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Innovative Food Science and Emerging Technologies
journal homepage: www.elsevier.com/locate/ifset
Comparing the effectiveness of natural and synthetic emulsifiers on
oxidative and physical stability of avocado oil-based nanoemulsions
Carla Arancibia
a,⁎
, Natalia Riquelme
a
, Rommy Zúñiga
b
, Silvia Matiacevich
a
a
Food Properties Research Group, Food Science and Technology Department, Facultad Tecnológica, Universidad de Santiago de Chile, Obispo Umaña 050, Estación
Central, Santiago, Chile
b
Bioprocess Engineering Laboratory, Department of Biotechnology, Universidad Tecnológica Metropolitana, Las Palmeras 3360, Ñuñoa, Santiago, Chile
ARTICLE INFO
Keywords:
Emulsifiers
Lecithin
Tween 80
Nanoemulsions
Physical properties
Lipid oxidation
ABSTRACT
Food industries search for replace synthetic surfactant with natural ingredients, but emulsifier type has an
impact on lipid oxidation and stability of emulsions. The aim of the study was to evaluate the emulsifier type and
concentration effect on avocado oil-based nanoemulsion development, specifically on physical and oxidative
stability. O/W nanoemulsions were prepared with 10% avocado oil using natural (lecithin) and synthetic (Tween
80) emulsifiers at different concentrations (2.5–10%). Results showed that nanoemulsions exhibited anionic (Z-
potential: −26 to −59 mV) lipid droplets with particle size between 103 and 249 nm. Emulsifier type and
concentration affected physical stability, being the most stable at 7.5–10% Tween 80 (15 days) and 7.5–10%
lecithin (10 days). Meanwhile, emulsifier concentration affected oxidative stability of nanoemulsions, being the
most unstable at 2.5% Tween 80 and 10% lecithin. Finally, although Tween 80 was more effective than lecithin,
it also could be used to develop natural nanoemulsions with good physical properties.
1. Introduction
The current demand of consumers on healthy and more natural food
products has led to an increasing interest for food industry by replacing
synthetic ingredients for others more naturals (McClements,
Bai, & Chung, 2017; Walker, Decker, & McClements, 2015). In addition,
driven by the need for edible systems able to encapsulate, protect, and
release functional compounds, the industry and researchers are fo-
cusing their efforts in nanotechnology to address issues relevant to food
and nutrition (Silva, Cerqueira, & Vicente, 2012).
Nanoemulsions are similar systems to conventional emulsions but
their particles and/or dispersed droplets are considerably smaller
(20–200 nm of diameter) (Acevedo-Fani, Soliva-Fortuny, & Martín-
Belloso, 2016; Mason, Wilking, Meleson, Chang, & Graves, 2006). In the
last decade, due to nanoemulsions can be fabricated entirely from
generally recognized as safe (GRAS) food ingredients, they are be-
coming increasingly popular because of their several advantages over
conventional emulsions, such as the following: (i) they increase water-
dispersibility of encapsulate oils, obtaining optically transparent or
slightly turbid emulsions and easy manufactured; and (ii) they have
good physical and chemical stability and besides, high bioavailability of
their lipid components (Shin, Kim, & Park, 2015; Walker et al., 2015).
However, from the thermodynamic point of view, nanoemulsions are
unstable colloidal dispersions that are formed from two immiscible
phases, so it is necessary to use an emulsifier to achieve stability
(McClements & Li, 2010).
One of the most critical aspects of forming emulsions is the selection
of the emulsifier, because they play two key roles: first, they facilitate
the emulsification, and secondly, they promote physical stability
(Krstonošić, Dokić, Dokić, & Dapčević, 2009) by adsorbing at the oil-
water interface, reducing the interfacial tension and improving the
protection of droplet from aggregation (Bai, Huan, Gu, & McClements,
2016). In addition, emulsifier type can have an impact on oxidative
stability, especially when prooxidants compounds (e.g. transition me-
tals) are into the aqueous phase, since metals can be attracted for the
anionic emulsion droplet interface and they can interacted with lipids
in the emulsion droplet core (Fomuso, Corredig, & Akoh, 2002; Uluata,
McClements, & Decker, 2015). There are numerous types of emulsifiers
that can be used in food industry, such as: proteins, polysaccharides,
phospholipids, and artificial emulsifiers (Kralova & Sjöblom, 2009),
where the last ones are the most common used due to the high effec-
tiveness (McClements, 2015; Raikos, Duthie, & Ranawana, 2016).
However, considering the request of changes for more natural food
products with “label friendly”ingredients (McClements et al., 2017), it
is necessary to increase the studies of natural alternatives in the for-
mulation of new emulsion-based products.
http://dx.doi.org/10.1016/j.ifset.2017.06.009
Received 1 February 2017; Received in revised form 20 May 2017; Accepted 15 June 2017
⁎
Corresponding author.
E-mail address: carla.arancibia@usach.cl (C. Arancibia).
