Food Chemistry 253 (2018) 179–184

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Optimization of mixed surfactants-based β-carotene nanoemulsions using T

response surface methodology: An ultrasonic homogenization approach
⁎
Tahir Mehmooda, , Anwaar Ahmeda, Asif Ahmada, Muhammad Sheeraz Ahmadb,
Mansur Abdullah Sandhuc
a
Institute of Food and Nutritional Sciences, PMAS-Arid Agriculture University, Rawalpindi 46300, Pakistan
b
Institute of Biochemistry and Biotechnology, PMAS-Arid Agriculture University, Rawalpindi 46300, Pakistan
c
Department of Veterinary Biomedical Sciences, PMAS-Arid Agriculture University, Rawalpindi 46300, Pakistan

A R T I C L E I N F O A B S T R A C T

Keywords: In the present study, food grade mixed surfactant-based β-carotene nanoemulsions were prepared without using
β-Carotene any co-surfactant. Response surface methodology (RSM) along with central composite design (CCD) was used to
Nanoemulsions investigate the effect of independent variables (surfactant concentration, ultrasonic homogenization time and oil
RSM content) on response variables. RSM analysis results revealed that experimental results were best fitted into a
Droplet size
quadratic polynomial model with regression coefficient values of more than 0.900 for all responses. Optimized
Mixed surfactant
β-Carotene retention
preparation conditions for β-carotene nanoemulsions were 5.82% surfactant concentration, 4 min ultrasonic
homogenization time and 6.50% oil content. The experimental values at optimized preparation conditions were
119.33 nm droplet size, 2.67p-Anisidine value and 85.63% β-carotene retention. This study will be helpful for
the fortification of aqueous products with β-carotene.

1. Introduction Nanoemulsions are kinetically stable systems with mean radii

β-Carotene is a member of the carotenoid family, which is mainly
found in fruits and vegetables. It provides a substantial proportion of
vitamin A in the human diet because of its retinol precursor and higher
conversion rate (Naves & Moreno, 1998). It is also useful in the pre-
vention of numerous diseases, such as heart diseases, cataracts and
cancer (Aherne, Daly, Jiwan, O’Sullivan, & O’Brien, 2010). Further-
more, it is also used in the food industry as a colorant and antioxidant
(Hou et al., 2012). Therefore, the food industry is interested in its in-
corporation into food products to take advantage of the above-men-
tioned benefits. However, its incorporation into beverages and various
other foods is challenging due to its poor water solubility, instability in
heat, oxygen and light and appearance in crystalline state at ambient
temperature (Mattea, Martín, Matías-Gago, & Cocero, 2009). To over-
come this problem, β- carotene can be dissolved in oil or another sui-
table medium in oil in water emulsions before its incorporation into
aqueous food products (Qian, Decker, Xiao, & McClements, 2012).
Stability of β-carotene in oil in water emulsion depends on the com-
position of emulsion and environmental conditions, e.g. heat, surfac-
tant, light, food systems, singlet oxygen and antioxidant addition (Hou
et al., 2010). The most convenient way to incorporate β- carotene into
food products is in a nanoemulsion based colloidal system.
of < 100 nm. Furthermore, these emulsions have higher stability, so-
lubility and bioavailability due to their smaller particle size as com-
pared to conventional emulsions (McClements & Rao, 2011). Nanoe-
mulsions can be produced using high energy and low energy methods.
During high energy methods, intense disruptive force is generated to
mechanically break the oil phase into tiny droplets, which can be dis-
persed into the aqueous phase. These high energy methods (sonication,
high pressure homogenization and microfluidization) are desirable for
the food industry because we can prepare nanoemulsions by using
lower surfactant to oil ratio as compared to low energy methods
(Ozturk, Argin, Ozilgen, & McClements, 2014). Previously, some studies
were carried out on the preparation of β-carotene nanoemulsions using
low energy methods, microfluidization and high pressure homo-
genization but no study has been carried out on the preparation of
nanoemulsions through the ultrasonic homogenization method. Hence,
the present study was designed to investigate the suitability of ultra-
sonic homogenization for development of β-carotene nanoemulsions.
β-Carotene nanoemulsions prepared through the ultrasonic homo-
genization method were influenced by multiple variables during our
laboratory experiments (unpublished data). So, there is a need for op-
timization of process or product in order to investigate the relationship
between independent variables and response variables. Response

