LWT - Food Science and Technology 117 (2020) 108690

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LWT - Food Science and Technology

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Improvement of calcium sulfate-induced gelation of soy protein via T

incorporation of soy oil before and after thermal denaturation
Haibo Zhaoa,b, Bin Yua, Yacine Hemara, Jie Chenb,∗∗, Bo Cuia,∗
a
State Key Laboratory of Biobased Material and Green Papermaking, College of Food Science and Engineering, Qilu University of Technology, Shandong Academy of
Sciences, Jinan, 250353, China
b
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China

A R T I C LE I N FO A B S T R A C T

Keywords: Soy protein-based foods have attracted extensive attention for their potential health benefits. In this study, soy
Soy protein isolate protein isolate (SPI) emulsions containing 10–70 mL/L of soy oil were primarily prepared either by adding the
Tofu oil, emulsifying and heat treatment (OEH), or by heat treatment of the soy protein dispersion, addition of oil and
Texture emulsification (HOE). After adding CaSO4, the rheological properties of emulsions during gelation and the
Viscoelasticity
textural properties of the resulting gels were investigated and compared. The results showed that the increase in
Heat denaturation
the volume fraction (Φ) of the soy oil from 10 to 70 mL/L resulted in the increase of the final elastic modulus (G′)
of HOE and OEH emulsion gels from 5216.7 to 5827.3 Pa, and 4184.3–4935.3 Pa, respectively. The gel hardness
and water-holding capacity gradually improved with increasing Φ. HOE gels exhibited greater performance of
gelling properties than OEH gels at the same Φ. Confocal laser scanning microscopy images demonstrated that
HOE gels, with smaller soy oil droplets dispersed within the networks, had more homogeneous and compact
network structures than OEH gels. Reinforced emulsifying capacity of denatured SPI could enhance the protein-
oil interactions and result in improvement of CaSO4-gelation of SPI.

1. Introduction chemical and structural characteristics, which are, in turn, affected by

Soy products have attracted interest worldwide because of their
potential health benefits, such as reducing the risk of heart and cardi-
ovascular diseases, lowering the cholesterol levels and preventing cer-
tain cancers (Chang, 2006; Rekha & Vijayalakshmi, 2010). Tofu, also
called soy curd, is a typical gel product normally prepared by coagu-
lating soy milk with acid (glucono-δ-lactone) or salt (e.g., CaSO4)
(Nishinari, Fang, Guo, & Phillips, 2014; Obatolu, 2008; Ringgenberg,
Alexander, & Corredig, 2013). The process is relatively complicated and
cumbersome, including soaking and grinding of soya beans, heat
treatment, separation of soy milk, coagulation, breaking of the curd and
pressing to reform the gel (Chang, 2006; Corzo-Martinez, Garcia-
Campos, Montilla, & Moreno, 2016). Conversely, soy protein, which is
free of cholesterol and contains less saturated fat, is a versatile source of
vegetable protein (Wang et al., 2018). The development of tofu-type gel
using soy protein as the basic material would be an alternative available
for the food manufacturers, restaurant, canteen, etc., which are not
equipped with tofu processing facilities.
The gel properties of soy protein are related to their physical,
treatment parameters, processing methods and interactions with other
ingredients (Zhao, Wang, Li, Qin, & Chen, 2017). Therefore, under-
standing the effects of specific processing factors and other ingredients
on the gelation properties of soy protein isolate (SPI) will be very useful
for the development of soy protein-based products, including tofu-type
gel.
Soy oil, the main constituent of soy milk in addition to soy protein,
plays an important role in the texture, flavor and nutritional value of
soy protein foods, including soy protein-based gels, such as tofu
(Fukushima, 1991). A gel containing soy oil droplets could be con-
sidered a protein gel matrix in which the soy oil droplets are embedded
(Kim, Renkema, & van Vliet, 2001). The soy oil droplets combine with
protein particles and participate to form a stable tofu–curd emulsion
network (Guo, Ono, & Mikami, 1999). Murekatete, Zhang,
Hategekimana, Karangwa, and Hua (2016) studied the effects of dif-
ferent soy oil volume fractions (50, 150, 250 and 350 mL/L) on the
rheological behavior of acid- or salt-induced SPI gels, and the results
showed that the storage modulus (G′) of acid-induced SPI gels de-
creased with increasing soy oil volume fraction, while the influence was

∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: chenjie@jiangnan.edu.cn (J. Chen), cuibopaper@163.com (B. Cui).

https://doi.org/10.1016/j.lwt.2019.108690
Received 11 June 2019; Received in revised form 24 September 2019; Accepted 24 September 2019
Available online 25 September 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
H. Zhao, et al.
opposite for salt-induced SPI gels. Kim et al. (2001) indicated that the
G′ and the loss modulus (G″) of SPI gels after the temperature cycle
increased as soy oil volume fractions increased from 100 to 300 mL/L.
(Tang et al., 2011) evaluated the effects of thermal treatment and
emulsification on the properties of CaCl2-or glucono-δ-lactone (GDL)-
induced SPI gels and concluded that the physicochemical properties of
the gels have a close relationship with their microstructures. These
findings strongly suggested that the incorporation of soy oil might be of
vital importance for the gelation behavior and network structures of
tofu-type gels.
However, it is surprising that in a model system close to traditional
soy milk and tofu, information about the effects of different soy oil
volume fractions on the textural properties and microstructures of
CaSO4-induced SPI gels is scant. Although the effects of soy protein's
thermal denaturation on the properties of tofu or tofu-type gels have
been widely reported (Liu, Chang, Li, & Tatsumi, 2004; Shin,
Yokoyama, Kim, Wicker, & Kim, 2015; Zhao, Li, Qin, & Chen, 2016),
the effects of thermal denaturation of SPI before or after the in-
corporation of different soy oil volume fractions via emulsification on
the gel structure and gelation behavior of CaSO4-induced SPI gels have
not been discussed in detail. Therefore, this study aimed to investigate
the effects of different volume fractions of additional soy oil (within the
scope of soy oil content in traditional tofu) and thermal denaturation of
SPI before and after incorporation of soy oil on the rheological and
textural properties of CaSO4-induced SPI gels. Based on the earlier
preliminary work (not published), the oil content in the tofu ranges
from 20 g/kg to 50 g/kg. In addition, Wang et al. (2018) and Luo et al.
(2019), who investigated the effects of pre-aggregation and pre-cross-
linking of SPI on the CaSO4-induced gelation properties of soy protein
emulsion, chose a 50 mL/L oil content. Therefore, the oil volume fac-
tions in this study varied from 10 to 70 mL/L. The dispersion of soy oil
droplets in the protein gel matrix and microstructures of the gels were
characterized by confocal laser scanning microscopy (CLSM).
2. Materials and methods
2.1. Materials
Soybeans (Xuelang Seed Station, Wuxi, China) were ground with a
15B grain mill (Minyi, Yongkang, China). After removal of the hull, the
flour was defatted using n-hexane/ethanol (10:1, mL:mL) at room
temperature (25 °C). SPI was extracted from the defatted soybean meal
following the method of Wang, Liu, Ma, and Zhao (2019) with some
modifications. Briefly, SPI was obtained by alkali extraction (pH 8.0)
with 2 mol/L NaOH and acid precipitation (pH 4.5) with 2 mol/L HCl.
The extract was then washed and resuspended with deionized water
and adjusted to pH 7.0 with 2 mol/L NaOH, followed by freeze-drying.
The protein content was 910 g/kg as measured by Kjeldahl method
(N × 6.25). Rhodamine B and Nile Blue were purchased from Sigma-
Aldrich (St. Louis, MO, USA), CaSO4·2H2O from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China), and soy oil from a local super-
market (Wuxi, China). All other chemicals were of analytical grade.
2.2. Emulsion preparation
SPI was dispersed in deionized water to give a protein concentration
of 75 g/L, and stirred using a magnetic stirrer for 2 h at room tem-
perature (25 °C). The pH value of the SPI dispersion was 7.0.
Subsequently, the SPI dispersion was divided into two parts. One SPI
dispersion was directly mixed with soy oil using a T18 basic high-speed
blender (IKA, Staufen, Germany) for 2 min. The added soy oil volume
fractions were 10, 30, 50 and 70 mL/L. The final protein concentration
