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Viognier is a warm climate grape variety prone to loss of acidity and accumulation of excessive sugars. The yeast

Anthocyanins are polyphenolic compounds present in grapes that are responsible for the initial colour of red wine and their incorporation into derived pigments leads to long term colour stability. The ability to predict the effect of process variables, either controlled by winemakers or that naturally change throughout fermentation, on the extraction of anthocyanins is vital to producing red wine of high quality. A 23 factorial experiment with additional points located at central factor conditions was used to determine the impact of temperature, sugar and ethanol concentrations on the mass transfer properties of anthocyanins from fresh Pinot noir grape solids. Factor conditions were chosen to replicate ethanol and sugar concentrations that would be found in a 14% red wine and its respective unfermented juice. A previously described mathematical model was applied to anthocyanin extraction curves to determine mass transfer coefficients and distribution constants for the generation of response surface models able to predict these mass transfer variables for dynamic fermentation scenarios. The coefficient of determination for the model solution exceeded 0.94 in all cases, demonstrating a good agreement between experimental and mathematically-derived anthocyanin concentrations. Following this, simulations of anthocyanin extraction under fermentation conditions were conducted using a previously developed wine fermentation model allowing for the prediction of extraction rates and anthocyanin concentrations under various winemaking scenarios. The extraction simulations predicted a previously observed but so far undescribed anthocyanin extraction pattern during fermentation.

This review focuses on the considerable amount of research that has been directed towards the improvement of efficiency and reliability of malolactic fermentation (MLF), which is important in winemaking. From this large body of work, it is clear that reliable MLF is essential for process efficiency and prevention of spoilage in the final product. Impediments to successful MLF in wine, the impact of grape and wine ecology and how this may affect MLF outcome are discussed. Further focus is given to how MLF success may be enhanced, via alternative inoculation strategies, MLF progress sensing technologies and the use of different bacterial species. An update of how this information may be used to enhance and improve sensory outcomes through metabolite production during MLF and suggestions for future research priorities for the field are also provided.

In Latin, 'pulcherrima' is a superlative form of an adjective that translates as beautiful. Apart from being 'the most beautiful' yeast, Metschnikowia pulcherrima has a remarkable potential in production of wines with lower ethanol content. The oenological performance of six M. pulcherrima strains was hereby tested in sequential cultures with Saccharomyces cerevisiae. The best-performing strain MP2 was further characterised in fermentations with different S. cerevisiae inoculation delays in both white grape juice and Chemically Defined Grape Juice Medium (CDGJM). The analysis of main metabolites, undertaken prior to sequential inoculations and upon fermentation completion, highlighted metabolic interactions and carbon sinks other than ethanol in MP2 treatments. Depending on the inoculation delay, MP2 white wines contained between 0.6% and 1.2% (v/v) less ethanol than the S. cerevisiae monoculture, with even larger decreases detected in the CDGJM. The MP2 treatments also contained higher concentrations of TCA cycle by-products (i.e. fumarate and succinate) and glycerol, and lower concentrations of acetic acid. The analysis of volatile compounds showed increased production of acetate esters and higher alcohols in all MP2 wines, alongside other compositional alterations arising from the S. cerevisiae inoculation delay.

Severe oenological conditions, such as limited assimilable nitrogen and high sugar contents restrict yeast's ability to successfully complete fermentation. In the absence of a comprehensive commercially available deletion collection in a wine yeast background, a screening approach was applied to a transposon library in a wine yeast derivative to identify clones with superior fermentation performance. Five candidate genes, when disrupted by Ty insertion, were identified as enabling yeast to efficiently complete a model oenological fermentation with limited nitrogen availability. Analogous single gene disruptions were subsequently constructed in the haploid wine yeast strain C911D, and the performance of these during fermentation was analysed. Deletion of ECM33 resulted in the shortest fermentation (up to 31% reduction) in both synthetic medium and grape juice. Interestingly, no significant differences were found in nitrogen utilization, cell viability or biomass yield between ∆ecm33 and the wild type. ∆ecm33 did, however, display growth hypersensitivity to the dyes Calcofluor White and Congo Red, suggesting a link to cell wall integrity. Transcriptional profiling of ∆ecm33 during fermentation demonstrated the up-regulation of SLT2 and HOG1, encoding mitogen activated protein kinases involved in the cell wall integrity (CWI) and high osmolarity glycerol (HOG) pathways, respectively. CHS3 a major chitin synthase gene was also found to be upregulated, and the transcript abundance of key genes of central nitrogen metabolism, GLN1, GLT1, GDH1 and GDH2 in mutant ∆ecm33 were also altered. The findings highlight the complexity of the robust fermentation phenotype and provide clues for further improvement of industrial strains.