⁎
Corresponding author at: Institute of Food and Nutritional Sciences, PMAS-Arid Agriculture University, Rawalpindi, Pakistan.
E-mail address: tahiraridian@gmail.com (T. Mehmood).

https://doi.org/10.1016/j.foodchem.2018.01.136
Received 8 October 2017; Received in revised form 4 January 2018; Accepted 22 January 2018
0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
T. Mehmood et al. Food Chemistry 253 (2018) 179–184

surface methodology is an effective mathematical and statistical tech- represents absorption before reaction and M denotes sample mass in
nique to investigate the effects of multiple independent variables and grams.
their interaction on response variables (Li, Wang, & Wang, 2017;
Mehmood, Ahmad, Ahmed, & Ahmed, 2017). Hence, in our study, we
2.5. β-Carotene retention
have used RSM for optimization of emulsifying conditions.
The present study was designed to prepare mixed surfactant-based,
The concentration of β-carotene in nanoemulsions was determined
co-surfactant free (due to irritation and toxic effects of co-surfactants)
after one week by a spectrophotometric method. Firstly, a 1 ml sample
β-carotene nanoemulsions using an ultrasonication approach. After
was extracted using a mixture of n-Hexane (3 ml) and ethanol (2 ml).
that, preparation conditions (surfactant concentration, homogenization
After that, this mixture was shaken well and the hexane phase was
time and oil content) for β-carotene nanoemulsions were optimized
removed. This extraction procedure was repeated two times more. At
using RSM in order to obtain smallest droplet size, lower p-anisidine
the end, all hexane phases were combined and their absorbance was
value and maximum β-carotene retention.
measured through an UV- spectrophotometer at 450 nm after desired
dilution with n-Hexane. The β-carotene concentration was determined
2. Material and methods
using a standard curve prepared under similar conditions. Vitamin re-
tention was calculated using Eq. (2):
2.1. Materials
VRBC = VBC,N/VBC,I × 100 (2)
Tween 80 and soya lecithin were obtained from Sigma-Aldrich (St.
Louis, USA). Purified β-carotene (powder form) was supplied by BASF where VRBC represents β-carotene retention, VBC,N is the concentration
(Lampertheim, Germany). Olive oil (refined, bleached and deodorized) of β-carotene in the nanoemulsion and VBC,I indicates initial con-
was purchased from Hamza Vegetable Oil Refinery and Ghee Mills centration of β-carotene (Yuan, Gao, Zhao, & Mao, 2008).
(Lahore, Pakistan). Double distilled water was used for the preparation
of nanoemulsions and solutions. 2.6. Experimental design

2.2. Nanoemulsions preparation
Nanoemulsions were prepared by mixing 10% dispersed phase and
90% continuous phase. The dispersed phase was prepared by dissolving
a pre-determined amount of β-carotene in olive oil (5.48–10.52%). The
continuous phase consisted of double distilled water carrying pre-de-
termined amount of surfactants (2.64–9.36%). These components were
mixed with polytron (KRH-I, KONMIX, Shanghai, China) at 8000 rpm
for 7 min to prepare coarse emulsions. For the preparation of nanoe-
mulsions, these coarse emulsions were subjected to ultrasonic homo-
genization by using a 20 kHz sonicator (230VAC, Cole-Parmer, USA).
Ultrasonic homogenization was performed by placing the tip horn
(20 mm diameter) of the sonicator in coarse emulsions and applying
ultrasonic powers for different times (2.98–8.02 min). The temperature
of the emulsions was controlled by placing them in ice bath during
homogenization. These nanoemulsions were stored at room tempera-
ture for further analysis.
2.3. Droplet size analysis
The droplet size of the nanoemulsions was measured by dynamic
light scattering using nanotrac (Microtrac, Tri-Blue, USA).
Nanoemulsion samples were diluted to 10% by using deionized water in
order to avoid multiple scattering effects.
Response surface methodology was used to investigate the effect of
independent variables, including surfactant concentration (X1), ultra-
sonic homogenization time (X2) and oil contents (X3) on response
variables, such as droplet size (Y1), p-Anisidine value (Y2) and retention
of β-carotene (Y3) in nanoemulsions. RSM design along with coded and
uncoded levels is presented in Table 1. Central composite design (Five
levels) and quadratic model was used to design this experiment. Twenty
treatments, including six axial points, eight fractional factorial points
and six central points were randomly performed according to CCD,
which is summarized in Table 1. Real levels of independent variables
were coded according to Eq. (3);
Z= Z0−−ZC/ΔZ (3)
where Z and Z0 indicate coded and real levels of independent variables,
respectively. ΔZ represents step change while ZC indicates actual value
at the central point. The specific equations for each independent vari-
able were derived from the above equation to code their actual values.
Specific equations for surfactant concentration (X1), ultrasonic homo-
genization time (X2) and oil contents (X3) are mentioned in below Eqs.
(4)–(6).
z1 = (MS−6)/2 (4)
z2 = (HT−5.5)/1.5 (5)

