Meeting the consumer challenge through genetically customized wine-yeast strains

Wine producers are facing intensifying competition brought about by a widening gap between wine production and wine consumption, a shift of consumer preferences away from basic commodity wine to top quality wine, and economic globalization. Consequently, they are calling for a total revolution in the 'magical' world of wine. The process of transforming the wine industry from a production- to a market-orientated industry results in an increasing dependence on, amongst others, biotechnological innovation. Market-orientated wine-yeast strains are currently being developed for the cost-competitive production of wine with minimized resource inputs, improved quality and low environmental impact. The emphasis is on the development of Saccharomyces cerevisiae strains with improved fermentation, processing and biopreservation abilities, and capacities for an increase in the wholesomeness and sensory quality of wine.     

Distribution and correlation of three oenological traits inSaccharomyces cerevisiae

The improvement of yeast starters for wine making, with classical genetic techniques, relays on the effective independence of the main oenological traits in order to combine them optimally. The analysis of three characters (ethanol production, volatile acidity and fermenting rate) in 787Saccharomyces cerevisiae strains, isolated from several parts of the world and substrates, shows moderate correlation between the volatile acidity and the other two traits, whereas ethanol production and fermenting rate appear more correlated. The minimum number of strains necessary to start selection or genetic improvement programs is discussed. The correlation between the oenological traits and the source of isolation showed that the best strains can be obtained from wineries and wine, whereas the isolates from grapes and must are less performing on the average.     

Selection of an autochthonous Saccharomyces strain starter for alcoholic fermentation of Sherry base wines

Several indigenous Saccharomyces strains from musts were isolated in the Jerez de la Frontera region, at the end of spontaneous fermentation, in order to select the most suitable autochthonous yeast starter, during the 2007 vintage. Five strains were chosen for their oenological abilities and fermentative kinetics to elaborate a Sherry base wine. The selected autochthonous strains were characterized by molecular methods: electrophoretic karyotype and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and by physiological parameters: fermentative power, ethanol production, sugar consumption, acidity and volatile compound production, sensory quality, killer phenotype, desiccation, and sulphur dioxide tolerance. Laboratory- and pilot-scale fermentations were conducted with those autochthonous strains. One of them, named J4, was finally selected over all others for industrial fermentations. The J4 strain, which possesses exceptional fermentative properties and oenological qualities, prevails in industrial fermentations, and becomes the principal biological agent responsible for winemaking. Sherry base wine, industrially manufactured by means of the J4 strain, was analyzed, yielding, together with its sensory qualities, final average values of 0.9 g/l sugar content, 13.4 % (v/v) ethanol content and 0.26 g/l volatile acidity content; apart from a high acetaldehyde production, responsible for the distinctive aroma of “Fino”. This base wine was selected for “Fino” Sherry elaboration and so it was fortified; it is at present being subjected to biological aging by the so-called “flor” yeasts. The “flor” velum formed so far is very high quality. To the best of our knowledge, this is the first study covering from laboratory to industrial scale of characterization and selection of autochthonous starter intended for alcoholic fermentation in Sherry base wines. Since the 2010 vintage, the indigenous J4 strain is employed to industrially manufacture a homogeneous, exceptional Sherry base wine for “Fino” Sherry production.     

Transgenic wine yeast technology comes of age: is it time for transgenic wine?

Saccharomyces cerevisiae is the main yeast responsible for alcoholic fermentation of grape juice during wine making. This makes wine strains of this species perfect targets for the improvement of wine technology and quality. Progress in winemaking has been achieved through the use of selected yeast strains, as well as genetic improvement of wine yeast strains through the sexual and pararexual cycles, random mutagenesis and genetic engineering. Development of genetically engineered wine yeasts, their potential application, and factors affecting their commercial viability will be discussed in this review.     

Wine yeasts for the future

International competition within the wine market, consumer demands for newer styles of wines and increasing concerns about the environmental sustainability of wine production are providing new challenges for innovation in wine fermentation. Within the total production chain, the alcoholic fermentation of grape juice by yeasts is a key process where winemakers can creatively engineer wine character and value through better yeast management and, thereby, strategically tailor wines to a changing market. This review considers the importance of yeast ecology and yeast metabolic reactions in determining wine quality, and then discusses new directions for exploiting yeasts in wine fermentation. It covers criteria for selecting and developing new commercial strains, the possibilities of using yeasts other than those in the genus of Saccharomyces, the prospects for mixed culture fermentations and explores the possibilities for high cell density, continuous fermentations.     

