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(nukamisozuke) (15), but no Lactococcus has ever been isolated from such Japanese
food. We isolated an organism from the drain pit of a sink in a home kitchen in
downtown Fukuoka. The organism was a homofermentative, L-lactate-producing
coccus (6, 7). As almost all lactate fermentation bacteria produce a mixture of
stereochemical isomers of lactic acid, L-lactate is one of the desired products in the
fermentation industry. However, the species used for such fermentation, L. cremoris,
needs temperatures as low as 27°C (1,8) and requires high utility consumption.
Since the optimal temperature of our isolated strain for growth and L-lactate
production is 37°C, it is valuable for use in industrial L-lactate fermentation.
Phenotypically, this organism did not resemble any established species of the genus
Streptococcus, Lactococcus, or Enterococcus (14,17,18). Thus, we characterized
the strain genetically.
Bacterial strain. Strain JO-1 was isolated in the middle of May, 1986, from
water in the drain pit of a kitchen sink in Fukuoka-shi, Japan. Water from the
drain was directly spread on the surface of a thioglycolate agar plate with dextrose
(TGC, Difco Laboratories, USA) and incubated anaerobically for 2 days. Colonies
were subcultured in TGC broth for 2 days at 30°C. The organism was purified
by repeating single colony isolation on TGC agar plate.
Type strains were obtained directly from NCFB (National Collection of Food
Bacteria, U.K.), ATCC (American Type Culture Collection, U.S.A.), and NCTC
(National Collection of Type Cultures, U.K.). Strains used were Lactococcus lactis
NCFB 604T, Lactococcus garvieae NCFB 2155T, Lactococcus raffinolactis NCFB
617T, Streptococcus dysgalactiae ATCC 27957T, Streptococcus bovis NCFB 597T,
Streptococcus salivarius ATCC 7073T, Enterococcus faecalis NCTC 775T,
Enterococcus faecium NCTC 7171T and Escherichia coli ATCC 12435. Lactococcus
lactis subspecies cremoris TUA 13446L (= ATCC 19257) is a gift from the
Department of Agricultural Chemistry, Tokyo University of Agriculture, Tokyo,
Japan.
Biochemical characterization. Strain TO-1 was biochemically characterized ac-
cording to the method of Facklam and Carey (4). Amino acid requirement was
determined according to the method described by Tsunoda (20). Lactate was
analyzed by enzymatic methods using L-lactate hydrogenase and D-lactate
hydrogenase. The enzymes were purchased from Boehringer Mannheim
Yamanouchi Co. Ltd. The optical density of the treated solution was measured by
a spectrophotometer at a wave length of 340 nm (16). L-lactate was also determined
with a YSI lactate analyzer (model 23L, YSI, U.S.A.). Other organic acids were
identified and determined by HPLC using a Hitachi Model 655A analyzer with a
Hitachi #2618 (cation exchange resin) 8 mm4 x 500 mm column eluted with 0.1%
phosphoric acid at 60°C. Optical absorbance was monitored at a wave length of
210 nm.
1990 High Temperature Lactococcus 3
Table 1. Carbon source assimilation and acid formation by Lactococcus lactis I0-1.
cremoris. In addition, the type strain hardly grew at 42°C and not at all at 45°C.
Strain I0-1 could survive after heating at 60°C for 30 min. In 1% glucose medium,
strain I0-1 decreased the pH of the medium lower than 4 within a day and finally
converted more than 90% of the glucose into L-lactic acid. In this respect, strain
TO-1 is better adapted than the type strain L. lactis for industrial application in
L-lactate fermentation.
This strain produced unidentified antibiotics whose behavior was similar to
nisin (5). The molecular weights of the unidentified antibiotics were smaller than
nisin and a few amino acids that occur among the nisin amino acids were lacking
1990 High Temperature Lactococcus 5
in the hydrolyzate of the antibiotics. The details of this subject will be reported
elsewhere.
REFERENCES
1) Bibal, B., Goma, G., Vayssier, Y., and Pareilleux, A., Influence of pH, lactose and lactic acid on the
growth of Streptococcus cremoris: a kinetic study. App!. Microbiol. Biotechnol., 28, 340-344 (1988).
2) Ezaki, T., Hashimoto, Y., Takeuchi, N., Yamamoto, H., Liu, S. L., Miura, H., Matsui, K., and
Yabuuchi, E., Simple genetic method to identify viridans group streptococci by colorimetric dot
hybridization and fluorometric hybridization in microdilution wells. J. Clin. Microbiol., 26, 1708-
1713 (1988).
3) Ezaki, T., Hashimoto, Y., and Yabuuchi, E., Fluorometric deoxyribonucleic acid-deoxyribonucleic
acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which
radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst.
Bacteriol., 39, 224-229 (1989).
4) Facklam, R. R. and Carey, R. B., Streptococci and Aerococci. In Manual of Clinical Microbiology.
4th ed., ed. by Lennette, E. H., Hausler Jr., W. J., and Shadomy, H. J., American Society for
Microbiology, Washington, D.C. (1985), p. 154-175.
S) Hurst, A., Nisin. In Advance in Applied Microbiology, Vol. 27, ed. by Perlman, D. and Laskin, A.
I., Academic Press Inc., London (1981), p. 85-123.
6) Ishizaki, A. and Ohta, T., Batch culture kinetics of L-lactate fermentation employing Streptococcus
sp. I0-1. J. Ferment. Bioeng., 67, 46-51 (1989).
7) Ishizaki, A., Ohta, T., and Kobayashi, G., Batch culture growth model for lactate fermentation. J.
Ferment. Bioeng., 68, 123-130 (1989).
8) Jorgensen, M. H. and Nikolajsen, K., Mathematic model for lactic acid formation with
Streptococcus cremoris from glucose. App!. Microbiol. Biotechnol., 25, 313-316 (1987).
9) Kaneko, T., Katoh, K., Fujimoto, M., Kumagai, M., Tamaoka, J., and Fujimura, Y. K.,
Determination of the nucleotide composition of a deoxyribonucleic acid by high-performance
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