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κ-carrageenan mixed gels induced by high pressure processing
Journal Pre-proof Modification of the structural and rheological properties of β-lactoglobulin/κcarrageenan mixed gels induced by high pressure processing Xiaoying Li, Xiaoye He, Like Mao, Yanxiang Gao, Fang Yuan PII:
S0260-8774(19)30494-7
DOI:
https://doi.org/10.1016/j.jfoodeng.2019.109851
Reference:
JFOE 109851
To appear in:
Journal of Food Engineering
Received Date: 4 July 2019 Revised Date:
28 November 2019
Accepted Date: 28 November 2019
Please cite this article as: Li, X., He, X., Mao, L., Gao, Y., Yuan, F., Modification of the structural and rheological properties of β-lactoglobulin/κ-carrageenan mixed gels induced by high pressure processing, Journal of Food Engineering (2019), doi: https://doi.org/10.1016/j.jfoodeng.2019.109851. 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
Modification
of
the
structural
and
rheological
properties
2
β-lactoglobulin/κ-carrageenan mixed gels induced by high pressure processing
of
3 4
Xiaoying Li1, Xiaoye He1, Like Mao, Yanxiang Gao, Fang Yuan*
5 6
Key Laboratory of Functional Dairy, Ministry of Education, College of Food Science
7
and Nutritional Engineering, China Agricultural University, Beijing 100083, P. R.
8
China
9 10
Running Title: β-Lactoglobulin/κ-carrageenan mixed gels induced by HPP
11 12
* Corresponding Author.
13
1
These authors contributed equally to this work.
14 15
Tel.: +86 10 6273 7034; fax: +86 10 6273 7986.
16
Address: Box 112, No.17 Qinghua East Road, Haidian District, Beijing 100083,
17
China
18
E-mail: [email protected]
1
19
Abstract
20
High pressure processing (HPP) is an emerging non-thermal processing technology
21
and a preparation method to induce protein-polysaccharide mixed gels, which can
22
encapsulate and delivery thermosensitive bioactive compounds. This study
23
investigated the properties of β-lactoglobulin (β-Lg)/ κ-carrageenan (κ-car) mixed
24
gels induced by HPP (0.1-600 MPa for 30 min, 25 °C) with different ratios of β-Lg to
25
κ-car at pH 3.0, 5.0 and 7.0. The results showed that the pressure required to form
26
β-Lg/κ-car mixed gels was at least 400 MPa when the β-Lg/κ-car mass ratio increased
27
to 14:1 at all tested pH values. The water holding capacity and textural properties
28
increased with the increase of pressure levels. The hydrophobic interaction was
29
dominant in the mixed gels induced by HPP at all pH values, and the network
30
structure was more compact and smoother with higher pressure.
31 32
Key words: β-lactoglobulin; κ-carrageenan; mixed gels; high pressure processing; gel
33
properties
2
34
1. Introduction
35
Gelation is a common phenomenon in foods, and the molecules responsible for the
36
gelation are typically proteins or polysaccharides (Ji et al., 2017). In case where two
37
types of biopolymers (e.g., proteins and polysaccharides, different types of proteins,
38
or different types of polysaccharides) are present during gelation can affect the final
39
properties of the gel network due to the interaction of molecules (Ersch et al., 2016).
40
Food gels can be induced by heat treatment (Zhou et al., 2014), acid (Rabiey &
41
Britten, 2009), Ca2+ (Phan-Xuan et al., 2014), enzyme (Wu et al., 2016), etc. In
42
addition, as a non-thermal technology, high pressure processing (HPP) is widely used
43
in food gel systems in recently years because it can enable the formation of unique
44
food gel structures without the use of heat or chemical additives (Cao et al., 2012; Ma
45
et al., 2013). HPP can prepare food gels with higher storage modulus and loss
46
modulus, and the gel structure has more pores (Saowapark et al., 2008). Compared
47
with heat treatment, HPP is more moderate and conducive to the encapsulation and
48
release of heat-sensitive substances, such as β-carotene (Mensi et al., 2013). In
49
addition, HPP could change protein conformation and influence denaturation,
50
aggregation and gelation, resulting in a modification of textural properties and
51
possible extension of shelf-life (Ma et al., 2013).
52
It had been known that the physicochemical conditions, such as pH, ionic
53
strength, temperature, pressure and holding time, have a great influence on the
54
interaction between proteins and polysaccharides, giving biopolymers some unique
55
gelling properties (Cao et al., 2012; Zhang et al., 2015). Previous studies have shown 3
56
that the polysaccharides can reduce the critical gelation concentration of proteins, so
57
protein-polysaccharide biopolymers were widely considered as a new gel system. The
58
composition, distribution, physical status, volume fraction of polysaccharides can
59
affect the interactions between them (Zhou et al., 2014; Turgeon & Beaulieu, 2001).
60
β-lactoglobulin (β-Lg) is a globular protein, which is made up of 162 amino acid
61
residues and 5 thiol groups; its molecular weight is 18.4 KD and isoelectric point is
62
about 5.2 (Wu et al., 2016). β-Lg is now widely applied in food systems because of its
63
good gelation property. κ-carrageenan (κ-car) is a linear anionic sulphated
64
polysaccharide extracted from red alga cell wall, which has been widely used in the
65
food industry as gelling, thickening and stabilizing agent (Piculell, 2006). Κ-car could
66
enhance the strength of milk protein gels. In addition, the appearance of β-Lg gel can
67
be also affected by the presence of κ-car. It was observed that the single β-Lg formed
68
transparent gels at pH 7.0 and opaque white gels over pH 4.0-6.0, however, when
69
κ-car was added, it formed opaque white gels for all pH (Eleya & Turgeon, 2000).
70
In our previous work, the effects of high pressure treatments, protein
71
concentrations and pH on gelation of β-Lg solutions have been studied systematically
72
(Li et al., 2018). Based on the work above, protein-polysaccharide mixed gels induced
73
by HPP directly without any heat treatment was reported in this research. The
74
objective of our current study was to investigate the effects of pressure (200-600
75
MPa), pH (3.0-7.0), β-Lg concentration (16-32%) and its ratio to κ-car (8:1-16:1) on
76
the structural and functional properties of β-Lg/κ-car mixed gels induced by HPP.
77
Furthermore, the mechanism of the β-Lg/κ-car mixed gels formed by HPP was 4
78
explored, which could be used as a food wall material to provide theoretical guidance
79
for the delivery system of thermosensitive bioactive compounds with better
80
microstructure.
81
2. Materials & methods
82
2.1 Materials
83
The β-Lg (97.7%, protein) was obtained from Davisco Food International (Le Sueur,
84
MN, USA). The κ-car (purity > 90%) was purchased from CP Kelco (Copenhagen,
85
Denmark). All other chemicals used were of analytical grade.
86
2.2 Solution preparation
87
Firstly, the β-Lg (16, 20, 24, 28, 32%, w/v) and κ-car (2%, w/v) were dissolved in
88
different buffer solutions at pH 3.0 (10 mM glycine-HCl buffer), pH 5.0 (10 mM
89
acetic acid buffer) and pH 7.0 (10 mM phosphate buffer), respectively. All samples
90
were magnetically stirred at 25 ℃ for 4 h, and then stored overnight at 4 ℃ for further
91
use. Secondly, different concentrations of β-Lg solution (16, 20, 24, 28, 32%, w/v)
92
were mixed with κ-car solution (2%, w/v) in equal volumes to prepare different mixed
93
solutions (8:1, 10:1, 12:1, 14:1, 16:1, w/w). The mixed samples were stirred at 25 ℃
94
for 4 h.
95
2.3 High pressure treatment
96
The β-Lg/κ-car mixed solutions were moved to centrifuge tubes (50 mL) and vacuum
97
sealed in polyethylene bags. The sealed samples were subjected to high-pressure
98
treatment at 200, 400 and 600 MPa for 30 min at 25 ℃ using HPP L2-700/1 ultra-high
99
pressure equipment (Tianjin Huatai Senmiao Biotechnology and Technique Co. Ltd, 5
100
Tianjin, China) with water as the medium. Untreated samples (0.1 MPa) were used as
101
controls. The rates of compression and decompression were 6.5 MPa/s and 20 MPa/s,
102
respectively. Compression was accompanied by increasing the temperature of about 3℃
103
100 MPa-1 (Balasubramaniam, Farkas, & Turek, 2008). After pressurization, the
104
sample temperature started to decrease during the holding time (30 min) due to the
105
heat transfer from samples to the stainless steel of pressure vessel. However, a quick
106
drop of sample temperature was happened during depressurization, which is even
107
lower after depressurization compared with initial temperature (Chen & Hoover,
108
2003). Considering that the lowest temperature is around 60 °C that can modify the
109
tertiary structure of β-lg (Rodrigues et al., 2019), as well as the maximum temperature
110
of sample is about 40 °C, which was calculated by heating rate mentioned above, the
111
degree of short-lived temperature increase in high-pressure process was not sufficient
112
to denature the protein samples. All samples were stored at 4 ℃ after treated and the
113
measurements of the properties were taken after 24 h.
114
2.4 Small deformation measurements
115
Small deformation oscillatory measurements were performed, on a controlled-strain
116
rheometer (AR-1500ex, TA Instruments, Delaware, USA) using a parallel plate
117
geometry (plate diameter, 40 mm; gap, 5 mm). After HPP treated, the cylindrical
118
samples (diameter is about 25 mm) were separated from the centrifuge tube with a
119
blade. Before the test, the gel samples were cut into slices of 5 mm uniform thickness,
120
and then transferred to the measuring geometry. The plate was equipped with a
121
circulating water system for the temperature control (25 ℃). Storage and loss modulus, 6
122
G' and G'' of mixed gels, were measured by this small deformation oscillation test. For
123
frequency sweep, measurements were performed from 0.1 to 100 rad/s under a shear
124
strain of 1% (Strain sweeps curves of β-Lg /carrageenan mixed gels were shown in
125
Supplementary Figure 1). Each time a new sample was used for the measurement.
