Bacteriocin from Streptococcus thermophilus Against Bifidobacterium

Full Paper
Bioscience Microflora Vol. 18 (2), 125-131, 1999
Bacteriocin
Produced
Bifidobacterium
Nagendra
P. SHAH* and
thermophilus
against
Species
Linh
LY
School of Life Sciences
and Technology,
Centre, VIC 8001, Australia
Received
by Streptococcus
April 26, 1999; Accepted
Victoria University
for publication,
August
of Technology,
Werribee
Campus,
PO Box 14428 , Melbourne
City Mail
24, 1999
This study was carried out to evaluate the antagonistic relationship between yogurt and probiotic bacteria and the
nature of the inhibitory compound produced by the organisms. Eight strains each of Streptococcus thermophilus,
Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus and bifidobacteria were isolated from eight
commercial AB (L. acidophilus and Bifidobacterium spp.) products containing these four groups of bacteria. The isolates
were screened for the production of bacteriocins against each of the 8 isolates of L. acidophilus and Bifidobacterium spp.
Twelve strains showed inhibitory activity against all the 8 strains of Bifidobacterium spp. and 5 L. acidophilus isolates
with the 'spot on lawn' assay. Of these, only one yogurt bacterium, S. thermophilus was identified to be a bacteriocinproducing organism. The S. thermophilus strain was found to specifically target 2 strains of bifidobacteria. The crude
antimicrobial compound was found to be heat stable, resistant over a wide range of pH, and sensitive to proteolytic
enzymes, but it retained activity after treatment with lipase. The compound was purified using ultrafiltration,
precipitation with ammonium sulfate and dialysis. The bacteriocin-fractionate was also subjected to SDS-PAGE analysis
and the molecular weight of the bacteriocin was estimated to be approximately 80 kDa.
Key words:
yogurt bacteria; probiotic bacteria; antagonism; viability
Bacteriocins are defined as 'proteinaceous compounds that show antibacterial activity against closely
related species' (17). While the definition holds true
for majority of bacteriocins, it is now evident that bacteriocins may act beyond closely related species or those
confined within the same ecological niche (9). The presence of bacteriocin producing organisms can influence
or alter the stability of a culture. Although bacteriocins
have been studied for many years, much of work has
focused on evaluating the performance of bacteriocins
as inhibitors for pathogens for food preservation. A recent study by Joseph et al. (6) reported that the viability of probiotic organisms was related to antagonism
between yogurt and probiotic bacteria.
The viability of probiotic bacteria has been a serious
problem. Several factors have appear to be responsible
for the viability of probiotic bacteria including acid produced during fermentation and storage, dissolved oxygen and antimicrobial substances produced by yogurt
bacteria against probiotic bacteria. Dave and Shah (3)
reported that a strain of bifidobacteria lost its viability
in yogurt made from starter culture containing S.
thermophilus, and bifidobacteria. This inhibition was
presumed to be due to production of antimicrobial substances produced by S. thermophilus against bifidobacteria.
The aims of this study were to (i) determine antago-
INTRODUCTION
The benefits derived from the consumption of
probiotics such as Lactobacillus acidophilus and
Bifidobacterium spp. (known as AB products) are well
documented (4, 8, 11, 15). Probiotic bacteria grow
slowly in milk, so the usual practice is to add yogurt
bacteria, Streptococcus thermophilus, and Lactobacillus delbrueckii subsp. bulgaricus to enhance the fermentation process to obtain a milk product of 'excellent therapeutic value' (10). Presently, over 90 products containing probiotics are available in the market
worldwide. To achieve health benefits, the suggested
minimum level of probiotic bacteria is 106viable cells
per gram of a product (3). Despite the importance of
the viability of these beneficial bacteria, various studies have shown poor survival of probiotic organisms,
especially bifidobacteria in fermented foods (12, 1416). The exact cause for loss of viability is uncertain;
however, bacteriocin produced by yogurt bacteria
against probiotic bacteria is likely to be one of the factors responsible for the loss of viability of probiotic
bacteria.
*Corresponding
author . Mailing address: School of Life Sciences and Techof Technology, Werribee Campus, PO Box 14428,
nology, Victoria University
Melbourne City Mail Centre, VIC 8001, Australia.
Fax: +61-3-9216-8284.
Phone: +61-3-9216-8289.
