Infrared Physics & Technology 123 (2022) 104149

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Infrared Physics and Technology

journal homepage: www.elsevier.com/locate/infrared

Evaluation of optical properties of tofu samples produced with different

coagulation temperatures and times using near-infrared
transmission spectroscopy
Yoshito Saito a, b, Tetsuhito Suzuki a, *, Naoshi Kondo a
a
Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, 6068267 Kyoto, Japan
b
Research Fellow of Japan Society for the Promotion of Science, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: To investigate the feasibility of using near-infrared (NIR) spectroscopy to evaluate the coagulation extent of tofu,
Tofu the optical properties of tofu samples obtained with different coagulation temperatures and times were measured
Coagulation in the wavelength range 1300–1800 nm. The apparent absorbance was measured using the NIR transmission
Near-infrared spectroscopy
spectrum, and then divided into absorption and scattering components by curve fitting. The apparent absorbance
Light scattering
Viscosity
increased over the entire wavelength range as the coagulation temperature increased, in which the scattering
Microstructure increase was dominant over that of the absorption. The viscosity was measured as an index of the physical
properties of tofu, which increased with the coagulation temperature and had a high correlation (R2 = 0.803)
with one of the fitting parameters of the scattering. The microstructure of the tofu became finer as the coagu­
lation temperature increased, which suggests that the increase in scattering might be due to changes in the
microstructure. Furthermore, the light scattering increased with the coagulation time as well as with the
coagulation temperature. The increase in scattering with the coagulation time followed the first-order reaction
kinetics. Both scattering intensity and increase speed were larger at shorter wavelengths. This can be explained
by the theory of Mie scattering and multiple scattering, indicating that scattering measurements at shorter
wavelengths are useful for monitoring of coagulation. Thus, light scattering measurements using NIR spectros­
copy are promising for investigating the physical properties of tofu.

1. Introduction
Tofu is one of the most important processed soybean products, which
has been consumed since ancient times, mainly in Asian countries. In
recent years, tofu has attracted worldwide attention as a highly nutri­
tious and low-calorie food because of the increasing demand for health.
Thus, there is a need for a stable production of a high-quality tofu.
The tofu production process consists of extraction of soymilk by
soaking, grinding, heating, and filtering of soybeans and coagulation of
soymilk [1]. During the coagulation process, protein particles in the
soymilk aggregate surrounding oil body particles, forming a micro­
structure referred to as curd holding water inside [2].
In general, the characteristics of the microstructures of foods with
porous structures similar to that of tofu, such as noodles and breads,
have a significant effect on the physical properties [3]. For tofu, the
physical properties are considered as one of the most important tofu
qualities, evaluated by the firmness, viscosity, elasticity, water holding
capacity (WHC), etc. Various factors affect the physical properties of
tofu, including the soybean variety characteristics [4], soymilk con­
centration [5], coagulant type [6,7], coagulant concentration [8,9],
coagulant stirring speed [1], and coagulation temperature [10]. In
particular, the coagulation speed of soymilk is largely affected by the
type of coagulant, stirring speed of the coagulant, and coagulation
temperature. Therefore, the instability of physical properties such as the
firmness, viscoelasticity, and WHC of tofu is an issue. Currently, the
coagulation process is controlled by the experience and intuition of
highly skilled people in many factories. A method for monitoring of the
coagulation is needed for its accurate control.
Electrical impedance spectroscopy can be used to monitor the
coagulation process of soymilk [11] as a simple and rapid method.

Abbreviations: NIR, near-infrared; SEM, scanning electron microscopy.

