The leading role of the steric hindrance effect and dipole-dipole interaction in superlattice nanostructures formed via the assembly of discotic liquid crystals

Article abstract of DOI:10.1039/c8nj05405c

To reveal the relationships among molecular structures, self-assembly behavior and the organic electronic properties of discotic liquid crystals, 2-(neopentyloxy)-3,6,7,10,11-pentakis(pentyloxy)triphenylene (T5ON1) and 2-pivalate-3,6,7,10,11-penta-pentylo

Full text of DOI:10.1039/c8nj05405c

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One-step assembly of zein/caseinate/alginate
nanoparticles for encapsulation and improved
Cite this: DOI: 10.1039/c8fo01614c
bioaccessibility of propolis†
Hao Zhang,a Yuying Fu,
*
a,b Yujuan Xu,c Fuge Niu,a Zeya Li,a Chujie Ba,a Bing Jin,a
Guowen Chena and Xiaomeng Lia
The design of zein-based nanoparticles to encapsulate bioactive molecules has gained great attention in
recent years. However, the use of ethanol to dissolve zein presents flammability concerns and the scale-
up production of zein-based nanoparticles is also a concern. In our study, propolis loaded zein/caseinate/
alginate nanoparticles were fabricated using a facile one-step procedure: a well-blended solution was
prepared containing deprotonated propolis, soluble zein, dissociated sodium caseinate micelles (NaCas)
and alginate at alkaline pH, and then this alkaline solution was added to 0.1 M citrate buffer (pH 3.8) to
fabricate composite nanoparticles without using organic solvents and sophisticated equipment. During
acidification, the alginate molecules adsorbed on the zein/NaCas surfaces by electrostatic complexation,
which improved the stability towards aggregation of zein/NaCas nanoparticles under gastrointestinal (GI)
or acidic pH. The nanoparticles prepared under the optimized method (method 3 sample) were of spheri-
cal morphology with a particle size around 208 nm and a negative zeta potential around −27 mV. The
encapsulation efficiency (EE) and loading capacity (LC) of propolis reached 86.5% and 59.6 μg mg−1 by
zein/NaCas/alginate nanoparticles, respectively. These nanoparticles were shown to be stable towards
aggregation over a wide range of pH values (2–8) and salt concentrations (0–300 mM NaCl). Compared
to free propolis, the bioaccessibility of propolis encapsulated with nanoparticles was increased to 80%.
Our results showed a promising clean and scalability strategy to encapsulate hydrophobic nutraceuticals
for applications in foods, supplements, and pharmaceuticals.
Received 12th August 2018,
Accepted 22nd December 2018
DOI: 10.1039/c8fo01614c
rsc.li/food-function
lated into zein nanoparticles. For example, β-carotene,3,4 cur-
1. Introduction
cumin,5,6 and quercetagetin7 have been successfully encapsu-
Zein is one of the few alcohol-soluble biopolymers that has lated into zein–polysaccharide composite nanoparticles.
more than 50% hydrophobic amino acids.1 As a highly hydro-
Traditional methods to fabricate zein nanoparticles usually
phobic protein, zein has a unique brick-like shape in an take advantage of the solubility of zein in 55–95% (v/v) alcohol
aqueous environment, thus, overcoming the disadvantage of solutions or other organic solvents.8 Specifically, due to the
hydrophilic protein-based vehicles that may require the inherent non-polar nature of its 50% amino acid sequence,
inclusion of additional steps like cross-linking and hydro- zein can be assembled into nanoparticles by simply mixing a
phobic modifications to harden the nanoparticles for con- non-solvent with the primary solvent (aqueous ethanol or
trolled drug release.2 Thanks to zein’s promising properties of other organic solvents) with dissolved zein; the process is com-
biodegradability, biocompatibility, and slower digestibility, monly known as the anti-solvent precipitation method.9
many lipophilic compounds have been sparked to be encapsu- However, the use of ethanol and other organic solvents pre-
sents a flammability hazard in industrial production and is
also not preferred in health foods.10 Although the organic sol-
aSchool of Food Science and Biotechnology, Zhejiang Gongshang University,
Hangzhou, 310018, China. E-mail: webfu@126.com
bHangzhou College of Commerce, Zhejiang Gongshang University, Hangzhou,
311508, China
vents can be removed by rotary evaporation, this protocol adds
complexity and additional costs.11 Thus, methods by which
food nutrients can be encapsulated in the zein nanoparticles
without using an organic solvent while maintaining the stabi-
lity of zein in aqueous systems are the major challenges. It is
worth noting that zein is soluble at high alkaline pH from 11.3
to 12.7.11 Recently, a pH-driven method has been reported to
cCollege of Food Science and Technology, Nanjing Agricultural University, Nanjing
210095, China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8fo01614c
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prepare zein–sodium caseinate (NaCas) nanoparticles based cause the assembling of zein with NaCas and electrostatic
on pH transition (pH ≥ 11.5 to neutral conditions) to disas- complexation to occur between anionic alginate molecules and
semble and reassemble the protein molecules without using cationic zein/NaCas nanoparticles. On the other hand, propo-
organic solvents and specialized equipment.2,11,12 Briefly, the lis becomes more hydrophobic and is in situ encapsulated in
reversible assembly can be described as follows: zein is solubil- zein/NaCas/alginate nanoparticles during acidification back to
ized and NaCas micelles are dissociated at sufficiently high acidic pH.
