Molecular methods for detection and identification of contaminants in brewery quality control

Contamination of brewing process by spoilage organisms may lead to production problems and quality defects in final product. Present quality control methods based on cultivation reveal the possible contamination only after several days or weeks of delay. Moreover, they do not discriminate between spoilage and non-spoilage microbes nor allow the detection of fastidious or injured organisms. 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 developed for contaminants present in brewing process, i.e. for lactic acid bacteria (Lactobacillus, Pediococcus), strictly anaerobic bacteria (Pectinatus, Megasphaera, Selenomonas, Zymophilus), enterobacteria, acetic acid bacteria and wild yeasts. Various PCR detection formats (PCR-ELISA, LightCyclerTM PCR, standard PCR) have been set-up for most of the beer spoilage organisms. Pre-PCR treatment methods have been developed for filterable samples, such as bright beer and process water, and for unfilterable samples, such as pitching yeast, fermenting wort and unfilterable beer. Due to the very low amounts of microbes in brewing samples, they need to be enriched in a suitable medium prior to the PCR. Practical DNA extraction methods and removal of PCR inhibitors have been developed for different sample types. For example, a commercial system containing magnetic particles for DNA collection and purification can be used for this purpose. Modular PCR kits for standard PCR and real-time PCR are available from several companies. The developed PCR applications are, despite of the enrichment step, more rapid and specific than conventional methods. Without prior enrichment, sensitivity of PCR methods is lower and they are at the moment expensive to use compared to cultivation methods. An alternative to PCR, a commercial fluorescent in situ hydridization (FISH) method has been developed for beer-spoilage bacteria. It is based on the use of fluorescently labelled gene probes and epifluorescence microscope. The automated ribotyping system (RiboPrinter®System DuPont Qualicon,USA) is rapid molecular biological method for identification and characterization of bacteria to species or even below species level. The comprehensive identification database using three different restriction enzymes has been created to the brewery contaminants including Lactobacillus spp., Pediococcus spp, Pectinatus spp., Megasphaera cerevisiae and Obesumbacterium proteus. In addition to identification purposes and renaming of bacteria, the database has been applied in tracing contamination sources and in detection of troublesome "house flora" in industrial processes.