* Corresponding author.
E-mail address: suzuki.tetsuhito.4u@kyoto-u.ac.jp (T. Suzuki).

https://doi.org/10.1016/j.infrared.2022.104149
Received 31 May 2021; Received in revised form 16 February 2022; Accepted 20 March 2022
Available online 22 March 2022
1350-4495/© 2022 Elsevier B.V. All rights reserved.
Y. Saito et al.
However, in principle, it is limited to contact measurements on the
sample. As the scale and style of tofu production are extremely diverse, a
nondestructive measurement method is desirable.
Optical measurements are widely applied for nondestructive mea­
surements of various agricultural products and foods. Among the
various techniques, near-infrared (NIR) spectroscopy has been widely
applied to the prediction of sugar content and acidity of fruits, detection
of internal or external defects, and nondestructive prediction of chem­
ical components in food [12]. For food processing, NIR spectroscopy can
be used for real-time monitoring of the milk coagulation [13,14].
Biological measurements using NIR spectroscopy are based on ab­
sorption derived from intermolecular stretching vibrations involving H
atoms, such as those of O–H, C–H, and N–H, and scattering derived from
structural sizes on the order of several hundreds of nanometers to
several tens of micrometers [15]. To evaluate the feasibility of NIR
spectroscopy for monitoring of the soymilk coagulation, it is important
to elucidate the basic optical properties, based on mainly absorption and
scattering. Absorption and reduced scattering coefficients at a wave­
length of 633 nm were measured for tofu samples with different hard­
ness, where the change in scattering was more dominant than that in the
absorption [16]. However, the absorption of chemical components is
rarely observed at a single wavelength of 633 nm. The absorption and
scattering characteristics in the NIR region, where the absorption of
chemical components is observed, have not been reported.
Therefore, the objective of this study was to evaluate the absorption
and scattering properties of tofu with different extent of coagulation
using NIR spectroscopy. Tofu samples were produced at different
coagulation temperatures, which were provided for the viscosity mea­
surement and microstructural images acquisition. The absorption and
scattering components were relatively separated by NIR transmission
measurement, and the optical properties were compared to the viscosity
and microstructure. Subsequently, to simulate the monitoring of the
soymilk coagulation, NIR transmission measurements were carried out
on tofu produced with different coagulation times and the changes in
optical properties were characterized with the coagulation time.
2. Materials
In this study, soymilk (Nagoya Seiraku Co., Ltd., Japan) and CaSO4･
1/2H2O (Nakajima Shoten Co., Ltd., Japan) were used. CaSO4･1/2H2O,
commonly used as a coagulant in tofu production, was used as the
soaking powder. The solid concentration of the soymilk was 10.2%, and
the pH was 6.6. A mixture of CaSO4･1/2H2O was added to 100 mL of the
soymilk at a concentration of 58.1 mM at room temperature (22 ℃),
well stirred, and then used as a sample. This concentration of CaSO4･1/
Fig. 1. Viscosity of the tofu samples produced at different coagulation
temperatures.
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Infrared Physics and Technology 123 (2022) 104149
2H2O was optimal for the same soymilk product produced by Nagoya
Seiraku Co., Ltd. [17].
3. Experimental devices and methods
3.1. Sample preparation
For viscosity measurement and scanning electron microscopy (SEM)
images acquisition, tofu samples were processed in a 100-mL glass
beaker at six different coagulation temperatures: 40, 50, 60, 70, 80, and
90 ℃ with 20 min of a coagulation time. The beaker containing the
sample was kept at the constant coagulation temperature in a thermo­
static bath. After coagulation, each sample was immediately cooled and
provided for viscosity and SEM image acquisition.
For NIR transmission measurement, each tofu sample was prepared
in a transmission cuvette with an optical path length of 0.30 mm con­
sisting of crown glass slides (thickness: 0.9 ± 0.1 mm) as windows. The
sample mixture was filled into the cuvette and coagulated in an oven
maintained at a constant temperature with the opening protected by a
tape to prevent water evaporation.
Two different conditions, different coagulation temperatures and
coagulation times, were adapted for transmission measurement. To
investigate the relationships between the viscosity and SEM images, the
samples were coagulated at the same patterns of temperatures as
described above (40, 50, 60, 70, 80, and 90 ℃). The coagulation time
was set to 10 min at which the transmission spectrum tended to stabilize.
The other tofu samples were produced with different coagulation times
to simulate the coagulation process monitoring. The samples were
coagulated with 10 different coagulation times of 0, 1, 2, 4, 7, 10, 15, 20,
25, and 30 min. The coagulation temperature was 80 ℃, which is
common for tofu production in Japan. After the coagulation, each
sample was cooled at 4 ℃ for 7 min and left at 22 ℃ for 7 min, and then
provided for the transmission measurement.
3.2. Viscosity measurement and SEM image acquisition
The viscosity was measured as an index of the physical quality of the
tofu. The 100 mL of tofu samples were measured using a tuning fork
vibration viscometer SV-10 (A&D Co., Ltd., Japan) at room temperature
(22 ℃). Considering the variation within the same sample, the viscosity
was measured three times for one sample and four different samples
were measured under the same conditions. In addition to each tofu
sample, the viscosity of soymilk was also measured as the original
properties before coagulation.
To investigate the differences in the microstructure of tofu, micro­
structural images were acquired using SEM. The coagulated tofu sam­
ples were pretreated before SEM image acquisition by the method
proposed by Onodera et al. [18]. The samples were cut out at 1 cm below
the surface with a thickness of 1 mm, and immersed in a 1% glutaral­
dehyde solution for 1 h for tissue fixation. The specimens were then
washed with distilled water, flash-frozen with liquid nitrogen, and
freeze-dried in a freeze dryer FDU-1200 (EYELA, TOKYO RIKAKIKAI
Co., Ltd., Japan) at a pressure below 50 Pa for 72 h. Each pretreated
specimen of tofu was coated with silver using a sputter and observed
using SEM (SEMTRAC mini, Nikkiso Co., Ltd., Japan). Three different
pieces were measured at each coagulation temperature and 10 images
were used for each piece, resulting in a total of 30 images for each
coagulation temperature. The acceleration voltage was 5 kV, and the
magnification was × 1000.
3.3. NIR transmission measurement
The tofu samples described in the section 3.1 were provided for the
NIR transmission measurement. The original mixture of soymilk and
CaSO4･1/2H2O was also measured.
The transmission spectra were measured using an
Y. Saito et al. Infrared Physics and Technology 123 (2022) 104149

