Food Hydrocolloids 44 (2015) 40e48

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

Nano-emulsification of orange peel essential oil using sonication and

native gums
Adel Mirmajidi Hashtjin, Soleiman Abbasi*
Food Colloids and Rheology Lab., Department of Food Science & Technology, Faculty of Agriculture, Tarbiat Modares University, P. O. Box 14115-336, Tehran,
Iran

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

Article history: Essential oils are widely used in food and pharmaceutical industries where they encounter major con-
Received 20 January 2014 cerns more likely insolubility and instability. Therefore, using the response surface methodology, the
Accepted 28 August 2014 influence of ultrasonication conditions as well as native gums on the mean droplets diameter (Z-average
Available online 17 September 2014
value), polydispersity index (PDI), and viscosity of the orange peel essential oil (OPEO) nanoemulsions
were evaluated. In addition, the flow behavior and stability of selected nanoemulsions was assessed
Keywords:
during storage at different temperatures. Results showed that the optimum conditions for producing
Nanoemulsions
OPEO nanoemulsions (12.68 nm) were determined as 94% (sonication amplitude), 138 s (sonication time)
Sonication
Orange peel essential oil
and 37
C (process temperature). Moreover, the soluble fraction of Persian and tragacanth gums at low
Rheology concentration showed significant effect on stability, particle size, and rheology. In addition, the flow
behavior of produced nanoemulsions was Newtonian, and the effect of storage conditions (time and
temperature) on the Z-average value was highly significant (P < 0.0001).

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction The popularity of OPEO is due to its pleasant aromatic scent as

Nowadays, due to increased level of knowledge, most con-
sumers prefer foods without synthetic chemicals. Accordingly, the
use of natural aromatic compounds and flavors such as essential
oils in food products is of utmost importance (Teixeira, Andrade,
Farina, & Rocha-Leao, 2004). Orange peel essential oil (OPEO) is
among the most common important essential oils used in food,
cosmetics, and pharmaceutical industries. The OPEO normally in-
cludes limonene (94%), myrcene (2%), linalool (0.5%), octanal (0.4%),
decanal (0.4%), neral (0.1%), geraniol (0.1%) and some others
(Ashurst, 1999). Moreover, the most constitutional components of
OPEO such as aldehydes (octanal, decanal), alcohols (linalool), es-
ters and terpenoids (limonene) are low molecular weight com-
pounds (Fisher & Scott, 1997), making the essential oil more
volatile. In addition, most of these compounds are highly lipophilic,
therefore they have lower solubility in aqueous and aqueous sugar-
containing beverages due to lower hydrogen bonding (Ashurst,
1999).
* Corresponding author. Tel.: þ98 21 48292321; fax: þ98 21 48292200.
E-mail address: sabbasifood@modares.ac.ir (S. Abbasi).
well as facilitating its acceptance by the individuals to benefit from
its therapeutic properties (Duke, Bogenschutz-Godwin, Cellier, &
Duke, 2002).
Due to their particular aromas and low costs, the use of such
compounds has always been considered by food industries, but
their applications have been associated with problems such as lack
of compatibility and solubility in most food environments, volatility
and instability during processing and storage. Therefore, finding
some methods to increase the stability of these essential oils in food
environments as well as its controlled release when needed is a
turning point in the production, trade and use of aromatic com-
pounds and flavors in foodstuffs and other non-food products. In
this regard, the emulsion technology seems to be one of the most
essential processes to enhance their solubility, nanocapsulation,
and protection (Peter & Given, 2009).
Nanoemulsions are a group of emulsions that have droplet sizes
mostly smaller than 100 nm, and have been used for many indus-
trial applications due to high kinetic stability, low viscosity, and
high transparency, which are not formed spontaneously
(McClements, 2005; Solans, Izquierdo, Nolla, Azemar, & Garcia-
Celma, 2005).
In emulsion systems, properties such as stability, rheology,
appearance, color, and texture depend on the size of emulsion

