Food Hydrocolloids 19 (2005) 269–278

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Cold gelation of b-lactoglobulin oil-in-water emulsions

Valérie Leung Sok Line, Gabriel E. Remondetto, Muriel Subirade*
Chaire de recherche du Canada sur les protéines, les bio-systèmes et les aliments fonctionnels, Centre de recherche en sciences et technologie du lait (STELA),
Institut des nutraceutiques et des aliments fonctionnels (INAF), Faculté des sciences de l’agriculture et de l’alimentation, Université Laval,
Pavillon Paul-Comtois, QC, Canada G1K 7P4
Received 20 February 2004; revised 7 June 2004; accepted 7 June 2004

Abstract
The cold gelation of b-lactoglobulin emulsion gels was investigated in this work. The effects of oil and calcium concentrations on the
rheological properties and structure of emulsion gels were studied using dynamic small strain rheometry and electron microscopy techniques.
Results show that the storage modulus (G 0 ) is only affected by the oil content, the effect of which is greater than that of calcium. Emulsion
gels with high G 0 and good water-holding capacity (WAC) were obtained by raising the oil concentration. In contrast, an increase in salt
concentration reduced the WHC and changed the structure of the emulsion gel from fine-stranded to random aggregates. Oil and calcium
regulated the process of gel network formation and modulated the functional properties of cold-set emulsion gels.
q 2004 Elsevier Ltd. All rights reserved.

