P-21
Combined applications of three typing methods for assessment of microflora in the brewery environments
Presenter: Asako Takeuchi, Asahi Breweries, LTD., Moriya, Ibaraki, JAPAN
Co-Authors: Koji Suzuki, Kazutaka Ozaki, and Hiroshi Yamashita, Asahi Breweries, LTD., Moriya, Ibaraki, JAPAN

The assessment of microflora is very important for microbiological control in brewery environments. This is especially true for the Japanese brewers who typically produce unpasteurized beer. Recently, we have developed a quality control (QC) system for assessment of microflora in brewery environments using RiboPrinter and the rDNA internal spacer analysis (RISA). The RiboPrinter is a fully automated ribotyping system for characterizing detected bacteria on the basis of the accumulated database consisting of a large number of DNA fingerprints. 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. As compared with RISA, Riboprinter provides more sophisticated information concerning precise species identification and detailed strain-level subtyping, but it is prohibitively expensive for each brewery to operate this ribotyping system independently. In contrast, RISA has clear advantages in terms of costs and time required for analysis, an ideal feature for brewery applications, but the typing potential is broadly limited to species level. At the annual meeting of ASBC in 2002, we proposed the use of RISA in breweries as a prescreening method for Riboprinter that is operated at the QC center. While this combined QC system is useful for keeping costs relatively inexpensive, it sometimes takes a week for the final results to be obtained, delaying the critical decisions at the brewery. This is especially the case when the brewery has to identify the exact contamination source of detected bacteria in a timely fashion. Therefore, a more-detailed method than RISA has been desired, which enables breweries to act promptly without being overly dependent on RiboPrinter. In this study, we evaluated the enterobacterial repetitive intergenic consensus (ERIC)-PCR, originally based on diversity of enterobacterial repetitive sequence, as an alternative method for brewery applications. As a result, almost all the species isolated from breweries were found to show distinctive DNA fingerprints. We further confirmed that the isolates from our breweries exhibiting identical RISA fingerprints were distinguishable by using ERIC-PCR, demonstrating that this method is more suitable for detailed analysis in the brewery than RISA. The disadvantages compared with RISA are that ERIC-PCR takes more than five hours and requires complicated protocol. Our preliminary applications to our QC system indicate that ribotyping, RISA, and ERIC-PCR have their own advantages and disadvantages for assessment of brewery environments. Therefore, these three methods should be selected depending on the situations. We have thus concluded that the combined use of three typing methods is a valuable tool for the assessment of microflora in brewery environments.

Asako Takeuchi received an 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.