in all SPI emulsions was adjusted to 70 g/L. Next, the coarse SPI
emulsions were homogenized using a homogenizer (ATS Engineering
Inc., Brampton, Canada) at a pressure of 20 MPa, heated at 90 °C for
15 min and subsequently cooled down to room temperature. The
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LWT - Food Science and Technology 117 (2020) 108690
generated SPI emulsions were denoted as OEH. The second SPI dis-
persion was initially heated at 90 °C for 15 min and cooled down to
room temperature (25 °C) before incorporating the same volume frac-
tions of soy oil, and then SPI emulsions were prepared according to the
above procedure. The resultant SPI emulsions were denoted as HOE. A
70 g/L SPI dispersion free of soy oil under the same conditions was used
as a control in this study.
2.3. Formation of CaSO4-induced SPI gels
A freshly prepared CaSO4 solution (100 g/L) was added to the SPI
emulsions to give a final coagulant concentration of 3.5 g/L, and the
solutions were stirred gently for 1 min. Next, they were heated at 90 °C
for 15 min and cooled down to room temperature with iced water to
allow the formation of the gel. The gels were stored at 4 °C for further
analysis.
2.4. Characteristics of SPI emulsion droplets
The droplet size distribution of freshly prepared SPI emulsions was
determined using a Microtrac S3500 Particle Size Analyser (Microtrac
Inc., Largo, FL, USA) according to the methods of Guo, Hu, Wang, and
Liu (2018) and Zhuang et al. (2019) with some modifications. The
range of measurement was from 0.01 to 2000 μm. After the preparation
of emulsions, each sample was dripped into the sample chamber and
directly diluted with deionized water. Afterwards, the measurement
was performed at 25 °C. The droplet size of the SPI emulsions, expressed
as the volume-average diameter, was calculated as an average of three
freshly prepared SPI emulsion samples. Measurements were taken in
duplicate. The refractive indices of soy oil and continuous phase used
were 1.456 and 1.33, respectively.
2.5. Dynamic rheological measurements
An AR-G2 Rheometer (TA Instruments, New Castle, DE, USA) was
used to perform the rheological measurements. The viscosity of the SPI
emulsions subjected to heat treatment before and after incorporation of
different soy oil volume fractions was measured using a cone plate
geometry (diameter = 40 mm; angle = 2°). Measurements were taken
in triplicate at room temperature (25 °C).
G′ and G″ of the SPI emulsions were determined using a parallel
plate (diameter = 40 mm). After adding a coagulant, each sample was
immediately loaded into the rheometer, and the excess solution around
the edge of a plate was removed using tissue paper. The exposed rim of
each sample was covered with silicon oil to prevent evaporation during
gelation. The strain applied was 0.01, which was within the linear
viscoelastic region, and the frequency was 1 Hz. The samples were
heated from 25 °C to 90 °C at a heating rate of 5 °C/min, followed by
incubation at 90 °C for 10 min and subsequently cooled to 25 °C at a
cooling rate of 5 °C/min. After the temperature cycle, the gels formed
were subjected to a strain sweep measurement at 25 °C. The strain was
varied from 0.001 to 1, and the frequency was 1 Hz.
2.6. Texture analysis of CaSO4-induced SPI gels
The gel hardness was measured using a TA-XT2 texture analyser
(Stable Micro Systems, Godalming, Surrey, UK) according to the
methods described by Zhao et al. (2016) and (Wang et al., 2019). The
gel size in the containers were 41 mm in diameter and 23 mm in height.
Before the measurement, the gels were equilibrated at room tempera-
ture (25 °C) for 30 min. Then, the gels were punctured to 30% of the
original height using a cylindrical plunger with a diameter of 10 mm at
a pre-test speed of 3 mm/s, test speed of 0.8 mm/s and post-test speed
of 5 mm/s. The trigger force was 9.81 × 10−3 N and the option of re-
turn to start was selected. The peak value in the force–distance curve
was referred to as the gel hardness.
H. Zhao, et al. LWT - Food Science and Technology 117 (2020) 108690