126
And each measurement was repeated for three times.
127
2.5 Texture profile analysis (TPA)
128
TMS-Pro Food Property Analyzer (Food Technology Corporation, Virginia, USA)
129
was used to analyze the textural properties at 25 ℃. After HPP treated, the cylindrical
130
samples (diameter is about 25 mm) were separated from the centrifuge tube with a
131
blade. Each gel sample was cut into six slices of 10 mm uniform thickness. Before the
132
test the sample was equilibrated for 2 h at room temperature and then subjected to a
133
compression test using a cylindrical probe (TMS-50mm) at a speed of 60 mm/min
134
with a 0.5 N trigger force and 30% deformation. The textural properties of the mixed
135
gels including hardness, cohesiveness, springiness and chewiness were gained directly
136
from the software (Chen et al., 2010).
137
2.6 Determination of water holding capacity (WHC)
138
The determination of WHC was according to Zhang's method (Zhang et al., 2015),
139
with minor modifications. The β-Lg/κ-car mixed gel (about 3 g) was centrifuged at a
140
speed of 10000 r/min for 20 min, then the surface water was removed, and the total
141
weight of centrifuge tube and mixed gel was weighed before and after centrifugation,
142
until the difference between two consecutive measurements was less than 0.05 g
143
(about 5-8 times). WHC was described as the ratio of final gel weight after 7
144
centrifugation to the initial gel weight.
145
WHC ( % ) =
146
W1 - W 0 W2 - W0
× 100 %
147
where W1 is the total weight (g) of centrifuge tube and mixed gel after centrifugation,
148
W2 is the initial total weight (g) of centrifuge tube and mixed gel, W0 is the weight (g)
149
of centrifuge tube. Each measurement was performed in triplicate.
150
2.7 Fourier transform infrared (FTIR) spectroscopy
151
The functional groups of the freeze-dried β-Lg gel (16%, w/v) and β-Lg/κ-car mixed
152
gel (16:1, w/w) at pH 5.0 before (0.1 MPa) and after high pressure treatment (200
153
MPa, 400 MPa, 600 MPa) for 30 min were determined by Spectrum 100 Fourier
154
transform spectrophotometer (PerkinElmer, UK). Before the measurement, 2.0 mg
155
samples were mixed with 200 mg potassium bromide (KBr) and tableted into pellet.
156
FTIR spectra were from 4000 cm-1 to 400 cm-1 at a resolution of 4 cm-1 and eleven
157
times scanning. In addition, pure KBr powder was used as a baseline. The data were
158
processed by Omnic v8.0 (Thermo Nicolet, USA).
159
2.8 Field emission scanning electron microscopy (FE-SEM)
160
In order to observe the microstructure of the mixed gels, field emission scanning
161
electron microscopy (FE-SEM, JSM-6701F, JEOL, Japan) was used at an accelerating
162
voltage of 5.0 kV. Prior to the observation, β-Lg/κ-car mixed gels were freeze-dried,
163
and the surfaces of the samples were coated with a gold layer to avoid charging under
164
the electron beam.
165
2.9 Molecular force of mixed gel 8
166
The preparation of β-Lg/κ-car solution (16:1, w/w) was according to the method
167
described in section 2.2. NaCl, urea and propylene glycol were added to the mixed
168
solution, respectively, which to make the additive concentrations of 0.4, 0.8, 1.2, 1.6
169
and 2.0 mol/L in resultant solution. Then all the samples were high-pressure treated at
170
400 MPa for 30 min. After that, the samples were stored at 4 ℃ overnight. Next, the
171
texture properties of the mixed gel were measured according to the method described
172
in section 2.5, to study the contribution of electrostatic force, hydrogen bonding and
173
hydrophobic interaction on the gel formation.
174
2.10 Statistical analysis
175
All the data obtained were repeated at least three times. Analysis of variance
176
(ANOVA) was performed using SPSS 18.0 software (SPSS Inc., Chicago, USA), and
177
significant differences between means were identified using Duncan’s multiple range
178
test (p < 0.05).
179
3. Results and discussion
180
3.1 Rheological properties of β-Lg/κ-car mixed gels induced by HPP
181
Fig. 1 showed the frequency sweep curves of β-Lg/κ-car mixed gels at different
182
conditions including protein-polysaccharide ratio, pressure level and pH value.
183
According to the result of preliminary experiments, the pressure required to form
184
β-Lg/κ-car mixed gels was at least 400 MPa when the β-Lg/κ-car mass ratio increased
185
to 14:1 at all tested pH values. Nevertheless, the mixed gels can only be formed until
186
the pressure level achieved at 600 MPa with the β-Lg/κ-car mass ratio of 8:1. Thus,
187
the data regarding β-Lg/κ-car mass ratios of 8:1, 10:1 and 12:1 under 400 MPa, as 9
188
well as β-Lg/κ-car mixed solutions under 200 MPa at all ratios, were not shown in Fig.
189
1.
190
When the elasticity modulus (G') is much larger than the viscous modulus (G''),
191
G' and G'' are paralleled and independent of frequency, it is regarded as a typical gel
192
system (Morris & Ross-Murphy, 1981; Winter & Chambon, 1986). Furthermore,
193
according to Morris et al. (2012), when G'' > G' at low frequency showing fluid
194
characteristics, while G' increase faster than G'' with increasing frequency and
195
consequently G' > G'' showing solid-like characteristics, that is to say G' and G'' have
196
an intersection, it is defined as weak gel. It can be seen from Fig. 1 that G' and G''
197
increased with increasing frequency when the β-Lg/κ-car mass ratio and pressure
198
increased at the same pH, which indicated a typical viscoelastic characteristic. It was
199
worth to mention that the mixed gels with β-Lg/κ-car mass ratio of 8:1 were weak
200
gels after treated by 600 MPa, 30 min at pH 3.0 and 7.0, while the others were typical
201
gels.
202
In the current study, it was found that β-Lg/κ-car mixed gels induced by HPP
203
were pH-dependent, and the order of gel strength affected by pH was pH 5.0 > pH
204
7.0 > pH 3.0 in terms of G'. It was possible that κ-car formed the main body of mixed
205
gels, which depended on the helix and twist, and β-Lg was filled into that. There was
206
weak electrostatic force between β-Lg and κ-car when the pH was 5.0, which was
207
close to the isoelectric point (pI ≈ 5.2) of β-Lg; but hydrophobic interaction
208
increased with higher pressure levels at this pH, therefore the gel strength was
209
maximum in this case (Eleya & Turgeon, 2000). For sulphated polysaccharides, a 10
210
soluble protein-polysaccharide complex may be formed at pH above the protein pI
211
(pH 7.0), which could be a result of the interaction between NH3+ (β-Lg) and SO3-
212
(κ-car). Such attraction is particularly strong and even possible when both protein and
213
sulphated polysaccharide are negatively charged (Rafe & Razavi, 2013). Furthermore,
214
κ-car fixed a large amount of water and thickened β-Lg solution because of the
215
exclusion function of the system, therefore promoted the formation of gel and
216
improved the gel strength. At more acidic pH (below pH 4.0), it may prevent κ-car
217
gelation due to acid hydrolysis (Mleko et al., 1997), which impeded its crosslinking
218
with β-Lg, therefore the gel strength was lowest at pH 3.0.
219
Protein and polysaccharide mixed gels can be classified into three types:
220
interpenetrating, coupled and phase-separated networks. Interpenetrating network is
221
the simplest case that there is no interaction but only a topological structure between
222
the two components, which form an independent but continuous network. Nguyen et
223
al. (2015) considered that the network of β-Lg/κ-car mixed gels induced by Ca2+ is
224
interpenetrating, they found that the single networks (β-Lg gels or κ-car gels) and the
225
interpenetrated networks became stiffer with higher Ca2+ concentration and the elastic
226
modulus of the mixed gels was closed to the sum of the two single gels. Coupled
227
networks involved two different molecules that formed the junction zones because of
228
synergistic reaction. Le & Turgeon (2013) found that β-Lg and xanthan gum (XG)
229
formed coupled gel induced by electrostatic attractive interaction, by which XG
230
provided a frame for gel organization and β-Lg aggregated along the XG chains.
231
Phase-separated networks mean that both biopolymers form separate network 11
232
independently or both biopolymers get in two different phases separately (Le &
233
Turgeon, 2015). In our research, β-Lg and κ-car formed mixed gels probably induced
234
by electrostatic attraction, hydrogen bonding and hydrophobic interaction, and the
235
combination of protein and polysaccharide greatly reduced the β-Lg concentration
236
required for gel formation, therefore it may be coupled network formed between β-Lg
237
and κ-car.
238
3.2 Textural properties of β-Lg/κ-car mixed gels induced by HPP
239
Texture is an important property that determines the organoleptic quality of gels. In
240
our research, the protein-polysaccharide ratio and pressure had large effects on the
241
textural properties of β-Lg/κ-car mixed gels. Hardness reflects the strength of gel
242
structure when it is compressed (Zhao et al., 2014). Springiness is a measure of how
243
much the gel structure is broken down by the initial compression; high springiness
244
will result when the gel structure is broken into few large pieces during the first
245
compression whereas low springiness results from the gel breaking into many small
246
pieces (Lau et al., 2000). Fig. 2 showed that the hardness and springiness increased
247
with higher β-Lg/κ-car ratio and pressure. This finding was attributed to the denser
248
networks of the mixed gels with higher β-Lg/κ-car ratio and pressure, which reinforce
249
the intermolecular interaction of β-Lg and κ-car, and therefore the hardness and
250
springiness were increased (Le & Turgeon, 2015).
