Comparative analysis of intracellular metabolites, proteins and their molecular functions in a flor yeast strain under two enological conditions.

Flor yeasts confer a wide range of organoleptic properties to Sherry-type wines during a process called "biological aging" that takes place after alcoholic fermentation. These kinds of yeasts adapt to a biological aging condition by forming a biofilm known as "flor velum" and by changing from fermentative to oxidative metabolism. It has been reported that some functions such as increase of cell surface hydrophobicity or changes to lipid metabolism are enhanced when yeasts switch to biofilm lifestyle. Here, we attempt to reveal intracellular metabolites and protein molecular functions not documented before that are relevant in biofilm formation and in fermentation by an endometabolome and proteome screening. We report that at early stages of biofilm formation, flor yeasts accumulate mannose, trehalose, glycerol, oleic and stearic acids and synthesize high amounts of GTPases, glycosylases and lipoproteins. On the other hand, in early fermentation, flor yeasts rapidly consume glucose and phosphoric acid; and produce abundant proteins related to chromatin binding, transcription factors and methyl transferases.

Assessment of the Brettanomyces bruxellensis metabolome during sulphur dioxide exposure.

Brettanomyces bruxellensis displays a high degree of genotypic and phenotypic polymorphism and is the main yeast species involved in wine spoilage. The innate resistance of 108 B. bruxellensis strains to the antimicrobial agent SO2 used in winemaking was investigated. Nineteen strains (17.6%) were sensitive to SO2 , failing to grow at the lowest concentration tested (0.1 mg L(-1) molecular SO2). Twenty-nine strains (26.8%) grew at 0.1 mg L(-1), 42 strains (38.9%) grew at 0.2 mg L(-1) , and 16 strains (14.8%) were able to grow as high as 0.4 mg L(-1) mol. SO2. Two strains able to grow in the presence of 0.6 mg L(-1) mol. SO2 were further studied by GCMS-TOF analysis to define the metabolic response to SO2 treatment. Two hundred and fifty-three intracellular metabolites were detected. The main effect observed was a decrease in cytoplasmic levels of polyols and an increase in levels of some amino acids, alanine, glutamic acid, glycine, proline, 5-oxoproline, serine and valine, which were significantly accumulated in the presence of SO2. No alteration in the pentose phosphate pathway was observed, suggesting NADPH usage could be diverted to other pathways. Finally, a change in metabolites involved in the glycerophospholipid pathway (glycerol-3-phosphate and myo-inositol) was also found.

Next-generation sequencing reveals significant bacterial diversity of botrytized wine.

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.