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| Author(s): |
Henrique Pereira Ramos
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2008-07-15 |
| Examining board members: |
Suraia Said;
Sérgio Ricardo Ambrosio;
Fernando Batista da Costa
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| Advisor: | Suraia Said |
| Field of knowledge: | Biological Sciences - Microbiology |
| Indexed in: | Banco de Dados Bibliográficos da USP-DEDALUS; Biblioteca Digital de Teses e Dissertações - USP |
| Location: | Universidade de São Paulo. Biblioteca Central do Campus de Ribeirão Preto; FCFRP/Ramos, Henrique Pereira |
| Abstract | |
The purpose of the current project was to optimize the conditions of culture of the endophytic fungus Arthrinium state of Apiospora montagnei Sacc. in order to increase the production of secondary metabolites with antibacterial, antifungal, antiparasitary and antitumor activities. The fungus was cultivated in a liquid medium in two different steps; first, in 200 mL of Jackson pre-fermentative medium for 48 hours at 30 °C, under constant agitation (120 rpm), and subsequently, reinoculating the mycelial mass aseptically obtained in the pre-fermentative step in 400 mL of Czapek fermentative medium, followed by reincubation at 30 °C under constant agitation (120 rpm). After filtration of the culture fluid and partition with organic solvents, the following extracts were obtained: acetyl ethylic, buthanolic, ethanolic and aqueous. The standard culture condition, initial culture in Czapek fermentative medium, was performed during 20 days of incubation. The extracts obtained from this culture condition were evaluated regarding the antibacterial and antifungal activities. The acetyl ethylic extract presented, in this culture condition, the best antibacterial activity, with a Minimum Inhibitory Concentration (MIC) of 270 ?g/mL for Escherichia coli; antifungal activity was not observed in this extracts. After the initial evaluation, modifications in the standard culture condition in fermentative medium were performed, aiming to optimize the previously reported antibacterial activity. The following variables were assessed: incubation period, carbon source, nitrogen source, pH, temperature and proportion of carbon/nitrogen in the medium. Each variable was evaluated using MIC assays of the extracts obtained from the different cultures performed, allowing the selection of the better condition for the variable optimization. The best condition for the production of secondary metabolites with antibacterial activity was obtained in 9 days of incubation, using sucrose (3.1%) as the carbon source and sodium nitrate (0.1%) as the nitrogen source, in pH 4 at 30 °C, with a MIC value of 90 ?g/mL for Escherichia coli and P. aeruginosa, which was superior to the activity detected under the standard culture condition. For the antifungal activity, previously undetected, the best culture condition was obtained using sucrose (3.1%) and sodium nitrate (0.1%) in pH 4 at 30 °C for Aspergillus fumigatus (MIC of 130 ?g/mL), and sucrose (3.0%) and sodium nitrate (0.2%), in pH 4 at 30 °C for Candida albicans (MIC of 140 ?g/mL). The best culture condition for the production of secondary metabolites with antitumor activity was obtained using sucrose (3.0%) as the carbon source and sodium nitrate (0.2%) as the nitrogen source, in pH 4 at 30 °C, for the three human tumor cell lines assessed (HCT-8, MDA-MB435 and SF295), in which 100% of cell growth inhibition was detected; this condition also accounted for the best inhibitory activity of the enzyme APRT of L. tarentolae, presenting 66,41% of inhibition, indicating possible antiparasitary activity. The optimization of the culture conditions for the fungus Arthrinium state of Apiospora montagnei Sacc. proved to be relevant for the production of bioactive secondary metabolites. (AU) | |
| FAPESP's process: | 05/58427-3 - Optimization of the cultivation conditions of endophytic VA 16 for production of secondary metabolites with biological activities |
| Grantee: | Henrique Pereira Ramos |
| Support Opportunities: | Scholarships in Brazil - Master |