Journal of Colloid and Interface Science 505 (2017) 1082–1092

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Journal of Colloid and Interface Science

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

Effect of surfactant concentration and solidification temperature on the

characteristics and stability of nanostructured lipid carrier (NLC)
prepared from rambutan (Nephelium lappaceum L.) kernel fat
Pimchanok Witayaudom, Utai Klinkesorn ⇑
Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, 50 Ngam Wong Wan Road, Chatuchak, Bangkok 10900, Thailand

g r a p h i c a l a b s t r a c t

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

Article history: Nanostructured lipid carrier (NLC) was fabricated from rambutan (Nephelium lappaceum L.) kernel fat sta-
Received 11 April 2017 bilized with Tween 80 in this present work. The influence of the Tween 80 concentration (0.025, 0.05, 0.1,
Revised 27 June 2017 0.2, 0.5 and 1.0 wt%) and solidification temperature (5 and 25 °C) on the characteristics and stability of
Accepted 2 July 2017
the NLC were investigated. The results showed that an increase in the Tween 80 concentration caused
Available online 4 July 2017
decreased zeta-potential (f-potential) and particle size (Z-average) with no significant effect on the poly-
dispersity index (PDI). Lipid particles in the NLC at all Tween 80 concentrations had a tendency to grow
Keywords:
and the PDI tended to increase due to Ostwald ripening upon storage over 28 days. At least 0.2 wt%
Nanostructure lipid carrier
Rambutan kernel fat
Tween 80 concentrations could be used to stabilize 1 wt% rambutan NLC. The solidification temperature
Surfactant concentration affected the microstructure, melting behavior and stability of rambutan NLC. Pre-solidification at 5 °C
Solidification temperature could create stable NLC with monodispersed-spherical lipid particles. Consequently, these stable NLC
Particle characteristics particles produced from rambutan kernel fat may serve as useful carriers for the delivery of bioactive
Physical stability lipophilic nutraceuticals.
Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction

Nowadays, functional and medical foods, which fortification of

⇑ Corresponding author. nutraceutical and pharmaceutical compounds, respectively, are of
E-mail addresses: nps_order@hotmail.com (P. Witayaudom), utai.k@ku.th
increasing interest of consumer. Due to the poor water solubility
(U. Klinkesorn).

http://dx.doi.org/10.1016/j.jcis.2017.07.008
0021-9797/Ó 2017 Elsevier Inc. All rights reserved.
 P. Witayaudom, U. Klinkesorn / Journal of Colloid and Interface Science 505 (2017) 1082–1092
and/or low permeability of nutraceutical and pharmaceutical com-
pounds, their incorporation into a food matrix may need a properly
designed delivery system. Important criteria that need to be con-
sidered when designing suitable delivery systems for food applica-
tions include the characteristics of the compounds (physical state,
lipophilicity, chemical stability and cell permeability) and the com-
position and structure of the target food matrix [1]. Recently, var-
ious colloidal delivery systems have been developed for the
encapsulation, protection and release of pharmaceuticals and
nutraceutical lipophilic bioactive compounds intended for food
fortification, including liposome, nanocrystal dispersion,
microemulsion, nanoemulsion, macroemulsion, multilayer emul-
sion, solid lipid nanoparticle and nanostructured lipid carriers
[2–9]. However, for food application, food-grade materials are
required. This limits the choices of materials for carriers.
A nanostructured lipid carrier (NLC) is considered as one of the
recent, efficient colloidal delivery systems. It is a lipid-in-water
emulsion, which contain lipid droplets that are partially crystal-
lized and have a less-ordered crystalline structure or an amorphous
solid structure. The dominant advantage of NLCs over other col-
loidal delivery systems may be a high loading capacity of bioactive
compounds as the lipophilic cores dissolve in the liquid lipid and
simultaneously encapsulate in the solid lipid network. Moreover,
NLCs have the potential to enhance the solubility and bioavailabil-
ity of the bioactive materials and improve the release of the encap-
sulated food ingredients [10].
Usually, the lipids phase used to prepare an NLC is a blend
between liquid and solid lipids, including mono-, di- and tri-
acylglycerols, fatty acids and waxes [11–15]. In the current study,
we examined the possibility of forming semi-crystalline lipid dro-
plets by using a single bulk lipid containing either liquid or solid
fractions at room temperature. Rambutan (N. lappaceum L.) kernel
fat (RKF) is extracted from rambutan seed kernels that are a waste
by-product of the canning industry. These kernels contain a high
amount of fat (33–37 wt%), which is a white, soft solid at room
temperature [16,17]. It has been reported that RKF at 25 °C has a
solid fat content around 45–60% [18,19]. These reports indicate
that RKF contains either solid or liquid lipid portions at ambient
temperature. Hence, RKF may be an alternative fat that if properly
used can fabricate a stable NLC for the delivery of various lipophilic
active compounds.
The particle characteristics and physical stability of an NLC are
dependent on the lipid phase composition [15,20–22] as well as
the concentration and chemical structure of the emulsifier
[13,14,23–25]. Moreover, other variables, e.g. cooling rate, solidifi-
cation temperature, have been reported to affect the characteristics
and physical stability of NLCs [12,22,26]. The present study evalu-
ated the effect of the concentration of Tween 80, food grade non-
ionic surfactant, and the solidification temperature on the particle
characteristics and stability of NLCs using rambutan kernel fat as
the lipid phase. Moreover, the ability of developed NLCs to entrap
b-carotene was also evaluated.
2. Materials and methods
2.1. Materials
Rambutan seeds (Nephelium lappaceumL.) variety ‘‘Rongrien”
were provided by Malee Sampran Public Company Limited
(Nakhon Pathom, Thailand). The brown shell of the seeds was man-
ually removed to obtain the kernel. Then, the kernels were dried in
a hot air dryer at 65 °C for 5 h. The dried kernels (moisture content,

