Journal of Colloid and Interface Science 411 (2013) 105–113

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Journal of Colloid and Interface Science

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Effect of glycerol on formation, stability, and properties of vitamin-E

enriched nanoemulsions produced using spontaneous emulsification
Amir Hossein Saberi a, Yuan Fang b, David Julian McClements a,⇑
a
Biopolymers and Colloids Laboratory, Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA
b
PepsiCo Global R&D, 100 Stevens Ave, Valhalla, NY 1059, USA

a r t i c l e i n f o a b s t r a c t

Article history: Oil-in-water nanoemulsions are finding increasing use as delivery systems to encapsulate lipophilic bio-
Received 11 May 2013 active components in functional food, personal care, and pharmaceutical products. We investigated the
Accepted 21 August 2013 influence of a water-soluble cosolvent (glycerol) on the formation, stability, and properties of vitamin
Available online 2 September 2013
E acetate-loaded nanoemulsions (VE-NEs) prepared by spontaneous emulsification. VE-NEs were formed
by titration of a mixture of vitamin E acetate, carrier oil (MCT) and non-ionic surfactant (Tween 80) into
Keywords: an aqueous glycerol solution with continuous mixing. Cosolvent concentration had an appreciable effect
Nanoemulsions
on the particle size produced, with the smallest mean droplet diameters (d < 50 nm) being formed at 40
Spontaneous emulsification
Vitamin E
and 50 wt% glycerol. Nanoemulsions (d < 100 nm) containing 10% vitamin E acetate could be produced at
Glycerol relatively low surfactant concentrations (5%) using these high glycerol levels. The turbidity of the NEs
Emulsion stability decreased at high glycerol concentrations due to the reduction in droplet size and refractive index con-
Optical properties trast. The long-term stability of the VE-NEs was strongly influenced by glycerol concentration and storage
temperature. VE-NEs containing 40% glycerol were relatively stable to droplet growth when stored at 5
and 20 °C, but a rapid increase in droplet size and turbidity occurred during storage at 37 °C. Temperature
scanning experiments (20–80–20 °C) indicated that a steep and irreversible increase in turbidity occurred
during heating, which was around 70 °C in the absence of glycerol and 60 °C in the presence of 40% glyc-
erol. Droplet instability was attributed to an increase in the rate of Ostwald ripening and/or coalescence
as the temperature was increased, associated with dehydration of the non-ionic surfactant head-group
leading to a reduction in phase inversion temperature. Dilution (100) of VE-NEs containing glycerol
with water considerably improved their stability to droplet growth, especially at high storage tempera-
tures. This study provides important information about the effect of glycerol on the formation, stability
and physical properties of VE-enriched NEs suitable for food, personal care, and pharmaceutical products.
Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction vitamins [11]. Emulsion-based systems can be fabricated using rel-

The term ‘‘Vitamin E’’ is used to describe a group of lipophilic
molecules that have related molecular structures and biological
properties, including the a, b, c, and d derivatives of tocopherol
and tocotrienol [1,2]. Epidemiological and experimental studies
have shown that a-tocopherol may play an important role in pre-
vention of chronic disease [2,3] and carcinogenesis [4–6]. However,
the utilization of a-tocopherol as a functional ingredient in food
and beverages is currently limited owing to its heat and oxygen
sensitivity, poor water-solubility, and low and variable bioavail-
ability [7–10]. A number of different a-tocopherol delivery systems
have been investigated as potential means to overcome these prob-
lems. Among them, emulsion-based systems have been identified
as being particularly suitable for the oral delivery of oil-soluble
⇑ Corresponding author. Fax: +1 (413) 545 1262.
E-mail address: mcclements@foodsci.umass.edu (D.J. McClements).
atively simple processing operations and commercially viable
ingredients.
Nanoemulsions (NEs) have similar compositions, structures,
and thermodynamic properties as the conventional emulsions that
are currently widely used as emulsion-based delivery systems in
many commercial applications [12,13]. However, these two types
of colloidal dispersion can be distinguished from each other based
on the size of the droplets they contain. Nanoemulsions are catego-
rized as having a mean droplet radius less than 100 nm (i.e., diam-
eter <200 nm), whereas conventional emulsions are categorized as
containing larger droplets [14,15]. These differences in droplet
dimensions can have a pronounced influence on the functional
properties of emulsion-based delivery systems [14,15]. Nanoemul-
sions can be produced with droplet dimensions appreciably
smaller than the wavelength of light (d  k) so that they do not
scatter light strongly, thereby making them either transparent or
only slightly turbid [14,16,17]. These systems can be used to

0021-9797/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jcis.2013.08.041
106 A.H. Saberi et al. / Journal of Colloid and Interface Science 411 (2013) 105–113