Fig. 2. Representative SEM images of the tofu samples with different coagulation temperatures of (a) 40, (b) 50, (c) 60, (d) 70, (e) 80, and (f) 90 ℃. The
magnification was × 1000.

Fig. 3. Geometric parameters obtained by SEM images of the tofu samples produced at different coagulation temperatures. (a) Porosity, (b) number of holes per unit
area, and (c) mean size of holes. ** indicates p < 0.01, * indicates p < 0.05 (significant difference).

ultraviolet–visible–NIR spectrophotometer V-670 (JASCO Co., Ltd.,
Japan) with a double-beam method in which the branched light paths
were transmitted through the reference and sample. The spectrum
wavelength range was 1300–1800 nm, the bandwidth was 4.0 nm, the
number of scans was four for one measurement, and four different
samples were measured for each coagulation temperature. An empty
cuvette was placed on the reference side. The measurement wavelength
was limited to 1300–1800 nm because the signal intensity of the sample
side became extremely low at wavelengths shorter than 1300 nm and
the linearity of the absorbance was not maintained. To exclude the effect
of the absorption due to the difference in the thickness of the window
material, baseline measurements were performed in an empty trans­
mission cell prior to each sample preparation.
4. Results and discussion
4.1. Viscosity and SEM images of the tofu samples produced at different
coagulation temperatures
Fig. 1 shows the results of viscosity of the tofu samples produced at
different coagulation temperatures, while Fig. 2 shows the representa­
tive SEM images of each sample. Fig. 2(a)–(f) show images of the
microstructures of the tofu samples with coagulation temperatures of 40
to 90 ℃, respectively.
As shown in Fig. 1, the viscosity of tofu increased with the coagu­
lation temperature, which is consistent with previous studies [16]. As
shown in Fig. 2, the microstructure of tofu tended to be denser as the
coagulation temperature increased up to 80 ℃ and became slightly
coarser at 90 ℃. To quantitatively evaluate the characteristics of the
microstructure, geometric parameters were calculated for the tofu mi­
crostructures shown in Fig. 2 by the method of Saito et al. [19]. The
results of the two-dimensional porosity, number of holes, and mean size
of the holes are shown in Fig. 3.
In Fig. 3, the error bars represent the standard deviations of a total of
30 SEM images. Fig. 3 shows that the porosity and mean size of the holes
significantly decreased, while the number of holes significantly
increased in the coagulation temperature range of 40 to 50 ℃. Their
tendencies were observed to the coagulation temperature of 80 ℃. This
indicates that the microstructure became denser up to 80 ℃, which is
consistent with the trend observed in Fig. 2. On the other hand, the
porosity and mean size of the holes significantly increased, while the
number of holes significantly decreased at coagulation temperatures of
80–90 ℃, indicating that the microstructure became coarser. When
CaSO4 has been used as a coagulant, protein texture changes occurred at

