**4.5. Characteristics of curd curdling by enzyme**

was not activated by metal ions, which indicates that the protease is not a metalloprotease: a

The mechanisms of curdling soybean milk protease were investigated. At first, The curdled soybean milk samples with added protease and without protease were treated with sample

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Soybean milk was poured into a glass vessel (inner diameter 32 mm, height 45 mm). After 0.1 mL of enzyme solution adding to the soybean milk, or without enzyme 0.1 mL D.W., the mixtures were incubated at 40°C, and they were sampled sequentially; between 4- and 24-h. samples (0.01 mL) were added to 0.01 mL of sample buffer and then heated at 100°C for 3 min. Then samples (10 μl) were added in each well. The samples were electrophoresed on a 12.5%

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They were subsequently examined using SDS–PAGE (Fig. 7) of curdled soybeans. The left side lane shows the standard of protein size. The next lane (0 h.) shows soybean milk protein without reaction of protease. The other lanes show soybean milk protein decomposed for 4, 8, 12, 16, and 24 h. Lane 0 h shows the α′- and α-subunits of β-conglycinin (approximately 84– 73 kDa), the A3 acidic subunit (approximately 40 kDa), other acidic subunits as A4, A1a, A1b, and A2 (approximately 30–42 kDa) of glycinin, the β subunit (approximately 50 kDa), and basic

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The two bands shown as α′ and α disappeared gradually after the reaction, showing the protein band from curd making the protease. In the glycinin subunits, the band of the A3 acidic subunit disappeared completely after 4 h. Furthermore, A4, A1a, A1b, and A2 disappeared to a partial

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**Figure 7.** Digestion of soy protein during curding by soybean milk curdling enzyme.

subunits as B3, B1a, B1b, and B4 (approximately 20 kDa) [40, 41].

metal-dependent enzyme. The amino acid of active site contains cysteine residue.

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94 Food Production and Industry

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> As mechanisms that are closely involved in curdling soybeans, curdled soybean milk by enzyme and glucono-δ-lactone (GDL, 3.0% solution) as control samples were dissolved in chemical solutions. The enzyme solution (0.1 mL) was added to the soybean milk. The mixture was incubated at 40°C for 4 h., or 0.1 mL of glucono-δ-lactone (GDL, 3.0% solution) was added to the soybean milk (1.0 g) and incubated at 80°C for 1 h as control sample. To each of the two curdled soybean milk samples, 9 mL of chemical solution as 2% SDS solution, 4 M urea solution, and 10 mM 2-mercaptoethanol were added. They were held for 1 h at room temper‐

ature and were then centrifuged at 4000×*g* for 10 min. Protein in the supernatant was assayed using the Lowry method.

Results show the relative ratio of protein (%); that is, 100% relative ratio represents the amount of protein dissolving no-curdling soy milk in each solution (Fig. 8).

### ▪, SCE; □, GDL; ▪, no-curdled soy milk.

**Figure 8.** Curdled soy bean milk dissolved in chemical solutions. SCE shows soymilk curdled by enzymes; GDL shows soymilk curdled by glucono δ-lactone.

After dissolving the solutions, curd produced by the enzyme dissolved 47.3% of relative ratio from curd to the 2-mercaptoethanol solutions. That curdled by GDL dissolved 41.6% of relative ratio from the curd to the urea solutions. With urea solution, the curd making the enzyme dissolved 93.8% of relative ratio and GDL dissolved 40.3% of relative ratio. The curd produced by the enzyme can dissolve with urea. Both the curd produced by the enzyme and GDL dissolved with SDS solution. Actually, 2-mercaptoethanol solution cleaves the disulfide bond in protein. The urea solution cleaves the hydrogen bond, and the SDS solution cleaves the hydrophobic bond. That inference agrees with results reported by Yasuda *et al.* [44] that serine protease from *Bacillus* sp. curdled soybean milk and produced a protein bond through mutual hydrophobic bonding.

Next, the effects of the proteolysis enzyme against two protein in soybean, glycinin and βconglycinin, were researched. Glycinin and β-conglycinin were extracted from commercial soy protein according a process described by Nagano *et al.* [44]. The soy protein (100 g) suspended 1500 mL of distilled water at pH 7.5 adjusted 0.1 M NaOH. From their extraction, the glycinin was carried out to do isoelectric precipitation at pH to 6.4. Moreover, β-conglycinin was also precipitated at pH 4.8. The two fractions were freeze-dried. Each fraction as glycinin fraction and β-conglycinin (50 mg) was resolved to 1 mL of 50 mM phosphate buffer (pH 7.5). Fur‐ thermore, enzyme solution (0.1 mL) was added. The mixture were incubated at 40°C. After reaction, soybean milk curdling activity was assayed according the preceding method. Curdling activity was assayed against two substrates: glycinin and β-conglycinin (Fig. 9). Glycinin was curdled strongly: soybean curdling activity was 86.9 (U⋅mL–1⋅min–1). However, β-conglycinin was curdled weakly: 38.0 (U⋅mL–1⋅min–1).

ature and were then centrifuged at 4000×*g* for 10 min. Protein in the supernatant was assayed

Results show the relative ratio of protein (%); that is, 100% relative ratio represents the amount

