J. Gen. Appl. Microbiol.

, 44, 193–200 (1998)

Lactic acid bacteria found in fermented fish in Thailand

Somboon Tanasupawat,* Sanae Okada,1 and Kazuo Komagata 2

Department of Microbiology, Faculty of Pharmaceutical Sciences,

Chulalongkorn University, Bangkok 10330, Thailand
1
Culture Collection Center and 2Department of Agricultural Chemistry, Tokyo University of Agriculture,
Setagaya-ku, Tokyo 156–8502, Japan

(Received February 19, 1998; Accepted June 4, 1998)

Forty-seven strains of homofermentative rod-shaped and 5 heterofermentative sphere-shaped lac-

tic acid bacteria were isolated from 4 kinds of fermented fish (pla-ra, pla-chom, kung-chom, and
hoi-dong) in Thailand. These bacteria were separated into four groups by phenotypic and chemo-
taxonomic characteristics, including fluorometric DNA-DNA hybridization. Five strains (Group I)
contained meso-diaminopimelic acid in the cell wall. Four strains were identified as Lactobacillus
pentosus, and one strain was L. plantarum. Tested strains of this group produced DL-lactic acid.
The rest of the rod-shaped bacteria, 23 strains (Group II) and 19 strains (Group III), lacked meso-di-
aminopimelic acid in the cell wall and were identified as L. farciminis and Lactobacillus species, re-
spectively. The tested strains of these groups produced L-lactic acid. The amount of cellular fatty
acids of C16:0 and C18:1, and the DNA base compositions were significant for differentiating the
strains in Groups II and III. Five strains of cocci in chains (Group IV) produced gas from glucose.
The tested strains of this group produced D-lactic acid. They were identified as a Leuconostoc
species. The distribution of these bacteria in fermented fish in Thailand is discussed.

Key Words——fermented fish; lactic acid bacteria; Lactobacillus farciminis; L. pentosus; L. plantarum;
Lactobacillus sp.; Leuconostoc sp.

Traditional fermented fish with salt—nam-pla, bu-
du, tai-pla, pla-ra, pla-chom, kung-chom, and hoi-
dong—are found in different parts of Thailand (Tana-
supawat and Komagata, 1995). Lactic acid bacteria
Tetragenococcus halophilus, Enterococcus faecalis,
and Enterococcus hirae, and the catalase positive
cocci Staphylococcus carnosus and Staphylococcus
piscifermentans were reported to be distributed in
these products (Tanasupawat and Daengsubha, 1983;
Tanasupawat et al., 1991, 1992a, b, c). However, lac-
tobacilli reported for the fermented fish are limited.
This work deals with the identification of the rod-
shaped lactic acid bacteria in pla-ra, pla-chom, kung-
chom, and hoi-dong based on phenotypic and chemo-
taxonomic characteristics, including fluorometric DNA-
DNA hybridization.
Materials and Methods
Bacterial cell count and isolation method. The
highly salted fermented fish, pla-ra, and the lowly
* Address reprint requests to: Dr. Somboon Tanasupawat, Depart-
ment of Microbiology, Faculty of Pharmaceutical Sciences, Chula-
longkorn University, Bangkok 10330,Thailand.
salted products, pla-chom, kung-chom, and hoi-dong,
were obtained at the markets in Thailand and ana-
lyzed for pH by use of a Beckman pH meter. The cell
numbers were measured by a plating method on a
half strength of De Man, Rogosa, and Sharpe agar
(MRS agar) (De Man et al., 1960) with 5% NaCl after
incubation at 30°C for 3–5 days. The rod-shaped lactic
acid bacteria were isolated and cultivated in a half
strength of MRS (MRSH) broth. The pH of samples
and bacterial cell count (cells/g) are shown in Table 1.
Bacterial cultures. Fifty-two isolates and each of
the strains of Lactobacillus pentosus NRIC 1069T,
Lactobacillus plantarum NRIC 1067T, Lactobacillus
salivarius subsp. salivarius TISTR 1112T, Lactobacillus
farciminis TISTR 1113T, Lactobacillus animalis TISTR
1115T, Leuconostoc mesenteroides subsp. mesen-
teroides NRIC 1541T, and Leuconostoc mesen-
teroides subsp. cremoris NRIC 1538T were used in
this study, and strain designations and their isolation
sources are listed in Table 2. All tests were carried out
by incubating the cultures at 30°C, except for the in-
vestigation of the effects of temperature.
Morphological and cultural characteristics. Cell
form, cell size, cell arrangement, and colonial appear-
ance were examined on the cells grown on MRSH
194 TANASUPAWAT, OKADA, and KOMAGATA Vol. 44

Table 1. Chemical properties of fermented fish and bacterial cell counts of lactic acid bacteria.

