Stability and release properties of double emulsions for food applications

Lanny Sapei, Muhammad Ali Naqvi, Drick Rousseau

*
Department of Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto, ON M5B 2K3, Canada
a r t i c l e i n f o
Article history:
Received 31 January 2011
Accepted 11 October 2011
Keywords:
Double emulsion
Salt
Gelatin
Stability
Controlled release
Modelling
a b s t r a c t
Water-in-oil-in-water (W
1
/O/W
2
) double emulsions (DEs) containing gelatin and sodium chloride (NaCl)
in the inner aqueous phase were developed for controlled release applications. Emulsions were prepared
with water and canola oil, as well as with polyglycerol polyricinoleate and polysorbate 80 as emulsiers
for the primary water-in-oil (W
1
/O) emulsion and secondary W
1
/O/W
2
emulsions, respectively. All DEs
containing both NaCl and gelatin were stable against sedimentation for the month-long study whereas
control emulsions (with either no NaCl or gelatin) showed visual phase separation. The average oil
globule size in freshly-prepared DEs grew from w45 to 70 mm with an increase in salt load from 2 to 8%
(w/w), and changed little after 1 month. Besides its role in stabilization, NaCl was also used as a marker
to evaluate DE release behaviour. The salt diffusion coefcient obtained using Fujitas model rose from
4.7 to 6.0 10
11
cm
2
/s with increasing NaCl concentration in the DEs from 2 to 8% (w/w). All stable DEs
showed a high salt retention in the inner aqueous phase (>94%) after 1 month of storage at 4

