P-18
Grouping of Lactobacillus brevis strains using the gyrB
gene. In breweries, Lactobacillus brevis is known as a representative
beer-spoilage microorganism. For quality control in the brewing plant, it is
necessary to develop methods for the accurate identification of beer-spoilage
microorganisms and the prediction of their beer-spoilage ability. Therefore, we
tried to develop a new method for grouping the L. brevis strains based on
their beer-spoilage ability. A total of 16 representative strains with different
isohumulone resistant abilities were used, and the gyrB gene fragments
(ca. 540 bp) of these strains were PCR amplified and sequenced. As a result, the
strains of L. brevis were divided into 4 groups, groups I, IIa, IIb and
III, on the basis of the gyrB fragment sequences. A relationship was
observed between the isohumulone resistant ability and the grouping based on the
sequences of the gyrB fragments. All the isohumulone (40 ppm) resistant
strains were classified into group IIb. Moreover, all of them, except only one
strain, belonging to IIb, could grow in beer (pH 4.5, 30 ppm isohumulone). From
these results, it was suggested that the beer-spoilage strains of L.
brevis form one group in the phylogenetic tree based on the gyrB
gene. For the easy grouping of the L. brevis strains, the PCR amplified
gyrB fragment was digested by three restriction enzymes, Hin1I, ApaLI and
HaeIII, and the digested fragments were then electrophoresed in polyacryamide
gels. Although the Hin1I and ApaLI digestion gave the corresponding fragments
for groups I, IIa/IIb and III on the basis of the gyrB fragment
sequences, groups IIa and IIb were not distinguished by these two enzymes. On
the other hand, the gyrB fragment of group IIa was not digested by
HaeIII. Consequently, the digestion analysis using these three enzymes could
give the same grouping like their gyrB fragment sequences. This
gyrB analysis would be useful for the discrimination of the L.
brevis strains with respect to beer-spoilage ability.
Yasukazu Nakakita, a researcher at the Brewing Research Laboratories of
Sapporo Breweries Ltd., graduated from the University of Osaka Prefecture. He
investigated the bioactive compounds produced by soil microorganisms at the
university and got a Ph.D in agricultural chemistry. After studying Actinomycete
at Bristol-Myers Research Institute in Tokyo for 5 years, he joined Sapporo
Breweries Ltd. in 1989 and carried out the exploration of microorganisms
producing bioactive compounds. Since 1994 he has studied beer-spoilage bacteria
from various aspects.
YASUKAZU NAKAKITA, Hideo Maeba, and Masachika Takashio. Sapporo Breweries Ltd.,
Shizuoka, Japan.