Rapid detection of Brettanomyces bruxellensis in wine and beer

Brettanomyces is well-known worldwide as a spoilage organism in various food and beverage products where it produces unpleasant aromas. The detection of Brettanomyces bruxellensis, currently, the prevalent species found in wine, among the five species belonging to the genera Brettanomyces (B. bruxellensis, B. anomalus, B. custersianus, B. nanus, and B. naardenensis) with traditional methods is slow and inaccurate, thus a method not requiring colony growth would be helpful for winemakers and brewers alike to quickly obtain information about the presence of yeast in their products. Specific, sensitive and fast methods developed over the last years including various PCR-based methods have been used for this purpose. In this work a PCR protocol using DNA probes designed within the ITS1 - ITS2 DNA region has been optimized. Specificity and sensitivity were tested both by an artificial contamination of wine and by a direct analyses of naturally cotaminated samples. These wine samples have been analysed also using a classical microbiological method for the presence of B. bruxellensis, and by Gas Chromatography - mass spectrometry (GC/MS) to evaluate the 4\u2011ethylphenol, 4\u2011ethylguaiacol concentration. Despite the high sensitivity achieved for the specific probes used, and the good sensitivity obtained for the artificially tainted samples, the application on naturally spoiled wines didn't give the expected result. In fact, a satisfactory result corresponding to a sensitivity of 102 CFU/mL was obtained only for the artificially contaminated wine. Moreover, comparing the classical method results obtained by the WLD agar plate used, with the data obtained by GC/MS, the absence of a linear correlation between the B. bruxellensis CFU/ mL and the 4\u2011ethylphenol, 4\u2011ethylguaiacol content was confirmed. The critical point in the molecular method could be the DNA extraction, as a DNA without wine constituent compounds inhibiting DNA polymerase must be obtained to increase the sensitivity of the method to the desired level