Abstract: The aim of this study is to test the

production of bacteriocin in vaginal

Lactobacilli and determining the
characterization and antibacterial activity of
this bacteriocin. For this reason 100
Lactobacillus strains were isolated and
identified from vaginal swab samples of 75
women who attended obstetric and
gynecology clinics. It was determined that six
of 100 Lactobacillus strains produced
bacteriocin. The antibacterial activity of
bacteriocins was examined and found to be
effective on vaginal L. gasseri, L. acidophilus,
Gardnerella vaginalis ATCC 14018 and
Pseudomonas aeroginosa ATCC 10145. We
also found that bacteriocins were sensitive to
proteinase enzymes and alkalinity, but
resistant to catalase. Two of them, TL059a
and TL080, were resistant to chloroform and
1 h boiling. It was observed that adding 1%
NaCl to medians increased bacteriocin
production and it was also found that
mitomycin C induced Rogosa SL media was
more suitable than MRS (Z = -2, p < 0.05).
Key Words: Lactobacillus, Bacteriocin,
Antibacterial activity
Introduction
Among lactic acid bacteria members, the lactobacilli
are composed of a diverse group of homofermentative
and heterofermentative species. The production of
bacteriocins by lactic acid bacteria has been known for
many years (1). According to the original definition, the
term bacteriocin refers to proteins of the colicin type,
characterized by lethal biosynthesis, intraspecific activity,
and adsorption to specific receptors. Those produced by
gram-positive bacteria fit closely to the classical colicin
model (2). Lactobacillus bacteriocins are found within
each of the four major classes of antimicrobial proteins
produced by lactic acid bacteria. Class I (lantibiotics) was
only recently discovered in the Lactobacillaceae by
Mordvedt et al. (3). Lantibiotics are small membrane-
active peptides (<5 kDa) containing the unusual amino
acid lanthionine. Class II; small heat-stable, non-
lanthionine containing membrane-active peptides (<10
kDa) characterized. The class III bacteriocins, which have
to date only been found in Lactobacillus, include heat-
labile proteins of large molecular mass. Class IV is a
complex bacteriocin group. These proteins are associated
with other lipid or carbohydrate moieties, which appear
to be required for activity. The bacteriocin was relatively
hydrophobic and heat stable (1).
Lactobacilli produce many different bacteriocins of
similar activity, and are usually predominant species in the
vaginas of healthy women. Lactobacilli play an important
role in maintaining vaginal health. They produce lactic
acid and H
2
O
2
, which can prevent the overgrowth of
other microorganisms in the vagina including E. coli and
G. vaginalis (4,5).
The purpose of this study was to investigate the
antibacterial activity of Lactobacillus strains of vaginal
origin. Furthermore, we sought to characterize
bacteriocins for their structural properties and determine
their antimicrobial activities against some common
human pathogens including Gardnerella vaginalis,
Pseudomonas aeroginosa, Proteus vulgaris, Escherichia
coli, Enterobacter cloacea, Streptococcus milleri,
Staphylococcus aureus and Candida albicans.
Turk J Med Sci
33 (2003) 7-13
TBTAK
7
Received: August 02, 2002
1
Department of Biology, Faculty of Art &
Sciences at Rize, Karadeniz Technical
University, Rize,
2
Department of Microbiology and Clinical
Microbiology, Faculty of Medicine, Karadeniz
Technical University, 61080 Trabzon,
3
Department of Clinical Microbiology and
Infectious Diseases , Faculty of Medicine, Frat
University, 23100 Elaz - Turkey
engl ALPAY KARAOLU
1
Faruk AYDIN
2
S. Srr KILI
3
Ali O. KILI
2
Antimicrobial Activity and Characteristics of
Bacteriocins Produced by Vaginal Lactobacilli
Materials and Methods
Bacterial strains, media and growth conditions:
Lactobacillus gasseri ATCC 9857, L. fermentum ATCC
14931, L. fermentum ATCC 23271, L. delbrueckii subsp.