pH, and the structural interactions between zein and NaCas
To verify the above hypothesis, zein/NaCas/alginate nano-
can be enabled by inter-chain hydrogen bonds. During neu- particles were first fabricated to encapsulate propolis using the
tralization, reassociation of interior structures may turn the pH-driven method. The effects of three diverse encapsulating
protein complexes into co-assembled zein/NaCas nano- approaches on the morphology of nanoparticles, the particu-
particles.13 Although this is a promising method for fabricat- late characteristics and the encapsulation efficiency of propolis
ing zein nanoparticles as delivery vehicles, the zein/NaCas were examined. Furthermore, the encapsulation mechanism
nanoparticles thus formed still have some drawbacks, such as was investigated by Fourier transform infrared (FTIR) spec-
the poor aggregation stability under gastrointestinal (GI) con- troscopy. Physical stability of nanoparticles against pH and
ditions or certain pH ranges (near the NaCas isoelectric pH). ionic strength, in vitro release profile under the simulation gas-
The method is time-consuming since it needs titration from trointestinal conditions and bioaccessibility were also evalu-
high alkaline pH to neutral pH using HCl, which limits their ated. The results of this study can be applied to other hydro-
applications as oral delivery vehicles while the scalability of phobic nutraceuticals containing hydroxyl groups, such as cur-
the pH-driven method is a concern as well. In order to improve cumin, rutin and thymol.
the stability of nanoparticles, coating the outer surface of the
zein/NaCas nanoparticles with the polysaccharide to modulate
the electrostatic and steric repulsion between the particles is a
2. Materials and methods
green and simple approach. For example, alginates are water-
2.1. Materials
soluble anionic polysaccharides which have been used as a
Zein (Lot C10079965) and sodium alginate (Lot M28618017)
were obtained from Macklin Reagent Co. Ltd (Shanghai,
China). Propolis was obtained from Bee Words Industry Co.
Ltd, Zhejiang Province, China. NaCas from bovine milk,
pepsin, pancreatin, bile extract and 2,2′-azinobis (3-ethylben-
zothiazoline-6-sulfonic acid) (ABTS) were purchased from
Sigma-Aldrich Corp. (St. Louis, MO, USA). Citric acid, sodium
citrate, sodium hydroxide and other reagents were all of
analytical grade.
good wall material for drug and bioactive food component
encapsulation.14,15 Alginate is a promising polysaccharide to
fabricate oral delivery nanoparticles, which can shrink and
convert into so-called insoluble alginic acid skin to protect
nanoparticles from enzymic digestion.16 Recently, partial alka-
line hydrolysis of propylene glycol alginate has been used to
reduce pH from 12.5 to 7.5, providing an interesting perspec-
tive in preparing zein-based nanoparticles without the tedious
process of titrating the sample using HCl.17 More effort should
be made to provide another acidification strategy to scale up
the pH-cycle method.
2.2. Preparation of propolis loaded zein/NaCas/alginate
nanoparticles
In addition, some polyphenols and essential oils can be
encapsulated in different delivery systems using the pH-driven Propolis nanoparticles were prepared using three different
method as previous studies reported.2,18–21 Propolis contains methods in this section, and the detailed information of the
numerous natural polyphenols with many potentially ben- procedures is shown in Fig. 1A. In Method 1, the zein/NaCas/
eficial health properties such as antioxidant, antimicrobial, alginate nanoparticles were prepared using a previous method
and antitumor activities.22–24 However, the application of pro- with a minor modification.14 Zein (1%, w/v) was dissolved in
polis in pharmaceuticals and food products is still challenging 80% (v/v) aqueous-ethanol solution. Sodium alginate powder
due to its undesirable sensorial characteristics, poor water and NaCas powder were dissolved in double distilled water
solubility, and limited bioaccessibility.25 It is inspired whether and stirred overnight to prepare the polysaccharide (1%, w/v)
this pH-driven method can be used to encapsulate propolis and protein (1%, w/v) stock solutions, respectively. Briefly,
using zein, NaCas, and alginate as building blocks. 1.0 mL of zein solution (1%, w/v) with dissolved propolis
How the nanoparticles influence the bioaccessibility of propo- (5 mg) was quickly poured into 9.0 mL of diluted NaCas solu-
lis is a question. Furthermore, it is worth investigating since tion under constant stirring (1000 rpm) using a syringe to
propolis containing hydroxyl groups become deprotonated at obtain the zein/NaCas solution at a mass ratio of 1 : 1. The
alkaline pH, and this results in a negative charge that resulting mixture was stirred at 1500 rpm for 30 min and
increases the water solubility. Therefore, propolis can be poured into 15.0 ml of diluted sodium alginate solution to
encapsulated into the zein/NaCas/alginate nanoparticles by obtain mixtures with a mass ratio of 1 : 1 : 1 of zein/NaCas/algi-
mixing a well-blended alkaline solution of propolis, zein, nate. The pH of the mixtures was then adjusted to 4.0 using 3
NaCas, and alginate with citrate buffer solution. When mixing M citric acid with stirring at 1500 rpm for 30 min. A rotating
the two solutions, the pH is reduced that leads to the insolubi- evaporator was used to remove ethanol (40 °C, −0.1 MPa) to
lity of zein. Meanwhile, reassociation of NaCas micelles may obtain zein/NaCas/alginate aqueous dispersions. Double dis-
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Fig. 1 (A) Schematic illustration of propolis loaded zein/NaCas/alginate nanoparticle fabrication using three different encapsulating approaches. (B)
Appearance of zein/NaCas without alginate and nanoparticles prepared using three different encapsulating approaches after incubation at pH 12.5
for 30 min followed by acidification to pH 4.0. (C) Size distributions and correlation coefficient of pH 4.1 dispersions prepared with three different
encapsulation approaches, after storage at 4 °C for 30 days.