incorporate lipophilic bioactive compounds into transparent aque-
ous-based products, such as some functional foods and beverages.
The small size of the droplets in nanoemulsions may also improve
their stability to gravitational separation, flocculation, and coales-
cence when compared to conventional emulsions [15]. Finally, it
has been suggested that the bioavailability of encapsulated
lipophilic compounds increases as the droplet size in emulsions
decreases [18–20], although this is likely to be highly system
dependent [21].
In general, nanoemulsions can be prepared using two main ap-
proaches: high-energy and low-energy. High-energy approaches
utilize mechanical devices that produce intense disruptive forces
that intermingle and breakup the oil and water phases, such as
high pressure homogenizers, microfluidizers, and ultrasound gen-
erators [14,22]. In contrast, low-energy approaches mainly rely
on the internal chemical energy of the system to form small drop-
lets [14,22]. As a consequence, low energy approaches are usually
much more energy efficient and require simpler equipment than
high energy approaches [14,22–24], and they are often more effec-
tive at producing small droplet sizes [14,22]. A number of different
low-energy approaches have been developed to form NEs, includ-
ing spontaneous emulsification (SE), phase inversion temperature
(PIT), phase inversion composition (PIC), and emulsion inversion
point (EPI) methods [14,22,23]. In the current study, we focused
on the utilization of the spontaneous emulsification method as
one of the easiest and most cost-effective methods for commercial
uses, since it simply involves titrating an oil-surfactant mixture
into an aqueous solution with constant mixing.
Previous studies have shown that addition of cosolvents (such
as alcohols and polyols) can greatly influence the formation and
properties of emulsion-based systems [25–31]. In this study, we
focus on the influence of glycerol on the formation, properties,
and stability of vitamin E-loaded nanoemulsions. Glycerol is a
small water-soluble co-solvent that modifies the bulk physico-
chemical properties of aqueous solutions, such as density, viscos-
ity, refractive index, and interfacial tension [32]. The addition of
glycerol to aqueous solutions has also been shown to alter the
interfacial tension, optimum curvature, and solubility characteris-
tics of both ionic and non-ionic surfactants [33–37]. The influence
of glycerol on the properties of aqueous solutions and surfactants
would be expected to impact the tendency for small droplets to
be formed by the spontaneous emulsification method [24]. A num-
ber of studies have investigated the influence of glycerol and sim-
ilar cosolvents on various types of emulsion-based systems. The
solubilization of limonene in microemulsions formed using non-io-
nic surfactants was significantly increased in the presence of glyc-
erol [29,30]. The addition of glycerol to the aqueous phase was
shown to reduce the amount of surfactant needed for solubiliza-
tion of vitamin A in microemulsions [28]. Increasing the glycerol
concentration in the aqueous phase was found to decrease the
droplet size of oil-in-water emulsions produced using a high shear
mixer or homogenizer [25,26,38,39]. Addition of small amounts of
glycerol (1.5 wt%) was found to significantly increase the storage
stability of oil-in-water emulsions [38,40].
Recently, we showed that the spontaneous emulsification
method can be used to form VE-NEs with small mean particle
diameters (d < 50 nm). An investigation of the influence of system
composition on the formation of these NEs showed that oil phase
composition, surfactant type, and surfactant concentration all
had an appreciable effect on particle size [41]. The purpose of the
present study was to investigate the possibility of using glycerol
as a cosolvent in formulation of VE-NEs produced using spontane-
ous emulsification. In particular, our aim was to understand how
glycerol influences the formation, properties, and stability of these
nanoemulsions. Further, since emulsion-based delivery systems
are often highly diluted in commercial applications (such as
functional foods and beverages), the influence of dilution on stabil-
ity of VE-NEs in presence and absence of glycerol was also
considered.
2. Materials and methods
2.1. Materials
Vitamin E acetate (VE) was kindly donated by BASF (Ludwigsha-
fen, Germany). Medium chain triglyceride (MCT) oil (MIGLYOLÒ
812) was purchased from Warner Graham Company (Sasol Ger-
many GmbH). Non-ionic surfactant (Tween 80) was purchased from
Sigma–Aldrich Co. (St. Louis, MO). Citric acid and sodium benzoate
were provided by PepsiCo (Valhalla, NY). Glycerol was purchased
from Fisher Scientific (Pittsburgh, PA). Double distilled water was
used in the preparation of all solutions and nanoemulsions. All con-
centrations are expressed as a mass percentage (wt/wt%).
2.2. Nanoemulsion preparation
Nanoemulsion formation was carried out using a method based
on a spontaneous emulsification procedure described in a previous
study [41]. In brief, spontaneous emulsification was performed by
addition of an organic phase to an aqueous phase while magneti-
cally stirring (600 rpm) the system at 25 °C. The organic phase con-
sisted of different amounts of VE, MCT and surfactant, while the
aqueous phase was a buffer solution (pH 3.0, 0.8% citric acid,
0.08% sodium benzoate) that contained different amounts of glyc-
erol (0 to 50 wt%). Unless otherwise stated, the experiments were
carried out using the following conditions: (i) 10% oil (80%
VE + 20% MCT or 100% VE); (ii) 10% surfactant (Tween 80); and
(iii) 80% aqueous phase (0 to 50% glycerol; 50 to 100% buffer solu-
tion); (iv) a magnetic stirrer speed of 600 rpm; and, (v) a prepara-
tion temperature of 25 °C. In these samples, the oil (10 g) and
surfactant (10 g) were first mixed together and then the mixture
was slowly poured into 80 g of aqueous phase over a 15 min period
with continuous stirring. In some experiments, the effect of differ-
ent surfactant concentrations on droplet size was investigated
using the following approach. The surfactant-to-emulsion ratio
(SER) was varied, while keeping the total oil content constant
(10%): %SER = 100  mS/(mO + mW + mS). Here, mS, mO and mW are
the masses of the surfactant, oil, and water, respectively. This
was achieved by varying the relative amounts of surfactant and
aqueous phase present in the system [41]. Diluted emulsions were
prepared by dilution of the concentrated VE-NEs (dilution factor:
100) using acidic buffer solution (pH 3).
2.3. Particle size measurements
Particle size distributions were measured using a dynamic light
scattering instrument (Zetasizer Nano ZS, Malvern Instruments,
Malvern, UK). This instrument determines the particle size from
intensity-time fluctuations of a laser beam (633 nm) scattered
from a sample at an angle of 173°. Each individual measurement
was an average of 13 runs. To avoid multiple scattering effects,
samples were diluted before the particle size measurements using
acidic buffer solution (pH 3.0). The mean particle diameter (Z-aver-
age) and polydispersity index (PDI) were calculated from the par-
ticle size distribution. All measurements were conducted at
ambient temperature immediately after emulsion preparation.
2.4. Turbidity measurements
A spectrophotometric method was used to monitor changes in
the turbidity of NEs throughout one month storage. The turbidity
 A.H. Saberi et al. / Journal of Colloid and Interface Science 411 (2013) 105–113 107

