Molecular methods for detection and identification of microbial contaminants in brewing quality control

Current cultivation-based methods used by most breweries for microbiological quality control reveal possible contamination only after several days or weeks of delay. Moreover, they do not discriminate between spoilage and nonspoilage microbes or allow the detection of viable but noncultivable cells or tracing of contamination sources. In recent years, several promising molecular biological applications have been described for the detection, characterization, and identification of brewery contaminants. Group-, genus-, and species-specific PCR tests have been designed and evaluated for brewery contaminants, i.e., for lactic and acetic acid bacteria (Lactobacillus, Pediococcus), strictly anaerobic bacteria (Pectinatus, Megasphaera, Selenomonas, Zymophilus), enterobacteria, and wild yeasts. Various PCR detection formats (PCR-ELISA, LightCycler(™) PCR, standard PCR) have been set up for most of these organisms. In order to detect the very low amounts of microbes in brewing samples, an enrichment method has been devised. Practical pre-PCR treatment methods, including collection of cells, DNA extraction, and removal of PCR inhibitors, have been developed for filterable (such as bright beer and process water) and nonfilterable (pitching yeast, wort, fermenting wort) samples. Modular PCR kits for standard PCR and real-time PCR have also become available from several companies. All developed PCR applications are, despite the enrichment step, more rapid and specific than the cultivation methods. Currently, PCR without prior cultivation is less sensitive and more expensive to use than cultivation. Denaturing gradient gel electrophoresis (DGGE) for separation of bacterial or eukaryotic ribosomal DNA amplicons is a valuable tool for characterizing microbial communities in specific environmental niches. This technique has also been applied to study and compare microbial communities during beer, wine, and whisky production. The automated ribotyping system (RiboPrinter® System DuPont Qualicon, U.S.A.) is a rapid molecular biological method for the identification and characterization of bacteria to species or even below species level. A comprehensive identification database has been created for brewery contaminants, such as Lactobacillus spp., Pediococcus spp., Pectinatus spp., M. cerevisiae, and Obesumbacterium proteus, using three different restriction enzymes. In addition to identification and renaming of bacteria, the database has been applied to tracing of contamination sources and detection of troublesome house flora in industrial processes.