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Encapsulation of -carotene within ferritin

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 Food Chemistry 149 (2014) 307312

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

Encapsulation of b-carotene within ferritin nanocages greatly increases

its water-solubility and thermal stability
Lingli Chen a, Guangling Bai a, Rui Yang a, Jiachen Zang a, Ting Zhou b, Guanghua Zhao a,
a
CAU & ACC Joint-Laboratory of Space Food, College of Food Science and Nutritional Engineering, China Agricultural University,
Beijing Key Laboratory of Functional Food from Plant Resources, Beijing 100083, PR China
b
College of Life and Environmental Sciences, HangZhou Normal University, HangZhou 310036, Zhejiang Province, PR China

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

Article history: Carotenoids may play a number of potential health benets for human. However, their use in food indus-
Received 20 August 2013 try is limited mostly because of their poor water-solubility and low thermal stability. Ferritins are widely
Received in revised form 20 September 2013 distributed in nature with a shell-like structure which offers a great opportunity to improve the water-
Accepted 21 October 2013
solubility and thermal stability of the carotenoids by encapsulation. In this work, recombinant human H-
Available online 1 November 2013
chain ferritin (rHuHF) was prepared and used to encapsulate b-carotene, a typical compound among
carotenoids, by taking advantage of the reversible dissociation and reassembly characteristic of apoferri-
Keywords:
tin in different pH environments. Results from high-performance liquid chromatography (HPLC), UV/Vis
Ferritin
b-Carotene
spectroscopy and transmission electron microscope (TEM) indicated that b-carotene molecules were suc-
Thermal stability cessfully encapsulated within protein cages with a b-carotene/protein molar ratio of 12.41. Upon such
Encapsulation encapsulation, these b-carotene-containing apoferritin nanocomposites were water-soluble. Interest-
ingly, the thermal stability of the b-carotene encapsulated within apoferritin nanocages was markedly
improved as compared to free b-carotene. These new properties might be favourable to the utilisation
of b-carotene in food industry.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Carotenoids are a class of natural pigments mainly found in
fruits and vegetables that typically have 40-carbon molecules
and multiple conjugated double bonds (Qian, Decker, Xiao, &
McClements, 2012). In the specic case of carrots, the most impor-
tant nutrient is b-carotene, which is a lipid-soluble carotenoid.
b-Carotene consists of a polyene system with 11 conjugated double
bonds and a b-ring at each end of the chain (Knockaert et al., 2012)
(Fig. 1A). There is a considerable interest in b-carotene because not
only it is one of the most effective vitamin A precursors, but also it
has a number of other potential health benets, e.g., it has been
proposed as a cancer prevention agent, ulcer inhibitor, life exten-
der, and heart attack inhibitor (Colditz et al., 1985; Cutler, 1984;
Gerster, 1993; Kritchevsky, 1999; Mozsik, Javor, Toth, Zsoldos, &
Tigyi, 1984; Peto, Doll, Buckley, & Sporn, 1981; von Lintig, 2010).
Nevertheless, its utilisation is currently limited mostly because of
its poor water-solubility and chemical instability. b-Carotene is
lipophilic and thus it has to be dissolved in oils or dispersed in
other suitable matrices before they can be utilised in foods (Soares
& Craft, 1992). b-Carotene is also highly prone to chemical degra-
dation during food processing and storage due to various environ-
Corresponding author. Tel./fax: +86 10 62738737.
E-mail addresses: gzhao@cau.edu.cn, gzhao1000@163.com (G. Zhao).
mental effects, such as common thermal stress (Tai & Chen, 2000).
Therefore, improving the thermal stability of b-carotene is an
important challenge.
