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INTRODUCTION
Lactic acid bacteria (LAB) are widely utilized in the production of various fermented products, dietary adjuncts, probiotics and even cosmetics ingredients in Japan. Since LAB
can inhibit growth of other bacteria in their environments,
they may contribute to the maintenance of hygienic quality of
products or host health. Bacteriocins are possible components
involved (Daeschell993). A number of bacteriocins produced
by LAB including Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Enterococcus and Carnobacterium (e.g. Lozano et al.
1992 ;Piard et al. 1992 ;Van Laack et al. 1992 ; Arihara et al.
1993 ; Ten Brink et al. 1994) have been reported in recent
years. However, most of them have a narrow spectrum of
activity, mainly against other members of LAB. Only a few
bacteriocins active against pathogenic bacteria, like nisin
(Delves-Broughton 1990), pediocin AcH (Yousef et al. 1991)
and sakacin A (Lewus et al. 1991), have industrial potential,
e.g. as natural food preservatives or probiotics.
Due to the scarcity and limited activity of existing bacteriocins and bacteriocin producers for industrial use, we
screened LAB strains for antimicrobial activity in this study.
Also, efforts were directed to preliminarily characterize a
bacteriocin (salivacin 140) produced by Lactobacillus salivarius subsp. salicinius T 140.
Three hundred and fifty-three LAB cultures screened for antimicrobial activity were recovered from food, plant, saliva or
animal faeces using MRS (Difco Laboratories Inc., Detroit, MI)
agar (1.5% w/v; Difco) containing 1% CaC03. After anaerobic
incubation at 37C (48 h), only colonies which formed clear
zones (due to destruction of CaC03by bacterial acid production)
that were Gram-positive and catalasenegative were retained.
Prior to use, all strains were passaged twice in screw-capped test
tubes containing MRS broth at 37C.
The bacterial strains used as indicator organisms are listed
in Table 2. Lactobacillus salivarius subsp. salicinius (JCM1040,
JCM1042, JCM1044, JCM1045, JCM1046, JCMl150') and
Lact. salivarius subsp. salivarius JCM1231T were all obtained
from the Japan Collection of Microorganisms (Wako, Japan).
Lactobacillus strains were propagated in MRS broth at 37C.
Listeria monocytogenes and Streptococcus mutans were grown
in BHI broth (Nissui, Tokyo, Japan) at 37C. Salmonella
enteritidis, Staphylococcus aureus and Esctierichia coli were cultivated in Nutrient broth (Oxoid, Basingstoke, UK) supplemented with 0.5% NaCl at 37C. Bacillus cereus and Yersinia
enterocolitica were propagated in Nutrient broth supplemented with 0.5% NaCl at 30C. Actinomyces viscosus and
Propionibacterium acnes were grown in GAM broth (Oxoid)
at 37C. Agar media were prepared by adding 1.5% agar
(Difco) to broth media.
0 1996 The Society for Applied Bacteriology
T o test antimicrobials for sensitivity to enzymes, filter-sterilized enzyme solutions were separately added (1 mg ml-
each in d H 2 0 ) to soft agar, when agar spot tests were performed.
To test for the presence of bacteriophage, putative bacteriocinogenic strains were streaked onto MRS agar plates
(0.2% glucose) and incubated overnight at 37C under anaerobic conditions. T h e agar was then inverted into the lid of
the Petri dish and the L . monocytogenes IID 579 was streaked
transversely across the original streak. Incubation for L. monocytogenes followed and inhibition was observed as a lack of
growth.
RESULTS AND DISCUSSION
Source
L. monocytogenes
Strep. mutans
Act. vtscosus
P.acnes
F1
F3
T27
T46
T56
T58
T93
T103
TI05
T113
TI40
T142
TI60
TI72
T176
pork
pork
hog faeces
cow faeces
grass
grass
tree leaves
tree leaves
grass
tree leaves
grass
9.0
2.5
NZD
NZD
2.5
NZD
7.5
9.0
10.5
7.0
10.0
NZD
NZD
NZD
NZD
NZD
NZD
NZD
NZD
NZD
NZD
NZD
NZD
NZD
NZD
9.0
NZD
NZD
NZD
NZD
NZD
NZD
NZD
NZD
1.o
2.0
NZD
NZD
NZD
NZD
7.5
2.0
3.0
3.0
NZD
NZD
NZD
2.5
7.5
NZD
NZD
NZD
NZD
NZD
NZD
7.0
NZD
NZD
NZD
2.0
grass
grass
grass
grass
* Zone size defined as the distance from the edge of the producer strains to the farthest part of the clear zone.
NZD, No zone detected.
0 1996 The Society for Applied Bacteriology, Letters in Applied Microbiology 22, 420-424
422 K . A R I H A R A E T A L .
Indicator strains*
Inhibition
Gram-positive bacteria
Lactobacillus acidophilus
Lact. casei subsp. casei
Lact. casei subsp. rhnmnosus
Lact. cellobiosus
Lact. delbrueckii subsp. bulgaricus
Luct. delbrueckii subsp. delbrueckii
Lact. delbrueckii subsp. lactis
Lact. gasseri
Lact. helveticus
Lact. plantarum
JCM 1132T
JCM 1 134T
JCMl 136T
JCM 1137T
JCM1002'
JCM 1012"'
JCMl148"'
JCM 1 131T
JCM1120'r
JCMl 149'r
Actinomyces viscosus
Bacillus cereus
Listeria monocytogenes
Staph,ylococcus aureus
Strept(icoccus mutans
Prcipionibacterium acnes
NIAH 1010
JCM2 152'r
IID579
JCM2413T
JCM5705
GAI5491
+
+
+
+
+
+
+
+
+
+
+
+
Gram-negative bacteria
Escherichia coli
Salmonella enteritidis
Yersinia enterocolitica
JCM5491
NIAH10590
IID98 1
0 1996 The Society for Applied Bacteriology, Letters in Applied Microbiology 22, 420-424
Test strains
10.0
NZD
2.0
2.0
NZD
NZD
NZD
*Zone size defined as the distance from the edge of the producer
strains to the farthest part of the clear zone.
NZD. No zone detected.
8 1996 The Society for Applied Bacteriology, Letters in Applied Microbiology22, 420-424
424 K . A R I H A R A ET A L .
@ 1996 The Society for Applied Bacteriology, Letters in Applied Microbiology 22, 420-424