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Interest in the use of non- Saccharomyces yeast in mixed cultures is increasing due to the perceived improvement in the quality and complexity of the resulting wines. The aim of the study was to determine the ability of monocultures and mixed yeast cultures for deacidification and improvement of the composition of cold climate grape wines. Fermentation of grape musts with increased total acidity was carried out with the use of monocultures of Saccharomyces cerevisiae MH020215 (Sc), Zygosaccharomyces bailii 749 (Zb) and Metschnikowia pulcherrima MG970690 (Mp), and their mixed cultures, inoculated simultaneously and sequentially. Oenological parameters, organic acids and volatile compounds profiles of obtained wines were characterized. The fermentation kinetics and analytical profiles of the obtained wines showed that the use of mixed yeast cultures contributed to the reduction of volatile acidity and acetic acid content in the wines, as well as obtaining a favorable aromatic profile of the wines. The dominant higher alcohols in all wines were 2-methyl-1-propanol, 3-methyl-1-butanol and 2-methyl-1-butanol. Significantly higher amounts of the first two compounds were found in wines obtained with M. pulcherrima MG070690, both in monoculture and in mixed cultures. The monocultures of M. pulcherrima MG070690 (Mp) compared with Z. bailli 749 (Zb) synthesized higher levels of esters in wines, including ethyl acetate, ethyl propionate, isobutyl acetate, ethyl pyroracemate and isoamyl acetate.

Current consumer preferences are determined by well-structured, full-bodied wines with a rich flavor and with reduced alcohol levels. One of the strategies for obtaining wines with reduced ethanol content is sequential inoculation of non-Saccharomyces and Saccharomyces cerevisiae yeasts. However, different factors affect the production of metabolites like ethanol, glycerol and acetic acid by inoculated yeasts. In order to obtain low alcohol wines without quality loss, the aims of our study were: i) to determine optimum conditions (fermentation temperature and time of permanence and initial inoculum size of the non-Saccharomyces population at the beginning of the process, prior to inoculation with S. cerevisiae); ii) to validate the optimized factors; and iii) to assess sensory quality of the wines obtained after validation. Two combinations of yeasts were used in this study: Hanseniaspora uvarum BHu9/S. cerevisiae BSc114 and Candida membranaefaciens BCm71/S. cerevisiae BSc114. Optimization of three fermentation factors that affect to non-Saccharomyces yeasts prior to S. cerevisiae inoculation was carried out using a Box-Behnken experimental design. Applying the models constructed by Response Surface Methodology, the lowest ethanol production by H. uvarum BHu9/S. cerevisiae BSc114 co-culture was obtained when H. uvarum BHu9 was inoculated 48 h 37 min prior to S. cerevisiae inoculation, at a fermentation temperature of 25 °C and at an initial inoculum size of 5 × 10 6  cells/mL. Lowest alcohol production with C. membranaefaciens BCm71/S. cerevisiae BSc114 was observed when C. membranaefaciens BCm71 was inoculated 24 h 15 min prior to S. cerevisiae at a fermentation temperature of 24.94 °C and at an initial inoculum size of 2.72 × 10 6  cells/mL. The optimized conditions of the two co-cultures were subsequently submitted to lab-scale validation. Both proposed strategies yielded ethanol levels that were significantly lower than control cultures (S. cerevisiae). Wines fermented with non-Saccharomyces/Saccharomyces co-cultures under optimized conditions were also associated with higher aromatic complexity characterized by the presence of red fruit aromas, whereas wines obtained with S. cerevisiae BSc114 were described by parameters linked with high ethanol levels., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Since sulphur dioxide (SO(2)) is associated with health risks, the wine industry endeavours to reduce SO(2) levels in wines with new innovative techniques. The aim of this study was, therefore, to investigate the efficacy of ultraviolet radiation (UV)-C (254 nm) as an alternative technology to inactivate microorganisms in grape juices and wines. A pilot-scale UV-C technology (SurePure, South Africa) consisting of an UV-C germicidal lamp (100 W output; 30 W UV-C output) was used to apply UV-C dosages ranging from 0 to 3672 J l(-1), at a constant flow rate of 4000 l h(-1) (Re > 7500). Yeasts, lactic and acetic acid bacteria were singly and co-inoculated into 20 l batches of Chenin blanc juice, Shiraz juice, Chardonnay wine and Pinotage wine, respectively. A dosage of 3672 J l(-1), resulted in an average log(10) microbial reduction of 4.97 and 4.89 in Chardonnay and Pinotage, respectively. In Chenin blanc and Shiraz juice, an average log(10) reduction of 4.48 and 4.25 was obtained, respectively. UV-C efficacy may be influenced by liquid properties such as colour and turbidity. These results had clearly indicated significant (p < 0.05) germicidal effect against wine-specific microorganisms; hence, UV-C radiation may stabilize grape juice and wine microbiologically in conjunction with reduced SO(2) levels., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In this study, the antimicrobial activity of a commercial beta-glucanase preparation against wine spoilage yeasts such as Cryptococcus albidus, Dekkera bruxellensis, Pichia membranifaciens, Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus has been evaluated. Yeast species tested showed different sensitivities to the enzyme preparation. In vitro assays in laboratory medium (GPY) showed inhibition by the beta-glucanase preparation of D. bruxellensis and Z. bailii growth with IC(50) and MIC values approximately 3 to 4-fold greater than the recommended dose for improving wine filtration. Wine spoilage experiments showed antimicrobial action against D. bruxellensis and Z. bailii although with reduced effectiveness to that showed in laboratory medium. Under the conditions tested, the addition of beta-glucanase did not affect wine enological parameters. Our data suggest the potential use of beta-glucanases as antimicrobial agents in wine and indicate that the application of antimicrobial enzymes for yeast spoilage control deserves further investigation.