Keywords: b-Lactoglobulin; Emulsion gel; Cold gelation; Rheology; Microstructure

1. Introduction by different rheological and microstructural properties, may

Whey proteins (WP) are widely used as food ingredients,
because of their high nutritional value and their remarkable
functional properties (de Wit, 1989; Morr & Ha, 1993).
Among these is their gel-forming ability, which is
particularly of great importance to the food industry.
Gelation of b-lactoglobulin (b-lg), the main WP, is
traditionally achieved through heat treatment. When con-
centrated b-lg solutions are heated above 65 8C, the globular
protein molecules partially unfold, expose their hydro-
phobic and sulfhydryl groups, and aggregate to create a
three-dimensional network that entraps water by capillary
forces (Twomey, Keogh, Mehra, & O’Kennedy, 1997).
In salt-free solutions, two types of heat-set b-lg gels (i.e.,
‘fine-stranded’ and ‘particulate’), which are characterized
Abbreviations: ANOVA, analysis of variance; b-lg, b-lactoglobulin; d,
phase angle; G 0 , storage modulus; G 00 , loss modulus; pI, isoelectric point;
SEM, scanning electron microscopy; TEM, transmission electron
microscopy; WHC, water-holding capacity; WP, whey proteins; WPI,
whey protein isolates.
* Corresponding author. Tel.: C1-418-656-2131x4278; fax: C1-418-
656-3353.
E-mail address: muriel.subirade@aln.ulaval.ca (M. Subirade).
0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2004.06.004
be produced depending on pH conditions (Stading &
Hermansson, 1990, 1991). Opaque particulate gels com-
posed of random aggregates occur at pH values near the
isoelectric point of b-lg (pI) (pH 4–6), whereas transparent
fine-stranded gels are obtained by shifting the pH far from
the pI. Both types of gels have been recently characterized
and differentiated at a molecular level (Lefèvre & Subirade,
2000).
WP, especially b-lg, also show good emulsifying
properties. Because of their flexibility and amphiphilic
nature, these proteins rapidly adsorb to the emulsion
interface where they self-aggregate and form continuous
and homogeneous membranes around the oil droplets
through intermolecular b-sheets interactions (Lefèvre &
Subirade, 2003). By coating the oil droplets with charged
layers, the protein films provide an electrostatic barrier
against flocculation and coalescence (Phillips, Whitehead,
& Kinsella, 1994). Oil-in-water emulsions stabilized by
whey protein isolates (WPI) or by b-lg can be converted into
heat-set gels. Moreover, over the past few years, there has
been a growing interest in these composite systems,
because of their practical application in food formulations
(Dickinson & Chen, 1999). In fact, several workers have
270 V.L. Sok Line et al. / Food Hydrocolloids 19 (2005) 269–278
investigated the microstructure and rheological properties of
such emulsion gels (Chen & Dickinson, 1998; Dickinson,
Hong, & Yamamoto, 1996; Jost, Baechler, & Masson, 1986;
Jost, Dannenberg, & Rosset, 1989; McClements, Monahan,
& Kinsella, 1993). They found that droplets covered by WP
become an integral part of the network and increase the gel
elastic modulus. This behavior is consistent with the
mechanical reinforcement of the gel matrix by strongly
interacting filler particles (Van Vliet, 1988). Other studies
have also revealed that the viscoelasticity of these materials
is mainly controlled by the type of interactions between the
gel matrix and the filler particles, although oil volume
fraction and average droplet size also play a role
in determining the texture of emulsion gels (Chen &
Dickinson, 1999a; Chen, Dickinson, Langton, &
Hermansson, 2000; Dickinson & Chen, 1999). The use of
WP emulsion gels extends the possibilities for creating
foods with new and improved organoleptic properties.
However, the thermal treatment needed to produce these
emulsion gels limits their application in formulations
containing heat-sensitive ingredients. An alternate gelation
method, involving low temperatures (i.e., i cold gelation),
could be judiciously exploited to overcome this limitation.
According to Barbut and Foegeding (1993), WPI or b-lg
gel production at 25 8C is achieved through two consecutive
steps: the preparation of a heat-denatured protein suspen-
