Killer Yeasts - Cause of Stuck Fermentations in a Wine Cellar

Sluggish fermentations in five fermenters in a wine cellar were investigated. Methylene blue-stains of yeast suspensions revealed that approximately 90% of the total yeast population in each of the fermenters were dead. The viable cells in each fermenter were killer yeasts. Polyacrylamide gel electrophoresis of total soluble cell proteins showed that the same killer yeast occurred in each of the five fermenters. The effect of killer yeast on viability and fermentation activity of the wine yeast was studied in an enriched grape juice medium at 20°C and 30°C. Death rate of the wine yeast was considerably higher in the presence of the killer yeast and fermentations were retarded at both temperatures. The killer yeast induced flocculation of the non-flocculent wine yeast

Malic Acid in Wine: Origin, Function and Metabolism during Vinification

The production of quality wines requires a judicious balance between the sugar, acid and flavour components of wine.  L-Malic and tartaric acids are the most prominent organic acids in wine and play a crucial role in the winemaking process, including the organoleptic quality and the physical, biochemical and microbial stability of wine.  Deacidification of grape must and wine is often required for the production of well-balanced wines. Malolactic fermentation induced by the addition of malolactic starter cultures, regarded as the preferred method for naturally reducing wine acidity, efficiently decreases the acidic taste of wine, improves the microbial stability and modifies to some extent the organoleptic character of wine. However, the recurrent phenomenon of delayed or sluggish malolactic fermentation often causes interruption of cellar operations, while the malolactic fermentation is not always compatible with certain styles of wine. Commercial wine yeast strains of Saccharomyces are generally unableto degrade L-malic acid effectively in grape must during alcoholic fermentation, with relatively minor modifications in total acidity during vinification. Functional expression of the malolactic pathway genes, i.e. the malate transporter (mae1) of Schizosaccharomyces pombe and the malolactic enzyme (mleA) from Oenococcus oeni in wine yeasts, haspaved the way for the construction of malate-degrading strains of Saccharomyces for commercial winemaking

Relationship between Ergosterol Concentrations in Wine Yeast and Sugar Fermentation at Different Temperatures

The effect of cellular ergosterol on the fermentation ability of four different Saccharomyces cerevisiae wine yeast strains was studied. F~rmentations were carried out at three temperatures and ergosterol concentrations determined at six fermentation stages. The lower ergosterol concentrations at the higher temperatures are ascribed to the higher growth rate of the yeast cells resulting in a more rapid dilution of the ergosterol. Growth of yeast during fermentation was linked with the ergosterol content of the cells. The ergosterol concentration of the yeast cells was not directly correlated to specific fermentation rates

Relationship Between Fatty Acid Concentrations in Wine Yeasts and Sugar Fermentation at Different Temperatures

The effects of temperature and fermentation stage on the cellular concentrations of unsaturated and saturated fatty acids on the fermentation abilities of four differentSaccharomyces cerevisiae wine yeasts were studied at three temperatures. The total unsaturated and saturated fatty acid concentrations were determined at six fermentation stages. Unsaturated fattyacid concentration decreased and the concentration of saturated fatty acids increased as fermentation proceeded. Saturated fatty acid concentration of the yeast cells correlated positively with high specific fermentation rates during the later stages of fermentation in contrast to the belief that fluid membranes with high unsaturated fatty acid concentrations enhance ethanol tolerance and, therefore, fermentation performance

Malic Acid Distribution and Degradation in Grape Must During Skin Contact: The Influence of Recombinant Malo-Ethanolic Wine Yeast Strains

Wine acidity plays an important role in determining wine quality and ensuring physiochemical and microbiological stability. In high-acid wines, the L-malic acid concentration is usually reduced through bacterial malolactic fermentation, while acidulation in low-acidity wines is usually done during final blending of the wine before bottling.  This study showed that skin contact did not influence the relative concentration of L-malic acid in the pulp and juice fractions from Colombard, Ruby Cabernet and Cabernet Sauvignon grape musts, with 32%-44% of the L-malic acid present in the pulp fraction. Four recombinant malo-ethanolic (ME) Saccharomyces wine yeast strains containing the malic enzyme (mae2) and malate transporter (mael) genes of Schizasaccharomyces pombe, effectively degraded the L-malic acid in both the juice and pulp fractions of all three cultivars, with a complete degradation of malic acid in the juice fraction within 2 days

Genetic Engineering of an Industrial Strain of Saccharomyces cerevisiae for L-Malic Acid Degradation via an Efficient Malo-Ethanolic Pathway

