Food Hydrocolloids xxx (2014) 1e10

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

Physicochemical characterization and antimicrobial activity of food-

grade emulsions and nanoemulsions incorporating essential oils
Laura Salvia-Trujillo, Alejandra Rojas-Graü, Robert Soliva-Fortuny, Olga Martín-Belloso*
Department of Food Technology, University of Lleida e Agrotecnio Center, Av. Alcalde Rovira Roure 191, 25198, Lleida, Spain

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

Article history: Coarse emulsions containing essential oils (lemongrass, clove, tea tree, thyme, geranium, marjoram,
Received 21 November 2013 palmarosa, rosewood, sage or mint) and stabilized with tween 80 and sodium alginate were prepared by
Accepted 13 July 2014 high shear homogenization. Nanoemulsions were obtained by microfluidization of coarse emulsions. In
Available online xxx
general, the average droplet size of coarse emulsions was dramatically reduced after microfluidization
down to a few nanometers, with the exception of palmarosa and rosewood oil emulsions, which were
Keywords:
already in the nano-range before being treated. The z-potential of nanoemulsions exhibited values more
Antimicrobial activity
negative than
30 mV, indicating a strong electrostatic repulsion of the dispersed oil droplets in the
Escherichia coli
Nanoemulsion
aqueous phase. The viscosity of nanoemulsions significantly decreased after microfluidization, with at
Essential oils least a 30% drop in their initial values. The whiteness index of nanoemulsions diminished after being
Microfluidization treated. In fact, nanoemulsions containing tea tree, geranium or marjoram essential oils became
Droplet size completely transparent after microfluidization. Lemongrass, clove, thyme or palmarosa-loaded nano-
emulsions were those with a higher in vitro bactericidal action against Escherichia coli, as they achieved
4.1, 3.6, 2.8 or 3.9 log-reductions after 30 min of contact time. In addition, a faster and enhanced inac-
tivation kinetic was observed in the case of nanoemulsions containing lemongrass or clove essential oils
in comparison with their respective coarse emulsions. Thus, the present work evidences the promising
advantages of using nanoemulsions as delivery systems of flavoring and preservative agents in the food
industry.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction the incorporation of antimicrobial essential oils to foods still pre-

Essential oils contain a complex mixture of non-volatile and
volatile compounds produced by aromatic plants as secondary
metabolites (Bakkali, Averbeck, Averbeck, & Idaomar, 2008; Burt,
2004). They are used in a wide variety of applications in food,
pharmaceutical and cosmetics industries due to their flavoring,
antioxidant and antimicrobial properties (Adorjan & Buchbauer,
2010; Brud, 2010; Tajkarimi, Ibrahim, & Cliver, 2010). In partic-
ular, the antimicrobial action of essential oils has been attributed to
their phenolic compounds and their interaction with microbial cell
membranes. They are known to penetrate through the microbial
membrane and cause the leakage of ions and cytoplasmatic content
thus leading to cellular breakdown (Burt, 2004). The interest of
incorporating essential oils in foods as preservatives is related to
their recognition as safe natural compounds, being a potential
alternative to produce foods free of synthetic additives. However,
* Corresponding author.
E-mail address: omartin@tecal.udl.es (O. Martín-Belloso).
sents several drawbacks due to their poor water solubility as well as
to toxicological and economical considerations (Burt, 2004;
Sa
nchez-Gonz
alez, Vargas, Gonza
lez-Martínez, Chiralt, & Cha
fer,
2011). In addition, their strong flavor makes incorporation at high
doses difficult in certain types of food products due to possible
objectionable sensory characteristics. Hence, there is a need to
investigate new delivery systems to encapsulate and release
essential oils in food products.
In the food sector, the incursion of nanotechnological advances
is still discreet but it is gaining more and more interest by both the
scientific and industrial community (Rashidi & Khosravi-Darani,
2011). Recently, emulsions with small droplet size, typically from
10 to 100 nm, also called nanoemulsions, are being investigated as
lipophilic drug delivery systems in food, cosmetic and pharma-
ceutical products (Bernardi et al., 2011; He et al., 2011). Due to their
intrinsic properties, they may present several advantages for
encapsulating functional lipophilic compounds over conventional
emulsions. On the one hand, their reduced droplet size might not
only enhance the transport of active molecules through biological

