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


The high pressure inactivation of lipoxygenase in soy milk and crude soybean extract was studied in the pressure range 0.1–650 MPa with temperature varying from 5 to 60 °C.

Table 2. Estimated inactivation rate constants (10-2 min-1) for the isothermal inactivation of lipoxygenase in soy milk and in crude soybean extract

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

High Pressure Treatments of Soybean and Soybean Products 75

lipoxygenase in green bean juice could be described by a two-fraction first-order inactivation model, referring to the existence of two fractions (isozymes) with different

 Van Loey et al. (1999) studies soybean lipoxygenase inactivation [0.4 mg/mL in Tris-HCl buffer (0.01 M, pH 9)] quantitatively under constant pressure (up to 650 MPa) and temperature (-15 to 68 °C) conditions and kinetically characterized by rate constants, activation energies, and activation volumes. The irreversible lipoxygenase inactivation followed a first-order reaction at all pressure-temperature combinations tested. In the entire pressure-temperature area studied, LOX inactivation rate constants increased with increasing pressure at constant temperature. On the contrary, at constant pressure, the inactivation rate constants showed a minimum around 30 °C and could be increased by either a temperature increase or decrease. On the basis of the calculated rate constants at 102 pressure temperature combinations, an iso-rate contour diagram was constructed as a function of pressure and temperature. The pressure-temperature dependence of the LOX inactivation rate constants was described successfully using a modified kinetic model (Van

Penas et al. (2011) reported that sprouts obtained from HHP-treated soybean seeds demonstrated an important reduction in immune-reactivity. Furthermore, they were a good source of proteins and essential amino acids, with Met and Cys corresponding to the limiting amino acids, as indicated by the chemical score (CS), and a high essential amino acid index (EAAI) (Table 4). These results suggested that HHP could constitute an important technological approach for the industrial production of hypoallergenic and nutritive

 The HHP treatment of raw seeds (PRS) produced lower Gly and Cys levels than raw seeds (RS), while no significant differences (*P* ≤ 0.05) were observed for total EAA (Table 4). The germination process resulted in a significant decrease in Glu, Trp and Cys, while no changes were observed in the other amino acids, compared to RS. The total EAA content showed a 4% reduction compared to RS. Pomeranz et al. (1977) found only minor differences in the amino acid composition of germinated and ungerminated soybean, while Mostafa et al. (1987) observed a marked increase in the relative contents of both EAA and NEAA after germination. The levels of sulphur amino acids in germinated soybean seeds remained almost constant, whereas Asp increased compared to raw seeds. Discrepancies between the data reported by other authors and those reported in the present work could be attributed to

The application of HHP treatment to seeds prior to germination (GPS) led to a reduction in Glu and Ala as NEAA and Trp, Met and Cys in EAA in comparison with GS, and also Pro, and Ile compared to PRS. GPS showed similar statistical (*P* ≤ 0.05) values of total EAA content to GS (32 and 34 g/100 g protein, respectively), whilst significant (*P* ≤ 0.05)

 In another study, the effects of HPP on soybean cotyledon as a cellular biological material were investigated from the viewpoints of the cell structure and enzyme reaction system (Ueno et al., 2010). Damage to cell structure was evaluated by measuring dielectric properties using the Cole−Cole arc, the radius of which decreased as pressure level increased. Results suggested that cell structure was damaged by HPP. The distribution of

differences were found compared to RS and PRS (34 g/100 g protein) (Table 4).

thermal stability. However, in their study, this phenomenon was not observed.

**5. Immunoreactivity and nutritional quality of soybean products** 

differences in the germination conditions and seed varieties.

Loey et al., 1999)

soybean sprouts.

pressure–temperature dependence of the lipoxygenase inactivation rate constants was successfully described either using an empirical mathematical model or using a thermodynamic kinetic model for both systems. On a kinetic basis, neither the reaction order of inactivation nor the pressure and temperature sensitivities of the inactivation rate constants were influenced by the different levels of food complexity between the two systems (Wang et al., 2008).

