Validação da metodologia para a determinação da atividade da fitase por espectrofotometria UV-Vis

Abstract

Phytase enzymes have the property of breaking the binding of the organic phosphorus bound to the phytic acid salts, making it available biologically. In plants, about two-thirds of phosphorus is bound to phytates. The released inorganic phosphate is determined by forming a yellow complex with the acidic molybdate / vanadate reaction. The complex is measured at the wavelength of 415 nm and the inorganic phosphate is quantified by the standard phosphate curve. The applied method measures the phytase activity (FTU) by the amount of micromoles of inorganic phosphorus released from sodium phytate in one minute at a temperature of 37 ° C and at pH 5.5. (1 FTU = 1μmol inorganic P). Validation is the performance of a study, proven that guarantees whatever method, process, equipment, material, activity or system is reliable and evident, is favorable to the intended results. In this study was determined figures of merit(linearity, sensitivity, limits of detection (LD) and quantification LQ), precision, accuracy and robustness) of the method used to determine the fitase activity. In the study the phytase Sigma® standard, phytase raw material and phytase inclusion rations were evaluated. The calibration curve was constructed with standard potassium dihydrogenphosphate (0.10-2.50 μmol / mL), where the mean linear equation obtained was y = 0.3694x - 0.0138. Phytase activity was unstable for variations in pH, time and reaction temperature, and remained stable when the volume of substrate varied. The method applied in this study presented linearity, precision, analytical sensitivity and adequate detection and quantification limits, over a wide concentration range, with results close to those described in the method.