C. These
results demonstrated the synergistic action of a gelling agent and electrolyte in stabilizing and modu-
lating the release behaviour of NaCl from W
1
/O/W
2
DEs.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
Water-in-oil-in-water (W
1
/O/W
2
) double emulsions (DEs)
consist of a water-in-oil (W
1
/O) emulsion dispersed as droplets
within an aqueous phase (Garti, 1997; Sagalowicz & Leser, 2010).
Due to the presence of two aqueous domains separated by an oil
layer, the inner aqueous compartment offers great potential for the
encapsulation and controlled release of hydrophilic bioactive
ingredients. However, such emulsions are more difcult to prepare
and control than simple emulsions as they typically consist of
relatively large droplets that coalesce either quiescently or due to
commonly-encountered processing regimes (e.g., shear, steriliza-
tion), and have a strong tendency to release entrapped compounds
in an uncontrolled manner (Garti, 1997). Furthermore,
commercially-sterile DEs may be difcult to manufacture, partic-
ularly when numerous ingredients are present (Dalgleish, 2001).
Usage of W
1
/O/W
2
DEs for food applications is further limited by
the lack of suitable food-grade emulsiers and stabilizers for the
inner and outer emulsions.
W
1
/O/W
2
emulsions have been used in cosmetics and pharma-
ceuticals for applications such as drug controlled release and tar-
geted delivery (Gallarate, Carlotti, Trotta, & Bovo, 1999; Laugel,
Chaminade, Baillet, Seiller, & Ferrier, 1996; Vaziri & Warburton,
1994; Vlaia, Vlaia, Miclea, Olariu, & Coneac, 2009). Other applica-
tions have included the removal of toxic materials via entrapment
and solubility enhancement of poorly-soluble materials (Yan & Pal,
2001). W
1
/O/W
2
DEs have also been investigated for various food
applications, including the encapsulation of vitamin/minerals
(Benichou, Aserin, & Garti, 2007; Bonnet et al., 2009; ORegan &
Mulvihill, 2010), aroma and avour release (Malone, Appelqvist, &
Norton, 2003) and the production of low-calorie foods (DeCindio
& Cacace, 1995), e.g., low-fat dressing (Taki, 2008).
Given their lack of kinetic stability, the widespread application
of W
1
/O/W
2
DEs in the food industry remains elusive. Gartis
group attempted to improve the stability and release character-
istics of W
1
/O/W
2
DEs using steric stabilizers (Garti, Aserin, &
Cohen, 1994; Lutz, Aserin, Wicker, & Garti, 2009), Pickering
stabilization with fat crystals (Garti, Binyamin, & Aserin, 1998) and
protein-polysaccharide hybrids (Benichou et al., 2007). Muschio-
liks group examined the incorporation of gelatin, NaCl and poly-
glycerol polyricinoleate (Pgpr) in the primary emulsion, the use of
sodium caseinate-dextran conjugates as an external emulsier,
and membrane emulsication, all to better harness DE stability
and controlled release patterns (Fechner, Knoth, Scherze, &
Muschiolik, 2007; Muschiolik, 2007; Muschiolik et al., 2006).
Others have focused on reducing Pgpr concentration by incorpo-
rating sodium caseinate in the inner aqueous phase (Su, Flanagan,
Hemar, & Singh, 2006) or by adding a highly-concentrated modi-
ed gum Arabic in the external aqueous phase (Su, Flanagan, &
Singh, 2008).
* Corresponding author. Tel.: 1 416 979 5000x2155; fax: 1 416 979 5044.
E-mail address: rousseau@ryerson.ca (D. Rousseau).
Contents lists available at SciVerse ScienceDirect
Food Hydrocolloids
j ournal homepage: www. el sevi er. com/ l ocat e/ f oodhyd
0268-005X/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2011.10.008
Food Hydrocolloids 27 (2012) 316e323
The objective of this research was to formulate a food-grade W
1
/
O/W
2
DE with good stability and controllable release properties.
The DEs herein developed were kinetically-stable for the duration
of the study (1 month) with the release of NaCl incorporated as
a marker triggered through changes in osmotic environment.
2. Materials and methods
2.1. Materials
Deionized water with a resistivity of >15 megohm-cm (Barn-
stead E-Pure, Ottawa, ON, Canada) was used for the aqueous phase.
Gelatin (porcine skin, type A, Bloom w300) and the water-tending
emulsier polysorbate 80 were purchased from SigmaeAldrich
(Oakville, ON, Canada). Sodiumchloride (NaCl) was purchased from
Fisher Scientic (Ottawa, ON, Canada). Canola oil (acid value < 0.2)
was purchased from a local supermarket (Toronto, ON, Canada).
The oil-tending emulsier polyglycerol polyricinoleate (Pgpr) was
obtained from Nealanders (Mississauga, ON, Canada). All chemicals
were used without further purication.
2.2. Double emulsion preparation
The inner aqueous phase (W
1
) containing gelatin (0%, 3% or 10%
w/w) and NaCl (0e8% w/w) was hydrated for 30 min followed by
mixing at 65

C for 25 min. The oil phase (O) containing 6% (w/w)
Pgpr was also mixed at 65

C for 25 min. Finally, the outer aqueous
phase (W
2
) containing 1% (w/w) polysorbate 80 was mixed 25

C
for 30 min.
Food-grade W
1
/O/W
2
DEs were prepared via a simple, repro-
ducible two-stage process. The primary water-in-oil emulsion (W
1
/
O) [40% w/w inner aqueous phase (W
1
) and 60% w/w oil phase (O)]
was homogenized at 65

C using a rotor-stator with a homogeni-
zation generator (d 1.2 cm) (Polytron

PT 10/35, Kinematic, CH-

6010, Switzerland) at 27,000 rpm for 3 min. The resulting emul-
sion was immediately quench-cooled to 4

C in a waterbath to
trigger the solegel transition of the gelatin present in the dispersed
aqueous phase. Prior to second-stage homogenization, the
temperature was slowly raised to 26

C to improve emulsication
efcacy, as preliminary results showed inefcient emulsication at
lower temperatures. The secondary emulsion (W
1
/O/W
2
) was
prepared by gradually adding the W
1
/O emulsion (20% w/w) to the
outer aqueous phase (W
2
) followed by mixing with the rotor-stator
at 10,000 rpm for 2 min and subsequent quench-cooling in a 4