10 wt%) were ground and stored in high-density polyethylene
plastic bags at 18 °C before fat extraction. Analytical-grade
Tween 80 (Polyoxyethylene (20) sorbitanmonooleate), all-trans-
1083
b-carotene (97% purity) and sodium azide (NaN3) were products
of Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Double-
distilled water was used for all solution preparation.
2.2. Rambutan kernel fat extraction
The frozen dried kernels were equilibrated at room temperature
overnight before being used for fat extraction. Extraction of rambu-
tan kernel fat (RKF) was carried out using a screw press manufac-
tured in Thailand. The pressing conditions were set at 25 rpm and
2.5 cm for the screw speed and compression clearance, respec-
tively. The screw-pressing process resulted in RKF slurry which
was a viscous mixture of crude fat and fine kernel particles. The
RKF was separated from the rambutan kernel particle by mixing
with warm water (
60 °C) and then centrifuging at 6000 rpm for
15 min. This separation step was performed twice to obtain crude
RKF. Crude RKF was transferred into an inert screw-cap bottle and
kept at -18 °C.
2.3. Nanostructured lipid carrier preparation
Nanostructured lipid carriers (NLCs) were prepared using a hot
homogenization method based on that described by Qian et al. [27]
with some modifications. A lipid phase was prepared by melting
crude RKF at 70 °C. The melted fat was then filtered under vacuum
through a filter paper (pore size of 11 mm) in a Buchner funnel. Var-
ious aqueous phases were prepared by dissolving Tween 80 at
0.025, 0.05, 0.1, 0.2, 0.5 and 1.0 wt% of the total NLCs into distilled
water and then heating to 70 °C. The aqueous phase (99 wt%) was
mixed separately with 1 wt% lipid phase at the same temperature
Ò Ò
using a hand-held homogenizer (Ultra-Turra xT25 basic, Ika -
Werke Gmbh& Co., Germany) at 9500 rpm for 2 min. Then, coarse
emulsion was passed three times through a two-stage high pres-
sure valve homogenizer (15MR-8TA, APV Gaulin Inc., Wilming,
MA, USA) at 5000 psi (4500 and 500 psi for the first and second
stages, respectively). The resulting hot emulsions were then cooled
to 25 °C by placing the sample containers in cold water (
20 °C).
Sodium azide (0.02 wt%) was added to prevent microbial growth,
after which the NLC samples were kept at 25 °C.
In order to study the effect of solidification temperature, the
NLCs were prepared as described above but using a lipid phase
content of 5 wt% and a 95 wt% aqueous phase containing 1 wt%
Tween 80. After cooling to 25 °C, the NLCs were divided into two
sets. The first set of samples was maintained at 25 °C, whereas
the second set was further cooled to 5 °C and kept at this temper-
ature for 1 h. After that, all samples were stored at 25 °C in a tem-
perature control incubator.
2.4. Zeta-potential measurement
The zeta-potential of the NLCs was analyzed at 25 °C using a
particle electrophoresis instrument (Zetasizer Nano series-Zen
3600, Malvern Instruments, Worcestershire, UK). Samples were
measured after dilution with distilled water to an appropriate con-
centration (
0.1 wt%) to prevent multiscattering effects. The
instrument measured the electrophoretic mobility of the particles,
and used the Henry equation to calculate the zeta-potential. The
desired parameters, including the reflective index, dielectric con-
stant and viscosity were set at 1.331, 78.5 and 0.8872, respectively.
2.5. Particle size measurement
The particle size of the NLCs was measured at 25 °C using a
dynamic light scattering instrument (Zetasizer Nano series-Zen
3600, Malvern Instruments, Worcestershire, UK). The measure-
ment was conducted after dilution of samples with distilled water
1084 P. Witayaudom, U. Klinkesorn / Journal of Colloid and Interface Science 505 (2017) 1082–1092
to an appropriate concentration (
0.1 wt%). The measurement
results were reported as the Z-average size or the volume-
intensity mean diameter and polydispersity index (PDI) according
to cumulants analysis. The reflective index and viscosity (1.331 and
0.8872, respectively) were used in the instrument to calculate the
particle size distributions.
2.6. Creaming stability evaluation
The creaming stability of the NLC samples (10 ml in a test tube)
was evaluated at 25 °C by visual observation of the height of the
cream layer (HC) on top and the height of the serum phase (HS)
at the bottom of the tube at 1, 3, 7, 10, 14, 21 and 28 days storage.
The creaming stability index (CSI) was then expressed as a percent-
age of the total height of NLCs in the tube (HT) which was calcu-
lated using the equation based on that described by Mirhosseini
et al. [28], with some slight modifications as follows:
ðHT  ðHC þ HSÞÞ
CSI ¼ 100  ð1Þ
HT
2.7. Microstructure observation
The microstructure of RKF and NLCs was observed using an
optical microscope (AxiolabÒ, Carl Zeiss Ply Ltd., Germany). The
NLC samples were gently agitated before analysis to ensure that
the particles were homogeneously distributed. A drop of sample
(
10 mL) was placed on a glass slide and glass cover slip was then
slowly placed over the samples to avoid creating any air bubbles.
An objective magnification of 40 was used to observe the
microstructure of the NLC samples under normal light mode. For
RKF crystal microstructure, a fat sample was made molten at
70 °C for 30 min to destroy any crystalline structure. The molten
