Co-inoculation with Dekkerea bruxellensis impacts the trancriptome of Saccharomyces cerevisiae during wine fermentation. Saccharomyces cerevisiae

The yeast Dekkera bruxellensis is as ethanol tolerant as Saccharomyces cerevisiae and may be found in bottled wine. It causes the spoilage of wine, beer, cider and soft drinks. In wines, the metabolic products responsible for spoilage by Dekkera bruxellensis are mainly volatile phenols. These chemical compounds are responsible for the taints described as ‘‘medicinal’’ in white wines (due to vinyl phenols) and as ‘‘leather’’, ‘‘horse sweat’’ and ‘‘stable’’ in red wines (due to ethyl phenols mainly 4-ethylphenol). Apart from the negative aroma nuances imparted by these yeasts, positive aromas such as ‘smoky’, ‘spicy’ and ‘toffee’ are also cited. Our goal was to identify the impact that the wine spoilage yeast Dekkera bruxellensis has on fermenting S. cerevisiae cells, especially on its gene expression level. To this end we co-inoculated both yeast species at the start of fermentation in a synthetic wine must, using S. cerevisiae-only fermentations without Dekkera bruxellensis as a control. All fermentations were employed in special membrane reactors (1.2 um pore size cut-off) physically separating Dekkera bruxellensis from wine yeast S. cerevisiae. Biomass separation with this membrane was done to abolish the possibility of hybridizing also D. bruxellensis probes on Agilent V2 (8x15K format) G4813 DNA microarrays designed just for S. cerevisiae ORF targets. The 1.2 um pore membrane separating both yeasts allowed the exchange of ethanol, metabolites and sugars during the fermentation. Overall design: Fermentations were carried out in synthetic wine must in duplicate for both the control S. cerevisiae (strain Lalvin EC1118) and mixed fermentation. Sampling of yeast S. cerevisiae for RNA extractions were performed at 22 h of fermentation, during the exponential growth phase of S. cerevisiae, at 92 h and 144 h of fermentation, during its early and late stationary growth phase and at 187 h of fermentation, during its phase of growth decline.

德克酵母（Dekkera bruxellensis）的乙醇耐受性与酿酒酵母（Saccharomyces cerevisiae）相当，可在瓶装葡萄酒中检出，且会引发葡萄酒、啤酒、苹果酒及软饮料的变质。在葡萄酒中，德克酵母导致变质的主要代谢产物为挥发性酚类物质。这类化合物会使白葡萄酒产生“药用”异味（源于乙烯基酚类），红葡萄酒则出现“皮革味”“马汗味”及“厩舍味”（主要源于4-乙基苯酚等乙基酚类）。除上述负面香气瑕疵外，相关研究也提及该酵母可赋予“烟熏”“辛香”及“太妃糖”等正面香气。
本研究旨在探究致葡萄酒变质的德克酵母（Dekkera bruxellensis）对酿酒酵母发酵细胞的影响，尤其是其基因表达水平。为此，我们在合成葡萄酒醪发酵初始阶段对两种酵母进行共接种，并以仅接种酿酒酵母、不含德克酵母的发酵体系作为对照。所有发酵实验均在特殊膜反应器（孔径截留值1.2 μm）中开展，该反应器可将德克酵母与酿酒酵母物理分隔。采用该孔径的膜进行菌体分离，目的是避免针对酿酒酵母开放读码框（ORF）靶点设计的安捷伦（Agilent）V2（8×15K格式）G4813型DNA微阵列（DNA microarray）出现德克酵母探针杂交的情况。1.2 μm孔径的分离膜可允许发酵过程中乙醇、代谢产物及糖类的交换。
实验总体设计：对照酿酒酵母（菌株Lalvin EC1118）与混合发酵体系均在合成葡萄酒醪中进行重复发酵。酿酒酵母的取样节点为：发酵22 h时（处于酿酒酵母指数生长阶段）、发酵92 h和144 h时（分别处于其早期和晚期稳定生长阶段），以及发酵187 h时（处于生长衰退阶段），取样后用于RNA提取。