**2. High pressure processing of soybean**

Due to the great variety of foods obtained from soybeans, different processing methods are required. Traditional methods include two types of processes: (1) fermentation, which uses fungi to produce fermented products; and (2) soaking and grinding of the soybeans to make bean curd and soymilk. During fermentation, protein is digested into peptides and later into amino acids for increasing digestibility of protein by the human body. Soaking and grinding are usually combined with thermal treatment to inactivate biologically active compounds such as trypsin inhibitors, lipoxygenase, and hemagglutinins, while increasing digestibility of proteins. In addition, thermal treatment (continued steaming) helps to diminish the characteristic beany flavor of raw soy products due to the volatilization of monocarbonyl compounds, which results from oxidation of fatty acids by the enzyme lipoxygenase. However, excessive heating may destroy certain amino acids that are sensitive to heat such as lysine, with losses of possibly more than 50% (Estrada-Giron et al., 2005).

 Until recently, little research was being done on the effects of HHP on soybean grains and their sub-products. This is because of the recent interest in the use of HHP as a potential technology to improve the quality of cereals and textured products. These studies include reduction in the microbial population of soymilk curd, commonly known as tofu, to obtain a product with longer shelf life and to avoid secondary contamination. The solubilization of protein from whole soybean grains subjected to different treatments of pressure, time, and temperature was also reported. Additional information is available in a more extensive context about the effects of this technology on the inactivation of pure soybean lipoxygenase and lipoxygenase from some legumes (Estrada-Giron et al., 2005).

#### **2.1 Microbial inactivation**

The effects of high hydrostatic pressure on microbial inactivation depend on several factors such as type of microorganism, extent and duration of the high pressure treatment, temperature, and composition of suspension media or food. Therefore, suitable pressure treatment should be applied taking into account these factors to assure microbial inactivation of pathogenic, spoilage, and vegetative cells present in foods. Prestamo et al. (2000) reported that the microbial population of tofu pressurized at 400 MPa and 5 °C for 5, 30, and 45 min decreases from an initial microorganism count of 5.54104 cfu/g to 0.31, 1.56, or 2.38 log units, respectively. Prestamo et al. (2000) also postulated that the effectiveness of HHP treatment to reduce microbial population at 400 MPa largely depends on the exposure time (Fig. 1). In the same study, after HHP treatment of tofu, psycrotrophs were reduced 2 log units from an initial population of 1103 cfu/g. Mesophilic microorganisms were reduced 1 log unit from an initial number of 1.6103 cfu/g, whereas yeast and molds decreased from an initial population of 2.64103 cfu/g to 1102 cfu/g. Other microorganisms such as *Pseudomonadaceae*, *Salmonella*, and Gram-negative bacteria (confirmed before HHP treatment) were not detected after HHP treatment of tofu. *Yersenia enterocolitica* and *Listeria monocytogenes*, which are more resistant to high pressure, were not found before and after HHP treatment. *Hafnia halvei* and *Bacillus cereus* remained active after high pressure treatment of tofu.

In addition to temperature and the extent and duration of high pressure treatment, a factor that significantly influences the effectiveness of HHP treatment on the inactivation and consequently the reduction in microbial population is the medium composition in which microorganisms are dispersed.

Fig. 1. Viable aerobic mesophilic population in tofu after treatment at 400 MPa and 5 °C for 5, 30, and 45 min (from Prestamo et al., 2000).

Food constituents such as sucrose, fructose, glucose, and salts affect the baro-resistance of microorganisms present in food (Oxen & Knorr, 1993). This effect is often observed since food constituents appear to protect microorganisms from the effects of high pressure. Therefore, a non-nutritive solution can reduce the microorganism's baro-tolerance. The presence of microorganisms such as *Hafnia halvei* and *Bacillus cereus* that remained active after HHP treatment could explain the baro-protective effect that food components exert over the extent on microbial reduction (Prestamo et al., 2000).

