Doctoral Degrees (Institute for Wine Biotechnology)

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Browsing Doctoral Degrees (Institute for Wine Biotechnology) by browse.metadata.advisor "Dicks, Leon Milner Theodore"

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    Strategies for the control of malolactic fermentation : characterisation of Pediocin PD-1 and the gene for the malolactic enzyme from Pediococcus damnosus NCFB 1832
    (Stellenbosch : Stellenbosch University, 2004-12) Bauer, Rolene; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Malolactic fermentation (MLF) is conducted by lactic acid bacteria (LAB) and entails the decarboxylation of L-malate to L-Iactate through a reaction catalysed by the malolactic enzyme (MLE). The consequence of this conversion is a decrease in total acidity. MLF plays a part in microbial stabilisation and due to the metabolic activity of the bacteria the organoleptic profile of the wine is modified. In some wines MLF is considered as spoilage, especially in warm viticultural regions with grapes containing less malic acid. In addition to undesirable organoleptic changes, MLF can alter wine colour, and biogenic amines may be produced. To induce MLF we provided s. cerevisiae with the enzymatic activities required for MLF, which is then conducted by the yeast during alcoholic fermentation. The malolactic enzyme-encoding gene (mieD) was cloned from Pediococcus damnosus NCFB 1832, characterised and expressed in S. cerevisiae. The activity of this enzyme was compared to two other malolactic genes, mieS from Lactococcus lactis MG1363 and mleA from Oenococcus oeni La11, expressed in the same yeast strain. All three recombinant strains of S. cerevisiae converted L-malate to L-Iactate in synthetic grape must, reaching L-malate concentrations of below 0.3 gIL within 3 days. However, a lower conversion rate and a significant lower final L-Iactate level were observed with the yeast expressing mieD. In order to inhibit MLF, we show that the growth of O. oeni, the main organism responsible for MLF, could be safely repressed with a ribosomaly synthesised antimicrobial peptide, pediocin PD-1, produced by P. damnosus NCFB 1832, 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 to homogeneity and sequence analysis suggests that the peptide is a member of the lantibiotic family of bacteriocins. The molecular mass was estimated by mass spectroscopy to be 2866.7 ± 0.4 Da. Pediocin PD-1 forms pores in sensitive cells, as indicated by the efflux of K+ from O. oeni, combined with inhibition of cell wall biosynthesis, 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 in the 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 stable between 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 preservatives such as S02.