Food Hydrocolloids 13 (1999) 303–316

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Composite structure formation in whey protein stabilized O/W emulsions.

I. Influence of the dispersed phase on viscoelastic properties
C.K. Reiffers-Magnani a, J.L. Cuq b, H.J. Watzke a,*
a
Nestec Ltd., Nestlé Research Center, Department of Food Science and Process Research, Vers-chez-les-Blanc, P.O. Box 44, CH-1000 Lausanne 26,
Switzerland
b
Université des Sciences et Techniques du Languedoc, Département de Génie Biologique et Sciences des Aliments, Place Eugène Bataillon,
34085 Montpellier Cedex 5, France
Received 10 June 1998; accepted 1 March 1999

Abstract
The viscoelastic properties of whey protein stabilized emulsions containing varying amounts of protein and oil were investigated during
heat treatment using oscillatory rheology. The emulsions were thoroughly characterized prior to gelation by measuring droplet size and
protein adsorption. This emulsion characterization permitted the concentration of protein remaining in the bulk phase of the emulsion to be
determined, by taking into account both oil exclusion effect and protein adsorption, and to prepare a cream and a whey protein solution
corresponding to the emulsion’s dispersed and bulk phases, respectively. Gelation of the phases was investigated separately and was used to
better understand how the oil droplets and bulk proteins contribute to gelation of the whole emulsion. The rheology of polymeric solutions,
giving the gel point as the G 0 – G 00 cross-over, could be applied to the whey protein solution during heat treatment. Regarding the cream, its
gelation revealed that the proteins adsorbed at the O/W interface gelled and were responsible for droplet–droplet interactions, leading to a
particle gelled network. The rheological behavior of the whole emulsion during heat treatment was in-between those of a polymeric gel and a
particle gel. The main parameters influencing the rheology were the bulk protein concentration and the aggregation state of the oil droplets.
The bulk protein concentration determined the formation of a polymeric network in the aqueous phase of the emulsion, and the aggregation
state of the droplets influenced their possible interactions and the formation of a particle network. q 1999 Elsevier Science Ltd. All rights
reserved.
Keywords: Whey proteins; Emulsion; Gelation; Composite; Polymer; Rheology