,QQRYDWLYH)RRG6FLHQFHDQG(PHUJLQJ7HFKQRORJLHV[[[[[[[[[[²[[[
(OVHYLHU/WG$OOULJKWVUHVHUYHG
3OHDVHFLWHWKLVDUWLFOHDV$UDQFLELD&,QQRYDWLYH)RRG6FLHQFHDQG(PHUJLQJ7HFKQRORJLHV
KWWSG[GRLRUJMLIVHW
Tween 80 is one of the most used synthetic emulsifier in the pre-
paration of nano/conventional emulsions due to good emulsifying
properties (Guttoff, Saberi, & McClements, 2015; Raikos et al., 2016;
Salvia-Trujillo, Rojas-Graü, Soliva-Fortuny, & Martín-Belloso, 2015;
Sari et al., 2015). This emulsifier has no charge (nonionic) and its hy-
drophilic part is considerably more important than lipophilic (hydro-
philic-lipophilic balance-HLB value is 15.0), which allows effectively
stabilize oil-in-water emulsions. It has very low toxicity as compared to
other synthetic emulsifiers, but its consume is limited at an acceptable
daily intake (ADI) of 25 mg/kg (172.840, U.S. FDA) (McClements,
2015).
On the other hand, lecithin is the most widely used natural emul-
sifying agent in the food industry (Klang & Valenta, 2011). Food le-
cithin consists in a mixture of phospholipids (phosphatidylcholine,
phosphatidylethanolamine and phosphatidylinositol) with different
head and tail groups, as well as other types of lipids, such as trigly-
cerides, glycolipids, and sterols (Guiotto, Cabezas, Diehl, & Tomas,
2013; Ozturk & McClements, 2016). Lecithin is soluble in water and can
be easily dispersed in the aqueous phase forming a colloidal suspension,
favoring oil-in-water emulsion stability due to that contain hydrophilic
and hydrophobic groups, which are easily oriented in the oil-water
interface (Mezdour, Desplanques, & Relkin, 2011), Few studies have
reported useful use of lecithin as an emulsifier on food nanoemulsions.
Ozturk, Argin, Ozilgen, and McClements (2014) indicated that lecithin-
coated droplets of vitamin E were stable to droplet aggregation at high
temperature due to the strong electrostatic repulsion between them.
Also, Bai et al. (2016) performed oil-in-water nanoemulsions by mi-
crofluidization technique using natural emulsifiers (whey protein, gum
arabic, quillaja saponin and soy lecithin), and they found that O/W
nanoemulsions could be produced for all emulsifier studied and that
mean particle diameter decreased as increasing emulsifier concentra-
tion.
In addition, in this study we examined the potential of using O/W
nanoemulsions to encapsulate avocado oil in order to develop healthier
and more natural emulsions, due to the good properties attributed to
this oil type. Avocado oil is rich in monounsaturated fatty acids and low
in saturated fatty acids, and contains no cholesterol
(Dreher & Davenport, 2013). Also, has a high content of phytosterols,
sterols and vitamins A, D and E; it is an oil easily absorbed, induces the
production of collagen which helps to slow the aging of the skin, it has
antioxidant compounds (Dreher & Davenport, 2013) and its fatty acid
composition, lowering LDL cholesterol (“bad”cholesterol) and in-
creasing HDL cholesterol (“good”cholesterol) in the blood, reducing
the incidence of cardiovascular disease (Rader & Hovingh, 2014 ).
However, the current use of this oil is for direct consumption, since
there are not healthy products based on avocado oil in the market.
Therefore, due to its good characteristic could be used to develop a
natural and healthy stable nanoemulsion.
In this context, the aim of the study was to evaluate the effect of
type and concentration of emulsifiers in the development of stable
nanoemulsions based on avocado oil, comparing a natural (lecithin)
and a synthetic (Tween 80) emulsifier and also, to establish the influ-
ence of physical properties on their physical and oxidative stability.
2. Materials and methods
2.1. Materials
Nanoemulsions were elaborated with avocado oil (Casta de Peteroa)
purchased from Terramater S.A. (Santiago-Chile) and two types of
emulsifiers: natural: soy lecithin (Emultop, Cargill, Decatur-USA) do-
nated by Blumos S.A. (Santiago-Chile) and synthetic: Tween 80 ob-
tained from Sigma-Aldrich S.A. (St. Louis-USA). Purified water (con-
ductivity: 28.07 μs/cm) from an inverse osmosis system (Vigaflow S.A.,
Santiago-Chile) was used as dispersant.
For oxidative stability measurements, the reagents used were:
thiobarbituric acid obtained from Merck Co. (Steinheim-Germany),
trichloroacetic acid and 1,1,3,3-tetraethoxypropane from Sigma-
Aldrich S.A. (St. Louis-USA) and chloride acid from J.T. Baker
(Xalostoc-Mexico).
