Different growth media were used for the optimal production of peptaibols. The media were (i) potato dextrose broth (PDB); (ii) alkaline media based on phosphate buffers (pH 10 (±0.2); and (iii) Chapek–Doks media according to the previous protocol [4 (link),14 (link),53 (link)]. The effect of pH on the growth rate was evaluated in triplicate on plates with several growth media based on citrate, phosphate, and carbonate buffers (pH 4, 5, 6, 7, 8, 9, 10 (±0.2)). The contents of the alkaline media and buffers composition were followed by Grum–Grzhimaylo et al. [1 (link)]. For the production of EmiA, homologous fungi were cultivated at 26 °C in Erlenmeyer flasks under stationary conditions for 14 days [8 (link)]. The culture fluid (CF) was separated by filtration through membrane filters on a Seitz funnel under a vacuum. To isolate the antibiotic substances, the CF of the producers was extracted three times with ethyl acetate in an organic solvent/CF ratio of 1:5. The obtained extracts were evaporated in a vacuum on a Rotavapor rotary evaporator (Buchi, Flawil, Switzerland) to dryness at 42 °C, the residue was dissolved in aqueous 70% ethanol, and the alcohol concentrates were obtained [14 (link)].
Rotavapor rotary evaporator
Manufactured by Büchi
Sourced in Switzerland
The Rotavapor rotary evaporator is a laboratory equipment used for the efficient removal of solvents from liquid samples through controlled evaporation. It consists of a rotating evaporation flask, a condensing system, and a vacuum system to facilitate the evaporation process.
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Lab products found in correlation
5 protocols using rotavapor rotary evaporator
1
Optimization of Peptaibol Production
2
Purification of Methotrexate-loaded Nanoparticles
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A solution of MTX-loaded NPs was prepared by dissolving 1.2 mg (±0.1) in 2.00 mL distilled water. To 1 mL of the MTX-loaded NP solution, 100 µL of 1% BSA solution was added. The solution then was incubated for 2 h at 38°C with shaking at 350 rpm. The sample was then collected and dried using a Rotavapor rotary evaporator (BUCHI, Flawil, Switzerland), weighed, labelled, and stored at 4°C.
The obtained white solid of (MTX-loaded) NPs was purified by dialysis. A 100-µm membrane was placed on each side of a 1,000-µL double-sided reservoir and was filled with 1 mL of NP solution (MTX-loaded NP solution); this was placed in 250 mL of 0.06 M Phoshphate Buffer Saline (PBS) with stirring. The PBS solution was changed every 3 h, a total of three times.
The obtained white solid of (MTX-loaded) NPs was purified by dialysis. A 100-µm membrane was placed on each side of a 1,000-µL double-sided reservoir and was filled with 1 mL of NP solution (MTX-loaded NP solution); this was placed in 250 mL of 0.06 M Phoshphate Buffer Saline (PBS) with stirring. The PBS solution was changed every 3 h, a total of three times.
3
Cultivation and Extraction of Antibiotic Compounds from Fungi
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Fungi were cultivated according to the previous protocol on a special alkaline medium, containing, per liter, the following: (1) Na2CO3—24 g, NaHCO3—6 g, NaCl—5 g, KNO3—1 g, and K2HPO4—1 g; and (2) malt extract—17 g, yeast extract—1 g. Components 1 and 2 were autoclaved separately for 20 min at 120 °C and mixed together after cooling, which resulted in a final pH of 10 [9 (link)]. Fungi were grown in 500 mL flasks for 14 days at 26 °C in Erlenmeyer flasks [15 (link)]. The culture liquid (CL) was separated by filtration through membrane filters MF-Millipore, Merk (Darmstadt, Germany) on a Seitz funnel under vacuum. To isolate the antibiotic substances, the CL of the producers was extracted three times with ethyl acetate in an organic solvent/CL ratio of 1:5. The extracts were evaporated under vacuum on a Rotavapor rotary evaporator (Buchi, Flawil, Switzerland) to dryness at 42 °C, the residue was dissolved in aqueous 70% ethanol, and the alcohol concentrates were obtained.
4
Fungal Antibiotic Extraction Protocol
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The fungi were cultivated according to the previous protocol on a special alkaline medium at 26 °C in Erlenmeyer flasks under stationary conditions for 14 days [21 (link)]. The culture fluid (CF) was separated by filtration through membrane filters on a Seitz funnel under a vacuum. To isolate the antibiotic substances, the CF of the producers was extracted three times with ethyl acetate in an organic solvent/CF ratio of 1:5. The obtained extracts were evaporated in a vacuum on a Rotavapor rotary evaporator (Buchi, Switzerland) to dryness at 42 °C, the residue was dissolved in aqueous 70% ethanol, and the alcohol concentrates were obtained.
5
Polyphenol Extraction from Pomegranate Peel
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The polyphenolic extract was obtained from Punica granatum L. peel (Mollar de Eche cultivar) delivered from Spain. The dried peel of pomegranates was shredded in a Thermomix domestic kitchen appliance. The resulting material (1 kg) was extracted and re-extracted twice with 50% ethanol. The extraction process was conducted in an ultrasonic bath for 25 min. The extract was subsequently concentrated with a Rotavapor rotary evaporator (BÜCHI Labortechnik, Flawil, Switzerland) in a water bath at 40°C. The concentrated ethanol extract from the pomegranate peel was passed through a column with Amberlite XAD-16 resin (Brenntag, Essen, Germany), and the column was washed with distilled water to rinse out the organic acids, sugars and other undesirable compounds. Polyphenols were eluted with 80% ethanol. The collected fractions were dried in an SPT-200 vacuum oven (Zeamil, Kraków, Poland).
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