Nanotechnology has become one of the most promising and
interesting research elds during the past few decades. However,
in the eld of food science, this technology is still relatively new
and not fully explored (Tan & Nakajima, 2005). It has been reported
that the water-insoluble bioactive compounds such as carotenoids,
phytosterols and natural antioxidants can become water-soluble
with higher dissolution velocity and saturation solubility, if they
are in the form of nanodispersions with a particle size in the nano-
metre range (Tan & Nakajima, 2005; Chu, Ichikawa, Kanafusa, &
Nakajima, 2007). Certain techniques such as solvent displacement,
emulsicationevaporation, emulsicationdiffusion and precipi-
tation have been developed to prepare such nanodispersions (Horn
& Rieger, 2001). However, these methods result in different particle
sizes. Moreover, organic solvents are required in the above-men-
tioned preparation processes, which can lead to sample contami-
nation, and environmental pollution from solvent waste.
Fortunately, the widespread occurrence of ferritins with a specic
nanocage in nature offers a good opportunity to convert these
water-insoluble carotenoids and other biologically functional com-
pounds into corresponding water-soluble nanocomposites by
nanotechnology.

0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.10.115
308 L. Chen et al. / Food Chemistry 149 (2014) 307312
Fig. 1. Chemical structure of b-carotene (A) and ferritin viewed down a four-fold
axis (B) (Toussaint et al., 2007).
Ferritins are a class of iron storage proteins that are widely dis-
tributed in animals, plants and bacteria (Zhao, 2010). They play a
key role in iron metabolism, manifested as they serve both to
sequester excess iron taken up by the cell and to release stored iron
to meet cells metabolic needs during iron scarcity. Ferritins usu-
ally consist of 24 identical/different subunits that are arranged in
an octahedral (432) symmetry to form a hollow protein shell (out-
side diameter is about 1213 nm, inside diameter is about 78 nm)
capable of storing up to 4500 iron atoms as a ferric inorganic com-
plex. The inner cavity of ferritin and outside solvent is connected
with 3-fold and 4-fold narrow channels (Zhao, 2010) (Fig. 1B). Apo-
ferritin cages can reversibly disassemble and reassemble depend-
ing on pH (Kim et al., 2011). This advantage has been used to
prepare nanoparticles (Li, Viravaidya, & Mann, 2007). Briey, apo-
ferritin was disassociated into subunits at pH 2 or pH 11 but not
denatured, and then reconstituted when pH was adjusted to pH
7.0. During this process, small molecules in solution can be
trapped within its interior (Liu, Wang, Lea, & Lin, 2006). Thus, by
using this interesting property, apoferritin has been used widely
as a protein cage to entrap inorganic or organic components (Iwa-
hori, Yoshizawa, Muraoka, & Yamashita, 2005; Li et al., 2007). All
the obtained nanoparticles have the same and narrow size distri-
bution because the apoferritin molecules all have the same size.
To the best of our knowledge, so far, there has been no report on
the application of apoferritin in encapsulation of any lipophilic
component. In this study, we have successfully encapsulated the
lipophilic component, b-carotene, within apoferritin shell. Associ-
ated with such encapsulation is a increase in the thermal stability
of b-carotene. More importantly, these newly prepared b-carotene-
ferritin composites become fully water-soluble. These new proper-
ties of the b-carotene molecules within ferritin nanocages may be
favourable for their application in food industry.
2. Materials and methods
2.1. Chemicals
The all-trans-b-carotene (>98% purity) was purchased from He-
fei Bomei Biotechnology Co., Ltd. Methanol and cyclohexane were
HPLC grade. N0 , N0 -bis-methyleneacrylamide, sodium dodecyl sul-
fate (SDS), Tris (hydroxymethyl) aminomethane (Tris), TEMED, b-
Mercaptoethanol, and Coomassie Bright Blue R-250 were pur-
chased from SigmaAldrich Co. (Beijing, P. R. China). The others
were of reagent grade or purer.
