The extraction of prodigiosin from the PU foam cubes of 500 mL cultures was performed with a Soxhlet extractor. The red-colored foam cubes were recovered from the cell culture by sieving, wrung out and charged into a 250 mL Soxhlet apparatus fitted with a reflux condenser and a 500 mL round bottom flask filled with 300 mL of acidified ethanol (4% v/v, 1 M HCl) or diethyl ether. The sample was continuously extracted by heating with the chosen solvent. For the extraction of prodigiosin from the cells of 500 mL cultures without PU, 50 mL portions of broth were centrifuged (15′, 4°C, 16,000 × g) to pellet the cells. Collected pellets (from 100 mL broth) were then extracted twice with 10 mL acidified ethanol. Cell debris was removed by centrifugation and extracts were combined. In both procedures (with or without PU), the solvent of extracts was removed under reduced pressure by rotatory evaporation and the residual material was extracted with water and dichloromethane (3 mL × 30 mL) to remove water soluble impurities. The combined organic layers were washed with brine (2 mL × 20 mL) and dried over MgSO4. The solvent was removed under reduced pressure yielding the prodigiosin extract as a red solid. The crude extracts were analyzed by UV-VIS spectroscopy, HPLC, HRMS, and compared to chemically synthesized prodigiosin (Supplementary Figures S6–S8 ). The quantity of prodigiosin was verified by quantitative 1H-NMR with 4-methoxyphenol as internal standard (Supplementary information 2).
Preparative flash column chromatography for further purification of the crude extract was performed using silica gel 60 (particle size 0.040-0.063 mm, 230-240 mesh) and dichloromethane providing prodigiosin as a deep red solid. Dichloromethane was distilled prior to use. Purification was monitored by thin layer chromatography (TLC) on pre-coated plastic sheets (Polygram® SIL G/UV254, Macherey–Nagel) with detection by ultraviolet irradiation at 254 nm and treatment with an acidic solution of p-anisaldehyde followed by brief heating with a heat gun.
Preparative flash column chromatography for further purification of the crude extract was performed using silica gel 60 (particle size 0.040-0.063 mm, 230-240 mesh) and dichloromethane providing prodigiosin as a deep red solid. Dichloromethane was distilled prior to use. Purification was monitored by thin layer chromatography (TLC) on pre-coated plastic sheets (Polygram® SIL G/UV254, Macherey–Nagel) with detection by ultraviolet irradiation at 254 nm and treatment with an acidic solution of p-anisaldehyde followed by brief heating with a heat gun.
Free full text: Click here
