high pressure treatment effects on proteins in soy milk

ARTICLE IN PRESS

Lebensm.-Wiss. u.-Technol. 38 (2005) 7–14

High-pressure treatment effects on proteins in soy milk
Hongkang Zhanga,b,*, Lite Lib, Eizo Tatsumic, Seiichiro Isobea
a

Food Processing Laboratory, Japan National Food Research Institute, 2-1-12 Kannondai, Tsukuba Science City, Ibaraki 305-8642, Japan
b
Food College, China Agriculture University, Beijing 100083, China
c
Japan International Research Center for Agricultural Sciences, Tsukuba Science city, Ibaraki 305-8642, Japan
Received 19 December 2003; received in revised form 14 April 2004; accepted 20 April 2004

Abstract
Effects of high-pressure treatment on the modifications of soy protein in soy milk were studied using various analytical
techniques. Blue shifts of lmax could be observed in the fluorescence spectra. Spectrofluorimetry revealed that the soy protein
exhibited more hydrophobic regions after high-pressure treatment. Electrophoretic analysis showed the change of soy protein clearly
and indicated that soy proteins were dissociated by high pressure into subunits, some of which associated to aggregate and became
insoluble. High-pressure denaturation occurred at 300 MPa for b-conglycinin (7S) and at 400 MPa for glycinin (11S) in soy milk.
High pressure-induced tofu gels could be formed that had gel strength and a cross-linked network microstructure. This provided a
new way to process soy milk for making tofu gels.
r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: High pressure; Soy milk; b-conglycinin (7S); Glycinin (11S); Tofu gel

1. Introduction
Soy protein is the most inexpensive source of highnutritional quality protein and therefore is the predominant commercially available vegetable protein in
the world. Food made from soy protein is very popular
and traditional in Asian countries. The United States
Food and Drug Administration authorized the Soy

Protein Health Claim on 26 October 1999 stating that
25 g of soy protein a day may reduce the risk of heart
disease. Soybean foods continue to penetrate rapidly
into western cultures and diets since the market is very
responsive to this health claim (Fukushima, 2001;
Hermansson, 1978).
Soy milk is a popular beverage in Asian countries. It
is a colloidal solution extracted from ground soybeans
and therefore almost all its components (protein, lipid,
and saccharides) of the soy seeds are present in soy milk
*Corresponding author. Food Processing Laboratory, Japan
National Food Research Institute, 2-1-12 Kannondai, Tsukuba
Science City, Ibaraki 3058642, Japan. Tel.: +81-29-838-8029; fax:
+81-29-838-8122.
E-mail address: (H. Zhang).

(Guo, Tomotada, & Masayuki, 1997). Soy milk and its
products are regarded as being nutritious and cholesterol-free health foods with considerable potential for
greater use in the future. Conventional processing
methods of soy milk and its products involve heating.
It is well known that thermal treatments induce
dissociation, denaturation and aggregation of soy
protein. Thermal denaturation and coagulation of soy
protein have been the subjects of numerous papers and
reviews (Kwok and Niranjan, 1995). However, heattreatment has negative effects on their solubility and
water absorption characteristics (Kinsella, 1979), but
mildly heat-treated products produce strong off-flavors,
which is the primary problem for developing soy protein
foods (Fukushima, 2000). Thus, it is important to
develop novel texturized soy foods or a range of new

food formulations through innovative technology.
High pressure denatures proteins, solidifies lipids,
destabilizes biomembranes, and inactivates microorganisms (Cheftel, 1992).This process is considered to be
energy-efficient and safe compared to some conventional processes. The observation in many cases that
pressure treatment does not cause any change in taste
and flavor of food materials is of special interest to the

0023-6438/$30.00 r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2004.04.007


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H. Zhang et al. / Lebensm.-Wiss. u.-Technol. 38 (2005) 7–14

food industry. A range of products in Japan, such as
jams and yoghurts, have been available on the market
for several years; the most recent innovations are variety
of rice products. In France, pressure-treated fruit juices
have been available for some time, and high pressureprocessed guacamole is a big seller in the United States
(Palou et al., 2000).
Molina, Defaye, and Ledward (2002) investigated the
functional and textural properties of pressure and heatinduced gels formed in soy protein isolates and its two
main fractions (7S and 11S). They reported and found that
high pressure-induced gels yielded significantly lower
values of adhesiveness and hardness when compared to
heat-treated gels. Zhang, Li, Tatsumi, and Kotwal (2003)
studied the influence of high pressure on conformational
changes of soybean glycinin. They found conformation of

glycinin could be changed after high-pressure treatment.
Many of these researches focused on purified proteins
systems. Researches about high-pressure effects on the
proteins in soy milk were little. The exact denaturation
pressures for soy protein fractions in soy milk are still
not confirmed. Therefore, the objective of this study was
to examine the effect of high-pressure treatment on the
modifications of soy proteins in soy milk, using various
analytical techniques, such as spectrofluorimetry, differential scanning calorimetry (DSC) and electrophoresis,
to explore an alternative processing for novel textural
soy foods.

