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Exploring the mechanism of hollow microcapsule formation by self-assembly of soy 11s protein upon heating
Journal Pre-proof Exploring the mechanism of hollow microcapsule formation by self-assembly of soy 11s protein upon heating Huanhuan Zhao, Mingming Guo, Tian Ding, Xingqian Ye, Donghong Liu PII:
S0268-005X(19)31271-8
DOI:
https://doi.org/10.1016/j.foodhyd.2019.105379
Reference:
FOOHYD 105379
To appear in:
Food Hydrocolloids
Received Date: 16 June 2019 Revised Date:
22 August 2019
Accepted Date: 10 September 2019
Please cite this article as: Zhao, H., Guo, M., Ding, T., Ye, X., Liu, D., Exploring the mechanism of hollow microcapsule formation by self-assembly of soy 11s protein upon heating, Food Hydrocolloids (2019), doi: https://doi.org/10.1016/j.foodhyd.2019.105379. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Exploring the mechanism of hollow microcapsule
2
formation by self-assembly of soy 11s protein upon
3
heating
4
Huanhuan Zhao a, Mingming Guo a,b, Tian Ding a,b, Xingqian Ye a,b, Donghong Liu a,b,c,
5
a
College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agro-Food
6
Processing, Zhejiang R&D Center for Food Technology and Equipment, Zhejiang University,
7
Hangzhou 310058, Zhejiang, China b
8 9
c
Fuli Institute of Food Science, Hangzhou 310058, Zhejiang, China
Ningbo Research Institute, Zhejiang University, Ningbo 315100, Zhejiang, China
10
Abstract: This study investigates the phenomenon of hollow microcapsule formation through
11
simple heat treatment of soy 11s protein solution. Microscopies were used to monitor the
12
morphological changes of proteins in the aqueous phase during fabrication; the corresponding
13
changes in zeta potential, protein solubility and composition were examined. In the presence of
14
0.05 M sodium chloride, the 11s protein self-assembled into irregular flocs, which transformed
15
into microgels or hollow microcapsules upon heating at 80
16
microcapsule transformation occurred within the first 60 s of heating and the microcapsules
17
formed their perfect hollow spherical structure in 4 min of heating. A mechanism was proposed
1
for 20 min. The key protein flocs-
18
to describe the hollow structure formation in microcapsules. The samples with protein
19
concentration of 2 g/L had more microgels formed, while those with 5 and 10 g/L protein was
20
mainly composed of hollow microcapsules. It was found that as the particle radius approached
21
the wall thickness, microgels formed instead of microcapsules.
22 23
Keywords: hollow microcapsule; microgel; soy 11s protein; self-assembly; protein aggregates
2
24
1 Introduction
25
Design of hollow microcapsules (or nanocapsules) has been receiving particular interest due to
26
their wide applicability; for example, hollow microcapsules can be developed as delivery
27
systems of active components, catalytic systems and absorbents for removal of hazardous
28
compounds in aqueous phase (Loiseau et al., 2017). Hollow microcapsules have the potential to
29
load more core materials compared to the solid (i.e. homogenous porous) spheres and can have a
30
more sustained release as the substrates are entrapped inside the hollow structure (Rivera,
31
Pinheiro, Bourbon, Cerqueira, & Vicente, 2015).
32
Hollow microcapsules are commonly prepared through self-assembly of wall materials onto
33
the surface of either a solid or a liquid template (Wan, Guo, & Yang, 2015). A layer-by-layer
34
method has been widely used, which permits the formation of microcapsules with engineered
35
features upon dissolution of the sacrificial solid core (Zhang, Guan, & Zhou, 2005). Another
36
approach is through fabrication of colloidosomes with the colloidal particles assembled around
37
the templating droplets (Lee & Weitz, 2009). In addition to the interfacial self-assembly of wall
38
materials at the solid-liquid or liquid-liquid boundaries, another strategy of hollow microcapsule
39
fabrication is through double emulsions with the wall materials incorporated into the middle
40
phase (Loiseau et al., 2017). In spite of a higher encapsulating capability and potential better
41
controlled release performance of hollow microcapsules, conventional approaches to assemble
42
such microcapsules are usually complex and time consuming and often involve addition of toxic
43
solvents to remove the sacrificial templates, performed as temporary cores, during the processes
44
(Feng & Lee, 2017; Rivera et al., 2015).
45
Natural biopolymer-based delivery systems have attracted much attention owning to their
46
advantages of biodegradability and biocompatibility (Larrañaga, Lomora, Sarasua, Palivan, &
3
47
Pandit, 2017). Soy protein, as a natural polymer, has been commonly used as a starting material
48
to prepare polymeric delivery systems for bioactive compounds; it is an abundant resource,
49
which has desirable water solubility and non-immunogenic and anti-carcinogenic properties
50
(Maltais, Remondetto, & Subirade, 2009). Recently, spontaneous formation of hollow
51
microcapsules through heating of pure plant-based protein solutions has been reported. To the
52
best of our knowledge, Chen, Zhao, Nicolai, and Chassenieux (2017) was the first found
53
spontaneous formation of hollow microcapsules under heating using pure legume protein
54
solutions. The authors demonstrated ion-induced microphase separation of native soy glycinin
55
solution, based on which, hollow microcapsules were produced by heating the dispersion above
56
60
57
and a wall thickness of approximately 1 µm. Later, Cochereau, Nicolai, Chassenieux, and Silva
58
(2019) also reported hollow microcapsule formation in pea protein isolate solution through pH-
59
induced phase separation and subsequent heating above 40
60
such self-assemble ability was not solely for specific types of plant proteins.
for more than 5 min; the resulting microcapsules had a diameter ranging from 1 to 40 µm
for 2 min and they suggested that
61
The aforementioned spontaneous formation of plant protein-based hollow microcapsules has
62
several advantages over the traditional methods such as a simpler fabrication routing and no
63
requirements of sacrificial core materials, chemical cross-linkers or emulsion templates. The
64
hollow microcapsules produced from the new method is stable and can keep their integrity
65
between a pH range from 1 to 11.5; the microcapsules has desirable pH responsive permeability
66
of FITC-dextran (Chen, Zhang, Mei, & Wang, 2018). Despite the above advantages and potential
67
applications, the driving forces and underlying mechanisms of the microcapsule fabrication are
68
unknown, more specifically, spontaneous formation of the shell and disappearance of proteins at
69
the center during heating. Chen et al. (2017) hypothesized that the increased attractive
4
70
interactions drove densification of soy protein within the protein microdomains as a result of the
71
configurational changes during heating, while Cochereau et al. (2019) attributed it to possible
72
redistribution of protein from the core of microdomains to solution and formation of permanent
73
protein-protein crosslinks at the shell. However, there is still a lack of study in the literature to
74
validate any of the hypotheses and to reveal the actual mechanisms of this new protein self-
75
assembly process.
76
Therefore, the objective of this research was to (1) explore the key phenomena involved at
77
each stage of hollow microcapsule formation using soy 11s protein and to (2) gain further
78
insights into the mechanisms through monitoring the changes in morphology of protein
79
flocs/particles and the corresponding changes in composition and properties of the aqueous
80
systems.
81 82 83
2 Materials and Methods
84 85
2.1 Isolation of soy 11s protein
86
Soy 11s protein was isolated from defatted soy flour, which was offered by Sinoglory Health
87
Food (Shangdong, China). The isolation method of Wu, Murphy, Johnson, Fratzke, and Reuber
88
(1999) was adopted with minor modifications. Firstly, the defatted soy flour was dispersed in
89
deionized water with a weight ratio of 1:20, using a magnetic stirrer (30 min, 1200 rpm). The pH
90
was then modified to 7.5 using 2 M sodium hydroxide, after which the mixture was centrifuged
91
by a Sigma 3K15 centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) at
92
9500 rpm for 30 min (4
). Then, sodium bisulfite powder was dissolved into the supernatant at
5
93
a concentration of 0.98 g/L, prior to pH adjustment to 6.4 using 2 M HCl. The turbid dispersion
94
was stored in a refrigerator (4
95
The precipitates obtained was quickly rinsed with deionized water for 3 times and then re-
96
dispersed in deionized water. The solution was finally freeze-dried and the dry powder of 11s
97
protein was stored in a refrigerator set to 4
98
.
99
2.2 Microcapsule preparation
100
) for 20 h and then centrifuged at 7000 rpm for 30 min (4
).
.
Hollow microcapsule dispersion was prepared following Chen et al. (2018) with some
101
modifications. Isolated soy 11s protein powder was firstly dissolved in deionized water at 20
.
102
Three batches were prepared with protein concentration of 4, 10 and 20 g/L, respectively, where
103
each batch was centrifuged at 9000 rpm (20
104
centrifuge (Keda Chuangxin, Hefei, China) to remove insoluble fraction. Each solution was then
105
combined with an equal amount of 0.1 M sodium chloride (NaCl) solution under magnetic string
106
at 700 rpm for 1 min; each new turbid dispersion has a final ionic strength value of 0.05 M and
107
protein concentration of approximately 2, 5 and 10 g/L, respectively. Thereafter, the dispersion
108
was immediately immerged in a hot water bath with a temperature of 80±2
109
the time of immersion. Hollow microcapsules or homogeneous microgels formed spontaneously
110
during heating.
