0.45 m ptfe syringe filters

For PLA quantification in microbial cultures in liquid Ym synthetic medium, 1 mL of culture media was withdrawn at each sampling time and filtrated through 0.45 µm PTFE syringe filters (Thermo Fisher Scientific) to eliminate microorganisms from the supernatant before injection to HPLC apparatus. Regarding micro-malting assays, PLA was quantified in the same extracts as for the T-2 toxin quantification. Analyses of PLA were performed using a Luna C18(2) column (5 µm, 250 × 4.6 mm) and a pre-column with the same characteristics (Phenomenex). The PLA was detected and quantified using HPLC-DAD according to the methodology previously described by Kawtharani et al. [20 (link)]. PLA quantification was calculated according to a standard calibration curve with concentrations ranging between 0.01 and 1 g/L. In micro-malting experiments, PLA concentrations are expressed in g/g.

At each sampling time, 1 mL of culture media was withdrawn and filtrated through 0.45 µm PTFE syringe filters (Thermo Scientific Fisher, Villebon-Sur-Yvette, France) to eliminate microorganisms from the supernatant prior to injection into HPLC apparatus. Analyses of PLA were performed using a Luna C18(2) column (5 µm, 250 × 4.6 mm) and a pre-column with the same characteristics (Phenomenex, Torrance, CA, USA). The detection of PLA was performed using a Dionex Ultimate 3000 UHPLC system coupled with a diode-array detector (DAD) set at 210 nm (Thermo Fisher Scientific, Illkirch, France). The analysis was performed in a gradient mode using acidified water (0.2% of acetic acid glacial) as solvent A and pure HPLC grade acetonitrile as solvent B. Flow was set at 1.2 mL/min with A/B ratios of 90:10, 50:50, 50:50, 0:100 and 90:10, with run times of 0.0, 4.0, 9.0, 10.0 and 15.0 min, respectively. Injection volume was set at 50 µL. PLA quantification was calculated according to a standard calibration curve with concentrations ranging between 10 and 1000 mg/L.