http://dx.doi.org/10.1016/j.foodhyd.2014.07.012
0268-005X/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Salvia-Trujillo, L., et al., Physicochemical characterization and antimicrobial activity of food-grade emulsions
and nanoemulsions incorporating essential oils, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.012
2 L. Salvia-Trujillo et al. / Food Hydrocolloids xxx (2014) 1e10
membranes but also increase the surface area/volume ratio, thus
leading to improved functionality. On the other hand, nano-
emulsions are kinetically stable and transparent colloidal disper-
sions, being suitable for a wide range of practical applications
(Solans, Izquierdo, Nolla, Azemar, & Garcia-Celma, 2005).
To produce nanoemulsions with droplet sizes in the nano-range
a high shear rate is required in order to generate a high interface
area (Delmas et al., 2011). Among the few methods that can pro-
duce nanoemulsions, microfluidization has been shown to generate
extremely fine emulsions with droplet sizes between 60 and
600 nm (Hatanaka et al., 2010; Hatanaka, Kimura, Lai-Fu, Onoue, &
Yamada, 2008; Jafari, He, & Bhandari, 2007a, 2007b; Qian &
McClements, 2011; Rao & McClements, 2011; Wooster, Golding, &
Sanguansri, 2008). The microfluidizer device uses a pump to force a
coarse emulsion pre-mix to an interaction chamber under high
pressure (Jafari et al., 2007a; McClements, 2011). In the interaction
chamber the coarse emulsion is split in two streams that further
impinge on each other at high velocity, providing an exceptionally
fine emulsion (Mahdi Jafari, He, & Bhandari, 2006; McClements,
2011).
The use of nanoemulsions as potential delivery systems of
lipophilic food ingredients is arising with promising expectations.
However, there is a lack of scientific evidence about the enhanced
functionality of nanoemulsions in comparison with similar non-
nano-formulated delivery systems (Bouwmeester et al., 2009).
Therefore, the purpose of the present research work was to char-
acterize microfluidized nanoemulsions incorporating essential oils
in terms of droplet size, size distribution, z-potential, viscosity and
color in comparison with conventional emulsions. Moreover, the
in vitro antimicrobial activity against Escherichia coli of nano-
emulsions was assessed and compared with that exhibited by
conventional emulsions.
2. Material and methods
2.1. Primary emulsion formation
Sodium alginate (FMC Biopolymers, UK) was dissolved in hot
water at 70  C (1% w/v) under continuous stirring until complete
homogenization. Coarse emulsions were prepared by mixing the
resulting sodium alginate aqueous solution with essential oils (1%
v/v). Sodium alginate was included in the aqueous phase as the
purpose of further investigations was to apply such nanoemulsions
as antimicrobial edible coatings in fresh-cut fruit. Lemongrass
(Cymbopogon citratus) essential oil was purchased at Laboratoris
Dicana (Spain) and clove (Eugenia caryophyllata), tea tree (Mela-
leuca alternifolia), thyme (Thymus vulgaris), geranium (Pelargonium
graveolens), marjoram (Origanum majorana), palmarosa (Cymbo-
pogon martinii), rosewood (Aniba rosaedora), sage (Salvia officinalis)
and mint (Mentha spicata) were purchased from Dietetica Intersa
(Spain). The composition of each type of oil is detailed in Table 1,
Table 1
Essential oil composition, oil refractive index and absorbance (l ¼ 633 nm) of essential oils.
Essential oil Plant
Lemongrass Cymbopogon citratus
Clove Eugenia caryophyllata
Tea Tree Melaleuca alternifolia
Thyme Thymus vulgaris
Geranium Pelargonium graveolens
Marjoram Origanum majorana
Palmarosa Cymbopogon martinii
Rosewood Aniba rosaedora
Sage Salvia officinalis
Mint Mentha spicata
Please cite this article in press as: Salvia-Trujillo, L., et al., Physicochemical characterization and antimicrobial activity of food-grade emulsions
and nanoemulsions incorporating essential oils, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.012
according to the information given by the supplier. Tween 80 (1% v/
v) (Scharlau, Spain) was added as surfactant with a laboratory T25
digital Ultra-Turrax mixer (IKA, Staufen, Germany) working at
3400 rpm for 2 min. All samples were prepared using ultra pure
water obtained from a Mili-Q filtration system.
2.2. Nanoemulsion formation
A microfluidization system (M110P, Microfluidics, Massachu-
setts, USA) was used to obtain nanoemulsions from the coarse
emulsions. Coarse emulsions were passed through the system 3
times at a constant pressure of 150 MPa. At the outlet of the
interaction chamber the product was refrigerated through an
external cooling coil immersed in an ice-water bath so that the
temperature of the product was always kept below 20  C.
2.3. Emulsion and nanoemulsion characterization
2.3.1. Particle size and z-potential
The oil droplet size was measured by dynamic light scattering
(DLS) with a Zetasizer NanoZS laser diffractometer (Malvern In-
struments Ltd, Worcestershire, UK) working at 633 nm at 25  C and
equipped with a backscatter detector (173 ), which is appropriate
to measure sub-micron particles (Brar & Verma, 2011). DLS mea-
sures the Brownian motion of nano-sized droplets and relates this
movement to an equivalent hydrodynamic diameter (nm). Average
droplet size, size distribution curves and polydispersity index were
used to characterize oil droplets dispersion in nanoemulsions. The
refractive index of essential oils and their absorbance at 633 nm,
were measured with a refractometer and a spectrophotometer,
respectively (Table 1).
The electrophoretic mobility of oil droplets, also reported as z-
potential, was measured by phase-analysis light scattering (PALS)
with a Zetasizer NanoZS laser diffractometer (Malvern Instruments
Ltd, Worcestershire, UK). It determines the surface electrical charge
of the droplets dispersed in the continuous phase.
2.3.2. Viscosity
Viscosity of emulsions and nanoemulsions was measured from
approximately 10 mL of sample using a SV-10 vibro-viscometer
(A&D Company, Tokyo, Japan) vibrating at 30 Hz and constant
amplitude. The viscosity of the sodium alginate solution (30 mPa s)
was considered the viscosity of the dispersant material with
respect to the dynamic light scattering measurements.
2.3.3. Whiteness index
The color of emulsions and nanoemulsions was measured with a
Minolta CR-400 colorimeter (Konica Minolta Sensing, Inc., Osaka,
Japan) at room temperature set up for illuminant D65 and 10
observer angle and calibrated with a standard white plate. CIE L*, a*
and b* values were determined and the Whiteness Index (WI) was
Composition Refractive index Abs633
Geranial, neral, geraniol 1.453 0.022
Eugenol, eugenil acetate 1.499 0.023
4-terpineol, terpinene 1.465 0.001
Timol, p-cimene 1.471 0.094
Citronelol, geraniol 1.462 0.157
4-terpineol, cis tuyan-4-ol 1.472 0.002
Geraniol, geranil acetate, linalol 1.461 0.003
a-terpineol, linalol 1.468 0.002
Canfor, 1,8-cineol, canfene, sabinil acetate 1.479 0.071
Mentol 1.477 0.002
 L. Salvia-Trujillo et al. / Food Hydrocolloids xxx (2014) 1e10 3

