Food Hydrocolloids 29 (2012) 326e334

Contents lists available at SciVerse ScienceDirect

Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Food-grade microemulsions and nanoemulsions: Role of oil phase composition on

formation and stability
Jiajia Rao, David Julian McClements*
Biopolymers and Colloids Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

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

Article history: Lemon oil is widely used as a flavoring component in beverages, foods, cosmetics, and household
Received 3 January 2012 products. Lemon oil comes in a variety of chemical compositions depending on its biological origin,
Accepted 11 April 2012 extraction methods, and purification procedures. At present, there is a relatively poor understanding of
the influence of lemon oil composition on its functional properties. In this study, we examined the
Keywords: influence of lemon oil fold (1
, 3
, 5
and 10
) on the formation and properties of oil-in-water
Emulsions
microemulsions and nanoemulsions. The concentration of both polar (high water solubility and low
Nanoemulsions
log P) and non-polar (low-water solubility and high log P) components increased with increasing oil fold.
Microemulsions
Solubilization
The nature of the colloid dispersions formed was established using an emulsion titration method that
Lemon oil involved titrating lemon oil droplets into a surfactant micelle solution (1% Tween 80). Oil fold affected
Tween 80 the rate and extent of solubilization, as well as the stability of lemon oil droplets to growth. The
Micelles maximum amount of lemon oil that could be solubilized within the micelles increased with increasing oil
fold, as did the stability of lemon oil droplets to growth. The results were interpreted in terms of the
ability of different lemon oil molecules to be incorporated within water or surfactant micelles, and the
influence of lemon oil polarity on Ostwald ripening. This study provides valuable information about the
relationship between lemon oil composition and its performance in colloidal delivery systems suitable
for use in the food and beverage industries.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Oil-in-water nanoemulsions and microemulsions are two types

There has been growing interest in the development of food-grade
colloidal delivery systems to encapsulate lipophilic functional ingre-
dients, such as flavors, colors, micronutrients, nutraceuticals, and
antimicrobials (Chen, Remondetto, & Subirade, 2006; McClements,
Decker, Park, & Weiss, 2009; Sanguansri & Augustin, 2006; Velikov
& Pelan, 2008). In particular, colloidal dispersions containing small
particles (r < 100 nm) have received particular attention because of
their potential advantages over other types of delivery systems for
certain applications: high optical clarity; good physical stability; and
enhanced bioavailability (Fanun, 2009; Gutierrez et al., 2008; Huang,
Yu, & Ru, 2010). In this study, we focused on the preparation and
properties of colloidal dispersions suitable for use in the beverage
industry to deliver lipophilic flavor molecules into aqueous based
products. Colloidal dispersions used for this purpose must have good
physical and chemical stability in both their concentrated and diluted
forms (Given, 2009).
* Corresponding author. Tel.: þ1 413 545 1019; fax: þ1 413 545 1262.
E-mail address: mcclements@foodsci.umass.edu (D.J. McClements).
of colloidal dispersion suitable for encapsulation and delivery of
lipophilic components within the beverage industry (McClements
& Rao, 2012). It is possible to fabricate both microemulsions and
nanoemulsions from exactly the same ingredients (oil, water, and
surfactant) but used in different ratios (Mason, Wilking, Meleson,
Chang, & Graves, 2006; Sonneville-Aubrun, Simonnet, & L’Alloret,
2004; Tadros, Izquierdo, Esquena, & Solans, 2004). The main
difference between these two types of colloidal dispersion is that
nanoemulsions are thermodynamically unstable whereas micro-
emulsions are thermodynamically stable (McClements, 2011).
However, it should be stressed that microemulsions are only
thermodynamically stable under a particular range of compositions
and environmental conditions, and may breakdown if these
conditions are changed (e.g., due to dilution, ingredient addition, or
temperature changes). Since nanoemulsions are thermodynami-
cally unstable systems they always tend to breakdown during
storage. The most commonly reported manifestation of beverage
emulsion deterioration is the formation of a whitish ring or shiny
oil slick around the neck of the container (Given, 2009;
McClements, 2005). These defects are the result of a variety of
physiochemical mechanisms that occur within beverage emulsions,

