LWT - Food Science and Technology 96 (2018) 58–65

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Optimization of palm oil in water nano-emulsion with curcumin using T

microfluidizer and response surface methodology
Revathi Raviadarana, Davannendran Chandranb,∗, Liew Huey Shina, Sivakumar Manickama
a
Department of Chemical and Environmental Engineering, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia
b
Graphene & Advanced 2D Materials Research Group (GAMRG), School of Science and Technology, Sunway University, No. 5, Jalan Universiti, Bandar Sunway, 47500
Subang Jaya, Selangor, Malaysia

A R T I C LE I N FO A B S T R A C T

Keywords: This study aims to produce and optimize palm oil-based nano-emulsion to encapsulate curcumin using micro-
Curcumin fluidizer and Response Surface Methodology (RSM). Encapsulation of curcumin is essential to overcome cur-
Nano-emulsion cumin's poor bioavailability through the formation of nano-sized droplets in order to harvest its outstanding anti-
Microfluidizer inflammatory and anti-cancer medicinal properties. Among the parameters of concern are microfluidizer's
Response surface methodology (RSM)
pressure, number of cycles and surfactant concentration (Tween 80). Optimisations were performed by em-
ploying RSM. Characterisations were conducted for the droplet size, poly-dispersity index (PDI), zeta potential
(ZP) and viscosity. Stable palm oil-based oil in water nano-emulsion encapsulating curcumin was achieved at a
droplet size of 275.5 nm, PDI of 0.257, ZP of −36.2 and viscosity of 446 cP using microfluidizer. The optimized
conditions were at 350 bar, 5 cycles and 1 wt% surfactant. Optimized microfluidizer with the aid of RSM is
deemed capable to produce palm oil-based oil in water nano-emulsion encapsulating curcumin with small
droplet size using low surfactant concentration and under optimum energy consumption.

1. Introduction bioavailability drawback in human body in order to benefit its out-

Curcumin, an active compound extracted from turmeric is reported
to exhibit several medicinal properties such as antioxidant (Fan, Yi,
Zhang, & Yokoyama, 2018), anti-inflammatory (Kocaadam & Şanlier,
2017), antibacterial (Izui et al., 2016), antifungal (Zhao et al., 2017),
anti-hypocholestrolemic (Rachmawati, Soraya, Kurniati, & Rahma,
2016), anti-parasitic (Mallo et al., 2017), anti-tumour (Teymouri, Pirro,
Johnston, & Sahebkar, 2017) and anti-proliferative (Panda,
Chakraborty, Sarkar, Khan, & Sa, 2017). Nevertheless, its potential is
yet to be fully exploited to date due to its poor bioavailability in human
body. For instance, only 50 ng/ml is detected in the serum for orally
consumed curcumin by human at 10–12 g/ml of concentration. Con-
trastingly, approximately 75% of the curcumin (dose) administered
orally by rats at 1000 mg/kg is being excreted in faeces and urine
(Wahlström & Blennow, 1978). The minimum presence of curcumin in
the blood circulation which is known as low bioavailability fails to
create the required therapeutic effects. The low bioavailability is sug-
gested to occur due to the prompt metabolism in the intestine into
several products (di-, tetra-, hexahydrocurcumin, and hexahy-
drocurcuminol) and their glucuronide or sulfate conjugates (Ireson
et al., 2001). Hence, it is critical to overcome curcumin's low
standing medicinal properties.
To date, several studies have reported improved bioavailability of
active compounds as a result of encapsulation through nano-emulsion
formulation. Nano-emulsion (oil-in- water) is capable to incorporate
hydrophobic and amphipathic drugs (Anuchapreeda, Fukumori,
Okonogi, & Ichikawa, 2012). It is of a nanoscale droplet of multiphase
colloidal dispersions which are optically transparent for size of
50 nm–200 nm and milky in appearance for size up to 500 nm (Mason,
Wilking, Meleson, Chang, & Graves, 2006). In a study by Zhao et al.
(2013), the authors encapsulated baicalin, a herb to treat inflammation
in nano-emulsion. The authors concluded that the baicalin nano-
emulsion enhanced the bioavailability by 7-folds. Similar outcome was
achieved by Shafiq et al. (2007) in encapsulating ramipil in nano-
emulsion in which the bioavailability was enhanced by 539.49% as
compared to normal suspension. In another study, there was a 1.6-fold
enhancement in bioavailability when vitamin E was encapsulated in
nano-emulsion compared with the marketed soft capsule (Gong et al.,
2012). Yu and Huang (2012) in a study reported that by encapsulating
curcumin in organogel nano-emulsion, the bioavailability of curcumin
increased by 9-folds as compared to the unformulated curcumin.
In line with the above, this study aims to produce palm oil-based