) using a HC 3018R high speed refrigerated
for 20 min from
111 112
2.3 Microscopic observations
113
Aliquots of soy protein solutions in the presence of 0.05 M NaCl were heated at 80
water
114
bath for 0, 15, 30, 45, 60, 120, 240 and 1200 s, respectively. A Leica TCS SP8 confocal laser
115
scanning microscopy (CLSM; Leica Microsystems CMS GmbH, Wetzlar, Germany) was used to
6
116
observe the difference in morphology of protein flocs or particles (microcapsules/microgels) in
117
aqueous phase. Approximately 3 mL of sample was taken and stained with Rhodamine B
118
(Yuanye Bio-Technology Co., Ltd., Shanghai, China) at a concentration of 1 ppm. A drop of
119
sample was then mounted onto a glass slide, which was viewed using a 63× water immersion
120
objective lens. A laser beam at 561 nm was used and the fluorescence intensity was recorded
121
between 560 to 660 nm. The measurements were carried out immediately after sample making.
122
A UPH 203i Phase Microscope (Aopu Photoelectric Technology Co., Ltd., Chongqing, China)
123
was also used for observation and image acquisition by a 10× objective lens.
124 125
2.4 Particle size characterization
126
ImageJ software (Schneider, 2012) was employed to separate particles from the CLSM images
127
of the samples after 20 min (1200 s) heat treatment. Holes of hollow particles were filled during
128
image processing, and particles smaller than 0.14 µm2 were eliminated for the aim of removing
129
redundant pixels or hazy particles. Equivalent diameter of each particle was calculated based on
130
the area obtained by the software, assuming the particles as perfect spheres. The size values were
131
then grouped into a continuous series of size bins with a bin width of 0.5 µm, using OriginPro
132
2018 software (OriginLab, Northampton, MA). The particle size distribution was represented
133
based on the relative frequency in percentage as a function of equivalent diameter (bin center
134
value). Coefficient of variation (CV) was calculated to evaluate the particle size monodispersity
135
using the following equation.
136 137 138
CV (%) =
× 100% (1)
Where standard deviation (STD) and average equivalent diameter (ADE) of each type of samples (2, 5 and 10 g/L) were obtained using the OriginPro software.
7
139 140
2.5 Protein concentration and solubility measurements
141
After the aforementioned 1:1 combination of protein solution and NaCl solution, the dispersion
142
became turbid, either prior to heating or after heating and there were significant phase separation
143
after 40 min of sample making (Figure 1). Accordingly, the turbid dispersions were centrifuged
144
immediately after sample preparation at 9000 rpm, 20
145
refrigerated centrifuge and the supernatants containing NaCl were collected and defined as
146
Solution Type 2 (ST2, prior to heating) and ST3 (after heating), respectively. The original
147
protein solutions with concentration of 4, 10 and 20 g/L were also diluted with an equal amount
148
of deionized water and the new solutions had final protein concentration of 2, 5 and 10 g/L,
149
respectively, without the presence of NaCl; these solutions were named as ST1.
using the HC 3018R high speed
150
The total protein concentration of the isolated soy 11s protein powder was determined by the
151
Kjeldahl method in duplicates based on the nitrogen content and a Kjeldahl factor of 6.25 was
152
used (Renkema, Gruppen, & van Vliet, 2002). Then, the protein content of ST1, ST2 and ST3
153
was measured based on Bradford (1976) using bovine serum albumin as the standard. The
154
solubility of protein in the solutions was estimated using nitrogen solubility index (NSI):
155
percentage of soy protein in solutions (the Bradford method) over the total protein content (the
156
Kjeldahl method). Moreover, the pH values of the three types of solutions were measured using a
157
digital pH meter (Five Easy Plus, Mettler Toledo, Columbus, USA).
158 159
2.6 Electrophoresis
160
SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed using
161
a 12% acrylamide resolving gel and a 5% acrylamide stacking gel. The composition of ST1, ST2
8
162
and ST3 was examined after proper dilution. Aliquots of 5 µL sample (a combination of protein
163
solution and buffer solution) per well were loaded on to the gel. The electrophoresis was run
164
with an electrophoresis cell connected to a Bio-Rad PowderPac Basic power supply (Hercules,
165
CA, USA) at 80 V and continued with 120 V when the protein reached the resolving gel. After
166
electrophoresis, the gel was stained with Coomassie brilliant blue R250 solution for 30 min on a
167
shaker and then it was rinsed with deionized water, after which it was destained using a mixture
168
of deionized water, methanol and acetic acid several times during a period of 24 h.
169 170
2.7 Changes in Zeta potential
171
Zeta potential was measured using a Malvern Zetasizer (Nano ZS90, Malvern Instruments,
172
Worcestershire, U.K). Samples heated for 0, 15, 30, 45, 60, 120, 240, 480 and 1200 s were used;
173
the first series of samples with protein concentration of 2 g/L were diluted with 0.05 M NaCl
174
buffer for 10 times, while the second series with protein concentration of 5 g/L were diluted for
175
25 times. The samples with protein concentration of 10 g/L was not measured as there were
176
particles or aggregates beyond the measurement range, i.e. >100 µm. Each sample was loaded
177
into a DTS1070 cuvette and the measurements were controlled by the Zetasizer software.
178 179
2.8 Statistical analysis
180
The measurements of zeta potential, protein concentration and solubility were carried out three
181
times with three replication for each individual measurement unless otherwise stated and the
182
values were reported as means ± standard error. Analyze of variance was performed in SPSS
183
software (SPSS 17.0, IBM Corp., NY, USA). The protein concentration and NSI values were
184
evaluated using one-way ANOVA with a post hoc Tukey test, at a significance level of 95%.
9
185 186 187
3 Results and Discussion
188 189
3.1 Effect of protein concentration on microcapsule formation
190
3.1.1 Microscopic observations
191
The process of microcapsule formation can be split into two procedures: ion-induced phase
192
separation and heat-induced particle formation. According to Figure 2 (A1, B1 and C1), it can be
193
seen that, prior to heating, soy protein flocs appeared in the presence of 0.05 M NaCl and with a
194
higher protein concentration (2, 5 and 10 g/L), the size of flocs became larger and there was
195
denser spatial distribution of the flocs. The phase separation phenomenon of protein solution
196
caused by ion addition has been extensively studied, commonly reported for gel formation
197
(Totosaus, Montejano, Salazar, & Guerrero, 2002). In this study, the surface charges were
198
screened by addition of Na+ and as the electrostatic repulsion decreased, proteins flocculated at
199
their native states (low ionic strength) through weak non-covalent links, i.e. hydrogen bonds, van
200
der Waals force or hydrophobic interactions; such aggregating state was reversible depending on
201
solution conditions (Kastelic, Kalyuzhnyi, Hribar-Lee, Dill, & Vlachy, 2015; Peng, Ren, & Guo,
202
2016).
203
The sizes of spherical particles after heat treatment also increased with the protein
204
concentration (Figure 2, A2, B2 and C2), which was consistent with Chen et al. (2017) using soy
205
glycinin concentration of 8-15 g/L. The hollow microcapsules had a single cavity or vacuoles at
206
the center, in which there could be smaller protein aggregates remained. In addition to the hollow
207
microcapsule formation, small homogeneous microgels presented in all samples. Particularly, in
10
208
Figure 2 (A2), hollow microcapsules were rarely identified and there was a significant amount of
209
microgel aggregates. The wall thickness of the hollow microcapsules were estimated to be
210
between approximately 0.7 and 2.2 µm based on the CLSM micrographs, where larger hollow
211
microcapsules had thicker walls; an example was shown in Figure 2 (C2). As such it was
212
reasonable to expect that the vacuoles can hardly form within a single spherical particle when the
213
size of the particles was small, e.g. with a radius smaller than 2.2 µm. This renders that wall
214
thickness is a crucial factor that determines the lower limit of the size of hollow microcapsules.
215
The morphology of the protein flocs in Figure 2 (A1, B1 and C1) was irregular. Chen et al.
216
(2017) had similar observations of irregular protein flocs in 10 g/L soy glycinin solution with
217
0.05 M NaCl and pH of 7.2, while spherical microdomains formed when the NaCl concentration
218
increased to 0.1 M; however, both types of dispersion had desirable formation of hollow
219
microcapsules under heating (Chen et al., 2018; Chen et al., 2017). In addition, as the protein
220
flocs were relatively unstable, their morphology could be mixing-speed dependent just before
221
heating (Cochereau et al., 2019). Thus, there was no evident correlation found between the
222
morphology of phase separated flocs and the formation of hollow microcapsules.
223 224
3.1.2 Particle size distribution
225
The size distribution of particles in each type of samples after 20 min heating at 80
were
226
presented in Figure 3. The majority of particles had equivalent diameter smaller than 15 µm;
227
however, there were 5.5% and 9.1% particles exceeded this size boundary in the 5 and 10 g/L
228
samples, respectively. Although there was presence of large microcapsule aggregates as
229
identified in Figure 2 (B3 and C3) and microgel aggregates as displayed in Figure 2 (A2), the
230
former was eliminated and the later was separated into individual particles with care during
11
231
image processing. The average equivalent diameter values of the three types of samples were 2.8
232
µm (2 g/L), 6.6 µm (5 g/L) and 6.6 µm (10 g/L), and their corresponding CV values were 47.4%,
233
65.9% and 89.5%, respectively. A higher CV value indicates less homogeneous or uniform
234
particles. This was in good agreement with the observation in Figure 3 that the size distribution
235
of particles in the 2 g/L samples exhibited narrower unimodal, while the size of particles in the 5
236
and 10 g/L samples had more significant variation. Despite the same mean size values of the 5
237
and 10 g/L samples, the size distributions were different and the 10 g/L sample had the highest
238
CV value.
