Strategies for the control of malolactic fermentation : characterisation of Pediocin PD-1 and the gene for the malolactic enzyme from Pediococcus damnosus NCFB 1832

[摘要] ENGLISH ABSTRACT:Malolactic fermentation (MLF) is conducted by lactic acid bacteria (LAB) and entailsthe decarboxylation of L-malate to L-Iactate through a reaction catalysed by themalolactic enzyme (MLE). The consequence of this conversion is a decrease in totalacidity. MLF plays a part in microbial stabilisation and due to the metabolic activity ofthe bacteria the organoleptic profile of the wine is modified. In some wines MLF isconsidered as spoilage, especially in warm viticultural regions with grapes containingless malic acid. In addition to undesirable organoleptic changes, MLF can alter winecolour, and biogenic amines may be produced. To induce MLF we provideds. cerevisiae with the enzymatic activities required for MLF, which is then conductedby the yeast during alcoholic fermentation. The malolactic enzyme-encoding gene(mieD) was cloned from Pediococcus damnosus NCFB 1832, characterised andexpressed in S. cerevisiae. The activity of this enzyme was compared to two othermalolactic genes, mieS from Lactococcus lactis MG1363 and mleA from Oenococcusoeni La11, expressed in the same yeast strain. All three recombinant strains ofS. cerevisiae converted L-malate to L-Iactate in synthetic grape must, reachingL-malate concentrations of below 0.3 gIL within 3 days. However, a lower conversionrate and a significant lower final L-Iactate level were observed with the yeastexpressing mieD. In order to inhibit MLF, we show that the growth of O. oeni, themain organism responsible for MLF, could be safely repressed with a ribosomalysynthesised antimicrobial peptide, pediocin PD-1, produced by P. damnosus NCFB1832, without effecting yeast growth. Pediocin PD-1 is stable in wine at 4°C-100°C,and ethanol or S02 does not affect its activity. The peptide was purified tohomogeneity and sequence analysis suggests that the peptide is a member of thelantibiotic family of bacteriocins. The molecular mass was estimated by massspectroscopy to be 2866.7 ± 0.4 Da. Pediocin PD-1 forms pores in sensitive cells, asindicated by the efflux of K+ from O. oeni, combined with inhibition of cell wallbiosynthesis, leading to cell lysis. Loss of cell K+was reduced at low temperatures,presumably as a result of the increased ordering of the lipid hydrocarbon chains inthe cytoplasmic membrane. Although pediocin PD-1 is active over a broad pH range,optimal activity was recorded at pH 5.0. The petide is, however, more stablebetween pH 2.0 and 5.0, with the best stability observed between pH 3.0 and 4.0.Pediocin PD-1 provides a safer biological alternative than chemical preservativessuch as S02.

[发布机构] Stellenbosch University