Background and Aims The choice of yeast strain(s) to conduct the fermentation can greatly affect wine chemical and sensory profile. Even though the use of non‐Saccharomyces co‐inocula to build complexity and diversify styles is increasingly in vogue, a limited number of such products are available to date, and more research is required to guide their use in the wine industry. This study evaluates the potential of commercial yeast inocula to modulate the quality of Shiraz wines at two maturity levels. Methods and Results Vinification outcomes of eight yeast treatments were compared in earlier (24°Brix) and later (29°Brix) harvested Shiraz fruit. Yeast treatments included five non‐Saccharomyces products with sequentially inoculated Saccharomyces cerevisiae, a commercial blend of non‐Saccharomyces and S. cerevisiae strains, and a S. cerevisiae inoculum. Fermentation monitoring, and comprehensive analytical profiling in terms of basic chemistry, volatile composition, phenolic measurements and descriptive sensory analysis, allowed for the comparison of the resulting wines. Both harvest date and yeast inoculation treatments had a significant impact on a range of compositional and, in turn, sensory parameters of the wines. Conclusions Certain non‐Saccharomyces sequential inoculation treatments led to increased appeal of earlier harvest wines compared to the S. cerevisiae Control. These treatments, however, were related to an increased risk of arrested fermentation in higher ripeness conditions. Significance of the Study This study contributes to a better understanding of yeast inoculum‐derived modulation of Shiraz wine quality parameters at different maturity levels.

Malolactic fermentation (MLF) is an important step in winemaking, which can be notoriously unreliable due to the fastidious nature of Oenococcus oeni. This study aimed to use directed evolution (DE) to produce a more robust strain of O. oeni having the ability to withstand high ethanol concentrations. DE involves an organism mutating and potentially adapting to a high stress environment over the course of extended cultivation. A continuous culture of O. oeni was established and exposed to progressively increasing ethanol content such that after approximately 330 generations, an isolate from this culture was able to complete MLF in high ethanol content medium earlier than its parent. The ethanol tolerance of a single isolate, A90, was tested to confirm the phenotype and its fermentation performance in wine. In order to investigate the genotypic differences in the evolved strain that led to the ethanol tolerance phenotype, the relative expression of a number of known stress response genes was compared between SB3 and A90. Notably, there was increase in hsp18 expression in 20% (v/v) ethanol by both strains with A90 exhibiting a higher degree of expression. This study is the first to use directed evolution for O. oeni strain improvement and confirms that this technique can be used successfully for the development of new candidate strains for the wine industry. This study also adds to the current knowledge on the genetic basis of ethanol tolerance in this bacterium.

Background The overall quality of red wine is well known to be influenced markedly by various phenolic compounds that are extracted from the grape solids during maceration. The concentration and composition of phenolics then impact the flavour and mouthfeel of wines. Scope and Approach This review analyses the available literature on specific process variables that influence the diffusive mass transfer and evolution of phenolic compounds during red wine maceration. These variables are discussed in terms of techniques and strategies used by winemakers to influence the extractive behaviour of phenolic compounds and control their concentration in the finished wine. Mathematical models used to describe extraction and evolution phenomena in wine are also examined and the potential for future models to predict phenolic behaviour is discussed. Key Findings and Conclusions The impact of various winemaking techniques aimed at improving the extraction of phenolic compounds during red winemaking, as well as the subsequent reactions that take place following extraction, are qualitatively well understood. Mathematically, many of these techniques can be described in terms of their changing process variables such as temperature, solvent conditions and sold-liquid contact. Despite this, non-steady state models for mass transport or reaction kinetics of phenolic compounds in wine fermentation are currently unavailable in published literature. Further research into the production of global models capable of accurately describing this behaviour would be a significant advancement for industry and would aid in the development of adaptive process control technologies for red wine phenolic composition.

An industrial wastewater treatment plant (WWTP) in Australia has long suffered from bulking problems associated with the proliferation of Thiothrix spp. The WWTP consists of a covered anaerobic lagoon (CAL) followed by a sequencing batch reactor (SBR). The CAL functions as both an anaerobic digester and surge lagoon for the irregular flow of wastewater generated from the production of seasonal products. Chemical analysis of the raw influent showed it was composed of a mixture of organic acids, phenols and alcohols. The CAL effluent was characterised by high acetic acid and phenolic concentrations. An attempt was made to manipulate the SBR microbial community to improve settling by direct feeding small volumes of raw influent into the SBR. After raw feeding, the plant ceased bulking as the settled sludge volume reduced from 930 to 200 mL L−1. 16S rRNA gene profiling and biovolumes of SBR samples revealed major changes in the microbial community. The Thiothrix spp. population decreased from 36.8% to 0.2%, and Zoogloea spp. dominated all samples after raw feeding. Therefore, direct feeding is proposed as a control method for industrial plants with surge/anaerobic lagoons in order to manage the bulking problems caused by Thiothrix spp. in downstream SBRs.