sion and the subsequent formation of a network through the
addition of Ca2C to the chilled solution. The preheating
step, during which no gelation occurs, is essential as it
causes the protein molecules to open their structure and
interact with each other (Barbut & Foegeding, 1993;
Hongsprabhas & Barbut, 1997b). Typically, a low ionic
strength solution of native WP is kept at a pH far from the
isoelectric point, usually at pH 7, and held for 5–60 min
between 70 and 90 8C. Protein concentration is also
maintained below that which is required to create a network
(Bryant & McClements, 1998). After the preheating
treatment, the protein molecules form aggregates without
gelation. These aggregates constitute the structural units
responsible for the three-dimensional network of cold-set
gels (Remondetto & Subirade, 2003). After cooling, salt is
added to screen the repulsive forces between the aggregated
protein molecules, which can then form a gel. Besides
charge dispersion, a divalent salt, such as CaCl2, induces
cross-linking of proteins and thus promotes gelation at much
lower concentrations than a monovalent salt (Bryant &
McClements, 2000; Hongsprabhas & Barbut, 1997a).
The amount of salt used to form a cold-set gel is likely to
be the major determinant of the structure and spatial
organization of protein aggregates (Hongsprabhas, Barbut,
& Marangoni, 1999). Many studies confirm that the network
formation processes are governed by salt concentration
resulting in different gelation mechanisms and in various
structure types (Hongsprabhas & Barbut, 1997b, 1998;
Hongsprabhas et al., 1999; Remondetto, Paquin, &
Subirade, 2002; Remondetto & Subirade, 2003), which
determine the water-holding capacity (WHC), permeability,
texture, and appearance of the gel (Bryant & McClements,
2000).
As for all WP gels, modulation of microstructure and
functional properties of WP emulsion gels is possible by
cold gelation. Nevertheless, to our knowledge, no exhaus-
tive reports on the cold-induced gelation of WP emulsion
gels are available, despite the fact that WP beads have been
successfully produced using an emulsification/cold-gelation
process in a previous study (Beaulieu, Savoie, Paquin, &
Subirade, 2002). Thus, our objective was to develop cold-set
b-lg emulsion gels and investigate the effects of oil and
Ca2C concentrations on their rheological and structural
properties.
2. Material and methods
2.1. Materials
b-Lactoglobulin (b-lg) was obtained from Davisco
International, Inc. (Le Sueur, MN). It contained 98.2%
protein determined by semi-micro Kjeldahl method
(AOAC, 1984) using N-factor 6.38. Sunflower oil was
purchased from a local retailer and calcium chloride (CaCl2)
was provided by Fisher Scientific (Fair Lawn, NJ).
2.2. Preparation of cold-set emulsion gels
Protein suspensions (9.5 wt% protein) were prepared by
dispersing the powdered b-lg in deionized water and stirring
for 1 h to ensure complete dissolution. After having adjusted
the pH at 7 with 1 N HCl or NaOH, the suspensions
were degassed for 30 min, heated at 85 8C during 45 min
in a water bath, and cooled to room temperature (w23 8C)
for 2 h.
Oil-in-water coarse emulsions (6.65 wt% protein) were
produced by mixing appropriate amounts of protein solution
and sunflower oil with a high speed blender operating at
20,000 rpm for 2 min (Ultra-Turrax, Janke and Kunkel,
IKA-Labortechnik, Staufen, Germany). The size of emul-
sion droplets was then reduced further using a high-pressure
homogenizer (Emulsiflex-C5, Avestin Inc., Ottawa,
Canada). A two-stage homogenization was performed: the
first pass was carried out at 100 MPa and the second at
10 MPa. Emulsions were then degassed for 30 min to
remove all the air bubbles entrapped during homogenization
as their presence can lead to irreproducibility in subsequent
rheological measurements (Dickinson & Hong, 1995).
To induce cold gelation, emulsions were mixed with
CaCl2 by vortexing at 23 8C. The levels of oil and CaCl2 to
be added were determined using a central rotatable
composite experimental design as shown in Table 1. The
final protein concentration in all formulations was adjusted
to 6.5 wt%.
 V.L. Sok Line et al. / Food Hydrocolloids 19 (2005) 269–278 271