In winemaking, spontaneous grape must fermentations have been replaced by inoculation of commercial active dry wine yeast (ADWY). Yeast rehydration is the key to avoiding stuck and sluggish fermentations. Despite the importance of this step, not enough is known about what this process implies for winemaking as a whole or about what kind of practices could help to improve it. The main aim of this study is to determine the best yeast rehydration conditions for ensuring good cell viability and vitality before inoculation into the must. The experimental rehydration media in this study can be divided into four groups: carbon and nitrogen compounds, metallic ions, oxidant and antioxidant agents, and membrane fluidity agents. We studied the biochemical and biophysical behaviour of ADWY after rehydration in the various media under oenological conditions, i.e. incubation at 37 degrees C for 30 min. The viability of rehydrated yeast cells was evaluated by plating, and assessed by fluorescence microscopy and flow cytometry. The vitality of rehydrated cells was estimated by indirect impedance. The rehydrating solution complemented with magnesium provided the best vitality rate because the time taken to reach the activity threshold was cut by two thirds. This improvement was also illustrated by the less time needed to stop the leakage of intracellular compounds during the rehydration process.

Aims: The toxicity of the fungicide pyrimethanil on the growth of wine yeasts was evaluated using in vivo and in vitro experimentation., Methods and Results: The effect of pyrimethanil in the must was studied during the spontaneous wine fermentation of three consecutive vintages and by the cultivation of Hanseniaspora uvarum and Saccharomyces cerevisiae yeasts in a liquid medium. The residues of the fungicide were measured using gas chromatography-mass spectrometry system and the sugar concentration in the must using HPLC-RI. Molecular and standard methods were used for identifying the yeast species. Although the pyrimethanil residues in grapes were below the maximum residue limits, they significantly affected the reduced utilization of sugars in the first days of fermentation. Its residues controlled the growth of H. uvarum during the fermentation and during in vitro cultivation as well., Conclusions: The fungicide pyrimethanil had an effect on the course and successful conclusion of spontaneous wine fermentation that was correlated with the initial concentration of yeasts in the must., Significance and Impact of the Study: The impact of pyrimethanil on the indigenous mixed yeast flora in fermenting must was investigated for the first time. The results showed that its residues might play an important role in the growth and succession of yeast during spontaneous wine fermentation.