2.4. p-Anisidine value z 3 = (OC−8)/1.5 (6)

p-Anisidine value is an important indicator of the stability of na- where MS, HT and OC represent surfactant concentration, homo-
noemulsions. The oxidative stability of β-carotene nanoemulsions was genization time and oil contents, respectively.
determined according to the method of Mehmood et al. (2017). Firstly, A second order polynomial equation was used to indicate the pre-
20 g of β-carotene nanoemulsions were incubated for one week at 50 °C. dicted responses (droplet size, p-Anisidine value and retention of β-
Then, 1 g of solution was dissolved in n-Hexane (HPLC Grade) and the carotene) as a function of an independent variable as follows (Eq. (7)):
absorbance of the solutions was measured using an UV-spectro-
photometer at 350 nm. After that, 1 ml p-Anisidine reagent (prepared Table 1
Independent variables and their corresponding levels for β- Carotene nanoemulsion.
by dissolving 2.5 g of p-Anisidine in one litre of acetic acid) was added
in 5 ml of solution and they were incubated for 10 min to allow their Independent variable Symbol Coded levels
reaction. The absorbance of the fat solution was also determined as a
blank in a reference cell. p-Aniside value was determined using Eq. (1): −α −1 0 +1 +α

25 × (1.2AAR−ABR) Surfactant Concentration (%) X1 2.64 4 6 8 9.36

p−Anisidine Value =
M (1) Homogenization Time (min) X2 2.98 4 5.5 7 8.02
Oil Content (%) X3 5.48 6.5 8 9.5 10.52
where AAR is the absorption of the solution after reaction, ABR

180
T. Mehmood et al. Food Chemistry 253 (2018) 179–184

Z= β0 + β1 Y1 + β2 Y2 + β3 Y3 + β11 Y12 + β22 Y22 + β33 Y32 + β12 Y1 Y2 + β13 Y1 Y3 Table 3