120 120

100 50% 100 50%

Relative Intensity (%)

80
Relative Intensity (%)

80 40% 40%

60 30%
60 30%

40 20%
40 20%

20 10%
20 10%

0 0%
0 0% 1 10 100 1000 10000
1 10 100 1000 10000 Particle Diameter (nm)
Particle Diameter (nm)
Fig. 1b. Effect of glycerol concentration on the particle size distribution of
Fig. 1a. Effect of glycerol concentration on the particle size distribution of nanoemulsions produced using 10% oil phase (100% VE) and 10% Tween 80.
nanoemulsions produced using 10% oil phase (80% VE + 20% MCT) and 10% Tween
80.
concentration was increased from 0% to 50% (Fig. 2), indicating that
smaller droplets with a narrower particle size distribution could be
was measured at 600 nm using a UV–visible spectrophotometer formed at high glycerol concentrations. The addition of glycerol to
(Agilent 7010 Particle Size Analyzer, Agilent Technologies Inc., aqueous solutions alters many physicochemical and molecular
CA). The influence of temperature on nanoemulsion turbidity was properties that may alter the size of the droplets produced by
determined using a UV–visible spectrophotometer with tempera- spontaneous emulsification. The density, refractive index, and vis-
ture scanning capabilities (Evolution Array, Thermo Scientific). cosity of aqueous solutions increase as the glycerol concentration
The absorbance at 600 nm was measured as the temperature was increases [32]. Changes in the viscosity of an aqueous solution
increased from 20 to 80 °C at 1 °C per minute, and then decreased would be expected to influence the mass transport kinetics of sur-
back to 20 °C. factant and oil molecules at the boundary between the organic and
aqueous phases, which may influence spontaneous droplet forma-
2.5. Statistical analysis tion [42]. The addition of high levels of glycerol to aqueous solu-
tions has been shown to decrease the oil–water interfacial
All experiments were carried out at least twice using freshly tension [43], increase the critical micelle concentration [35],
prepared samples and the results are reported as the calculated decrease the cloud point [25,26], reduce the HLB number [25],
mean and standard deviation of these measurements. and alter the optimum monolayer curvature of surfactants [36].
In general, three different physicochemical or molecular phe-
3. Results and discussions nomena may account for the ability of water-soluble cosolvents
to alter the properties of surfactants in aqueous solutions [37].
3.1. Effect of glycerol on particle size
60 0.25

Initially, the influence of glycerol concentration on the initial Mean Particle Diameter
size of the oil droplets formed in NEs produced using spontaneous Polydispersity Index
emulsification was investigated. In a recent study, we showed that 0.20
Mean Particle Diameter (nm)

