Soybeans are a traditional food in eastern Asia, particularly in Japan and China. They were eaten in 100 BC in China. The beans can be processed into Tofu, soy milk, fermented seasonings, soy sauce or Miso paste, and Natto and green beans. Soybeans have rich nutrition, protein lipid, and other functional substances such as isoflavones. However, soybeans are difficult to process for use as food because of tissue and cell wall hardness. Therefore, soybeans are conducted to do some treatments, e.g., boiling, steaming, roasting, crushing/grinding, and some enzyme treating, to eat soy protein easily. Soy storage proteins mainly comprise two proteins as 7S globulin composed with β-conglycinin and 11S globulin containing glycinin composed of 5 subunits. β-Conglycinin, included in 7S globulin, is composed of three subunits.
- Soybean milk
- Saccharomyces bayanus
1.1. Utility of soybeans
Soybeans have been used as traditional foods from ancient times. They are rich in nutrients such as rich proteins, lipids, and others. Furthermore, soybeans can be eaten after processing in various ways.
Eastern Asian people and particularly Japanese people have eaten soybeans after various stages of processing. Soybean seedlings are eaten in many dishes as bean sprouts. Furthermore, soybeans are eaten as green beans in the pod after boiling, as
After hard tissues in boiled soybeans are crushed and ground physically, the soluble fraction is extracted as soy milk. Soy milk is processed as
1.2. History of soybeans
Theories about the origin of soybean use and cultivation remain controversial in their details. By some accounts, soybeans used as food originated in the area of Manchuria in China and Siberia in Russia. Alternatively, their use as food originated in southern China . Yet another possible history holds that soybeans were bred from wild soybeans as
In China, the first literal record can be found in a Chinese dictionary published in 100 B.C. The dictionary inserted “
In Japan also, “
1.3. Consumption worldwide and in Japan
Soybeans were produced only in eastern Asia for a long time. In contrast, other cereals such as rice, barley, wheat, and corn have diffused throughout the world. Moreover, it is considered that some other endemic bean or pea or pulse had become cultivated in each area already .
For instance, in central Asia, broad beans were cultivated, as were chick peas in India, shell peas in western Asia, and kidney beans and ground peas in North America. Nevertheless, soybeans have been cultivated in the United States as oil seed crops since the 1920s. Furthermore, the crop has begun to be cultivated in Canada and South America. In 2012, approximately 82 million tons of soybeans were harvested, with the United States accounting for 34% of the world production. Brazil harvested approximately 66 million tons that year, accounting for 27% of world production. Argentina harvested approximately 40 million tons, or 17% of world production. Therefore, most soybeans (over 80%) consumed worldwide are now produced and harvested in North and South America  (Table 1).
In Japan, soybean production was sufficient to provide for domestic consumption until the Taisho Era . Soybean consumption in Japan has been high, but it decreased after the Taisho Era. In 2013, 30 million tons were consumed, but only 240 thousand tons were harvested domestically. That figure is less than 0.1% of the world production amount. Soybeans used domestically account for 104 thousand tons for feed, 6 thousand tons for seed, and 1.9 million tons for oilseeds, all together accounting for 70% of the 30 million tons consumed. Furthermore, those figures indicate that only 30% of soybeans are used as food. The self-sufficiency ratio of soybeans was 97% in 1947. It decreased gradually to 28% in 1959, 11% in 1965, and 7% in 2013 . The ratios of soybeans used for food are 49% used for
|Delicatessen of soybean||33||30|
|Kinako soy powder||17||18|
2.1. Soybean protein
Soybeans contain 35% protein as storage protein, which is used for nutrition during germination. That storage protein is stored in granules, called protein bodies, of about 5–8 μm diameter. Soluble soy protein is extracted from insoluble protein bodies that are burst during soy milk and
Protein digestibility-corrected amino acid score (PDCAAS) values are used to evaluate the protein quality based on the amino acid requirements of humans and their ability to digest it. It was long thought that the amount of amino acid requirements of humans dictate a low score for soybeans because methionine and cysteine residues, sulfur amino acids, in soybean storage proteins have a low composition. The score was only 86 points based on the amino acid requirements of a developing rat. However, in 1985, the score was modified to 100 points, the same as milk and eggs, based on the amino acid requirements of humans. Soy storage proteins are rich in nutrition for human needs .
