Genistein

The following flavonoids were used in this study: biochanin A (>98%, Tokyo Chemical Industry, B4098), (+)-catechin hydrate (98%, Tokyo Chemical Industry, C0705), chrysin (98%, Alfa Aesar, L14178), cyanidin chloride (98%, Fujifilm Wako Chemicals, 030-21961), daidzein (DAI; ≥98%, Nagara Science, NH010102), desmethylglycitein (DMG; >95%, Tokyo Chemical Industry, T3473), S-equal (≥97%, Merck, SML2147), fisetin (>96%, Tokyo Chemical Industry, T0121), genistein (>98%, Tokyo Chemical Industry, G0272), 2′-hydroxyflavanone (>98%, Tokyo Chemical Industry, H1024), kaempferol (≥98%, Cayman Chemical Company, 11852), luteolin (95%, Fujifilm Wako Chemicals, 127-06241), myricetin (>97%, Tokyo Chemical Industry, M2131), naringenin (>98%, Alomone Labs, N-110), petunidin (≥98%, Cayman Chemical Company, 19755), quercetin (96.5%, Fujifilm Wako Chemicals, 512-58344), tamarixetin (>99%, Extrasynthese, 1140S), 5,3′,4′-trihydroxyflavone (>85%, Toronto Research Chemicals, T896685), and 7,3′,4′-trihydroxyflavone (>85%, Toronto Research Chemicals, T896780).

The preparation of miso and PFM extracts and UPLC analysis were performed essentially as described elsewhere [22 (link),23 (link)]. One gram of miso or pre-fermented miso was extracted with 80% methanol at 37 °C for 2 h and then centrifuged at 1000× g for 5 min to collect 1 mL of supernatant samples. After drying in an evaporator, these supernatant samples were extracted with ethyl acetate to collect the extraction phase. The extraction phase samples were dried with nitrogen gas and dissolved in 200 µl of mobile phase consisting of 16 mM acetic acid (pH 4.6) and acetonitrile (70:30 v/v). After filtration using 0.22 µm PTFE membrane filters (Merck Millipore), samples were analyzed by ultra-performance liquid chromatography (UPLC) as described below.
The UPLC analysis was performed using an ACQUITY UPLC H-class system (Waters, Milford, MA, USA) with a turnable UV (TUV) detector. An ACQUITY UPLC^®® BEH C18 column (Waters, 2.1 × 100 mm, 1.7 µm) was used to analyze samples. The samples were separated using an isocratic mobile phase at a flow rate of 0.45 mL/min. Purified daidzein (Wako Pure Chemical) and genistein (Tokyo Chemical Industry, Tokyo, Japan) were used as the standards. Daidzein and genistein were monitored by UV absorption at 260 nm.

The following compounds were purchased to carry out the micronization process: poly(vinyl acetate) (PVA), purity > 98.0%, Tokyo Chemical Industries (Portland, OR, USA); genistein, purity > 98.0%, Tokyo Chemical Industries (Portland, OR, USA); ethyl acetate was purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland).
The description of the procedure for obtaining genistein in nano- and microforms was presented in the publication of Banyś et al. [9 (link)]. genistein in macroform was used as received. In order to study the mean size of genistein particles and their zeta potential, the dynamic light scattering (DLS) method was used. A Zetasizer Nano ZS analyzer (Malvern Instruments, Westborough, MA, USA) equipped with a red laser, with a wavelength of 633 nm and a scattering angle of 173° at 25 °C, was used for the tests. The sample was suspended in distilled water (zeta potential measurement) or in aqueous glycerol (particle size measurement). The obtained test results are presented in Table 1.

To analyze WT and NU19 susceptibility to plant-derived antimicrobials, WT and NU19 were grown at 28 °C on KB medium. The bacterial suspensions were standardized to an OD₆₀₀ of 0.01 with KB, and after 6 h incubation, 200 µM antimicrobials, including brassinin (Merck KGaA), sulforaphane (Funakoshi), camalexin (Merck KGaA), daidzein (INDOFINE Chemical Company, Hillsborough, NJ, USA), genistein (Tokyo Chemical Industry, Tokyo, Japan), indole (Tokyo Chemical Industry), and phloretin (Funakoshi), was added to each sample. Bacterial growth was measured at OD₆₀₀ after 24 h incubation.

Trans-RSVT was purchased from Caymen Chemicals. Apigenin, chrysin, luteolin, and quercetin were purchased from Alfa Aesar. Myricetin, piceatannol, and pterostilbene were purchased from Enzo Life Sciences. Genistein and doxycycline were purchased from Tokyo Chemical Industry. All drugs were dissolved in DMSO.