delbrueckii ATCC 9649, L. delbrueckii subsp. lactis ATCC
4749, L. jensenii ATCC 25258, L. casei subsp. casei ATCC
27139, L. vaginalis ATCC 49540 and L. acidophilus ATCC
4356, Streptococcus milleri IS51 and G. vaginalis ATCC
14018 were provided by Tao at the University of Illinois
in Chicago. Lactobacillus type strains were used for
species identification as control strains. Escherichia coli
ATCC 25922, Proteus vulgaris ATCC 13315,
Pseudomonas aeruginosa ATCC 10145, Enterobacter
cloacae ATCC 13047, Staphylococcus aureus ATCC
25923, and Candida albicans ATCC 60193 were obtained
from the Hifzisihha Institute of Refik Saydam (Ankara,
Turkey).
The vaginal swab samples transported in
Thioglycollate Broth (Difco, Detroit, MI) medium were
inoculated onto Rogosa SL agar (Difco) and incubated at
37 C in a candle jar for 48 h. The lactobacilli were
identified on the basis of growth on selective Rogosa SL
agar (pH 5.2), cell morphology, gram staining, and
catalase activity. Further identification of the species of
these lactobacilli was performed according to
carbohydrate fermentation patterns, esculin hydrolysis
and growth at 15 C and 45 C in the Lactobacilli DeMan
Rogosa Sharpe (MRS) broth (Difco), as described in
Bergeys Manual of Systematic Bacteriology (6). The
vaginal Lactobacilli strains isolated in this study were
called TL (Trkiye Lactobacilli). Purified cultures were
maintained at 80 C in MRS broth with 10% glycerol.
Preparation of culture supernatants: The
bacteriocin-producing strains were grown in MRS broth
(pH 5.5) at 37 C for 18-20 h. The lactobacilli culture
was centrifuged at 10,000 rpm for 5 min, and then the
supernatant was adjusted to pH 6.5-7.0 with 1N NaOH
(7).
Bacteriocin assay: Bacteriocin activity was detected
by the agar-spot test (7). The test was performed as
follows: 200 l of each Lactobacillus culture at the early
exponential growth phase (OD600 of 0.2-0.3) in MRS
broth was mixed with 4 ml of MRS soft agar (0.6% agar,
prewarmed to 48 C) and poured on an MRS agar plate.
Then, 3 l of each culture supernatant was dropped onto
the solidified soft agar. The plates were incubated for 48
h in a candle jar. Bacteriocin inhibition was indicated by a
clear zone in the soft agar layer.
The antagonistic effects of culture supernatants of
bacteriocin producing Lactobacillus strains on various
gram positive, gram negative organisms and C. albicans
were tested by the agar-well-diffusion assay as described
by Toba et al. (8). In brief, 0.5 mm-diameter wells were
made on preinoculated agar media and each well was
filled with 100 l of culture supernatant of bacteriocin-
producing Lactobacillus strains after neutralization with
NaOH. G. vaginalis was grown on Columbia agar (Difco),
C. albicans was grown on Saboraud Dextrose agar
(Becton Dickinson) and S. aureus, S. milleri IS51, E. coli,
E. cloacea and P. aeruginosa were grown on Brain Heart
Infusion agar (Difco). Aside from G. vaginalis, which was
grown in the candle jar, all cultures were grown
aerobically at 37 C for 48 h. Inhibition zones around the
wells were measured and recorded.
Sensitivity to heat: To test heat sensitivity, 100 l of
culture supernatant was heated for 10 min at 60 C, 70
C, 80 C and 90 C. The agar spot test was performed
to detect residual activity. The resistant culture
supernatants were further heated for 10, 30 and 60 min
at 100 C, and the residual activity was assayed (9).
Sensitivity to different pH values: The pH of culture
supernatants was adjusted to 3.0, 4.5, 7.0 and 9.0 and
then kept at room temperature for 4 h. Residual activity
was determined by the agar-spot method as described
(9).
