Production and Characterization of Fungal Secretomes

Based on previous studies [12 (link),13 (link)], fungal cultures were grown in 250 mL baffled Erlenmeyer flasks with 100 mL medium containing 2.5 g/L⁻¹ of maltose as a starter, 15 g/L⁻¹ (based on dry matter) of an autoclaved MB fraction (provided by ARD, Pomacle, France), Avicel® (Avicel PH-101, Sigma-Aldrich), WS and WS-R as a carbon source, 1.842 g/L⁻¹ of diammonium tartrate as a nitrogen source, 0.5 g/L⁻¹ yeast extract, 0.2 g/L⁻¹ KH₂PO₄, 0.0132 g/L⁻¹ CaCl₂/2H₂O and 0.5 g/L⁻¹ MgSO₄/7H₂O. In parallel, a reference culture was made with 20 g/L⁻¹ of maltose as a carbon source. Cultures were incubated in the dark at 30°C with shaking at 120 rpm. The cultures were stopped 10 days after inoculation in all the inducing conditions, and the culture broths (secretomes) were filtered (using 0.2 μm polyethersulfone membrane, Vivaspin, Sartorius), diafiltered with 50 mM acetate solution buffer pH 5.2, concentrated (Vivaspin with a 10 kDa cut-off polyethersulfone membrane, Sartorius) and then stored at −20°C until use.
The total amount of proteins was assessed using Bradford assays (Bio-Rad Protein Assay Dye Reagent Concentrate, Ivry, France) with a BSA standard that ranged from 0.2 to 1 mg/mL⁻¹. Gel electrophoresis and carboxymethyl cellulose zymograms were performed as described elsewhere [49 (link)].