http://dx.doi.org/10.1016/j.foodhyd.2014.08.017
0268-005X/© 2014 Elsevier Ltd. All rights reserved.
 A.M. Hashtjin, S. Abbasi / Food Hydrocolloids 44 (2015) 40e48
droplets and particle size distribution (Gutierrez et al., 2008;
Tadros, Izquierdo, Esquena, & Solans, 2004). The characteristics of
emulsions are also dependent upon the techniques used to prepare
the emulsion and emulsification process. There are also many
techniques for producing nanoemulsions, each of which with its
own advantages and disadvantages generating emulsions with
different properties (Gutierrez et al., 2008; Qian & McClements,
2011; Silva, Cerqueira, & Vicente, 2012; Tadros et al., 2004).
As mentioned earlier, nanoemulsions are unbalanced systems
which are not formed spontaneously. Therefore, some kinds of
energy (mechanical or chemical) are required for their formation
(Gutierrez et al., 2008; Silva et al., 2012; Solans et al., 2005; Tadros
et al., 2004). As a result, their optimization would be achievable
through optimizing the composition and variables related to the
preparation methods and conditions. In addition, the ultimate
objective in optimizing is often to reach the minimum droplet size,
minimum polydispersity, and maximum stability (Gutierrez et al.,
2008; Tadros et al., 2004).
In the food industry, nanoemulsions are usually produced using
high-energy emulsification methods, such as high pressure ho-
mogenization, microfluidization, and high intensity ultrasonication
(Rao & McClements, 2011a). Among these, the use of ultra-
sonication to produce nanoemulsions is a recent development (Li &
Chiang, 2012). Lower energy and surfactant consumption, smaller
droplet size, lower polydispersity, and higher stability of nano-
emulsions are among the major advantages of this technique over
the other methods (Kentish et al., 2008; Li & Chiang, 2012; Tadros
et al., 2004). It should be noted that in the production of nano-
emulsions using ultrasonication, some variables such as amplitude,
sonication time, as well as temperature can be effective upon the
characteristics of nanoemulsions (Kaltsa, Michon, Yanniotis, &
Mandala, 2013; Kentish et al., 2008; Li & Chiang, 2012; Mirmajidi
Hashtjin & Abbasi, 2014).
Moreover, the type and concentration of the components of
emulsions play significant roles in determining their characteris-
tics. Therefore, to improve the physical and rheological properties
and stability of nanoemulsions, selecting the type of compounds
and their proportions in the formulation is an important matter.
Water, oil phase and emulsifier form the basic structure of nano-
emulsions (Silva et al., 2012). In this regard, the type and concen-
tration of emulsifier are the most important factors affecting
nanoemulsion systems. There are a number of diverse emulsifiers
such as Spans and Tweens which just play the role of emulsifier
(Marie, Perrier-Cornet, & Gervais, 2002), and some such as gums,
modified starches and milk proteins which have both emulsifying
and stabilizing roles (Dickinson, 2009; Mohan & Narsimhan, 1997).
On the other hand, because of the willingness and propensity of
consumers to reduce food additives in formulated food products,
recently the feasibility of replacing additives with natural in-
gredients has become especially important. In this regard, it has
already been reported that native gums (e.g. gum tragacanth and
Persian gum) can function as emulsifier and emulsion stabilizer
(Abbasi & Mohammadi, 2013; Abbasi & Rahimi, 2014; Azarikia &
Abbasi, 2010), therefore, they might be able to improve some
properties of nanoemulsion systems as well.
Creaming, flocculation and coalescence are the most common
examples of emulsion instability during storage. It is known that
the presence of hydrocolloids in an emulsion strongly influence
emulsion stability (Garti & Leser, 2001; Kaltsa et al., 2013). Mean-
while, the presence of an appropriate amount of hydrocolloids in
the emulsions can lead to a reduction in the size of the droplets.
This is possibly due to covering more interfacial area, higher rate of
surface coverage and lower rate of droplet collisions because of the
increase in emulsifier concentration and continuous phase viscos-
ity. All these reasons will lead to a lower re-coalescence and
41
consequently, smaller emulsion droplet size (Jafari, He, & Bhandari,
2007; Qian & McClements, 2011). Preliminary results obtained
during course of this investigation showed that a proper concen-
tration of soluble phases of gum tragacanth and Persian gum
individually and in combined forms had a favorable effect on
certain characteristics such as reducing the mean droplet size and
PDI. Consequently, the aims of this study were to investigate the
effect of different sonication conditions as well as the presence of
native gums on some physical and rheological properties of OPEO
nanoemulsions using response surface methodology (RSM).
2. Materials and methods
2.1. Materials
Natural orange peel essential oil (OPEO), without any purifica-
tion, was supplied by a local manufacturer (Giah Essance, Gorgan,
Iran). Tween 80 was purchased from Merck Chemicals Company
(Darmstadt, Germany). Persian gum (PG) and gum tragacanth (GT)
were bought from a local herbal store. Sodium azide (analytical
grade) was purchased from Sigma Chemicals Company (St. Louis,
USA). Deionized water (Electrical resistivity ¼ 18 MU$cm) was used
for the production of nanoemulsions.
2.2. Preparation of gum tragacanth and Persian gum powder
Gum tragacanth ribbons (GT) as well as Persian gum hunks (PG)
were pulverized using domestic grinder (Moulinex, France), and
after passing through a number of sieves, the powders (mesh
size < 60) were collected and kept in a closed container (Abbasi &
Mohammadi, 2013; Azarikia & Abbasi, 2010).
2.3. Preparation of PG and GT dispersions
In order to prepare dispersions, powders (PG 3, GT 0.5% w/w)
were weighted (Tecator, Swiss) and gradually added to a beaker
containing deionized water on a magnetic stirrer (Heidolph-MR
3001, Germany) to obtain uniform dispersions. Sodium azide
(0.004 wt %) was added to the dispersions as an antimicrobial
agent. Afterwards, to assure fully hydration, the dispersions were
incubated in water bath (Kottermann, Germany) for 30 min at
50
C, and were then kept for 24 h at ambient temperature (Abbasi
& Mohammadi, 2013; Azarikia & Abbasi, 2010).
2.4. Separation of soluble and insoluble fractions of PG and GT
At this step, the dispersions were transferred to 50 ml plastic
tubes. Then, the soluble and insoluble fractions were separated by
centrifugation (Sigma, model K 30-3, Germany) at 20,000 g for
20 min at 25
C. Next, the fractions were manually separated, and
after weighting the phases, the amount of their dry matter was
determined by oven (EHRET, model TKL 4105, Germany) at a tem-
perature of 105
C (Abbasi & Mohammadi, 2013; Azarikia & Abbasi,
2010).
2.5. Preparation of nanoemulsions
The oil-in-water (O/W) nanoemulsions were prepared using
OPEO (1% w/w), as the oil phase, and mixture of Tween 80 (2% w/
w), combined soluble fractions of PG (SFPG) and GT (SFGT) (0.25%
w/w) and deionized water (96.75% w/w), as the aqueous phase. So
that, formulation of all samples was as follows: 1% OPEO þ 2%
Tween 80 þ 0.25% gum (SFPG:SFGT, with a mixing ratio of 75:25).
All emulsions were prepared through a two-stage process. At first,
the oil and aqueous phases (total of 100 g) were placed in a glass
42 A.M. Hashtjin, S. Abbasi / Food Hydrocolloids 44 (2015) 40e48