125
126
N.P. SHAH and L. LY
nism
between
commercial
yogurt
bacteria
yogurts,
and
and
(ii)
crobial
substance
produced
against
probiotic
organism(s).
probiotic
bacteria
characterise
by
the
yogurt
in
tine
antimi-
bacterial
cocci,
organism(s)
(MRS-C)
MATERIALS
subsp.
bifidobacteria
AB
Thomastown,
ucts
Pty.
Ltd.,
N.
Foods
Ltd.,
Ltd.,
S. Brisbane,
Ltd.,
Lidcom,
QLD),
Gardens,
Products,
of
in
isolation
of
tified
their
Eve
Balance
and
Manual
cies
and
Shah
(5,
used
(Table
for
codes.
names
Maintenance
of
bacterial
in
skim
milk
(RSM).
was
additionally
teria
isolates
the
and
and
18
hr.
at
cryogenic
20•Ž
All
use,
frozen
cultures
and
maximum
ture
vials
as
was
were
made
10
were
subsp.
bulgaricus,
the
ST
agar
cul-
hydrochloride
into
RSM
for
RSM
the
growth
was
incubated
of
inoculated
at 37°C
for
were
then
(Iwaki
Glass,
Canada)
RSM
the
on
before
frozen
stock
a new
to
for
(4,500
15R
centrifuge
USA).
The
basis
For
for
a
(1)
rpm,
untreated,
and,
catalase
(3)
filter
sterilised
rou-
filter
and
dispensed
of
incu-
inhibition
agar
for
inhibition
purple
was
to
as-
was
used
used
for
of
the
of
the
set
broth
min,
na-
organ-
employed
acids
and
were
wells
collected
using
seeded
organism.
poured
in
cut
described.
4•Ž)
hydrogen
was
were
was
me-
activity.
indicator
previously
the
liquid
at 45°C
agar
by
in
the
and
initial
the
was
held
of
the
(18)
bacteriocin
(0.9%)
to
in
determine
McGiven
of organic
sterile
into
the
Cell-free
by
su-
centrifuga-
a Beckman
CS-
Instruments,
Palo
Alto,
CA,
extract
was
into
three
por-
(2)
neutralised
mg/ml).
by
and
examined
For
positive
neutralised
(0.05-0.1
cul-
were
thermophilus
were
wells.
study
(Beckman
crude
at
1.0%
All
tested
ml
15
the
plates
S.
that
culture
left
over
solidify
in this
agar
and
room
(2).
presence
the
working
MRS
the
25
plates
tion
NaOH
and
and
from
before
bromocresol
diffusion
pernatant
stored
of
Tagg
with
into
plates
the
well
MRS
cultures
duplicates.
the
overnight
at
the
with
for
around
of effects
1.0%
or-
incubated
organisms.
in
addition
left
spotted
aerobically
by
as
1.0%
the
other
out
agar
broth
cultures.
left
solidified
and
a weekly
were
anaerobic
tions:
in
Plates
petri
transferred
twice
seeded
used
Approximately
cultures.
propagated
agar
potential
approximately
media
into
bifidobac-
oxidation-reduction
0.9%
technique
screen
with
Finally,
of
agar
peroxide.
(0.05%)
3 hr.
ml
organisms
Elimination
(12%)
plates
produced
The
Bacterial
sealed
and
substance
described
cultures.
were
inhibitory
dia
with
wells
then
the
was
producer
then
the
isms.
the
the
organisms
organisms
were
by
of
and
wells
of
devoid
of
the
followed
were
borer
of
were
thermophilus
group
of
7 mm
migration
incubation,
says,
ture
a sterile
bottom
Wells
Plates
for
inhibition
S.
solidify.
was
allow
to
carried
the
other
to
culture
at 37°C,
of
com-
correlate
reconstituted
hr
zones
screening
product
of
72
Producer
spe-
to the
for
After
S.
necessarily
sterile
subcultures
from
assays
for
Bifidobacterium
reputation
enhance
stock
subcultured
of
not
the
to
each
coagulated
anaerobically
to
ST
The
and
bio-
follows:
agar.
producer
bated
according
according
incorporated
of
2 ml
for
L-Cysteine
Sterile
The
their
comparing
wells.
ac-
to
bifidobacteria.
isolate
and
by
left
using
2 hr
ssp.
iden-
and
the
strains.
for
BB
do
lower
medium
test
as
the
the
maintained
were
organpoured
for
indicator
inhibitory
agar
agar
for
of
1.0%
sterile
10
lawn
some
of
overnight
further
on
with
millilitres
agar
the
spot
used
probiotic
0.9%
for
un-
and
the
into
The
was
detection
fresh
37°C
(2),
yogurt
of
of
broth
the
with
layer
strepto-
spp.