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.

Learning to objectively evaluate wine sensory properties (such as appearance, aroma, flavour, taste and mouthfeel attributes) features prominently in wine education programs. Formal, structured sensory classes that involve recording detailed observations and perceptions is the traditional approach to build perceptual and linguistic learning. This research explored students’ behaviour in maintaining wine tasting notes and their perceptions of wine sensory classes by survey across four Australian institutions (n=109) and by focus groups (n=24). International students were not as confident in class room discussions or describing wine, and did not perform as well in sensory exams, suggesting that language ability and/or cultural/life experience is important for technical wine assessment. Given that 98% of students surveyed owned a smartphone, mobile learning may provide an opportunity to enhance and facilitate learning of wine sensory analysis outside of the classroom. The My Wine WorldTM App (developed for smartphones) was evaluated by academic staff and students as a potentially valuable e-learning tool for the development of perceptual and linguistic memory of sensory attributes.

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.

The yeast Lachancea thermotolerans (previously Kluyveromyces thermotolerans) is a species of large, yet underexplored, oenological potential. This study delivers comprehensive oenological phenomes of 94 L. thermotolerans strains obtained from diverse ecological niches worldwide, classified in nine genetic groups based on their pre-determined microsatellite genotypes. The strains and the genetic groups were compared for their alcoholic fermentation performance, production of primary and secondary metabolites and pH modulation in Chardonnay grape juice fermentations. The common oenological features of L. thermotolerans strains were their glucophilic character, relatively extensive fermentation ability, low production of acetic acid and the formation of lactic acid, which significantly affected the pH of the wines. An untargeted analysis of volatile compounds, used for the first time in a population-scale phenotyping of a non-Saccharomyces yeast, revealed that 58 out of 90 volatiles were affected at an L. thermotolerans strain level. Besides the remarkable extent of intra-specific diversity, our results confirmed the distinct phenotypic performance of L. thermotolerans genetic groups. Together, these observations provide further support for the occurrence of domestication events and allopatric differentiation in L. thermotolerans population.

Recent studies have suggested a strong niche adaptation for Brettanomyces bruxellensis strains according to human-related fermentation environments, including beer, wine and bioethanol. This is further supported by a correlation between B. bruxellensis genetic grouping and tolerance to SO2, the main antimicrobial used in wine. The allotriploid AWRI1499-like cluster, in particular, shows high SO2 tolerance suggesting that the genetic configuration observed for these strains may confer a selective advantage in winemaking conditions. To test this hypothesis, we evaluated the relative selective advantage of representatives of the three main B. bruxellensis genetic groups in presence of SO2. As a proof-of-concept and using recently developed transformation cassettes, we compared strains under different SO2 concentrations using pairwise competitive fitness experiments. Our results showed that AWRI1499 is specifically adapted to environments with high SO2 concentrations compared to other B. bruxellensis wine strains, indicating a potential correlation between allotriploidisation origin and environmental adaptation in this species. Additionally, our findings suggest different types of competition between strains, such as coexistence and exclusion, revealing new insights on B. bruxellensis interactions at intraspecies level.

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.

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.

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.

Here, we report the first sequenced genome of an indigenous Australian wine isolate of Torulaspora delbrueckii using the Oxford Nanopore MinION and Illumina HiSeq sequencing platforms. The genome size is 9.4 Mb and contains 4,831 genes.

Oenococcus oeni is crucial for winemaking, bringing stabilization, deacidification, and sensory impacts through malolactic fermentation (MLF) to most wine styles. The poor nutritional make-up of wine together with typically low processing temperatures and pH and high ethanol content and sulfur dioxide (SO2) hinder O. oeni growth and activity. Production delays and interventions with starter cultures and nutritional supplements have significant cost and quality implications; thus, optimization of O. oeni has long been a priority. A range of optimization strategies, some guided by detailed characterization of O. oeni, have been exploited. Varying degrees of success have been seen with classical strain selection, mutagenesis, gene recombination, genome shuffling, and, most recently, directed evolution (DE). The merits, limitations, and future prospects of each are discussed.

