1220 infinity 2

The LC system (Agilent 1220 Infinity II) is equipped with an Ultraviolet/Visible (UV/Vis) detector, and ChemStation software is used. Hettich UNIVERSAL 320 R centrifuge and Ika Vortex Genius were used during the analysis. Microcon^® centrifugal filters (<3 kD) were purchased from EMD Millipore.

P. igniarius was cultured in MMM medium for seven days at 28 °C. An agar patch (1 cm × 1 cm) was added into a 250 mL conical bottle containing 150 mL liquid medium (the above MMM without agar). The culture conditions were 28 °C, 180 rpm. Then, 15 days later, the TL, which was dissolved in double distilled water, was added into the fermentation liquor at a final concentration of ~ 0.1 mg/mL. The co-culture was then allowed to grow for another 9 days under the same conditions. Samples (2.0 mL) were taken at 1, 3, 5, 7, and 9 days after the addition of TL, and were extracted by EtOAc (3 × 2.0 mL). The organic layer was removed by volatilization and the residue was dissolved in 2 mL methanol for HPLC detection. HPLC was conducted using Agilent 1220 Infinity II equipment with Agilent Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm) as the separation column. Two-phase gradient elution methods were used in this work including 0.2% formic acid (an aqueous solution) as phase A, and MeOH as phase B. The MeOH fraction in phase B was gradually increased from 40% (V/V) to 80% (V/V) over 25 min. The spectra were recorded by a DAD detector at 380 nm. The flow velocity was set to 1 mL/min, and the injection volume was 10 μL.

The concentration of mycotoxins (DON and two acetylated DONs) and their metabolites in the supernatant from incubation samples was analyzed and quantified. The mycotoxin stock solutions were diluted in acetonitrile and freshly prepared. Calibration curves were built from stock solutions by creating nine standards as follows: 1, 2.5, 5, 10, 25, 50, 100, 250, and 500 µM. The analysis was undertaken using an Agilent 1220 Infinity II system connected to a photodiode array detector (Agilent Technologies, Palo Alto, California, CA, USA). The column was an Agilent TC-C18 (250 mm × 4.6 mm). The flow rate was 1.0 mL/min. 20 μL of each sample was injected with a mobile phase composed of nanopure water (A) and acetonitrile (B). The gradient elution program was as follows: starting condition 100% A, 1.0 min 20% A, 20.0 min 20% A, 25.0 min 100% A, 30.0 min 100% A. The concentration of mycotoxins and their metabolites was obtained by comparing the peak areas to the corresponding built calibration curves at a wavelength of 237 nm.