C
waterbath.
2.3. Stability evaluation
2.3.1. Sedimentation
Freshly-made DEs were poured into 40 ml glass vials
(ID 25 mm; length 95 mm; Fisher Scientic, Nepean, ON,
Canada) to a height of w6 cm and stored at 4

C. The height of the
opaque emulsion phase was measured weekly over 1 month (h
t
)
and compared to the initial emulsion height (h
0
) to determine
sedimentation stability (S):
S

h
0
h
t
h
0

100% (1)
2.3.2. Microscopy
Brighteld light microscopy was used to examine DE emulsion
morphology. Samples were placed onto microscope slides (Fisher
Scientic, Nepean, ON, Canada) and gently covered with a cover slip
(Fisher Scientic, Nepean, ON, Canada). A Zeiss Axiovert 200M
inverted light microscope (Zeiss Inc., Toronto, ON, Canada) with
a 20 objective (combined with a 1.6 magnier lens) was used.
Images captured with a Q-Imaging CCD camera were analyzed
using Northern Eclipse software (version 7.0, Empix Imaging,
Mississauga, ON, Canada). Emulsions were characterized at room
temperature (25

C).
2.3.3. Oil globule size determination
The mean oil globule size distribution in the DEs (W
1
/O) was
characterized for 1 month with a Malvern particle size analyzer
(Mastersizer 2000S, Malvern Instruments Ltd., Malvern, Worces-
tershire, UK), equipped with a HeeNe laser (l 633 nm). The
optical parameters selected were: dispersed phase refractive index
of 1.47; globule absorbance of 0.01 and a dispersant liquid
(deionized water) refractive index of 1.33. Measurements were
carried out in triplicate on each emulsion and the results are re-
ported as the typical globule size distribution (in mm), and the
volume-weighted mean globule size D (4,3):
D4; 3
P
n
i
d
4
i
P
n
i
d
3
i
(2)
where n
i
is number of particle i and d
i
is diameter of particle i (mm).
The oil globule size distribution was determined from the best
t between the experimental measurements and prediction using
the Mie light scattering theory. As the inner aqueous droplets were
considered encapsulated within the oil globules, their contribution
to scattering was not signicant.
2.4. Release properties
2.4.1. NaCl release and kinetics
NaCl release fromthe inner aqueous phase towards the external
aqueous phase was measured using a conductivity meter (model HI
98188, Hanna Instrument, Romania) equipped with an integrated
data logger and associated transfer software (HI 92000 version
5.0.7). To carry out measurements, the conductivity meter was
dipped into a 15 ml aliquot of W
1
/O/W
2
DE placed in a 50 ml Falcon
tube (Fisher Scientic, Nepean, ON, Canada). The time-dependent
conductivity values were converted into NaCl concentration using
a calibration curve (not shown). The fraction of NaCl released (FR) in
the external aqueous phase was dened as the ratio of NaCl
released into W
2
at a specic time (M
t
) relative to the total amount
present in the external aqueous phase if all NaCl were released
M
N
.
FR%
M
t
M
N
100% (3)
Release experiments for all samples containing salt and gelatin
were conducted in triplicates, whereas for the control samples (no
gelatin), the experiments were conducted in duplicates due to lack
of emulsion stability. Experiments were conducted at 4