Chemical properties
Bacterial counts
Thai name of products
(cells/g)
NaCl (%)a pH

Pla-ra (fermented fish) 11.5–23.9 4.33–5.64 1.02
107–2.84
1010

Pla-chom (fermented small fish) 3.2– 9.4 4.19–6.20 4.20
107–2.72
1010
Kung-chom (fermented shrimp) 3.2– 9.4 3.85–4.67 7.30
107–9.56
1011
Hoi-dong (pickled mussel) 3.5– 4.4 3.98–4.74 1.92
107–3.60
1010

a
Data cited from Phithakpol et al., 1995.

Table 2. Strains studied and their distribution in fermented fish.

Province
Fermented fish Strain number Group Identification
where collected

Pla-ra Bangkok FS7-1, FS7-2(PCU 203), FS7-3, FS49-2 II L. farciminis

Chaiyaphoom FS45-2
Ubonratchathani FS73-1, FS73-3
Bangkok FS49-1 III Lactobacillus sp.
Nakhonsawan FS53-2, FS53-3, FS53-4
Chaiyaphoom FS55-1, FS55-2, FS56-1(PCU 206), FS56-2
Chiangrai FS58-1, FS58-2, FS62-1, FS62-2, FS62-3
Chiangmai FS60-1(PCU 207), FS60-2
Chaiyaphoom FS45, FS45-1(PCU 209), FS46-2 IV Leuconostoc sp.
Chiangmai FS61-1(PCU 210), FS61-2
Pla-chom Surin FS100(PCU 201), FS101 I L. pentosus
Surin FS103, FS104, FS105 II L. farciminis
Chaiyaphoom FS44-1(PCU 204), FS44-2, FS44-3, FS47-3
Surin FS109, FS110, FS111(PCU 208) III Lactobacillus sp.
Nonthaburi FS4-1
Kung-chom Surin FS42-1, FS42-2 I L. pentosus
Bangkok FS31-1(PCU 202) I L. plantarum
Surin FS9-1, FS9-3, FS40-1, FS40-2 II L. farciminis
Bangkok FS67-1
Hoi-dong Bangkok FS24-3(PCU 205), FS50-3 II L. farciminis
Nonthaburi FS5-2, FS5-3

PCU, Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.

agar incubated for 3 days. Hucker-Conn modification
(Hucker and Conn, 1923) was used for gram stain.
Spore formation was examined in gram-stained speci-
mens. Motility was detected by the appearance of
stab cultures in soft agar (Whittenbury, 1963).
Biochemical and physiological characteristics.
Catalase activity was detected on cells grown on
MRSH agar with and without 5% heat blood. Nitrate
reduction was tested in medium composed of 1.0 g
KNO3, 3.0 g yeast extract, 5.0 g peptone, 0.2 g beef
extract, 5.0 g NaCl, 0.25 g Tween 80, 1.0 g agar, and
1,000 ml distilled water adjusted to pH 6.8, after incu-
bation for 7 days. A hydrolysis of esculin, arginine, and
starch and reactions in litmus milk were detected by
the methods previously described (Tanasupawat et
al., 1992a). The effects of temperature (15, 42, and
45°C), different starting pHs (3.5, 4.0, 4.5, 7.5, 8.0,
and 8.5), and different concentrations of NaCl (2.0,
4.0, 6.0, 8.0, 10.0, and 12.0%) were tested by using
MRSH broth. The production of gas from glucose
was examined in MRS broth with a Durham tube. Acid
formation from carbohydrates was tested by the use
of a basal medium of GYPB broth with the omission
of glucose. The GYPB medium contained 10.0 g
glucose, 5.0 g yeast extract, 5.0 g peptone, 2.0 g
beef extract, 2.0 g sodium acetate, 0.25 g Tween 80,
200 mg MgSO4 · 7H2O, 10 mg MnSO4 · 4H2O, 10 mg
FeSO4 · 7H2O, 5.0 g NaCl, and 1,000 ml distilled water
and was adjusted to pH 6.8. Bromcresol purple was
used as an indicator, or the acid produced in 3 ml
broth was titrated with 0.1 N NaOH. Vitamin require-
ments were assayed by the method of Kihara and
Snell with a modification (Kihara and Snell, 1960).
Isomer of lactic acid. All tested strains were culti-
vated in GYPB broth for 3 days. Lactic acid was ex-
tracted from the supernatant of the fermentation broth
1998 Lactic acid bacteria in fermented fish 195

