Evidence for viable but nonculturable yeasts in botrytis-affected wine

Divol, Benoit; Lonvaud‐Funel, Aline

doi:10.1111/j.1365-2672.2005.02578.x

Cited by 122 publications

(67 citation statements)

References 35 publications

(63 reference statements)

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“…The detection of R. mucilaginosa, which was never found since berries studies, confirmed previous works (Ubeda et al, 1999;Nisiotou and Gibson, 2005) reporting its ability to acquire progressively strong resistance. That may implicated the passage by a viable but nonculturable state at very low population during the winemaking process (Divol and Lonvaud-Funel, 2005). Zygosaccha-romyces bailii was also detected in oldest wines.…”

Section: Pcr-dgge Analysis Of Old Bottled Winementioning

confidence: 96%

Yeast and bacteria analysis of grape, wine and cellar equipments by PCR-DGGE

Renouf¹,

Strehaiano

Lonvaud-Funel

2007

OENO One

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Aims:Microorganisms play an important role in winemaking. Most of their metabolisms are beneficial to the wine quality but several species have spoilage effects. Microbiological knowledge are crucial for improving species and strains identification and avoiding contamination by spoilage agents. Our objective was to follow wine microbial consortia at each stage of the process from grapes to bottles, and also on cellar equipment surface: barrels for the yeast analysis and tanks for the bacteria analysis. Methods and results:For a global microbial monitoring, molecular tools are particularly efficient. We chose the PCR-DGGE for direct analyses without cultivation step. We used the rpoB gene as target for the bacteria, and the 26S rRNA gene for the yeast. Species inventories were made during the whole winemaking process, which had never been done before. Conclusion:From the grape surface to storage in bottles, the microbial diversity decreased. Only the most resistant species were able to survive to the ethanol, SO2, low oxygen and low pH constraints. Among them, Pediococcus parvulus for the bacteria and Brettanomyces bruxellensis for the yeast were the most resistant. Significance and impact of study:The use of a molecular method independent of the culture stage of the microorganisms like the PCR-DGGE has allowed to have a more exhaustive vision of the diversity of the bacteria and yeasts species and to follow their evolution during the winemaking and in the cellar environment . These follow-ups could be extended to other estates in order to study the causes and the consequences of certain variations. The detection of these two prejudicial species was an argument for the PCR-DGGE approach in wine microbial studies.Keys words: winemaking, yeast, bacteria, species, diversity, PCR-DGGE Objectif : Les microorganismes jouent un rôle déterminant dans l'élaboration des vins. La plupart de leurs métabolismes sont bénéfiques pour la qualité du vin (fermentations alcoolique et malolactique), mais certaines espèces ont une activité préjudiciable, comme la production des phénols volatils par la levure Brettanomyces bruxellensis. Une meilleure connaissance des populations microbiennes est cruciale pour améliorer l'identification des espèces et éviter la multiplication des espèces d'altération. Notre objectif était de suivre le consortium microbien du vin à chaque étape du procédé de vinification depuis la baie de raisin jusqu'à la bouteille, mais aussi au chai, à la surface des cuves pour l'analyse des bactéries, et, des barriques pour celle des levures.Méthodes and résultats : Pour avoir une vision globale des suivis microbiologiques, les méthodes de biologie moléculaire sont particulièrement efficaces. Nous avons utilisé les analyses directes par PCR-DGGE sans étape de culture, en ciblant le gène rpoB pour l'étude des bactéries et le gène de l'ARN 26S pour l'identification levures. Des inventaires des espèces sont proposés à chaque étape du procédé de vinification, ce qui n'avait jamais été réalisé auparavant par des méthodes...

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Section: Pcr-dgge Analysis Of Old Bottled Winementioning

confidence: 96%

Yeast and bacteria analysis of grape, wine and cellar equipments by PCR-DGGE

Renouf¹,

Strehaiano

Lonvaud-Funel

2007

OENO One

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“…The effect was more drastic with green cider fermentation, due to a synergistic effect of SO 2 and other inhibitors, such as ethanol. Actually, SO 2 has been reported to lead wine yeast and bacteria to adopt VBNC states rather than to undergo cell death (6,7,15).Determination of malolactic activities of viable and VBNC subpopulations. In order to quantify the malolactic activity of VBNC cells, experimental data for cell subpopulations and malate uptake were fitted (MicroMath Scientist version 2.0) to a segregated kinetic model developed previously (20).…”

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confidence: 99%

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confidence: 99%

Quantitative Approach to Determining the Contribution of Viable-but-Nonculturable Subpopulations to Malolactic Fermentation Processes