calculated with equation (1) (Vargas, Ch

afer, Albors, Chiralt, & and nanoemulsions containing different essential oils at a 5% sig-
Gonz
alez-Martínez, 2008): nificance level.


0:5
WI ¼ 100
ð100
LÞ2 þ a2 þ b2 (1) 3. Results

3.1. Particle size and particle size distribution

2.3.4. Antimicrobial activity assay
The antimicrobial activity of essential oil-alginate emulsions The results of the average droplet size and polydispersity index
and nanoemulsions was assessed evaluating the in vitro inhibition of essential oil-loaded emulsions and nanoemulsions are shown in
of E. coli, following the method described previously by other au- Table 2, and their droplet size distribution is presented in Fig. 1. The
thors with some modifications (Ferreira et al., 2010). E. coli 1.107 method used to prepare the mixtures significantly influenced the
(Laboratoire de Re
pression des Fraudes, Montpellier, France) was emulsion droplet size. The further processing of blends by micro-
cultured in tryptone soy broth (Bioakar Diagnostic; Beauvais, fluidization after a previous high shear homogenization rendered
France) and incubated at 37  C with continuous agitation at nanoemulsions with a reduced droplet size. Moreover, strong dif-
120 rpm for 11 h to obtain cells in stationary growth phase. The ferences among emulsion droplet size and size distribution were
final concentration reached in the culture was 108e109 colony observed depending on the essential oil used in the formulation of
forming units/millilitre (CFU/mL). A 0.5 mL-aliquot of overnight nanoemulsions. Namely, microfluidized lemongrass oil-loaded
bacterial culture was mixed with 0.5 mL of the essential oil-alginate nanoemulsions exhibited a dramatic reduction of their average
emulsion or nanoemulsion and 4.5 mL of sterile Mili-Q water. Ali- droplet size, down to 5.5 ± 0.3 nm, in comparison with the values of
quots of 0.1 mL were taken after 5, 15 and 30 min and were serially the coarse emulsion, which averaged 1120 ± 230 nm. Oppositely,
diluted and spread on McConkey agar (Biokar Diagnostics, Beau- emulsions incorporating palmarosa and rosewood oils exhibited
vais, France) plates to determine the inactivation kinetics. A blank average droplet sizes already in the nano-scale range (33 ± 7 or
determination was performed with the same method, replacing the 13.8 ± 0.5 nm, respectively) before microfluidization and with a
emulsion or nanoemulsion by sterile Mili-Q water. Colony counts narrow size distribution. Therefore, in both cases the micro-
were determined after incubation of agar plates at 37  C for 24 h. fluidization treatment had a slight effect on the average droplet
size, which was confirmed by size distribution data (Fig. 1).
2.4. Kinetic modeling Regarding the rest of essential oils (clove, tea tree, thyme, gera-
nium, marjoram, sage or mint), the average droplet size of coarse
The inactivation kinetics of essential oil-alginate emulsions or emulsions was also in the nano-scale range, with values between
nanoemulsions against E. coli was adjusted to a Weibull distribu- 13.2 ± 0.9 and 87 ± 4 nm. Nonetheless, a multimodal size distri-
tion model, by Eq. (2) (Peleg, 2006): bution was observed with major intensity peaks positioned at
 a 1000 nm and with minor peaks at the nano-range. Those minor
t peaks might be surfactant micelles that were not adsorbed in the
ln SðtÞ ¼
(2)
b oil droplets interface, which are typically around 10 nm
(Heydenreich, Westmeier, Pedersen, Poulsen, & Kristensen, 2003;
where S is the survival fraction (N/N0) of E. coli, t is the reaction time Me
nard et al., 2011).
(min), b is the scale factor, and a is the shape factor. The scale factor In fact, the particle size distribution graphs of microfluidized
represents the contact time (min) necessary to inactivate the first emulsions (Fig. 1) show a significant decrease in the intensity peaks
log-cycle of the microbial population, whereas the shape factor is of particles above 100 nm. Consequently, the intensity peaks of
related to the trend of the inactivation curve. A shape factor below 1 smaller particles increased, indicating a clear particle size
indicates a concavity upwards while a value greater than 1 means a
concavity downwards.
The model was fitted to experimental data by non-linear Table 2
Average droplet size (z-average) (nm) and polydispersity index (PDI) of essential oil-
regression using the Statgraphics Plus v.5.1 Windows package.
alginate emulsions or nanoemulsions produced by high shear homogenization
The adjusted regression coefficients (R2 adj) and accuracy factor (Af, (3400 rpm, 2 min) or microfluidization (150 MPa, 3 cycles).
Eq. (3)) (Ross, 1996) of the model were calculated.
Essential oil Homogenization system z-average (nm) PDI
  
