Preparation of 9 Z-β-Carotene and 9 Z-β-Carotene High-Loaded Nanostructured Lipid Carriers: Characterization and Storage Stability

Article abstract of DOI:10.1021/acs.jafc.0c02342

Cis (Z)-β-carotenes with 25.3% 9Z-β-carotene were prepared for nanostructured lipid carriers (NLCs). The optimal conditions for NLC preparation using an orthogonal experimental method were as follows: the total lipid concentration was 9% (w/v), the surfac

Full text of DOI:10.1021/acs.jafc.0c02342

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Article
Preparation of 9Z‑β-Carotene and 9Z‑β-Carotene High-Loaded
Nanostructured Lipid Carriers: Characterization and Storage
Stability
Cheng Yang, Hongxiao Yan, Xin Jiang, Huaneng Xu, Rong Tsao, and Lianfu Zhang*
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ABSTRACT: Cis (Z)-β-carotenes with 25.3% 9Z-β-carotene were prepared for nanostructured lipid carriers (NLCs). The optimal
conditions for NLC preparation using an orthogonal experimental method were as follows: the total lipid concentration was 9% (w/
v), the surfactant concentration was 1.4% (w/v), the solid to liquid lipid ratio was 3:1 (w/w), and the homogenization pressure was
set at 500 bar for three cycles. Under these conditions, the encapsulation efficiency (%) of the NLC was 95.64%, and the total β-
carotene in NLCs was 2.9 mg/mL, which was significantly higher than those reported by others. The proportion of total Z-β-
carotenes was as high as 53.3%, the particle size was 191 6.46 nm, and the polydispersity index was 0.2 0.03. Storage stability
results indicated that the β-carotene-loaded NLC stabilizes both 9Z-β-carotene and total β-carotene from leakage and degradation
during 21 days of storage at pH 3.5−7.5 at low temperatures (4 °C), especially for the more bioactive 9Z-β-carotene. The technique
with an improved ratio of 9Z-β-carotene, loading capacity, water solubility, and bioaccessibility of the β-carotene NLC provides an
effective strategy for β-carotene applications in functional foods or beverages and in nutraceutical preparations.
KEYWORDS: 9Z-β-carotene, nanostructured lipid carriers, loading capacity, stability, bioaccessibility
carotene in inhibiting atherogenesis in low-density lipoprotein
receptor knockout mice14 and in further inhibiting the
progression of established atherosclerosis in old male apoE-
deficient mice.15
INTRODUCTION
■
β-Carotene is one of the most important carotenoids that
occurs widely in vegetables (e.g., yellow pepper, carrots, and
pumpkins) and fruits (e.g., mangoes) with a deep orange-
yellow color.1,2 As a cyclic carotenoid containing 11
conjugated double bonds, β-carotene exhibits provitamin A
and antioxidant activities and many human health benefits
including reducing the risk of developing chronic diseases (e.g.,
cancer, cardiovascular diseases) and age-related degenerative
diseases.3,4 However, applications of β-carotene in food and
beverage systems and in pharmaceutical formulations are still
challenging because of its high hydrophobicity, chemical
instability, and high crystallinity, and consequently its low
bioavailability.5,6
Like most carotenoids, β-carotene exists predominantly in
all-trans configuration in unprocessed fruits and vegetables.7,8
However, higher ratios of 9Z-, 13Z-, and 15Z-β-carotenes
(25% of total β-carotene) were present in human tissues.9
Studies showed that trans-cis isomerization not only improved
the solubility in solvents of all-E-β-carotene, but also boosted
its physiological functions such as antioxidant and anticancer
activities.10,11 9Z-β-Carotene showed higher antioxidant
activity than all-E-β-carotene against external oxidants and
other reactive oxygen species in different tests.12 Even though
the antioxidant activities of β-carotene isomers were found
similar in a peroxyl radical (ROO•) scavenging test,3 according
to the electroaccepting and electrodonating powers, they
followed a decreasing order of 9,13′-dicis >13Z- ≳ 9Z- ≳ 15Z-
> 7Z- > all-E- > 11Z-β-carotene.13 Moreover, a diet containing
the alga Dunaliella bardawil rich in 9Z-β-carotene (50%) was
found to be more effective than that containing all-E-β-
The abovementioned observations indeed point to the fact
that cis isomers such as 9Z-β-carotene could be a more
effective functional ingredient than its all-E-β-carotene
counterpart. A technology that can increase the ratio of Z-β-
carotene will therefore lead to the development of β-carotene
products with enhanced health benefits. Several methods have
been developed to obtain Z-β-carotene; however, most of these
methods are still limited to low conversion efficiency and a
long reaction period or require complicated removal of the
catalyst after the reaction.16−18 On the other hand, the pigment
extract from D. bardawil is shown to contain 38% 9Z-β-
carotene, and the extract from D. salina contains 3.2% 9Z-β-
carotene and 10% all-E-β-carotene.19 However, the cultivation
of D. bardawil requires high salinity and intense illumination,
and the purification process also costs significantly, making it
uneconomical to produce from this natural source. An efficient
method to prepare 9Z-β-carotene with a high ratio is necessary.