Throughout the world, the recently improving healthy image of soy protein is interesting. In particular, the health benefits of soy foods attract attention in the United States. Health claims are authorized by the Food and Drug Administration (FDA) in the United States: foods containing 6.25 g of soy protein or more can be said by manufacturers to reduce the risk of heart disease if a consumer ingests 25 g/day of soy protein . In Japan, some soybean containing foods are manufactured as
The taxonomy of soybean storage protein has been conducted according to the sedimentation coefficient by an ultracentrifugal fraction as 2S globulin, 7S globulin, 11S globulin, and 15S globulin. Yamauchi  reported details of soybean proteins: 2S globulin contained α-conglycinin, 7S globulin composed with β-conglycinin and γ-conglycinin, and 11S globulin-containing glycinin. In addition, the 11S globulin composes hexamer. It is a 350,000 Da protein. Furthermore, their proteins are composed with five subunits as G1–G5; their subunit was 10 polypeptides as A1a, A2, A1b, A3, A5A4, A4, B2, B1b, B4, and B3. Their polypeptides are combined specifically as A1aB1b, A1bB2, A2B1a, A3B4, and A5A4B3. In fact, β-conglycinin, called 7S globulin, combines a dimer protein and a monomer protein, which are 150,000–200,000 Da, or an average of 180,000 Da. They are composed of three subunits: an α-subunit of 63,000 Da, an α′-subunit of 67,000 Da, and a β-subunit of 48,000 Da. The protein has a low concentration of sulfur amino acids. In particular, the β-subunit does not contain methionine, cysteine, and tryptophan .
Soybeans have 20% lipids. The lipid concentration varies among harvested regions. Soybeans harvested in the United States have more lipids than those in China . A main reason is that soybeans there have long been bred and modified to contain high oil concentrations as oilseed.
Components of fatty acids in foods shows Table 3. Soy oil comprises a small amount of saturated fatty acids, such as palmitic acid and stearic acid, and large amount of unsaturated fatty acids such as oleic acid, linoleic acid, and linolenic acid. Polyunsaturated fatty acids (PUFAs) containing more than unsaturated bonds are important nutrition as necessary lipids for humans. Soybeans have over 60% PUFA. In particular, one kind of PUFA as linoleic acid contained approximately 54.5%.
Actually, PUFAs in animal lipids have low concentration. Therefore, they are insufficient nutritionally. Saturated fats and unsaturated fats are ideally in the following ratio: saturated–unsaturated (1:2) . Soybean lipids were well known to be much stable against oxidation because they are covered as oil body particle by oleosin and other proteins.
Generally, soybeans have totally 12 isoflavones in 3 aglycones, and they have three types of glycosides as glucoside, acetyl-glycoside and malonyl-glycosides: genistein, daidzein, glystein, genistin, daidzin, glycitin, acetyl-genistin, acetyl-daidzin, acetyl-glycitin, malonyl-genistin, malonyl-daidzin and malonyl-glycitin . After soybean consumption, glycoside isoflavone, which is contained in food as soy milk or
Many Japanese and throughout eastern Asia intake isoflavone from soybeans. Some researchers have reported negative opinions about plant estrogen . Isoflavones are produced via phenyl–propanoid pathway from phenylalanine in plants. Two intermediate substances, naringenins, are converted to genistein by two specific enzymes in soybeans: isoflavone synthetase and dehydrogenase. Chalcones are converted to daidzein by three specific enzymes in soybean: chalcone reductase, chalcone isomerase, and isoflavone synthetase. Isoflavone and a similar substance, phytoalexin, are used as antibacterial substances against phytopathogenic fungi and bacteria. In addition, they grow well as root nodule bacteria at the root for nitrogen fixation [9, 10].
Isoflavones in many plants store glucosyl, malonyl-glucosyl, and acetyl-glucosyl conjugate as hydrophilic substances. After invasion of phytopathogenic fungus and bacteria, the glucosyl conjugate isoflavone is transferred to infested wounds, where it hydrolyzes for phytoalexin . Isoflavone has health functions against climacteric disturbances and type 2 diabetes. In Japan, soybean isoflavones are a
3. Food processing
3.1. Formation of
Soy proteins have properties that produce curd to add specific metal ions. The property is applied for
Soy milk is an emulsion composing lipid and soy protein, mainly glycinin and β-conglycinin. Actually, 60% of the protein in soy milk protein is composed of these two proteins, and a large size of the protein body was constructed with their proteins and others. Lipid is a triacylglyceride composed of linoleic acid, oleic acid, and phospholipids. Before treating, triacylglyceride is stored in oil bodies in soybeans. During soy milk production, and of course during
This phenomenon induces the charge of protein to dissipate by a combination of phytic acid and bivalent ion. Repulsion among their proteins is decreased. Moreover, the immobilized protein combines with outside layer protein easily around an oil drop . Consequently, the steric network structure of soy protein is formed to gather oil drops with intermediary soy protein. Becoming low pH in soy milk, soluble soy protein becoming dissoluble is taken into the network. The curd produces a hard gel that is water retentive, such that moisture is trapped in the network.