sample was dropped onto a preheated glass slide and a preheated
glass cover slip was placed over the molten sample, taking care to
avoid creating any air bubbles. The slides were then cooled to 25 °C
and several slides were maintained at 25 °C, whereas other slides
were further cooled to 5 °C and kept at this temperature for 1 h.
After that, all slides were stored at 25 °C in a temperature control
incubator for 24 h to allow the fat to crystallize. The fat crystal
microstructure images were then recorded under polarized light
mode at an objective magnification of 5. At least five images of
each slide were recorded using digital image processing software
(Image-Pro PlusTM, version 6).
2.8. Differential scanning calorimetry analysis
The melting behaviors of RKF and the NLC were determined
using differential scanning calorimetry (DSC1 STARe System, Met-
tler Toledo, Switzerland). Nitrogen gas (99.999% purity) was
purged at a rate of 50 mL/min. Approximately 5–6 mg of each sam-
ple were weighed and hermetically sealed in an aluminum pan
with an empty pan used as the reference. Each sample was heated
from 25 to 80 °C at a rate of 10 °C/min to record the melting
behavior.
2.9. Encapsulation efficiency and loading of b-carotene
The pure b-carotene at 0.1 wt% of lipid phase was accurately
weighed and mixed with molten rambutan fat (
70 °C). This lipid
phase (5 wt%) was added to 95 wt% aqueous phase containing
Tween 80 (1 wt%) at the same temperature. The mixture was then
homogenized using a hand-held and a two-stage high pressure
valve homogenizers as described in Section 2.3. The resulting hot
emulsions were then cooled to 25 °C by placing the sample con-
tainers in cold water (
20 °C). Then, they were further cooled to
5 °C and kept at this temperature for 1 h. After that, NLC samples
were stored at 25 °C in a temperature control incubator for 24 h.
To calculating the encapsulation efficiency (EE) and b-carotene
loading (CL), the free b-carotene was determined according to the
method describe by Nik et al. [29] with some modifications. A 0.5 g
NLC dispersion was placed in a centrifugal filter tube with a molec-
Ò
ular weight cutoff of 30 kDa (Amicon Ultra-15; Merck Millipore
Ltd., Ireland) and centrifuged (MX-305; TOMY SEIKO Co., Ltd.,
Japan) at 7000 rpm (
4445g) for 10 min. The supernatant phase
was collected and then mixed with ethanol, acetone, and distilled
water (0.5, 3.0 and 1.0 ml, respectively). The free b-carotene in
supernatant was extracted with 2.0 mL hexane three times. The
organic phase was then dried under nitrogen at 35 °C. The residual
was resolubilized in hexane and the absorbance at 450 nm was
measured using a spectrophotometer (GENESYS 10S UV–Vis spec-
trophotometer; Thermo Fisher Scientific, USA). The measurements
were carried out in triplicate. The b-carotene concentration was
computed by using the calibration curve of pure b-carotene in hex-
ð1Þ ane (R2  0.999). The EE and CL were calculated based on the equa-
tions describe by Fathi et al. [30] with some modifications.
ðCaroteneT  CaroteneF Þ
EEð%Þ ¼  100 ð2Þ
CaroteneT
ðCaroteneT  CaroteneF Þ
CLðmg=gÞ ¼  1000 ð3Þ
LipidU
where CaroteneT is the total content of applied b-carotene in NLCs,
CaroteneF is the amount of free b-carotene in supernatant phase,
and LipidU is the content of the used lipid in preparation of the NLCs.
2.10. Statistical analysis
All experiments were performed in triplicate replications and
triplicate analyses were conducted on each replication. The vari-
ance of data results was analyzed using the SPSS statistical soft-
ware program. Duncan’s Multiple Range Test was used to
determine mean differences at the P < 0.05 level to denote statisti-
cal significance.
3. Results and discussion
3.1. Influence of surfactant concentration on characteristics and
stability of NLCs
3.1.1. Zeta-potential
All NLCs had a negative charge (
30 to 65 mV) even though
the particles were stabilized using a non-ionic surfactant, Tween
80 (Fig. 1). This negative charge was attributed to preferential
adsorption of OH- ions from water by the lipid particles [31]. The
same phenomenon has been reported by a number of researchers
[32–36]. In addition, it is also possible that the electrical charge
of the lipid particles used in the present study arose from
anionic-endogenous surface active compounds present within the
crude RKF including polyphenols, free fatty acids, partial glyc-
erides, and phospholipids [37–38], which are always found in
pressed oil [39–41]. The increase in the Tween 80 concentration
from 0.025 to 1.0 wt% caused a decrease in the value of the particle
zeta-potential from 64.17 to 32.11 mV (P  0.05). This result
suggested that Tween 80 did adsorb on the surfaces of the particles
by displacing the endogenous surface active compounds due to the
higher surface activity of Tween 80 [42]. Since Tween 80 is a non-
ionic surfactant, the increase in the Tween 80 concentration
adsorbed on the surface of particles resulted in a reduction in the
net charge at the particle surface. These results were consistent
with the previous work of Kheradmandnia et al. [43] who prepared
 P. Witayaudom, U. Klinkesorn / Journal of Colloid and Interface Science 505 (2017) 1082–1092
0.025
-10
Zeta-potential (mV)
-20
-30
-40
-50
-60
-70
1 3
Fig. 1. Zeta potential of NLC dispersions prepared from rambutan kernel fat at various surfactant concentrations (0.025, 0.05, 0.1, 0.2, 0.5 and 1.0 wt%) during 28 days storage