∗
Corresponding author.
E-mail address: dchandran@sunway.edu.my (D. Chandran).

https://doi.org/10.1016/j.lwt.2018.05.022
Received 15 March 2018; Received in revised form 5 May 2018; Accepted 7 May 2018
Available online 08 May 2018
0023-6438/ © 2018 Elsevier Ltd. All rights reserved.
R. Raviadaran et al.
nano-emulsion encapsulating curcumin from an optimized micro-
fluidizer. This is achieved by investigating different parameters of a
microfluidizer for nano-emulsion formation, optimizing the formation
of nano-emulsion by using Response Surface Methodology (RSM),
characterizing the nano-emulsion formed and finally investigating the
stability of the nano-emulsion formed. High-energy emulsification
method (microfluidization) was employed in this study due to its cap-
ability in reducing the droplet size effectively in a short process time
and it also provides more flexibility control over the physical properties
of the nano-emulsion. Tween 80 was used as the surfactant based on
their Hydrophilic Lipophilic Balance values and its ability to solubilize
curcumin (Anjana et al., 2012). Palm oil was used since limited studies
have reported on using palm oil to produce nano-emulsion en-
capsulating curcumin. The minor phytonutrients in palm oil, such as
carotenes, tocopherols and tocotrienols provide health benefits in-
cluding antioxidant and anticancer properties (Olafisoye, Oguntibeju, &
Osibote, 2017). Furthermore, the availability in abundance and cheap
cost as Malaysia is among the largest producer of palm oil in the world
also encouraged the use of palm oil in this study. RSM was employed to
optimize the amount of surfactant used, the pressure and the number of
cycles used in the microfluidizer to produce the nano-emulsion.
2. Materials and methods
2.1. Materials
Curcumin (94% curcuminoid content) from Sigma-Aldrich
(Malaysia), refined palm olein (food grade) was purchased from Sime
Darby Foods (Malaysia), ethanol from Merck (Germany), poly-
oxythelyene sorbitan monooleate (Tween 80) from R&M chemicals
(UK) and water from Milli-Q Plus apparatus (USA) were used in this
study.
2.2. Preparation of coarse emulsion
Oil-in-water (O/W) type emulsions were prepared for this experi-
ment. The following methodology is utilized to prepare pre-mixed O/W
emulsion at 1 L. The dispersed phase consists of refined palm olein and
curcumin dissolved in ethanol solution (1 mg/ml), while continuous
phase consists of water. For the formulation of emulsion, 78 wt% of
water, 20 wt% of refined palm olein, 1 wt% of curcumin in ethanol
solution and (0.25 wt% - 1 wt%) of Tween 80 were used. The con-
centration of the surfactant would be optimized in order to produce a
stable nano-emulsion with the least amount of surfactant. The mixture
was homogenized with high-speed homogenizer (ULTRA-TURRAX's
model T18 Basic, Germany) at approximately 7000 rev/min for 1 h with
a sample volume of 1000 mL for each batch to form coarse emulsion.
The final temperature at the end of the homogenization process was at
31 ± 2 °C.
2.3. Microfluidization
Nano-emulsion was prepared with an air driven bench-top micro-
fluidizer (Microfluidics, Newton, MA, USA) which consisted of an air-
driven intensifier pump, an interaction chamber (200 μm) and a filter.
Individual batches of coarse emulsion were fed into the microfluidizer
through a glass reservoir inlet and pumped through the interaction
chamber (H30Z, ceramic, 200 μm), with a variation of pressure (150 bar
- 450 bar), concentration of surfactant (0.25 wt% - 1.0 wt%) and
number of cycle (1–12). Nano-emulsions were collected at the product
outlet.
2.4. Droplet size and zeta potential measurement