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. Lg2T 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.

High concentrations of ethanol, low pH, the presence of sulfur dioxide and some polyphenols have been reported to inhibit Oenococcus oeni growth, thereby negatively affecting malolactic fermentation (MLF) of wine. In order to generate superior O. oeni strains that can conduct more efficient MLF, despite these multiple stressors, a continuous culture approach was designed to directly evolve an existing ethanol tolerant O. oeni strain, A90. The strain was grown for ∼350 generations in a red wine-like environment with increasing levels of stressors. Three strains were selected from screening experiments based on their completion of fermentation in a synthetic wine/wine blend with 15.1% (v/v) ethanol, 26 mg/L SO2 at pH 3.35 within 160 h, while the parent strain fermented no more than two thirds of l -malic acid in this medium. These superior strains also fermented faster and/or had a larger population in four different wines. A reduced or equivalent amount of the undesirable volatile, acetic acid, was produced by the optimised strains compared to a commercial strain in Mouvedre and Merlot wines. These findings demonstrate the feasibility of using directed evolution as a tool to generate more efficient MLF starters tailored for wines with multiple stressors.

Defluviicoccus vanus -related glycogen accumulating organisms (GAO) regularly proliferate in industrial wastewater treatment plants handling high carbon but nitrogen deficient wastes. When GAO dominate, they are associated with poor performance, characterised by slow settling biomass and turbid effluents. Although their ecophysiology has been studied thoroughly in domestic waste treatment plants, little attention has been paid to them in aerobic industrial systems. In this study, the effect of nitrogen addition on GAO carbon metabolism was investigated during an 8 h cycle. Activated sludge dominated by GAO from a winery wastewater sequencing batch reactor was incubated under different carbon to nitrogen (COD:N) ratios (100:1, 60:1 and 20:1) using 13 C — acetate and 15 N — urea. GAO cell assimilation was quantified using FISH-NanoSIMS. The activated sludge community was assessed by 16S rRNA gene profiling, DNA and storage polymer production. Carbon and nitrogen quantification at the cellular level by NanoSIMS revealed that low (COD:N of 100:1) or null nitrogen concentrations enhanced GAO carbon uptake. COD:N ratios of 60:1 and 20:1 reduced GAO carbon uptake and promoted whole microbial community DNA production. Nitrogen dosing at COD:N ratios of 60:1 or higher was demonstrated as feasible strategy for controlling the excessive GAO growth in high COD waste treatment plants.

Previous work has shown that Oenococcus oeni produces esterases that are capable of hydrolysing artificial substrates. Using SPME-GCMS, this study provides evidence that purified O. oeni esterases have the ability to both synthesise and hydrolyse esters. Two purified esterases (EstA2 and EstB28) synthesised ethyl butanoate and ethyl hexanoate to varying degrees. Both purified esterases hydrolysed ethyl butanoate, ethyl hexanoate and ethyl octanoate. Once this dual activity was confirmed, malolactic fermentation (MLF) trials were conducted in wine with O. oeni strains that had been previously observed to have either high or low esterase activity against artificial substrates. Strain specific differences were observed and strains with low esterase hydrolysis activity against artificial substrates had a higher level of total esters measured after MLF. The results demonstrate the impact that O. oeni has on wine aroma and relates this to the ester hydrolysis and synthesis abilities of O. oeni strains.

Oenococcus oeni is a multiple physical stress-tolerant lactic acid bacterium that plays an important role in wine making. It is often added as a starter culture to carry out malolactic fermentation (MLF). In this study, a total of 22 out of 127 lactic acid bacteria, isolated from Chinese wines undergoing MLF, were identified as O. oeni by species-specific PCR and 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis showed that all strains could be typed under these conditions, and three main groups were determined by cluster analysis, which showed intraspecific homology higher than 69 %. Eight strains, representative of SE-AFLP clusters, were tested for malolactic activity. Significant differences were observed among strains with regard to the amount of malic acid consumed. Seventeen amino acids in different wines that were inoculated by 4 O. oeni strains, respectively, were analyzed before and after MLF. The results indicated that the amino acid metabolism of the 4 strains was significantly different between each strain.

Microbial spoilage of wine can lead to significant economic loss. At present sulfur dioxide is the main additive to juice/must/wine to prevent and control microbial spoilage. As an alternative, or complement to SO(2), high power ultrasonics (HPU) may be used to control microbes. Several wine spoilage yeasts and bacteria were treated with HPU in saline (0.9% w/v NaCl), juice and red wine to assess their susceptibility to HPU. Significant killing was seen across several yeasts and bacteria commonly associated with winemaking and wine spoilage. In general the viability of yeast was more affected than that of bacteria.