2.2. Recombinant human H-chain ferritin (rHuHF) isolation and
purication
rHuHF was puried as previously described with some modi-
cation (Deng et al., 2010; Masuda, Goto, Yoshihara, & Mikami,
2010a). The Escherichia coli strain BL21 (DE3) which contained rHu-
HF expression plasmids was grown at 37 C on LB medium supple-
mented with 50.0 mg/L of ampicillin sodium. Protein expression
was induced with 200 lM IPTG (isopropyl b-D-1-thiogalactopyra-
noside) when the cell density reached an A600 of 0.6. The cells were
harvested by centrifugation after 6 h of induction and resuspended
in buffer A (10.0 mM TrisHCl pH 7.5, 1.0 mM EDTA, 0.15 M NaCl)
to the concentration of about 40 g (fresh weight bacteria)/L, fol-
lowed by disruption by sonication. The supernatant of the resulting
crude extract was collected by centrifugation and incubated at
60 C for 10 min to eliminate proteases and other heat-sensitive
proteins. The heat stable supernatants were further puried by
ammonium sulfate fractionation (60% saturated fraction). Next
the solution was centrifuged at 12,000g for 10 min, and then pre-
cipitate was dissolved in buffer B (20.0 mM TrisHCl pH 7.5,
1.0 mM EDTA), followed by dialysis (100 kD cutoff) against the
same buffer (buffer B) three times to remove the ammonium sul-
fate. The protein was further puried by ion exchange chromatog-
raphy (IEC) in a DEAE-Sepharose Fast Flow column previously
equilibrated in 20.0 mM TrisHCl buffer (pH 7.5). Fractions con-
taining rHuHF were pooled and concentrated, nally loaded to
Sephacryl S-300 gel ltration chromatography (GFC) equilibrated
in 20.0 mM TrisHCl buffer (pH 7.5, 0.15 M NaCl) before use. A
UVVis detector was used to monitor protein fraction at 280 nm,
and nally polyacrylamide gel electrophoresis was applied to
determine protein purity. Protein concentrations were determined
according to the Lowry method (Lowry, Rosebrough, Farr, & Ran-
dall, 1951) with bovine serum albumin (BSA) as standard.
2.3. Protein gel electrophoresis
Determination of the molecular weight of the rHuHF was
achieved with native PAGE by using a 10% polyacrylamide gel
run at 2 mA for 15 h at 4 C. The buffer system of the gel was
TrisHCl (0.025 M, pH 8.3). Gels were stained with coomassie blue
R-250. Electrophoresis of proteins under denaturing conditions
was done in 15% SDSpolyacrylamide gel (Laemmli, 1970). Tested
protein solution was added equal volume sample buffer containing
25% glycerol, 12.5% 0.5 M TrisHCl, pH 6.8, 2% SDS, 1% bromophe-
nol blue and 5% b-mercaptoethanol. After the solution was boiled
for 5 min, the supernatant was isolated by centrifugation at
10,000g for 10 min.
2.4. Preparation of rHuHF-encapsulated b-carotene
b-Carotene was dissolved in ethanol to make a nal concentra-
tion of 30.0 mg/L. rHuHF solution pH was raised slowly to pH 11.0
with NaOH (1.0 M). After 30 min, resulting solution was added
with b-carotene solution slowly. Upon adjusting the pH of resul-
tant solution to 7.5 with HCl (1.0 M), the nal concentration of
rHuHF was 0.5 lM with a rHuHF/b-carotene ratio of 140. It
needed to state that the reaction solution was stirred slowly during
all the previous steps. Finally the resulting solution was dialyzed
(100 kD cut-off) against 20.0 mM TrisHCl buffer (pH 7.5) three
times at 6.0 h intervals and then ultra-ltrated (Millipore with a
100 kD cutoff) to remove the free b-carotene. The rHuHF-encapsu-
lated b-carotene solution was stored at 4 C for later use.
 L. Chen et al. / Food Chemistry 149 (2014) 307312
2.5. TEM experiments
Liquid samples were diluted with 20.0 mM TrisHCl buffer (pH
7.5) to make the required concentration of sample prior to being
placed on carbon-coated copper grids, and excess solution was re-
moved with lter paper. Resulting samples were stained using 2%
uranyl acetate for 10 min. TEM were imaged at 30 kV through a
Hitachi S-5500 scanning electron microscope (Li, Jia, Yang, Deng,
& Zhao, 2012).
