Stability mechanisms of liquid water-in-oil emulsions

F.Y. Ushikubo, R.L. Cunha

*
Faculty of Food Engineering, University of Campinas, 13.083-862 Campinas, SP, Brazil
a r t i c l e i n f o
Article history:
Received 2 June 2012
Accepted 12 November 2012
Keywords:
Water-in-oil emulsion
Emulsion stability
Polyglycerol polyricinoleate
Sorbitan monooleate
Lecithin
a b s t r a c t
Although the stability of emulsions is widely discussed in the literature, most of them dealt with oil-in-
water emulsions or water-in-oil systems with solid and semi-solid structures, which are easier to
stabilize. In this study, the stability mechanism of liquid water-in-oil emulsions was investigated in
different systems. The combination of two different oils (soybean oil and hexadecane) and three
emulsiers (PGPR, Span 80 and lecithin) at two water:oil ratios was investigated. Emulsions with higher
kinetic stability e water and soybean oil emulsion stabilized with PGPR and water and hexadecane with
Span 80 e presented an interface with low initial interfacial tension and practically constant complex
viscoelastic modulus with time. Therefore, small droplets were formed and their coalescence was
hindered by a stable elastic interface. The molecular structure of both oil and emulsier were important
to dene the emulsion stability. Better chemical afnity of the hydrophobic moieties of the emulsier
and the oil led to more stable interface. Steric stability was obtained in more viscous systems, such as
those at higher water volume fraction content. However, the water incorporation capacity into the
emulsion depended on the molecular structure of hydrophilic portion of the emulsier. Moreover, the
water e soybean oil systems with Span 80 or lecithin emulsiers did not form a macroemulsion, but
a gelled structure. This study discussed the many factors that affect the emulsion formation and stability,
which can contribute to the development of new water-in-oil emulsion based products with higher
stability.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
There are many products in the pharmaceutical, cosmetic and
food industry that contain water-in-oil (W/O) emulsions. The
stability mechanism of those emulsions differs from the oil-in-
water (O/W) emulsions, which can be stabilized by both steric
and electrostatic repulsion. In the case of W/O emulsions, only
steric forces are expected to stabilize the emulsion, because of the
low electrical conductivity of the continuous phase (Claesson,
Blomberg, & Poptoshev, 2004). In fact, most of the W/O emulsion
based products are in the solid or semi-solid state, such as butter
and margarine, and are stabilized by fat crystallization, which
prevents the sedimentation of water droplets by the formation of
a three-dimensional network (McClements, 2005; Rousseau, 2000).
However, uid W/O emulsions generally present low stability
because of the high mobility of water droplets, which can easily
sediment, occulate or coalesce. A number of studies about emul-
sions focuses on O/Wsystems, but there are fewworks about liquid
W/O emulsions. A better understanding about the interactions
between water, oil and emulsier at the interface and the factors
that affect the emulsion stability would allow producing stable
liquid W/O emulsions and, therefore, would encourage the devel-
opment of new products and applications.
The emulsiers used to prepare W/O emulsions have low
hydrophileelipophile balance (HLB). Among the hydrophobic food-
grade emulsiers, lecithin is the only natural one (Dickinson, 1993).
It is an emulsier with amphoteric character contained in different
natural sources, such as soybean and egg, and is composed by
a mixture of phospholipids. Its main component is the phosphati-
dylcholine (PC) (Fig. 1a), followed by phosphadylethanolamine (PE)
(Fig. 1b), phosphatidylinositol (PI), phosphatidic acid (PA) and other
minorities. The fatty acid composition of soybean lecithin is similar
to that of the soybean oil (Bergensthl & Fontell, 1983). Lecithin is
soluble in mineral oils, but practicable insoluble in non-heated
vegetable oils. It is also insoluble but highly dispersible in water
(Wendel, 2000). Moreover, a powerful hydrophobic emulsier used
in food industry is the polyglycerol polyricinoleate (PGPR), an
oligomeric and non-ionic emulsier (Fig. 2a) produced by the
esterication of castor oil fatty acid (Fig. 2b) with polyglycerol. With
lecithin, PGPR is widely used to reduce the viscosity in chocolate
products (Weyland & Hartel, 2008). Other food-grade emulsier
* Corresponding author. Tel.: 55 19 3521 4047; fax: 55 19 3521 4027.
E-mail address: rosiane@fea.unicamp.br (R.L. Cunha).
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http://dx.doi.org/10.1016/j.foodhyd.2012.11.016
Food Hydrocolloids 34 (2014) 145e153
with low HLB is sorbitan fatty acid esters, which are monomeric
and non-ionic emulsiers. Particularly, sorbitan monooleate (Span
80) is obtained from a reaction between sorbitol and fatty acids
(Fig. 3). PGPR and Span 80 are soluble in mineral and vegetable oils,
but insoluble in water. All those emulsiers are commonly used in
the food industry, such as in bakery, dairy, confectionary and oil
processing (Bueschelberger, 2006; Cottrell & van Peij, 2006), in
the cosmetic industry, such as skin care creams, lip balms,
sunscreens, shampoos and soaps, and in pharmaceuticals (Tadros,
2008; Wendel, 2000).
Furthermore, the bulk physicochemical characteristics are also
important for the formation and stability of the emulsion. For
example, the density is decisive in the stability to sedimentation,
the viscosity has inuence in the droplets breakup and the mobility
of droplets, and the oil polarity can affect the interfacial tension
with the aqueous phase and the partitioning of the components at
the interface. Besides, the chemical structure, such as fatty acids
chain length, number of unsaturations, molecule conguration
have also effect in the stability of the emulsion. Many studies use
model systems with highly puried oils and with known chemical
composition, but the behavior of those systems is different fromthe
complex food-grade oils (McClements, 2005).
Thus, this study is aimed to evaluate the stability of W/O
emulsions formulated with different types of oil and emulsiers at
two water:oil ratios. In order to understand the stability mecha-
nisms, the interface of the systems was studied regarding the
interfacial tension and the viscoelastic properties. Besides, the
structure of the emulsions was analyzed microscopically through
images and particle size distribution, and macroscopically, by the
rheology of the emulsions.
2. Materials and methods
Oils with different characteristics were chosen to prepare the
emulsions: n-hexadecane (C
16
H
34
) (Sigma Aldrich, USA), a linear
chain hydrocarbon (molecular weight, MW 226.4 g mol
1
), with
very low polarity and viscosity (3 mPa s); and soybean oil (Soya,
Bunge Alimentos S.A., Brazil), a triglyceride composed mainly by
43e56% linoleic (C
18:2
), 15e33% oleic (C
18:1
), 7e11% palmitic (C
16:0
)
and 5e11% a-linolenic (C
18:3
) acids (Poth, 2001), (average
MW 278.2 g mol
1
) with higher polarity and viscosity (50 mPa s)
than the former oil. Deionized water was used as aqueous phase.
The emulsiers were sorbitan monooleate (Span 80), HLB
4.3, MW 428.6 g mol
1
, kindly provided by Croda do Brasil
(Brazil); natural lipophilic lecithin (Solec SG), HLB 4, average
MWy750 g mol
1
, with phospholipid composition of 16% (w/w)
phosphadidylcholine (PC), 14% phosphatidylethanolamine (PE), 9%
phosphatidylinositol (PI) and 5% phosphatidic acid (PA), according
to the supplier (Solae, Brazil) and polyglycerol polyricinoleate
(GRINDSTED