One of the weaknesses of the encapsulation technology for
carotenoids is the loading capacity (LC), which is strongly
Received: April 13, 2020
Revised: August 11, 2020
Accepted: October 28, 2020
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A

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by high-pressure liquid chromatography (HPLC) at 450 nm. The
method has been previously studied and confirmed to be repeatable.30
The isomerization products were reproducible, and the ratio of Z-
isomers was stable during the reaction.29 All experiments were
performed in triplicate.
affected by the solubility of carotenoids in the oil phase; thus
the quantity of the oil phase is a key determinant factor.20 It
has been reported that lipid nanoparticles improve drug
absorption and bioavailability because of their nanosize
diameter and enhancing effect of lipids.21,22 Nanostructured
lipid carriers (NLCs) have attracted considerable attention as
an effective delivery system with sustained-release effects and
capability for industrial-scale production.23 The NLC system is
generally from a mixture of solid and liquid lipids and is
normally first heated until melted down and then cooled to
form a large amount of imperfect crystals and amorphous lipid
cores and incorporate more drugs.24 Schjoerring-Thyssen et al.
reported solid lipid nanoparticles (SLNs) loaded with a high
concentration of all-E-β-carotene (37.5%, w/w in the lipid
phase) with Z-isomers; however, they used a concentration of
surfactant as high as 10% of the SLN system and a temperature
as high as 165 °C, which are not good for the encapsulation
and stability of β-carotene inside the SLN product.25
Moreover, a surfactant with high concentration was used in
most studies. Tween 80 was used at 3% (w/v) in the NLC
containing β-carotene26 and lycopene,27 and at as high as 10−
40% (w/v) in a milk-fat-based NLC for β-carotene.28 On the
other side, Ono et al. developed an NLC containing Z-β-
carotene, while the major Z-isomer was a 13Z-isomer, a less
stable cis-isomer than 9Z-β-carotene. In this system, the β-
carotene loaded was less than 1 mg/mL, and the NLC was less
stable than the all-E-isomer.6
The objectives of the present study were therefore to
establish an effective method to produce 9Z-β-carotene and to
develop an NLC system using β-carotene with a high ratio of
9Z-isomer to improve the water-solubility, functionality,
stability, and bioavailability of a β-carotene-containing product
with a smaller amount of surfactant. The physicochemical
properties, that is, the LC, morphology, particle size, and
polydispersity index (PDI), of the 9Z-β-carotene high-loaded
NLC were determined. In addition, the effects of temperature
and pH on the retention and stability of both β-carotene
isomers and the NLC were also investigated during storage.
The pH and the homogeneous pressure were chosen according
to the processing conditions in the beverage industry. Results
of this study will provide fundamental information on the
production, stability of Z-β-carotene, and application of Z-β-
carotene loaded-NLCs in functional foods and/or pharma-
ceutical products.
Analysis and Identification of β-Carotene Isomers. β-
Carotene isomers in the isomerized product and β-carotene-loaded
NLCs were separated and analyzed using a polymeric C30 column
(YMC Carotenoids, 250 mm × 4.6 mm, 2.6 μm, Phenomenex Inc.,
Torrance, CA, USA) according to a previous report with slight
modifications.31 The column temperature was set at 25 °C. The
detection wavelength was 450 nm, the injection volume was 20 μL,
and the flowing rate was 1.0 mL/min for a total run time of 30 min.
The binary mobile phase consisted of A: 25% methanol mixed with
75% acetonitrile (v/v) and B: 100% MTBE. The solvent gradient was
as follows: 0−20 min, 100−50% A; 20−30 min, 50% A. Peaks were
detected at 450 nm. The DAD scan range was 250 to 600 nm.
Different β-carotene isomers were identified through matching
retention times and UV/vis spectral data with those of the standard
and data reported in the literature and further identified using a high-
pressure liquid chromatography-mass spectrometry (HPLC-MS)
system.32−34 Quantification was performed using a calibration curve
of the all-E-β-carotene standard. Concentrations of Z-β-carotenes
were expressed in all-E-β-carotene equivalents.
A Waters HPLC system (Alliance 2695) coupled with an Esquire
6000 ion-trap mass spectrometer (Bruker-Daltronics, Bremen,
Germany) was used to perform the HPLC-MS analysis. The
optimized APCI conditions were as follows: APCI, normal mode;
spray voltage, 4.0 kV; nebulizer, 45 psi; desolvation temperature, 300
°C; vaporizer temperature, 400 °C; flow rate of desolvation gas, 5.0
L/min; and scanning range, m/z 310 to 650.
