You are on page 1of 6

J. Gen. App!. Microbiol.

, 36, 1-6 (1990)





' Department of Food Science and Technology, 2Institute of Genetic Resources,

Faculty of Agriculture, Kyushu University,
46-09, Hakozaki Higashi-ku, Fukuoka-shi, 812, Japan
3 Department of Microbiology , School of Medicine, Gifu University,
40 Tsukasamachi, Gifu 500, Japan

(Received September 1, 1989)

An L-lactate producing strain whose optimal temperature for growth and

L-lactate production was 37°C, previously labeled as Streptococcus species
I0-1, was characterized biochemically and genetically and identified as a
variant strain of Lactococcus lactis. This strain tolerated 6.5°c NaC1 and
grew on bile esculin agar. Thus the strain resembled enterococci biochemi-
cally. However, there was no Lancefield's group D antigen in the strain,
but group N antigen was present. Quantitative DNA-DNA hybridi-
zation experiments showed that the strain did not resemble enterococci
and should be assigned to the genus Lactococcus. Strain JO-1 was
identified as a strain of Lactococcus lactic because the homology values
between it and the type strain of the L. lactis, NCFB 604Twere in a range
of 62-77%. The growth of L. lactis is usually suppressed and very slow at
42°C but strain JO-1 grew confluently and very quickly at that tempera-
ture. It was also different from the type strain of L. lactis in that it
produced antibiotics, which were not nisin but were similar to nisin to
which other lactococci and some species of genus Bacillus and Clostridium
were sensitive. This strain has been deposited in the Japan Collection of
Microorganisms as L. lactis I0-1 JCM 7638.

Lactic acid-producing bacteria are important organisms in the food industry.

The role of lactic acid bacteria in Japanese traditional fermentation products such
as sake brewery (10, 11), miso and shoyu are well known. Some Lactobacillus are
active in the traditional Japanese process of fresh vegetable pickle fermentation
* Address reprint requests to: 1)r. A. Ishizaki, Department of Food Science and Technology,
Faculty of Agriculture, Kyushu University, 46-09, Hakozaki, Higashi-ku, Fukuoka 812, Japan.

2 ISHIZAKI et al. VOL. 36

(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,
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

D and N antigen were identified with a Slidex Strepto-kit (BioMerieux,

France) and antiserum from Wellcome Laboratory.
The G + C mol% was determined by HPLC using a Hitachi Model 638-30
with a column packed with 10C18. DNA extracted by Marmur's method (12) was
hydrolyzed by Pl nuclease and then alkaline phosphatase according to the method
described by Tamaoka and Komagata (19). Four equimolar mixtures of nucleotides
(Yamasa, Japan) was used as the standard (9).
Quantitative hybridization was carried out in a microdilution plate, as
previously described (2, 3), in experiments at 37°C in 2 x SSC (0.3 M NaCI and
0.03 M sodium citrate) containing 50% formamide.


Strain I0-1 grew in both anaerobic and microaerophilic conditions. The

organism was a gram-positive ovoid coccus, and catalase negative. It produced
L-lactic acid as a major metabolic product from glucose and no other significant
lower fatty acids. So the strain was first assigned to streptococci. The strain, however,
grew on bile esculin agar and in 6.5% NaCI. Those characteristics indicated the
strain might belong to group D streptococci, most of whose members are currently
assigned to the genus Enterococcus. However, the strain did not carry Lancefield
group D antigen which the members of the genus Enterococcus invariably have. It
carried Lancefield group N antigen. The antigen is carried by Lancefield group N
streptococci and most members have been transferred to the genus Lactococcus.
The I0-1 strain required glutamic acid, leucine and valine as essential amino acids
for growth and L-lactate production. This agreed well with the results for L. lactis
reported by Marshall et al. (13). Carbon source assimilation and acid production
of strain TO-1 indicated in Table 1 did not resemble any established species of the
genus Lactococcus. Moreover, the strain produced nisin-like antibiotic, which
suppresses the growth of the members of the genus Lactococcus. These phenotypic
characters were not enough to assign the strain to a taxonomic position. Thus we
considered genetic characterization of strain I0-1 by determining the mol% of
G + C in the DNA and quantitative DNA-DNA hybridization among selected
strains of the genus Enterococcus, Streptococcus and Lactococcus. The mol% of
G+ C of the DNA was 38% which resembled 38.6 of the L. lactis ATCC 19435T(14).
Quantitative hybridization among the selected strains clearly indicated that strain
I0-1 was genetically more related to lactococci than to enterococci. The homology
value between the type strain of L, lactis NCFB 604T and TO-1 strain was in a
range from 62 to 77% under optimal conditions (Table 2). So we identified the
strain as L, lactis.
L. lactis I0-1 strain had several characteristics different from the type strain
of the L. lactis. It grew between l0°C and 45°C. The optimal temperature was
37°C with a highest specific growth rate of 1.2 h- (6). The point to emphasize
is that strain TO-1 is different from the strains of L. lactis and L. lactis subspecies
4 ISHIZAKI et al. VOL. 36