2.2. Preparation of nanoemulsions
Oil in water (O/W) nanoemulsions were prepared with avocado oil
at 10% w/w, and emulsifiers (lecithin and Tween 80) at four con-
centrations (2.5, 5, 7.5 and 10% w/w). The aqueous phase for the
formation of nanoemulsions was prepared dispersing each emulsifier in
the purified water using a magnetic stirrer (Arex, VelpScientifica,
Usmate Velate MB-Italy) at 200 rpm for 10 min at room temperature.
Then, avocado oil was slowly added to the aqueous phase, whilst pre-
homogenizing to 16,800 rpm using a high-speed homogenizer (Hauser
D130, Wiggen, Berlin-Germany). After adding all oil phase, pre-emul-
sion was continued pre-homogenizing for 15 min more in a water bath
at 5 ± 1 °C, to prevent overheating of the samples. Subsequently, in
order to decrease the particle size, pre-emulsions were homogenized
using a homogenizer by ultrasound (VCX500, Sonics, Newtown-USA)
with a 13 mm (diameter) stainless steel ultrasound probe. The process
of ultrasonic homogenization was performed for 20 min at 90% am-
plitude, in a pulsed mode of 15 s and 5 s of rest and at frequency of
20 kHz. Finally, before measurements, the nanoemulsions were stored
at 5 ± 1 °C for 24 h. Notably, under these conditions of storage, no
phenomena of destabilization of the nanoemulsions were observed.
2.3. Interfacial tension
The interfacial tension between the continuous and dispersed
phases was determined using an optical tensiometer (Ramé-Hart Inc.,
model 250-F4, Roxbury-USA) at room temperature. The method used to
determine the interfacial tension was the “pendant drop”(drop of
emulsifier dispersion in oil), which consists in capturing an image of the
dispersion drop by a digital high-speed camera and analyzing their
dimensions. An axisymmetric drop (3–8μL) of emulsifier dispersion
was delivered and allowed to stand at the tip of the needle inside a
quartz container with 30 mL of avocado oil for 12 min to achieve
emulsifier adsorption at the oil-water interface. The interfacial tension
(mN/m) was calculated by DropImage software (DropImage Advanced,
Roxbury-USA) by fitting the Laplace equation to the drop shape and
each sample was measured in triplicate. Data reported is the average of
triplicate with their corresponding standard deviation.
2.4. Particle characterization
2.4.1. Particle size and polydispersity index
The particle size and polydispersity index of different nanoemul-
sions were determined by Dynamic Light Scattering (DLS) using a
Zetasizer (NanoS90, Malvern Instruments, Malvern-UK). In order to
perform measurements, each nanoemulsion was deposited into a cuv-
ette and diluted with milli-Q water to obtain a clear solution (ap-
proximately 8% v/v concentration), in order to obtain a concentration
detectable by the equipment and to avoid interferences in the mea-
surement. Refractive indices of 1.47 for the dispersed phase (avocado
oil) and 1.33 for the continuous phase (water) were used, which were
determined by a refractometer (RA-130, Kyoto Electronics, Tokyo-
Japan). The particle size of samples was described by the zeta-average
particle size (PS) and the size distribution was described by the poly-
dispersity index (PdI). The values reported are an average of 10 de-
terminations and each sample was measured in triplicate.
2.4.2. Zeta potential
Zeta potential (mV) of different nanoemulsions was determined by
Electrophoretic Light Scattering using a Zetasizer (Nano-ZS, Malvern
Instruments, Malvern-UK). Nanoemulsions were diluted (20 μL of
C. Arancibia et al. ,QQRYDWLYH)RRG6FLHQFHDQG(PHUJLQJ7HFKQRORJLHV[[[[[[[[[[²[[[
nanoemulsion in 1 mL of milli-Q water) and deposited in capillary cells
equipped with two electrodes. Particle charge data was collected over
30 continuous readings and all measurements were made in triplicate.
2.5. Stability of nanoemulsions
2.5.1. Physical stability
The stability of nanoemulsions was analyzed using a vertical scan
analyzer (Turbiscan MA2000, Formulation, L'Union-France). The
reading head is composed of a pulsed near-IR light source
(λ= 850 nm) and two synchronous detectors: transmission (T) and
backscattering (BS), which can detect the change of the particle size of
the nanoemulsions due to coalescence and/or flocculation phenomena,
and the gravitational separation of the phases by sedimentation or
creaming processes, as a function of the sample height into the cy-
lindrical glass tube. For the measurements, a volume of 8 mL of dif-
ferent samples were added to a glass tube of 14 cm high and 1.5 cm in
diameter and were storage at 20 °C during 25 days. Measurements were
made at days: 0, 5, 10, 15, 20 and 25.
Stability was evaluated as Turbiscan stability index (TSI), which is a
statistical parameter used to estimate the suspension stability
(Wiśniewska, 2010), where a low TSI value indicate high stability of the
system (Wiśniewska, Urban, Nosal-Wiercińska, Zarko, & Gunko, 2014;
Xu, Zhang, Cao, Wang, & Xiao, 2016 ). The TSI value was calculated
using Eq. (1) reported for Xu et al. (2016):
∑
=∑−−
scan h scan h
H
T
SI |() ()|
i
hii1
(1)
where: scan i(h) is the average backscattering for each time (i) of
measurement, scan i −1(h) is the average backscattering for the i−1
time of measurement and His the number of scan for each sample.
