Letters in Applied Microbiology 1996,22, 420-424

Salivacin 140, a novel bacteriocin from Lactobaci//us

salr'varius subsp. salicinius T I 40 active against pathogenic
bacteria
K. Arihara, S. Ogihara, T. Mukai, M. ltoh and Y. Kondo
School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada-shi, Japan
GWG/326: received 24 November 1995 and accepted 25 November 1995
K. ARIHARA, s. OGIHARA, T. MUKAI, M. ITOH A N D Y . KONDO. 1996. Fifteen of 353
environmental isolates of lactic acid bacteria consistently showed activity against
Listeria monocytogenes, Streptococcus mutans, Actinomyces viscosus, a n d / o r
Propionibacterium acnes. Strain T140, isolated from the surface of Japanese pampas
grass leaves and identified as Lactobacillus salivarius subsp. salicinius, also had activity
against several Lactobacillus species, Staphylococcus aureus a n d Yersinia enterocolitica. Since
the antagonistic factor(s) produced b y T140 was sensitive to a proteolytic enzyme, it
was concluded that a bacteriocin (named salivacin 140) was involved in the inhibition activity.
Strain TI40 required a high initial pH (7.5-8.5) in agar plates for bacteriocin production.

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.

Correspondence to :Keizo .4rihara, PhD, School of' Veterinary Medicine

and Animal Sciences, Kitasatv Universitji, Towada-shi 034. Japan.

MATERIALS AND METHODS

Bacteria

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

BACTERIOCIN FROM LACTOBACILLI 421

Identification of the isolated LAB (strain T140) was carried

out according to the description of Bergeys Manual of Systematic Bacteriology (Kandler and Weiss 1986). For references, the phenotypic characteristics of type strains of Lact.
salivarius subsp. salicinius JCMl 150T and Lact. salivarius
subsp. salivarius JCM1231T were also tested.
Detection of antimicrobial activity

An agar spot test (Fleming et al. 1975) was employed to

screen and characterize for antibacterial activity. Freshlygrown cultures (5 pl) were spotted onto the surface of agar
plates (MRS containing 0.2% glucose to restrict the extent
of acid production) and incubated anaerobically overnight at
37C. T h e plates were overlaid with 5 ml of respective soft
agar (0.7% w/v) seeded with 50 pl of freshly-grown indicator
cells. After overnight incubation at the appropriate temperature, inhibition was recorded if a clear zone extended laterally
from the border of producer colonies.
The agar well diffusion method (Schillinger and Liicke
1989) was used to measure the culture fluid activity. Briefly,
agar plates were overlaid with 5 ml of soft agar containing
sensitive indicator cells. Wells (5 mm diam.) were cut into
the agar surface, and 50 p1 of culture supernatant fluid or
extract from agar plates were separately placed into each well.
Plates were incubated overnight at the appropriate temperature and examined for clear zones.
Putative bacteriocinogenic strains were propagated in
MRS broth for 24 h at 37C and resulting cells were removed

by centrifugation (10 000 g, 10 min, 4C). The supernatant

fluid was sterilized by filtration (0.45 pm pore size, Dismic25CS, Toyo Roshi Kaisha, Tokyo, Japan) and stored at
-20C until used.
Characterization of antimicrobial substances

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

Preliminary screening of 353 strains by an agar spot test

identified 15 LAB that inhibited four pathogenic bacteria
(Table 1). T h e inhibitory activities of all 15 strains were
eliminated by the addition of pronase E to the soft agar
overlay used in agar spot tests, suggesting the antimicrobial
substance(s) was proteinaceous. In contrast, the activity was
not diminished by the addition of catalase to soft agar over-

Table 1 Antimicrobial activity of putative bacteriocinogenicstrains in agar spot tests

Inhibition zone size (mm)*

Producer strain no.

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 .

Table 2 Inhibitory spectrum of the bacteriocin produced by

Lactobacillus salivarius subsp. salicinius in agar spot tests

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

*A superscript T after the strain number indicates that the strain

is the type strain of the species. JCM, Japan Collection of
Microorganisms (Wako, Japan) ; NIAH, the National Institute
of Animal Hygiene (Tsukuba,Japan) ; GAI, Institute of
Anaerobic Bacteriology, Gifu Medical School of Medicine (Gifu,
Japan) ; IID, the Institute of Medical Science, University of Tokyo
(Tokyo, Japan).
+, Inhibition ; -, no inhibition.

lays, thus eliminating the possibility that H202contributed

significantly to the activity. Bacteriophage induction and subsequent cell lysis was dismissed, since the inhibitory substance could diffuse through agar (data not shown). T h e
antimicrobial activity of the strain T140, which was isolated
from the surface of Japanese pampas grass leaves close to an
animal barn on our campus, was consistently greater than the
activity of the other 14 strains and thus, only T140 was
retained for further experiments. However, subsequent
experiments using culture supernatant fluids revealed that
T140 did not exhibit antimicrobial activity in agar well diffusion assays. In this regard, it is not uncommon to observe
greater bacteriocin activity in the presence of producer cells
(i.e. on solid media; agar spot tests) than in the absence of
producer cells (i.e. liquid media; well diffusion assays ; West
and Warner 1988 ;Harris et al. 1989).