the smallest droplet sizes could be produced using this approach 55

Polydispersity Index

when the oil phase contained 80% VE and 20% MCT [41], and so
we used this oil phase composition in the present study. Neverthe- 0.15
less, we also used an oil phase consisting of pure vitamin E acetate
(100% VE) to compare with this system, since commercially it is of- 50
ten important to increase the loading capacity of emulsion-based
0.10
delivery systems.
The influence of glycerol on the particle size distributions of
nanoemulsions prepared using the two different types of oil phases 45
is shown in Figs. 1a and 1b. The nanoemulsions prepared from a 0.05
mixed oil phase (80% VE & 20% MCT) contained droplets that could
be characterized by a monomodal distribution that was fairly sim-
ilar at all glycerol levels (Fig. 1a). On the other hand, nanoemul- 40 0.00
sions prepared from pure vitamin E acetate (100% VE) contained 0 10 20 30 40 50
droplets that were characterized by distributions whose location Glycerol (%)
and width varied much more with glycerol concentration Fig. 2. Effect of glycerol concentration on mean particle diameter and polydisper-
(Fig. 1b). The mean particle diameter and polydispersity index of sity index of emulsions produced using 10% oil phase (80% VE + 20% MCT) and 10%
the mixed oil nanoemulsions decreased slightly when the glycerol Tween 80.
108 A.H. Saberi et al. / Journal of Colloid and Interface Science 411 (2013) 105–113
First, the presence of the cosolvent may change the solubility of the
surfactant monomers in aqueous solutions by altering the magni-
tude of the hydrophobic effect, e.g., by making the aqueous phase
a less thermodynamically unfavorable environment for non-polar
groups [43]. Second, cosolvent molecules may partially dehydrate
the hydrophilic head-groups of surfactants through their ability
to compete with water molecules, thereby altering the packing
and optimum curvature of surfactant monolayers [36]. Third, some
cosolvents can penetrate into the hydrophilic head group region
(‘‘palisade layer’’) of surfactant monolayers, thereby also altering
their packing and optimum curvature [37]. Previous studies sug-
gest that the main effects of glycerol are on the first and second
mechanisms listed above, i.e., on surfactant monomer solubility
in aqueous solutions and on surfactant head-group dehydration
[37]. The solubility and optimum curvature of surfactants in sur-
factant-oil–water mixtures are known to play an important role
in the spontaneous formation of oil droplets [22,24,44]. The fact
that glycerol can modulate these properties may therefore account
for its ability to influence the size of the droplets produced using
the spontaneous emulsification method. Nevertheless, further
work is clearly required to identify the precise molecular and phys-
icochemical mechanisms involved.
The influence of glycerol and surfactant concentration on the
mean particle diameter and polydispersity index (PDI) of nano-
emulsions prepared from pure vitamin E acetate (100% VE) is
shown in Figs. 3a and 3b. The main purpose of these experiments
was to determine whether very small VE droplets could be formed
at reduced surfactant levels by adding glycerol. Qualitatively, fairly
similar behavior was observed at all three surfactant levels: the
mean particle diameter decreased when the glycerol content in-
creased from 0% to 50% (Fig. 3a), while the PDI increased and then
decreased (Fig. 3b). Interestingly, the effect of surfactant concen-
tration on the mean particle diameter depended on glycerol con-
centration. At relatively low glycerol concentrations (620%), the
mean particle diameter increased with increasing surfactant
concentration, but at higher glycerol concentrations the opposite
effect was seen (Fig. 3a). An increase in particle size of emulsions
prepared by the low-energy method with increasing surfactant
concentration has previously been reported by our group as well
as others [41,45–47]. It has been postulated that the droplet size
may increase above a certain surfactant level due to the formation
of a highly viscous liquid crystalline phase, which makes spontane-
ous breakup of the oil–water interface more difficult [45].
180
Tween 80= 10%
160 Tween 80= 7.5%
Tween 80= 5%
Mean Particle Diameter (nm)
140
120
100
80
60
40
20
0 10 20 30 40
Glycerol (%)
Fig. 3a. Effect of glycerol concentration on mean particle diameter of nanoemul-
sions produced using 10% oil phase (VE) and different surfactant concentrations (5%,
7.5%, and 10% Tween 80).
0.45
Tween 80= 10%
0.40 Tween 80= 7.5%
Tween 80= 5%
0.35
Polydispersity Index
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0 10 20 30 40 50
Glycerol (%)
Fig. 3b. Effect of glycerol concentration on polydispersity of nanoemulsions
produced using 10% oil phase (VE) and different surfactant concentrations (5%,
7.5%, and 10% Tween 80).
At high glycerol concentrations (50%) and similar surfactant
concentrations (10%), the droplets produced using an oil phase
containing 80% VE and 20% MCT were fairly similar in size
(d = 45 nm) to those produced using 100% VE (d = 45 nm). These
experiments show that VE-enriched nanoemulsions containing
100% VE with very small droplets (d < 50 nm) can be produced
using a combination of high surfactant and high glycerol concen-
trations. They also show that the amount of surfactant required
to produce small droplets can be reduced if high levels of glycerol
are present in the aqueous phase during the spontaneous emulsi-
fication process (Fig. 3a).
3.2. Effect of glycerol on transparency of vitamin E-loaded
nanoemulsion
The optical properties of delivery systems are of particular
importance for their application in certain types of functional food
and beverage products. In some situations, the final product should
be optically transparent or only slightly turbid (e.g., vitamin-forti-
fied waters and clear juices), so the addition of any delivery system
should not make them look cloudy or opaque [14]. The influence of
glycerol concentration on the appearance of VE-NEs was therefore
investigated by preparing a series of nanoemulsions from an oil
phase containing either 80% VE & 20% MCT (Fig. 4a) or 100% VE
(Fig. 4b). For both oil phases, the optical clarity of the VE-NEs in-
creased with increasing glycerol concentration, which may be
due to two different phenomena. First, it can be attributed to the
ability of glycerol to decrease the size of the droplets in VE-NEs
(Figs. 1a–3b), thereby decreasing their scattering efficiency
[14,16,17]. Second, it can be attributed to the ability of glycerol
to increase the refractive index of the aqueous phase, which also
reduces the droplet scattering efficiency [48]. The refractive index
of aqueous glycerol solutions has been reported to increase from
1.33 to 1.40 as the solute concentration increases from 0% to
50%, whereas the refractive index of vitamin E acetate has been re-
ported to be around 1.50 [32]. Therefore, the refractive index con-
trast decreases as the glycerol concentration increases, which
would reduce the scattering efficiency of the droplets.
50 The nanoemulsions containing the mixed oil phase (80% VE &
20% MCT) appeared clearer than those containing pure vitamin E
acetate (100% VE), particularly at lower glycerol levels (Fig. 4).
The most likely origin of this difference in visual appearance is
the fact that the droplets in the mixed oil nanoemulsions
 A.H. Saberi et al. / Journal of Colloid and Interface Science 411 (2013) 105–113 109

200
a 180
5°C
20°C
37°C

Mean Particle Diameter (nm)

160

140

120

100

80

60
0 10 20 30 40 50
Glycerol (%) 40

20

b 0
Fresh 1 month (UD) 1 month (D)

Fig. 5a. Influence of storage time and temperature on mean particle diameter of
undiluted (UD) and diluted (D) nanoemulsions produced using 10% oil phase (80%
VE & 20% MCT), 10% surfactant phase (Tween 80), and 80% aqueous phase (buffer).

200

180 5°C
20°C

Mean Particle Diameter (nm)

0 10 20 30 40 50 160 37°C
Glycerol (%) 140
Fig. 4. Effect of glycerol concentration on optical transparency of nanoemulsions 120
produced using (a) 10% oil phase (80% VE and 20% MCT) and 10% surfactant (Tween
80) and (b) 10% oil phase (100% VE) and 10% surfactant (Tween 80). 100

80
(Fig. 3a) were appreciably smaller than those in the pure VE nano-
emulsions (Fig. 2) at low glycerol concentrations. For example, at 60
10% glycerol the mean droplet diameter was >170 nm in the pure 40
VE nanoemulsions, but <55 nm in the mixed oil nanoemulsions.
20
There may also have been a small contribution from differences
in the refractive index, since MCT (n = 1.44) has a lower refractive 0
Fresh 1 month (UD) 1 month (D)
index than vitamin E acetate (n = 1.50), hence the refractive index
of the mixed oil phase would be expected to be around n = 1.49. Sample
Nevertheless, this effect is expected to be quite small since in both Fig. 5b. Influence of storage time and temperature on mean particle diameter of
cases the oil phase was predominantly vitamin E acetate (either 80 undiluted (UD) and diluted (D) nanoemulsions produced using 10% oil phase (80%
or 100%), and so the refractive indices were fairly similar. At higher VE & 20% MCT), 10% surfactant phase (Tween 80), and 80% aqueous phase (40%
glycerol concentrations, both types of nanoemulsion appeared glycerol + 40% buffer).