Instability of nanoemulsions during storage, was characterized by
the level of creaming, which is measured by the creaming index (H);
where at high creaming index value is representative of high instability
of nanoemulsion. Creaming index was calculated according Eq. (2)
described by Petrovic, Sovilj, Katona, and Milanovic (2010):
=×
C
reaming Index (%) HS
HE 100
%
(2)
where, HE is the total height of the nanoemulsion (mm) and HS is the
height of cream layer (mm), which was visually measured as a function
of time. All measurements were performed in triplicate for each
emulsion.
2.5.2. Oxidative stability
Lipid oxidation was measured during storage time; where 30 mL of
each nanoemulsion were placed in plastic centrifuge tubes and stored
for 20 days at 50 °C (to accelerate oxidation). Oxidative stability was
evaluated by determination of thiobarbituric acid reactive substances
(TBARs). TBARs were measured according to the methodology of
McDonald and Hultin (1987). First, TBA (thiobarbituric acid) solution
was prepared by mixing 15% w/v trichloroacetic acid, 0.375% w/v
TBA in 0.25 M HCl. Then, 250 μL of each nanoemulsion, 2 mL of TBA
solution and 1 mL of distilled H
2
O were added to a test tube and
homogenized in a vortex mixer (ZX3, VelpScientifica, Usmate Velate
MB-Italy). After that, the mixture was heated in a water bath (WB 14,
Memmert, Schwabach-Germany) at 90 °C for 15 min, cooled at room
temperature, and centrifuged (Mini Spin Plus, Eppendorf, Hamburg-
Germany) at 14,000 rpm for 15 min. Finally, the supernatant was
measured at 580 and 520 nm in a microplate-reader (Multiskan Go,
Thermo Fisher, Waltham-USA). The absorbance was calculated as
A
532 nm
−A
580 nm
, according to Mei, McClements, and Decker (1999),
where absorbance at 580 nm was used to correct for any potential light
scattering, since 580 nm represents the closest non-TBARS absorbing
Fig. 1. Interfacial tension (A–B) and time-dependent surface pressure (C–D) of lecithin and Tween80 at different concentrations. Error bars indicate standard deviation of mean of
triplicates.
C. Arancibia et al. ,QQRYDWLYH)RRG6FLHQFHDQG(PHUJLQJ7HFKQRORJLHV[[[[[[[[[[²[[[
wavelength to 532 nm. Concentrations of TBARs were determinated
from a standard curve prepared using solution of 1,1,3,3-tetra-
ethoxypropane (12.5 μM) with an increasing concentration from 0 to
1μM/2 mL of TBA solution.
2.6. Statistical analysis
An analysis of variance (ANOVA) of two factors (type and con-
centration of emulsifier) was carried out for physical parameter values:
particle size and polydispersity index, zeta potential, creaming index
and oxidative stability. Tukey's test (α= 0.05) was used to calculate
the minimum significant difference among samples. All calculations
were carried out with XLSTAT Pro software 2015 (Addinsoft, Paris-
France).
3. Results and discussions
3.1. Interfacial tension
Interfacial properties of emulsifiers play an important role in their
ability to form and stabilize nanoemulsions. Interfacial tension profiles
of lecithin and Tween 80 measured at the oil-water interface are shown
in Fig. 1A and B. It was observed that lecithin showed higher interfacial
tension values than Tween 80, due probably to differences in orienta-
tion and configuration of the emulsifiers at the interface. This result is
in agreement with a study realized by Nash and Kendra (2017) where it
was found that Tween 20 was adsorbed in the interface more rapidly
than lecithin; however, the O/W interface containing lecithin displayed
much higher viscoelasticity than interface with Tween 20, which it was
correlated well with kinetic stabilization properties. The emulsifier
concentration also had an effect on interfacial tension profiles, since the
interfacial tension values decreased as the emulsifier concentration
increased (Fig. 1A and B). The decrease of interfacial tension as in-
creasing emulsifier concentration can be related to a faster emulsifier
adsorption to the oil-droplets surface. However, it was observed that
the interfacial tension reached relatively constant values at the highest
Tween 80 (7.5 and 10%) and lecithin (10%) concentrations, which
could indicate the saturation of the oil-water interface by emulsifier
molecules (Bai et al., 2016).