In 63 to 71 °C temperature range, isothermal inactivation of soybean lipoxygenase followed first-order kinetics, allowing inactivation rate constants (*k*) to be determined from plots of

the natural logarithm of relative residual activity, as a function of inactivation time. The estimated *k* values, together with standard errors and regression coefficients, are summarized in Table 2. Over the entire temperature domain studied, lipoxygenase was less thermostable in crude soybean extract than in soy milk and the temperature sensitivity of the rate constants for lipoxygenase inactivation in both systems could be estimated using the Arrhenius relation (Wang et al., 2008). First-order kinetics for thermal inactivation of soybean lipoxygenase has been frequently reported in the literature (Indrawati et al., 1999; Ludikhuyze et al., 1998b). Ludikhuyze et al. (1998a, 1998c) investigated the thermal inactivation kinetics of commercial soybean lipoxygenase in Tris–HCl buffer (0.01 M, pH 9) at two different concentrations (0.4 and 5 mg/ml) over the temperature range 60–70 °C (Table 3).


Table 3. Inactivation rate constants (10-2 min-1) for the isothermal inactivation of commercial soybean lipoxygenase in 0.01 M, pH 9 Tris–HCl buffer

Lipoxygenase in soy milk or in crude soybean extract exhibited a higher thermal stability with the corresponding smaller inactivation rate constants. The two activation energy values derived from the plots of the natural logarithm of inactivation rate constants, as a function of the reciprocal of the absolute temperature were larger, pointing to higher temperature sensitivity of the k values. Likewise, kinetic inactivation of lipoxygenase from many different sources, such as green peas, green beans, potatoes, asparagus, wheat germ, and germinated barley, have also been studied (Bhirud & Sosulski, 1993; Ganthavorn et al., 1991; Guenes & Bayindirli, 1993; Hugues et al., 1994; Indrawati et al., 1999; Park et al., 1988; Svensson & Eriksson, 1974). Indrawati et al. (1999) reported that thermal inactivation of

pressure–temperature dependence of the lipoxygenase inactivation rate constants was successfully described either using an empirical mathematical model or using a thermodynamic kinetic model for both systems. On a kinetic basis, neither the reaction order of inactivation nor the pressure and temperature sensitivities of the inactivation rate constants were influenced by the different levels of food complexity between the two

In 63 to 71 °C temperature range, isothermal inactivation of soybean lipoxygenase followed first-order kinetics, allowing inactivation rate constants (*k*) to be determined from plots of the natural logarithm of relative residual activity, as a function of inactivation time. The estimated *k* values, together with standard errors and regression coefficients, are summarized in Table 2. Over the entire temperature domain studied, lipoxygenase was less thermostable in crude soybean extract than in soy milk and the temperature sensitivity of the rate constants for lipoxygenase inactivation in both systems could be estimated using the Arrhenius relation (Wang et al., 2008). First-order kinetics for thermal inactivation of soybean lipoxygenase has been frequently reported in the literature (Indrawati et al., 1999; Ludikhuyze et al., 1998b). Ludikhuyze et al. (1998a, 1998c) investigated the thermal inactivation kinetics of commercial soybean lipoxygenase in Tris–HCl buffer (0.01 M, pH 9) at two different concentrations (0.4 and 5 mg/ml) over the temperature range 60–70 °C

T (ºC) Lipoxygenase concentration

68 15.5 ± 0.52 r2 = 0.992

commercial soybean lipoxygenase in 0.01 M, pH 9 Tris–HCl buffer

 0.4 mg/mlb 5 mg/mlc 60 2.09 ± 0.13 r2 = 0.978 62 2.02 ± 0.09a 4.86 ± 0.21 r2 = 0.987 r2 = 0.991 64 4.94 ± 0.16 10.8 ± 0.61 r2 = 0.993 r2 = 0.984 66 9.18 ± 0.32 29.1 ± 3.37 r2 = 0.992 r2 = 0.949

Ea (kJ/mol) 319.8 ± 27.3 408.2 ± 14.7

Lipoxygenase in soy milk or in crude soybean extract exhibited a higher thermal stability with the corresponding smaller inactivation rate constants. The two activation energy values derived from the plots of the natural logarithm of inactivation rate constants, as a function of the reciprocal of the absolute temperature were larger, pointing to higher temperature sensitivity of the k values. Likewise, kinetic inactivation of lipoxygenase from many different sources, such as green peas, green beans, potatoes, asparagus, wheat germ, and germinated barley, have also been studied (Bhirud & Sosulski, 1993; Ganthavorn et al., 1991; Guenes & Bayindirli, 1993; Hugues et al., 1994; Indrawati et al., 1999; Park et al., 1988; Svensson & Eriksson, 1974). Indrawati et al. (1999) reported that thermal inactivation of

r2 = 0.986 r2 = 0.997 a Standard error. b Ludikhuyze et al. (1998c). c Ludikhuyze et al. (1998a). (From Wang et al., 2008).

