Ultrasonic-assisted extraction and automated determination of catalase and lipase activities in bovine and poultry livers using a digital movie-based flow-batch analyzer.

An ultrasonic reactor (UR) was developed and coupled to a digital movie-based flow-batch analyzer (DM-FBA) for the ultrasonic-assisted extraction (UAE) and fast determination of catalase and lipase activities in bovine and poultry livers. The lab-made UR mainly consisted of a borosilicate glass container and a piezoelectric disc. The DM-FBA mainly consisted of a webcam, an ultrasonic actuator controller, a peristaltic pump, six solenoid valves, a valve driver, a mixing chamber, a magnetic stirrer, an Arduino Mega 2560, and a personal computer. This setup, named UR-DM-FBA, was controlled by custom software. Ultrasound (US) frequency, US power, sonication time, and concentration of extraction agent were optimized using the Taguchi method. Experiments at silent conditions (mechanical stirring at 1500 rpm) were carried out to evaluate extraction efficiency. Optimized parameters for the UAE of catalase were US frequency of 30 kHz, 2.0 mL of Triton X-100, sonication time of 270 s, and US power of 10.8 W. For the UAE of lipase, the optimized parameters were US frequency of 20 kHz, 0.30 mL of triethanolamine, sonication time of 270 s, and US power of 18 W. Catalase and lipase activities obtained with the UR were, on average, 1.9 × 103% and 2.0 × 103% higher than those obtained at silent conditions, respectively, which indicates that that the lab-made UR was capable of extracting these enzymes more efficiently. Determinations using the UR-DM-FBA were highly accurate (relative error ranging from -1.98% to 1.96% for bovine catalase, -0.65% to 0.76% for bovine lipase, -2.03 to 2.08% for poultry catalase, and -0.55% to 0.64% for poultry lipase) and precise (overall coefficient of variation <0.02% for bovine and poultry catalase and <0.2% for bovine and poultry lipase). Results obtained with the proposed system and reference methods were in good agreement according to the paired t-test (95% confidence level). High sampling rates (>69 h-1) and low sample/reagent consumption (<1.6 mL) were also obtained. Due to the highly efficient UAE, the proposed system can be applied for fast and accurate quantification of lipase and catalase in biological samples with low waste generation.

Digital movie-based on automatic titrations.

This study proposes the use of digital movies (DMs) in a flow-batch analyzer (FBA) to perform automatic, fast and accurate titrations. The term used for this process is "Digital movie-based on automatic titrations" (DMB-AT). A webcam records the DM during the addition of the titrant to the mixing chamber (MC). While the DM is recorded, it is decompiled into frames ordered sequentially at a constant rate of 26 frames per second (FPS). The first frame is used as a reference to define the region of interest (ROI) of 28×13pixels and the R, G and B values, which are used to calculate the Hue (H) values for each frame. The Pearson's correlation coefficient (r) is calculated between the H values of the initial frame and each subsequent frame. The titration curves are plotted in real time using the r values and the opening time of the titrant valve. The end point is estimated by the second derivative method. A software written in C language manages all analytical steps and data treatment in real time. The feasibility of the method was attested by application in acid/base test samples and edible oils. Results were compared with classical titration and did not present statistically significant differences when the paired t-test at the 95% confidence level was applied. The proposed method is able to process about 117-128 samples per hour for the test and edible oil samples, respectively, and its precision was confirmed by overall relative standard deviation (RSD) values, always less than 1.0%.