- Email: [email protected]
The effect of adding NaCl on thermal aggregation and gelation of soy protein isolate
Accepted Manuscript The effect of adding NaCl on thermal aggregation and gelation of soy protein isolate Nannan Chen, Mouming Zhao, Christophe Chassenieux, Taco Nicolai PII:
S0268-005X(16)30601-4
DOI:
10.1016/j.foodhyd.2017.03.024
Reference:
FOOHYD 3835
To appear in:
Food Hydrocolloids
Received Date: 14 October 2016 Revised Date:
24 February 2017
Accepted Date: 19 March 2017
Please cite this article as: Chen, N., Zhao, M., Chassenieux, C., Nicolai, T., The effect of adding NaCl on thermal aggregation and gelation of soy protein isolate, Food Hydrocolloids (2017), doi: 10.1016/ j.foodhyd.2017.03.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT The effect of adding NaCl on thermal aggregation and gelation of soy protein isolate
AC C
EP
TE D
M AN U
SC
RI PT
Nannan Chen, Mouming Zhao, Christophe Chassenieux, Taco Nicolai
ACCEPTED MANUSCRIPT 1
The effect of adding NaCl on thermal aggregation and gelation of soy
2
protein isolate
3
Nannan Chen1,2, Mouming Zhao1, Christophe Chassenieux2, Taco Nicolai2
5
1
6
Guangzhou, China
7
2
8
72085 Le Mans cedex 9, France
9
Email : [email protected]
School of Food Science and Engineering, South China University of Technology, 510640,
Tel : (33)-243833139
AC C
EP
TE D
11
SC
LUNAM Université du Maine, IMMM UMR-CNRS 6283, Polymères, Colloïdes et Interfaces,
M AN U
10
RI PT
4
1
ACCEPTED MANUSCRIPT Abstract
13
Thermal aggregation and gelation of soy protein isolate (SPI) was studied at fixed charge
14
density over a wide range of protein concentrations (1-95 g/L), NaCl concentrations (0 - 0.5
15
M) and temperatures (30-85 oC). Gelation was studied with oscillatory shear measurements
16
and aggregation with light scattering. At all conditions, self-similar aggregates were formed
17
by random association of elementary units consisting of dense SPI particles with radii
18
between 30 nm and 50 nm that were formed in a first step of the thermal aggregation process.
19
Two distinct irreversible aggregation processes were identified: one dominating between 30
20
and 45°C and one dominating above 65°C. Addition of salt led to faster aggregation and
21
gelation, but did not influence the gel stiffness at steady state. The size and density of the
22
elementary units of the aggregates increased with increasing NaCl concentration and confocal
23
laser scanning microscopy images showed increasingly heterogeneous gels structures. The
24
effect of varying the ionic strength on thermal aggregation is compared with that of varying
25
the net charge density of the proteins.
26
Key words: soy, protein, aggregation, gelation, rheology, structure
28
Introduction
TE D
27
M AN U
SC
RI PT
12
Soy globulin in the form of soy protein isolate (SPI) is an important food ingredient
30
and has been widely used in the food industry for its good functional and nutritional
31
properties. It contains two main components: glycinin (11S) and β-conglycinin (7S), having
32
molar masses of about 200 kg/mol and 360 kg/mol, respectively (Nishinari, Fang, Guo and
33
Phillips, 2014) . In aqueous solutions, these proteins associate or dissociate to different
34
extents depending on the concentration, pH and ionic strength (Nishinari et al., 2014) .
AC C
35
EP
29
At high temperatures, irreversible aggregation of soy globulin is observed, which is
36
generally considered to be caused by denaturation of the proteins (Nishinari et al., 2014) . The
37
rate of thermal aggregation increases strongly with increasing temperature and SPI
38
concentration and has therefore been mostly investigated at elevated temperatures (T>80°C)
39
where it is rapid (Hua, Cui, Wang, Mine and Roysa, 2005; Renkema and van Vliet, 2002b) .
40
In the absence of salt, the aggregation rate was found to have an Arrhenius temperature
41
dependence characterized by an activation energy (Ea) of 180 kJ/mol (Chen, Zhao,
42
Chassenieux and Nicolai, 2016b) in the range 65°C – 85°C. 2
ACCEPTED MANUSCRIPT A detailed investigation of the influence of the pH between pH 7 and pH 5.8 on
44
thermal aggregation of SPI indentified a two step aggregation process (Chen, Zhao,
45
Niepceron, Chassenieux and Nicolai, 2016c) . In the first step, dense approximately spherical
46
particles are formed that in the second step randomly associate into self-similar aggregates.
47
Decreasing the pH caused an increase of the radius of the primary particles formed in the first
48
step, but did not modify the fractal dimension of the aggregates on larger length scales.
49
Growth of the self similar aggregates led to the formation of a system spanning network at a
50
critical gel time. The aggregation and gelation rate of SPI increased with decreasing pH, but
51
did not significantly influence the activation energy nor the elastic modulus of the gels.
RI PT
43
The effect of salt on thermal aggregation of SPI has also been investigated in the past.
53
The denaturation temperature of glycinin and β-conglycinin was found to increase with
54
increasing NaCl concentration implying that soy proteins have a higher thermal stability in the
55
presence of salt (Bikbov et al., 1983; Jiang, Xiong and Chen, 2010; Renkema, Gruppen and
56
van Vliet, 2002a) . Nevertheless, (Jiang et al., 2010) observed that the turbidity of SPI
57
solutions at pH 7.0 increased more strongly during a heating ramp if 0.1 M or 0.6 M NaCl
58
was added than in deionized water, indicating that aggregation is favored by screening of
59
electrostatic repulsion. (Hua et al., 2005) reported that the stiffness of the gels at C=80 g/L
60
after a given heat treatment (95°C, 15 min) increased with increasing NaCl concentration until
61
approximately 0.2 M, but further increase of the salt concentration led to a slight decrease of
62
the gel stiffness. On the other hand, (Renkema et al., 2002a) did not find a systematic effect
63
of adding 0.2 M or 0.5 M NaCl on the stiffness of SPI gels (C=12 wt%) that were heated at
64
95°C during 1 h. These literature results give some indication about the effect of salt on
65
thermal SPI aggregation, but, as far as we are aware, no detailed systematic investigation has
66
yet been reported.
M AN U
TE D
EP
AC C
67
SC
52
Here, we present an investigation of the effect of salt on the aggregation and gelation
68
of SPI over a wide range of NaCl concentrations (0-0.5 M), protein concentrations (1-95 g/L)
69
and heating temperatures (30-85°C). We have studied the effect of adding salt on the kinetics
70
of the thermal aggregation process, the structure of the aggregates, the elastic modulus of the
71
gels and the microstructure of the gels as a function of the temperature and concentration. The
72
net charge density was fixed at α=-175, where α is expressed in number of charges per protein
73
unit with molar mass 3.0x105 g/mol. It was shown that SPI in pure water forms stable
74
suspensions for α<-150 and are only weakly associated because the electrostatic repulsion is
75
strong (Chen, Zhao, Chassenieux and Nicolai, 2016a) . Notice that if the net charge density is 3
ACCEPTED MANUSCRIPT fixed, the pH decreases with increasing NaCl concentration or protein concentration (Chen et
77
al., 2016a) . The pH of the samples studied here varied between pH 7.2 at low SPI
78
concentrations without added salt to pH 6.4 at 0.5 M NaCl. The effect of screening
79
electrostatic repulsion by adding salt for SPI with high charge density studied here will be
80
compared with the effect of reducing the charge density of SPI in the absence of salt reported
81
elsewhere (Chen et al., 2016c)
RI PT
76
82
Materials and Method
84
Materials
SC
83
Soy protein isolate (SPI) was obtained from defatted soybean flakes (Yuwang Group,
86
China) by precipitation at pH 4.5 using the procedure detailed elsewhere (Chen, Lin, Sun and
87
Zhao, 2014) . The SPI powder contained 94% protein (Kjeldahl, N×6.25). It has been argued
88
that a smaller nitrogen correction factor (5.5) should be used for soy proteins (Mariotti, Tomé
89
and Mirand, 2008) , but we have used 6.25 here for easier comparison with previous results
90
reported by us and others in the literature. Analysis by SDS-page showed that the sample
91
contained about 60% glycinin and 40% β-conglycinin. Protein solutions were prepared in salt-
92
free Milli-Q water with 3 mM NaN3 to avoid bacterial growth. They were centrifuged at room
93
temperature with an Allegra 64R centrifuge (Beckman Coulter, USA) at 5x104 g for 4 h. The
94
ionic strength of the protein solutions was set by adding aliquots of a concentrated NaCl
95
solution (2 or 4 M). After setting of the ionic strength, the solution was put in air-tight glass
96
tubes with diameter of about 10 mm and heated in a temperature control water bath
97
immediately. After heating, the solutions were cooled to room temperature with tap water.
98
Light Scattering.
TE D
EP
AC C
99
M AN U
85
Static (SLS) and dynamic (DLS) light scattering measurements were done using a
100
commercial apparatus (ALV-Langen). The light source was a He–Ne laser with wavelength
101
λ= 632 nm. The temperature was controlled by a thermostatic bath to within ± 0.2 °C.
102
Measurements were made at angles of observation (θ) between 13° and 140°. The relative
103
scattering intensity (Ir) was calculated as the intensity minus the solvent scattering divided by
104
the scattering intensity of toluene at 20°C. In dilute solutions, Ir is related to the weight
105
average molar mass (Mw) and the structure factor (S(q)) of the solute (Brown, 1996; Higgins
106
and Benoit, 1994) 4
ACCEPTED MANUSCRIPT 107
ூೝ
= ܯ௪ ܵ()ݍ
(1)
108 109
with K an optical constant. The initial dependence of S(q) on the scattering wave vector (q)
110
was used to obtain the z-average radius of gyration (Rg).
