I've recently used a kit to measure alcohol quantities during apple fermentation by yeasts. Results showed that, at the very start of the experiment, we obtained a high amount of alcohol in the solution by quantifying it with a kit. These results are not very logical. I wonder if the enzyme used by the kit, the alcohol dehydrogenase, could interact with the hydroxyl, functional group of the polyphenols, contained in a high amount in the apple juice.
Dear Bertrand, I do not think that substrate specificity of ADH includes also polyphenols (do you have any information regarding origin of the enzyme?). I suppose that you measured absorbance (or fluorescence?) of NADH produced during asssay, right? High absorbance can be caused for instance by turibidity of the sample...
ADH belongs to broad spectrum enzyme families owing to its position in metabolism. However, the substrate specificities spray across a rather wide range. For a living plant like apple riches in polyphenols I expect you may find your ADH in apple juice has higher polyphenol activities, if you are using ADH from fungal sources when measuring EtOH. Because, fungal in its natural environments are closely related to wood decomposition, kp
Thanks for your replies. We decided to stop using this kit. We'll probably set an other protocol to measure EtOH in our samples
1. It just reminds me from very beginning to view your problem. There are two approaches in alcohol determination, kinetic method and equilibrium method(static state method). In general measuring NAD(P)H change at 340nm is kinetic approach. High polyphenol background might or might not interfere the kinetic readings depends upon how big polyphenol Abs at the wavelength? However, it would be rather difficult to us static method(colorimetric) to perform the assay owing to brownish background from cider. That leaves not much room to make clear distinction. 2. There are plenty of polyphenols from lignin degradation and other sources, of different sizes. Some of which may eventually work with ADH, a key enzyme in evolution. 3. MeOH from pectic substance degradation might interfere with ADH assay of EtOH, since the majority of ADH carries MeOH activities. 4. MeOH in fermented fruit sources usually derived from esterases cleave methyl group from pectic acids.
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