C.
2.4.2. Encapsulation efciency
Encapsulation efciency (EE) was dened as the percentage of
NaCl still entrapped within the inner aqueous phase (W
1
):
EE% 100 FR% (4)
2.4.3. Modelling NaCl release
Ficks second law in a spherical coordinate system was used to
describe NaCl transport in the DEs (Eq. (5)). The system was
simplied mathematically into a two-phase system consisting of
the NaCl reservoir (in the inner W
1
/O emulsion) and the
L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323 317
continuous phase (W
2
). The mass transfer was explained using
concentration-dependent diffusivities where C
t
is the NaCl
concentration in the continuous phase at time t, r is the radial
coordinate discretized along reservoir radius (specied using oil
globule size data) into 50 intervals and D is the concentration-
dependent diffusion coefcient of NaCl from the inner emulsion.
vC
t
vt

v
vr

D
vC
t
vr

(5)
Fujitas model of free volume was used to model salt release
kinetics (Eq. (6)) (Fujita, 1952).
D D
eq
exp

1
C
t
C
eq

(6)
where D
eq
is the diffusion coefcient of NaCl in an equilibrated DE,
b is a dimensionless scaling parameter that relates the initial and
equilibrium diffusivities and C
eq
is the equilibrium NaCl concen-
tration in the continuous phase. The equations were integrated
using a nite-difference method in Athena Visual Studio modeling
software v.14 (Athena Visual Software, Inc, 2009, Evanston, IL, USA).
Shown below are the initial and boundary conditions.
Initial conditions:
t 0; C
t
0
Boundary conditions:
r 0;
vC
t
vr
0
r R
eq
; C
t
1
where R
eq
is the equilibrium oil globule size based on light scat-
tering experiments. The sum of NaCl released from the emulsion
at each discretized radial interval was equal to the NaCl increase in
the continuous phase. The model was only concerned with the
excipient NaCl content and hence the release concentration as
well as rate of release was zero at t 0 and r 0. The spherical
coordinate system in Athena Visual Studio considers only a single
plane of the sphere to integrate the diffusion model equation
according to a nite-difference method. Sink conditions were
assumed to exist at the surface of the reservoirs. Emulsion
decomposition, swelling and contraction were ignored in the
model.
2.5. Statistics
All experiments were carried out in triplicate and the results are
expressed as mean standard deviation. Statistical analysis was
conducted using SigmaStat 4 integrated in SigmaPlot 11 (Systat
Software, Chicago, IL, USA). The average oil globule size and
encapsulation efciency were analyzed using t-tests or one-way
analyses of variance (ANOVA) and the Holm Sidak post hoc test.
The goodness of t of the NaCl release kinetics modelled with
Fujitas model was based on correlation coefcients (r
2
) and their
95% condence interval. Statistical analyses were deemed signi-
cant at p < 0.05.
3. Results
3.1. Sedimentation
The DEs prepared with both NaCl (Fig. 1bee) and gelatin (Fig. 2b
and c) were stable against sedimentation for the month-long study.
In contrast, control DEs with either no NaCl (Fig. 1a) or gelatin
(Fig. 2a) showed signs of phase separation immediately after
preparation. The sedimentation stability (S) of emulsions contain-
ing gelatin but no NaCl decreased fromw45% on day 1 to w30% on
day 29 whereas those with only NaCl showed a higher sedimen-
tation stability (w90% on day 1 and w80% on day 29) (Table 1). All
DEs containing salt and gelatin were visually more viscous than the
control DEs, but still pourable.
3.2. Double emulsion morphology
The morphology of all formulations was similar after 1 day
(Fig. 3I) and 1 month (Fig. 3II), with numerous internal aqueous
droplets present within oil globules themselves dispersed in
a continuous aqueous phase. The internal aqueous droplets in the