Table 3. Inactivation rate constants (10-2 min-1) for the isothermal inactivation of

systems (Wang et al., 2008).

(Table 3).

lipoxygenase in green bean juice could be described by a two-fraction first-order inactivation model, referring to the existence of two fractions (isozymes) with different thermal stability. However, in their study, this phenomenon was not observed.

 Van Loey et al. (1999) studies soybean lipoxygenase inactivation [0.4 mg/mL in Tris-HCl buffer (0.01 M, pH 9)] quantitatively under constant pressure (up to 650 MPa) and temperature (-15 to 68 °C) conditions and kinetically characterized by rate constants, activation energies, and activation volumes. The irreversible lipoxygenase inactivation followed a first-order reaction at all pressure-temperature combinations tested. In the entire pressure-temperature area studied, LOX inactivation rate constants increased with increasing pressure at constant temperature. On the contrary, at constant pressure, the inactivation rate constants showed a minimum around 30 °C and could be increased by either a temperature increase or decrease. On the basis of the calculated rate constants at 102 pressure temperature combinations, an iso-rate contour diagram was constructed as a function of pressure and temperature. The pressure-temperature dependence of the LOX inactivation rate constants was described successfully using a modified kinetic model (Van Loey et al., 1999)

#### **5. Immunoreactivity and nutritional quality of soybean products**

Penas et al. (2011) reported that sprouts obtained from HHP-treated soybean seeds demonstrated an important reduction in immune-reactivity. Furthermore, they were a good source of proteins and essential amino acids, with Met and Cys corresponding to the limiting amino acids, as indicated by the chemical score (CS), and a high essential amino acid index (EAAI) (Table 4). These results suggested that HHP could constitute an important technological approach for the industrial production of hypoallergenic and nutritive soybean sprouts.

 The HHP treatment of raw seeds (PRS) produced lower Gly and Cys levels than raw seeds (RS), while no significant differences (*P* ≤ 0.05) were observed for total EAA (Table 4). The germination process resulted in a significant decrease in Glu, Trp and Cys, while no changes were observed in the other amino acids, compared to RS. The total EAA content showed a 4% reduction compared to RS. Pomeranz et al. (1977) found only minor differences in the amino acid composition of germinated and ungerminated soybean, while Mostafa et al. (1987) observed a marked increase in the relative contents of both EAA and NEAA after germination. The levels of sulphur amino acids in germinated soybean seeds remained almost constant, whereas Asp increased compared to raw seeds. Discrepancies between the data reported by other authors and those reported in the present work could be attributed to differences in the germination conditions and seed varieties.

The application of HHP treatment to seeds prior to germination (GPS) led to a reduction in Glu and Ala as NEAA and Trp, Met and Cys in EAA in comparison with GS, and also Pro, and Ile compared to PRS. GPS showed similar statistical (*P* ≤ 0.05) values of total EAA content to GS (32 and 34 g/100 g protein, respectively), whilst significant (*P* ≤ 0.05) differences were found compared to RS and PRS (34 g/100 g protein) (Table 4).

 In another study, the effects of HPP on soybean cotyledon as a cellular biological material were investigated from the viewpoints of the cell structure and enzyme reaction system (Ueno et al., 2010). Damage to cell structure was evaluated by measuring dielectric properties using the Cole−Cole arc, the radius of which decreased as pressure level increased. Results suggested that cell structure was damaged by HPP. The distribution of

High Pressure Treatments of Soybean and Soybean Products 77

such as trypsin and chymotrypsin. Both compounds are important animal digestive enzymes for splitting proteins to render dipeptides and tripeptides (Scheider, 1983). However, the specificity of these inhibitors is not necessarily restricted to trypsin and chymotrypsin but also to elastase and serine proteases for which serine constitutes the active site. Nevertheless, the literature reports two main types of soybean PIs, specifically called trypsin inhibitors (TIs). The Kunitz soybean inhibitor, with a molecular weight of 20,000 and two disulfide bridges, exhibits specificity to inhibit trypsin. The Bowman–Birk inhibitor, on the other hand, with a molecular weight ranging from 6000 to 10,000 and seven disulfide

