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- Wine Spoilage Control: Impact of Saccharomycin on Brettanomyces bruxellensis and Its Conjugated Effect with Sulfur DioxidePublication . Branco, Patricia; Coutinho, Rute; Malfeito-Ferreira, Manuel; Prista, Catarina; Albergaria, HelenaABSTRACT: The yeast Brettanomyces bruxellensis is one of the most dangerous wine contaminants due to the production of phenolic off-flavors such as 4-ethylphenol. This microbial hazard is regularly tackled by addition of sulfur dioxide (SO2). Nevertheless, B. bruxellensis is frequently found at low levels (ca 10(3) cells/mL) in finished wines. Besides, consumers health concerns regarding the use of sulfur dioxide encouraged the search for alternative biocontrol measures. Recently, we found that Saccharomyces cerevisiae secretes a natural biocide (saccharomycin) that inhibits the growth of different B. bruxellensis strains during alcoholic fermentation. Here we investigated the ability of S. cerevisiae CCMI 885 to prevent B. bruxellensis ISA 2211 growth and 4-ethylphenol production in synthetic and true grape must fermentations. Results showed that B. bruxellensis growth and 4-ethylphenol production was significantly inhibited in both media, although the effect was more pronounced in synthetic grape must. The natural biocide was added to a simulated wine inoculated with 5 x 10(2) cells/mL of B. bruxellensis, which led to loss of culturability and viability (100% dead cells at day-12). The conjugated effect of saccharomycin with SO2 was evaluated in simulated wines at 10, 12, 13 and 14% (v/v) ethanol. Results showed that B. bruxellensis proliferation in wines at 13 and 14% (v/v) ethanol was completely prevented by addition of 1.0 mg/mL of saccharomycin with 25 mg/L of SO2, thus allowing to significantly reduce the SO2 levels commonly used in wines (150-200 mg/L).
- Dominance of Saccharomyces cerevisiae in wine fermentations: secretion of antimicrobial peptides and microbial interactionsPublication . Albergaria, Helena; Branco, Patricia; Francisco, Diana; Coutinho, Rute; Monteiro, Margarida; Malfeito-Ferreira, Manuel; Arneborg, Nils; Almeida, M. Gabriela; Caldeira, JorgeIn present work we investigated the antagonistic effect by S. cerevisiae against several wine-related microbial species vis-à-vis the secretion of antimicrobial peptides (AMPs).
- Wine Microbial Consortium: Seasonal Sources and Vectors Linking Vineyard and Winery EnvironmentsPublication . Camilo, Sofia; Chandra, Mahesh; Branco, Patricia; Malfeito-Ferreira, ManuelABSTRACT: Winemaking involves a wide diversity of microorganisms with different roles in the process. The wine microbial consortium (WMC) includes yeasts, lactic acid bacteria and acetic acid bacteria with different implications regarding wine quality. Despite this technological importance, their origin, prevalence, and routes of dissemination from the environment into the winery have not yet been fully unraveled. Therefore, this study aimed to evaluate the WMC diversity and incidence associated with vineyard environments to understand how wine microorganisms overwinter and enter the winery during harvest. Soils, tree and vine barks, insects, vine leaves, grapes, grape musts, and winery equipment were sampled along four seasons. The isolation protocol included: (a) culture-dependent microbial recovery; (b) phenotypical screening to select fermenting yeasts, lactic acid, and acetic acid bacteria; and (c) molecular identification. The results showed that during all seasons, only 11.4% of the 1424 isolates presumably belonged to the WMC. The increase in WMC recovery along the year was mostly due to an increase in the number of sampled sources. Acetic acid bacteria (Acetobacter spp., Gluconobacter spp., Gluconoacetobacter spp.) were mostly recovered from soils during winter while spoilage lactic acid bacteria (Leuconostoc mesenteroides and Lactobacillus kunkeii) were only recovered from insects during veraison and harvest. The fermenting yeast Saccharomyces cerevisiae was only isolated from fermented juice and winery equipment. The spoilage yeast Zygosaccharomyces bailii was only recovered from fermented juice. The single species bridging both vineyard and winery environments was the yeast Hanseniaspora uvarum, isolated from insects, rot grapes and grape juice during harvest. Therefore, this species appears to be the best surrogate to study the dissemination of the WMC from vineyard into the winery. Moreover, the obtained results do not evidence the hypothesis of a perennial terroir-dependent WMC given the scarcity of their constituents in the vineyard environment along the year and the importance of insect dissemination.
- Saccharomyces cerevisiae and Dekkera bruxellensis interactions in alcoholic fermentations: growth and 4-ethylphenol productionPublication . Coutinho, Rute; Branco, Patricia; Monteiro, Margarida; Malfeito-Ferreira, Manuel; Albergaria, HelenaThe yeast Dekkera/Brettanomyces bruxellensis can cause enormous economic losses both in wine industry and fuel-ethanol processes due to production of phenolic off-flavour compounds and low ethanol productivities. In winemaking this microbial hazard is usually tackled by the use of chemical preservatives such as sulphur dioxide. In spite of this, D. bruxellensis strains are frequently found in wines at low levels (ca 103 cells/ml) where they can metabolise residual sugars producing phenolic off-flavours compounds, such as 4-ethyl phenol. In the present work we investigated S. cerevisiae and D. bruxellensis interactions during alcoholic fermentations and evaluated the effectiveness of antimicrobial peptides secreted by S. cerevisiae to prevent growth of the main wine spoilage yeast and the production of 4-ethylphenol. Several fermentations were performed with single cultures of D. bruxellensis and mixed cultures of S. cerevisiae and D. bruxellensis, both in synthetic grape juice (SGJ) and grape must. Yeast growth (culturability and viability) and fermentation performance (i.e. sugars consumption, ethanol and 4-ethylphenol production) of those fermentations was accessed by different methods, namely by florescence in situ hybridization and flow cytometry. Results showed that S. cerevisiae significantly reduced the growth of D. bruxellensis and the production of 4-ethylphenol both in SGJ and grape must fermentations performed with mixed cultures. Moreover, our work also showed that antimicrobial peptides secreted by S. cerevisiae are effective to prevent growth of D. bruxellensis and production of phenolic off-flavor compounds in wine.