Systems-based approaches enable identification of gene targets which improve the flavour profile of low-ethanol wine yeast strains.

Metabolic engineering has been vital to the development of industrial microbes such as the yeast Saccharomyces cerevisiae. However, sequential rounds of modification are often needed to achieve particular industrial design targets. Systems biology approaches can aid in identifying genetic targets for modification through providing an integrated view of cellular physiology. Recently, research into the generation of commercial yeasts that can produce reduced-ethanol wines has resulted in metabolically-engineered strains of S. cerevisiae that are less efficient at producing ethanol from sugar. However, these modifications led to the concomitant production of off-flavour by-products. A combination of transcriptomics, proteomics and metabolomics was therefore used to investigate the physiological changes occurring in an engineered low-ethanol yeast strain during alcoholic fermentation. Integration of 'omics data identified several metabolic reactions, including those related to the pyruvate node and redox homeostasis, as being significantly affected by the low-ethanol engineering methodology, and highlighted acetaldehyde and 2,4,5-trimethyl-1,3-dioxolane as the main off-flavour compounds. Gene remediation strategies were then successfully applied to decrease the formation of these by-products, while maintaining the 'low-alcohol' phenotype. The data generated from this comprehensive systems-based study will inform wine yeast strain development programmes, which, in turn, could potentially play an important role in assisting winemakers in their endeavour to produce low-alcohol wines with desirable flavour profiles.

Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma.

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.

Impacts of variations in elemental nutrient concentration of Chardonnay musts on Saccharomyces cerevisiae fermentation kinetics and wine composition.

Chardonnay, being the predominant white wine-grape cultivar in the Australian wine sector, is subject to widely varying winemaking processes with the aim of producing a variety of wine styles. Therefore, juice composition might not always be ideal for optimal fermentation outcomes. Our aim was to better understand the composition of Chardonnay juice and how compositional parameters impact on fermentation outcomes. This was achieved through a survey of 96 commercially prepared Chardonnay juices during the 2009 vintage. Common juice variables were estimated using near infrared spectroscopy, and elemental composition was determined using radial view inductively coupled plasma optical emission spectrometry. The influence of elemental composition on fermentation outcomes was assessed by fermentation of a defined medium formulated to reflect the composition and range of concentrations as determined by the juice survey. Yeast (Saccharomyces cerevisiae) strain effects were also assessed. Key parameters influencing fermentation outcomes were verified by laboratory scale fermentation of Chardonnay juice. This exploration of Chardonnay juice identified interactions between juice pH and potassium concentration as key factors impacting on fermentation performance and wine quality. Outcomes differed depending on yeast strain.

Occurrence of hydrogen sulfide in wine and in fermentation: influence of yeast strain and supplementation of yeast available nitrogen.

Hydrogen sulfide (H2S) is a powerful aroma compound largely produced by yeast during fermentation. Its occurrence in wines and other fermented beverages has been associated with off-odors described as rotten egg and/or sewage. While the formation of hydrogen sulfide (H2S) during fermentation has been extensively studied, it is the final H2S content of wine that is actually linked to potential off-odors. Nevertheless, factors determining final H2S content of wine have received little attention, and it is commonly assumed that high H2S-forming fermentations will result in high final concentrations of H2S. However, a clear relationship has never been established. In this report, we investigated the contribution of yeast strain and nitrogen addition to H2S formation during fermentation and its consequent occurrence the resulting wines. Five commercial Saccharomyces cerevisiae wine yeast strains were used to ferment a Chardonnay juice containing 110 mg/l of YAN (yeast assimilable nitrogen), supplemented with di-ammonium phosphate (DAP) to increase YAN concentration to moderate (260 mg/l) and high (410 mg/l) levels. In contrast to the widely reported decrease in H2S production in response to DAP addition, a non-linear relationship was found such that moderate DAP supplementation resulted in a remarkable increase in H2S formation by each of the five wine yeasts. H2S content of the finished wine was affected by yeast strain, YAN, and fermentation vigor. However, we did not observe a correlation between concentration of H2S in the finished wines and H2S produced during fermentation, with low-forming fermentations often having relatively high final H2S and vice versa. Management of H2S in wine through nitrogen supplementation requires knowledge of initial YAN and yeast H2S characteristics.

Comparison of inorganic and organic nitrogen supplementation of grape juice - Effect on volatile composition and aroma profile of a Chardonnay wine fermented with Saccharomyces cerevisiae yeast.

Inorganic nitrogen salts, and to a growing extent organic nitrogen preparations, are widely used to ameliorate a nitrogen deficiency in wine fermentation, but the impact of nitrogen supplementation on perceived wine sensory profile is essentially unknown. Supplementation of a low nitrogen Chardonnay grape juice with either ammonium nitrogen or combined amino acid and ammonium nitrogen showed that the type of nitrogen and concentration in the range 160-480mgN/l had a substantial impact on the formation of yeast volatile compounds and perceived wine aroma. Addition of amino acid and ammonium nitrogen increased both acetate and medium chain fatty acid esters to a greater extent and decreased higher alcohols to a lesser extent than ammonium nitrogen alone whereas ammonium nitrogen substantially increased ethyl acetate and acetic acid. Low nitrogen wines were rated relatively low in floral/fruity aroma descriptors, while moderate nitrogen wines showed a good balance between desirable and less desirable attributes, whereas high nitrogen produced either an acetic/solvent character or highest ratings for floral/fruity attributes, depending on nitrogen type. These results show that amount and type of nitrogen supplement can substantially modulate Chardonnay wine volatiles composition and perceived aroma.

Hanseniaspora vineae and the Concept of Friendly Yeasts to Increase Autochthonous Wine Flavor Diversity.

