**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).

Initial pH was 6.70. ++, coagulating very well; +, coagulating; –, noncoagulating.

**Table 4.** Curdling soybean milk condition by screened yeast

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 [24].

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 *Saccharomyces* sp.

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, Dmannitol, 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 *Saccharomyces bayanus*.

For researching molecular biological characteristics of the strain, primers were used for amplification and sequencing of 18S-rRNA-encoding genes. The PCR products were se‐ quenced 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 *S. bayanus* (accession no. AY046227). It was identified as *S. bayanus* through homology research and phenotypic testing.

## **4.3. Purification of the protease as a soybean milk curdling enzyme**

at pH greater than 5.90. The media were pH 5.90 (SCY 001), pH 6.05 (SCY 002), and pH 6.38

≥6.50 0 0 7 6.49–5.90 1 2 43 5.89–5.50 0 17 17 5.49–5.00 2 11 0 ≤4.99 3 0 0

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

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 *Saccharomyces* sp.

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, Dmannitol, salicin, inositol, dulcitol, ethanol, D-sorbitol, disodium succinate, and trisodium citrate. The yeast was inoculated into yeast carbon base medium (Difco Laboratories) adding

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,

For researching molecular biological characteristics of the strain, primers were used for amplification and sequencing of 18S-rRNA-encoding genes. The PCR products were se‐ quenced 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/).

and molecular characteristics, it was identified as *Saccharomyces bayanus*.

**Curdling soy milk** (++) (+) (–)

(SCY003) (Table 4).

88 Food Production and Industry

sodium nitrate solution.

**pH**

Initial pH was 6.70. ++, coagulating very well; +, coagulating; –, noncoagulating.

**Table 4.** Curdling soybean milk condition by screened yeast

according to methods described in earlier studies [24].

Enzyme extraction, intercellular, in *S. bayanus* SCY003, soybean milk curdling test/activity was conducted using the method described [25-27] for modified soybean milk from bovine milkcurdling activity. The mixture was centrifuged at 400×*g* for 10 min. The supernatant was removed gently using a Pasteur pipette. The weight of the precipitate was measured using a chemical balance.

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 precip‐ itate is produced from commercial soybean milk by low centrifugal gravity as 400×*g*. However, precipitation ratios increase with the enzyme reaction period.

The precipitation ratio was related with the reaction period, and with the enzyme solution volume. They are mutually correlated: *R*<sup>2</sup> = 0.9978. Therefore, one curdling unit expressed the ratio of curdling (%) from 1 mL of soybean milk at 40°C for 1 h. The precipitation ratio from soybean milk was assayed efficiently to propose a new curdling method. The precipitation ratio was assayed (Fig. 3).

**Figure 3.** Curdling soybean milk enzyme activity.

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). Fluores‐ cence 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 *Candida albicans* [30, 31, 32], *Candida humicola* [33], and *Saccharomyces cerevisiae* [34]. Extracellular proteases produced by yeasts as *Candida* spp. are 42–45 kDa [35, 32]; those by bacteria are 21 kDa [36].

By contrast, few reports describe intracellular protease producing *Saccharomyces cerevisiae*, although many intracellular proteases in the vacuole or other organelles are known to be related to proteinase A, which is 42 k Da [34]. The molecular weight of curdling soy protein enzyme protease agreed with protease produced by other yeast as Ascomycota. However, the *Mucor* sp. enzyme, which curdles bovine milk, produced a 49-kDa protease [37], which is larger than those produced by these yeasts.