Optimization of the Preparation of the 9Z-β-Carotene
High-Loaded NLC. The 9Z-β-carotene high-loaded NLC was
prepared by high-pressure homogenization without organic solvents
according to previous reports with modifications.27,35 Briefly, β-
carotene with a high ratio of 9Z-isomer (5%, w/w total lipids) was
added to the mixture of GMS (melting point: ∼81 °C) and MCTs at
85 °C under the protection of nitrogen. An aqueous phase (100 mL)
containing Tween 80 was also preheated at 85 °C and mixed with the
above lipid phase, stirred for 1 min, and then sheared by high-shear
homogenization (IKA Instruments, Germany) at 16000 rpm for 2
min. After pre-emulsification, the mixture was subjected to high-
pressure homogenization (ATS Instruments, Canada) under different
conditions. After that, the dispersion was cooled down in an ice bath
for 20 min to obtain a 9Z-β-carotene high-loaded NLC. The samples
were made freshly and kept at 4 °C in a refrigerator before analysis.
A systematic investigation into the optimization and effects of the
various factors including lipid concentration, percentage of the
surfactant, ratio of solid to liquid lipids, and homogeneous conditions
on the particle size, PDI, and encapsulation efficiency (EE) of the 9Z-
β-carotene high-loaded NLC was performed. The orthogonal
experimental design was applied for the optimization of NLC
preparation (Table S1). The EE of the NLC was set as the evaluation
index. A brief description of the experimental design is as follows:
Effect of Total Lipid Concentration. Five lipid concentrations
(2.5%, 5.0%, 7.5%, 10.0%, and 12.5%, w/v) were tested under the
following fixed conditions: homogeneous pressure of 500 bar for three
cycles; 1.2% (w/v) Tween 80 as the aqueous phase; and a solid−
liquid lipid ratio at 3:1 (w/w). The particle size and PDI of each
sample were monitored.
MATERIALS AND METHODS
■
Materials. All-E-β-carotene (HPLC >96%) was purchased from
Sigma-Aldrich (Shanghai, China). Glyceryl monostearate (GMS),
Tween 80, citric acid, iodine, sodium phosphate monobasic, and
sodium phosphate dibasic were purchased from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China). Medium-chain triglycerides
(MCTs) were obtained from Shanghai General Pharmaceutical Co.,
Ltd. (Shanghai, China). HPLC-grade solvents, including acetonitrile,
methanol, and methyl tert-butyl ether (MTBE), were purchased from
TEDIA High Purity Solvents (Ohio, USA). Distilled and deionized
water was used for the preparation of solutions. All the other
chemicals were of analytical grade.
Effect of Surfactant Concentration. The nonionic surfactant
Tween 80 with a hydrophilic−lipophilic balance number of 15 was
suitable to stabilize the O/W solutions and had been widely used in
NLC and beverage products.36 The critical micelle concentration of
Tween 80 was 13−15 mg/L, which was good for it to stabilize the
emulsion in lower concentration. The surfactant concentrations of
Tween 80 were therefore selected as 0.4, 0.8, 1.2, 1.6, and 2.0% (w/v)
according to reported studies.26,27,35 These emulsions were analyzed
for the particle size, PDI, and EE under the same pressure and
Rapid Conversion of all-E-β-Carotene to the 9Z-Isomer. The
isomerization was performed according to a previous report with
slight modifications.29 All-E-β-carotene was dissolved in ethyl acetate
(1 mg/mL) and heated at 50 °C for 2 h, or heating at 50 °C for 2 h
with an I-TiO2 catalyst or I2 (5%, I2/β-carotene, w/w). The reaction
with I2 was terminated by adding Na2S2O3 solution (1 mL, 1 mol/L)
to each vial to wash out the I2 by vortexing. After reaction, samples
were centrifuged, filtered through a 0.22-μm membrane, and analyzed
B
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C0 − C
C0
number of cycles as stated above. The total lipid concentration was
10% (w/v), and the ratio of solid−liquid lipids was set at 3:1 (w/w).
Ratio of Solid to Liquid Lipids. Different ratios of solid to liquid
lipids (1:1, 2:1, 3:1, 4:1, and 5:1, w/w) were selected to investigate
their effects on the particle size, PDI, and EE under the same pressure
and number of cycles as above. Tween 80 was 1.2% (w/v), and the
total lipid concentration was 10% (w/w).
A = Degradation I (%) =
× 100
(4)
where c0 and c (m/v) are the concentrations of total β-carotene before
and after the isomerization reaction, respectively.
B. Percent degradation of total β-carotene during pre-emulsifica-
tion:
Effects of Homogeneous Pressure. The concentration of the
surfactant was set at 1.2% (w/v). Other conditions were the same as
those described in the “Effect of surfactant concentration” section.
The homogeneous pressure was set at 200, 300, 400, 500, and 600 bar
each for three cycles. The particle size and PDI of the NLC were
investigated.
m0 − m
m0
B = Degradation II (%) =
× 100
(5)
where m0 and m (g) separately represent the amount of total β-
carotene before and after the mixing and shearing process during the
preparation of the NLC.
Characterization of the 9Z-β-Carotene High-Loaded NLC.
Particle Size and PDI Measurements. The particle size and PDI of
the NLC were determined using a Zetasizer Nano-ZS90 (Malvern
Instruments, Worcester, UK). The appearances of the 9Z-β-carotene
high-loaded NLC were compared at 1, 10, 100, and 1000 times
dilutions. The original preparation was diluted by 1000 times with
deionized water for the measurement of the particle size and PDI.