tions of pressure and time at room temperature in a
hydraulic-type cell (Yamamoto Suiatsu Co., Ltd, Osaka,
Japan: Model S7K-4-15; maximum pressure 700 MPa).
The cell had an inner capacity of 40 mm  200 mm
(diameter  height) and a water jacket for temperature
control. Filtered water was used as the pressure medium.
The pressure build-up time was less than 1 min and the
depressurization time were less than 30 s. Temperature
changes in the pressure transferring medium were
measured by a K-type thermocouple (nickel–copper).
The change of temperature caused by adiabatic compression and expansion was found to be within 74 C of
the starting temperature when the pressure increased to
300 MPa.
2.4. pH value, density, viscosity and phase
The pH value and density of the soy milks were
measured by a pH meter (Model F-23, HORIBA Ltd.,
Japan) and a densitometer (Model DA-110, Kyoto
Denshi Seizou Ltd., Japan), respectively, after high

pressure treatment. The viscosity was measured with a
No. 2 spindle (+21 mm) of the viscometer (Model
NDJ-8S, Shanghai Balance Instrument Factory) at
60 rpm for a 175 ml sample in a 200 ml beaker at room
temperature. Each measurement time was preset to
5 min. All measurements were done in triplicate.
2.5. Spectrofluorimetry

2. Materials and methods
2.1. Preparation of soy milk
Soybeans of the Proto cultivar (Kefeng No. 6) used in
this study were obtained from the Institute of Genetics
and Developmental Biology, Chinese Academy of
Sciences, harvested in October 1999.
Soybeans were thoroughly washed and soaked overnight at room temperature with 10 times their weight of
distilled water. Soaked soybeans were homogenized with a
homogenizer (PH91, SMT Company) at 10,300 rpm for
5 min in an incubator (4 C). Soy milks were obtained from
the slurry after centrifugation (1200 Â g, at 4 C) for 5 min.
2.2. Proximate analysis
The moisture content was measured by a vacuum
oven method. Crude protein was determined by the
Kjeldahl method using a conversion factor of 6.25
(AACC, 2000).
2.3. High-pressure treatment
The soy milks without air bubbles were sealed in
polyethylene bags and subjected to different combina-

Takagi, Akashi, and Yasumatsu (1979) established a
method to determine the hydrophobic region in soy

globulin using 8-anilino-1-naphthalene sulfonic acid
(ANS). This method was also used to detect a change
of the hydrophobic region of soy protein in soy milk by
Obata and Matsuura (1993). Since the major component
in soy milk aside from soy protein is lipid as mentioned
above, Kajiyama, Isobe, Uemura, and Noguchi (1995)
compared the hydrophobicity change of protein in soy
milk and defatted soy milk to investigate the effects of
lipids on the hydrophobicity change of soy protein.
They found that defatted soy milk exhibited a clearer
change in hydrophobicity than nondefatted soy milk;
although nondefatted soy milk did display some change
in hydrophobicity. The hydrophobic region in the
nondefatted soy milk was determined by this method,
in our experiment, using ANS. The fluorescence
intensity is directly proportional to the range of protein
concentration in soy milk from 0 to 0.05 g/100 g;
therefore each soy milk sample was diluted with
0.1 mol/l phosphate buffer (pH 6.8) to yield a final
concentration of 0.05 g/100 g and 4.5 ml of a diluted
sample was mixed with 0.5 ml of 1.25 Â 10À3 mol/l ANS
phosphate-buffered solution. The fluorescence intensity
was measured after standing for 2 h at room temperature using a Hitachi-850 spectrofluorimeter (excitation


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H. Zhang et al. / Lebensm.-Wiss. u.-Technol. 38 (2005) 7–14