P predicated  Lemongrass HSH 1121.43 ± 228.93 0.49 ± 0.08
 log
observed  MF 5.49 ± 0.28 0.51 ± 0.04
Af ¼ 10 n (3) Clove HSH 23.23 ± 10.51 0.94 ± 0.09
MF 20.88 ± 5.10 0.82 ± 0.07
Tea Tree HSH 16.20 ± 5.24 1.91 ± 0.70
where log (predicted/observed) is the logarithmic relation between
MF 2.65 ± 0.68 0.10 ± 0.01
the predicted and the experimental values, and n is the number of Thyme HSH 13.16 ± 0.92 0.23 ± 0.03
observations. MF 12.86 ± 0.25 0.36 ± 0.01
The higher the values of R2 adj and the nearer the values of Af to Geranium HSH 29.24 ± 9.73 0.38 ± 0.18
1, the better the fitting of the model to experimental data. MF 2.88 ± 0.16 0.28 ± 0.09
Marjoram HSH 35.14 ± 8.26 0.21 ± 0.18
MF 7.71 ± 0.64 0.10 ± 0.01
2.5. Statistics Palmarosa HSH 32.91 ± 6.92 1.02 ± 0.01
MF 13.82 ± 0.31 0.71 ± 0.08
Rosewood HSH 13.82 ± 0.48 1.02 ± 0.32
All the experiments were assayed in duplicate, and two replicate
MF 12.26 ± 0.09 1.08 ± 0.11
analyses of each sample were carried out in order to calculate a Sage HSH 86.49 ± 4.31 0.27 ± 0.09
mean value. Statgraphics Plus v.5.1 Windows package (Statistical MF 8.56 ± 1.29 0.34 ± 0.04
Graphics Co., Rockville, MD) was used to perform the analysis of Mint HSH 13.95 ± 1.97 0.78 ± 0.01
variance. The least significant difference (LSD) test was run to MF 2.22 ± 0.06 0.12 ± 0.01

determine significant differences (p  0.05) between emulsions Values are expressed as mean ± standard deviation.

Please cite this article in press as: Salvia-Trujillo, L., et al., Physicochemical characterization and antimicrobial activity of food-grade emulsions
and nanoemulsions incorporating essential oils, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.012
4 L. Salvia-Trujillo et al. / Food Hydrocolloids xxx (2014) 1e10

Fig. 1. Droplet size distribution of essential oil-alginate emulsions or nanoemulsions produced by high shear homogenization (3400 rpm, 2 min) (continuous line) or micro-
fluidization (150 MPa, 3 cycles) (dotted line).

reduction. The average droplet size (z-average) of emulsions cor-
responds to the mean hydrodynamic diameter and is calculated
according to the ISO-13321 (1996), but it may lead to misinter-
pretation in the case of non-monodispersed emulsions. In general,
from the results described above it can be observed a clear droplet
size disruption caused by microfluidization treatment, leading to
nanoemulsions with small droplet sizes. During the micro-
fluidization treatment, the process stream is pressurized and
passed through an interaction chamber thus creating shear and
impact forces as well as cavitation phenomena within the product
that reduce the emulsion droplet size (Maa & Hsu, 1999). However,
the final droplet size of nanoemulsions is a complex interplay be-
tween the processing conditions and the adsorption of surfactant
onto oil droplets (Wooster et al., 2008). On the one hand, the ability
of flavor oil molecules to be incorporated into surfactant micelles
depends on their molecular characteristics, such as molecular
weight, polarity and conformation (Djekic & Primorac, 2008). It has
been reported that essential oils with higher concentration of polar

