*2.4. Application of DGAS to IFA-Rich Extract from Soybeans*

Ultrasound-assisted extraction is known by its high efficiency, high productivity, lower consumption of solvents, and eco-friendly process [42]. In addition, in this study, to prevent protein interference after the extraction of soybeans with organic solvent, proteins were precipitated by dehydration and aggregation using ice-cold acetone [43]. After the successful conversion of IFAs into transglycosylated IFAs, optimal reaction conditions established for the DGAS process were used for the transglycosylation of IFA-rich soybean extract (SBE) as the acceptor. IFAs in SBE existed at lower concentrations than the other isoflavones such as isoflavone glucosides (Figure 5a and Table 2).

**Table 2.** Content (mg/g powder) of isoflavones in soybean extract (SBE), cellulase (CE)-treated SBE (CE-SBE; isoflavone aglycone-rich extract), and CE-SBE transglycosylated using DGAS (CE-SBE-DGAS).


<sup>1</sup> Data are expressed as means ± standard deviations (*<sup>n</sup>* <sup>=</sup> 3). Means with different superscripted letters in the same row differ significantly by the Tukey-Kramer honestly significant difference test (*p* < 0.05).

Isoflavones in soybeans are mainly present in malonyl forms when extracted under normal conditions such as neutral solvent, ambient temperature, and short extraction time [44]. Similarly, malonyl forms were found to be the major isoflavones in SBE in this study (Figure 5a). Peaks labeled MD, ML, and MG are malonyldaidzin, malonylglycitin, and malonylgenistin, respectively (Figures 4a and 5a,b). Therefore, 7-β-*O*-glucoses (with acetyl and malonyl moieties) bound to IFA should be hydrolyzed using cellulase (CE) prior to DGAS transglycosylation. Extraction under harsh conditions (low pH and high temperature) and microbial fermentation has been used previously to obtain IFAs from soybeans [44]. However, acid-modified extraction is not suitable for enzymatic glycosylation reactions and is not environmentally friendly. Conjugated isoflavones (malonylisoflavones, acetylisoflavones, and non-acylated isoflavone glucosides) are converted into their non-conjugated counterparts using enzymes such as glycosylase [45]. In this study, we obtained IFAs by removing β-1,4-linked glucose moiety from conjugated isoflavones using CE. After CE processing, malonylisoflavones were degraded into their corresponding aglycones. As the concentrations of conjugated daidzeins (peaks GD, MD, and AD), conjugated glyciteins (peaks GL, ML, and AL), and conjugated genisteins (peaks GG and MG) in SBE decreased (Figure 5a), the concentrations of their corresponding IFAs increased after CE treatment (Figure 5b). CE-treated soybean extract (CE-SBE; IFA-rich extract) had the highest content of genistein (53.9% of total), daidzein (42.2% of total), and glycitein (4.0% of total) compared with SBE and CE-SBE transglycosylated using DGAS (CE-SBE-DGAS) (Table 2). Consistent with the results

of this study, the content of glycitein derivatives in soybeans and their processed products has been reported to be less than 5% of the total isoflavone content [5].

**Figure 5.** HPLC traces (254 nm) of IFA-rich soybean extract after transglycosylation using DGAS. (**a**) Soybean extract, (**b**) cellulase-treated soybean extract, (**c**) and cellulase-treated soybean extract transglycosylated using DGAS. Peaks: GD, daidzin; GL, glycitin; GG, genistin; MD, malonyldaidzin; ML, malonylglycitin; AD, acetyldaidzin; AL, acetylglycitin; MG, malonylgenistin; DN, daidzein; LN, glycitein; GN, genistein. Refer to Table 3 for the identification of each numbered peak of the 19 compounds.

Soy isoflavones have different ultraviolet-visible spectra depending on their aglycones [3,46]. The peaks newly produced by DGAS-treated transglycosylation were tentatively confirmed by comparison with the patterns of ultraviolet-visible spectra of isoflavone standards and molecular weight (Table 3). As results of mass spectrometry (MS), 19 products including four unknown compounds of DGAS-treated CE-SBE were tentatively identified (Table 3). The malonylisoflavones in SBE were not changed by DGAS (peaks 15, 16, and 17 of Figure 5c) in this study. In contrast, IFAs were not detected in CE-SBE-DGAS (Figure 5c and Table 2), indicating that IFAs in CE-SBE are successfully transglycosylated using DGAS. In previous studies, DGAS has been reported to transglycosylate a sugar moiety to the −OH group of the flavonoid aglycone [27,30,31]. It is also reported that glucose moiety was transferred to the isoflavone monoglucosides such as daidzin [36].

Among the peaks newly generated using DGAS, the peaks 10, 12, and 13 were identified as daidzein, glycitein, and genistein monoglucosides with different stereospecificity and retention time from daidzin, glycitin, and genistin, respectively (Figure 5c and Table 3). The generated isoflavone monoglucosides can be seen to have a similar polarity compared to the isoflavone 7-β-*O*-glucosides (peaks GD, GL, and GG) in the SBE (Figure 5a). The MS results of CE-SBE-DGAS indicate that the more polar components, which are eluted earlier than isoflavone monoglucosides, are transglycosylated with two or more glucose moieties (Figure 5c and Table 3). The treatment of IFA standards and IFA-rich extract with DGAS resulted in higher amounts of isoflavone polyglucosides than isoflavone monoglucosides (Figures 4b–d and 5c). As mentioned earlier, DGAS creates various transglycosylated isoflavones from IFAs. Studies of enzyme-modified flavonoids have reported binding of one or more glucose moieties to aglycones [24,27]. It has also been reported that glucose-added products, which are more polar than daidzin, are produced when transglycosylation is carried out using daidzin as the acceptor [36]. Hence, by using DGAS for the transglycosylation of CE-SBE, various isoflavone polyglucosides with two and three glucose moieties bound to their backbones, as well as isoflavone monoglucosides, can be generated.


**Table 3.** Identification of isoflavone derivatives of CE-SBE-DGAS.

<sup>1</sup> n.d Out of the set molecular weight in mass conditions.