4. Soy milk curdling yeast and characteristics of curdling enzymes
4.1. Soy milk curdling
As discussed above, soybean proteins provide rich nutrition [4, 19]. In fact, soymilk consumption is increasing quickly throughout the world because of its health benefits. Moreover, differently from bovine milk, it contains no cholesterol. Yogurt-like foods and cheese-like foods made from soybeans can be consumed by people who are concerned about health issues or allergies related to bovine milk.
It is not yet a viable alternative to dairy foods. For that reason, the commercial use of enzymes such as bromelain, ficin, and papain for the curdling of soybean milk has been unsuccessful [20–22]. The authors have reported that physical properties of soy protein modified by enzyme reactions such as germinated proteolysis in soybeans. Therefore, in this section, along with a report of yeast containing soybean curdling enzyme , this investigation was undertaken to screen and identify specific food yeast strains (
4.2. Screening of yeast producing curdling soybean milk enzyme
The yeast strains (1345 strains) stored in the laboratory were screened using soybean milk agar plate medium. The strains were inoculated by streaking on a plate surface. Then they were incubated at 30°C for 7 days. After cultivation, the clear zone diameter was measured using calipers. Yeast strains that produced a clear zone were selected. Results show that 1242 yeast strains among all 1345 yeast strains produced no clear zone on the plate medium. The yeast strains (42 strains) produced less than 1 mm of a clear zone. Also, 57 yeast strains produced 1.0–5.0 mm; 4 yeast strains produced more than 6 mm.
In the second screening of curdling soybean milk enzyme-producing yeast, the screened strains (103 strains) were inoculated to the soybean milk liquid medium. Purchased soybean milk was added to them aseptically.
The soybean milk medium was incubated at 30°C for 24 h. When curdling occurred, the pH of whey was measured using a pH meter (Horiba Ltd.). Results show that three yeasts curdled at pH greater than 5.90. The media were pH 5.90 (SCY 001), pH 6.05 (SCY 002), and pH 6.38 (SCY003) (Table 4).
The curd activity of strain SCY003 was the highest among the strains. Therefore, the SCY003 strain was finally screened. Isolated yeasts were classified taxonomically and were identified according to methods described in earlier studies .
The morphology was observed by microscope. Their 1.5- to 6.5-μm-long cells were short and ovaloid. The yeast, which buds by multibudding reproduction, does not form pseudomycelia or pellicles on the liquid medium. It forms ascospores. It was identified as
For researching physiological characteristics of the strain, the yeast was inoculated into a yeast nitrogen base medium (Difco Laboratories) adding 0.5% of each carbon source: sugar or organic acid as glucose, galactose, sucrose, maltose, raffinose, trehalose, lactose, melibiose, cellobiose, melezitose, starch, D-xylose, L-arabinose, D-ribose, L-rhamnose, erythritol, D-mannitol, salicin, inositol, dulcitol, ethanol, D-sorbitol, disodium succinate, and trisodium citrate. The yeast was inoculated into yeast carbon base medium (Difco Laboratories) adding sodium nitrate solution.
The glucose, galactose, sucrose, maltose, and raffinose in the medium were fermented as carbon sources using strain SCY003. The yeasts grew in a vitamin-free medium. Furthermore, the strain did not grow in 0.01% cycloheximide. According to the morphological, physiological, and molecular characteristics, it was identified as
For researching molecular biological characteristics of the strain, primers were used for amplification and sequencing of 18S-rRNA-encoding genes. The PCR products were sequenced using a kit (ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction; Applied Biosystems). Analyses of DNA sequence reactions were performed using a sequencer (3130; Applied Biosystems). The 18S rRNA coding DNA was sequenced. Homology was assessed using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/BLAST/).