The optimal ratio of L-malic and L-tartaric acid in relation to other wine components is one of the most important aspects that ultimately determine wine quality during winemaking. Winemakers routinely rely on the judicious use of malolactic fermentation (MLF) after alcoholic fermentation to deacidify and stabilise their wines. However, due to theunreliability of the process and unsuitable sensory modifications in some grape cultivars, especially for fruity-floral wines, MLF is often regarded as problematic and undesirable. Alternative methods for reducing the amounts of L-malic acid in wine will contribute to improving the production of quality wines in the future, especially in coolclimate regions. Most wine yeast strains of Saccharomyces are unable to effectively degrade L-malic acid, whereas the fission yeast Schizosaccharomyces pombe efficiently degrades high concentrations of L-malic acid by means of malo-ethanolic fermentation. However, strains of S. pombe are not suitable for vinification due to the production of undesirable off-flavours. Previously, the 5. pombe malate permease (mael) and malic enzyme (mae2) genes were  successfully expressed under the 3-phosphoglycerate kinase (PGK1) regulatory elements in 5. cerevisiae, resulting in a recombinant laboratory strain of S. cerevisiae with an efficient malo-ethanolic pathway. Stable integration of the S. pombe malo-ethanolic pathway genes has now been obtained through the construction of a unique integration strategy in a commercial wine yeast strain. Co-transformation of the linear integration cassette containing the mael and mae2 genes and PGK1 regulatory elements and a multi-copy plasmid containing the phleomycin-resistance marker into a commercial Saccharomyces cerevisiae strain resulted in the successful transformation and integration of the malo-ethanolic genes. The recombinant 5. cerevisiae strain was successfully cured of phleomycin-resistance plasmid DNA in order to obtain malo-ethanolic yeast containing only yeast-derived DNA. The integrated malo-ethanolic genes were stable in 5. cerevisiae and during synthetic and grape must fermentation, L-malic acid was completely fermented to ethanol without any negative effect on fermentation kinetics and wine quality

Differentiation of Leuconostoc species by nicotinamide adenine dinucleotide-dependent D(-)-lactic dehydrogenase profiles

The electrophoretic mobility of the D(-)-nLDH's of Leuconostoc oenos (nine strains), Leuconostoc mesenteroides subsp. mesenteroides (one strain), Leuconostoc mesenteroides subsp. dextranicum (one strain), Leuconostoc lactis (three strains), Leuconostoc paramesenteroides (two strains) and a Leuconostoc sp. were compared using conventional and stacked polyacrylamide gels. A single diffused D(-)-nLDH band was obtained for each strain on a 7% acrylamide gel. However, several well defined D(-)-nLDH bands were obtained for each strain when a polyacrylamide stacked gel was used. Based on D(-)-nLDH profiles, strains of L. oenos were divided into two subgroups. The overall D(-)-nLDH profiles of strains of L. oenos were different from those obtained for the non-acidophilic Leuconostoc spp. Leuconostoc mesenteroides subsp. mesenteroides and L. mesenteroides subsp. dextranicum had similar D(-)-nLDH profiles. The overall D(-)-nLDH profiles of L. lactis corresponded well. Different D(-)-nLDH profiles were obtained for L. paramesenteroides DSM 20201 and the type strain of L. paramesenteroides (DSM 20288(T)). The D(-)-nLDH profile of Leuconostoc sp. ATCC 21436 corresponded well with the profile obtained for the D(-)-nLDH electrophoretic marker of Lactobacillus leichmannii.Articl

Characterization of Killer Yeast Isolates from Chenin blanc Grapes and Grape Skins

Wild-type killer yeast strains isolated from six South African wineries were identified using classical taxonomic methods. They were further characterized according to their cross-reactions with reference killer yeasts (K1-Kn) and by electrophoresis of their double-stranded RNA molecules. All isolates belonged to the K2 phenotype and were identified as strains of Saccharomyces cerevisiae and Saccharomyces bayanus. The killer strains differed substantially in their ability to kill a sensitive wine yeast (Geisenheim GS-1). This phenomenon may be attributed to strain differences among the killer yeasts as was shown by electrophoresis of total soluble cell proteins and gas chromatographic analysis of cellular fatty acids

Malo-ethanolic fermentation in Saccharomyces and Schizosaccharomyces

Yeast species are divided into the K(+) or K(-) groups, based on their ability or inability to metabolise tricarboxylic acid (TCA) cycle intermediates as sole carbon or energy source. The K(-) group of yeasts includes strains of Saccharomyces, Schizosaccharomyces pombe and Zygosaccharomyces bailii, which is capable of utilising TCA cycle intermediates only in the presence of glucose or other assimilable carbon sources. Although grouped together, these yeasts have significant differences in their abilities to degrade malic acid. Typically, strains of Saccharomyces are regarded as inefficient metabolisers of extracellular malic acid, whereas strains of Sch. pombe and Z. bailii can effectively degrade high concentrations of malic acid. The ability of a yeast strain to degrade extracellular malic acid is dependent on both the efficient transport of the dicarboxylic acid and the efficacy of the intracellular malic enzyme. The malic enzyme converts malic acid into pyruvic acid, which is further metabolised to ethanol and carbon dioxide under fermentative conditions via the so-called malo-ethanolic (ME) pathway. This review focuses on the enzymes involved in the ME pathway in Sch. pombe and Saccharomyces species, with specific emphasis on the malate transporter and the intracellular malic enzyme.Revie

A method for the detection of Issatchenkia orientalis in a baker's yeast factory

A method for the detection of Issatchenkia orientalis (Candida krusei), a contaminant of bakers' yeast, is described. Nine different liquid medium types were compared and maximum specific growth rates for the yeast contaminant were determined in each medium. Issatchenkia orientalis grew fastest in malt extract broth (0,37 h-1) and potato dextrose broth (0,36 h-1). Five antibiotics were tested for selective inhibition of seven different bakers' yeast strains in malt extract broth. Nystatin was the only antibiotic tested that inhibited the growth of all the bakers' yeast strains used, but did not affect the growth of I. orientalis. © 1984 Springer-Verlag.Articl