electrostatic interaction
depending on the nature of the molecular species
involved, for example, ion±ion, ion±dipole or dipole±
dipole (in decreasing order of strength). The sign, mag-
nitude and distribution of the charge on a protein
molecule is governed principally by the pH of the sur-
rounding aqueous solution [19]. Above their isoelectric
point (pI) proteins are negatively charged; below it they
are positively charged, and at it they have no net charge
(although they still have some regions of positive charge
and other regions of negative charge) [18].
Electrostatic interactions between charged species are
particularly sensitive to the ionic strength of the inter-
vening medium. The magnitude and range of the inter-
actions can be reduced appreciably in the presence of
electrolytes because of electrostatic screening by the
counter-ions [14, 18, 20, 21]. Electrostatic interactions
between charged species in solution are principally
entropic in origin and, therefore, they increase in
strength with increasing temperature [14].
Ion bridging is another type of molecular interaction
which involves electrostatic interactions [22]. It occurs
when a polyvalent ion simultaneously binds to the sur-
face of two molecules which have an opposite charge to
the ion. These polyvalent ions may be low molecular
weight species, such as Ca2+, Mg2+, or Al3+, or high
molecular weight biopolymers, such as polysaccharides
or other proteins. The ability of polyvalent ions to form
ion bridges is superimposed on their ability to reduce
the magnitude of the electrostatic interactions through
electrostatic screening [22].
The sensitivity of electrostatic interactions to pH and
ionic strength gives food scientists a simple and con-
venient method of manipulating the interactions
between protein molecules and, therefore, of controlling
their functional properties.
Hydrogen bonds
Hydrogen bonds are formed between a lone pair of
electrons on an electronegative atom (such as oxygen),
and a hydrogen atom on a neighbouring group, i.e.
O-H‡-Oÿ [24±26]. The major contribution to hydrogen
bonds is electrostatic (dipole±dipole), but van der Waals
forces and steric repulsion also make a signi®cant con-
tribution [13]. Typically, they have bond strengths
between about 10 and 40 kJ molÿ1 and lengths of about
0.18 nm [9]. The actual strength of a particular hydrogen
bond depends on the electronegativity and orientation
of the donor and acceptor groups [24]. Hydrogen bonds
are stronger than most other dipole±dipole interactions
because hydrogen atoms have a strong tendency to
become positively polarized and have a small radius.
Hydrogen bonds are so strong that they cause appreci-
able alignment of the molecules involved. The strength
and directional character of hydrogen bonds are
145
responsible for many of the unique properties of
water [27].
Although hydrogen bonds are not usually the major
driving force determining the conformation and aggre-
gation of globular proteins, they do play an important
role in stabilizing the structures once formed [2].
Hydration interactions
Hydration interactions are fairly short range repulsive
interactions that arise when two hydrated molecules
closely approach each other [9]. They are the result of
the energy required to disrupt the hydrogen bonds
between the molecule and the water molecules in its
immediate vicinity [28]. The strength of this type of
interaction depends on the degree of hydration of the
molecule: the greater the hydration, the stronger the
repulsion and longer the range of the interaction. This
type of interaction may play an important role in pre-
venting protein molecules from aggregating in solution,
particularly at high salt concentrations when the binding
of the ions to the proteins is likely to be greatest. It is
possible for food scientists to regulate hydration inter-
actions by incorporating highly hydrated ionic species
that bind to the surface of the protein molecules [9].
Disul®de bonds
Cysteine is an amino acid, which is found in whey
protein molecules [1, 29]. It has a thiol group (-SH),
which is capable of forming disul®de bonds (-S-S-) with
other thiol groups by an oxidation reaction [30]. Free
thiol groups can also participate in thio±disul®de inter-
changes with disul®de bonds. These reactions usually
occur under alkaline conditions. Proteins are capable of
forming both intramolecular and intermolecular dis-
ul®de bonds under the appropriate conditions. Intra-
molecular bonds form when a pair of cysteine residues
are brought into close proximity by the folding of a
protein molecule, whereas intermolecular disul®de
bonds form when cysteine residues on di€erent mole-
cules are brought into close proximity [19].
It should be noted that the free thiol and disul®de
groups in native whey proteins are located in the inter-
ior of the folded molecule and are, therefore, unavail-
able for interaction, even under conditions which would
normally favor thiol/disul®de interchange. For this rea-
son, it is often necessary for the proteins to unfold so
that the reactive sulfhydryl groups are exposed to the
aqueous phase before any reactions can occur [29].