111
S(q)=(1+q2.Rg2/3)-1
RI PT
112 113
(2)
The normalized intensity autocorrelation functions measured with DLS could be
115
described by a monomodal relaxation time distribution. An apparent q-dependent diffusion
116
coefficient was calculated from the average relaxation rate (Γ): Da=Γ.q-2 (Berne and Pecora,
117
2000) . In dilute solutions and for q→0, Da is equal to the translational diffusion coefficient of
118
the solute (D) and is related to the hydrodynamic radius (Rh):
M AN U
SC
114
119 120
121
=ܦ
୩ ୖ
with η the viscosity, k Boltzmann’s constant, and T the absolute temperature.
124
TE D
122 123
(3)
Rheology
Oscillatory shear measurements were done with a stress imposed rheometer (AR2000,
Instruments)
using
a
cone–plate geometry
(diameter
44
mm).
126
The temperature was controlled by a Peltier system and the geometry was covered
127
with paraffin oil to prevent water evaporation. The storage (G') and loss (G'') moduli
128
were determined in the linear response regime.
129
Confocal Laser Scanning Microscopy (CLSM)
AC C
TA
EP
125
130
CLSM was used in the fluorescence mode. Observations were made at 20°C with
131
a Leica TCS-SP2 (Leica Microsystems Heidelberg, Germany). A water immersion
132
objective lens was used (HCxPL APO 63× NA = 1.2) with theoretical resolution of 75
133
nm in the x–y plane. Soy protein was physically labeled with the fluorochrome rhodamine
134
B, by adding a small amount of a concentrated rhodamine solution to the 95 g/L soy
5
ACCEPTED MANUSCRIPT 135
protein solutions with a final concentration of 5 ppm. The solutions were inserted between a
136
concave slide and a cover slip and hermetically sealed and heated at 80oC during 16 h.
138
3 Results
139
3.1 Influence of the NaCl concentration on gelation
RI PT
137
Figure 1 shows the evolution of the storage shear modulus during heating at 80oC for
141
SPI solutions at C= 95 g/L containing different NaCl concentrations between 0 and 0.4 M. G'
142
increased sharply when SPI gelled. For the gels, G' was almost independent of the oscillation
143
frequency in the range tested (0.01 - 1 Hz) and much larger than the loss modulus (results not
144
shown). The gel time decreased with increasing [NaCl] up to a factor 10 at 0.1 M, but adding
145
more salt did not have an effect on the kinetics. Remarkably, addition of salt did not have a
146
significantly influence on the stiffness of extensively heated gels. This is in agreement with
147
the results by Renkema et al. (2002a) , but appears to contradict the results by Hua et al. (2005)
148
who observed an increase of G' up to 0.2 M NaCl. However, the results can be reconciled if
149
we consider that Hua et al. (2005) compared G' of the gels after a fixed relatively short heat
150
treatment, which confounds the effects on the gelation rate and the gel stiffness.
TE D
M AN U
SC
140
The microstructure of gels obtained after heating for 16 h at 80oC was observed by
152
CLSM. Images of the gels show that the gels were increasingly heterogeneous for [NaCl] ≥
153
0.05 M, see Fig. 2. We note that CLSM images of the samples before heating were
154
homogeneous. The more heterogeneous structure was formed during gelation, i.e. much
155
sooner than 16 h. Further heating did not have a significant influence on the structure once the
156
systems had gelled even though G' continued to increase.
157
3.2 Influence of the temperature on aggregation and gelation
AC C
EP
151
Fig. 3a shows the evolution of G' for SPI solutions at C=95 g/L in the presence of 0.1
158 159
M NaCl during heating at different temperatures between 65°C and 85°C. The gelation rate
160
increased strongly with increasing temperature. The results obtained at different temperatures
161
superimpose when they are plotted as a function of time normalized by the gel time (tg)
162
estimated as the time at which G'=0.1 Pa, see fig. 3c. This shows that the heating temperature
163
did not influence the stiffness of the gels if the difference in the kinetics is properly accounted
164
for. 6
ACCEPTED MANUSCRIPT As was discussed elsewhere for thermal aggregation of SPI in the absence of salt
166
(Chen et al., 2016c) gelation is preceded by the formation of self-similar aggregates that can
167
be characterized by light scattering. Fig. 3b shows the evolution with time of the average
168
molar mass of the aggregates during heating at temperatures between 30 and 70°C. Mw
169
increased with increasing heating time and diverged at tg when a space filling network was
170
formed. The aggregation rate increased with increasing temperature, but, curiously, tg varied
171
little between 45 and 60°C. When Mw is plotted as a function of t/tg, we find that the results
172
obtained at 30, 35 and 40°C superimposed, but the results at higher temperatures deviated for
173
smaller t/tg, see fig. 3c.
RI PT
165
Similar results were obtained for solutions at other NaCl concentrations between 0 and
175
0.4 M NaCl. The temperature dependence of tg could be obtained over a broad range by
176
combining the results of rheology and light scattering, see fig. 4. In the narrow temperature
177
range where both techniques could be used (65-70°C) the same value of tg was obtained. In
178
the absence of salt, a continuous strong decrease of tg with increasing temperature was
179
observed over the whole temperature range where it could be investigated (50-85°C). As was
180
mentioned in the introduction, tg was found to have an Arrhenius temperature dependence
181
between 65°C and 85°C in the absence of salt with Ea=180 kJ/mol independent of the protein
182
concentration and the pH, at least between pH 5.8 and pH 6.8 (Chen et al., 2016c) .
TE D
M AN U
SC
174
It was already shown in fig. 1 for SPI solutions heated at 80°C that addition of NaCl
184
up to 0.1 M increased the gelation rate, but further increase of [NaCl] had no effect on tg. The
185
effect of salt was similar in the temperature range between 65°C and 85°C. However, a
186
remarkably different temperature dependence of tg was observed at lower temperatures. tg was
187
almost independent in the temperature range 40-55°C, 45-60°C and 50-65°C for 0.05, 0.1 and
188
0.4 M NaCl, respectively. At temperatures below this range, tg increased again with
189
decreasing temperature. It appears that addition of salt induces a different aggregation process
190
at low temperatures.
192
AC C
191
EP
183
3.3 Influence of the protein concentration on gelation
193
The effect of the protein concentration on the evolution of G' during heating at 90°C is
194
shown in fig. 5 for SPI solutions at [NaCl] = 50 mM between C=50 g/L and C=95 g/L. For
195
C<50 g/L, very weak gels or precipitates were formed that could not be properly characterized 7
ACCEPTED MANUSCRIPT with the rheometers at our disposal. G' increased steeply when the gel was formed and
197
reached a steady state value at long heating time. It is clear that the rate of gelation and the
198
stiffness of extensively heated gels increased with increasing protein concentration. The
199
steady state values of G' and the gel time are plotted as a function of C in fig 6. G' increased
200
by a factor of 30 between C=50 g/L and C=95 g/L and in the same concentration range, tg
201
decreased from 20 min to 3 min. Similar results were reported elsewhere for heated SPI
202
solutions in the absence of salt at pH 6.0 (Chen et al., 2016c) and are reproduced in fig 6 for
203
comparison. G' is the same within the experimental uncertainty at all concentrations, but the
204
gel time is systematically shorter at the conditions studied here.
3.6 Structure of the aggregates
M AN U
206
SC
205
RI PT
196
SPI aggregates were formed by heating solutions at 95°C during 0.5 h at different
208
protein and salt concentrations. The dependence of Mw on [NaCl] is shown in fig. 7a for
209
different protein concentrations and the dependence on C is shown in fig. 7b for different
210
ionic strengths. At a given protein concentration larger than 28 g/L, Mw increased with
211
increasing [NaCl] until gels were formed. For C<28 g/L, gels were not formed and Mw
212
reached a maximum at [NaCl]=0.2 M. The decrease of the aggregation rate for [NaCl]>0.2 M
213
is strikingly illustrated by the observation that at C= 28 g/L a gel was formed at 0.2 M NaCl,
214
but not at 0.5 M NaCl. At a given ionic strength, Mw increased with increasing C until gels
215
were formed at a critical concentration. The latter decreased from about 85 g/L in the absence
216
of salt to about 30 g/L at 0.2 M NaCl. However, it should be realized that the critical gel
217
concentration depends on the intensity of the heat treatment and decreases with increasing
218
heating time. It was shown elsewhere for salt free SPI solutions that gels can be formed at
219
least down to C=50 g/L if the systems are heated much longer (Chen et al., 2016b) .
EP
AC C
220
TE D
207
The dependence of Ir/KC on the scattering wave vector of highly diluted solutions
221
characterizes the structure of the aggregates. Figure 8a shows the results for aggregates
222
formed at 0.1 M NaCl at different SPI concentrations. Similar results were obtained at other
223
ionic strengths (see supplementary information). The q-dependence increases with increasing
224
concentrations, because the aggregates become larger. Close to the gel point, a power law
225
dependence is observed over the whole accessible q-range, indicating that the aggregates have
226
a self-similar structure (Sorensen, 2001) . The power law exponent is equal to the fractal
227
dimension of the aggregates and was found here to be df=1.7. The results superimpose if S(q) 8
ACCEPTED MANUSCRIPT is plotted vs q.Rg see figure 8b, which means that the aggregates have the same overall
229
structure independent of their size. Master curves obtained at other ionic strengths were very
230
close, see supplementary information. The master curves could be well described by eq. 2,
231
which implies df=2.0, except for the results at the highest values of q.Rg that suggests a
232
slightly smaller df. No significant effect of the salt concentration on the structure factor was
233
found.
RI PT
228
The hydrodynamic radius (Rh) of the aggregates was determined as briefly described
235
in the materials and methods section. As was shown elsewhere (Chen et al., 2016b) Rh is
236
proportional to Rg, but was systematically between 10% and 30% smaller for aggregates
237
formed at the different salt concentrations investigated here. It is more time consuming to
238
determine Rh than Rg, but it can be determined down to smaller sizes. Mw is plotted as a
239
function of Rh for aggregates obtained at different concentrations and ionic strengths in figure
240
9a. For a given ionic strength, Mw increased steeply with increasing Rh for small aggregates
241
formed at low protein concentrations, but for larger aggregates the data were consistent with a
242
power law dependence. A power law dependence between Mw and Rh is characteristic for
243
self-similar aggregates and the power law exponent is equal to the fractal dimension:
244
Mw∝Rhdf (Sorensen, 2001) . Here we find df between 1.7 and 2.0, but the data are not
245
sufficient to give a more precise value or to conclude if df depends on the salt concentration.
