Study of the Citrate Metabolism of Lactococcus lactis subsp. lactis Biovar Diacetylactis by Means of 13 C Nuclear Magnetic Resonance

Abstract

The metabolic fate of citrate and pyruvate in four strains of Lactococcus lactis subsp. lactis biovar diacetylactis has been studied by means of 13 C nuclear magnetic resonance, using as a substrate either [3- 13 C]pyruvic acid or custom-synthesized citric acid that is 13 C labeled either at carbons 2 and 4 or at carbon 3. The fermentations were carried out batchwise in modified M17 broth. For the actual conversions of the 13 C-labeled substrates, cells at the end of their logarithmic growth phase were used to minimize the conversion to lactic acid. A mass balance of the main citric acid metabolites was obtained; the four strains produced from 50 to 70% (on a molar basis) lactic acid from either citrate or pyruvate. The remaining 50 to 30% was converted mainly to either α-acetolactic acid (for one strain) or acetoin (for the other three strains). One of the strains produced an exceptionally high concentration of the diacetyl precursor α-acetolactic acid. Another strain (SDC6) also produced α-acetolactic acid, but this was decarboxylated to acetoin at a high rate. The 13 C nuclear magnetic resonance method confirmed that the biosynthesis of α-acetolactic acid occurs via condensation of pyruvate and “active” acetaldehyde. Diacetyl was not found as a direct metabolite of citrate or pyruvate metabolism.