2.6. HPLC analysis
The b-carotene content was determined using a standard curve
created from a series of b-carotene solutions at different concen-
trations. pH of samples (500 lL) was risen to 11.0 to make
encapsulated b-carotene release from ferritin. After the released
b-carotene was extracted with cyclohexane (1.0 mL), HPLC was
used to determine its concentration with 455 nm as a detection
wavelength (Zhu, Margulis-Goshen, Magdassi, Talmon, & Macosko,
2010). Individual working standard solutions were freshly pre-
pared from individual stock standard solutions by diluting in cyclo-
hexane. Shimadzu SPD-20A Series HPLC system (Shimadzu, Kyoto,
Japan) consisted of two LC-10AT pump, a PuXiang 320 column
thermostat (Tianjin, P. R. China), and a SPD-10A UVVis detector.
A Shim-pack VP-ODS C18 column (5 lm, 150  4.6 mm; Shimadzu,
Kyoto, Japan) coupled with a Shim-pack VP-ODS guard column
(10  4.6 mm; Shimadzu, Kyoto, Japan) was employed to analyse
samples at 25 C. Methanol was used as mobile phase. The ow
rate was 1.0 mL/min; The 20 lL of a sample was injected.
2.7. Stability of rHuHF-encapsulated b-carotene
rHuHF-encapsulated b-carotene samples were heated at 37 C
in a dark tube. UVVis spectra of the samples were scanned at
1.5 h intervals over the range of 7.5 h by using a Cary 50 Bio ultra-
violet spectrometer (Varian Palo Alto, USA) at room temperature,
with the scanning wavelength from 320 nm to 600 nm. b-Carotene
solution diluted with 20.0 mM TrisHCl buffer (pH 7.5) was used as
a control sample.
2.8. Statistical analysis
All data analysis was performed using Origin 8.0 software
(Micro Cal Inc.) and the structural formula was processed by
ChemDraw 7.0. All experiments were carried out at least two
times.
Fig. 2. Native PAGE and reducing SDSPAGE analyses of recombinant human H-
chain ferritin (rHuHF). (A) Native PAGE; (B) SDSPAGE. Lane M represents protein
markers and their corresponding molecular masses.
309
3. Results and discussion
3.1. Preparation and characterization of rHuHF
Apo recombinant human H-chain ferritin (rHuHF) was prepared
as previously described (Masuda et al., 2010a). Characterization of
rHuHF was carried out by polyacrylamide gel electrophoresis
(PAGE), and results are shown in Fig. 2. Non-denaturing polyacryl-
amide gel electrophoresis (native PAGE) showed that apo rHuHF
was puried to homogeneity with an apparent molecular weight
(MW) of around 480 kDa at pH 7.5 (Fig. 2A). SDSPAGE showed
that apo rHuHF was composed of just only one subunit with MW
of 21.0 kDa (Fig. 2B), being in good agreement with previous nd-
ings (Masuda et al., 2010a). These results indicated that we had
puried rHuHF successfully, and it can be used for following
experiments.