The present article aims to review research papers that focus on the parietal adsorption activity of wine yeast and on its contribution to the enhancement of wine safety and quality. There is a great diversity among yeasts for their parietal adsorption activity: the outermost layer of the cell wall has a chemical composition that notably varies from yeast to yeast. Parietal mannoproteins can contain phosphate, pyruvate, or glucuronic acid, which impart negative charges, modifying the electrostatic and ionic interactions with wine components. The following could give a good reason to propose a specific selection of wine yeasts based on their parietal adsorption activity to improve wine safety and quality: (a) ochratoxin A content of wines is greatly reduced by expressly selected yeasts, sequestering the toxin during winemaking; (b) yeast influences concentration and composition of phenolic compounds in wine, above all by adsorbing them on cell wall; (c) among grape pigments, anthocyanins, in particular, may be adsorbed by yeast cell wall; and (d) yeast can also interact with wine colour producing anthocyanin-beta-d-glucosidase, pyruvic acid, and acetaldehyde or releasing mannoproteins and different polysaccharides. Genomic strategies could also be used to obtain a further enhancement of the adsorption/non-adsorption activity of wine yeasts. Based on winemaking requirements and on parietal adsorption activity, a specific selection of yeasts might be performed: (a) to protect wine colour during red winemaking, (b) to remove residual wine colour during white winemaking, (c) to selectively remove ochratoxin A, and (d) to protect phenolic compounds responsible for antioxidant activity.

Although the use of starter cultures in winemaking has improved the reproducibility and predictability of wine quality, the main drawback to this practice is the lack of the typical traits of wines produced by spontaneous fermentation. In this study, we identified for the first time the yeast population occurring during spontaneous fermentation of the Catalanesca white grape, a variety from Campania (Italy). Yeasts were identified using molecular tools: PCR-DGGE and partial sequence analysis of the 26S rRNA gene from isolates. Eighteen different species belonging to 11 different genera were identified. Hanseniaspora spp., Issatchenkia spp. and Candida spp. were the dominant yeasts during the early stages of fermentation, while the middle and end phases were dominated by Saccharomyces cerevisiae. Four species of Issatchenkia spp., rarely isolated from wine fermentation, were found in this study accounting for the 33.5% of the total isolates. The RAPD-PCR screening of the isolates followed by partial rRNA gene sequencing proved to be a very effective approach to first differentiate the isolates and then identify yeast species involved in a wine making procedure. The results show very high yeast diversity in this natural wine fermentation and also highlight the possibility of considering interesting autochthonous strains for starter selection.

Evolution of the microbial composition during the production of Vinsanto wine was investigated under different fermentation conditions to determine their impact on the yeast population and the wine sensorial characteristics. Fermentations were carried out according to the traditional process in 50-l barrels. Different fermentation conditions were applied (yeast inoculum, "mother sediment" addition and temperature) to standardise the Vinsanto production process. Fermentations and products were monitored over the aging period by chemical, microbial and sensory evaluation. The low temperature at the beginning of the fermentations under traditional cellar conditions results in prolonged survival of the non-Saccharomyces yeast. In contrast, Saccharomyces yeast populations dominated throughout the fermentation when the temperature of the cellar was maintained at a constant 16-18 degrees C. Results indicate that inoculation with a commercial yeast strain and fermentation temperature strongly influence the evolution of Vinsanto wine. The "mother sediment" seems to have no direct role as a microbiological starter in Vinsanto production but, despite this, it does have a strong influence on the sensory attributes of the Vinsanto wine. Our work highlights the importance of managing the fermenting microflora to improve the sensorial characteristics of Vinsanto wine.