Con®gurational entropy
The major force opposing the folding of globular
proteins is the entropy associated with the greater num-
ber of con®gurational states that an unfolded protein
can take up relative to the folded protein [13, 31]. Pro-
teins have both local and non-local contributions to
their overall entropy. The local entropy depends on the
146 C.M. Bryant and D.J. McClements/Trends in Food Science & Technology 9 (1998) 143±151

number of conformations that can be assumed by which explains why slight changes in environmental
amino acids connected to one another along the poly- conditions can cause a protein to undergo a conforma-
peptide. The formation of secondary structure, such as tional change [5, 8].
-helix and -sheet, is an example of a decrease in local
entropy. The non-local entropy depends on the possible
con®gurations the whole polypeptide chain can assume.
Excluded volume e€ects play an important role in
determining the non-local entropy due to the fact that
two segments in a chain cannot occupy the same space
simultaneously [13]. A chain that occupies a large
volume in space can take up a large number of possible
conformations, but if the volume is reduced the number
of possible conformations decreases, largely because of
steric overlap. The folding of a protein is therefore
unfavorable because of the loss of con®gurational free-
dom. The entropy gained by unfolding increases with
temperature, which explains why proteins unfold as
their temperature is increased, even though the hydro-
phobic e€ect strongly favors the folded state [31].
An important consequence of the non-local entropy
contribution to protein stability is the e€ect of cova-
lently cross-linking protein chains. Introducing a cova-
The major contribution to the forces responsible for
maintaining the folded state of whey proteins in solu-
tion is the hydrophobic e€ect, whilst the major force
favoring the unfolded state is con®gurational entropy.
At relatively low temperatures (<65 C), the hydro-
phobic e€ect dominates and so the globular state is
favored, whereas at higher temperatures the con®gura-
tional entropy term dominates and so the unfolded state
is favored [13, 31, 32]. At the temperatures normally
used for the preparation of whey protein ingredients
(<100 C), the globular proteins only partially unfold
into a `molten globule' state rather than undergoing
complete unfolding into a random-coil con®guration
[21, 33, 34]. The molten-globule state of the whey pro-
tein molecule retains much of the native-like secondary
structural architecture of the native molecule, although
unfolding leads to the formation of hydrophobic patches
on the surface of the molecule [33, 34]. A knowledge of
the molecular characteristics of native and denatured
lent cross-link (e.g., a disul®de bond) severely restricts
the number of conformations that the unfolded protein
can take up and therefore reduces the entropic driving
force, which favors protein unfolding. The stability of
proteins can, therefore, be modi®ed by introducing or
removing disul®de bonds [1].
Protein unfolding
The thickening of solutions or formation of gels
depends on the ability of whey proteins to form aggre-
gates. In their native state, the attractive forces (mainly
van der Waals and hydrophobic) between the protein
molecules are not suciently strong to overcome the
repulsive forces (mainly electrostatic, hydration and
entropic) and, therefore, the molecules tend to exist
either as individual entities or as small aggregates [2].
Many of the reactive amino-acids (non-polar and
cysteine residues) are located in the interior of the glob-
ular proteins and so it is usually necessary to promote
some degree of protein unfolding before aggregation
will occur [4].
In aqueous solution, whey proteins exist in equili-
brium between two states: native and denatured [8]. The
former being the compact, organized globular con-
formation and the latter being a more randomly orien-
ted, disordered state. The conformation that a protein
adopts under a particular set of environmental condi-
tions (pH, ionic strength, temperature, solvent compo-
sition) depends on a delicate balance between forces
that favor the folded state and forces that favor the
unfolded state. These two sets of forces are large and
approximately equal in magnitude, but opposite in sign.
As a consequence, the di€erence in the free energy
between the folded and unfolded states is quite small,
-lactoglobulin is essential for understanding the nature
of the aggregates it forms in solution, as it is this com-
ponent which dominates whey protein behavior [2].
Aggregation under heat-setting conditions
Once the whey proteins have been heated to a tem-
perature where they unfold, they may either aggregate
or remain as individual molecules depending on the
balance of attractive and repulsive interactions between
them. At pH values signi®cantly above or below the
isoelectric point, the native whey proteins exist as elec-
trically charged globular molecules with their hydro-
phobic groups predominantly located in the interior.
