Transcriptomics unravels the adaptive molecular mechanisms of Brettanomyces bruxellensis under SO2 stress in wine condition.

Sulfur dioxide is generally used as an antimicrobial in wine to counteract the activity of spoilage yeasts, including Brettanomyces bruxellensis. However, this chemical does not exert the same effectiveness on different B. bruxellensis yeasts since some strains can proliferate in the final product leading to a negative sensory profile due to 4-ethylguaiacol and 4-ethylphenol. Thus, the capability of deciphering the general molecular mechanisms characterizing this yeast species' response in presence of SO2 stress could be considered strategic for a better management of SO2 in winemaking. A RNA-Seq approach was used to investigate the gene expression of two strains of B. bruxellensis, AWRI 1499 and CBS 2499 having different genetic backgrounds, when exposed to a SO2 pulse. Results revealed that sulphites affected yeast culturability and metabolism, but not volatile phenol production suggesting that a phenotypical heterogeneity could be involved for the SO2 cell adaptation. The transcriptomics variation in response to SO2 stress confirmed the strain-related response in B. bruxellensis and the GO analysis of common differentially expressed genes showed that the detoxification process carried out by SSU1 gene can be considered as the principal specific adaptive response to counteract the SO2 presence. However, nonspecific mechanisms can be exploited by cells to assist the SO2 tolerance; namely, the metabolisms related to sugar alcohol (polyols) and oxidative stress, and structural compounds.

Cloning the putative gene of vinyl phenol reductase of Dekkera bruxellensis in Saccharomyces cerevisiae.

Vinylphenol reductase of Dekkera bruxellensis, the characteristic enzyme liable for "Brett" sensory modification of wine, has been recently recognized to belong to the short chain dehydrogenases/reductases family. Indeed, a preliminary biochemical characterisation has conferred to the purified protein a dual significance acting as superoxide dismutase and as a NADH-dependent reductase. The present study aimed for providing a certain identification of the enzyme by cloning the VPR gene in S. cerevisiae, a species not producing ethyl phenols. Transformed clones of S. cerevisiae resulted capable of expressing a biologically active form of the heterologous protein, proving its role in the conversion of 4-vinyl guaiacol to 4-ethyl guaiacol. A VPR specific protein activity of 9 ± 0.6 mU/mg was found in crude extracts of S. cerevisiae recombinant strain. This result was confirmed in activity trials carried out with the protein purified from transformant cells of S. cerevisiae by a his-tag purification approach; in particular, VPR-enriched fractions showed a specific activity of 1.83 ± 0.03 U/mg at pH 6.0. Furthermore, in agreement with literature, the purified protein behaves like a SOD, with a calculated specific activity of approximatively 3.41 U/mg. The comparative genetic analysis of the partial VPR gene sequences from 17 different D. bruxellesis strains suggested that the observed polymorphism (2.3%) and the allelic heterozygosity state of the gene do not justify the well described strain-dependent character in producing volatile phenols of this species. Actually, no correlation exists between genotype membership of the analysed strains and their capability to release off-flavours. This work adds valuable knowledge to the study of D. bruxellensis wine spoilage and prepare the ground for interesting future industrial applications.

The vintage effect overcomes the terroir effect: a three year survey on the wine yeast biodiversity in Franciacorta and Oltrepò Pavese, two northern Italian vine-growing areas.

A three year survey on the dominant yeast populations in samples of air, must and wine in different vineyards and cellars of two northern Italian vine-growing territories (six sites in Franciacorta and eight sites in Oltrepò Pavese areas) was carried out. A total of 505 isolates were ascribed to 31 different species by RFLP analysis of the ITS1-5.8SrRNA-ITS2 region and partial sequence analysis of the 26S rRNA gene. The most commonly found species were Saccharomyces cerevisiae (frequency, F' = 58.7%; incidence, I' = 53.5%), Hanseniaspora uvarum (F' = 14.3%; I' = 5.3%), Metschnikowia fructicola (F' = 11.1%; I' = 5.0%) and Torulaspora delbrueckii (F' = 10.3%; I' = 3.8%). Among 270 S. cerevisiae new isolates, 156 (57.8%) revealed a different genetic pattern through polymorphism analysis of the interdelta regions by capillary electrophoresis, while 47 isolates (17.4 %) were clones of starter cultures. By considering the Shannon-Wiener index and results of principal component analysis (PCA) analyses, the year of isolation (vintage) proved to be a factor that significantly affected the biodiversity of the yeast species, whereas the geographical site (terroir) was not. Seventy-five per cent of S. cerevisiae isolates gathered in a unique cluster at a similarity level of 82%, while the remaining 25% were separated into minor groups without any evident relationship between δ-PCR profile and territory, year or source of isolation. However, in six cases a similar strain appeared at the harvesting time both in Franciacorta and Oltrepò Pavese areas, whereas surprisingly no strain was reisolated in the same vineyard or cellar for consecutive years.

