Changes in Isoflavone Profile from Soybean Seeds during Cheonggukjang Fermentation Based on High-Resolution UPLC-DAD-QToF/MS: New Succinylated and Phosphorylated Conjugates

In this study, thirty-eight isoflavone derivatives were comprehensively identified and quantified from the raw, steamed and fermented seeds of four selected soybean cultivars based on UPLC-DAD-QToF/MS results with reference to the previously reported LC-MS library and flavonoid database, and summarized by acylated group including glucosides (Glu), malonyl-glucosides (Mal-Glu), acetyl-glucosides (Ac-Glu), succinyl-glucosides (Suc-Glu) and phosphorylated conjugates (Phos) in addition to aglycones. Among them, Suc-Glu and Phos derivatives were newly generated due to fermentation by B. subtilis AFY-2 (cheonggukjang). In particular, Phos were characterized for the first time in fermented soy products using Bacillus species. From a proposed roadmap on isoflavone-based biotransformation, predominant Mal-Glu (77.5–84.2%, raw) decreased rapidly by decarboxylation and deesterification into Ac-Glu and Glu (3.5–8.1% and 50.0–72.2%) during steaming, respectively. As fermentation continued, the increased Glu were mainly succinylated and phosphorylated as well as gradually hydrolyzed into their corresponding aglycones. Thus, Suc-Glu and Phos (17.3–22.4% and 1.5–5.4%, 36 h) determined depending on cultivar type and incubation time, and can be considered as important biomarkers generated during cheonggukjang fermentation. Additionally, the changes of isoflavone profile can be used as a fundamental report in applied microbial science as well as bioavailability research from fermented soy foods.


Introduction
Isoflavones are mainly distributed as O-glycosyl or C-glycosyl derivatives with acylated moieties (e.g., none-, acetyl-and malonyl-) in soybean, kudzu, astragalus and red clover belonging to Leguminosae Family [1,2] and have been reported to have potential preventive effects in a wide range of human diseases such as cancer, hypertension, atherosclerosis, diabetes, hyperlipidemia, menopausal symptoms, osteoporosis, neurodegeneration and multiple sclerosis [1,3,4]. Particularly, soybean (Glycine max L.), commonly known suggest that the presence of succinylated and phosphorylated isoflavones can be considered as important biomarkers during fermentation with Bacillus species, and can be used as a fundamental report in advanced microbial science as well as human bioavailability research after consumption of fermented soy foods.

Identification of 38 Isoflavone Derivatives in Raw, Steamed and Fermented Soybean Seeds
As shown in Figure 1 and Table 1, a total of thirty-eight isoflavone derivatives were tentatively identified from the raw, steamed and fermented seed samples of four selected soybean cultivars (KLS 87248, Nongrim 51, GNU-2007-14613 and Daewon) by comparing elution order, UV spectra and MS fragments pattern provided from the present UPLC-DAD and QToF-MS spectral interpretation as well as previously reported LC-MS library and NMR-elucidated results, and mainly composed of glucosides (four), malonyl-glucosides (nine), acetyl-glucosides (seven), succinyl-glucosides (nine) and phosphorylated conjugates (four) based on daidzein, genistein and glycitein aglycones. These derivatives with good separation on UPLC-DAD chromatograms (KLS 87248, wavelength at 254 nm) ( Figure 1) were fragmented in detail through positive ionization (+ESI, m/z [M + H] + ) of high-resolution QToF-MS (Table 1).
Through recent high-resolution MS technologies, the positive ionization makes it easier to distinguish the parent molecules than conventional negative ionization due to adductive sodium (Na + , 23 Da) and potassium (K + , 39 Da) ions with hydrogen (H + , 1 Da), when some mixed flavonoid and phenolic acid derivatives are complexly fragmented at low concentrations [24,27,28].

New Succinyl-Glucosides and Phosphorylated Conjugates in Fermented Seeds
During cheonggukjang fermentation by B. subtilis AFY-2, succinylated and phosphorylated derivatives were newly generated from the steamed seeds [12,13], and detailed as succinyl-glucosides (Suc-Glu, 262 Da; peaks 15, 18, 20, 21, 23 [14,25], thus far, there are no experimental reports on the qualitative and quantitative determination of these derivatives via microbial phosphorylation from fermented soy products.   [14,25], thus far, there are no experimental reports on the qualitative and quantitative determination of these derivatives via microbial phosphorylation from fermented soy products.