Please cite this article in press as: Salvia-Trujillo, L., et al., Physicochemical characterization and antimicrobial activity of food-grade emulsions
and nanoemulsions incorporating essential oils, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.012
 L. Salvia-Trujillo et al. / Food Hydrocolloids xxx (2014) 1e10
compounds might reduce the interfacial tension and facilitate
droplet disruption during high pressure homogenization (Ziani,
Fang, & McClements, 2012). In this sense, low molecular weight
or high polarity compounds tend to be solubilized in the aqueous
phase rather than in the micelle interior. On the other hand, the
emulsifier type and concentration are important in determining the
emulsion droplet size, since they should adsorb to oil droplet sur-
face to prevent re-coalescence (Jafari, Assadpoor, He, & Bhandari,
2008). Moreover, viscosity and concentration of the dispersed
phase are determining factors when considering the formation of
nano-sized emulsions (Jafari et al., 2008; McClements, 2005; Qian
& McClements, 2011; Seekkuarachchi, Tanaka, & Kumazawa,
2006; Wooster et al., 2008).
It has been observed that the higher the viscosity of the oil
phase, the higher the emulsion droplet size (Qian & McClements,
2011). In fact, as the oil viscosity increases, the disruption forces
needed to deform the oil droplets during homogenization are
higher (Håkansson, Tra €gårdh, & Bergenståhl, 2009; McClements,
2005). Namely, Wooster et al. (2008) observed droplet sizes of
around 120 nm in nanoemulsions formulated with high viscosity
oils, whereas nanoemulsions containing low viscosity oils pre-
sented droplet sizes of around 80 nm. Usually, plant essential oils
are constituted of low molecular weight compounds and present
low viscosity values (Sell, 2010). In agreement to our results, Rao
and McClements (2012) and Ziani, Chang, McLandsborough, and
McClements (2011) have used microfluidization approaches for
the generation of lemon oil and thyme oil-loaded nanoemulsions,
respectively. In addition, Donsì, Annunziata, Sessa, and Ferrari
(2011) obtained D-limonene nanoemulsions stabilized with tween
20 and glycerol monooleate through high pressure homogenization
(10 cycles at 300 MPa) with an average droplet size of
154.6 ± 1.4 nm. Differences in droplet size observed in the present
work between various essential oil-loaded nanoemulsions might
be attributed to their molecular structure, concentration of volatile
compounds, viscosity, interfacial tension or surfactant affinity to
each type of flavor oil.
3.2. z-potential
The surface charge of oil droplets in emulsions and nano-
emulsions incorporating several essential oils is shown in Fig. 2.
Particles with z-potential more positive than þ30 mV or more
negative than
30 mV are usually considered to be stable, since
electrical charge of droplets is strong enough to assume that
repulsive forces between droplets are predominant in the
Fig. 2. z-potential of essential oil-alginate emulsions or nanoemulsions produced by
high shear homogenization (3400 rpm, 2 min) (HSH) or microfluidization (150 MPa, 3
cycles) (MF).
Please cite this article in press as: Salvia-Trujillo, L., et al., Physicochemical characterization and antimicrobial activity of food-grade emulsions
and nanoemulsions incorporating essential oils, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.012
5
nanoemulsion system (Heurtault, Saulnier, Pech, Proust, & Benoit,
2003). On one hand, coarse emulsions formed by high shear ho-
mogenization exhibited a weak surface charge with values less
negative than
30 mV. In fact, the coarse emulsion incorporating
mint essential oil exhibited the weaker z-potential (
10.0 mV). In
contrast, clove, tea tree or marjoram-loaded emulsions treated by
high shear homogenization exhibited a strong electrical charge
of
52.6,
30.8 and
37.4 mV, respectively. On the other hand,
nanoemulsions obtained by microfluidization showed a significant
decrease in their z-potential regardless of the oil type used in the
formulation. In fact, most of the nanoemulsions had z-potential
values more negative than
30 mV, with the exception of
palmarosa-loaded nanoemulsions (
20.9 mV). According to