The rate at which an emulsifier adsorbs to an interface is one of the
important factors to consider during emulsion formation (McClements,
2015; Ozturk & McClements, 2016). If the diffusion of surfactant at the
interface controls the adsorption process, a plot of surface pressure
against time
1/2
can be used to calculate diffusion rate (k
diff
), which
corresponds at the slope of the plot (Martinez, Carrera,
Rodríguez, & Pilosof, 2009; McClements, 2015; McClements & Gumus,
2016; Mezdour et al., 2011). The plots of surface pressure versus time
1/
2
and the values of diffusion rate (k
diff
) for samples containing lecithin
and Tween 80 at different concentrations are shown in the Fig. 1C–D
and Table 1, respectively. In the case of surface pressure plot, it was
observed that at early stages of adsorption, the diffusion of Tween 80
from the bulk onto the interface was faster than lecithin ones, and at the
later stages, the diffusion of Tween 80 remained almost constant, in-
dicating that the interface becomes saturated with emulsifier molecules
because there were less sites available to adsorb into the interface
(McClements, 2015). As at the early stages surface pressure was linearly
related to the square root of time, diffusion rate (k
diff
) was calculated in
this period (2–11 s
1/2
). The coefficient of determination (R
2
) showed
the good of fit (R
2
> 0.97) of these data to the adsorption kinetic
model for all samples. ANOVA results showed that emulsifier con-
centration affected diffusion rate values (Table 1), but this effect de-
pended on emulsifier type. Tween 80 exhibited a higher diffusion rate
than lecithin, but an increasing of concentration varied slightly diffu-
sion rate (from 0.21 to 0.17), whilst k
diff
of lecithin samples decreased
significantly (p < 0.05) (from 0.27 to 0.10) as increasing its con-
centration. The diminish of diffusion rate by increasing lecithin con-
centration was attributed to a higher viscosity of continuous phase as
increasing lecithin concentration (data not shown). This increase of
viscosity could be attributed to the formation of phospholipid vesicles
in the aqueous phase (McClements & Gumus, 2016; Pan,
Tomas, & Anon, 2004). Finally, results showed that the emulsifier ef-
fectiveness to reduce interfacial tension depended on both emulsifier
type, being more effective Tween 80, and concentration, which affect
the diffusion rate. However, although the same diffusion rate is possible
to obtain with lecithin, the interfacial tension decrease could not be
enough to reduce the particle size during emulsion formation, and
therefore have an impact on physical stability of nanoemulsions.
3.2. Particle characterization
Table 2 shows the values of particle size (PS), polydispersity index
(PdI) and zeta potential (ZPot) of nanoemulsions with different type
and concentration of emulsifier. In the case of particle size, it was ob-
served that both factors studied (type and concentration of emulsifier)
and its interaction had a significant effect (p < 0.05) on PS values
(data not shown). At the same level of emulsifier, nanoemulsions with
Tween 80 presented significantly (p < 0.05) lower PS values than ones
with lecithin. This result was attributed to the rapid adsorption of the
Tween 80 on the surface of the oil droplets during homogenization
process, observed as higher k
diff
(Table 1), causing a lower interfacial
tension, which also was observed in Fig. 1a–b, that therefore, facilitate
rupture of the oil droplets (McClements & Gumus, 2016). As expected,
by increasing emulsifier concentration PS values decreased significantly
(p < 0.05), except in the nanoemulsions with the highest Tween 80
concentrations (7.5 and 10%). The dependence between emulsifier
concentration and the droplet size is known (Bai et al., 2016;
McClements et al., 2017; Surh, Decker, & Mcclements, 2017;), since the
minimum droplet size that can be produced is mainly determined by the
Table 1
Effect of emulsifier type and concentration on diffusion rate (K
diff
) obtained by adsorption
kinetic model.
Emulsifier type Emulsifier concentration (% w/w) K
diff
R
2
Lecithin 2.5 0.27 ± 0.01
e
0.99
5.0 0.22 ± 0.02
d
0.99
7.5 0.17 ± 0.01
b
0.98
10 0.10 ± 0.004
a
0.98
Tween 80 2.5 0.21 ± 0.01
cd
0.98
5.0 0.21 ± 0.01
cd
0.99
7.5 0.19 ± 0.001
bc
0.98
10 0.17 ± 0.01
b
0.97
a–d
Means values within a column with different superscripts differ significantly
(p < 0.05).
Table 2
Mean values and significant differences of particle size (PS), polydispersity index (PdI)
and zeta potential (ZPot) for nanoemulsions with different type and concentration of
emulsifier.
Emulsifier type Emulsifier
concentration
(% w/w)
PS (nm) PdI (−) ZPot (mV)
Lecithin 2.5 249 ± 7
a
0.17 ± 0.02
b
−57.7 ± 2.1
d
5.0 209 ± 12
b
0.17 ± 0.01
b
−59.7 ± 1.0
d
7.5 180 ± 10
c
0.17 ± 0.02
b
−57.9 ± 0.4
d
10 152 ± 3
d
0.18 ± 0.01
b
−59.4 ± 1.4
d
Tween 80 2.5 208 ± 17
b
0.17 ± 0.03
b
−35.7 ± 1.4
c
5.0 167 ± 1
cd
0.19 ± 0.04
b
−32.0 ± 2.2
b
7.5 115 ± 4
e
0.26 ± 0.04
a
−29.4 ± 2.0
ab
10 103 ± 3
e
0.29 ± 0.01
a
−26.6 ± 0.8
a
a-d
Means values within a column with different superscripts differ significantly
(p < 0.05).
