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P. ROMANO, G. SUZZI, G. COMI AND R. ZIRONI. 1992. Ninety‐six strains of apiculate wine yeasts were investigated for their ability to produce higher alcohols and acetic acid in synthetic medium. Less isoamyl alcohol and more n‐propanol and isobutanol were formed by Hanseniaspora guilliermondii than by Kloeckera apiculata. The latter produced twice as much acetic acid as H. guilliermondii. The production of higher alcohols and acetic acid was found to be a characteristic of individual strains and was statistically significant. In a multivariate analysis of higher alcohol production two main groupings were formed at 86%S, corresponding to the taxa H. guilliermondii and K. apiculata. Strains that produced low amounts (50 mg/1) of acetic acid, comparable with that of Saccharomyces cerevisiae, were found in both species of apiculate yeasts.

Secondary products in wines obtained bv pure. mixed and sequential cultures of Saccharomyces cerevisiae, Plansentaspora guillierrnondii or Kloeckera apiculata were studied. Consistent differences in the composition were determined in wines fermented by sequential cultures. When S.cerevisiae was added to musts partially fermented by apiculate yeasts, its metabolism was significantly affected. In particular it synthesized high amounts of n-propanol and metabolized high quantities of acetoin, produced by apiculate yeasts

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In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B. anomalus. Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from other Brettanomyces spp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B. bruxellensis and B. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection of B. bruxellensis and B. anomalus in wines suspected to be spoiled by Brettanomyces spp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.A wide variety of yeast species have been implicated in wine spoilage. Of the spoilage yeasts, species of Brettanomyces (imperfect state, Dekkera) are probably the most serious (21) and controversial. These organisms are most frequently found in red wine 6 to 10 months after barrelling (4), but they have also been isolated from other fermented beverages, such as beer and cider. Brettanomyces species can survive, multiply, and contaminate wines from transfer piping or cooperage that has been insufficiently cleaned and disinfected after use. These potential spoilage yeasts have been identified in almost every wine-producing area of the world (12). Wines infected with Brettanomyces/Dekkera yeasts develop off-flavors, which are described as animal, stable, barnyard, horse blanket, and burnt plastic (5,12,13,16,18). Brettanomyces can produce a distinct haziness when the concentration of cells present in the wine is Ͻ3 ϫ 10 3 cells/ml (4) or higher (13). Brettanomyces species can synthesize volatile phenolic compounds, including phenol, syringol (16), and several ethylphenols (6). Control of these organisms is difficult due to their relative resistance to normally used concentrations of sulfur dioxide (15). The physiology and ecology of this spoilage genus are still unclear, and little is known about it (14).Current methods for identification and enumeration of Brettanomyces contamination take 1 to 2 weeks and rely on growth on semiselective culture media or selective culture media, followed by final identification by biochemical and physiological analysis and morphology as determined by microscopic examination (19). Newer techniques for rapid detection and identification of Brettanomyces, such as a nested PCR...