s, Vargas, and Chiralt (2012), differences observed in
Bonilla, Atare
the z-potential of emulsions and nanoemulsions formulated with
different essential oils might be attributed to differences in the
dissociation degree and number of ionizable compounds of oils.
Moreover, it is generally considered that the competitive adsorp-
tion of the different emulsifiers is based on principle that the more
surface-active material replaces the less active compound
(Hasenhuettl & Hartel, 2008; Kralova & Sjo € blom, 2009), but this
might depend on the affinity with the type of oil. Therefore, the
differences in the z-potential observed among essential oils might
be due to differences between the adsorption of the surface-active
compounds in the oilewater interface.
Tween 80 is a non-ionic low-mass surfactant that rapidly coats
the newly created droplets during emulsification (Kralova &
Sjo€ blom, 2009). It is preferably adsorbed on the oil surface in the
co-presence of polymeric stabilizers (Nambam & Philip, 2012),
giving a neutral (Kralova & Sjo €blom, 2009) or slightly negative
electrical charge on oilewater interface (Hsu & Nacu, 2003). Thus,
the negative charge of oil droplets observed in the present work
might be due to the anionic sodium alginate molecules dispersed in
the aqueous phase. Even though alginates, as well as most food
hydrocolloids, do not possess hydrophobic moieties (Dickinson,
2003, 2009), it has recently been observed that they can present
surface activity under certain conditions (Garti & Leser, 2001; Yang,
Jiang, He, & Xia, 2012). The mechanical stress during micro-
fluidization can lead to break up sodium alginate (see viscosity
discussion 3.3) thus increasing the number of molecules to be
potentially adsorbed on the oilewater interface. This fact could
explain the z-potential strengthening observed in microfluidized
nanoemulsions.
3.3. Viscosity
The viscosity of emulsions or nanoemulsions incorporating
different essential oils before and after microfluidization at
150 MPa for 3 cycles is presented in Fig. 3. The viscosity of coarse
emulsions ranged between 22.95 and 36.05 mPa s depending on
the type of essential oil used in the formulation. Mint oil led to
more viscous emulsions, whereas thyme oil rendered the lowest
viscosity values. The characteristics of the dispersed phase have a
significant impact on the rheological properties of emulsions
(McClements, 2005; Pal, 2011; T. F. Tadros, 1994). Similarly to our
results, Bonilla et al. (2012) found that thyme oil-loaded emulsions
stabilized with chitosan showed lower viscosity values in com-
parison with emulsions containing basil essential oil. They attrib-
uted these variations to differences in the dissociation degree and
number of ionizable compounds of the different essential oils, since
biopolymeric molecules would be adsorbed at the oil droplet sur-
face and lead to changes in the effective thickening concentration
in the aqueous phase.
In addition, a dramatic reduction of emulsion viscosity has been
observed after microfluidization, regardless of the essential oil used
6 L. Salvia-Trujillo et al. / Food Hydrocolloids xxx (2014) 1e10
Fig. 3. Viscosity (mPa s) of essential oil-alginate emulsions or nanoemulsions pro-
duced by high shear homogenization (3400 rpm, 2 min) (HSH) or microfluidization
(150 MPa, 3 cycles) (MF).
in the formulation. Coarse emulsion viscosity values decreased
down to a 60.66% after microfluidization. In this sense, nano-
emulsions presented viscosities ranging between 9.77 and
14.95 mPa s. Furthermore, there were no significant differences in
viscosity among microfluidized nanoemulsions with different
essential oils. The droplet size influences the emulsion viscosity, as
it has been demonstrated in template oil-in-water emulsions
(Chanamai & McClements, 2000; Corte
s-Mun~ oz, Chevalier-Lucia, &
Dumay, 2009; Derkach, 2009; Pal, 2011). However, in the present
work the thickening properties of the continuous phase play a
fundamental role to determine the rheological behavior of the
nanoemulsion system. It is known that microfluidization process-
ing may induce conformational changes on the molecular structure
of biopolymers, as well as a significant reduction on their molecular
weight (Lagoueyte & Paquin, 1998).
Recently, Villay et al. (2011) observed a viscosity reduction on
sodium alginate solutions when they were treated with high
pressure homogenization at 195 MPa and several cycles. They
attributed this effect to a decrease in the molar mass of sodium
alginate due to a depolymerization of chains caused by covalent
bond disruption. Similarly, Harte and Venegas (2010) reported a
75e85% viscosity reduction in alginate, k-carrageenan and xanthan
gum solutions processed with high pressure homogenization from
1 to 5 cycles up to 300 MPa. Hence, in basis of the results observed
in the present work, it can be assumed that sodium alginate may
undergo molecular changes in its molecular structure during
microfluidization process, leading to a decrease in its thickening
properties and therefore in the viscosity of nanoemulsions.
3.4. Whiteness index
The type of oil used in the formulation significantly influenced
the whiteness index of coarse emulsions and nanoemulsions
(Fig. 4). Coarse emulsions containing lemongrass, thyme, geranium
or palmarosa essential oils were those presenting higher whiteness
index, with values of 47.9, 49.6, 57.8 or 51.3, respectively. On the
contrary, coarse emulsions formulated with clove, tea tree or
marjoram essential oils exhibited the lowest whiteness index, with
values of 24.4, 29.9 or 27.8, respectively. In general, micro-
fluidization significantly affected the whiteness index of mixtures
generating nanoemulsions with lower whiteness index. Nonethe-
less, the extent of whiteness index decrease after microfluidization
depended on the oil type used in the formulation. For instance,
nanoemulsions incorporating lemongrass, sage or geranium
essential oils showed a drastic decrease in their whiteness index
from 47.9, 36.0 and 57.8 down to 32.2, 27.7 and 49.6, respectively.
Please cite this article in press as: Salvia-Trujillo, L., et al., Physicochemical characterization and antimicrobial activity of food-grade emulsions
and nanoemulsions incorporating essential oils, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.012
Fig. 4. Whiteness index (WI) of essential oil-alginate emulsions or nanoemulsions
produced by high shear homogenization (3400 rpm, 2 min) (HSH) or microfluidization
(150 MPa, 3 cycles) (MF).
Nevertheless, emulsions incorporating thyme essential oil exhibi-
ted an opposite behavior, as the whiteness index of microfluidized
nanoemulsions was higher in comparison with the coarse emul-
sion. It is known that oil droplet size significantly influences the
optical properties of emulsions (McClements, 2002). Large particles
scatter the light more intensely than smaller ones, which causes an
increase in the lightness, opacity and whiteness index of emulsions
(McClements, 2011). In general, nanoemulsions are described as
transparent systems due to their reduced droplet size (Mason,
Wilking, Meleson, Chang, & Graves, 2006; McClements, 2011;
Solans et al., 2005; T. Tadros, Izquierdo, Esquena, & Solans, 2004).
However, in spite that all coarse emulsions of the present study
exhibited a significant decrease in their average droplet size and
size distribution after being microfluidized, not all of them became
completely transparent (Fig. 5). In fact, only tea tree, geranium and
marjoram nanoemulsions became completely transparent after 3
microfluidization cycles, which is consistent with the light scat-
tering measurements showing that these nanoemulsions presented
the lowest average droplet size and narrowest size distribution
(Table 2, Fig. 1). Moreover, some essential oils might be water-
soluble due to their polar compounds, thus being able to solubi-
lize in the aqueous phase and leading also to transparent systems
(Ziani et al., 2012). In line with our results, Wooster et al. (2008)
obtained transparent nanoemulsions containing peanut oil by
microfluidization (100 MPa and 5 cycles), with a droplet size lower
than 60 nm.
Fig. 5. Images of essential oil-alginate emulsions or nanoemulsions produced by high
shear homogenization (3400 rpm, 2 min) (1) or microfluidization (150 MPa, 3 cycles)
(2). Lemongrass (L), clove (C), tea tree (T), thyme (Th), geranium (G), marjoram (M),
palmarosa (Pr), rosewood (R), sage (S), mint (Mi).
 L. Salvia-Trujillo et al. / Food Hydrocolloids xxx (2014) 1e10 7

Fig. 6. Survival fraction of Escherichia coli through 30 min of reaction time with essential oil-alginate emulsions or nanoemulsions produced by high shear homogenization
(3400 rpm, 2 min) (-) or microfluidization (150 MPa, 3 cycles) (:). A control with water is represented (A). The plotted lines of log S vs. reaction time (min) correspond to the data
fitted to a Weibull equation. Data shown are a mean ± standard deviation.