with ether and was analyzed by HPLC as described gas-liquid chromatography.

by Otsuka et al. (1994).
Peptidoglycan type of cell wall. Diaminopimelic
acid in the cell wall was detected by hydrolysis of 3
mg dried cells grown in GYPB broth with 1 ml 6 N HCl
at 100°C for 18 h, and the hydrolysate was applied on
cellulose TLC plate (Merck no. 5716). The TLC plate
was developed with the solvent system of methanol-
water-6 N HCl-pyridine (80 : 26 : 4 : 10) (v/v) (Komagata
and Suzuki, 1987). Spots were visualized by spraying
with 0.5% ninhydrin solution in n-butanol (Tokyo Kasei
Kogyo Co., Ltd., Tokyo, Japan), followed by heating at
100°C for a few minutes.
Cellular fatty acid composition. Methyl esters of
fatty acids were prepared by using a 5% methanolic
hydrochloric acid solution (Kokusan Chemical Works,
Ltd., Tokyo, Japan) from dried cells (40 mg), which
were cultivated in GYPB broth as described by Ike-
moto et al. (1978). The samples were analyzed by
DNA base composition. DNAs were isolated from
cells grown in GYPB broth for 18–48 h and purified by
the method of Saito and Miura (1963). For strains with
difficulty in isolation of DNA, the medium was supple-
mented with 0.8–1.5% glycine (Yamada and Koma-
gata, 1970). The purified DNA was hydrolyzed to nu-
cleotides and nucleosides as described by Tamaoka
and Komagata (1984), and the hydrolysate was ap-
plied to reversed phase high-performance liquid chro-
matography (HPLC).
DNA-DNA hybridization. Photobiotin labeling DNA-
DNA homologies were carried out in 2
SSC (saline
trisodium citrate) and 50% formamide solution at
50°C, 2 h, for the strains in Group I, and at 40°C, 12 h,
for the strains in Group II, Group III, and Group IV, as
reported previously (Ezaki et al., 1988, 1989; Tanasu-
pawat et al., 1992a).

Table 3. General characteristics of strains.

Group Group Group Group

Characteristics I II III IV
(5)a (23) (19) (5)

Cell form rods rods cocci

Cell size (mm) 0.4–0.6
0.8–2.0 0.4–0.6
1.2–5.0 0.4–0.5
0.6–0.8
Cell arrangement singly, in pairs or in chains in pairs or in chains
Colony circular, white, low convex
Colony size (mm) 1.6–2.0 0.5–1.8 0.7–2.1
Catalase (with blood)    
Gas from glucose    
Nitrate reduction    
Arginine hydrolysis    
Esculin hydrolysis    
Reaction in litmus milk
Acidification   (12)  (4) 
Coagulation  (2)  (1)  (1) 
Liquefaction    
Reduction  (2)  (1)  (2) 
Final pH in broth 3.70–3.84 3.67–3.78 3.87–4.34 4.32–4.55
Growth at 15°C   (5)   (1)
42°C   (8)  
45°C    
pH 3.5  (3)   
pH 4.0    
pH 4.5    (1) 
pH 7.5    (2)  (1)
pH 8.0   (1)   (1)
pH 8.5   (10)  
Growth in 4% NaCl    
6% NaCl    
8% NaCl  (1)  (1)  
10% NaCl  (2)  (5)  (1) 
12% NaCl   (5)  (1)  (2)
Peptidoglycans type
meso-Diaminopimelic acid    
Isomer of lactic acid DL(3) L (19) L (15) D (4)

a
Number of tested strains. Numbers in parentheses indicate the number of strains showing positive or negative reaction.
196 TANASUPAWAT, OKADA, and KOMAGATA Vol. 44