The mean droplet diameter, poly dispersity index (PDI), zeta po-
tential (ZP) were measured using Zetasizer Nano ZS (Malvern
59
LWT - Food Science and Technology 96 (2018) 58–65
Instruments, U.K.). The diameter of the emulsion droplets was mea-
sured using dynamic light scattering mechanism, at a wavelength of
633 nm and scattering angle of 173° at 25 °C ± 0.1 °C. PDI is di-
mensionless, ranging from 0 to 1. ZP is measured by the principle of
electrophoresis using Doppler Effect and calculated based on
Smoluchowski model. The samples were diluted with water (2 drops to
100 ml) before analysing to avoid multiple scattering effects.
2.5. Stability test
The physical stability of nano-emulsion was assessed by measuring
the change in nano-emulsion size, PDI value, ZP value and viscosity
value (Lovelyn & Attama, 2011; Raviadaran, Ng, Muthoosamy, &
Manickam, 2018). The samples were assessed on the first, seventh,
fourteenth, twenty first and thirtieth day. All the samples were stored in
glass bottles at dark room condition (25 °C) prior to analysis. Apparent
viscosity of the nano-emulsions was measured at 25 °C using a Brook-
field Digital Rheometer (model DVIII, Brookfield Engineering Labora-
tories Inc., MA).
2.6. Optimization of microfluidization of nano-emulsion by RSM and
statistical analysis
An optimization study for the parameters involved in the micro-
fluidization of nano-emulsion was carried out in two stages. In the first
stage, a single-factor experiment was conducted to select the range of
each of the emulsification factors such as surfactant concentration,
number of cycles and operating pressure towards the formation of
smallest droplet size. The analysis of variance (ANOVA) and regression
analysis were performed. The ranges used were microfluidizer oper-
ating pressure, 300 bar-400 bar; number of cycles, 5–7; and con-
centration of surfactant, 0.8%–1%.
In the second stage, three-level-three-factor Box–Behnken Design
(BBD) was employed to optimize the parameters achieved in the first
stage. The Design expert (Version 8.0, Stat-Ease Inc., Minneapolis, MN,
USA) software was used here. The independent variables (surfactant
concentration, number of cycles and operating pressure) and their le-
vels have been shown in Table 1. The BBD design consists of 15 factorial
points as given in Table 2. The response variable could be fitted into the
general form of a quadratic polynomial model (Eq. (1)), where Y is the
response variable measured for each combination of factorial level;
β0, βi , βii , and βij are terms are regression coefficients for intercept,
linearity, square and interaction; and X1 and X2 are codes of the in-
dependent variables.
3 3 2 3
y = β0 + ∑ βi Xi + ∑ βii Xi2 + ∑ ∑ βij Xi Xj + ε
i=1 i=1 i=1 j=i+1 (1)
The coefficients of second order polynomial model were determined
by a multiple regression analysis for the values of responses obtained
from the experiments. The degree of fitness of the model could be in-
dicated by the coefficient of determination (R2), adjusted coefficient of
determination (R2 adj), p-value and lack-of-fit. When the coefficient of
determination (R2) approaches the integrity (≈1) and the lack-of-fit
test has a p-value > 0.05 (not significant), the model is expressed to
have fitted well. F-test and t-test were used to check the statistical
Table 1
Independent variables used in Response Surface Methodology design.
Independent variables Codes Factor levels
−1 0 1
Surfactant concentration (wt.%) A (X1) 0.8 0.9 1
Number of cycle B (X2) 5 6 7
Pressure (bar) C (X3) 300 350 400
R. Raviadaran et al. LWT - Food Science and Technology 96 (2018) 58–65