#### **2.2 Proteins**

68 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

Due to the great variety of foods obtained from soybeans, different processing methods are required. Traditional methods include two types of processes: (1) fermentation, which uses fungi to produce fermented products; and (2) soaking and grinding of the soybeans to make bean curd and soymilk. During fermentation, protein is digested into peptides and later into amino acids for increasing digestibility of protein by the human body. Soaking and grinding are usually combined with thermal treatment to inactivate biologically active compounds such as trypsin inhibitors, lipoxygenase, and hemagglutinins, while increasing digestibility of proteins. In addition, thermal treatment (continued steaming) helps to diminish the characteristic beany flavor of raw soy products due to the volatilization of monocarbonyl compounds, which results from oxidation of fatty acids by the enzyme lipoxygenase. However, excessive heating may destroy certain amino acids that are sensitive to heat such

 Until recently, little research was being done on the effects of HHP on soybean grains and their sub-products. This is because of the recent interest in the use of HHP as a potential technology to improve the quality of cereals and textured products. These studies include reduction in the microbial population of soymilk curd, commonly known as tofu, to obtain a product with longer shelf life and to avoid secondary contamination. The solubilization of protein from whole soybean grains subjected to different treatments of pressure, time, and temperature was also reported. Additional information is available in a more extensive context about the effects of this technology on the inactivation of pure soybean lipoxygenase

The effects of high hydrostatic pressure on microbial inactivation depend on several factors such as type of microorganism, extent and duration of the high pressure treatment, temperature, and composition of suspension media or food. Therefore, suitable pressure treatment should be applied taking into account these factors to assure microbial inactivation of pathogenic, spoilage, and vegetative cells present in foods. Prestamo et al. (2000) reported that the microbial population of tofu pressurized at 400 MPa and 5 °C for 5, 30, and 45 min decreases from an initial microorganism count of 5.54104 cfu/g to 0.31, 1.56, or 2.38 log units, respectively. Prestamo et al. (2000) also postulated that the effectiveness of HHP treatment to reduce microbial population at 400 MPa largely depends on the exposure time (Fig. 1). In the same study, after HHP treatment of tofu, psycrotrophs were reduced 2 log units from an initial population of 1103 cfu/g. Mesophilic microorganisms were reduced 1 log unit from an initial number of 1.6103 cfu/g, whereas yeast and molds decreased from an initial population of 2.64103 cfu/g to 1102 cfu/g. Other microorganisms such as *Pseudomonadaceae*, *Salmonella*, and Gram-negative bacteria (confirmed before HHP treatment) were not detected after HHP treatment of tofu. *Yersenia enterocolitica* and *Listeria monocytogenes*, which are more resistant to high pressure, were not found before and after HHP treatment. *Hafnia halvei* and *Bacillus cereus* remained active after high pressure

In addition to temperature and the extent and duration of high pressure treatment, a factor that significantly influences the effectiveness of HHP treatment on the inactivation and consequently the reduction in microbial population is the medium composition in which

as lysine, with losses of possibly more than 50% (Estrada-Giron et al., 2005).

and lipoxygenase from some legumes (Estrada-Giron et al., 2005).

**2.1 Microbial inactivation** 

treatment of tofu.

microorganisms are dispersed.

**2. High pressure processing of soybean** 

Unlike allergenic proteins in cereals such as rice, soybeans contain a large number of proteins with important functional properties (Wolf & Cowan, 1975). Eighty percent of the proteins in soybeans are glycinin and β- and γ-conglycinin, which are globular salt-soluble proteins. On the basis of their sedimentation constants at pH 7.6 and ionic strength buffer of 0.5, the globulins are characterized as 11S or glycinin and 7S or β- and γ-conglycinin (Fukushima, 1991), with other less abundant globulins including 2S or α-conglycinin, 9S globulins, and 15S globulins. Functional properties associated with these kinds of soybean proteins:


Therefore, the method of processing intact soybeans is important since the retention of proteins in the soybean seed is of special interest because of the high-quality vegetable protein, which also contains most of the essential amino acids (Steinke et al., 1992). When soybeans are immersed in hot water at 50–60 °C for 1 h, a considerable amount of protein solubilized from the soybean seeds is released to the surrounding water (Asano et al., 1989). Later studies identified these solubilized proteins as 7S globulins, which accounted for about 3% of the total protein in mature soybean seeds (Hirano et al., 1992).