2-Mercaptoethanol Urea SDS

**Figure 8.** Curdled soy bean milk dissolved in chemical solutions. SCE shows soymilk curdled by enzymes; GDL shows

After dissolving the solutions, curd produced by the enzyme dissolved 47.3% of relative ratio from curd to the 2-mercaptoethanol solutions. That curdled by GDL dissolved 41.6% of relative ratio from the curd to the urea solutions. With urea solution, the curd making the enzyme dissolved 93.8% of relative ratio and GDL dissolved 40.3% of relative ratio. The curd produced by the enzyme can dissolve with urea. Both the curd produced by the enzyme and GDL dissolved with SDS solution. Actually, 2-mercaptoethanol solution cleaves the disulfide bond in protein. The urea solution cleaves the hydrogen bond, and the SDS solution cleaves the hydrophobic bond. That inference agrees with results reported by Yasuda *et al.* [44] that serine protease from *Bacillus* sp. curdled soybean milk and produced a protein bond through mutual

Next, the effects of the proteolysis enzyme against two protein in soybean, glycinin and βconglycinin, were researched. Glycinin and β-conglycinin were extracted from commercial soy protein according a process described by Nagano *et al.* [44]. The soy protein (100 g) suspended 1500 mL of distilled water at pH 7.5 adjusted 0.1 M NaOH. From their extraction, the glycinin was carried out to do isoelectric precipitation at pH to 6.4. Moreover, β-conglycinin was also precipitated at pH 4.8. The two fractions were freeze-dried. Each fraction as glycinin fraction and β-conglycinin (50 mg) was resolved to 1 mL of 50 mM phosphate buffer (pH 7.5). Fur‐

of protein dissolving no-curdling soy milk in each solution (Fig. 8).

using the Lowry method.

96 Food Production and Industry

0

▪, SCE; □, GDL; ▪, no-curdled soy milk.

soymilk curdled by glucono δ-lactone.

hydrophobic bonding.

20

40

60

Ratio of protein dissolution (%)

80

100

The data agree with reports in the relevant literature [45, 46, 47]. Bromelain decomposes 11S globulin to curdling. The entire band of acidic subunits and most basic subunits disappeared [46]. Glycinin-rich soybean milk was curdled strongly [47]. However, the enzyme made glycinin curdle without metal ion or GDL. Generally, glycinin is known to contain more sulfur amino acid than β-conglycinin does. According to Fig. 7, soymilk was curdled by a hydrogen bond or hydrophobic bond. Furthermore, some alkaline protease as subtilisin and chymo‐ trypsin cleaves hydrophobic amino acid residue.

**Figure 9.** Curdling activity against two soy protein as glycinin and β-conglycinin.

The enzyme made curd from soymilk mainly by an enzyme reaction against glycinin. Choi *et al.* [48] reported that the α-subunit in β-conglycinin contains a hydrophobic sequence.

Soybean milk (10 mL) with 0.01 mL of anti-foam (KM-72F) was poured into a glass cup (32 mm inner diameter, 45 mm height). Then 1.5 mL of enzyme solution was added to the soybean milk. The mixture was incubated at 40°C for 4 h. As a control sample, glucono-δ-lactone (GDL, 0.3%) was added to soybean milk (10 mL) with 0.01 mL of anti-foam (KM-72F) added. Then the mixture was incubated at 80°C for 1 h. The curdled soybean milk samples were held at room temperature for 30 min. The rheological characteristics of enzyme curdling soybean milk were measured directly using a creep meter with a 16-mm-diameter plunger compressing 1 mm s–1 with 80.0%. As a control sample, soybean milk was curdled by GDL, 0.3% at 80°C for 1 h.

The rupture strength of the curdled soybean milk in the cups was measured directly using a creep meter (RE-3305; Yamaden Co. Ltd., Tokyo, Japan) with a 16-mm-diameter plunger compressing 1 mm s–1 with 80.0%.

The stress–strain curves of curdled soybean milk are presented in Fig. 10. The vertical axis shows stress (N⋅m–2), which represents internal forces of the sample curd pushing back against the strain. The horizontal axis shows the strain of the curd. The sample curd strained by the plunger is broken by a force that exceeds a certain force: the breaking point. The breaking load represents the hardness or softness of the curd sample. The breaking strain represents the resilience of the sample curd: a large value signifies a high-resilience sample. Pressure by the plunger was loaded more. Then the curd was broken more heavily. After strain loading, the stress value decreased partly. The brittleness shows a different breaking point with the local minimal value. A large brittleness load shows brittle sample curd. The soybean milk was poured into a glass vessel and then curdled using the respective methods. After curdling, rheological analysis of the sample was conducted using a creep meter without taking out. UKHRORJLFDODQDO\VLVRIWKHVDPSOHZDVFRQGXFWHGXVLQJDFUHHSPHWHUZLWKRXWWDNLQJRXW

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times smaller brittleness than that of GDL. Great breaking point shows hard curd and elasticity. It showed that the curd produced by SCE had hard, but soft, springy and sticky curd.

Heretofore, Yasuda *et al.* [45] reported soy milk curdled by bacteria protease. As a result, the curd produced using the bacteria protease is too soft to measure rheological characteristics, but they also reported that curd produced using the bacteria protease with calcium ion had 2– 3×105 (N⋅m–2) of the breaking stress. Their curd was as hard as 20–30 times of the curd produced using this yeast enzyme. The curd produced using the bacteria protease with calcium was broken during fermentation and aging for long time. However, the curd produced using the bacteria protease had not springy and sticky texture.

It is considered that the curd produced using yeast enzymes was not like *Tofu* or the curd produced using the bacteria protease of its main characteristics or rheology.

Guo and Ono [47] and Toyokawa *et al.* [49] reported that the breaking stress of normal *Tofu* showed a relationship to soy milk conditions, such as their concentrations of glycinin, proteins, or temperature. Future investigations will examine if the condition of soy milk has a relation‐ ship with the breaking stress for the enzyme curdling.

According to this report, the new protease from *S. bayanus* SCY 003 produced a new texture of soy food that is applicable for new healthy foods, anti-milk allergy foods, and others.