1. Introduction Shimuzi & Kamiya, 1980; Dickinson, Rolfe & Dalgleish,

A wide range of food products exist as emulsions, either
liquid or gelled, for instance dairy products, mayonnaise and
sauces. The understanding of structure formation is a key
issue in their production because it allows the creation of
desired textures.
In this context, gellable whey protein stabilized emul-
sions are especially suitable models to investigate structure
formation. The work presented here concentrates on the
importance of the dispersed phase in structure formation
of a whey protein stabilized emulsion during heat treatment,
focusing on the importance of the adsorbed protein layer at
the oil–water interface.
Whey protein isolates exhibit interesting functional prop-
erties for the food industry. Their emulsifying (Yamauchi,
* Corresponding author. Tel.: 1 41-21-785-8026; fax: 1 41-21-785-
8554
E-mail address: heribert.watzke@rdls.nestle.com (H.J. Watzke)
0268-005X/99/$ - see front matter q 1999 Elsevier Science Ltd. All rights reserved.
PII: S0268-005 X( 99)00 013-2
1989; Klemaszewski, Das & Kinsella, 1992; Suttiprasit, Al-
Malah & McGuire, 1993) and gelling (Gault & Fauquant,
1992; Matsudomi, Oshita, Sasaki & Kobayashi, 1992; Hines
& Foegeding, 1993; McSwiney, Singh & Campanella,
1994; Boye, Alli & Ismail, 1996) have been reviewed.
They adsorb at the O/W interface and contribute to the
formation of stable emulsions, due to lowering of the inter-
facial tension and to their almost irreversible adsorption
(Tornberg, 1978).
Their gelling properties come from their ability to dena-
ture and unfold during heat treatment. Intermolecular inter-
actions are favored over intramolecular interactions and this
can result in formation of a network. The mechanisms
involved in protein gelation have been extensively studied
(Hermansson, 1979; Steventon, Gladden & Fryer, 1991;
Aguilera, 1995; Hoffmann, van Mil & de Kruif, 1995), espe-
cially in terms of the kinetics (Dannenberg & Kessler, 1988;
Taylor & Fryer, 1993) and gel structure (Stading, 1993;
Harwalkar & Kalab, 1985).
304 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316
Table 1
Whey protein isolate composition
g/100 g powder
Total proteins 89.3
Soluble proteins
(Total nitrogen × 6.38)
a -lactalbumin (% total protein)
b -lactoglobulin (% total protein)
Serum albumin (% total protein)
Fat 0.6
Lactose 0.4
Ash 1.9
Na 1(mg/100 g)
Ca 21(mg/100 g)
K 1(mg/100 g)
Mg 21(mg/100 g)
Humidity 6.0
Sum known 98.2
The development of viscoelastic properties, which occurs
during gel formation, can be detected by oscillatory rheol-
ogy. The sol–gel transition is of great importance because it
indicates a change in structure. Various methods to detect
this point have been published (Farris & Lee, 1983;
Apicella, Masi & Nicolais, 1984; Winter & Chambon,
1986; Chambon & Winter, 1987; Winter, 1987) and a rheo-
logical method, derived from synthetic polymer science and
which takes the cross-over of elastic and viscous moduli as
the gel point, is well accepted (Winter & Chambon, 1986;
Chambon & Winter, 1987; Winter, 1987). At this point G 0 ˆ
G 00 and the phase angle d is 458.
Whey protein stabilized emulsions gel during heat treat-
ment under suitable conditions. A material science approach
can be used to study the mechanical properties of the gel
formed. The gelled emulsion has been described as a
composite material where oil droplets play the role of filler
particles and free proteins form the gel matrix (Langley,
1990). The classical approach to study these emulsions
involves gelation of the whole emulsion under either a
large (Aguilera & Kessler, 1988, 1989; Langley & Green,
1989; Aguilera & Kinsella, 1991; Xiong, Aguilera &
Kinsella, 1991) or small deformation (Van Vliet, 1988;
Aguilera, Xiong & Kinsella, 1993; Dickinson & Yamamoto,
1996). Oil droplets are found to modify the mechanical
properties of the protein gel. They reinforce it by increasing
the bulk protein concentration due to excluded volume;
consequently the proteins strengthen the gel in the bulk
phase of the emulsion (Dickinson & Hong, 1995). They
also modify the viscoelastic properties of the gel, by provid-
ing an interface capable of interacting with the matrix (Van
Vliet, 1988). These interactions depend on the emulsifier
adsorbed at the interface, and also on the interfacial area
which in turn determines the number of connections
between the filler and the matrix (Xiong et al., 1991).
When proteins are adsorbed, droplets are well incorporated
within the matrix and the adsorbed proteins form a colloidal
network around them (Jost, Dannenberg & Rosset, 1989).
When other emulsifiers like Tween or lecithin are used, no
interaction occurs between droplets and matrix and no gel
strengthening is observed (Dickinson & Yamamoto, 1996;
Matsumara, Kang, Sakamoto, Motoki & Mori, 1993;
McClements, Monahan & Kinsella, 1993).
The work presented here contributes to a better under-
82.6
15.0 standing of the viscoelastic properties of food composites.
79.0 By studying separately gelation of the dispersed and the
5.0 bulk phases of a whey protein stabilized emulsion, the
contribution of both bulk proteins and oil droplets to gela-
tion of the whole emulsion is evaluated. In particular, gela-
500 tion of the adsorbed proteins is studied in detail and explains
150 the particle gel structure of the heated emulsion’s dispersed
100 phase.
25
2. Materials and methods
2.1. Materials
A commercial whey protein isolate (Le Sueur Isolates, Le
Sueur, MN, USA) was used as the protein source, without
further purification. The composition of the protein isolate is
given in Table 1. The pH of the powder in deionized water
was between 7.1 and 7.3 depending on concentration. Soya
oil was used as received from Morgia (Switzerland). Its
density was 915 kg m 23 at 208C. Tween 40 was purchased
from Fluka (Switzerland) and was used as received.
2.2. Preparation of whey protein solutions
The whey protein isolate was dissolved in deionized
water at room temperature under moderate magnetic agita-
