Evaluation of an optimized enzymatic biosensor for ethanol used in apple storage management with low oxygen stress

Ethanol has been proposed to be one of the target molecules to monitor the dynamic controlled atmosphere (DCA) technique during apple storage, measured in the squeezed juice or in the air of the storage chamber. One of the proposed commercial sensors for ethanol in apple juice is based on amperometry, after a two-step enzyme-based reaction that involves a diaphorase and an alcohol-dehydrogenase. Even though this method has been reported to overestimate ethanol, this difference is fairly fixed and it is industrially used to check the correct application of the treatment and to set the gas composition protocols when the maximum acceptable ethanol is reached. During the 2018 harvest, the ethanol concentration in juices measured with the commercial sensor appeared much higher than those usually reported in precedent years, particularly for the lower concentrations. Laboratory experiments suggested that differences between years could be due to the presence of a secondary enzyme activity present in the commercial diaphorase employed. In order to increase the sensitivity and accuracy, it has been evaluated the performance of the biosensor emploting a further diaphorase. The performances of both sensors were compared with those obtained with a gaschromatophy mass spectrometry approach after head space extraction (HS-GC-MS) in which the mass spectra was acquired in selected-ion monitoring mode. Samples belonging to ‘Red Delicious’ cv. were picked up at different temporary points from industrial storage rooms following the application of low oxygen stress. The new biosensor reduced 97% the mean difference respect to the values obtained with the GC-MS method. The difference between sensors was even clearer for samples with concentrations up to 100 mg/L, that could be used as a discriminating value for the evaluation of the technique success in ‘Red Delicious’ apple juice. The increased sensitivity of the sensor allowed a more accurate monitoring of the DCA at industrial conditions, limiting the risks linked to a false positive on the monitoring during storage.