Please cite this article in press as: Salvia-Trujillo, L., et al., Physicochemical characterization and antimicrobial activity of food-grade emulsions
and nanoemulsions incorporating essential oils, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.012
8 L. Salvia-Trujillo et al. / Food Hydrocolloids xxx (2014) 1e10

3.5. Antimicrobial activity
The inactivation kinetics of emulsions and nanoemulsions
against E. coli is shown in Fig. 6. A Weibull equation was fitted to
experimental data of the survival curves, with accuracy factors near
to 1 and R2 values higher than 86 (Table 3).
Emulsions and nanoemulsions containing lemongrass, clove,
thyme or palmarosa essential oil showed the strongest antimicrobial
activity. Namely, lemongrass or clove-loaded emulsions exhibited
3.98 ± 0.01 or 3.52 ± 0.02 log reductions after being in contact 30 min
with the inoculated microorganism, respectively. On the contrary,
coarse emulsions containing tea tree, geranium, marjoram, rose-
wood, sage or mint essential oils presented less than 1 log reduction
of E. coli after 30 min of contact time. It is known that the antimi-
crobial activity of each essential oil is due to their phenolic com-
pounds and it largely depends on their concentration as well as on
their chemical structure (Dorman & Deans, 2000). In general, the
mechanism of action of essential oils against microorganisms in-
volves the interaction of phenolic compounds with the proteins
(porins) in the cytoplasmatic membrane that can precipitate and lead
to leakage of ions and other cell content causing the cell breakdown
(Nychas, Skandamis, & Tassou, 2000). Those essential oils containing
carvacrol and thymol terpenes, such as thyme essential oil, are re-
ported to have a strong bactericidal action (Lambert, Skandamis,
Coote, & Nychas, 2001). Nevertheless, eugenol and citral, the major
components of clove and lemongrass or rosewood essential oil,
respectively (Friedman, Henika, Levin, & Mandrell, 2004), have been
found to inactivate a broad spectrum of microorganisms (Hammer,
Carson, & Riley, 1999). Other aromatic compounds such as linalool
(tea tree), pinene (mint), geraniol (geranium), or borneol (sage),
which can be found in essential oils, exhibit a lower inhibitory in-
fluence against bacteria (Zachariah & Leela, 2006). Therefore, the
final antimicrobial activity of a determined essential oil is a combi-
nation of composition and concentration of each volatile compound,
which in turn are governed by the plant variety, growing conditions
and method of extraction of the essential oil.
In our experiments it was observed that microfluidization of
coarse emulsions significantly affected their antimicrobial activity,
since nanoemulsions exhibited an increased and faster inactivation
of E. coli. This behavior was clearly noticeable in the case of
lemongrass or clove oil-loaded nanoemulsions. In particular, while
the coarse emulsion formulated with lemongrass essential oil pre-
sented a b of 22.30, its respective microfluidized nanoemulsion
exhibited a b of 6.97 (Table 3). Similarly, the b factor of the inacti-
vation kinetics of E. coli with clove oil decreased from 19.71 with the
coarse emulsion to 5.97 with the nanoemulsion. In addition, a
concavity upwards was observed in the inactivation kinetics pre-
sented by both microfluidized nanoemulsions with a lower than 1,
whereas coarse emulsions presented inactivation curves with a
values higher than 1, which implies a slow release of phenolic
compounds at early contact times. Therefore, microfluidized nano-
emulsions containing lemongrass or clove essential oils showed a
faster release of antimicrobial compounds in comparison with
coarse emulsions, presenting a faster inactivation of inoculated
E. coli population. The rest of microfluidized nanoemulsions con-
taining other essential oils did not exhibit a significant enhancement
of their antimicrobial potential compared to their corresponding
coarse emulsion, since the b values of the inactivation curves were
slightly reduced or even were increased in some cases. It is generally
assumed that nanoencapsulated lipophilic antimicrobials would be
able to penetrate more easily though microbial membranes thus
leading to an improved bactericidal action. In the case of Gram-
negative bacteria, such as E. coli, the presence of proteins (porins)
that form channels (pores) in the outer membrane allow the influx
of hydrophilic particles, being a barrier for lipophilic compounds
and also restricting the entry of compounds by size (Schweizer,
2012; Taber, 2008). Therefore, it would be reasonable to assume
that the solubilization of lipophilic antimicrobials within nano-
emulsions would lead to an increase of binding sites able to interact
with porins. In agreement with our results, recent research papers
report an enhancement on the antimicrobial activity of nano-
emulsions containing flavor oils such as thyme oil (Chang,
McLandsborough, & McClements, 2012), peppermint oil (Liang
et al., 2012), limonene (Donsì et al., 2011) or eugenol and cinna-
maldehyde (Gomes, Moreira, & Castell-Perez, 2011). In line with our
results, Buranasuksombat, Kwon, Turner, and Bhandari (2011) found
that emulsion droplet size had no influence on the antimicrobial
properties of lemon myrtle oil. These apparently controversial re-
sults indicate that a significant droplet size reduction does not
necessarily imply an increase in the functionality of essential oil
nanoemulsions, as their antimicrobial activity might be given by the
whole delivery system and its particular ability to react with