PGPR), HLB 1.5e2.0, average MW y 3000 g mol

1
,
donated by Danisco Brasil Ltda (Brazil).
2.1. Emulsion preparation
Emulsions were prepared at 30:70 and 60:40 (w
water
/w
oil
) ratios,
aiming to evaluate the effect of the volume fraction of water in
the W/O emulsion stability. In order to observe the interaction
between the emulsier and the oil, each emulsier (Span 80, leci-
thin and PGPR) was used to stabilize water-in-oil (W/O) emulsions
composed by water as aqueous phase and n-hexadecane or soybean
oil as the oil phase.
For the emulsion preparation, the emulsier was previously
dissolved into the oil. Then, the aqueous phase was added dropwise
to the oil phase while homogenizing in a rotor-stator system (Ultra
Turrax T18, IKA, Germany) at 14,000 rpm. When the aqueous phase
was completely incorporated into the emulsion (after 10 min at
30:70 water:oil ratio and after 20 min at 60:40), the rotational
speed was decreased to 11,000 rpm and the system was homoge-
nized for 4 min. The emulsions were prepared at roomtemperature
(25 1

C).
2.2. Interfacial tension and dilational rheology
The interfacial tension was measured at ambient temperature
(25 1

C) using the pendant drop method using a Tracker-S
tensiometer (Teclis Sarl, France), in which the deionized water
was injected with a syringe at a volume of 5 ml and 1.5 ml for soybean
oil and hexadecane systems with emulsiers, respectively, and 25 ml
for oil pure systems. For the calculation of the viscoelastic proper-
ties, an oscillation of 10% of the volume was set at a frequency of
0.2 Hz.
2.3. Evaluation of kinetic stability
The phase separation of the emulsions was observed in a 25 mL
cylindrical tube (internal diameter 17 mm), sealed with a plastic
cap and stored for 14 days at 25

C. The oil phase separation was
expressed in sedimentation index (SI), which is calculated as the
(a) (b)
O
O
R
O
R
O
O P O
N
+
(CH
3
)
3
O-
OH
O
O
R
O
R
O
O P O
O-
OH
NH
3
+
Fig. 1. Formula of (a) phosphatidylcholine and (b) phosphatidylethanolamine, in which RCOOe are fatty acid residues.
(a) (b)
RO O
R
OR
n
OH
O
OH
Fig. 2. (a) Formula of PGPR, in which R is a hydrogen, ricinoleic acid or polyricinoleic acid. (b) Formula of ricinoleic acid.
F.Y. Ushikubo, R.L. Cunha / Food Hydrocolloids 34 (2014) 145e153 146
relation between the upper oil phase height (H) and the initial
height (H
0
), as described by Eq. (1):
SI % H=H
0
*100 (1)
In the case of emulsions in which the aqueous phase separated
from the emulsion, the percentage of aqueous phase volume in
relation to the total volume was calculated.
2.4. Optical microscopy
The emulsion microstructure was observed through an optical
microscope (Axio Scope.A1, Carl Zeiss, Germany) at 100 magni-
cation for qualitative evaluation. The images were captured with
the software AxioVision Rel. 4.8 (Carl Zeiss, Germany).
2.5. Particle size distribution
The particle size distribution was determined by the laser
diffraction method using a Mastersizer 2000 (Malvern Instruments
Ltd., UK). The emulsions were dispersed in their respective
continuous phase oil: soybean oil and hexadecane (refractive
indexes 1.467 and 1.435, respectively). In order to avoid the pres-
ence of bubbles, ultrasound was applied for 5 min in the dispersion
medium. The measurements were done at laser obscuration
between 5 and 15% and at 1750 rpm agitation speed. The particle
size distribution was presented in volume fraction, considering the
parameters of water droplets (refractive index 1.33 and
absorption 0.01).
The particle size was represented as volume-weighted mean
diameter (d
43
), dened by Eq. (2):
d
43