at 25 °C.
lipid nanoparticles using a mixture of lecithin and Tween 80 at dif-
ferent ratios as the surfactant. They reported that there was a
decrease in the magnitude of the particles’ zeta-potential with
increasing Tween 80 concentration.
The zeta-potential is an indirect measure of the physical stability
of the NLCs; therefore, variation was monitored in the particles’
zeta-potential as a function of the storage time for various Tween
80 concentrations. After 28 days storage at 25 °C, the zeta-
potential of lipid particles slightly decreased for the NLCs stabilized
with a Tween 80 concentration less than 0.5 wt%, being 60.51 ±
0.76, 55.27 ± 1.72, 45.60 ± 1.80 and 37.91 ± 2.05 mV from
64.17 ± 0.44, 59.39 ± 0.99, 49.34 ± 0.99 and 42.62 ± 1.58 mV
for 0.025, 0.05, 0.1 and 0.2 wt% Tween 80, respectively. For NLCs
stabilized with Tween 80 at concentrations of 0.5 and 1.0 wt%, the
zeta-potential of the particles remains unchanged during storage
for 28 days (P > 0.05). Reduction in the particles’ zeta-potential dur-
ing storage was also observed for NLCs stabilized with sodium
dodecyl sulfate [12] and polyglycerol 6-distearate [14]. The
decrease of zeta-potential during storage is probably due to crys-
talline re-orientation of fat crystals which can result in changes of
the charges on the particle surface. Moreover, the crystal transfor-
mation to stable form of saturated fatty glycerides in fat might
occurred. Transformation of crystals changes the surface ratio of
differently charged crystal sides and consequently the measured
zeta potential changes [44].
3.1.2. Particle size and PDI
At 1 day of storage, the mean particle size (Z-average) of the
NLCs decreased significantly (P  0.05) with increasing surfactant
concentration (Fig. 2A). However, the polydispersity index (PDI)
was not significantly impacted (P > 0.05) by an increase in the sur-
factant concentration (Fig. 2B). This result was in agreement with
that of Kheradmandnia et al. [43] who reported that an increasing
concentration of surfactant from 0.5 to 1.5% resulted in a reduced
mean particle size of lipid nanoparticles from around 100 to
65 nm with a similar PDI of around 0.23–0.26. This decrease in par-
ticle size with increasing surfactant concentration can be attribu-
ted to the fact that there was more surfactant available to cover
the lipid-water interfaces formed during homogenization [45].
The ability of surfactant to rapidly adsorb on the particle surface
and to reduce the interfacial tension between the lipid and water
1085
0.05 0.1 0.2 0.5 1.0
7 10 14 21 28
Storage Time (Day)
phase stabilized the newly developed surfaces during homogeniza-
tion and the production of small particles [46–47].
The measurement of particle size changes against storage time
is one of the preferred methods used to assess the dispersion sta-
bility. Therefore, in the present study, the NLCs were stored at
25 °C over a period of 28 days and the lipid particle size and the
PDI were measured. During storage, the particle size (Fig. 2A) of
the NLCs stabilized with Tween 80 at all concentrations signifi-
cantly increased (P  0.05). The PDI (Fig. 2B) showed a slight
increase (P > 0.05) for the NLCs prepared using 0.025 and 0.05 wt
% Tween 80; however, the PDI of the NLCs stabilized with higher
concentrations of Tween 80 (0.1–1.0 wt%) significantly increased
during 28 days storage (P  0.05). The increase in particle size of
all samples may have been related to a number of possible mech-
anisms including flocculation, coalescence and Ostwald ripening.
Usually, flocculation and coalescence lead to a wide particle size
distribution with a high PDI [47–48]. From our results, all NLC dis-
persions had a PDI around 0.2–0.3, indicating a narrow particle size
distribution [10]. Therefore, the main unstable physicochemical
mechanism of the NLCs in the present work was possibly due to
Ostwald ripening more than flocculation and coalescence. Ostwald
ripening is the process whereby there is growth of larger particles
at the expense of smaller particles due to the mass transport of the
dispersed phase through the intervening continuous phase driven
by Laplace pressure [49]. However, if Ostwald ripening is the pre-
dominant mechanism for the increase in particle size then a linear
relationship between the average droplet radius (r3) and storage
time (t) should be observed according to the Lifshitz–Slesov and
Wagner (LSW) theory [45]. Therefore, in this study, the r3 data
were plotted as a function of time over a 28-day period for the
NLCs at various Tween 80 concentrations (Fig. 3). The results
showed that the NLCs at all surfactant concentrations had linear
plots of r3 versus t, except for the NLCs stabilized with 1.0 wt%
Tween 80. As can be seen (Fig. 3), considerable Oswald ripening
occurred in the NLC dispersions stabilized with 0.025–0.5 wt%
Tween 80 in which the ripening rate increased with the surfactant
concentration due to the smaller particle sizes. For NLCs stabilized
with 1.0 wt% Tween 80, the particle size tended to increase expo-
nentially with time, suggesting that particle flocculation is the pos-
sible mechanism for particle growth. This may have been to the
higher amount of surfactant having appreciable amounts of free
1086 P. Witayaudom, U. Klinkesorn / Journal of Colloid and Interface Science 505 (2017) 1082–1092

