Development of a Novel Culture-Independent Method for Comprehensive Detection and Identification of Beer-Spoilage Microorganisms
Culture method is often used to detect beer-spoilage microorganisms in microbiological quality control in breweries. It is a time-consuming process, requiring 3-14 days for cultivation. Therefore, culture-independent methods have been developed for direct detection of beer-spoilage microorganisms in beer. Conventionally, polymerase chain reaction (PCR) is combined with the DNA extraction method from the cells trapped on polycarbonate membranes. Since the cells are trapped on the surface of polycarbonate membranes, they can be released easily from the membranes for subsequent DNA extraction. However, filtration volume by polycarbonate membranes is rather limited because of its poor filterability, making it difficult to detect low concentrations of contaminated microorganisms in beer. In our new method, cellulose membranes were selected for recovery of the cells because of its high filterability, although microorganisms are trapped within the membrane matrix. To overcome the difficulty, we developed a novel direct DNA extraction and recovery method from trapped cells within cellulose membranes using pressure cycling technology. Pressure cycling technology using Barocycler allows rapid cycles between extremely high (up to 235 MPa) and ordinary pressure conditions in the reaction vessel. The rapid cycles of high- and low-pressure conditions enhance the penetration of DNA-extracting solution into membrane matrix and disrupt cells effectively. However, this approach alone enables the detection of 101-103 cells/membrane for beer-spoilage microorganisms. To improve the sensitivity, carrier DNA was added to the DNA-extracting solution as an adsorption competitor to prevent extracted DNA from adsorbing to the membrane matrix. In addition, an ethanol precipitation process was added to concentrate DNA and even higher pressure (300 MPa) was adopted to enhance DNA recovery. In our modified protocol, the detection limits of major beer-spoilage lactic acid bacteria, including Lactobacillus brevis and Pediococcus damnosus, were found to be as low as 100 cells/membrane (up to 3,000 mL of beer). It was also shown that this protocol is applicable to wild yeast (Saccharomyces and Dekkera/Brettanomyces) species and the identical detection limits were accomplished. In contrast, the optimization of pore sizes for the cellulose membrane is needed to achieve the detection limits of 100 cells/membrane for some of the Pectinatus strains. This modified approach was found to be applicable to all of the beer-spoilage Pectinatus species, as well as beer-spoilage Megasphaera species. This series of measures in combination finally allows the detection of over 20 species of beer-spoilage microorganisms with the detection limit of 100 cells/membrane and enables the comprehensive culture-independent identification of beer-spoilage species within 8 hr. Taken collectively, our new method is considered as an extraordinarily rapid and highly sensitive culture-independent detection and identification method and represents a significant step forward in the brewing industry.