375 nm, slit 2.5 nm; emission 380–580 nm, slit 2.5 nm;
scanning speed 200 nm/min). The hydrophobic regions

in the soy milk were expressed as the fluorescence
intensity with a 0.05 g/100 g protein concentration.
2.6. DSC
A 0.75 g soy milk sample was hermetically sealed into
a sample vessel of a Micro DSC III microcalorimeter
(SETARAM company, France), which was suitable to
investigate the denaturation of protein solution. The
same amount of distilled water was used as a reference
in a reference vessel. Calorimetric measurements were
carried out at a scanning rate of 1 C/min under nitrogen
at 2 bars from 20 C to 105 C.
2.7. Native polyacrylamide gel electrophoresis
Sodium dodecyl sulfate (SDS) breaks the hydrophobic interactions and reducing reagent (e.g. beta-mercaptoethanol) breaks the disulfide bonds among protein
molecules. Therefore, the soluble proteins in samples
were analysed by native polyacrylamide gel electrophoresis (PAGE) according to the method of Laemmli
(1970), but without adding SDS and reducing reagent,
to investigate effects of high pressure on the noncovalent
interactions (i.e. electrostatic interactions, hydrophobic
interactions and hydrogen bonds) and disulfide bonds
among the soy protein subunits. The protein samples for
electrophoresis were prepared by diluting each sample in
sample buffer (400 ml/l glycerol and 0.02 g/100 g bromophenol blue) to yield different final protein concentrations of 1, 2, and 3 g/100 g. The samples were not
heated before application. The separating gel was 7.5 g/
100 g acrylamide; the stacking gel was 2.5 g/100 g
acrylamide. The gels were stained with Coomasie
brilliant blue (R-250) (Neuhoff, Arold, Taube, &
Ehrhardt, 1988) and destained with a 75 ml/l acetic acid
/50 ml/l methanol solution.
2.8. High pressure-induced tofu gel
Tofu is a traditional soy food for Asian, and is a

protein gel-like product. The procedure for making tofu
generally includes soaking, grinding the soybeans in
water, filtering, boiling, coagulation and pressing.
Numerous works have been done on these subjects.
Various types of coagulants such as glucono-deltalactone (GDL), calcium chloride (CaCl2) and magnesium chloride have been used with different concentrations (Deman & Gupta, 1986). CaCl2 with low
concentration was chosen here as the coagulant to
investigate the denaturation and gel formation ability of
soy protein in soy milk by high pressure. The soy milk
was uniformly mixed with the coagulant, CaCl2 until the
concentration was 10 mM; the mixture was then

9

transferred into a plastic tube (+30 mm  50 mm) and
sealed without any air bubbles. Prepared samples were
subjected to high pressure for gelling.
2.9. Gel strength
The gel strength of high pressure-induced tofu gel was
measured with a Rheometer using a 3 mm diameter
plunger at 60 mm/min. The maximum broken gel
strength (s) was calculated as the following function:
F is the maximum broken force (g) of the gel; r (mm) is
the radius of the plunger.
s¼

F ðgÞ Â 9:81
ðkPaÞ:
p  r2

2.10. Scanning electron microscopy (SEM)

Pressurized gels left for 24 h were cut with a razor
blade and soaked in potassium phosphate buffer
(0.05 mol/l, pH 7.0) containing 40 ml/l glutaraldehyde
at 4 C for 16 h. The gel pieces were rinsed with
phosphate buffer, dehydrated in a graded series of
ethanol/water solutions from 500 to 1000 ml/l with a
residence time of at least 2 h in each solution, and then
critical-point dried with carbon dioxide. Dried samples
were fractured, mounted on aluminum stubs with silver
( by a sputtering
lacquer, coated with gold (80–100 A)
apparatus and examined using SEM (Hitachi S-2360N)
as described by Dumay, Laligant, Zasypkin, and Cheftel
(1999).
2.11. Statistical analysis
All data were the average of three-independent trials
except the electrophoretic work. The results were
reported as mean values with a standard deviation.
ANOVA and Duncan’s multiple tests were used to
determine whether or not a significant difference existed
in the means. All tests of significance were at the 0.05
significance level.

3. Results and discussions
3.1. pH value, density, viscosity and phase
The crude protein content of soy milk was 4.4 g/100 g.
Table 1 shows phase changes of pressurized soy milk.
Pressure treated beyond 500 MPa for 30 min, the
products were transformed to sol but below this
pressurized level the products displayed in liquid phase.

There were no significant differences in the pH values
and densities of soy milk after high-pressure treatments
(data not shown here).


