2.6. Sterol Detection and Quantification

Lipid extraction from fungal cultures was done as described before [41]. Briefly, this involved inoculation of 200 mL of malt extract broth (MEB; 2% Bacto™ malt extract [BD BioSciences, San Jose, CA, USA]) with a small block of agar overgrown with mycelium and incubation for 14 days at 25 °C in the darkness with shaking at 150 rpm (222DS Benchtop Shaking Incubator; Labnet international, Edison, NJ, USA). Fungal tissue was then collected by filtration through filter paper (Whatman, Maidstone, United Kingdom), washed with distilled water, and freeze-dried. Sterols were then extracted from 50 mg lyophilized mycelium by saponification at 80 °C for 90 min in a 500 µL solution containing 10% (w/v) KOH in methanol. To this mixture, 250 µL distilled water was added and left to cool to 25 °C. The solution was then subjected to three rounds organic extraction using 1 mL hexane. The pooled hexane fractions were evaporated to dryness under a stream of nitrogen and dissolved in 100 µL methanol.

The samples were derivatized using 50 µL N-methyl-N-trimethylsilyl-trifluoroacetamide (Macherey-Nagel, Dueren, Germany) for 2 h at 40 °C before analysis using a gas chromatograph attached to a quadrupole mass spectrometer with an electron impact ion source (Agilent 5973 6890 GC MS, Agilent, Santa Clara, USA). A standard 30 m HP-5ms capillary column (Agilent) was used with a constant flow of helium of 1 mL min⁻¹. Sample (1 µL) was injected with a 1:10 split ratio. The injector temperature was kept at 230 °C. The oven temperature was ramped from an initial 70 °C to 320 °C at a constant rate of 6 °C min⁻¹, with a final hold time of 3 min. The mass spectrometer was operated in full scan mode with a mass range of 50–350 m z⁻¹. The ion source was kept at 70 eV at 250 °C.

Compounds were identified by spectral comparisons with the NIST library version 12 (National Institute for Standards and Technology, Boulder, USA), retention indices and when available, retention times of pure standards. Peaks were integrated and quantified relative to a calibration curve produced for cholesterol. All experiments were done in three independent replications. Data were imported into the open-source R-based statistics program Metaboanalyst (https://www.metaboanalyst.ca/faces/home.xhtml accessed on 5 January 2021), normalized using the natural logarithm. Hierarchical clustering was performed with the hclust function in the R package using Euclidean distance as the similarity measure and the Ward’s linkage clustering algorithm.