2.7. Water-holding capacity of CaSO4-induced SPI gels

The water-holding capacity (WHC) measurement of the gels was

conducted according to the method of Maltais, Remondetto, Gonzalez,
and Subirade (2005) with some modifications. Equal volumes (3 mL) of
emulsions was transformed into centrifuge tubes and the gels are al-
lowed to form following the process mentioned in section 2.3. The gels
were stored at 4 °C overnight, and centrifuged at 10,000 g for 10 min at
4 °C. WHC (%) was calculated using the following formula:

(Weight of water remaining in the gels after centrifugation/Weight

of the total water in the samples) × 100

2.8. Confocal scanning laser microscopy analysis

The microstructure of the SPI gels was evaluated using a Leica TCS
SP8 confocal laser scanning microscope (Leica Microsystems,
Mannheim, Germany). The SPI gels were stained with the fluorescent
dyes Rhodamine B and Nile blue, which stain the protein and oil phases,
respectively, at excitation wavelengths of 488 and 633 nm, respectively.
Both dye solutions (0.1 g/L) were mixed well with the SPI emulsions
prior to gel formation. After adding CaSO4, the mixtures were heated at
90 °C for 15 min to allow gel formation. Subsequently, the gels formed
were placed at the bottom of petri dishes and covered with coverslips.
Images were taken at a certain depth below the coverslip surface. The
protein stained with Rhodamine B appeared red, and the soy oil dro-
plets stained with Nile blue were green.