P

n
i
$d
4
i

n
i
$d
3
i
; (2)
in which n is the number of d diameter droplets.
The polydispersion of the size distribution as evaluated through
the span value, shown in Eq. (3):
span
d
90
d
10

d
50
; (3)
in which d
10
d
50
and d
90
are the particle diameter belowwhich 10%,
50% and 90% of the sample lies, respectively.
2.6. Rheological essays
Flow curves were obtained by a stress-controlled rheometer
(Physica MCR301, Anton Paar, UK) at 25

C. The shear rate varied in
the range between 0 and 300 s
1
and the ow curves were
obtained in sequential three steps: up-down-up. The second up
curve was tted to the HerscheleBulkley model:
s s
0
k _ g
n
; (4)
in which s is the shear stress (Pa), s
0
is the yield stress (Pa), _ g is the
shear rate (s
1
), k is the consistency index (Pa s
n
) and n is the ow
index.
2.7. Statistical analysis
The difference in the results of particle size distribution and
rheological parameters was statistically analyzed through the
Tukey test, using the software Statistica 7.0 (Statsoft Inc., USA).
3. Results
3.1. Interfacial tension
The interfacial tension measured at the waterepure soybean oil
interface was about 22 mN/m, while at waterehexadecane inter-
face was 40 mN/m. When the emulsiers were added, all of them
showed a considerable reduction of the interfacial tension (Fig. 4).
At wateresoybean oil interface, the initial interfacial tension was
around 6.7 mN/m with PGPR and 2.9 mN/m with Span 80. In the
latter system, an opaque lm formed at the interface during the
tension measurement. The rigid interface of wateresoybean oil
stabilized with Span 80 could have contributed to lower the
interfacial tension. The interfacial tension of both systems
decreased with time, reaching values of 2.7 mN/m with PGPR
and 2.3 mN/m with Span 80 in the stability. The initial interfacial
tension at waterehexadecane interface was approximately
5.9 mN/m with PGPR and 6.1 mN/m with Span 80 and after 1 h
decreased to 3.2 and 3.6, respectively. Lecithin showed low inter-
facial tension at wateresoybean oil interface (1.2 mN/m) at
stability, although the initial tension was higher (9.2 mN/m) than in
other emulsiers systems. At waterehexadecane interface with
lecithin, the initial interfacial tensionwas even higher (24.1 mN/m),
thus the mobility of lecithin was much lower. The interfacial
tension of that system at the stability was 2.8 mN/m. Comparing all
the systems, the interfacial tension of waterehexadecane in the
stability condition was higher than that of wateresoybean inter-
face, due to the less polar character of hexadecane.
3.2. Interfacial dilational rheology
The complex viscoelastic modulus of the wateresoybean oil
with PGPR showed a slight decrease along time, keeping a value of
Fig. 3. Formula of span 80.
1
2
3
4
5
6
7
0 500 1000 1500 2000 2500 3000 3500 4000
I
n
t
e
r
f
a
c
i
a
l

T
e
n
s
i
o
n
,

m
N
/
m

Time, s
0
5
10
15
20
25
0 500 1000 1500 I
n
t
.