Acid neutralization test: This test was performed by
agar well diffusion assay (10). In addition to 100 l of
supernatants buffered with NaOH to 7.0, 75 l of
Lactobacillus suspension and 25 l of 10% CaCO
3
solution
were mixed and placed into the well. The original culture
supernatants were used as control samples. When the
inhibition zone was determined around the wells of both
the control and buffered samples, the inhibitory effect
was assumed to be due to bacteriocin or H
2
O
2
, and other
tests were performed.
Sensitivity to catalase: Eighteen-hour cultures of
strains showing antimicrobial activity were diluted at
1:10 in 10 mM Tris HCl (pH 7.0) and 2 l of the
suspension (about 10
6
cell/ml) was inoculated on Rogosa
SL agar in tissue culture plate and incubated (10). Eight-
hour growing cultures of indicator strains were diluted at
a 1:10 ratio in 10 mM Tris HCl (pH 7.0) and mixed with
Antimicrobial Activity and Characteristics of Bacteriocins Produced by Vaginal Lactobacilli
8
. ALPAY KARAOLU, F. AYDIN, S. S. KILI, A. O. KILI
9
Rogosa SL soft agar (48 C). Catalase enzyme was then
added at a final concentration of 0.5 mg/ml. The mixture
was poured onto the tissue culture plate wells. One well
having no catalase enzyme was used as the control. The
tissue culture plates were examined after 18-24 h of
incubation. The presence of an inhibition zone around
wells both with and without catalase was determined to
be the effect of bacteriocin.
Sensitivity to proteolytic enzymes and chloroform:
Culture supernatants were treated with proteinase K,
pronase E and trypsin, each at a final concentration of 0.1
mg/ml (11). The samples with and without proteases
were incubated at 30 C for 1 h and residual activity was
determined. To test for chloroform sensitivity, the
culture supernatant was mixed with an equal volume of
chloroform and kept at room temperature for 4 h before
antimicrobial activity testing.
The effect of growth media on bacteriocin
production: Two commercial media, Rogosa SL and MRS
media, were tested for their ability to support the
production of bacteriocin. Five milliliters of both broth
media supplemented with 0.2 g/ml mitomycin C
7
and
without mitomycin C inoculated with bacteriocin-
producing Lactobacillus strains and incubated at 37 C for
18 h. The bacteriocin activity of the culture supernatants
was detected by agar-spot assay.
The effect of NaCl on bacteriocin production: After
sterilization by autoclaving, MRS agars with (1%, 2%,
3% and 4% NaCl) and without NaCl were aseptically
transferred onto a tissue culture plate. Bacteriocin-
producing Lactobacillus strains (about 2 x 10
3
cells) in 2
l were dropped onto the MRS agar and grown
overnight. The fresh culture of the indicator strain was
mixed with 0.6% MRS agar and spread onto the surface
of the MRS agar containing the bacteriocin-producing
Lactobacillus colonies followed by incubation for 24-48 h.
Bacteriocin activity was assayed according to inhibition
zones surrounding the colonies. The diameters of
bacteria-free zones were measured and the differences
were found by the Mann-Whitney U test.
Results
One hundred Lactobacillus strains were isolated from
the vaginal samples of 75 reproductive women admitted
to obstetrics and gynecology departments. The culture
supernatants obtained from 100 lactobacilli isolates were
tested for antibacterial activity against the same group of
lactobacilli. Among them, six strains of lactobacilli were
observed to have bacteriocin activity against eight of 10
different Lactobacillus species as well as S. milleri, P.
aeruginosa, E. coli, P. vulgaris, E. cloacea and G.
vaginalis. However, none of the six bacteriocins had
inhibitory effects on S. aureus and C. albicans strains. All
of the six bacteriocins showed inhibitory activity against
G. vaginalis ATCC 14018 and P. aeruginosa ATCC 10145
among the sensitive bacteria tested (Table 1).