tion, to avoid foaming, for 1–2 h. The pH was adjusted to
7.0 with 1 N HCl.
Final protein concentrations were 11.2–16.3% w/w.
2.3. Preparation of emulsions
Emulsions were prepared by mixing the protein solution
or the Tween 40 solution with the oil under vigourous
magnetic agitation. The final concentrations were 20–
40% w/w of oil and 9.8–13% w/w of protein or 2% w/w
of Tween 40. The protein ensured the formation of a stable
emulsion (Magnani-Reiffers, 1997; Hunt & Dalgleish,
1994). Both the aqueous solution and the oil were preheated
to 308C. The mixtures were then prehomogenized in a
laboratory mixer (Warring Blender, Ismatec SA., Switzer-
land) for 15 s and immediately homogenized by two
passages through a Microfluidizer with a head pressure of
800 bars (Model 110 T, Microfluidics Corporation, MA,
USA).
All the emulsions were used within two days of their
preparation.
2.4. Preparation of creams
Creams (fat globules and adsorbed emulsifier, i.e. whey
 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316
proteins or Tween 40) were obtained after centrifugation of
20% w/w oil emulsions containing either 10% w/w protein
or 2% w/w Tween 40, according to the procedure of Hunt
and Dalgleish (1994). The emulsions were centrifuged at
20 000 g and 208C for 1 h. The cream formed was removed
and washed with deionized water. The optimal number of
washing cycles was determined by observing changes in the
average particle diameter and the presence of free oil on the
top of the cream layer due to coalescence of the droplets.
After three cycles the particle size increased drastically and
free oil appeared, therefore, two washings of the cream were
adopted as the most appropriate procedure. The whey
protein and Tween 40 stabilized creams contained 66% w/
w oil and 2.3% w/w protein for the former and 74% w/w oil
for the latter.
For some experiments, an aliquot of the whey protein
stabilized cream was taken and split into two parts, which
were diluted with deionized water. The first contained
60% w/w oil and 1.9% w/w protein and the second
contained 33% w/w oil and 1.1% w/w protein. /
2.5. Particle size measurements
Particle size in the emulsions and in the creams was
routinely checked by Photon Correlation Spectroscopy
(PCS 100, Malvern Instruments, England). This method
has already been used to determine particle size in micro-
fluidized systems (Pouliot, Paquin, Robin & Giasson, 1991;
Robin, Kalab, Britten & Paquin, 1996) and its principle was
previously reported (Schurtenberger & Newman, 1993;
Ricka, 1995). Measurements were performed at a scattering
angle of 908 at 258C. The sample was dispersed in ultrapure
water. The viscosity of the dispersing medium was
0.89 mPa s and the refractive index of the oil was 1.33.
The determination of the hydrodynamic diameter
permitted to calculate the interfacial area AT and the number
of fat droplets per 100 g of emulsion NT, according to Eqs
(1) and (2)
M0 3
AT ˆ × …1†
r0 R0
M0 3 Measurements were performed with an oscillatory
NT ˆ × …2†
r0 4pR30
with M0 being the oil mass in the emulsion, r 0 the oil density
and R0, the average hydrodynamic diameter measured in the
emulsion.
2.6. Protein content in the emulsion bulk phase
The protein concentration in the bulk phase of the emul-
sion was calculated by taking into account (A) the oil exclu-
sion effect and (B) both the oil exclusion effect and protein
adsorption. All products were prepared by weighing the
appropriate mass of oil, protein, and water, so that the
following calculations were based on mass values, and
expressed for 100 g of emulsion. In the former case (A),
305
the corrected bulk protein MPB(A) was given by Eq. (3),
100
MPB…A† ˆ MP × …3†
100 2 M0
with MP the total mass of protein in the emulsion.
In the latter case (B), the corrected bulk protein was
calculated from the quantity of protein (g) adsorbed per
gram oil kmass determined by the Kjeldahl method (Associa-
tion of Analytical Chemists, 1980). To validate the calcula-
tions, the protein recovery was evaluated. The sum of the
protein measured in the cream and in the serum was
compared to the original protein in the emulsion and the
recovery was 106%. This value was in the range reported
previously, between 96–108% (Hunt & Dalgleish, 1994),
which ensured that adsorbed proteins are not substantially
removed from the interface during the washing procedure.
The mass of oil in the cream was calculated by determining
the dry mass at 1358C (Mettler PM100 balance, coupled
with a Mettler LP16 desiccator, Mettler Toledo AG.,
Switzerland) and correcting for the adsorbed protein
mass.The protein mass adsorbed in the emulsion MPA
could thus be calculated:
MPA ˆ Kmass × M0 : …4†
The mass of unadsorbed protein in the emulsion MPF was
determined from the following equation:
MPF ˆ MP 2 MPA : …5†
To obtain the protein concentration in the bulk phase of
the emulsion, the value obtained from Eq. (5) was corrected
by the mass of the dispersed phase MDP as follows:
MDP ˆ M0 1 MPA : …6†
The corrected protein mass in the aqueous phase MPB(B) of
the emulsion was obtained from Eqs. (5) and (6) as
100
MPB…B† ˆ MPF × : …7†
100 2 MDP
2.7. Oscillatory rheology
rheometer (Carri-Med CSL 100, TA Instruments, Germany)
fitted with cone and plate geometry (cone diameter 6 cm,
angle 38 59 min, gap 107 mm). The samples were poured
onto the plate of the rheometer at 258C and the edge covered
with paraffin oil to prevent dehydration. Whey protein solu-
tions, emulsions and creams were heated directly on the
rheometer plate, using an integrated Peltier temperature
regulation system. The typical heating protocol was from
25 to 808C at 1 or 5.58C/min, holding at 808C for 15 min,
cooling to 258C at 5.58C/min and then holding at 258C for
15 min. The measurements were performed at a frequency
of 1 Hz and a strain amplitude of 0.01. In the case of creams,
this strain value was found to damage the structure and so a
strain of 0.005 was used. The geometry chosen allowed a
306 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316