TE D
M AN U
SC
234
However, the onset of the power law dependence started at larger Rh and Mw when
247
more salt was present. This means that Rh and Mw of the elementary unit of the fractal
248
aggregates increased with increasing ionic strength between Rh≈30 nm, Mw≈3x106 g/mol and
249
Rh≈50 nm, Mw≈ 3x107 g/mol. Mw increased slightly more than Rh3, which suggests that the
250
density of the elementary unit increased weakly with increasing ionic strength. An increase of
251
the size of the elementary unit was also found for aggregates formed by SPI with different
252
charge densities in the absence of salt and we reproduced the figure here, see fig. 9b (Chen et
253
al., 2016c) . In that case, the size and density of the elementary units increased with
254
decreasing |α|, i.e. decreasing pH and were shown by cryo-TEM to be spherical and rather
255
polydisperse in size.
AC C
EP
246
256
The effect of heating temperature on the structure of the aggregate was studied for
257
aggregates formed at C=95 g/L in the presence of 0.05, 0.1 and 0.4 M NaCl. Aggregates of
258
different sizes were formed by heating the SPI solutions during different times at fixed
259
temperatures between 30°C and 70°C. The dependence of Mw on Rg of aggregates formed at 9
ACCEPTED MANUSCRIPT 0.1 M NaCl are shown in fig.10. At all temperatures a steep increase of Mw with increasing Rg
261
was followed by a power law dependence with approximately the same exponent. The results
262
at different temperatures for T≥60°C were the same within the experimental error. The results
263
obtained at different temperatures for T≤45°C were also the same, but for a given value of Rg
264
Mw was systematically about 50% smaller than for T≥60°C. The behavior at 50°C was
265
intermediate. A similar behavior was observed at the other two ionic strengths, see
266
supplementary information.
267
Discussion
RI PT
260
The present investigation of the influence of salt (0-0.5 M) on thermal aggregation and
269
gelation of SPI at a fixed charge density (α=-175) complements the investigation of the
270
influence of the net charge density (α=-100 to -175; pH 5.8 to 7.2) in the absence of salt that
271
was reported elsewhere (Chen et al., 2016c) . At all α and [NaCl] investigated, irreversibly
272
cross-linked aggregates were formed that have a self-similar structure on large length scales
273
with a fractal dimension close to two. The aggregates grow with heating time until they
274
percolate into a system spanning network. This behavior is similar to that reported for various
275
other types of globular proteins.
M AN U
SC
268
At steady state, elastic modulus of the gels obtained after extensive heat treatment
277
does not depend on the pH nor on the ionic strength in the range investigated. This is
278
remarkable, because the structure of the elementary units and the microscopic structure do
279
vary when the strength of the electrostatic interactions is varied either by varying the pH or by
280
adding salt. It suggests that differences in structure are compensated by differences in bond
281
strength, but in the absence of detailed knowledge about which and how many bonds are
282
deformed under stress it is not possible to go beyond this observation. As expected, G'
283
decreases with decreasing SPI concentration and for C≤40 g/L the gels either break when the
284
vials are turned upside down or the solutions become macroscopically heterogeneous.
EP
AC C
285
TE D
276
The principal effects of varying [NaCl] or α are on the aggregation kinetics and the
286
structure. If [NaCl] is increased or |α| is decreased, aggregation and gelation is faster, the
287
microstructure of the gels is more heterogeneous, and the elementary units of the fractal
288
aggregates are larger and denser. The strikingly similar effects of increasing [NaCl] or
289
decreasing |α| are obviously related to the decrease of the repulsive electrostatic interactions
290
either by screening or by reducing the charge. The screening length of electrostatic
291
interactions decreases with increasing ionic strength and becomes very small for [NaCl]>0.1. 10
ACCEPTED MANUSCRIPT 292
This may explain why tg no longer depends on [NaCl] for [NaCl]>0.1. However, the structure
293
of the gels is coarser at 0.4 M NaCl than at 0.1 M NaCl, see fig. 2, which is perhaps caused by
294
the effect of salt on hydration of the proteins. The formation of larger and denser elementary units of the fractal aggregates with
296
decreasing |α| or increasing [NaCl] may be attributed to the reduction of electrostatic
297
repulsion. It was shown by (Chen et al., 2016c) that roughly spherical particles are formed in
298
the first step of the process that subsequently randomly associate into fractal aggregates and
299
eventually form a network if the SPI concentration is sufficiently high. The size and density
300
of the elementary units is clearly determined by the strength of the electrostatic interactions.
301
The particles grow until the repulsion between the accumulated charges render further growth
302
unfavorable. However, these particles can still randomly associate into larger clusters or a
303
network. A similar aggregation mechanism was reported for thermal aggregation of whey
304
proteins (Phan-Xuan et al., 2013) .
M AN U
SC
RI PT
295
The temperature appears to have only a kinetic effect for T>60°C, as the structure of
306
the aggregates and the elastic modulus of the gels was independent of the temperature for all
307
systems. The aggregation and gelation rate increased with increasing temperature following
308
an Arrhenius dependence with the same activation energy independent of the pH, the ionic
309
strength and the SPI concentration. However, a remarkably different temperature dependence
310
of tg behavior was observed at lower temperatures in the presence of salt. Below a
311
characteristic temperature, tg become almost independent of the temperature over a range of
312
temperatures before increasing again. The characteristic temperature increased with
313
increasing ionic strength from 55°C at 50 mM NaCl to 65°C at 0.5 M NaCl. It is tempting to
314
relate the characteristic temperature with the denaturation temperature determined by DSC
315
that was reported by Bikbov et al. (1983) to increase with increasing NaCl concentration from
316
68°C without salt to 78°C at 0.5M NaCl for β-conglycinin and from 85°C to 95°C for
317
glycinin. These values are close to those reported by Renkema et al. (2002a) who also
318
showed that it depended only weakly on the pH between 6 and 8. It is likely that the
319
aggregation process at high temperatures characterized by Ea=180 kJ/mol is controlled by
320
denaturation of β-conglycinin. Denaturation of glycinin, which becomes fast above 75°C
321
(Zhao, Li, Qin and Chen, 2016) , does not appear to influence the aggregation process as the
322
kinetics is characterized by a single activation energy between 60°C and 85°C and the
323
aggregate structure is the same.
AC C
EP
TE D
305
11
ACCEPTED MANUSCRIPT The irreversible aggregation process at T≤45°C, which can only be observed in the
325
presence of salt, leads to the formation of aggregates with the same fractal dimension, but
326
with elementary units that are less dense than those formed at higher temperatures. This
327
observation together with the change in the temperature dependence of tg shown in Fig. 4 and
328
the different dependence of Mw on t/tg shown in Fig. 3, suggest that there are two different
329
thermal aggregation process of SPI. One dominates at T≥60°C and one dominates at T≤45°C.
330
We speculate that the temperature dependence of tg is weak at intermediate temperatures,
331
because in this range both processes occur with varying relative importance. As was
332
mentioned above, it appears that fast denaturation of β-conglycinin plays a major role in the
333
process at T≥60°C, denaturation of β-conglycinin is extremely slow below 45°C so the
334
driving force for irreversible aggregation at 30°C≤T≤45°C is unclear. Unfortunately, the
335
experimental techniques used in this study do not reveal the crosslinking mechanism of the
336
proteins on the molecular scale.
338
Conclusion
M AN U
337
SC
RI PT
324
During heating SPI solutions, relatively dense spherical particles are formed first that
340
in a second step randomly associate into larger fractal aggregates. If the protein concentration
341
is sufficiently high, the aggregates connect into a system spanning network. Addition of NaCl
342
causes an increase in the rate of thermal aggregation and gelation, but does not modify the
343
stiffness of the gels at steady state. The size and density of the elementary units increases with
344
increasing NaCl concentration and the microstructure of the gels becomes increasingly
345
heterogeneous. In many aspects, increasing the ionic strength is equivalent to decreasing the
346
net charge density of the protein, i.e. decreasing the pH towards the isoionic point. In both
347
cases the effect on aggregation is caused by the reduction of electrostatic repulsion.
EP
AC C
348
TE D
339
For T>60°C, the effect of heating temperature on the aggregation process is
349
principally on the kinetics. The gelation rate has an Arrhenius temperature dependence
350
between 60°C and 85°C with an activation energy of 180kJ/mol that does not depend on the
351
ionic strength, the pH or the protein concentration. A different irreversible aggregation
352
process dominates at temperature between 30°C and 45°C, which leads to fractal aggregates
353
with less dense elementary units. There are indications that the high temperature process is
354
controlled by denaturation of β-conglycinin. The driving force for irreversible aggregation at
355
lower temperatures is not yet elucidated. 12
ACCEPTED MANUSCRIPT Supplementary information
357
Measurements of the dependence of the molar mass on the radius on the gyration of the
358
aggregates formed during heating at different temperatures are shown for 0.05 M NaCl and
359
0.4 M NaCl. Also the structure factor of the aggregates formed at different ionic strengths is
360
shown.
