Strains of A. niger were cultivated on MEA (Malt Extract Agar, HiMedia Laboratories, Mumbai, India) agar plates (thickness of the solid medium 0.5 cm) at room temperature for 14 days.

Rings of the fungal cultures (d = 1 cm) were placed onto the start line on the TLC silica gel plate (Merck TLC aluminium silica gel, 20 × 20 cm, Merck KGaA, Darmstadt, Germany). The medium side of the rings placed onto the silica gel enables diffusion of the fungal exotoxins (excreted by the fungus into its growing medium, here MEA) into the sorbent (here silica gel), while the biomass side soaked with two drops of extraction mixture methanol/water (80:20, disrupting the fungal cell wall and extracting the toxicants present inside the cell) was used to detect fungal endotoxins (kept in fungal cells). Griseofulvin (10 μL of a solution 100 μg/ml chloroform) (Sigma Aldrich, St. Louis, United States) was applied as the standard. Dried TLC plates were eluted in a TEF mixture (toluene/ethyl acetate/formic acid 90%, 5:4:1) (Merck KGaA, Darmstadt, Germany) at the exotoxin side and in a CAP solution (chloroform/acetone/isopropanol, 85:15:20) at the side of endotoxins in preparation cuvettes (Camag, Muttenz, Switzerland) (Nielsen et al., 2020).

Upon completion of both elutions, the spots of extrolites (metabolites), including potentially present mycotoxins, were visualised. The production of OTA was confirmed, as presented by Filtenborg et al. (1989). The dried TLC plate was exposed to NH3 (Lachema, Brno, Czechia) vapour, and the spots were observed under UV light (254 nm) in the TLC cabinet Camag TLC Visualiser 2 (Camag, Muttenz, Switzerland). OTA spots gave blue-green and the griseofulvin spot blue fluorescence.

FB1 was detected using the method developed by Nelson et al. (1993). TLC plates were eluted in a mixture of chloroform/methanol/acetic acid (6:3:1). Dried plates were sprayed with 0.5% p-anisaldehyde in a mixture of ethanol/acetic acid/sulphuric acid (17:2:1) and heated at 140°C for 2–3 min. FB1 appears as lilac-deep lilac spots, the standard as a blue spot.

All the spots observed were characterised by their retention factors (RF). The ratio of RF of the spot analysed to the RF of the standard griseofulvin (always considered equal to one) was proving the particular mycotoxin production by comparison to the database of these ratio factors according to Filtenborg et al. (1989). The complex method is graphically documented in Figure 3.

Diagram of the TLC analysis of mycotoxins synthesised by the tested Aspergillus niger strains. MEA, malt extract agar; CAP, chloroform/acetone/isopropanol, 85:15:20; TEF, toluene/ethyl acetate/formic acid 90%, 5:4:1; OTA, ochratoxin A; FB1, fumonisin B1; Rf, retention factor.

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