251
Fig. 3 showed comparison of textural properties of β-Lg gels and β-Lg/κ-car
252
mixed gels at pH 5.0 after treated by 600 MPa, 30 min. It can be seen that the addition
253
of κ-car obviously improved the mixed gels strength (p < 0.05), which suggested that 12
254
HPP could promote the intermolecular interaction of β-Lg and κ-car. In addition, the
255
springiness of the mixed gels was significantly higher than the single β-Lg gels (p <
256
0.05), which indicated that there were differences in the structure of these gels. These
257
findings were consistent with the results of Dickinson & James (2000) studies on the
258
effects of high pressure on β-Lg and pectin mixtures.
259
3.3 The WHC of β-Lg/κ-car mixed gels
260
Water holding capacity (WHC) refers to the water retention capacity of raw materials
261
during the manufacturing process of foods, which is directly related to the gel texture,
262
structure and state (Urbonaite et al., 2016). Fig. 4A exhibited the changes of WHC of
263
different β-Lg/κ-car mixed gels under 600 MPa. The ratio of β-Lg and κ-car had
264
significant effects on the WHC of the mixed gels (p < 0.05), the WHC increased with
265
higher content of β-Lg. It was attributed to the increase of β-Lg strengthening the
266
intermolecular interaction between β-Lg and κ-car, as well as β-Lg and water.
267
WHC of a gel has been reported to be determined by both gel microstructure and
268
gel stiffness (Urbonaite et al., 2015). Fig. 4B showed that the effects of pressure and
269
pH on WHC of the mixed gels. It can be seen that water holding capacity was higher
270
under 600 MPa than 400 MPa at the same ratio of β-Lg and κ-car, and the WHC was
271
the highest at pH 5.0 under the same β-Lg/κ-car ratio and pressure, which were in
272
accordance with the changes of G' value, gel hardness and gel springiness. It was
273
probably that a more cross-linked network structure was formed with higher pressure,
274
and the pore diameter of the mixed gels was reduced, which was beneficial to the
275
increase of WHC. 13
276
3.4 The FTIR of β-Lg/κ-car mixed gels
277
In order to investigate the interaction between β-Lg and κ-car, FTIR spectroscopic
278
analysis of β-Lg (16%, w/v) and mixed gels (16:1, w/w) were carried out as shown in
279
Fig. 5. The peak around 3000-3500 cm-1 represents the water peak of amide A, which
280
could be used for to evaluate the interaction between protein and water molecules and
281
reflect the changes of hydrogen bonds. It can be seen that the wavenumber of β-Lg
282
hydrogen bond was at 3283.62 cm-1. The wavenumbers of β-Lg/κ-car mixtures under
283
0.1, 200, 400 and 600 MPa HPP treatments were 3314.68, 3314.80, 3298.48 and
284
3298.48 cm-1, respectively, which indicated that the addition of κ-car resulted in the
285
change of hydrogen bond compared with the single β-Lg. The wavenumbers increased;
286
the hydrogen bonding ability enhanced. The changes of interaction between protein
287
and polysaccharide were related to peak stretching vibration. In our study, it was
288
speculated that the shift of hydrogen bonds could be attributed to sulfate group of
289
κ-car, which enhanced intermolecular forces and hydrogen bonding ability after
290
pressurization, therefore the G' of mixed gels increased (Liu et al., 2014).
291
From Fig. 5, compared with β-Lg, the peak intensity of β-Lg/κ-car mixture at
292
about 2960 cm-1 increased firstly and then decreased. The wavenumbers of β-Lg/κ-car
293
mixture under 400 and 600 MPa were 2958.04 cm-1 and 2956.78 cm-1, respectively. It
294
may be due to the increase of the esterification and acetylation degree of κ-car
295
induced by high pressure. The C-H stretches associated with the ring methine
296
hydrogen atoms in k-car might also contribute to the changes at 2960 cm-1 (Fang et al.,
297
2002; Chen et al., 2014). 14
298
The amide I band between 1600 and 1700 cm-1 was commonly used to analyze
299
the secondary structure of proteins (Carbonaro & Nucara, 2010). From Fig. 5, the
300
typical absorption peaks of amide I and amide II region were at 1648.66 cm-1 and
301
1533.66 cm-1, respectively. The amide I region was mainly C=O stretching vibration
302
and the amide II region was attributed to C-N stretching vibration and in-plane
303
bending vibration (Bhattacharjee et al., 2005; Perisic et al., 2011). In addition, the
304
peak wavenumbers decreased with the increase of pressure, which also showed that
305
the interactions between β-Lg and κ-car enhanced.
306
Compared with β-Lg, there were more two peaks appeared at 1075 cm-1 and 930
307
cm-1 in β-Lg/κ-car mixtures, which were the characteristic peaks of -SO3- and free
308
sulfate in κ-car, respectively. In addition, it can be seen from Fig. 5, the peaks
309
intensity decreased after pressurization, which showed that the pressure promoted the
310
transformation between -SH and -S-S- (Gómez-Ordóñez & Rupérez, 2011).
311
3.5 The SEM images of β-Lg/κ-car mixed gels
312
Fig. 6 showed the SEM images of β-Lg/κ-car mixtures (16:1, w/w) treated with
313
different pressures at pH 5.0. Fig. 6E was the image of single β-Lg. It can be seen that
314
κ-car was attached to β-Lg when β-Lg/κ-car mixtures formed at atmospheric pressure
315
(Fig. 6A). β-Lg and κ-car did not form regular internal structures, showing
316
fragmented patterns under 200 MPa (Fig. 6B). However, the microstructure of the
317
mixed gel under 400 MPa was similar to sponge, and the pore size was larger than the
318
mixed gel under 200 MPa (Fig. 6C), which also confirmed that the WHC of the gel
319
was poor. When treated by 600 MPa pressure, the microstructure of the mixed gel was 15
320
similar to that of honeycomb which was very dense and homogeneous (Fig. 6D).
321
3.6 Effects of molecular interaction on the formation of β-Lg/κ-car mixed gels
322
The gelation is the outcome of protein denaturation, which intermolecular covalent
323
and noncovalent interactions are involved (Wijaya et al., 2017). Electrostatic
324
interactions, hydrogen bonds and hydrophobic interactions are the major non-covalent
325
interaction responsible for the structural organization of food components
326
(McClements et al., 2009). Sodium salts affect protein-polysaccharides interactions,
327
either by ionic strength effects, or binding to the protein charged groups (Uruakpa &
328
Arntfield, 2006). The electrostatic shielding effect caused by high concentration of
329
NaCl can reduce electrostatic interaction mentioned above. Hence, the addition of
330
NaCl to β-Lg/κ-car dispersions is an effective method to evaluate the contribution of
331
electrostatic interactions to the gel network formation. As to hydrogen bonds, urea can
332
hinder the formation of hydrogen bonds by affecting the structure of water molecules,
333
which can be used to evaluated the contribution of hydrogen bonds in the gel
334
formation (Le´ger & Arntfield, 1993). Propylene glycol is another reagent that affects
335
water structure and it can disrupt hydrophobic forces and promote hydrogen and
336
electrostatic bonds (Bernal et al., 1987).
337
Fig. 7 showed that the relationship between the hardness and springiness of the
338
mixed gels with different concentrations of NaCl, urea and propylene glycol, which
339
can reveal the strength of the three kinds of interaction. It was found that low
340
concentration (0.4 mol/L) of NaCl improved the gel hardness and springiness,
341
however, as the concentration of NaCl increased, the hardness and springiness of all 16
342
gel samples decreased quickly. It was mainly attributed to the neutralization of
343
electrostatic interaction between charged amino acids by intense salty ions screening
344
that reduced the electrostatic interaction. The decrease of hardness and springiness of
345
gels caused by addition of urea and propylene glycol at different concentrations
346
suggested that hydrogen bonding and hydrophobic interaction had effects on the
347
mixed gel formation. However, the contribution of hydrogen bonding was less than
348
the electrostatic interaction at pH 3.0 and 7.0. At pH 5.0, which is close to the
349
isoelectric point of β-Lg, the contribution of hydrogen bonding was greater than the
350
electrostatic interaction. Furthermore, because adding propylene glycol has the most
351
obvious effects on the hardness and springiness of β-Lg/κ-car mixed gels, it was
352
believed that hydrophobic interaction was dominant in the mixed gels induced by
353
HPP.
354
4. Conclusion
355
This work evaluated the properties of β-Lg/κ-car mixed gels induced by HPP at pH
356
3.0, 5.0 and 7.0. In the current study, it was found that the pressure required to form
357
β-Lg/κ-car mixed gels was at least 400 MPa when the β-Lg/κ-car mass ratio increased
358
to 14:1 at all tested pH values. The results of rheological test showed that the network
359
of β-Lg/κ-car mixed gels induced by HPP was coupled. There were hydrophobic,
360
electrostatic and hydrogen bonding interactions between β-Lg and κ-car, and the
361
hydrophobic interaction was dominant in the mixed gels induced by HPP. The G'
362
value, WHC, hardness, springiness, and chewiness of the mixed gels with higher
363
pressure and higher concentration of β-Lg were better, and the network structure of 17
364
the mixed gels was more compact and uniform. The gel properties of β-Lg/κ-car
365
mixed gels were better than the single β-Lg gels induced by HPP. The results of this
366
study can provide a theoretical guidance for the development of a novel food wall
367
material which can encapsulate and delivery thermosensitive compounds.
368 369
Acknowledgements
370
This research was funded by the National Natural Science Foundation of China (No.
371
31371836), National Key R&D Program of China (No. 2016YFD0400804), and
372
Natural Science Foundation of Beijing (No. 6192015).