(A) 0.025 0.05 0.1 0.2 0.5 1.0

200
180
160
Particle size (nm) 140
120
100
80
60
40
20
0
1 3 7 10 14 21 28
Storage Time (Day)

(B) 0.025 0.05 0.1 0.2 0.5 1.0

0.5
Polydispersity index (PDI)

0.4

0.3

0.2

0.1

0.0
1 3 7 10 14 21 28
Storage Time (Day)
Fig. 2. Particle size (A) and PDI (B) of NLCs dispersions prepared from rambutan kernel fat at various surfactant concentrations (0.025, 0.05, 0.1, 0.2, 0.5 and 1.0 wt%) during
28 days storage at 25 °C.

0.025 0.05 0.1 0.2 0.5 1

3.1.3. Creaming stability index
100
The physical stability of the NLCs was observed during storage
by visual observing the cream and serum layers at the top and bot-
80
tom of the NLCs, respectively. The effects of Tween 80 on the phys-
r3 × 102 (nm3)

ical stability of NLCs are presented in Fig. 4. After preparation, all

60
NLC dispersion samples appeared homogeneous with no phase
separation. Phase separation was observed in the NLCs containing
40
0.025 and 0.05 wt% Tween 80 after storage for 3 days and after
14 days for NLCs containing 0.1 wt% Tween 80. The most stable
20 emulsions were observed in the NLCs stabilized with 0.2, 0.5 and
1.0 wt% Tween 80, which exhibited no visible phase separation
0 over 28 days of storage. The layer of cream and/or serum phase
0 5 10 15 20 25
increased with decreasing surfactant concentration leading to a
Storage Time × 105 (s) reduction in the creaming stability index (CSI). After 28 days stor-
3
age, the CSI was 94.04 ± 3.41, 95.48 ± 3.71, 97.75 ± 2.49 and
Fig. 3. The plots of r as a function of time over a 28 days period for the NLCs at
100 ± 0.00 for 0.025, 0.05, 0.1 and 0.2–1.0 wt% Tween 80, respec-
various Tween 80 concentrations (0.025, 0.05, 0.1, 0.2, 0.5 and 1.0 wt%) at 25 °C.
tively. From these results, NLCs prepared with 0.025–0.1 wt%
Tween 80 concentration were not stable because of insufficient
surfactant micelles which promoted particle flocculation through a surfactant molecules with incomplete cover of the particle
depletion mechanism [46,50]. surface [47,51].
 P. Witayaudom, U. Klinkesorn / Journal of Colloid and Interface Science 505 (2017) 1082–1092
0.025
100
Creaming stability index (CSI)
98
96
94
92
90
1 3
Fig. 4. Creaming stability of NLC dispersions prepared from rambutan kernel fat at various surfactant concentrations (0.025, 0.05, 0.1, 0.2, 0.5 and 1.0 wt%) during 28 days
storage at 25 °C.
3.2. Influence of solidification temperatures on particle characteristics
and stability of rambutan NLCs
This experiment studied the dependence of particle characteris-
tics and physical stability of NLCs prepared from RKF on solidifica-
tion temperature. The solidification temperature of 5 and 25 °C
were selected based on the solid fat content of RKF which is found
to be approximately 60 and 35%, respectively (data not shown).
Solidification of NLCs at these selected temperatures might provide
the different fat crystallization which may affect the characteristics
and stability of NLCs.
3.2.1. Microstructure
It has been reported that conventional oils and fats are mixtures
of different triacylglycerols (TAGs), with either high- or low-
melting TAGs. When oils or fats are cooled, the highest-melting
TAGs will crystallize as a solid in a liquid phase of the lower-
melting TAGs [52]. The crystal microstructure under a polarized
light microscope is shown in Fig. 5A and B of rambutan kernel
fat (RKF) which had pre-solidified at 5 and 25 °C, respectively,
and then had been further stored at 25 °C for 24 h. Microscopy of
the 5 °C-solidified RKF showed an even spatial distribution of small
crystals in a network (Fig. 5A), while the 25 °C-solidified RKF
showed a small number of larger fat crystals within the crystalline
structures (Fig. 5B). At the lower temperature, the degree of super-
cooling was observed to be higher than at the higher holding tem-
perature. The high degree of supercooling led to a faster nucleation
rate than the crystal growth rate, which caused the formation of a
large number of small, fat crystals. In contrast, a small number of
