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Development of diagnosis tools for Brettanomyce monitoring (Brett Monitoring)

Final Report Summary - BRETT MONITORING (Development of Diagnosis Tools for Brettanomyce Monitoring)

The yeast genus Dekkera (anamorph: Brettanomyces) plays a significant role in flavour production in beer and wine. Low amount may be necessary for typical 'flavours', but oversynthesis of volatile phenols (4-ethyl phenol (4-EP) and 4-ethyl guaicol (4EG)) or tetrahydropyridines, results in production with 'animal' or 'mousy' type odour. To avoid the development of Dekkera, winegrowers can only use preventive measures. Rigorous hygiene in wine cellar contributes to avoid the development of the micro-organisms.

Problems due to Brettanomyces appear mostly during storage and aging of wine. The problem is especially relevant in large wood barrels because they are difficult to clean and some species can use cellobiose as carbon source. Furthermore, the present trends in quality wine making, involving the production of very concentrated wines with high alcoholic degree, combined with the use of micro-oxygenation techniques, favour the development of Brettanomyces sp.

Because of the ubiquitous presence and high spoilage potential of Dekkera (Brettanomyces), there is a demand for a simple, fast and reproducible monitoring method. The earlier the detection in the wine, the better the chances for winegrowers to prevent further growth and spread to other wine batches. It is difficult to obtain a fast diagnosis of the presence and amount of Dekkera yeats because methodologies of analysis are complex and poorly diffused among wine-makers. A few specialised laboratories are able to carry out analyses. The most widespread methods of analysis use customary media and techniques, where it can take a week or longer before growing colonies become visible, a far longer incubation time compared to other yeast, which may overgrow Dekkera colonies and prohibit proper detection. Strictly selective media are not available commercially. Other detection methods based on molecular techniques have been developed in the recent years (e.g. RAPD-PCR, in situ hybridisation with fluorescence detection, fatty acid profiling). These techniques are powerful but can only be implemented by specialised laboratories. The project aims at improving methodologies for Brettanomyces monitoring.

The consortium, has conceived, for the wine and drinks fields, an innovating system which can detect a Brettanomyces contamination. Thanks to its innovation, the consortium is marketing three revolutionary detection tests: immunological, genomic and micro biological.

NKS Brettanomyces and agrar culture media
The NKS culture media or agar Brettanomyces is based on a decolouration of its colour into yellow, due to the environment acidification. The kit includes the specific culture media put in a sterile Petri dishes, with the adequate filtrating membranes. Culture media on NKS dehydrated cardboard discs meet all the needs of controls imposed by health regulations.

Real time PCR -the polymerase chain reaction is a method of biology molecular
The PCR remains an in vitro targeted application technique. It makes it possible to obtain, from a complex and small sample, large quantities of specific and defined-length DNA from a DNA fragment. This kit presents a lot of advantages, such as, sensitivity, simplicity of implementation and easy preparation, results obtained in less than 4 hours, and the possibility of automation.

ELISA method
The ELISA method relies on the performance of an immunological test used for detection of Brettanomyces in wine. This immunological test is intended to detect and/or dose a protein in an organic liquid. This kit presents a lot of advantages: time saving, selectivity, ease of use and preparation, rapidity, reliability, possibility of automation.

These three kits make it possible for the producers to carry out analyses of Brettanomyces detection by themselves. In addition to the speed of analysis, guarantee safety, profitability, flexibility and savings. Today, the results of these kits are very promising but in the current state of the results, none of the three methods is usable and commercially viable. There still remains a lot of research and development (R&D) to finalise the product.

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