(a) 1000 0.08

2*Rh
kmass
800
0.06

600
0.04
400

0.02
200

0 0
0 2 4 6 8 10 12 14
Proteins (% w/w)

(b)
16

14

12

10

0
0 2 4 6 8 10 12 14
Total proteins (% w/w)
Fig. 1. Characteristics of a whey protein stabilized emulsion containing 20% w/w, oil and increasing protein content at pH 7.0. (a) Average hydrodynamic
diameter (2 × Rh) and protein/oil ratio (kmass). (b) Bulk protein concentration. no correction (—); correction for the oil exclusion effect (–-); correction for the
oil exclusion effect and the protein adsorption (…).

fast heat transfer from the plate to the sample, even at a
heating rate of 5.58C/min. The gap size fulfilled the
requirements of a ratio of at least 10 between the gap
and the maximum particle diameter (Roberts, 1996).
Elastic (G 0 ) and viscous (G 00 ) moduli were followed as
indicators of viscoelastic properties. Tan d , obtained as
the ratio between viscous and elastic moduli, was also
used to evaluate the material’s viscoelasticity. The
precision of the gel temperature was ^0.58C and
^2.58C, respectively for heating rates of 1 and 5.58C/
min, due to the temperature regulation of the Peltier
system (Magnani-Reiffers, 1997).
 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316
Table 2
Characteristics of whey protein stabilized emulsions at pH 7.0. The total protein content in the emulsions was 9.8% w/w
Oil (% w/w) D (nm) a Nb
20 196 6 × 10 21
30 223 6 × 10 21
40 300 3 × 10 21
a
D: average hydrodynamic diameter of the fat globules.
b
N: number of fat globules per 100 g emulsion.
c
A: interfacial area per 100 g emulsion (m 2).
d
kmass: gram protein adsorbed per gram oil.
e
A: the concentration is corrected for the oil exclusion effect. B: The concentration is corrected for the oil exclusion effect and the protein adsorption.
2.8. Transmission electron microscopy
The samples were encapsulated in agar gel tubes (Allan-
Wojtas & Kalab, 1984; Kalab, 1988). They were fixed in
2.5% glutaraldehyde in phosphate buffer. This procedure
was followed by fixation in 2% osmium tetroxide (Geis-
singer & Stanley, 1981). Dehydratation was performed in
a graded alcohol series starting with 30% alcohol. Spurr
resin was used to embed the samples. Ultra-thin sections
(60–80 nm) were stained with uranyl-lead citrate and exam-
ined by transmission electron microscopy (Philips CM12,
The Netherlands) at an acceleration voltage of 60 kV.
3. Results and discussion
3.1. Emulsion characterization
Whey protein stabilized emulsions consist of oil droplets
stabilized by whey proteins, dispersed in a bulk continuous
phase containing non-adsorbed proteins. The size of the oil
droplets depends on the homogeneization conditions and
also on the quantity of protein used to prepare the emulsion,
which determines also the protein adsorption at the O/W
interface (Hunt & Dalgleish, 1994) and consequently, the
remaining unadsorbed proteins in the continuous phase of
the emulsion. Gelation of a whey protein stabilized emul-
sion under appropriate conditions leads to the formation of a
composite gel in which the dispersed and the continuous
phases influence the viscoelastic properties (Dickinson &
Hong, 1995; Jost et al., 1989). The microstructural features
of these emulsions, i.e. particle size, protein adsorption and
partitioning, were therefore investigated to understand
better how the dispersed droplets and the bulk protein solu-
tion participate in the gelation process.
Fig. 1 depicts the droplet diameter and the quantity of
protein adsorbed per g of oil of a 20% w/w oil emulsion
containing increasing total protein concentrations. For
protein contents below 1% w/w a sharp decrease of the
diameter can be observed, while the curve flattens between
1 and 2% w/w. Above this concentration, the total protein
concentration in the emulsion has no influence on droplet
307
Ac kmass d Protein in bulk phase e (% w/w)
A B
6.7 × 10 5 0.048 12.3 11.2
8.8 × 10 5 0.059 14.0 11.8
8.7 × 10 5 0.081 16.3 11.6
size (Fig. 1(a)). The protein adsorption increased as the
diameter decreased. The increased size of the droplets at
low protein concentrations was due to droplet recoalescence
resulting from the insufficient protein coverage of the origi-
nal surface area generated (Hunt & Dalgleish, 1994). Above
2% w/w the protein content was sufficient to ensure a
complete coverage of the interface, leading to an accumula-
tion of the proteins in the bulk phase of the emulsion.
The bulk protein concentration of the emulsions, calcu-
lated from the quantity of adsorbed proteins and the oil
exclusion effect, is depicted in Fig. 1(b). For comparison,
the total protein concentration in the emulsion (full line) and
the bulk protein content corrected only for the oil exclusion
effect (dashed line) are also plotted.
The bulk protein content depended on the total protein
concentration, but also on the quantity of protein adsorbed at
the O/W interface and on the oil exclusion effect. Below
4.5% w/w, total protein, the bulk protein content corrected
for the oil exclusion effect and the protein adsorption was
lower than the total protein concentration, suggesting that
protein adsorption mainly determined the bulk protein
concentration. The ratio adsorbed protein/unadsorbed
protein was high under these conditions. At 4.5% w/w
protein, the effect of protein adsorption and oil exclusion
effect canceled out each other. Above this value, the bulk
protein concentration was mainly determined by the oil
exclusion effect; the ratio adsorbed protein/unadsorbed
protein decreased. In the protein concentration range
where whey protein can gel, typically around 11% w/w
(Fernandes, 1994), the oil content determined the bulk
protein concentration, which is an important result for the
following emulsion gelation study.
Emulsions containing a constant protein concentration
(9.8% w/w) and increasing amounts of oil (from 20 to
40% w/w) were also characterized (Table 2). The average
hydrodynamic diameter and the protein/oil ratio increased
with oil concentration, which suggests a poor stability of the
emulsions with increased oil concentrations, by analogy
with Fig. 1(a). Having determined the bulk protein concen-
tration, an insufficient protein coverage to stabilize the
droplets was improbable as the bulk content was between
11.2 and 11.8% w/w. An explanation may be the increased
308 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316
100
10
1
0.1
0.01
72 73
Fig. 2. Determination of the gel point with the G 0 –G 00 cross-over method for a 13% w/w whey protein solution at pH 7.0. G 0 (black symbols) and G 00 (open
symbols). Variation of phase angle with frequency is plotted in the inserted graph. (X, W) 0.5 Hz; (O, e) 1.0 Hz; (V, S) 5.0 Hz. The heat treatment, performed
in the rheometer, was 18C min 21 from 25 to 808C, at a strain of 0.01.
emulsion viscosity at higher oil contents and, therefore, a
different behavior of the product during emulsification,
leading to different emulsion characteristics. The interfacial
area of the 40% w/w oil emulsion did not increase as
compared to the 20 and 30% w/w oil emulsions because
the higher oil concentration may be compensated by the
increased droplet diameter. The bulk phase protein concen-
tration was roughly equivalent for the 20, 30 and 40% w/w
oil emulsions, once corrected for the oil exclusion effect and
protein adsorption. This result will be considered after-
wards, when studying emulsion gelation.
3.2. Gelation of whey protein solutions and emulsions
The following study deals with the gelation of whey
protein solutions, emulsions and creams. At this point, it
is useful to differentiate between polymeric and particle
gels. Both can be distinguished by their microstructure
and rheological properties. The former consists of fine-
stranded molecular networks whose behavior follows the
synthetic polymer theory (Winter & Chambon, 1986;
Chambon & Winter, 1987; Winter, 1987) while the latter
consists of particle-aggregated networks, whose rheological
behavior depends on particle–particle interactions.
3.2.1. Gel point determination
In solution, whey proteins denature and aggregate upon
heat treatment, leading to the formation of an infinite cluster
at the gel point above the gel concentration threshold. The
sol–gel transition corresponds to an increased connectivity
200
Gel point 150
100
50
0
72 73 74 75 76 77 78
Temperature (˚C)
74 75 76 77 78
Temperature (˚C)
and the formation of a network. It indicates that the system
has undergone transition from a viscous state, where
protein–solvent interactions are favored, to an elastic state
where protein–protein interactions take place. The gel point
is of great importance because it indicates transition from a
liquid to a solid state.
In the field of synthetic polymers, the rheological
measurement of the elastic and viscous moduli of a given
material as a function time or temperature, for example, is
used to determine the gel point. This corresponds to the
point where the two moduli cross-over (Winter & Chambon,
1986; Chambon & Winter, 1987; Winter, 1987). Fig. 2
depicts the G 0 and G 00 variations with temperature at various
frequencies for a 13% w/w protein solution. The average
gelation temperature for a 13% w/w whey protein solution
was 74.5 ^ 0.38C, which is below the precision of the
apparatus at a heating rate of 18C/min ( ^ 0.58C). The
corresponding phase angle was 45 ^ 28 and does not depend
on the frequency. At pH 7.0 the whey protein solution
behaved rheologically like a polymeric solution.
Fig. 3 shows the effect of emulsion composition on G 0
and G 00 variations with temperature. Fig. 3(a) depicts the
case for a 20% w/w oil emulsion containing 13% w/w
protein and Fig. 3(b) the case for a 40% w/w oil emulsion
containing 9.8% w/w protein. It was technically not possible
to produce an emulsion with comparable droplet size with
9.8% w/w protein and 40% w/w oil as with 13% w/w
protein and 20% w/w. In the latter, the increased product
viscosity led to a substantially higher droplet size after
emulsification. As droplet size could also influence the
 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316 309

