**4.3 Comparison of isoflavone concentrations in soybeans fermented with mycelia versus non-fermented soybeans**

## *4.3.1 Methods: high-performance liquid chromatography (HPLC) analysis*

We followed the methods described in Kudou et al. [55] for measuring isoflavone concentrations in fermented and non-fermented soybeans. An LC-6A system (Shimadzu, Kyoto, Japan) equipped with a PEGASIL-ODS (4.6 mm i.d. × 250 mm) HPLC column (Senshu Scientific, Tokyo, Japan) was used for analysis. HPLC parameters used for the measurement of different isoflavones, such as genistein, daidzein, glycitein, daidzin, and glycitin, both in the glycosylated and in aglycone forms, are detailed in Suruga et al. [10].

### *4.3.2 Methods: liquid chromatography/mass spectrometry (LC/MS) analysis*

An ACQUITY UPLC apparatus (Waters MS Technologies, Manchester, UK) equipped with a reversed-phase Acquity UPLC CHS C18 column with a particle size of 2.1 mm × 100 mm × 1.7 μm (Waters MS Technologies) was used for LC/MS analysis. Parameters of analysis are documented in detail in Suruga et al. [10].

#### **Figure 7.**

*Inhibition of alpha-glucosidase activity soybeans fermented by mushroom mycelia. (A) Yeast alpha-glucosidase inhibition using pNP-glucoside as substrate, (B) mammalian alpha-glucosidase inhibition using maltose as substrate, and (C) mammalian alpha-glucosidase inhibition using sucrose as substrate. Results are expressed as mean ± SD (n = 3). N.D.: not detectable. 1: p < 0.01 vs. control, 2: p < 0.01 vs.* G. lucidum*, 3: p < 0.01 vs.*  H. erinaceum*, 4: p < 0.01 vs.* H. ramosum*, and 5: p < 0.05 vs.* H. ramosum *[10].*
