Abstract: A continuous system for the determination of fish freshness with double enzyme reactors was developed and applied to the determination of the freshness indicator K K = 100(HxR + Hx)/(IMP + HxR + Hx), where IMP, HxR and Hx are Inosine monophosphate, Inosine and Hypoxanthine, respectively. The system was assembled with a three electrode screen-printed element (graphite as working electrode, silver as counter and silver, silver chloride as reference electrode) placed in a flow cell, a sample injection valve and two enzyme reactors. The determination of the total amt. of HxR and Hx is realized by flowing the sample through two reactors in series: one reactor was packed with nucleoside phosphorylase (Np) and the other with xanthine oxidase (XO) immobilized on aminopropyl glass. Similarly, the other term of the equation was evaluated by flowing through the two reactors the sample treated by Alkaline phosphatase (AlP) for 5-10 min at 45°C. One assay could be completed within 5 min. The system for the determination of fish freshness was reproducible within 2-3% (n = 4). The immobilized enzymes were fairly stable for at least three months at 4°C. More than 200-300 samples could be analyzed in about one month by using these enzyme reactors provided the disposable screen-printed electrode should be changed every 30-40 real samples. The results obtained suggest that the proposed sensor system provides a simple, rapid and economical method for the determination of fish freshness (K). We applied the present system with two reactors for the determination of K values in fish samples and compared the results with those obtained by the XO-reactor. Correlation factor and regression line between the two methods were 0.992 and Y = -3.14+1.03X respectively. We concluded that the present flow injection analysis (FIA) system with XO and Np reactors was suitable as a simple, easy to handle and reliable instrument for quality control in the fish industry.

Abstract: Glycerol, the most important secondary product of alcohol fermentation, contributes to the smoothness and viscosity of wines with favorable effects on taste. An electrochemical biosensor was developed for online monitoring of glycerol biosynthesis. Anal. parameters as pH, buffer composition, and cofactor concentration. were optimized. The lifetime of the sensor in working conditions was also evaluated. The biosensor was assembled by immobilizing glycerol-3-phosphate oxidase on polymeric membranes and glycerokinase on aminopropyl glass beads. The enzymatic membrane was placed in contact with the surface of a Pt electrochemical transducer for H2O2 detection. Glass beads with immobilized glycerokinase were packed to form a reactor connected with the enzyme electrode in a flow system. A cell with a wall-jet configuration was used to monitor glycerol levels by flow-through and flow injection analysis (FIA) in a 10^-5 to 10^-3 mol/L concentration. range. This system was used to measure glycerol levels during alcohol fermentation at different temperatures and pH values.