C. Arancibia et al. ,QQRYDWLYH)RRG6FLHQFHDQG(PHUJLQJ7HFKQRORJLHV[[[[[[[[[[²[[[
initial emulsifier concentration added, thus an increase of emulsifier
concentration increases the molecules of emulsifier available to coat the
surface of oil droplets and decreases the interfacial tension during
homogenization, which it ease droplet disruption
(McClements & Gumus, 2016). However, the droplet size of nanoe-
mulsion also depend on homogenization process, since in this case an
increase of Tween 80 concentration from 7.5% to 10% almost did not
vary PS values, probably because the limit for droplets disruption by the
sonication process used was already reached.
Regarding to PdI, results showed that the effect of emulsifier con-
centration on PdI values was different depending on emulsifier type.
Lecithin-based nanoemulsions did not differ significantly (p > 0.05)
on PdI values ranging between 0.17 and 0.18, whilst the nanoemulsions
with the highest concentrations of Tween 80 (7.5 and 10%) showed a
higher polydispersity of particle size with values between 0.26 and
0.29. In general, it is considered that PdI values below 0.2 indicate
uniformity among droplet sizes or monomodal distributions and
therefore good physical stability (Guerra-Rosas, Morales-Castro, Ochoa-
Martinez, Salvia-Trujillo, & Martín-Belloso, 2016). In this case, most of
samples showed PdI values below 0.2 which could suggested a good
stability during storage, although this effect not only depended on
particle size distribution, but also on continuous phase viscosity, elec-
trostatic and steric repulsive interactions, density of each phase, etc.
(McClements, 2015).
Results of ZPot for both emulsifiers (lecithin and Tween 80) at the
different concentrations studied are also shown in Table 2. In general, it
was observed that all nanoemulsions exhibited a negative electrical
charge, being samples with lecithin significantly (p < 0.05) more
electronegative than ones with Tween 80. This difference between
emulsifiers can be due to electrical charge differences between both
emulsifiers, Tween 80 is a non-ionic emulsifier, whilst lecithin is an
amphiphilic phospholipid that at neutral pH of the samples (pH be-
tween 6.5 and 7) presents negatively charged the phospholipid head
groups (Ozturk & McClements, 2016; Uluata et al., 2015). On the other
hand, all Tween 80-based nanoemulsions showed negative charge,
which it was not an expected result. These negative values of ZPot may
be due to the ability of oil-water interfaces to preferentially adsorb
hydroxyl ions from water or due to the presence of anionic impurities in
the oil or surfactant (McClements, 2015; Troncoso,
Aguilera, & McClements, 2012). The emulsifier concentration effect was
also dependent on emulsifier type. Lecithin-based nanoemulsions did
not differ significantly (p > 0.05) among them on ZPot values,
showing values approximately of −58 mV (Table 2), whilst Tween 80-
based samples showed an increase significantly (p < 0.05) of ZPot
absolute values from −26 to −36 mV as Tween 80 concentration in-
creased. Considering that large absolute surface charge values higher
than ± 30 mV can lead to oil droplet repulsion and thus a better sta-
bility against coalescence (Guerra-Rosas et al., 2016; Klang & Valenta,
2011); in this study, most of samples showed ZPot absolute values over
30 mV, so these nanoemulsions could be considered stable; however,
these results cannot guarantee nanoemulsion stability, since there are
other factors that affect the emulsion stability, as droplet size and dis-
tribution, interfacial tension reduction, environmental conditions, etc.
(Ozturk & McClements, 2016).
3.3. Physical stability
In Fig. 2 can be observed the backscattering (BS) profiles during
storage at 20 °C for 25 days of storage for nanoemulsions with lecithin
and Tween 80 at the lowest (2.5%) and the highest (10%) concentra-
tions studied. First, in all samples at zero time, a linear BS is observed,
indicating that initial emulsions were homogenous. In general, nanoe-
mulsions with 2.5% showed a BS over 70%, whilst nanoemulsions with
10% emulsifier presented lower %BS that varied between 30 and 50%
(Fig. 2), obtaining the lowest BS on Tween 80 samples. The differences
on the %BS were attributed to the particle size differences among these
samples (Table 2), since larger particles scatter the light more intensely
than smaller ones (Salvia-Trujillo et al., 2015). On the other hand,
comparing BS profile during storage time at the lowest concentration
(2.5%) for both emulsifiers, it was observed that %BS decreased in the
bottom of the glass tube (between 5 and 15 mm) and increased in the
top (55 and 65 mm) as increasing storage time (Fig. 2a and c). This
profile is indicative of destabilization mechanism of clarification and
formation of a cream layer, mainly due to the migration of oil droplets
by action of gravity force, which it can be attributed to poor coverage of
droplets by surfactants. The amount of emulsifier required to stabilize
an emulsions depends on the surface load, which is determined by the
mass of surfactant per unit surface area at saturation
(Ozturk & McClements, 2016). In this case, there was more surface load
than surfactant available to cover the droplets, therefore a higher
droplet aggregation and creaming were observed. This creaming de-
stabilization mechanism was observed in all samples independently of
emulsifier type and its concentration. However, it is also observed that
at highest concentration (10%) for both emulsifiers, a slight sedi-
mentation destabilization mechanism is observed in the bottom of the
tube (approx. at 10 mm) after 20 days of storage, possible attributed to
sedimentation of oil droplets.