239
Due to the limited resolution of CLSM, only the visible particles were taken into account
240
during image processing. The 2 g/L sample had the largest proportion of small particles with a
241
size range between 0 and 4.5 µm, approximately 89.9%. Hence, the 2 g/L sample was dominated
242
by microgels, different form the 5 and 10 g/L samples composed of both microgels and
243
microcapsules. The size of hollow microcapsules were generally larger than the microgels. It can
244
be proposed that when the protein concentration was low, there were less opportunities of
245
protein-protein interactions and therefore, smaller “solid” protein particles can form upon
246
heating.
247 248
3.2 Microstructural changes of protein particles upon heating
249
Samples with protein concentration of 2 g/L formed numerous small microgels, while those
250
with protein concentration of 10 g/L had more large microcapsules and microcapsule aggregates.
251
As a result, samples with protein concentration of 5 g/L was selected and heated at 80
252
different time duration. The CLSM micrographs in Figure 4 illustrate the continuous changes in
253
particle morphology during the protein flocs-microcapsule transformation in the presence of 0.05
12
for
254
M NaCl upon heating. It has to be noted, however, that there could be changes in the
255
particle/aggregate morphology due to cooling once the samples were taken for CLSM
256
measurements; therefore, the particle/aggregate morphology shown in Figure 4 may be different
257
from the scenario of 20 min heating at 80
258
function of time.
with continuous changes in protein structure as a
259
The soy protein flocs (Figure 4, A1) exhibited random shapes prior to heating, similar to those
260
in Figure 2 (A1, B1 and C1). Interestingly, however, “hollow” structure or cavities inside the
261
flocs sporadically appeared as presented in Figure 4 (A2), different to those in Figure 4 (A1),
262
although the samples were independently prepared with the same solution conditions. This was
263
ascribed to the strong sensitivity of the morphology of such protein system to solution conditions
264
as suggested by Cochereau et al. (2019), and Chen et al. (2017) also had similar inconsistent
265
observations in soy glycinin system with pH between 4-6.4 and NaCl concentration between 0.4-
266
0.5 M. This was an implication that the protein particles tent to interact with each other forming
267
hollow structure although the driving forces and trigger conditions currently remained unclear.
268
Despite the slight divergent findings in the morphology of protein flocs during the first 15 s of
269
heating (Figure 4, A1-B1 or A2-B2), hollow microcapsules started to form into their shapes from
270
30 s of heating. As heating proceeded, the main changes were within each single microcapsules:
271
single vacuoles formation at the center and perfection of the spherical shapes (Figure 4, C-E).
272
From 4 min (240 s) of heating there were minimal changes (Figure 4, F-G). Therefore, the most
273
important transformation from ion-induced flocs to hollow microcapsules occurred rapidly
274
within 30 s of heating at 80
275
worth mentioning that the variations in particle size of microcapsules among the micrographs
, followed by post-heating for microcapsule perfection. It was
13
276
was due to the difference of position in focus during image acquisition as there could be phase
277
separation.
278
Figure 5 (A) showed CLSM cross-sectional images of a particulate type floc (P1), focused at
279
varying depth after heating for 15 s and strand type flocs and cavities within them were also
280
indicated using blue arrows. Each floc was composed of individual spherical protein clusters,
281
which were self-associated with each other and there were also a few free clusters suspended in
282
the peripheral solution; the diameter of protein clusters were estimated to be approximately 2-5
283
µm. P1 was composed of loosely packed protein clusters with cavities within it; however, it was
284
not a hollow sphere. Figure 5 (B) showed microstructural details of the intermediate-state hollow
285
microcapsules after heating for 30 s, where the particles became more spherical in shape and
286
vacuoles presented at the center, although some particles inter-connected with each other as
287
indicated (P2 and P3). These inter-connected particles could separate or merge into one
288
microcapsule during subsequent heating.
289 290
3.3 Changes in protein solubility during microcapsule formation
291
The total protein concentration of the isolated soy 11s protein was determined as 94.34±0.47
292
wt %; therefore, it was shown in Table 1 that the actual protein concentration (C0) of the 2, 5 and
293
10 g/L samples were calculated as approximately 1.89, 4.72 and 9.43 g/L, respectively. With the
294
removal of insoluble protein fraction by centrifugation, the protein concentrations in ST1 (C1)
295
decreased to 1.48, 3.71 and 7.68 g/L, respectively. It is noticeable that the concentration values
296
of ST2 (C2: 043, 1.76 and 2.96) were significantly (P<0.05) smaller than those of ST3 (C3: 0.6,
297
2.45 and 4.64) for each type of sample, which suggested that the soy proteins in ion-induced
298
flocs partially re-dispersed/dissolved into the solution upon heating, resulting in higher protein
299
concentration values in ST3.
14
300
In Figure 6, the NSI1 values of ST1 solutions, being estimated between 0.79 and 0.81, were not
301
statically different (P>0.05) among the three types of samples. This was because ST1 solutions
302
had pH values ranging from 7.34 to 7.44 which contributed to high solubility of soy globulins in
303
water as the pH was away from the isoelectric point, approximately 4.9-5.2 (Golubovic, van
304
Hateren, Ottens, Witkamp, & van der Wielen, 2005; Wolf, 1970). For each type of samples, the
305
changes in NSI values had a similar trend as the changes in concentrations, i.e. a decrease by
306
adding NaCl and then a slight increase because of heating. These results reveal that the proteins
307
had the lowest solubility due to ion-induced phase separation and the solubility increased slightly
308
due to the conformational changes of protein during heating; the pH values of ST2 and ST3
309
solutions varied between 6.72 and 6.85. The NSI2 and NSI3 values of the 2 g/L sample were both
310
significantly (P<0.05) lower than those of the 5 and 10 g/L samples and it meant higher
311
productivity of microgels than the microcapsules.
312 313
Table 1. Protein concentration (C) of each type of solutions and supernatants.
Sample
Actual concentration
C1
C2
C3
ST1
ST2
ST3
g/L
314 315
2 g/L
1.89
1.48±0.01 a
0.43±0.03 b
0.60±0.11 c
5 g/L
4.72
3.71±0.01 a
1.76±0.02 b
2.45±0.04 c
10 g/L
9.43
7.68±0.01 a
2.96±0.02 b
4.64±0.05 c
Note: different superscript letters in each row denote significant differences at P<0.05. Values are means ± standard error (n=3).
316 317
3.4 Changes in zeta potential during microcapsule formation
15
318
Zeta potential values of the 2 and 5 g/L samples containing 0.05 M NaCl were measured at
319
different stages of heating. It was seen from Figure 7 (the full diagram, Figure A1, has been
320
provided in the Supplementary data) that both samples had an initial value of approximately -
321
11.8 mV, which was followed by a sharp decrease within the first 15 to 30 s. Then it increased
322
quickly until approximately 60 s of heating. The zeta potential value continued to change and
323
after approximately 4 min, it almost kept constant with time. Overall, the curve of 5 g/L sample
324
had a similar trend as the 2 g/L sample in spite of slight shifts. It has to be aware that the
325
morphological difference of the particles in these two samples may influence the zeta potential
326
results. The redistribution of protein from flocs to solution during heating was possibly occur
327
within the first 60 s of heating as indicated by on our CLSM observations and the changes in zeta
328
potential. In addition, the zeta potential values were in a range of aggregation threshold of -11 to
329
-20 (Schramm, 2014); hence, the dispersions were relatively unstable as being observed in
330
Figure 1.
331
Heat treatment generally induced conformational changes of native soy proteins and soy 11s
332
protein could undergo dissociation and then association/aggregation reactions (Nishinari, Fang,
333
Guo, & Phillips, 2014). Therefore, the protein composition of ST1, ST2 and ST3 were compared
334
using SDS-PAGE analysis. The 11s fraction of soy proteins can generally dissociate into smaller
335
2s, 3s or 7s forms depending on pH, ionic strength and heating conditions (Hashizume &
336
Watanabe, 1979; Nishinari et al., 2014). However, in Figure 8, no marked changes in protein
337
electrophoretic patterns was detected apart from the differences in the color intensity, which was
338
ascribed to varying dilution levels. Note that here the protein composition of ST2 and ST3 were
339
similar by comparing A and D, B and E, or C and F in Figure 8, which elucidated in return that
340
the protein composition in flocs prior to heating and particles formed after heating were also
16
341
similar. The solutions were dominant in soy 11s acidic and basic subunits and there were minor
342
amount of 7s β subunits. The molecular mass of the 11s acidic subunits was approximately 35
343
kDa and the 11s basic subunits were approximately 19-20 kDa, which was in line with the values
344
reported in the literature (Hsiao, Yu, Li, & Hsieh, 2015); there were no additional pattern
345
exhibited between the range of 10-25 kDa (not shown). These results rendered that the
346
phenomena of ion-induced phase separation, partial redistribution of proteins during heating and
347
heat-induced microcapsule formation were probably irrelevant to specific protein components.