large fat crystals formed at a high solidified temperature due to
the nucleation rate being slower than the crystal growth rate
[53–54]. The crystal modification using the tempering process in
this bulk rambutan kernel fat merits further study as the informa-
tion may be useful for the production of specific fats for use in var-
ious products, e.g. fat for shortening, margarine or confectionery
products.
These results were consistent with the microstructure of NLCs
observed using the optical light microscope (Fig. 5C and D). At a
solidification temperature of 5 °C, the NLCs appeared to homoge-
nously dispersion with a spherical particle shape (Fig. 5C), whereas
there were aggregated, ellipsoid-shaped fat particles with the NLCs
solidified at 25 °C (Fig. 5D). It has been reported that when melted
fat droplets are initially cooled to a temperature below their
1087
0.05 0.1 0.2 0.5 1.0
7 10 14 21 28
Storage Time (Day)
melting point, they tend to crystallize in an a-polymorphic phase
and form spheroid, solid particles. However, these fat particles
may change to ellipsoid or non-spherical shapes during storage,
which leads to extensive particle aggregation through hydrophobic
patches on the particle surfaces. There is probably a polymorphic
transformation from an a to a b in the particles [54–55]. In the pre-
sent study, it was surprising that the fat particles of the NLCs pre-
solidified at 5 °C could maintain their initial spheroid shape during
storage. This indicated that polymorphic transformation could be
retarded. The possible reason to explain these results is the tem-
pering effect which similar conducts for cocoa butter in chocolate
[56]. Cooling of hot melted fat dispersion at 5 °C enhanced the
nucleation and induced the formation of nuclei. Holding at this
temperature for 1 h could promote crystal formation. Subse-
quently, when the temperature was increased to 25 °C followed
by storage for 24 h, this step could destroy the unstable crystals
and preserve the stable ones. The inhibition of polymorphic trans-
formation by controlling the storage temperature was also
reported by Awad et al. [57]. Other approaches have been reported,
including the addition of oil-soluble inhibitors, the addition of
excess emulsifier to cover the hydrophobic patches formed on
the particle surfaces, and the utilization of emulsifiers that increase
the repulsive interactions between particles [54]. For example, the
a-polymorphcan be preserved for a considerable period of time in
tristearin nanoparticles when the particles are stabilized with a
blend of saturated long-chain phospholipids and the bile salt
sodium glycocholate [55].
3.2.2. Differential scanning calorimetry
The DSC heating curves (from 25 to 80 °C at a rate of
10 °C min1) of RKF and NLCs are presented in Fig. 6. The signs of
phase transition that occurred suggested that RKF either in bulk
form or in lipid dispersions contained solid fat components at a
storage temperature of 25 °C. These results were consistent with
previous studies which reported that RKF at 25 °C had a solid fat
content around 45 to 60% [18,19]. When 25 °C-solidified RKF was
heated in the DSC, it showed three main endothermic peaks
(Fig. 6A). The first had a peak temperature of 27.6 ± 0.4 °C and an
endset temperature of 29.4 ± 0.6 °C. The second appeared as broad-
ened with a shoulder at a lower temperature with peak and endset
temperatures of 42.0 ± 1.2 and 45.3 ± 2.0 °C, respectively. Another
small peak at high temperature had peak and endset temperatures
of 51.6 ± 0.9 and 55.6 ± 1.6 °C, respectively. Similarly, RKF
1088 P. Witayaudom, U. Klinkesorn / Journal of Colloid and Interface Science 505 (2017) 1082–1092

Fig. 5. Polarized light microscopic images of RKF and optical light microscopic images of NLC pre-solidified at different temperatures. (A): RKF at 5 °C; (B): RKF at 25 °C; (C):
NLC at 5 °C and (D): NLC at 25 °C.

Fig. 6. DSC heating curves of RKF (A) and NLC (B) pre-solidified at 5 °C and 25 °C.

