Abstract: The simultaneous determination of inosine monophosphate (IMP) and l-glutamate (GL), by enzyme-supported flow injection analysis (FIA), was performed using two enzyme reactors in parallel. Inosine monophosphate dehydrogenase (IMPDH) was extracted and partially purified from Bacillus cereus (JCM 2152). The reaction of IMP with NAD+ was catalyzed by IMPDH and the NADH produced was further converted to NAD+ and hydrogen peroxide by NADH oxidase. GL was determined by using the enzyme glutamate oxidase. Thus, IMP and GL were indirectly measured by monitoring hydrogen peroxide electrochemically. The ghost response caused by the presence of interferences in real sample were reduced by covering the platinum electrode surface with a diaminobenzene polymer membrane which was generated by electropolymn. Linear relations between sensor response and each species were observed in the ranges of 0.1-1.0 mM (IMP) and 0.05-1.0 mM (GL) with correlation coefficients >0.997 for each species. The proposed method was used for seasoning analyzes and the results showed good agreement with those obtained using a liquid chromatography method (for IMP) or a conventional method (for GL with a kit), suggesting that this system may be applicable to the quality evaluation of food seasoning.

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.