Regression coefficients values for β- Carotene nanoemulsions.
+ β23 Y2 Y3 (7)
Regression Droplet size p-Anisidine β- Carotene
where Z represents response values, βj βjj βjk indicates the values of linear, coefficients (nm) Value Retention (%)
quadratic and interactive coefficients, respectively and β0 is constant.
Intercept (α0) 114.85 3.66 79.43
Design expert software (version. 6.0.11) was used to calculate the va- A-Surfactant (α1) −5.44*** −1.48*** 9.82***
lues of coefficients of determinations. B-Time (α2) −8.01*** 0.64** −2.42*
C-Oil (α3) 2.55* 1.31*** −3.21**
A2 (α11) 6.13*** 0.41* −3.21**
2.7. Statistical analysis B2 (α22) −3.95** 0.43* −4.27***
C2 (α33) 0.82 0.69*** 2.09*
AB (α12) −2.88* −0.038 −2.37*
Experimental data were statistically analyzed using Design Expert AC (α13) −1.38 0.19 −0.87
Software (version 6.0.11). Numerous statistical parameters (lack-of-fit, BC (α23) −0.88 −0.037 0.38
predicted and adjusted multiple correlation coefficients and coefficient R2 0.9456 0.9580 0.9604
of variation) of different polynomial models were compared to select
the best fitting polynomial model. Significant difference was de-
termined through analysis of variance by calculating F-value at the p−Anisidine Value = +29.78−2.40Y1−1.46Y2−4.34Y3 + 0.10Y12 + 0.19Y22
probability of 0.5, 0.1 and 0.01. To understand the effect of emulsifying + 0.31Y32−0.013Y1Y2 + 0.063Y1Y3−0.017Y2Y3 (9)
conditions on response variables, response plots were generated using
Design Expert Software (version 6.0.11). All these experiments were β−Carotene Retention = +16.43 + 21.23Y1 + 22.68Y2−16.19Y3−0.80Y12
performed in triplicate. −1.90Y22 + 0.93Y32−0.79Y1Y2−0.29Y1Y3 + 0.17Y2Y3
(10)
3. Results and discussion Statistical analysis (ANOVA) results revealed that the experimental
data could be represented well with a quadratic polynomial model with
3.1. Fitting the model coefficient of determination (R2) values for droplet size (Y1), p-
Anisidine value (Y2) and retention of β-carotene (Y3) being 0.9456,
Response surface methodology (RSM) is a statistical, theoretical and 0.9580 and 0.9604, respectively (Table 3).
mathematical technique for model building in order to optimize the Lack of fit was non-significant (p ≤ 0.05) relative to pure error for
level of independent variables (Homayoonfal, Khodaiyan, & Mousavi, all variables, which indicates that our model is statistically accurate. If
2015). The effect of independent variables (β-carotene nanoemulsions) the value of R2 is closer to unity then it is the indication of better model
on droplet size (Y1), p-Anisidine value (Y2) and retention of β-carotene fitting to actual data. On the other end, lower values of R2 indicate that
(Y3) are given in Table 2. Coefficients of polynomial equation were response variables were not appropriate to explain the variation in
computed from experimental data to predict the values of the response behaviour (Myers, Montgomery, & Anderson-Cook, 2016). In our study,
variable. Regression equations for each response variable, obtained proximity to unity R2 demonstrates that the influence of surfactant
from response surface methodology are mentioned in Eqs. (8)–(10): concentration (X1), ultrasonic homogenization time (X2) and oil con-
tents (X3) on response variables could be adequately described through
Droplet Size = +101.66−12.16Y1 + 22.83Y2 + 0.74Y3 + 1.53Y12−1.76Y22 a quadratic polynomial model. Significance level for coefficients of the
+ 0.37Y32−0.96Y1Y2−0.46Y1Y3−0.39Y2Y3 (8) quadratic polynomial model were determined through analysis of var-
iance (ANOVA). Smaller P-value and larger F-value is the indication of a
highly significant effect of any term on the response variable
Table 2 (Quanhong & Caili, 2005).
Experimental design for β- Carotene nanoemulsions with independent variables, experi-
mental and predicted values of responses.
3.2. Effect of independent variables on response variables
Run Independent Variables Response Values
β-Carotene nanoemulsions were successfully prepared by using
Surfactant (%) Time Oil Droplet p-Anisidine β- Carotene
(min) Content Size (nm) Value retention
different levels of independent variables (Fig. 1). The effect of in-
(%) (%) dependent variables on droplet size, p-anisidine value and β-carotene
retention are given in Table 2. Regression coefficients for independent
1 8 4 6.50 121 1.5 94 variables are summarized in Table 3.
2 6 5.50 8 110 3.2 77
3 4 7 6.50 115 6.3 64
4 6 5.50 5.48 111 3.3 92 3.2.1. Droplet size
5 8 4 9.50 125 5.2 84 The droplet size of β-carotene nanoemulsions depended on surfac-
6 6 8.02 8 89 5.6 66 tant concentration due to its significant effect on droplet size at a linear
7 9.36 5.50 8 122 2.1 86
(p < 0.001), quadratic (p < 0.001) and interaction level (p < 0.05)
8 6 5.50 10.52 124 7.1 82
9 6 5.50 8 114 4.1 80 with homogenization time. Surfactants lower the interfacial tensions
10 6 5.50 8 110 3.7 76 between disperse and continuous phase, which leads to smaller droplet
11 8 7 9.50 101 5.9 75 formation (Mehmood et al., 2017). Other independent variables, which
12 8 7 6.50 104 3.1 82 had significant effect on droplet size were linear term of homogeniza-
13 6 5.50 8 117 3.6 83
14 6 5.50 8 121 3.5 80
tion time (p < 0.001) and oil content (p < 0.05), and quadratic terms
15 4 4 6.50 124 5.3 65 of homogenization time (p < 0.001).
16 4 4 9.50 130 7.5 60 The influence of homogenization time and surfactant concentration
17 6 5.50 8 117 4 80 on droplet size of β-carotene nanoemulsions is illustrated in Fig. 2 (A).
18 2.64 5.50 8 143 6.7 58
Both these variables exert quadratic effect on droplet size. At higher
19 6 2.98 8 119 3.3 72
20 4 7 9.50 121 9.1 59 surfactant concentration, decrease in droplet size of nanoemulsions was
observed with the increase of homogenization time. This downward