beaker and mixed at room temperature (z25
C) using a magnetic Table 1

stirrer (700 rpm for 15 min). Afterwards, they were sonicated Uncoded and coded independent variables used in RSM design.

(Sonicator 4000, 20 kHz, high gain cylindrical titanium sonotrode Symbol Independent variable Coded levels
of 19.1 mm in diameter, Misonix, Inc, New York) in a double-walled 1.68 1 0 1 1.68
cylindrical glass cell where the sonotrode was immersed 1 cm
X1 Amplitude (%) 69.86 76 85 94 100.14
below the surface of the emulsion. The sonication was carried out at
X2 Time (s) 89.73 102 120 138 150.27
various amplitudes (70e100%) for different times (90e150 s) X3 Temperature (
C) 4.82 13 25 37 45.18
controlled by the software of the device. The total input energy (J)
was also recorded for every treatment. The sonication was carried
out at various temperatures that were kept constant at intended
Yi ¼ a0 þ a1 X1 þ a2 X2 þ a3 X3 þ a11 X12 þ a22 X22 þ a33 X32
temperature (5e45
C) throughout sonication by circulating
coolant fluid through the jacket of chamber. In order to control the þ a12 X1 X2 þ a13 X1 X3 þ a23 X2 X3 (1)
temperature during the sonication process, a double-walled cylin-
drical glass container (inner diameter 65, depth 90 mm) was where a0 is a constant, ai, aii and aij are the linear, quadratic and
designed. Due to the continuous circulation of the antifreeze interactive coefficients, respectively. The coefficients of the
(propylene glycol) liquid through the jacket and the embedded response surface equation were determined using Design-Expert
spiral coil, the temperature of premix was kept constant (with an 7.1.1 software.
accuracy of ±1
C) at intended temperature throughout sonication
(Mirmajidi Hashtjin & Abbasi, 2014). 2.10. Statistical analysis

