*4.4. Mycotoxin Extraction and HPLC Measurements*

The amount of mycotoxin was determined by high performance liquid chromatography (HPLC) analysis using a YL9100 HPLC system equipped with a YL9150 autosampler (YL Instruments, Gyeonggi, Korea). For the measurement, the mycotoxin was extracted from the samples by the following steps. For the extraction of the mycotoxin from the biomass, 1.8 mL of dichloromethane and 0.2 mL of methanol were added to the Falcon tube containing the biomass, using the ratio that gave best results in preliminary experiments. The mixture was pipetted into Eppendorf tubes. The tubes were vortexed in a horizontal shaker for 20 min in the dark and then centrifuged at 3000 rpm for 10 min. One ml of the supernatant was taken out and the solvent was evaporated to the dryness in a clean Eppendorf tube at 45 ◦C under a fume hood in a Thermo Shaker (TS-100, Biosan, Riga, Latvia). MRS broth was extracted similarly, but 1 mL of dichloromethane was shaken with one milliliter of supernatant for 20 min. From the dichloromethane phase, 0.5 mL was taken out and concentrated in a clean Eppendorf tube at 45 ◦C under a fume hood. The residues were solved in 1 mL of eluent (see below) and the sample was filtered through a 0.45 µm hydrophilic polytetrafluoroethylene (PTFE) syringe filter (Labex Ltd., Budapest, Hungary) prior to HPLC determination.

The mycotoxin content of the samples was determined by UV detection (HPLC-UV) after an isocratic liquid chromatographic separation. UV detector signals were recorded at λ = 365 nm or λ = 240 and 325 nm for AFB1 and ST, respectively. The separation was performed on a Brisa (Technochroma, Barcelona, Spain) C18 column (5 µm, 15 cm × 0.46 cm) at 30 ◦C. The eluent flow rate was set to 1.0 mL/min and 30 µl of samples were injected. The eluent consisted of 60:20:20 = A:B:C eluents, and 40:30:30 = A:B:C eluents (A = 90% water: 10% MeOH, B = MeOH, C = Acetonitrile), held till 8 and 12 min for AFB1 and ST, respectively. Extracts of blank non-spiked control biomass did not contain interfering matrix components, therefore quantitation was based on instrumental (external) calibration with standard solutions in the range between 0.010 and 2.00 µg/mL. Recoveries at concentration of 0.2 µg/mL in the spiked samples were determined by adding a known concentration of AFB1 or ST to the liquid of blank samples. Peak purities were systematically checked by recording absorption at two wavelengths, and peak area ratios at those wavelengths were compared to the ratios characteristic to standard solutions of the analyte (ST). Binding capacities (%) were calculated on the basis of analyte concentrations in the extracted biomass samples related to the initial MRS broth levels considering the corresponding concentration factor applied (see sample preparation, above). RSD values calculated from the three parallel injections of standard solutions ranged between 0.2 and 1.4%.

**Author Contributions:** Conceptualization, J.K. (József Kukolya); methodology, J.K. (Judit Kosztik), I.B.-V., M.M. and A.S.; validation, M.M.; investigation, J.K. (Judit Kosztik) and I.B-V.; writing—original draft preparation, J.K. (Judit Kosztik), I.B.-V. and M.M.; writing—review and editing, I.B.-V. and A.S.; supervision, J.K. (József Kukolya) and A.S.; funding acquisition, J.K. (József Kukolya). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Research, Development and Innovation Office in Hungary, projects NVKP-16-1-2016-0009, NVKP-16-1-2016-0049 and the Hungarian National Research Fund, OTKA K116631.

**Conflicts of Interest:** The authors declare no conflict of interest.