optically clear because they both had very small droplets

(d < 50 nm).
3.3. Effect of glycerol on storage stability of vitamin E-loaded
nanoemulsions
For commercial applications, it is often important that emul-
sion-based delivery systems remain physically stable during stor-
age, transport, and utilization. Nanoemulsions may become
unstable due to a number of different physicochemical processes,
including flocculation, coalescence, Ostwald ripening, and gravita-
tional separation [49]. The rate at which a nanoemulsion breaks
down, and the mechanism by which this process occurs, depends
on its composition and microstructure, as well as on the environ-
mental conditions it experiences during its lifetime. We therefore
examined the influence of storage time and temperature on the
stability of VE-NEs. Nanoemulsions were prepared using a mixed
oil phase (80% VE & 20% MCT) or a pure vitamin E acetate oil phase
(100% VE), using either 0% or 40% glycerol in the aqueous phase.
They were then stored for a month at three different temperatures
(5, 20, and 37 °C), which were selected to represent refrigeration
conditions, ambient storage in mild climates, and ambient storage
in hot climates, respectively.
In the absence of glycerol, there was only a slight increase in
the mean particle diameter in the NEs produced using the mixed
oil phase when stored at different temperatures (Fig. 5a): d in-
creased from 58 nm initially to 62 nm at 5 °C (about 9% in-
crease), 65 nm at 20 °C (about 14% increase), and 65 nm at
37 °C (about 14% increase) after one month storage. The particle
size distribution of the nanoemulsions remained monomodal
and did not change appreciably at all storage temperatures
(Fig. 6a). If the glycerol-free nanoemulsions were diluted with
water (1-to-100) prior to storage the overall increase in mean
particle diameter after storage was appreciably less at all storage
temperatures (<5% increase). These results suggest that the glyc-
erol-free nanoemulsions were susceptible to slow droplet growth
over time, with the rate of droplet growth increasing with
increasing storage temperature, and that dilution with water
prior to storage improved their stability.
In the presence of 40% glycerol, the stability of the nanoemul-
sions to droplet growth was much more dependent on storage
temperature and dilution (Fig. 5b and Fig. 6b). At the lower storage
temperatures, there was only a slight increase in the mean particle
diameter after one month: 8% increase at 5 °C and 19% increase
at 20 °C. On the other hand, at the highest storage temperature the
110 A.H. Saberi et al. / Journal of Colloid and Interface Science 411 (2013) 105–113

80 that addition of 40% glycerol to VE-NEs remarkably decreased their

stability to droplet growth at elevated storage temperatures. Parti-
70 cle size distribution measurements showed monomodal distribu-
tions for all the VE-NEs after one month storage at different
60 37°C temperatures (Fig. 6b). The width of the particle size distributions
was also affected by storage time and temperature. For example,
Relative Intensity (%)

50 for the undiluted nanoemulsions containing 100% VE in the oil

phase and 40% glycerol in the aqueous phase, the polydispersity in-
40 20°C dex changed from 0.12 initially to 0.14, 0.13 and 0.06 after stor-
age for one month at 5, 20 and 37 °C, respectively. This result
suggests that there was a narrowing of the particle size distribu-
30
tion during storage in the nanoemulsions containing high glycerol
concentrations when they were stored at elevated temperatures.
20 5°C
We also examined the influence of dilution (1-to-100 with water)
on the stability of the nanoemulsions initially containing 40% glyc-
10
erol in the aqueous phase, since emulsion-based delivery systems
are often highly diluted in commercial applications (such as
0 FRESH
functional foods and beverages). The diluted emulsions had much
1 10 100 1000 10000
better stability to droplet growth than the undiluted emulsions,
Particle Diameter (nm)
particularly at the highest storage temperature (Fig. 5b and
Fig. 6a. Influence of storage temperature on particle size distribution of nano- Table 1). For example, the mean particle diameter of the nano-
emulsions produced using 10% oil phase (80% VE & 20% MCT), 10% surfactant phase emulsion containing 40% glycerol after one month storage was
(Tween 80), and 80% aqueous phase (buffer) immediately after formation (‘‘Fresh’’) around 181 nm when stored in the undiluted form and 51 nm
and after storage at different temperatures for one month.
when stored in the diluted form (Fig. 5b).
The influence of storage at different temperatures on the optical
80
properties of undiluted and diluted VE-NEs was also investigated
(Figs. 7a and 7b). Similar results were obtained for nanoemulsions
70
prepared using mixed oil (80% VE & 20% MCT) and pure vitamin E
acetate (100% VE), and therefore we only show the data for the
60 37°C mixed oil system here. Similar trends were observed in the depen-
dence of nanoemulsion turbidity on temperature, cosolvent type,
Relative Intensity (%)