2.6. Droplet size and size distribution measurements
The mean droplet diameter (Z-average) and polydispersity in-
dex (PDI) of OPEO nanoemulsions were determined by dynamic
light scattering (Nano-ZS90, Malvern Instruments, Worcestershire,
UK) using a Zetasizer (Tang, Manickam, Wei, & Nashiru, 2012).
2.7. Rheological measurements
The analysis of variance (ANOVA), regression coefficient calcu-
lation, performance stepwise procedure to simplify the models and
generating of three-dimensional surface plots were carried out
using Design-Expert 7.1.1 software. The significance of the equation
parameters for each response was assessed by F-value at (P) of 0.05.
In addition, the adequacy of the models was determined using
model analysis, lack-of-fit test and coefficient of determination (R2)
analysis.

The rheological properties of nanoemulsions were determined 3. Results and discussion

at 25
C with a Brookfield rheometer (LV DV-ІІІ Ultra, Brookfield
Engineering Laboratories Inc., MA, USA), equipped with an
enhanced UL Adapter. In order to determine the shear stress and
viscosity as functions of shear rate, as well as the flow behavior of
nanoemulsions, the shear rate (0.01e171.2 s1 in 5 min and
171.2e0.01 s1 within next 5 min) was changed. The data were
fitted with the Newtonian model using linear regressions. Mean-
while, their time-dependency (constant shear rate of 91.73 s1,
temperature of 25
C, 2 min) were assessed (Abbasi & Mohammadi,
2013; Barnes, 2000; Bourne, 2002; Mirmajidi Hashtjin & Abbasi,
3.1. Fitting the models
The total energy input during ultrasonication, viscosity, Z-
average and polydispersity index of the OPEO nanoemulsions are
Table 2
Effect of different ultrasonication conditions (time, amplitude, temperature) on
energy consumption, viscosity, Z-average, and PDI of nanoemulsion (1 wt% OPEO,
2 wt% Tween 80 and 0.25 wt% gum (SFPG:SFGT, ratio of 75:25)).

2014). Runa Independent variableb Responsec

X1 X2 X3 Y1 Y2 Y3 Y4
2.8. Stability at different storage temperature 1 76 138 37 13,193 2.22 17.24 0.545
2 85 89.73 25 9672 2.05 34.75 0.607
For this purpose, some 20 g of nanoemulsions were transferred 3 85 150.27 25 16,289 1.81 20.28 0.475
into a 25 ml glass bottles, tightly sealed with plastic caps, wrapped 4 94 138 37 16,981 2.10 12.68 0.439
5 76 138 13 12,529 2.19 22.71 0.549
with aluminum foil, and stored at 5, 25 and 45
C for 12 weeks.
6 94 102 13 12,703 2.19 20.48 0.512
Then, their stability was assessed by measuring the mean droplet 7 94 138 13 16,471 2.06 15.33 0.477
diameter and size distribution at 4 week intervals. 8 76 102 37 9787 2.38 22.14 0.611
9 69.86 120 25 10,534 2.02 26.24 0.570
10 85 120 25 12,755 1.91 26.08 0.544
2.9. Experimental design 11 85 120 25 12,819 1.90 22.65 0.549
12 85 120 45.18 12,837 2.28 13.76 0.471
Response surface methodology (RSM) was used to evaluate the 13 85 120 25 12,991 1.90 22.10 0.547
effects of independent variables [sonication amplitude 76e94% 14 85 120 25 13,065 1.89 21.31 0.538
15 85 120 25 12,954 1.92 21.59 0.553
(X1), sonication time 102e138 s (X2), and process temperature 16 85 120 25 12,972 1.92 23.03 0.576
13e37
C (X3) ] as well as their interactions on responses [energy 17 94 102 37 12,204 2.36 19.16 0.571
input during ultrasonication (Y1), viscosity (Y2), Z-average (Y3) and 18 76 102 13 9588 2.29 25.62 0.620
PDI (Y4)] of OPEO nanoemulsions. The coded and uncoded inde- 19 100.14 120 25 15,753 1.83 18.83 0.486
20 85 120 4.82 13,210 2.14 20.58 0.553
pendent variables used in the RSM design are shown in Table 1. The
a
experiments were designed according to the central composite In runs 9 and 19, amplitude of sonication was considered 70 and 100%,
design (CCD) using a 23 factorial and star design with six central respectively. Moreover, in runs 12 and 20, process temperature was considered 45
and 5
C, respectively.
points as shown in Table 2. A second-order polynomial equation b
X1, sonication amplitude (%); X2, sonication time (s); X3, process temperature
was used to express the responses as a function of the independent (
C).
c
variables as follows: Y1, energy input (J); Y2, viscosity (mPa$s); Y3, Z-average (nm); Y4, PDI.
 A.M. Hashtjin, S. Abbasi / Food Hydrocolloids 44 (2015) 40e48 43

