Investigation of beer-spoilage ability of Dekkera/Brettanomyces yeasts and development of multiplex PCR method for beer-spoilage yeasts

Microbiology Session
Satoshi Shimotsu, Asahi Breweries, Ltd., Research & Development Laboratories for Brewing, Ibaraki, Japan
Co-author(s): Shizuka Asano, Kazumaru Iijima, Koji Suzuki, Hiromi Yamagishi, and Masayuki Aizawa, Asahi Breweries, Ltd., Research & Development Laboratories for Brewing, Ibaraki, Japan

ABSTRACT: In the brewing industry, microbiological control of wild yeasts is very important to produce high quality beers. Among the wild yeasts, Saccharomyces cerevisiae, including S. cerevisiae var. diastaticus, Dekkera anomala, and D. bruxellensis, have been reported to cause turbidity and off-flavors in beer. Brettanomyces custersianus and B. nanus, which are closely related to the established beer-spoilage Dekkera species, have been isolated from beers, but their beer-spoilage ability remains poorly characterized. In this study, we therefore investigated the beer-spoilage ability of Brettanomyces yeasts and developed a rapid and simple method to identify beer-spoilage yeasts. To evaluate beer-spoilage ability, the strains of B. custersianus, B. nanus, and B. naardenensis were inoculated in pilsner-type beers. The inoculated beers were incubated at room temperature and examined regularly for visible growth for up to three months. Sediment was observed in all beers inoculated with B. custersianus strains. On the other hand, it was found that B. nanus and B. naardenensis strains were unable to grow in beer. These results indicate that B. custersianus should be treated as a beer-spoilage yeast in addition to the established beer-spoilage species S. cerevisiae, D. anomala, and D. bruxellensis. Subsequently, we developed a multiplex PCR method for detecting and identifying beer-spoilage yeasts, S. cerevisiae, and three beer-spoilage Dekkera/Brettanomyces species by designing PCR primers and optimizing PCR conditions. PCR primers were designed in the 26S rDNA region to amplify the different sizes of PCR product from each target yeast to make it possible to identify individual target species simultaneously. Specificity, reactivity, and sensitivity of the designed primer pairs were evaluated by conducting multiplex PCR. It was found that amplicons were obtained from only target species, while no false positive reactions were detected for other non-target species tested. Reactivity was investigated using several strains of S. cerevisiae, D. bruxellensis, D. anomala, and B. custersianus, and it was verified that all of the tested strains could be detected. Sensitivity was examined by comparing the detection limit of spoilage wild yeasts. Because the detection limits of the multiplex primer mix were identical to those of the universal primer pairs with 103 cells/tube detection limits, the sensitivity of this method was sufficient for detection. These results indicate that the developed multiplex PCR method has high specificity and reactivity. Taken together, the developed multiplex PCR method is considered an effective tool to detect beer-spoilage yeast, contributing to microbiological quality assurance in breweries.

In 2008, Satoshi Shimotsu received a master’s degree in food hygienics from Kyushu University, Japan, where he majored in biofilm of food-poisoning bacteria. He joined Asahi Breweries, Ltd. in April 2008. He is now engaged in microbiological quality assurance in breweries and alcoholic beverages.