181
T. Mehmood et al.
trend was observed due to reduction of interfacial tension with the
increase in surfactant concentration (Polychniatou & Tzia, 2018). At
lower surfactant concentration, droplet size increased with increasing
homogenization time. This increase was observed because not enough
emulsifier is present to cover newly formed smaller droplets, which
initiates a coalescence process (Anarjan, Mirhosseini, Baharin, & Tan,
2010). Fig. 2(B) represented the combined effect of oil and surfactant
concentration on the droplet of β-carotene nanoemulsions. Oil content
exerts a linear effect while surfactant concentrations have a quadratic
effect on droplet size of nanoemulsions. Droplet size increased with
rising oil concentration due to increase in viscosity. As a result of this,
higher energy is required to break the droplet, which results in larger
droplet size. Additionally, higher oil concentration encourages ag-
gregation and collision of nanoemulsion droplets, which increased the
droplet size (Mehmood, 2015; Zhang, Fan, & Smith, 2009). Initially,
droplet size decreased with the increase of surfactant concentration due
to reduction in surface tension. But, after a minimal level, higher con-
centration of surfactant caused increased width of the diffusion layer
due to excessive coverage of crystalline particles by surfactant. This
mechanism lowers zeta potential value and encourages agglomeration
tendency, which increased droplet size of β-carotene nanoemulsions
(Mehmood et al., 2017; Tan, Billa, Roberts, & Burley, 2010).
3.2.2. p-Anisidine value
p-Anisidine value is an important indicator for measurement of
oxidation products (Cho, Kim, Bae, Mok, & Park, 2008). As the p-Ani-
sidine value of β-carotene nanoemulsions was concerned, oil content
had a pronounced effect on the p-Anisidine value of β-carotene na-
noemulsions due to its significant effect on p-Anisidine value at a linear
(p < 0.001) and quadratic level (p < 0.001). Other factors which
significantly contribute toward p-Anisidine value were linear term of
surfactant concentration (p < 0.001) and homogenization time
(p < 0.001), and quadratic term of surfactant concentration
(p < 0.05) and homogenization time (p < 0.05). The lipid oxidation
mechanism is remarkably different in nanoemulsions as compared with
bulk oily phase due to the presence of interface and aqueous phase. In
nanoemulsions, lipid oxidation depends on many factors, which include
oxygen concentration, pH and ionic strength of aqueous phase, droplet
size, thickness and interfacial properties (Waraho, McClements, &
Decker, 2011; Öztürk, Urgu, & Serdaroğlu, 2017).
The combined effects of homogenization time and surfactant con-
centration on p-Anisidine value are illustrated in Fig. 2(C), which ex-
plicated the linear effect of both independent variables on p-Anisidine
value. p-Anisidine value of β-carotene nanoemulsions increased with
182
Food Chemistry 253 (2018) 179–184
Fig. 1. (A) Particle size distribution of β-carotene nanoe-
mulsions (B) Visual appearance of β-carotene nanoemul-
sions.
the increase in homogenization time while higher concentration of
surfactants result in a lower p-Anisidine value. During this study, β-
carotene nanoemulsions were developed using mixed surfactant
(Tween 80 and soya lecithin), which act as an interfacial barrier against
oxidation. These surfactants built a protective membrane at the inter-
face of the aqueous and oily phase, which remarkably reduces proox-
idant accessibility into oil droplets, which results in lower p-Anisidine
value (Hwang et al., 2017). Fig. 2(D) depicts the interactive effect of oil
content and surfactant concentration on p-Anisidine value. Both vari-
ables have a linear effect on the p-Anisidine value of β-carotene na-
noemulsions. The downward trend was observed in p-Anisidine value
with the increase of surfactant and oil concentration. Oil concentrations
have an inverse effect on p-anisidine value because droplet size in-
creases when oil concentration is high, which results in lower p-Anisi-
dine value due to reduced surface area for oxidation (Mehmood et al.,
