Investigation the Molecular Mechanisms of Survival in Brettanomyces Bruxellensis


Project Number
IWBT W 13-01

Project title
Investigation the molecular mechanisms of survival in Brettanomyces bruxellensis

Project leader
Divol, B

Institution
Stellenbosch University. Department of Viticulture and Oenology

Team members
Divol, B
Louw, M
Jacobson, D
Alexandre, H

Project description
Brettanomyces bruxellensis occurs as a spoilage yeast in red wines. The typical off-flavours produced by this yeast include volatile phenols as well as acetic, isovaleric and isobutric acids. The interactions between these compounds in sensorial terms render the off-flavours that describe typical Brett character complex. Generally, spoilage by Brettanomyces bruxellensis suppresses berry like character, fruitiness and enhances the atypical sick sweet, confected character. Elastoplast, medicinal, savoury and pungent are the off-flavour terms used by experienced tasters .

South African wines are not free of Brettanomyces spoilage as seen in 2009 where 9% of the taints detected in the wines tasted at the Stellenbosch Wine Festival were associated with the presence of Brettanomyces. It is extremely difficult to obtain statistics on the amount of wines still contaminated by Brettanomyces today. Improved cellar hygiene and management of preservatives (sulphur dioxide in particular) definitely help to limit spoilage. As long as Brettanomyces response to sulphur dioxide exposure will not be understood, one might always be surprised by its development at an unexpected time. According to VinLab, it would indeed be denial to say that there are no more spoiled wines in South Africa as they still find volatile phenols during chemical analyses of South African wine samples on a regular basis. Indeed, over a 2 month period, 15 May 2012 – 15 July 2012, 13% of the wines analysed by this laboratory showed concentrations of 4-ethyl phenols above the perception threshold (i.e. >0.6 mg/L).

In this context, a very accurate detection and enumeration technique for Brettanomyces cells was optimised in a previous project (IWBT W 10-01). This technique (i.e. quantitative PCR) by using mRNA as a template, showed that it was powerful enough to distinguish between fully active (i.e. viable and culturable), dead and viable but nonculturable cell. It could be used by winemakers to detect Brettanomyces cells in their wines before the appearance of olfactory faults.

The impact of sulphur dioxide exposure on Brettanomyces bruxellensis was investigated in project IWBT W 10-01. This study showed that Brettanomyces bruxellensis seems to respond to sulphur dioxide exposure using similar mechanism as Saccharomyces cerevisiae and that sulphur dioxide resistance was highly strain dependant. It was found difficult to link sulphur dioxide tolerance and specific mechanism such as active efflux. Acetaldehyde was found to be a marker of stress rather than an active mechanism of resistance of sulphur dioxide. This needs to be confirmed using more strains. The viable but nonculturable state seems to be a critical strategy for Brettanomyces bruxellensis to survive sulphur dioxide exposure, but this needs to be further investigated, in terms of amount of sulphur dioxide needed to induce entry into the VBNC state, the identification of factors inducing exit from this state and the ability of B bruxellensis to spoil wine in a VBNC state. The latter aspect was reported a few times in the literature and shows that a more in-depth
knowledge of the VBNC state is critical for winemakers to optimally control the development of Brettanomyces bruxellensis.

As sulphur dioxide exposure and entry into the VBNC state are closely linked and this physiological state is poorly understood in eukaryotes, we propose to investigate this state at a molecular level in order to better comprehend the survival mechanisms of Brettanomyces bruxellensis and to help predict the behaviour of this yeast species during wine ageing.

Poster(s)
Fracassetti, D, Louw, M, Vigentini, I, Du Toit, M and Divol, B. 2014. Impact of different nitrogen sources on Brettanomyces bruxellensis growth in the presence of sulphur dioxide. Poster presented at 5th International Symposium on Macromolecules and Secondary Metabolites of Grapevine and Wines: MacroWine 2014. 7-10 September 2014, Stellenbosch, South Africa.

Presentation(s)
Louw, M, Alexandre, H and Divol, B. 2013. Management of spoilage yeasts – the case of Brettanomyces. Paper presented at the 35th South African Society for Enology and Viticulture Conference. 13-15 November, Somerset West, South Africa.

Louw, M, Du Toit, , Alexandre, H and Divol, B. 2014. Effect of sulphur dioxide exposure on the cell morphology and growth kinetics of Brettanomyces bruxellensis. Paper presented at the 36th South African Society for Enology and Viticulture Conference. 12-14 November 2014, Somerset West, South Africa.

Louw, M, Du Toit, M, Alexandre, H and Divol, B. 2014. Impact of sulphur dioxide on the cell morphology and viability of the yeast Brettanomyces bruxellensis. Microscopy Society of Southern Africa Conference 2014. 2-5 December 2014, Stellenbosch, South Africa.

FinalReport.pdf

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