EXPERIMENTS ON THE ENZYMATIC ONLINE DETER MINATION OF DIACETYL AND 2-ACETOLACTATE IN BEER

In regard to an automated and continuous control of beer ripening: a f low injection analysis (FIA) system with immobilized enzymes has been conceived and tested. As to the enzymatic determination of free and to tal diacetyl using NADH fluorescence it turned out that none of the th ree available ''diacetyl reductases'' had a sufficient specificity, al l of them reducing in addition 2,3-pentanedione and acetoine. The dice tones could be preseparated from the latter compound by continuous per vaporation, and in the pervaporate satisfactory results of diacetyl de termination could be obtained, provided the concentration of acetoine in the primary analyte solution was below 8 ppm. The reaction time for the oxidative decarboxylation of 2-aceto-lactate to diacetyl could be reduced from 60 min. at 90 degrees C to 2 min. at 25 degrees C, using Fe3+ as oxidant; this would permit to integrate a corresponding conve rsion device in a FIA-system for the analysis of ''total diacetyl''. A s an alternative for the enzymatic determination of 2-acetolactate the reductive isomerization of this compound was tested. The correspondin g enzyme from enterobacter aerogenes catalyzed the conversion of the s ubtrate, but also of its homologue 2-aceto-2-hydroxybutyrate. Neverthe less the determination of 2-acetolactate was possible in batch systems , however, in an on-line FIA-system with immobilized enzyme the sensit ivity of the enzyme (K-m for acetolactate = 0.45 mM) did not meet the demands needed for the problem in question. The potential of screening or genetic engineering for the provision of more selective and sensit ive enzymes is discussed.