348 349
3.5 Mechanism of microcapsule formation
350
Relying on the obtained results, we can describe the two procedures of hollow microcapsule
351
formation as illustrated in Scheme 1: ion-induced phase separation and heat-induced
352
microcapsule formation. The presence of sodium ions neutralized the surface charges of proteins
353
(Mulvihill & Kinsella, 1988) and promoted protein-protein association forming protein clusters,
354
which stacked together yielding a particulate type or strand type protein floc; there were single
355
clusters remained as shown in Figure 5. Peng et al. (2016) had similar observations in ion or acid
356
induced soymilk prior to gel formation and the morphology of flocs were pH and ion-strength
357
dependent. Thereafter, when the phase separated system was heated at 80 oC, soy proteins started
358
to denature (Nagano, Akasaka, & Nishinari, 1994), accompanied by exposure of hydrophobic
359
groups and aggregate/gel formation (Wang et al., 2017). As mentioned by Ferry (1948), protein
360
gelling involved unfolding or dissociation of proteins and then association or aggregation
361
reactions, where the rate of the first step was faster than the second. The ongoing denaturation
362
allowed protein-protein interactions mainly driven by hydrophobic interactions (Hashizume &
363
Watanabe, 1979; Peng et al., 2016).
17
364
Regarding to the structural evolution of microcapsules or microgels, we propose the following
365
procedures as illustrated in Scheme 1, based on our results of microscopic observations,
366
solubility tests and zeta potential measurements. (1) Ion-induced protein flocs partially re-
367
dispersed into the periphery solution at the start of heating within 60 s, possibly due to
368
dissociation reaction or an increase of protein solubility, contributing to higher NSI values in
369
ST3. (2) the gelling or aggregation process occurred simultaneously and can be described by the
370
LENP model (Andrews & Roberts, 2007) following a process of nucleation and nucleation-
371
dependent aggregation (polymerization and then condensation). Permanent crosslinks between
372
proteins formed, e.g. intermolecular disulfide bonds (Lakemond, de Jongh, Hessing, Gruppen, &
373
Voragen, 2000) and the shell became rigid, although the driving force of the hollow structure
374
development in microcapsules remained unclear and more efforts were required. (3) The strand
375
type protein flocs may be split into several smaller particles or rearrange into one large particle.
376
(4) Microgels formation was favored when the flocs had a diameter smaller than 4.4 µm, because
377
there could be more protein gelled than re-dispersed.
378
Cochereau et al. (2019) heated 2 g/L pea protein isolate solution (pH 6.3) at 40 oC for 2 min
379
and found pH-induced phase separated microdomains (spherical) transforming to hollow
380
microcapsules, where the former had a mean diameter of 4.9±1.1 µm and the later was 7.2±1.9
381
µm. Although the authors failed to explain why the later was larger than the former, they also
382
proposed that formation of the hollow pea protein microcapsules was due to the vacuoles growth
383
within the spherical protein microdomains when protein partially redistributed during heating,
384
which was followed by formation of permanent crosslinks between pea proteins. However, our
385
results were different as it was found that there were no evident relevance between the
386
morphology of protein flocs, i.e. either spherical or not, and hollow microcapsule formation.
18
387
Nishinari et al. (2014) mentioned that globular proteins can convert into varying intermediate
388
states when denatured and their actual structure or format were still unclear. As a result, due to
389
the complexity of protein structure and high aqueous-environment dependence of the
390
morphologies, continuous efforts are required to clarify the microcapsule formation process and
391
to further explore the kinetics of protein flocs-microcapsule transformation.
392 393 394
4 Conclusions
395
The present work has shown the morphological evolution of protein aggregates during protein
396
flocs-microcapsule transformation upon heating at 80 oC. A mechanism of microcapsule
397
formation has been proposed based on the microscopic observations and solubility and zeta
398
potential measurements at different stages of the transformation process. It was found that phase
399
separation of soy 11s fraction, induced by the presence of 0.05 M NaCl, contributed to the
400
formation of protein clusters and irregular protein flocs. During the first 30 to 60 s of heating, the
401
protein partially re-dispersed into the solution, yielding higher NSI values, where the remaining
402
protein gradually gelled and formed either spherical hollow microcapsules or microgels upon the
403
subsequent heating. The phenomena of ion-induced phase separation and heat-induced gelation
404
were both irrelevant to specific protein components. The 2 g/L sample was mainly composed of
405
small microgels after heating and those with protein concentration of 5 and 10 g/L were
406
dominated in hollow microcapsules. The microgels formed when the radius was close to the wall
407
thickness between approximately 0.7 and 2.2 µm. A better understanding of the process of
408
hollow microcapsule formation using pure plant-based protein can benefit in future size
19
409
reduction, bio-conjugation and tailored structure design of this type of hollow microcapsules,
410
allowing their broader applications in industry.
411 412
Supporting Information: The following file is available free of charge. Appendix A: Changes
413
in zeta potential values of samples during heating. Appendix B: Representative original and
414
processed images (ImageJ processing).
415 416 417
Funding Sources: This work was a part of the research project of China Postdoctoral Science Foundation [grant No. 2019M652091].
418 419
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Cochereau, R., Nicolai, T., Chassenieux, C., & Silva, J. V. C. (2019). Mechanism of the
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Feng, Y., & Lee, Y. (2017). Microfluidic Fabrication of Hollow Protein Microcapsules for Ratecontrolled Release. RSC Advances, 7(78), 49455-49462. Ferry, J. D. (1948). Protein Gels. In M. L. Anson & J. T. Edsall (Eds.), Advances in Protein Chemistry (Vol. 4, pp. 1-78): Academic Press.
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Kastelic, M., Kalyuzhnyi, Y. V., Hribar-Lee, B., Dill, K. A., & Vlachy, V. (2015). Protein
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Lakemond, C. M. M., de Jongh, H. H. J., Hessing, M., Gruppen, H., & Voragen, A. G. J. (2000).
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Heat Denaturation of Soy Glycinin: Influence of pH and Ionic Strength on Molecular
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Chloride and Calcium Chloride on the Rheological and Structural Properties of Gels.
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Nagano, T., Akasaka, T., & Nishinari, K. (1994). Dynamic Viscoelastic Properties of Glycinin
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Biomolecules, 34(10), 1303-1309.
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Nishinari, K., Fang, Y., Guo, S., & Phillips, G. O. (2014). Soy Proteins: A Review on
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Composition, Aggregation and Emulsification. Food Hydrocolloids, 39, 301-318.
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Peng, X., Ren, C., & Guo, S. (2016). Particle Formation and Gelation of Soymilk: Effect of Heat.
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Renkema, J. M. S., Gruppen, H., & van Vliet, T. (2002). Influence of pH and Ionic Strength on
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Heat-Induced Formation and Rheological Properties of Soy Protein Gels in Relation to
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Denaturation and Their Protein Compositions. Journal of Agricultural and Food
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Chemistry, 50(21), 6064-6071.
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Rivera, M. C., Pinheiro, A. C., Bourbon, A. I., Cerqueira, M. A., & Vicente, A. A. (2015).
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Hollow Chitosan/alginate Nanocapsules for Bioactive Compound Delivery. International
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Journal of Biological Macromolecules, 79, 95-102. Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature methods, 9(7), 671. Schramm, L. L. (2014). Emulsions, Foams, Suspensions, and Aerosols: Microscience and Applications (2 ed.). Weinheim, Germany: Wiley-VCH.
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Totosaus, A., Montejano, J. G., Salazar, J. A., & Guerrero, I. (2002). A Review of Physical and
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Chemical Protein-gel Induction. International Journal of Food Science & Technology,
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37(6), 589-601.
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Wan, Z., Guo, J., & Yang, X. (2015). Plant Protein-based Delivery Systems for Bioactive Ingredients in Foods. Food & Function, 6(9), 2876-2889.
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Wang, X., He, Z., Zeng, M., Qin, F., Adhikari, B., & Chen, J. (2017). Effects of the Size and
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Content of Protein Aggregates on the Rheological and Structural Properties of Soy
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Protein Isolate Emulsion Gels Induced by CaSO4. Food Chemistry, 221, 130-138.
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Wolf, W. J. (1970). Soybean Proteins. Their Functional, Chemical, and Physical Properties.
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Journal of Agricultural and Food Chemistry, 18(6), 969-976.
493
Wu, S., Murphy, P. A., Johnson, L. A., Fratzke, A. R., & Reuber, M. A. (1999). Pilot-plant
494
Fractionation of Soybean Glycinin and β-conglycinin. Journal of the American Oil
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Chemists' Society, 76(3), 285-293.
496 497
Zhang, Y., Guan, Y., & Zhou, S. (2005). Single Component Chitosan Hydrogel Microcapsule from a Layer-by-Layer Approach. Biomacromolecules, 6(4), 2365-2369.
498 499
23
500
Figure Captions
501 502
Figure 1. Sedimentation of protein flocs or particles in samples with varying protein
503
concentration after 40 min of sample making. The red arrows indicate precipitates at the bottom.
504
A: after heating; B: prior to heating.
505 506
Figure 2. Representative images showing protein flocs formed prior to heating (A1-C1: CLSM)
507
and protein particles formed after heating (A2-C2: CLSM; A3-C3: phase microscopy).
508 509
Figure 3. Particle size distribution of microcapsules or microgels formed in samples with
510
varying protein concentration after 20 min heating at 80
511
the value of bin center was displayed on x-axis.