50
40
30
20
10
0
1 10 100 1000
Particle Diameter (nm)
Fig. 6b. Influence of storage temperature on particle size distribution of nano-
emulsions produced using 10% oil phase (80% VE & 20% MCT), 10% surfactant phase
(Tween 80), and 80% aqueous phase (40% glycerol + 40% buffer) immediately after
formation (‘‘Fresh’’) after storage at different temperatures for one month.
nanoemulsions were highly unstable to droplet growth, with the
mean particle diameter increasing by about 320% after one month
storage at 37 °C. Similar trends were also observed for NEs pre-
pared using 100% VE as the oil phase and 40% glycerol in the aque-
ous phase: the mean particle diameter increased by about 2% at
5 °C, 7% at 20 °C, and 250% at 37 °C (Table 1). These results indicate
and dilution as were observed in the particle size measurements.
In the absence of glycerol, the turbidity of the nanoemulsions in-
20°C
creased slightly after storage, with this effect becoming greater
at higher storage temperatures (Fig. 7a). In the presence of 40%
glycerol, there was a slight rise in turbidity after storage at 5 and
20 °C, but a steep rise after storage at 37 °C (Fig. 7b). Dilution of
5°C
the NEs prior to storage inhibited the observed increase in turbid-
ity (especially at 37 °C) in both the absence and presence of
glycerol (Figs. 7a and 7b). The close correlation between the
appearance and particle size measurements can be attributed to
Fresh
the fact that the turbidity of a nanoemulsion tends to increase as
10000 the particle size increases [49].
There are a number of possible mechanisms that might account
for the observed increase in mean particle size in the nanoemul-
sions during storage, including flocculation, coalescence, and Ost-
wald ripening [15,49,50]. We do not believe that flocculation was
important in the nanoemulsions used in this study since floccu-
lated droplets often dissociate upon dilution for light scattering
measurements, and because we did not observe flocs using an opti-
cal microscope. In addition, very fine droplets stabilized by non-io-
nic surfactants tend to be stable to flocculation due to the strong
steric repulsion between the hydrophilic surfactant head groups
[14]. We therefore postulate that either coalescence or Oswald rip-
ening was responsible for the droplet growth observed in this
study.

Table 1
Influence of storage time and temperature on mean particle diameter and polydispersity index of undiluted and diluted nanoemulsions produced using 10% oil phase (100% VE),
10% surfactant phase (Tween 80), and 80% aqueous phase (40% glycerol in buffer).

Z-Average (nm) Polydispersity index

5 °C 20 °C 37 °C 5 °C 20 °C 37 °C
Fresh 48.9 ± 4.8 48.9 ± 4.8 48.9 ± 4.8 0.19 ± 0.07 0.19 ± 0.07 0.19 ± 0.07
1 month (undiluted) 49.7 ± 3.3 52.4 ± 2.4 171.1 ± 2.3 0.17 ± 0.05 0.15 ± 0.03 0.07 ± 0.02
1 month (diluted) 49.2 ± 6.6 50.6 ± 3.9 50.1 ± 4.6 0.22 ± 0.07 0.22 ± 0.05 0.18 ± 0.05
 A.H. Saberi et al. / Journal of Colloid and Interface Science 411 (2013) 105–113
0.6
5°C
20°C
0.5 37°C
Turbidity (cm-1 g-1 Oil)
0.4
0.3
0.2
0.1
0.0
Fresh (UD) 1 month (UD) Fresh (D) 1 month (D)
Fig. 7a. Influence of storage time and temperature on turbidity of undiluted (UD)
and diluted (D) nanoemulsions produced using 10% oil phase (80% VE & 20% MCT),
10% surfactant phase (Tween 80), and 80% aqueous phase (buffer).
0.6
5°C > 5 cm-1 g-1
20°C
0.5 37°C
Turbidity (cm-1 g-1 Oil)
0.4
0.3
0.2
0.1
0.0
Fresh (UD) 1 month (UD) Fresh (D) 1 month (D)
Fig. 7b. Influence of storage time and temperature on turbidity of undiluted (UD)
and diluted (D) nanoemulsions produced using 10% oil phase (80% VE & 20% MCT),
10% surfactant phase (Tween 80), and 80% aqueous phase (40% glycerol in buffer).
The rate of droplet coalescence in oil-in-water emulsions stabi-
lized by non-ionic surfactants usually increases as the temperature
is raised towards the phase inversion temperature (PIT) of the sys-
tem [24,51]. This effect has been attributed to dehydration of the
surfactant head-groups leading to changes in the optimum curva-
ture and interfacial tension at elevated temperatures [52]. The
addition of glycerol is known to decrease the PIT of emulsions sta-
bilized by non-ionic surfactant molecules, which is also attributed
to its ability to dehydrate the surfactant head groups [25,26]. The
rapid droplet growth and steep turbidity increase observed in the
nanoemulsions during storage at 37 °C in the presence of high
glycerol levels may therefore have been due to the ability of this
cosolvent to reduce the PIT of the system thereby accelerating
droplet coalescence. It should be noted that a more detailed insight
into the molecular and physicochemical origins of emulsion insta-
bility in these systems may be obtained by applying a variety of
additional analytical tools, such as electrical conductivity [53,54],
interfacial tension [55], spectroscopy, scattering, and microscopy
methods.
The rate of droplet growth due to Ostwald ripening (OR) may
also increase with increasing temperature. The change in mean
droplet diameter (d) with time (t) due to Ostwald ripening of an
emulsified oil in the steady-state regime is given by [56]:
111
3 3 64cV m cD
d  d0 ¼ t ð1Þ
9RT
where c is the interfacial tension, Vm is the molar volume of the oil,
R is the gas constant, and T is the absolute temperature, c is the sol-
ubility of the oil in the aqueous phase, and D is the translational dif-
fusion coefficient of the oil molecules through the aqueous phase.
This equation shows that the rate of droplet growth due to OR in-
creases as the solubility of the oil in the aqueous phase increases.
The presence of glycerol in an oil-in-water emulsion will increase
the solubility of the non-polar oil molecules in the surrounding
aqueous phase since a glycerol solution is less polar than pure
water. This phenomenon may therefore increase the droplet growth
rate due to Ostwald ripening. On the other hand, the interfacial ten-
sion and diffusion coefficient should decrease with increasing glyc-
erol concentration [57], which may partly counteract this effect.
It is usually difficult to precisely establish the physicochemical
origin of droplet growth in oil-in-water emulsions because both
coalescence and OR lead to a net increase in mean droplet size
[58]. In addition, both of these instability mechanisms may lead
to either a monomodal or bimodal particle size distribution
depending on the initial nature of the oil(s) and surfactant(s) used.
In this study, we found that the particle size distribution of the
nanoemulsions after storage remained monomodal (Figs. 6a and
6b), which is consistent with either Ostwald ripening or a homoge-
neous coalescence process [58]. A homogenous coalescence process
is one in which the rate of droplet growth is not strongly depen-
dent on droplet size.
3.4. Effect of glycerol on thermal stability of vitamin E-loaded
nanoemulsions
Knowledge of the thermal stability of nanoemulsion-based
delivery systems is important for many commercial applications
since they are often exposed to elevated temperatures during their
manufacture, storage, or utilization, e.g., hot filling, pasteurization,
or cooking. We therefore studied the influence of glycerol on the
thermal stability of the vitamin E-loaded nanoemulsions. Changes
in the turbidity of selected VE-NEs were monitored when they
were subjected to a controlled heating and cooling cycle, i.e., 20
to 80 to 20 °C (Figs. 8a and 8b). Experiments were carried out using
nanoemulsions prepared from a mixed oil phase (80% VE & 20%
MCT) and from pure vitamin E acetate (100% VE). The results for
both oil phases were qualitatively similar and therefore we only
show them for the mixed oil system, and highlight any differences
observed for the 100% VE system.
In the absence of glycerol, the turbidity of the VE-NEs remained
relatively low and constant during heating from 20 to 70 °C, but
then it increased steeply when the temperature was further in-
creased from around 70 to 80 °C (Fig. 8a). Upon cooling, the turbid-
ity remained relatively high indicating that the turbidity increase
that occurred during heating was irreversible. The increase in tur-
bidity can be attributed to an increase in droplet size during heat-
ing. The physicochemical origin of this effect can be related to
progressive dehydration of the polar head group of the non-ionic
surfactant molecules (Tween 80) as the temperature is raised. This
process reduces the hydration repulsion between the droplets
thereby allowing them to get closer together. In addition, head-
group dehydration means that the optimum curvature of the sur-
factant monolayer moves closer to unity, which favors an ultralow
interfacial tension and promotes droplet coalescence [14,59]. The
fact that the turbidity remained high during cooling (and the
samples appeared visibly cloudy) suggests that the oil droplets re-
mained large after coalescence had occurred.
In the presence of 40% glycerol, the turbidity – temperature
profile of the VE-NEs during heating and cooling was much more
112 A.H. Saberi et al. / Journal of Colloid and Interface Science 411 (2013) 105–113