2017).
3.2.3. β-Carotene retention
β-Carotene retention of nanoemulsion mainly depended on surfac-
tant concentration as it had a significant effect on vitamin retention at
linear (p < 0.001), quadratic (p < 0.01) and interactive level
(p < 0.05). Surfactant prevents the degradation of β-carotene by
forming a membrane like structure around new surfaces (Hejri,
Khosravi, Gharanjig, & Hejazi, 2013). Other factors which significantly
contributed to β-carotene retention were linear effect of homogeniza-
tion time (p < 0.05) and oil content (p < 0.01), quadratic effect of
homogenization time (p < 0.001) and oil content (p < 0.05) and in-
teractive effect of homogenization time (p < 0.05).
A contour plot in Fig. 2(E) illustrates the retention of β-carotene as a
function of homogenization time and surfactant concentration. Sur-
factant concentrations have a linear effect while homogenization time
exerts a quadratic effect on the retention of β-carotene. At a lower level
of surfactant, β-carotene retention is significantly reduced with in-
creasing homogenization time due to the formation of smaller droplets,
which are not covered with surfactant molecule. Hence, surface area of
droplet significantly increases, which encourages β-carotene degrada-
tion (Waraho et al., 2011). Additionally, pre-existence of peroxides in
surfactant molecules may also cause β-carotene degradation. These
peroxides breakdown into reactive radicals at elevated temperature and
significantly degrade β-carotene during storage (Liu & Wu, 2010).
Fig. 2(F) represents the interactive effect of oil content and surfactant
concentration on the β-carotene retention in nanoemulsions. Both
emulsifying conditions have a linear effect on β-carotene retention.
With the increase in surfactant concentration, degradation of β-
T. Mehmood et al.
carotene reduced due to the formation of a rigid surfactant shell at the
water–oil interface. This shell increases the stability of β-carotene by
preventing repulsion of β-carotene and avoiding new surface formation
(Hejri et al., 2013). Higher oil content also increases the stability of β-
carotene nanoemulsions by formation of larger droplets, which have
lower surface area (Liu & Wu, 2010).
3.3. Optimization of independent variables
To illustrate the effects of surfactant concentration, homogenization
time and oil content on response variables, response surface graphs
were drawn using design expert software. These graphs were generated
by varying two independent variables within experimental ranges while
183
Food Chemistry 253 (2018) 179–184
Fig. 2. 3D graphic surface optimization of
(A) droplet size (nm) versus surfactant con-
centration (%) and homogenization time
(min) (B) droplet size (nm) versus oil content
(%) and surfactant concentration (%) (C) p-
Anisidine value versus surfactant concentra-
tion (%) and homogenization time (min) (D)
p-Anisidine value versus oil content (%) and
surfactant concentration (%) (E) β-carotene
retention (%) versus surfactant concentra-
tion (%) and homogenization time (min) (F)
β-carotene retention (%) versus oil content
(%) and surfactant concentration (%).
keeping the third variable at central point. Fig. 2(A, C and E) were
generated by varying the surfactant concentration and homogenization
time at 8% oil content, while Fig. 2(B, D and F) were drawn by chan-
ging the concentration of oil and surfactant at a central value of
homogenization time (5.5 min). These graphs illustrated complex in-
teraction among independent variables.
After that, numerical optimization was executed by desirability
function using Design Expert Software. The goals selected for the op-
timization of β-carotene nanoemulsions were minimum level of sur-
factant concentration, homogenization time and oil content in order to
obtain smaller droplet size, lower p-Anisidine value and maximum re-
tention of β-carotene. Ten different solutions were found which contain
different levels of independent variables. The solution with maximum
T. Mehmood et al.
Table 4
Optimum conditions, experimental and predicted value of response at optimized condi-
