The flux of carbon into lactic acid, diacetyl and acetoin during the co-metabolism of glucose and citrate by Lactococcus lactis subsp. lactis biovar. diacetylactis has been determined using natural abundance isotopic ratio analysis. During fermentation in the conditions used (glucose, 27.8 mM; citric acid, 13.9 mM; initial pH 6.2-6.4, anaerobic) it is shown that approximately 65% of the carbon source used for the aroma compounds is derived from the carbohydrate. Equally, citrate contributes approximately 30% of the carbon recovered in lactic acid. Thus, there is no evidence for a metabolic separation of the catabolism of these two carbon sources.

The use of solid-phase microextraction (SPME) coupled with isotope ratio mass spectrometry (IRMS) for the analysis of flavor compounds produced by lactic acid bacteria has been evaluated using both liquid and headspace sampling modes. Initially, it was necessary to optimize the conditions for the SPME extraction of flavors-diacetyl and acetoin-in standard aqueous solutions. The effects of salt, headspace versus liquid sampling, and coating phase were tested. Second, the suitability of the coupling of SPME and gas chromatography-combustion interface-IRMS (GC-C-IRMS) for the determination of delta(13)C values was assessed. It is shown that neither the analyte concentration nor the period of fiber exposure has an effect on the delta(13)C values. Finally, having verified that there are no matrix effects from the fermentation medium, it is reported for the first time that flavor compounds can be extracted directly from culture supernatant by SPME and their delta(13)C values can be obtained by GC-C-IRMS.

The simultaneous catabolism of citrate and glucose by growing Lactococcus lactis subsp. lactis biovar. diacetylactis to obtain energy was followed quantitatively, using a non-enrichment isotopic technique. Both citrate and glucose are precursors of pyruvate, which may either be reduced to lactate, the principle product that accumulates, or be converted to diacetyl and acetoin. Under suitable conditions, both routes regenerate NAD+. Until recently, however, the quantitative relationships between the two substrates and these three products were poorly defined. It was recently shown, by exploiting differences in natural abundance 13C/12C ratios in the two substrates, that there is no metabolic separation of the catabolism of these two carbon sources. In this study, it is shown that the relative consumption rates change throughout the growth phase, citrate being preferentially metabolised at the onset of a culture of energy-depleted cells, with a subsequent evolution towards a metabolism dominated by glucose consumption. Additionally, it is shown that the relative consumption rates are influenced by environmental factors, notably initial pH and temperature.