In this perspective, we will explain the concept of "friendly" yeasts for developing wine starters that do not suppress desirable native microbial flora at the initial steps of fermentation, as what usually happens with Saccharomyces strains. Some non-Saccharomyces strains might allow the development of yeast consortia with the native terroir microflora of grapes and its region. The positive contribution of non-Saccharomyces yeasts was underestimated for decades. Avoiding them as spoilage strains and off-flavor producers was the main objective in winemaking. It is understandable, as in our experience after more than 30 years of wine yeast selection, it was shown that no more than 10% of the isolated native strains were positive contributors of superior flavors. Some species that systematically gave desirable flavors during these screening processes were Hanseniaspora vineae and Metschnikowia fructicola. In contrast to the latter, H. vineae is an active fermentative species, and this fact helped to build an improved juice ecosystem, avoiding contaminations of aerobic bacteria and yeasts. Furthermore, this species has a complementary secondary metabolism with S. cerevisiae, increasing flavor complexity with benzenoid and phenylpropanoid synthetic pathways practically inexistent in conventional yeast starters. How does H. vineae share the fermentation niche with other yeast strains? It might be due to the friendly conditions it creates, such as ideal low temperatures and low nitrogen demand during fermentation, reduced synthesis of medium-chain fatty acids, and a rich acetylation capacity of aromatic higher alcohols, well-known inhibitors of many yeasts. We will discuss here how inoculation of H. vineae strains can give the winemaker an opportunity to develop ideal conditions for flavor expression of the microbial terroir without the risk of undesirable strains that can result from spontaneous yeast fermentations.

Acetic acid bacteria spoilage of bottled red wine -- a review.

Acetic acid bacteria (AAB) are ubiquitous organisms that are well adapted to sugar and ethanol rich environments. This family of Gram-positive bacteria are well known for their ability to produce acetic acid, the main constituent in vinegar. The oxidation of ethanol through acetaldehyde to acetic acid is well understood and characterised. AAB form part of the complex natural microbial flora of grapes and wine, however their presence is less desirable than the lactic acid bacteria and yeast. Even though AAB were described by Pasteur in the 1850s, wine associated AAB are still difficult to cultivate on artificial laboratory media and until more recently, their taxonomy has not been well characterised. Wine is at most risk of spoilage during production and the presence of these strictly aerobic bacteria in grape must and during wine maturation can be controlled by eliminating, or at least limiting oxygen, an essential growth factor. However, a new risk, spoilage of wine by AAB after packaging, has only recently been reported. As wine is not always sterile filtered prior to bottling, especially red wine, it often has a small resident bacterial population (<10(3) cfu/mL), which under conducive conditions might proliferate. Bottled red wines, sealed with natural cork closures, and stored in a vertical upright position may develop spoilage by acetic acid bacteria. This spoilage is evident as a distinct deposit of bacterial biofilm in the neck of the bottle at the interface of the wine and the headspace of air, and is accompanied with vinegar, sherry, bruised apple, nutty, and solvent like off-aromas, depending on the degree of spoilage. This review focuses on the wine associated AAB species, the aroma and flavour changes in wine due to AAB metabolism, discusses the importance of oxygen ingress into the bottle and presents a hypothesis for the mechanism of spoilage of bottled red wine.

The role of lysine amino nitrogen in the biosynthesis of mousy off-flavor compounds by Dekkera anomala.

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.

Hydrolysis and transformation of grape glycosidically bound volatile compounds during fermentation with three Saccharomyces yeast strains.

The ability of three Saccharomyces wine yeasts (S. cerevisiae AWRI 838, S. cerevisiae AWRI 1537, and S. bayanus AWRI 1375) to liberate volatile compounds from sugar-bound aroma precursors was investigated using synthetic and grape glycosides under different experimental conditions. In model systems involving the incubation of yeast cells with either synthetic or grape-derived glycosides under conditions more favorable for glycosidase activities and less favorable for acid-catalyzed hydrolysis (pH 5.0 and 30 degrees C), all yeast strains studied proved to be capable of hydrolyzing glycosides, with S. bayanus AWRI 1375 displaying greater hydrolytic activity than S. cerevisiae AWRI 838 and AWRI 1537. During the fermentation of a chemically defined grape juice-like medium containing glycosidic precursors extracted from Vitis vinifera cv. White Frontignac (synonym Muscat à Petit Grains Blanc), all yeasts promoted a significant hydrolysis of different precursors, which varied according to the chemical structures of both the sugar and the aglycon moieties, as determined by GC-MS analysis of trifluoroacetylated derivatives. Hydrolysis of the White Frontignac derived glycosidic precursors during fermentation resulted in the release of monoterepene alcohols, terpene oxides, terpene diols, and 3-oxo-alpha-ionol, demonstrating the significant potential of these yeast strains to contribute to wine varietal volatile composition during alcoholic fermentation.

De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts.

This paper reports the production of monoterpenes, which elicit a floral aroma in wine, by strains of the yeast Saccharomyces cerevisiae. Terpenes, which are typical components of the essential oils of flowers and fruits, are also present as free and glycosylated conjugates amongst the secondary metabolites of certain wine grape varieties of Vitis vinifera. Hence, when these compounds are present in wine they are considered to originate from grape and not fermentation. However, the biosynthesis of monoterpenes by S. cerevisiae in the absence of grape derived precursors is shown here to be of de novo origin in wine yeast strains. Higher concentration of assimilable nitrogen increased accumulation of linalool and citronellol. Microaerobic compared with anaerobic conditions favored terpene accumulation in the ferment. The amount of linalool produced by some strains of S. cerevisiae could be of sensory importance in wine production. These unexpected results are discussed in relation to the known sterol biosynthetic pathway and to an alternative pathway for terpene biosynthesis not previously described in yeast.