Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation

The multi-yeast strain composition of wine fermentations has been well established. However, the effect of multiple strains of Saccharomyces spp. on wine flavour is unknown. Here, we demonstrate that multiple strains of Saccharomyces grown together in grape juice can affect the profile of aroma compounds that accumulate during fermentation. A metabolic footprint of each yeast in monoculture, mixed cultures or blended wines was derived by gas chromatography - mass spectrometry measurement of volatiles accumulated during fermentation. The resultant ion spectrograms were transformed and compared by principal-component analysis. The principal-component analysis showed that the profiles of compounds present in wines made by mixed-culture fermentation were different from those where yeasts were grown in monoculture fermentation, and these differences could not be produced by blending wines. Blending of monoculture wines to mimic the population composition of mixed-culture wines showed that yeast metabolic interactions could account for these differences. Additionally, the yeast strain contribution of volatiles to a mixed fermentation cannot be predicted by the population of that yeast. This study provides a novel way to measure the population status of wine fermentations by metabolic footprinting.     

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.     

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.     

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.     

Simultaneous yeast–bacteria inoculum. A feasible solution for the management of oenological fermentation in red must with low nitrogen content

The simultaneous inoculum of yeasts and bacteria is a feasible solution for improving fermentation in wines with a harsh chemical composition, capable of inhibiting microbial activity. Considering the risk of wine spoilage due to lactic bacteria, co-inoculum is suggested in white wines with a low pH. However, climate change has also caused problems in achieving malolactic fermentation in red wines, due to the high concentration of ethanol and the low nutrient content. In this work, 5 pairs of commercial oenological starters were tested in simultaneous fermentation, using 4 red musts with a low nitrogen content, and compared with a traditional winemaking process. The simultaneous inoculum caused a slowdown in the activity of yeasts, although no problems in the accomplishment of alcoholic fermentations were observed. More reliable malolactic fermentation was performed in the co-inoculum trials, while, in traditional winemaking, some failures in the degradation of malic acid were observed. Microbiological analyses agreed with these observations. No differences were found in yeast density during alcoholic fermentation, demonstrating the absence of negative interaction between the yeast and the bacteria. However, simultaneous fermentation is not without risks; the highest increases of acetic acid were noted in the co-inoculum trials. The addition of yeast and bacteria to must with a serious lack of nutrients would appear to be a promising alternative to traditional fermentation; however, careful control of the chemical composition of must is mandatory to obtain reliable microbiological activity in the first stages of winemaking.     

Molecular analysis of red wine yeast diversity in the Ribera del Duero D.O. (Spain) area

Molecular characterization of wine yeast population during spontaneous fermentation in biodynamic wines from Ribera del Duero D.O. located at northern plateau of Spain has been carried out during two consecutive years. A total of 829 yeast strains were isolated from the samples and characterized by electrophoretic karyotype. The results show the presence of three population of yeast differentiated by their electrophoretic karyotypes, (1) non-Saccharomyces yeast dominant in the initial phase of the fermentations (NS); (2) Saccharomyces bayanus var uvarum detected mainly mid-way through the fermentation process at 20–25 °C; and (3) Saccharomyces cerevisiae which remained dominant until the end of the fermentation. This is the first study showing the population dynamic of S. bayanus var. uvarum in red wines produced in Ribera del Duero that could represent an important source of autochthonous wine yeasts with novel oenological properties.     

An inverse correlation between stress resistance and stuck fermentations in wine yeasts. A molecular study.

During alcoholic fermentations yeast cells are subjected to several stress conditions and, therefore, yeasts have developed molecular mechanisms in order to resist this adverse situation. The mechanisms involved in stress response have been studied in Saccharomyces cerevisiae laboratory strains. However a better understanding of these mechanisms in wine yeasts could open the possibility to improve the fermentation process. In this work an analysis of the stress response in three wine yeasts has been carried out by studying the expression of several representative genes under several stress conditions which occur during fermentation. We propose a simplified method to study how these stress conditions affect the viability of yeast cells. Using this approach an inverse correlation between stress-resistance and stuck fermentations has been found. We also have preliminary data about the use of the HSP12 gene as a molecular marker for stress-resistance in wine yeasts.     