Intensity distribution was analyzed to evaluate the grain size. Three
separate measurements were taken for PDI and particle diameter data
collection.
C. Percent degradation of total β-carotene during homogenization:
M0 − M
M0
C = Degradation III (%) =
× 100
(6)
where M0 and M (g) represent the amount of total β-carotene before
and after homogenization, respectively.
The overall percent degradation of total β-carotene during the
entire preparation process of the 9Z-β-carotene high-loaded NLC was
calculated based on the following equation:
Transmission Electron Microscopy (TEM). The morphological
characteristics of the 9Z-β-carotene high-loaded NLC was determined
by TEM. Deionized water was added to dilute the NLC by 1000
times. One drop of the diluted sample was placed on carbon-coated
copper grids and dried at room temperature.37 The sample was dyed
using phosphotungstic acid and observed using a FEI Tecnai G2 F20
S-TWIN TEM at 200 kV (FEI Company, Hillsboro, USA).
Fourier Transform Infrared (FTIR) Spectroscopy. All-E-β-carotene
powder, the freeze-dried 9Z-β-carotene high-loaded NLC, and a
blank-NLC were scanned using a Fourier transform infrared (FTIR)
spectrophotometer (Nicolet Instruments, USA), in a frequency range
between 4000 and 400 cm−1 at 2 cm−1 measuring resolution by the
KBr pellet method.35
Total β-Carotene Loading in the NLC. The free amount of total β-
carotene in the NLC was analyzed following a reported method with
slight modifications.23,35 A mixture of 100 μL of the 9Z-β-carotene
high-loaded NLC and 3 mL hexane was vortexed for 1 min at room
temperature and centrifuged at 2500 × g for 2 min. The upper organic
phase was collected, and the aqueous phase was extracted two times
more. The organic phase was pooled, filtered using a 0.22-μm
membrane, and subjected to HPLC analysis as described above. The
total amount of β-carotene in the NLC was evaluated in the same way
as described above with ethyl acetate as the solvent.
Degradation (%) = 1 − (1 − A) × (1 − B) × (1 − C)
(7)
Storage Test of β-Carotene Isomers and the 9Z-β-Carotene-
Loaded NLC. The particle size, PDI, and retention of 9Z-β-carotene
and total β-carotene of the NLC were assessed during storage under
different temperature and pH conditions.
Effects of Temperature. The freshly made NLC was filled with
nitrogen gas and stored in the dark for 21 days, at 4 °C, 25 °C, and 37
°C. The original isomerized β-carotene product with a high ratio of
9Z-isomer was used as the negative control (NC). All experiments
were performed in triplicate.
Effects of pH. The stability of the 9Z-β-carotene high-loaded NLC
was examined at pH 3.5, 4.5, 5.5, 6.5, and 7.5, a pH range of most
food systems, in disodium hydrogen phosphate-citric acid buffer
solutions. A total amount of the 1 mL NLC dispersion was mixed with
2 mL of the buffer solution, filled with nitrogen, and then stored in a
refrigerator at 4 °C for 21 days. The retention percentages of total β-
carotene and 9Z-β-carotene were analyzed by HPLC in different time
intervals during storage. All experiments were performed in triplicate.
Morphology of the NLC after Storage. The morphology of the
9Z-β-carotene high-loaded NLC was examined at the end of the
storage test, using an inverted biological microscope at 1000×
magnification (ZEISS Axio Vert A1, Carl Zeiss Microscopy,
Germany).
The retention percentage (RP, %) of β-carotene isomers, EE (%),
and LC (%) were calculated according to the following equations:
Statistical Analysis. All experiments were performed in triplicate,
and the data were presented as mean value
standard deviation
(mean SD). One-way analysis of variance followed by Duncan’s
total amount of β‐carotene − free β‐carotene
EE(%) =
× 100
(1)
multiple range test was used to analyze data. Differences were
considered significant at p < 0.05. Statistical analyses were performed
using SPSS (Version 18.0, Chicago, IL, USA).
total amount of β‐carotene
incorporated amount of β‐carotene
RESULTS AND DISCUSSION
■
LC(%) =
× 100
total of the used lipid
(2)
Preparation of Z-β-Carotene. Refluxing all-E-β-carotene
with an iodine-doped TiO2 catalyst in ethyl acetate for 2 h
significantly enhanced the conversion rate, resulting in 25.3%
9Z-β-carotene and 47.3% of total Z-β-carotene after the
reaction (Table 1A), significantly higher than 0.39% 9Z-β-
carotene by heating in ethyl acetate at 50 °C for 2 h. The UV/
RP(%)
amount of encapsulated β‐carotene isomer after storage
initial amount of encapsulated β‐carotene isomer
=
(3)
× 100
Table 1. Ratios of β-Carotene Isomers in Isomerization
Degradation of β-Carotene. Degradation of total β-carotene can
be affected by the conditions of 9Z-isomer preparation, the process of
shearing during pre-emulsification, and homogenization during the
preparation of the 9Z-β-carotene high-loaded NLC.
