Study of the citrate metabolism of Lactococcus lactis subsp. lactis biovar diacetylactis by means of 13C 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 13C nuclear magnetic resonance, using as a substrate either [3-13C]pyruvic acid or custom-synthesized citric acid that is 13C 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 13C-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 13C 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.