2.9. Statistical analysis

All results were expressed as the mean ± standard deviation of

three independent tests, each with at least triplicate analyses. Data were
subjected to nonparametric test using IBM SPSS Statistical 22.0 soft-
ware (Armonk, New York, USA). Significant differences (P < 0.05)
between individual means within OEH or HOE group were identified by
the Kruskal-Wallis test followed by the Dunn-Bonferroni post hoc test.
The comparisons between OEH and HOE groups at given volume
fractions were performed using Wilcoxon signed-rank test.
Fig. 1. Oil droplet size distribution of OEH emulsions (A) and HOE emulsions
(B) containing different soy oil volume fractions: 0 mL/L (●); 10 mL/L (◯);
3. Results and discussion
30 mL/L (▼); 50 mL/L (△); 70 mL/L (■). OEH emulsions: soy protein emul-
sions prepared following the process of addition of oil, emulsification and heat
3.1. Soy oil droplet size distribution of SPI emulsions treatment; HOE emulsions: soy protein emulsions prepared following the pro-
cess of heat treatment, addition of oil and emulsification.
The droplet size distribution of the freshly formed SPI emulsions
was measured using laser diffraction and the results were shown in
emulsions is larger compared to HOE emulsions.
Fig. 1. Generally, the volume mean diameter (VMD) of all the SPI
emulsions was in the range of 1.60–3.09 μm. As the soy oil volume
fraction gradually increases from 10 to 70 mL/L, the mean droplet
3.2. Viscosity of SPI emulsions
diameter of OEH and HEO emulsions increases correspondingly. This
might be because of the increased hydrophobic interactions between
Fig. 2 illustrates the viscosities of OEH and HOE emulsions at soy oil
individual soy oil droplets (Wong & Kitts, 2003). In addition, at a given
volume fractions of 10, 30, 50 and 70 mL/L. The results showed that the
protein concentration in the continuous phase, a possible coalescence of
viscosity decreased with increasing shear rate for all emulsions, in-
soy oil droplets in the SPI emulsion system would occur more easily at a
dicating a typical shear-thinning (pseudoplastic) behavior (Li et al.,
higher soy oil volume fraction (Yang, Liu, & Tang, 2013).
2011). The apparent increase in viscosity could also be observed at a
The mean droplet diameter of the HOE emulsions ranged from 1.60
specific shear rate with increasing soy oil volume fractions, exhibiting a
to 1.92 μm. In contrast, the mean droplet size of the OEH emulsions was
parallel trend like the droplet size. This phenomenon could be attrib-
slightly higher (1.96–3.09 μm). Thermal denaturation of SPI before the
uted to the interactions between proteins and soy oil droplets promoted
addition of soy oil can cause unfolding of protein molecules and ex-
by the thermo-denatured protein molecules (Taha & Mohamed, 2004).
posure of hydrophobic groups initially located in the interior of SPI
Moreover, the viscosity of HOE emulsions is higher than that of OEH
molecules. The emulsifying properties of denatured proteins would
emulsions at the same soy oil volume fraction and shear rate. Based on
improve accordingly (Dissanayake & Vasiljevic, 2009; Keerati-u-rai &
the results shown in Fig. 1, HOE emulsions with a smaller droplet size at
Corredig, 2009). In contrast, heat treatment of SPI dispersions after
a given soy oil volume fraction (compared to OEH emulsions) comprise
incorporation of soy oil and emulsification usually leads to flocculation
a larger number of droplets. Therefore, the probability that any two soy
of soy oil droplets (Iordache & Jelen, 2003), possibly from interactions
oil droplets enter the attraction region increases, leading to an increase
between adsorbed proteins at a droplet interface or between droplets in
in viscosity of HOE emulsions.
emulsions (Li, Kong, Zhang, & Hua, 2011). Therefore, the VMD of OEH