A significant amount of capital and maintenance is invested in oak barrels, which contribute greatly to wine quality. If not cleaned correctly, barrels can accumulate tartrate deposits and wine spoilage organisms, which can be detrimental to wine quality. We examined the efficacy of using high-power ultrasonics (HPU) for sanitization of wine barrels using the spoilage yeast Dekkera (Brettanomyces) bruxellensis as the test organism and indirectly compared it to standard hot water washing practices. Both 1- and 3-year-old oak were investigated and no culturable cells were detected on the surface (0 to 2 mm) or subsurface (2 to 4 mm) of the oak after treatment with HPU in hot water (60°C). Additionally, wines stored over a 12-month period in barrels initially cleaned with hot water, cold water, or HPU did not differ in their extraction of oak compounds, nor could these wines be differentiated by a sensory panel. Thus, HPU did not adversely affect oak extraction into wine.

This review focuses on the considerable amount of research directed at defining the accumulation of esters during fermentation and their contribution to aromas in foods and beverages. From this research it is clear that esters are extremely important for the aroma profile of fermented beverages and various dairy products. A large amount of this research is focused on wine and has yielded the genes involved in ester synthesis and hydrolysis in organisms such as Saccharomyces sp. It is also clear from recent research in both the fermented beverage and dairy context that lactic acid bacteria possess an extensive collection of ester synthesising and hydrolysing activities. This review describes the major esters reported in wine and the enzymes responsible for their hydrolysis and synthesis. Ester impact on wine aroma and formation during primary and malolactic fermentation is also evaluated. Finally the potential applications of current knowledge are outlined.

The quality of fermented foods and beverages is affected in part by their composition of aroma compounds. In winemaking, the malolactic fermentation is used to deacidify wine and is typically carried out by Lactobacillus spp., Pediococcus spp., and particularly Oenococcus oeni (10, 7). Numerous reports clearly show that outside of this core function, lactic acid bacteria (LAB) can also bring about significant changes of sensorial importance (2, 5, 12, 32, 40, 49, 55). Such studies typically examined the action of active cultures or whole cells; however, the promise of LAB as a source of purified enzymes for use as additives in winemaking has recently been highlighted (43). Oenococcus oeni is acidophilic and indigenous to wine and similar environments. While the genome of the commercial PSU-1 strain has been sequenced and analyzed (44), there is limited information on the genes or their potential contribution to food and beverage aroma. The only such genes which have been cloned and partially characterized are alsS and alsD (24), which are thought to be responsible for the production of diacetyl, the principle compound conferring “buttery” aroma and flavor in wine (reviewed in reference 4). Analogous characterization of other flavor-related genes and enzymes not only may have practical implications for processes using LAB but also may be of fundamental interest. As a group, esters are a quantitatively significant constituent of beverages such as wine (total of >100 mg·liter−1) (15). Included in this group are the C4 to C10 ethyl esters of organic acids, ethyl esters of straight-chain fatty acids (and branched-chain fatty acids to a lesser degree), and acetates of higher alcohols which are largely, if not exclusively, responsible for the fruity aroma of wine (13, 14). Some volatile esters are frequently found in fermented beverages in only trace amounts, often below threshold concentrations (3, 21, 25, 29, 50). However, they are extremely important for the flavor profile of these products, with different esters often having a synergistic effect to collectively affect aroma when their individual threshold concentrations are not exceeded. The fact that most esters are present in wine at concentrations around the threshold value implies that minor concentration changes might have a dramatic effect on the wine's flavor (3, 21, 25, 29, 50). For this reason, an understanding of the hydrolysis and synthesis of esters in winemaking and how these may be manipulated is essential. A large amount of esters is formed during the primary fermentation by yeast; after this, LAB can contribute by increasing and decreasing the ester concentration (2, 5, 12, 40, 49, 55). Ester hydrolysis and synthesis can be catalyzed by esterases (6, 35, 38, 54). These enzymes commonly contain a catalytic triad composed of Ser, His, and Asp/Glu residues and a nucleophilic elbow structural motif (GXSXG), which contains the active-site serine residue (1, 31, 36, 48). They also contain an oxyanion hole, of which two residues donate their backbone amide protons to stabilize the substrate in the transition state. The oxyanion hole residues (in bold) have been divided into two groups termed GX and GGGX, with the glycine and a hydrophobic residue (X) being highly conserved (48). While extensive research has been carried out on the enzymes responsible for ester formation by wine strains of Saccharomyces cerevisiae (22, 23, 45, 51), esterase activity for wine-related LAB is not well documented. Most characterization of esterases in LAB has focused on dairy isolates (9, 16-18, 20). Parallel work in a wine context is limited despite general acceptance of the importance of esters in wine. Until recently, most evidence that wine LAB possess esterase activity came from wine volatile profiling studies which investigated the changes in concentration of individual esters during malolactic fermentation (12, 40, 55). Such changes in ester concentration were strain specific and had the potential to greatly affect the final aroma of wine. Our survey of the esterase activities of whole LAB cells found variations within species and even greater variation between the genera (42), with O. oeni showing greatest activity toward the p-nitrophenyl (pNP)-linked substrates tested. More recently (41), the esterase activities of whole O. oeni, lactobacillus, and pediococcus cells was determined under conditions with some relevance to wine. At least partial resistance to the harsh conditions used was observed, thereby demonstrating a necessary requirement of any enzyme intended for application in analogous environments. To more completely characterize esterases of LAB, the enzymes and their structural genes must be fully investigated. This study represents an effort to dissect the complex array of ester synthesis and hydrolysis activities in whole cells by cloning, heterologous expression, partial purification, and biochemical characterization of a single esterase from O. oeni. With a view to applying such an esterase under conditions found in wine and perhaps other industrial settings, enzyme function under the harsh physicochemical conditions frequently encountered in wine was examined.