pre-solidified at 5 °C had three endothermic peaks. There was no
significant difference (P  0.05) for the low and high melting peaks,
but there were slight lower (P  0.05) of peak and endset temper-
atures for the intermediate melting peak of RKF pre-solidified at
5 °C compared to at 25 °C. The peak and endset temperatures,
respectively, presented at 27.6 ± 0.1 and 29.4 ± 0.1 °C, at
39.7 ± 0.5 and 43.0 ± 0.8 °C, and at 51.4 ± 0.1 and 54.3 ± 0.04 for
the low, intermediate and high melting regions, respectively.
The DSC melting curves of NLC samples are provided in Fig. 6B.
The NLCs solidified at 25 °C had only one main endothermic signal
at a peak temperature of 63.3 ± 0.9 °C and an endset temperature
of 65.5 ± 1.1 °C, while the 5 °C pre-solidified NLC showed two
endothermic peaks in low and high melting regions. The low melt-
ing region had a small signal with peak and endset temperatures of
31.4 ± 1.1 and 34.0 ± 1.3 °C, respectively. The high melting fraction
had a peak temperature of 50.2 ± 1.0 °C and an endset temperature
of 53.4 ± 0.8 °C, which were significant lower (P  0.05) than the
high melting component of NLCs prepared at 25 °C. Sonwai and
Ponprachanuvut [19] reported that RKF solidified at 25 °C for
24 h had diffraction peaks from X-ray diffractometry (XRD) at
3.85, 4.15, 4.24 and 4.55 Å which corresponded to b0 , pseudo-b0 ,
b0 and b structures, respectively. However, they suggested that if
RKF were solidified at low temperature, the diffraction peaks at
4.15 Å could corresponded to an a structure. Therefore, in the pre-
sent work the lower melting temperature of fat crystals in the NLCs
pre-solidified at 5 °C compared to at 25 °C was probably due to dif-
ferences in the fat crystal polymorphic structure. The DSC results
together with the microstructure (Fig. 5) suggested that the a
structure of the fat crystals in the NLCs pre-solidified at 5 °C might
form instead of b0 or b structures in the NLCs solidified at 25 °C.
However, to elucidate this phenomenon, the crystal polymorphic
form of rambutan fat in NLCs needs further analysis using XRD.
 P. Witayaudom, U. Klinkesorn / Journal of Colloid and Interface Science 505 (2017) 1082–1092
According to widespread belief, the crystallization of fat in its
bulk state occurs differently from its emulsified state. A shift of
the melting transition to a lower temperature was observed for
lipid dispersions compared to bulk lipid in previous works
[25,47,58–59], which was attributed to the small size of the NLCs.
In the present work, the NLCs prepared using pre-solidification at
5 °C (Fig. 6) had a completed melting point (endset temperature)
at around 53.4 °C which was slight lower that for bulk RKF under
the same conditions (
54.3 °C). However, an unexpected result
was observed for NLCs solidified at 25 °C, which had a higher com-
pleted melting temperature (
65.5 °C) than bulk fat (
55.6 °C).
Similar results have been reported for anhydrous milk
fat-in-water emulsion, which had a higher melting temperature
(39.3–39.6 °C) than bulk anhydrous milk fat (37.3–37.6 °C) [60].
The one possible reason to explain this result is that the small size
of the fat crystals in the NLCs caused a faster polymorphic transi-
tion than in the bulk phase. Therefore, the hard fat in the NLCs
seemed to transform more rapidly into the stable polymorph
which had the higher melting temperature [61].
3.2.3. Particle characteristics and physical stability
The zeta-potential of all RKF dispersion particles had values
around 36.3 to 38.5 mV, which did not change during storage
for 28 days (data not shown). After preparation and being kept
(A)
180
160
Particle size (nm)
140
120
100
1 7
(B)
Fig. 7. Particle size and PDI at 1, 7, 14, 21 and 28 day (A) and size distribution at 1 (B) and 28 day (C) of NLC dispersions prepared from rambutan kernel fat and pre-solidified
at 5 and 25 °C.
1089
for 1 day at 25 °C, the mean particle diameter (Z-average) and
PDI of NLCs pre-solidified at 5 °C (148.5 ± 6.5 nm and
0.06 ± 0.01, respectively) were not significant different (P > 0.05)
compared to NLC solidified at 25 °C (144.8 ± 2.4 nm and
0.06 ± 0.02, respectively). These results were not consistent with
the optical microscopic images described in Section 3.2.1 (Fig. 5)
probably due to the dilution effect which occurred in the sample
preparation step before the analysis of the particle size [62].
Dilution may have an effect on the original structure of disper-
sion samples as reported by Müller et al. [63]. The concentrated
lipid nanoparticle dispersion showed the dense packing of the
particles. After dilution of this lipid nanoparticle with water to
be a diluted dispersion, individual and freely diffusible lipid
nanoparticle are obtained. In the present work, the microstruc-
ture of diluted emulsion could not clearly observe due to the
limited resolution of optical light microscope. To observe the
microstructure of these diluted NLC samples, the other type of
microscopes with higher resolution, e.g. transmission electron
microscope (TEM) or cryo-scanning electron microscope
(Cryo-SEM) may be more suitable. The mean particle size and
PDI of fat particles in the NLCs increased significantly
(P  0.05) with increasing storage time (Fig. 7) regardless of
the solidification temperature as a result of the Ostwald ripening
as described in Section 3.1.2.
25°C (Particle size) 5°C (Particle size)
25°C (PDI) 5°C (PDI)
0.5
0.4
Polydispersity index (PDI)