373 374
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Fig. 1 Frequency sweep curves (G' and G'') of β-Lg/κ-car mixed gels at different pH values (A1 and A2, pH 3; B1 and B2, pH 5; C1 and C2, pH 7). Fig. 2 Textural properties of β-Lg/κ-car mixed gels (mean ± SD, n=6) (A, B, C & D). Different superscript letters indicate significant differences in the same graph (p < 0.05). Fig. 3 Comparison of textural properties of β-Lg gels and β-Lg/κ-car mixed gels at pH 5.0 and 600 MPa. Different superscript letters indicate significant differences in the same graph (p < 0.05). Fig. 4 Water holding capacity (WHC) of β-Lg/κ-car mixed gels under 600 MPa HPP treatment with different mass ratios at different pH values (A) and the WHC of β-Lg/κ-car mixed gels under 400 and 600 MPa HPP treatment with the mass ratio of 14:1 and 16:1 at different pH values (B). Different superscript letters indicate significant differences in the same graph (p < 0.05). Fig. 5 FTIR spectra of β-Lg (16%, w/v) and β-Lg/κ-car mixed gels with the mass ratio of 16:1 at pH 5.0. Fig.6 SEM images of β-Lg/κ-car mixtures (16:1, w/w) at pH 5.0 under different pressures: (A) 0.1 MPa; (B) 200 MPa; (C) 400 MPa; (D) 600 MPa, and (E) β-Lg (16%, w/v) at pH 5.0 under 0.1 MPa. Fig. 7 The effects of different concentrations (0.4, 0.8, 1.2, 1.6 and 2.0 mol/L) of NaCl, urea and propylene glycol on hardness and springiness of β-Lg/κ-car mixed gels at different pH values (A1 and A2, pH 3; B1 and B2, pH 5; C1 and C2, pH 7).
Fig. 1
1000
1000
100
100
G'' (Pa)
10000
G' (Pa)
10000
10 pH 3.0-14:1-400 MPa pH 3.0-16:1-400 MPa pH 3.0- 8:1-600 MPa pH 3.0-10:1-600 MPa pH 3.0-12:1-600 MPa pH 3.0-14:1-600 MPa pH 3.0-16:1-600 MPa
1 0.1
A1 0.01 0.1
1
10
10 1
pH 3.0-14:1-400 MPa pH 3.0-16:1-400 MPa pH 3.0- 8:1-600 MPa pH 3.0-10:1-600 MPa pH 3.0-12:1-600 MPa pH 3.0-14:1-600 MPa pH 3.0-16:1-600 MPa
0.1
A2 0.01
100
0.1
1
ω (rad/s)
1000
100
100
10 pH 5.0-14:1-400 MPa pH 5.0-16:1-400 MPa pH 5.0- 8:1-600 MPa pH 5.0-10:1-600 MPa pH 5.0-12:1-600 MPa pH 5.0-14:1-600 MPa pH 5.0-16:1-600 MPa
B1 0.01 0.1
1
10
G'' (Pa)
1000
G' (Pa)
10000
0.1
10 pH 5.0-14:1-400 MPa pH 5.0-16:1-400 MPa pH 5.0- 8:1-600 MPa pH 5.0-10:1-600 MPa pH 5.0-12:1-600 MPa pH 5.0-14:1-600 MPa pH 5.0-16:1-600 MPa
1 0.1
B2 0.01 0.1
100
1
10000
10000
1000
1000
100
100
10 pH 7.0-14:1-400 MPa pH 7.0-16:1-400 MPa pH 7.0- 8:1-600 MPa pH 7.0-10:1-600 MPa pH 7.0-12:1-600 MPa pH 7.0-14:1-600 MPa pH 7.0-16:1-600 MPa
0.1
C1 0.01 0.1
1
10
ω (rad/s)
10
100
ω (rad/s)
100
G'' (Pa)
G' (Pa)
ω (rad/s)
1
100
ω (rad/s)
10000
1
10
10 pH 7.0-14:1-400 MPa pH 7.0-16:1-400 MPa pH 7.0- 8:1-600 MPa pH 7.0-10:1-600 MPa pH 7.0-12:1-600 MPa pH 7.0-14:1-600 MPa pH 7.0-16:1-600 MPa
1 0.1
C2 0.01 0.1
1
10
ω (rad/s)
100
Chewiness (mJ) 18
b
0 abc
C
9
f
3
a
8
6 h h
4 g
de f
2 ef
a bc bc
pH 3.0
h d ef
ab
pH 5.0
12
6 i
ab o
c ef
cd
hi
Springiness (mm)
pH 5.0
l
k
j
e 14 16:1-4 10:1-400 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a 14 Pa : 1 16 -4 10:1-400 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a 14 Pa : 1 16 -4 10:1-400 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a Pa
Hardness (N)
pH 3.0
Cohesiveness
14 : 16 1-4 : 0 10 1-4 0 M :1 00 P 12 -6 M a : 0 14 1-6 0 MPa : 0 16 1-6 0 MPa :1 00 P -6 M a 00 P M a Pa 14 :1 16 -4 : 0 10 1-4 0 M :1 00 P 12 -6 M a : 0 14 1-6 0 MPa : 0 16 1-6 0 MPa :1 00 P -6 M a 00 P M a Pa 14 :1 16 -4 : 0 10 1-4 0 M :1 00 P 12 -6 M a : 0 14 1-6 0 MPa : 0 16 1-6 0 MPa :1 00 P -6 M a 00 P M a Pa
A
14 16:1-4 10:1-400 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a 14 Pa : 1 16 -4 : 0 10 1-4 0 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a 14 Pa : 1 16 -4 : 0 10 1-4 0 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a Pa
14 : 16 1-4 : 0 10 1-4 0 M : 0 12 1-6 0 MPa :1 0 0 P 14 -6 M a : 0 16 1-6 0 MPa :1 0 0 P -6 M a 00 P M a Pa 14 : 16 1-4 : 0 10 1-4 0 M : 0 12 1-6 0 MPa :1 0 0 P 14 -6 M a : 0 16 1-6 0 MPa :1 0 0 P -6 M a 00 P M a Pa 14 : 16 1-4 : 0 10 1-4 0 M : 0 12 1-6 0 MPa : 0 14 1-6 0 MPa :1 0 0 P 16 -6 M a :1 0 0 P -6 M a 00 P M a Pa
Fig. 2
pH 7.0
3.5
k j
i
ab
0
pH 7.0 0.9
15
m
0.4
B
3.0
n
pH 3.0
2.5
1.0
D
0.8 f fg
0.7
0.6
0.5
pH 5.0 i
gh
d
hij
fg
f
e
0.5
pH 3.0
e
pH 7.0
gh
d h
f
f
2.0 e
a
pH 5.0
ghij ij fghi ghij
f
1.5
c d
bc ab
0.0
pH 7.0 hij j
fgh fgh
d e
d c
g b
a
c
0.00
Fig. 3
8
d
A
B d
3
c
c
4
2
Springiness (mm)
Hardness (N)
6
b
a
0 14% β-Lg
16
14:1 β-Lg/κ-car 16% β-Lg
1 a
0
16:1 β-Lg/κ-car
14% β-Lg
14:1 β-Lg/κ-car 16% β-Lg
16:1 β-Lg/κ-car
d
C
D bc
0.8
14 12
c b
c
Cohesiveness
Chewiness (mJ)
b
2
10 8 6 b
4
0.6
0.4
a
0.2
2 a
0.0
0 14% β-Lg
14:1 β-Lg/κ-car 16% β-Lg
16:1 β-Lg/κ-car
14% β-Lg
14:1 β-Lg/κ-car 16% β-Lg
16:1 β-Lg/κ-car
Fig. 4
100
A
pH 3.0
pH 5.0 gh
i
pH 7.0
g
90
ef
h
f
e
80
d
d
WHC (%)
70
c
60
b
b
b
50 40
a
a
30
8: 1 10 :1 12 :1 14 :1 16 :1
8: 1 10 :1 12 :1 14 :1 16 :1
8: 1 10 :1 12 :1 14 :1 16 :1
0
ratios of β-Lg/κ-car
100 B
400 MPa 600 MPa
WHC (%)
pH 3.0 d c
pH 5.0 f e
e
d
d
c c
b
80
pH 7.0
b
a
60 14:1 16:1
14:1 16:1 ratios of β-Lg/κ-car
14:1 16:1
Fig. 5
1644.50 1527.44 3298.48 2956.78
1075.36 932.00
600 MPa
1648.44 1532.67 3298.48 2958.04
1075.27 931.72
400 MPa
1648.69 1533.16 3314.80
2960.29
1075.36 931.80
200 MPa
1650.05 1533.16 3314.68
3283.62
2960.61
1075.85
931.98
0.1 MPa
1648.66 1533.66 2958.72
3500 3000 2500 2000 1500 1000 Wavenumber (cm-1)
β-Lg
500
Fig.6
A
B
C
D
E
Fig. 7
pH 3.0
pH 3.0
NaCl urea propylene glycol
Hardness (N)
3
2
1
A1 0 0.0
2.0 1.5 1.0
A2
0.5 0.5
1.0
1.5
NaCl urea propylene glycol
2.5
Springiness (mm)
4
0.0
2.0
0.5
Concentration (mol/L)
pH 5.0
pH 5.0
2
1
B1
2.0 1.5 1.0
B2
0.5 0.5
1.0
1.5
0.0
2.0
0.5
Concentration (mol/L)
pH 7.0
2
1
C1
pH 7.0
1.0
1.5
Concentration (mol/L)
2.0
2.0
NaCl urea propylene glycol
2.0 1.5 1.0 0.5
0.5
1.5
2.5
Springiness (mm)
Hardness (N)
3
0 0.0
1.0
Concentration (mol/L)
NaCl urea propylene glycol
4
2.0
NaCl urea propylene glycol
2.5
Springiness (mm)
Hardness (N)
3
0 0.0
1.5
Concentration (mol/L)
NaCl urea propylene glycol
4
1.0
0.0
C2 0.5
1.0
1.5
Concentration (mol/L)
2.0
Highlights
It can form mixed gels by HPP directly at β-Lg: κ-car≥8: 1 (P=600 MPa for 30 min).
HPP can improve gel strength, WHC and textural properties of β-Lg/κ-car mixed gels.
The hydrophobic interaction was dominant in the mixed gels.
The network structure was more compact and smoother with higher pressure.
