P-15
Application of ribotyping and RISA to assessment of microflora in brewery
environments. The assessment of microflora is very important for microbiological control in
brewery environments. This is especially true for Japanese brewers who typically
produce unpasteurized beer. Microbial characterization in breweries has been
routinely carried out, using conventional methods, such as morphological
examination and Gram staining. But the interpretation of the results is often
difficult and inconclusive. Therefore a more reliable and accurate method has
been desired. In this study, we evaluated two methods for potential applications
to microflora assessment in breweries. The RiboPrinter is a fully automated
ribotyping system for characterizing bacteria. This system has powerful library
and query functions, which allowed us to analyze approximately 2,000 brewery
isolates by ribotyping. In the course of our study, we acquired DNA
fingerprints, called ribopatterns, and identified the isolates with the
unencountered ribopatterns by 16S rDNA analysis. As a result, it was
demonstrated that ribotyping was able to differentiate among closely related
species found in the brewery environments. In addition, a variety of
ribopatterns were obtained from strains belonging to the same species. These
results indicate that this approach is useful for assessing the microflora on
the strain level and monitoring the sanitary status of brewery environments. The
rDNA internal spacer analysis (RISA) is based on the polymorphism of the spacer
regions between 16S rDNA and 23S rDNA, which can be easily characterized by a
PCR method. We evaluated RISA as a more rapid and inexpensive alternative to
ribotyping. The application of RISA to brewery isolates revealed that the DNA
fingerprints obtained from this method were broadly species-specific. Although
RISA was found to have a rather limited discriminatory potential compared with
ribotyping, this method has a distinct advantage in terms of costs and,
depending on the situations, provides a means of more rapid and simple
microbiological analysis. Taken together, ribotyping and RISA are well suited to
microflora analysis on the strain level and the species level, respectively.
Both methods have advantages on their own and are complementary to each other.
Therefore we have concluded that the combined use of ribotyping and RISA is a
valuable tool for the assessment of microflora in brewery environments.
Asako Takeuchi received a M.S. in applied microbiology from Mie
University, Japan. She began employment with Asahi Breweries, LTD. in April
1998. She was transferred to the research section in October 2000, and has been
working on brewing microbiology at Analytical Technology Laboratory.
ASAKO TAKEUCHI, Kazumaru Iijima, Koji Suzuki, Yutaka Miyamoto and Hiroshi
Yamashita. Asahi Breweries, Ltd., 1-1-21 Midori, Moriya-machi, Kitasoma-gun,
Ibaraki 302-0106, Japan.