Physiology and metabolism of Dekkera/Brettanomyces yeast in relation to mousy taint production

Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture, and Oenology, 199

Mousy off-flavor: a review

Although mousy off-flavor occurs infrequently in wine, it can be economically disastrous to the wine producer as, at worst, it can render the wine unpalatable or, at best, decrease the quality of the wine resulting in a lower sale price. Wines infected with either lactic acid bacteria (LAB) (particularly heterofermentative strains) or Dekkera/Brettanomyces yeast can potentially produce mousy off-flavor. There are three known compounds that cause mousy off-flavor: 2-ethyltetrahydropyridine, 2-acetyltetrahydopyridine, and 2-acetylpyrroline. Dekkera/Brettanomyces have been shown to be capable of producing at least two of these compounds, whereas LAB are capable of producing all three. The reason as to why mousy off-flavor forms in some wines and not in others is still not fully understood. The issue is further complicated by the fact that the compounds that have thus far been identified as necessary for off-flavor formation are all potentially available in wine (e.g., ethanol, L-lysine, L-ornithine, and metal ions). For these reasons, the microbe's metabolism probably plays a key role in mousy off-flavor formation. In the case of Dekkera/Brettanomyces-induced mousy off-flavor, it appears that oxygen may play a key role. Thus, a wine infected with Dekkera/Brettanomyces in the absence of oxygen may not become mousy unless exposed to oxygen via a processing or handling procedure

Isolation and Characterization of High-Ethanol-Tolerance Lactic Acid Bacteria from Australian Wine

Lactic acid bacteria are very important in winemaking. In this study, 108 lactic acid bacteria isolates were obtained from high-ethanol-content (~17% (v/v)) Grenache wines during uninoculated malolactic fermentation (MLF). The 16S rRNA and species-specific PCR showed that 104 of these were Oenococcusoeni, three were Lactobacillus hilgardii, and one was Staphylococcus pasteuri. AFLP of HindIII and MseI digests of the genomic DNA of the O. oeni strains was developed for the first time to discriminate the strains. The results showed that the method was a suitable technique for discriminating the O. oeni strains. Based on the cluster analysis, nine O. oeni strains were chosen for inclusion in an ethanol tolerance assay involving monitoring of optical density (ABS600nm) and viable plating. Several O. oeni strains (G63, G46, G71, G39) survived and grew well in MRS-AJ with 17% (v/v) ethanol, while the commercial O. oeni reference strain did not. Strain G63 could also survive and grow for 168 h after inoculation in MRS-AJ medium with 19% (v/v) ethanol. These results suggest that O. oeni G63, G46, G71, and G39 could potentially be used as MLF starters for high-ethanol-content wines. All three L. hilgardii strains could survive and grow in MRS-AJ with 19% (v/v) ethanol, perhaps also indicating their suitability as next-generation MLF starter cultures

Mass Transfer of Anthocyanins during Extraction from Pre-Fermentative Grape Solids under Simulated Fermentation Conditions: Effect of Convective Conditions

The colour of red wine is largely determined by the concentration of anthocyanins that are extracted from grape skins during fermentation. Because colour is a key parameter in determining the overall quality of the finished product, understanding the effect of processing variables on anthocyanin extraction is critical for producing a red wine with the desired sensorial characteristics. In this study, the effect of convective conditions (natural and forced) on the mass transfer properties of malvidin-3-glucoside (M3G) from pre-fermentative grape solids was explored at various liquid phase conditions representing stages of fermentation. A mathematical model that separates solid and liquid phase mass transfer parameters was applied to experimental extraction curves, and in all cases, provided a coefficient of determination exceeding 0.97. Calculated mass transfer coefficients indicated that under forced convective conditions, the extraction process was controlled by internal diffusion whereas under natural convection, both internal diffusion and liquid-phase mass transfer were relevant in determining the overall extraction rate. Predictive simulations of M3G extraction during active fermentation were accomplished by incorporating the current results with a previously developed fermentation model, providing insight into the effect of a dynamic liquid phase on anthocyanin extraction

Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid Bacterium Oenococcus oeni▿ †

We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C2 to C18. EstB28 exhibited greatest specificity for C2 to C4 pNP-linked substrates

Genomic insights into the metabolism of 'Candidatus Defluviicoccus seviourii', a member of Defluviicoccus cluster III abundant in industrial activated sludge