These molecules do not undergo extensive aggregation
because of the relatively strong electrostatic repulsion
between them [10]. When the proteins are heated above
their denaturation temperature, they partially unfold
which leads to the exposure of non-polar amino-acids,
thereby increasing the hydrophobic attraction between
them (Table 2). In the absence of electrolyte, the unfol-
ded proteins may remain separate due to the strong
electrostatic repulsion, however, zero salt conditions are
unlikely to occur outside the laboratory due to the
inherent mineral content of whey proteins. As the salt
concentration is increased, the electrostatic repulsion
between the charged molecules is progressively screened.
At relatively low salt levels, most of the surface of the
protein molecules is incapable of forming bonds with
other proteins because of the residual electrostatic
repulsion between them. Nevertheless, bonds may be
formed between a non-polar patch on one protein and a
non-polar patch on another due to the attractive
hydrophobic interaction. The molecules are thought to
aggregate in an ordered `string-of-beads' or ®lament
 C.M. Bryant and D.J. McClements/Trends in Food Science & Technology 9 (1998) 143±151 147

structure (Fig. 1), suggesting that they come together at tured monomer to produce a dimer. This results in pro-
®xed sites on opposing ends of the molecule. However, duction of an intermolecular disul®de bond, and a new
there is no concrete evidence that such sites exist in reactive free sulfhydryl is made available to further
heat-denatured whey protein molecules [21]. At rela- propagate the polymer chain. It has also been suggested
tively high salt concentrations, the electrostatic repul-
sion between the protein molecules is completely
screened and, consequently, they can form bonds at any
point on their surface. This leads to the formation of
fairly large spherical aggregates. At intermediate salt
concentrations a mixture of these two types of structure
is formed [21, 35].
Following initiation of aggregation by hydrophobic
forces, disul®de bonding is known to play an important
role in the strengthening of the aggregates. This is
accomplished through inter- and intramolecular dis-
ul®de bonding via sulfhydryl±disul®de interchange or
that the conformation of -lactoglobulin lends itself to
the formation of linear aggregates because it appears
that only one of the two intramolecular disul®de bonds
and only one sulfhydryl group per monomer is reactive
[36].
At low protein concentrations, the aggregation of
protein molecules leads to an increase in the solution
viscosity because the aggregates have an e€ective
volume fraction that is larger than the actual volume
fraction of the individual molecules. This is due to the
increased axial ratio of the linear aggregates upon for-
mation of the `string-of-beads' network allowing them
sulfhydryl oxidation reactions [33]. Studies of -lacto-
globulin polymerization suggest that heating causes a
free sulfhydryl group to become reactive resulting in a
sulfhydryl/disul®de bond exchange reaction with one of
the two intramolecular disul®de bonds of an undena-
to trap water within their structure [21, 22]. A gel is
formed when the protein concentration exceeds some
critical level, because the protein forms a three-dimen-
sional network of aggregated or entangled molecules
that ®lls the entire volume of the container [21]. The

Fig. 1. Development of particulate and ®lamentous gel structures.

148 C.M. Bryant and D.J. McClements/Trends in Food Science & Technology 9 (1998) 143±151
appearance and properties of the solution or gel formed
depends on the nature of the protein aggregates. A
solution of ®lamentous aggregates appears transparent
because the width of the ®laments is so small that they
do not scatter light strongly. On the other hand, a solu-
tion of particulate aggregates appears optically opaque
because the particles are of a size that strongly scatters
light. Gels with ®lamentous type structures tend to have
better water-holding capacity than those containing
particulate type structures because they contain smaller
and more homogenous pore sizes and, therefore, hold
the water more strongly due to capillary forces [37±39].
The bulk physicochemical properties of foods contain-
ing whey proteins can be controlled somewhat by alter-
ing either the preparation conditions (temperatures,
heating or cooling rate, holding time) and/or the solu-
tion conditions (pH, ionic strength, and protein con-
centration). However, the tendency for whey proteins to
form opaque gels with low water-holding capacity at
high salt concentrations limits their application in cer-
tain foods. In addition, it would be useful in many foods
to have a whey protein ingredient that thickened or
gelled in a refrigerator or at ambient temperatures,
rather than having to be heated. The functional prop-
erties of whey proteins can be improved by modi®cation
of their molecular structure, for example, genetically,
chemically, enzymatically or physically [2, 7, 38, 40, 41±
43]. Many of these techniques have proved useful for
studying fundamental relationships between the mole-
cular properties of proteins and their functional prop-
erties. Nevertheless, they are often too expensive to be
adapted for large-scale production of food ingredients
or are not GRAS. For this reason there has been con-
siderable interest in modifying the functional properties
of whey proteins by using simple physical techniques,
such as heat-denaturation. It should be noted that whey
protein gels can be formed at low temperature using
high pressure techniques [41], although this is not the
focus of the current paper.
