Microbiological investigation, growing conditions and metabolic potential in the search for bioactive natural products in endophytic fungus AC-1 isolated from Annona Cacans

Abstract

This project of investigation aims to investigate the conditions for growth and production of secondary metabolites in biotechnological study with endophytic fungus strain AC1 isolated from the leaves of Annona cacans (Annonaceae), with a view to obtaining bioactive substances from the chemical / biological study of the crude extract of these endophytes. To do so, shall isolating and identifying the fungi using solid culture media: malt extract agar (MEA), synthetic nutrient agar (SNA), oatmeal agar (OA), Sabouraud agar and potato dextrose agar (PDA). The cultivation should be carried out initially on a small scale in liquid media (400 ml) in Czapek, malt extract, yeas and malt (YM), potato dextrose and Nutrient) and solids media (maize and rice) at 25 ° C for 28 days, under stirring and static mode, respectively. After the incubation period, for liquid media, the broth will be separated of the mycelium by filtration and subjected to partition liquid/liquid with ethyl acetate (EtOAc) providing the crude extract after solvent evaporation. For solid medium, an extraction will be performed with AcOEt and MeOH (3 times), followed by filtration and evaporation of the solvent providing the crude extract. The study will be monitored by the antimicrobial activity (against gram-positive and gram-negative bacteria and Candida species) and antioxidant activity (²-carotene, DPPH and ORAC-FL methods) and were selected for fractionation, isolation and identification of substances bioactive of the extracts with better results in terms of bioactivity and mass yield. The extract selected for chemical / biological study that show more promising will be cultivated on a large scale, in the midst selected after screening, providing the crude extract, which will be fractionated by standard chromatographic techniques. The pure compounds isolated by the use of different chromatographic methods (column chromatography, preparative thin layer chromatography and HPLC) will have their structures specific and/o elucidated by spectroscopic methods (MRN 1H, RMN 13C and ESI-MS) and will be subjected to testing to evaluate the antimicrobial and antioxidant activities. This work aims to search for new potential bioactive agents and the implementation of rational studies of the chemistry of micro-organisms associated with plants (endophytes).