Isotopic characterization of the bioconversion of lactose into ethanol

In order to compare natural abundance isotopic properties of lactose with those of other carbohydrates such as glucose, fructose, sucrose and starch, the accessibility to ethanol obtained by fermentation, as a common probe, has been investigated. Only strictly reproducible isotopic fractionation will ensure that the fermentation products are always representative of their lactose precursor. An analytical strategy has therefore been designed that avoids discriminating isotope effects in the fermentation of pure lactose or whey permeates by Kluyveromyces fragilis. Appropriate experimental conditions ensure nearly full consumption of lactose and a high yield of conversion into ethanol. A lactose concentration of the order of magnitude of that found in whey permeates (50 g l-1) is convenient and an inoculum grown for 24 h is selected. Whereas sodium ions exert an inhibitory effect, added peptones optimize the fermentation process without introducing isotopic perturbations. The overall carbon isotope parameter of ethanol, delta 13C, is measured by isotope ratio mass spectrometry with a reproducibility of 0.2 per thousand and the site-specific natural isotope ratios of the methyl, (D/H)I, and methylene, (D/H)II, sites of ethanol are obtained with a reproducibility of about 1.5 ppm. These parameters provide useful criteria for comparing various starting materials such as lactoses (alpha or beta) from different commercial producers, milk powders, ordinary or skimmed milks and lactoserum. The methyl parameter, (D/H)I, mainly reflects the deuterium content of the lactose precursor, which is itself expected to provide information on the feeding of the animal and on the metabolism. The methylene isotope ratio is connected to that of the fermentation medium and may be influenced by isotopic fractionation introduced in the exchangeable sites of lactose by the extraction process.