Results All tested strains required niacin and Ca-pantothenate

Morphological and cultural characteristics
All the strains were gram-positive. Forty-seven
strains were rods, 0.4–0.6
0.8–5.0 mm in size, and
they appeared singly, in pairs, or in chains. Five
strains were cocci, 0.4–0.5
0.6–0.8 mm in size, and
appeared in pairs or in chains. Cells were nonmotile
and nonsporing. Colonies on MRSH agar plate were
circular, low convex with entire margin, and nonpig-
mented (Table 3).
Biochemical and physiological characteristics
All the strains were microaerophilic. The rod-shaped
strains were homofermentative, and the cocci were
heterofermentative. All showed negative reactions to
catalase and nitrate reduction except, the isolates of
Group I. All isolates in Group II hydrolyzed arginine
and esculin. Not all liquefied litmus milk and hy-
drolyzed starch, and not all grew at 45°C. The isolates
of Group I grew at pH 8.5 and at 15°C, but most iso-
lates of Group II and Group III did not. The isolates of
Group III could not grow at pH 8.0. All the isolates
grew in 6% NaCl, and most of them in Groups II, III,
and IV could grow in 10% NaCl. The final pH of iso-
lates in Groups III and IV were higher than in other
groups. Variable reactions were shown in Table 3. All
produced acid from D-glucose, D-fructose, and D-man-
nose, and variable reactions were shown in Table 4.
for growth, as shown in Table 5.
Isomer of lactic acid
The major products from glucose of the tested iso-
lates were DL-lactic acid, L-lactic acid, and D-lactic acid
in Group I, in Groups II and III, and in Group IV, re-
spectively, as shown in Table 3.
Peptidoglycan type of cell wall
The isolates in Group I contained meso-diamino-
pimelic acid in the whole cell hydrolysate. All the other
isolates had no meso-diaminopimelic acid, as shown
in Table 3.
Cellular fatty acid composition
The tested strains in Groups I and III contained
straight chain acids of C18:1 and C16:0 as major
components, and the strains in Group II contained
C18:1 as a major component. The straight chain acids
of C12:0, C14:1, C14:0, C16:1, C18:0, C20:1, and cy-
clopropane acids of Cy17 and Cy19 were also found,
as shown in Table 6.
DNA base composition
DNA base compositions of tested strains were 45.4
to 47.0, 35.0 to 37.0, 38.6 to 41.5, and 38.0 to 41.2
mol% of guanine plus cytosine (GC) content in
Group I, Group II, Group III, and Group IV, respec-

Table 4. Acid from carbohydrates.

Group Group Group Group

Carbohydrates I II III IV
(5)a (23) (19) (5)

D-Amygdalin   (3)  (5) 

L-Arabinose    (2) 
D-Cellobiose   (11)  
D-Fructose    
D-Galactose   (2)  
D-Glucose    
Gluconate     (2)
Glycerol  (1)   (8) 
Lactose  9  (w9)  (2)
D-Maltose   (5)  (3) 
D-Mannitol    
D-Mannose    
D-Melibiose    
Methyl-a-D-glucoside  (1)    (1)
Raffinose    
L-Rhamnose  (1)   (1) 
D-Ribose    (1) 
Salicin   (2)  
D-Sorbitol  (1)   
Sucrose   (4)  (8)  (1)
D-Trehalose   (10)  (6)  (1)
D-Xylose  (1)   (8) 

a
Number of tested strains. Numbers in parentheses indicate the number of strains showing positive, weak positive, or negative reaction.
1998 Lactic acid bacteria in fermented fish 197

Table 5. Vitamin requirements of strains tested.

Group I Group II Group III Group IV

Vitamin
A B A B A B C
(2)a (1) (6) (2) (8) (1) (1) (1)

Thiamine        
Riboflavin        
Biotin        
Niacin        
p-Aminobenzoic acid        
Ca-pantothenate        
Folic acid        
Pyridoxine-HCl        

a
Number of tested strains: , required; , not required.
Group I A: FS100 and FS31-1; B: FS42-1. Group II A: FS5-2, FS7-2, FS9-3, FS24-3, FS47-3, and FS73-1; B: FS103 and FS105. Group III:
FS4-1, FS111, FS49-1, FS53-4, FS55-1, FS56-1, FS60-1, and FS62-1. Group IV A: FS45-1; B: FS46-2; C: FS61-2.