tions.
Optimum Conditions Coded Levels Actual Levels
Surfactant Concentration (%) −0.09 5.82
Homogenization Time (min) −1.00 4
Oil Contents (%) −1.00 6.50
Response Predicted Values Experimental Values
Droplet Size (nm) 116.46 119.33 ± 2.5
p-Anisidine Value 2.937 2.67 ± 0.9
β- Carotene Retention (%) 82.085 85.63 ± 1.5
desirability value was selected as the optimized emulsifying condition.
Combined optimized preparation conditions for β-carotene nanoemul-
sions were 5.82% surfactant concentration, 4 min. ultrasonic homo-
genization time and 6.50% oil content. The response values at opti-
mized preparation conditions were 116.46 nm droplet size, 2.937p-
Anisidine value and 82.085% β-carotene retention (Table 4).
3.4. Verification of RSM model
Optimized emulsifying conditions were used to check the suitability
of the model for prediction of response values. Optimized preparation
conditions were validated by performing experiments under optimized
conditions. The response values at optimized preparation conditions
were 116.46 nm droplet size, 2.937p-Anisidine value and 82.085% β-
carotene retention. On the other hand, the experimental values at op-
timized preparation conditions were 119.33 nm droplet size, 2.67p-
Anisidine value and 85.63% β-carotene retention. Experimental re-
sponse values were well in agreement with predicted response values
(Table 4).
4. Conclusions
In this study, we have evaluated the preparation conditions of β-
carotene nanoemulsions using ultrasonic homogenization techniques
and incorporated β-carotene in mixed surfactant-based nanoemulsions
in order to protect β-carotene from harsh environmental conditions
during food fortification. Mixed surfactant-based β-carotene nanoe-
mulsions were successfully prepared using an ultrasonic homogeniza-
tion approach. This study illustrates that response surface methodology
is a useful tool to optimize the emulsifying conditions of β-carotene
nanoemulsions and explore the relationship between independent and
response variables. The results of this study showed that emulsifying
condition and ingredients have significant effect on the properties of
nanoemulsions. The current study illustrates that the quadratic model
was sufficient to describe and predict the responses of droplet size, p-
Anisidine value and β-carotene retention, with the change of in-
dependent variables (surfactant concentration, homogenization time
and oil content). The optimum condition was obtained through nu-
merical optimization using desirability function. Optimized preparation
conditions for β-carotene nanoemulsions were 5.82% surfactant con-
centration, 4 min. ultrasonic homogenization time and 6.50% oil con-
tent.
References
Aherne, S. A., Daly, T., Jiwan, M. A., O’Sullivan, L., & O’Brien, N. M. (2010).
184
Food Chemistry 253 (2018) 179–184
Bioavailability of β-carotene isomers from raw and cooked carrots using an in vitro
digestion model coupled with a human intestinal Caco-2 cell model. Food Research
International, 43(5), 1449–1454.
Anarjan, N., Mirhosseini, H., Baharin, B. S., & Tan, C. P. (2010). Effect of processing
conditions on physicochemical properties of astaxanthin nanodispersions. Food
Chemistry, 123(2), 477–483.
Cho, Y. H., Kim, S., Bae, E. K., Mok, C., & Park, J. (2008). Formulation of a cosurfactant-
free O/W microemulsion using nonionic surfactant mixtures. Journal of Food Science,
73(3).
Hejri, A., Khosravi, A., Gharanjig, K., & Hejazi, M. (2013). Optimisation of the formula-
tion of β-carotene loaded nanostructured lipid carriers prepared by solvent diffusion
method. Food Chemistry, 141(1), 117–123.
Homayoonfal, M., Khodaiyan, F., & Mousavi, M. (2015). Modelling and optimising of
physicochemical features of walnut-oil beverage emulsions by implementation of
response surface methodology: Effect of preparation conditions on emulsion stability.
Food Chemistry, 174, 649–659.
Hou, Z., Gao, Y., Yuan, F., Liu, Y., Li, C., & Xu, D. (2010). Investigation into the physi-
cochemical stability and rheological properties of β-carotene emulsion stabilized by