A. Percent degradation of total β-carotene during the isomerization
of all-E-β-carotene:
Product (A) and NLC Suspension (B)
ratio (%) 15Z
13Z
9Z,13Z All-E
9Z
total Z-β-carotene
A
B
3.0
3.7
14.0
17.2
5.0
5.4
52.6
46.7
25.3
27.0
47.3
53.3
C
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Figure 1. Influence of total lipid concentration (A), surfactant concentration (B), the ratio of solid to liquid lipids (C), and homogenization
pressure (D) on the particle size and the PDI of 9Z-β-carotene high-loaded NLC. Data were means SD of three replicated treatments. Different
letters indicated a significant (p < 0.05) difference for the particle size (in black) or PDI (in blue).
vis spectrum and Q-ratio were used to identify the isomerized
products.38 HPLC-MS was applied to further identify the β-
carotene isomers.39 The protonated molecular ion of each β-
carotene isomer was 537 [M + H]+.39 The percentage of the Z-
β-carotene isomers in the mixture was in the following order:
9Z > 13Z > 9Z, 13Z > 15Z-β-carotene as shown in Table 1A.
This isomerization product had the same total content of Z-β-
carotenes as that from D. bardawil extract and a higher ratio of
9Z-β-carotene than that from D. salina extract.19
I2 alone had a similar catalytic effect to I-TiO2 on both the
reaction rate and the isomer composition at equilibrium (data
not shown); however, because of the lack of reusability of
iodine and ease in separation and recycling of iodine-doped
TiO2, the latter was selected as the catalyst for the laboratory
process and potentially for industrial production of the Z-β-
carotene isomers. The percent degradation of total β-carotene
after isomerization (degradation I) was 12.6%, which was
similar to that of astaxanthin after isomerization.29,39 The
catalyst I-TiO2 could be reused five times, and the composition
of the isomerization products was consistent after each
reaction under the same conditions. This method has been
validated and confirmed to be reproducible.30
Optimization of 9Z-β-Carotene High-Loaded NLC
Preparation. Total Lipid Concentration. Figure 1A shows
that as the total lipid concentration increases, the particle size
of the NLC also increases, to above 300 nm at the highest
concentration of 12.5% used in the study. This is similar to
reports by others.37 Increased lipid concentration promotes
collision and aggregation among the molecules that ultimately
results in a larger particle size.40 Meanwhile, the PDI of the
NLC decreased at first and then increased with the increasing
lipid concentration. The PDI was the lowest (0.2 0.03) at an
intermediate lipid concentration of 7.5%, at which the average
particle size was 194 5.43 nm.
Concentration of the Surfactant. The particle size of the
NLC gradually decreased as the concentration of surfactant
increased (Figure 1B); however, the decrease was only
significant at concentrations lower than 1.2%. The changes
of the particle size were not significant at higher surfactant
concentrations (p > 0.05). A similar phenomenon was reported
by others. For example, the particle size of the ifosfamide-
loaded NLC showed a negative correlation with the increase in
the surfactant from 0.25 to 1%.41 It was also found that once
the surfactant reached certain concentrations, such as 1.2% for
Tween 80 in this study, the surfactant concentration could no
longer affect the particle size.36 The PDI showed a similar
trend to that of the particle size. The EE also increased
gradually with increased surfactant concentration (Figure
S1A), but stopped at 94.23% when the latter reached 1.2%.
Elevated surfactant concentration increases the surfactant
adsorbed on the interface and improves the strength of the
oil−water interface film formed on the surface of the NLC,
resulting in a beneficial inhibition of the precipitation of β-
carotene and a better dispersion of the particles in water.37 At
surfactant concentrations higher than 1.2%, the strength of the
oil−water interface film on the surface of the NLC reached the
maximum, which explains why there was no significant further
improvement in EE (Figure S1A).
Ratio of Solid to Liquid Lipids. As the ratio of solid lipid to
liquid lipid increased, the particle size and PDI of the NLC
increased gradually (Figure 1C). When the ratio reached 3:1,
the particle size and PDI reached 188 5.79 nm and 0.2
0.03, respectively. Once the ratio exceeded 3:1, the particle size
and PDI both increased significantly (p < 0.05). A PDI of 0.5
means that the dispersion of NLC was unstable. A further
increase in solid lipid to liquid lipid ratio was therefore not
beneficial in lowering the particle size or enhancing the
stability of the system.
D
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When the ratio of solid lipid to liquid lipid was 3:1, the EE
reached the highest (94%, Figure S1B). In fact, any higher or
lower ratios led to EE less than 90%, suggesting that a balance
between the content of solid and liquid lipids is important for
obtaining the highest EE in the NLC. Imbalanced ratios are
not beneficial for the maximum formation of lipid carriers and
can have imperfect crystals. A balanced ratio allows for a large
amount of space to accommodate more bioactive substances
and the ability to avoid leaking.23
Effects of Homogeneous Pressure. As shown in Figure 1D,
the particle size of the NLC showed an inverse association with
the homogenization pressure between 200 bar and 600 bar,
similar to other reports.42 When the homogenization pressure
was at 500 bar, the particle size was 198 6.65 nm. However,
there was no significant change in the particle size beyond 500
bar. The PDI showed a similar trend to the particle size
(Figure 1D). When the homogenization pressure was 500 bar,
the PDI was 0.26 0.03, and the particles of the NLC were
dispersed uniformly.