Table 2 Siah Ying, and Manickam (2013) in a study highlighted that extreme
Experimental values of droplet size obtained from Box Behnken Design. shear and high-energy input are required during the emulsification
Run X1 X2 X3 Y (Actual values) Y (Predicted values) process for the fragmentation of droplets in overcoming the Laplace
pressure. With regards to the above, a range of operating pressure be-
1 0.8 5 350 308.6 307.9 tween 300 bar and 400 bar was chosen for use in RSM study.
2 1 5 350 270.4 271.8
3 0.8 7 350 274.5 273.0
4 1 7 350 263.9 264.5 3.2. Effect of number of cycle on droplet diameter
5 0.8 6 300 316.1 317.4
6 1 6 300 313.6 312.8 As shown in Fig. 1, at 150 bar of operating pressure, the droplet
7 0.8 6 400 297.8 298.5
8 1 6 400 260.1 258.7
diameter decreased throughout the 12 cycles. At 250 bar, the droplet
9 0.9 5 300 318.5 317.8 diameter decreased until the 6th cycle and plateaued. At 350 bar and
10 0.9 7 300 256.8 256.9 450 bar, independently, the droplet diameter plateaued after the 5th
11 0.9 5 400 241.6 241.5 cycle. This shows that the size decreases will increasing number of
12 0.9 7 400 259.6 260.2
cycles. This phenomenon is related to the emulsion entering the inter-
13 0.9 6 350 275.0 281.9
14 0.9 6 350 284.5 281.9 action chamber in the microfluidizer. In the interaction chamber, the
15 0.9 6 350 286.3 281.9 droplets near to the wall of the channel experiences a smaller shear flow
as compared to the droplets away from the wall. Hence, a uniform
shearing could not be achieved in all regions of interaction chamber
significance of R2 and the regression coefficients respectively. (Lee & Norton, 2013). By increasing the number of cycles, emulsion was
subjected to continuous shearing which resulted in the larger droplets
3. Results and discussion to eventually break up in the subsequent cycles forming smaller dro-
plets.
3.1. Effect of microfluidizer operating pressure on droplet diameter Besides, lesser number of cycles were required to achieve smaller
droplet diameter at higher operating pressure. This phenomenon is
As shown in Fig. 1, at 150 bar of operating pressure, the droplet size explained by relating the energy density to the droplet size. In relating
decreased throughout the 12 cycles without attaining plateau. At the energy dissipation to droplet size, the mean droplet diameter is
250 bar, the droplet size decreased until the 6th cycle and attained given as a function of energy density, Ev (Karbstein & Schubert, 1995);
plateau. While, at 350 bar and 450 bar, independently, the plateau was where d is the mean sauter diameter of the droplet, C is the constant
achieved from the 5th cycle onwards. A droplet size of ∼270 nm was depending on the dispersed phase viscosity, b is a constant depending
attained at plateau for 250 bar, 350 bar and 450 bar, independently. on the degree of turbulence, εave is the mean power density and tave is
Similar outcomes as the above were reported in several existing studies the mean residence time of droplets in the dispersing zone as shown in
(Donsì, Sessa, & Ferrari, 2012; Maali & Mosavian, 2013; Tan & Eqs. (3) and (4).
Nakajima, 2005). The formed smaller droplet size at higher operating
d = C (Ev )−b (3)
pressure can be explained using the Laplace equation for a spherical
droplet of radius R as given in Eq. (2). This equation relates the re- Ev = εave x tave (4)
sistance of the droplet deformation with the internal pressure of the
droplet, ΔP (Lee, Niknafs, Hancocks, & Norton, 2013; Walstra, 1993): By relating Eqs. (3) and (4), the mean residence time of droplets in
the interaction chamber has the same weightage as the mean energy
ΔP = 2γ / R (2)
density. Therefore, the mean power density is proportional to the op-
Based on the equation above, an increase in the interfacial tension erating pressure. Thus, at higher operating pressure, much shorter
(γ) , and/or a decrease in the droplet radius (R) would result in an in- contact time was required to attain small droplet size.
crease in the droplet's internal pressure. Hence, as the droplet size re- By further analysing the results presented in Fig. 1, an increase in
duces, more energy is required to break the droplet. In the attempt to the droplet size by ∼8% was observed from the 8th to 12th cycle for the
further break the droplet, the shearing induced in the interaction operating pressure at 450 bar. This is referred to as ‘over processing’
chamber must exceed the Laplace pressure of a droplet. Parthasarathy, due to higher rate of droplet re-coalescence than droplet disruption