Aggregation under cold-setting conditions
The production and application of cold-setting whey
protein ingredients involves two stages: (i) the prepara-
tion of a heat-denatured protein solution; and (ii) the
induction of gelation at low temperatures.
Preparation of heat-denatured protein solution
The objective is to produce a solution that contains
proteins that are aggregated into ®lamentous type
structures, but that does not gel. This requires careful
control of the initial solution conditions (pH, mineral
content and protein concentration) and heating condi-
tions (temperature and holding time).
A solution of native whey protein must be heated
above a temperature of at least 70 C before the mole-
cules unfold and aggregate into the required structure
[7]. Typically, solutions are held for between 5 and
60 min at temperatures between 70 and 90 C, to ensure
the correct degree of protein unfolding and aggregation
[7, 43]. The pH of the initial solution should be su-
ciently di€erent from the isoelectric point of the proteins
to ensure that they do not immediately aggregate upon
heating. Most experiments have been carried out at pH
7 (2 pH units above the isoelectric point of the pro-
teins) [7, 21, 36±39, 43]. It may also be possible to create
heat-denatured protein solutions with di€erent char-
acteristics at other pH values, but more research is still
required in this area. The salt concentration of the
initial solution must be low enough to prevent excessive
aggregation of the protein molecules. At pH 7, this
typically means NaCl concentrations of less than
50 mM [45] or CaCl2 concentrations of less than
10 mM [7]. In addition, the protein concentration
must be low enough to prevent the molecules from
forming a three-dimensional network that extends
through out the volume of the container, i.e., gel [7].
Induction of cold-setting
After a solution of heat denatured whey proteins has
been prepared it can be cooled and made to thicken or
gel by adding salt [7, 21, 36±39, 43]. The salt shields the
electrostatic repulsion between the ®lamentous-type
protein aggregates, which causes them to come together
and form strands (Fig. 1). The turbidity of the system
increases because the aggregated ®laments in the strands
scatter light more e€ectively than the individual ®la-
ments [43]. The solution becomes more viscous and may
eventually gel due to an increase in the number and
thickness of contact points between the ®laments. The
result is a stronger, more transparent gel than that pre-
pared by the heat-set method using a protein solution
with the same composition [43]. The properties of the
solution or gel formed depend on its environmental
conditions:
Salt type and concentration. Salts alter the interactions
between protein ®laments in a number of di€erent ways.
Both monovalent and divalent salts act as counter-ions,
which shield the electrostatic repulsion between the
electrically charged ®laments and cause them to aggre-
gate. Divalent cations, such as Ca2+ may also induce
aggregation because of their ability to act as bridges
between the negatively charged carboxylic groups on
neighboring whey protein molecules [23, 38, 39]. Much
lower concentrations of divalent ions are needed to
cause aggregation than monovalent ions because they
are much more e€ective at screening electrostatic inter-
actions and because of their ability to form salt bridges.
Increasing the concentration of salt used to promote
gelation of the protein ®laments has a number of e€ects
on the properties of the gels formed (Table 3). Firstly, it
causes an increase in the rate at which gelation occurs
[45, 46]. Secondly, it causes an increase in the turbidity
 C.M. Bryant and D.J. McClements/Trends in Food Science & Technology 9 (1998) 143±151 149

Table 3. Changes in whey protein gel texture with increasing CaCl2 concentrationa
CaCl2 conc. Strand WHCc Shear stressd Shear straind Fracture forcee G0 f
(mM) thicknessb (nm) (%) at fracture (kPa) at fracture (m/m) (N) (kPa)
10 25±75 50 30 1.8 55 9.6
30 increased 52 44 1.6 69 16
50 18
90 120±160 51 46 1.5 83
100 17
150 95
180 300 47 49 1.4
a
Denatured protein suspensions were prepared by heating at 80 C for 30 min. After references, [7] [35] [36].
b
Strand thickness=protein stand diameter as measured from TEM.
c
WHC=water holding capacity: determined by compression of gel between parallel plates at 200 psi for 30 s. % WHC calculated from
the loss of water into ®lter paper.
d
Shear stress and strain at fracture: cylindrical gel samples were ground to a minimum diameter of 10 mm and then twisted to fracture at
2.5 rpm with a Torsion Gelometer. Stress and strain were calculated from the torque and angular displacement.
e
Fracture force: cylindrical gel samples compressed between plates to fracture, the ®rst peak indicating force to fracture.
f 0
G : storage modulus=ratio of in-phase stress to strain.