0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2012.04.008
 J. Rao, D.J. McClements / Food Hydrocolloids 29 (2012) 326e334 327

including creaming, flocculation, coalescence, and Ostwald 2.3. Solubilization experiments

ripening (McClements, 2005; Taherian, Fustier, & Ramaswamy,
2006). There is therefore a need to understand the major factors The underlying principle of the solubilization experiments is
that impact the formation of food-grade nanoemulsions and shown schematically in Fig. 1. A series of samples was prepared by
microemulsions, and to determine the influence of system titrating different amounts of stock nanoemulsion into surfactant
composition on their stabilities. solutions (1 wt% Tween 80) to give a range of final lemon oil
In this study, we focused on the influence of lemon oil compo- concentrations (0.1e2 wt%). The resulting colloidal dispersions
sition on the fabrication and stability of microemulsions and were then stored overnight at ambient temperature and neutral pH
nanoemulsions stabilized by a non-ionic surfactant (Tween 80). (23  C, pH 7), and samples were withdrawn for turbidity and
Lemon oil is one of the most widely used flavoring agents in the food particle size analysis.
and beverage industries (González-Molina, Domínguez-Perles,
Moreno, & García-Viguera, 2009; Mei et al., 2009; Reineccius,
2.4. Turbidity measurements
Reineccius, & Peppard, 2005). Commercially, lemon oils are avail-
able with a variety of different compositions that are suitable for
A spectrophotometric method was used to monitor changes in
different applications. These lemon oils vary due to differences in
the characteristics of lemon oil droplets after they were added to
their biological origins, isolation methods, and purification proce-
micelle solutions. The turbidity of the colloidal dispersions was
dures. The lemon oil obtained directly by cold pressing of lemon peel
measured using a UVevisible spectrophotometer at 600 nm (Ultra-
is called single-fold (1
) oil. This crude oil can be fractionated to
spec 3000 pro, Biochrom Ltd Cambridge, UK). Turbidity measure-
various degrees by distillation under vacuum to obtain higher fold
ments were carried out over time after an aliquot of stock nano-
oils (Misharina, Terenina, Krikunova, & Medvedeva, 2010). Different
emulsion (10 wt% lemon oil, 1 wt% Tween 80, 89 wt% buffer
lemon oils may be blended with each other or lemon oils may be
solution) was added to a surfactant solution (1 wt% Tween 80).
mixed with other components to create higher fold oils (Veriotti &
Sacks, 2001), such as the three-fold (3
), five-fold (5
), and ten-
fold (10
) oils used in this study. The main components of single-
fold lemon oils are terpenes, such as limonene, a and b-pinene, g-
terpinene, and sabinene (Gamarra, Sakanaka, Tambourgi, & Cabral,
2006). The concentration of terpenes remaining in lemon oils nor-
mally decreases after distillation, which changes their chemical
composition and physicochemical characteristics, and therefore
their functionality.
At present, there is a relatively poor understanding of the role of
lemon oil composition on its ability to form stable colloidal
dispersions suitable for application in food and beverage products.
The purpose of this study was therefore to systematically examine
the relationship between the composition of lemon oils and their
ability to form and stabilize microemulsions and nanoemulsions.
This information should facilitate the rational design of food-grade
colloidal delivery systems for different applications based on lemon
oil composition.

2. Materials and methods

2.1. Materials

Lemon oils (1
, 3
, 5
, 10
) and information about their
composition were kindly supplied by Citrus & Allied Essences (Lake
Success, NY). Sorbitan monooleate (Tween 80) was purchased from
the SigmaeAldrich Co. (St. Louis, MO). Monobasic phosphate,
sodium chloride, sodium hydroxide and hydrochloric acid were
purchased from Fisher Scientific (Pittsburgh, PA). Double distilled
water was used in the preparation of all solutions and colloidal
dispersions. All other solvents and reagents used were of analytical
grade.

2.2. Stock emulsion preparation

A stock emulsion was formed by weighing 10 wt% oil phase

(lemon oil), 1 wt% surfactant (Tween 80), and 89 wt% buffer solu-
tion (10 mM phosphate buffer, pH 7.0) into a container and then
blending them together using a high-speed stirrer (Tissue-Tearor,
Biospec Products, Bartlesville, OK) for 2 min at ambient tempera-
ture. The resulting coarse emulsion was then passed through a high
pressure homogenizer for three passes at 9000 psi (Model M-110L
Microfluidizer Processor, Microfluidics, Newton, MA) to form
a stock nanoemulsion.
Fig. 1. Schematic representation of emulsion solubilization experiment: lemon oil
nanoemulsion droplets were titrated into in a surfactant micelle solution. A: Stock
lemon oil nanoemulsion (1 wt% Tween 80, 10 wt% lemon oil, 89 wt% aqueous phase). B:
Surfactant micelle solution (1% Tween 80). Regime I: lemon oil concentration < CSat e
all lemon oil droplets solubilized in swollen micelles; Regime II: lemon oil
concentration z CSat e mixture of swollen micelles and nanoemulsion droplets;
Regime III: lemon oil concentration > CSat e most of lemon oil remains in nano-
emulsion droplets.
328 J. Rao, D.J. McClements / Food Hydrocolloids 29 (2012) 326e334

2.5. Particle size determination

The mean particle diameter and particle size distribution of the

colloidal dispersions were measured using dynamic light scattering
(NanoZS, Malvern Instruments, Malvern, U.K.). The samples were
diluted 20
in phosphate buffer solution at ambient temperature
prior to analysis to avoid multiple scattering effects.