3
Y. Saito et al.
Fig. 4. (a) Apparent absorbance spectra of the tofu samples produced at different coagulation temperatures and (b) an example of absorption and scattering obtained
by the apparent absorbance of tofu (coagulation temperature: 80 ℃, coagulation time: 10 min).
coagulation temperatures higher than 90 ℃ and the cohesiveness,
springiness, and chewiness decreased simultaneously [20]. In this
experiment, the viscosity reached its maximum at a coagulation tem­
perature of 90 ℃. However, the microstructure was densest at a coag­
ulation temperature of 80 ℃, as shown in Fig. 3. This suggests that the
homogeneity of the microstructure was impaired at a coagulation tem­
perature of 90 ℃, probably because of the textural changes of proteins at
the high temperature.
4.2. Optical properties of the tofu samples and the relationships to
viscosity and microstructural properties
Fig. 4(a) shows the apparent absorbance spectrum of each sample. In
Fig. 4(a), the vertical axis shows the absorbance calculated by the in­
tensity ratio of incident light (I0) to transmitted light (I), expressed by
-log(I/I0), while the horizontal axis shows the wavelength. The change
in absorbance of the crown glass due to heating and effect of multiple
reflections inside the cuvette were sufficiently small compared to the
change in absorbance of the sample.
As shown in Fig. 4(a), the absorption peak was observed at 1450 nm
in the spectrum of each sample. The apparent absorbance increased in
the entire wavelength range as the coagulation temperature increased.
The absorption peak at 1450 nm is derived from the first overtone of the
O–H stretching vibration [21], which represents mainly the absorption
by water. In addition, the baseline of the spectrum tends to increase at
shorter wavelengths, which is considered to originate from the attenu­
ation of straight light by multiple scattering. In the transmission spectra
Fig. 5. (a) Scattering spectra of the tofu samples produced at different coagulation temperatures and (b) correlation between β and viscosity of tofu.
4
Infrared Physics and Technology 123 (2022) 104149
of soymilk and tofu in the NIR region, it is necessary to analyze the
attenuation of straight light due to multiple scattering in addition to the
absorption mainly by water.
The attenuation of light in transmission measurements of highly
scattering media can be expressed as the sum of two components, ab­
sorption and scattering [22], which can be expressed as.
− log(I/I0 ) = Aα + G (1)
where I is the intensity of the transmitted light, I0 is the intensity of
the incident light, A is the weighting factor for the absorption coefficient
of the sample, α is the absorption coefficient of the sample, and G is the
attenuation due to scattering. If α can be considered as the absorption
coefficient of water, the parameter A approximately corresponds to the
product of the optical path length and concentration of water based on
the Lambert–Beer law.
Furthermore, assuming that G, the attenuation due to scattering, is
linear with respect to the reduced scattering coefficient, which repre­
sents the intensity of multiple scattering, G can be expressed by Eq. (2)
using the Mie approximation [23],
G ≃ Bλ− β
(2)
where B reflects the concentration of the scatterer, β is a parameter
that includes the size information of the scatterer, and λ is the wave­
length. Based on Eq. (2), Eq. (1) can be expressed as.
− log(I/I0 ) = Aα + Bλ− β
(3)
According to Eq. (3), the measured absorbance can be decomposed
Y. Saito et al.
Table 1
Pearson correlation coefficient between scattering properties and geometric
parameters of the tofu microstructure.
Parameter Correlation coefficient
Porosity Number of holes Mean size of holes
0.792* − 0.506 0.659
β
G at 1300 nm − 0.876* 0.675 − 0.778*
*p < 0.05 (significant correlation).
into absorption (Aα) and scattering (G ≃ Bλ− β ) by fitting with wave­
length as an independent variable, by which the three parameters, A, B,
and β, are obtained. Fig. 4(b) shows an example of the decomposition of
the apparent absorbance spectrum into absorption and scattering com­
ponents for a sample with a coagulation temperature of 80 ℃. In Fig. 4
(b), the solid gray line represents the measured apparent absorbance,
while the red and blue dotted lines represent the scattering and ab­
sorption spectra after decomposition, respectively. As shown in Fig. 4