RI PT
356
361
References
363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400
Berne, B. & Pecora, R. (2000), Dynamic light scattering With Applications to Chemistry, Biology, and physics. New York: Dover Publications. Bikbov, T., Grinberg, V., Danilenko, A. N., Chaika, T. S., Vaintraub, I. A. & Tolstoguzov, V. (1983), Studies on gelation of soybean globulin solutions Part 3., Colloid & Polymer Sci., 261, 346-358. Brown, W. (1996), Light scattering: principles and development. Oxford: Clarendon Press. Chen, N., Lin, L., Sun, W. & Zhao, M. (2014), Stable and pH-Sensitive Protein Nanogels Made by SelfAssembly of Heat Denatured Soy Protein, Journal of Agricultural and Food Chemistry, 62, 9553-9561. Chen, N., Zhao, M., Chassenieux, C. & Nicolai, T. (2016a), Structure of self-assembled native soy globulin in aqueous solution as a function of the concentration and the pH, Food Hydrocolloids, 56, 417-424. Chen, N., Zhao, M., Chassenieux, C. & Nicolai, T. (2016b), Thermal aggregation and gelation of soy globulin at neutral pH, Food Hydrocolloids, 61, 740-746. Chen, N., Zhao, M., Niepceron, F., Chassenieux, C. & Nicolai, T. (2016c), The effect of the pH on thermal aggregation and gelation of soy proteins, Food Hydrocolloids, 61, 740-746. Higgins, J. S. & Benoit, H. C. (1994), Polymers and neutron scattering. Oxford: Clarendon Press. Hua, Y., Cui, S. W., Wang, Q., Mine, Y. & Roysa, V. (2005), Heat induced gelling properties of soy protein isolates prepared from different defatted soybean flours, Food Research International, 38, 377-385. Jiang, J., Xiong, Y. L. & Chen, J. (2010), pH Shifting alters solubility characteristics and thermal stability of soy protein isolate and its globulin fractions in different pH, salt concentration, and temperature conditions, J Agric Food Chem, 58, 8035-42. Mariotti, F., Tomé, D. & Mirand, P. P. (2008), Converting nitrogen into protein - beyond 6.25 and Jones' factors, Critical reviews in food science and nutrition, 48, 177-184. Nishinari, K., Fang, Y., Guo, S. & Phillips, G. O. (2014), Soy proteins: A review on composition, aggregation and emulsification, Food Hydrocolloids, 39, 301-318. Phan-Xuan, T., Durand, D., Nicolai, T., Donato, L., Schmitt, C. & Bovetto, L. (2013), Tuning the structure of protein particles and gels with calcium or sodium ions, Biomacromolecules, 14, 1980– 1989. Renkema, J. M. S., Gruppen, H. & van Vliet, T. (2002a), Influence of pH and ionic strength on heatinduced formation and rheological properties of soy protein gels in relation to denaturation and their protein compositions, Journal of Agricultural and Food Chemistry, 50, 6064-6071. Renkema, J. M. S. & van Vliet, T. (2002b), Heat-induced gel formation by soy proteins at neutral pH, Journal of Agricultural and Food Chemistry, 50, 1569-1573. Sorensen, C. (2001), Light scattering by fractal aggregates: a review, Aerosol Science & Technology, 35, 648-687. Zhao, H., Li, W., Qin, F. & Chen, J. (2016), Calcium sulphate-induced soya bean protein tofu-type gels: influence of denaturation and particle size, International Journal of Food Science and Technology, 51, 731-741.
AC C
EP
TE D
M AN U
SC
362
13
ACCEPTED MANUSCRIPT 401 402
AC C
EP
TE D
M AN U
SC
RI PT
403
14
ACCEPTED MANUSCRIPT 404
Figure captions
405
Figure 1 Influence of the ionic strength on the evolution of G' during heating at 80oC for SPI
407
solutions at C=95 g/L.
408
Figure 2 CLSM images (40 x 40 µm) of SPI gels at C=95g/L and different NaCl
409
concentrations. The gels were formed by heating at 80oC for 16 h.
410
Figure 3 G' (a) and Mw (b) as a function of time during heating at different temperatures
411
([NaCl]= 0.1M, C=95g/L). Figure 3c shows the same data after normalizing the heating time
412
by gel time (tg).
413
Figure 4 Comparison of the gelation time as a function of temperature at different salt
414
concentration obtained by light scattering (T<70oC) and rheology (T≥65oC).
415
Figure 5 Storage shear modulus of SPI solutions at 0.05 M NaCl and different protein
416
concentrations as a function of time during heating at 90oC.
417
Figure 6 Dependence of G' (a) and the gel time (b) on the protein concentration for the SPI
418
gels by heating either without salt at pH 6.0 at 85 oC (triangles) or at 0.05 M NaCl at 90oC
419
(circles).
420
Fig. 7 Mw as a function of [NaCl] at different SPI concentrations (a) and as a function of SPI
421
concentration at different ionic strengths (b) for SPI aggregates obtained after 0.5 h heating at
422
95°C.
423
Figure 8 Dependence of of Ir/KC on the scattering wave vector for aggregates formed at 0.1
424
M NaCl at different SPI concentrations (a) and the master curve obtained by plotting the same
425
data as S(q) vs qRg (b). The solid line in figure 8b represents a fit to equation 2.
426
Figure 9 Mw as a function of Rh for SPI aggregates formed at different salt concentrations (a).
427
and pH (b). The aggregates were formed by heating SPI solutions for 0.5 h at 95°C at
428
different concentrations between 1.8 g/L and the gel concentration.
429
Figure 10 Mw as a function of Rg for aggregates formed during heating SPI solutions at C=95
430
g/L and [NaCl]= 0.1 M at different temperatures.
AC C
EP
TE D
M AN U
SC
RI PT
406
431
15
ACCEPTED MANUSCRIPT 432
Figure 1
433 434
G ' (Pa)
RI PT
0 mM 20 mM 50 mM 0.1M 0.2 M 0.4 M
102
SC
101
101
M AN U
100
102
t (min)
435 436
AC C
EP
TE D
437
16
103
ACCEPTED MANUSCRIPT 438
Figure 2
M AN U
SC
RI PT
439
440 441
AC C
EP
TE D
442
17
ACCEPTED MANUSCRIPT 443
Figure 3 103
a
85 oC 80 oC
101
RI PT
G' (Pa)
102
75 oC 70 oC
100
68 oC 65 oC
10-1 102
103
SC
101
t (min)
M AN U
444 109
Mw (g/mol)
b
108
TE D
107
101
103
104
EP
t (min)
Mw (g/mol)
109
103
c 102
108 101
107
100
10-1
10
446
-3
10
-2
10
-1
10
t/tg
18
0
10
1
10
2
G' (Pa)
AC C
445
102
70 °C 65 °C 60 °C 50 °C 45 °C 40 °C 35 °C 30 °C
ACCEPTED MANUSCRIPT 447
Figure 4
448
104
RI PT
103
101 0M 20 mM 50 mM 0.1 M 0.4 M
100 10-1 10-2
40
50
60
70
80
90
M AN U
30
SC
Tg (h)
102
o T( C)
449
AC C
EP
TE D
450
19
ACCEPTED MANUSCRIPT 451
Figure 5
M AN U
SC
RI PT
452
453 454
AC C
EP
TE D
455
20
ACCEPTED MANUSCRIPT 456
Figure 6
457
103
RI PT
G'(Pa)
a
101 60
70
80
90 100
M AN U
50
SC
102
C (g/L)
458
100
b
TE D
50
tg (min)
30 20 10
EP
5 3
459 460 461
AC C
2
50
60
70
C (g/L)
21
80
90 100
ACCEPTED MANUSCRIPT 462
Figure 7
463
109
a
RI PT
1.8 g/L 4.5 g/L 9 g/L 18 g/L 28 g/L 37 g/L 50 g/L 60 g/L 70 g/L
107
SC
Mw (g/mol)
108
106 10-2
10-1
100
M AN U
10-3
NaCl (M)
464
109
107
3 mM 10 mM 35 mM 70 mM 0.1 M 0.2 M 0.5 M
EP
Mw (g/mol)
108
TE D
b
AC C
106
0
465 466
20
40 C (g/L)
467
22
60
ACCEPTED MANUSCRIPT 468
Figure 8
469
Ir/KC(g/mol)
108
107 107 q (m-1)
470
b
TE D
AC C
10-2 10-1
472
b
10-1
EP
S (q)
100
471
108
M AN U
106
SC
1.8 g/L 4.5 g/L 9 g/L 18 g/L 28 g/L 37 g/L
a
RI PT
109
100 qRg
23
101
ACCEPTED MANUSCRIPT 473
Figure 9
109 a
RI PT
Mw (g/mol)
108
3 mM 10 mM 35 mM 70 mM 0.1M 0.2M 0.5M
107
10
100
109
M AN U
Rh (nm)
474
SC
106
b
pH 6.8 pH 6.6 pH 6.4 pH 6.3 pH 6.2 pH 6.1 pH 6.0 pH 5.9 pH 5.8
TE D
Mw (g/mol)
108
107
EP
106
10
476
Rh (nm)
AC C
475
100
24
ACCEPTED MANUSCRIPT 477
Figure 10
478
109
RI PT
108
o
70 C o 65 C 60 oC 50 oC 45 oC o 40 C o 35 C o 30 C
107
M AN U
100
SC
Mw (g/mol)
0.1 M
Rg (nm)
479 480
AC C
EP
TE D
481
25
ACCEPTED MANUSCRIPT 482 483
Supplementary information
RI PT
484
SC
10-1
0.003 M 0.01 M 0.035 M 0.07 M 0.1 M 0.2 M 0.5 M
10-2 10-1
100
101 qRg
485
M AN U
S (q)
100
Figure S1 Structure factor of SPI aggregates obtained at different NaCl concentrations. The
487
solid line represents a fit to equation 2.
EP
108
AC C
Mw (g/mol)
0.05M
107
109 0.4M
Mw (g/mol)
109
TE D
486
60 oC 55 oC
108 65 oC 60 oC o 50 C o 45 C 40 oC
50 oC 40 oC
107
35 oC
o 35 C
106
100
488
100
Rg (nm)
Rg (nm)
489
Figure S2 Mw as a function of Rg for aggregates formed during heating SPI solutions at C=95
490
g/L and [NaCl]= 0.05 M or 0.4 M at different temperatures.
491 26
ACCEPTED MANUSCRIPT Self similar SPI aggregates are formed during heating. Local density of the aggregates increases with increasing [NaCl]. The gel stiffness does not depend on the ionic strength. Gelation is faster at higher ionic strength.