(a) 10000

1000

100

200
10
150
Gel point
1 100

50

0.1 0
68 69 70 71 72 73 74

Temperature (˚C)

0.01
70 71 72 73 74 75
Temperature (˚C)

(b) 1000

100

10

G'
G''
1
25 35 45 55 65
Temperature (˚C)

Fig. 3. Elastic and viscous moduli of whey protein stabilized emulsions at pH 7.0 as a function of temperature. The phase angle is plotted in the inserted graph.
(X, W) 0.5 Hz; (O, e) 1.0 Hz; (V, S) 5.0 Hz. (a) Whey protein emulsion (13% w/w protein and 20% w/w oil). The heat treatment, performed in the rheometer,
was 18C min 21 from 25 to 808C, at a strain of 0.01. (b) Whey protein emulsion (9.8% w/w protein and 40% w/w oil). The heat treatment, performed in the
rheometer, was 5.58C min 21 from 25 to 808C, at an oscillation frequency of 1 Hz and a strain of 0.01.

composite rheological properties, it was, therefore, chosen
to produce an emulsion containing less protein but having a
comparable droplet size.
For the 20% w/w oil emulsion, a G 0 –G 00 cross-over could
be determined. The gelation temperature was 70.8 ^ 0.48C,
irrespective of the applied frequency. The phase angle was
45 ^ 28 (Fig. 3(a)). The emulsion behaved qualitatively like
the protein solution, thus like a polymeric solution.
However, the gelation temperature was lower for the emul-
sion than for the solution, even though both contained the
same total protein concentration.
Increasing the oil concentration to 40% w/w led to a
different rheological behavior (Fig. 3(b)); the system had
elastic properties before any heat treatment and no gel
310 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316

Table 3 3.2.2. Comparison of whey protein solution and emulsion

Composition of emulsions prepared at pH 7.0
Oil (% w/w) Protein (% w/w)
Total Aa Bb
20 9.8 12.3 11.2
30 9.8 14.0 11.8
40 9.8 16.3 11.6
a
A: the concentration is corrected for the oil exclusion effect.
b
B: the concentration is corrected for the oil exclusion effect and protein
adsorption.
point could be determined with the classical polymeric
approach (G 0 –G 00 cross-over). Comparing this behavior
with that of the protein solution presented above, and taking
into account the corrected bulk protein content of
11.6% w/w (Table 2), the bulk protein may not be respon-
sible for the observed elastic properties before and during
heat treatment. The increased oil concentration may be
rather responsible for the deviation in behavior of the emul-
sion from that of a polymeric solution. However, the bulk
protein concentration (11.6% w/w) was enough to form a
network and it was felt necessary to define a method which
allowed the detection of the gel point independently of the
elastic behavior observed. Fig. 3(b) shows that between 45
and 658C, the curve went through a minimum. As the
proteins represented the only gelling components in the
system, the increased viscoelasticity can be attributed to
their gelation. The gel point was then determined as the
point where G 0 increased significantly. In the case corre-
sponding to Fig. 3(b), the gelation temperature was 56 ^
28C.
gelation parameters
To obtain a better understanding of the influence of the
dispersed phase on the rheological behavior of whey protein
stabilized emulsions, three emulsions containing 9.8% w/w
total protein and increasing amounts of oil from 20 to
40% w/w were prepared at pH 7.0 and their gelation was
compared to those of whey protein solutions. The composi-
tion of the protein solutions corresponded to the emulsion
bulk phase concentrations after correction for (A) the oil
exclusion effect and for (B) the oil exclusion effect and
protein adsorption (Table 3).
Fig. 4 shows the elastic modulus of the emulsions
(composites) and of the protein solutions corresponding
to the emulsion bulk phases as a function of the oil concen-
tration in the emulsions. A sharp increase of the composite
elastic modulus was observed with increasing oil concentra-
tion, the effect appearing markedly for the most concen-
trated emulsion.
Previous studies have already mentioned the reinforce-
ment of protein gels by the inclusion of oil droplets (Xiong
et al., 1991; Aguilera et al., 1993; Dickinson & Hong, 1995;
Jost et al., 1989). This reinforcement was explained by the
oil exclusion effect (Aguilera et al., 1993; Jost et al., 1989),
which led to an increased protein concentration in the bulk
phase of the emulsion. However, taking into account the oil
exclusion effect (matrix A), the protein solutions had a
higher elastic modulus than the corresponding emulsions
(Fig. 4), suggesting that the oil exclusion effect alone did
not fully explain the reinforcement of the protein gel.
Taking into account both the oil exclusion effect and the
protein adsorption (matrix B), the protein solutions having
an essentially constant protein concentration (Table 3), had