DEs with 8% (w/w) NaCl were somewhat larger after 1 month
Fig. 1. Role of salt concentration in the inner aqueous phase on the sedimentation of
W
1
/O/W
2
double emulsions stabilized with 3% gelatin. (a) 0% NaCl; (b) 2% NaCl; (c) 4%
NaCl; (d) 6% NaCl; (e) 8% NaCl. All double emulsions are 1 month old.
Fig. 2. Role of gelatin concentration in the inner aqueous phase on the sedimentation
of W
1
/O/W
2
double emulsions containing 2% NaCl. (a) 0% gelatin; (b) 3% gelatin; (c)
10% gelatin. All double emulsions are 1 month old.
L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323 318
(Fig. 3c I vs. II) compared to the other formulations [i.e, 2e6% (w/w)
NaCl], where no obvious size differences were apparent. The
internal aqueous droplets in the DEs sans gelatin were slightly
larger than in gelatin-containing emulsions (Fig. 3d vs. aec). Oil
globule occulation was pronounced in the DEs without NaCl
(Fig. 3a), with a high dispersed oil globule packing density in all
salt-containing systems (Fig. 3bed).
3.3. Droplet size characteristics
There were no changes in the oil globule size distribution of any
DE after 1 month of storage (not shown). The initial oil globule size
distributions (Fig. 4) of all DEs containing NaCl were bimodal with
a dominant w15e200 mmdistributionand a small distributionin the
w2e15 mm range. In the DEs prepared without NaCl, the dominant
mode was at 2e20 mm, with several smaller modes. Thus, these DE
droplet size distributions were not bimodal. The initial average oil
globule size [D (4,3)] increased from w45 mm to w70 mm with an
increase in salt concentration from 2% (w/w) to 8% (w/w) (Table 1),
which was corroborated by the oil globule size distributions also
shifting to larger values with more added NaCl (Fig. 4a) (p < 0.05).
DEs containing 2% (w/w) NaCl stabilized with varying amounts of
gelatin demonstrated similar oil globule size distributions, with
a dominant 10e100 mm distribution and a small distribution in the
2e10 mm range (Fig. 4b). In all DEs, the average oil globule sizes after
days 1 and 29 were not statistically signicantly different (p > 0.05),
except for the sample with 2% NaCl and 10% gelatin (p < 0.05)
(Table 1). Thus, all DEs were considered kinetically-stable.
3.4. NaCl release and kinetics
NaCl release for 48 h after DE preparation showed an initially
rapid ux followed by a gradual rise towards a plateau (Fig. 5).
Release after 30 min was 2.6e2.9% with plateau values lower at 8%
than 2% (w/w) salt (4.5 vs. 5.1%) (Fig. 5a). Fig. 5b shows that
increasing the gelatin concentration within the internal aqueous
phase slowed salt release. However, release from these DEs was
only recorded for 20 h as the gelatin-free DEs phase-separated.
The release proles of all salt-containing DEs stabilized with
gelatin were normalized and tted using Fujitas model (curve
tting examples shown in Fig. 6) to determine the diffusion
parameters D
eq
and b (Table 2). Application of this model yielded
correlation coefcients (r
2
) > 0.99 for all release curves. The NaCl
diffusion coefcient increased from 4.7 10
11
cm
2
/s for the DE
with 2% (w/w) NaCl to 6.0 10
11
cm
2
/s in the 8% (w/w) NaCl DE.
The impact of gelatin concentration on NaCl release rate was slight
with D
eq
diminishing from4.7 10
11
cm
2
/s for the DE with 3% (w/
w) gelatin to 4.1 10
11
cm
2
/s in the 10% (w/w) gelatin DE.
b parameter estimates were very similar and could not be used to
distinguish differences in release proles between the various DEs.
3.5. NaCl encapsulation efciency
The NaCl encapsulation efciency of all DEs remained at 94e95%
during the month-long study irrespective of NaCl load and gelatin
concentration in the inner aqueous phase (Table 3). However, the
small variations observed (DEE < 1%) still proved to be statistically