Table 3
Estimated parameters of Weibull distribution function proposed to describe the inactivation kinetics of Escherichia coli by essential oil-alginate emulsions and nanoemulsions
processed by high shear homogenization (HSH) or microfluidization (MF).

Essential oil Homogenization b a R2 (%) Af

system

Lemongrass HSH 22.30 ± 0.66 4.68 ± 0.48 99.89 1.29

MF 6.97 ± 0.84 0.98 ± 0.07 97.91 1.23
Clove HSH 19.71 ± 0.38 3.00 ± 0.12 99.03 1.10
MF 5.97 ± 0.62 0.82 ± 0.07 97.63 1.16
Tea Tree HSH 35.56 ± 2.80 1.91 ± 0.70 99.40 1.35
MF 363.01 ± 135.37 0.10 ± 0.01 99.70 1.02
Thyme HSH 0.82 ± 0.22 0.23 ± 0.03 99.01 1.04
MF 1.87 ± 0.09 0.36 ± 0.01 97.99 1.06
Geranium HSH 41.56 ± 13.67 0.38 ± 0.18 96.91 1.06
MF 38.31 ± 9.75 0.28 ± 0.09 99.67 1.01
Marjoram HSH 52.45 ± 21.26 0.21 ± 0.18 97.64 1.04
MF 122.19 ± 24.48 0.10 ± 0.01 99.91 1.00
Palmarosa HSH 7.34 ± 0.03 1.02 ± 0.01 96.22 1.47
MF 4.39 ± 0.17 0.71 ± 0.08 99.66 1.03
Rosewood HSH 149.21 ± 87.16 1.02 ± 0.32 96.21 1.25
MF 72.35 ± 6.71 1.08 ± 0.11 92.93 1.29
Sage HSH 10,050.06 ± 12,587.12 0.27 ± 0.09 86.55 1.22
MF 3361.82 ± 38.27 0.34 ± 0.04 98.54 1.61
Mint HSH 38.69 ± 3.21 0.78 ± 0.01 94.94 1.17
MF 631.13 ± 30.14 0.12 ± 0.01 99.92 1.01

Values are expressed as mean ± standard deviation, b, is the scale factor; a, is the shape factor; Af, is the accuracy factor.

Please cite this article in press as: Salvia-Trujillo, L., et al., Physicochemical characterization and antimicrobial activity of food-grade emulsions
and nanoemulsions incorporating essential oils, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.012
 L. Salvia-Trujillo et al. / Food Hydrocolloids xxx (2014) 1e10
microbial membranes. In this sense, it is also known that the
membrane permeability and cellular absorption is surfactant-
dependent since the droplet charge is known to influence the effi-
cacy of nanoemulsified antimicrobials. Our results show that
nanoemulsions with different essential oils had significantly
different droplet charge, which indicates a distinct interaction be-
tween oil and surfactant or biopolymer molecules. In our case,
negative droplet charges could have blocked the entrance of anti-
microbial molecules though the microbial pores. Ziani et al. (2011)
reported an antagonistic effect of cationic and anionic surfactants
on the antimicrobial activity of thyme oil nanoemulsions, which was
attributed to the partition of surfactant molecules between the oil
droplet and microbial surfaces.
4. Conclusions
In the present work, a significant influence of microfluidization
on the physicochemical properties of nanoemulsions with essential
oils was evidenced. On the one hand, there was an appreciable
decrease in the droplet size of emulsions after microfluidization,
down to values below 20 nm. The droplet size of nanoemulsions
could explain the whiteness index results, being transparent those
nanoemulsions with the smallest droplets. Nonetheless, the optical
properties of nanoemulsions were highly dependent on the oil type
used in the formulation. On the other hand, the mechanical stress
during microfluidization caused a decrease in the nanoemulsions
viscosity. This fact could be related with the stronger z-potential of
nanoemulsion in comparison with coarse emulsions. The antimi-
crobial activity of nanoemulsions was rather determined by the
essential oil type used in the formulation than by their droplet size.
Only in the case of nanoemulsions with lemongrass or clove
essential oil an enhanced antimicrobial activity against E. coli could
be observed. These results evidence the potential advantages of
nanoemulsions as delivery systems of essential oils as preservatives
in foods but also indicate the need to elucidate the mechanism of
interaction of lipid nanoparticles with biological membranes.
Acknowledgments
This study was supported by the Ministerio de Ciencia e
Innovacio
n (Spain) throughout the project AGL2009-11475. Laura
Salvia-Trujillo thanks the Ministry of Science and Education (Spain)
for the predoctoral fellowship. Prof. Olga Martín-Belloso thanks the

Catalana de Recerca i Estudis Avançats (ICREA) for the
Institucio
Academia 2008 Award.
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