Volatile phenols are produced by Dekkera yeasts and are of organoleptic importance in alcoholic beverages. The key compound in this respect is 4-ethylphenol, responsible for the medicinal and phenolic aromas in spoiled wines. The microbial synthesis of volatile phenols is thought to occur in two steps, beginning with naturally occurring hydroxycinnamic acids (HCAs). The enzyme phenolic acid decarboxylase (PAD) converts HCAs to vinyl derivatives, which are the substrates of a second enzyme, postulated to be a vinylphenol reductase (VPR), whose activity results in the formation of ethylphenols. Here, both steps of the pathway are investigated, using cell extracts from a number of Dekkera and Brettanomyces species. Dekkera species catabolise ferulic, caffeic and p-coumaric acids and possess inducible enzymes with similar pH and temperature optima. Brettanomyces does not decarboxylate HCAs but does metabolise vinylphenols. Dekkera species form ethylphenols but the VPR enzyme appears to be highly unstable in cell extracts. A partial protein sequence for PAD was determined from Dekkera anomala and may indicate the presence of a novel enzyme in this genus.

Industrial scale food and beverage processes that utilize microorganisms are typically faced with issues related to the exclusion, suppression or elimination of spoilage organisms. Yet the use of traditional anti-microbial treatments such as heat, chemical biocides or sterile filtration may themselves be restricted by regulations or else be undesirable due to their adverse sensory impacts on the product. High power ultrasound (HPU) is a technology whose application has been evaluated if not exploited in several food and beverage processes but has yet to be introduced into the wine industry. This review examines the research findings from related industries and highlights possible applications and likely benefits of the use of HPU in winemaking.

During barrel maturation, volatile compounds are extracted from oak wood and impart aroma and flavor to wine, enhancing its character and complexity. However, barrels contain a finite pool of extractable material, which diminishes with time. As a consequence, most barrels are decommissioned after 5 or 6 years. This study investigated whether or not decommissioned barrels can be "reclaimed" and utilized as a previously untapped source of quality oak for wine maturation. Oak battens were prepared from staves of decommissioned French and American oak barrels, and their composition analyzed before and after toasting. The oak lactone glycoconjugate content of untoasted reclaimed oak was determined by liquid chromatography-tandem mass spectrometry, while the concentrations of cis- and trans-oak lactone, guaiacol, 4-methlyguaiacol, vanillin, eugenol, furfural, and 5-methylfurfural present in toasted reclaimed oak were determined by gas chromatography-mass spectrometry. Aroma potential was then evaluated by comparing the composition of reclaimed oak with that of new oak. Comparable levels of oak lactone glycoconjugates and oak volatiles were observed, demonstrating the aroma potential of reclaimed oak and therefore its suitability as a raw material for alternative oak products, i.e., chips or battens, for the maturation of wine. The temperature profiles achieved during toasting were also measured to evaluate the viability of any yeast or bacteria present in reclaimed oak.