(b), the measured absorbance spectrum can be separated into absorption
and scattering spectra by fitting Eq. (3).
Only the scattering spectra obtained from the transmission mea­
surement for tofu at different coagulation temperatures are shown in
Fig. 5(a).
As shown in Fig. 5(a), the scattering spectra of tofu increased with
the coagulation temperature over the entire measurement wavelength
range, while the variation in the absorption component was small (not
shown in Fig. 3(a)). In addition, the parameter β in Eq. (2) tends to
decrease. Fig. 5(b) shows the correlation between the viscosity of the
tofu and size parameter β, where the vertical axis represents the vis­
cosity of tofu, the horizontal axis represents β, and the error bars
represent the standard deviations of the four samples. As shown in Fig. 5
(b), there was a significant negative correlation (R2 = 0.803, p < 0.01)
between the viscosity of tofu and β. A previous study demonstrated that
β is decreased after milk coagulation, i.e., when the average size of the
particles is increased, consistent with the trend in Fig. 3(b) [24]. This
suggests that light scattering measurements by NIR spectroscopy may be
useful for evaluation of the differences in the physical properties of tofu.
The relationship between the light scattering properties of tofu and
microstructure was evaluated, as shown in Table 1. The simple Pearson
correlation coefficients were determined between the two parameters, β
and scattering G at 1300 nm, obtained by Eq. (2), and three geometric
parameters. We selected the wavelength of 1300 nm as a representative
wavelength because the correlation coefficients between G and each
geometrical parameter were almost the same for each wavelength.
As shown in Table 1, β exhibited a significant positive correlation
with the porosity. In the coagulation of particles in soymilk, soluble
proteins are less likely to coagulate than particle proteins [2]. Thus,
when the coagulation temperature is low, mainly particle proteins
Fig. 6. (a) Apparent absorbance spectra and (b) scattering spectra of the tofu samples produced with different coagulation times.
5
Infrared Physics and Technology 123 (2022) 104149
coagulate. As the coagulation temperature increases, soluble proteins
coagulate, followed by particle proteins. This implies that, as the coag­
ulation temperature increased, the number of aggregated particles
increased, which resulted in a decrease in porosity. As the parameter β
also decreased with the increase in coagulation temperature, a signifi­
cant positive correlation was observed between β and porosity.
The scattering G at 1300 nm exhibited significant negative correla­
tions with the porosity and mean size of the holes. The negative corre­
lation between G and porosity suggests the aggregation of soluble
proteins due to the increase in the coagulation temperature, resulting in
a decrease in porosity and increase in the volume of scatterers. The
negative correlation between G and mean size of the holes may be
explained by light scattering at interfaces with different refractive
indices. The mean size of the holes decreases with the increase in
coagulation temperature, as shown in Fig. 3 (c), which increases the
frequency of light encountering the interface between the curd walls and
holes, resulting in the light scattering increase. Furthermore, the mois­
ture content of the tofu was also considered as a possible factor in the
scattering increase in NIR region, but there was no significant difference
in moisture content of the tofu sample coagulated under 60 ℃. There­
fore, it is suggested that the increase in scattering of tofu in NIR region is
mainly due to the microstructural change.
4.3. Optical properties of the tofu samples produced with different
coagulation times
Fig. 6(a) shows the apparent absorbance spectra of the tofu samples
produced with different coagulation times. The vertical and horizontal
axes are the same as those in Fig. 4(a). Fig. 6(b) shows the spectra of G
extracted from the absorbance spectrum by fitting Eq. (3). For a clear
data visualization, only the results at coagulation times of 0, 1, 2, 4, 10,
and 20 min are shown.
The apparent absorbance increased with the coagulation time (Fig. 6
(a)). The scattering also increased (Fig. 6(b)). Its tendency was similar to