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Table 1
High-pressure effect on the phase changes of soymilk

70

Pressure (MPa)

Time (min)

Phase

60

Control
100
200
300
400
500
500

500
600
600
600

0
10
10
10
10
10
20
30
10
20
30

Liquid
Liquid
Liquid
Liquid
Liquid
Liquid, sol
Liquid, sol
Sol
Sol
Sol
Sol

5

Relative Fluorescence Intensity

4

6
50

7

40

3
30

20

2

10

1
0
400

0.16

440

460

480

500

520

540

560

580

600

Wavelength (nm)

0.14

Fig. 2. Fluorescence spectra of ANS in various high pressure-treated
soy milks. (1) Control; (2) 100 MPa; (3) 200 MPa; (4) 300 MPa; (5)
400 MPa; (6) 500 MPa; (7) 600 MPa (pressurization 10 min, room
temperature).

0.12
Viscosity (Pa.s)

420

0.1

0.08
0.06
0.04
0.02
0
0

100

200
300
400
Pressure (MPa)

500

600

Fig. 1. Viscosity of soy milk after pressurization at room temperature
for 10 min.

The viscosity of soy milk was found to increase with
increasing pressure treatments (Fig. 1). The same
phenomenon happened with heated samples. Soy
protein dispersion increases in viscosity after heating
and undergoes an irreversible change to the progel state
(Yamauchi, Yamagishi, & Iwabuchi, 1991). The changes
in viscosity and phase of soy milk after high-pressure
treatments indicated that the soy proteins in soy milk
had been modified to form colloidal phase.

3.2. Hydrophobicity
Hydrophobic interactions play substantial role in
stabilization of the tertiary structure and in protein–
protein interactions. Denaturation of protein by heating
increases the surface hydrophobicity (Sorgentini,
Wagner, & Anon, 1995). Studies carried out with
purified soy protein fractions indicated that heating
causes an increase in surface hydrophobicity for 7S and
11S globulins: particularly significant differences were
observed at their respective denaturation temperatures,
about 70 C (Kato, Osako, Matsudomi, & Kobayashi,

1983) and 85 C (Belyakova, Semenova, & Antipova,
1999). Hydrophobic interaction can also be affected by
high pressure (Ohmiya, Kajino, Shimizu, & Gekko,
1989). The increase in surface hydrophobicity after highpressure treatment was also observed for both purified
11S and 7S globulins (Galazka, Dickinson, & Ledward,
1999; Pedrosa & Ferreira, 1994; Zhang et al., 2003).
ANS was used here as a fluorescence probe to detect the
change of hydrophobicity of soy protein in soy milk.
Fig. 2 illustrates the fluorescence intensity of ANS in
various high pressure-treated soy milks. The intensity of
fluorescence increased sharply with increasing pressure
up to 300 MPa, but higher pressure (500 MPa) yielded a
decrease in fluorescence. Blue shifts of lmax with the
increasing pressure could be observed at the same time.
These results indicated that 300 MPa is a transition
pressure to some protein fractions. These fractions were
completely denatured under this pressure as more
hydrophobic regions were exposed, resulting in a

sharply increase of the fluorescence intensity. The
decrease of fluorescence intensity could be due to the
lower number of hydrophobic groups binding to the
ANS because of intermolecular interactions (Hayakawa,
Kajihara, Morikawa, Oda, & Fujio, 1992), or the
pressure treated sample has refolded into a slightly
different conformation, obscuring some of the hydrophobic groups.
The fluorescence spectra of ANS in soy milks after
treatment at 300 MPa for different durations are
presented in Fig. 3. The fluorescence intensity increased
with an increase in treatment time; the maximum
relative fluorescence intensity value increased significantly when the time reached 15 min. A longer treatment


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70

6
5

Relative Fluorescence Intensity

60

7

50

3

30

2

Endothermic Heat flow (mW)

6

4

40

11

5

4
3

2

Peak A

20

Peak B
1

10

Peak C

1

0

25 30 35 40

360 380 400 420 440 460 480 500 520 540 560 580 600 620 640

Wavelength (nm)
Fig. 3. Fluorescence spectra of ANS in soy milks after treated under
different time. (1) Control; (2) 5 min; (3) 10 min; (4) 15 min; (5) 20 min;
(6) 25 min; (7) 30 min (pressure 300 MPa, room temperature).

time led to an insignificant increase of that value. The
relative fluorescence intensity decreased when the
treated time reached 30 min. The degree of denaturation
depends on the treatment time under certain pressurizations.