As a result, the yeast showed homology of 99% with
4.3. Purification of the protease as a soybean milk curdling enzyme
Enzyme extraction, intercellular, in
Generally, commercial soy milk has dispersion stability attributable to the presence of oleosomes or forming aggregate formation of soy proteins on it [28, 29]. Therefore, no precipitate is produced from commercial soybean milk by low centrifugal gravity as 400×
The precipitation ratio was related with the reaction period, and with the enzyme solution volume. They are mutually correlated:
After crude extraction of the enzyme, the enzyme protein was purified using chromatography. After crushing cells and extracting the enzyme, the enzyme was precipitated to between 30% and 40% saturation of ammonium sulfate. After redissolving the precipitate, the solution was dialyzed overnight at 4°C, and they carried out ion-exchange chromatography using a column (25 mm × 300 mm) of DEAE gel. The protein was eluted using a 0- to 1-M NaCl linear gradient. Proteolytic activity and curdling were assayed each fraction. Proteolytic activity was measured in duplicate using a commercial kit fluorescein isothiocyanate-labeled casein (FTC). Fluorescence was measured using a 485-nm excitation wave and a 535-nm emission wave. The proteolytic activity was decided for one unit expressed equal amount of trypsin (1 ng⋅mL–1) doing proteolysis of FTC solution.
After ion chromatography, fractions containing the highest level of activity were pooled and reprecipitated using 80% saturation of ammonium sulfate. After redissolving the precipitate, gel filtration chromatography (10 mm × 350 mm, P-100 gel; Bio-Rad Laboratories Inc., CA, USA) was carried out. Then the molecular weight of the enzyme was analyzed using also the chromatography as various molecular weight standards (myosin, 200 kDa; serum albumin, 66.2 kDa; ovalbumin, 45.0 kDa; trypsin inhibitor, 21.5 kDa).
The results are portrayed in Figs. 4a and 4b. One main curdling activity peak was identified using ion-exchange chromatography. The peak (fraction no. 49) agreed with protein and activity. Furthermore, curdling activity agreed with the same fractions presenting protease activity (fraction no. 49).
As a result, the larger peak of proteolysis activity was found around fraction number 25, and a small peak was found at fraction number 49. The fraction of the proteolysis enzyme around number 25 was not representative of curdling activity. It is considered that the former fractions are attributable to intense proteolysis enzymes and that the latter fractions are attributable to soybean milk-curdling enzymes.
After reprecipitation, the sample was analyzed using gel filtration chromatography. A peak was found at fraction numbers 11–14. Their fractions agreed with soybean milk curdling activity, proteolytic activity, and protein. This result on their chromatograms demonstrates that protease and soybean milk curdling enzyme have some mutual relation of activity.
After purification, the enzyme protein band was approximately 45 kDa (Fig. 5a), which agrees with data of other proteases. The protease molecular weight was measured using gel filtration chromatography (Fig. 5b). The molecular mass is about 45 kDa. The protease was inferred.
The soy milk curdling enzyme has proteolytic activity. Results suggest that the soy milk curdling enzyme was a proteolysis enzyme. Many researchers have reported protease produced by yeasts as
By contrast, few reports describe intracellular protease producing
4.4. Characteristics of the protease as a soybean milk curdling enzyme
Optimum pH, temperature, and stability of the enzyme are presented in Figs. 6a and 6b. Optimum pH was assayed at pH 4.0–8.0 using 50 mM phosphate-citric buffer or phosphate-NaOH. For optimum pH of the enzyme assaying, 0.1 mL of a fluorescein isothiocyanate-labeled casein (FTC) solutions, which dissolved in each phosphate buffer (50 mM, pH 4.0–8.0), and 20 μL of enzyme solution were reacted at 40°C for 60 min. For optimum temperature of the enzyme assaying, FTC solution at pH 7.5 (0.1 mL) and 20 μL of enzyme solution were reacted at 15°C–70°C for 60 min to find optimum temperature. After reaction, fluorescence was measured using a 485-nm excitation wave and a 535-nm emission wave.
The optimum pH for the protease activity as curdling was pH 7.5; the optimum temperature was 50°C. The optimum pH of a bovine milk curdling protease,
Effects of metal ions and inhibitors on protease are presented in Table 5. In fact, zinc, copper, and mercury all inhibit protease activity. The amino acid of the active site contains cysteine residue because of inhibition by mercury . Furthermore, EGTA (10 mM) inhibited protease activity (62.0% of relative activity).
|Iodo acetate||1 mM||74.1|
The activity was not activated by metal ions, but it was inactivated by mercury. Soybean milk-curdling enzyme  was inhibited by zinc ions and mercury ions. These results agree with our data related to zinc and mercury. Its survival activity was 18% by mercury. The protease was not activated by metal ions, which indicates that the protease is not a metalloprotease: a metal-dependent enzyme. The amino acid of active site contains cysteine residue.
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 buffer solution.