2.6. Determination of emulsion instability

An optical microscope (C1 Digital Eclipse, Nikon, Tokyo, Japan)

with a 40
objective lens (NA 0.75) was used to capture images of
selected colloidal dispersions. Samples were sandwiched between
slide and cover slip, the slide was loaded onto the microscope stage,
and then images were recorded periodically.

2.7. Statistical analysis

All measurements were performed at least twice using freshly

prepared samples and are reported as calculated means and stan-
dard deviations.

3. Results and discussion

3.1. Composition, molecular, and physicochemical properties of

lemon oils

In a recent paper, we reported the chemical composition and

physicochemical properties of the same lemon oils used in this
study (Rao & McClements, 2012). We also tabulated the molecular
characteristics of the major constituents of the lemon oils, such as
molar volume, boiling point, oil-water partition coefficient, and
water solubility. Our analysis of this data led to the following
general conclusions: the viscosity, refractive index, density, and
interfacial tension of lemon oils tend to increase as the oil fold
increased from 1
to 10
(Table 1). We also found that there was an
increase in the concentrations of both polar (high water solubility,
low log P) components and non-polar (low-water solubility, high
log P) components with increasing oil fold (Fig. 2). This effect was
attributed to the reduction in the limonene content of the lemon
oils with increasing degrees of distillation. Limonene has an inter- Fig. 2. a. Influence of lemon oil fold on the percentage of constituents that can be
mediate polarity and water solubility and so when it is removed categorized as low, medium or high water solubility. b. Influence of lemon oil fold on
the percentage of constituents that can be categorized as having low, medium or high
there is an increase in the concentrations of both low and high
oil-water partition coefficients (log P).
polarity substances in the remaining oils. In our previous paper, we
showed that emulsions prepared from relatively low fold oils (1
,
3
, 5
) were highly unstable to Ostwald ripening during storage, on the influence of lemon oil composition on the formation of
whereas those prepared from the highest fold oil (10
) were stable microemulsions and nanoemulsions.
to Ostwald ripening (Rao & McClements, 2012). This effect was
attributed to the relatively high concentration (>14%) of compo- 3.2. Emulsion solubilization experiments
nents with low-water solubility in the 10
lemon oil, which
inhibited Ostwald ripening through an entropy of mixing effect The ability of the different lemon oils to form either micro-
known as “compositional ripening”. In the present study, we focus emulsions or nanoemulsions was determined using an emulsion
solubilization protocol (McClements & Dungan, 1995). In this
Table 1
protocol, a pre-formed nanoemulsion was titrated into a surfactant
Measured physicochemical properties of lemon oils with different folds (Rao & micelle solution and the change in turbidity and particle size
McClements, 2012). Measurements were made at ambient temperature on bulk distribution was measured (Fig. 1). Measurements of the turbidity
oils. The interfacial tension (g) of the lemon oils was measured against distilled versus time provide information about the rate and extent of
water.
solubilization, whereas measurements of the particle size distri-
Oil type Viscosity (mPa s) Density (kg m3) Refractive index g (mN m1) bution at the end of the process provide information about the type
(fold) of structures remaining in the colloidal dispersion. The experi-
1
1.41  0.01 851.63  0.03 1.47  0.01 12.9  0.2 ments performed using one-fold lemon oil are reported in this
3
1.29  0.01 858.18  0.01 1.48  0.01 12.8  0.1 section to highlight the principles behind this method.
5
1.85  0.02 864.82  0.04 1.48  0.01 8.3  0.1
10
5.27  0.08 909.86  0.06 1.49  0.01 11.2  0.3
Initially, information about solubilization kinetics was obtained
by measuring the change in turbidity after a fixed amount of lemon
 J. Rao, D.J. McClements / Food Hydrocolloids 29 (2012) 326e334 329