tilled water adjusted to pH 4.0 was added to achieve a final stored in a refrigerator at 4 °C for further analysis or were
volume of 25.0 mL. For Method 2, zein powder and NaCas freeze-dried for 24 h to obtain powders of nanoparticle disper-
powder were mixed with double distilled water with the pH sions for further characterization.
adjusted to 12.5 using 3.0 M sodium hydroxide solution
2.3. Determination of total phenolic contents
(NaOH) to prepare the zein (1%, w/v) and NaCas (1%, w/v)
stock solutions, respectively. Sodium alginate (1%, w/v) was
dissolved in double distilled water. Briefly, 1 mL of zein solu-
tion and 1 mL of NaCas solution were added into a glass vial
containing 8 mL of double distilled water with the pH adjusted
to 12.5 using 1.0 M NaOH to prepare a well-blended solution
by stirring at 600 rpm for 30 min. Powdered propolis (5 mg)
was then added into the mixture and stirred for 10 min. The
resulting mixture was then poured in 15.0 mL of diluted
sodium alginate solution (pH 3.8, 0.1 M citrate buffer) and
stirred at 1500 rpm for 30 min, corresponding to
zein : NaCas : alginate mass ratios of 1 : 1 : 1. For Method 3, dis-
persions with equal amounts of propolis, zein, NaCas, and
alginate were also prepared, and all procedures were similar to
Method 2 except that alginate solution was directly mixed with
zein and NaCas under alkaline pH conditions for 30 min,
instead of prediluting in 0.1 M citrate buffer at pH 3.8. The
For the determination of total polyphenol contents in the pro-
polis extract (PE) and nanoparticles, the Folin–Ciocalteu
method26 was employed with gallic acid as a standard. For
this, appropriately diluted samples (1.0 mL) were mixed with
1.0 mL of the Folin–Ciocalteu reagent. After 5 min, 5.0 mL of
Na2CO3 (1.0 M) solution was added and diluted to 10 mL
using double distilled water. After shaking well, the mixture
was incubated in the dark for 1 h, and the absorbance was
read at 765 nm using
UV-2550). Quantification was performed using a standard
curve of gallic acid at a concentration of 5–30 μg mL−1 (R2
a spectrophotometer (Shimadzu
=
0.998). The total polyphenol content was expressed as milli-
gram of gallic acid equivalent per gram of sample.
2.4. Nanoparticle characterization
The average particle size, zeta potential, and polydispersity
index (PDI) of freshly prepared formulations were determined
liquid dispersions with a final pH value of 4.1
0.1 were
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using a combined dynamic light scattering and particle elec- gastric fluid (SGF) and simulated intestinal fluid (SIF), as
trophoresis instrument (Zetasizer Nano-ZS90, Malvern described in previous reports with slight modifications.25,27,28
Instruments Ltd, Worcestershire, UK). The particle size was The composition of the simulated gastric and intestinal fluids
calculated using the Stokes–Einstein equation. The zeta poten- is presented in Table 2. Ten milliliters of freshly prepared pro-
tial of nanoparticles was obtained using the Smoluchowski polis loaded nanoparticle samples were first incubated in an
model through electrophoretic mobility measurements by equal volume of SGF. The mixture was maintained at constant
laser Doppler velocimetry (Zetasizer Nano ZS90, Malvern, UK). pH and temperature (pH 3.0, 37 °C) with mild stirring for 2 h.
The refraction index used here was 1.330. Samples were After 120 min, the pH of the digestive production was raised to
diluted 10 times using 0.1 M citrate buffer (pH 4.1) before 7.0 using 2.0 M NaOH, 20 mL of preheated intestinal fluid was
measurement to avoid multiple particle effects. All samples added and digested for 4 h at 37 °C under mild stirring. To cal-
were analyzed in triplicate at room temperature (25 °C). The culate the accumulative percentage of released propolis in
FT-IR spectra of the samples were obtained at room tempera- designated digestion times, the digesta was withdrawn (1 mL)
ture (25 °C) using an ATR-FTIR spectrophotometer (Nicolet at different time intervals (10, 30, 60, 90, 120, 150, 180, 240,
iS10, Thermo-Scientific, WI, USA). The spectra were acquired 300, 360), 4.0 mL of PBS was added and subjected to centrifu-
at a wavenumber range from 400 to 4000 cm−1 with a 4 cm−1 gation (4000g, 50 min) using an ultrafiltration device. Then,
resolution and accumulation of 32 scans. The microstructures the polyphenol content in the filtrate which represented
of nanoparticles were determined using a transmission elec- released propolis was measured using the Folin–Ciocalteu
tron microscope (TEM, H-7650, Hitachi, Japan) operating at 80 method after diluting appropriately with PBS. In addition, pro-
kV and the sample was stained with 1% uranyl acetate.