Author Contributions Section Xiaoying Li: Conceptualization; Investigation; Methodology; Formal analysis; Writing review & editing. Xiaoye He: Conceptualization; Investigation; Methodology; Formal analysis; Writing-review & editing. Like Mao: Methodology; Writing-review & editing. Yanxiang Gao: Assist Project administration. Fang Yuan: Supervision; Project administration; Resources.
Conflict of interest The author declares no conflict of interest.
S0260-8774(19)30494-7
DOI:
https://doi.org/10.1016/j.jfoodeng.2019.109851
Reference:
JFOE 109851
To appear in:
Journal of Food Engineering
Received Date: 4 July 2019 Revised Date:
28 November 2019
Accepted Date: 28 November 2019
Please cite this article as: Li, X., He, X., Mao, L., Gao, Y., Yuan, F., Modification of the structural and rheological properties of β-lactoglobulin/κ-carrageenan mixed gels induced by high pressure processing, Journal of Food Engineering (2019), doi: https://doi.org/10.1016/j.jfoodeng.2019.109851. 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
Modification
of
the
structural
and
rheological
properties
2
β-lactoglobulin/κ-carrageenan mixed gels induced by high pressure processing
of
3 4
Xiaoying Li1, Xiaoye He1, Like Mao, Yanxiang Gao, Fang Yuan*
5 6
Key Laboratory of Functional Dairy, Ministry of Education, College of Food Science
7
and Nutritional Engineering, China Agricultural University, Beijing 100083, P. R.
8
China
9 10
Running Title: β-Lactoglobulin/κ-carrageenan mixed gels induced by HPP
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* Corresponding Author.
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1
These authors contributed equally to this work.
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Tel.: +86 10 6273 7034; fax: +86 10 6273 7986.
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Address: Box 112, No.17 Qinghua East Road, Haidian District, Beijing 100083,
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China
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E-mail: [email protected]
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Abstract
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High pressure processing (HPP) is an emerging non-thermal processing technology
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and a preparation method to induce protein-polysaccharide mixed gels, which can
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encapsulate and delivery thermosensitive bioactive compounds. This study
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investigated the properties of β-lactoglobulin (β-Lg)/ κ-carrageenan (κ-car) mixed
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gels induced by HPP (0.1-600 MPa for 30 min, 25 °C) with different ratios of β-Lg to
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κ-car at pH 3.0, 5.0 and 7.0. The results showed that the pressure required to form
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β-Lg/κ-car mixed gels was at least 400 MPa when the β-Lg/κ-car mass ratio increased
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to 14:1 at all tested pH values. The water holding capacity and textural properties
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increased with the increase of pressure levels. The hydrophobic interaction was
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dominant in the mixed gels induced by HPP at all pH values, and the network
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structure was more compact and smoother with higher pressure.
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Key words: β-lactoglobulin; κ-carrageenan; mixed gels; high pressure processing; gel
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properties
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1. Introduction
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Gelation is a common phenomenon in foods, and the molecules responsible for the
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gelation are typically proteins or polysaccharides (Ji et al., 2017). In case where two
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types of biopolymers (e.g., proteins and polysaccharides, different types of proteins,
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or different types of polysaccharides) are present during gelation can affect the final
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properties of the gel network due to the interaction of molecules (Ersch et al., 2016).
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Food gels can be induced by heat treatment (Zhou et al., 2014), acid (Rabiey &
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Britten, 2009), Ca2+ (Phan-Xuan et al., 2014), enzyme (Wu et al., 2016), etc. In
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addition, as a non-thermal technology, high pressure processing (HPP) is widely used
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in food gel systems in recently years because it can enable the formation of unique
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food gel structures without the use of heat or chemical additives (Cao et al., 2012; Ma
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et al., 2013). HPP can prepare food gels with higher storage modulus and loss
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modulus, and the gel structure has more pores (Saowapark et al., 2008). Compared
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with heat treatment, HPP is more moderate and conducive to the encapsulation and
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release of heat-sensitive substances, such as β-carotene (Mensi et al., 2013). In
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addition, HPP could change protein conformation and influence denaturation,
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aggregation and gelation, resulting in a modification of textural properties and
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possible extension of shelf-life (Ma et al., 2013).
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It had been known that the physicochemical conditions, such as pH, ionic
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strength, temperature, pressure and holding time, have a great influence on the
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interaction between proteins and polysaccharides, giving biopolymers some unique
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gelling properties (Cao et al., 2012; Zhang et al., 2015). Previous studies have shown 3
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that the polysaccharides can reduce the critical gelation concentration of proteins, so
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protein-polysaccharide biopolymers were widely considered as a new gel system. The
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composition, distribution, physical status, volume fraction of polysaccharides can
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affect the interactions between them (Zhou et al., 2014; Turgeon & Beaulieu, 2001).
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β-lactoglobulin (β-Lg) is a globular protein, which is made up of 162 amino acid
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residues and 5 thiol groups; its molecular weight is 18.4 KD and isoelectric point is
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about 5.2 (Wu et al., 2016). β-Lg is now widely applied in food systems because of its
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good gelation property. κ-carrageenan (κ-car) is a linear anionic sulphated
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polysaccharide extracted from red alga cell wall, which has been widely used in the
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food industry as gelling, thickening and stabilizing agent (Piculell, 2006). Κ-car could
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enhance the strength of milk protein gels. In addition, the appearance of β-Lg gel can
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be also affected by the presence of κ-car. It was observed that the single β-Lg formed
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transparent gels at pH 7.0 and opaque white gels over pH 4.0-6.0, however, when
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κ-car was added, it formed opaque white gels for all pH (Eleya & Turgeon, 2000).
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In our previous work, the effects of high pressure treatments, protein
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concentrations and pH on gelation of β-Lg solutions have been studied systematically
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(Li et al., 2018). Based on the work above, protein-polysaccharide mixed gels induced
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by HPP directly without any heat treatment was reported in this research. The
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objective of our current study was to investigate the effects of pressure (200-600
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MPa), pH (3.0-7.0), β-Lg concentration (16-32%) and its ratio to κ-car (8:1-16:1) on
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the structural and functional properties of β-Lg/κ-car mixed gels induced by HPP.
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Furthermore, the mechanism of the β-Lg/κ-car mixed gels formed by HPP was 4
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explored, which could be used as a food wall material to provide theoretical guidance
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for the delivery system of thermosensitive bioactive compounds with better
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microstructure.
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2. Materials & methods
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2.1 Materials
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The β-Lg (97.7%, protein) was obtained from Davisco Food International (Le Sueur,
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MN, USA). The κ-car (purity > 90%) was purchased from CP Kelco (Copenhagen,
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Denmark). All other chemicals used were of analytical grade.
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2.2 Solution preparation
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Firstly, the β-Lg (16, 20, 24, 28, 32%, w/v) and κ-car (2%, w/v) were dissolved in
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different buffer solutions at pH 3.0 (10 mM glycine-HCl buffer), pH 5.0 (10 mM
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acetic acid buffer) and pH 7.0 (10 mM phosphate buffer), respectively. All samples
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were magnetically stirred at 25 ℃ for 4 h, and then stored overnight at 4 ℃ for further
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use. Secondly, different concentrations of β-Lg solution (16, 20, 24, 28, 32%, w/v)
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were mixed with κ-car solution (2%, w/v) in equal volumes to prepare different mixed
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solutions (8:1, 10:1, 12:1, 14:1, 16:1, w/w). The mixed samples were stirred at 25 ℃
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for 4 h.
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2.3 High pressure treatment
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The β-Lg/κ-car mixed solutions were moved to centrifuge tubes (50 mL) and vacuum
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sealed in polyethylene bags. The sealed samples were subjected to high-pressure
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treatment at 200, 400 and 600 MPa for 30 min at 25 ℃ using HPP L2-700/1 ultra-high
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pressure equipment (Tianjin Huatai Senmiao Biotechnology and Technique Co. Ltd, 5
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Tianjin, China) with water as the medium. Untreated samples (0.1 MPa) were used as
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controls. The rates of compression and decompression were 6.5 MPa/s and 20 MPa/s,
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respectively. Compression was accompanied by increasing the temperature of about 3℃
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100 MPa-1 (Balasubramaniam, Farkas, & Turek, 2008). After pressurization, the
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sample temperature started to decrease during the holding time (30 min) due to the
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heat transfer from samples to the stainless steel of pressure vessel. However, a quick
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drop of sample temperature was happened during depressurization, which is even
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lower after depressurization compared with initial temperature (Chen & Hoover,
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2003). Considering that the lowest temperature is around 60 °C that can modify the
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tertiary structure of β-lg (Rodrigues et al., 2019), as well as the maximum temperature
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of sample is about 40 °C, which was calculated by heating rate mentioned above, the
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degree of short-lived temperature increase in high-pressure process was not sufficient
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to denature the protein samples. All samples were stored at 4 ℃ after treated and the
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measurements of the properties were taken after 24 h.
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2.4 Small deformation measurements
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Small deformation oscillatory measurements were performed, on a controlled-strain
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rheometer (AR-1500ex, TA Instruments, Delaware, USA) using a parallel plate
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geometry (plate diameter, 40 mm; gap, 5 mm). After HPP treated, the cylindrical
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samples (diameter is about 25 mm) were separated from the centrifuge tube with a
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blade. Before the test, the gel samples were cut into slices of 5 mm uniform thickness,
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and then transferred to the measuring geometry. The plate was equipped with a
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circulating water system for the temperature control (25 ℃). Storage and loss modulus, 6
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G' and G'' of mixed gels, were measured by this small deformation oscillation test. For
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frequency sweep, measurements were performed from 0.1 to 100 rad/s under a shear
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strain of 1% (Strain sweeps curves of β-Lg /carrageenan mixed gels were shown in
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Supplementary Figure 1). Each time a new sample was used for the measurement.
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And each measurement was repeated for three times.
