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.
Highlights
During the aging of red wines, mainly if they are stored in barrels, undesirable metabolites can appear due to the growth of contaminating yeasts, such as Dekkera/Brettanomyces bruxellensis species (Silva et al, 2004)
Wine spoilage by D./B. bruxellensis has increased in frequency because of the use of less-severe processing conditions, the great variety of diverse vinification techniques and the tendency to reduce the use of preservatives, such as sulfur dioxide
The capability of D./B. bruxellensis to survive and to grow in wine can be partially ascribed to its high resistance to SO2; one of the main research question that can be addressed regarding the prevention of this spoilage yeast species is: “how the SO2 addition can be managed in order to counteract the yeast occurrence during winemaking and in the final product?” since the active form of SO2 against microbial proliferation depends on pH, ethanol concentration, and temperature (Usseglio-Tomasset and Bosia, 1984; RibéreauGayon et al, 2006), the answer has to take into consideration that wine is an extremely heterogeneous environment
Summary
During the aging of red wines, mainly if they are stored in barrels, undesirable metabolites (off-flavors) can appear due to the growth of contaminating yeasts, such as Dekkera/Brettanomyces bruxellensis species (Silva et al, 2004). This sensory modification resulting in wine defect is termed “Brett character” and it is described by “leather,” “horse sweat,” “medicinal,” “barnyard,” and “bacon” descriptors (Chatonnet et al, 1995). Vinyl phenols are reduced into their corresponding ethyl derivatives (4-ethylphenol, 4-ethylguaiacol, and 4-ethylcatechol) in a step catalyzed by a vinylphenol reductase (VPR) that represents the key enzyme designating D./B. bruxellensis species as the spoilage yeast able to produce ethyl phenols. The cloning of DbVPR gene in Saccharomyces cerevisiae, a species not producing ethyl phenols, has recently confirmed its role in the off-flavor production (Romano et al, 2017)
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