AC C
EP
TE D
M AN U
SC
RI PT
Influence of decreasing pH and increasing [NaCl] is similar.
S0268-005X(16)30601-4
DOI:
10.1016/j.foodhyd.2017.03.024
Reference:
FOOHYD 3835
To appear in:
Food Hydrocolloids
Received Date: 14 October 2016 Revised Date:
24 February 2017
Accepted Date: 19 March 2017
Please cite this article as: Chen, N., Zhao, M., Chassenieux, C., Nicolai, T., The effect of adding NaCl on thermal aggregation and gelation of soy protein isolate, Food Hydrocolloids (2017), doi: 10.1016/ j.foodhyd.2017.03.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT The effect of adding NaCl on thermal aggregation and gelation of soy protein isolate
AC C
EP
TE D
M AN U
SC
RI PT
Nannan Chen, Mouming Zhao, Christophe Chassenieux, Taco Nicolai
ACCEPTED MANUSCRIPT 1
The effect of adding NaCl on thermal aggregation and gelation of soy
2
protein isolate
3
Nannan Chen1,2, Mouming Zhao1, Christophe Chassenieux2, Taco Nicolai2
5
1
6
Guangzhou, China
7
2
8
72085 Le Mans cedex 9, France
9
Email : [email protected]
School of Food Science and Engineering, South China University of Technology, 510640,
Tel : (33)-243833139
AC C
EP
TE D
11
SC
LUNAM Université du Maine, IMMM UMR-CNRS 6283, Polymères, Colloïdes et Interfaces,
M AN U
10
RI PT
4
1
ACCEPTED MANUSCRIPT Abstract
13
Thermal aggregation and gelation of soy protein isolate (SPI) was studied at fixed charge
14
density over a wide range of protein concentrations (1-95 g/L), NaCl concentrations (0 - 0.5
15
M) and temperatures (30-85 oC). Gelation was studied with oscillatory shear measurements
16
and aggregation with light scattering. At all conditions, self-similar aggregates were formed
17
by random association of elementary units consisting of dense SPI particles with radii
18
between 30 nm and 50 nm that were formed in a first step of the thermal aggregation process.
19
Two distinct irreversible aggregation processes were identified: one dominating between 30
20
and 45°C and one dominating above 65°C. Addition of salt led to faster aggregation and
21
gelation, but did not influence the gel stiffness at steady state. The size and density of the
22
elementary units of the aggregates increased with increasing NaCl concentration and confocal
23
laser scanning microscopy images showed increasingly heterogeneous gels structures. The
24
effect of varying the ionic strength on thermal aggregation is compared with that of varying
25
the net charge density of the proteins.
26
Key words: soy, protein, aggregation, gelation, rheology, structure
28
Introduction
TE D
27
M AN U
SC
RI PT
12
Soy globulin in the form of soy protein isolate (SPI) is an important food ingredient
30
and has been widely used in the food industry for its good functional and nutritional
31
properties. It contains two main components: glycinin (11S) and β-conglycinin (7S), having
32
molar masses of about 200 kg/mol and 360 kg/mol, respectively (Nishinari, Fang, Guo and
33
Phillips, 2014) . In aqueous solutions, these proteins associate or dissociate to different
34
extents depending on the concentration, pH and ionic strength (Nishinari et al., 2014) .
AC C
35
EP
29
At high temperatures, irreversible aggregation of soy globulin is observed, which is
36
generally considered to be caused by denaturation of the proteins (Nishinari et al., 2014) . The
37
rate of thermal aggregation increases strongly with increasing temperature and SPI
38
concentration and has therefore been mostly investigated at elevated temperatures (T>80°C)
39
where it is rapid (Hua, Cui, Wang, Mine and Roysa, 2005; Renkema and van Vliet, 2002b) .
40
In the absence of salt, the aggregation rate was found to have an Arrhenius temperature
41
dependence characterized by an activation energy (Ea) of 180 kJ/mol (Chen, Zhao,
42
Chassenieux and Nicolai, 2016b) in the range 65°C – 85°C. 2
ACCEPTED MANUSCRIPT A detailed investigation of the influence of the pH between pH 7 and pH 5.8 on
44
thermal aggregation of SPI indentified a two step aggregation process (Chen, Zhao,
45
Niepceron, Chassenieux and Nicolai, 2016c) . In the first step, dense approximately spherical
46
particles are formed that in the second step randomly associate into self-similar aggregates.
47
Decreasing the pH caused an increase of the radius of the primary particles formed in the first
48
step, but did not modify the fractal dimension of the aggregates on larger length scales.
49
Growth of the self similar aggregates led to the formation of a system spanning network at a
50
critical gel time. The aggregation and gelation rate of SPI increased with decreasing pH, but
51
did not significantly influence the activation energy nor the elastic modulus of the gels.
RI PT
43
The effect of salt on thermal aggregation of SPI has also been investigated in the past.
53
The denaturation temperature of glycinin and β-conglycinin was found to increase with
54
increasing NaCl concentration implying that soy proteins have a higher thermal stability in the
55
presence of salt (Bikbov et al., 1983; Jiang, Xiong and Chen, 2010; Renkema, Gruppen and
56
van Vliet, 2002a) . Nevertheless, (Jiang et al., 2010) observed that the turbidity of SPI
57
solutions at pH 7.0 increased more strongly during a heating ramp if 0.1 M or 0.6 M NaCl
58
was added than in deionized water, indicating that aggregation is favored by screening of
59
electrostatic repulsion. (Hua et al., 2005) reported that the stiffness of the gels at C=80 g/L
60
after a given heat treatment (95°C, 15 min) increased with increasing NaCl concentration until
61
approximately 0.2 M, but further increase of the salt concentration led to a slight decrease of
62
the gel stiffness. On the other hand, (Renkema et al., 2002a) did not find a systematic effect
63
of adding 0.2 M or 0.5 M NaCl on the stiffness of SPI gels (C=12 wt%) that were heated at
64
95°C during 1 h. These literature results give some indication about the effect of salt on
65
thermal SPI aggregation, but, as far as we are aware, no detailed systematic investigation has
66
yet been reported.
M AN U
TE D
EP
AC C
67
SC
52
Here, we present an investigation of the effect of salt on the aggregation and gelation
68
of SPI over a wide range of NaCl concentrations (0-0.5 M), protein concentrations (1-95 g/L)
69
and heating temperatures (30-85°C). We have studied the effect of adding salt on the kinetics
70
of the thermal aggregation process, the structure of the aggregates, the elastic modulus of the
71
gels and the microstructure of the gels as a function of the temperature and concentration. The
72
net charge density was fixed at α=-175, where α is expressed in number of charges per protein
73
unit with molar mass 3.0x105 g/mol. It was shown that SPI in pure water forms stable
74
suspensions for α<-150 and are only weakly associated because the electrostatic repulsion is
75
strong (Chen, Zhao, Chassenieux and Nicolai, 2016a) . Notice that if the net charge density is 3
ACCEPTED MANUSCRIPT fixed, the pH decreases with increasing NaCl concentration or protein concentration (Chen et
77
al., 2016a) . The pH of the samples studied here varied between pH 7.2 at low SPI
78
concentrations without added salt to pH 6.4 at 0.5 M NaCl. The effect of screening
79
electrostatic repulsion by adding salt for SPI with high charge density studied here will be
80
compared with the effect of reducing the charge density of SPI in the absence of salt reported
81
elsewhere (Chen et al., 2016c)
RI PT
76
82
Materials and Method
84
Materials
SC
83
Soy protein isolate (SPI) was obtained from defatted soybean flakes (Yuwang Group,
86
China) by precipitation at pH 4.5 using the procedure detailed elsewhere (Chen, Lin, Sun and
87
Zhao, 2014) . The SPI powder contained 94% protein (Kjeldahl, N×6.25). It has been argued
88
that a smaller nitrogen correction factor (5.5) should be used for soy proteins (Mariotti, Tomé
89
and Mirand, 2008) , but we have used 6.25 here for easier comparison with previous results
90
reported by us and others in the literature. Analysis by SDS-page showed that the sample
91
contained about 60% glycinin and 40% β-conglycinin. Protein solutions were prepared in salt-
92
free Milli-Q water with 3 mM NaN3 to avoid bacterial growth. They were centrifuged at room
93
temperature with an Allegra 64R centrifuge (Beckman Coulter, USA) at 5x104 g for 4 h. The
94
ionic strength of the protein solutions was set by adding aliquots of a concentrated NaCl
95
solution (2 or 4 M). After setting of the ionic strength, the solution was put in air-tight glass
96
tubes with diameter of about 10 mm and heated in a temperature control water bath
97
immediately. After heating, the solutions were cooled to room temperature with tap water.
98
Light Scattering.
TE D
EP
AC C
99
M AN U
85
Static (SLS) and dynamic (DLS) light scattering measurements were done using a
100
commercial apparatus (ALV-Langen). The light source was a He–Ne laser with wavelength
101
λ= 632 nm. The temperature was controlled by a thermostatic bath to within ± 0.2 °C.
102
Measurements were made at angles of observation (θ) between 13° and 140°. The relative
103
scattering intensity (Ir) was calculated as the intensity minus the solvent scattering divided by
104
the scattering intensity of toluene at 20°C. In dilute solutions, Ir is related to the weight
105
average molar mass (Mw) and the structure factor (S(q)) of the solute (Brown, 1996; Higgins
106
and Benoit, 1994) 4
ACCEPTED MANUSCRIPT 107
ூೝ
= ܯ௪ ܵ()ݍ
(1)
108 109
with K an optical constant. The initial dependence of S(q) on the scattering wave vector (q)
110
was used to obtain the z-average radius of gyration (Rg).