soybean soluble polysaccharides and chitosan. Journal of Agricultural and Food
Chemistry, 58(15), 8604–8611.
Hou, Z., Zhang, M., Liu, B., Yan, Q., Yuan, F., Xu, D., & Gao, Y. (2012). Effect of chitosan
molecular weight on the stability and rheological properties of β-carotene emulsions
stabilized by soybean soluble polysaccharides. Food Hydrocolloids, 26(1), 205–211.
Hwang, J. Y., Ha, H. K., Lee, M. R., Kim, J. W., Kim, H. J., & Lee, W. J. (2017).
Physicochemical property and oxidative stability of whey protein concentrate mul-
tiple nanoemulsion containing fish oil. Journal of Food Science, 82(2), 437–444.
Li, X., Wang, L., & Wang, B. (2017). Optimization of encapsulation efficiency and average
particle size of Hohenbuehelia serotina polysaccharides nanoemulsions using re-
sponse surface methodology. Food Chemistry, 229, 479–486.
Liu, C.-H., & Wu, C.-T. (2010). Optimization of nanostructured lipid carriers for lutein
delivery. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 353(2),
149–156.
Mattea, F., Martín, Á., Matías-Gago, A., & Cocero, M. J. (2009). Supercritical antisolvent
precipitation from an emulsion: β-carotene nanoparticle formation. The Journal of
Supercritical Fluids, 51(2), 238–247.
McClements, D. J., & Rao, J. (2011). Food-grade nanoemulsions: formulation, fabrication,
properties, performance, biological fate, and potential toxicity. Critical Reviews in
Food Science and Nutrition, 51(4), 285–330.
Mehmood, T. (2015). Optimization of the canola oil based vitamin E nanoemulsions
stabilized by food grade mixed surfactants using response surface methodology. Food
Chemistry, 183, 1–7.
Mehmood, T., Ahmad, A., Ahmed, A., & Ahmed, Z. (2017). Optimization of olive oil based
O/W nanoemulsions prepared through ultrasonic homogenization: A response sur-
face methodology approach. Food Chemistry, 229, 790–796.
Myers, R. H., Montgomery, D. C., & Anderson-Cook, C. M. (2016). Response surface
methodology: Process and product optimization using designed experiments. John Wiley &
Sons.
Naves, M. M. V., & Moreno, F. S. (1998). Β-Carotene and cancer chemoprevention: From
epidemiological associations to cellular mechanisms of action. Nutrition Research,
18(10), 1807–1824.
Ozturk, B., Argin, S., Ozilgen, M., & McClements, D. J. (2014). Formation and stabiliza-
tion of nanoemulsion-based vitamin E delivery systems using natural surfactants:
Quillaja saponin and lecithin. Journal of Food Engineering, 142, 57–63.
Öztürk, B., Urgu, M., & Serdaroğlu, M. (2017). Egg white powder-stabilised multiple
(water-in-olive oil-in-water) emulsions as beef fat replacers in model system meat
emulsions. Journal of the Science of Food and Agriculture, 97(7), 2075–2083.
Polychniatou, V., & Tzia, C. (2018). Evaluation of surface-active and antioxidant effect of
olive oil endogenous compounds on the stabilization of water-in-olive-oil nanoe-
mulsions. Food Chemistry, 240, 1146–1153.
Qian, C., Decker, E. A., Xiao, H., & McClements, D. J. (2012). Physical and chemical
stability of β-carotene-enriched nanoemulsions: Influence of pH, ionic strength,
temperature, and emulsifier type. Food Chemistry, 132(3), 1221–1229.
Quanhong, L., & Caili, F. (2005). Application of response surface methodology for ex-
traction optimization of germinant pumpkin seeds protein. Food Chemistry, 92(4),
701–706.
Tan, S., Billa, N., Roberts, C., & Burley, J. (2010). Surfactant effects on the physical
characteristics of Amphotericin B-containing nanostructured lipid carriers. Colloids
and Surfaces A: Physicochemical and Engineering Aspects, 372(1), 73–79.
Waraho, T., McClements, D. J., & Decker, E. A. (2011). Mechanisms of lipid oxidation in
food dispersions. Trends in Food Science & Technology, 22(1), 3–13.
Yuan, Y., Gao, Y., Zhao, J., & Mao, L. (2008). Characterization and stability evaluation of
β-carotene nanoemulsions prepared by high pressure homogenization under various
emulsifying conditions. Food Research International, 41(1), 61–68.
Zhang, J., Fan, Y., & Smith, E. (2009). Experimental design for the optimization of lipid
nanoparticles. Journal of Pharmaceutical Sciences, 98(5), 1813–1819.