signicant (p < 0.05).
4. Discussion
The presence of gelatin and salt played a key role in DE stability
and release (Fig. 7). Salt (Fig. 7a) likely enhanced emulsier efcacy
and affected Laplace pressure. Aronson and Petko (Aronson &
Petko, 1993) reported that electrolytes could increase emulsier
adsorption density at the oil/water interface and reduce interfacial
tension in W/O emulsions. Scherze et al. showed that addition of
NaCl to the dispersed phase of Pgpr-stabilized W/O emulsions was
essential in preventing dispersed water droplet coalescence
(Scherze, Knoth, & Muschiolik, 2006). Parallel experiments in our
lab (results not shown) also established a synergistic effect
between NaCl and Pgpr on canola oil-based W/O emulsions, with
these demonstrating remarkable sedimentation and droplet size
stability. Such stable primary (W/O) emulsions were crucial to
obtaining stable double W
1
/O/W
2
emulsions.
Rosano et al. found that electrolytes could stabilize W/O emul-
sions by counter-balancing the Laplace pressure differences
between water droplets, thus preventing Ostwald ripening
(Rosano, Gandolfo, & Hidrot, 1998). Similarly, Kanouni et al.
(Kanouni, Rosano, & Naouli, 2002) found that DE stability required
a balance between the Laplace and osmotic pressures (between W
1
droplets in O and between W
1
droplets and the external aqueous
phase W
2
). Thus, the presence and concentration of salt in W
1
played a critical role in balancing these effects, with excess salt
resulting in water migration from W
2
to W
1
and subsequent
swelling of the W
1
/O droplets (Lutz & Garti, 2006; Rosano et al.,
1998).
In DEs with gelatin and no salt (Fig. 7b), gelation of the inner
aqueous phase enhanced DE stability (Fechner et al., 2007;
Muschiolik et al., 2006) and increased NaCl encapsulation ef-
ciency. Instability of ungelled DEs was also evident based on NaCl
release behaviour, which was much more rapid in comparison to
gelatin-stabilized DEs (Fig. 5b).
The amount of gelatin present also altered DE release behaviour
and stability. The overall fraction release of NaCl for the rst 20 h
was much slower in DEs stabilized with 10% gelatin compared to
with 3% gelatin (Fig. 5b), likely due to a rmer inner aqueous gelled
phase that retarded possible salt release during secondary
homogenization. The encapsulation efciency of the DEs with 10%
(w/w) gelatin was slightly higher than with 3% (w/w) gelatin
(p < 0.05).
Table 1
Double emulsion sedimentation stability and average oil globule sizes after days 1 and 29.
Double emulsion Sedimentation stability (S), % Average oil globule size (D (4,3)), mm
Day 1 Day 29 Day 1 Day 29
0% NaCl, 3% gelatin 44.8 0.4 28.5 0.4 12.35 0.34
a,1
13.08 0.91
a,1
2% NaCl, 3% gelatin 100 100 46.37 1.27
a,2
46.02 0.53
a,2
4% NaCl, 3% gelatin 100 100 56.64 0.82
a,3
57.28 1.40
a,3
6% NaCl, 3% gelatin 100 100 65.79 0.87
a,4
65.66 1.53
a,4
8% NaCl, 3% gelatin 100 100 70.50 0.77
a,5
70.73 0.90
a,5
2% NaCl, 10% gelatin 100 100 47.70 0.35
a,6
48.56 0.44
b,6
2% NaCl, 0% gelatin 90.2 0.4 78.5 0.5 41.81 0.74
a,7
41.98 1.55
a,7
a,b
Means within the same row without a common letter are signicantly different (p < 0.05).
1,2
Means within the same column without a common number are signicantly different (p < 0.05).
L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323 319
Salt-gelatin interactions also affected DE stability and release
behaviour (Fig. 7c). For example, inner water droplets containing
both salt and gelatin were densely-packed within the oil droplets
due to enhanced gelatin swelling in the presence of salt.
Kawashima et al. noted that W
1
/O/W
2
emulsions with a hyper-
tonic inner aqueous phase were stable due to a swelling-induced
increase in viscosity compared to emulsions with an isotonic or