Identifying the Main Drivers in Microbial Diversity for Cabernet Sauvignon Cultivars from Europe to South Africa: Evidence for a Cultivar-Specific Microbial Fingerprint.

Microbial diversity in vineyards and in grapes has generated significant scientific interest. From a biotechnological perspective, vineyard and grape biodiversity has been shown to impact soil, vine, and grape health and to determine the fermentation microbiome and the final character of wine. Thus, an understanding of the drivers that are responsible for the differences in vineyard and grape microbiota is required. The impact of soil and climate, as well as of viticultural practices in geographically delimited areas, have been reported. However, the limited scale makes the identification of generally applicable drivers of microbial biodiversity and of specific microbial fingerprints challenging. The comparison and meta-analysis of different datasets is furthermore complicated by differences in sampling and in methodology. Here we present data from a wide-ranging coordinated approach, using standardized sampling and data generation and analysis, involving four countries with different climates and viticultural traditions. The data confirm the existence of a grape core microbial consortium, but also provide evidence for country-specific microbiota and suggest the existence of a cultivar-specific microbial fingerprint for Cabernet Sauvignon grape. This study puts in evidence new insight of the grape microbial community in two continents and the importance of both location and cultivar for the definition of the grape microbiome.

Use of Native Yeast Strains for In-Bottle Fermentation to Face the Uniformity in Sparkling Wine Production.

The in-bottle fermentation of sparkling wines is currently triggered by few commercialized Saccharomyces cerevisiae strains. This lack of diversity in tirage yeast cultures leads to a prevalent uniformity in sensory profiles of the end products. The aim of this study has been to exploit the natural multiplicity of yeast populations in order to introduce variability in sparkling wines throughout the re-fermentation step. A collection of 133 S. cerevisiae strains were screened on the basis of technological criteria (fermenting power and vigor, SO2 tolerance, alcohol tolerance, flocculence) and qualitative features (acetic acid, glycerol and H2S productions). These activities allowed the selection of yeasts capable of dominating the in-bottle fermentation in actual cellar conditions: in particular, the performances of FX and FY strains (isolated in Franciacorta area), and OX and OY strains (isolated in Oltrepò Pavese area), were compared to those of habitually used starter cultures (IOC18-2007, EC1118, Lalvin DV10), by involving nine wineries belonging to the two Consortia of Appellation of Origin. The microbiological analyses of samples have revealed that the indigenous strains showed an increased latency period and a higher cultivability along the aging time than the commercial starter cultures do. Results of chemical analyses and sensory evaluation of the samples after 18 months sur lies have shown that significant differences (p < 0.05) were present among the strains for alcoholic strength, carbon dioxide overpressure and pleasantness, whereas they were not observed for residual sugars content, titratable acidity or volatile acidity. Indigenous S. cerevisiae exhibited comparable values respect to the commercial starter cultures. The ANOVA has also proven that the base wine formulation is a key factor, by significantly affecting (p < 0.01) some oenological parameters of wine, like alcoholic strength, volatile acidity, carbon dioxide overpressure, titratable acidity and dry extract. The use of native yeast strains for the re-fermentation step can be considered a convenient way for introducing differentiation to the final product without modifying the traditional technology. In a perspective of "precision enology," where the wine is designed on specific vine cultivars and microorganisms, this work underlines that exploring yeast biodiversity is a strategic activity to improve the production.

Wild Grape-Associated Yeasts as Promising Biocontrol Agents against Vitis vinifera Fungal Pathogens.