once the PIT temperature is reached the optimum curvature of the

0.40 surfactant monolayer is close to unity and the interfacial tension
becomes ultralow, which favors the formation of a microemulsion
0.35
phase containing structures that are too small to scatter light
Heating strongly, thereby leading to a decrease in turbidity (‘‘clearing’’)
Turbidity (cm-1 g -1 Oil)

0.30
Cooling [24]. Upon further heating, the surfactant head group becomes
0.25 more dehydrated, which again changes the monolayer curvature
and solubility of the surfactant. At these high temperatures the
0.20 surfactant is relatively lipophilic and has an optimum curvature
that favors the formation of reverse micelles and water-in-oil
0.15 emulsions. As a consequence, water-in-oil emulsions containing
relatively large water droplets are formed above the PIT, which
0.10
again leads to strong light scattering and a high turbidity. Upon
0.05
cooling, the opposite process occurs: the system goes from a
water-in-oil emulsion containing relatively large droplets (highly
0.00 turbid), to a microemulsion containing small structures (clear), to
20 30 40 50 60 70 80 an oil-in-water emulsion containing relatively large droplets
Temperature (°C) (highly turbid). The reason that the oil-in-water emulsions in our
study contained relatively large droplets after cooling may be be-
Fig. 8a. Influence of temperature on turbidity of undiluted nanoemulsions
cause we used a relatively slow cooling rate, and so substantial
produced using 10% oil phase (80% VE & 20% MCT), 10% surfactant phase (Tween
80), and 80% aqueous phase (buffer solution). droplet coalescence probably occurred just below the PIT. Anton
and Vandamme (2009) have also suggested that nanoemulsions
can be formed from surfactant–oil–water mixtures by quench
cooling them well below the PIT with continuous stirring [24].
0.40 The reason that the clear region (where the systems had low tur-
bidity) was different during the heating and cooling cycles may
0.35 have been because of hysteresis effects associated with the time
required for the measurement cell to heat up or cool down in the
Heating
Turbidity (cm-1 g-1 Oil)