High Pressure Treatments of Soybean and Soybean Products 71

In soybean products, off-flavor development is highly dependent on the action of lipoxygenase since subsequent decomposition of the resulting hydroperoxides yields especially rancid flavor and beany aroma. Nevertheless, lipoxygenase is sensitive to heat and is destroyed at 82 °C when processed for 15 min (Baker & Mustakas, 1972). It is well known that thermal processing methods reduce considerably or completely inactivate unwanted enzyme activity, which limits or largely determines the conditions of storage needed to extend shelf life of food products. Although the behavior of enzymes under the influence of heat has been extensively studied, the effects of HHP treatment on enzyme

P/T treatment (MPa/ºC) Total time (min) Cycling time Activity retention 350/40 40 1×40 0.709 350/40 40 4×10 0.314 400/35 40 4×10 0.324 400/40 40 4×10 0.300 450/40 40 1×40 0.481 475/10 40 1×40 0.652 475/25 60 4×15 0.160 475/30 60 4×15 0.373 500/15 30 1×30 0.367 500/15 30 3×10 0.018 525/25 20 1×20 0.099 525/25 20 2×10 0.110

Table 1. Influence of multi-cycling on the inactivation of lipoxygenase in Tris–HCl at pH 9 Thermal inactivation of enzymes at atmospheric pressure occurs in the temperature range 60–70 °C. In contrast, pressure–temperature inactivation occurs in the pressure range 50–650 MPa at temperatures between 10 and 64 °C. Also, depending on the objectives of the research, pressure treatment may be applied in a single cycle or multi-cycles. Multi-cycling is the multiple application of pressure alone or in combination with temperature for the same total treatment time but with various numbers of cycles. Ludikhuyze et al. (1998a) reported the multi-cycling application of pressure to inactivate lipoxygenase. These authors found that in the pressure range 350–525 MPa and thermal treatment at 10–40 °C, the use of multi-cycles exerted an additional inactivation effect on lipoxygenase, compared to single cycle treatments (Table 1). Furthermore, temperature treatments at 10 °C caused an enhanced inactivation of lipoxygenase because the temperature inside the vessel dropped

In crude green bean extract, irreversible lipoxygenase inactivation was reported in the temperature range 55–70 °C at ambient pressure, whereas at room temperature, pressures around 500 MPa were required to inactivate lipoxygenase. High pressure treatment at 200 MPa and 50 °C resulted in 10% inactivation, while at least 50%, lipoxygenase inactivation occurred at pressures greater than 500 MPa and thermal treatment between 10 and 30 °C (Indrawati et al., 1999). The effect of HHP on enzyme inactivation in food systems is different compared to its effects on pure components dissolved in buffer solutions. As an example, solutions of commercial soybean lipoxygenase type I (100 mg/ml) dissolved in 0.2 M citrate-phosphate (pH range of 4.0–9.0) and 0.2 M Tris buffer (pH range of 6.0–9.0) were

inactivation are not clearly understood.

Adapted from Ludikhuyze et al. (1998a).

below zero upon depressurization.

In soybean seeds immersed in distilled water and treated at 300 MPa and 20 °C for 0–180 min, the solubilized proteins accounted for 0.5–2.5% of the total seed proteins. No apparent changes in shape, color, and size between treated and untreated soybeans were reported. The solubility of protein in surrounding water increased with increasing pressure, reaching a maximum value at 400 MPa (Fig. 2). Similar to what occurs when heat treatment is applied, SDS-PAGE patterns of high pressure treated seeds exhibited solubilization of 7S globulin, consisting of 27 and 16 KDa bands with staining intensity increasing as pressure increased to 400 MPa. The increase in staining intensity is indicative of the amount of release protein; thus, at higher intensity larger amounts of protein are released. At 700 MPa, 11S glycinin and 2S β-conglycinin also increased their staining intensity (Omi et al., 1996).

Fig. 2. Effect of high-pressure treatment on the release of proteins from soybean seeds. Water-immersed soybean seeds were pressurized at (A) 0-700 MPa and 20 °C for 25 min and at (B) 300 MPa and 20 °C for 0-180 min (From Omi et al., 1996).