Reduction of volatile acidity of wines by selected yeast strains

Herein, we isolate and characterize wine yeasts with the ability to reduce volatile acidity of wines using a refermentation process, which consists in mixing the acidic wine with freshly crushed grapes or musts or, alternatively, in the incubation with the residual marc. From a set of 135 yeast isolates, four strains revealed the ability to use glucose and acetic acid simultaneously. Three of them were identified as Saccharomyces cerevisiae and one as Lachancea thermotolerans. Among nine commercial S. cerevisiae strains, strains S26, S29, and S30 display similar glucose and acetic acid initial simultaneous consumption pattern and were assessed in refermentation assays. In a medium containing an acidic wine with high glucose-low ethanol concentrations, under low oxygen availability, strain S29 is the most efficient one, whereas L. thermotolerans 44C is able to decrease significantly acetic acid similar to the control strain Zygosaccharomyces bailii ISA 1307 but only under aerobic conditions. Conversely, for low glucose-high ethanol concentrations, under aerobic conditions, S26 is the most efficient acid-degrading strain, while under limited-aerobic conditions, all the S. cerevisiae strains studied display acetic acid degradation efficiencies identical to Z. bailii. Moreover, S26 strain also reveals capacity to decrease volatile acidity of wines. Together, the S. cerevisiae strains characterized herein appear promising for the oenological removal of volatile acidity of acidic wines.     

Field-flow fractionation as analytical technique for the characterization of dry yeast: correlation with wine fermentation activity

Important oenological properties of wine depend on the winemaking yeast used in the fermentation process. There is considerable controversy about the quality of yeast, and a simple and cheap analytical methodology for quality control of yeast is needed. Gravitational field flow fractionation (GFFF) was used to characterize several commercial active dry wine yeasts from Saccharomyces cerevisiae and Saccharomyces bayanus and to assess the quality of the raw material before use. Laboratory-scale fermentations were performed using two different S. cerevisiae strains as inocula, and GFFF was used to follow the behavior of yeast cells during alcoholic fermentation. The viable/nonviable cell ratio was obtained by flow cytometry (FC) using propidium iodide as fluorescent dye. In each experiment, the amount of dry wine yeast to be used was calculated in order to provide the same quantity of viable cells. Kinetic studies of the fermentation process were performed controlling the density of the must, from 1.071 to 0.989 (20/20 density), and the total residual sugars, from 170 to 3 g/L. During the wine fermentation process, differences in the peak profiles obtained by GFFF between the two types of commercial yeasts that can be related with the unlike cell growth were observed. Moreover, the strains showed different fermentation kinetic profiles that could be correlated with the corresponding fractograms monitored by GFFF. These results allow optimism that sedimentation FFF techniques could be successfully used for quality assessment of the raw material and to predict yeast behavior during yeast-based bioprocesses such as wine production.     

Evaluation of gene modification strategies for the development of low-alcohol-wine yeasts

Saccharomyces cerevisiae has evolved a highly efficient strategy for energy generation which maximizes ATP energy production from sugar. This adaptation enables efficient energy generation under anaerobic conditions and limits competition from other microorganisms by producing toxic metabolites, such as ethanol and CO(2). Yeast fermentative and flavor capacity forms the biotechnological basis of a wide range of alcohol-containing beverages. Largely as a result of consumer demand for improved flavor, the alcohol content of some beverages like wine has increased. However, a global trend has recently emerged toward lowering the ethanol content of alcoholic beverages. One option for decreasing ethanol concentration is to use yeast strains able to divert some carbon away from ethanol production. In the case of wine, we have generated and evaluated a large number of gene modifications that were predicted, or known, to impact ethanol formation. Using the same yeast genetic background, 41 modifications were assessed. Enhancing glycerol production by increasing expression of the glyceraldehyde-3-phosphate dehydrogenase gene, GPD1, was the most efficient strategy to lower ethanol concentration. However, additional modifications were needed to avoid negatively affecting wine quality. Two strains carrying several stable, chromosomally integrated modifications showed significantly lower ethanol production in fermenting grape juice. Strain AWRI2531 was able to decrease ethanol concentrations from 15.6% (vol/vol) to 13.2% (vol/vol), whereas AWRI2532 lowered ethanol content from 15.6% (vol/vol) to 12% (vol/vol) in both Chardonnay and Cabernet Sauvignon juices. Both strains, however, produced high concentrations of acetaldehyde and acetoin, which negatively affect wine flavor. Further modifications of these strains allowed reduction of these metabolites.     