Fig. 4. Elastic moduli of whey protein stabilized emulsion gels (composite) and whey protein gels (matrixes) as a function of oil concentration. The emulsions
were prepared with 9.8% w/w protein at pH 7.0 and they were heated in the rheometer at 18C min 21 from 25 to 808C, held at 808C during 15 min and cooled to
258C at 5.58C min 21 and held at 258C during 15 min. The frequency was 1 Hz and the strain 0.01. The elastic moduli represented on the graph are those
obtained after the mentioned heat treatment. Matrix A: the protein solution concentration corresponds to that in the emulsion bulk phase after correction for the
oil exclusion effect. Matrix B: the protein solution concentration corresponds to that in the emulsion bulk phase after correction for the oil exclusion effect and
protein adsorption.
 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316 311

Table 4
Comparison of gelation temperature and elastic modulus of whey protein stabilized emulsions (composites) and whey protein solutions (matrixes) at pH 7.0.
The samples were heated in the rheometer at 18C min 21 from 25 to 808C. The frequency was 1 Hz and the strain 0.01 (NB: The standard deviations for Tgel and
G 0 gel correspond to the determination deviations of the gel point with the G 0 –G 00 cross-over)

Tgel(8C) a G 0 gel(Pa)

Oil (% w/w) 20 30 40 20 30 40

Emulsion 79.2 ^ 0.5 71.9 ^ 0.5 57.3 ^ 2.0 8.5 ^ 3.1 11.3 ^ 4.1 35.1 ^ 6.2
Matrix A b 78.3 ^ 0.5 72.8 ^ 0.5 70.9 ^ 0.5 5.5 ^ 2.1 5.0 ^ 2.1 16.1 ^ 5.2
Matrix B c 80.0 ^ 0.5 80.0 ^ 0.5 80.0 ^ 0.5 7.2 ^ 2.3 5.4 ^ 2.3 7.5 ^ 2.1
a
Tgel: Gelation temperature.
b
Matrix A: The protein solution concentration corresponds to that in the emulsion bulk phase after correction for the oil exclusion effect.
c
Matrix B: The protein solution concentration corresponds to that in the emulsion bulk phase after correction for the oil exclusion effect and the adsorbed
protein.