#### **2.3 Enzymes**

At present, research regarding the inactivation of enzymes in intact grains and their subproducts is scarce. However, it is well known that high pressure modifies the activities of a whole range of unwanted food enzymes, which can result in a reduction in food quality or cause spoilage during storage. Recent investigations have reported the effect of combined pressure and temperature on soybean lipoxygenase. Lipoxygenase is one of the main antinutritional factors in soybean processing, which is also known to occur in other legume seeds, some cereal grains, and oil seeds. At least three types of lipoxygenase are well identified in soybeans as lipoxygenase I, II, and III. These enzymes catalyze the oxidation of unsaturated fatty acids in the presence of molecular oxygen. The presence of lipoxygenase can have detrimental effects on foods, for example:


In soybean seeds immersed in distilled water and treated at 300 MPa and 20 °C for 0–180 min, the solubilized proteins accounted for 0.5–2.5% of the total seed proteins. No apparent changes in shape, color, and size between treated and untreated soybeans were reported. The solubility of protein in surrounding water increased with increasing pressure, reaching a maximum value at 400 MPa (Fig. 2). Similar to what occurs when heat treatment is applied, SDS-PAGE patterns of high pressure treated seeds exhibited solubilization of 7S globulin, consisting of 27 and 16 KDa bands with staining intensity increasing as pressure increased to 400 MPa. The increase in staining intensity is indicative of the amount of release protein; thus, at higher intensity larger amounts of protein are released. At 700 MPa, 11S glycinin and 2S β-conglycinin also increased their staining intensity (Omi et al., 1996).

Fig. 2. Effect of high-pressure treatment on the release of proteins from soybean seeds. Water-immersed soybean seeds were pressurized at (A) 0-700 MPa and 20 °C for 25 min and

At present, research regarding the inactivation of enzymes in intact grains and their subproducts is scarce. However, it is well known that high pressure modifies the activities of a whole range of unwanted food enzymes, which can result in a reduction in food quality or cause spoilage during storage. Recent investigations have reported the effect of combined pressure and temperature on soybean lipoxygenase. Lipoxygenase is one of the main antinutritional factors in soybean processing, which is also known to occur in other legume seeds, some cereal grains, and oil seeds. At least three types of lipoxygenase are well identified in soybeans as lipoxygenase I, II, and III. These enzymes catalyze the oxidation of unsaturated fatty acids in the presence of molecular oxygen. The presence of lipoxygenase

1. Degradation of the essential fatty acids linoleic, linolenic, and arachidonic acid to yield

2. Degradation of formed hydroperoxides, resulting in the formation of volatile compounds such as aldehydes, ketones, and alcohols, which cause the development of

3. Production of free radicals that can damage other compounds, including vitamins and

at (B) 300 MPa and 20 °C for 0-180 min (From Omi et al., 1996).

can have detrimental effects on foods, for example:

fatty acid hydroperoxides.

proteins (Whitaker, 1972).

off-flavors.

**2.3 Enzymes** 

In soybean products, off-flavor development is highly dependent on the action of lipoxygenase since subsequent decomposition of the resulting hydroperoxides yields especially rancid flavor and beany aroma. Nevertheless, lipoxygenase is sensitive to heat and is destroyed at 82 °C when processed for 15 min (Baker & Mustakas, 1972). It is well known that thermal processing methods reduce considerably or completely inactivate unwanted enzyme activity, which limits or largely determines the conditions of storage needed to extend shelf life of food products. Although the behavior of enzymes under the influence of heat has been extensively studied, the effects of HHP treatment on enzyme inactivation are not clearly understood.


Adapted from Ludikhuyze et al. (1998a).

Table 1. Influence of multi-cycling on the inactivation of lipoxygenase in Tris–HCl at pH 9

Thermal inactivation of enzymes at atmospheric pressure occurs in the temperature range 60–70 °C. In contrast, pressure–temperature inactivation occurs in the pressure range 50–650 MPa at temperatures between 10 and 64 °C. Also, depending on the objectives of the research, pressure treatment may be applied in a single cycle or multi-cycles. Multi-cycling is the multiple application of pressure alone or in combination with temperature for the same total treatment time but with various numbers of cycles. Ludikhuyze et al. (1998a) reported the multi-cycling application of pressure to inactivate lipoxygenase. These authors found that in the pressure range 350–525 MPa and thermal treatment at 10–40 °C, the use of multi-cycles exerted an additional inactivation effect on lipoxygenase, compared to single cycle treatments (Table 1). Furthermore, temperature treatments at 10 °C caused an enhanced inactivation of lipoxygenase because the temperature inside the vessel dropped below zero upon depressurization.