Modulation of volatile sulfur compounds by wine yeast

Sulfur compounds in wine can be a ‘double-edged sword’. On the one hand, certain sulfur-containing volatile compounds such as hydrogen sulfide, imparting a rotten egg-like aroma, can have a negative impact on the perceived quality of the wine, and on the other hand, some sulfur compounds such as 3-mercaptohexanol, imparting fruitiness, can have a positive impact on wine flavor and aroma. Furthermore, these compounds can become less or more attractive or repulsive depending on their absolute and relative concentrations. This presents an interesting challenge to the winemaker to modulate the concentrations of these quality-determining compounds in wine in accordance with consumer preferences. The wine yeast Saccharomyces cerevisiae plays a central role in the production of volatile sulfur compounds. Through the sulfate reduction sequence pathway, the HS^- is formed, which can lead to the formation of hydrogen sulfide and various mercaptan compounds. Therefore, limiting the formation of the HS^- ion is an important target in metabolic engineering of wine yeast. The wine yeast is also responsible for the transformation of non-volatile sulfur precursors, present in the grape, to volatile, flavor-active thiol compounds. In particular, 4-mercapto-4-methylpentan-2-one, 3-mercaptohexanol, and 3-mercaptohexyl acetate are the most important volatile thiols adding fruitiness to wine. This paper briefly reviews the metabolic processes involved in the production of important volatile sulfur compounds and the latest strategies in the pursuit of developing wine yeast strains as tools to adjust wine aroma to market specifications.     

A breeding strategy to harness flavor diversity of Saccharomyces interspecific hybrids and minimize hydrogen sulfide production

Industrial food-grade yeast strains are selected for traits that enhance their application in quality production processes. Wine yeasts are required to survive in the harsh environment of fermenting grape must, while at the same time contributing to wine quality by producing desirable aromas and flavors. For this reason, there are hundreds of wine yeasts available, exhibiting characteristics that make them suitable for different fermentation conditions and winemaking practices. As wine styles evolve and technical winemaking requirements change, however, it becomes necessary to improve existing strains. This becomes a laborious and costly process when the targets for improvement involve flavor compound production. Here, we demonstrate a new approach harnessing preexisting industrial yeast strains that carry desirable flavor phenotypes - low hydrogen sulfide (H(2) S) production and high ester production. A low-H(2) S Saccharomyces cerevisiae strain previously generated by chemical mutagenesis was hybridized independently with two ester-producing natural interspecies hybrids of S.?cerevisiae and Saccharomyces kudriavzevii. Deficiencies in sporulation frequency and spore viability were overcome through use of complementary selectable traits, allowing successful isolation of several novel hybrids exhibiting both desired traits in a single round of selection.     

Wine yeast typing by MALDI-TOF MS

For the production of wine, the most important industrially used yeast species is Saccharomyces cerevisiae. Years of experience have shown that wine quality and property are significantly affected by the employed strain conducting the fermentation. Consequently, the ability of a strain level differentiation became an important requirement of modern winemaking. In our study, we showed that the differentiation by time-consuming and laborious biochemical and DNA-based methods to enable a constant beverage quality and characteristics can be replaced by matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), accompanied by the additional benefit of an application prediction. Mass fingerprints of 33 Saccharomyces strains, which are commonly used for varying wine fermentations, were generated by MALDI-TOF MS upon optimized sample preparation and instrument settings and analyzed by a cluster analysis for strain or ecotype-level differentiation. As a reference method, delta-PCR was chosen to study the genetic diversity of the employed strains. Finally, the cluster analyses of both methods were compared. It could be shown that MALDI-TOF MS, acting at proteome level, provides valuable information about the relationship between yeast strains and their application potential according to their MALDI mass fingerprint.     

沙棘果酒酿造中优良酵母菌的筛选及鉴定

从成熟的沙棘果皮中筛选得到9株酵母茵,通过产气性能、产酒精能力和发酵力测试试验及对发酵所得的沙棘果酒的感官评价,选出R1为最佳的沙棘果酒酿酒用茵株.该茵株适合在酸度较高的沙棘果汁中进行正常的酒精发酵,起酵快、发酵能力强,发酵所得沙棘果酒品质优,而且酒体澄清透明、色泽红润,具有沙棘果酒的典型风味,适用于果酒生产以及下一步...     

