The application of non-Saccharomyces yeast in fermentations with limited aeration as a strategy for the production of wine with reduced alcohol content.

High alcohol concentrations reduce the complexity of wine sensory properties. In addition, health and economic drivers have the wine industry actively seeking technologies that facilitate the production of wines with lower alcohol content. One of the simplest approaches to achieve this aim would be the use of wine yeast strains which are less efficient at transforming grape sugars into ethanol, however commercially available wine yeasts produce very similar ethanol yields. Non-conventional yeast, in particular non-Saccharomyces species, have shown potential for producing wines with lower alcohol content. These yeasts are naturally present in the early stages of fermentation but in general are not capable of completing alcoholic fermentation. We have evaluated 48 non-Saccharomyces isolates to identify strains that, with limited aeration and in sequential inoculation regimes with S. cerevisiae, could be used for the production of wine with lower ethanol concentration. Two of these, Torulaspora delbrueckii AWRI1152 and Zygosaccharomyces bailii AWRI1578, enabled the production of wine with reduced ethanol concentration under limited aerobic conditions. Depending on the aeration regime T. delbrueckii AWRI1152 and Z. bailii AWRI1578 showed a reduction in ethanol concentration of 1.5% (v/v) and 2.0% (v/v) respectively, compared to the S. cerevisiae anaerobic control.

Evaluation of non-Saccharomyces yeasts for the reduction of alcohol content in wine.

Over recent decades, the average ethanol concentration of wine has increased, largely due to consumer preference for wine styles associated with increased grape maturity; sugar content increases with grape maturity, and this translates into increased alcohol content in wine. However, high ethanol content impacts wine sensory properties, reducing the perceived complexity of flavors and aromas. In addition, for health and economic reasons, the wine sector is actively seeking technologies to facilitate the production of wines with lower ethanol content. Nonconventional yeast species, in particular, non-Saccharomyces yeasts, have shown potential for producing wines with lower alcohol content. These yeast species, which are largely associated with grapes preharvest, are present in the early stages of fermentation but, in general, are not capable of completing alcoholic fermentation. We have evaluated 50 different non-Saccharomyces isolates belonging to 24 different genera for their capacity to produce wine with a lower ethanol concentration when used in sequential inoculation regimes with a Saccharomyces cerevisiae wine strain. A sequential inoculation of Metschnikowia pulcherrima AWRI1149 followed by an S. cerevisiae wine strain was best able to produce wine with an ethanol concentration lower than that achieved with the single-inoculum, wine yeast control. Sequential fermentations utilizing AWRI1149 produced wines with 0.9% (vol/vol) and 1.6% (vol/vol) (corresponding to 7.1 g/liter and 12.6 g/liter, respectively) lower ethanol concentrations in Chardonnay and Shiraz wines, respectively. In Chardonnay wine, the total concentration of esters and higher alcohols was higher for wines generated from sequential inoculations, whereas the total concentration of volatile acids was significantly lower. In sequentially inoculated Shiraz wines, the total concentration of higher alcohols was higher and the total concentration of volatile acids was reduced compared with those in control S. cerevisiae wines, whereas the total concentrations of esters were not significantly different.

Effect of oxygen and lipid supplementation on the volatile composition of chemically defined medium and Chardonnay wine fermented with Saccharomyces cerevisiae.

Oxygen or lipids are required to complete stressful alcoholic fermentation. Lack of these nutrients can inhibit sugar uptake and growth, which leads to incomplete or 'stuck' fermentation. Oxygen or lipids supplementation not only restores yeast fermentative activity and also affects formation of yeast volatile metabolites. To clarify the effect of oxygen and lipid supplementation on the formation of flavour active metabolites during wine fermentation, we evaluated the addition of these two nutrients to chemically defined grape juice and filter clarified Chardonnay must. Lipid addition increased the concentration of esters, higher alcohols and volatile acids, whereas oxygen increased the concentration of higher alcohols and altered the proportion of acetate to ethyl esters and the proportion of branch-chain acids to medium-chain fatty acids. Combined addition of lipids and oxygen showed an additive effect on concentration of higher alcohols whereas oxygen suppressed the enhancing effect of lipids on formation of esters and volatile acids. Our results demonstrate the potential of lipid and oxygen supplementation for the manipulation of wine aroma in white wine fermentation.

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.

Genotype-dependent sulphite tolerance of Australian Dekkera (Brettanomyces) bruxellensis wine isolates.