Yeast biodiversity and dynamics during sweet wine production as determined by molecular methods

In this study we investigated yeast biodiversity and dynamics during the production of a sweet wine obtained from dried grapes. Two wineries were selected in the Collio region and grapes, grape juices and wines during fermentations were analyzed by culture-dependent methods (plating on WLN medium) and culture-independent methods (PCR-DGGE). Moreover, the capability of the Saccharomyces cerevisiae starter cultures to take over the fermentation was assessed by RAPD-PCR. On WLN agar several species of non-Saccharomyces yeasts (Hanseniaspora, Metschnikowia, Pichia, Candida, Torulaspora and Debaryomyces), but also strains of S. cerevisiae, were isolated. After inoculation of the starter cultures, only colonies typical of S. cerevisiae were observed. Using PCR-DGGE, the great biodiversity of moulds on the grapes was underlined, both at the DNA and RNA level, while the yeast contribution started to become important only in the musts. Here, bands belonging to species of Candida zemplinina and Hanseniaspora uvarum were visible. Lastly, when the S. cerevisiae isolates were compared by RAPD-PCR, it was determined that only in one of the fermentations followed, the inoculated strain conducted the alcoholic fermentation. In the second fermentation, the starter culture was not able to promptly implant and other populations of S. cerevisiae could be isolated, most likely contributing to the final characteristics of the sweet wine produced.     

A low-cost procedure for production of fresh autochthonous wine yeast

A low-cost procedure was designed for easy and rapid response-on-demand production of fresh wine yeast for local wine-making. The pilot plant produced fresh yeast culture concentrate with good microbial quality and excellent oenological properties from four selected wine yeasts. The best production yields were obtained using 2% sugar beet molasses and a working culture volume of less than 60% of the fermenter capacity. The yeast yield using 2% sugar grape juice was low and had poor cell viability after freeze storage, although the resulting yeast would be directly available for use in the winery. The performance of these yeasts in commercial wineries was excellent; they dominated must fermentation and improved its kinetics, as well as improving the physicochemical parameters and the organoleptic quality of red and white wines.     

Scaling-up in industrial winemaking using low electric current as an alternative to sulfur dioxide addition

AIMS: To better understand the outcome of employing low electric current (LEC) technology as a new preservation and alternative in wine technology, and to contribute to its development. It is used in industrial-scale winemaking with commercial yeast (Saccharomyces cerevisiae) during the grape must fermentation. METHODS AND RESULTS: LEC (200 mA, time 16 days) was applied to fresh grape must as an alternative method to the usual sulfur dioxide addition used in the industrial process; two tanks, each 30,000 l, were employed for parallel fermentations. The results show that LEC decreased the survival time and increased the death rate of apiculate yeasts, whereas it did not affect the growth and survival of S. cerevisiae. A comparison was made of the main chemical and sensory parameters of the wines obtained. CONCLUSIONS: The results have demonstrated that the low-voltage treatment had a positive effect on the grape juice fermentation (yeast microflora) during the early stages of winemaking. SIGINIFICANCE AND IMPACT OF THE STUDY: These results could be of significant importance in developing, for 'biological wine', new winemaking technologies for an innovative control process of yeast fermentation.     

Patagonian wines: implantation of an indigenous strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in fermentations conducted in traditional and modern cellars