Table 1 2.5. Rheology of cold-set emulsion gels

Experimental range and levels of the independent variables established
according to the Box–Wilson central composite design for cold gelation of
b-lactoglobulin emulsionsa
Small deformation viscoelasticity of emulsion gels was
investigated using dynamic oscillatory measurements per-
Treatment Variables formed by a controlled-stress rheometer equipped with
Oil concentration (%) Calcium concentration (mM) 40 mm diameter parallel plates (SR-5000, Rheometric
Coded X1 Uncoded Coded X2 Uncoded Scientific, Piscataway, NJ). The phase angle d and the
[Oil] [Ca2C] storage and loss moduli, G 0 and G 00 , were monitored as a
1 1 28 1 60 function of time in the linear viscoelastic region at 23 8C and
2 1.414 31.1 0 40 at a frequency of 1 Hz. All measurements were conducted for
3 0 20.5 K1.414 11.7 15 h and a solvent trap was used to prevent sample drying.
4 K1.414 9.9 0 40
5 0 20.5 0 40
6 0 20.5 0 40 2.6. Scanning and transmission electron microscopy
7 0 20.5 0 40 (SEM and TEM) of cold-set emulsion gels
8 1 28 K1 20
9 0 20.5 0 40 The microstructure of cold-set emulsion gels was
10 K1 13 K1 20
11 K1 13 1 60
analyzed by transmission electron microscopy (TEM) and
12 0 20.5 1.414 68.3 scanning electron microscopy (SEM). Samples (1!1!
13 0 20.5 0 40 2 mm) were fixed in 2.5% glutaraldehyde C1.5%
a paraformaldehyde in 0.1 M cacodylate buffer for 24 h,
The experimental runs were performed in random order.
rinsed and post-fixed in 1% OsO4 for 90 min. For a better
2.3. Particle size analysis penetration of the fixative solution in the floating gel
samples, vacuum was applied for 1 h during the fixation
The droplet size distribution of freshly made emulsions step. Dehydration was accomplished in a graded series of
was determined by photon correlation spectroscopy ethanol. For TEM, the samples were immersed in propylene
(Nicomp Submicron Particle Sizer System, Model C370, oxide, propylene oxide/Epon solution (1:1), and finally pure
Pacific Scientific, Manlo Park, CA) with the method of Epon. After infiltration overnight at room temperature, they
Robin and Paquin (1991). Emulsion samples were dispersed were embedded in Epon, with polymerization at 60 8C,
in a dissociating buffer composed of urea (8 M), EDTA thinly sectioned, stained with uranyl and lead acetate, and
(50 mM), and b-mercaptoethanol (10 mM). Dilute emul- viewed at 80 kV (JEOL Model 1200EX, JEOL Ltd.
sions, placed into the measurement cell of the instrument, Akishima, Japan). For SEM, the samples were critical
were illuminated by a focused laser beam and fluctuations in point dried, sputter coated with 30 nm of gold/palladium,
the intensity of the scattered light were measured by a and examined at 15 kV using a JEOL JSM-6360 LV
photomultiplier detector at an angle of 908. Particle instrument.
diffusivity was computed from the autocorrelation function
of the scattered light by the method of cumulants and 2.7. Experimental design
translated into a particle diameter value using the Stokes–
Einstein relation for spheres (Nicoli, McKenzie, & Wu, A response surface methodology study was conducted to
1991). Each determination was performed in triplicate. determine the simultaneous effects of oil and Ca2C
concentrations on the rheological and water-holding proper-
ties of cold-set emulsion gels. A 22 factorial Box-Wilson
2.4. Water-holding capacity (WHC) central composite design with four-star points and five
replicates at the center point, leading to a set of 13
The WHC was evaluated as described by Remondetto experiments performed in random order was used. Five
et al. (2002). Emulsion gels were formed in an ultrafiltration levels of each factor (oil and Ca2C concentrations) were
unit (Ultrafree-CL Filters, Millipore Co., Billerica, MA.) incorporated into the design. Maximum and minimum ranges
consisting of a collection tube (5 ml) and a filter cup (2 ml) of independent variables investigated were chosen by
with an ultrafiltration membrane of 5000 NMWL. After carrying out preliminary tests. The full experimental plan,
24 h, the unit was centrifuged at 4000 g for 20 min. WHC with respect to the variable values in their actual and coded
was calculated as: forms, is listed in Table 1. Assessment of error was derived
WT K WF from replication of the central point treatment conditions.
WHC Z !100%
WT
2.8. Statistical analysis
where WT is the total amount (g) of water in the sample and
WF is the quantity of water (g) released. Each determination Regression analysis was performed on the obtained data.
was carried out in five replicates. The following second-order equation was fitted for each
272 V.L. Sok Line et al. / Food Hydrocolloids 19 (2005) 269–278

