Impact of volatile phenols and their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts

Volatile phenols are aromatic compounds and one of the key molecules responsible for olfactory defects in wine. The yeast genus Brettanomyces is the only major microorganism that has the ability to covert hydroxycinnamic acids into important levels of these compounds, especially 4-ethylphenol and 4-ethylguaiacol, in red wine. When 4-ethylphenols reach concentrations greater than the sensory threshold, all wine’s organoleptic characteristics might be influenced or damaged. The aim of this literature review is to provide a better understanding of the physicochemical, biochemical, and metabolic factors that are related to the levels of p-coumaric acid and volatile phenols in wine. Then, this work summarizes the different methods used for controlling the presence of Brettanomyces in wine and the production of ethylphenols

De-Novo Assembly and Analysis of the Heterozygous Triploid Genome of the Wine Spoilage Yeast Dekkera bruxellensis AWRI1499

Despite its industrial importance, the yeast species Dekkera (Brettanomyces) bruxellensis has remained poorly understood at the genetic level. In this study we describe whole genome sequencing and analysis for a prevalent wine spoilage strain, AWRI1499. The 12.7 Mb assembly, consisting of 324 contigs in 99 scaffolds (super-contigs) at 26-fold coverage, exhibits a relatively high density of single nucleotide polymorphisms (SNPs). Haplotype sampling for 1.2% of open reading frames suggested that the D. bruxellensis AWRI1499 genome is comprised of a moderately heterozygous diploid genome, in combination with a divergent haploid genome. Gene content analysis revealed enrichment in membrane proteins, particularly transporters, along with oxidoreductase enzymes. Availability of this assembly and annotation provides a resource for further investigation of genomic organization in this species, and functional characterization of genes that may confer important phenotypic traits

Modeling the effect of ethanol vapor on the germination time of Penicillium chrysogenum

The influence of ethanol vapor on germination of Penicillium chrysogenum was determined on yeast nitrogen base plus glucose agar medium at 25°C. Ethanol vapors were generated by 0 to 6% (wt/wt) ethanol solutions at the bottom of hermetically closed petri dishes. The logistic equation was used to describe the data as the percentage of germination versus time and to estimate the germination time. The effect of ethanol concentration on germination time was described by a new reparameterized equation, resulting in an estimated limiting ethanol concentration of 4.3%. Up to 3% ethanol, all spores germinated, and the germination time increased with increasing ethanol concentration. At 3.5 and 4%, some spores formed abnormal germ tubes and others were inhibited at the swelling stage. The inhibiting effect of ethanol was reversible under these experimental conditions

New insights on the features of the vinyl phenol reductase from the wine-spoilage yeast Dekkera/Brettanomyces bruxellensis

Vinyl phenol reductase activity was assayed in extracts from 19 strains of Dekkera bruxellensis isolated from wine. In all strains, vinyl phenol reductase activity was insensitive to the presence/absence of 4-vinyl guaiacol, confirming that expression is not related to the presence of the substrate. D. bruxellensis CBS 4481 showed the highest vinyl phenol reductase activity toward 4-vinyl guaiacol. Vinyl phenol reductase from D. bruxellensis CBS 4481 was purified to mass spectrometric homogeneity, and sequenced by trypsinolysis and mass spectrometry. The sequence of the purified protein showed convincing homology with a Cu/Zn superoxide dismutase in the D. bruxellensisAWRI 1499 genome, and indeed it was found to possess both vinyl phenol reductase and superoxide dismutase activities. A bioinformatics analysis of the sequence of vinyl phenol reductase/superoxide dismutase from D. bruxellensis CBS 4481 reveals the presence in this protein of cofactor-binding structural features, that are absent in sequences of superoxide dismutases from related microorganisms, that do not display vinyl phenol reductase activity

Hydroxycinnamic acid ethyl esters as precursors to ethylphenols in wine

A method for determining ethyl coumarate and ethyl ferulate in wine using GC-MS with deuterium-labeled analogues has been developed and used to measure the evolution of these two esters during the production of two commercial monovarietal red wines, cv. Grenache and Shiraz. During fermentation, the concentration of ethyl coumarate rose from low levels to 0.4 mg/L in Grenache and 1.6 mg/L in Shiraz wines. These concentrations then increased further during barrel aging to 1.4 and 3.6 mg/L, respectively. The concentration of ethyl ferulate was much lower, reaching a maximum of only 0.09 mg/L. Conversion of ethyl coumarate and ethyl ferulate to their corresponding ethylphenols was observed during fermentations of a synthetic medium with two strains of Dekkera bruxellensis (AWRI 1499 and AWRI 1608), while a third (strain AWRI 1613) produced no ethylphenols at all from these precursors. Strains AWRI 1499 and 1608 produced 4-ethylphenol from ethyl coumarate in 68% and 57% yields, respectively. The corresponding yields of 4-ethylguaiacol from ethyl ferulate were much lower, 7% and 3%. Monitoring of ethyl coumarate and ethyl ferulate concentration during the Dekkera fermentations showed that the selectivity for ethylphenol production according to yeast strain and the precursor was principally a result of variation in esterase activity. Consequently, ethyl coumarate can be considered to be a significant precursor to 4-ethylphenol in wines affected by these two strains of Brettanomyces/Dekkera yeast, while ethyl ferulate is not an important precursor to 4-ethylguaiacol.Josh L. Hixson, Nicola R. Sleep, Dimitra L. Capone, Gordon M. Elsey, Christopher D. Curtin, Mark A. Sefton, and Dennis K. Taylo

Implications of new research and technologies for malolactic fermentation in wine

The initial conversion of grape must to wine is an alcoholic fermentation (AF) largely carried out by one or more strains of yeast, typically Saccharomyces cerevisiae. After the AF, a secondary or malolactic fermentation (MLF) which is carried out by lactic acid bacteria (LAB) is often undertaken. The MLF involves the bioconversion of malic acid to lactic acid and carbon dioxide. The ability to metabolise L-malic acid is strain specific, and both individual Oenococcus oeni strains and other LAB strains vary in their ability to efficiently carry out MLF. Aside from impacts on acidity, LAB can also metabolise other precursors present in wine during fermentation and, therefore, alter the chemical composition of the wine resulting in an increased complexity of wine aroma and flavour. Recent research has focused on three main areas: enzymatic changes during MLF, safety of the final product and mechanisms of stress resistance. This review summarises the latest research and technological advances in the rapidly evolving study of MLF and investigates the directions that future research may take.Krista M. Sumby, Paul R. Grbin, Vladimir Jirane