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

Table 2. Determination of homology values for the strain I0-1 and

Lactococcus lactis NCFB 604T against the selected strains.

in the hydrolyzate of the antibiotics. The details of this subject will be reported


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
6 ISHIZAKI et al. VOL. 36

liquid chromatography of its enzymatic hydrolysate: a review. J. Microbiol. Methods, 4, 229-240

10) Kitahara, K., Kaneko, T., and Goto, 0., Taxonomic studies on the hiochi-bacteria, specific
saprophytes of sake: I. Isolation and grouping of bacterial strains. J. Gen. Appl. Microbiol., 3, 102-
110 (1957).
11) Kitahara, K., Kaneko, T., and Goto, 0., Taxonomic studies on the hiochi-bacteria, specific
saprophytes of sake: II. Identification and classification of hiochi-bacteria. J. Gen. App!. Microbiol.,
3, 111-120 (1957).
12) Marmur, J., A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol.
Biol., 3, 208-218 (1961).
13) Marshall, V. M. E. and Law, B. A., The physiology and growth of dairy lactic acid bacteria. In The
Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk, ed. by Davies, F.
L. and Law, B. A., Elsevier Applied Science Publishers, Essex (1984), p. 70.
14) Mundt, J. 0., Enterococci, Lactic acid streptococci. In Bergey's Manual of Systematic Bacteriology,
Vol. 2, ed. by Sneath, P. H. A., Mair, N. S., Sharpe, M. E., and Holt, J. G., William & Wilkins,
Baltimore (1986), p. 1063, 1065.
15) Nakano, M., Miso, shoyu and tsukernono (Japanese). In Hakkoushokuhin, Korin Shoin, Tokyo
(1967), p. 48, 68, 165.
16) Okada, S., Toyoda, T., and Kosaki, M., An easy method for the determination of the optical types
of lactic acid produced by lactic acid bacteria. Agric. Biol. Chem., 42, 1781-1783, (1978).
17) Schleifer, K. H. and Kilpper-Balz, R., Transfer of Streptococcus faecalis and Streptococcus faecium
to the genus Enterococcus nom. rev, as Enterococcus faecalis comb. nov. and Enterococcus faecium
comb. nov. Int. J. Syst. Bacteriol., 34, 31-34 (1984).
18) Schleifer, K. H., Kraus, J., Dvorak, C., Kilpper-Balz, R., Collins, M. D., and Fischer, W., Transfer
of Streptococcus lactis and related Streptococci to the genus Lactococcus gen. nov. System. App!.
Microbiol., 6, 183-195 (1985).
19) Tamaoka, J. and Komagata, K., Determination of DNA base composition by reversed-phase high-
performance liquid chromatography. FEMS Microbiol. Lett., 25, 125-128 (1984).
20) Tsunoda, T., Biseibutsu niyoru aminosan no teiryouhou (in Japanese). In The Chemistry of
Proteins, Vol. 1, ed. by Akabori, S. and Mizushima, S., Kyoritsu Shuppan, Tokyo (1954), p. 282.