a lower and more constant elastic modulus than the corre-
sponding emulsions (Fig. 4), suggesting that protein adsorp-
tion played a role in the reinforcement of the protein gel.
These results are consistent with those of Dickinson and
Hong (1995), who used b -lactoglobulin stabilized emul-
sions containing N-tetradecane and b -lactoglobulin solu-
tion.
Gel reinforcement with increasing oil content was
also explained by an increased number of particles
(oil droplets), leading to a higher connectivity between
filler and matrix (Xiong et al., 1991). The calculation of
the interfacial area in the 20, 30 and 40% w/w oil emul-
sions indicated that this area increased until 30% w/w
oil and then decreased again as a function of the oil
content (Table 2). Consequently, the increase of the
composite viscoelastic properties observed with the
increased oil concentration could not be fully explained
by an increased filler–matrix connectivity. The major
difference observed in the three emulsions was in the
protein adsorption, which nearly doubled when the oil
concentration increased from 20 to 40% w/w.
The influence of the dispersed phase on the initial
stage of the network formation was also studied for
the three emulsions. Table 4 gives the gelation tempera-
ture and the elastic modulus at the gel point for the
emulsions and for their corresponding bulk phases.
The results obtained for the gel point parameters
(Table 4) followed the same trends as those observed
for the final viscoelastic properties (Fig. 4): taking into
account the oil exclusion effect (matrix A), the protein
solutions had a lower gelation temperature and a higher
elastic modulus than the corresponding emulsions
(Table 4). Taking into account both the oil exclusion
effect and protein adsorption (matrix B), the protein
solution had a higher gelation temperature and an
equal elastic modulus than the corresponding emulsions,
except for the 40% w/w oil emulsion. These results
would suggest that the adsorbed proteins rather than
the oil exclusion effect influenced the initial stage of
gel formation, by decreasing the time until the gel
point and enhancing the network connectivity.
3.2.3. Heating behavior of emulsion dispersed phase
The behavior during heat treatment of the proteins
adsorbed at the O/W interface was studied further by
using creams prepared from whey or Tween 40 stabilized
emulsions. The preparation of the creams was described in
the Materials and Methods (Section 2).
The results presented above showed that whey protein solu-
tions and whey protein stabilized emulsions containing
20% w/w oil could be regarded as classical polymeric solu-
tions. Their gelation was dictated by the bulk proteins which
formed a polymeric network. Increasing the oil concentration
led to a non-polymeric behavior, due to the interference of the
dispersed phase in the gelation process. The whey protein
stabilized creams could be regarded as a particle network
where the dispersed phase dictated the network formation.
Fig. 5(a) showed the variations of the elastic modulus of a
whey protein and a Tween 40 stabilized cream as a function
of time during three cycles of heating at 808C and cooling at
258C (called 1, 2 and 3 in Fig. 5(a)). At the beginning of the
experiment, the moduli of both creams were high, around
5000 Pa. The viscous moduli were around 700 and 50 Pa,
respectively, for the whey and Tween 40 stabilized creams
(data not shown), suggesting that the creams were in a gel
state (G 0 . G 00 ). Fig. 6 shows the arrangement of the oil
globules in the whey protein cream. Close packing of the
droplets, together with the measured elastic properties,
suggest hindering of the oil globules in the two creams.
However, the elasticity of the Tween 40 stabilized creams
is between two and four times lower than the elasticity of the
whey protein stabilized cream, irrespective of the applied
strain and throughout the whole temperature cycle (Fig.
5(a)). As the only difference between the two creams was
the nature of the emulsifier adsorbed at the O/W interface, it
could be concluded that the emulsifier influenced the heat-
ing behavior of the whole cream.
The Tween 40 stabilized cream also did not respond in
the same way as the whey protein stabilized cream to varia-
tions of applied strain. The behavior of the Tween 40 stabi-
lized cream with heat treatment was not influenced by
strain. An explanation could be breakage of the structure,
due to free movement of the droplets around each other.
312 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316

Fig. 5. Viscoelastic behavior of whey protein or Tween 40 stabilized creams (a) and whey protein solution (b) during heat treatment at pH 7.0. The whey
protein stabilized cream contained 66% w/w oil and 2.3% w/w protein. The Tween 40 contained 74% w/w oil and the whey protein solution was prepared with
13% w/w protein. The heat treatment, performed in the rheometer at an oscillation frequency of 1 Hz, is symbolized by the full line on the graph: three
temperature cycles were made (noted 1, 2 and 3 on the graph), each of them from 25 to 808C.

In the case of the whey protein stabilized cream, elastic
properties developed during both the heating and the cool-
ing periods of the first temperature cycle. However the elas-
tic modulus increase which occurred on cooling was
reversible (cycle 2 and 3 in Fig. 5(a)). To understand further
this behavior and to find an explanation, the elastic proper-
ties of the whey protein cream were compared to those of a
whey protein solution (Fig. 5(b)). Qualitatively, the whey
protein solution behaved similarly to the cream, and the
heating behavior of the whey protein cream was, therefore,
attributed to gelation of the proteins adsorbed at the O/W
interface. An increase in the elastic properties could be
observed during both the heating and the cooling periods
of the first temperature cycle. This observation is consistent
with previous studies dealing with the whey protein heat
gelation (Dickinson & Hong, 1995). Moreover, reversibility
 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316
Fig. 6. Microphotograph obtained by TEM of a whey protein stabilized
cream at pH 7.0 before heat treatment. (F: fat globule. P: protein).
could be observed in the present study; suggesting that the
interactions established during the cooling period are rever-
sible.
The results reported in this study showed that the heating
behavior of the cream depended on the strain applied. With
the lowest strain, namely 0.005, the maximal elastic modu-
lus was observed at 258C for the three temperature cycles.
While the highest, namely 0.01, the maximal elastic modu-
lus is observed during the cooling period of the three
temperature cycles (Fig. 5(a)). These observations suggest
breakage of the structure at the highest strain.
Finally, the above observations on the heating behavior of
the whey protein and Tween 40 stabilized creams permit to
explain better the role of interfacial proteins in the gelation
process: during heating, the proteins adsorbed at the inter-
face form a gelled structure which contributes, together with
the close packing of the oil droplets, to the formation of a
particle network. The sensitivity of the whey protein cream
to the applied strain suggested that the network surrounding
the oil droplets is fragile.
The heating behavior of the adsorbed protein was also
investigated in diluted creams (Fig. 7), which could simulate
a whey protein stabilized emulsion in which the bulk phase
contained no proteins. With 60% w/w oil the diluted cream
behaved qualitatively like the non-diluted cream, with a G 0
value approximately 10 times lower (Fig. 7(a)). The diluted
313
cream containing 33% w/w oil had an elastic behavior, but
the steep decrease in the elastic modulus suggested break-
down of the structure (arrows in Fig. 7(b)).
Flocculation of the diluted cream could explain this beha-
vior. Gelation of the adsorbed proteins in the interior of flocs
could generate the elasticity found in the 33% w/w diluted
cream. It is, however, not possible from the results reported
in this study to say if flocculation took place before or during
protein gelation. It is possible that either the absence of
proteins in the bulk phase of the emulsion or gelation of
the adsorbed proteins promoted droplet flocculation.
3.2.4. Models of whey protein stabilized emulsion gels
From the results obtained in this study, different micro-
structures can be proposed for whey protein stabilized emul-
sion gels. The models presented in Fig. 8 depict the case of
polymeric, mixed and particle composites.
Polymeric composites consist of gelled emulsions in
which the bulk phase proteins dictate the gelation behavior.
The protein content in the bulk phase is above the protein
gelation threshold, typically 11% w/w (case 1 in Fig. 8).
Polymeric composites are not prone to flocculation of
their dispersed phase, either before or during bulk protein
gelation. These proteins form a polymeric network in which
the oil droplets are entrapped.
Mixed composites consist of gelled emulsions in which
both the bulk phase proteins and the dipersed phase proteins
dictate the gelation behavior. The protein content in the bulk
phase of the emulsion is above the protein gelation threshold
and the oil droplets tend to interact with each other. Inter-
actions between oil droplets can be favored by a high oil
concentration which promotes close packing of droplets or
by flocculation of the droplets prior to or during heat treat-
ment. Polymeric gelation of the bulk protein and particle
gelation of the dispersed phase both contribute to the beha-
vior of the emulsion upon heating and to the viscoelastic
properties of the final composite (case 2 in Fig. 8). This
model was already suggested by van Vliet (Aguilera et al.,
1993), but no experimental work has so far been performed
to support it.
Particular composites are formed in the absence of bulk
protein matrix formation (cases 3 and 4 in Fig. 8). Depend-
ing on the oil concentration or on emulsion flocculation, the
formation of a whole particle gel (case 3 in Fig. 8) or the
formation of cohesive flocs (case 4 in Fig. 8) may arise. In
the former, close packing of the droplets permits the
adsorbed proteins to form a network occupying the whole
volume of the sample (case 3 in Fig. 8). In the latter, gelation
of adsorbed proteins around a limited number of oil droplets
leads to the formation of dispersed units of gels in the whole
volume of the sample (case 4 in Fig. 8). In the present study,
this case arise with diluted cream and was attributed to the
absence of protein in the bulk phase. From a macroscopic
point of view, the sample had elastic properties, as measured
by rheology, but appeared visually like a liquid.
314 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316