0.3
0.2
0.1
0.0
14 21 28
Storage Time (Day)
(C)
1090 P. Witayaudom, U. Klinkesorn / Journal of Colloid and Interface Science 505 (2017) 1082–1092
25°C 5°C
Creaming stability index (CSI)
100
98
96
94
92
90
1 7 14 21
Storage Time (Day)
Fig. 8. Emulsion stability index of NLC dispersions prepared from rambutan kernel
fat and pre-solidified at different temperatures (5 and 25 °C) at 25 °C stored for 1, 7,
14, 21 and 28 day.
Physical stability of the NLCs was observed during storage time
of 28 days. All samples showed homogeneity and no phase separa-
tion after preparation and storage for less than 14 days (Fig. 8).
However, after 14 days storage, the NLC solidified at 25 °C had a thin
cream layer on the top. The creaming stability index (CSI) of these
NLCs decreased to 98.44 ± 0.21% after 28 days storage, in the NLCs
solidified at 5 °C were stable to creaming over 28 day storage
(CSI  100%). In fact, crystalline reorientation or polymorphic tran-
sition of lipids can result in changes on the particle surface leading
to an increased surface-to-volume ratio through the formation of
needle-shaped crystals [51] as seen for NLCs solidified at 25 °C.
There may not have been sufficient surfactant to cover the reformed
surface. NLC dispersion would be very susceptible to aggregation
due to hydrophobic interactions and prone to creaming.
3.3. Encapsulation efficiency and loading of b-carotene
b-Carotene is recognized as an important functional material in
foods. It shows the effective antioxidant activity which has the
promising preventive effects against some types of chronic disorders,
such as cancer and cardiovascular diseases. Moreover, b-carotene
has been implicated as a most active precursor of vitamin A which
is essential for human health [64,65]. However, b-carotene is very
sensitive to heat, light and oxygen and has very low bioavailability
[65,66]. There were reported that lipid-based encapsulation systems,
in particularly solid lipid nanoparticles (SLNs) and NLCs can be
used to improve the stability and bioavailability of b-carotene
[13,29,67–70]. Therefore, the ability of NLC prepared from rambutan
fat to entrap b-carotene was evaluated in the present work.
The mean particle diameter (150.08 ± 1.43 nm), PDI (0.10 ±
0.02) and zeta-potential (-39.99 ± 0.84 mV) of unloaded NLC were
not significant different (P > 0.05) compared to b-carotene loaded
NLC, which being 151.54 ± 1.96 nm, 0.10 ± 0.02 and -40.06 ±
1.23 mV for mean particle diameter, PDI and zeta-potential,
respectively (Table 1). The insignificant impact on particle
Table 1
Particle characteristics of unloaded and b-carotene loaded NLCs,
Particle characteristics Formulations
Unloaded NLC b-Carotene loaded NLC
Mean particle diameter (nm)ns 150.08 ± 1.43 151.54 ± 1.96
Polydispersity index (PDI)ns 0.10 ± 0.02 0.10 ± 0.02
Zeta-potential (mV)ns 39.99 ± 0.84 40.06 ± 1.23
Encapsulation efficiency (%) – 83.6 ± 0.7
b-Carotene loading (mg/g) – 0.836 ± 0.007
ns
Data in the same row are not significantly different (P > 0.05).
characteristics of NLC with incorporating of b-carotene was also
previously found in other works [29,69].
Developed rambutan fat NLCs had encapsulation efficiency and
b-carotene loading of 83.6 ± 0.7% and 0.836 ± 0.007 mg/g,
respectively. The values of EE and b-carotene loading was previous
found ranging from 65 to 75% [67,69] and 0.880 to 14.870 mg/g
[29,67–70], respectively. These variations occurring are probably
related to the different composition and concentration of lipid
phase, production method and concentration of applied
b-carotene. In future studies, it would be useful to investigate the
influence of different amounts of lipid phase and incorporated
b-carotene on EE and loading capacity of rambutan fat NLCs.
28
4. Conclusions
This study showed that rambutan kernel fat could be used as
the lipid core for preparing NLCs. The surfactant concentration
was one of the important factors that influenced the particle
characteristics and stability of the NLCs. Use of the proper concen-
tration is necessary to produce stable NLCs with desirable charac-
teristics. For rambutan fat NLCs prepared in this work, the suitable
concentration ratio of lipid to surfactant was approximately 1 to
0.2 which showed small monodispersed lipid particles and had a
high stability to creaming during storage. Another prominent fac-
tor that affected the fat crystal formation in the NLCs and impacted
the NLC storage stability was the solidification temperature. The
rambutan NLCs pre-solidified at 5 °C provided a small particle size
(
148 nm), good dispersion (PDI0.06) and high stability with no
phase separation (CSI  100%). These stable NLCs prepared from
rambutan kernel fat have potential use as an encapsulation and
delivery system for lipophilic bioactive components. Blending of
rambutan kernel fat with other solid lipids for using as lipid phase
may be useful for developing NLC systems with various character-
istics. However, further work needs to be done in this area.
Acknowledgements
This research is supported in part by the Graduate Program
Scholarship from the Graduate School, Kasetsart University, Thai-
land. We are grateful to Assoc. Prof. Dr. Parichat Hongsprabhas
for her great suggestions. In addition, the authors would like to
thank Malee Sampran Public Company Limited (Nakorn Pathom,
Thailand) for providing rambutan seeds in this work.
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