A Response Surface Methodology Approach to Investigate the Effect of Sulfur Dioxide, pH, and Ethanol on DbCD and DbVPR Gene Expression and on the Volatile Phenol Production in Dekkera/Brettanomyces bruxellensis CBS2499

Abstract

Dekkera/Brettanomyces bruxellensis, the main spoilage yeast in barrel-aged wine, metabolize hydroxycinnamic acids into off-flavors, namely ethylphenols. Recently, both the enzymes involved in this transformation, the cinnamate decarboxylase (DbCD) and the vinylphenol reductase (DbVPR), have been identified. To counteract microbial proliferation in wine, sulfur dioxide (SO₂) is used commonly to stabilize the final product, but limiting its use is advised to preserve human health and boost sustainability in winemaking. In the present study, the influence of SO₂ was investigated in relation with pH and ethanol factors on the expression of DbCD and DbVPR genes and volatile phenol production in D. bruxellensis CBS2499 strain under different model wines throughout a response surface methodology (RSM). In order to ensure an exact quantification of DbCD and DbVPR expression, an appropriate housekeeping gene was sought among DbPDC, DbALD, DbEF, DbACT, and DbTUB genes by GeNorm and Normfinder algorithms. The latter gene showed the highest expression stability and it was chosen as the reference housekeeping gene in qPCR assays. Even though SO₂ could not be commented as main factor because of its statistical irrelevance on the response of DbCD gene, linear interactions with pH and ethanol concurred to define a significant effect (p < 0.05) on its expression. The DbCD gene was generally downregulated respect to a permissive growth condition (0 mg/L mol. SO₂, pH 4.5 and 5% v/v ethanol); the combination of the factor levels that maximizes its expression (0.83-fold change) was calculated at 0.25 mg/L mol. SO₂, pH 4.5 and 12.5% (v/v) ethanol. On the contrary, DbVPR expression was not influenced by main factors or by their interactions; however, its expression is maximized (1.80-fold change) at the same conditions calculated for DbCD gene. While no linear interaction between factors influenced the off-flavor synthesis, ethanol and pH produced a significant effect as individual factors. The obtained results can be useful to improve the SO₂ management at the grape harvesting and during winemaking in order to minimize the D./B. bruxellensis spoilage.

Keywords: D./B. bruxellensis; cinnamate decarboxylase gene; gene expression; response surface methodology; vinylphenol reductase gene; volatile phenols.

FIGURE 1

Standardized Pareto charts for each analyzed variable (A) DbCD and (B) DbVPR gene expression, (C) vinyl phenol, (D) vinyl guaiacol, (E) ethyl phenol, and (F) ethyl guaiacol yields. The color of the bars shows whether an effect is positive (pink) or negative (red). A line is drawn on the chart beyond which an effect is statistically significant at the specified significance level of 5%.

FIGURE 2

Response surface fitted to experimental data points corresponding to: (A) DbCD expression as function of SO₂ and pH interaction (AB); (B) DbCD expression as function of SO₂ and ethanol interaction (AC).

FIGURE 3

Interaction plots for the expression of DbCD gene. (A) SO₂ and pH (AB); (B) SO₂ and ethanol concentration (AC). Lines represent the predicted responses at further experimental combinations among the analyzed factors. Continuous line (), 0 mg/L mol. SO₂; long-dashed line (), 0.125 mg/L mol. SO₂; short-dashed line (), 0.25 mg/L mol. SO₂.