(a) 1500 100

80

1000
60

40
500

20

0 0
0 20 40 60 80
1 2 3
Time (min)

(b) 15 100

80

10
60

40
5

20

0 0
0 20 40 60 80
1 2 3
Time (min)

Fig. 7. Viscoelastic behavior during heat treatment of diluted whey protein stabilized creams at pH 7.0. (a) The diluted cream contained 60% w/w oil and
1.9% w/w protein. The diluted cream contained 33% w/w oil and 1.1% w/w protein. The heat treatment, performed in the rheometer at an oscillation frequency
of 1 Hz and a strain of 0.005, is symbolized by the full line on the graph: Three temperature cycles were made (noted 1, 2 and 3 on the graph), each of them from
25 to 808C.

4. Conclusion interface and then to calculate the remaining protein

The heat gelation of whey protein stabilized emulsions
and their viscoelastic properties after heat treatment were
investigated. As a first step, we thoroughly characterized the
emulsions prior to heating. This characterization permitted
to determine the quantity of proteins adsorbed at the O/W
concentration in the emulsion’s bulk phase. Then a cream
equivalent to the emulsion dispersed phase (whey protein
stabilized oil droplets) and a whey protein solution equiva-
lent to the emulsion bulk phase were prepared and their
gelation was investigated.
Bulk phase proteins form a polymeric network while the
 Before heat treatment After heat treatment

"Polymeric composite"
1
B ulk p ha se p ro te in a b o ve g e la tio n thre sho ld P o lym e ric n e tw o rk

C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316

O il d ro p le ts d o no t inte ra ct with e a ch o the r
Bulk proteins G e lle d a d so rb e d
p ro te in s
Fat globule

Adsorbed proteins

P a rticle n e tw o rk
2 "Mixed composite"
B ulk p ha se p ro te in a b o ve g e la tio n thre sho ld
O il d ro p le ts inte ra ct with e a ch o the r
P o lym e ric n e tw o rk
(clo se p a cking , flo ccula tio n)

"Particle composite"
3
P a rticle n e tw o rk
B ulk p ha se p ro te in b e lo w g e la tio n thre sho ld
D ro p le t clo se p a cking

4 "Particle composite" F lo c
B ulk p ha se p ro te in b e lo w g e la tio n thre sho ld
D ro p le t flo ccula tio n

315
Fig. 8. Propositions for gelled microstructures in heated whey protein stabilized emulsions at pH 7.0. Cases 1, 2, 3 and 4 are described in the text.
316 C.K. Reiffers-Magnani et al. / Food Hydrocolloids 13 (1999) 303–316
dispersed phase forms a particle network. The viscoelastic
properties of the gelled emulsion after heat treatment were
in between those of a whey protein solution and a whey
protein cream, depending on the protein concentration in
the bulk phase and the aggregation state of droplets prior
to and during the heat treatment. Close packing or floccula-
tion of the droplets promoted droplet–droplet interactions
and lead to the emulsion’s behavior changing from poly-
meric to particle gel behavior.
Acknowledgements
This work is part of a Ph.D Thesis, which was supported
by the Nestlé Research Center, Switzerland. The Center is
gratefully acknowledged for technical and financial support.
The authors gratefully acknowledge M.-L. Dillmann and
M.-F. Clerc for their skilled microscopic measurements.
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