3.3. Analysis of DSC
DSC has been used extensively to study the thermodynamics and kinetic properties of protein denaturation,
both in solution and solid states (Sessa, 1993).
Thermal studies of soy proteins using DSC showed
thermal transitions occurring at around 70 C and 90 C
for 7S and 11S globulins (German, Damodaran, &
Kinsella, 1982). DSC scanning can be used to follow the
degree of denaturation by high pressure since completely
denatured proteins will have no endothermic peaks

during such scanning.
Fig. 4 shows DSC curves of soy milk protein after
treatments under different pressure conditions. The
control sample (No.1) displays three endothermic peaks,
peaks A–C, with peak temperature of 58 C, 71 C, and
92 C, respectively, peaks B and C were more discernible. The soy proteins in soy milk are composed of three
major components, 2S, 7S, and 11S, accounting for
about 8–22 g/100 g, 35 g/100 g, and 31–52 g/100 g of the
total proteins, respectively (Yamauchi et al., 1991;
Utsumi, Gidamis, Kanamori, Kang, & Kito, 1993). It
can be assumed that peaks B and C are the endothermic
peaks of 7S and 11S globulins, respectively . Peak A is
probable the endothermic peak of the 2S globulin. These
discernible peaks are attributable to a combination of
endothermic reactions, such as rupture of hydrogen

45 50 55 60 65 70 75 80

85 90 95 100 105

Temperature (˚C)

Fig. 4. DSC curves of soy milk protein with different pressure
treatments. (1) Control; (2) 100 MPa; (3) 200 MPa; (4) 300 MPa; (5)
400 MPa; (6) 500 MPa (heating rate: 1 C/min, protein content: 4.4 g/
100 g, pressurization 10 min, room temperature).

bonds and formation of hydrophobic interactions
during DSC scanning (Molina et al., 2002).
All discernible peaks became smaller with an increase

of pressure. Peaks A and B disappeared beyond pressure
treated at 200 and 300 Mpa, respectively, all endothermic peaks disappeared at pressure 400 MPa or higher.
The absence of all endothermic peaks in the DSC curves
of the pressure-treated samples suggests complete
denaturation of the proteins in soy milk after highpressure treatments.
The 7S fraction was denatured after treatment at
300 MPa; this agrees with the hydrophobicity data,
which exhibited a significant improvement after treatment at 300 MPa. The 7S fraction was very sensitive to
both pressure and heat. This is in good agreement with
the fact that the 7S fraction is trimer without any
disulfide bonds, in which the subunits are associated
mainly via hydrophobic interactions (Yamauchi et al.,
1991) and hence are sensitive to pressure. The denaturation of the 11S fraction, indicated by disappearance of
the peak C, seems to occur at 400 MPa. The mechanism
of denaturation may involve in the rupture of disulfide
bonds. Since the 11S globulin has 12 sub-units linked by
a number of disulfide bonds, it is possible for the
disulfide bonds to be reduced if the pressure is
sufficiently high, as in this case of 400 MPa or higher,
leading to denaturation. Kajiyam et al. (1995) reported
the creation of free new sulfhydryl residues resulting
from the reduction of the disulfide bonds after high
pressure-treated soy proteins. This can be concluded
that the different soy protein fractions in soy milk have
different levels of denaturated pressure. High-pressure
denaturation for 7S and 11S globulins in soy milk
occurred at 300 and 400 Mpa, respectively.


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3.4. Electrophoretic analysis
The native PAGE was applied without adding SDS
and reducing reagent. The samples were not heated
before application. The 7S and 11S bands could not be
distinguished in the native PAGE patterns, unlike SDSPAGE. Electrophoresis under these conditions would
provide information regarding the relative charge for
molecules with the same size and shape or regarding the
relative size of molecules with the same charge. Therefore, the native PAGE patterns can detect the pressure
affects on the change of soy protein subunits without
interference from SDS, reducing reagent or heat
treatment. Fig. 5 depicts the native PAGE patterns of
high pressure-treated protein in soy milk. It clearly
indicates some changing of the bands. The bands near
the middle area (Nos. 5 and 6) disappeared after
treatment at 300 MPa (lanes d, h and m). While
bands (Nos. 2 and 3) disappeared and band No. 6
appeared again at 400 MPa (lanes c, g, and k) and
500 MPa (lanes b, f, and j). These results indicate
that the soy proteins were denatured and dissociated by
high pressure into subunits, some of which may
associate to aggregate and become insoluble. These
results are also in accord with the hydrophobicity and
DSC analysis.
3.5. High pressure-induced tofu gel
The conventional process of making tofu is to
denature the soy protein by heating, and then to

