Whatman 1ps phase separator

Pre-cultures of strains MACI46 (NRRL 66708), MACI219 (NRRL 66709), MACI254 (NRRL 66710) and MACI264 (NRRL 66711) incubated in the dark at 27 °C for seven days. For metabolite profile characterization, isolates were cultured in four different media: MEA, CYA, YES agar, and Potato Dextrose Agar medium (PDA) (Sigma-Aldrich, Saint Quentin Fallavier, France). For each medium, three biological replicates were inoculated centrally with 10 µL of calibrated spore suspensions (10⁵ spores/mL) prepared from seven-day cultures on 7.5 cm Petri dishes. The samples were incubated in the dark at 27 °C for seven days.
To perform extrolite extractions, culture media were macerated and placed in 50 mL sterilized tubes, and thereafter 35 mL of ethyl acetate was added to each sample. Samples were agitated 48 h in an orbital incubator at 170 rpm at room temperature. Ethyl acetate was filtered through a Whatman 1PS phase separator (GE Healthcare Life Sciences, Vélizy-Villacoublay, France) and evaporated at 60 °C until dry. Samples were then dissolved in 400 µL of methanol. To eliminate possible impurities, each sample was filtered through a 0.45 µm disk filter (ThermoFisher Scientific, Illkirch, France) [81 (link)].

AFB1 analysis was done as described by El Khoury et al. [19 (link)]. In brief, AFB1 extraction was done with 30 mL of HPLC-grade absolute chloroform. Next, supernatants were filtered through a Whatman 1PS phase separator (GE Healthcare Life Sciences, Vélizy-Villacoublay, France). Two milliliters of filtrate were evaporated to dryness in a STUART SBH200D/3 sample concentrator (Stuart equipment, Paris, France) at 45 °C, and the samples were re-solubilized in 2 mL of acetonitrile. Finally, the samples were filtered through 0.45 μm disk filters (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) and placed in HPLC vials. AFB1 was analyzed by using an Ultimate 3000 UPLC (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) with an EvoC18 column (3 µm, 150 × 3.2, Phenomenex, Le Pecq, France) conditioned at 27 °C. The elution program used for separation consisted of forming an isocratic mixture composed of acetonitrile and water (25:75 v:v). The mobile phase had a flow rate of 1.2 mL/min. Ten microliters of sample were injected. AFB1 was detected by using a fluorescent detector at 365 (430) nm excitation (emission) wavelengths. The identity of the molecule was confirmed by analyzing the UV absorption spectrum by an additional diode matrix detector (DAD) coupled to the system. AFB1 production levels were calculated based on a standard calibration curve (0.16 to 20 mg/L).

Quantitative determination of aflatoxin B1 production by HPLC-FLD was adapted from Caceres et al. [31 (link)]. Culture media were first mixed with 30 mL of chloroform and agitated for 2 h on a horizontal shaking table (150 rpm at room temperature). Chloroform extracts were then filtered through a Whatman 1PS phase separator (GE Healthcare Life Sciences, Vélizy-Villacoublay, France) and evaporated to dryness at 60 °C. The residue was dissolved in a water–acetonitrile–methanol (65:17.5:17.5; v:v:v) mixture, then filtered (0.45 µm PTFE disks). Ten microliters were finally injected into the HPLC system, a Dionex Ultimate 3000 UHPLC system (Thermo Scientific, Illkirch, France) and analyzed using a LC column Luna^® C18 (125 × 2 mm, 5 μm, 100 Å) (Phenomenex, Torrance, CA, USA) at 30 °C. The eluent flow was 0.2 mL/min in an isocratic program with eluent A: eluent B (82.5%:17.5%) where eluent A is a mixture of acidified water (0.2% of acetic acid): acetonitrile (79:21; v:v) and eluent B is pure methanol. AFB1 was detected by FLD at 365 nm/430 nm as excitation and emission wavelengths, respectively. AFB1 peaks were confirmed using a coupled diode array detector (DAD) and sample concentrations were calculated based on a linear calibration curve of standards.