. Each bin has a bin size of 0.5 µm and
512 513
Figure 4. CLSM images illustrating evolution in morphology of protein flocs or particles
514
distributed in the 5 g/L sample during heating at 80 oC for 0 s (A1 and A2), 15 s (B1 and B2), 30
515
s (C), 45 s (D), 60 s (E), 240 s (F) and 1200 s (G).
516 517
Figure 5. CLSM images showing the morphology of protein aggregates distributed in the 5 g/L
518
sample after heating at 80 oC for 15 s (A) and 30 s (B). Cross-sectional images of P1 (P: particle)
519
from top to bottom (focusing at different depth) were shown.
520
24
521
Figure 6. Changes in NSI values (NSI1, NSI4 and NSI3) of the solutions (ST1, ST2 and ST3,
522
respectively) during sample preparation. Different superscript letters denote significant
523
differences at P<0.05. Values are means ± standard error (n=3).
524 525
Figure 7. Changes in zeta potential values of samples with protein concentration of 2 and 5 g/L
526
during heating. Points are means ± standard error (n=3).
527 528
Figure 8. Differences in electrophoretic patterns of the solutions (ST1, ST2 and ST3) with
529
varying protein concentration.
530 531
Scheme 1. Diagram illustrating the changes in protein-protein interactions during microcapsule/
532
microgel formation. R: re-dispersion; G: gelation.
533 534
25
Highlights
Morphological evolution of protein aggregates in the aqueous phase was shown.
The key protein flocs-microcapsule transformation occurred within 60 s of heating.
Microgels formed instead of microcapsules when the radius was smaller than 2.2 µm.
Hollow microcapsule formation were irrelevant to specific protein components.
A potential microcapsule formation routine was proposed based on the results.
Declarations of Interest: None.
S0268-005X(19)31271-8
DOI:
https://doi.org/10.1016/j.foodhyd.2019.105379
Reference:
FOOHYD 105379
To appear in:
Food Hydrocolloids
Received Date: 16 June 2019 Revised Date:
22 August 2019
Accepted Date: 10 September 2019
Please cite this article as: Zhao, H., Guo, M., Ding, T., Ye, X., Liu, D., Exploring the mechanism of hollow microcapsule formation by self-assembly of soy 11s protein upon heating, Food Hydrocolloids (2019), doi: https://doi.org/10.1016/j.foodhyd.2019.105379. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Exploring the mechanism of hollow microcapsule
2
formation by self-assembly of soy 11s protein upon
3
heating
4
Huanhuan Zhao a, Mingming Guo a,b, Tian Ding a,b, Xingqian Ye a,b, Donghong Liu a,b,c,
5
a
College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agro-Food
6
Processing, Zhejiang R&D Center for Food Technology and Equipment, Zhejiang University,
7
Hangzhou 310058, Zhejiang, China b
8 9
c
Fuli Institute of Food Science, Hangzhou 310058, Zhejiang, China
Ningbo Research Institute, Zhejiang University, Ningbo 315100, Zhejiang, China
10
Abstract: This study investigates the phenomenon of hollow microcapsule formation through
11
simple heat treatment of soy 11s protein solution. Microscopies were used to monitor the
12
morphological changes of proteins in the aqueous phase during fabrication; the corresponding
13
changes in zeta potential, protein solubility and composition were examined. In the presence of
14
0.05 M sodium chloride, the 11s protein self-assembled into irregular flocs, which transformed
15
into microgels or hollow microcapsules upon heating at 80
16
microcapsule transformation occurred within the first 60 s of heating and the microcapsules
17
formed their perfect hollow spherical structure in 4 min of heating. A mechanism was proposed
1
for 20 min. The key protein flocs-
18
to describe the hollow structure formation in microcapsules. The samples with protein
19
concentration of 2 g/L had more microgels formed, while those with 5 and 10 g/L protein was
20
mainly composed of hollow microcapsules. It was found that as the particle radius approached
21
the wall thickness, microgels formed instead of microcapsules.
22 23
Keywords: hollow microcapsule; microgel; soy 11s protein; self-assembly; protein aggregates
2
24
1 Introduction
25
Design of hollow microcapsules (or nanocapsules) has been receiving particular interest due to
26
their wide applicability; for example, hollow microcapsules can be developed as delivery
27
systems of active components, catalytic systems and absorbents for removal of hazardous
28
compounds in aqueous phase (Loiseau et al., 2017). Hollow microcapsules have the potential to
29
load more core materials compared to the solid (i.e. homogenous porous) spheres and can have a
30
more sustained release as the substrates are entrapped inside the hollow structure (Rivera,
31
Pinheiro, Bourbon, Cerqueira, & Vicente, 2015).
32
Hollow microcapsules are commonly prepared through self-assembly of wall materials onto
33
the surface of either a solid or a liquid template (Wan, Guo, & Yang, 2015). A layer-by-layer
34
method has been widely used, which permits the formation of microcapsules with engineered
35
features upon dissolution of the sacrificial solid core (Zhang, Guan, & Zhou, 2005). Another
36
approach is through fabrication of colloidosomes with the colloidal particles assembled around
37
the templating droplets (Lee & Weitz, 2009). In addition to the interfacial self-assembly of wall
38
materials at the solid-liquid or liquid-liquid boundaries, another strategy of hollow microcapsule
39
fabrication is through double emulsions with the wall materials incorporated into the middle
40
phase (Loiseau et al., 2017). In spite of a higher encapsulating capability and potential better
41
controlled release performance of hollow microcapsules, conventional approaches to assemble
42
such microcapsules are usually complex and time consuming and often involve addition of toxic
43
solvents to remove the sacrificial templates, performed as temporary cores, during the processes
44
(Feng & Lee, 2017; Rivera et al., 2015).
45
Natural biopolymer-based delivery systems have attracted much attention owning to their
46
advantages of biodegradability and biocompatibility (Larrañaga, Lomora, Sarasua, Palivan, &
3
47
Pandit, 2017). Soy protein, as a natural polymer, has been commonly used as a starting material
48
to prepare polymeric delivery systems for bioactive compounds; it is an abundant resource,
49
which has desirable water solubility and non-immunogenic and anti-carcinogenic properties
50
(Maltais, Remondetto, & Subirade, 2009). Recently, spontaneous formation of hollow
51
microcapsules through heating of pure plant-based protein solutions has been reported. To the
52
best of our knowledge, Chen, Zhao, Nicolai, and Chassenieux (2017) was the first found
53
spontaneous formation of hollow microcapsules under heating using pure legume protein
54
solutions. The authors demonstrated ion-induced microphase separation of native soy glycinin
55
solution, based on which, hollow microcapsules were produced by heating the dispersion above
56
60
57
and a wall thickness of approximately 1 µm. Later, Cochereau, Nicolai, Chassenieux, and Silva
58
(2019) also reported hollow microcapsule formation in pea protein isolate solution through pH-
59
induced phase separation and subsequent heating above 40
60
such self-assemble ability was not solely for specific types of plant proteins.
for more than 5 min; the resulting microcapsules had a diameter ranging from 1 to 40 µm
for 2 min and they suggested that
61
The aforementioned spontaneous formation of plant protein-based hollow microcapsules has
62
several advantages over the traditional methods such as a simpler fabrication routing and no
63
requirements of sacrificial core materials, chemical cross-linkers or emulsion templates. The
64
hollow microcapsules produced from the new method is stable and can keep their integrity
65
between a pH range from 1 to 11.5; the microcapsules has desirable pH responsive permeability
66
of FITC-dextran (Chen, Zhang, Mei, & Wang, 2018). Despite the above advantages and potential
67
applications, the driving forces and underlying mechanisms of the microcapsule fabrication are
68
unknown, more specifically, spontaneous formation of the shell and disappearance of proteins at
69
the center during heating. Chen et al. (2017) hypothesized that the increased attractive
4
70
interactions drove densification of soy protein within the protein microdomains as a result of the
71
configurational changes during heating, while Cochereau et al. (2019) attributed it to possible
72
redistribution of protein from the core of microdomains to solution and formation of permanent
73
protein-protein crosslinks at the shell. However, there is still a lack of study in the literature to
74
validate any of the hypotheses and to reveal the actual mechanisms of this new protein self-
75
assembly process.
76
Therefore, the objective of this research was to (1) explore the key phenomena involved at
77
each stage of hollow microcapsule formation using soy 11s protein and to (2) gain further
78
insights into the mechanisms through monitoring the changes in morphology of protein
79
flocs/particles and the corresponding changes in composition and properties of the aqueous
80
systems.
81 82 83
2 Materials and Methods
84 85
2.1 Isolation of soy 11s protein
86
Soy 11s protein was isolated from defatted soy flour, which was offered by Sinoglory Health
87
Food (Shangdong, China). The isolation method of Wu, Murphy, Johnson, Fratzke, and Reuber
88
(1999) was adopted with minor modifications. Firstly, the defatted soy flour was dispersed in
89
deionized water with a weight ratio of 1:20, using a magnetic stirrer (30 min, 1200 rpm). The pH
90
was then modified to 7.5 using 2 M sodium hydroxide, after which the mixture was centrifuged
91
by a Sigma 3K15 centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) at
92
9500 rpm for 30 min (4
). Then, sodium bisulfite powder was dissolved into the supernatant at
5
93
a concentration of 0.98 g/L, prior to pH adjustment to 6.4 using 2 M HCl. The turbid dispersion
94
was stored in a refrigerator (4
95
The precipitates obtained was quickly rinsed with deionized water for 3 times and then re-
96
dispersed in deionized water. The solution was finally freeze-dried and the dry powder of 11s
97
protein was stored in a refrigerator set to 4
98
.