 Residual trypsin was measured in soymilk subjected to selected pressures, temperatures and holding times. Treatment combination at higher pressures and temperatures, for selected holding times resulted in an increased inhibition rate of trypsin inhibitors in soymilk. It was not possible to obtain inactivation rate parameters for treatments at 550 MPa and 80 °C because the data did not fit a first order kinetics model. However, a clear increase of residual trypsin was observed as treatment times increased. Soaking of soybeans in sodium bicarbonate solution, prior to preparation of soymilk, resulted in smaller inhibition rates of trypsin at the working selected pressures, combined with thermal treatment and holding times, than in soybeans soaked in distilled water. The use of sodium bicarbonate, as soaking medium of soybeans, did not result in a significant increase in the percentage of residual trypsin in soymilk treated at 550 MPa and 80 °C for the selected holding times

Ven et al. (2005) also evaluated HPP as an alternative for the inactivation of TIs in soy milk and also studied the effect of HPP on in whole soybeans and soy milk. For complete lipoxygenase inactivation either very high pressures (800 MPa) or a combined temperature/pressure treatment (60 °C/600 MPa) was needed. Pressure inactivation of TIs was possible only in combination with elevated temperatures. For TIs inactivation, three process parameters, temperature, time, and pressure, were optimized using experimental design and response surface methodology. A 90% TIs inactivation with treatment times of <2 min can be reached at temperatures between 77 and 90 °C and pressures between 750

High hydrostatic pressure (HHP) processing is an innovative technology for processing of soybean which is an important food from nutritional point of view. HHP enables the inactivation of pathogenic bacteria at ambient temperatures. It also showed an increase in protein solubility and staining intensity due to the release of more protein after application of HHP. It also favored the inactivation of quality deteriorating enzymes such as lipoxygenase at room temperatures. Treatment of soybean with HHP improved the bioavailability of nutrients such as amino acids and the reduction of immune-reactivity. HHP also favored the activity of proteases, probably by reducing the activity of their inhibitors. It can be inferred that soybean and its products which are valuable food commodities can be effectively processed using this innovative processing technology, however more research needs to be done on HHP optimization and its effects on various physicochemical properties

bonds, exhibits specificity to inhibit chymotrypsin (Liener, 1994).

(Fig. 4).

and 525 MPa.

**7. Conclusions** 

of soybean and different soy-foods.

free amino acids was measured after HPP (200 MPa) of soybean soaked in water or sodium glutamate (Glu) solution. HPP resulted in high accumulation of free amino acids in watersoaked soybean, due to proteolysis. HPP of soybean in Glu solution caused higher accumulation of γ-aminobutyric acid, suggesting that both proteolysis and specific Glu metabolism were accelerated by HPP. They concluded that HPP partially degraded cell structure and accelerated biochemical reactions by allowing enzyme activities to remain. These events were described as "high-pressure induced transformation" of soybean.


statistically significant differences (*P* ≤ 0.05). PRS: HHP-treated soybean seeds; RS: raw soybean seeds; GPS: germinated PRS, HHP-treated soybean seeds; GS: germinated soybean seed. EAA: essential amino acid; CS: chemical score; EAAI: essential amino acid index. B (FAO/WHO/UNU, 1985). (From Penas et al., 2011).

Table 4. Effect of HHP and/or germination on the total protein and amino acids (g/100 g protein) content of soybean seeds and sprouts during germination.A

#### **6. Inactivation of soymilk trypsin inhibitors**

Protease inhibitors (PIs) are generally considered the main anti-nutritional factors in soybeans. Soybean PIs belong to a broad class of proteins that inhibit proteolytic enzymes,

free amino acids was measured after HPP (200 MPa) of soybean soaked in water or sodium glutamate (Glu) solution. HPP resulted in high accumulation of free amino acids in watersoaked soybean, due to proteolysis. HPP of soybean in Glu solution caused higher accumulation of γ-aminobutyric acid, suggesting that both proteolysis and specific Glu metabolism were accelerated by HPP. They concluded that HPP partially degraded cell structure and accelerated biochemical reactions by allowing enzyme activities to remain.

RS PRS GS GPS Whole egg protein

B

These events were described as "high-pressure induced transformation" of soybean.