3.2. Encapsulation of b-carotene by apoferritin
Apoferritin is ferritin devoid of the iron core, which consists of a
protein shell composed of 24 polypeptide subunits that interact to
form a cage structure of 12 nm diameter. The subunits in apoferri-
tin are very tightly packed to give a roughly spherical molecule
with rhombic dodecahedral geometry (Toussaint, Bertrand, Hue,
Crichton, & Declercq, 2007) (Fig. 1B). The apoferritin shell was still
kept intact upon heating at 80 C for 10 min, indicative of its rela-
tively high thermostability (Stefanini et al., 1996). A marked disso-
ciation occurs only at pH higher than 10.0 (Kim et al., 2011). The
dissociation and reassembly characteristic of apoferritin at differ-
ent pH values provides a facile route for preparing nanocomposites
loaded with carotenoids such as b-carotene. Scheme 1 illustrates
the process involved in the encapsulation of b-carotene into apo-
ferritin during its reassembly (Liu et al., 2006). In this case, the pro-
tein nanocage of apoferritin is dissociated into individual subunits
at pH 11.0, and the subunits reconstitute into a cage-like structure
at pH 7.5. b-Carotene molecules were encapsulated in the core dur-
ing the reassembly process. The encapsulated b-carotene will be
retained within the apoferritin shell because its size (28.4 in
length and 6.5 in width) is larger than that of the channel of
about 34 (Masuda, Goto, Yoshihara, & Mikami, 2010b). This
encapsulation approach provides an alternative route that is not
available by diffusing through the channels of apoferritin.
Although ferritin is widely distributed in plants, especially in le-
gume seeds, the content of ferritin is much lower as compared to
typical food proteins such as BSA and beta-lactoglobulin. For exam-
ple, the content of ferritin in dried legume seeds is usually about
0.3% (m/m) (Zhao, 2010). Therefore, recombinant ferritin was used
in this study based on two major reasons: (1) the objective of this
study was to elucidate whether or not apoferritin can be used as a
biomaterial to trap beta-carotene; (2) the yield of recombinant fer-
ritin during isolation and purition is much higher than that of
natural one, and this is very important for ferritin to be used in
food industry in the future. Additionally, it was shown that the
structure and other chemical properties of recombinant ferritin
are the same as the natural one.
3.3. Characterization of b-carotene-containing apoferritin
nanocomposites
To conrm whether b-carotene had been encapsulated within
the inner cavity of rHuHF, the b-carotene-loaded apoferritin nano-
particles were examined by transmission electron microscopy
(TEM). For a control sample (apoferritin alone), black uranium-con-
taining cores can be seen in the cavity due to uranium owed into
ferritin cavity via channels and then formed uranium-containing
310 L. Chen et al. / Food Chemistry 149 (2014) 307312

Scheme 1. Illustration of the process involved in the encapsulation of b-carotene into apoferritin during its reassembly. In this case, the protein nanocage of apoferritin is
dissociated into individual subunits at pH 11.0, and the subunits reconstitute into a cage-like structure at pH 7.5. About 12 molecules of b-carotene were encapsulated within
one ferritin nanocage after the reassembly process (Liu et al., 2006). The encapsulated b-carotene will be retained within the apoferritin shell because its size (0.6 nm) is
larger than that of the channel of about 0.4 nm.

Fig. 3. Transmission electron micrographs of apoferritin (A), and b-carotene-containing apoferritin nanocomposites (B). Samples were stained using 2% uranyl acetate. Bar in
TEM is 100 nm. UVVis spectra of apoferritin (C), and b-carotene-containing apoferritin nanocomposites (D, spectrum a) and free b-carotene (D, spectrum b).

cores after negatively stained with uranyl acetate (Fig. 3A), which
is in good agreement with previous observation (Meldrum, Wade,
Nimmo, Heywood, & Mann, 1991). If b-carotene molecules were
encapsulated within protein nanocages, we would expect that no
uranium-containing cores would form within the cavity, because
such encapsulation prevents the entrance of uranyl acetate. As ex-
pected, compare with TEM image of rHuHF alone (Fig. 3A), the dis-
crete electron-dense cores disappeared; instead, white
nanoparticles were observed with most of protein molecules
(Fig. 3B). This is because b-carotene occupied the inner cavity, pre-
venting the uranyl acetate entry into the inner cavity of ferritin
(Meldrum et al., 1991). Thus, these results demonstrated that
b-carotene molecules were successfully encapsulated within the
protein inner cavity. Hydrophobic or van der Waals interactions
are possibly involved in the interactions between b-carotene mol-
ecules and the inner surface of rHuHF.