As excessive pressure may increase the particle size. The
higher the pressure was, the more intense was the collision
between particles, resulting in agglomeration of the particles,
increased particle size, and poor stability of the carrier
system.43 The temperature of the solution could also increase
during the increment of the pressure, which affects the
emulsification effect of the surfactant and intensifies the
degradation of β-carotene. For these reasons, the optimal
homogenization pressure of the present study was set at 500
bar. Additionally, the homogenization time was also tested for
the optimization of NLC preparation (data not shown).
Homogenization for three cycles was set for the optimal
particle size and PDI of the NLC.
Based on the preliminary single-factor tests, the optimized
preparation of the 9Z-β-carotene high-loaded NLC was carried
out at total lipid concentrations of 6, 7.5, and 9% (w/v), at
surfactant concentrations of 1, 1.2, and 1.4% (w/v), and at the
ratios of solid to liquid lipids of 5:2, 3:1, and 7:2, respectively
(Table S1). The results of the orthogonal experiment showed
that the influence of the three factors on the EE of the NLC
followed the order of: the solid lipid to liquid lipid ratio > the
surfactant concentration > total lipid concentration (Table
S2). As shown in Table S2, the final conditions for the optimal
NLC preparation were as follows: the total lipid concentration
was 9% (w/v), the surfactant concentration was 1.4% (w/v),
the solid to liquid lipid ratio was 3:1 (w/w), and the
homogenization pressure was set at 500 bar for three cycles.
Under these conditions, the EE of the NLC was 95.64%, which
was higher than the EE (91.2%) of the β-carotene-loaded NLC
based on palm stearin and palm olein in a single factor test
reported by others.44 Moreover, the overall percent degrada-
tion of total β-carotene was 35.2% after the preparation of the
NLC as calculated using equation 7, and the temperature and
homogeneous pressure during the production could be the
major reasons for the degradation of total β-carotene.37 The
NLC can be scaled up in food production systems by
autoclaving, spray-drying, and lyophilizing because the particle
diameter is below 200 nm.20,23
Figure 2. HPLC chromatogram of β-carotene isomers in the 9Z-β-
carotene high-loaded NLC. CA was short for carotene.
carotene high-loaded NLC, and that of 9Z- and total Z-isomers
increased from 25.31% and 47.41% in the isomerized product
to 27.02% and 53.26% in the NLC, respectively (Table 1). The
result suggests that the homogenization treatment of the NLC
increased the content of the desirable 9Z-β-carotene, 13Z-β-
carotene, and total Z-isomers during the processing.
Particle Size, PDI, and 9Z-β-Carotene Load of the NLC.
The 9Z-β-carotene high-loaded NLC had a good aqueous
solubility with a bright orange-yellow color and good color
stability at different dilutions (Figure 3A). Similar preparations
from milk fat were reported to be in yellowish color with thick
texture.28 The 9Z-β-carotene high-loaded NLC of the present
study contained 2.9 mg/mL of β-carotene, far much higher
than that of similar preparations with high oleic sunflower oil
(25 μg/mL) obtained by the solvent displacement method and
the reported β-carotene nanosuspension (∼1 mg/mL).6
The mean particle size and the PDI are key characteristics
for nanodispersions that determine the physical stability,
solubility, biological performance, release rate, and chemical
stability of a preparation.20,45 The particle size distribution of
the NLC is shown in Figure 3B. The 9Z-β-carotene high-
loaded NLC showed a uniform particle size distribution with
an average particle size of 191 6.46 nm and PDI of 0.2
0.03, indicating potential higher bioavailability (bioaccessi-
bility) than conventional emulsions (1−10 μm).46
Morphology of the 9Z-β-Carotene-Loaded NLC. TEM
reflects the apparent morphology and dispersion of β-carotene-
loaded particles. Particles of the 9Z-β-carotene high-loaded
NLC were found to be spherical and uniform in shape (Figure
3C), which was consistent with particle size distribution shown
in Figure 3B.
FTIR Analysis. The FTIR spectrum (Figure 4A) of all-E-β-
carotene powder revealed the absorption bands at 3027 cm−1
(C−H stretching vibration), 2950 cm−1 (asymmetric methyl
stretching vibration), 2916 cm−1 (C−H stretching vibration),
2860 cm−1 (symmetric methylene stretching vibration), 1560
cm−1 (aromatic ring skeletal stretching vibration), and 965
cm−1 (R1HCCHR2 wagging vibration).47 FTIR spectra of
the 9Z-β-carotene high-loaded NLC (Figure 4B) and the blank
NLC (Figure 4C) were almost the same, and the characteristic
peaks of β-carotene isomers were not present. The results
indicated that β-carotene isomers were well incorporated into
the NLC.
