Lactic acid bacteria are very important in winemaking. In this study, 108 lactic acid bacteria isolates were obtained from high-ethanol-content (~17% (v/v)) Grenache wines during uninoculated malolactic fermentation (MLF). The 16S rRNA and species-specific PCR showed that 104 of these were Oenococcusoeni, three were Lactobacillus hilgardii, and one was Staphylococcus pasteuri. AFLP of HindIII and MseI digests of the genomic DNA of the O. oeni strains was developed for the first time to discriminate the strains. The results showed that the method was a suitable technique for discriminating the O. oeni strains. Based on the cluster analysis, nine O. oeni strains were chosen for inclusion in an ethanol tolerance assay involving monitoring of optical density (ABS600nm) and viable plating. Several O. oeni strains (G63, G46, G71, G39) survived and grew well in MRS-AJ with 17% (v/v) ethanol, while the commercial O. oeni reference strain did not. Strain G63 could also survive and grow for 168 h after inoculation in MRS-AJ medium with 19% (v/v) ethanol. These results suggest that O. oeni G63, G46, G71, and G39 could potentially be used as MLF starters for high-ethanol-content wines. All three L. hilgardii strains could survive and grow in MRS-AJ with 19% (v/v) ethanol, perhaps also indicating their suitability as next-generation MLF starter cultures.
Keywords: amplified fragment length polymorphism; ethanol tolerance; lactic acid bacteria; malolactic fermentation.