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% uniform gel at 20 mA.
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 subunits as B3, B1a, B1b, and B4 (approximately 20 kDa) [40, 41].
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 degree same as A3 acidic subunit. Peptides smaller than 20 kDa were detected on the gel. The β-conglycinin as α, α′, and part of glycinin as A3 A4, A1b, and A2 were decomposed. Soybean protein became loosely curdled with the addition of other proteases from microorganisms or plants. The protein was decomposed. The low-molecular-weight peptides increased on the polyacrylamide gel. Generally, 11S glycinin was related to the formation of a stiffer gel. Furthermore, Ono
According to our data, after the curdling soy milk by enzyme, α- and α′-subunits cleaved by the protease easily, whereas basic and β-subunit remained. It is considered that surface proteins as α- and α′-subunits were decomposed easily. Some decomposed subunits such as α, α′, A3, acidic, and basic subunits are regarded as related to the curdling soybean milk.
The enzyme of mechanisms for proteolysis was searched that the enzyme had the peptidase activity as exotype proteolysis activity and protease as endotype proteolysis activity. The synthesis peptide substrates, Z-glutamyl-tyrosine, and casein, FTC, were reacted by the enzymes. Peptidase (carboxypeptidase) activity was determined by the increase in ninhydrin after hydrolysis of benzyloxycarbonyl-glutamyl-tyrosine (pH 8.0) at 40°C.
The results show that the enzyme had 0.14 U⋅mg–1 protein as peptidase activity and 0.55 U⋅mg–1 protein protease as endotype proteolysis activity (data not shown). Results also show that the soybean milk-curdling enzyme as a proteolysis enzyme had endotype proteolysis activity.
4.5. Characteristics of curd curdling by enzyme
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 temperature and were then centrifuged at 4000×
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).
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
Next, the effects of the proteolysis enzyme against two protein in soybean, glycinin and β-conglycinin, were researched. Glycinin and β-conglycinin were extracted from commercial soy protein according a process described by Nagano
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 . Glycinin-rich soybean milk was curdled strongly . 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 chymotrypsin cleaves hydrophobic amino acid residue.
The enzyme made curd from soymilk mainly by an enzyme reaction against glycinin. Choi
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.
The breaking point of curd curdled using GDL, which is used by many Japanese
By contrast, the breaking point of curd curdled by soy curdling enzymes (SCE) was 58.4% strain. Their breaking stress was 10,930 (N⋅m–2). The brittleness point is 81.2% and 10,200 (N⋅m–2). The brittleness of this curd produced using the enzyme was 730 (N⋅m–2). The curd that used SCE had 1.4 times greater breaking point, shown hardness, than that of the curd produced using the GDL same as the Japanese
It is considered that the curd produced using yeast enzymes was not like
Guo and Ono  and Toyokawa
According to this report, the new protease from
Soybeans are a traditional food in eastern Asia, particularly in Japan and China. They were eaten in 100 BC in China. The beans can be processed into
Soy storage proteins mainly comprise two proteins as 7S globulin composed of β-conglycinin and γ-conglycinin and 11S globulin containing glycinin composed of 5 subunits. β-Conglycinin, included in 7S globulin, is composed of three subunits.
To modify the physical properties of soy protein, a new type of enzyme for curdling soybean milk enzyme was purified as an extract from yeast. Yeast producing curdling soybean milk enzyme, the SCY003 strain, was isolated from 1345 yeast strains. According to the morphology, physiology, and molecular and characteristics, SCY003 was identified as
The enzyme cleaved the β-conglycinin as α and α′, and part of glycinin as A3 A4, A1b, and A2 in soy protein by endoproteolysis. Soybean protein became loosely curdled with the addition of other proteases from microorganisms or plants. Soybean milk curdled after cleaving endoproteolysis enzyme in SCY003 strain. The rheological characteristics of enzyme curdling soybean milk, the breaking point, was 58.4% strain; their breaking stress was 10,900 (N⋅m–2); the brittleness point is 81.2% and 10,200 (N⋅m–2). The brittleness of the curd produced using the enzyme was 727 (N⋅m–2). The curd had a sticky and chewy texture and did not resemble
In this way, some properties of soy protein were modified by enzymes, such as decomposing specific subunits in soybeans and making soy milk curdle, which are expected to be applicable for new healthy foods, anti-milk allergy foods, and others.
We thank A-STEP, Adaptable and Seamless Technology, Transfer Program through Target Driven R&D by Japan Science and Technology Agency (JST) (AS231Z00291E) for financial support.