Characterization of the 9Z-β-Carotene High-Loaded
NLC. Isomeric Profiles of β-Carotene in the NLC. The
chromatogram and ratios of β-carotene isomers in the 9Z-β-
carotene high-loaded NLC are presented in Figure 2 and Table
1. The proportion of the all-trans-β-carotene isomer decreased
from 52.59% in the isomerized product to 46.74% in the 9Z-β-
Storage Test of the 9Z-β-Carotene High-Loaded NLC.
The particle size and PDI of NLC stored at 25 °C were close
to those stored at 4 °C during the 21 days storage period, and
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Figure 3. Characterization of the 9Z-β-carotene high-loaded NLC. A: Appearances of the NLC with different dilution multiples containing 2.9 mg/
mL (a), 0.29 mg/mL (b), 0.029 mg/mL (c), and 0.0029 mg/mL (d) of total β-carotene; B: Size distribution of the 9Z-β-carotene high-loaded
NLC; C: TEM of 9Z-β-carotene high-loaded NLC in μm and nm scales.
Figure 4. FTIR spectra of all-E-β-carotene powder (A), 9Z-β-carotene high-loaded NLC (B), and blank-NLC (C).
the NLC remained stable and uniform at both temperatures
(Figure 5A,B). The morphology of the NLC stored at 4 °C
showed that the particles were intact, and little agglomeration
was found after the storage (Figure S2). However, the particle
size and PDI of the system increased significantly at 37 °C,
from 168 4.24 nm to 594 5.37 nm and 0.2 0.02 to 0.6
0.01, respectively, and the system became less stable over the
same period (p < 0.05) (Figure 5A,B). This may be attributed
to the higher kinetic energy at 37 °C than the other two
temperatures tested for the carrier system. The high kinetic
energy accelerates the collision of the nanoparticles and
thereby increases the probability of agglomeration between
nanoparticles.48 These data suggest that the β-carotene-NLC
should be stored at lower temperatures such as 4 °C and 25
°C, which are common conditions for the storage of foods and
beverages.
21 days period (Figure 5C,D). Both 9Z-β-carotene and total β-
carotene in either the NLC or the isomerized powder were
most stable at 4 °C compared with those at other tested
temperatures (25 °C and 37 °C), suggesting again that the
storage temperature had a great influence on the stability and
degradation of compounds in the NLC.37,49 The slopes of the
retentions of 9Z-β-carotene and total β-carotene in the NLC
and the isomerized product among the treatments during
storage were determined (Table S3). Fitting results showed
that there were significant differences for the retention rates of
9Z-β-carotene stored in different temperatures (Table S3).
The original percentage of 9Z-β-carotene in the isomerized
product decreased to half in 54 days at 25 °C during the
storage by the fitting equation (Table S3); however, the
original amount of 9Z-β-carotene was reduced to half in 101
days under the same conditions with the protection of NLC,
which was almost twice that of 9Z-β-carotene in the isomerized
product. There were also significant differences of the
retention rates of total β-carotene stored at different temper-
atures in each system. However, there was no significant
difference between the retention rate of total β-carotene in the
NLC stored at 37 °C and that of total β-carotene in the
The retention of 9Z-β-carotene and total β-carotene in the
NLC was compared with that of the isomerized product under
different storage conditions. In general, the retention
percentages of 9Z-β-carotene and total β-carotene in the
NLC were higher than those of the nonformularized
isomerized β-carotene powder at all temperatures over the
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Figure 5. Effect of temperature on the particle size (A) and PDI (B) of the 9Z-β-CA high-loaded NLC and the retention of 9Z-β-CA (C) and total
β-CA (D) in the 9Z-β-CA high-loaded NLC and isomerized product (NC). CA was short for carotene. Each value represented the mean SD (n =
3). The degradation kinetics of β-carotene is shown in Table S3.
isomerized product stored at 25 °C. All the results indicated a
protective effect of the NLC. 9Z-β-Carotene could be
efficiently protected by this formulation at lower temperatures
during the tested storage period of the present study.
period (data not shown). There was no significant trend of the
PDI or particle size of the NLC stored from 3.5 to 7.5. The
overall result indicated that the NLC suspension was relatively
more stable under acidic conditions than in an alkaline
environment. This supports the use of the NLC product in
beverages, which are mostly kept under acidic conditions.
pH also showed little effects on the retention of β-carotene
isomers; however, the retention decreased over a period of 21
days at pH 3.5 but was relatively stable in a neutral
environment (pH 6.5 and pH 7.5) (Figure 7). The retention
The differences in retention between 9Z-β-carotene and
total β-carotene (Figure 5C,D) and the final balance among
different isomers may be attributed to the relatively higher
stability of 9Z-β-carotene than that of other cis-isomers13,50
and degradation and transformation of all-E-β-carotene to
13Z-, 15Z-, and the 9Z-isomers, among which the 13Z-isomer
is known to be relatively less stable.3 The retention of 9Z-β-
carotene was similar to what has been reported for lycopene.35
The effects of pH on the particle size and PDI of the NLC
are shown in Figure 6. In general, the PDI was lower than 0.33,
and the particle size distribution remained uniform around 300
nm after storage for 21 days at pH 3.5 − 7.5 (Figure 6). In fact,
these parameters did not vary significantly during the 21 days
Figure 7. Effects of pH on the retentions of 9Z-β-carotene (A) and
total β-carotene (B) in the 9Z-β-carotene high-loaded NLC during
the storage in the dark at 4 °C for 21 days. Each value represented the
mean SD (n = 3).
of 9Z-β-carotene decreased by 7.94% after 21 days of storage at
pH 6.5 and pH 7.5, whereas it was by 15.61% at pH 3.5
(Figure 7A). A similar trend was found for the total β-carotene.