AIMS: The aim of this study was to determine sulphite tolerance for a large number of Dekkera bruxellensis isolates and evaluate the relationship between this phenotype and previously assigned genotype markers. METHODS AND RESULTS: A published microplate-based method for evaluation of yeast growth in the presence of sulphite was benchmarked against culturability following sulphite treatment, for the D. bruxellensis type strain (CBS 74) and a reference wine isolate (AWRI 1499). This method was used to estimate maximal sulphite tolerance for 41 D. bruxellensis isolates, which was found to vary over a fivefold range. Significant differences in sulphite tolerance were observed when isolates were grouped according to previously assigned genotypes and ribotypes. CONCLUSIONS: Variable sulphite tolerance for the wine spoilage yeast D. bruxellensis can be linked to genotype markers. SIGNIFICANCE AND IMPACT OF THE STUDY: Strategies to minimize risk of wine spoilage by D. bruxellensis must take into account at least a threefold range in effective sulphite concentration that is dependent upon the genotype group(s) present. The isolates characterized in this study will be a useful resource for establishing the mechanisms conferring sulphite tolerance for this industrially important yeast species.

Adaptive evolution of Saccharomyces cerevisiae to generate strains with enhanced glycerol production.

The development of new wine yeast strains with improved characteristics is critical in the highly competitive wine market, which faces the demand of ever-changing consumer preferences. Although new strains can be constructed using recombinant DNA technologies, consumer concerns about genetically modified (GM) organisms strongly limit their use in food and beverage production. We have applied a non-GM approach, adaptive evolution with sulfite at alkaline pH as a selective agent, to create a stable yeast strain with enhanced glycerol production; a desirable characteristic for wine palate. A mutant isolated using this approach produced 41% more glycerol than the parental strain it was derived from, and had enhanced sulfite tolerance. Backcrossing to produce heterozygous diploids revealed that the high-glycerol phenotype is recessive, while tolerance to sulfite was partially dominant, and these traits, at least in part, segregated from each other. This work demonstrates the potential of adaptive evolution for development of novel non-GM yeast strains, and highlights the complexity of adaptive responses to sulfite selection.

Assimilable nitrogen utilisation and production of volatile and non-volatile compounds in chemically defined medium by Saccharomyces cerevisiae wine yeasts.

Surveys conducted worldwide have shown that a significant proportion of grape musts are suboptimal for yeast nutrients, especially assimilable nitrogen. Nitrogen deficiencies are linked to slow and stuck fermentations and sulphidic off-flavour formation. Nitrogen supplementation of grape musts has become common practice; however, almost no information is available on the effects of nitrogen supplementation on wine flavour. In this study, the effect of ammonium supplementation of a synthetic medium over a wide range of nitrogen values on the production of volatile and non-volatile compounds by two high-nitrogen-demand wine fermentation strains of Saccharomyces cerevisiae was determined. To facilitate this investigation, a simplified chemically defined medium that resembles the nutrient composition of grape juice was used. Analysis of variance revealed that ammonium supplementation had significant effects on the concentration of residual sugar, L-malic acid, acetic acid and glycerol but not the ethanol concentration. While choice of yeast strain significantly affected half of the aroma compounds measured, nitrogen concentrations affected 23 compounds, including medium-chain alcohols and fatty acids and their esters. Principal component analysis showed that branched-chain fatty acids and their esters were associated with low nitrogen concentrations, whereas medium-chain fatty esters and acetic acid were associated with high nitrogen concentrations.

Microsatellite PCR profiling of Saccharomyces cerevisiae strains during wine fermentation.

AIMS: Use of microsatellite PCR to monitor populations of Saccharomyces cerevisiae strains during fermentation of grape juice. METHOD AND RESULTS: Six commercial wine strains of S. cerevisiae were screened for polymorphism at the SC8132X locus using a modified rapid PCR identification technique. The strains formed four distinct polymorphic groups that could be readily distinguished from one another. Fermentations inoculated with mixtures of three strains polymorphic at the SC8132X locus were monitored until sugar utilization was complete, and all exhibited a changing population structure throughout the fermentation. CONCLUSIONS: Rapid population quantification demonstrated that wine fermentations are dynamic and do not necessarily reflect the initial yeast population structure. One or more yeast strains were found to dominate at different stages of the fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The population structure of S. cerevisiae during mixed culture wine fermentation is dynamic and could modify the chemical composition and flavour profile of wine.

Spoilage of bottled red wine by acetic acid bacteria.