shown in Table 2. The experimental data was used to compute the Table 4
coefficients of the quadratic polynomial equations, and the derived ANOVA of the regression coefficients of the fitted quadratic equations for the Z-
average (Y3) and polydispersity index (Y4) of the nanoemulsions (1 wt% OPEO, 2 wt%
equations (Eqs. (2)e(5)) were used to predict the values of Tween 80 and 0.25 wt% gum (SFPG:SFGT, ratio of 75:25)).
dependent variables of the nanoemulsions. According to Tables 3
and 4, analysis of variance (ANOVA) showed that the coefficient Variable Z-average (Y3) Polydispersity index (Y4)

of multiple determinations (R2) of the models for the dependent Regression F-value P-value Regression F-value P-value
variables was 0.99, 0.80, 0.88 and 0.81, respectively. This indicates coefficient coefficient

that the quadratic polynomial models were only adequate to a0 22.89 0.54
describe and predict the influence of the independent variables on Linear
a1 2.38 13.93 0.0039 0.034 28.26 <0.0001
the energy input and Z-average. The estimated regression co-
a2 3.21 25.24 0.0005 0.039 35.81 <0.0001
efficients of the polynomial response surface models along with the a3 1.79 7.84 0.0188 0.009512 2.18 0.1589
corresponding R2 values and lack of fit tests are also shown in Interaction
Tables 3 and 4. The significance of each term was evaluated using a12 0.48 0.33 0.5794 e e e
the F-value and P-value where a small P-value and a large F-value a13 0.62 0.56 0.4724 e e e
a23 0.41 0.25 0.6294 e e e
would indicate a more significant effect on the respective inde-
Quadratic
pendent variables. Thus, during ultrasonication the variables with a11 0.72 1.33 0.2754 e e e
the largest effect on the energy input were the linear terms of a22 1.04 2.83 0.1237 e e e
sonication time and amplitude (P < 0.0001). The interaction be- a33 2.61 17.71 0.0018 e e e
tween amplitude and sonication time as well as sonication time Model 7.91 0.0017 22.08 <0.0001
and process temperature also had a significant effect (P < 0.05) on Lack of fit 2.70 0.1495 4.29 0.0600
the energy input. Furthermore, none of the quadratic terms had a R2 0.88 0.81
significant effect (P > 0.05) on the energy input (Table 3). In terms of
the viscosity (Table 3), the linear term of sonication time (P < 0.05)
and the quadratic term of process temperature had a significant Y3 ¼ 22:30  2:38X1  3:21X2  1:79X3 þ 1:12X22  2:54X32 (4)
effect (P < 0.001).
For the Z-average of the nanoemulsions (Table 4), the variables Y4 ¼ 0:54  0:034X1  0:039X2 (5)
having the largest effect on this response were the linear terms of
sonication time (P < 0.001), amplitude (P < 0.01) and process
temperature (P < 0.05), followed by the quadratic term of process 3.2. Analysis of response surfaces
temperature (P < 0.01), while the effect of the other terms was
insignificant (P > 0.05). In order to recognize the effect of the independent variables on
Regarding the polydispersity index (Table 4), only the linear the dependent ones, surface response plots of the quadratic poly-
terms of amplitude and sonication time had a significant effect nomial models were generated by varying two of the independent
(P < 0.0001), whereas the effect of the other terms was insignificant variables, within the experimental range, while holding another
(P > 0.05). one constant at the central point.