oil droplets was added to aqueous solutions containing either 0 or
1 wt% Tween 80 (Fig. 3a). In the absence of surfactant micelles (0 wt
% Tween 80), the turbidity remain constant over time indicating
that the nanoemulsion droplets were not solubilized by the
aqueous solution. In the presence of surfactant micelles (1 wt%
Tween 80), the turbidity decreased appreciably during the first
200 s, decreased slightly from 200 to 500 s, and then remained
relatively constant at longer storage times (close to zero). The
decrease in turbidity is indicative of a decrease in the amount of
light scattering by the lemon oil droplets, which can be attributed
to a reduction in their concentration and/or size when lemon oil
molecules moved from the droplets into the micelles. These
experiments indicated that the solubilization process was a rela-
tively rapid one, which can be partly attributed to the small initial
dimensions (and therefore large surface area) of the lemon oil
droplets and the relatively high water solubility of the lemon oil
molecules.
After establishing the kinetics of the solubilization process, we
carried out a series of experiments to determine the influence of
surfactant-to-oil ratio (SOR) on the distribution of lemon oil
between droplets and micelles. A series of colloidal dispersions was
prepared by adding increasing amounts of lemon oil droplets to
aqueous solutions (0 or 1 wt% Tween 80), and then storing them
overnight at ambient temperature. The turbidity (Fig. 3b) and
particle size (Fig. 3c and d) of the resulting colloidal dispersions was
then measured.
In the absence of surfactant micelles (0 wt% Tween 80), the
turbidity increased approximately linearly as the concentration of
lemon oil droplets added to the aqueous solution increased
(Fig. 3b). The increase in turbidity can be attributed to an increase in
the number of non-solubilized lemon oil droplets present in the
colloidal dispersions. The measured particle size remained fairly
constant with increasing oil concentration, and was close to the
value in the original nanoemulsion after 24 h storage (Fig. 3c).
These experiments suggest that the nanoemulsion droplets were
not appreciably solubilized within pure water, which can be
attributed to the relatively low concentration of surfactant micelles
present and the fact that the major components in lemon oil have

Fig. 3. a. Change in turbidity over time when lemon oil (1
) droplets are titrated into either buffer solution or 1% surfactant (Tween 80) solution. b. Influence of lemon oil
concentration on the turbidity of samples prepared by titrating different amounts of lemon oil (1
) droplets into either buffer solution or 1% surfactant (Tween 80) solution. c.
Influence of lemon oil concentration on the mean particle diameter of samples prepared by titrating different amounts of lemon oil (1
) droplets into either buffer solution or 1%
surfactant (Tween 80) solution. d. Influence of lemon oil concentration on the particle size distribution of samples prepared by titrating different amounts of lemon oil (1
) droplets
into 1% surfactant (Tween 80) solution. The data has been offset on the y-axis so as to distinguish different curves.
330 J. Rao, D.J. McClements / Food Hydrocolloids 29 (2012) 326e334
a relatively low-water solubility. For example, the major compo-
nent of single-fold oil (limonene) has a water solubility of about
5 mg/L, which corresponds to 0.0005 wt%. Consequently, very little
lemon oil is likely to move from the oil droplets and into the pure
water phase. On the other hand, we did observe some increase in
the mean particle diameter upon storage: from around 217 nm soon
after preparation to around 296 nm after overnight storage. A
bimodal distribution was formed after overnight storage, which
indicated that these systems were unstable to Ostwald ripening as
has been discussed in a recent study using similar oils (Rao &
McClements, 2012).
Appreciably different behavior was observed when the nano-
emulsion droplets were titrated into an aqueous solution contain-
ing surfactant micelles (Fig. 4). In the presence of surfactant
micelles (1 wt% Tween 80), the turbidity remained very low
(<0.01 cm1) from 0 to 0.2 wt% surfactant, increased gradually from
0.2 to 0.4 wt% surfactant, and then increased steeply when the
Fig. 4. a. Influence of lemon oil concentration on the turbidity of samples prepared by
titrating different amounts and types of lemon oil (1, 3, 5, and 10
) droplets into either
buffer solution or 1% surfactant solution. b. Influence of lemon oil concentration on the
mean particle diameter of samples prepared by titrating different amounts and types
of lemon oil (1, 3, 5, and 10
) droplets into either buffer solution or 1% surfactant
solution.
droplet concentration was increased further (Fig. 3b). As the lemon
oil concentration was increased, the appearance of the systems
went from transparent to turbid to opaque. Presumably, all of the
lemon oil initially present within the nanoemulsion droplets was
able to be incorporated within micelles for lemon oil < 0.2 wt%, but
at higher concentrations some of the lemon oil remained within the
nanoemulsion droplets. This information is important for the
formulation of products such as soft drinks, fortified waters, and
other beverages containing lemon oil. To form a transparent
colloidal dispersion containing single-fold lemon oil it would be
necessary to utilize <0.2 wt% lemon oil per 1 wt% of Tween 80,
which corresponds to a surfactant-to-oil ratio of 5-to-1.
Solubilization involves the transport of oil molecules from
nanoemulsion droplets to micelles, and therefore one would
expect a change in the size of the various colloidal particles present
in the system: micelles should grow as lemon oil is incorporated
within them, whereas nanoemulsion droplets should shrink as
lemon oil leaves them. We therefore measured the dependence of
the mean volume-weighted droplet diameter (Fig. 3c) and particle
size distribution (Fig. 3d) on lemon oil concentration using
dynamic light scattering. The mean particle diameter remained
relatively low (<30 nm) from 0 to 0.2 wt% lemon oil, increased
steeply from 0.3 to 0.6 wt% lemon oil, and then increased more
gradually at higher lemon oil concentrations (Fig. 3c). The
measured increase in particle diameter was consistent with the
observed increase in turbidity, as would be expected since larger
particles scatter light more strongly in this regime (d < l). In the
absence of lemon oil, the effective diameter of the Tween 80
micelles was determined to be about 9.9 nm. There was an
appreciable increase in the mean particle diameter when the
lemon oil concentration was increased from 0 to 0.2 wt% (Fig. 3b),
while the particle size distribution remained monomodal (Fig. 3b).
For example, the particle diameter grew from around 9.9 to
12.8 nm when 0.2 g of lemon oil was solubilized per 1 g of Tween
80. These results are indicative of the formation of a micro-
emulsion containing swollen micelles, i.e., micelles with lemon oil
molecules incorporated within them. The fact that there was only
a modest increase in particle size within this region suggests that
the lemon oil molecules may have been incorporated between the
non-polar tails of the surfactant molecules, rather than forming
a separate core in the interior. Nevertheless it is difficult to
ascertain the precise location of the different molecules in lemon
oil within the micelles, especially when lemon oil has such
a complex composition.
Once the surfactant micelles become fully saturated with lemon
oil, any further emulsion droplets added to the system are no longer
dissolved. Consequently there would be a mixture of swollen
micelles and oil droplets in the resulting colloidal dispersions. Since
larger particles scatter light much more efficiently than smaller
ones, the overall signal in the dynamic light scattering instrument
will tend to be dominated by the presence of the larger oil droplets.
Consequently, at higher lemon oil concentrations there was an
increase in the measured mean particle diameter. In our previous
study we found that emulsions prepared using single-fold oil were
unstable to Ostwald ripening after preparation (Rao & McClements,
2012), which explains why the final lemon oil droplets had larger
mean diameters than the initial nanoemulsion used to carry out the
titration (d z 296 nm).
The influence of lemon oil concentration on the particle size
distributions of colloidal dispersions containing 1 wt% Tween 80 is
shown in Fig. 3d. In the absence of lemon oil, a single population of
relatively small particles was observed with a diameter around
10 nm, which we attribute to the presence of surfactant micelles.
The particle size distribution was fairly similar for the system
containing 0.2 wt% lemon oil, which suggests that all the emulsion
 J. Rao, D.J. McClements / Food Hydrocolloids 29 (2012) 326e334 331