1800

1600

1400
Mean droplet diameter (nm)

1200

1000
150 bar
800 250 bar
350 bar
600
450 bar
400

200

0
0 2 4 6 8 10 12 14
Number of cycles

Fig. 1. Effect of microfluidizer operating pressure and number of cycles on droplet diameter.

60
R. Raviadaran et al. LWT - Food Science and Technology 96 (2018) 58–65

7000

6000

Mean droplet diameter (nm)

5000

4000
0.25% surfactant
3000 1% surfactant
0.75% surfactant
2000
0.5% surfactant
1000

0
0 2 4 6 8 10 12 14
Number of cycles

Fig. 2. Effect of surfactant concentration and number of cycles on mean droplet diameter.

0.9

0.8

0.7
Polydispersity Index

0.6
150 bar
0.5
250 bar
0.4 350 bar

0.3 450 bar

0.2

0.1

0
0 2 4 6 8 10 12 14
Number of cycles

Fig. 3. Effect of microfluidizer operating pressure and number of cycles on polydispersity index.

-20
0 2 4 6 8 10 12 14

-25
Zeta potential/ (mV)

-30
150 bar
-35 250 bar
350 bar
-40 450 bar

-45

-50
Number of cycles
Fig. 4. Effect of microfluidizer operating pressure and number of cycles on Zeta-potential.

reported in the existing studies (Mahdi Jafari, He, & Bhandari, 2006; 3.3. Effect of surfactant concentration on droplet diameter
Olson, White, & Richter, 2004; Schulz & Daniels, 2000). Thus, a range
of 5–7 number of cycles was considered for further investigation in As shown in Fig. 2, the mean droplet diameter decreases with in-
RSM. creasing surfactant concentration. The smallest droplets were obtained

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R. Raviadaran et al. LWT - Food Science and Technology 96 (2018) 58–65

Table 3 increasing number of cycles. Therefore, a range of surfactant con-

Stability of nanoemulsion. centration at 0.8 wt% to 1 wt% would be used in RSM.
Duration Size (nm) PDI Zeta potential Viscosity (cP)
(day) 3.4. Effect of microfluidization pressure on polydispersity index