that with the increase in coagulation temperature. As the spectral
changes of the absorption component were small, the increase in the
apparent absorbance with the increase in coagulation time was due to
the increase in scattering.
The increase in viscoelasticity of tofu during the coagulation obeys
the first-order reaction kinetic equation [25],
y = C(1 − e− kt ) (4)
where y is the measured value, k is the rate constant, t is the reaction
time, and C is the equilibrium value. To verify whether the increase in
scattering shown in Fig. 6(b) follows the first-order reaction kinetics
with respect to the coagulation time, we calculated the increment in the
scattering G from 0 min of coagulation, ΔG. And then ΔG at 1300 nm
was fitted with Eq. (4), where ΔG at 1300 nm is y and the coagulation
Y. Saito et al.
Fig. 7. Fitting result of ΔG at 1300 nm with the first-order reaction ki­
netic equation.
Table 2
Results of the equilibrium value and rate constant obtained by fitting the first-
order reaction kinetic equation at each selected wavelength.
Wavelength (nm) C k (min− 1)
1300 0.73 0.208
1400 0.66 0.202
1500 0.59 0.197
1600 0.54 0.193
1700 0.49 0.189
1800 0.45 0.185
time is t. The result of fitting ΔG at 1300 nm using Eq. (4) is shown in
Fig. 7. The plots represent the measured values, the solid line represents
the fitting curve, and the error bars represent the standard deviations of
the measurements of the four samples.
Fig. 7 shows that ΔG can be fitted to the first-order reaction kinetic
equation, where the equilibrium value C was 0.73 and the rate constant
k was 0.208 min− 1. Furthermore, the differences in the parameters were
investigated depending on the selected wavelength. The results of C and
k obtained by fitting with Eq. (4) at six different wavelengths in the
range of 1300 to 1800 nm are shown in Table 2.
As shown in Table 2, both C and k are larger at shorter wavelengths.
This indicates that, as the wavelength becomes shorter, both scattering
intensity and increase rate increase with the solidification time. This
tendency can be explained by the scattering and coagulation mechanism
of soymilk.
In Mie scattering, the scattering intensity reaches its maximum when
the wavelength of light and particle diameter are almost equal and ap­
proaches a constant value as the particle size increases in relation to the
wavelength [26]. In the early stage of soymilk coagulation, small protein
particles with diameters of approximately 80–100 nm aggregate around
oil body particles with diameters of approximately 400 nm. Further
aggregation of the oil body particles surrounded by proteins leads to the
formation of a curd structure with a periodic size on the order of several
to tens of micrometers [2]. This suggests that, as the size of the aggregate
increases, the wavelength at which scattering appears changes from
shorter to longer in turn, resulting in a difference in the rate constant k.
5. Conclusion
In this study, the absorption and scattering properties of tofu with
different coagulation temperatures and times were evaluated by NIR
spectroscopy. The apparent absorbance increased with the coagulation
6
Infrared Physics and Technology 123 (2022) 104149
temperature, where the increase in scattering was more dominant than
that in the absorption. The viscosity increased with the coagulation
temperature and had a high correlation (R2 = 0.803) with the fitting
parameter β in the Mie approximation. Additionally, the porosity and
mean size of the holes in the microstructure had significant correlations
with the light scattering, indicating that the denseness of the micro­
structure of tofu possibly contributes to light scattering. Furthermore,
from NIR transmission measurement of tofu samples with different
coagulation times, the increment in light scattering could be expressed
by the first-order reaction kinetic equation with the coagulation time,
which increased faster at shorter wavelengths. These results indicate
that light scattering measurements in the NIR region can be used to
monitor the coagulation process of tofu.
Funding
This study was supported by Japan Society for the Promotion of
Science KAKENHI [20J10925] and Fuji Foundation for Protein
Research.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We appreciate Kaheitofu Co., Ltd., Niigata, Japan, for their profes­
sional suggestions.
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