111
S(q)=(1+q2.Rg2/3)-1
RI PT
112 113
(2)
The normalized intensity autocorrelation functions measured with DLS could be
115
described by a monomodal relaxation time distribution. An apparent q-dependent diffusion
116
coefficient was calculated from the average relaxation rate (Γ): Da=Γ.q-2 (Berne and Pecora,
117
2000) . In dilute solutions and for q→0, Da is equal to the translational diffusion coefficient of
118
the solute (D) and is related to the hydrodynamic radius (Rh):
M AN U
SC
114
119 120
121
=ܦ
୩ ୖ
with η the viscosity, k Boltzmann’s constant, and T the absolute temperature.
124
TE D
122 123
(3)
Rheology
Oscillatory shear measurements were done with a stress imposed rheometer (AR2000,
Instruments)
using
a
cone–plate geometry
(diameter
44
mm).
126
The temperature was controlled by a Peltier system and the geometry was covered
127
with paraffin oil to prevent water evaporation. The storage (G') and loss (G'') moduli
128
were determined in the linear response regime.
129
Confocal Laser Scanning Microscopy (CLSM)
AC C
TA
EP
125
130
CLSM was used in the fluorescence mode. Observations were made at 20°C with
131
a Leica TCS-SP2 (Leica Microsystems Heidelberg, Germany). A water immersion
132
objective lens was used (HCxPL APO 63× NA = 1.2) with theoretical resolution of 75
133
nm in the x–y plane. Soy protein was physically labeled with the fluorochrome rhodamine
134
B, by adding a small amount of a concentrated rhodamine solution to the 95 g/L soy
5
ACCEPTED MANUSCRIPT 135
protein solutions with a final concentration of 5 ppm. The solutions were inserted between a
136
concave slide and a cover slip and hermetically sealed and heated at 80oC during 16 h.
138
3 Results
139
3.1 Influence of the NaCl concentration on gelation
RI PT
137
Figure 1 shows the evolution of the storage shear modulus during heating at 80oC for
141
SPI solutions at C= 95 g/L containing different NaCl concentrations between 0 and 0.4 M. G'
142
increased sharply when SPI gelled. For the gels, G' was almost independent of the oscillation
143
frequency in the range tested (0.01 - 1 Hz) and much larger than the loss modulus (results not
144
shown). The gel time decreased with increasing [NaCl] up to a factor 10 at 0.1 M, but adding
145
more salt did not have an effect on the kinetics. Remarkably, addition of salt did not have a
146
significantly influence on the stiffness of extensively heated gels. This is in agreement with
147
the results by Renkema et al. (2002a) , but appears to contradict the results by Hua et al. (2005)
148
who observed an increase of G' up to 0.2 M NaCl. However, the results can be reconciled if
149
we consider that Hua et al. (2005) compared G' of the gels after a fixed relatively short heat
150
treatment, which confounds the effects on the gelation rate and the gel stiffness.
TE D
M AN U
SC
140
The microstructure of gels obtained after heating for 16 h at 80oC was observed by
152
CLSM. Images of the gels show that the gels were increasingly heterogeneous for [NaCl] ≥
153
0.05 M, see Fig. 2. We note that CLSM images of the samples before heating were
154
homogeneous. The more heterogeneous structure was formed during gelation, i.e. much
155
sooner than 16 h. Further heating did not have a significant influence on the structure once the
156
systems had gelled even though G' continued to increase.
157
3.2 Influence of the temperature on aggregation and gelation
AC C
EP
151
Fig. 3a shows the evolution of G' for SPI solutions at C=95 g/L in the presence of 0.1
158 159
M NaCl during heating at different temperatures between 65°C and 85°C. The gelation rate
160
increased strongly with increasing temperature. The results obtained at different temperatures
161
superimpose when they are plotted as a function of time normalized by the gel time (tg)
162
estimated as the time at which G'=0.1 Pa, see fig. 3c. This shows that the heating temperature
163
did not influence the stiffness of the gels if the difference in the kinetics is properly accounted
164
for. 6
ACCEPTED MANUSCRIPT As was discussed elsewhere for thermal aggregation of SPI in the absence of salt
166
(Chen et al., 2016c) gelation is preceded by the formation of self-similar aggregates that can
167
be characterized by light scattering. Fig. 3b shows the evolution with time of the average
168
molar mass of the aggregates during heating at temperatures between 30 and 70°C. Mw
169
increased with increasing heating time and diverged at tg when a space filling network was
170
formed. The aggregation rate increased with increasing temperature, but, curiously, tg varied
171
little between 45 and 60°C. When Mw is plotted as a function of t/tg, we find that the results
172
obtained at 30, 35 and 40°C superimposed, but the results at higher temperatures deviated for
173
smaller t/tg, see fig. 3c.
RI PT
165
Similar results were obtained for solutions at other NaCl concentrations between 0 and
175
0.4 M NaCl. The temperature dependence of tg could be obtained over a broad range by
176
combining the results of rheology and light scattering, see fig. 4. In the narrow temperature
177
range where both techniques could be used (65-70°C) the same value of tg was obtained. In
178
the absence of salt, a continuous strong decrease of tg with increasing temperature was
179
observed over the whole temperature range where it could be investigated (50-85°C). As was
180
mentioned in the introduction, tg was found to have an Arrhenius temperature dependence
181
between 65°C and 85°C in the absence of salt with Ea=180 kJ/mol independent of the protein
182
concentration and the pH, at least between pH 5.8 and pH 6.8 (Chen et al., 2016c) .
TE D
M AN U
SC
174
It was already shown in fig. 1 for SPI solutions heated at 80°C that addition of NaCl
184
up to 0.1 M increased the gelation rate, but further increase of [NaCl] had no effect on tg. The
185
effect of salt was similar in the temperature range between 65°C and 85°C. However, a
186
remarkably different temperature dependence of tg was observed at lower temperatures. tg was
187
almost independent in the temperature range 40-55°C, 45-60°C and 50-65°C for 0.05, 0.1 and
188
0.4 M NaCl, respectively. At temperatures below this range, tg increased again with
189
decreasing temperature. It appears that addition of salt induces a different aggregation process
190
at low temperatures.
192
AC C
191
EP
183
3.3 Influence of the protein concentration on gelation
193
The effect of the protein concentration on the evolution of G' during heating at 90°C is
194
shown in fig. 5 for SPI solutions at [NaCl] = 50 mM between C=50 g/L and C=95 g/L. For
195
C<50 g/L, very weak gels or precipitates were formed that could not be properly characterized 7
ACCEPTED MANUSCRIPT with the rheometers at our disposal. G' increased steeply when the gel was formed and
197
reached a steady state value at long heating time. It is clear that the rate of gelation and the
198
stiffness of extensively heated gels increased with increasing protein concentration. The
199
steady state values of G' and the gel time are plotted as a function of C in fig 6. G' increased
200
by a factor of 30 between C=50 g/L and C=95 g/L and in the same concentration range, tg
201
decreased from 20 min to 3 min. Similar results were reported elsewhere for heated SPI
202
solutions in the absence of salt at pH 6.0 (Chen et al., 2016c) and are reproduced in fig 6 for
203
comparison. G' is the same within the experimental uncertainty at all concentrations, but the
204
gel time is systematically shorter at the conditions studied here.
3.6 Structure of the aggregates
M AN U
206
SC
205
RI PT
196
SPI aggregates were formed by heating solutions at 95°C during 0.5 h at different
208
protein and salt concentrations. The dependence of Mw on [NaCl] is shown in fig. 7a for
209
different protein concentrations and the dependence on C is shown in fig. 7b for different
210
ionic strengths. At a given protein concentration larger than 28 g/L, Mw increased with
211
increasing [NaCl] until gels were formed. For C<28 g/L, gels were not formed and Mw
212
reached a maximum at [NaCl]=0.2 M. The decrease of the aggregation rate for [NaCl]>0.2 M
213
is strikingly illustrated by the observation that at C= 28 g/L a gel was formed at 0.2 M NaCl,
214
but not at 0.5 M NaCl. At a given ionic strength, Mw increased with increasing C until gels
215
were formed at a critical concentration. The latter decreased from about 85 g/L in the absence
216
of salt to about 30 g/L at 0.2 M NaCl. However, it should be realized that the critical gel
217
concentration depends on the intensity of the heat treatment and decreases with increasing
218
heating time. It was shown elsewhere for salt free SPI solutions that gels can be formed at
219
least down to C=50 g/L if the systems are heated much longer (Chen et al., 2016b) .
EP
AC C
220
TE D
207
The dependence of Ir/KC on the scattering wave vector of highly diluted solutions
221
characterizes the structure of the aggregates. Figure 8a shows the results for aggregates
222
formed at 0.1 M NaCl at different SPI concentrations. Similar results were obtained at other
223
ionic strengths (see supplementary information). The q-dependence increases with increasing
224
concentrations, because the aggregates become larger. Close to the gel point, a power law
225
dependence is observed over the whole accessible q-range, indicating that the aggregates have
226
a self-similar structure (Sorensen, 2001) . The power law exponent is equal to the fractal
227
dimension of the aggregates and was found here to be df=1.7. The results superimpose if S(q) 8
ACCEPTED MANUSCRIPT is plotted vs q.Rg see figure 8b, which means that the aggregates have the same overall
229
structure independent of their size. Master curves obtained at other ionic strengths were very
230
close, see supplementary information. The master curves could be well described by eq. 2,
231
which implies df=2.0, except for the results at the highest values of q.Rg that suggests a
232
slightly smaller df. No significant effect of the salt concentration on the structure factor was
233
found.
RI PT
228
The hydrodynamic radius (Rh) of the aggregates was determined as briefly described
235
in the materials and methods section. As was shown elsewhere (Chen et al., 2016b) Rh is
236
proportional to Rg, but was systematically between 10% and 30% smaller for aggregates
237
formed at the different salt concentrations investigated here. It is more time consuming to
238
determine Rh than Rg, but it can be determined down to smaller sizes. Mw is plotted as a
239
function of Rh for aggregates obtained at different concentrations and ionic strengths in figure
240
9a. For a given ionic strength, Mw increased steeply with increasing Rh for small aggregates
241
formed at low protein concentrations, but for larger aggregates the data were consistent with a
242
power law dependence. A power law dependence between Mw and Rh is characteristic for
243
self-similar aggregates and the power law exponent is equal to the fractal dimension:
244
Mw∝Rhdf (Sorensen, 2001) . Here we find df between 1.7 and 2.0, but the data are not
245
sufficient to give a more precise value or to conclude if df depends on the salt concentration.