of the gels because of a thickening of the strands of
aggregated ®laments [7, 37, 47]. Thirdly, above a certain
salt concentration it causes a decrease in the water-
holding capacity of the gels because the size of the pores
between the protein ®laments increases [37, 47].
Fourthly, the rheological characteristics of the gels are
altered. As the salt concentration increases, the shear
stress at fracture increases, whilst the shear strain at
fracture decreases, which means a greater force is
required to rupture the gels but they are more brittle
[37±39]. Experimental studies have indicated that there
is a relationship between the structural properties of the
gels and their rheology. Gels that fracture at low strain
values have networks composed of relatively thin
strands and small, homogenous pores, whereas gels
which fracture at high strain values have thicker strands
and relatively large, inhomogeneous pores [47]. The
dependence of the strength and brittleness of cold-set
gels on salt concentration allows food manufacturers to
create products with a variety of di€erent textural
characteristics by carefully controlling the mineral con-
tent.
pH. The fact that electrostatic interactions play a
dominant role in determining the aggregation of the
®laments in heat-denatured protein solutions suggests
that pH should have a large in¯uence. The authors
could ®nd no evidence of previous work carried out in
this area. Nevertheless, we would expect that cold-set-
ting could be induced in a heat-denatured protein solu-
tion by altering the pH towards the isoelectric point of
the proteins, because this would reduce the electrostatic
repulsion between the ®laments and, therefore, promote
aggregation.
Protein concentration. The concentration of protein in
the heat-denatured protein solution has a major in¯u-
ence on the properties of the viscous solutions or gels
formed by adding salt. At relatively low protein con-
centrations, the heat-denatured protein will tend to
form a viscous solution rather than a gel. As the protein
concentration is increased, the viscosity of this solution
increases. Above a critical protein concentration, the
heat-denatured protein solution forms a gel rather than
a viscous solution. A further increase in the protein
concentration then leads to an increase in the Young's
modulus and water-holding capacity of the gel [38].
Temperature. The temperature at which the gelation
process is carried out also has an important in¯uence on
the rate of gelation and the structure of the gels formed.
The gelation rate of cold-set gels formed by adding
either NaCl or CaCl2 to heat-denatured WPI solutions
has been found to increase with increasing temperature
[38, 44, 45]. It has been suggested that this is due to the
importance of hydrophobic interactions in determining
the initial aggregation of the protein ®laments [43]. The
hydrophobic attraction is relatively strong and long
range, and its magnitude increases as the temperature is
raised [9, 16]. Consequently, it is believed to play an
important role in determining the aggregation rate of
the protein ®laments. Regardless, once the gels have
formed their strength tends to increase as the tempera-
ture is decreased [44]. This suggests that hydrophobic
interactions play a dominant role in the initial stages of
aggregation, but that non-hydrophobic interactions
play a more important role in determining the ®nal gel
strength.
Conclusions
The availability of a wide variety of whey protein
ingredients, each tailored for a speci®c application, has
arisen because of the systematic application of knowl-
edge of the relationship between the molecular and
functional properties of proteins. Scientists from a vari-
ety of di€erent disciplines have contributed to our
understanding of this area, including physicists, chemists,
150 C.M. Bryant and D.J. McClements/Trends in Food Science & Technology 9 (1998) 143±151
biologists and engineers. Information about the properties
of proteins has come from advances in computer simu-
lations, theoretical developments and experimental
techniques. The development of whey protein ingredients
clearly highlights the need to use a fundamental and
multidisciplinary approach in order to make advances
in the food industry.
Cold-setting ingredients may be particularly useful
for replacing biopolymers that are currently used as
thickening or gelling agents in foods; for example, gela-
tin in comminuted meat and ®sh products, or poly-
saccharides in deserts, sauces and dips. Fundamental
research is still needed to clarify the factors that in¯u-
ence the properties of cold-set gels and their incorpora-
tion into such systems. Work is presently underway in
our laboratory to improve the mode of gelation and to
develop a cold-set powder, further enhancing the appli-
cation of this ingredient.
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