droplets had dissolved and the lemon oil had been incorporated
into the micelles to form swollen micelles (microemulsion drop-
lets). In the presence of 0.5 wt% lemon oil, a very broad particle size
distribution was observed around 170 nm, which can be attributed
to the presence of a high concentration of non-dissolved emulsion
droplets. It is likely that there was still a population of small
particles (swollen micelles) present, but that it could not be
observed in the particle size distribution because the overall signal
in the dynamic light scattering instrument was dominated by the
light scattered from the larger particles. At higher lemon oil
concentrations, a very broad peak was observed that was larger
than the original nanoemulsion droplets indicating that they were
unstable to droplet growth, which has previously been attributed to
Ostwald ripening (Rao & McClements, 2012).
The physicochemical origin of the movement of lemon oil
molecules from emulsion droplets to micelles can be attributed to
thermodynamic effects. The droplets in the lemon oil-in-water
emulsions are thermodynamically unstable because of the posi-
tive free energy associated with the oil-water interface. The
micelles in the initial Tween 80 solution are thermodynamically
stable mainly because of the free energy decrease associated with
the removal of non-polar surfactant tail groups from water
Fig. 5. Influence of lemon oil type on maximum amount of lemon oil that could be
(hydrophobic effect). However, the monolayer formed by the solubilized in 1 wt% of Tween 80 surfactant solution.
non-swollen surfactant micelles may not be at the optimum
curvature, which is determined by the relative size of the
surfactant head group and tail group (Israelachvili, 2011). When
the lemon oil nanoemulsion is brought into contact with the
surfactant solution, lemon oil molecules may therefore move
from the droplets to the micelles until the micelles reach their
optimum curvature and become saturated. Above this lemon oil
concentration, any excess oil molecules remain in the emulsion
droplets.