In this work we evaluate the implantation capacity of the selected S. cerevisiae indigenous strain MMf9 and the quality of the produced wines in a traditional (T) and a modern (M) cellar with different ecological and technological characteristics in North Patagonia (Argentina). Red musts were fermented in 10,000 l vats using the indigenous strain MMf9 as well as the respective controls: a fermentation conducted with a foreign starter culture (BC strain) in M cellar and a natural fermentation in T cellar. Since commercial S. cerevisiae starters are always used for winemaking in M cellar and in order to compare the results, natural fermentations and fermentations conducted by the indigenous strain MMf9 were performed at pilot (200 l) scale in this cellar, concomitantly. Thirty indigenous yeasts were isolated at three stages of fermentation: initial, middle and end. The identification of the yeast biota associated to vinifications was carried out using ITS1-5.8S-ITS2 PCR-RFLP. The intra-specific variability of the S. cerevisiae populations was evaluated using mtDNA-RFLP analysis. Wines obtained from all fermentations were evaluated for their chemical and volatile composition and for their sensory characteristics. A higher capacity of implantation of the indigenous MMf9 strain was evidenced in the fermentation carried out in M cellar (80% at end stage) than the one carried out in T cellar (40%). This behaviour could indicate that each cellar differs in the diversity of S. cerevisiae strains associated to wine fermentations. Moreover a higher capacity of implantation of the native starter MMf9 with regard to the foreign (BC) one was also found in M cellar. The selected indigenous strain MMf9 was able to compete with the yeast biota naturally present in the must. Additionally, a higher rate of sugar consumption and a lower fermentation temperature were observed in vinifications conducted by MMf9 strain with regard to control fermentations, producing wines with favourable characteristics. Even when its implantation in T fermentation was lower than that observed in M one, we can conclude that the wine features from MMf9 fermentations were better than those from their respective controls. Therefore, MMf9 selected indigenous strain could be an interesting yeast starter culture in North Patagonian wines.     

Study of beta-glucosidase production by wine-related yeasts during alcoholic fermentation. A new rapid fluorimetric method to determine enzymatic activity

AIMS: The beta-glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of beta-glucosidase in a number of wine-related yeast strains and to measure and identify this activity over the course of grape juice fermentation. METHODS AND RESULTS: beta-glucosidase activity was measured as the amount of 4-methylumbelliferone released from 4-methylumbelliferyl-beta-d-glucopyranoside substrate. Intact cells of some grape and wine-spoilage yeasts showed beta-glucosidase activity much higher than those observed in wine yeasts "sensu stricto". During fermentation, three Saccharomyces cerevisiae strains, one Hanseniaspora valbyensis strain and one Brettanomyces anomalus strain showed beta-glucosidase activity both intra- and extracellularly. CONCLUSIONS: In the studied strains, beta-glucosidase activity was at its maximum when the cells were in the active growth phase. However, a lowering of medium pH to values around 3 during fermentation led to total loss of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: During the course of this study, a new, rapid and reproducible method to assay beta-glucosidase activity was developed. The fact that Saccharomyces and non-Saccharomyces yeast strains are able to express beta-glucosidase activity during the alcoholic fermentation sheds new light on the contribution of these yeasts in the aroma expression of wines.     

Newly generated interspecific wine yeast hybrids introduce flavour and aroma diversity to wines

Increasingly, winemakers are looking for ways to introduce aroma and flavour diversity to their wines as a means of improving style and increasing product differentiation. While currently available commercial yeast strains produce consistently sound fermentations, there are indications that sensory complexity and improved palate structure are obtained when other species of yeast are active during fermentation. In this study, we explore a strategy to increase the impact of non-Saccharomyces cerevisiae inputs without the risks associated with spontaneous fermentations, through generating interspecific hybrids between a S. cerevisiae wine strain and a second species. For our experiments, we used rare mating to produce hybrids between S. cerevisiae and other closely related yeast of the Saccharomyces sensu stricto complex. These hybrid yeast strains display desirable properties of both parents and produce wines with concentrations of aromatic fermentation products that are different to what is found in wine made using the commercial wine yeast parent. Our results demonstrate, for the first time, that the introduction of genetic material from a non-S. cerevisiae parent into a wine yeast background can impact favourably on the wine flavour and aroma profile of a commercial S. cerevisiae wine yeast.     

