Developing a Fast and Reliable Technique for Detecting Brettanomyces/Dekkera spp. and Investigating their Response to the Presence of Sulphur Dioxide


Project Number
IWBT W 10-02

Project title
Developing a fast and reliable technique for detecting Brettanomyces/Dekkera spp. and investigating their response to the presence of sulphur dioxide

Project leader
Divol, B

Institution
University of Stellenbosch. Faculty of AgriSciences. Institute for Wine Biotechnology

Team members
Du Toit, M

Project description
The Brettanomyces problem is well identified in the wine industry. The off-flavours associated with this microbiological spoilage are mainly associated to the yeast genus Dekkera/Brettanomyces. Three species occur in wine but D. bruxellensis is the most common. In red wines, the yeast Dekkera bruxellensis (teleomorph of Brettanomyces bruxellensis) is indeed able to be metabolically active and to build populations able to affect wine composition, in spite of the very low concentrations of sugars and the presence of numerous microbiological inhibitors. This species has the ability to degrade hydroxycinnamic acids. Unfortunately for the winemaker and the wine connoisseur, the products of this catabolism are volatile phenols, released in the wine and responsible for undesired off-flavours, whose best known descriptors are animal, horse sweat and urine, stables, barnyard, medicinal, clove, etc. The biological reason for these yeasts to degrade hydroxycinnamic acids is still unknown, but the most relevant hypotheses include the production of energy and the medium detoxification. Spoilage by D.bruxellensis leads to the apparition of other undesired compounds such as acetic acid, unpleasant fatty acids, mousy off flavours and biogenic amines. The metabolic pathways leading to the production of these compounds are not always well known. After many years of extensive studies worldwide, the behaviour (i.e. ability to grow and survival in wine) of D. bruxellensis in wine is still poorly understood. A partial sequencing of its genome has revealed a large plasticity and demonstrated how much it differs from that of Saccharomyces cerevisiae. The genes responsible for the catabolism of hydroxycinnamic acids are still unknown. Moreover, its survival capacity in the presence of sulphur dioxide has been partially explained by its ability to enter into and exit from a physiological state known as viable but not cultivable (VBNC), but the intracellular mechanisms lying behind this phenomenon are still far from understood. This physiological state is well known in bacteria and has been described for wine yeasts a few years ago. The main characteristics of this state are an inability to grow, a decrease in size and a decrease in vitality. This state is reversible and believed to be an intermediate state giving time to the cells to adapt to a stress that could potentially be lethal. In wine, it seems that sulphur is the main factor triggering the entry into the VBNC state. The ability of D. bruxellensis to use the VBNC state as a survival strategy has been reported by many authors but intracellular mechanisms are still unknown. In this state, the yeast is still active and high levels of volatile phenols can be found in wine, even without recovering a single cell, by using common techniques of microbiology. This latter result suggests that cells in the VBNC state could degrade hydroxycinnamic acid. Oxygen seems to play a large role in the exit from the VBNC state, growth as well as the production of acetic acid by D. bruxellensis, but once again, the molecular mechanisms lying behind these phenomena are unknown.

The early detection of D. bruxellensis in wine remains the best preventive way for the winemakers to take action against this yeast (e.g. addition of sulphur dioxide, dimethyl dicarbonate, sorbic acid, filtration, etc.). The use of classic microbiology techniques provides inaccurate cell counts, due to this species entering the VBNC state. New molecular tools, including nested PCR, PCRDGGE and quantitative real-time PCR, allow detecting and quantifying cells of Dekkera in wine. Nevertheless, these techniques have not been implemented in South Africa and the local wine industry does therefore not benefit yet from them.

This study will focus on two main points:
investigating the intracellular mechanisms of the metabolic and physiological responses of these yeasts to the presence of sulphur
dioxide implementing a fast and reliable technique for detecting Dekkera spp. in wine

Presentation(s)
Duckitt, E J, Du Toit, M and Divol, B. 2012. Investigation the response to sulphur dioxide exposure in Brettanomyces bruxellensis: A preliminary study. Paper presented at the 34th National Congress of the South African Society for Enology and Viticulture. 14-16 November, Somerset West, South Africa

Article
Divol B, Du Toit M, Duckitt E. 2012. Surviving in the presence of sulphur dioxide: strategies developed by wine yeasts, Applied Microbiology and Biotechnology, Mnth Aug v. 95 (3) (p. 601-613)
http://dx.doi.org/10.1007/s00253-012-4186-x

Willenburg, E, Divol, B. 2012. Quantitative PCR: An appropriate tool to detect viable but not culturable Brettanomyces bruxellensis in wine, International Journal of Food Microbiology, Mnth Nov v. 160 (2) (p. 131-136)
DOI: 10.1016/j.ijfoodmicro.2012.09.012

Thesis
Duckitt, E. 2012. Investigating the impact of sulphur dioxide on Brettanomyces bruxellensis at a molecular and cellular level MSc. University of Stellenbosch, Stellenbosch. South Africa.

FinalReport.pdf

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