factor assessed: Table 2

Results of storage modulus (G 0 ) and WHC for each condition of
Y Z b0 C b1 X1 C b2 X2 C b11 X12 C b22 X22 C b12 X1 X2 experimental design

where Y is the estimated response, b0, b1, b2, b11, b22, b12 Treatment G 0 (Pa) WHC (%)
are constant and regression coefficients of the model, and 1 28,870 90
X1, X2 are the independent variables. Analysis of variance 2 29,680 92
3 10,048 94
(ANOVA) with Fisher’s test for significance was carried out
4 3693 78
to evaluate linear, quadratic, and interactive effects of oil 5 13,884 87
and Ca2C concentrations, and to determine the adequacy of 6 9797 86
the experimental model. P%0.05 was considered to be 7 11,044 85
significant. Proportion of variance explained by the model 8 31,184 94
9 14,839 87
was given by the multiple coefficient of regression R2.
10 8543 85
Statgraphics Plus Professional version 4.1 (Manugistics 11 5096 79
Inc., Rockville, MD) was used to generate and analyze the 12 11,850 87
experimental design. 13 10,972 87

3. Results
3.1. Emulsion properties
The particle size distribution of freshly prepared emul-
sions containing various concentrations of oil was
measured. The volume frequency of oil droplets as a
function of size is presented in Fig. 1. All emulsions had a
mean particle diameter ranging from 0.68 to 0.80 mm and
a monomodal particle size distribution with an almost
symmetrical peak. Fig. 1 reveals that the droplet size
distribution was not significantly different between the
emulsions, suggesting here a negligible effect of the amount
of oil added. These preliminary results ensured that
subsequent changes observed in emulsion gel oil droplet
size were attributed only to the cold gelation process.
that the significant terms are both the linear and quadratic
components of oil and Ca2C concentrations. Fig. 2 displays
the response surface plot. It can be seen that oil and Ca2C
had antagonistic effects on the ability of emulsion gels to
physically hold water. Indeed, salt addition decreased the
WHC values by promoting the expulsion of water, while the
presence of oil improved the water retention of the emulsion
gels. In this case, the regression model correctly described
the collected data (lack of fit, pO0.05). Its coefficients are
listed in Table 4. According to the R2 value, 98% of the
variability in the response can be attributed to the model. It
is then possible to use this model to estimate the cold-set
b-lg emulsion gel WHC values when experimenting with
different oil and calcium concentrations.
3.3. Rheological measurements of emulsion gels

