Molecular Identification of Brettanomyces yeasts
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Egli, Christoph; Mitrakul, Craig; Henick-Kling, Thomas
Brettanomyces/Dekkera yeasts grow in wine mainly during barrel aging. Their presence is often associated with formation of off-flavors. This potential spoilage generates a high demand for a fast, sensitive and reliable identification procedure in the food industry. Presently, these exigences are only fulfilled by the use of genetic techniques. In addition to yeasts from type culture collections Brettanomyces yeast strains isolated from Cabernet Sauvignon wines characterized by winemakers panels as having 'bretty' aromas were extensively analyzed in this regard. Brettanomyces/Dekkera reference strains from a type culture collection were included for comparison. Whereas karyotyping and chromosomal RFLP resulted in clear distinctions it did not allow for relatedness studies due to the lack of pattern conservation among species and strains. Conversely, i) ribosomal DNA restriction fragments length polymorphism, ii) comparative sequence analysis of the two internal transcribed spacer (ITS) regions located between the ribosomal RNA genes and iii) RAPD-PCR allowed for species discrimination within the genus Brettanomyces and strain discrimination within the species B. bruxellensis. All wine-isolated Brettanomyces/Dekkera yeasts belonged to the species B. bruxellensis. Cabernet Sauvignon wines were chosen from the seven vintages 1989 and from 1991 to 1996. Merlot, Chardonnay, and Sauvignon blanc were taken from vintage 1995. All wines originated from the same winery. Whereas populations of one single or two strains were found, one strain always dominated in the examined wines. Populations of two clone A and B were found in 1989 in the ratio of 90% to 10%. Clone B was not found in 1991 but clone A completely dominated. Populations derived from clone C were found in 1992 (100%), 1993 (90%), 1994 (100%), 1995 (90%), and 1996 (70%). Clone D started to evolve in 1995 (10%) and was present in the following year 1996 to 30%. Clone B which was present in 1989 (10%) was not found any more in the following vintages. Brettanomyces yeasts were found in Merlot, but no yeasts were found in the two white wines. This established procedure for the first time makes the determination of Brettanomyces yeast possible thus allowing to learn about their successions in wine.