Anaerobic Metabolism of Organic and Amino Acids in Grape. I. A Device for Measuring the Decarboxylating and Ethanol-releasing Kinetics from a Single ¹⁴C-labeled Berry

Abstract

This paper describes a radio-isotopic method for studying the anaerobic metabolism of grapes. With the proposed system, one grape berry labeled with ¹⁴C-aspartate, malate, or succinate was continuously flushed at 32°C with nitrogen gas, and the diffused CO₂ and ethanol were continuously trapped. This allowed the reliable determination of the kinetics of their respective diffusions as a function of the elapsed time under anaerobic conditions. The derived carbon dioxide curves generally showed three steps: a lag phase and a steady-state metabolic period followed by a decreasing phase when the label was completely decarboxylated. The slope of the steady-state period was interpreted as the rate of the in vivo metabolism of the label, and its value was a proportional function of the total number of translocated nmoles (the consumed nmol/min/berry = 7.10^-5 x translocated nmol). Stoichiometry of the reaction and relative CO₂-ethanol diffusional characteristics were also obtained; one translocated molecule gave two CO₂ and one ethanol, and the releasing rate for CO₂ was 10-fold higher than for ethanol. The partition coefficient between total diffused ethanol and berry ethanol at the end of the experiment was ca 0.1, showing that ethanol accumulated more slowly inside the berry than under the usual conditions of carbonic maceration. Good interberry reproducibility and high radioactivity recoveries (95.8%) support this method as being a reliable and versatile tool for assessing, through the use of ¹⁴C-labeled molecules which undergo decarboxylation, the main pathways and enzymes occurring in the anaerobic metabolism of grapes.