3.2. Water-holding capacity The small deformation rheological properties of emul-

Results of WHC for all the experimental conditions are
summarized in Table 2. Variance analysis (Table 3) shows
sion gels can be characterized by the storage modulus (G 0 ),
the loss modulus (G 00 ), and the phase angle (d). G 0 is a
measure of the energy stored per cycle of deformation and
its value reflects the solid-like or elastic behavior of the
tested material, while G 00 refers to the energy dissipated by
the sample and is associated with the fluid-like or viscous

Table 3
Analysis of variance of storage modulus (G 0 ) and WHC

Source of Degrees of Sum of squares

variation freedom G0 WHC
8*
X1:[Oil] 1 8.65!10 1.98!102*
X2:[Ca2C] 1 1.29!106 4.95!101*
X1$X1 1 8.24!107** 5.48**
X1$X2 1 3.21!105 1.00
X2$X2 1 2.28!106 2.41!101*
Lack of fit 3 6.27!107 1.13
Error 4 1.84!107 3.20
Total (corr.) 12 1.03!109 2.86!102
R2 92.12 98.49
Fig. 1. Effect of oil on particle size distribution of 6.65 wt% b-lactoglobulin
oil-in-water emulsions (pHZ7). *p!0.05; **p!0.01.
 V.L. Sok Line et al. / Food Hydrocolloids 19 (2005) 269–278 273

Fig. 2. Response surface graph of the effect of oil and Ca2C concentrations
on the water-holding capacity of cold-set b-lg emulsion gels containing
6.5 wt% protein (pHZ7).

characteristics of the material (Bell, 1989; Tadros, 1996).

The phase angle, which is the arc tangent of the ratio G 00 /G,
corresponds to the response delay of strain to the applied
stress and indicates the relative importance of viscous and
elastic elements in the emulsion gel. For example, d values
of 0 and 908 are typical for perfect elastic solids and viscous
liquids, respectively. When the value of G 0 is far larger than
the value of G 00 , d is very small and the material is said to be
predominantly elastic (Chen & Dickinson, 1999a).
These three parameters were recorded during the
emulsion gel formation. All samples showed the same
type of gelation curves: both G 0 and G 00 increased similarly
to reach a maximum value, while d decreased to a plateau
(Fig. 3). Because G 0 values were considerably greater in
magnitude than G 00 values and because they suggested the
formation of elastic emulsion gels, only the results of G 0 at
the end of the gelation process are presented and discussed
hereafter (Table 2).
The analysis of variance, presented in Table 3, indicates
that, among the variables studied, only the oil concen-
tration significantly affected the rheological properties of
the cold-set emulsion gels. Varying oil levels had a strong
positive effect on the storage modulus, the linear and
quadratic components both being significant. As shown in
Fig. 4, the greater was the oil content, the more important
was the value of G 0 . Such increase in the modulus of the
emulsion gels suggests that the dispersed oil droplets act as
active fillers that interact with the gel matrix. This is
confirmed by Fig. 5 where the storage modulus of cold-set
b-lg emulsions gels is compared with that of an equivalent
gel without oil droplets. As it can be observed,
Fig. 3. Evolution of G 0 , G 00 , and tan d during cold-set b-lg emulsion gel
formation (6.5 wt% protein, 28 wt% oil, and 20 mM Ca2C) at 23 8C
and 1 Hz.
the incorporation of oil droplets significantly improved
the elasticity of cold-set b-lg gels at any given Ca2C
concentration. For example, adding up to 30 wt% oil in the
protein network enhanced about 10-fold the gel storage
modulus. Although Ca2C was necessary to build up the
three-dimensional network, it had a negligible effect on the
elasticity of the emulsion gels. The regression coefficients
of the model used to describe the evolution of the storage
modulus as a function of oil and Ca2C concentrations are
displayed in Table 4. Since the lack of fit test was not
significant (pO0.05), the model appears to be adequate for
the observed data at the 95% confidence level and can
explain 92% of the variability in the response, as indicated
by the R2 value. Thus, this model can be used to predict the
rheological behavior of cold-set b-lg emulsion gels at
various oil concentrations.
3.4. Microstructure analysis
Electron micrographs were taken using TEM and SEM in
order to get insight into the structure of cold-set emulsion
gels obtained with different levels of added oil and Ca2C.