hypotonic inner aqueous phase (Kawashima, Hino, Takeuchi, &
Fig. 3. Double emulsion microstructure prepared using various NaCl and gelatin concentrations incorporated in the inner aqueous phase. (a) 0% NaCl and 3% gelatin; insets are
higher magnication regions of the oil globules; (b) 2% NaCl and 3% gelatin; (c) 8% NaCl and 3% gelatin; (d) 2% NaCl and 0% gelatin. (I) day 1; (II) day 29. The W
1
/O/W
2
double
emulsions prepared using 3% gelatin combined with 4% and 6% NaCl or with 10% gelatin and 2% NaCl are not shown due to their similar appearance to the samples shown. Bar is
40 mm.
L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323 320
Niwa, 1992). The increase in oil globule size with more NaCl
added resulted from the higher osmotic pressure gradient
experienced between the two aqueous phases, implying greater
water diffusion from the external to the internal aqueous phase
(Mezzenga, Folmer, & Hughes, 2004). This water ux resulted in
the swelling and expansion of the oil globules and a consequent
increase in their packing density (Chanamai & McClements,
2000) because the polydispersed aqueous droplets effectively
lled the available space within the oil globules (Das & Ghosh,
1990). In this regard, NaCl screening of the charged sites
present on the gelatin polymer chains allowed them to more
freely re-organize and swell further (Chatterjee & Bohidar, 2006).
Yet, this mechanism applied only to DEs containing 3% gelatin
and 2e8% (w/w) NaCl [which all showed unchanging oil globule
distributions over time (p > 0.05) (Table 1)]. For DEs with 10% (w/
w) gelatin, there was a signicant difference in D (4,3) between
days 1 and 29 (p < 0.05), suggesting that an increase in gelatin
beyond a critical concentration resulted in an osmotic pressure
imbalance between the two aqueous phases and oil globule
coalescence during storage.
Fig. 4. Initial oil globule size distributions [D (4,3)] of W
1
/O/W
2
double emulsions. (a)
W
1
/O/W
2
double emulsions stabilized with 3% gelatin combined with various NaCl
concentrations entrapped in the inner aqueous phase. (C-C) 0% NaCl; (V-V) 2% NaCl;
(---) 4% NaCl; (>->) 6% NaCl; (:-:) 8% NaCl. (b) W
1
/O/W
2
double emulsions
containing 2% NaCl stabilized with various gelatin concentrations. (C-C) 0% gelatin;
(V-V) 3% gelatin; (---) 10% gelatin.
Fig. 5. Release patterns of W
1
/O/W
2
double emulsions examined via conductivity. (a)
W
1
/O/W
2
double emulsions stabilized with 3% gelatin combined with various NaCl
concentrations entrapped in the inner aqueous phase. (C-C) 2% NaCl; (V-V) 4% NaCl;
(---) 6% NaCl; (>->) 8% NaCl. (b) W
1
/O/W
2
double emulsions containing 2% NaCl in
the inner aqueous phase stabilized with various gelatin concentrations. (C-C) 0%
gelatin; (V-V) 3% gelatin; (---) 10% gelatin.
Fig. 6. Normalized release prole of W
1
/O/W
2
double emulsions tted with Fujitas
model. (C-C) 2% NaCl and 10% gelatin; (B-B) 8% NaCl and 3% gelatin. (d) Fit from
Fujitas model.
L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323 321
The incorporation of NaCl as a marker in the internal aqueous
phase induced an osmotic gradient that led to the migration of
external aqueous phase into the internal droplets. The osmotic
gradient increased from w685 to 2735 mOsmol by the
incorporation of 2e8% (w/w) NaCl. This was 4e15higher than the
recommended osmotic gradient range (180e200 mOsmol) for
obtaining stable DEs (Muschiolik et al., 2006). However, these
emulsions still showed long-term stability and no oil globule
breakdown or phase separation was visible upon swelling. This
indicated that the Pgpr likely provided an expandable and
compressible interfacial layer that covered the internal aqueous
droplets after swelling.
Initial NaCl release from the DEs increased from 2.6% to 2.9%