TE D
M AN U
SC
234
However, the onset of the power law dependence started at larger Rh and Mw when
247
more salt was present. This means that Rh and Mw of the elementary unit of the fractal
248
aggregates increased with increasing ionic strength between Rh≈30 nm, Mw≈3x106 g/mol and
249
Rh≈50 nm, Mw≈ 3x107 g/mol. Mw increased slightly more than Rh3, which suggests that the
250
density of the elementary unit increased weakly with increasing ionic strength. An increase of
251
the size of the elementary unit was also found for aggregates formed by SPI with different
252
charge densities in the absence of salt and we reproduced the figure here, see fig. 9b (Chen et
253
al., 2016c) . In that case, the size and density of the elementary units increased with
254
decreasing |α|, i.e. decreasing pH and were shown by cryo-TEM to be spherical and rather
255
polydisperse in size.
AC C
EP
246
256
The effect of heating temperature on the structure of the aggregate was studied for
257
aggregates formed at C=95 g/L in the presence of 0.05, 0.1 and 0.4 M NaCl. Aggregates of
258
different sizes were formed by heating the SPI solutions during different times at fixed
259
temperatures between 30°C and 70°C. The dependence of Mw on Rg of aggregates formed at 9
ACCEPTED MANUSCRIPT 0.1 M NaCl are shown in fig.10. At all temperatures a steep increase of Mw with increasing Rg
261
was followed by a power law dependence with approximately the same exponent. The results
262
at different temperatures for T≥60°C were the same within the experimental error. The results
263
obtained at different temperatures for T≤45°C were also the same, but for a given value of Rg
264
Mw was systematically about 50% smaller than for T≥60°C. The behavior at 50°C was
265
intermediate. A similar behavior was observed at the other two ionic strengths, see
266
supplementary information.
267
Discussion
RI PT
260
The present investigation of the influence of salt (0-0.5 M) on thermal aggregation and
269
gelation of SPI at a fixed charge density (α=-175) complements the investigation of the
270
influence of the net charge density (α=-100 to -175; pH 5.8 to 7.2) in the absence of salt that
271
was reported elsewhere (Chen et al., 2016c) . At all α and [NaCl] investigated, irreversibly
272
cross-linked aggregates were formed that have a self-similar structure on large length scales
273
with a fractal dimension close to two. The aggregates grow with heating time until they
274
percolate into a system spanning network. This behavior is similar to that reported for various
275
other types of globular proteins.
M AN U
SC
268
At steady state, elastic modulus of the gels obtained after extensive heat treatment
277
does not depend on the pH nor on the ionic strength in the range investigated. This is
278
remarkable, because the structure of the elementary units and the microscopic structure do
279
vary when the strength of the electrostatic interactions is varied either by varying the pH or by
280
adding salt. It suggests that differences in structure are compensated by differences in bond
281
strength, but in the absence of detailed knowledge about which and how many bonds are
282
deformed under stress it is not possible to go beyond this observation. As expected, G'
283
decreases with decreasing SPI concentration and for C≤40 g/L the gels either break when the
284
vials are turned upside down or the solutions become macroscopically heterogeneous.
EP
AC C
285
TE D
276
The principal effects of varying [NaCl] or α are on the aggregation kinetics and the
286
structure. If [NaCl] is increased or |α| is decreased, aggregation and gelation is faster, the
287
microstructure of the gels is more heterogeneous, and the elementary units of the fractal
288
aggregates are larger and denser. The strikingly similar effects of increasing [NaCl] or
289
decreasing |α| are obviously related to the decrease of the repulsive electrostatic interactions
290
either by screening or by reducing the charge. The screening length of electrostatic
291
interactions decreases with increasing ionic strength and becomes very small for [NaCl]>0.1. 10
ACCEPTED MANUSCRIPT 292
This may explain why tg no longer depends on [NaCl] for [NaCl]>0.1. However, the structure
293
of the gels is coarser at 0.4 M NaCl than at 0.1 M NaCl, see fig. 2, which is perhaps caused by
294
the effect of salt on hydration of the proteins. The formation of larger and denser elementary units of the fractal aggregates with
296
decreasing |α| or increasing [NaCl] may be attributed to the reduction of electrostatic
297
repulsion. It was shown by (Chen et al., 2016c) that roughly spherical particles are formed in
298
the first step of the process that subsequently randomly associate into fractal aggregates and
299
eventually form a network if the SPI concentration is sufficiently high. The size and density
300
of the elementary units is clearly determined by the strength of the electrostatic interactions.
301
The particles grow until the repulsion between the accumulated charges render further growth
302
unfavorable. However, these particles can still randomly associate into larger clusters or a
303
network. A similar aggregation mechanism was reported for thermal aggregation of whey
304
proteins (Phan-Xuan et al., 2013) .
M AN U
SC
RI PT
295
The temperature appears to have only a kinetic effect for T>60°C, as the structure of
306
the aggregates and the elastic modulus of the gels was independent of the temperature for all
307
systems. The aggregation and gelation rate increased with increasing temperature following
308
an Arrhenius dependence with the same activation energy independent of the pH, the ionic
309
strength and the SPI concentration. However, a remarkably different temperature dependence
310
of tg behavior was observed at lower temperatures in the presence of salt. Below a
311
characteristic temperature, tg become almost independent of the temperature over a range of
312
temperatures before increasing again. The characteristic temperature increased with
313
increasing ionic strength from 55°C at 50 mM NaCl to 65°C at 0.5 M NaCl. It is tempting to
314
relate the characteristic temperature with the denaturation temperature determined by DSC
315
that was reported by Bikbov et al. (1983) to increase with increasing NaCl concentration from
316
68°C without salt to 78°C at 0.5M NaCl for β-conglycinin and from 85°C to 95°C for
317
glycinin. These values are close to those reported by Renkema et al. (2002a) who also
318
showed that it depended only weakly on the pH between 6 and 8. It is likely that the
319
aggregation process at high temperatures characterized by Ea=180 kJ/mol is controlled by
320
denaturation of β-conglycinin. Denaturation of glycinin, which becomes fast above 75°C
321
(Zhao, Li, Qin and Chen, 2016) , does not appear to influence the aggregation process as the
322
kinetics is characterized by a single activation energy between 60°C and 85°C and the
323
aggregate structure is the same.
AC C
EP
TE D
305
11
ACCEPTED MANUSCRIPT The irreversible aggregation process at T≤45°C, which can only be observed in the
325
presence of salt, leads to the formation of aggregates with the same fractal dimension, but
326
with elementary units that are less dense than those formed at higher temperatures. This
327
observation together with the change in the temperature dependence of tg shown in Fig. 4 and
328
the different dependence of Mw on t/tg shown in Fig. 3, suggest that there are two different
329
thermal aggregation process of SPI. One dominates at T≥60°C and one dominates at T≤45°C.
330
We speculate that the temperature dependence of tg is weak at intermediate temperatures,
331
because in this range both processes occur with varying relative importance. As was
332
mentioned above, it appears that fast denaturation of β-conglycinin plays a major role in the
333
process at T≥60°C, denaturation of β-conglycinin is extremely slow below 45°C so the
334
driving force for irreversible aggregation at 30°C≤T≤45°C is unclear. Unfortunately, the
335
experimental techniques used in this study do not reveal the crosslinking mechanism of the
336
proteins on the molecular scale.
338
Conclusion
M AN U
337
SC
RI PT
324
During heating SPI solutions, relatively dense spherical particles are formed first that
340
in a second step randomly associate into larger fractal aggregates. If the protein concentration
341
is sufficiently high, the aggregates connect into a system spanning network. Addition of NaCl
342
causes an increase in the rate of thermal aggregation and gelation, but does not modify the
343
stiffness of the gels at steady state. The size and density of the elementary units increases with
344
increasing NaCl concentration and the microstructure of the gels becomes increasingly
345
heterogeneous. In many aspects, increasing the ionic strength is equivalent to decreasing the
346
net charge density of the protein, i.e. decreasing the pH towards the isoionic point. In both
347
cases the effect on aggregation is caused by the reduction of electrostatic repulsion.
EP
AC C
348
TE D
339
For T>60°C, the effect of heating temperature on the aggregation process is
349
principally on the kinetics. The gelation rate has an Arrhenius temperature dependence
350
between 60°C and 85°C with an activation energy of 180kJ/mol that does not depend on the
351
ionic strength, the pH or the protein concentration. A different irreversible aggregation
352
process dominates at temperature between 30°C and 45°C, which leads to fractal aggregates
353
with less dense elementary units. There are indications that the high temperature process is
354
controlled by denaturation of β-conglycinin. The driving force for irreversible aggregation at
355
lower temperatures is not yet elucidated. 12
ACCEPTED MANUSCRIPT Supplementary information
357
Measurements of the dependence of the molar mass on the radius on the gyration of the
358
aggregates formed during heating at different temperatures are shown for 0.05 M NaCl and
359
0.4 M NaCl. Also the structure factor of the aggregates formed at different ionic strengths is
360
shown.