99
2.2 Microcapsule preparation
100
) for 20 h and then centrifuged at 7000 rpm for 30 min (4
).
.
Hollow microcapsule dispersion was prepared following Chen et al. (2018) with some
101
modifications. Isolated soy 11s protein powder was firstly dissolved in deionized water at 20
.
102
Three batches were prepared with protein concentration of 4, 10 and 20 g/L, respectively, where
103
each batch was centrifuged at 9000 rpm (20
104
centrifuge (Keda Chuangxin, Hefei, China) to remove insoluble fraction. Each solution was then
105
combined with an equal amount of 0.1 M sodium chloride (NaCl) solution under magnetic string
106
at 700 rpm for 1 min; each new turbid dispersion has a final ionic strength value of 0.05 M and
107
protein concentration of approximately 2, 5 and 10 g/L, respectively. Thereafter, the dispersion
108
was immediately immerged in a hot water bath with a temperature of 80±2
109
the time of immersion. Hollow microcapsules or homogeneous microgels formed spontaneously
110
during heating.
) using a HC 3018R high speed refrigerated
for 20 min from
111 112
2.3 Microscopic observations
113
Aliquots of soy protein solutions in the presence of 0.05 M NaCl were heated at 80
water
114
bath for 0, 15, 30, 45, 60, 120, 240 and 1200 s, respectively. A Leica TCS SP8 confocal laser
115
scanning microscopy (CLSM; Leica Microsystems CMS GmbH, Wetzlar, Germany) was used to
6
116
observe the difference in morphology of protein flocs or particles (microcapsules/microgels) in
117
aqueous phase. Approximately 3 mL of sample was taken and stained with Rhodamine B
118
(Yuanye Bio-Technology Co., Ltd., Shanghai, China) at a concentration of 1 ppm. A drop of
119
sample was then mounted onto a glass slide, which was viewed using a 63× water immersion
120
objective lens. A laser beam at 561 nm was used and the fluorescence intensity was recorded
121
between 560 to 660 nm. The measurements were carried out immediately after sample making.
122
A UPH 203i Phase Microscope (Aopu Photoelectric Technology Co., Ltd., Chongqing, China)
123
was also used for observation and image acquisition by a 10× objective lens.
124 125
2.4 Particle size characterization
126
ImageJ software (Schneider, 2012) was employed to separate particles from the CLSM images
127
of the samples after 20 min (1200 s) heat treatment. Holes of hollow particles were filled during
128
image processing, and particles smaller than 0.14 µm2 were eliminated for the aim of removing
129
redundant pixels or hazy particles. Equivalent diameter of each particle was calculated based on
130
the area obtained by the software, assuming the particles as perfect spheres. The size values were
131
then grouped into a continuous series of size bins with a bin width of 0.5 µm, using OriginPro
132
2018 software (OriginLab, Northampton, MA). The particle size distribution was represented
133
based on the relative frequency in percentage as a function of equivalent diameter (bin center
134
value). Coefficient of variation (CV) was calculated to evaluate the particle size monodispersity
135
using the following equation.
136 137 138
CV (%) =
× 100% (1)
Where standard deviation (STD) and average equivalent diameter (ADE) of each type of samples (2, 5 and 10 g/L) were obtained using the OriginPro software.
7
139 140
2.5 Protein concentration and solubility measurements
141
After the aforementioned 1:1 combination of protein solution and NaCl solution, the dispersion
142
became turbid, either prior to heating or after heating and there were significant phase separation
143
after 40 min of sample making (Figure 1). Accordingly, the turbid dispersions were centrifuged
144
immediately after sample preparation at 9000 rpm, 20
145
refrigerated centrifuge and the supernatants containing NaCl were collected and defined as
146
Solution Type 2 (ST2, prior to heating) and ST3 (after heating), respectively. The original
147
protein solutions with concentration of 4, 10 and 20 g/L were also diluted with an equal amount
148
of deionized water and the new solutions had final protein concentration of 2, 5 and 10 g/L,
149
respectively, without the presence of NaCl; these solutions were named as ST1.
using the HC 3018R high speed
150
The total protein concentration of the isolated soy 11s protein powder was determined by the
151
Kjeldahl method in duplicates based on the nitrogen content and a Kjeldahl factor of 6.25 was
152
used (Renkema, Gruppen, & van Vliet, 2002). Then, the protein content of ST1, ST2 and ST3
153
was measured based on Bradford (1976) using bovine serum albumin as the standard. The
154
solubility of protein in the solutions was estimated using nitrogen solubility index (NSI):
155
percentage of soy protein in solutions (the Bradford method) over the total protein content (the
156
Kjeldahl method). Moreover, the pH values of the three types of solutions were measured using a
157
digital pH meter (Five Easy Plus, Mettler Toledo, Columbus, USA).
158 159
2.6 Electrophoresis
160
SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed using
161
a 12% acrylamide resolving gel and a 5% acrylamide stacking gel. The composition of ST1, ST2
8
162
and ST3 was examined after proper dilution. Aliquots of 5 µL sample (a combination of protein
163
solution and buffer solution) per well were loaded on to the gel. The electrophoresis was run
164
with an electrophoresis cell connected to a Bio-Rad PowderPac Basic power supply (Hercules,
165
CA, USA) at 80 V and continued with 120 V when the protein reached the resolving gel. After
166
electrophoresis, the gel was stained with Coomassie brilliant blue R250 solution for 30 min on a
167
shaker and then it was rinsed with deionized water, after which it was destained using a mixture
168
of deionized water, methanol and acetic acid several times during a period of 24 h.
169 170
2.7 Changes in Zeta potential
171
Zeta potential was measured using a Malvern Zetasizer (Nano ZS90, Malvern Instruments,
172
Worcestershire, U.K). Samples heated for 0, 15, 30, 45, 60, 120, 240, 480 and 1200 s were used;
173
the first series of samples with protein concentration of 2 g/L were diluted with 0.05 M NaCl
174
buffer for 10 times, while the second series with protein concentration of 5 g/L were diluted for
175
25 times. The samples with protein concentration of 10 g/L was not measured as there were
176
particles or aggregates beyond the measurement range, i.e. >100 µm. Each sample was loaded
177
into a DTS1070 cuvette and the measurements were controlled by the Zetasizer software.
178 179
2.8 Statistical analysis
180
The measurements of zeta potential, protein concentration and solubility were carried out three
181
times with three replication for each individual measurement unless otherwise stated and the
182
values were reported as means ± standard error. Analyze of variance was performed in SPSS
183
software (SPSS 17.0, IBM Corp., NY, USA). The protein concentration and NSI values were
184
evaluated using one-way ANOVA with a post hoc Tukey test, at a significance level of 95%.
9
185 186 187
3 Results and Discussion
188 189
3.1 Effect of protein concentration on microcapsule formation
190
3.1.1 Microscopic observations
191
The process of microcapsule formation can be split into two procedures: ion-induced phase
192
separation and heat-induced particle formation. According to Figure 2 (A1, B1 and C1), it can be
193
seen that, prior to heating, soy protein flocs appeared in the presence of 0.05 M NaCl and with a
194
higher protein concentration (2, 5 and 10 g/L), the size of flocs became larger and there was
195
denser spatial distribution of the flocs. The phase separation phenomenon of protein solution
196
caused by ion addition has been extensively studied, commonly reported for gel formation
197
(Totosaus, Montejano, Salazar, & Guerrero, 2002). In this study, the surface charges were
198
screened by addition of Na+ and as the electrostatic repulsion decreased, proteins flocculated at
199
their native states (low ionic strength) through weak non-covalent links, i.e. hydrogen bonds, van
200
der Waals force or hydrophobic interactions; such aggregating state was reversible depending on
201
solution conditions (Kastelic, Kalyuzhnyi, Hribar-Lee, Dill, & Vlachy, 2015; Peng, Ren, & Guo,
202
2016).
203
The sizes of spherical particles after heat treatment also increased with the protein
204
concentration (Figure 2, A2, B2 and C2), which was consistent with Chen et al. (2017) using soy
205
glycinin concentration of 8-15 g/L. The hollow microcapsules had a single cavity or vacuoles at
206
the center, in which there could be smaller protein aggregates remained. In addition to the hollow
207
microcapsule formation, small homogeneous microgels presented in all samples. Particularly, in
10
208
Figure 2 (A2), hollow microcapsules were rarely identified and there was a significant amount of
209
microgel aggregates. The wall thickness of the hollow microcapsules were estimated to be
210
between approximately 0.7 and 2.2 µm based on the CLSM micrographs, where larger hollow
211
microcapsules had thicker walls; an example was shown in Figure 2 (C2). As such it was
212
reasonable to expect that the vacuoles can hardly form within a single spherical particle when the
213
size of the particles was small, e.g. with a radius smaller than 2.2 µm. This renders that wall
214
thickness is a crucial factor that determines the lower limit of the size of hollow microcapsules.
215
The morphology of the protein flocs in Figure 2 (A1, B1 and C1) was irregular. Chen et al.