In crude green bean extract, irreversible lipoxygenase inactivation was reported in the temperature range 55–70 °C at ambient pressure, whereas at room temperature, pressures around 500 MPa were required to inactivate lipoxygenase. High pressure treatment at 200 MPa and 50 °C resulted in 10% inactivation, while at least 50%, lipoxygenase inactivation occurred at pressures greater than 500 MPa and thermal treatment between 10 and 30 °C (Indrawati et al., 1999). The effect of HHP on enzyme inactivation in food systems is different compared to its effects on pure components dissolved in buffer solutions. As an example, solutions of commercial soybean lipoxygenase type I (100 mg/ml) dissolved in 0.2 M citrate-phosphate (pH range of 4.0–9.0) and 0.2 M Tris buffer (pH range of 6.0–9.0) were

High Pressure Treatments of Soybean and Soybean Products 73

Tofu froze during pressurization at 100 or 686 MPa; conversely, tofu did not freeze between 200 and 600 MPa and -20 °C, but it froze rapidly when the pressure was released. It was found that tofu frozen at 0.1, 100 or 686 MPa had larger ice crystals and was firmer (less like unfrozen tofu) than tofu frozen at 200–600 MPa. In the sensory evaluation, results showed that mouth feel (texture. of tofu frozen at 400 MPa) was more like the control when 2.5%

The micro structure of the tofu gel network high pressure frozen at 686 MPa was compared with untreated tofu (Fig. 3). Tofu (0% trehalose) frozen at 0.1–500 MPa maintained a comparatively coarse network (data not shown), but tofu gel frozen at 686 MPa was compressed. Compression of the protein gel network might have occurred above 600–686 MPa; however, the gel network in tofu frozen at 686 MPa became coarse with the addition of trehalose. This indicates that trehalose with high-pressure-freezing appears to protect against compression (effects of concentration of protein and coagulants on frozen tofu)

The high pressure inactivation of lipoxygenase in soy milk and crude soybean extract was

T (ºC) Soy milk Crude soybean extract 63 0.55 ± 0.02a 0.68 ± 0.03 r2 = 0.993 r2 = 0.995 65 1.35 ± 0.05 1.54 ± 0.05 r2 = 0.995 r2 = 0.996 67 3.57 ± 0.09 4.31 ± 0.12 r2 = 0.998 r2 = 0.997 69 13.25 ± 0.50 14.43 ± 0.42 r2 = 0.994 r2 = 0.997 71 47.72 ± 4.64 53.40 ± 2.74 r2 = 0.972 r2 = 0.987 Ea (kJ/mol) 538.78 ± 29.04 526.94 ± 29.54

studied in the pressure range 0.1–650 MPa with temperature varying from 5 to 60 °C.

r2 = 0.991 r2 = 0.991 a Standard error. (From Wang et al., 2008).

lipoxygenase in soy milk and in crude soybean extract

Table 2. Estimated inactivation rate constants (10-2 min-1) for the isothermal inactivation of

For both systems, the isobaric–isothermal inactivation of lipoxygenase was irreversible and followed a first-order reaction at all pressure–temperature combinations tested. In the entire pressure–temperature area studied, the lipoxygenase inactivation rate constants increased with increasing pressure at constant temperature for both systems; the rate constants were somewhat smaller in soy milk system than in crude soybean extract. At constant elevated pressure, lipoxygenase exhibited the greatest stability around 20 °C in both systems, indicating that the Arrhenius equation was not valid over the entire temperature range. For both systems, the temperature dependence of the lipoxygenase inactivation rate constants at high temperature decreased with increasing pressure, while the highest sensitivity of the lipoxygenase inactivation rate constants to pressure was observed at about 30 °C. The

trehalose was added (Fuchigami et al., 2002).

**4. High pressure inactivation of soybean lipoxygenase** 

(Fuchigami et al., 2002).

subjected to pressures of 0.1, 200, 400, and 600 MPa for 20 min. Under these conditions, lipoxygenase in citrate-phosphate buffer lost more than 80% of its activity at alkaline pH, whereas it was completely inactivated at acidic conditions and pressure treatment of 400 and 600 MPa (Tangwongchai et al., 2000). In Tris buffer, lipoxygenase activity was significantly inactivated at pH 9.0 and 400 MPa and lost all activity at 600 MPa and all pH values. Similar results were observed by Seyderhelm et al. (1996) who reported that lipoxygenase in Tris buffer pH 7.0 was completely inactivated at 600 MPa and temperatures 45 and 50 °C for 10 min and 5–10 min, respectively.