AIMS: To determine the bacterial species associated with an outbreak of spoilage in commercially bottled red wine where the bottles had been stored in an upright vertical compared with horizontal position. METHODS AND RESULTS: Bottled wines comprising Cabernet Sauvignon, Pinot Noir, Shiraz, Merlot and blended red varieties were examined for visible spoilage. Analysis of visibly affected and non-affected wines revealed a spectrum of aroma and flavour defects, ranging from loss of fruity aroma, staleness, oxidized character to overt volatile acidity. Only acetic acid bacteria, and not yeast or lactic acid bacteria, could be isolated from both spoiled and unspoiled wines and were found to grow only on Wallerstein Nutrient (WL) medium supplemented with 10% red wine or 1-2% ethanol. Analysis of the 16S rRNA region and RAPD-PCR analysis showed the isolates to be a closely related group of Acetobacter pasteurianus, but this group was differentiated from the group comprising beer, vinegar and cider strains. CONCLUSIONS: A. pasteurianus was the species considered responsible for the spoilage but the isolates obtained had atypical properties for this species. In particular, they failed to grow on WL nutrient medium without ethanol or wine supplementation. Storage of the bottles of wine containing A. pasteurianus in an upright vertical position specifically induced growth and spoilage in a proportion of the bottles under conditions that were inhibitory for horizontally stored bottles. We hypothesize that the upright position created a heterogeneous environment that allowed the growth of bacteria in only those bottles sealed with cork closures that had upper limit for the natural permeability to oxygen. Such a heterogeneous environment would not exist in horizontally stored bottles as the larger volume of wine adjacent to the cork would strongly compete with the bacteria for the oxygen as it diffuses through the cork closure. SIGNIFICANCE AND IMPACT OF THE STUDY: A low level of bacteria (acetic acid bacteria) in wine can proliferate and cause wine spoilage in bottles stored in an upright vertical as opposed to an horizontal position under conditions that would normally limit bacterial development.

The incidence of killer activity of non-Saccharomyces yeasts towards indigenous yeast species of grape must: potential application in wine fermentation.

Fourteen killer yeasts were assayed for their ability to kill species of yeast that are commonly associated with fermenting grape must and wine. A total of 147 of a possible 364 killer-sensitive interactions were observed at pH 4.5. Of the killer yeasts studied, Pichia anomala NCYC 434 displayed the broadest killing range. At a pH value comparable with those of wine ferments, pH 3.5, the incidence of killer-sensitive interactions was reduced by 700% across all the yeasts. Williopsis saturnus var. mrakii CBS 1707 exhibited the broadest killing range at the lower pH, killing more than half of the tester strains. Intraspecific variation in sensitivity to killer yeasts was observed in all species where more than one strain was tested. Also, in strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens, the three species in which more than one killer yeast was analysed, intraspecific variation in killer activity was observed.

AFLP fingerprinting for analysis of yeast genetic variation.

Amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in commercial strains, type strains and winery isolates from a number of yeast species. AFLP was shown to be effective in discriminating closely related strains. Furthermore, sufficient similarity in the fingerprints produced by yeasts of a given species allowed classification of unknown isolates. The applicability of the method for determining genome similarities between yeasts was investigated by performing cluster analysis on the AFLP data. Results from two species, Saccharomyces cerevisiae and Dekkera bruxellensis, illustrate that AFLP is useful for the study of intraspecific genetic relatedness. The value of the technique in strain differentiation, species identification and the analysis of genetic similarity demonstrates the potential of AFLP in yeast ecology and evolutionary studies.

Differentiation and species identification of yeasts using PCR.

A PCR-based method has been developed that permits both intraspecies differentiation and species identification of yeast isolates. Oligonucleotide primers that are complementary to intron splice sites were used to produce PCR fingerprints that display polymorphisms between different species of indigenous wine yeasts. Although polymorphisms existed between isolates of the same species, the banding patterns shared several amplification products that allowed species identification. Importantly, the method was able to distinguish between species of the closely related Saccharomyces sensu stricto yeasts. In two cases where isolates could not be positively identified there was discrepancy between the phenetic and phylogenetic species concept. The method has applications in yeast ecological studies, enabling the rapid grouping of isolates with related genomes and the investigation of population dynamics of strains of the same species.

PCR differentiation of commercial yeast strains using intron splice site primers.