216
(2017) had similar observations of irregular protein flocs in 10 g/L soy glycinin solution with
217
0.05 M NaCl and pH of 7.2, while spherical microdomains formed when the NaCl concentration
218
increased to 0.1 M; however, both types of dispersion had desirable formation of hollow
219
microcapsules under heating (Chen et al., 2018; Chen et al., 2017). In addition, as the protein
220
flocs were relatively unstable, their morphology could be mixing-speed dependent just before
221
heating (Cochereau et al., 2019). Thus, there was no evident correlation found between the
222
morphology of phase separated flocs and the formation of hollow microcapsules.
223 224
3.1.2 Particle size distribution
225
The size distribution of particles in each type of samples after 20 min heating at 80
were
226
presented in Figure 3. The majority of particles had equivalent diameter smaller than 15 µm;
227
however, there were 5.5% and 9.1% particles exceeded this size boundary in the 5 and 10 g/L
228
samples, respectively. Although there was presence of large microcapsule aggregates as
229
identified in Figure 2 (B3 and C3) and microgel aggregates as displayed in Figure 2 (A2), the
230
former was eliminated and the later was separated into individual particles with care during
11
231
image processing. The average equivalent diameter values of the three types of samples were 2.8
232
µm (2 g/L), 6.6 µm (5 g/L) and 6.6 µm (10 g/L), and their corresponding CV values were 47.4%,
233
65.9% and 89.5%, respectively. A higher CV value indicates less homogeneous or uniform
234
particles. This was in good agreement with the observation in Figure 3 that the size distribution
235
of particles in the 2 g/L samples exhibited narrower unimodal, while the size of particles in the 5
236
and 10 g/L samples had more significant variation. Despite the same mean size values of the 5
237
and 10 g/L samples, the size distributions were different and the 10 g/L sample had the highest
238
CV value.
239
Due to the limited resolution of CLSM, only the visible particles were taken into account
240
during image processing. The 2 g/L sample had the largest proportion of small particles with a
241
size range between 0 and 4.5 µm, approximately 89.9%. Hence, the 2 g/L sample was dominated
242
by microgels, different form the 5 and 10 g/L samples composed of both microgels and
243
microcapsules. The size of hollow microcapsules were generally larger than the microgels. It can
244
be proposed that when the protein concentration was low, there were less opportunities of
245
protein-protein interactions and therefore, smaller “solid” protein particles can form upon
246
heating.
247 248
3.2 Microstructural changes of protein particles upon heating
249
Samples with protein concentration of 2 g/L formed numerous small microgels, while those
250
with protein concentration of 10 g/L had more large microcapsules and microcapsule aggregates.
251
As a result, samples with protein concentration of 5 g/L was selected and heated at 80
252
different time duration. The CLSM micrographs in Figure 4 illustrate the continuous changes in
253
particle morphology during the protein flocs-microcapsule transformation in the presence of 0.05
12
for
254
M NaCl upon heating. It has to be noted, however, that there could be changes in the
255
particle/aggregate morphology due to cooling once the samples were taken for CLSM
256
measurements; therefore, the particle/aggregate morphology shown in Figure 4 may be different
257
from the scenario of 20 min heating at 80
258
function of time.
with continuous changes in protein structure as a
259
The soy protein flocs (Figure 4, A1) exhibited random shapes prior to heating, similar to those
260
in Figure 2 (A1, B1 and C1). Interestingly, however, “hollow” structure or cavities inside the
261
flocs sporadically appeared as presented in Figure 4 (A2), different to those in Figure 4 (A1),
262
although the samples were independently prepared with the same solution conditions. This was
263
ascribed to the strong sensitivity of the morphology of such protein system to solution conditions
264
as suggested by Cochereau et al. (2019), and Chen et al. (2017) also had similar inconsistent
265
observations in soy glycinin system with pH between 4-6.4 and NaCl concentration between 0.4-
266
0.5 M. This was an implication that the protein particles tent to interact with each other forming
267
hollow structure although the driving forces and trigger conditions currently remained unclear.
268
Despite the slight divergent findings in the morphology of protein flocs during the first 15 s of
269
heating (Figure 4, A1-B1 or A2-B2), hollow microcapsules started to form into their shapes from
270
30 s of heating. As heating proceeded, the main changes were within each single microcapsules:
271
single vacuoles formation at the center and perfection of the spherical shapes (Figure 4, C-E).
272
From 4 min (240 s) of heating there were minimal changes (Figure 4, F-G). Therefore, the most
273
important transformation from ion-induced flocs to hollow microcapsules occurred rapidly
274
within 30 s of heating at 80
275
worth mentioning that the variations in particle size of microcapsules among the micrographs
, followed by post-heating for microcapsule perfection. It was
13
276
was due to the difference of position in focus during image acquisition as there could be phase
277
separation.
278
Figure 5 (A) showed CLSM cross-sectional images of a particulate type floc (P1), focused at
279
varying depth after heating for 15 s and strand type flocs and cavities within them were also
280
indicated using blue arrows. Each floc was composed of individual spherical protein clusters,
281
which were self-associated with each other and there were also a few free clusters suspended in
282
the peripheral solution; the diameter of protein clusters were estimated to be approximately 2-5
283
µm. P1 was composed of loosely packed protein clusters with cavities within it; however, it was
284
not a hollow sphere. Figure 5 (B) showed microstructural details of the intermediate-state hollow
285
microcapsules after heating for 30 s, where the particles became more spherical in shape and
286
vacuoles presented at the center, although some particles inter-connected with each other as
287
indicated (P2 and P3). These inter-connected particles could separate or merge into one
288
microcapsule during subsequent heating.
289 290
3.3 Changes in protein solubility during microcapsule formation
291
The total protein concentration of the isolated soy 11s protein was determined as 94.34±0.47
292
wt %; therefore, it was shown in Table 1 that the actual protein concentration (C0) of the 2, 5 and
293
10 g/L samples were calculated as approximately 1.89, 4.72 and 9.43 g/L, respectively. With the
294
removal of insoluble protein fraction by centrifugation, the protein concentrations in ST1 (C1)
295
decreased to 1.48, 3.71 and 7.68 g/L, respectively. It is noticeable that the concentration values
296
of ST2 (C2: 043, 1.76 and 2.96) were significantly (P<0.05) smaller than those of ST3 (C3: 0.6,
297
2.45 and 4.64) for each type of sample, which suggested that the soy proteins in ion-induced
298
flocs partially re-dispersed/dissolved into the solution upon heating, resulting in higher protein
299
concentration values in ST3.
14
300
In Figure 6, the NSI1 values of ST1 solutions, being estimated between 0.79 and 0.81, were not
301
statically different (P>0.05) among the three types of samples. This was because ST1 solutions
302
had pH values ranging from 7.34 to 7.44 which contributed to high solubility of soy globulins in
303
water as the pH was away from the isoelectric point, approximately 4.9-5.2 (Golubovic, van
304
Hateren, Ottens, Witkamp, & van der Wielen, 2005; Wolf, 1970). For each type of samples, the
305
changes in NSI values had a similar trend as the changes in concentrations, i.e. a decrease by
306
adding NaCl and then a slight increase because of heating. These results reveal that the proteins
307
had the lowest solubility due to ion-induced phase separation and the solubility increased slightly
308
due to the conformational changes of protein during heating; the pH values of ST2 and ST3
309
solutions varied between 6.72 and 6.85. The NSI2 and NSI3 values of the 2 g/L sample were both
310
significantly (P<0.05) lower than those of the 5 and 10 g/L samples and it meant higher
311
productivity of microgels than the microcapsules.
312 313
Table 1. Protein concentration (C) of each type of solutions and supernatants.
Sample
Actual concentration
C1
C2
C3
ST1
ST2
ST3
g/L
314 315
2 g/L
1.89
1.48±0.01 a
0.43±0.03 b
0.60±0.11 c
5 g/L
4.72
3.71±0.01 a
1.76±0.02 b
2.45±0.04 c
10 g/L
9.43
7.68±0.01 a
2.96±0.02 b
4.64±0.05 c
Note: different superscript letters in each row denote significant differences at P<0.05. Values are means ± standard error (n=3).
316 317
3.4 Changes in zeta potential during microcapsule formation
15
318
Zeta potential values of the 2 and 5 g/L samples containing 0.05 M NaCl were measured at
319
different stages of heating. It was seen from Figure 7 (the full diagram, Figure A1, has been
320
provided in the Supplementary data) that both samples had an initial value of approximately -
321
11.8 mV, which was followed by a sharp decrease within the first 15 to 30 s. Then it increased
322
quickly until approximately 60 s of heating. The zeta potential value continued to change and
323
after approximately 4 min, it almost kept constant with time. Overall, the curve of 5 g/L sample
324
had a similar trend as the 2 g/L sample in spite of slight shifts. It has to be aware that the
325
morphological difference of the particles in these two samples may influence the zeta potential
326
results. The redistribution of protein from flocs to solution during heating was possibly occur
327
within the first 60 s of heating as indicated by on our CLSM observations and the changes in zeta
328
potential. In addition, the zeta potential values were in a range of aggregation threshold of -11 to
329
-20 (Schramm, 2014); hence, the dispersions were relatively unstable as being observed in
330
Figure 1.
