*3.4. Preparation and Transglycosylation of SBE*

Soybean seeds (cv. Daewon) were obtained from the Department of Southern Area Crop Science, National Institute of Crop Science, Rural Development Administration (Dalseong, Korea). Soybean seeds (10 g) were pulverized using a cutting mill (Tube Mill 100 control; IKA®, Staufen, Germany). Extraction was carried out with 250 mL of 70% (*v*/*v*) aqueous ethanol using an ultrasonic bath (Sonifier 450; Branson Ultrasonics Corp.) at ambient temperature for 1 h. The aqueous ethanol extract of soybean seeds was filtered through Whatman no. 4 filter paper (Whatman Inc., Clifton, NJ, USA). The filtrate was evaporated using a rotary evaporator (N-1000; Eyela, Tokyo, Japan) in a water bath at 40 ◦C. Five hundred milliliters acetone was added to dry SBE, and this mixture was placed at −20 ◦C for 24 h. The mixture was filtered through Whatman no. 4 filter paper (Whatman Inc.) and evaporated (N-1000; Eyela). To obtain IFA-rich extract by enzymatically hydrolyzing conjugated isoflavones in SBE, 200 mg of SBE powder and 20 mg of CE (EC 3.2.1.6) (Vision Corp., Seoul, Korea) were reacted in water at 40 ◦C for 24 h. The reactant from the CE-SBE was concentrated in the methanol phase by removing the matrix using an ODS Sep-Pak cartridge (Oasis HLB; Waters, Milford, MA, USA). Purified CE-SBE reactant was evaporated using a rotary evaporator (N-1000; Eyela) in a water bath at 40 ◦C. For transglycosylation, CE-SBE powder (10 g/L) was used as an acceptor, and sucrose (100 mM) was used as the donor. The reaction temperature, time, buffer, and pH conditions of SBE were the same as those described in Section 3.3. After termination of the enzyme reaction, the pretreatment process for HPLC analysis proceeded in the same manner described in Section 3.3. The final samples were stored in a freezer at −20 ◦C until further analysis.

#### *3.5. Analysis of Transglycosylated Isoflavones by HPLC and MS*

SBE, CE-SBE, and CE-SBE-DGAS were analyzed using a Waters HPLC system (Alliance e2998; Waters) equipped with a ProntoSIL ace-EPS-C18 column (120 Å, 5 μm, 4.6 × 250 mm; Bischoff, Leonberg, Germany) and a photodiode array detector (2695; Waters) at 254 nm. Gradient elution was performing using 0.1% (*v*/*v*) formic acid in water (solvent A) and acetonitrile (solvent B). All solvents were filtered, degassed, and kept under pressure. The initial mobile phase was 100% solvent A. The gradient of mobile phase B was as follows: 0–2.5 min, 0% B; 2.5–5 min, 0–12% B; 5–7 min, 12–18% B; 7–10 min, 18–18% B; 10–13 min, 18–26% B; 13–19 min, 26–26% B; 19–23 min, 26–46% B; 23–26 min, 46–46% B; 26–30 min, 46–75% B; 30–32 min, 75–0% B; 32–35 min, 0% B. Flow rate, column oven temperature, and injection volume were 1.0 mL/min, 30 ◦C, and 5 μL, respectively.

The mass detection was measured using the modified method used in our previous study [36]. The quadrupole Dalton-based (QDa) detector (Waters) was used to obtain the MS data. An isocratic solvent manager system split the analyte phase into acetonitrile at a ratio of 8:2. The QDa parameters in positive ion mode were as follows: capillary voltage, 0.8 kV; cone voltage, 5 V; source temperature, 600 ◦C; desolvation gas flow, 800 L/h. The mass values to identify the transglycosylated isoflavones were used for daidzein glucosides (255.1, 417.1, 579.1, 741.1, and 903.1 *m*/*z*), glycitein glucosides (285.1, 447.1, 609.1, 771.1, and 933.1 *m*/*z*), and genistein glucosides (271.2, 433.2, 595.2, 757.2, and 919.2 *m*/*z*), respectively. Empower 3 (Waters) was used to control the HPLC-QDa system and analyze the data obtained.