T
e
n
s
i
o
n
,

m
N
/
m

Time, s
Fig. 4. Interfacial tension at soybean oilewater (full symbols) and hexadecaneewater
(open symbols) interfaces with (A) PGPR, (-) Span 80 and (C) lecithin. The detail
shows the curve with larger scale related to the hexadecaneewater interface with
lecithin.
F.Y. Ushikubo, R.L. Cunha / Food Hydrocolloids 34 (2014) 145e153 147
approximately 5 mN/m (Fig. 5). This value was the highest among
the evaluated systems. At waterehexadecane interface with PGPR,
the complex modulus increased with time from about 1.5 mN/m to
4 mN/m. On the other hand, Span 80 at wateresoybean oil interface
led to a steep decrease in complex viscoelastic modulus with time,
reaching values near zero. At waterehexadecane interface, a low
viscoelastic modulus (about 2 mN/m) was kept constant along
time. The modulus at the wateresoybean oil interface with lecithin
decreased from a high value (about 35 mN/m) to decrease up to
values near 2 mN/m. The results of the waterehexadecane interface
with lecithin oscillated between 20 and 100 mN/m, although those
results are not so reliable, since the surface area of the drop
extensively changed with time (data not shown).
3.3. Kinetic stability
The stability of the emulsions was observed for 14 days. Most
emulsions destabilized only through sedimentation mechanism.
However, lecithin formulations with high water content showed
aqueous phase separation, resulting in a three-phase system. The
appearance of the emulsion and the sedimentation index (SI) and
aqueous phase separation at the equilibrium are presented
in Table 1. The oil phase separation along time is shown in Fig. 6a
and b, which presents the data of emulsions prepared with soybean
oil and hexadecane, respectively.
After the 14-day storage, each oilesurfactant combination
resulted in products with distinct appearance. Those prepared with
PGPR were a homogeneous liquid. Similar aspect was observed in
all waterehexadecane systems. However, the wateresoybean oil
emulsions with Span 80 or lecithin showed a gel-like structure. In
the Span 80 system at 60:40 W:O ratio the structure was a rmer
gel (Table 1).
The emulsions produced with PGPR and soybean oil were the
most stable, resulting in low SI at both W:O ratios through the
whole observation time (Table 1, Fig. 6a). The systems with Span 80
and soybean oil presented a faster phase separation followed by
a tendency to stabilize with time. The phase separation was more
accentuated at 30:70 than at 60:40 W:O ratio. In turn, lecithin
emulsions produced with soybean oil showed slower phase sepa-
ration rate than emulsions with Span 80, mainly at 30:70 W:Oratio.
However, differently from Span 80 emulsions, the phase separation
in lecithin systems did not stabilize with time. In the last observa-
tion day, the 30:70 sample had higher SI than the equivalent Span
80 system(Table 1). Moreover, at 60:40 W:O ratio, the separation of
the aqueous phase indicated that lecithin could not stabilize the
water droplets, oppositely to PGPR and Span 80, which showed
higher stability in emulsions at higher water content.
In general, the phase separation of emulsions prepared with
hexadecane was much faster than in the case of the emulsions
prepared with soybean oil, mainly at 30:70 W:O ratio, in which the
sedimentation index was higher than 40% in less than one day
(Fig. 6b). The emulsions with hexadecane and lecithin were the
most unstable, since practically instantaneous phase separation
was observed. Besides, similarly to the soybean oil emulsion with
the same emulsier, there was aqueous phase separation in the
emulsion at 60:40 W:O ratio. The PGPR emulsion with hexadecane
at 60:40 W:O ratio showed better stability than the respective
system at 30:70, but much worse than the soybean oil systems.
On the other hand, the emulsion prepared with water and
hexadecane stabilized by Span 80 at 60:40 W:O ratio showed better
kinetic stability than the respective system with soybean oil. After
two days of storage, only 0.8% of the hexadecane separated from
the emulsion, while in the wateresoybean oil system, 8.0% of the oil
had already come apart from the emulsion.
In order to understand the stabilization mechanism and the
characteristics of each watereoil systemformed using different oils
and emulsiers, the structure and viscosity of the emulsions was
studied in microscopic and rheological approaches.
3.4. Optical microscopy imaging and particle size distribution
The microscopic images of the emulsions with soybean oil just
after the homogenization are shown in Fig. 7. It is noted that
emulsions prepared with PGPR are composed of spherical small
droplets. On the other hand, the systems composed of Span 80
presents a different structure than an emulsion, with some non-
spherical particles. Similar appearance was observed at both W:O
ratios. In the case of wateresoybean oil systems with lecithin, some
big gel-like structures were formed in the continuous phase. Those
structures agglomerated during the observation of the microscopy
image, showing the high instability of the system.
Differently from the soybean oil emulsions, the microscopic
images of emulsions produced with hexadecane showed the
formation of spherical droplets in all samples (Fig. 8). In general, the
waterehexadecaneePGPR emulsions showed larger droplets than
emulsions with soybean oil and the same emulsier. However,
waterehexadecaneeSpan 80 emulsions showed smaller droplets
than PGPR emulsions. At 30:70 W:O ratio, the droplets seem to be
occulated. The droplets of the emulsion with lecithin were very
Fig. 5. Complex dilational viscoelastic modulus at soybean oilewater (full symbols)