Bacteriocin sensitivity to physical conditions and
chemical substances was also evaluated (Table 2). All six
bacteriocins were completely destroyed by proteinase K,
pronase E and trypsin treatment. However, the activity of
six bacteriocins was maintained after catalase treatment,
indicating that antibacterial activity was due to bacteriocin
not H
2
O
2
.
Among the six bacteriocins, L. gasseri TL093c and
TL143a strains were identified as lipid-containing
bacteriocins because of their sensitivity to chloroform.
These two bacteriocins were also inactivated by heating at
60 C for 10 min. On the other hand, L. gasseri TL029,
L. delbrueckii subsp. delbrueckii TL059a, L. gasseri
TL080 and L. acidophilus TL099a were resistant to
chloroform and resistant to boiling for at least 10 min.
These bacteriocins belong to low-molecular weight non-
lipid containing bacteriocins. All six bacteriocins were
stable between pH 4.5 and 7.0, but sensitive to pH 9.0.
Two bacteriocins isolated from L. delbrueckii subsp.
delbrueckii TL059a and L. acidophilus TL099a were
found to be active at pH 3.0 (Table 2).
The effects of media composition and mitomycin C on
the production of bacteriocin were also evaluated. We
found no significant difference between spontaneous
induced MRS and Rogosa SL medians in terms of
bacteriocin production (Mann-Whitney U test, Z = -0.66,
p > 0.05). However, we found a significant difference
between mitomycin C induced medians (Z = -2, p <
0.05). As seen in Table 3, Rogosa SL broth with
mitomycin C had greater stimulatory effects particularly
on the production of bacteriocins compared to those of
MRS broth. Bacteriocin from L. gasseri TL029 was only
produced after mitomycin C induction in both media.
Among the six bacteriocins, bacteriocin produced by L.
delbrueckii subsp. delbrueckii TL059a was not produced
in Rogosa SL broth in the absence of mitomycin C.
Antimicrobial Activity and Characteristics of Bacteriocins Produced by Vaginal Lactobacilli
10
Table 1. Effects of six bacteriocins on the growth of some bacteria on agar plates.
Bacteriocin-producing Lactobacillus strains
Indicator Strains TL029 TL059a TL080 TL093c TL099a TL143a
(L.g) (L.dd) (L.g) (L.g) (L.a) (L.g)
L. gasseri 1*/44** 2/44 1/44 10/44 1/44 13/44
L. acidophilus 1/11 1/11 0/11 2/11 1/11 3/11
L. delbrueckii subsp. delbrueckii 0/14 0/14 0/14 1/14 0/14 1/14
L. delbrueckii subsp. lactis 0/9 0/9 0/9 1/9 0/9 1/9
L. fermentum 0/4 0/4 0/4 0/4 0/4
1
/
4
L. vaginalis 0/4 0/4 0/4 0/4 0/4
1
/
4
L. jensenii 0/2 0/2 0/2 1/2 0/2
1
/
2
L. coryniformis 0/1 0/1 0/1 1/1 0/1 1/1
L. agilis 0/1 0/1 0/1 0/1 0/1 0/1
L. casei 0/2 0/2 0/2 0/2 0/2 0/2
G. vaginalis ATCC 14018 1/1 1/1 1/1 1/1 1/1 1/1
E. coli ATCC 25922 0/1 1/1 0/1 0/1 0/1 0/1
P. vulgaris ATCC 13315 1/1 0/1 0/1 0/1 0/1 0/1
P. aeruginosa ATCC 10145 1/1 1/1 1/1 1/1 1/1 1/1
E. cloacea ATCC 13047 0/1 0/1 0/1 0/1 1/1 1/1
S. milleri IS51 0/1 0/1 1/1 0/1 0/1 0/1
S. aureus ATCC 25923 0/1 0/1 0/1 0/1 0/1 0/1
C. albicans ATCC 60193 0/1 0/1 0/1 0/1 0/1 0/1
* The number of sensitive TL and ATCC strains, ** The number of strains tested
L. g: L. gasseri, L. dd: L. delbrueckii subsp. delbrueckii, L.a: L. acidophilus
Table 2. Effect of catalase, protease, chloroform and heat treatment on bacteriocin activity.