Strain T140 is a catalase-negative facultative anaerobic

Gram-positive rod and produces lactic acid as the major
fermentation product. Most of the phenotypic characteristics
of T140, such as growth temperature and carbohydrate fermentation, agreed with those of Lact. salivarius listed by
Kandler and Weiss (1986) and with type strains (Lact. salivarius subsp. salivarius JCM12311, Lact. salivarius subsp.
salicinius JCMl 150T). Since strain T140 fermented salicin
and aesculin but not rhamnose, it was identified as Lactobacillus salivarius subsp. salicinius. T h e inhibitory factor(s)
produced by the strain T140 was designated as a bacteriocin
or bacteriocin-like inhibitory substance (Tagg et al. 1976) and
as a consequence, named salivacin 140.
The inhibitory effects of Lact. salivarius subsp. salicinius
T140 against different test organisms are shown in Table 2.
T h e strain demonstrated a relatively broad inhibitory spectrum including against several pathogenic bacteria. As shown
in Table 3, of the other seven strains of Lact. salivarius,
only two strains exhibited weak antimicrobial activity against
Listeria monocytogenes. Thus salivacin 140 production is a
strain-specific phenomenon like most cases of the bacteriocin
synthesis by lactic acid bacteria. Recently, Ten Brink et al.
(1994) reported a bacteriocin, salivaricin B produced by
Lactobacillus salivarius M7. However, the spectrum of salivaricin B is apparently different from that of salivacin 140 in
this study.
The antimicrobial activity of the strain T140 was significantly affected by the agar plate p H (Fig. l), preferring a
high initial pH (7.5-8.5) for bacteriocin production, although
its growth was lowered at p H values above 8.5. Since the pH
(between 4 and 9) did not affect the stability of crude salivacin
140 preparation from agar plates (data not shown), the lower
antimicrobial activity at lower p H values was not due to

Fig. 1 Effect of the initial pH of agar plates on the bacteriocin

activity of Lactobacillus salivarius subsp. salicinius T140 against
Listeria monocytogenes IID579 in agar spot tests. The initial pH
of MRS plates was adjusted with 1 moll-' HCl or NaOH

0 1996 The Society for Applied Bacteriology, Letters in Applied Microbiology 22, 420-424

BACTERIOCIN FROM LACTOBACILLI 423

Table 3 Inhibitory activity of Lactobacillus salivarius strains

against Listeria monocytogenes IID579 in agar spot tests

Test strains

Lact. salivarius subsp. salicinius

T140
JCM 1040
JCM1044
JCM1045
JCM1046
JCM1150T
Lact. salivarius subsp. salivarius
JCM1231T

Inhibition zone size

(mm)*

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.

inactivation of the bacteriocin. Also, since this strain grew

better at pH 5.5-7.5 than at 7.5-8.5, the bacteriocin activity
was not related to its growth rate. Several reports have mentioned that pH appears to be a critical factor for production
of bacteriocins (Barefoot and Klaenhammer 1984 ; Muriana
and Klaenhammer 1987 ; Wrtvedt-Abildgaard et al. 1995).
Lactobacillus salivarius is a gastrointestinal species of lactobacilli. Lactobacilli are generally recognized as safe and usually give health benefits to host animals and humans (Arihara
and Luchansky 1994). In gastrointestinal tracts, bacteriocin
production may contribute to colonization, since the production of antimicrobials may enhance competitive ability
(Tannock 1990). Fortunately, strain T140 did not inhibit
Lact. acidophilus and Lact. gassevi, which are predominant
lactobacilli in intestinal tracts. In these respects, strain T140
seems to be potentially suitable for probiotic products. Also,
the strain and the bacteriocin produced may have significant
potential for natural biopreservative applications in foods. In
other respects, considering its antimicrobial activity against
pathogenic bacteria in the oral cavity (Streptococcus mutans
and Actinomyces viscosus) and on skin (Propionibacterium
acnes), strain T140 and/or salivacin 140 may be applicable
in oral hygiene systems (mouthwash or toothpaste), facial
cleaning products and cosmetics.
ACKNOWLEDGEMENT

This work was supported in part by Kitasato Research Fund

(Grant) No. H7-2.
REFERENCES
Arihara, K. and Luchansky, J.B. (1994) Dairy lactobacilli. In Food