Further UV/Vis measurements conrmed that the b-carotene
molecules were encapsulated within the apoferritin interior. A con-
trol experiment was carried out, which involved the direct mixing
of b-carotene (the molecular ratio of rHuHF to b-carotene was
1:40) with the apoferritin solution at pH 7.5 while stirring for 2 h.
There is no visible absorption in UV/Vis spectrum with the resulting
apoferritin solution (Fig. 3C) after a 24 h dialysis against TrisHCl
buffer solution at pH 7.5, suggesting that b-carotene molecules
physically absorbed to the external surface of the apoferritin nano-
cages were not able to stay intact after the rigid dialysis process. In
contrast, under the same experimental conditions except that
b-carotene molecules were mixed with apo ferritin at pH 11.0,
 L. Chen et al. / Food Chemistry 149 (2014) 307312
Fig. 4. A typical HPLC chromatogram of b-carotene extracted from b-carotene-
containing apoferritin nanocomposites with cyclohexane. Inset: a standard curve of
b-carotene in cyclohexane.
followed by adjusting the pH value of the solution to pH 7.5, and a
rigorous dialysis against TrisHCl buffer, ferritin solution shows
two maximal absorptions at about 455 nm and 480 nm (Fig. 3D,
spectrum a), which are characteristic of b-carotene. However it
has some blue shift at these two maximal absorptions when com-
pared with free b-carotene (Fig. 3D, spectrum b), that maybe be-
cause the inner cavity has a mass of carboxyl which is an
electron-withdrawing group to b-carotene. Indeed, the b-caro-
tene-containing ferritin solution has a yellow colour (Fig. 3D, Inset),
while the control sample lacks such colour (Fig. 3C, Inset). These re-
sults further conrmed the above conclusion that b-carotene mole-
cules were trapped within ferritin nanocages. After encapsulation,
the solubility of b-carotene was greatly changed; namely, a natu-
rally water-insoluble compound becomes a water-soluble one.
Such change might facilitate its application in food industry.
The above results demonstrated that b-carotene was encapsu-
lated in the inner cavity of rHuHF successfully. This raises an inter-
esting question of how many b-carotene molecules were locked in
each protein shell. To answer this question, HPLC was applied to de-
tect the amount of b-carotene in the protein shell with 455 nm as a
detection wavelength. A typical HPLC spectrum is shown in Fig. 4, in
which b-carotene appears at 31 min. The inset of Fig. 4 shows a
standard curve (concentration versus peak area) of b-carotene dis-
solved in cyclohexane. The calibration of peak area versus b-caro-
tene concentration was linear in the concentration range of
Fig. 5. Decay curves of b-carotene encapsulated within apo rHuHF (A) and free b-carotene (B) due to heating treatment at 37 C as a function of time. Conditions: [apo
rHuHF] = 1.04 lM, [b-carotene] = 12.9 lM in 20 mM TrisHCl, pH 7.5. Values are means SD (n = 3).
311
1.010.0 lM (R2 = 0.9974). After calculating the concentration of
rHuHF and b-carotene, a molar ratio of 12.4:1 between b-carotene
and apoferritin was obtained under the current conditions. The
concentration of b-carotene encapsulated within the apoferritin
shell was 12.9 lM, while the encapsulation efciency and loading
efciency were 32.25% and 1.37%, respectively. Recently, interlock-
ing of b-carotene in beta-lactoglobulin aggregates produced under
high pressure has been investigated by Mensi et al. (2013), and such
interlocking, results in a higher entrapment efciency. However,
ferritin nanoparticles containing b-carotene reported here were
monodisperse because b-carotene molecules were encapsulated
within each ferritin shell (Fig. 3B). The size of the b-carotene-
ferritin composites was about 12 nm in diameter as shown in
Fig. 3B, and consequently, they were water-soluble and transparent
as shown in Fig. 3D, inset. In contrast, the b-carotene and beta-
lactoglobulin particles aere polydisperse, and the size of these
particles in diameter varied from 6.5, 24, to 255 nm (Mensi et al.,
2013).