Quirós

Herrero

Garcı́a

et al. 2009

Appl Environ Microbiol

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Different sizes of viable-but-nonculturable cell subpopulations of a lactic acid bacterium strain were induced by adding increasing amounts of SO 2 . The experimental data obtained here were fitted to a segregated kinetic model developed previously. This procedure allowed us to determine in quantitative terms the contribution of this physiological state to malolactic fermentation.The persistence of stressed, damaged, or viable-but-nonculturable (VBNC) cells during microbial fermentation underlines the requirement of alternative methods for detecting and characterizing these emergent states not otherwise detectable by traditional culture-based methods (13). Flow cytometry (FC) has evolved as an outstanding tool in bioprocesses due to its usefulness in cell physiology monitoring (5, 12). The persistence of nonculturable cells during microbial fermentation has been attributed to changes in water activity, acidity, redox potential, nutrient availability, and starvation (14,17,18,24,25) or to the use of preserved starter cultures (20). Additionally, the quantification of catalytic activity is critical to bioprocess optimization, as it measures the individual contributions of different cell subpopulations to the global process (2, 13). Despite the loss of culturability under standard conditions, it is strongly suspected that VBNC cells remain alive, maintain the transport system and biosynthesis, and are able to metabolize substrates (16,26). Gene expression has also been demonstrated previously (9, 23). However, although the physiology, biochemistry, and genetics of the VBNC state have been studied over the years, its functionality and biological implication are still issues under intense debate (1,21,22).In this work, cider malolactic fermentation (MLF) was selected as a model system to clarify the role played by VBNC cells in bioprocesses. MLF was carried out under different SO 2 concentrations (0, 30, and 60 ppm total) for inducing VBNC states. The fermentation medium was sterile apple must or "green" cider (obtained just after alcoholic fermentation and containing 5.6% [vol/vol] ethanol), obtained as previously described (11). Sodium bisulfite was used for SO 2 treatments. MLF was carried out in duplicate at 22°C statically in 250-ml bottles. An indigenous strain of Lactobacillus hilgardii was inoculated at an optical density at 600 nm of ϳ0.1 to start MLF. Flasks were shaken just before sampling in order to homogenize the biomass content. Samples were taken aseptically at time intervals until malic acid was consumed (Յ0.5 g liter Ϫ1 ), and cells were collected and processed for further analysis as described previously (20). Supernatants were filtered (0.45 m pore size) and frozen (Ϫ20°C) until chemical analysis. The amount of malic acid was determined by highpressure liquid chromatography (Alliance 2690; Waters) with a photodiode array detector (Waters 996), as reported previously (19).Evolution of bacterial subpopulations during MLF. Viable cells (measured as CFU ml Ϫ1 ) were monitored by a plate counting metho...

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“…The following FastDigest® restriction enzymes were used for the ITS amplification product of yeasts: HhaI (interface 5′-GC^GC-3′), HaeIII (interface 5′-GG^CC-3′) and HinfI (interface 5′-G^ANTC-3′; N stands for a random base) (Divol & Lonvaud-Funel, 2005). From the PCR product, 8.7 µL were digested with 0.3 µL restriction endonuclease in a final volume of 10 µL and incubated at 37 °C for 20 min and then at 65 °C for 20 min.…”

Section: Restriction Enzyme Digestionmentioning

confidence: 99%

An Exemplary Model Study for Overcoming Stuck Fermentation during Spontaneous Fermentation with the Aid of a Saccharomyces Triple Hybrid

Christ

Kowalczyk²,

Zuchowska³

et al. 2015

JAS

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Sluggish or stuck fermentations cause significant financial losses for winemakers each year. In order to investigate the reasons for problems during spontaneous fermentation of Riesling must in a well-known German vineyard of the lower Moselle, yeast strains involved in must fermentation were identified during winemaking in the two years 2011 and 2012. Identification of the yeast isolates was performed by applying analyses of the ITS-1-5.8-ITS2 region and restriction fragment analyses of different gene sequences. It revealed that Saccharomyces (S.) bayanus and not Saccharomyces cerevisiae was the main fermenting yeast. Either S. bayanus finished the fermentation or led to stuck fermentation. After about four weeks of stuck, fermentation continued spontaneously S. bayanus was replaced by the triple hybrid S. cerevisiae × S. kudriavzevii × S. bayanus. The triple hybrid strain HL 78 was able to utilize fructose more efficiently than S. bayanus strain HL 77. The fructophilic character of the triple hybrid strain correlated with an enhanced uptake of radiolabeled fructose compared to glucose.In contrast to the usual starter culture S. cerevisiae, both isolates, S. bayanus strain HL 77 and the triple hybrid strain HL 78, could grow in the absence of ammonium when amino acids were present. However, the triple hybrid was able to consume glucose and especially fructose at lower amino acid concentrations. Thus, the triple hybrid strain HL 78 was a suitable strain to overcome stuck fermentation without changing the fermentation conditions and the aroma profile desired by the selected winery. It has already been successfully used to restart stuck fermentation. The procedure described here could be a useful approach for wine makers facing problems during spontaneous fermentation. Since the application of genetically modified yeast strains is not allowed for starter culture, based on these studies we suggest the generation of hybrid strains with desired phenotypical features from mother yeasts strains/species of a certain winery and their application in case that during a spontaneous fermentations a sluggish or stuck fermentation is observed.

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Evidence for viable but nonculturable yeasts in botrytis-affected wine

Cited by 122 publications

References 35 publications

Yeast and bacteria analysis of grape, wine and cellar equipments by PCR-DGGE

Yeast and bacteria analysis of grape, wine and cellar equipments by PCR-DGGE

Quantitative Approach to Determining the Contribution of Viable-but-Nonculturable Subpopulations to Malolactic Fermentation Processes

An Exemplary Model Study for Overcoming Stuck Fermentation during Spontaneous Fermentation with the Aid of a Saccharomyces Triple Hybrid

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