The fermentation medium for primary screening was FM 3 [67 ]: soluble starch, 20 g; soy powder, 15 g; yeast extract, 5 g; peptone, 2 g; CaCO3, 4 g; sea salts, 18 g in 1 L distilled water, pH 7.2~7.4. Two media were used for re-screening, namely FM 2 [68 ]; Glucose, 10 g; soluble starch, 30 g; yeast powder, 2 g; casein, 4 g; K2SO4, 8 g; MOPS (3-[N-morpholino]propanesulfonic acid), 5 g; sea salts, 18 g; distilled water, 1 L; pH 7.2–7.4; and FM 17 [69 ]; Glucose, 20 g; yeast powder, 5 g; casein, 5 g; KNO3, 15 g; CaCO3, 4 g; sea salts, 18 g; distilled water, 1 L; pH 7.2–7.4. All three media were autoclaved at 121 °C for 20 min.
Each of 2,041 purified isolates were transferred to a test tube (30 mm×200 mm) which contained 20 mL of the relevant fermentation medium, and cultured at 200 rpm, at an angle of 45º, for 7–10 days at 28 °C. Crude extracts were prepared by adding 60 mL of methanol to each of the cultures and the extraction allowed to proceed for 2 weeks. One mL fractions of the resultant extracts were transferred to wells in deep 96-well plates, vacuum dried at 60 °C, dissolved in 200 μL DMSO and used in the biochemical screens and for cytotoxicity assay against HCT-116 cells.
Each of 2,041 purified isolates were transferred to a test tube (30 mm×200 mm) which contained 20 mL of the relevant fermentation medium, and cultured at 200 rpm, at an angle of 45º, for 7–10 days at 28 °C. Crude extracts were prepared by adding 60 mL of methanol to each of the cultures and the extraction allowed to proceed for 2 weeks. One mL fractions of the resultant extracts were transferred to wells in deep 96-well plates, vacuum dried at 60 °C, dissolved in 200 μL DMSO and used in the biochemical screens and for cytotoxicity assay against HCT-116 cells.
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