Bacteriocin Resistance to Resistance to Sensitivity
Producing Sensitivity heating (10 min) boiling Different pH
Lactobacillus Temperature (C) (min) values
Strains
P-K P-E Trp Cat Chl 60 70 80 90 10 30 60 3.0 4.5 7.0 9.0
TL029 R/S S S R R R R R R R/S S S S R R S
TL059a S S S R R R R R R R R R R R S
TL080 S S S R R R R R R R R R S R R S
TL093c R/S S S R S R/S S S S S S S S R R S
TL099a R/S S R/S R R R R R R R S S R R R S
TL143a R/S S S R S S S S S S S S S R R S
R= Resistance = Un inhibited bacteriocin activity, S= Sensitive = Inhibited bacteriocin activity
R/S= Moderate sensitivity, P-K= Proteinase K, P-E= Pronase E, Trp= Trypsin
The effects of NaCl on the production of the six
bacteriocins were studied. In both Rogosa SL and MRS
broth, 1% NaCl increased the production of bacteriocins
from 25 to 75% isolated from Lactobacillus strains of
TL059a, TL080, TL093c and TL099a. Among them,
bacteriocin from L. gasseri TL080 showed activity in the
presence of 3% NaCl concentration, but this activity
ended at 4% NaCl. Two bacteriocins from L. casei TL029
and L. gasseri 143a saw no increase in their activity, but
were inhibited by more than 1% NaCl in both Rogosa SL
and MRS media.
Discussion
Because viable lactobacilli can inhibit food-borne and
enteric pathogenic microorganisms by producing lactic
acid and other antimicrobial substances, yogurt and
acidophilus milk have been considered to be healthy
probiotic diets (4).
In this study, of 100 Lactobacillus culture
supernatants examined for acid and bacteriocin
production, six had inhibitory effects on sensitive bacteria
including Lactobacillus strains of human origin and some
common pathogenic bacteria colonized in the human
intestine and vagina.
Among the bacteriocins tested, bacteriocins from L.
gasseri TL93a and TL143a strains had a broader host
range. We observed that all six bacteriocins had an
inhibitory effect on G. vaginalis and P. aeruginosa. In
addition, L. casei TL029 had an inhibitory effect on P.
vulgaris, L. delbrueckii subsp. delbrueckii TL059a had an
inhibitory effect on E. coli, and L. acidophilus TL099a,
and L. gasseri TL143a had an inhibitory effect on E.
cloace. However, none of them affected S. milleri, S.
aureus or C. albicans.
An expanded host range has been noted recently for a
number of Lactobacillus bacteriocins which kill
Enterococcus faecalis, Listeria monocytogenes,
Clostridium botulinum, Candida tyrobutyricum, S. aureus
and Aeromonas hydrophila (1). Toba et al. determined
bacteriocins in six L. gasseri, L. acidophilus JCM 1132 and
L. acidophilus LAPT 1060 strains from infant feces active
against other Lactobacillus stains (12). Kanatani et al.
identified a bacteriocin (acidocin A) from L. acidophilus
TK9201 (13). This bacteriocin had inhibitory effects on
closely related lactic acid bacteria and food-borne
pathogens including Listeria monocytogenes. Itoh et al.
indicated that gassericin A produced by L. gasseri LA39
was one of the most active bacteriocins for use against
enteric pathogens (14).
Silva et al. isolated a low-molecular weight substance
from the Lactobacillus GC strain from the feces of a
healthy person with potent inhibitory activity against a
wide range of gram positive and negative bacteria (15).