and hexadecaneewater (open symbols) interfaces with (A) PGPR, (-) Span 80 and
(C) lecithin. Complex elastic modulus of the hexadecaneewaterelecithin system is
not shown because it is out of the axis range.
Table 1
Appearance of W/O emulsions and Sedimentation Index (SI) and the aqueous phase separation (AS) after 14-day storage.
Type of oil Parameter 30:70 60:40
PGPR Span 80 Lecithin PGPR Span 80 Lecithin
Soybean oil Appearance Liquid Gel Gel Liquid Gel Gel
SI 2.8% 42.0% 49.6% 2.4% 16.0% 29.6%
AS e e e e e 38.4%
Hexadecane Appearance Liquid Liquid Liquid Liquid Liquid Liquid
SI 68.4% 62.8% 72.4% 24.4% 9.2% 46.0%
AS e e e e e 18.0%
F.Y. Ushikubo, R.L. Cunha / Food Hydrocolloids 34 (2014) 145e153 148
large. At 60:40 W:O ratio, the droplets are coalescing, forming
a bicontinous phase.
The particle size distribution of emulsions prepared with
soybean oil and hexadecane is shown in Fig. 9a and b, respectively.
The volume-weighted mean diameter (d
43
) and the span value are
presented in Table 2.
From those data, it is clearly seen that emulsions produced with
PGPR and soybean oil presented the smallest particles among the
systems, showing a bimodal distribution. In PGPR samples at both
W:O ratios, the rst peak mode was around 0.1e0.2 mm, which
could represent the excess of emulsier that is free or as small
aggregates in the continuous phase. This interpretation is corrob-
orated by the size distribution at lower PGPR concentration, in
which the rst peak decreases with the lower emulsier concen-
tration (data not shown). The second peak, in the range between 1
and 3 mm, represents the water droplets of the emulsions, which
matches the droplet size observed in microscopy images.
Overall, the particle size distribution of wateresoybean oil
systems with Span 80 or lecithin at both W:O ratios were similar,
with the rst peak mode in the range of 0.9e1.3 mmand other peaks
at larger particle sizes (Fig. 9a). Although the frequency of peaks
of small size is low, they cannot be neglected, since the distribution
was plotted in volume frequency. Fromthe particle size distribution
and the values of d
43
and span, it was observed that the particles
of samples at 30:70 were smaller and less polydisperse than at
60:40 W:O ratio (Table 2). Based on the optical microscopy
images, in which no spherical droplets were observed, in both
samples with Span 80 and lecithin, the particle size distribution
probably represents the distribution of aggregated water droplets
and/or small particles of the gel.
Regarding the emulsions with hexadecane, the PGPR and Span
80 emulsions showed a monomodal distribution with low poly-
dispersity (Fig. 9b). The mean diameter (d
43
) matched the droplet
size qualitatively observed in the microscopic images, in which
droplets of emulsions at 30:70 ratio were larger than those at
60:40, and droplets of Span 80 emulsions were smaller than those
of PGPR ones. In the case of lecithin emulsions, a bimodal distri-
bution was observed at both W:O ratios, with one peak mode of
5 mm and a second one near 40 mmin the 30:70 ratio emulsion, and
13 mm and 91 mm at 60:40 ratio. The bimodal distribution,
Fig. 6. Sedimentation index (SI) of W/O emulsions composed of (a) soybean oil and (b) hexadecane as oil phase stabilized by (A) PGPR, (-) span 80 and (C) lecithin. Full symbols
are related to 30:70 W:O and open symbols, to 60:40 ratios.
Fig. 7. Optical microscopy images of W/O emulsions produced with soybean oil as oil phase and different emulsiers at 30:70 and 60:40 W:O ratios. The scale bar is 10 mm length.
F.Y. Ushikubo, R.L. Cunha / Food Hydrocolloids 34 (2014) 145e153 149
combined with the microscopy images, indicates the occurrence of
occulation and coalescence of the droplets in the emulsions with
lecithin.
3.5. Rheological essays
The ow curves obtained for the emulsions with soybean oil or
hexadecane were tted to the HerscheleBulkley (HB) model and
the rheological parameters are presented in Table 3. The emulsions
produced with soybean oil and PGPR showed a Newtonian behavior
at 30:70 W:O ratio. When the water content was increased (60:40
ratio), the higher volume of water droplets became susceptible to
the shear deformation, characterizing a shearethinning uid.
Besides, an increase in the apparent viscosity was observed.
Furthermore, just after the homogenization, low viscosity uids
were obtained with water and soybean oil with lecithin, with
Newtonian behavior at 30:70 ratio and shear-thinning at 60:40
W:O ratio. Those uids were unstable and phase separated. In one
day, the systems presented yield stress and higher apparent
viscosity. That characterizes the formation of a gel network, which
was intensied along the observation time.
On the other hand, the wateresoybean oil systemwith Span 80,
mainly at 60:40 W:Oratio, presented yield stress and high apparent
viscosity just after the homogenization. The systems presented
Bingham plastic and HB uid behavior at 30:70 and 60:40 W:O
ratios, respectively. After one day, at 30:70 ratio, the rheological
characteristics practically did not change with time. At 60:40, the
apparent viscosity at 3 s
1
decreased, probably due to a change into
a weaker structure.
The waterehexadecane emulsions presented a shearethinning
behavior in most of the conditions, except at 30:70 W:O ratio
using lecithin as emulsier, which showed a Newtonian behavior.
The shearethinning behavior indicates that the droplets of water
formed in the hexadecane are affected by the shear deformation.
Most of the emulsions presented lower apparent viscosity than the
wateresoybean oil systems, since the viscosity of pure hexadecane