polis (5 mg) dissolved in ethanol (0.5 mL, 10 mg mL−1) was
add to digestion solution (9.5 mL gastric fluid) as a blank
control. After intestinal digestion was completed, the raw
digesta from each sample was centrifuged at 15 000g for 1 h
and the mixed micelle phases (clear supernatant) were
collected in which propolis was solubilized. The propolis con-
centration was measured according to section 2.3. The propo-
lis concentration in the mixed micelle phases (Cmicell) as a per-
centage of the propolis concentration in the overall digesta at
the end of the simulated GIT model (Coverall digesta) was defined
as the in vitro bioaccessibility of propolis using the following
equation:
2.5. Encapsulation efficiency (EE) and loading capacity (LC)
According to our previous study,25 the EE (%) of propolis in
the nanoparticles was calculated using eqn (1):
EE ð%Þ ¼ ð1 ꢀ free propolis=total propolisÞ ꢁ 100
ð1Þ
where total propolis is the total content of polyphenol con-
tained in the nanoparticle suspension and free propolis is the
content of polyphenol obtained in the filtrate receiver after
ultrafiltration (4000g for 50 min) using centrifugal filters
(Ultrafree®-M 10 000 NMWL Filter Unit, Millipore, Cork,
Ireland). The total and free polyphenol content was measured
using the Folin–Ciocalteu method as described in section 2.3.
The freeze-dried samples were weighed, and the LC was calcu-
lated as follows:
Bioaccessibility ð%Þ ¼ Cmicell=Coverall digesta ꢁ 100
ð3Þ
2.8. Statistical analysis
All experiments were performed in triplicate and the results
were represented by mean standard error. Experimental stat-
istics were performed by Duncan’s test and differences with
P < 0.05 were defined as statistically significant.
LC ¼ ðencapsulated propolis=weight of nanoparticlesÞ ꢁ 100:
ð2Þ
2.6. Stability to environmental conditions
Influence of sodium alginate concentration. Different
sodium alginate concentrations may tailor particular charac-
teristics of the propolis loaded nanoparticles. A series of nano-
particle suspensions with alginate concentrations ranging
from 0 to 1.50 (mg mL−1) were prepared and measured for par-
ticle size using dynamic light scattering.
3. Results and discussion
3.1. Preparation and characterization of propolis loaded
nanoparticles
According to our previous study, encapsulation of propolis
into zein/carboxymethyl chitosan nanoparticles offered
a
Influence of pH and ionic strength. The pH (2.0, 3.0, 4.0,
4.5, 5.0, 6.0, 7.0, 8.0) and/or ionic strength (0, 100, 200, 300,
400 mM) of the nanoparticle suspensions were adjusted to
various values and the stability of the nanoparticles to aggrega-
tion was examined using dynamic light scattering
measurements.
promising approach to improve their solubility and bio-
availability.25 However, the different loading techniques that
would affect the particulate characteristics of zein-based com-
posite nanoparticles have not been reported yet. As shown in
Fig. 1A, the method 1 loading technique is similar to the con-
ventional anti-solvent precipitation method to fabricate zein/
caseinate/alginate nanoparticles. In method 2 and method 3,
an innovative pH-induced one-step assembly method was
2.7. In vitro digestion profile and bioaccessibility
The in vitro release profiles and bioaccessibility of free and employed to encapsulate propolis into zein/caseinate/alginate
encapsulated propolis were evaluated using in vitro simulated nanoparticles; however the sequence of adding alginate was
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different. The photographic images presented in Fig. 1B show a smaller PDI value than the method 1 sample. These obser-
that zein/NaCas nanoparticles without the addition of alginate vations corresponded to the intensity weighted particle size
were completely unstable and precipitated at pH 4.0. The dis- distributions of all dispersions after storage at 4 °C for 30 days
persions prepared with alginate using the three methods did (Fig. 1C). All the three dispersions were monomodal, while the
not show visible precipitates after one month of storage at method 1 sample showed majority of the particles around
4 °C. The phenomenon indicated that alginate interacted with larger particle sizes than the ones prepared using method 2
zein/NaCas nanoparticles and enhanced electrostatic and and method 3. Thus, the pH-induced one-step assembly
steric repulsion against aggregation.15 It was noted that the method could be an effective method for the preparation of
dispersions prepared using method 2 and method 3 had an zein/NaCas/alginate nanoparticles with a smaller particle size
orange-yellow color. A similar observation was reported by a and narrower particle size distribution. This speculation is in
previous study, showing that the color of clove bud oil became accordance with previous studies which have shown that the
darker after heating and alkaline treatment.29 The effect of the pH-driven loading method has resulted in curcumin loaded
pH cycle (alkaline dissolution and acidification) on the change solid lipid nanoparticles with a smaller particle size and accep-
of biological activity and stability of propolis components is table homogeneity.30 It is worth mentioning that the zeta
