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 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: zhk@affrc.go.jp (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 ARTICLE IN PRESS 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 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 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 All measurements were done in triplicate 2.5 Spectrofluorimetry 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 in an incubator (4 C) Soy milks were obtained from the slurry after centrifugation (1200  g, at 4 C) for 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 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  10À3 mol/l ANS phosphate-buffered solution The fluorescence intensity was measured after standing for h at room temperature using a Hitachi-850 spectrofluorimeter (excitation ARTICLE IN PRESS 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 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 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 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 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 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 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 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) ARTICLE IN PRESS H Zhang et al / Lebensm.-Wiss u.-Technol 38 (2005) 7–14 10 Table 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 10 10 10 10 10 20 30 10 20 30 Liquid Liquid Liquid Liquid Liquid Liquid, sol Liquid, sol Sol Sol Sol Sol Relative Fluorescence Intensity 50 40 30 20 10 400 0.16 440 460 480 500 520 540 560 580 600 Wavelength (nm) 0.14 Fig 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 0 100 200 300 400 Pressure (MPa) 500 600 Fig Viscosity of soy milk after pressurization at room temperature for 10 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 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 The fluorescence intensity increased with an increase in treatment time; the maximum relative fluorescence intensity value increased significantly when the time reached 15 A longer treatment ARTICLE IN PRESS H Zhang et al / Lebensm.-Wiss u.-Technol 38 (2005) 7–14 70 Relative Fluorescence Intensity 60 50 30 Endothermic Heat flow (mW) 40 11 Peak A 20 Peak B 10 Peak C 25 30 35 40 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 Wavelength (nm) Fig Fluorescence spectra of ANS in soy milks after treated under different time (1) Control; (2) min; (3) 10 min; (4) 15 min; (5) 20 min; (6) 25 min; (7) 30 (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 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 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 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 ARTICLE IN PRESS 12 H Zhang et al / Lebensm.-Wiss u.-Technol 38 (2005) 7–14 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 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 and 6) disappeared after treatment at 300 MPa (lanes d, h and m) While bands (Nos and 3) disappeared and band No 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 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 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 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: g/100 g, g/100 g and g/100 g) 60 50 40 30 20 10 0 200 400 600 800 Pressure (MPa) Fig Gel strength of high pressure-induced CaCl2 (0.01 mol/l) tofu gel (pressurization 10 min, room temperature) ARTICLE IN PRESS H Zhang et al / Lebensm.-Wiss u.-Technol 38 (2005) 7–14 Fig 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 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 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 References AACC (2000) Approved methods of the American association of cereal chemists (10th ed.) 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