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 the unreliability 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.

Ten Cryptococcus strains were screened for phytase activity, of which the Cryptococcus laurentii ABO 510 strain showed the highest level of activity. The cell wall‐associated enzyme displayed temperature and pH optima of 62°C and 5.0, respectively. The enzyme was thermostable at 70°C, with a loss of 40% of its original activity after 3 h. The enzyme was active on a broad range of substrates, including ATP, d‐glucose 6‐phosphate, d‐fructose 1,6‐diphosphate and p‐nitrophenyl phosphate (p‐NPP), but its preferred substrate was phytic acid (Km of 21 μM). The enzyme activity was completely inhibited by 0.5 mM inorganic phosphate or 5 mM phytic acid, and moderately inhibited in the presence of Hg2+, Zn2+, Cd2+ and Ca2+. These characteristics suggest that the Cry. laurentii ABO 510 phytase may be considered for application as an animal feed additive to assist in the hydrolysis of phytate complexes to improve the bioavailability of phosphorus in plant feedstuff.

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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.

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