3.3. Influence of oil type on nanoemulsion solubilization

Previous studies have shown that the rate and extent of solu-
bilization depend on the molecular structure of the oils, as well as
surfactant type, surfactant concentration, and the surface area of oil
exposed to the aqueous phase (Evilevitch, Jonsson, Olsson, &
Wennerstrom, 2001; Evilevitch, Olsson, Jonsson, & Wennerstrom,
2000). In this section, we therefore examined the influence of
lemon oil type (1
, 3
, 5
and 10
) on the solubilization of lemon
oil droplets by Tween 80 micelles.
The influence of lemon oil type on the solubilization process was
determined using the emulsion titration method described in the
previous section. Different amounts of lemon oil droplets were
titrated into 1 wt% Tween 80 solutions, the resulting samples were
stored overnight, and then their turbidities (Fig. 4a) and particle
sizes (Fig. 4b) were measured. All of the lemon oil types showed
similar qualitative behavior: turbidity and mean particle diameter
remained low up to a critical lemon oil concentration and then
increased appreciably. Nevertheless, there were some quantitative
differences in the behaviors of the various lemon oils. In general,
the increase in turbidity with increasing lemon oil concentration
was smaller for higher fold oils, i.e., more lemon oil had to be added
to the surfactant solution to cause an appreciable increase in
turbidity for the 10
oil than for the lower fold oils (Fig. 4a). On the
other hand, a lower concentration of 10
oil needed to be added
before there was an appreciable increase in mean particle diameter
(Fig. 4b). These apparently contradictory results may be because
the turbidity of a colloidal dispersion depends on both particle
concentration and particle size. The droplets in the colloidal
dispersions containing relatively high concentrations of 10
lemon Fig. 6. a. Time-dependence of the particle size distribution during storage at 20  C of
oil may have scattered light less efficiently than those in lower fold lemon oil-in-water emulsions (10 wt% lemon oil, 1 wt% Tween 80 and 89 wt% phos-
oils due to differences in particle size. phate buffer solution, pH 7.0): 1
oil. b. 10
oil.
332 J. Rao, D.J. McClements / Food Hydrocolloids 29 (2012) 326e334
From a practical point of view, it is important to establish the
maximum amount of lemon oil that can be incorporated into an
aqueous solution before the turbidity increases appreciably and the
product becomes cloudy. We therefore determined the maximum
amount of lemon oil (CSat) that could be incorporated into the
different systems before the turbidity exceeded an arbitrary
threshold level of 0.05 cm1. The amount of lemon oil solubilized in
the Tween 80 micelles increased as the lemon oil fold increased
(Fig. 5), which may be explained in terms of the molecular and
physiochemical properties of lemon oils. The ability of a surfactant
micelle to incorporate non-polar molecules depends on the size
and shape of its hydrophobic interior relative to the non-polar
molecules. Oil molecules may pack between the tails of surfactant
molecules or they may form a separate hydrophobic core in the
micelle interior (Israelachvili, 2011). For example, experiments
have shown that relatively large non-polar molecules (such as
triglycerides) cannot be incorporated into Tween micelles because
their dimensions are too large, whereas relatively small non-polar
molecules (such as hydrocarbons) can (Weiss, Coupland,
Brathwaite, & McClements, 1997). It is therefore possible that
some of the molecules within the lemon oils could easily be
incorporated within the surfactant micelles (e.g., those with rela-
tively small molar volumes and an appropriate distribution of
functional groups), whereas others could not (e.g., those that were
Fig. 7. Optical microscopy images of coarse 1
lemon oil emulsions (10 wt% lemon oil, 1 wt% Tween 80, 89 wt% phosphate buffer). The emulsions were diluted 1:1 with a 1 wt%
Tween 80 solution. The scale bars indicate 20 mm.
too large or that had a distribution of polar and non-polar groups
that could not be easily accommodated within the micelles). In
addition, it is possible that some of the lemon oil components (the
more water-soluble ones) were directly solubilized in the aqueous
phase, rather than in surfactant micelles. Measurement of the size
of Tween 80 micelles before and after addition of lemon oil indi-
cated that there was an appreciable increase in their diameter
(Fig. 4b), which suggests that oil molecules were incorporated into
the micelle interior. For example, the particle diameter increased
from around 10.0 to 12.3 nm when the lemon oil concentration was
increased from 0 to 0.2 wt%, while the particle size distribution only
contained a single monomodal peak. Our results suggest that there
was a higher fraction of molecules within the 10
lemon oil that
were capable of being incorporated within the structure of the
Tween 80 micelles than for the other lemon oils.
3.4. Stability of systems to droplet growth