(0.003 Pa s) is lower than that of soybean oil (0.050 Pa s) and no
structured system was formed. Most of the samples showed an
Fig. 8. Optical microscopy images of W/O emulsions produced with hexadecane as oil phase and different emulsiers at 30:70 and 60:40 W:O ratios. The scale bar is 10 mm length.
Fig. 9. Particle size distribution of W/O emulsions composed of water and (a) soybean oil or (b) hexadecane stabilized by (A) PGPR, (-) span 80 and (C) lecithin. Full symbols are
related to 30:70 and open symbols, to 60:40 W:O ratios.
F.Y. Ushikubo, R.L. Cunha / Food Hydrocolloids 34 (2014) 145e153 150
increase in the apparent viscosity after one day because of the oil
phase separation. The exceptions were the PGPR and Span 80
emulsions at 60:40 W:O ratio, which were the most stable among
the hexadecane systems.
3.6. Discussion
Among the systems in which emulsions were formed, 60:40
W:O ratio emulsions were more stable than 30:70 ones. The main
reason is that, at 60:40 ratio, with larger volume and number of
water droplets, the apparent viscosity increased, intensifying the
hydrodynamic interaction, which led to a lower sedimentation rate.
In the same way, a faster destabilization of emulsions with hex-
adecane occurred because this oil has lower viscosity than the
soybean oil, which decreases steric stabilization. Besides, the higher
interfacial tension of the waterehexadecane interface and the
bigger difference between the water and hexadecane densities led
to a faster phase separation in the emulsions with hexadecane in
relation to those with soybean oil.
The wateresoybean oil emulsions stabilized by PGPR showed
highkinetic stability. The goodemulsifying properties of PGPRinW/
O emulsions with triglycerides as the oil phase was reported by
a number of studies (Garti, Aserin, Tiunova, & Binyamin, 1999;
Mrquez, Medrano, Panizzolo, & Wagner, 2010; Pawlik, Cox, &
Norton, 2010; Su, Flanagan, Hemar, & Singh, 2006). The high
stability is supported by the small mean droplet size distribution,
shown by both microscopic images and particle size distribution
measurements. Small water droplets are more stable in oil phase
because of the lower sedimentation speed. The droplet breakup was
favored by the fast interfacial tension drop, from 22 mN/m (watere
pure oil) to 6.7 mN/m (wateresoybean oil with PGPR) (Fig. 4).
Furthermore, the coalescence of the droplets was avoided by the
viscoelastic characteristic of the interface, shown by the relatively
high complex modulus (Fig. 5), which was kept practically constant
alongtime. The relationof the complexviscoelastic modulus andthe
stability of the emulsions was discussed in many studies in the
literature (Bouyer et al., 2011; Laza-Knoerr, Huang, Grossiord,
Couvreur, & Gref, 2011; Santini, Liggieri, Sacca, Clausse, & Ravera,
2007). The good elasticity of the interface could be attributed to
the anchoring of the PGPR hydrophobic chains into the branched
structure of soybean oil, formed by oleic fatty acids (Benichou,
Aserin, & Garti, 2001). In addition, the hydrophilic portion could
also have playedanimportant role inthe anchoringof the emulsier
molecule into the aqueous phase. The hydrophilic portion of PGPR is
constituted by polyglycerol moieties, which are relatively large. The
hydroxyl groups bond to the water molecules by hydrogen bonds,
resulting in a structured interface. The good xation of the hydro-
philic part of the emulsier withwater contributed to the stability of
emulsions with higher water volume fraction (60:40 W:O ratio).
On the other hand, the emulsions prepared with hexadecane
and PGPR were not as stable as the soybean oil emulsions. The
lower stability may be related to the linear structure of hexadecane,
which did not promote a good xation of the PGPR on the interface.
With that, a weaker oileemulsier interaction occurred at the
interface, leading to a faster destabilization.
However, Span 80 was a good emulsier in a system composed
of hexadecane at 60:40 W:O ratio. The high stability at high water
content was supported by the hydrogen bonds formed between the
hydroxyls of the hydrophilic moieties of Span 80 (sorbitan) and the
water molecules. In addition, the emulsion at this condition
showed enhanced stability due to the small droplets, as observed in
the particle size distribution and the images obtained through
optical microscopy. Those droplets were formed favored by the
rapid migration of Span 80 to the waterehexadecane interface, as
indicated by tension drop between the waterehexadecane inter-
facial tension (40 mN/m) to the initial tension with Span 80 in this
system (6.1 mN/m) (Fig. 4). Regarding the complex modulus,
although it was low (Fig. 5), it was constant with time. This indi-
cates that the interface was stable evenwith the perturbation of the
system. This stable elastic network could avoid the droplet coa-
lescence. The explanation of the structured interface can be given in
terms of the hydrophobic chainechain interaction between Span
80 and hexadecane, which was more compatible than interactions
between PGPR and hexadecane chains. As it is observed in Fig. 3,
the hydrophobic part of Span 80 molecule has only one unsatura-
tion in its hydrophobic chain, while PGPR hydrophobic components
have one unsaturation and one hydroxyl (Fig. 2), which has
a hydrophilic character. Since hexadecane is highly hydrophobic,
with no unsaturation in its structure, this would create a better
interaction with Span 80, which has a more hydrophobic chain. On
the other hand, PGPR showed more compatibility with soybean oil,
since this oil is less hydrophobic. The chain compatibility between
surfactant and oil was described by Chattopadhyay, Shah, and