3
H. Zhao, et al.
Fig. 2. Viscosity of OEH emulsions (A) and HOE emulsions (B) containing
different soy oil volume fractions (0 mL/L: solid line; 10 mL/L: dotted; 30 mL/L:
dash-dot; 50 mL/L: short dash; 70 mL/L: dash-dot-dot) as a function of shear
rate. OEH emulsions: soy protein emulsions prepared following the process of
addition of oil, emulsification and heat treatment; HOE emulsions: soy protein
emulsions prepared following the process of heat treatment, addition of oil and
emulsification.
3.3. Viscoelastic measurement
According to a previous report (Zhao et al., 2016), CaSO4-induced
gelation of SPI emulsions is considered a two-step process. Step 1 is the
thermal denaturation of soy proteins, during which hydrophobic re-
gions and –SH groups initially located inside the protein molecules are
exposed to the outside (Guo et al., 2015). Meanwhile, protein ag-
gregates are formed via hydrophobic interactions and disulfide bonds
among the SPI molecules. After the addition of a coagulant, negative
charges on the surface of protein molecules are neutralized and elec-
trostatic repulsion among them is reduced, leading to further ag-
gregation with Ca2+ acting as a bridge between the charged carboxylic
groups on adjacent SPI molecules (Kao, Su, & Lee, 2003). In step 2, the
SPI aggregates interact more strongly with one another through pro-
tein–protein interactions, eventually promoting the formation of a
three-dimensional gel network. Because of the addition of soy oil, the
potential interactions could be more complicated in the SPI emulsions.
To investigate the effects of thermal denaturation of SPI before and/
or after incorporation of soy oil with different Φ values on the
4
LWT - Food Science and Technology 117 (2020) 108690
Fig. 3. Storage modulus (G′) and temperature (T) as a function of time for OEH
emulsions (A) and HOE emulsions (B) containing different soy oil volume
fractions (0 mL/L: solid line; 10 mL/L: dotted; 30 mL/L: dash-dot; 50 mL/L:
short dash; 70 mL/L: dash-dot-dot) during CaSO4-induced gelation. OEH
emulsions: soy protein emulsions prepared following the process of addition of
oil, emulsification and heat treatment; HOE emulsions: soy protein emulsions
prepared following the process of heat treatment, addition of oil and emulsi-
fication.
viscoelastic properties of CaSO4-induced SPI gels, G′ and G″ were ob-
tained by applying a temperature-time profile during the rheological
measurements. The typical profiles of G′ after the addition of CaSO4 are
presented in Fig. 3. The curves of G″ which are similar, to those of G′ are
not shown. The final G′ and G″ of the SPI gels after the temperature
cycles are summarized in Fig. 4. A CaSO4-induced SPI gel free of soy oil
was used as the control.
After the addition of a coagulant, all the SPI emulsions underwent
sol to gel transition as the temperature increased. During the heating
period and holding time at 90 °C, the rigidity (elastic modulus) gradu-
ally increased for all the SPI gels among which the differences were not
obvious along with the gels made from SPI emulsions with a 70 mL/L
soy oil level. The increase in G′ during the heating period could be
attributed to the protein–oil and protein–protein physical interactions
combined with the structural rearrangements of the SPI gel network
(Chen & Dickinson, 2000). During the cooling period, a sharp increase
in the elasticity of all the SPI gels is observed, and the differences in G′
resulting from the incorporation of different soy oil volume fractions
were distinguishable on the curves. The dramatic escalation of this ri-
gidity during the cooling stage indicated that the gel structures could be
further enhanced by the formation of stronger bonds. Typically, hy-
drogen bond formation is generally considered more likely at lower
temperatures. Fig. 4 indicates that the viscoelastic properties of the SPI
gels improved after the addition of soy oil. For CaSO4-induced OEH
H. Zhao, et al.
Fig. 4. Final storage modulus (G′) (A) and loss modulus (G″) (B) of CaSO4-
induced soy protein gels made from OEH emulsions (□) and HOE emulsions
(■) as a function of oil volume fraction. Means with different letters sig-
nificantly differ (P < 0.05). The means between OEH and HOE groups (not
shown) differ significantly (P < 0.05). OEH emulsions: soy protein emulsions
prepared following the process of addition of oil, emulsification and heat
treatment; HOE emulsions: soy protein emulsions prepared following the pro-
cess of heat treatment, addition of oil and emulsification.
emulsion gels containing 10–70 mL/L of soy oil, the increase in the final
G′ ranged from 20.4% to 42.0% compared to the control. In contrast,