*Non-essential amino acids*

*Essential amino acids* Leu 6.10a 6.18a 6.52a 5.68a 8.6 Lys 4.32a 4.40a 4.18a 4.13a 7.0 Val 4.19a 4.26a 4.26a 4.11a 6.6 Phe 4.13bc 4.17c 3.70ab 3.90abc 9.3 (Phe+Tyr) Ile 4.12b 4.22b 3.78ab 3.75ab 5.4

Trp 2.26c 2.29c 1.99b 1.71a 1.7 Thr 2.89b 2.52ab 2.50ab 2.32a 4.7 His 2.03a 1.93a 1.96a 1.85a 2.2 Met 1.32b 1.36b 1.32b 1.16a 5.7 (Met+Cys)

EAAI 72.4 70.9 68.5 65.6 A Data are the mean of three independent results. Different superscripts in the same row mean statistically significant differences (*P* ≤ 0.05). PRS: HHP-treated soybean seeds; RS: raw soybean seeds; GPS: germinated PRS, HHP-treated soybean seeds; GS: germinated soybean seed. EAA: essential amino acid; CS: chemical score; EAAI: essential amino acid index. B (FAO/WHO/UNU, 1985). (From Penas et

Table 4. Effect of HHP and/or germination on the total protein and amino acids (g/100 g

Protease inhibitors (PIs) are generally considered the main anti-nutritional factors in soybeans. Soybean PIs belong to a broad class of proteins that inhibit proteolytic enzymes,

d.w.) 45.0a 44.3a 47.3b 48.6c

Glu 15.9c 15.7c 14.8b 14.0a Asp 7.25a 7.26a 7.09a 6.80a Arg 5.44ab 5.60b 5.05a 5.27ab Pro 4.26b 4.25b 3.79ab 3.71ab Ala 4.05b 3.97ab 4.04b 3.71a Ser 3.87a 3.94a 3.92a 3.68a Gly 3.50b 3.07a 3.15ab 3.11a

Tyr 2.91c 2.67abc 2.81bc 2.57ab

Cys 1.24c 1.08b 1.06b 0.94a Total EAA 35.5b 35.1b 34.1ab 32.1a CS 45.0 42.8 41.8 37.0

protein) content of soybean seeds and sprouts during germination.A

**6. Inactivation of soymilk trypsin inhibitors** 

Protein (%

al., 2011).

such as trypsin and chymotrypsin. Both compounds are important animal digestive enzymes for splitting proteins to render dipeptides and tripeptides (Scheider, 1983). However, the specificity of these inhibitors is not necessarily restricted to trypsin and chymotrypsin but also to elastase and serine proteases for which serine constitutes the active site. Nevertheless, the literature reports two main types of soybean PIs, specifically called trypsin inhibitors (TIs). The Kunitz soybean inhibitor, with a molecular weight of 20,000 and two disulfide bridges, exhibits specificity to inhibit trypsin. The Bowman–Birk inhibitor, on the other hand, with a molecular weight ranging from 6000 to 10,000 and seven disulfide bonds, exhibits specificity to inhibit chymotrypsin (Liener, 1994).

 Residual trypsin was measured in soymilk subjected to selected pressures, temperatures and holding times. Treatment combination at higher pressures and temperatures, for selected holding times resulted in an increased inhibition rate of trypsin inhibitors in soymilk. It was not possible to obtain inactivation rate parameters for treatments at 550 MPa and 80 °C because the data did not fit a first order kinetics model. However, a clear increase of residual trypsin was observed as treatment times increased. Soaking of soybeans in sodium bicarbonate solution, prior to preparation of soymilk, resulted in smaller inhibition rates of trypsin at the working selected pressures, combined with thermal treatment and holding times, than in soybeans soaked in distilled water. The use of sodium bicarbonate, as soaking medium of soybeans, did not result in a significant increase in the percentage of residual trypsin in soymilk treated at 550 MPa and 80 °C for the selected holding times (Fig. 4).

Ven et al. (2005) also evaluated HPP as an alternative for the inactivation of TIs in soy milk and also studied the effect of HPP on in whole soybeans and soy milk. For complete lipoxygenase inactivation either very high pressures (800 MPa) or a combined temperature/pressure treatment (60 °C/600 MPa) was needed. Pressure inactivation of TIs was possible only in combination with elevated temperatures. For TIs inactivation, three process parameters, temperature, time, and pressure, were optimized using experimental design and response surface methodology. A 90% TIs inactivation with treatment times of <2 min can be reached at temperatures between 77 and 90 °C and pressures between 750 and 525 MPa.
