Escalamiento del Cultivo de Células en Suspensión de Azadirachta Indica (NEEM) en Biorreactor tanque agitado para la producción de Azadiractina
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Azadirachta indica is a tree known for its ability to synthesize secondary metabolites. Among these metabolites highlights azadirachtin (Aza), triterpenoid widely used as a potent natural insecticide. The production of this compound by traditional methods do not meet market demand and chemical synthesis is not yet possible due to the complexity of this molecule. Because of this, has been sought new biotechnological tools that make possible the production of azadirachtin. The objective of this project was to evaluate azadirachtin production in cell culture of Azadirachta indica in stirred tank bioreactor type. For this, cell suspensions were established from friable callus, induced from seeds collected in Bagaces, Guanacaste, Costa Rica, using as a growth regulator TDZ supplemented with hydrolyzed casein, allowing maximum growth of biomass 10,71mg / L in 18 days of culture. In addition the cell suspensions were scaled to a volume of 3L stirred tank bioreactor type. It also evaluated the best solvent and extraction method. Methanol and stirring with temperature showed the best extraction coefficients. Through high-performance liquid chromatography (HPLC) field samples, plants in vitro and cell suspensions (in flask and bioreactor) were analyzed to quantify the amount of azadirachtin produced in different systems. There was a high production of azadirachtin in seed, reaching values of 18,4mg/g dry sample, whereas in field sheet was obtained 8,8mg / g dry. Additionally, in vitro leaf occurred 0.8mg / g. In vitro roots did not show quantifiable amounts of Aza by the method.ompounds quantifiable by the method employed. Cell suspensions in flasks, reached a maximum production of 167,85mg/L of culture, while in bioreactor was obtained 42,13mg/L. In both cases Aza production was directly related to biomass growth. The concentrations obtained by analyzing the material, suggesting the presence of elite material and, therefore, good production rates of Aza in vitro material. This allows to consider that the use of this biotechnological tool is viable to meet the demand of the compound on the market, considering that the process requires adjustments and optimizations.
Proyecto de Graduación (Licenciatura en Ingeniería en Biotecnología) Instituto Tecnológico de Costa Rica, Escuela de Biología, 2016.