RI PT
356
361
References
363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400
Berne, B. & Pecora, R. (2000), Dynamic light scattering With Applications to Chemistry, Biology, and physics. New York: Dover Publications. Bikbov, T., Grinberg, V., Danilenko, A. N., Chaika, T. S., Vaintraub, I. A. & Tolstoguzov, V. (1983), Studies on gelation of soybean globulin solutions Part 3., Colloid & Polymer Sci., 261, 346-358. Brown, W. (1996), Light scattering: principles and development. Oxford: Clarendon Press. Chen, N., Lin, L., Sun, W. & Zhao, M. (2014), Stable and pH-Sensitive Protein Nanogels Made by SelfAssembly of Heat Denatured Soy Protein, Journal of Agricultural and Food Chemistry, 62, 9553-9561. Chen, N., Zhao, M., Chassenieux, C. & Nicolai, T. (2016a), Structure of self-assembled native soy globulin in aqueous solution as a function of the concentration and the pH, Food Hydrocolloids, 56, 417-424. Chen, N., Zhao, M., Chassenieux, C. & Nicolai, T. (2016b), Thermal aggregation and gelation of soy globulin at neutral pH, Food Hydrocolloids, 61, 740-746. Chen, N., Zhao, M., Niepceron, F., Chassenieux, C. & Nicolai, T. (2016c), The effect of the pH on thermal aggregation and gelation of soy proteins, Food Hydrocolloids, 61, 740-746. Higgins, J. S. & Benoit, H. C. (1994), Polymers and neutron scattering. Oxford: Clarendon Press. Hua, Y., Cui, S. W., Wang, Q., Mine, Y. & Roysa, V. (2005), Heat induced gelling properties of soy protein isolates prepared from different defatted soybean flours, Food Research International, 38, 377-385. Jiang, J., Xiong, Y. L. & Chen, J. (2010), pH Shifting alters solubility characteristics and thermal stability of soy protein isolate and its globulin fractions in different pH, salt concentration, and temperature conditions, J Agric Food Chem, 58, 8035-42. Mariotti, F., Tomé, D. & Mirand, P. P. (2008), Converting nitrogen into protein - beyond 6.25 and Jones' factors, Critical reviews in food science and nutrition, 48, 177-184. Nishinari, K., Fang, Y., Guo, S. & Phillips, G. O. (2014), Soy proteins: A review on composition, aggregation and emulsification, Food Hydrocolloids, 39, 301-318. Phan-Xuan, T., Durand, D., Nicolai, T., Donato, L., Schmitt, C. & Bovetto, L. (2013), Tuning the structure of protein particles and gels with calcium or sodium ions, Biomacromolecules, 14, 1980– 1989. Renkema, J. M. S., Gruppen, H. & van Vliet, T. (2002a), Influence of pH and ionic strength on heatinduced formation and rheological properties of soy protein gels in relation to denaturation and their protein compositions, Journal of Agricultural and Food Chemistry, 50, 6064-6071. Renkema, J. M. S. & van Vliet, T. (2002b), Heat-induced gel formation by soy proteins at neutral pH, Journal of Agricultural and Food Chemistry, 50, 1569-1573. Sorensen, C. (2001), Light scattering by fractal aggregates: a review, Aerosol Science & Technology, 35, 648-687. Zhao, H., Li, W., Qin, F. & Chen, J. (2016), Calcium sulphate-induced soya bean protein tofu-type gels: influence of denaturation and particle size, International Journal of Food Science and Technology, 51, 731-741.
AC C
EP
TE D
M AN U
SC
362
13
ACCEPTED MANUSCRIPT 401 402
AC C
EP
TE D
M AN U
SC
RI PT
403
14
ACCEPTED MANUSCRIPT 404
Figure captions
405
Figure 1 Influence of the ionic strength on the evolution of G' during heating at 80oC for SPI
407
solutions at C=95 g/L.
408
Figure 2 CLSM images (40 x 40 µm) of SPI gels at C=95g/L and different NaCl
409
concentrations. The gels were formed by heating at 80oC for 16 h.
410
Figure 3 G' (a) and Mw (b) as a function of time during heating at different temperatures
411
([NaCl]= 0.1M, C=95g/L). Figure 3c shows the same data after normalizing the heating time
412
by gel time (tg).
413
Figure 4 Comparison of the gelation time as a function of temperature at different salt
414
concentration obtained by light scattering (T<70oC) and rheology (T≥65oC).
415
Figure 5 Storage shear modulus of SPI solutions at 0.05 M NaCl and different protein
416
concentrations as a function of time during heating at 90oC.
417
Figure 6 Dependence of G' (a) and the gel time (b) on the protein concentration for the SPI
418
gels by heating either without salt at pH 6.0 at 85 oC (triangles) or at 0.05 M NaCl at 90oC
419
(circles).
420
Fig. 7 Mw as a function of [NaCl] at different SPI concentrations (a) and as a function of SPI
421
concentration at different ionic strengths (b) for SPI aggregates obtained after 0.5 h heating at
422
95°C.
423
Figure 8 Dependence of of Ir/KC on the scattering wave vector for aggregates formed at 0.1
424
M NaCl at different SPI concentrations (a) and the master curve obtained by plotting the same
425
data as S(q) vs qRg (b). The solid line in figure 8b represents a fit to equation 2.
426
Figure 9 Mw as a function of Rh for SPI aggregates formed at different salt concentrations (a).
427
and pH (b). The aggregates were formed by heating SPI solutions for 0.5 h at 95°C at
428
different concentrations between 1.8 g/L and the gel concentration.
429
Figure 10 Mw as a function of Rg for aggregates formed during heating SPI solutions at C=95
430
g/L and [NaCl]= 0.1 M at different temperatures.
AC C
EP
TE D
M AN U
SC
RI PT
406
431
15
ACCEPTED MANUSCRIPT 432
Figure 1
433 434
G ' (Pa)
RI PT
0 mM 20 mM 50 mM 0.1M 0.2 M 0.4 M
102
SC
101
101
M AN U
100
102
t (min)
435 436
AC C
EP
TE D
437
16
103
ACCEPTED MANUSCRIPT 438
Figure 2
M AN U
SC
RI PT
439
440 441
AC C
EP
TE D
442
17
ACCEPTED MANUSCRIPT 443
Figure 3 103
a
85 oC 80 oC
101
RI PT
G' (Pa)
102
75 oC 70 oC
100
68 oC 65 oC
10-1 102
103
SC
101
t (min)
M AN U
444 109
Mw (g/mol)
b
108
TE D
107
101
103
104
EP
t (min)
Mw (g/mol)
109
103
c 102
108 101
107
100
10-1
10
446
-3
10
-2
10
-1
10
t/tg
18
0
10
1
10
2
G' (Pa)
AC C
445
102
70 °C 65 °C 60 °C 50 °C 45 °C 40 °C 35 °C 30 °C
ACCEPTED MANUSCRIPT 447
Figure 4
448
104
RI PT
103
101 0M 20 mM 50 mM 0.1 M 0.4 M
100 10-1 10-2
40
50
60
70
80
90
M AN U
30
SC
Tg (h)
102
o T( C)
449
AC C
EP
TE D
450
19
ACCEPTED MANUSCRIPT 451
Figure 5
M AN U
SC
RI PT
452
453 454
AC C
EP
TE D
455
20
ACCEPTED MANUSCRIPT 456
Figure 6
457
103
RI PT
G'(Pa)
a
101 60
70
80
90 100
M AN U
50
SC
102
C (g/L)
458
100
b
TE D
50
tg (min)
30 20 10
EP
5 3
459 460 461
AC C
2
50
60
70
C (g/L)
21
80
90 100
ACCEPTED MANUSCRIPT 462
Figure 7
463
109
a
RI PT
1.8 g/L 4.5 g/L 9 g/L 18 g/L 28 g/L 37 g/L 50 g/L 60 g/L 70 g/L
107
SC
Mw (g/mol)
108
106 10-2
10-1
100
M AN U
10-3
NaCl (M)
464
109
107
3 mM 10 mM 35 mM 70 mM 0.1 M 0.2 M 0.5 M
EP
Mw (g/mol)
108
TE D
b
AC C
106
0
465 466
20
40 C (g/L)
467
22
60
ACCEPTED MANUSCRIPT 468
Figure 8
469
Ir/KC(g/mol)
108
107 107 q (m-1)
470
b
TE D
AC C
10-2 10-1
472
b
10-1
EP
S (q)
100
471
108
M AN U
106
SC
1.8 g/L 4.5 g/L 9 g/L 18 g/L 28 g/L 37 g/L
a
RI PT
109
100 qRg
23
101
ACCEPTED MANUSCRIPT 473
Figure 9
109 a
RI PT
Mw (g/mol)
108
3 mM 10 mM 35 mM 70 mM 0.1M 0.2M 0.5M
107
10
100
109
M AN U
Rh (nm)
474
SC
106
b
pH 6.8 pH 6.6 pH 6.4 pH 6.3 pH 6.2 pH 6.1 pH 6.0 pH 5.9 pH 5.8
TE D
Mw (g/mol)
108
107
EP
106
10
476
Rh (nm)
AC C
475
100
24
ACCEPTED MANUSCRIPT 477
Figure 10
478
109
RI PT
108
o
70 C o 65 C 60 oC 50 oC 45 oC o 40 C o 35 C o 30 C
107
M AN U
100
SC
Mw (g/mol)
0.1 M
Rg (nm)
479 480
AC C
EP
TE D
481
25
ACCEPTED MANUSCRIPT 482 483
Supplementary information
RI PT
484
SC
10-1
0.003 M 0.01 M 0.035 M 0.07 M 0.1 M 0.2 M 0.5 M
10-2 10-1
100
101 qRg
485
M AN U
S (q)
100
Figure S1 Structure factor of SPI aggregates obtained at different NaCl concentrations. The
487
solid line represents a fit to equation 2.
EP
108
AC C
Mw (g/mol)
0.05M
107
109 0.4M
Mw (g/mol)
109
TE D
486
60 oC 55 oC
108 65 oC 60 oC o 50 C o 45 C 40 oC
50 oC 40 oC
107
35 oC
o 35 C
106
100
488
100
Rg (nm)
Rg (nm)
489
Figure S2 Mw as a function of Rg for aggregates formed during heating SPI solutions at C=95
490
g/L and [NaCl]= 0.05 M or 0.4 M at different temperatures.
491 26
ACCEPTED MANUSCRIPT Self similar SPI aggregates are formed during heating. Local density of the aggregates increases with increasing [NaCl]. The gel stiffness does not depend on the ionic strength. Gelation is faster at higher ionic strength.
AC C
EP
TE D
M AN U
SC
RI PT
Influence of decreasing pH and increasing [NaCl] is similar.