discussed in ESI Fig. S1.† Interestingly, as shown in Table 1, potential of nanoparticles prepared using the pH-induced one-
the zein/NaCas/alginate nanoparticles fabricated by method step assembly method (method 2 and method 3) was signifi-
1 had a larger particle size than those fabricated by pH- cantly less negative than that of those prepared using the con-
induced one-step assembly methods (method 2 and method 3) ventional anti-solvent precipitation method (method 1) while
without using ethanol as a loading solvent for zein and propo- the method 3 sample exhibited the lowest zeta potential
lis. Meanwhile, the method 2 and method 3 samples exhibited (Table 1). These observations indicated that the structure of
nanoparticles prepared using method 2 or method 3 changed
significantly compared to nanoparticles prepared using
method 1. Similar phenomena have also been observed by
Table 1 Comparisons of size, PDI, count rate, zeta potential and
other researchers.12,13 This could be due to the complete
encapsulation efficiency of colloidal particles produced with equal
mixing of the protein and alginate and coalition to form dis-
masses of zein, NaCas and alginate using three different methods. The
propolis concentration was 0.25 mg mL−1
crete nanoparticles with clear partition and part of the nega-
tively charged polysaccharide chains buried in the core of the
zein/NaCas/alginate nanoparticles.13 Therefore, compared to
other nanoparticles, the zeta potential of the nanoparticles
(method 3) significantly decreased.
The count rate plays a vital role in indicating the number
and concentration of colloidal nanoparticles, which is pro-
portional to the sixth power of particle size as well as their
number.30,31 Interestingly, the method 3 sample exhibited the
highest count rate as well as the smallest particle size among
the formulations (Table 1). According to a previous study,31,32
these results implied that smaller nanoparticles were formed
in greater number by method 3. The NTA data (Fig. S2†)
further supported the count rate data in DLS (Table 1). Most
Treatment
Method 1
Method 2
Method 3
Size (nm)
PDI
362.2 1.5c
0.26 0.01b
175.6 0.4b
−31.7 0.8a
76.2 0.6a
43.6 2.2a
267.4 1.5b
0.24 0.00a
144.9 0.3a
−28.0 0.8b
82.4 1.3b
54.2 1.2b
208.1 2.1a
0.23 0.02a
268.2 1.2c
−26.9 0.5c
86.5 0.7c
59.6 1.5c
Count rate (kcps)
Zeta potential (mV)
EE (%)
LC (μg mg−1
)
Numbers are mean standard deviation (n = 3). Different superscript
letters in the same row indicate significant differences (P < 0.05).
Please refer to Fig. 1A for detailed information on the different
methods.
Table 2 Final concentrations of constituents before addition to the notably, the nanoparticles prepared by method 3 exhibited the
sample of the simulated gastric and intestinal fluids
highest encapsulation efficiency compared to the other two
methods while the corresponding loading capacity increased
Simulated gastric
fluid (pH 3.0)
mmol L−1
Simulated intestinal
fluid (pH 7.0)
from around 43.6 μg mg−1 to around 59.6 μg mg−1 (Table 1).
All of these are compelling evidence to support the fact that
the pH-induced one-step assembly method is a promising
approach to encapsulate propolis into zein nanoparticles
modified by NaCas and alginate, and that interaction of algi-
nate with the protein (zein and NaCas) under alkaline con-
ditions is critical to formulate the most compact and homo-
geneous propolis loaded zein/NaCas/alginate nanoparticles
(method 3 sample).
Constituent
KCl
5.52
0.72
20.00
37.76
0.08
0.40
5.44
0.64
68.00
30.72
0.26
KH2PO4
NaHCO3
NaCl
MgCl2(H2O)6
(NH4)2CO3
NaOH
3.73
6.72
0.30
HCl
CaCl2(H2O)2
22.48
0.07
3.2. Effect of NaCas/alginate mass ratio
mg mL−1
0.5
10
Tween 20
Pepsin
Pancreatin
Bile extract
Fig. 2 shows the effect of the mass ratio of NaCas/alginate
(8 : 1, 4 : 1, 2 : 1, 4 : 3, 1 : 1, 4 : 5, and 2 : 3) on the particle size
and PDI at fixed concentrations of zein (1.0 mg mL−1) and
30
6.25
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the method 1 sample showed a larger size than the method 2
and method 3 samples. The nanoparticles (method 2 sample)
were clumped to each other, which might be attributed to the
alginate coating on the surface of zein/NaCas nanoparticles.33
Interestingly, the nanoparticles (method 3 sample) appeared
spherical and exhibited smooth surfaces with clearer partition
among each other. Overall, nanoparticles formed with method
3 were more homogeneous, more discrete, and smaller than
those of method 2, suggesting that most alginates were prob-
ably involved in nanoparticle fabrication in method 3 but
adsorbed on the surface of zein/NaCas nanoparticles in
method 2.17 The TEM technique can also distinguish the local-
ization of different materials in the sample according to the
difference in the material density without using fluorescent
tagging.34 As shown in Fig. 3 (M1), the core–shell structure was
clearly observed with the shell being more electron dense as
compared to the core (marked with red arrows), which was in
agreement with published results.35
Fig. 2 Effect of NaCas/alginate mass ratio on the Z-average diameter
and PDI of zein/NaCas/alginate nanoparticles (method 3 sample). Data
represent the mean standard deviation (SD, n = 3).