Ghaicha (1992).
Table 2
Volume-weighted mean diameter (d
43
) and span value of the particle size distri-
bution of W/O emulsions. Different letters between the samples with same oil and
emulsier, at different W:O ratio, indicate signicant difference at p < 0.05.
Emulsier Soybean oil Hexadecane
30:70 60:40 30:70 60:40
d
43
(mm) Span d
43
(mm) Span d
43
(mm) Span d
43
(mm) Span
PGPR 1.15
a
1.15 1.19
a
3.54 5.29
a
1.20 4.99
b
1.14
Span 80 42.54
a
1.72 59.55
b
3.91 3.74
a
0.99 2.86
b
1.19
Lecithin 35.73
a
1.48 73.37
b
1.83 38.23
a
0.91 82.41
b
0.99
Table 3
Rheological parameters (s
0
: yield stress, Pa; k: consistency index, Pa s
n
; n: ow index; h
3
: apparent viscosity at 3 s
1
, Pa s) of W/O emulsions at two measurement times: just
after the emulsication and one day after in samples with phase separation (lower phase). Soybean oilePGPR emulsions did not showphase separation after one day. Different
letters between the same sample at different measured times indicate signicant difference at p < 0.05.
Emulsier 30:70 60:40
s
0
k n h
3
s
0
k n h
3
Soybean oil PGPR 0 0.16 1 0.16 0 2.36 0.79 1.25
Span 80 (t 0) 7.66 0.13 1 1.77
a
10.30 4.45 0.79 20.40
a
Span 80 (t 1 day) 9.64 0.12 1 2.06
b
24.70 3.49 0.69 8.16
b
Lecithin (t 0) 0 0.16 1 0.16
a
0 0.07 0.77 0.11
a
Lecithin (t 1 day) 3.00 0.29 0.87 1.21
b
2.91 0.19 0.83 0.95
b
Hexadecane PGPR (t 0) 0 0.07 0.72 0.06
a
0 0.30 0.73 0.46
a
PGPR (t 1 day) 0 0.69 0.55 0.60
b
0 0.37 0.71 0.46
a
Span 80 (t 0) 0 0.03 0.82 0.04
a
0 0.30 0.67 0.50
a
Span 80 (t 1 day) 0 0.10 0.74 0.18
b
0 0.33 0.66 0.56
b
Lecithin (t 0) 0 0.01 1 0.01
a
0 0.07 0.79 0.03
a
Lecithin (t 1 day) 0 0.30 0.71 0.23
b
0 0.93 0.57 0.67
b
F.Y. Ushikubo, R.L. Cunha / Food Hydrocolloids 34 (2014) 145e153 151
Regarding the lecithin systems, the kinetic stability was very
low, mainly inwaterehexadecane systems. The microscopic images
of the systems with hexadecane showed the formation of an
emulsion with very large and polydisperse water droplets, which
explains the fast emulsion destabilization. The reason for the big-
sized droplets was the slow migration of lecithin molecules to the
interface, which hindered the droplet breakup. The low mobility of
the emulsier was supported by the short decrease of the interfa-
cial tension of the waterepure oil interface (22 and 40 mN/m for
wateresoybean oil and waterehexadecane, respectively) to the
interface with lecithin (9.2 mN/m for soybean oil and 24.1 mN/m
for hexadecane) (Fig. 4). The slow mobility of lecithin molecules is
probably due to the presence of charged groups in the hydrophilic
moiety, which have low mobility in the hydrophobic medium. The
mobility is even lower in hexadecane, which is more hydrophobic
than soybean oil. Another reason for the low stability of lecithin
emulsions is the fact that the lecithin tested in this study did not
have its phospholipids concentration modied. As it is already
known, PC enriched lecithin is better for oil-in-water emulsions,
while lecithin with higher concentration of PE and PI are more
appropriated to produce water-in-oil emulsions (Wu & Wang,
2003). Furthermore, the aqueous phase separation observed in
emulsions with lecithin at high water content (60:40 W:O ratio)
(Table 1) suggests the weak interaction of the hydrophilic part of
the emulsier with water molecules. Lecithin hydrophilic moiety
has only one hydroxyl and ionic groups (Fig. 1), which cannot form
a structured interface as suggested in PGPR and Span 80 emulsions.
Moreover, considering the difference in the visual appearance
and in the microscopic images of the Span 80 systems prepared
with soybean oil and hexadecane, it can be seen that distinct
structures were formed. A macroemulsion was formed in the
waterehexadecane system, while a gel-like structure was observed
in the wateresoybean oil system. This gelled structure is probably
an organogel formed by self-assembly structures of Span 80
molecules in oil in the presence of water. Sorbitan esters, such as
Span 20 (Yu, Shi, Liu, & Huang, 2012), Span 60 (Murdan, van den
Bergh, Gregoriadis, & Florence, 1999) and Span 80 (Bhattacharya,
Kumar, Sagiri, Pal, & Ray, 2012) have been reported to form orga-
nogels. Similar results were observed in emulsions prepared with
lecithin. The systems with soybean oil presented a structure of
a gel, although with a different aspect from the Span 80esoybean
oil system. The microscopic images showed the formation of many
irregular-shaped aggregates, similarly as observed by Knoth,
Scherze, and Muschiolik (2005) in an emulsion prepared with
sunower oil and phosphadidylcoline-depleted lecithin. As well as
in the Span 80esoybean oil system, those aggregates are thought to
be an auto-organized structure. In fact, lecithin can form a very
wide range of structures, such as monomolecular layers, bimolec-
ular lms, liposomes and vesicles, liquid crystals, besides emul-
sions, microemulsions and organogels (Shchipunov, 1997).
According to Vintiloiu and Leroux (2008), in the presence of organic
solvents, reverse micelles are formed, which convert in cylindrical
reverse micelles with the addition of a polar solvent. Those micelles
form a gel network, resulting in a weak organogel.
4. Conclusion
This study showed that different mechanisms act on the stabi-
lization of water-in-oil emulsions. The main stability mechanism
against the sedimentation of a water-in-oil emulsion was the steric
stability, which depends on the hydrodynamic interactions
between the droplets that, in turn, is dependent on the apparent
viscosity. Higher water internal volume resulted in more viscous
emulsions, increasing the stability. On the other hand, higher