Y1 ¼ 12965:35 þ 1613:79X1 þ 1905:30X2 þ 18:06X3 3.2.1. Energy input

The effect of changes in sonication time and amplitude at con-
þ 274:75X1 X2 þ 184:25X2 X3 (2) stant temperature (25
C) on the amount of consumed energy is
shown in Fig. 1a. As can be seen, with increasing sonication time
and amplitude, the consumed energy was increased and its
Y2 ¼ 1:97  0:077X2 þ 0:041X3 þ 0:14X32 (3)
maximum value was achieved at 94% (amplitude) and 138 s (son-
ication period).
The effect of amplitude and temperature at a constant time
Table 3 (120 s) on the energy consumption is shown in Fig. 1b. It can be seen
ANOVA of the regression coefficients of the fitted quadratic equations for the energy
that the impact of amplitude was much higher than temperature.
input (Y1), viscosity (Y2) of the nanoemulsions (1 wt% OPEO, 2 wt% Tween 80 and
0.25% gum (SFPG:SFGT, ratio of 75:25)). Moreover, at constant amplitude (85%), the effect of process time on
energy consumption was much higher than temperature (Fig. 1c).
Variable Energy input (Y1) Viscosity (Y2)

Regression F-value P-value Regression F-value P-value 3.2.2. Viscosity

coefficient coefficient
Three-dimensional response surface plots of viscosity are shown
a0 12,930.02 1.9 in Fig. 2. As can be observed, the effect of independent variables on
Linear
viscosity had a downward trend up to the central point, after which
a1 1613.79 914.98 <0.0001 0.050 2.82 0.1242
a2 1905.30 1275.40 <0.0001 0.077 6.58 0.0282
the trend is changed. In other words, around the central point,
a3 18.06 0.11 0.7419 0.041 1.89 0.1988 minimum viscosity is reached. Meanwhile, in comparison to other
Interaction variables, the effect of the quadratic term of process temperature
a12 274.75 15.54 0.0028 0.016 0.17 0.6880 has been significantly high (P < 0.001).
a13 106.50 2.33 0.1575 0.011 0.082 0.7806
a23 184.25 6.99 0.0246 0.024 0.37 0.5591
Quadratic 3.2.3. Z-average
a11 50.60 0.95 0.3529 0.048 2.65 0.1347 The effect of sonication time and amplitude at constant tem-
a22 7.03 0.018 0.8950 0.049 2.85 0.1224 perature (25
C) on Z-average value of nanoemulsions is shown in
a33 8.17 0.025 0.8781 0.15 25.68 0.0005
Fig. 3a. As can be seen, with increasing intensity and duration of
Model 246.26 <0.0001 4.45 0.0144 sonication, Z-average value is decreased, and its minimum was
Lack of fit 4.79 0.0553 167.54 <0.0001 reached at 94% (sonication amplitude) and 138 s (sonication time).
R2 0.99 0.80
The droplet disruption, as the major mechanism of emulsion
44 A.M. Hashtjin, S. Abbasi / Food Hydrocolloids 44 (2015) 40e48

significantly decreased (P < 0.01). However, higher intensities may

lead to an increased rate of droplet coalescence (Li & Chiang, 2012).
On the other hand, in an O/W emulsion system, sonication time,
owing to thermodynamic equilibrium, has paramount effect upon
the adsorption rate of surfactant to the surface of the droplets and

Fig. 1. Response surface plots of the energy input during ultrasonication as a function
of a) time and amplitude, b) temperature and amplitude, c) temperature and time.

formation, is depended on the type and quantity of applied shear

force (Kentish et al., 2008; Li & Chiang, 2012; Tadros et al., 2004). As Fig. 2. Response surface plots of the viscosity of the nanoemulsions (1 wt% OPEO, 2 wt
a consequence, in this study with increasing the intensity or % Tween 80 and 0.25 wt% gum (SFPG:SFGT, ratio of 75:25)) as a function of a) time and
amplitude of sonication (up to 94%), the size of emulsion droplets amplitude, b) temperature and amplitude, c) temperature and time.
 A.M. Hashtjin, S. Abbasi / Food Hydrocolloids 44 (2015) 40e48 45

size distribution of newly formed droplets. Therefore, longer son-

ication (above 138 s) may lead to formation of larger droplets
mainly because of coalescence (Li & Chiang, 2012).
The effect of amplitude and temperature in the fixed time
(120 s) on the Z-average value of nanoemulsions is also shown in
Fig. 3b. As can be seen, by increasing the temperature (up to 25
C),
the Z-average is increased, whereas it is decreased at higher tem-
peratures. These findings mean that in constant temperature