Table 4
Coefficients of the regression models for storage modulus (G 0 ) and WHC

Treatment G0 WHC
4
b0 1.40!10 8.13!101
b1 –1.20!103* 1.18*
b2 K1.73!102 K5.65!10K1*
b1$b1 6.12!101** K1.58!10K2**
b1$b2 1.89 3.33!10K3
b2$b2 1.43 4.66!10K3* Fig. 4. Response surface graph of the effect of oil and Ca2C concentrations
on the storage modulus of cold-set b-lg emulsion gels containing 6.5 wt%
*
p!0.05; **p!0.01. protein (pHZ7).
274 V.L. Sok Line et al. / Food Hydrocolloids 19 (2005) 269–278

that increasing the oil volume fraction, while keeping Ca2C

Fig. 5. Storage modulus of cold-set b-lg gels containing 6.5 wt% protein,
with or without oil, and at different calcium concentrations after 15 h.
A selection of conditions was tested rather than the full
experimental design. Four samples were then produced by
combining the central point of a given factor with the
extreme levels of the other factor. The microstructure of the
analyzed samples is presented in Figs. 6 and 7. TEM shows
concentration constant, led to emulsion gels with higher
density and smaller pores (Fig. 6a and b), mainly due to
space filling by the oil droplets and their interaction with the
protein matrix. At 9.9% oil and 40 mM Ca2C (Fig. 6a),
the gelled emulsion has an inhomogeneous and porous
structure. However, when the oil content was elevated to
31.1%, the matrix, in which the oil droplets were evenly
distributed, was denser (Fig. 6b). Micrographs from TEM
agreed with those from SEM: when the oil concentration
was more important, the network structure became more
compact (Fig. 6c and d). Fig. 6c and d also show that
increasing the oil volume fraction leads to changes in the
structure of emulsion gels containing the same Ca2C
concentration from a particulate to a mixed-type gel,
where both fine-stranded and random aggregates are found.
On the other hand, raising Ca2C concentration and
maintaining the oil volume fraction constant resulted in
larger pores and larger protein aggregates. For example, at
11.7 mM Ca2C, fine protein aggregates were uniformly
dispersed in the matrix and linked together by thin strands
(Fig. 7a). At 68 mM Ca2C, the structural difference is
obvious as the aggregates clumped together to form larger
particles, which were well separated from each other by

Fig. 6. Transmission (TEM) and scanning (SEM) electron micrographs of cold-set b-lg emulsion gels containing 6.5 wt% protein, prepared at pH 7 and
constant Ca2C concentration of 40 mM in the presence of: 9.9 wt% oil, TEM (a); 31.1 wt% oil, TEM (b); 9.9 wt% oil, SEM (c); and 31.1 wt% oil, SEM (d).
 V.L. Sok Line et al. / Food Hydrocolloids 19 (2005) 269–278 275

Fig. 7. Transmission (TEM) and scanning electron micrographs (SEM) of cold-set b-lg emulsion gels containing 6.5 wt% protein, prepared at pH 7 and
constant oil concentration of 20.5 wt% in the presence of: 11.7 mM Ca2C, TEM (a); 68 mM Ca2C, TEM (b); 11.7 mM Ca2C, SEM (c); 68 mM Ca2C, SEM (d).

