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1984 - 198911990 - 20001

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Subject: search.filters.subject.Wine and wine making -- Microbiology

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    Studies on the non-specific esterases of Saccharomyces cerevisiae : a thesis in partial fulfillment [sic.] of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1984) Dick, Bruce
    Twenty wine-making and three laboratory strains of Saccharomyces cerevisiae were examined for non-specific esterases by Polyacrylamide Gel Electrophoresis. All wine-making strains contained the fast alleles of the Est 1 and Est 2 loci, confirming there is a selective advantage for the Est 1f and Est 2f genes in these strains. Only one wine-making strain carried the Est 3 and Est 4 genes, which was a much lower frequency than that published. The three laboratory strains all contained the Est 1f and Est 2s genes. A new non-specific esterase band, labelled Est 5, was identified by using a modified staining technique, which was apparently of low molecular weight as it travelled with the tracking dye front. Fast and slow alleles of Est 1 and Est 2 were determined to be charge allozymes. Est 2 proteins were considered to be polymeric, probably dimeric, and the Est 1 proteins to undergo post-translational modification. Difficulty in resolving the Est 4 band was overcome by adding Triton X-100 to cell suspensions before disruption, indicating this esterase protein may be particulate bound. Molecular weights were determined by Ferguson Plots to be 51,000 ± 10,000 daltons (Est 2), 60.000 ± 12,000 daltons (Est 3), 73,000 ± 15,000 daltons (Est l), and 113,000 ± 23,000 daltons (Est 4). No isolates of S. cerevisiae for comparison of allele frequencies could be made from mature locally-grown grapes, indicating that this species is rare in the New Zealand environment, which is in accordance with published studies. No "inducible" non-specific esterases were found in strains examined at different stages in the life cycle, or by growth in different media. The level of esterase activity in cells increased throughout aerobic growth in liquid media, but was quickly lost during fermentation. Esterase activity during sporulation also decreased. A non-specific esterase mutant was induced by ethyl methane-sulfonate and detected by the hydrolysis of α-naphthyl acetate incorporated into solid medium. This mutant lost expression of both Est If and Est 2s , as did subsequent mutants produced by hybridisation. Segregation of esterase-deficient to esterase-proficient spores after hybridisation, showed that two unlinked loci were involved in esterase suppression, both genes being unlinked to ade 1, Est 1 and mating type locus MAT. It is hypothesised these genes are a suppressor (SUP) and a mutated regulator (Reg Est− ). Gas Liquid Chromatography was used to quantitatively determine volatile ester concentrations produced during fermentation. Selected wine-making strains and diploid strains produced by micromanipulation and having different non-specific esterase compositions were fermented to the limit of their ethanol tolerance in Reisling Sylvaner grape juice and Complete Defined Medium. Ethyl acetate, ethyl propanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, 2-phenethyl acetate, n-hexyl acetate and iso-pentyl acetate were all quantitated. A maximum error of ±30% was determined for differences in ester concentration between two fermentations using the same strain. Correction for differences in fermentation ability by different strains was attempted, and the resulting ester concentrations compared qualitatively. Results indicate that differences in volatile ester concentrations between strains are not due to the esterase composition. The non-specific esterases probably have little if any influence on wine bouquet as the majority of ester production is late in fermentation when esterase activity has ceased.
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    Acetaldehyde metabolism by wine lactic acid bacteria and its oenological implications : a thesis presented in fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 2000) Osborne, James
    Acetaldehyde is one of the most important sensory carbonyl compounds formed during vinification. Excess acetaldehyde can adversely affect the flavour of wine and acetaldehyde plays a role in the colour development of red wines. Excess acetaldehyde is usually masked by the addition of sulphur dioxide (SO2) to the wine (SO2 is also used as an antimicrobial and antioxidant agent in wine and acetaldehyde bound SO2 is less effective in these roles). To date there has been no definitive study of the impact of wine LAB on free and bound acetaldehyde. Therefore, this study investigated the metabolism of free and bound acetaldehyde and its oenological implications. A survey of 11 commercial malolactic starter cultures (mostly Oenococcus oeni strains) showed that 9 out of 11 were able to metabolise acetaldehyde (in a resting state) with the corresponding formation of ethanol and acetic acid as products. SO2 bound acetaldehyde was also metabolised by the two strains tested (Lactobacillus buchneri CUC-3 and Oenococcus oeni MCW). This is the first evidence that LAB can indeed catabolise SO2 bound acetaldehyde, therefore releasing free SO2. During growth Oenococcus oeni EQ54 and Oenococcus oeni VFO were able to metabolise free acetaldehyde in wine at pH 3.3 and pH 3.6. In wine containing SO2 bound acetaldehyde, Oenococcus oeni EQ54 and Oenococcus oeni VFO were able to metabolise SO2 bound acetaldehyde at pH 3.6 after a period of sluggish growth. At pH 3.3 there was no metabolism of SO2 bound acetaldehyde by Oenococcus oeni EQ54 and Oenococcus oeni VFO during the incubation period. Results from growth experiments showed that in broth there was inhibition of growth at 300 mg/L concentration of acetaldehyde for all strains. In wine, no significant inhibition or stimulation of the cultures examined was found at any acetaldehyde concentrations up to 300 mg/L. In a simultaneous resting cell incubation of Saccharomyces bayanus Première Cuvée and Oenococcus oeni Lol11, acetaldehyde produced by the yeast was metabolised by the wine LAB. The metabolism of acetaldehyde by wine LAB is expected to influence wine flavour as small amounts of ethanol and acetic acid are produced and acetaldehyde is removed. This removal of acetaldehyde by wine LAB suggests that less SO2 will need to be added to the wine to mask excess acetaldehyde when malolactic fermentation is performed. Inhibition of wine LAB growth in broth by high levels of acetaldehyde suggests a role for acetaldehyde in stuck or sluggish MLF. Sluggish growth in wine containing SO2 bound acetaldehyde also suggests a possible role of SO2 bound acetaldehyde in stuck and sluggish MLF. This is due to the release of free SO2 through the metabolism of the acetaldehyde moiety of SO2 bound acetaldehyde.