The influence of six fungicides (famoxadone, fenhexamid, fluquinconazole, kresoxim-methyl, quinoxyfen, trifloxystrobin) on the yeast content in harvested grapes has been studied along with their effect on it during the wine-making process. Two treatments were carried out with authorized formulates at the manufacturer doses. The first was carried out under good agricultural practices, obeying the security times, and the second one under critical conditions, applied on the day of harvesting. The grapes were harvested two hours after the application. During the wine-making process, samples were taken at 1, 5, 12 and 20 days after the start. The levels observed in the control sample (1.9 log CFU/cm2) were similar to previous studies. The counts in all treated samples were higher than the control sample, without adverse effect of the pesticides on the yeast count even in the grapes treated on the day of harvest. As with the observations on grapes, no effect of the pesticides on the fermentation count was observed. All counts on the treated samples were higher than the control sample. The treatment on the day of harvest did not have any effect on the fermentation count, with superior results being obtained for all the pesticides.

We report the detection of several fungicide residues (Dichlofluanid, Benomyl, Iprodione, Procymidone and Vinclozolin) in red and white bottled wines from two Portuguese wine-producing zones. Studies were done in order to evaluate the active compound transfer percentage from grapes to the final product along fermentation process. We also investigated their effects in Saccharomyces and non-Saccharomyces yeasts as well as their influence on the physical, chemical and organoleptic wine properties. All the tested fungicides had a negative effect on in vitro yeast growth, with Dichlofluanid and Benomyl being the most toxic. Zygosaccharomyces rouxii and Saccharomyces cerevisiae were the most resistant yeasts while Rhodotorula glutinis was the most susceptible. Microvinification experiments carried out in the presence of fungicides produced wine samples with unaltered physical, chemical and organoleptic properties, though Benomyl, a slight delay in the initiation of fermentation process was observed.

To analyse the yeast population diversity during wine fermentations, specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in this environment were designed and tested with reference strains. The probes were then used to identify wine must isolates and to follow, in combination with plate counts, the evolution of yeast populations in two winery fermentations of white and red grape musts. In both cases, a high diversity of non-Saccharomyces yeast species was detected, including Candida stellata, Hanseniaspora uvarum, H. guilliermondii, Kluyveromyces marxianus, K. thermotolerans and Torulaspora delbrueckii. Some of these species (e.g., K. marxianus, K. thermotolerans and T. delbrueckii) were present in significant amounts during the tumultuous fermentation stage, despite the predominance of Saccharomyces cerevisiae cells following the inoculation of the wine musts with a starter strain. To further clarify the yeast population dynamics at the late phase of the fermentations, and because winery conditions do not allow a reliable control of experimental variables, strains isolated from the industrial musts were used to conduct two laboratory microvinifications in synthetic grape juice, using different ratios of S. cerevisiae/non-Saccharomyces in the inocula. Under these conditions, the results were similar to those obtained in the winery, showing a yeast profile with mixed species throughout the first fermentation stage, i.e. until about 40-50% of the total sugar was consumed. Non-Saccharomyces yeasts were outgrown by S. cerevisiae only after ethanol reached concentrations around 4-5% (v/v), which argues in favour of a potential important role of non-Saccharomyces in the final organoleptic characteristics of the wine.

In agricultural sciences, breeding strategies have historically been used to select new, optimized plant varieties or animal breeds. Similar strategies are possible for genetic improvement of wine yeasts. We optimized 11 relevant enological traits in a single clone using successive hybridization and segregation steps. A hybrid obtained by crossing two parent strains derived from commercial wine yeasts showed that some of the traits were readily optimized. Dominance/recessivity, heterosis and transgression were observed among 51 segregating progeny. On the basis of this information, all the optimal characters from both parents were combined in a single strain following two targeted sexual crosses. This article presents a powerful methodology for obtaining a single wine strain with numerous fermentative qualities that does not produce off-flavors.

Methods for immobilizing champagne yeasts, physiological and biochemical characteristics of the immobilized cells, and problems of their utilization in the production of quality champagne wines are reviewed. Studies aimed at the development of efficient biocatalysts for champaignizing wines using bottle fermentation (method champenoise) and tank processing (bulk, or Charmat process), based on the use of immobilized yeast cells, are described. Data on the industrial use of such biocatalysts in countries manufacturing champagne wines are presented. Problems and prospects of further research in this field are discussed.