Table 6. Cellular fatty acid compositions of strains tested.

Straight chain acids

Unknown %
Species Strain
(No. of peaks)
C12:0 C14:1 C14:0 C16:1 C16:0 C18:1 C18:0 C20:1 Cy17 Cy19

Group I FS100 T T 3.2 2.6 25.2 40.6 5.6 6.5 T 4.6 10.9 (7)
FS42-1 T T 1.7 1.5 36.2 43.2 4.5 4.0 T 3.0 3.8 (4)
FS31-1 T T 4.1 3.9 34.1 27.2 3.3 10.7 T 8.7 7.1 (3)

L. pentosus NRIC 1069T T T 3.9 5.6 20.8 45.1 4.3 8.0 T 5.7 5.7 (3)

Group II FS105 T T 3.4 5.1 7.7 53.2 6.9 6.7 1.3 4.6 10.0 (4)
FS5-2 T T 3.0 6.6 7.2 43.3 6.0 11.2 1.4 7.6 12.6 (5)
FS7-2 T T 3.1 4.8 7.2 51.3 4.9 8.4 1.3 5.3 12.6 (5)
FS9-3 T T 3.4 6.1 8.2 45.2 6.1 9.6 1.4 7.2 11.8 (5)
FS47-3 T T 3.0 4.8 7.3 56.3 4.3 6.0 1.4 4.2 11.3 (5)
FS73-1 T T 3.0 4.7 6.4 54.0 4.9 7.1 1.5 5.3 10.0 (4)

L. farciminis TISTR 1113T T T 3.6 4.7 9.3 45.5 6.3 9.6 1.2 6.5 10.3 (3)

Group III FS4-1 2.2 2.9 12.3 3.7 29.0 22.1 4.8 8.5 T 6.2 8.0 (5)
FS53-4 1.9 4.2 12.5 6.4 19.3 21.1 4.9 12.2 T 8.4 8.4 (4)
FS60-1 1.5 4.3 10.7 6.3 18.1 20.4 5.9 11.6 T 8.2 11.0 (5)
FS62-1 T 2.2 6.6 7.3 17.0 33.4 4.8 8.9 T 5.7 12.4 (7)

Group IV FS45-1 T 4.0 3.8 27.5 9.8 10.7 19.5 T 13.7 10.1 (5)
FS61-1 T 7.3 8.3 24.1 17.7 6.6 18.7 T 12.0 4.0 (1)

Cy, cyclopropane acid; T, trace of acid less than 1%. NRIC, Culture Collection Center, Tokyo University of Agriculture, Tokyo, Japan.
TISTR, Thailand Institute of Scientific and Technological Research, Bangkok, Thailand.

tively, as shown in Tables 7, 8, and 9.
DNA-DNA hybridization
The isolates were separated into 4 groups on the
basis of DNA relatedness as shown in Tables 7, 8,
and 9. The tested strains of Group I showed high de-
grees of DNA homology (over 98.9%) with L. pento-
sus NRIC 1069T, and strain FS31-1 showed 86.2%
homology with L. plantarum NRIC 1067T. The strains
of Group II exhibited high degrees of homology (over
78.0%) with L. farciminis TISTR 1113T and showed
high degrees of homology (over 75.3%) with strains
FS7-2 and FS105. The strains in Group III showed
low degrees of homology (less than 22.9%) with L.
farciminis TISTR 1113T and L. animalis TISTR 1115T.
Strain FS111 showed low degrees of homology (27.3,
15.7, and 26.7%) with L. salivarius subsp. salivarius
TISTR 1112T, L. farciminis TISTR 1113T, and L. ani-
malis TISTR 1115T, respectively. The strains of Group
IV showed low degrees of homology (less than
198 TANASUPAWAT, OKADA, and KOMAGATA Vol. 44

Table 7. DNA base compositions and DNA relatedness of strains in Group I and Group II.