the final G′ of HOE emulsion gels containing soy oil in the same range of
volume fraction increased by 50.1%–67.7% compared to the control.
The reinforced protein–oil interactions with increasing soy oil volume
fractions led to a higher degree of cross-linking in the gel matrix. As
depicted in Fig. 1, the droplet size of the HOE emulsions was less than
that of the OEH emulsions at the same volume fraction of soy oil.
Therefore, the contact area between protein molecules and soy oil
droplets in HOE emulsions tended to be more predominant, and the
resultant interactions corresponding to the gel network were stronger.
In this study, a strain sweep test was performed after the formation
of the SPI gels, and the results are shown in Fig. 5. For all SPI gels, a
linear response at strain less than 10% was observed wherein G′ was
independent of strain. This implies that gelation measurements con-
ducted at 1% strain fall within the linear viscoelastic region. When the
strain was increased further, G′ decreased sharply, indicating a
breakage of bonds in the gel network and transition from linear to non-
linear behavior (Eleya, Ko, & Gunasekaran, 2004; Maltais, Remondetto,
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LWT - Food Science and Technology 117 (2020) 108690
Fig. 5. Strain sweep of storage modulus (G′) of CaSO4-induced gels made from
OEH emulsions (A) and HOE emulsions (B) with different soy oil volume
fractions: 0 mL/L (●); 10 mL/L (◯); 30 mL/L (▼); 50 mL/L (△); 70 mL/L (■).
The measurement was performed at room temperature (25 °C). OEH emulsions:
soy protein emulsions prepared following the process of addition of oil, emul-
sification and heat treatment; HOE emulsions: soy protein emulsions prepared
following the process of heat treatment, addition of oil and emulsification.
& Subirade, 2008). The strain value at which G′ decreased to 95% of its
maximum value was considered the critical strain of the gel (Bi, Li,
Wang, & Adhikari, 2013). The results demonstrated all SPI gels exhibit
similar behavior (Fig. 5). Furthermore, at the critical strain, G′ showed
an upward trend with increasing soy oil volume fraction, and the ri-
gidity of the HOE emulsion gels was stiffer compared to OEH emulsion
gels, in good agreement with the results shown in Figs. 3 and 4.
3.4. Hardness and WHC of CaSO4-induced SPI gels
Fig. 6 show the hardness and WHC of CaSO4-induced SPI gels made
from OEH and HOE emulsions containing different soy oil volume
fractions. The results indicated that incorporation of soy oil at a volume
fraction of 10–70 mL/L increases both the hardness and WHC of the SPI
gels to different degrees. In addition, the SPI gels made from HOE
emulsions have significantly greater (P < 0.05) hardness and WHC
compared to OEH emulsion gels at the same soy oil volume fraction.
The results of hardness and WHC of CaSO4-induced SPI gels are
consistent with the results of G′ discussed earlier. The increase in
hardness may arise from the formation of a greater number of cross-
links in the system of emulsion gel at higher oil concentrations.
H. Zhao, et al.
Fig. 6. Hardness (A) and water holding capacity (B) of CaSO4-induced soy
protein gels made from OEH emulsions (□) and HOE emulsions (■) as a
function of oil volume fraction. Means with different letters significantly differ
(P < 0.05). The means between OEH and HOE groups (not shown) differ sig-
nificantly (P < 0.05). OEH emulsions: soy protein emulsions prepared fol-
lowing the process of addition of oil, emulsification and heat treatment; HOE
emulsions: soy protein emulsions prepared following the process of heat
treatment, addition of oil and emulsification.
Furthermore, the viscous properties of the interfacial layers in the
system could dissipate energy. Hence, the emulsion gels require more
force to break at the higher oil volume fractions (Xi, Liu, McClements, &
Zou, 2019). In addition, because of the role that soy oil plays in en-
hancing SPI elasticity with increasing volume fraction, soy oil droplets
are normally recognized as active fillers (Dickinson, 2001; Xi et al.,
2019). Soy oil interacts with the gel matrix, with stronger interactions
at higher volume fractions, resulting in reinforcement of the capacity of
the gel to resist compression and puncture. The relatively lower mean
droplet size of HOE emulsions (compared to OEH emulsions at the same
soy oil volume fractions) could improve the filling effect of the active
particles and reduce the interstitial spaces of particles within the gel
matrix. Considering an earlier work (Zhao et al., 2016), the denatura-
tion of soy protein could improve the compactness of gel network due