3.4. FTIR analysis
NaCas (1.0 mg mL−1), and pH 4.1. Notably, extensive particle
aggregation occurred when the NaCas/alginate mass ratio was
higher than the ratio of 4 : 1, suggesting that insufficient algi-
nate molecules covered the zein/NaCas nanoparticles.14 The
particle size remained relatively constant at higher alginate
concentration, corresponding to a NaCas/alginate mass ratio
of 1 : 1 to 2 : 3. This phenomenon indicated that the zein/
NaCas nanoparticles were saturated with alginate at these
higher alginate levels.14 For the effect of alginate on the par-
ticle count rate, the particle count rate at a NaCas/alginate
mass ratio of 4 : 1 was the lowest (146.75 kcps). As alginate
increased to a higher concentration, the count rate of particles
increased and was higher at a higher alginate/NaCas mass
ratio (data not shown). Because the NaCas concentration was
kept unaltered in sample fabrication and the amount of algi-
nate was higher at a higher alginate/NaCas mass ratio, the
higher count rate suggests the formation of a greater popu-
lation of nanoparticles. A previous study by Luo et al.18 has
also described this phenomenon. The nanoparticles (NaCas/
alginate mass ratio of 4 : 1–2 : 3) exhibited a small PDI
(0.21–0.24), suggesting that particles may have a more uniform
size. For the reason of small particle size, high stability and
ease of fabrication, NaCas/alginate with a mass ratio of 1 : 1
was selected to develop propolis loaded zein/NaCas/alginate
nanoparticles in further experiments using the pH-induced
one-step assembly method (method 3).
In this study, the intermolecular interaction between propolis
and nanoparticles was revealed by FTIR (Fig. 4). The pure pro-
polis spectrum showed a peak at 3362 cm−1 related to phenolic
compounds and carbohydrates36 (Fig. 4A). Propolis exhibited
two characteristic peaks at 2927 and 2854 cm−1, respectively,
which are attributed to hydrocarbon stretching vibration. The
two peaks at 1639 and 1513 cm−1 are generated by ν(CvO),
ν(CvC), and δas(N–H), which are shown in aromatic com-
pounds, for instance, flavonoids.36,37 The band at 1500 to
1000 cm−1 is mostly contributed by the presence of flavonoids,
lipid, and alcohols.36 For zein (Fig. 4A), the peak at 3317 cm−1
corresponded to –OH stretching. Two peaks located at
1655 cm−1 and 1535 cm−1 corresponded to CvO stretching
(amide I) and N–H bending and stretching of C–N (amide II),
respectively.25 The FTIR spectrum of NaCas displayed a peak
for –OH stretching at 3316 cm−1, and two characteristic peaks
for amide
I and amide II bonds in the range of
1500–1700 cm−1 (1654 cm−1 and 1535 cm−1).38 The peaks in
the spectrum of alginate at around 3500–3000, 1609, 1413, and
1032 cm−1 represented the –OH stretch, the asymmetrical
stretch of –COOH, the symmetrical stretch of –COOH, and the
COH stretch, respectively.39 Characteristic aromatic rings of
flavonoids existed in propolis absorption peaks at 1162 cm−1
and 1031 cm−1, which were shifted to 1159 cm−1 and
1021 cm−1 in the spectrum of propolis loaded zein nano-
particles (NPs) (Fig. 4B). This indicated that hydrophobic inter-
actions occurred between zein and propolis.25 By comparing
the spectra of zein and propolis loaded zein NPs (Fig. 5B), it
3.3. Morphological observation
To observe the morphology of the zein/NaCas complex and was found that the adsorption peak of amide II (C–N stretch-
propolis loaded zein/NaCas/alginate nanoparticles, the TEM ing and N–H bending) was shifted from 1535 cm−1 in zein to
images of four samples were obtained and are presented in 1516 cm−1 in propolis loaded zein NPs which was the sign of
Fig. 3. The zein/NaCas complex without alginate formed large the formation of hydrogen bonds between zein and propolis
aggregates due to their poor aggregation stability near the via the N–H bond in zein.40 After the formation of zein/NaCas/
NaCas isoelectric pH, which agreed with the photographic alginate NPs, the characteristic peaks of the asymmetrical
images presented in Fig. 1B. When alginate was added, the stretch at 1609 cm−1 (–COOH) of alginate and the –NH3
+
three nanoparticle samples showed spherical appearance, and bending vibrations (1535 cm−1) of the protein (zein, NaCas)
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Fig. 3 TEM images of zein/NaCas without alginate and nanoparticles produced using three different loading methods.