0 275.5 ± 5.3 0.257 ± 0.011 −32.8 ± 0.3 446 ± 6

As shown in Fig. 3, the PDI values decreased with increasing pres-
7 264.2 ± 3.4 0.243 ± 0.014 −34.8 ± 0.2 542 ± 8
14 272.4 ± 4.8 0.238 ± 0.016 −31.6 ± 0.4 481 ± 4
sure and number of cycles. At 150 bar, the produced samples have PDI
21 258.1 ± 10.1 0.244 ± 0.011 −33.4 ± 0.3 562 ± 8 value of 0.5 and above. At 250 bar, the PDI values were in the range of
30 260.1 ± 8.4 0.240 ± 0.015 −36.2 ± 0.6 640 ± 10 0.3–0.5. This indicates that the degree of polydispersity of the droplet
size is high in the nano-emulsion. At 350 bar and 450 bar, the obtained
PDI values were between 0.2 and 0.3. The formed uniform turbulence
Table 4 in the interaction chamber with increasing operating pressure and
Estimated regression model between response variable and independent vari- number of cycles induced droplet breakup causing uniformity in droplet
ables.
size. Considering the minimal difference of less than 2% in the range of
Source SSa DFb MSc F-value p-value obtained PDI values between 350 bar and 450 bar of operating pressure,
independently, the maximum droplet break up was already achieved at
X1 990.13 1 990.13 58.36 0.0006
X2 888.31 1 888.31 52.36 0.0008
350 bar itself. This is also highlighted in a study by Cheong, Tan, Man,
X3 2660.85 1 2660.85 156.83 < 0.0001 and Misran (2008) that towards arriving to the smallest droplet size, the
X1X2 190.44 1 190.44 11.22 0.0203 force and energy required to break the droplets would seem to reach its
X1X3 309.76 1 309.76 18.26 0.0079 droplet break up limit.
X2X3 1588.02 1 1588.02 93.60 0.0002
X12 585.803 1 585.80 34.52 0.0020
X22 850.73 1 850.73 50.14 0.0009 3.5. Zeta potential of nano-emulsion droplets
X32 20.75 1 20.75 1.2 0.3191
As shown in Fig. 4, the ZP ranged between −31 mV and −43 mV
a b c
Sum of squares; Degree of freedom; Mean square. regardless of the pressure applied (150 bar–450 bar) and number of
cycles (2–12). ZP is an electro kinetic potential in colloidal systems
Table 5 (Preetz, Rübe, Reiche, Hause, & Mäder, 2008). It is an indicator of the
Analysis of variance (ANOVA) for the quadratic polynomial model. degree of repulsion between the charged droplets in dispersion which
Source Sum of Degree of Mean F-value p-value indirectly denotes the stability of colloidal dispersion (Silva, Cerqueira,
squares freedom square & Vicente, 2012). An arbitrary value of 30 mV is recognised as the
border line between low-charged surface and high-charged surface
Model 8206.23 9 911.80 53.74 0.0002
Residual 84.83 5 16.97 – – (Silva et al., 2012). Positive or negative sign indicates the charge pre-
Lack of Fit 11.11 3 3.70 0.10 0.9526 sent at the particle surface. Therefore, it can be assumed that a ZP of
Pure Error 73.73 2 36.86 – – more than ± 30 mV has a higher resistance towards particles aggrega-
Cor. Total 8291.06 14 – – – tion (Silva et al., 2012).
Since the obtained ZP were beyond −30 mV, stable nano-emulsion
R2 = 0.9898; R2adj = 0.9714.
was obtained. The negative charge obtained is related to the surfactant
tween 80 used in this study. Ahmed, Li, McClements, and Xiao (2012)
when surfactant concentration was of 1 wt%. This phenomenon is re-
indicated that the droplets stabilized by non-ionic surfactants such as