to the enhanced protein-protein interactions. In the present study, the
larger value of WHC corresponding to higher oil volume fractions (more
oil droplets) could be caused by the enhanced density of gel structure,
leading to the water to be trapped within the interior of the pores by
capillary forces. Moreover, the HOE emulsion gels exhibit more
6
LWT - Food Science and Technology 117 (2020) 108690
homogeneous microstructures with smaller pores than OEH gels, as will
be discussed later, causing an increase of the capillary forces and hence
resulting in gel matrix retained more water (Xi et al., 2019).
3.5. CLSM analysis of CaSO4-induced SPI gels
Microstructures of CaSO4-induced SPI gels made from OEH and/or
HEO emulsions containing different soy oil volume fractions were
characterized by CLSM. Fig. 7 reveals that the network structure is
somewhat loose and heterogeneous for the gel prepared by SPI dis-
persion without soy oil (control). The addition of soy oil before thermal
denaturation of soy protein improves the structure of CaSO4-induced
SPI gels to some extent after gelation. As shown in Fig. 7B–E, the SPI
gels formed exhibit a slightly dense structure because of the enrichment
of soy oil droplets, which could act as active fillers (Xi et al., 2019).
Moreover, larger soy oil droplets coated with protein molecules on the
surface are more likely to be observed on the images of former gels
(Fig. 7B–E) with increasing soy oil volume fractions, further demon-
strating the occurrence of flocculation within the gel matrix
(Murekatete et al., 2016; Xi et al., 2019). Fig. 7F–I presents the network
structure of SPI gels made from HOE emulsions, featuring a more
homogeneous and compact structure than that of SPI gels made from
OEH emulsions. In addition, the distribution of soy oil droplets within
the SPI gel structure resulting from HOE emulsions appears more uni-
form than that of the soy oil droplets within the SPI gels structure re-
sulting from OEH emulsions, and the visible droplet sizes are consistent
with the results of the VMD described in Fig. 1. The system could be
regarded as a network of particles formed by chemical and physical
bonds. The oil droplets will not only act as filler particles but will be
also component of the gel network (Xi et al., 2019). The enhanced
emulsifying properties resulting from the thermal denaturation of SPI
before incorporation of oil could strengthen the interaction force be-
tween emulsion droplets and between the emulsion droplets and the
protein matrix and explain the improvement of the gel microstructures.
In brief, the microstructure results further explain the differences of the
WHC and the viscoelastic properties among various gels.
4. Conclusions
The rigidity (final G′), hardness and WHC of the gels were all gra-
dually enhanced with the increase of soy oil from 10 mL/L to 70 mL/L.
The HOE emulsion gel displayed greater performance of gelation be-
havior and textural properties and exhibited more compact and
homogeneous structures compared to OEH emulsion gels at the same
volume fraction of soy oil, possibly resulting from the reinforced pro-
tein-oil interactions and active filler effect of smaller oil droplets within
the network structures. The results of this study could potentially fa-
cilitate the development of soy protein-based tofu-type gels with a
firmer texture and finer microstructure, thereby providing new alter-
natives for the making of tofu. However, detailed study is still required
to further understand the structural alteration of SPI aggregate under
various thermal treatment conditions on the characteristics of SPI
emulsion and CaSO4-induced SPI gels.
Declaration of competing interest
None.
Acknowledgement
The authors would like to thank the Special Funds for Taishan
Scholars Project, the Innovation Team of Jinan City (No.
2018GXRC004), the Nature Science Foundation of China (No.
31901645) and the Natural Science Foundation of Shandong Province
(ZR2019BCE088) for their financial support.
H. Zhao, et al.
Fig. 7. CLSM images of CaSO4-induced gels made from emulsions with different soy oil volume fractions. (A) Gel made without soy oil. (B–E) SPI gels made from
OEH emulsions containing 10 mL/L, 30 mL/L, 50 mL/L and 70 mL/L soy oil, respectively. (F–I) SPI gels made from HOE emulsions containing 10 mL/L, 30 mL/L,
50 mL/L and 70 mL/L soy oil, respectively. OEH emulsions: soy protein emulsions prepared following the process of addition of oil, emulsification and heat
treatment; HOE emulsions: soy protein emulsions prepared following the process of heat treatment, addition of oil and emulsification.
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