Program Book - Master Brewers Association of the Americas

O-46
Rapid detection and identification of beer spoilage lactic acid
bacteria by microcolony method
SHIZUKA ASANO (1), Kazumaru Iijima (1), Koji Suzuki (1), Yasuo
Motoyama (2), Tomoo Ogata (1), Yasushi Kitagawa (1)
(1) Research Laboratories of Brewing Technology, Asahi breweries,
Ltd., Ibaraki, Japan; (2) Department of Research & Development
Strategy, Asahi Breweries, Ltd., Tokyo, Japan
In the brewing industry, microbiological quality of beer products has
been traditionally ensured by culture methods, which detect colonies
grown on selective media. Although regarded as a reliable approach
for microbiological quality control (QC) in breweries, these methods
are time-consuming and require additional confirmatory tests
before any corrective actions are taken. In order to reduce the time
for microbiological QC tests, several rapid alternatives have been
proposed, including the fluorescence in situ hybridization (FISH)
technique. Since the FISH method directly detects beer-spoilage
bacteria with a species-specific fluorescein-labeled probe targeted
to rRNA, it enables us to identify contaminants in a speciesspecific
manner without culturing. The FISH method also shows
sensitivity sufficiently high to detect a single beer-spoilage bacterial
cell. Nevertheless, due to the presence of false-positive noise
signals, it may require further confirmatory tests for the definitive
interpretation of test results to be made. To solve this problem, we
evaluated a microcolony method for the detection and identification
of beer-spoilage lactic acid bacteria (LAB). In this approach,
bacterial cells were trapped onto a polycarbonate membrane filter
and then cultured on ABD medium, a medium that allows highly
specific detection of beer-spoilage LAB strains. After a short-time
incubation, viable cells forming microcolonies were stained with
CFDA and counted with the µFinder inspection system. As a result,
all of the beer-spoilage LAB strains examined in this study were able
to be detected within three days of incubation. The specificity of this
method was found to be exceptionally high and even discriminate
intra-species differences in the beer-spoilage ability of LAB strains.
These results indicate that our microcolony approach allows rapid
and specific detection of beer-spoilage LAB strains with inexpensive
CFDA staining. For further confirmation of the species status of
detected strains, subsequent treatment with species-specific FISH
probes was also shown as effective for identifying CFDA-detected
microcolonies. In addition, no false-positive results arising from
noise signals were recognized for the CFDA-staining and FISH
methods, because of the comparatively much stronger signals
obtained from microcolonies. Taken together, the developed
microcolony method was demonstrated to be a rapid and highly
specific countermeasure against beer-spoilage LAB and compared
favorably with the conventional culture methods.
Shizuka Asano received a M.S. degree in microbiology from Tokyo
University, Japan, in 2004, where she majored in fungal genetics
under Professor Kitamoto’s guidance. She joined Asahi Breweries,
Ltd. in April 2004. Since September 2005, she has been working
on microbiological quality assurance in breweries and developing
detection technology for beer-spoilage microorganisms.
88
O-47
Agar gradient-plate technique for determining beer-spoilage
ability of Lactobacillus and Pediococcus isolates
MONIQUE HAAKENSEN (1), Alison Schubert (1), Barry Ziola (1)
(1) University of Saskatchewan, Saskatoon, SK, Canada
To date, identification of beer-spoilage bacteria has largely taken two
approaches: identification of specific species of bacteria regardless
of ability to grow in beer or the identification of spoilage-associated
genes. The dilemma with these methods is that they are either overly
inclusive (i.e., detect all bacteria of a given species regardless of
spoilage potential) or overly selective (i.e., rely upon individual,
putative spoilage-associated genes). As such, our goal was to
design a method to assess the ability of bacteria to spoil beer that
is independent of speciation or genetic background. Our solution
to this problem is an agar gradient-plate technique. A gradient
is created by pouring a base layer of MRS agar containing hopcompounds
on a slant in a square Petri plate. Once solidified, the
Petri plate is laid flat, and MRS agar is poured on top to create a layer
through which the hop-compounds must diffuse. Bacterial isolates
are stamped onto the plate using the side of a glass microscope
slide and growth of isolates along the gradient of hop-compounds
is measured to determine resistance. Through the development of
this assay, we have made the additional finding that the basis for
the ability to grow in beer differs for Lactobacillus and Pediococcus
isolates. In contrast to Pediococcus isolates, hop-resistance alone
is not optimal for identification of Lactobacillus beer-spoilage
ability (76–82% accuracy in isolates tested). Instead, the presence of
ethanol (added to a concentration of 5% to both layers of MRS agar),
in addition to hop-compounds, is necessary for accurate prediction
of the ability of Lactobacillus isolates to grow in beer (100% accuracy
in isolates tested). In several instances, addition of ethanol to the
agar gradient plates produced an enhanced resistance of beerspoilage
Lactobacillus isolates to hop-compounds, possibly due to
an induced change in membrane permeability. The opposite effect
of ethanol was also observed, but only for bacteria unable to grow in
beer. Testing of the gradient plate technique was performed on 85
Lactobacillus and 50 Pediococcus isolates and was highly accurate
in differentiating between isolates capable of growing in beer and
benign bacteria (chi-square P < 0.0005) in only 36 hours. Our agar
gradient-plate technique provides a rapid and simple solution to the
dilemma of assessing the ability of Lactobacillus and Pediococcus
isolates to grow in beer and provides new insights into the different
strategies used by these bacteria to survive under the stringent
conditions of beer.
Monique Haakensen received a B.S. (Hon.) degree in microbiology
and immunology from the University of Saskatchewan, Canada,
in 2004. In 2006, she completed the Certification Program in
Bioinformatics hosted by the Canadian Genetic Diseases Network.
Monique is currently at the University of Saskatchewan pursuing a
Ph.D. degree in health sciences, with a focus on the various aspects of
the ability of lactic acid bacteria to grow in beer.