In order to compare between samples, the Turbiscan stability index
(TSI) of different nanoemulsions was calculated using Eq. (1) as a global
destabilization parameter. In general, TSI values vary in a range be-
tween 0 and 100, where the high TSI value corresponds to the more
unstable system (Wiśniewska et al., 2014). In this case, it was observed
that TSI values only increased until ~ 2.4 during the first 5 days and
then decreased slightly to remain constant (TSI = 1.0 ± 0.2) during
the following 20 days of storage (Fig. 3). Besides, the increase of
emulsifier concentration had not significant (p > 0.05) effect on TSI
values, except at day 5 in Tween 80-based nanoemulsions where the
samples with the highest concentration (10% Tween 80) showed the
lowest TSI values (0.95 ± 0.1). The TSI values near to zero as observed
in this samples (lower than 2) can indicate a great stability of different
nanoemulsions; however, nanoemulsions showed higher creaming
index values during storage time at 20 °C (Fig. 4). Therefore, this global
destabilization parameter was not useful to describe stability of this
kind of emulsions, for this reason, creaming index was calculated.
Regarding to creaming index (CI), it depended of type and con-
centration of emulsifier. It was observed that nanoemulsions with
Tween 80 were more stable than lecithin-based ones, exhibiting at
higher concentrations creaming formation from day 15 (Fig. 4), whilst
lecithin-based nanoemulsions showed cream formation from day 10. It
is know that emulsions containing Tween 80 as emulsifier present a
good physical stability during storage (Arancibia, Navarro-Lisboa,
Zúñiga, & Matiacevich, 2016; Raikos et al., 2016; Züge, Haminiuk,
Maciel, Silveira, & de Paula Scheer, 2013), because this surfactant can
rapidly adsorb to oil droplet surfaces during homogenization and
quickly reduce the interfacial tension, producing small droplets during
emulsion formation and giving a better stability to gravitational se-
paration. This result is in agreement with the interfacial tension and
particle size results observed in this study (Fig. 1 and Table 2), since
Tween 80 samples showed the lowest values of these parameters. Be-
sides, although the lowest Z-potential and PdI values of lecithin samples
(Table 2) could indicate higher stability of samples, the physical sta-
bility was more attributed to the final particle size than those para-
meters. On the other hand, it was observed that emulsifier concentra-
tion had a significant effect (p < 0.05) on CI values and, as expected,
an increase of emulsifier concentration decreased CI values significantly
(p < 0.05). The highest stability of nanoemulsions with Tween 80 can
be related to its smaller particle size. The creaming rate is proportional
to the square of droplet radius according to Stokes' law, and so a re-
duction in droplet size decreases the rate of gravitational separation
(Bai et al., 2016; McClements, 2009) and therefore increases the sta-
bility. Finally, the most stable nanoemulsions were those that contained
high emulsifier concentration (7.5 and 10% w/w) for both emulsifiers,
C. Arancibia et al. ,QQRYDWLYH)RRG6FLHQFHDQG(PHUJLQJ7HFKQRORJLHV[[[[[[[[[[²[[[
010 20 30 40 50 60 70
0
20
40
60
80
100 A: 2. 5 % w / w Le c i th i n
Tube length (mm)
%Back Scattering
010 20 30 40 50 60 70
0
20
40
60
80
100
0
5
10
15
20
Time (days)
B: 10% w/w Lecithin
Tube length (mm)
%Back Scattering
010 20 30 40 50 60 70
0
20
40
60
80
100 C: 2 .5% w /w Twee n 80
Tube length (mm)
%BackScattering
010 20 30 40 50 60 70
0
20
40
60
80
100 0
5
10
15
20
Time (days)
D: 10 % w/w Twe en 80
Tube length (mm)
%BackScattering
Fig. 2. Backscattering profiles as a function of the tube length after 20 days in quiescent conditions for nanoemulsions stored at 20 °C with: 2.5% w/w (A) and 10% w/w (B) of soy
lecithin, and 2.5% w/w (C) and 10% w/w (D) of Tween 80.