Fig. 3. Response surface plots of the Z-average of the nanoemulsions (1 wt% OPEO,
2 wt% Tween 80 and 0.25 wt% gum (SFPG:SFGT, ratio of 75:25)) as a function of a) time Fig. 4. Response surface plots of the polydispersity of the nanoemulsions (1 wt% OPEO,
and amplitude, b) temperature and amplitude, c) temperature and time. 2 wt% Tween 80 and 0.25 wt% gum (SFPG:SFGT, ratio of 75:25)) as a function of a) time
and amplitude, b) temperature and amplitude, c) temperature and time.
46 A.M. Hashtjin, S. Abbasi / Food Hydrocolloids 44 (2015) 40e48

(25
C), increasing the intensity of sonication can result in smaller
particle size. Furthermore, our results showed that the Z-average
value of nanoemulsions was minimum at 37
C. Thus, it appears
that the temperature can be very effective on the emulsion droplet
size because of its impact on viscosity and interfacial tension of oil
and water phases (Yuan, Gao, Mao, & Zhao, 2008).
Fig. 3c also shows the effect of sonication time and temperature
(constant sonication intensity of 85%) on the Z-average value. At
constant temperature (25
C), increasing sonication time can result
in smaller particle sizes.
3.2.4. Polydispersity
Three-dimensional response surface plots for polydispersity
index variable are shown in Fig. 4. As can be seen, with increasing
intensity and duration of sonication, PDI is decreased. Meanwhile,
in comparison to process temperature, the effect of other variables
has been significantly higher (P < 0.0001).
3.3. Optimization of conditions for producing nanoemulsions
One of the main objectives of the present study was to deter-
mine the optimal values of the independent variables to generate
nanoemulsions with minimum droplet size and PDI. Hence, nu-
merical optimization method was used to achieve the optimum
process conditions. In this method, the optimization goals were
specified, the levels of the responses (Z-average and PDI) and in-
dependent variables were adjusted, and the best responses were
obtained using fine tuning technique. The optimum range of

Fig. 5. Flow curves a) shear stress/shear rate, b) viscosity/shear rate and c) viscosity/time of nanoemulsions (1 wt% OPEO, 2 wt% Tween 80 and 0.25 wt% gum (SFPG:SFGT, ratio of
75:25)) prepared by sonication (94%, 138 s, 37
C).
 A.M. Hashtjin, S. Abbasi / Food Hydrocolloids 44 (2015) 40e48 47

Table 5 behavior of nanoemulsions (Fig. 5a). As can be seen, there is a

ANOVA of the storage time and temperature for the Z-average and PDI value of linear relationship between shear rate and shear stress. In
nanoemulsions (1 wt% OPEO, 2 wt% Tween 80 and 0.25 wt% gum (SFPG:SFGT, ratio of
75:25)) prepared by sonication (94%, 138 s, 37
C).
addition, in the laminar flow range, the viscosity is independent
of shear rate, which confirms its Newtonian behavior (Fig. 5b).
Source Z-average PDI Meanwhile, no hysteresis loop was observed in shear stress vs.
F-value P-value F-value P-value shear rate curves (Barnes, 2000; Bourne, 2002). Fig. 5c also
Time 290.066 <0.0001 10.924 0.001 shows that in constant shear rate, viscosity remains constant
Temperature 1288 <0.0001 110.254 <0.0001 despite increased shear time. It should be noted that such
Time*Temperature 269.462 <0.0001 13.772 <0.0001 behavior was observed in all the nanoemulsions even after three
<0.0001 <0.0001
Model 460.290 30.538
months of storage at 5, 25 and 45
C.
R2 0.998 0.966

3.5. Storage stability

independent variables including sonication amplitude, sonication
time and process temperature to achieve the best-predicted values
of Z-average (~13 nm) and PDI (0.457) were 94%, 138 s, and 37
C,
respectively. It should be noted that the desirability value of the
predicted zone was equivalent to 0.934 to 0.942. On the other hand,
according to Table 2, the Z-average and PDI value of nanoemulsions,
produced under the optimum conditions, was equivalent to
12.68 nm and 0.439, respectively. Thus, this indicates that a very
good correlation existed between the practical results and esti-
mated values of models.
3.4. Flow behavior
Based on our rheological measurements, the Newtonian
model was the most appropriate model to determine the flow
Colloidal dispersions and emulsions normally need to be stored at
different temperatures for long time for commercial and industrial
applications. Accordingly, in order to evaluate the effect of storage
time and temperature on the stability, nanoemulsions (including 1%
OPEO, 2% Tween 80 and 0.25 wt% gum (SFPG:SFGT, ratio of 75:25))
were produced at optimum emulsification conditions (sonication
amplitude 94%, sonication time 138 s, and temperature 37
C). Then,
they were kept at 5, 25 and 45
C for 12 weeks, and their mean
droplet size, particle size distribution and the flow behavior were
evaluated at 4-week intervals. It should be noted that the droplet
diameter and size distribution are important parameters affecting
emulsion stability (Cheong, Tan, Che Man, & Misran, 2008). Based on
statistical analysis (Table 5), the effect of storage time, storage tem-
perature and their interaction on the Z-average value was significant
(P < 0.0001). Regarding the PDI, the storage period showed a