3.4. The thermal stability of the b-carotene-ferritin nanoparticles
As we know, b-carotene is prone to thermal degradation, result-
ing in a loss of its health-related properties. Therefore, to nd a
way to improve its thermal stability appears to be very important.
Encapsulation of b-carotene within protein cages presented in this
study might improve the thermal stability of this carotenoid. To
conrm this idea, the degradation kinetics of b-carotene encapsu-
lated within protein cages was investigated at 37 C with b-caro-
tene alone as a control sample, and results were shown in Fig. 5.
Generally, the degradation of both ferritin-encapsulated and free
b-carotene was fast at the rst 4.5 h and then became slow. After
we tried several kinetic models, the data of both ferritin-encapsu-
lated and free b-carotene are best tted to the rst-order reaction
model as shown in Eq. (1). The regression coefcients (R2) were ob-
tained as 0.9871 and 0.9813, respectively, indicating an excellent
correlation between b-carotene degradation and treatment time.
This is in good agreement with a previous report that b-carotene
degradation during thermal processing was also best described
by a rst-order reaction (Knockaert et al., 2012).
C C f C 0  C f expkt 1
where C represents the b-carotene content, Cf the b-carotene con-
tent in equilibrium state, C0 the initial b-carotene content, k the
reaction rate constant (h1), and t the reaction time (h). Curve-t-
ting gave tted values of the apparent degradation rst-order rate
312 L. Chen et al. / Food Chemistry 149 (2014) 307312
constants for ferritin-encapsulated b-carotene of 0.472 0.041 h1
and free b-carotene of 0.621 0.062 h1. It is evident that the
apparent rate constant of b-carotene within protein cages is
signicantly smaller than that of free b-carotene. The half-life
(t1/2) of b-carotene within protein cages under current conditions
is 1.468 h, a value markedly longer than that of free b-carotene
(1.112 h), demonstrating that the thermal stability of b-carotene
molecules within ferritin nanocages can be greatly improved.
It has been reported that b-carotene degradation is mainly
attributed to oxidation (Knockaert et al., 2012). The above ob-
served increase in the thermal stability of b-carotene molecules
within ferritin nanocages may stem from several reasons. First,
apoferritin would not be denatured when heated at 80 C for
10 min (Stefanini et al., 1996), so its spheroidal crust may act as
a physical barrier that isolated temperature, and prevented any
prooxidants in the aqueous phase from contacting the b-carotene
present within the cavity. Second, ferritin contains cysteine resi-
dues and other functional groups which may serve as effective
antioxidants, either by chelating transition metals or by acting as
free radical scavengers. Third, ferritin may form molecular com-
plexes with b-carotene through hydrophobic interactions or van
der Waals interactions which may help protect b-carotene mole-
cules inside ferritin from degradation (Qian et al., 2012). Detailed
mechanism is under investigation.
4. Conclusion
b-Carotene is one of the most important food additives because
of its various health-related properties. However, its application in
food industry is, at least partly, limited due to much low water-sol-
ubility and thermal stability. In this study, we have successfully
encapsulated b-carotene within ferritin nanocages by taking
advantages of the reversible dissociation and reassembly charac-
teristic of apoferritin at different pH values. Each ferritin nanocage
contains 12.4 molecules of b-carotene within its inner cavity in
average. Compared to lipid-soluble b-carotene, these b-carotene-
containing apoferritin nanocomposites are water-soluble. More
importantly, the thermal stability of the b-carotene encapsulated
within protein cages was pronouncedly improved with respect to
free b-carotene. All these new properties might facilitate its appli-
cation in food industry. This is the rst report that ferritin was used
to encapsulate the lipid-soluble compound. The procedure pre-
sented in this study is also suitable for encapsulation of other li-
pid-soluble compounds encapsulated within protein shell.
Acknowledgements
This work was supported by China High-Tech (863) Project
(2013AA102208-4), and Project of Education Department of Zhe-
jiang Province (Y201225407).
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