331
Heat treatment generally induced conformational changes of native soy proteins and soy 11s
332
protein could undergo dissociation and then association/aggregation reactions (Nishinari, Fang,
333
Guo, & Phillips, 2014). Therefore, the protein composition of ST1, ST2 and ST3 were compared
334
using SDS-PAGE analysis. The 11s fraction of soy proteins can generally dissociate into smaller
335
2s, 3s or 7s forms depending on pH, ionic strength and heating conditions (Hashizume &
336
Watanabe, 1979; Nishinari et al., 2014). However, in Figure 8, no marked changes in protein
337
electrophoretic patterns was detected apart from the differences in the color intensity, which was
338
ascribed to varying dilution levels. Note that here the protein composition of ST2 and ST3 were
339
similar by comparing A and D, B and E, or C and F in Figure 8, which elucidated in return that
340
the protein composition in flocs prior to heating and particles formed after heating were also
16
341
similar. The solutions were dominant in soy 11s acidic and basic subunits and there were minor
342
amount of 7s β subunits. The molecular mass of the 11s acidic subunits was approximately 35
343
kDa and the 11s basic subunits were approximately 19-20 kDa, which was in line with the values
344
reported in the literature (Hsiao, Yu, Li, & Hsieh, 2015); there were no additional pattern
345
exhibited between the range of 10-25 kDa (not shown). These results rendered that the
346
phenomena of ion-induced phase separation, partial redistribution of proteins during heating and
347
heat-induced microcapsule formation were probably irrelevant to specific protein components.
348 349
3.5 Mechanism of microcapsule formation
350
Relying on the obtained results, we can describe the two procedures of hollow microcapsule
351
formation as illustrated in Scheme 1: ion-induced phase separation and heat-induced
352
microcapsule formation. The presence of sodium ions neutralized the surface charges of proteins
353
(Mulvihill & Kinsella, 1988) and promoted protein-protein association forming protein clusters,
354
which stacked together yielding a particulate type or strand type protein floc; there were single
355
clusters remained as shown in Figure 5. Peng et al. (2016) had similar observations in ion or acid
356
induced soymilk prior to gel formation and the morphology of flocs were pH and ion-strength
357
dependent. Thereafter, when the phase separated system was heated at 80 oC, soy proteins started
358
to denature (Nagano, Akasaka, & Nishinari, 1994), accompanied by exposure of hydrophobic
359
groups and aggregate/gel formation (Wang et al., 2017). As mentioned by Ferry (1948), protein
360
gelling involved unfolding or dissociation of proteins and then association or aggregation
361
reactions, where the rate of the first step was faster than the second. The ongoing denaturation
362
allowed protein-protein interactions mainly driven by hydrophobic interactions (Hashizume &
363
Watanabe, 1979; Peng et al., 2016).
17
364
Regarding to the structural evolution of microcapsules or microgels, we propose the following
365
procedures as illustrated in Scheme 1, based on our results of microscopic observations,
366
solubility tests and zeta potential measurements. (1) Ion-induced protein flocs partially re-
367
dispersed into the periphery solution at the start of heating within 60 s, possibly due to
368
dissociation reaction or an increase of protein solubility, contributing to higher NSI values in
369
ST3. (2) the gelling or aggregation process occurred simultaneously and can be described by the
370
LENP model (Andrews & Roberts, 2007) following a process of nucleation and nucleation-
371
dependent aggregation (polymerization and then condensation). Permanent crosslinks between
372
proteins formed, e.g. intermolecular disulfide bonds (Lakemond, de Jongh, Hessing, Gruppen, &
373
Voragen, 2000) and the shell became rigid, although the driving force of the hollow structure
374
development in microcapsules remained unclear and more efforts were required. (3) The strand
375
type protein flocs may be split into several smaller particles or rearrange into one large particle.
376
(4) Microgels formation was favored when the flocs had a diameter smaller than 4.4 µm, because
377
there could be more protein gelled than re-dispersed.
378
Cochereau et al. (2019) heated 2 g/L pea protein isolate solution (pH 6.3) at 40 oC for 2 min
379
and found pH-induced phase separated microdomains (spherical) transforming to hollow
380
microcapsules, where the former had a mean diameter of 4.9±1.1 µm and the later was 7.2±1.9
381
µm. Although the authors failed to explain why the later was larger than the former, they also
382
proposed that formation of the hollow pea protein microcapsules was due to the vacuoles growth
383
within the spherical protein microdomains when protein partially redistributed during heating,
384
which was followed by formation of permanent crosslinks between pea proteins. However, our
385
results were different as it was found that there were no evident relevance between the
386
morphology of protein flocs, i.e. either spherical or not, and hollow microcapsule formation.
18
387
Nishinari et al. (2014) mentioned that globular proteins can convert into varying intermediate
388
states when denatured and their actual structure or format were still unclear. As a result, due to
389
the complexity of protein structure and high aqueous-environment dependence of the
390
morphologies, continuous efforts are required to clarify the microcapsule formation process and
391
to further explore the kinetics of protein flocs-microcapsule transformation.
392 393 394
4 Conclusions
395
The present work has shown the morphological evolution of protein aggregates during protein
396
flocs-microcapsule transformation upon heating at 80 oC. A mechanism of microcapsule
397
formation has been proposed based on the microscopic observations and solubility and zeta
398
potential measurements at different stages of the transformation process. It was found that phase
399
separation of soy 11s fraction, induced by the presence of 0.05 M NaCl, contributed to the
400
formation of protein clusters and irregular protein flocs. During the first 30 to 60 s of heating, the
401
protein partially re-dispersed into the solution, yielding higher NSI values, where the remaining
402
protein gradually gelled and formed either spherical hollow microcapsules or microgels upon the
403
subsequent heating. The phenomena of ion-induced phase separation and heat-induced gelation
404
were both irrelevant to specific protein components. The 2 g/L sample was mainly composed of
405
small microgels after heating and those with protein concentration of 5 and 10 g/L were
406
dominated in hollow microcapsules. The microgels formed when the radius was close to the wall
407
thickness between approximately 0.7 and 2.2 µm. A better understanding of the process of
408
hollow microcapsule formation using pure plant-based protein can benefit in future size
19
409
reduction, bio-conjugation and tailored structure design of this type of hollow microcapsules,
410
allowing their broader applications in industry.
411 412
Supporting Information: The following file is available free of charge. Appendix A: Changes
413
in zeta potential values of samples during heating. Appendix B: Representative original and
414
processed images (ImageJ processing).
415 416 417
Funding Sources: This work was a part of the research project of China Postdoctoral Science Foundation [grant No. 2019M652091].
418 419
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Wang, X., He, Z., Zeng, M., Qin, F., Adhikari, B., & Chen, J. (2017). Effects of the Size and
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Content of Protein Aggregates on the Rheological and Structural Properties of Soy
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Protein Isolate Emulsion Gels Induced by CaSO4. Food Chemistry, 221, 130-138.
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Wolf, W. J. (1970). Soybean Proteins. Their Functional, Chemical, and Physical Properties.
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Fractionation of Soybean Glycinin and β-conglycinin. Journal of the American Oil
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Zhang, Y., Guan, Y., & Zhou, S. (2005). Single Component Chitosan Hydrogel Microcapsule from a Layer-by-Layer Approach. Biomacromolecules, 6(4), 2365-2369.
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Figure Captions
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Figure 1. Sedimentation of protein flocs or particles in samples with varying protein
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concentration after 40 min of sample making. The red arrows indicate precipitates at the bottom.
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A: after heating; B: prior to heating.
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Figure 2. Representative images showing protein flocs formed prior to heating (A1-C1: CLSM)
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and protein particles formed after heating (A2-C2: CLSM; A3-C3: phase microscopy).
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Figure 3. Particle size distribution of microcapsules or microgels formed in samples with
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varying protein concentration after 20 min heating at 80
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the value of bin center was displayed on x-axis.
. Each bin has a bin size of 0.5 µm and
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Figure 4. CLSM images illustrating evolution in morphology of protein flocs or particles
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distributed in the 5 g/L sample during heating at 80 oC for 0 s (A1 and A2), 15 s (B1 and B2), 30
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s (C), 45 s (D), 60 s (E), 240 s (F) and 1200 s (G).
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Figure 5. CLSM images showing the morphology of protein aggregates distributed in the 5 g/L
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sample after heating at 80 oC for 15 s (A) and 30 s (B). Cross-sectional images of P1 (P: particle)
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from top to bottom (focusing at different depth) were shown.
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Figure 6. Changes in NSI values (NSI1, NSI4 and NSI3) of the solutions (ST1, ST2 and ST3,
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respectively) during sample preparation. Different superscript letters denote significant
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differences at P<0.05. Values are means ± standard error (n=3).
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Figure 7. Changes in zeta potential values of samples with protein concentration of 2 and 5 g/L
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during heating. Points are means ± standard error (n=3).
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Figure 8. Differences in electrophoretic patterns of the solutions (ST1, ST2 and ST3) with
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varying protein concentration.
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Scheme 1. Diagram illustrating the changes in protein-protein interactions during microcapsule/
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microgel formation. R: re-dispersion; G: gelation.
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Highlights
Morphological evolution of protein aggregates in the aqueous phase was shown.
The key protein flocs-microcapsule transformation occurred within 60 s of heating.
Microgels formed instead of microcapsules when the radius was smaller than 2.2 µm.
Hollow microcapsule formation were irrelevant to specific protein components.
A potential microcapsule formation routine was proposed based on the results.
Declarations of Interest: None.