The cultures were centrifuged, re-suspended in saline, and set to achieve an optical density of 1.3 at a wavelength of 546 nm. In the case of minimal medium

(MM1), cultures were washed one time with saline to get rid of complex media used for inoculation. Two hundred ml R428 of complex medium (DSMZ 1, KM 1, and KM 5) containing agar were inoculated with 2 ml of this defined suspension of organisms (OD = 1.3). Ten ml of inoculated agar were poured into each Petri dish. Streptomyces pure culture filtrate (10 μl) or organic extract (10 μl) was applied on paper discs (diameter: 6 mm) and air dried. The paper discs were then placed on the previously prepared agar media. After 24 h, microbial growth inhibition was recorded by measuring the diameter of the inhibition zone. Fermentation of streptomycetes for the analysis of secondary metabolites The strains AcM9, AcM11, AcM20, AcM29 and AcM30 were cultivated in 100 ml ISP-2-medium at 120 rpm and 27 °C for 3 days. Of these cultures, four ml were used to inoculate 100 ml SGG, OM and MMN medium in 500 ml-Erlenmeyer flasks with one baffle. SGG-medium consisted of 10 g soluble starch, 10 g glucose, 10 g glycerol, 2.5 g cornsteep powder (Marcor, Hartge Ingredients, Hamburg), 5 g Bacto peptone (Difco), 2 g yeast extract (Ohly Kat, Deutsche Hefewerke, Hamburg), 1 g NaCl and 3 g CaCO3 per liter of tap water. The pH was adjusted to pH 7.3 prior to sterilization.

OM medium consisted of 20 g oat meal (Holo Hafergold, Selleckchem Osimertinib Neuform, Zarrentin) selleck screening library and 5 ml of the following micronutrient solution: 3 g CaCl2x2 H2O, 1 g iron-III-citrat, 200 mg MnSO4 x 1 H2O, 100 mg ZnCl2, 25 mg CuSO4 x 5H2O, 20 mg Na2B4O7 x 10 H2O, 4 mg CoCl2 x 6H2O, and 10 mg Na2MoO4 x 2 H2O per liter of deionized water. The pH

was adjusted to pH 7.3 prior to sterilization. Modified MMN medium was prepared according to Molina and Palmer [49]. Mocetinostat supplier Fermentations were carried out on a rotary shaker at 120 rpm and 27°C. After 2, 4 and 6 days (24, 48 and 72 hours) 10 ml of bacterial culture were centrifuged (3800 rpm, 10 min) and bacterial biomass was determined (volume percent). The culture filtrate – separated from the bacterial mycelium by centrifugation – was used for further analyses of secreted bacterial metabolites. Extraction and HPLC-UV-visible spectral analysis of Streptomyces secondary metabolites Culture filtrates (5 ml) of AcM 9, AcM11, AcM20, AcM29 and AcM30 were adjusted to pH 5 and extracted with 5 ml ethyl acetate for 30 min under shaking conditions. The organic extracts were concentrated to dryness using vacuum evaporator and resuspended in 0.5 ml of methanol. The 10-fold concentrated extracts were centrifuged (3 min, 13 000 rpm) and 5 μl of each sample was subjected to HPLC on a 5 μm Nucleosil C18-column (Maisch, Ammerbuch, Germany, 125 mm x 3 mm, fitted with a guard-column: 20 mm x 3 mm) with 0.1% -o-phosphoric acid as solvent A and acetonitrile as solvent B at a linear gradient (from 4.