coagulate the protein by coagulants and heating
(Yamauchi et al., 1991). However, the nature of high
pressure-induced gels is very different from those
induced by heat, since heat primarily affects hydrogen
bonded networks. While pressure more effectively

disrupts some type of hydrophobic and electrostatic
interactions.
The ability to form gel network structures by high
pressure was demonstrated first by Bridgman (1914),
who coagulated liquid egg white at 600 MPa without
additional heat supply to the pressure vessel. It was
found that a minimum pressure of 300 MPa with
holding time for 10–30 min is necessary to induce highpressure set soy protein gels (Matsumoto & Hayashi,
1990; Okamoto, Kawamura, & Hayashi, 1990). However, most of the works focused on the high pressureinduced gelation of purified protein with high concentrations. The gelation of low concentrations of protein
combined with coagulant under high pressure has been
seldom reported.
Fig. 6 illustrates the gel strength of high pressureinduced tofu gel with a coagulant (CaCl2). The soy milk
could not form a gel at less than 300 MPa. Gel was
formed with pressures up to 400 MPa, but these gels
displayed very little strength. Higher pressures formed
gels with greater strength.
Saio (1981) reviewed the microstructure of heatinduced tofu which exhibited a clear honeycomb-like
structure. The micrograph illustrates the cross-linked
network of high pressure-induced gel (Fig. 7). Gelation
of tofu may be formed by soy protein denaturation,
caused primarily by high pressure; coagulation is also
promoted by cations. The hydrophobic regions of the
native protein molecules are exposed to the solvent by
high-pressure treatment. As the denatured soy protein is

negatively charged (Kohyama & Nishinari, 1995), the
protons produced by calcium ions neutralize the net
charge of the protein. Thus, the hydrophobic interaction
of the neutralized soy proteins becomes more dominant
and induces aggregation to form a cross-linked network.
Some other interactions such as the oxidation of
sulfhydryl groups are also involved (Apichartsrangkoon, 2003). Preste´mo, Lesmes, Otero, and Arroyo
(2000) found that high-pressure treatment of tofu
reduced the microbial population leading to a safer

Gel Strength (KPa)

70

Fig. 5. Native-PAGE patterns of high pressure-treated protein in
soymilk. (a) Control (2 g/100 g); (b) 500 MPa (2 g/100 g); (c) 400 MPa
(2 g/100 g); (d) 300 MPa (2 g/100 g); (e) control (3 g/100 g); (f) 500 MPa
(3 g/100 g); (g) 400 MPa (3 g/100 g ); (h) 300 MPa (3 g/100 g); (i) control
(1 g/100 g); (j) 500 MPa (1g/100 g); (k) 400 MPa (1 g/100 g); (m)
300 MPa (1 g/100 g ) (pressurization 10 min, room temperature, protein
content: 1 g/100 g, 2 g/100 g and 3 g/100 g).

60
50
40
30
20
10
0
0

200

400

600

800

Pressure (MPa)
Fig. 6. Gel strength of high pressure-induced CaCl2 (0.01 mol/l) tofu
gel (pressurization 10 min, room temperature).


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H. Zhang et al. / Lebensm.-Wiss. u.-Technol. 38 (2005) 7–14

Fig. 7. SEM picture of high pressure-induced tofu gel (CaCl2
(0.01 mol/l) 500 MPa, 10 min).

product acceptable to consumers. High pressure inactivates microorganisms. This study indicated the potential
to provide a new way to process soy milk for making
tofu gels.

4. Conclusions
Our investigations revealed that high-pressure treatment can increase the viscosity of soy milk. The phase of
soy milk changed from liquid to sol after high-pressure
treatment at 500 MPa for 30 min. Blue shifts of lmax of
fluorescence intensity with the increasing pressure could
be observed in the fluorescence spectra. Viscosity,

spectrofluorimetry and DSC analysis revealed that high
pressure could denature soy protein completely and
exposed hydrophobic regions. Denaturation occurred at
300 and 400 MPa for 7S and 11S globulins in soy milk,
respectively. Native-PAGE patterns show the highpressure effects on the change of soy protein clearly.
These indicated that soy proteins were dissociated by
high pressure into subunits, some of which aggregated
and became insoluble. High pressure-induced tofu gels
were formed with pressure and coagulant; these gels had
strength and a cross-linked network. This investigation
indicated the potential to provide a new way to process
soy milk for making tofu gels.

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