with an increase in NaCl concentration from2% to 8% (w/w) (Fig. 5),
which was presumably due to loss of NaCl from the internal
aqueous phase during second-stage homogenization. Additionally,
it has been reported that low molecular weight compounds may
undergo rapid release as a result of a high osmotic and/or increased
concentration gradient (Lakkis, 2007), so that an initial release of
solute was foreseeable in this system. However, the DEs still
retained >94% salt after 1 month (Table 3).
Salt diffusion from the inner aqueous droplets to the external
aqueous phase was successfully modelled with the Fujita model
(r
2
> 0.99 at all salt loads), which is based on Ficks second law. As
the internal aqueous droplet and oil globule size and number
hardly changed over a month, this suggested that NaCl release was
governed by diffusion and not by droplet rupture or erosion
(Bonnet et al., 2009; Magdassi & Garti, 1984).
The DEs with the higher salt loads reached lower plateau
concentrations, despite their higher initial release (Fig. 5). As the
gelatin concentration was constant [3% (w/w)], this may have been
due to an increased osmotic gradient that induced water droplet
swelling, and consequently lengthened the diffusion path of the
entrapped salt. The DEs with no gelatin showed a higher release
Table 2
Diffusion parameter estimates based on the Fujita model. Error reported as 95%
condence intervals.
Double emulsion D
eq
10
11
(cm
2
/s) eb r
2
2% NaCl, 3% gelatin 4.68 0.13 2.28 0.997
4% NaCl, 3% gelatin 5.25 0.11 2.28 0.998
6% NaCl, 3% gelatin 5.29 0.18 2.45 0.994
8% NaCl, 3% gelatin 5.97 0.22 2.38 0.992
2% NaCl, 10% gelatin 4.12 0.05 2.50 1.000
Table 3
NaCl encapsulation efciency after days 2 and 29.
Samples Encapsulation Efciency, %
Day 2 Day 29
2% NaCl, 3% gelatin 94.86 0.02
a,1
94.70 0.06
b,1
4% NaCl, 3% gelatin 94.95 0.02
a,2
94.10 0.04
b,2
6% NaCl, 3% gelatin 95.07 0.03
a,3
94.35 0.11
b,3
8% NaCl, 3% gelatin 95.30 0.00
a,4
94.22 0.02
b,2,3
2% NaCl, 10% gelatin 95.75 0.06
a,5
95.07 0.14
b,4
a,b
Means within the same row without a common letter are signicantly different
(p < 0.05).
1,2
Means within the same column without a common number are signicantly
different (p < 0.05).
Fig. 7. Proposed (de-)stabilization mechanisms of W
1
/O/W
2
double emulsions. (a) no gelatin and with NaCl; (b) no NaCl and with gelatin; (c) with gelatin and with NaCl.
L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323 322
rate that was highly variable due to phase separation. Conversely,
DEs stabilized with 10% gelatin showed a highly-reproducible
lower initial release and a more delayed release prole than DEs
stabilized with 3% gelatin (Fig. 5b; Table 2).
5. Conclusions
We designed food-grade W
1
/O/W
2
DEs with long-term stability
and desirable controlled release behaviour using a simple, repro-
ducible approach. DE stability was dependent on the presence of
NaCl and gelatin in the inner aqueous phase, with the diffusion-
controlled release behaviour of these DEs governed by Fickian
diffusion. These results clearly demonstrated that the presence of
a gelling agent and electrolyte could be used to modulate the
release behaviour of incorporated compounds from W
1
/O/W
2
DEs.
With their high encapsulation efcacy, the potential use of these
DEs for food applications appears promising. However, shelf-life
stability at higher temperatures, exposure to typical unit opera-
tions (e.g. heat treatment) and organoleptic properties must be
ascertained prior to their successful usage in foods.
Acknowledgements
Financial support from the Natural Science and Engineering
Research Council (NSERC) of Canada, the Advanced Foods and
Materials Network (AFMNet), and Ryerson University is
acknowledged.
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