Effect of the addition of inert cellulose substrates to grape must on Saccharomyces cerevisiae diversity and the evolution of alcoholic fermentation

AIMS: To study the addition of cellulose-based adjuvant as a resource to offset the negative effects produced by grape juice clarification during alcoholic fermentations. METHODS AND RESULTS: The effect of the addition of two kinds of inert cellulose substrates in white wine vinification was investigated in two different musts. In one of these musts, stuck fermentations were detected. One of the types of cellulose examined had a fining effect, which caused a decrease in the number of viable yeasts in the medium and altered the distribution and frequency of the clones, which performed the fermentation. The other cellulose substrate made the medium cloudier but did not alter the distribution of yeasts in comparison with the control. CONCLUSIONS: The behaviour of the inert cellulose substrates on vinification depends on its physical characteristics and its capacity for making the must cloudy. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of inert cellulose substrates in white wine vinification improves the fermentation process and the quality of wines obtained. This effect is more noticeable in difficult fermentations. One variety of cellulose showed an inhibitory effect on Torulaspora delbrueckii yeasts.     

Monitoring of Saccharomyces and Hanseniaspora populations during alcoholic fermentation by real-time quantitative PCR

Real-time, or quantitative, PCR (QPCR) was developed for the rapid quantification of two of the most important yeast groups in alcoholic fermentation (Saccharomyces spp. and Hanseniaspora spp.). Specific primers were designed from the region spanning the internal transcribed spacer 2 (ITS2) and the 5.8S rRNA gene. To confirm the specificity of these primers, they were tested with different yeast species, acetic acid bacteria and lactic acid bacteria. The designed primers only amplified for the intended group of species and none of the PCR assays was positive for any other wine microorganisms. This technique was performed on reference yeast strains from pure cultures and validated with both artificially contaminated wines and real wine fermentation samples. To determine the effectiveness of the technique, the QPCR results were compared with those obtained by plating. The design of new primers for other important wine yeast species will enable to monitor yeast diversity during industrial wine fermentation and to detect the main spoilage yeasts in wine.     

Monitoring of killer yeast populations in mixed cultures: influence of incubation temperature of microvinifications samples

Killer yeasts are frequently used to combat and prevent contamination by wild-type yeasts during wine production and they can even dominate the wine fermentation. Stuck and sluggish fermentations can be caused by an unbalanced ratio of killer to sensitive yeasts in the bioreactor, and therefore it is important to determine the proportion of both populations. The aim of this study was to provide a simple tool to monitor killer yeast populations during controlled mixed microvinifications of killer and sensitive Saccharomyces cerevisiae. Samples were periodically extracted during vinification, seeded on Petri dishes and incubated at 25 and 37?°C; the latter temperature was assayed for possible inactivation of killer toxin production. Colonies developed under the described conditions were randomly transferred to killer phenotype detection medium. Significant differences in the killer/sensitive ratio were observed between both incubation temperatures in all microvinifications. These results suggest that 37?°C seems a better option to determine the biomass of sensitive yeasts, in order to avoid underestimation of sensitive cells in the presence of killer yeasts during fermentations. Incubation at a toxin-inhibiting temperature clearly showed the real ratio of killer to sensitive cells in fermentation systems.     

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.     

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.     

Use of flow cytometry with fluorescent antibodies in real-time monitoring of simultaneously inoculated alcoholic-malolactic fermentation of Chardonnay

AIMS: To monitor in real-time the changes in microbial populations and chemistry of grape juice simultaneously inoculated with Saccharomyces cerevisiae and Oenococcus oeni. METHODS AND RESULTS: Viable populations of S. cerevisiae and O. oeni in Chardonnay fermentations were identified and quantified using fluorescent dyes and fluorescently labelled antibodies in a flow cytometric assay. Fermentation chemistry was monitored using Fourier transform infrared (FTIR) spectroscopy, except for malic acid which was measured enzymatically. Malic acid utilization by O. oeni was greatest in the presence of the yeast Cepage. Growth of O. oeni was substantially slower in the presence of the yeast VL1. The three yeasts had similar fermentation rates in the presence and absence of O. oeni. CONCLUSIONS: Viable and nonviable yeast and bacterial populations can be rapidly discriminated in simultaneous malolactic-alcoholic wine fermentations using antibodies, fluorescent dyes and flow cytometry. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study using fluorescently labelled antibodies to discriminate and monitor yeast and bacterial populations in wine fermentations and offers a new approach to investigating microbial interactions in wine fermentations.