were not observable (Fig. 4B). It was revealed that zein, NaCas electrostatic repulsion between them due to effective coverage
and alginate can form stable polyelectrolyte complex nano- of alginate on the surface of zein/NaCas.15,42 However, the par-
particles through electrostatic interactions.16 More interest- ticle size increased when the pH was reduced to 3.0 and 2.0
ingly, the propolis loaded zein/NaCas/alginate NPs showed the (Fig. 5A). Meanwhile, the magnitude of the zeta potential
same FTIR spectrum as that of the blank nanoparticles around decreased significantly from −29.5 0.32 mV at pH 4.0 to −6.6
1718, 1599, 1401, and 1234 cm−1. This finding suggested that
0.25 mV at pH 2.0 (p < 0.05) (Fig. 5B). The particle size
the primary structure of the zein/NaCas/alginate NPs was increased at pH 2 due to the reduction in the electrostatic
virtually unchanged after propolis
encapsulation.41 repulsion between the particles.14 Indeed, carboxylic groups
Encapsulation of propolis was revealed by the result that the on the alginate molecules have pKa values around pH 3.5.
majority of the characteristic peaks of propolis were over- Consequently, at pH 2, protonation of the carboxyl groups on
lapped by the absorption peaks of the nanoparticle matrix.25
the alginate molecules occurred and they lose their negative
charge (–COO−).14 On the other hand, at low pH values, the
net charge on the biopolymer particles becomes less negative
3.5. Stability of propolis nanoparticles
Fig. 5A shows that the propolis loaded zein/NaCas/alginate also due to net-positively charged protein components (pH <
nanoparticles appeared to be stable against aggregation across pI, protonation of some amino acids of the protein) complex-
the entire pH range studied, especially near the isoelectric ing with free alginate.43 At pH between 6 and 8, the particle
point pH of the NaCas (pI, around pH 4.5) and zein (pI, size decreased appreciably (Fig. 5A). This result should be
around pH 6.2). The good aggregation stability of the nano- ascribed to the dissociation of the nanoparticles at high pH
particles can be attributed to a strong steric stabilization and values where both the protein (zein and NaCas) and alginate
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Fig. 4 Fourier transform infrared spectrum of (A) individual polymers
and (B) propolis loaded zein nanoparticles (NPs); zein/NaCas/alginate
nanoparticles; and propolis loaded zein/NaCas/alginate nanoparticles
(method 3 sample).
Fig. 5 Effect of pH on the (A) Z-average diameter, (B) zeta potential,
and appearance of propolis loaded zein/NaCas/alginate nanoparticles
(method 3 sample).
have the same charge (negative charge) and repel each other.15
Therefore, zein/NaCas/alginate nanoparticles may be a promis-
ing vehicle for pH-triggered release of propolis. Interestingly, anionic propolis nanoparticles and shield the electrostatic
when the pH was increased from 4.0 to 8.0, the surface charge repulsion between them, thereby leading to aggregation.19 The
of propolis loaded nanoparticles decreased from −29.5 0.32 conclusion was supported by zeta potential analysis (Fig. 6B),
to −21.7 0.65 mV. This phenomenon might be related to the which indicated that the zeta potential value decreased with
shielding effect caused by the excess free biopolymers (dis- the rise of ionic strength (from −34 to −20 mV for propolis
sociation of the nanoparticles) around the nanoparticles nanoparticles). Surprisingly, the propolis nanoparticles were
(Fig. 5B).44 The influence of ionic strength on the stability of unstable to aggregation at high salt levels, which is in contrast
the propolis loaded zein/NaCas/alginate nanoparticles to earlier studies on NaCas coated zein nanoparticles (stability
(method 3 sample) is presented in Fig. 6. It could be observed at 1.5 M NaCl).42 This discrepancy may be attributed to the
from Fig. 6A that the propolis nanoparticles had no visible difference in the structure of nanoparticles (core–shell vs. well
changes in their visual appearance and Z-average diameter at mixing and hybrids).12
low NaCl concentration (≤200 mM), but aggregation was
observed at high NaCl concentration (≥400 mM NaCl). The
3.6. In vitro release profile and bioaccessibility of propolis
finding suggested that the nanoparticles were unstable at As shown in Fig. 7A, only 33% of free propolis diffused into
higher ionic strength. This phenomenon was due to the ability the simulated gastrointestinal tract (SGI) within 6 h of incu-
of the sodium cations to accumulate around the surface of bation due to the poor solubility of propolis in an aqueous
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Fig. 7 (A) In vitro dissolution profiles of free and encapsulated propolis
in SGF and SIF. (B) In vitro bioaccessibility of free propolis and encapsu-
lated propolis in three different nanoparticles.
Fig. 6 Effect of NaCl concentration on the (A) Z-average diameter, (B)
zeta potential, and appearance of propolis loaded zein/NaCas/alginate
nanoparticles (method 3 sample).
Subsequently, the bioaccessibility of propolis after being
exposed to the small intestine phase was determined (Fig. 7B).
solution. This kinetics was in accordance with the release of Mixing propolis with an aqueous solution would cause the for-
resveratrol, as shown in a previous study.28 Nevertheless, pro- mation of precipitates immediately, which might result in pro-
polis released approximately 60% (method 1 sample) and 90% polis exhibiting lower bioaccessibility (around 30%).
