No abstract

The N-heterocyclic bases, 2-ethyltetrahydropyridine (1), 2-acetyl-1-pyrroline (2), and 2-acetyltetrahydropyridine (3) are associated with the occurrence of mousy off-flavor in wine. The biosynthesis of these N-heterocycles by the wine lactic acid bacterium, Lactobacillus hilgardii DSM 20176, was studied by high-cell-density incubation in combination with a minimal chemically defined N-heterocycle assay medium. The key components of the defined N-heterocycle assay medium included D-fructose, ethanol, L-lysine, L-ornithine, and mineral salts. N-heterocycle formation was quantitatively determined by gas chromatography-mass spectrometry. The formation of 2 and 3 required the concomitant availability of a fermentable carbohydrate (D-fructose), ethanol, and iron (Fe(2+)). In addition, L-ornithine stimulated the formation of 2 and repressed 3 formation, whereas L-lysine stimulated the formation of 3 and repressed 2 formation. Incorporation of d(6)-ethanol into the acetyl side chain of 2 and 3, and of d(4)-acetaldehyde into the acetyl side chain of 3, confirmed that ethanol and acetaldehyde could serve as major side chain precursors. A pathway for the formation of 2 and 3 by heterofermentative lactic acid bacteria is proposed involving the interaction of accumulated C-2 intermediates from the heterolactic pathway and N-heterocyclic intermediates derived from the metabolism of L-ornithine and L-lysine.

Thirty-four strains of the genera and species representative of the wine lactic acid bacteria (LAB) were screened in an ethanolic grape juice medium for the production of mousy off-flavour as determined by an alkaline test strip sensory method. Most Lactobacillus spp., Oenococcus oeni and Leuconostoc mesenteroides produced mousy off-flavour, whereas Pediococcus spp. were generally incapable of detectable off-flavour formation. To verify these results, selected LAB were tested for the ability to produce the three sensorially potent N-heterocycles, 2-ethyltetrahydropyridine (ETPY), 2-acetyltetrahydropyridine (ACTPY) and 2-acetyl-1-pyrroline (ACPY), which are associated with the mousy off-flavour in wine. N-heterocycle formation was determined by incubating a high density of bacterial cells in a chemically defined N-heterocycle assay medium, and quantifying the 'mousy' compounds by gas chromatography-mass spectrometry. Most strains produced each of the three N-heterocycles, with the general order for this capability being Lactobacillus (heterofermentative) > Oenococcus > Pediococcus. Strains of Lactobacillus hilgardii and Lactobacillus brevis were characterised as producing the highest concentration of ACTPY (328-580 µg/L, sensory threshold in water = 1.6 µg/L ), whereas ACPY and ETPY were produced at a moderate concentration (< 50 and < 10 µg/L, respectively). In addition to the formation of ACPY and ACTPY, three of five strains of the commercially important wine malolactic bacterium, O. oeni, produced the highest concentration of ETPY (87-162 µg/L). Strains of the homofermentative LAB, Pediococcus sp. and Lactobacillus plantarum L11a, however, produced only a relatively low concentration of each mousy N-heterocycle (≤ 37 µg/L), suggesting that the ability of LAB to produce mousy N-heterocycles is linked to the pathway of sugar catabolism. The importance of selecting LAB for induction of malolactic fermentation which have limited ability to produce mousy N-heterocycles is emphasised.Abbreviations LAB lactic acid bacteria; L. Lactobacillus; O. Oenococcus; P. Pediococcus; MLF malolactic fermentation; ETPY 2-ethyltetrahydropyridine; ACTPY 2-acetyltetrahydropyridine; ACPY 2-acetyl-1-pyrroline;GC-MS gas chromatography-mass spectrometry; GJ medium grape juice medium; MRSA medium de Man, Rogosa and Sharpe medium supplemented with 20% v/v preservative-free apple juice;MRS-CT medium de Man, Rogosa and Sharpe medium modified by the inclusion of casamino acids and trypticase peptone; m/z mass to charge ratio