Measurements of the particle size over time indicated that
nanoemulsions containing relatively low folds of lemon oil (1
, 3
,
5
) were highly unstable to droplet growth during storage,
whereas nanoemulsions containing the highest fold oil (10
) were
relatively stable. To highlight this effect the change in particle size
distribution of 1
and 10
lemon oil nanoemulsions during storage
 J. Rao, D.J. McClements / Food Hydrocolloids 29 (2012) 326e334
are compared in Fig. 6. The particle size distribution of nano-
emulsions containing 10
lemon oils showed little change during
storage over a 1 week period (Fig. 6b). On the other hand, there was
a pronounced change in the particle size distribution of nano-
emulsions containing 1
lemon oils. Soon after homogenization
the particle size distribution was bimodal suggesting that rapid
droplet growth had already occurred, and during storage there was
an increase in the fraction of large droplets present and an increase
in the size of the larger droplet population (Fig. 6a).
The growth in droplet size observed in the lower fold lemon oil
nanoemulsions during storage may be attributed to either droplet
aggregation (i.e., flocculation or coalescence) or Ostwald ripening
(i.e., diffusion of oil molecules from small to large droplets). We
therefore carried out some additional experiments to provide
further information about the possible physicochemical origin of
nanoemulsion instability in these systems. The change in droplet
size distribution over time was monitored using optical micros-
copy. Optical microscopy cannot reliably resolve particles that are
smaller than about 500 nm in diameter, and therefore we used
coarse lemon oil-in-water emulsions for this part of the study.
These emulsions were prepared by blending the lemon oil and
aqueous surfactant solution together using a high shear mixer.
Small aliquots of these emulsions were then placed onto a micro-
scope slide and the change in their microstructure over time was
observed (Fig. 7). For 10
lemon oil emulsions there was no
observable change in the particle size distribution during storage.
On the other hand, for 1
lemon oil emulsions there was evidence
of droplet growth during storage. Visual observation of the
microstructure throughout the storage period indicated that
droplet growth occurred without the necessity for collisions
occurring between different oil droplets, which indicated that
Ostwald ripening was an important growth mechanism.
In a recent study, we proposed that the stability of the higher
fold oil (10
) to droplet growth was due to the presence of rela-
tively high levels of lemon oil constituents with low-water solu-
bility, which inhibited droplet growth by generating
a compositional ripening effect that opposed Ostwald ripening
effect (Rao & McClements, 2012).
4. Conclusions
This study focused on establishing the influence of the compo-
sition and physicochemical properties of lemon oils on the forma-
tion and stability of microemulsions and nanoemulsions. An
emulsion titration method was used to study the solubilization of
lemon oil droplets by surfactant micelles. For all four lemon oils,
microemulsions were formed at relatively low lemon oil concen-
trations but nanoemulsions persisted at higher concentrations,
which were attributed to saturation of the micelle core with lemon
oil. The ability to form transparent solutions (considered to be
microemulsions) depended on lemon oil type: a higher concen-
tration of 10
oil could be incorporated into the surfactant solution
while maintaining optical clarity than lower fold oils. This differ-
ence was mainly attributed to differences in the molecular struc-
ture of the oil molecules in the different lemon oils. Higher fold oils
contained more water-soluble components (which may have been
more easily dispersed directly in pure water) as well as more water-
insoluble components (which may have been incorporated within
the hydrophobic interior of the surfactant micelles).
The nanoemulsions formed with relatively low fold lemon oils
(1
, 3
, 5
) were not stable during storage, which was attributed
to Ostwald ripening and possibly some coalescence. On the other
hand, the 10
lemon oil had good stability to droplet growth
during storage, which was attributed to the presence of highly non-
polar substances, which inhibited Ostwald ripening. In this study,
333
we used a commercial non-ionic surfactant (Tween 80) to form
lemon oil microemulsions or nanoemulsions. It should be noted
that commercial surfactants may vary considerably in their
compositions, which may also affect the properties and stability of
colloidal dispersions. The information generated in this study will
be useful for designing stable colloidal dispersions by using flavor
oils for applications in foods and beverages.
Acknowledgments
This material is based upon work supported by the United States
Department of Agriculture (AFRI program). We also thank Compass
Foods and Citrus & Allied Essences for providing some of the
materials used in this study, and for useful discussions about
