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

Abstract

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...

Effects of Oxygen Availability on Acetic Acid Tolerance and Intracellular pH in Dekkera bruxellensis

“…We performed a new set of cultures exposing the cells to the same concentration of acetic acid used under shaking conditions (120 mM) but incubating them under static conditions, which limits the dissolved oxygen. These conditions are similar to those encountered by D. bruxellensis in wine fermentation and in natural environments where yeast can be isolated, such as soils and plant surfaces (fruits, leaves, and trunks) (38,39).…”

Effect of fermentation temperature on microbial population evolution using culture-independent and dependent techniques

“…DGGE has been reported as a powerful technique for the study of the ecology of wine (Cocolin, Bisson, & Mills, 2000;Mills et al, 2002). However, as also reported in previous studies (Andorrà et al, 2008;Renouf et al, 2007), the main drawback of this technique is its low sensitivity. Minor species were barely detected, especially when the best adapted species constituted an overwhelming majority.…”

“…These studies based on culture-dependent tools are likely to produce biased results based on unrepresentative cultivation conditions (Renouf, Strehaiano, & Lonvaud-Funel, 2007). Minor populations and stressed or weakened cells, which need specific 1.82 ± 0.08 Â 10 7 9.05 ± 1.70 Â 10 5 2.08 ± 0.41 Â 10 7 1.18 ± 0.14 Â 10 7 1.75 ± 0.08 Â 10 7 6.62 ± 0.58 Â 10 6 Hanseniaspora 2.54 ± 0.85 Â 10 6 5.51 ± 0.22 Â 10 5 1.42 ± 0.13 Â 10 6 3.00 ± 1.79 Â 10 5 2.69 ± 0.62 Â 10 5 1.77 ± 0.22 Â 10 5 Saccharomyces 1.10 ± 0.14 Â 10 4 8.49 ± 0.43 Â 10 3 1.83 ± 0.18 Â 10 6 1.63 ± 0.07 Â 10 7 1.19 ± 0.11 Â 10 7 9.82 ± 1.35 Â 10 6 C + H + S a 2.08 Â 10 7 1.46 Â 10 6 2.41 Â 10 7 2.84 Â 10 7 2.97 Â 10 7 1.66 Â 10 7…”

Evaluation of Non-Saccharomyces Yeasts for the Reduction of Alcohol Content in Wine

“…The natural microflora present on grapes and harvesting and winemaking equipment includes many non-Saccharomyces yeasts, which are present at least during the early stages of fermentation (12,13,19,20). However, non-Saccharomyces species are generally not capable of completing alcoholic fermentation; coinoculation or sequential inoculation with S. cerevisiae is needed to achieve this (21)(22)(23)(24)(25).…”