Investigação do potencial antimicrobiano e químico de Penicillium spp aquático da Amazônia

Abstract

Fungi of the genus Penicillium produce secondary metabolites used as a model for synthesizing and developing antibiotics such as penicillin, penicillin V, ampicillin, amoxicillin, and griseofulvin. The production of such compounds can be explored through changes in the physical and chemical parameters of the crop. These changes stimulate silenced biosynthetic pathways, increasing the diversity of bioactive compounds produced by the fungus. Therefore, the present study aims to investigate the metabolomic profile and biological potential of four species of Penicillium isolated in the Amazon. The strains studied in this work were deposited in the Collection of Fungi from the Amazon (CFAM) of the Instituto Leônidas and Maria Deane (ILMD). They were reactivated in PDA medium for seven days. A spore suspension was used at a concentration of 1 x 10-6 CFU/mL to produce extracts. 50 µL of this suspension was inoculated into erlenmeyer containing 300 mL of BDL, SB, YES, and ISP2 media. The experiment was carried out in triplicate, in static mode, and incubated at 28 ºC for 15 days. After this period, the fermented broth was extracted with ethyl acetate and the mycelium with methanol. The extracts' minimum inhibitory concentration (MIC) against the pathogens Candida albicans, Candida tropicalis, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus were determined. The extracts of P. purpurogenum strains in ISP2, SB, and YES against Candida albicans showed a minimum inhibitory concentration of 500 ug.mL-1, 31.25 ug.mL-1, and 62.5 ug.mL-1, respectively. In the test against Candida tropicalis and Staphylococcus aureus, the MIC for P. purpurogenum extracts grown in SB was 250 ug.mL-1and 125 ug.mL-1, respectively. Other relevant results were observed for extracts of P. oxalicum and P. citrinum cultivated in BDL and SB with MIC of 250 ug.mL-1and 62.5 ug.mL-1, respectively. Due to the good performance of P. purpurogenum, P. oxalicum, and P. citrinum extracts in SB, largescale cultivation was carried out, followed by broth extraction and fractionation on C18 silica with a MeOH/H2O gradient of 10%, 50 %, and 100%. This procedure led to the isolation of two undescribed substances in P. purpurogenum, whose structures were determined by spectroscopic methods such as 1D NMR (1H NMR and 13C NMR) and 2D NMR (HMBC and HSQC), HRMS and MS/MS. The isolated substances were identified as two citrinin derivatives, dihydrocitrinin (1) and 1-methyldihydrocitrinin (2). The other substances present in the fractions of P. purpurogenum, P. oxalicum, and P. citrinum were characterized by LC-MS/MS and, when possible, HRMS. The compounds characterized belong to the class of terpenes, polyketides, oleamides, benzophenones, and alkaloids, thus revealing the potential of Penicillium spp to obtain compounds with high chemical and biotechnological importance.