At the end of the 21 days storage, percent retention of total β-
carotene was reduced by 20% under neutral conditions, but by
30.58% at pH 3.5 (Figure 7B). Mehrad et al. used the color
fading of β-carotene SLNs as an indicator for the chemical
degradation of β-carotene and also found that degradation was
faster at pH 3.5 than at neutral pH.51 A similar result was
Figure 6. Effects of pH on the particle size and PDI of the 9Z-β-
carotene high-loaded NLC after storage of 21 days. Data were means
SD of three replicated treatments. Different letters indicated a
significant (p < 0.05) difference for the particle size (in black) or PDI
(in blue).
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found in our previous study on the stability of astaxanthin
isomers that it was significantly affected by highly acidic
conditions, that is, pH 2.0 and 3.5.29 Our results suggest that
the 9Z-β-carotene high-loaded NLC would be stable when
used in common food systems. The stability of β-carotene
isomers under the tested pH may be due to the protection in
intact nanoparticles of the NLC (Figure 6).
Health, Beijing Technology and Business University, Beijing
100048, China; orcid.org/0000-0002-2109-8663
Hongxiao Yan − State Key Laboratory of Food Science and
Technology and School of Food Science and Technology,
Jiangnan University, Wuxi, Jiangsu 214122, China
Xin Jiang − State Key Laboratory of Food Science and
Technology and School of Food Science and Technology,
Jiangnan University, Wuxi, Jiangsu 214122, China; Beijing
Advanced Innovation Center for Food Nutrition and Human
Health, Beijing Technology and Business University, Beijing
100048, China
Huaneng Xu − State Key Laboratory of Food Science and
Technology and School of Food Science and Technology,
Jiangnan University, Wuxi, Jiangsu 214122, China;
orcid.org/0000-0002-1714-5105
Rong Tsao − Guelph Research and Development Centre,
Agriculture and Agri-Food Canada, Guelph, Ontario N1G
5C9, Canada; orcid.org/0000-0001-6537-1820
In this study, Z-β-carotenes with 25.3% 9Z-β-carotene were
prepared for the first time as an NLC. Using an orthogonal
experimental method, the following optimal conditions were
obtained for the NLC preparation: total lipid concentration,
9% (w/v); surfactant concentration, 1.4% (w/v); solid to
liquid lipid ratio, 3:1 (w/w); and homogenization pressure,
500 bar for three cycles. Under these conditions, the EE of the
NLC was 95.64%, and the total β-carotene in the NLC was 2.9
mg/mL which was significantly higher than that reported by
others. The proportion of total Z-β-carotenes was as high as
53.3%, the particle size was 191 6.46 nm, and the PDI was
0.2 0.03. The processing method also helped to maintain the
profiles of the β-carotene isomers loaded in the NLC without
causing any major conformational transformations. Storage
stability analysis indicated that the β-carotene-loaded NLC
stabilizes both 9Z-β-carotene and total β-carotene from leakage
and degradation during 21 days of storage at pH 3.5 − 7.5 at
low temperatures (4 °C), especially for the biologically more
interesting 9Z-β-carotene. The results on the improved ratio of
9Z-β-carotene, LC, water solubility, and bioaccessibility of the
β-carotene NLC provide an effective strategy for β-carotene
applications in functional foods or beverages and in
nutraceutical preparations.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c02342
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by grants from The Key Research
and Development Program of Jiangsu Province (BE2017374),
National Key Technology R&D Program for the 13th five-year
plan (2017 BAD33B05), the Natural Science Foundation of
Jiangsu Province (BK20190592), the Natural Sciences
Foundation of China (31901654), and the Jiangsu Province
“Collaborative Innovation Center for Food Safety and Quality
Control” industry development program.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c02342.
■
sı
REFERENCES
■
The orthogonal experimental design (Table S1), results
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different time intervals (Figure S2) (PDF)
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AUTHOR INFORMATION
Corresponding Author
■
Lianfu Zhang − State Key Laboratory of Food Science and
Technology, School of Food Science and Technology, and
Collaborative Innovation Center of Food Safety and Quality
Control in Jiangsu Province, Jiangnan University, Wuxi,
Jiangsu 214122, China; Beijing Advanced Innovation Center
for Food Nutrition and Human Health, Beijing Technology
and Business University, Beijing 100048, China;
Phone: +86-510-85917025; Email: lianfu@
jiangnan.edu.cn; Fax: +86-510-85917025
Authors
Cheng Yang − State Key Laboratory of Food Science and
Technology and School of Food Science and Technology,
Jiangnan University, Wuxi, Jiangsu 214122, China; Beijing
Advanced Innovation Center for Food Nutrition and Human
H
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