Fig. 6. Effect of storage (time and temperature) on a) Z-average and b) PDI of nanoemulsions (1 wt% OPEO, 2 wt% Tween 80 and 0.25 wt% gum (SFPG:SFGT, ratio of 75:25)) prepared
by sonication (94%, 138 s, 37
C).
48 A.M. Hashtjin, S. Abbasi / Food Hydrocolloids 44 (2015) 40e48
significant effect (P ¼ 0.001) where the storage temperature and
their interaction was more effective (P < 0.0001).
The influence of storage on Z-average and PDI is shown in Fig. 6.
As can be seen, the Z-average value of the samples stored at 5 and
25
C within the first 4 weeks showed a downward trend, after
which it was increased with a much lower slope. It seems that the
downward trend of Z-average during the first 4 weeks is due to a
gradual reduction in kinetic energy barrier in the system, subse-
quent to which the samples gradually reach kinetic equilibrium. In
the systems including oil, water and surfactant at ambient tem-
perature, there is a kinetic energy barrier preventing these systems
from reaching the highest kinetic equilibrium status (Rao &
McClements, 2011a). Reduction of this barrier over time probably
enhances the kinetic equilibrium of the system. The upward trend
of Z-average after the first 4 weeks is likely due to droplets collision
and phenomena such as coalescence and Ostwald ripening, which
subsequently lead to an increase in the size of the droplets (Rao &
McClements, 2011a). In contrast, at 45
C the OPEO nanoemulsions
were highly unstable and faced with an increasing trend in the
average size of the droplets. This trend could be due to the collision
rate and coalescence of droplets at higher temperatures, which
subsequently lead to an increase in emulsion droplet size (Rao &
McClements, 2011b). The difference which was observed on the
Z-average value at 5 and 25
C can be likely related to higher
droplets collision rate at higher temperatures (Li & Chiang, 2012).
That is why the Z-average value at 25
C was slightly higher than
one at 5
C.
In addition, during the first 8 weeks of storage (at 25 and 45
C),
the PDI showed a downward and then an almost steady trend
(Fig. 6b). It appears that during this storage period, the nano-
emulsion system has progressed towards more kinetically balance
due to the interactions occurring in the system. It is needs to be noted
that during the first 4 weeks of storage at 5
C, unlike trend was
clearly seen. But during the following weeks, it seems that low
temperature has decreased the coalescence rate and the subsequent
increase in kinetic equilibrium led to the further decrease of PDI.
4. Conclusions
The results of this study showed that the soluble fraction of
Persian and tragacanth gums (in combination) along with emulsi-
fier (Tween 80) could be used in the structure of stable nano-
emulsions. Moreover, the application of response surface method
in finding optimum conditions of ultrasonic emulsification process
to produce OPEO nanoemulsions is a very effective and reliable
method (Desirability ¼ 0.942). According to our findings, the son-
ication amplitude, sonication time and their interaction as well as
the interaction of sonication time and process temperature were
effective on energy consumption during emulsification process.
Regarding the Z-average value, the sonication time, sonication
amplitude and process temperature were effective. Meanwhile, the
effect of sonication amplitude and time on PDI was significant.
Furthermore, the optimum condition for production of nano-
emulsion (1% OPEO, 2% Tween 80 and 0.25% gum (SFPG:SFGT,
weight ratio of 75:25) Z-average 13 nm) was equivalent to 94%,
sonication amplitude, 138 s, sonication time, and 37
C, tempera-
ture. In addition, the nanoemulsions showed Newtonian behavior
and were physically stable at 5 and 25
C over three months of
storage. These results elucidate the potential capability of ultra-
sonic technique for production of nano-scale emulsions of essential
oils with reasonably long term kinetic stability which can be used in
food and pharmaceutical products.
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