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2.5 Texture profile analysis (TPA)
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TMS-Pro Food Property Analyzer (Food Technology Corporation, Virginia, USA)
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was used to analyze the textural properties at 25 ℃. After HPP treated, the cylindrical
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samples (diameter is about 25 mm) were separated from the centrifuge tube with a
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blade. Each gel sample was cut into six slices of 10 mm uniform thickness. Before the
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test the sample was equilibrated for 2 h at room temperature and then subjected to a
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compression test using a cylindrical probe (TMS-50mm) at a speed of 60 mm/min
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with a 0.5 N trigger force and 30% deformation. The textural properties of the mixed
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gels including hardness, cohesiveness, springiness and chewiness were gained directly
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from the software (Chen et al., 2010).
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2.6 Determination of water holding capacity (WHC)
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The determination of WHC was according to Zhang's method (Zhang et al., 2015),
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with minor modifications. The β-Lg/κ-car mixed gel (about 3 g) was centrifuged at a
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speed of 10000 r/min for 20 min, then the surface water was removed, and the total
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weight of centrifuge tube and mixed gel was weighed before and after centrifugation,
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until the difference between two consecutive measurements was less than 0.05 g
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(about 5-8 times). WHC was described as the ratio of final gel weight after 7
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centrifugation to the initial gel weight.
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WHC ( % ) =
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W1 - W 0 W2 - W0
× 100 %
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where W1 is the total weight (g) of centrifuge tube and mixed gel after centrifugation,
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W2 is the initial total weight (g) of centrifuge tube and mixed gel, W0 is the weight (g)
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of centrifuge tube. Each measurement was performed in triplicate.
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2.7 Fourier transform infrared (FTIR) spectroscopy
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The functional groups of the freeze-dried β-Lg gel (16%, w/v) and β-Lg/κ-car mixed
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gel (16:1, w/w) at pH 5.0 before (0.1 MPa) and after high pressure treatment (200
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MPa, 400 MPa, 600 MPa) for 30 min were determined by Spectrum 100 Fourier
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transform spectrophotometer (PerkinElmer, UK). Before the measurement, 2.0 mg
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samples were mixed with 200 mg potassium bromide (KBr) and tableted into pellet.
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FTIR spectra were from 4000 cm-1 to 400 cm-1 at a resolution of 4 cm-1 and eleven
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times scanning. In addition, pure KBr powder was used as a baseline. The data were
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processed by Omnic v8.0 (Thermo Nicolet, USA).
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2.8 Field emission scanning electron microscopy (FE-SEM)
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In order to observe the microstructure of the mixed gels, field emission scanning
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electron microscopy (FE-SEM, JSM-6701F, JEOL, Japan) was used at an accelerating
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voltage of 5.0 kV. Prior to the observation, β-Lg/κ-car mixed gels were freeze-dried,
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and the surfaces of the samples were coated with a gold layer to avoid charging under
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the electron beam.
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2.9 Molecular force of mixed gel 8
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The preparation of β-Lg/κ-car solution (16:1, w/w) was according to the method
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described in section 2.2. NaCl, urea and propylene glycol were added to the mixed
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solution, respectively, which to make the additive concentrations of 0.4, 0.8, 1.2, 1.6
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and 2.0 mol/L in resultant solution. Then all the samples were high-pressure treated at
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400 MPa for 30 min. After that, the samples were stored at 4 ℃ overnight. Next, the
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texture properties of the mixed gel were measured according to the method described
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in section 2.5, to study the contribution of electrostatic force, hydrogen bonding and
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hydrophobic interaction on the gel formation.
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2.10 Statistical analysis
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All the data obtained were repeated at least three times. Analysis of variance
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(ANOVA) was performed using SPSS 18.0 software (SPSS Inc., Chicago, USA), and
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significant differences between means were identified using Duncan’s multiple range
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test (p < 0.05).
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3. Results and discussion
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3.1 Rheological properties of β-Lg/κ-car mixed gels induced by HPP
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Fig. 1 showed the frequency sweep curves of β-Lg/κ-car mixed gels at different
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conditions including protein-polysaccharide ratio, pressure level and pH value.
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According to the result of preliminary experiments, the pressure required to form
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β-Lg/κ-car mixed gels was at least 400 MPa when the β-Lg/κ-car mass ratio increased
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to 14:1 at all tested pH values. Nevertheless, the mixed gels can only be formed until
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the pressure level achieved at 600 MPa with the β-Lg/κ-car mass ratio of 8:1. Thus,
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the data regarding β-Lg/κ-car mass ratios of 8:1, 10:1 and 12:1 under 400 MPa, as 9
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well as β-Lg/κ-car mixed solutions under 200 MPa at all ratios, were not shown in Fig.
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1.
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When the elasticity modulus (G') is much larger than the viscous modulus (G''),
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G' and G'' are paralleled and independent of frequency, it is regarded as a typical gel
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system (Morris & Ross-Murphy, 1981; Winter & Chambon, 1986). Furthermore,
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according to Morris et al. (2012), when G'' > G' at low frequency showing fluid
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characteristics, while G' increase faster than G'' with increasing frequency and
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consequently G' > G'' showing solid-like characteristics, that is to say G' and G'' have
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an intersection, it is defined as weak gel. It can be seen from Fig. 1 that G' and G''
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increased with increasing frequency when the β-Lg/κ-car mass ratio and pressure
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increased at the same pH, which indicated a typical viscoelastic characteristic. It was
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worth to mention that the mixed gels with β-Lg/κ-car mass ratio of 8:1 were weak
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gels after treated by 600 MPa, 30 min at pH 3.0 and 7.0, while the others were typical
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gels.
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In the current study, it was found that β-Lg/κ-car mixed gels induced by HPP
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were pH-dependent, and the order of gel strength affected by pH was pH 5.0 > pH
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7.0 > pH 3.0 in terms of G'. It was possible that κ-car formed the main body of mixed
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gels, which depended on the helix and twist, and β-Lg was filled into that. There was
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weak electrostatic force between β-Lg and κ-car when the pH was 5.0, which was
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close to the isoelectric point (pI ≈ 5.2) of β-Lg; but hydrophobic interaction
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increased with higher pressure levels at this pH, therefore the gel strength was
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maximum in this case (Eleya & Turgeon, 2000). For sulphated polysaccharides, a 10
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soluble protein-polysaccharide complex may be formed at pH above the protein pI
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(pH 7.0), which could be a result of the interaction between NH3+ (β-Lg) and SO3-
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(κ-car). Such attraction is particularly strong and even possible when both protein and
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sulphated polysaccharide are negatively charged (Rafe & Razavi, 2013). Furthermore,
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κ-car fixed a large amount of water and thickened β-Lg solution because of the
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exclusion function of the system, therefore promoted the formation of gel and
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improved the gel strength. At more acidic pH (below pH 4.0), it may prevent κ-car
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gelation due to acid hydrolysis (Mleko et al., 1997), which impeded its crosslinking
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with β-Lg, therefore the gel strength was lowest at pH 3.0.
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Protein and polysaccharide mixed gels can be classified into three types:
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interpenetrating, coupled and phase-separated networks. Interpenetrating network is
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the simplest case that there is no interaction but only a topological structure between
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the two components, which form an independent but continuous network. Nguyen et
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al. (2015) considered that the network of β-Lg/κ-car mixed gels induced by Ca2+ is
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interpenetrating, they found that the single networks (β-Lg gels or κ-car gels) and the
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interpenetrated networks became stiffer with higher Ca2+ concentration and the elastic
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modulus of the mixed gels was closed to the sum of the two single gels. Coupled
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networks involved two different molecules that formed the junction zones because of
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synergistic reaction. Le & Turgeon (2013) found that β-Lg and xanthan gum (XG)
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formed coupled gel induced by electrostatic attractive interaction, by which XG
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provided a frame for gel organization and β-Lg aggregated along the XG chains.
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Phase-separated networks mean that both biopolymers form separate network 11
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independently or both biopolymers get in two different phases separately (Le &
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Turgeon, 2015). In our research, β-Lg and κ-car formed mixed gels probably induced
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by electrostatic attraction, hydrogen bonding and hydrophobic interaction, and the
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combination of protein and polysaccharide greatly reduced the β-Lg concentration
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required for gel formation, therefore it may be coupled network formed between β-Lg
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and κ-car.
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3.2 Textural properties of β-Lg/κ-car mixed gels induced by HPP
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Texture is an important property that determines the organoleptic quality of gels. In
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our research, the protein-polysaccharide ratio and pressure had large effects on the
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textural properties of β-Lg/κ-car mixed gels. Hardness reflects the strength of gel
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structure when it is compressed (Zhao et al., 2014). Springiness is a measure of how
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much the gel structure is broken down by the initial compression; high springiness
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will result when the gel structure is broken into few large pieces during the first
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compression whereas low springiness results from the gel breaking into many small
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pieces (Lau et al., 2000). Fig. 2 showed that the hardness and springiness increased
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with higher β-Lg/κ-car ratio and pressure. This finding was attributed to the denser
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networks of the mixed gels with higher β-Lg/κ-car ratio and pressure, which reinforce
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the intermolecular interaction of β-Lg and κ-car, and therefore the hardness and
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springiness were increased (Le & Turgeon, 2015).
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Fig. 3 showed comparison of textural properties of β-Lg gels and β-Lg/κ-car
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mixed gels at pH 5.0 after treated by 600 MPa, 30 min. It can be seen that the addition
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of κ-car obviously improved the mixed gels strength (p < 0.05), which suggested that 12
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HPP could promote the intermolecular interaction of β-Lg and κ-car. In addition, the
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springiness of the mixed gels was significantly higher than the single β-Lg gels (p <
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0.05), which indicated that there were differences in the structure of these gels. These
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findings were consistent with the results of Dickinson & James (2000) studies on the
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effects of high pressure on β-Lg and pectin mixtures.
