The G. lucidum strain 5.1 from the collection of the Laboratory of Biosynthesis of Biologically Active Substances, Gauze Institute of New Antibiotics was used in this study. This strain was used in phylogenetic studies and the studied RNA sequence was deposited in the GenBank. Stock cultures were grown on potato glucose agar (PGA) at 26 °C for 7 days and then stored at 4 °C.
The liquid seed culture of G. lucidum was grown in 750 mL Erlenmeyer flasks containing 100 mL of liquid nutrient medium at 26 °C for 6 days. Unhopped beer wort (4° according to Balling scale) and pH 6.0 before sterilization was used as a liquid seed medium. The seed liquid medium was inoculated with mycelial agar plugs (3 mm diam.) of a seven-day culture of basidiomycete on PGA at the rate of 1/4 tube per flask.
Submerged cultivation of G. lucidum was carried out on a rotary shaker at 220 rpm and at 26 °C in 750 mL Erlenmeyer flasks containing 100 mL of nutrient medium inoculated with 10 mL of liquid seed culture for 7 days. The liquid nutrient medium for submerged cultivation was contained (g/l of water): anhydrous glucose 20.0, yeast extract (Serva) 10.0, potassium dihydrogen phosphate 2.0, and magnesium sulfate 0.2. All ingredients of the culture medium used were water-soluble, which ensured that no non-fungal compounds presence in the target films.
All nutrient media used in this work were sterilized at 1.2 atm for 30 min. The microbiological purity of cultures at all stages of work was controlled by using a light microscope BX41 (Olympus, Tokyo, Japan). The morphological characteristics of the submerged mycelium of the G. lucidum were observed by SEM using a high-resolution scanning electron microscope Mira II (Tescan, Warrendale, PA, USA).
After cultivation, the G. lucidum submerged mycelium was separated from the culture liquid by filtration, washed twice with distilled water, and lyophilized (Figure 2 ). The dry mycelium was ground with a T 25 ULTRA-TURRAX digital homogenizer (IKA, Staufen, Germany) at 10,000 rpm for 5 min in 48% aqueous ethanol. The ratio of mycelium to aqueous ethanol was 12.5 mg/mL. As a result, crushed mycelium of the G. lucidum in a solvent system (MEGl) was obtained. After that, the resulting suspension was centrifuged at 3500 rpm for 5 min and the supernatant was pipetted. As a result, the supernatant of a water/ethanol extract of the G. lucidum mycelium extract (EGl) was obtained.
The liquid seed culture of G. lucidum was grown in 750 mL Erlenmeyer flasks containing 100 mL of liquid nutrient medium at 26 °C for 6 days. Unhopped beer wort (4° according to Balling scale) and pH 6.0 before sterilization was used as a liquid seed medium. The seed liquid medium was inoculated with mycelial agar plugs (3 mm diam.) of a seven-day culture of basidiomycete on PGA at the rate of 1/4 tube per flask.
Submerged cultivation of G. lucidum was carried out on a rotary shaker at 220 rpm and at 26 °C in 750 mL Erlenmeyer flasks containing 100 mL of nutrient medium inoculated with 10 mL of liquid seed culture for 7 days. The liquid nutrient medium for submerged cultivation was contained (g/l of water): anhydrous glucose 20.0, yeast extract (Serva) 10.0, potassium dihydrogen phosphate 2.0, and magnesium sulfate 0.2. All ingredients of the culture medium used were water-soluble, which ensured that no non-fungal compounds presence in the target films.
All nutrient media used in this work were sterilized at 1.2 atm for 30 min. The microbiological purity of cultures at all stages of work was controlled by using a light microscope BX41 (Olympus, Tokyo, Japan). The morphological characteristics of the submerged mycelium of the G. lucidum were observed by SEM using a high-resolution scanning electron microscope Mira II (Tescan, Warrendale, PA, USA).
After cultivation, the G. lucidum submerged mycelium was separated from the culture liquid by filtration, washed twice with distilled water, and lyophilized (
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