the aqueous phase (Fig. 7b). As shown by TEM, exclusion
of some oil droplets from the protein matrix and their
tendency to flocculate together were also observed at higher
Ca2C concentration (Fig. 7b). Such a phenomenon is likely
related to the excessive calcium bridging between the
protein molecules and to the subsequent development of
tightly aggregated structures. Varying Ca2C concentrations
had a notable effect on the aspect of the emulsion gels. This
effect can be appreciated in SEM micrographs, where a
filamentous network, with small pore size, is evident at low
calcium concentrations (Fig. 7c) and where the formation of
particulate structure composed of random aggregates is
observed upon further salt addition (Fig. 7d).
The microstructure study indicates that the network
formation processes are governed by Ca2C concentration
and by oil volume fraction during the cold gelation of b-lg
emulsions.
4. Discussion
Composite gels consisting of a protein matrix and a
dispersed lipid phase can be classified as either interactive
or non-interactive systems (Mor, Shoemaker, & Rosenberg,
1999). It has been suggested that, in non-interactive
systems, lipid globules only fill pores in the gel matrix
without interacting with the protein-based network. Such
particles are termed ‘inactive fillers’ and are known to act as
structure breakers because they have little or no affinity for
the gel matrix and cause its disruption and weakening.
Increasing the volume fraction of these inactive fillers
generally results in lower gel strengths. By contrast,
interactive systems are characterized by strong interactions
between the lipid globules and the gel matrix. These
so-called ‘active fillers’ greatly enhance gel strength as they
are fully incorporated as a part of the network. This
reinforcement effect becomes more pronounced with greater
amounts of active fillers (Brownsey, Ellis, Ridout, & Ring,
1987; Jost et al., 1989; McClements et al., 1993; Ring &
Stainsby, 1982).
Cold-set b-lg emulsion gels produced in this work belong
to the category of interactive systems. In fact, the difference
observed between the elasticity of protein-only gel and that
of emulsion gels, and the steep increase in emulsion gel
storage modulus, along with higher oil concentration, mean
that the dispersed oil droplets not only function as space
276 V.L. Sok Line et al. / Food Hydrocolloids 19 (2005) 269–278
fillers, but also help build up the gel structure. This is
consistent with reported effects of WP-covered oil droplets
on the rheological properties of heat-set emulsion gels
(Chen & Dickinson, 1998, 1999b; Dickinson & Chen,
1999). Incorporation of oil globules into the matrix of these
heat-set gels is achieved through interactions between the
protein molecules adsorbed at a droplet surface as well as
those of the gel network during thermal treatment, resulting
from a combination of disulfide, hydrophobic, and hydrogen
bonds (McClements et al., 1993). However, for the
formation of cold gels, it has been shown that physical
interactions play a major role in the first stage of the process
while disulfide bonds are mainly involved in the second
stage of cold-set gel formation either for acid-induced cold
gelation (Alting, Hamer, de Kruif, Paques, & Visschers,
2003, Alting, Hamer, de Kruif, & Visschers, 2003) or for
cation-induced cold gelation (Remondetto & Subirade,
2003). Since the gelation of our emulsions was conducted
at room temperature (23 8C) upon calcium salt addition,
inclusion of dispersed oil droplets in the protein network is
thought to be realized via these interactions.
Increasing oil content in the emulsion led to a greater
number of oil globules fitted into the gel matrix, which
served as many anchor points that strengthened the three-
dimensional network. This explains, in part, the high
values of G 0 obtained on elevating the quantity of oil
added in emulsion gels as the mechanical properties of
this type of composite material may also originate from
the packing of oil droplets in the network. Indeed, when
the oil globules are concentrated above the random close
packing limit, they behave as perfectly elastic solids
(Bressy, Hébraud, Schmitt, & Bibette, 2003; Hemar &
Horne, 2000). Although the critical oil volume fraction
was not reached in this work, shifting from the lowest to
the highest oil concentration was sufficient for the droplets
to sustain more stress and possess more elastic
characteristics.
The elasticity provided by the oil droplets to the
emulsion gels appears precisely to be the main reason for
the greater WHC observed at higher oil content as it enabled
the samples to withstand the mechanical forces during
centrifugation. As suggested by other studies (Alzagtat &
Alli, 2002), the improvement of WHC could also be
attributed, to a certain degree, to an enhancement of the gel
structure density by the presence of oil droplets, which
causes the water to be trapped inside the pores by capillary
forces.
Changes in the emulsion gel microstructures and in the
WHC with the addition of calcium salt confirmed that Ca2C
was also an integral part of the protein network building
process. Emulsion gels obtained at low salt concentrations
were fine-stranded and retained more water than those
containing particulate type structures and formed at high
Ca2C concentrations. The pattern observed for the cold-set
emulsion gels agrees with previous studies on
divalent cation-induced WP gels (Barbut, 1995; Barbut &
Foegeding, 1993; Hongsprabhas & Barbut, 1997b, 1998;
Remondetto et al., 2002), where raising the mineral
content resulted in increasing protein aggregate size,
in forming a looser and more porous structure, and in
diminishing the WHC. Since the ability of gels to physically
hold water varies, among other, with the size of inner
structure spaces, gels with large and heterogeneous pores
tended to bind water to a lesser extent, because of low
capillary forces (Hermansson, 1986). High protein–protein
interactions caused by an increase in Ca2C level also
explain why water was more easily extracted from
particulate gels.
In this work, excessive Ca2C bridging between the
protein molecules led not only to the expulsion of water but
also to the exclusion of some oil droplets that flocculated
afterwards. Studies on heat-set emulsion gels have revealed
that the degree of incorporation of oil droplets into the
protein network affect the gel rheological properties
(McClements et al., 1993; Chen & Dickinson, 1999b). It
seems therefore reasonable to infer that aggregated droplets,
obtained at high Ca2Cconcentrations, have disruptive
effects on the protein matrix and hence weaken cold-set
emulsion gels. One would then expect a decrease in G 0
values by adding more Ca2C, especially as particulate gels
are also known to be less elastic than fine-stranded ones
(Stading & Hermansson, 1991). However, as seen earlier,
the effect of calcium on the emulsion gel storage modulus
was not significant. A possible explanation for the
rheological behavior at high Ca2C concentrations is the
greater effective volume fraction for a system of aggregated
droplets when compared to the sum of the volumes of
individual oil droplets (Van Vliet, 1988). A consequence of
this is a higher emulsion gel storage modulus that
overcomes the negative effect of aggregated structures
created at high Ca2C levels.
Oil and calcium are both clearly involved in the cold-set
b-lg emulsion gel network building process. Thus, it is
possible to manipulate the final gel characteristics by
adjusting the proportion of oil included in the matrix and
the amount of added calcium. In fact, for given calcium
concentration, emulsion gel elasticity and water retention
can be enhanced by increasing the oil volume fraction. On
the other hand, for given oil concentration, emulsion gel
structure type and water-holding abilities can be modified
by varying the calcium level. If a filamentous emulsion gel
with superior water retention capabilities is required, a low
calcium concentration should be used whereas for a
syneresing particulate emulsion gel, the use of high calcium
concentration should be considered.
5. Conclusion
This study demonstrates that gelation of b-lactoglobu-
lin emulsion gels can be achieved at room temperature
and that modulation of their microstructure and functional
 V.L. Sok Line et al. / Food Hydrocolloids 19 (2005) 269–278
properties is possible by varying the oil and calcium
concentrations. Emulsion gels with a high storage
modulus and a good WHC are produced by increasing
the oil content from 9.9 to 31.1 wt%. However, raising
Ca2C levels from 11.7 to 68.3 mM causes the structure of
emulsion gels to shift from filamentous to particulate type
and leads to a decrease in the water retention capacity
without affecting the gel rheological properties. The
balance between these two antagonistic effects results in
emulsion gels of unique properties that can be tailored to
specific food applications.
Acknowledgements
The authors acknowledge Anne-Françoise Allain for her
technical assistance. This work was supported by a Natural
Sciences and Engineering Research Council of Canada
(NSERC) fellowship (Valérie Leung Sok Line) and by the
NSERC Canada Research Chairs Program (Muriel
Subirade).
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