Natural Abundance Isotopic Fractionation in the Fermentation Reaction: Influence of the Fermentation Medium

Abstract

The isotopic parameters of fermentation products provide reliable criteria for characterizing their carbohydrate precursors on the condition that the isotopic coefficients which relate individual sites in the products and in the reactants have strictly reproducible or predictible values. Since fermentation may be performed either in natural media (such as fruit juices) which exhibit variable compositions or in “synthetic” media prepared from appropriate nutrients, it is necessary to estimate the influence of the medium on the carbon and hydrogen isotope ratios. By combining isotope ratio mass spectrometry and nuclear magnetic resonance investigations it has been shown that variations in the reaction rates induced by the medium are not connected with isotopic fractionation effects. At natural concentrations the nutrients present in juices, for instance, ensure a significant acceleration of the bioconversion with respect to pure water without introducing undesirable isotopic effects. The role of different nutrients added in relatively large amounts to synthetic media has been analyzed. It has been shown in particular that a significant decrease in the isotope ratio of the methylenic site of ethanol is due to the combined influence of both phosphates and assimilable nitrogen which are also the key components governing the growth and activity of yeasts. For a given nutrient composition the effect of the medium on the isotopic coefficients which connect the individual sites of ethanol to the starting water can be estimated. The observed behavior can be explained by modifications in the percentages of intra- and intermolecular hydrogen transfers (methyl site) and by variations in the kinetic isotope effects and exchange rates involving the hydrogens originating from water. Carbohydrates from different origins (beet and cane) have been fermented in different “natural” media prepared by eliminating sugars from orange, apple, and grape juices. In spite of very different fermentation rates the isotopic ratios of the products are then nearly independent of the composition of the medium. These results support the use of the ethanol probe for characterizing endogenous or exogenous sugars contained in different fruit juices.