Production of fermentation aroma compounds by Saccharomyces cerevisiae wine yeasts: effects of yeast assimilable nitrogen on two model strains

The contribution of yeast fermentation metabolites to the aromatic profile of wine is well documented; however, the biotechnological application of this knowledge, apart from strain selection, is still rather limited and often contradictory. Understanding and modeling the relationship between nutrient availability and the production of desirable aroma compounds by different strains must be one of the main objectives in the selection of industrial yeasts for the beverage and food industry. In order to overcome the variability in the composition of grape juices, we have used a chemically defined model medium for studying yeast physiological behavior and metabolite production in response to nitrogen supplementation so as to identify an appropriate yeast assimilable nitrogen level for strain differentiation. At low initial nitrogen concentrations, strain KU1 produced higher quantities of esters and fatty acids whereas M522 produced higher concentrations of isoacids, gamma-butyrolactone, higher alcohols and 3-methylthio-1-propanol. We propose that although strains KU1 and M522 have a similar nitrogen consumption profile, they represent useful models for the chemical characterization of wine strains in relation to wine quality. The differential production of aroma compounds by the two strains is discussed in relation to their capacity for nitrogen usage and their impact on winemaking. The results obtained here will help to develop targeted metabolic footprinting methods for the discrimination of industrial yeasts.     

The impact of acetate metabolism on yeast fermentative performance and wine quality: reduction of volatile acidity of grape musts and wines

Acetic acid is the main component of the volatile acidity of grape musts and wines. It can be formed as a by-product of alcoholic fermentation or as a product of the metabolism of acetic and lactic acid bacteria, which can metabolize residual sugars to increase volatile acidity. Acetic acid has a negative impact on yeast fermentative performance and affects the quality of certain types of wine when present above a given concentration. In this mini-review, we present an overview of fermentation conditions and grape-must composition favoring acetic acid formation, as well the metabolic pathways leading to its formation and degradation by yeast. The negative effect of acetic acid on the fermentative performance of Saccharomyces cerevisiae will also be covered, including its role as a physiological inducer of apoptosis. Finally, currently available wine deacidification processes and new proposed solutions based on zymological deacidification by select S. cerevisiae strains will be discussed.     

Patagonian wines: the selection of an indigenous yeast starter

The use of selected yeasts for winemaking has clear advantages over the traditional spontaneous fermentation. The aim of this study was to select an indigenous Saccharomyces cerevisiae yeast isolate in order to develop a regional North Patagonian red wine starter culture. A two-step selection protocol developed according to physiological, technological and ecological criteria based on killer interactions was used. Following this methodology, S. cerevisiae isolate MMf9 was selected among 32 indigenous yeasts previously characterized as belonging to different strains according to molecular patterns and killer biotype. This isolate showed interesting technological and qualitative features including high fermentative power and low volatile acidity production, low foam and low sulphide production, as well as relevant ecological characteristics such as resistance to all indigenous and commercial S. cerevisiae killer strains assayed. Red wines with differential volatile profiles and interesting enological features were obtained at laboratory scale by using this selected indigenous strain.     

Effects of GPD1 overexpression in Saccharomyces cerevisiae commercial wine yeast strains lacking ALD6 genes

The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP+-dependent Mg2+-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.     

Selection of Ethanol-Tolerant Yeast Hybrids in pH-Regulated Continuous Culture

Hybrids between naturally occurring wine yeast strains and laboratory strains were formed as a method of increasing genetic variability to improve the ethanol tolerance of yeast strains. The hybrids were subjected to competition experiments under continuous culture controlled by pH with increasing ethanol concentrations over a wide range to select the fastest-growing strain at any concentration of ethanol. The continuous culture system was obtained by controlling the dilution rate of a chemostat connected to a pH-meter. The nutrient pump of the chemostat was switched on and off in response to the pH of the culture, which was thereby kept near a critical value (pH_c). Under these conditions, when the medium was supplemented with ethanol, the ethanol concentration of the culture increased with each pulse of dilution. A hybrid strain was selected by this procedure that was more tolerant than any of the highly ethanol-tolerant wine yeast strains at any concentration of ethanol and was able to grow at up to 16% (vol/vol) ethanol. This improvement in ethanol tolerance led to an increase in both the ethanol production rate and the total amount of ethanol produced.