Filamentous cluster III Defluviicoccus (DF3) are known to proliferate and cause bulking issues in industrial wastewater treatment plants. Members of the genus Defluviicoccus are also known to exhibit the glycogen accumulating organism (GAO) phenotype, which is suggested to be detrimental to enhanced biological phosphorus removal (EBPR). Despite the reported negative impact members of the DF3 have on activated sludge wastewater treatment systems, limited research has focused on understanding the physiological traits that allow them to compete in these environments. In this study, a near complete genome of an abundant filamentous DF3 named 'Candidatus Defluviicoccus seviourii' was obtained from a full-scale sequencing batch reactor (SBR) treating winery wastewater. Annotation of the 'Ca. D. seviourii' genome revealed interesting metabolic features that help to understand the abundance of this microorganism in industrial wastewater treatment plants. Their potential for the storage of polyhydroxyalkanoates (PHA) is suggested to favour these organisms with the intermittent availability of carbon in these systems. An ability to fix nitrogen and take up urea may provide them with an additional advantage with the characteristically high carbon to nitrogen content of industrial waste. The genome and preliminary findings of this study provide a foundation for further research into these biotechnologically relevant organisms

Modelling the Mass Transfer Process of Malvidin-3-Glucoside during Simulated Extraction from Fresh Grape Solids under Wine-Like Conditions

Extraction of grape components is a key consideration for red winemaking. The impact of changing process variables on mass transfer properties of anthocyanins from fresh pre-fermentative red grape solids under forced convective conditions was explored using the dominant red grape anthocyanin, malvidin-3-glucoside (M3G) as a model solute. A two level full factorial design was implemented to investigate effects of temperature, sugar and ethanol on mass transfer properties. Factor levels were chosen to simulate conditions found at various points during the maceration and fermentation steps of the red winemaking process. A rigorous mathematical model was developed and applied to experimental extraction curves, allowing the separation of mass transport properties in liquid and solid phases in a wine-like system, for the first time. In all cases, the coefficient of determination exceeded 0.92, indicating good agreement between experimental and mathematically-solved M3G concentrations. For the conditions studied, internal mass transfer was found to limit M3G extraction and changes to the liquid phase composition and temperature influence the distribution constant. Surface response models of mass transfer parameters were developed to allow future simulations of fermentation scenarios aimed at maximising the extraction potential of M3G

Evaluation of indigenous non-Saccharomyces yeasts isolated from a South Australian vineyard for their potential as wine starter cultures

The use of non-Saccharomyces yeast in conjunction with Saccharomyces cerevisiae in wine fermentation is a growing trend in the wine industry. Non-Saccharomyces, through their distinctive production of secondary metabolites, have the potential to positively contribute to wine sensory profile. To discover new candidate strains for development as starter cultures, indigenous non-Saccharomyces were isolated from un-inoculated fermenting Shiraz musts from a South Australian vineyard (McLaren Vale wine region) and characterised. Among the 77 isolates, 7 species belonging to 5 genera (Kazachstania, Aureobasidium, Meyerozyma, Wickerhamomyces and Torulaspora) were identified by sequencing the internal transcribed spacer regions of the 5.8S rRNA gene (ITS1-5.8S-ITS2 region). The indigenous isolates were evaluated for oenological properties, namely, ethanol tolerance, enzyme activity, and H2S production. To determine their potential industrial use as starter cultures, representative isolates of each species were assessed in a sterile chemically defined grape juice and Viognier grape juice to evaluate their contribution to fermentation kinetics and production of key metabolites, including volatile compounds.</p

Impact of Lachancea thermotolerans on Chemical Composition and Sensory Profiles of Viognier Wines

Viognier is a warm climate grape variety prone to loss of acidity and accumulation of excessive sugars. The yeast Lachancea thermotolerans can improve the stability and balance of such wines due to the partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in co-inoculations and sequential inoculations with Saccharomyces cerevisiae in high sugar/pH Viognier fermentations. The results highlighted the dichotomy between the non-acidified and the bio-acidified L. thermotolerans treatments, with either comparable or up to 0.5 units lower pH relative to the S. cerevisiae control. Significant differences were detected in a range of flavour-active yeast volatile metabolites. The perceived acidity mirrored the modulations in wine pH/TA, as confirmed via &ldquo;Rate-All-That-Apply&rdquo; sensory analysis. Despite major variations in the volatile composition and acidity alike, the varietal aromatic expression (i.e., stone fruit aroma/flavour) remained conserved between the treatments

Genomic and in situ analyses reveal the Micropruina spp. as abundant fermentative glycogen accumulating organisms in enhanced biological phosphorus removal systems

Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the activity of polyphosphate accumulating organisms (PAOs). However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the Micropruina spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on Micropruina glycogenica str. Lg2(T) and compared to the in situ physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The Micropruina spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus in situ, but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal-as they compete for substrates with the Tetrasphaera PAO and stored carbon is not made available to the "Candidatus Accumulibacter" PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of potential carbon storage by fermentative organisms