(method 2 and method 3 samples) under SGI conditions, Nevertheless, the bioaccessibility of propolis was still around
respectively. Propolis in method 2 and method 3 samples pre- 30%. It can be explained that polyphenols interact with the
sented a similar kinetic release profile throughout SGI. protein (such as enzymes) present in the gastric and intestinal
Notably, compared to the method 1 sample, a more significant fluids increasing their solubility in the micelle phase.45 As
burst effect in the method 2 and method 3 samples was expected, the bioaccessibility of propolis encapsulated in zein/
detected after 4 h of SGI digestion. These findings may be due NaCas/alginate nanoparticles was markedly improved by 70%
to the intestinal pH value (around pH 7.0) leading to the dis- and 80% compared to free propolis (around 30%). The
sociation of the nanoparticles, which was in favour of the enzy- binding between propolis and the nanoparticles through
matic degradation of nanoparticles during intestinal digestion. hydrogen bonds and electrostatic and hydrophobic inter-
This phenomenon was also observed in section 3.5. actions enhanced the content of propolis in the micellar
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phase. These results indicate that zein/NaCas/alginate nano-
particles are a promising oral delivery system for propolis.
by emulsification-evaporation method, Food Hydrocolloids,
2018, 81, 149–158.
5 A. Patel, Y. Hu, J. K. Tiwari and K. P. Velikov, Synthesis and
characterisation of zein–curcumin colloidal particles, Soft
Matter, 2010, 6, 6192–6199.
6 L. Dai, R. Li, Y. Wei, C. Sun, L. Mao and Y. Gao, Fabrication
of zein and rhamnolipid complex nanoparticles to enhance
the stability and in vitro release of curcumin, Food
Hydrocolloids, 2018, 77, 617–628.
4. Conclusions
In summary, propolis loaded zein/NaCas/alginate nano-
particles were successfully prepared using a pH-induced one-
step assembly method to enhance its bioaccessibility. Three
different methods to load propolis into zein/NaCas/alginate
nanoparticles were advised and compared for their effects on
the physicochemical properties of the resultant nanoparticles.
Compared to the nanoparticles prepared using method 1,
zein/NaCas/alginate nanoparticles prepared using method 3
show smaller average particle size and higher encapsulation
efficiency and loading capacity. These nanoparticles were
shown to be stable against aggregation over a wide range of pH
values (2–8) and salt concentrations (0–300 mM NaCl). This
study suggested that a low-energy, pH-induced one-step assem-
bly method could potentially be used to fabricate zein-based
vehicles without using organic solvents and specific equip-
ment to broaden the applications of propolis and other bio-
active compounds in the food, cosmetic, and pharmaceutical
industries.
7 C. Sun, Y. Wei, R. Li, L. Dai and Y. Gao, Quercetagetin-
Loaded Zein-Propylene Glycol Alginate Ternary Composite
Particles
Induced
by
Calcium
Ions:
Structure
Characterization and Formation Mechanism, J. Agric. Food
Chem., 2017, 65, 3934–3945.
8 K. Hu, X. Huang, Y. Gao, X. Huang, H. Xiao and
D. J. McClements, Core-shell biopolymer nanoparticle delivery
systems: synthesis and characterization of curcumin fortified
zein-pectin nanoparticles, Food Chem., 2015, 182, 275–281.
9 Q. Zhong and M. Jin, Zein nanoparticles produced by
liquid–liquid dispersion, Food Hydrocolloids, 2009, 23,
2380–2387.
10 H. Chen and Q. Zhong, A novel method of preparing stable
zein nanoparticle dispersions for encapsulation of pepper-
mint oil, Food Hydrocolloids, 2015, 43, 593–602.
11 C. Sun, Y. Gao and Q. Zhong, Effects of acidification by
glucono-delta-lactone or hydrochloric acid on structures of
zein-caseinate nanocomplexes self-assembled during a pH
cycle, Food Hydrocolloids, 2018, 82, 173–185.
12 K. Pan and Q. Zhong, Low energy, organic solvent-free co-
assembly of zein and caseinate to prepare stable disper-
sions, Food Hydrocolloids, 2016, 52, 600–606.
13 T. Wang, M. Yue, P. Xu, R. Wang and Z. Chen, Toward
water-solvation of rice proteins via backbone hybridization
by casein, Food Chem., 2018, 258, 278–283.
14 K. Hu and D. J. McClements, Fabrication of biopolymer
nanoparticles by antisolvent precipitation and electrostatic
deposition: Zein-alginate core/shell nanoparticles, Food
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Financial support from the Natural Science Foundation of
Zhejiang Province (LY18C200003) and Zhejiang Provincial Top
Key Discipline of Food Science and Biotechnology is acknowl-
edged. The authors thank Dr Rituja Upadhyay for proofreading
of the research article.
15 W. Liu, J. Wang, D. J. McClements and L. Zou,
Encapsulation of β-carotene-loaded oil droplets in caseinate/
alginate microparticles: Enhancement of carotenoid stability
and bioaccessibility, J. Funct. Foods, 2018, 40, 527–535.
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