The increased use of pure starter cultures in the wine industry has made it necessary to develop a rapid and simple identification system for yeast strains. A method based upon the PCR using oligonucleotide primers that are complementary to intron splice sites has been developed. Since most introns are not essential for gene function, introns have evolved with minimal constraint. By targeting these highly variable sequences, the PCR has proved to be very effective in uncovering polymorphisms in commercial yeast strains. The speed of the method and the ability to analyze many samples in a single day permit the monitoring of specific yeast strains during fermentations. Furthermore, the simplicity of the technique, which does not require the isolation of DNA, makes it accessible to industrial laboratories that have limited molecular expertise and resources.

Determination of sulphite reductase activity and its response to assimilable nitrogen status in a commercial Saccharomyces cerevisiae wine yeast.

The liberation of H2S is a common problem afflicting wine fermentation. Sulphite reductase activity of a commercial wine yeast was investigated to define its involvement in this process. The activity studied here differed from those characterized previously from cider and bakers' yeasts by displaying a greater sensitivity to cold, low ionic strength and possibly, proteolytic action. These differences necessitated the development of a new method of quantification. Through this method, the onset of H2S liberation was shown not to be a result of variations in the levels of sulphite reductase activity. Thus, high levels of activity which occurred during the exponential phase of growth were not necessarily accompanied by the liberation of H2S. Similarly, nitrogen-starved cultures which liberated H2S showed no corresponding increase in sulphite reductase activity from prestarvation levels. In fact, rates of H2S liberation from cultures and in enzyme assays agreed closely. A short-term independence of sulphite reductase activity from culture nitrogen status was therefore evident. The only influence of nitrogen was achieved in its absence when enzyme activity decayed with a half-life (4.25 h) which was comparable to that induced by the presence of cycloheximide (5.75 h). A proposed transcriptional control mechanism mediated by methionine derivatives was only partly effective in this strain although an in vitro inhibitory effect of methionine was implicated. These data therefore support the notion that H2S liberation in response to nitrogen starvation stems from a failure of metabolism to sequester H2S which continues to be formed, at least initially, at prestarvation rates.

Regulation of hydrogen sulfide liberation in wine-producing Saccharomyces cerevisiae strains by assimilable nitrogen.

Saccharomyces cerevisiae wine-producing yeast cultures grown under model winemaking conditions could be induced to liberate hydrogen sulfide (H2S) by starvation for assimilable nitrogen. The amount of H2S produced was dependent on the yeast strain, the sulfur precursor compound, the culture growth rate, and the activity of the sulfite reductase enzyme (EC 1.8.1.2) immediately before nitrogen depletion. Increased H2S formation relative to its utilization by metabolism was not a consequence of a de novo synthesis of sulfite reductase. The greatest amount of H2S was produced when nitrogen became depleted during the exponential phase of growth or during growth on amino acids capable of supporting short doubling times. Both sulfate and sulfite were able to act as substrates for the generation of H2S in the absence of assimilable nitrogen; however, sulfate reduction was tightly regulated, leading to limited H2S liberation, whereas sulfite reduction appeared to be uncontrolled. In addition to ammonium, most amino acids were able to suppress the liberation of excess H2S when added as sole sources of nitrogen, particularly for one of the strains studied. Cysteine was the most notable exception, inducing the liberation of H2S at levels exceeding that of the nitrogen-depleted control. Threonine and proline also proved to be poor substitutes for ammonium. These data suggest that any compound that can efficiently generate sulfide-binding nitrogenous precursors of organic sulfur compounds will prevent the liberation of excess H2S.

Determination of killer yeast activity in fermenting grape juice by using a marked Saccharomyces wine yeast strain.

The Escherichia coli beta-glucuronidase gene has been used as a marker gene to monitor a killer Saccharomyces cerevisiae strain in mixed-culture ferments. The marked killer strain was cured of its M-dsRNA genome to enable direct assessment of the efficiency of killer toxin under fermentation conditions. Killer activity was clearly evident in fermenting Rhine Riesling grape juice of pH 3.1 at 18 degrees C, but the extent of killing depended on the proportion of killer to sensitive cells at the time of inoculation. Killer activity was detected only when the ratio of killer to sensitive cells exceeded 1:2. At the highest ratio of killer to sensitive cells tested (2:1), complete elimination of sensitive cells was not achieved.

A microprocessor-controlled photometer for monitoring microbial growth in multi-welled plates.

Multi-welled microtitre plates provide a convenient means of handling 'large block' multifactorial experiments with microbial cultures. An inexpensive instrument, termed a 'Biophotometer', has been designed to monitor microbial growth in each well, by transmitted light measurements. Optional microcomputer control is employed to facilitate scanning of plates and data handling. A unique method for agitating cultures is incorporated into the system. Typical results are presented to illustrate the versatility of this system.