Dual ajs esi

Characterization of the components of JBT was performed by a 1260 series HPLC instrument (Agilent, Waldbronn, Germany) connected to an Agilent 6530 Q-TOF mass spectrometer (Agilent Corp., USA) equipped with Dual Agilent Jet Stream Electrospray Ionization (Dual AJS ESI). The operating parameters were optimized in both positive and negative modes, as follows: capillary voltage, 3,500 V for ESI mode and 4000 V for ESI+ mode; nozzle voltage, 500 V; fragmentor, 110 V; nebulizer, 45 psi; drying gas temperature, 300°C; drying gas flow rate, 6 L/min; sheath gas temperature, 320°C; and sheath gas flow rate, 12 L/min. Separation of compounds in JBT was carried out on an ACE Excel 3 Super C18 column (100 mm × 2.1 mm; Advanced Chromatography Technologies Ltd., Aberdeen, Scotland). The mobile phase was composed of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid), with a flow rate of 0.35 ml/min. Gradient elution was performed as follows: 0–30 min, 95%–23% A; 30–40 min, 23%–15% A; 40–45 min, 15%–10% A; 45–50 min: 10%–5% A; 50–55 min, 5% A; 55–56 min: 5%–95% A; and 56–66 min, 95% A. The column temperature was maintained at 40°C. System operations and data analysis were conducted on Masshunter Workstation software (Agilent Technologies, USA).

The fermentation product is added to 80% ethanol at a ratio of 1:30, sonicated at 200 W for 30 min, and filtered. The filtrate was filtered by adding 80% ethanol at a ratio of 1:20 and sonicated at 200 W for 30 min. The filtrate was filtered by adding 80% ethanol at a ratio of 1:10 and sonicating at 200 W for 30 min. The filtrate was combined to obtain the crude extract of flavonoids, which was left at 4°C overnight and filtered. The filtrate was concentrated by a rotary evaporator (−0.1 MPa, 80°C) to obtain the flavonoid extract. The fermentation liquid was analyzed by HPLC-Q-TOF-MS on a Waters HSS-T3 column (150 $\times$ 2.1 mm, 3.5 μm) using 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B. The extract was centrifuged at 10,000 r/min for 10 min and the supernatant was filtered through a 0.22 μm membrane. Gradient elution (0–3 min, 0% B; 3–10 min, 0–10% B; 10–20 min, 10–40% B; 20–30 min, 40–80% B; 30–32 min, 80% B; 32.1–35 min, 0% B). After Agilent Dual AJS ESI ion source, positive and negative ion scanning (drying gas (N₂) temperature 300°C, flow rate 8 L/min; nebulizing gas (N₂) pressure 35 psi; sheath gas temperature 350°C; sheath gas flow rate 11 l/min; elect rospray voltage 3,500 V; capillary outlet voltage 150 V; cone hole voltage 65 V; octopole voltage 750 V; scan range:m/z 100-1,000; collision energies 10, 20, 40 eV) to obtain results.