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3.3 The WHC of β-Lg/κ-car mixed gels
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Water holding capacity (WHC) refers to the water retention capacity of raw materials
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during the manufacturing process of foods, which is directly related to the gel texture,
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structure and state (Urbonaite et al., 2016). Fig. 4A exhibited the changes of WHC of
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different β-Lg/κ-car mixed gels under 600 MPa. The ratio of β-Lg and κ-car had
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significant effects on the WHC of the mixed gels (p < 0.05), the WHC increased with
265
higher content of β-Lg. It was attributed to the increase of β-Lg strengthening the
266
intermolecular interaction between β-Lg and κ-car, as well as β-Lg and water.
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WHC of a gel has been reported to be determined by both gel microstructure and
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gel stiffness (Urbonaite et al., 2015). Fig. 4B showed that the effects of pressure and
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pH on WHC of the mixed gels. It can be seen that water holding capacity was higher
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under 600 MPa than 400 MPa at the same ratio of β-Lg and κ-car, and the WHC was
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the highest at pH 5.0 under the same β-Lg/κ-car ratio and pressure, which were in
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accordance with the changes of G' value, gel hardness and gel springiness. It was
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probably that a more cross-linked network structure was formed with higher pressure,
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and the pore diameter of the mixed gels was reduced, which was beneficial to the
275
increase of WHC. 13
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3.4 The FTIR of β-Lg/κ-car mixed gels
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In order to investigate the interaction between β-Lg and κ-car, FTIR spectroscopic
278
analysis of β-Lg (16%, w/v) and mixed gels (16:1, w/w) were carried out as shown in
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Fig. 5. The peak around 3000-3500 cm-1 represents the water peak of amide A, which
280
could be used for to evaluate the interaction between protein and water molecules and
281
reflect the changes of hydrogen bonds. It can be seen that the wavenumber of β-Lg
282
hydrogen bond was at 3283.62 cm-1. The wavenumbers of β-Lg/κ-car mixtures under
283
0.1, 200, 400 and 600 MPa HPP treatments were 3314.68, 3314.80, 3298.48 and
284
3298.48 cm-1, respectively, which indicated that the addition of κ-car resulted in the
285
change of hydrogen bond compared with the single β-Lg. The wavenumbers increased;
286
the hydrogen bonding ability enhanced. The changes of interaction between protein
287
and polysaccharide were related to peak stretching vibration. In our study, it was
288
speculated that the shift of hydrogen bonds could be attributed to sulfate group of
289
κ-car, which enhanced intermolecular forces and hydrogen bonding ability after
290
pressurization, therefore the G' of mixed gels increased (Liu et al., 2014).
291
From Fig. 5, compared with β-Lg, the peak intensity of β-Lg/κ-car mixture at
292
about 2960 cm-1 increased firstly and then decreased. The wavenumbers of β-Lg/κ-car
293
mixture under 400 and 600 MPa were 2958.04 cm-1 and 2956.78 cm-1, respectively. It
294
may be due to the increase of the esterification and acetylation degree of κ-car
295
induced by high pressure. The C-H stretches associated with the ring methine
296
hydrogen atoms in k-car might also contribute to the changes at 2960 cm-1 (Fang et al.,
297
2002; Chen et al., 2014). 14
298
The amide I band between 1600 and 1700 cm-1 was commonly used to analyze
299
the secondary structure of proteins (Carbonaro & Nucara, 2010). From Fig. 5, the
300
typical absorption peaks of amide I and amide II region were at 1648.66 cm-1 and
301
1533.66 cm-1, respectively. The amide I region was mainly C=O stretching vibration
302
and the amide II region was attributed to C-N stretching vibration and in-plane
303
bending vibration (Bhattacharjee et al., 2005; Perisic et al., 2011). In addition, the
304
peak wavenumbers decreased with the increase of pressure, which also showed that
305
the interactions between β-Lg and κ-car enhanced.
306
Compared with β-Lg, there were more two peaks appeared at 1075 cm-1 and 930
307
cm-1 in β-Lg/κ-car mixtures, which were the characteristic peaks of -SO3- and free
308
sulfate in κ-car, respectively. In addition, it can be seen from Fig. 5, the peaks
309
intensity decreased after pressurization, which showed that the pressure promoted the
310
transformation between -SH and -S-S- (Gómez-Ordóñez & Rupérez, 2011).
311
3.5 The SEM images of β-Lg/κ-car mixed gels
312
Fig. 6 showed the SEM images of β-Lg/κ-car mixtures (16:1, w/w) treated with
313
different pressures at pH 5.0. Fig. 6E was the image of single β-Lg. It can be seen that
314
κ-car was attached to β-Lg when β-Lg/κ-car mixtures formed at atmospheric pressure
315
(Fig. 6A). β-Lg and κ-car did not form regular internal structures, showing
316
fragmented patterns under 200 MPa (Fig. 6B). However, the microstructure of the
317
mixed gel under 400 MPa was similar to sponge, and the pore size was larger than the
318
mixed gel under 200 MPa (Fig. 6C), which also confirmed that the WHC of the gel
319
was poor. When treated by 600 MPa pressure, the microstructure of the mixed gel was 15
320
similar to that of honeycomb which was very dense and homogeneous (Fig. 6D).
321
3.6 Effects of molecular interaction on the formation of β-Lg/κ-car mixed gels
322
The gelation is the outcome of protein denaturation, which intermolecular covalent
323
and noncovalent interactions are involved (Wijaya et al., 2017). Electrostatic
324
interactions, hydrogen bonds and hydrophobic interactions are the major non-covalent
325
interaction responsible for the structural organization of food components
326
(McClements et al., 2009). Sodium salts affect protein-polysaccharides interactions,
327
either by ionic strength effects, or binding to the protein charged groups (Uruakpa &
328
Arntfield, 2006). The electrostatic shielding effect caused by high concentration of
329
NaCl can reduce electrostatic interaction mentioned above. Hence, the addition of
330
NaCl to β-Lg/κ-car dispersions is an effective method to evaluate the contribution of
331
electrostatic interactions to the gel network formation. As to hydrogen bonds, urea can
332
hinder the formation of hydrogen bonds by affecting the structure of water molecules,
333
which can be used to evaluated the contribution of hydrogen bonds in the gel
334
formation (Le´ger & Arntfield, 1993). Propylene glycol is another reagent that affects
335
water structure and it can disrupt hydrophobic forces and promote hydrogen and
336
electrostatic bonds (Bernal et al., 1987).
337
Fig. 7 showed that the relationship between the hardness and springiness of the
338
mixed gels with different concentrations of NaCl, urea and propylene glycol, which
339
can reveal the strength of the three kinds of interaction. It was found that low
340
concentration (0.4 mol/L) of NaCl improved the gel hardness and springiness,
341
however, as the concentration of NaCl increased, the hardness and springiness of all 16
342
gel samples decreased quickly. It was mainly attributed to the neutralization of
343
electrostatic interaction between charged amino acids by intense salty ions screening
344
that reduced the electrostatic interaction. The decrease of hardness and springiness of
345
gels caused by addition of urea and propylene glycol at different concentrations
346
suggested that hydrogen bonding and hydrophobic interaction had effects on the
347
mixed gel formation. However, the contribution of hydrogen bonding was less than
348
the electrostatic interaction at pH 3.0 and 7.0. At pH 5.0, which is close to the
349
isoelectric point of β-Lg, the contribution of hydrogen bonding was greater than the
350
electrostatic interaction. Furthermore, because adding propylene glycol has the most
351
obvious effects on the hardness and springiness of β-Lg/κ-car mixed gels, it was
352
believed that hydrophobic interaction was dominant in the mixed gels induced by
353
HPP.
354
4. Conclusion
355
This work evaluated the properties of β-Lg/κ-car mixed gels induced by HPP at pH
356
3.0, 5.0 and 7.0. In the current study, it was found that the pressure required to form
357
β-Lg/κ-car mixed gels was at least 400 MPa when the β-Lg/κ-car mass ratio increased
358
to 14:1 at all tested pH values. The results of rheological test showed that the network
359
of β-Lg/κ-car mixed gels induced by HPP was coupled. There were hydrophobic,
360
electrostatic and hydrogen bonding interactions between β-Lg and κ-car, and the
361
hydrophobic interaction was dominant in the mixed gels induced by HPP. The G'
362
value, WHC, hardness, springiness, and chewiness of the mixed gels with higher
363
pressure and higher concentration of β-Lg were better, and the network structure of 17
364
the mixed gels was more compact and uniform. The gel properties of β-Lg/κ-car
365
mixed gels were better than the single β-Lg gels induced by HPP. The results of this
366
study can provide a theoretical guidance for the development of a novel food wall
367
material which can encapsulate and delivery thermosensitive compounds.
368 369
Acknowledgements
370
This research was funded by the National Natural Science Foundation of China (No.
371
31371836), National Key R&D Program of China (No. 2016YFD0400804), and
372
Natural Science Foundation of Beijing (No. 6192015).
373 374
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Fig. 1 Frequency sweep curves (G' and G'') of β-Lg/κ-car mixed gels at different pH values (A1 and A2, pH 3; B1 and B2, pH 5; C1 and C2, pH 7). Fig. 2 Textural properties of β-Lg/κ-car mixed gels (mean ± SD, n=6) (A, B, C & D). Different superscript letters indicate significant differences in the same graph (p < 0.05). Fig. 3 Comparison of textural properties of β-Lg gels and β-Lg/κ-car mixed gels at pH 5.0 and 600 MPa. Different superscript letters indicate significant differences in the same graph (p < 0.05). Fig. 4 Water holding capacity (WHC) of β-Lg/κ-car mixed gels under 600 MPa HPP treatment with different mass ratios at different pH values (A) and the WHC of β-Lg/κ-car mixed gels under 400 and 600 MPa HPP treatment with the mass ratio of 14:1 and 16:1 at different pH values (B). Different superscript letters indicate significant differences in the same graph (p < 0.05). Fig. 5 FTIR spectra of β-Lg (16%, w/v) and β-Lg/κ-car mixed gels with the mass ratio of 16:1 at pH 5.0. Fig.6 SEM images of β-Lg/κ-car mixtures (16:1, w/w) at pH 5.0 under different pressures: (A) 0.1 MPa; (B) 200 MPa; (C) 400 MPa; (D) 600 MPa, and (E) β-Lg (16%, w/v) at pH 5.0 under 0.1 MPa. Fig. 7 The effects of different concentrations (0.4, 0.8, 1.2, 1.6 and 2.0 mol/L) of NaCl, urea and propylene glycol on hardness and springiness of β-Lg/κ-car mixed gels at different pH values (A1 and A2, pH 3; B1 and B2, pH 5; C1 and C2, pH 7).