Biotechnology : Microorganisms ed. Hui, Y.H. and Khachatourians,

G.G. pp. 609-643. New York, NY: VCH Publishers.
Arihara, K., Cassens, R.G. and Luchansky, J.B. (1993) Characterization of bacteriocins from Enterucoccus faecium with activity
against Listeria monocytogenes. International Journal o f Food
Microbiology 19, 123-134.
Barefoot, S.F. and Klaenhammer, T.R. (1984) Purification and
characterization of the Lactobacillus acidophilus bacteriocin lactacin B. Antimicrobial -4gents und Chemotherap.y 26, 328-334.
Daeschel, M.A. (1993) Applications and interactions of bacteriocins
from lactic acid bacteria in foods and beverages. In Bacteriocins qf
Lactic Acid Bacteria ed. Hoover, D. and Steenson, L.R. pp. 6391. New York, NY : Academic Press.
Delves-Broughton, J. (1990) Nisin and its uses as a food preservative. Food Technology 44( lo), lO(tl17.
Fleming H.P., Etchells, J.L. and Costilow, R.N. (1975) Microbial
inhibition by an isolate of Pediococcus from cucumber brines.
Applied Microbiology 30, 104&1042.
Harris, L.J., Daeschel, M.A., Stiles, M.E. and Klaenhammer, T.R.
(1989) Antimicrobial activity of lactic acid bacteria against Listeria
monocytogenes. Journal of Food Protection 52, 384-387.
Kandler, 0. and Weiss, N. (1986) Genus Lactobucillus Beijerindk
1901, 212"I.. In Bergty i Manuul of Systematic Bacteriology ed.
Sneath, P.H.A., Mair, N.S. and Sharpe, M.E. Vol. 2, pp. 12091234. Baltimore, M D : Williams and Wilkins.
Lewus, C.B., Kaiser, A. and Montville, T.J. (1991) Inhibition of
foodborne bacterial pathogens by bacteriocins from lactic acid
bacteria isolated from meat. Applied and Environmental Microbiology 57, 1683-1688.
Lozano, J.C.N., Meyer, J.N., Sletton, K., Pelaz, C. and Nes, I.F.
(1992) Purification and amino acid sequence of a bacteriocin
produced by Pediococcus acidilactici. Journal of General Microbiology 138, 1985-1990.
Wrtvedt-Abildgaard, C.I., Nissen-Meyer, J., Jelle, B., Grenov, B.,
Skaugen, M. and Nes, I.F. (1995) Production and pH-dependent
bactericidal activity of Lactocin S, a lantibiotic from Luctobucillus
sake L45. Applied and Environmental Microbiology 61, 175-179.
Muriana, P. and Klaenhammer, T.R. (1987) Conjugal transfer of
plasmid-encoded determinants for bacteriocin production and
immunity in Lactobacillus ucidophilus 88. Applied and Environmental Microbiology 53, 553-560.
Piard, J.-C., Muriana, P.M., Desmazeaud, M.J. and Klaenhammer,
T.R. (1992) Purification and partial characterization of lacticin
481, a lanthionine-containing bacteriocin produced by Lnctococcus
lactis subsp. lactis CNRZ 481. .4pplied and Environmental Microbiology 58, 279-284.
Schillinger, U. and Liicke, F.-K. (1989) Antibacterial activity of
Lactobacillus sake isolated from meat. Applied and Environmental
Microbiology 55, 1901-1 906.
Tagg, J.R., Dajani, A.S. and Wannamaker, L.W. (1976) Bacteriocins
of Gram-positive bacteria. Bacteriological Reviews 30, 722-756.
Tannock, G.W. (1990) The microecology of lactobacilli inhabiting
the gastrointestinal tract. Advances in Microbial Ecology 11, 147171.
Ten Brink, B., Minekus, M., Van der Vossen, J.M.B.M., Leer,
R.J. and Huis in't Veld, J.H.J. (1994) Antimicrobial activity of
lactobacilli : preliminary characterization and optimization of production of acidocin B, a novel bacteriocin produced by Lacto-

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 .

bacillus acidophilus M46. Journal of Applied Bacteriology 77, 14&

148.
Van Laack, R.L.J.M.,Schillinger, U. and Holzapfel, W.H. (1992)
Characterization and partial purification of a bacteriocin produced
by Leuconostoc carnosurn LA44A. International Journal of Food
Microbiology 16, 183-195.
West, C.S. and Warner, P.J. (1988) Plantacin B, a bacteriocin pro-

duced by Lactobacillus plantarum NCDO 1193. F E M S Microbiology Letters 49, 163-165.

Yousef, A.E., Luchansky, J.B., Degnan, A.J. and Doyle, M.P. (1991)
Behavior of Listeria monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin AcH during storage
at 4 or 25C. Applied and Environmental Microbiology 57, 14611467.

@ 1996 The Society for Applied Bacteriology, Letters in Applied Microbiology 22, 420-424