volume of less viscous oil showed rapid phase separation.
Furthermore, the rapid decrease in the interfacial tension with the
emulsier addition was also important for the formation of small
droplets, which resulted in higher stability against gravity force.
Other factor was the viscoelasticity of the lm at the watereoil
interface, which can prevent the coalescence of droplets. Moreover,
depending on the composition of the system, a gel network can be
formed, resulting in a product with different rheological charac-
teristics and distinct stability mechanisms.
Acknowledgment
The authors would like to thank FAPESP (2009/54.137-1, 2010/
16.708-4 and 2011/06.083-0) and CNPq (304611/2009-3) for the
nancial support.
References
Benichou, A., Aserin, A., & Garti, N. (2001). Polyols, high pressure, and refractive
indices equalization for improved stability of W/O emulsions for food
applications. Journal of Dispersion Science and Technology, 22, 269e280.
Bergensthl, B., & Fontell, K. (1983). Phase equilibria in the system soybean lecithin/
water. Progress in Colloid & Polymer Science, 68, 48e52.
Bhattacharya, C., Kumar, N., Sagiri, S. S., Pal, K., & Ray, S. S. (2012). Development of
span 80 e tween 80 based uid-lled organogels as a matrix for drug delivery.
Journal of Pharmacy and Bioallied Sciences, 4, 155e163.
Bouyer, E., Mekhlou, G., Le Potier, I., de Kerdaniel, T., du, F., Grossiord, J. L., et al.
(2011). Stabilization mechanism of oil-in-water emulsions by beta-lactoglobulin
and gum arabic. Journal of Colloid and Interface Science, 354, 467e477.
Bueschelberger, H. G. (2006). Lecithins. In R. J. Whitehurst (Ed.), Emulsiers in food
technology (pp. 1e39). Oxford: Blackwell Publishing, (chapter 1).
Chattopadhyay, A. K., Shah, D. O., & Ghaicha, L. (1992). Double-tailed surfactants
and their chain length compatibility in water-in-oil emulsions. Langmuir, 8,
27e30.
Claesson, P. M., Blomberg, E., & Poptoshev, E. (2004). Surfaces forces and emulsion
stability. In S. E. Friberg, K. Larsson, & J. Sjblom (Eds.), Food emulsions (4th ed.).
New York: Marcel Dekker, (chapter 7) (e-book version).
Cottrell, T., & van Peij, J. (2006). Sorbitan esters and polysorbates. In R. J. Whitehurst
(Ed.), Emulsiers in food technology (pp. 162e185). Oxford: Blackwell Publishing.
Dickinson, E. (1993). Towards more natural emulsiers. Trends in Food Science &
Technology, 4, 330e334.
Garti, N., Aserin, A., Tiunova, I., & Binyamin, H. (1999). Double emulsions of
water-in-oil-in-water stabilized by a-form microcrystals. Part 1: selection of
emulsiers and fat microcrystalline particles. Journal of the American Oil
Chemists Society, 76, 383e389.
Knoth, A., Scherze, I., & Muschiolik, G. (2005). Stability of water-in-oil emulsions
containing phosphatidylcoline-depleted lecithin. Food Hydrocolloids, 19, 635e
640.
Laza-Knoerr, A., Huang, N., Grossiord, J.-L., Couvreur, P., & Gref, R. (2011). Interfacial
rheologyas a tool to study the potential of cyclodextrinpolymers to stabilize oile
water interfaces. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 69,
475e479.
Mrquez, A. L., Medrano, A., Panizzolo, L. A., & Wagner, J. R. (2010). Effect of calcium
salts and surfactant concentration on the stability of water-in-oil (w/o)
emulsions prepared with polyglycerol polyricinoleate. Journal of Colloid and
Interface Science, 341, 101e108.
McClements, D. J. (2005). Food emulsions: Principles, practices, and techniques (2nd
ed.). Boca Raton: CRC Press (e-book version).
Murdan, S., van den Bergh, B., Gregoriadis, G., & Florence, A. T. (1999). Water-in-sor-
bitan monostearate organogels. Journal of Pharmaceutical Sciences, 88, 615e619.
Pawlik, A., Cox, P. W., & Norton, I. T. (2010). Food grade duplex emulsions designed
and stabilized with different osmotic pressures. Journal of Colloid and Interface
Science, 352, 59e67.
Poth, U. (2001). Drying oils and related products. In Ullmanns encyclopedia of
industrial chemistry (pp. 621e636). Weinheim: Wiley-VCH.
Rousseau, D. (2000). Fat crystals and emulsion stability e a review. Food Research
International, 33, 3e14.
Santini, E., Liggieri, L., Sacca, L., Clausse, D., & Ravera, F. (2007). Interfacial rheology
of Span 80 adsorbed layers at parafn oilewater interface and correlation with
the corresponding emulsion properties. Colloids and Surfaces A: Physicochemical
and Engineering Aspects, 309, 270e279.
Shchipunov, Y. A. (1997). Self-organising structures of lecithin. Russian Chemical
Reviews, 66, 301e322.
Su, J., Flanagan, J., Hemar, Y., & Singh, H. (2006). Synergistic effects of polyglycerol
ester of polyricinoleic acid and sodium caseinate on the stabilization of
water-oil-water emulsions. Food Hydrocolloids, 20, 261e268.
Tadros, T. F. (2008). Applied surfactants: Principles and applications (2nd ed.).
Weinheim: Willey-VCH.
Vintiloiu, A., & Leroux, J.-C. (2008). Organogels and their use in drug delivery e
a review. Journal of Controlled Release, 125, 179e192.
F.Y. Ushikubo, R.L. Cunha / Food Hydrocolloids 34 (2014) 145e153 152
Wendel, A. (2000). Lecithin. In Kirk-Othmer encyclopedia of chemical technology.
New York: John Wiley & Sons.
Weyland, M., & Hartel, R. (2008). Emulsiers in confectionery. In
G. L. Hasenhuettl, & R. W. Hartel (Eds.), Food emulsiers and their applications
(2nd ed.). (pp. 285e305) New York: Springer.
Wu, Y., & Wang, T. (2003). Soybean lecithin fractionation and functionality. Journal
of the American Oil Chemists Society, 80, 319e326.
Yu, H., Shi, K., Liu, D., & Huang, Q. (2012). Development of a food-grade orga-
nogel with high bioaccessibility and loading of curcuminoids. Food Chemistry,
131, 48e54.
F.Y. Ushikubo, R.L. Cunha / Food Hydrocolloids 34 (2014) 145e153 153