% Homology with labeled strains

G+C
Species Strain
(mol%) NRIC NRIC FS42-1
1069T 1067T

Group I FS100 47.0 98.9 9.4 82.3

FS42-1 45.4 100.6 3.2 100.0
FS31-1 45.5 2.3 86.2 11.1
L. pentosus NRIC 1069T 46.0a 100.0 5.9 81.8
L. plantarum NRIC 1067T 44.0a 3.6 100.0 9.0

TISTR
FS7-2 FS105 1113T FS111

Group II FS103 36.2 106.3 80.4 96.7 7.9

FS105 — 82.5 100.0 100.1 2.0
FS5-2 37.0 101.0 77.7 94.6 4.8
FS7-2 37.0 100.0 74.0 100.3 8.2
FS9-3 36.6 — 83.9 94.5 6.3
FS24-3 36.9 94.8 75.4 96.7 6.7
FS40-1 35.6 98.0 75.3 92.5 9.5
FS44-1 35.0 — 89.7 98.3 30.4
FS45-2 35.1 — 91.6 91.8 22.5
FS47-3 36.0 100.3 91.9 96.2 39.5
FS67-1 35.6 85.8 — 78.0 11.6
FS73-1 35.2 87.2 91.6 96.4 —

Group III FS111 40.3 10.4 3.0 15.7 100.0

L. farciminis TISTR 1113T 36.0a 91.8 97.8 100.0 15.7

—, not tested.
a
Data cited from Embley et al., 1989; Tanasupawat et al., 1992a.

Table 8. DNA base compositions and DNA relatedness of strains in Group III.

% Homology with labeled strains

G+C
Species Strain
(mol%) TISTR TISTR
FS105 FS111 1113T 1115T

Group III FS109 41.5 5.0 101.0 4.6 20.0

FS111 40.3 3.0 100.0 16.6 21.5
FS4-1 40.3 4.0 92.8 22.4 19.1
FS49-1 40.6 — 99.1 4.5 15.9
FS53-4 39.5 23.6 93.0 17.6 13.8
FS56-1 39.3 24.0 92.1 22.9 14.3
FS58-1 38.6 20.5 92.9 22.7 10.5
FS60-1 38.7 23.1 94.6 5.0 9.3
FS62-1 38.7 15.6 95.7 20.1 11.5
L. salivarius subsp.
salivarius TISTR 1112T 34.7a 28.0 27.3 28.7 —
L. farciminis TISTR 1113T 36.0a 97.8 15.7 100.0 5.8
L. animalis TISTR 1115T 42.4a 27.1 26.7 25.9 100.0

—, not tested.
a
Data cited from Embley et al., 1989; Nakase, 1995.

