Letters in Applied Microbiology 1991,12,228-231 ADONIS 0266825491OOO61 L

Potential of Lactobacillus gasseri isolated from infant faeces to

produce bacteriocin
T A K A H I RTOBA,
O EMIKO Y O S H I O K&A TAKATOSH I
ITOH Laboratory ofAnima2
Products Chemistry, Faculty of Agriculture, Tohoku University, 1-I Tsutsumidori-
Amamiyamachi, Aobaku, Sendai 981, Japan

Received 12 February I991 and accepted 14 February 1991

Paper number: J R N f I I I

T O B A,T., Y O S H I O K A ,E. & ITOH, T. 1991. Potential of Lactobacillus gasseri

isolated from infant faeces to produce bacteriocin. Letters in Applied Microbiology
12,228-231.
Thirty strains of Lactobacillus gasseri isolated from infant faeces were examined for
production of an antimicrobial agent against 10 strains of the seven species of the
genus Lactobacillus. Each of the strains inhibited the growth of at least one of the
indicator strains. The agent was sensitive to proteolytic enzymes and stable for 20
min at 120°C. It is a bacteriocin designated as gassericin A.

Bacteriocins are proteins produced by bacteria
which inhibit the growth of other closely related
bacteria (Hardy 1975). Many Gram-positive and
-negative bacteria have been known to produce
a wide range of bacteriocins (Hardy 1975; Tagg
et al. 1976). Bacteriocins are also formed by
species of the genus Lactobacillus (Klaen-
hammer 1988).
It has long been believed that L. acidophilus is
an important natural inhabitant in the intestinal
tract of humans and other animals. However,
recent investigation has shown that faecal ‘L.
acidophilus’ should be identified as L. gasseri
(formerly classified as groups B-1 and B-2 of L.
acidophilus by Johnson et al. 1990) (Benno et al.
1989). There is no report on its antagonistic
activity. This paper describes detection of a
heat-stable bacteriocin and its prevalence in L.
gasseri strains freshly isolated from infant faeces.
For reference L. acidophilus strains were also
tested for bacteriocin production.
modified lactobacillus selective (LBS) agar
(Mitsuoka et al. 1965) at 37°C with the Gas-Pak
system (BBL Microbiology Systems, Cockys-
ville, USA). They were maintained in MRS
broth (De Man et al. 1960). To identify isolates,
the Minitek system (BBL) was used according
to the manufacturer’s instructions. Gas pro-
duction from glucose and gluconate, fermenta-
tion of ribose, and growth at 15°C were also
tested in MRS broth. The configuration of lactic
acid was determined enzymatically with test-
combination L-lactic acid (Boehringer Mann-
heim GmbH, Mannheim, FRG) and D-(-)-
lactate dehydrogenase (Bohringer Mannheim
GmbH). Fermentation products were analysed
by gas chromatography (Smibert & Krieg 1981).
To complete the identification of isolates, the
electrophoretic mobility of the lactic dehydro-
genases (LDH) was determined by polyacryl-
amide gel electrophoresis of crude cell-free
extracts as described previously (Toba et al.
1991b).

Materials and Methods BACTERIAL S T R A I N S A N D C U L T U R E

I S O L A T I O N A N D I D E N T I F I C A T I O N OF
L. gasseri FROM I N F A N T FAECES
Thirty strains of L. gasseri were isolated from
the faeces of a 2 month-old breast-fed infant on
CONDITIONS
All bacterial strains other than the isolates were
obtained from the Japan Collection of Micro-
organisms (JCM), Wako, Japan with the excep-
tion of L. acidophilus 13952 which was obtained
 Table 1. Inhibition of various lactobacilli by L. yasseri and L. acidophilus strains when tested
Inhibition of indicator strain
1. delhrueckii
I,. acidophilus 1,. delhrueckii subsp. lacris L
subsp. s
JCM JCM JCM hulyaricus JCM JCM L. helveticus c
Test strain 1132' 5342 2125 JCM 1002' 1248' 1148* JCM 1120' !CM
L. yusseri
LA 32, LA 33, LA 38 + - + + + + +
LA 39
LA 34, LA 40, LA 41 + - + + + + +
LA 46
LA 4 8 + - + + + + +
LA 12, LA 13, LA 14 + - + + + + +
LA IS, LA 35, LA 36
LA 37, LA 42, LA 43
LA 44, LA 45, LA 49
LA 50
LA 47 + - + + + ND +
LA 2, LA 3, LA 4 - - + + + + -
LA 5
LA 8, LA 9, LA I 1 - - + - - - -
I.. aridophifus
JCM 1028, JCM 1132' - + + -k 4- +
JCM 5342, I F 0 13952 - - + + + + +-
JCM 2125 + + + + - +
JCM 1229 + +- +- t
JCM 5811 ~ -
+ - -
JCM 1034, JCM 5808 - ~- - - - -