lated to the surfactant surface excess. A higher concentration of sur-
Tween 80 would have a negative charge due to the presence of free
factant has a higher surfactant surface excess which results in the
fatty acids.
production of nano-emulsion with smaller droplet size (Azeem et al.,
2012). At 0.25 wt% of surfactant concentration, since the surface excess
3.6. Stability test
is small, the coalescence in the nano-emulsion droplets are dominant.
Hence, this causes the formation of emulsion with larger droplet dia-
As shown in Table 3, after 30 days, the droplets were still mono-
meter (beyond nano-range). Generally, droplet disruption during mi-
dispersed, the size was only changed by a maximum of 5% and the
crofluidization increases the specific surface area of the droplets dras-
viscosity only changed by a maximum of 15%. The ZP increased from
tically. Therefore, adequate surfactant concentration is required to
−32.8 mV to −36.2 mV in the 30 days. This shows that a stable nano-
cover the fresh interface and prevent coalescence with other droplets.
emulsion was formed.
Prevention of coalescence of the newly formed droplet is presumably
due to Gibbs-Maragoni effect in which it will only work if the surfactant
3.7. Response surface methodology study
is in the continuous phase and non-equilibrium condition (Tadros,
Izquierdo, Esquena, & Solans, 2004). As the system reaches equili-
3.7.1. Optimization of emulsification conditions by RSM-BBD (ANOVA,
brium, the colloidal interaction forces primarily determine the coales-
regression analysis and model fitting)
cence stability (Walstra, 1993; Wooster, Golding, & Sanguansri, 2008).
The estimated regression model has been shown in Table 4. From
Thus, the droplet diameter would be smaller and stable at higher sur-
Table 4, X1X2, X1X3 X2X3, X12 and X22 have positive effects on the re-
factant concentration.
sulting responses (p < 0.05). Of all the progressive parameters, the
From Fig. 2, at a constant 1 wt% of surfactant concentration, the
interaction of number of cycles and pressure has the most positive effect
number of cycles has minimal effect (∼3%) on the produced droplet
in minimizing the droplet size (p < 0.05). The independent parameter
diameter. This is because of the re-coalesce of the newly formed dro-
of pressure has the most negative effect (p > 0.05). Precisely, the en-
plets due to the limitation of surfactant concentration despite con-
ergy density is the dominating factor in optimizing the production of
tinuous energy supply with increasing number of cycles. Besides, the
nano-emulsion. This does not indicate that the role of surfactant is in-
duration for the surfactant to adsorb on the surface of the oil droplets
significant, but comparatively, energy input possesses more weightage
also influences the droplet diameter. For instance, if the time taken for
than surfactant concentration.
the surfactant to adsorb on the droplet's surface is longer than the re-
The results of multiple regression analysis based on the results ob-
coalescing rate, smaller droplet size could not be attained even with
tained from the experiments indicated that the response variable (Y)