ingredient functionality.
References
Chen, L., Remondetto, G. E., & Subirade, M. (2006). Food protein-based materials as
nutraceutical delivery systems. Trends in Food Science & Technology, 17(5),
272e283.
Evilevitch, A., Jonsson, B. T., Olsson, U., & Wennerstrom, H. (2001). Molecular
transport in a nonequilibrium droplet microemulsion system. Langmuir, 17(22),
6893e6904.
Evilevitch, A., Olsson, U., Jonsson, B., & Wennerstrom, H. (2000). Kinetics of oil
solubilization in microemulsion droplets. Mechanism of oil transport. Langmuir,
16(23), 8755e8762.
Fanun, M. (2009). Microemulsions with nonionic surfactants and mixed oils. Soft
Materials, 7(4), 258e276.
Gamarra, F. M. C., Sakanaka, L. S., Tambourgi, E. B., & Cabral, F. A. (2006). Influence
on the quality of essential lemon (Citrus aurantifolia) oil by distillation process.
Brazilian Journal of Chemical Engineering, 23(1), 147e151.
Given, P. S. (2009). Encapsulation of flavors in emulsions for beverages. Current
Opinion in Colloid & Interface Science, 14(1), 43e47.
González-Molina, E., Domínguez-Perles, R., Moreno, D. A., & García-Viguera, C.
(2009). Natural bioactive compounds of Citrus limon for food and health. Journal
of Pharmaceutical and Biomedical Analysis, 51(2), 327e345.
Gutierrez, J. M., Gonzalez, C., Maestro, A., Sole, I., Pey, C. M., & Nolla, J. (2008). Nano-
emulsions: new applications and optimization of their preparation. Current
Opinion in Colloid & Interface Science, 13(4), 245e251.
Huang, Q. R., Yu, H. L., & Ru, Q. M. (2010). Bioavailability and delivery of nutra-
ceuticals using nanotechnology. Journal of Food Science, 75(1), R50eR57.
Israelachvili, J. (2011). Intermolecular and surface forces (3rd ed.). London, UK:
Academic Press.
Mason, T. G., Wilking, J. N., Meleson, K., Chang, C. B., & Graves, S. M. (2006).
Nanoemulsions: formation, structure, and physical properties. Journal of
Physics: Condensed Matter, 18, R635.
McClements, D. (2005). Food emulsions: Principles, practices, and techniques. CRC.
McClements, D. J. (2011). Edible nanoemulsions: fabrication, properties, and func-
tional performance. Soft Matter, 7(6), 2297e2316.
McClements, D. J., Decker, E. A., Park, Y., & Weiss, J. (2009). Structural design
principles for delivery of bioactive components in nutraceuticals and functional
foods. Critical Reviews in Food Science and Nutrition, 49(6), 577e606.
McClements, D. J., & Dungan, S. R. (1995). Light-scattering study of solubilization of
emulsion droplets by nonionic surfactant solutions. Colloids and Surfaces A e
Physicochemical and Engineering Aspects, 104(2e3), 127e135.
McClements, D. J., & Rao, J. (2012). Food-grade nanoemulsions: formulation, fabri-
cation, properties, performance, biological fate, and potential toxicity. Critical
Reviews in Food Science & Nutrition, 51(4), 285e330.
Mei, L., Choi, S. J., Alamed, J., Henson, L., Popplewell, M., McClements, D. J., et al.
(2009). Citral stability in oil-in-water emulsions with solid or liquid octadecane.
Journal of Agricultural and Food Chemistry, 58(1), 533e536.
Misharina, T. A., Terenina, M. B., Krikunova, N. I., & Medvedeva, I. B. (2010).
Autooxidation of a mixture of lemon essential oils, methyl linolenoate, and
methyl oleinate. Applied Biochemistry and Microbiology, 46(5), 551e556.
Rao, J., & McClements, D. J. (2012). Impact of lemon oil composition on formation
and stability of model food and beverage emulsions. Food Chemistry, 134,
749e757.
Reineccius, T. A., Reineccius, G. A., & Peppard, T. L. (2005). The effect of solvent
interactions on a-, b-, and g-cyclodextrin/flavor molecular inclusion complexes.
Journal of Agricultural and Food Chemistry, 53(2), 388e392.
Sanguansri, P., & Augustin, M. A. (2006). Nanoscale materials development e a food
industry perspective. Trends in Food Science & Technology, 17(10), 547e556.
Sonneville-Aubrun, O., Simonnet, J. T., & L’Alloret, F. (2004). Nanoemulsions: a new
vehicle for skincare products. Advances in Colloid and Interface Science, 108,
145e149.
Tadros, T., Izquierdo, P., Esquena, J., & Solans, C. (2004). Formation and stability of
nano-emulsions. Advances in Colloid and Interface Science, 108, 303e318.
334 J. Rao, D.J. McClements / Food Hydrocolloids 29 (2012) 326e334

Taherian, A. R., Fustier, P., & Ramaswamy, H. S. (2006). Effect of added oil and
modified starch on rheological properties, droplet size distribution, opacity and
stability of beverage cloud emulsions. Journal of Food Engineering, 77(3),
687e696.
Velikov, K. P., & Pelan, E. (2008). Colloidal delivery systems for micronutrients and
nutraceuticals. Soft Matter, 4(10), 1964e1980.
Veriotti, T., & Sacks, R. (2001). High-speed GC and GC/time-of-flight MS of lemon
and lime oil samples. Analytical Chemistry, 73(18), 4395e4402.
Weiss, J., Coupland, J. N., Brathwaite, D., & McClements, D. J. (1997). Influence of
molecular structure of hydrocarbon emulsion droplets on their solubilization in
nonionic surfactant micelles. Colloids and Surfaces A: Physicochemical and
Engineering Aspects, 121(1), 53e60.