20.9%) with L. mesenteroides subsp. mesenteroides Discussion

NRIC 1541T and L. mesenteroides subsp. cremoris
NRIC 1538T, but showed a high degree of homology On the basis of phenotypic characteristics, isomer
(over 88.7%) with a strain FS45 as shown in Table 9. of lactic acid, peptidoglycan type of cell wall, cellular
1998 Lactic acid bacteria in fermented fish
Table 9. DNA base compositions and DNA relatedness of strains in Group IV.
Species Strain
(mol%)
Group IV FS45
FS45-1
FS46-2
FS61-1
FS61-2
L. mesenteroides subsp.
mesenteroides NRIC 1541T
L. mesenteroides subsp.
cremoris NRIC 1538T
—, not tested.
a
Data cited from Schillinger et al., 1989.
fatty acid composition, DNA base composition, and
DNA relatedness as shown in Tables 3 to 9, forty-
seven isolates were included in the genus Lactobacil-
lus, and 5 were Leuconostoc (Hammes et al., 1992;
Kandler and Weiss, 1986; Schillinger et al., 1989).
Group I contained 5 isolates, as shown in Table 2.
These strains contained meso-diaminopimelic acid in
the cell wall, and tested strains produced DL-lactic
acid. All produced acids from glycerol and D-xylose,
except strain FS31-1. The strains FS100 and FS42-1
showed a high degree of homology (over 98.9%) with
L. pentosus NRIC 1069T. Strains FS100, FS101,
FS42-1, and FS42-2 were identified as L. pentosus
(Hammes et al., 1992; Tanasupawat et al., 1992a;
Zanoni et al., 1987). Strain FS31-1 showed a high de-
gree of homology (86.2%) with L. plantarum NRIC
1067T. This strain was identified as L. plantarum
(Hammes et al., 1992; Tanasupawat et al., 1992a;
Zanoni et al., 1987).
Group II contained 23 isolates, as shown in Table 2.
They did not contain meso-diaminopimelic acid in the
cell wall. All hydrolyzed arginine and esculin the same
as L. farciminis did. The tested strains produced L-lac-
tic acid, contained a DNA base composition that
ranged from 35.0 to 37.0 mol%, and showed a high
degree of homology (over 78.0%) with L. farciminis
TISTR 1113T. They were identified as L. farciminis
(Hammes et al., 1992; Kandler and Weiss, 1986). Pre-
viously, only a few strains of L. farciminis were iso-
lated from fermented sausage (Dellaglio et al., 1975,
Reuter, 1970, 1983). In this study we reported their
characterization and distribution in fermented fish. The
details of this lactobacilli described are mentioned in
RESULTS and in the tables.
Group III contained 19 isolates, as shown in Table
2. These strains did not contain meso-diaminopimelic
acid in the cell wall. The tested strains produced L-lac-
tic acid. They were separated from the strains in
199
% Homology with labeled strains
G+C
NRIC NRIC
1541T 1538T FS45
40.0 12.9 16.8 100.0
38.0 15.5 18.3 96.4
39.8 11.3 20.1 88.7
41.2 16.5 18.8 90.8
— 16.7 20.9 92.1
39.9a 100.0 102.5 26.7
40.1a 96.6 100.0 21.7
Group II by the hydrolysis of arginine, acid from D-ri-
bose, not growing at pH 8.0, the amount of cellular
fatty acids of C16:0 and C18:1, and the DNA base
compositions ranging from 38.6 to 41.5 mol% of GC,
as shown in Tables 3–6 and 8. They showed low de-
grees of homology with L. farciminis TISTR 1113T, L.
animalis TISTR 1115T, and L. salivarius subsp. salivar-
ius TISTR 1112T, as shown in Table 8. They were con-
firmed to form one species, but were left unidentified.
Group IV contained 5 isolates (FS45, FS45-1,
FS46-2, FS61-1, and FS61-2). They produced gas
from glucose and grew in 10% NaCl. The tested
strains produced D-lactic acid. The DNA base compo-
sitions of the tested strains ranged from 38.0 to 41.2
mol% of GC. All showed low degrees of homology
with L. mesenteroides subsp. mesenteroides NRIC
1541T and L. mesenteroides subsp. cremoris NRIC
1538T, but they exhibited a high degree of homology
with strain FS45. They were Leuconostoc, but also left
unnamed (Schillinger et al., 1989). In this study, L.
mesenteroides subsp. mesenteroides NRIC 1541T
and L. mesenteroides subsp. cremoris NRIC 1538T
showed a high degree of homology with each other.
They should be the same species as reported previ-
ously (Schillinger et al., 1989).
In fermented fish in Thailand, pla-ra, pla-chom,
kung-chom, and hoi-dong contained high concentra-
tions of NaCl as shown in Table 1. The strains in L.
farciminis were isolated from all kinds of these prod-
ucts, but we found Lactobacillus sp. (Group III) only in
pla-ra and pla-chom. L. pentosus and L. plantarum
strains were found in pla-chom and kung-chom, which
contained less NaCl than pla-ra did. The strains of
Leuconostoc sp. (Group IV) were isolated from pla-ra.
The concentration of NaCl in these fermented fish
controls the growth of these bacteria (Tanasupawat
and Daengsubha, 1983; Tanasupawat et al., 1993).
The strains in Group III produced L-lactic acid from
200 TANASUPAWAT, OKADA, and KOMAGATA
glucose and could grow in 12% NaCl. They are differ-
ent from L. farciminis and the other lactobacilli
(Hammes et al., 1992; Kandler and Weiss, 1986). On
the other hand, Group IV strains grew in 10% NaCl,
but failed to grow below pH 4.5 and were thus distin-
guished from the strains in genera Weissella and
Oenococcus (Collins et al., 1993; Dicks et al., 1995).
These strains in Group III and Group IV required fur-
ther studies on the 16S rRNA sequence to reveal their
taxonomic positions.
We wish to thank the Ministry of Education, Science, Sports and
Culture, Japan, for the research grant; Dr. T. Yoshida and Dr. T.
Seki, International Center of Cooperative Research in Biotechnol-
ogy, Osaka University, the respective leader and coordinator of the
project, and Mr. M. Uchino and the students of the Tokyo University
of Agriculture for technical assistance.
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