Masaki Shimokawa (1), Kazumaru Iijima (1), Yasuo Motoyama (1), Koji Suzuki (1), Hiromi Yamagishi (1); (1) Asahi Breweries, Ltd., Moriya, Japan
BCOJ Symposium
Sunday, August 14 • 2:00–3:15 p.m.
Plaza Buiding, Concourse Level, Governor's Square 14
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.
Masaki Shimokawa received an M.E degree in environmental
engineering from Hokkaido University, Japan, in March 2008, where he
majored in environmental microbiology. He joined Asahi Breweries, Ltd.
in April 2008. Since September 2011, he has been working on
microbiological quality assurance in breweries and developing detection
technology for beer spoilage microorganisms in the Quality Control
Center.
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