The increasing level of hazardous residues in the environment and food chains has led the European Union to restrict the use of chemical fungicides. Thus, exploiting new natural antagonistic microorganisms against fungal diseases could serve the agricultural production to reduce pre- and post-harvest losses, to boost safer practices for workers and to protect the consumers' health. The main aim of this work was to evaluate the antagonistic potential of epiphytic yeasts against Botrytis cinerea, Aspergillus carbonarius, and Penicillium expansum pathogen species. In particular, yeast isolation was carried out from grape berries of Vitis vinifera ssp sylvestris populations, of the Eurasian area, and V. vinifera ssp vinifera cultivars from three different farming systems (organic, biodynamic, and conventional). Strains able to inhibit or slow the growth of pathogens were selected by in vitro and in vivo experiments. The most effective antagonist yeast strains were subsequently assayed for their capability to colonize the grape berries. Finally, possible modes of action, such as nutrients and space competition, iron depletion, cell wall degrading enzymes, diffusible and volatile antimicrobial compounds, and biofilm formation, were investigated as well. Two hundred and thirty-one yeast strains belonging to 26 different species were isolated; 20 of them, ascribed to eight species, showed antagonistic action against all molds. Yeasts isolated from V. vinifera ssp sylvestris were more effective (up to 50%) against B. cinerea rather than those isolated from V. vinifera ssp vinifera. Six strains, all isolated from wild vines, belonging to four species (Meyerozyma guilliermondii, Hanseniaspora uvarum, Hanseniaspora clermontiae, and Pichia kluyveri) revealed one or more phenotypical characteristics associated to the analyzed modes of antagonistic action.

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.

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.

Indigenous Georgian Wine-Associated Yeasts and Grape Cultivars to Edit the Wine Quality in a Precision Oenology Perspective.

In Georgia, one of the most ancient vine-growing environment, the homemade production of wine is still very popular in every rural family and spontaneous fermentation of must, without addition of chemical preservatives, is the norm. The present work investigated the yeast biodiversity in five Georgian areas (Guria, Imereti, Kakheti, Kartli, Ratcha-Lechkhumi) sampling grapes and wines from 22 different native cultivars, in 26 vineyards and 19 family cellars. One hundred and eighty-two isolates were ascribed to 15 different species by PCR-ITS and RFLP, and partial sequencing of D1/D2 domain 26S rDNA gene. Metschnikowia pulcherrima (F' = 0.56, I' = 0.32), Hanseniaspora guilliermondii (F' = 0.49, I' = 0.27), and Cryptococcus flavescens (F' = 0.31, I' = 0.11) were the dominant yeasts found on grapes, whereas Saccharomyces cerevisiae showed the highest prevalence into wine samples. Seventy four isolates with fermentative potential were screened for oenological traits such as ethanol production, resistance to SO2, and acetic acid, glycerol and H2S production. Three yeast strains (Kluyveromyces marxianus UMY207, S. cerevisiae UMY255, Torulaspora delbrueckii UMY196) were selected and separately inoculated in vinifications experiments at a Georgian cellar. Musts were prepared from healthy grapes of local varieties, Goruli Mtsvane (white berry cultivar) and Saperavi (black berry cultivar). Physical (°Brix) and microbial analyses (plate counts) were performed to monitor the fermentative process. The isolation of indigenous S. cerevisiae yeasts beyond the inoculated strains indicated that a co-presence occurred during the vinification tests. Results from quantitative GC-FID analysis of volatile compounds revealed that the highest amount of fermentation flavors, such as 4-ethoxy-4-oxobutanoic acid (monoethyl succinate), 2-methylpropan-1-ol, ethyl 2-hydroxypropanoate, and 2-phenylethanol, were significantly more produced in fermentation conducted in Saperavi variety inoculated with K. marxianus, whereas other aromatic compounds like 3-methylbutyl acetate, ethyl hexanoate and dihydrofuran-2(3H)-one (γ- butyrolactone) showed a higher content in Goruli Mtsvane variety samples fermented by S. cerevisiae. The selected yeast strains have proved to be promising for enhancing the flavor potential in low aromatic Georgian cultivars. This work intends to be a knowledge contribution for a precision oenology toward the strategic concept of "one grape variety-one yeast".