Coconnier et al. showed that the L. acidophilus LB strain
was able to kill intracellular S. thyphimurium in the
human intestinal Caco-2 cell culture model, and it
decreased the colonization of bacteria in a mouse model
due to its antibacterial activity (16).
In this study, six bacteriocins were characterized and
tested for their in vitro antimicrobial activity against a
group of microorganisms. All six bacteriocins were
identified and their antimicrobial activity was
differentiated from pH 3.0 to 9.0 and H
2
O
2
by standard
methods. Bacteriocin isolated from L. gasseri TL080 is
considered to be a class IV bacteriocin with its resistance
to chloroform and boiling. However, the other five
bacteriocins shared several common features with other
known Lactobacillus bacteriocins, and their classification
deserves further study.
. ALPAY KARAOLU, F. AYDIN, S. S. KILI, A. O. KILI
11
Spontaneous Inducibility Mitomycin C Inducibility
Bacteriocin Producing Strains
MRS Rogosa-SL MRS Rogosa SL
L. gasseri TL029 0 0 1 1
L. d. subsp. delbrueckii TL059a 1 0 4 4
L. gasseri TL080 1 1 1 2
L. gasseri TL093c 0 3 2 4
L. acidophilus TL099a 2 2 2 3
L. gasseri TL143a 2 3 2 3
Table 3. Inducible characteristics of
bacteriocins in different growth
media (mm).
Bacteriocins characterized in this study were found to
show antibacterial activity at a pH range of 4.0 to 7.0. In
addition, two bacteriocins from L. delbrueckii subsp.
lactis TL059a and L. acidophilus TL099a were active at as
low as pH 3.0 and up to pH 7.0. Tagg et al. reported that
most bacteriocins are resistant to acidic pH more than
basic pH (12). The inhibitory activity of the bacteriocin
isolated from L. acidophilus LB strain occurred between
pH 3.0 and 5.0, and the inhibitory activity was lost when
the pH was raised to 5.3 (17). Plantaricin S produced by
L. plantarum LPC010 showed inhibitory activity from pH
3.0 to 7.0 (11).
To determine the effects of media composition on the
production of bacteriocins, we used commercial Rogosa
SL and Lactobacilli MRS media for culturing lactobacilli
and in bacteriocin assays.
Sphelhaug et al. reported that MRS agar was suitable
for bacteriocin assays of lactobacilli (18). We compared
two media for their effects on bacteriocin production
with or without mitomycin C, which has been shown to
induce bacteriocin production. We found that Rogosa SL
broth increased a bacteriocins of some Lactobacillus
strains significantly compared to MRS broth in the
absence of mitomycin. It was evident that Rogosa SL
medium stimulated high-molecular weight and lipid-
containing bacteriocin production more than MRS broth
did. This effect may partly be due to the presence of high
concentrations of tryptophane in Rogosa SL broth, which
may constitute an important part of the proteinous part
of such bacteriocin. Furthermore, we observed that 1%
NaCl enhanced the bacteriocin production of six
bacteriocins except for two bacteriocins isolated from L.
casei TL029 and L. gasseri 143a strains. Larsen et al.
detected bavaricin A from L. bavaricus M1401 (9). The
production of this bacteriocin showed no changes at 1%
NaCl, but production was inhibited with increaseing
amounts of NaCl.
The natural inhibition of vaginal lactobacilli and some
common pathogenic bacteria by bacteriocins may be
important in understanding the initiation of vaginal
infections or bacterial vaginosis associated with an
unexplained decrease vaginal lactobacilli.
Acknowledgments
The authors would like to thank Drs. Murat Ertrk,
Lin Tao and Sylvia Pavlova for their technical support and
help. This project was partly supported by the Research
Fund of Karadeniz Technical University (97.114.001.15).
Corresponding author
engl ALPAY KARAOLU
Department of Biology,
Rize Faculty of Art & Sciences
Karadeniz Technical University, Rize - Turkey
Antimicrobial Activity and Characteristics of Bacteriocins Produced by Vaginal Lactobacilli
12
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