Changes of 38 Isoflavone Derivatives during Steaming and Fermentation in Soybean Seeds
In the present study, the internal standard material (ISTD, 6-methoxyflavone) was used to evaluate the changes of isoflavones during steaming and fermentation (after 21, 36 and 60 h with B. subtilis AFY-2) in four selected soybean cultivars. The contents (mg/100 g, dry weight) of thirty-eight isoflavone derivatives are classified into acylated group (aglycone, Glu, Mal-Glu, Ac-Glu, Suc-Glu and Phos) according to their aglycones (D, Gn and Gy) in Table 2. TIFs could be distributed differently depending on cultivar type and processed condition of soybean seeds without their loss as follows: Daewon (199

Proposed Roadmap on Biotransformation of Soy Isoflavones during Steaming and Cheonggukjang Fermentation
Biotransformation on soy isoflavones during steaming and fermentation (by B. subtilis AFY-2) could be proposed through a roadmap concluded from the changes of present 38 derivatives (aglycone, Glu, Api-Glu, Mal-Glu, Ac-Glu, Suc-Glu and Phos) and related previous reports [14,23,25] (Figure 4(Bd)) were newly formed by phosphorylation at the 7-OH or 4 -OH position of the converted aglycones. Therefore, isoflavone glycosides (Mal-Glu, Ac-Glu and Glu) were gradually hydrolyzed into their corresponding aglycones by thermal procedure (steaming) and β-glucosidase of B. subtilis AFY-2 (fermentation). After conversion to aglycones, it was found that the enhanced succinylation and phosphorylation varied depending on cultivar type and fermentation time. To enhance Suc-Glu and Phos additionally, different Bacillus species should be further characterized by means of their catalytic properties. In the future, it will be also possible to evaluate bioconversion for representative glycosidic type from other isoflavone-rich resources such as kudzu, astragalus and red clover by considering this roadmap.

Identification and Quantification of Isoflavone Derivatives
The previously constructed LC-MS library [27] and flavonoid database ('RDA DB 1.0-Flavonoids' completed in 2016, Korea) [2] (pp. 99-103) were used to clearly and efficiently identify isoflavone derivatives from the soybean seed samples (raw, steamed and cheonggukjang-fermented), and composed of twenty compounds information including positive and negative fragment ions. The purposed isoflavones were tentatively determined by comparing the positive fragmentation, UV spectra and elution order presented in the library and database. In particular, some derivatives of them were further confirmed through consistence with 13 types of reference standards provided in Table 1. Nevertheless, since it was impossible to obtain all available standards in relation to the identified derivatives, the quantification for each compound was calculated as 1:1 without considering the relative response factor for pre-inserted ISTD (based on UV detection at 254 nm).

Statistical Analysis
Data were expressed as means ± standard deviations in their triplicated results. Significant differences were determined with one-way analysis of variance (ANOVA) followed by Duncan's multiple-range test using SPSS version 27.0 software (SPSS Inc., Chicago, IL, USA). The p-values < 0.05 were considered statistically significant.

Conclusions
In this work, thirty-eight isoflavone derivatives were comprehensively identified and quantified from the raw, steamed and fermented seed samples of four selected soybean cultivars based on high-resolution UPLC-DAD-QToF/MS results with reference to previously reported LC-MS library and flavonoid database (RDA DB 1.0 completed in 2016), and categorized into Glu, Api-Glu, Mal-Glu, Ac-Glu, Suc-Glu and Phos as acylated group along with D, Gn and Gy. Especially, Suc-Glu and Phos derivatives were newly generated during cheonggukjang fermentation by B. subtilis AFY-2, and among them, Phos were characterized for the first time in fermented soy products using Bacillus species. Most of the isoflavone quantification have been accurately evaluated in variation according to variety, cropping environment, storage duration and extraction solvent system of soybeans based on only 12 types of available standards (Glu, Mal-Glu, Ac-Glu and aglycones) [37,39,43]. However, since Suc-Glu (17.3-22.4%) and Phos (1.5-5.4%) have a significant proportion in 36 h-fermented cheonggukjang products, their absence can lead to a reduction of the TIFs. Additionally, the role of these derivatives should be elucidated in leading to higher isoflavone bioavailability after cheonggukjang consumption on human clinical studies [26]. Thus, the succinylated and phosphorylated isoflavones can be considered as important biomarkers generated newly during fermentation with Bacillus species, and a roadmap on isoflavone-based biotransformation proposed in this study can contribute to the enhanced production of these conjugates through metabolites regulation at each bioconversion stage. In the future, it is also necessary to carry out metabolomics approach to how isoflavone derivatives change according to type and concentration of microorganisms during fermentation as well as discover potential pharmacological activities from the Suc-Glu and Phos derivatives.