Isolates from wine-associated genera of lactic acid bacteria (LAB), including Lactobacillus, Oenococcus and Pediococcus, were examined for enzymatic properties that could be of value in the production and processing of wine. Up to 438 combinations of bacterial isolates and substrates were examined. Across the 50 isolates investigated, all were found to hydrolyse esters, with the greatest activity being seen amongst the oenococci followed by the lactobacilli and pediococci. Lipase activity was restricted to three Lactobacillus isolates, while all tested isolates displayed tannase activity. Cellulase, β-glucanase and lichenase activities were observed for almost all Pediococcus and Lactobacillus isolates, while such activities were seen for only a minority of oenococci. No degradation of the xylan substrate was detected. Once confirmed under winelike conditions, our findings will help define potentially interesting genera of LAB for winemaking.

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.

Comparative cellular metabolism can be a difficult area of biochemistry to teach in the undergraduate laboratory class. Student practicals involving animal tissues generally require approval from animal ethic committees, and the relevance for students whose primary interest in biochemistry is in the area of food and wine sciences, is often questioned. In this report, we present an undergraduate practical exercise in which glucose catabolism via the pentose phosphate pathway is compared in two types of yeast with direct relevance to the wine and food industries, Saccharomyces cerevisiae and Kloeckera apiculata. The exercise is carried out as a demonstration to second year undergraduate students, studying metabolic biochemistry. It is of some value in that it illustrates comparative cellular metabolism in wine yeasts and introduces the students to the safe use of radioisotopes.

The ability of four known species of Dekkera and Brettanomyces yeasts to produce mousy off-flavour in grape juice and wine was investigated for the first time. Using a sensory assessment technique the twelve type strains of Dekkera and Brettanomyces, representing four species, were found to grow and to be capable of producing mousy off-flavour in a grape juice medium; however, differences between strains were apparent. Four strains representing the two species of Dekkera (D. bruxellensis and D. anomala) known to be associated with the spoilage of wine and other fermented beverages were further investigated for mousy off-flavour production in a red and white wine supplemented with nutrients. D. anomala and only one of the three D. bruxellensis strains tested grew in both wines and generally produced a moderate level of off-flavour, whereas the remaining two strains of D. bruxellensis, despite slowly losing viability (10- to 100-fold) over the 52-55 day period, produced detectable off-flavour. This work demonstrates the general ability of Dekkera and Brettanomyces yeasts to produce mousy off-flavour and confirms the importance of these yeasts for off-flavour production in grape juice and wine.

The potent flavour compound 2-acetyl-1-pyrroline (1) was identified for the first time in wines spoiled by mousy taint. Identification was verified by comparison of GC and GC-MS data with those of authentic reference material. GC-SNEFF analysis of (1) further confirmed its intense mousy aroma.

Aim To clone and characterize two related intracellular esterases from Oenococcus oeni and Lactobacillus hilgardii under wine-like conditions. Methods and Results The published genome sequences for O. oeni and Lact. hilgardii were used to identify, clone and purify putative esterase genes from these species designated EstCOo8 and EstC34, respectively. Both esterases are members of family V of lipolytic enzymes. However, EstC34 contains an SGSLG nucleophilic elbow structural motif instead of the usual GGSLG motif which is conserved in other lactic acid bacteria. Both esterases exhibited greatest specificity for C2–C4 pNP-linked substrates and retained activity under wine-like conditions. EstCOo8 had an optimum temperature, pH, and ethanol concentration of 40°C, 5·5 and 6% (v/v), respectively. Whereas EstC34 had an optimum temperature, pH and ethanol concentration of 50°C, 5·0 and 10% (v/v), respectively. Conclusions Both esterases were stable and retained activity under conditions that would be encountered in wine. They have the potential to reduce short-chain ethyl esters such as ethyl acetate. Significance and Impact of the Study This study provides information that might help improve the performance of LAB during malolactic fermentation in wine in the future, either by strain selection, optimization or direct enzyme addition.

Spoilage of red wine by the yeast species Dekkera bruxellensis is a common problem for the global wine industry. When conditions are conducive for growth of these yeasts in wine, they efficiently convert non-volatile hydroxycinnamic acids into aroma-active ethylphenols, thereby reducing the quality of the wine. It has been demonstrated previously that dissolved oxygen is a key factor which stimulates D. bruxellensis growth in wine. We demonstrate that whereas the presence of oxygen accelerates the growth of this species, oxygen-limited conditions favour 4-ethylphenol production. Consequently, we evaluated wine spoilage potential of three D. bruxellensis strains (AWRI1499, AWRI1608 and AWRI1613) under oxygen-limited conditions. Each strain was cultured in a chemically-defined wine medium and the fermentation products were analysed using HPLC and HS-SPME–GC/MS. The strains displayed different growth characteristics but were equally capable of producing ethylphenols. On the other hand, significant differences were observed for 18 of the remaining 33 metabolites analysed and duo-trio sensory analysis indicated significant aroma differences between wines inoculated with AWRI1499 and AWRI1613. When these wines were spiked with low concentrations of 4-ethylphenol and 4-ethylguaiacol, no sensorial differences could be perceived. Together these data suggest that the three predominant D. bruxellensis strains previously isolated during a large survey of Australian wineries do not differ substantively in their capacity to grow in, and spoil, a model wine medium.