A superscript T after strain no. indicated that the strain IS the type strain of the species.
* Type strain of 'L. [eichmanii'.
Indicator strain inhibited + , not inhibited - ; ND, not done.
230 Takahiro Toba et al.
Table 2. Effect of catalase, protease and heat treatment on antibacterial activity of Lactobacillus acidophilus and
L. gasseri strains against L. delbrueckii subsp. bulgaricus JCM 1002Twhen tested by agar well diffusion assay
Heating at
Supernatant
Test strain fluid Catalase Trypsin Actinase 100°C 30min 120°C l0min 120°C 2 h i n
L. acidophilus
JCM 1 132T + + - - + + +
L. gasseri
LA 12 + + - - +
LA 14 + + - - +
LA 33 + + - - +
LA 39 + + - - +
LA 48 + + - - +
LA 50 + +
-~~~ ~ ~ ~
- - +
A superscript T after strain no. indicated that the strain is the type strain of the species.
Indicator strain inhibited +.
not inhibited - .

from the Institute for Fermentation (IFO),
Osaka, Japan. Lactobacillus strains were propa-
gated in MRS broth at 37°C. Escherichia coli,
Bacillus subtilis and Staphylococcus aureus were
grown in Trypticase soy (TS) broth (BBL) at
37°C. Agar media were prepared by adding
1.5% agar no. 1 (Oxoid, Basingstoke, UK) to
the broth media listed above. Soft agars were
prepared with 0.7% agar.
D E T E C T I O N OF I N H I B I T O R Y A C T I V I T Y
Detection of inhibitory activity of L. gasseri and
L. acidophilus cultures was performed by the
direct (Barefoot & Klaenhammer 1983) and the
agar well diffusion method (Toba et al. 1991a).
C H A R A C T E R I Z A T I O N OF T H E I N H I B I T O R Y
AGENT
To test for heat sensitivity, culture supernatant
fluids were heated for 10, 30 and 60 min at
100°C or 10 and 20 min at 120°C and the
residual activity was assayed. Sensitivity to the
proteolytic enzymes actinase E, trypsin, and
catalase was tested as previously described
(Toba et al. 1991a).
Results and Discussion
The phenotypic characteristics of all of 30
strains isolated from the infant faeces agreed
with those of L. acidophilus, L. gasseri and L.
crispatus (Kandler & Weiss 1986). The D- and
L-LDHs from all of the isolates have the same
electrophoretic mobility as those of the type
strain of the L. gasseri. Therefore, all of the iso-
lates were identified as L. gasseri.
To screen producers of inhibitory agent, 30 L.
gasseri isolates from infant faeces and nine L.
acidophilus strains were tested against 10 strains
of the seven species of the genus Lactobacillus
by the direct method. As shown in Table 1, all
of the L. gasseri strains and seven of the nine L.
acidophilus strains inhibited the growth of at
least one of the indicator strains. Inhibitory
spectra of the L. gasseri and L. acidophilus
strains differed significantly. Only one test strain
showed inhibitory activity against L. acidophilus
JCM 5342 and none of the test strains inhibited
the growth of L. plantarurn JCM 1149. Lacto-
bacillus gasseri LA 32, LA 33, LA 38 and LA 39
showed a broad inhibitory spectrum by inhibit-
ing most of the indicator strains.
To identify the agent responsible for inhibi-
tion, six L. gasseri strains and L. acidophilus
JCM 1132 were subjected for further study as
shown in Table 2. Neutralized cell-free super-
*
natant fluids (pH 6.8 0.1) from MRS broth
cultures of these seven strains were inhibitory to
L. delbrueckii subsp. bulgaricus JCM 1002 when
tested by agar well diffusion method. However,
they did not inhibit the growth of the indicator
lawn of E. coli JCM 1649, B. subtilk JCM 1465
and Staph aureus JCM 2413. The inhibitory
activity was not affected by catalase, indicating
that it was not caused by the action of hydrogen
peroxide. The inhibitory activity was not
reduced after heating for 20 min at 120"C, but it
was completely destroyed by treatment with
actinase E 500 pg per ml for 1 h or trypsin 500
pg per ml for 12 h at 37°C. These results show
 Bacteriocin from L. gasseri
that the inhibitory agents produced by six L.
gaseri strains and L. acidophilus JCM 1132 were
heat-stable proteins. The agents inhibit only
strains of Lactobacillus spp., indicating that the
inhibitory substances in the culture supernatant
fluids of L. gasseri LA 12, LA 14, LA 33, LA 39,
LA 48 and LA 50 are bacteriocins. Although
these six strains showed a different spectrum of
inhibitory activity (Table l), their inhibitory
agents have the same heat stability (Table 2).
We have called the bacteriocin from L. gasseri
LA 33 and 39, gassericin A.
Gassericin A appears similar in its heat-
stability to lactacin B from L. acidophilus N2
(Barefoot & Klaenhammer 1983, 1984) and lac-
tacin F from L. ucidophilus 88 (Muriana &
Klaenhammer 1987). Additional studies are
needed to make direct comparisons of physical,
compositional and structural characteristics
among these bacteriocins. Muriana & Klaen-
hammer (1 987) reported that plasmid-encoded
determinants for bacteriocin production were
transferred by intragenic conjugation in L. aci-
dophilus 88. The high incidence of bacteriocin
production in faecal L. gasseri suggests in uiuo
conjugational transfer of determinants of bacte-
riocin production and the important role of
bacteriocin in control of intestinal microflora
ecosystems,
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