Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments

Abstract

In the context of research for new cytotoxic compounds, obtaining bioactive molecules from renewable sources remain a big challenge. Microorganisms and more specifically Actinobacteria from original sources are well known for their biotechnological potential and are hotspots for the discovery of new bioactive compounds. The strain DP94 studied here had shown an interesting cytotoxic activity of its culture broth (HaCaT: IC50 = 8.0 ± 1.5 μg/mL; B16: IC50 = 4.6 ± 1.8 μg/mL), which could not been explained by the compounds isolated in a previous work. The increase of the cytotoxic activity of extracts was investigated, based on a Taguchi L9 orthogonal array design, after DP94 culture in TY medium using two different vessels (bioreactor or Erlenmeyer flasks). Various culture parameters such as temperature, pH and inoculum ratio (%) were studied. For experiments conducted in a bioreactor, stirring speed was included as an additional parameter. Significant differences in the cytotoxic activities of different extracts on B16 melanoma cancer cell lines, highlighted the influence of culture temperature on the production of cytotoxic compound(s) using a bioreactor. A culture in Erlenmeyer flasks was also performed and afforded an increase of the production of the active compounds. The best conditions for the highest cytotoxicity (IC50 on B16: 6 ± 0.5 μg/mL) and the highest yield (202.0 mg/L) were identified as: pH 6, temperature 37°C and 5% inoculum.

Fig 1. Main-effect plots for optimization of culture conditions in bioreactor.

Fig 2

Comparison of chemical profiles of B9 (red) and E9 (blue). A. Resin extracts, B. Supernatant extracts. All samples were analysed at 220 nm on Prevail^® reversed phase C18 column with a gradient of H₂O (A)/acetonitrile (B) (10 min 100% of A, 30 min from 0% of B to 100% of B, 10 min 100% of B).

Fig 3. Comparison of the chemical profiles of the most active extracts.

SE of the large scale culture in TY shown in preliminary assays (red), SE of B3 (blue) and RE of E7 (black). All samples were analysed at 220 nm on Prevail^® reversed phase C18 column with a gradient of H₂O (A)/acetonitrile (B) (10 min 100% of A, 30 min from 0% of B to 100% of B, 10 min 100% of B).