0 5 10 15 20 25
0
2
4
6
8
10
2.5% Lecit hin
5% Lecit hin
7.5% Lecit hin
10% Lecit hin
A
Time (days)
TSI
0 5 10 15 20 25
0
2
4
6
8
10
2.5% Twe en 80
5% Twe en 80
7.5% Twe en 80
10% Twe en 80
B
Time (days)
TSI
Fig. 3. Turbiscan stability index (TSI) of nanoemulsions with different emulsifiers: A) soy lecithin and B) Tween 80, stored during 20 days at 20 °C. Error bars indicate standard deviation
of mean of triplicates.
0 5 10 15 20
0
10
20
30 2.5% Lecith in
5% Lecith in
7.5% Lecith in
10% Lecith in
A
Storage time (days)
Creaming index (%)
0 5 10 15 20
0
10
20
30 2.5% Twe en 8 0
5% Twe en 80
7.5% Twe en 8 0
10% Twe en 80
B
Storage time (days)
Creaming index (%)
Fig. 4. Creaming index of nanoemulsions with different emulsifiers: A) soy lecithin and B) Tween 80, during 20 days of storage at 20 °C. Error bars indicate standard deviation of mean of
triplicates.
C. Arancibia et al. ,QQRYDWLYH)RRG6FLHQFHDQG(PHUJLQJ7HFKQRORJLHV[[[[[[[[[[²[[[
being not statistically different (p > 0.05) between them; showing CI
values of 7.0 ± 0.4% (lecithin-based samples) and 5.6 ± 0.3%
(Tween 80-based samples) after 20 days of storage.
3.4. Oxidative stability
The formation of thiobarbituric acid reactive substances (TBARs) of
different nanoemulsions stored at 50 °C for 20 days is shown in the
Fig. 5. In general, it was observed that lecithin-based nanoemulsions
were less stable to lipid oxidation than Tween 80-based ones. This
difference can be due to emulsifier nature, since phospholipids (soy
lecithin) themselves are susceptible to lipid oxidation (Cui & Decker,
2015), autoxidation and photosensitized lipid oxidation in oil-in-water
emulsions (Uluata et al., 2015).
In relation to emulsifier concentration, significant differences
(p < 0.05) were found among nanoemulsions with lecithin, being the
samples with 10% of emulsifier the least stable to lipid oxidation. The
existence of a greater amount of phospholipids in this nanoemulsion
can increase the extent of oxidation; however, that is not completely
clear because some studies have found that soy lecithin as emulsifier
delays lipid oxidation of emulsions (García-Moreno, Frisenfeldt-
Horn, & Jacobsen, 2014), but there are also other studies where it was
observed an antagonistic behavior of lecithin on oxidative stability of
emulsions (Yang & Xiong, 2015). Besides, it is important to consider
that the promotion or inhibition of the lipid oxidation by natural
emulsifiers could also depend on other factors, such as molecular
properties, location and environmental conditions
(McClements & Gumus, 2016). In the case of Tween 80-based nanoe-
mulsions, it was obtained a contrary behavior (Fig. 5), since at the
highest concentrations of emulsifier (7.5 and 10% w/w) there was a
greater oxidative stability, which can be due to the fact that Tween 80
has a low critical micelle concentration (CMC: < 0.1 mM) (Walker
et al., 2015); therefore, at higher concentrations, there may be an ex-
cess of emulsifier, forming micelles that have been shown to decrease
lipid oxidation (Richards, Chaiyasit, McClements, & Decker, 2002).
4. Conclusions
This study compared the effectiveness of two (natural and synthetic)
food-grade emulsifiers on develop of healthy nanoemulsions and on
their physical and oxidative stability. Results showed that emulsifier
type and concentration affected physical characteristics of nanoemul-
sions. Tween 80 was more effective in decrease interfacial tension than
soy lecithin, which it eased the droplet disruption and the decrease of
particle size of theses nanoemulsions. In addition, an increase of sur-
factant concentrations gave rise higher amount of available surfactant
to cover oil droplet surface, which boosted their physical stability.
Nanoemulsions with 7.5 and 10% of emulsifier were the most stable to
gravitational separation for both emulsifiers, but showing instability by
creaming after storage at 20 °C for 10 days for samples containing le-
cithin and 15 days using Tween 80. Besides, nanoemulsions containing
Tween 80 were more stable against to lipid oxidation, and as increasing
emulsifier concentration oxidative stability increased. Finally, these
results have demonstrated that although physical properties such as PS,
PdI and ZPot could infer a better physical stability of emulsions con-
taining lecithin, this one was less effective than the synthetic emulsifier,
at the same concentration, to prevent lipid oxidation and gravitational
separation of nanoemulsions. However, natural surfactant could be
used to develop edible nanoemulsions with considerable physical
properties due to Tween 80 concentrations used in this study are over
the acceptable daily intake (ADI) (25 mg/kg of body weight), whilst soy
lecithin concentration depend on Good Manufacturing Practice.
Acknowledgements
The authors acknowledge financial support from CONICYT for
Project FONDECYT POSTDOCTORADO No. 3150537, USACH (VRIDEI
and Technological Faculty) for Project Basal MECESUP-USA1555LD
and BLUMOS S.A. for providing free samples of soy lecithin.
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