Fig. 1
1000
1000
100
100
G'' (Pa)
10000
G' (Pa)
10000
10 pH 3.0-14:1-400 MPa pH 3.0-16:1-400 MPa pH 3.0- 8:1-600 MPa pH 3.0-10:1-600 MPa pH 3.0-12:1-600 MPa pH 3.0-14:1-600 MPa pH 3.0-16:1-600 MPa
1 0.1
A1 0.01 0.1
1
10
10 1
pH 3.0-14:1-400 MPa pH 3.0-16:1-400 MPa pH 3.0- 8:1-600 MPa pH 3.0-10:1-600 MPa pH 3.0-12:1-600 MPa pH 3.0-14:1-600 MPa pH 3.0-16:1-600 MPa
0.1
A2 0.01
100
0.1
1
ω (rad/s)
1000
100
100
10 pH 5.0-14:1-400 MPa pH 5.0-16:1-400 MPa pH 5.0- 8:1-600 MPa pH 5.0-10:1-600 MPa pH 5.0-12:1-600 MPa pH 5.0-14:1-600 MPa pH 5.0-16:1-600 MPa
B1 0.01 0.1
1
10
G'' (Pa)
1000
G' (Pa)
10000
0.1
10 pH 5.0-14:1-400 MPa pH 5.0-16:1-400 MPa pH 5.0- 8:1-600 MPa pH 5.0-10:1-600 MPa pH 5.0-12:1-600 MPa pH 5.0-14:1-600 MPa pH 5.0-16:1-600 MPa
1 0.1
B2 0.01 0.1
100
1
10000
10000
1000
1000
100
100
10 pH 7.0-14:1-400 MPa pH 7.0-16:1-400 MPa pH 7.0- 8:1-600 MPa pH 7.0-10:1-600 MPa pH 7.0-12:1-600 MPa pH 7.0-14:1-600 MPa pH 7.0-16:1-600 MPa
0.1
C1 0.01 0.1
1
10
ω (rad/s)
10
100
ω (rad/s)
100
G'' (Pa)
G' (Pa)
ω (rad/s)
1
100
ω (rad/s)
10000
1
10
10 pH 7.0-14:1-400 MPa pH 7.0-16:1-400 MPa pH 7.0- 8:1-600 MPa pH 7.0-10:1-600 MPa pH 7.0-12:1-600 MPa pH 7.0-14:1-600 MPa pH 7.0-16:1-600 MPa
1 0.1
C2 0.01 0.1
1
10
ω (rad/s)
100
Chewiness (mJ) 18
b
0 abc
C
9
f
3
a
8
6 h h
4 g
de f
2 ef
a bc bc
pH 3.0
h d ef
ab
pH 5.0
12
6 i
ab o
c ef
cd
hi
Springiness (mm)
pH 5.0
l
k
j
e 14 16:1-4 10:1-400 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a 14 Pa : 1 16 -4 10:1-400 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a 14 Pa : 1 16 -4 10:1-400 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a Pa
Hardness (N)
pH 3.0
Cohesiveness
14 : 16 1-4 : 0 10 1-4 0 M :1 00 P 12 -6 M a : 0 14 1-6 0 MPa : 0 16 1-6 0 MPa :1 00 P -6 M a 00 P M a Pa 14 :1 16 -4 : 0 10 1-4 0 M :1 00 P 12 -6 M a : 0 14 1-6 0 MPa : 0 16 1-6 0 MPa :1 00 P -6 M a 00 P M a Pa 14 :1 16 -4 : 0 10 1-4 0 M :1 00 P 12 -6 M a : 0 14 1-6 0 MPa : 0 16 1-6 0 MPa :1 00 P -6 M a 00 P M a Pa
A
14 16:1-4 10:1-400 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a 14 Pa : 1 16 -4 : 0 10 1-4 0 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a 14 Pa : 1 16 -4 : 0 10 1-4 0 M 12:1-600 MPa 14:1-600 MPa 16:1-600 MPa :1 00 P -6 M a 00 P M a Pa
14 : 16 1-4 : 0 10 1-4 0 M : 0 12 1-6 0 MPa :1 0 0 P 14 -6 M a : 0 16 1-6 0 MPa :1 0 0 P -6 M a 00 P M a Pa 14 : 16 1-4 : 0 10 1-4 0 M : 0 12 1-6 0 MPa :1 0 0 P 14 -6 M a : 0 16 1-6 0 MPa :1 0 0 P -6 M a 00 P M a Pa 14 : 16 1-4 : 0 10 1-4 0 M : 0 12 1-6 0 MPa : 0 14 1-6 0 MPa :1 0 0 P 16 -6 M a :1 0 0 P -6 M a 00 P M a Pa
Fig. 2
pH 7.0
3.5
k j
i
ab
0
pH 7.0 0.9
15
m
0.4
B
3.0
n
pH 3.0
2.5
1.0
D
0.8 f fg
0.7
0.6
0.5
pH 5.0 i
gh
d
hij
fg
f
e
0.5
pH 3.0
e
pH 7.0
gh
d h
f
f
2.0 e
a
pH 5.0
ghij ij fghi ghij
f
1.5
c d
bc ab
0.0
pH 7.0 hij j
fgh fgh
d e
d c
g b
a
c
0.00
Fig. 3
8
d
A
B d
3
c
c
4
2
Springiness (mm)
Hardness (N)
6
b
a
0 14% β-Lg
16
14:1 β-Lg/κ-car 16% β-Lg
1 a
0
16:1 β-Lg/κ-car
14% β-Lg
14:1 β-Lg/κ-car 16% β-Lg
16:1 β-Lg/κ-car
d
C
D bc
0.8
14 12
c b
c
Cohesiveness
Chewiness (mJ)
b
2
10 8 6 b
4
0.6
0.4
a
0.2
2 a
0.0
0 14% β-Lg
14:1 β-Lg/κ-car 16% β-Lg
16:1 β-Lg/κ-car
14% β-Lg
14:1 β-Lg/κ-car 16% β-Lg
16:1 β-Lg/κ-car
Fig. 4
100
A
pH 3.0
pH 5.0 gh
i
pH 7.0
g
90
ef
h
f
e
80
d
d
WHC (%)
70
c
60
b
b
b
50 40
a
a
30
8: 1 10 :1 12 :1 14 :1 16 :1
8: 1 10 :1 12 :1 14 :1 16 :1
8: 1 10 :1 12 :1 14 :1 16 :1
0
ratios of β-Lg/κ-car
100 B
400 MPa 600 MPa
WHC (%)
pH 3.0 d c
pH 5.0 f e
e
d
d
c c
b
80
pH 7.0
b
a
60 14:1 16:1
14:1 16:1 ratios of β-Lg/κ-car
14:1 16:1
Fig. 5
1644.50 1527.44 3298.48 2956.78
1075.36 932.00
600 MPa
1648.44 1532.67 3298.48 2958.04
1075.27 931.72
400 MPa
1648.69 1533.16 3314.80
2960.29
1075.36 931.80
200 MPa
1650.05 1533.16 3314.68
3283.62
2960.61
1075.85
931.98
0.1 MPa
1648.66 1533.66 2958.72
3500 3000 2500 2000 1500 1000 Wavenumber (cm-1)
β-Lg
500
Fig.6
A
B
C
D
E
Fig. 7
pH 3.0
pH 3.0
NaCl urea propylene glycol
Hardness (N)
3
2
1
A1 0 0.0
2.0 1.5 1.0
A2
0.5 0.5
1.0
1.5
NaCl urea propylene glycol
2.5
Springiness (mm)
4
0.0
2.0
0.5
Concentration (mol/L)
pH 5.0
pH 5.0
2
1
B1
2.0 1.5 1.0
B2
0.5 0.5
1.0
1.5
0.0
2.0
0.5
Concentration (mol/L)
pH 7.0
2
1
C1
pH 7.0
1.0
1.5
Concentration (mol/L)
2.0
2.0
NaCl urea propylene glycol
2.0 1.5 1.0 0.5
0.5
1.5
2.5
Springiness (mm)
Hardness (N)
3
0 0.0
1.0
Concentration (mol/L)
NaCl urea propylene glycol
4
2.0
NaCl urea propylene glycol
2.5
Springiness (mm)
Hardness (N)
3
0 0.0
1.5
Concentration (mol/L)
NaCl urea propylene glycol
4
1.0
0.0
C2 0.5
1.0
1.5
Concentration (mol/L)
2.0
Highlights
It can form mixed gels by HPP directly at β-Lg: κ-car≥8: 1 (P=600 MPa for 30 min).
HPP can improve gel strength, WHC and textural properties of β-Lg/κ-car mixed gels.
The hydrophobic interaction was dominant in the mixed gels.
The network structure was more compact and smoother with higher pressure.
Author Contributions Section Xiaoying Li: Conceptualization; Investigation; Methodology; Formal analysis; Writing review & editing. Xiaoye He: Conceptualization; Investigation; Methodology; Formal analysis; Writing-review & editing. Like Mao: Methodology; Writing-review & editing. Yanxiang Gao: Assist Project administration. Fang Yuan: Supervision; Project administration; Resources.
Conflict of interest The author declares no conflict of interest.