The study of gene expression and accurate quantitation of target genes in any organism depends on correct normalisation. Due to the increase in studies on Oenococcus oeni gene expression, there is a clear need for alternative reference genes in order to reliably measure expression levels. In this manuscript, we propose the approach of using multiple reference genes to provide a more robust basis for establishing a reference gene set. The identification and evaluation of a panel of nine reference genes, including the commonly used ldhD, for real-time PCR normalisation was performed in O. oeni. Expression levels of these reference genes were then measured by real-time qPCR in an independent set of O. oeni samples (n = 30). The nine genes were ranked according to their stability of gene expression measure (M) using geNorm to identify the most consistently expressed reference genes. This approach resulted in the identification of multiple reference genes that may be used for a screening and more robust normalisation of target gene expression measured by real-time RT-qPCR. Expression of esterase genes was then measured in these O. oeni samples in the presence of known esterase substrates. The results give an indication of how these genes may be involved in ester synthesis and hydrolysis in O. oeni.

Simple phenolic components of wine, hydroxycinnamic acids (HCAs) are known to have antimicrobial properties. This study sought to determine the potential of ferulic acid as an antifungal agent for the control of Dekkera. Growth was inhibited by all HCAs examined in this study, with ferulic acid being the most potent at all concentrations. In the presence of ethanol, the inhibitory effects of ferulic acid were amplified. Scanning electron microscopy images reveal cellular damage upon exposure to ferulic acid. Thus, manipulation of ferulic acid concentrations could be of industrial significance for control of Dekkera and may be the basis for differences in susceptibility of wines to Dekkera spoilage.

Mousy off-flavor is an insidious and economically disastrous microbiologically derived spoilage characteristic of wine and other fermented beverages. Tainted wines are rendered unpalatable and there is currently no satisfactory procedure for removal of the off-flavor. Here we report the confirmation of that both d- and l-lysine can act as a precursor for the formation of mousy off-flavor N-heterocycles. Further, through the use of stable isotope feeding experiments, we could establish that a pentylamine group from lysine is incorporated into the piperideine moiety of two off-flavor N-heterocycles. A biochemical pathway for the formation of mousy off-flavor compounds is proposed.

Dekkera and Brettanomyces yeast are important spoilage organisms in a number of food and beverage products. Isolates of both genera were cultured in a defined medium and supplemented with hydroxycinnamic acids and vinylphenols to investigate their influence on growth and the formation of ethyl phenol derivatives. The growth rate of Brettanomyces species in the presence of acids was reduced, and no significant conversion to vinyl or ethyl derivatives was observed. The growth rate and substrate utilisation rates of Dekkera anomala and Dekkera bruxellensis yeast differed depending on strain and the acid precursor present. Growth of D. bruxellensis was slowed by the presence of ferulic acid with the addition of 1 mM ferulic acid completely inhibiting growth. This study provides an insight into the spoilage potential of these organisms and possible control strategies involving hydroxycinnamic acids.

Esters are an important group of volatile compounds that can contribute to wine flavour. Wine lactic acid bacteria (LAB) have been shown to produce esterases capable of hydrolysing ester substrates. This study aims to characterise the esterase activities of nine LAB strains under important wine conditions, namely, acidic conditions, low temperature (to 10 degrees C) and in the presence of ethanol (2-18% v/v). Esterase substrate specificity was also examined using seven different ester substrates. The bacteria were generally found to have a broad pH activity range, with the majority of strains showing maximum activity close to pH 6.0. Exceptions included an Oenococcus oeni strain that retained most activity even down to a pH of 4.0. Most strains exhibited highest activity across the range 30-40 degrees C. Increasing ethanol concentration stimulated activity in some of the strains. In particular, O. oeni showed an increase in activity up to a maximum ethanol concentration of around 16%. Generally, strains were found to have greater activity towards short-chained esters (C2-C8) compared to long-chained esters (C10-C18). Even though the optimal physicochemical conditions for enzyme activity differed from those found in wine, these findings are of potential importance to oenology because significant activities remained under wine-like conditions.