0.30
Cooling
0.25
0.20
0.15
0.10
0.05
0.00
20 30 40 50 60
Temperature (°C)
Fig. 8b. Influence of temperature on turbidity of undiluted nanoemulsions
produced using 10% oil phase (80% VE & 20% MCT), 10% surfactant phase (Tween
80), and 80% aqueous phase (40% glycerol in buffer solution).
complex than in its absence. During heating, the turbidity re-
mained relatively low and constant from 20 to 55 °C, suggesting
that the nanoemulsions were relatively stable to droplet growth
in this temperature range (Fig. 8b). There was then a steep increase
in turbidity from 55 to 67 °C, followed by a sharp drop from 70
to 73 °C, followed by another steep increase from 76 to 80 °C.
During cooling, the turbidity was relatively high at 80 °C, fell stee-
ply from 80 to 74 °C, and then increased sharply from 70 to 68 °C.
The turbidity-temperature profile of these nanoemulsions can
be attributed to a transitional phase inversion of the surfactant–
oil–water system around the PIT. Initially, the nanoemulsion has
a relatively low turbidity at room temperature because it contains
very fine droplets that do not scatter light efficiently [60]. At these
low temperatures the surfactant is relatively hydrophilic and has
an optimum curvature that favors the formation of micelles and
oil-in-water emulsions. Upon heating, the surfactant head groups
become progressively dehydrated which promotes droplet coales-
cence leading to an increase in droplet size and turbidity. However,
spectrophotometer.
We hypothesize that the PIT of the nanoemulsions heated in the
absence of glycerol was above the highest temperature (80 °C)
used during the thermal scanning experiment, and so we only ob-
served the initial droplet coalescence phenomenon (Fig. 8a), but
not the full phase inversion from O/W to microemulsion to W/O.
The presence of glycerol in the aqueous phase of the nanoemul-
sions reduced the PIT of the system so that it could be observed
within the temperature range used in the scanning experiment
(Fig. 8b). This effect can be attributed to the ability of glycerol to
dehydrate the polar head-groups of the non-ionic surfactant mole-
cules, thereby altering their optimum curvature and decreasing
70 80 their cloud point [25,26] and PIT [61].
Similar trends were also observed for nanoemulsions contain-
ing pure vitamin E acetate (data not shown). However, the temper-
atures where the nanoemulsions become cloudy and then clear
during heating were slightly less for 100% VE than for 80% VE &
20% MCT. For example, the clearing temperature was around
67 °C for nanoemulsion containing 100% VE, but around 73 °C for
nanoemulsion containing 80% VE & 20% MCT. These results indi-
cate that the nature of the oil phase also alters the phase inversion
temperature.
Finally, we examined the influence of dilution (1-to-100 with
water) of the VE-NEs prior to thermal cycling on their heat stabil-
ity. Diluting the nanoemulsions decreases the droplet concentra-
tion, but it also changes the composition of the aqueous phase so
that it is mainly comprised of water (0.4% glycerol +99.6% buffer).
All the diluted nanoemulsions had similar turbidity-temperature
profiles as the undiluted nanoemulsions containing no glycerol
(Fig. 8a), i.e., the turbidity remained relatively low from 20 to
73 °C, then increased steeply from 73 to 80 °C, and then remained
high when the samples were cooled back to 20 °C (data not
shown). This effect can be attributed to the fact that the aqueous
phase of the diluted nanoemulsions was predominantly water,
and so the PIT of the surfactant–oil–water system was increased.
Consequently, we only observed the initial droplet coalescence
stage during heating, but did not reach temperatures high enough
to observe the clearing stage. These results suggest that dilution of
 A.H. Saberi et al. / Journal of Colloid and Interface Science 411 (2013) 105–113
the nanoemulsions with water prior to heating may improve their
thermal stability.
4. Conclusions
In this study, we examined the influence of glycerol on the
properties of vitamin E acetate-loaded nanoemulsions prepared
by spontaneous emulsification. Previous studies have shown that
glycerol can influence the performance of non-ionic surfactants
in aqueous solutions due to its ability to alter their solubility char-
acteristics and optimum curvature [33–37]. Therefore, our aim was
specifically to investigate the influence of glycerol on the forma-
tion, properties, and stability of vitamin E-loaded nanoemulsions.
Our study has shown that the ability of glycerol to modulate sur-
factant properties has a considerable effect on the formation,
stability, and physical properties of nanoemulsions. The initial
droplet size and turbidity of nanoemulsions formed using the
spontaneous emulsification approach decreased with increasing
glycerol concentration, which was attributed to changes in surfac-
tant curvature and solubility. We showed that glycerol addition
could be used to reduce the total amount of surfactant required
to form vitamin E acetate nanoemulsions by spontaneous emulsi-
fication which was in line with a previous study [28]. We also
found that the storage stability of the nanoemulsions was depen-
dent on glycerol concentration, with rapid droplet growth being
observed at elevated temperatures in systems containing high
glycerol concentrations. This effect was attributed to increased
droplet coalescence and/or Ostwald ripening at higher tempera-
tures resulting from changes in surfactant curvature and oil solu-
bility in the presence of glycerol. However , other researchers
have reported that glycerol actually increased emulsion storage
stability [38,40]. This apparent discrepancy may be attributed to
differences in system composition, storage conditions, and analyt-
ical tools used in different studies. Finally, we found that diluting
nanoemulsions prepared in the presence of high glycerol levels
with water prior to heating improved their thermal stability by
raising the PIT. This study has important implications for develop-
ing stable vitamin E-fortified nanoemulsions suitable for use as
delivery systems in transparent foods and beverages. For example,
it suggests that high levels of glycerol should be used during the
formation of nanoemulsions to produce small droplets, but that
dilution of the nanoemulsions with water after preparation will
improve their long term stability to droplet growth. Nevertheless,
further studies are required (i) to gain a more detailed insight into
the molecular and physicochemical origin of the instability of vita-
min E nanoemulsions containing glycerol to droplet growth at ele-
vated storage temperatures, and (ii) to develop effective strategies
for increasing the storage stability of vitamin E nanoemulsions
containing glycerol.
Disclaimer
The opinions expressed in this manuscript are those of the
authors and do not represent statements of position, intention or
strategy of PepsiCo Inc.
Acknowledgments
This material is based upon work supported by the Cooperative
State Research, Extension, Education Service, United State Depart-
ment of Agriculture, Massachusetts Agricultural Experiment Sta-
tion and a United States Department of Agriculture, CREES, NRI
and AFRI Grants. The authors also thank William Mutilangi of Peps-
iCo for useful advice and discussions on this research.
113
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