62
R. Raviadaran et al. LWT - Food Science and Technology 96 (2018) 58–65

Fig. 5. (a) Effect of number of cycles and surfactant concentration, (b) Effect of microfluidizer operating pressure and surfactant concentration, (c) Effect of
microfluidizer operating pressure and number of cycles on droplet diameter using Response Surface Methodology.

could be predicted by a quadratic polynomial model expressed in Eq.
(5), where Y is nano-emulsion droplet size, whereas, X1, X2 and X3
represent the variables of surfactant concentration, number of cycles
and operating pressure, respectively.
Y = 281.93 − 11.13x1 − 10.54x2 − 18.24x3 + 6.90x1 x2 − 8.80x1 x3
+ 19.93x2 x3 + 12.60x12 − 15.18x 22 + 2.37x 32 (5)
The degree of fitness of the model could be indicated by the

63
R. Raviadaran et al.
coefficient of determination (R2), adjusted coefficient of determination
(R2 adj), p-value and lack-of-fit given in Table 5. The value of R2 cal-
culated from the quadratic regression model was 0.9898, which implied
a high degree of fitness of the model and closer agreement between the
experimental and predicted values, i.e. 98.9% of the variation could be
explained by the fitted model. This implies that only 1.02% of the
variations were not justified by the fitted model. For a good statistical
model, R2adj should be close to R2. The R2adj obtained was 0.9714, which
shows that only 1.84% of the total variations were not explained by the
model. This confirms that the model is extremely significant.
The F-test and analysis of variance (ANOVA) for the response sur-
face quadratic model showed that the p-value of the model is 0.0002 as
shown in Table 5. This suggests that the model is significant and ade-
quate for predicting the droplet size of nano-emulsion. Through the lack
of fit test, the obtained p-value of > 0.05 shows that the model suc-
cessfully predicted the variance occurrence in the test and well fitted to
the experimental data (Bezerra, Santelli, Oliveira, Villar, & Escaleira,
2008; Lu et al., 2017).
3.7.2. Response surface methodology-3D and contour plots analyses
Fig. 5 (a), (b) and (c) shows the 3D contour generated by the model.
In all the figures, the generated contours do not have strong curvature
or distortion as compared to the literature (Muralidhar, Chirumamila,
Marchant, & Nigam, 2001; Sood, Jain, & Gowthamarajan, 2014). This is
due the narrow range of parameters selected, causing the results to not
vary much with the variation of the parameters.
Fig. 5 (a) shows that by increasing both parameters, i.e. the number
of cycles and concentration of surfactant, the droplet size decreased.
However, the number of cycles had significant effect in reducing the
droplet diameter only at lower concentration of surfactant. This in-
dicates that at lower surfactant concentration, higher residence time
between the surfactant and the droplets were favourable in coating the
droplets. Thus, at higher surfactant concentration, lesser number of
cycles are sufficient to obtain smaller droplets since longer contact time
is not required to coat the droplets with surfactant.
Fig. 5 (b) shows that by increasing operating pressure and surfactant
concentration, the droplet diameter decreases. At lower concentration
of surfactant, the influence of pressure was not significant as sufficient
surfactant was unavailable to coat the droplets in obtaining smaller
droplet size. Thus, the use of higher operating pressure is most appro-
priate with the presence of sufficient emulsifier at higher surfactant
concentration to obtain smaller droplet size.
Fig. 5 (c) shows that there is a maximum distortion in the contours
observed at lower pressure and number of cycles. It indicates that at
lower pressure, the obtained droplet diameter depends heavily on the
number of cycles. With increasing operating pressure, the droplet dia-
meter decreases linearly until the 5th cycle. However, when the oper-
ating pressure was increased beyond the 5th cycle, a plateau was ob-
served indicating that there is no effect on the droplet diameter size.
Hence, lesser number of cycles are sufficient for small droplet size
formation at higher operating pressure.
3.7.3. Verification of the model
The predicted optimized parameters by RSM were 377.59 bar of
operating pressure, 5.04 number of cycles and 0.97 wt% of surfactant
concentration. The reliability of the optimum condition proposed was
tested by conducting experiment with the predicted parameters. Due to
experimentation constraints/limitations, 350 bar of operating pressure,
5 number of cycles and 1 wt% of surfactant concentration were utilized.
From the experiment, a mean droplet size of 275.5 nm, PDI of 0.256
and zeta potential of −32.8 mV were obtained. The obtained droplet
size from the experimentation was found to be larger by 11% to the
predicted value. This is reasonable since lower experimental pressure
was used as compared to the provided pressure from RSM due to the
equipment's sensitivity. This demonstrates that RSM is a capable tool to
aid in the optimization of nano-emulsion production using
64
LWT - Food Science and Technology 96 (2018) 58–65
microfluidizer.
4. Conclusions
A stable palm oil-based O/W nano-emulsion encapsulating cur-
cumin was successfully obtained using microfluidizer at 275.5 nm in
size, 0.257 of PDI, −32.8 of ZP and 446 cP of viscosity with a minimum
of 1 wt% of surfactant concentration (Tween 80). This demonstrates the
capability of palm oil in the formulation of a stable O/W nano-emulsion
which requires least amount of surfactant. The microfluidizer was
found capable to produce nano-emulsion with droplet size ranging
200 nm–300 nm at operating pressure of 350 bar and beyond 5 cycles.
The droplet diameter decreased with increased operating pressure until
reached an optimum level. The number of cycles had no significant
effects on droplet diameter once it has reached its minimum size due to
the limitation of surfactant concentration. RSM was successfully uti-
lized in optimizing the microfluidizer with only a 11% of difference in
the mean droplet diameter predicted using RSM as compared to the
obtained experimental results.
Abbreviations
BBD, Box-Behnken Design; Eq., equation; O/W, oil-in-water; PDI,
polydispersity index; RSM, response surface methodology; wt., weight;
ZP, zeta potential.
Acknowledgements
The authors would like to acknowledge the financial and facilities
support provided by the Faculty of Engineering, University of
Nottingham Malaysia Campus for this research work.
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