(17)
preliminary
by
plates
with
used
the
et al.
The
50 ƒÊl
for
MRS
hydrochloride
activity.
Tagg
petri
overlaid
were
patterns
protect
designated
were
(0.15%)
delbrueckii
confirmed
1-8
the
tures
cultures.
isolates
L. delbrueckii
pany,
of
temperature
appropriately
The
catalase
and
To
to
Dairy
media
L.
designated
numbered
brand
ganism
(Nestle
medium
(2).
and
L. acidophilus,
1).
Foods
7, 13).
were
were
Vale
was
bifidobacteria
were
LB
for
of
bacterial
and
fermentation
thermophilus,
sealed
used
and
Bifidobacterium
discarded.
through
of
thermophilus,
carbohydrate
LA
Nestle
selective
staining
isolates
Foods
was
L-cysteine
inhibitory
by
into
Foods,
Natural
(Dairy
sample
characteristics
The
Co-operative
The
Gram
Bergey's
portion
plating.
and
by
cut
cut
water
to Dave
chemical
then
and
S.
sterile
(National
(Aust.
of
Twenty-five
Industries
(Aust.
L. acidophilus
cording
into
for
for
(QUF
peptone
pour
bulgaricus,
Pty.
Prod-
Cream
used
produced
Yoplus
Natural
representative
by
Foods
isms.
0.05%
devised
VIC),
identification
sterile
followed
activity
broth
lactobacilli
stated.
modification
VIC).
and
gram
diluted
Ski
and
commercial
Dairy
(Regal
method
Vaalia
So
NSW),
Mulgrave,
Isolation
(Jalna
VIC),
NSW),
eight
Bulla
Melbourne,
NSW),
Tarenpoint,
the
Jalna
Morwell,
acidophilus
from
VIC),
L.
for
with
was
otherwise
Detection
thermophilus,
L.
isolated
yogurts:
Ltd.,
Cla.
S.
bulgaricus,
were
probiotic
One
METHODS
products.
delbrueckii
Ltd.,
AND
ST
broth
supplemented
less
Commercial
culturing,
MRS
passing
into
divided
to
pH
All
the
through
sterile
to pH
6.0
6.0
treated
with
portions
were
and
three
a 0.45 ƒÊm
Eppendorf
using
2
membrane
tubes.
The
BACTERIOCIN
tubes
were
allow
for
the
incubated
enzyme
with
200
,u1 of
each
room
temperature
Plates
were
zones
of
of
enzymes,
by
the
agar
diffusion
carried
out
in duplicate.
the
each
of
the
formed
to
isolates
(LA)
as
produced
(which
will
isolate
be
referred
Bifidobacterium
(ST-1)
The
from
the
by
the
supernatant
cooled
in
sayed
for
antimicrobial
sterile
Effect
an
4
by
the
the
isolate
as
ST-1
against
(BB)
of
L.
per-
pH
Partial
by
30
min
was
isolated
fur-
bath
6.0.
at 50,
60,
at
the
tubes
and
the
121°C
used
bacteriocin
of
the
of
as
in
activity
using
was
as-
BB-2
as
was
inoculated
mophilus-1
ST-1
with
and
and
estimation
bacteriocin.
Sterile
1%
incubated
of
the
at
ST
active
37•Ž.
The
were
acid
ammoat 0•Ž)
was
was
The
by
resulting
containing
using
am-
a dialysis
the
their
inhibitory
to
stirred
collected
0•Ž).
6.0)
for
tubing
24
hr
at 4•Ž.
solutions
activity
were
using the
technique.
protein
the
was
was
first
mini
boiled
containing
materials
to SDS-
protein
II dual
Hercules,
CA,
for
at
5 min
USA).
100•Ž
2-mercaptoethanol
required
the
subjected
Bio-Rad
Laboratories,
buffer
Eight
for
procedures
or
water
isolates
the
in
and
preparation
of Walker
spp.
were
dia
protocols
and
Kailasapathy
duce
therapeutic
counts
should
(19),
of
unless
the
oth-
be
found
of
thermophilus
of
molecular
(500
strain
S.
the
ml)
ther-
pH
old.
of
to be
average
observed
was
Dave
be
viable
of
Shah
that
in
counts
cycles
in
most
of
between
the
suggested
the
of
bifidobacteria
lower
count
(data
to
gram
of
a product.
and
bifidobacteria
per
log
not
shown).
meRybka
probiotic
cells
two
(2).
of
number
L. delbrueckii
selective
order
the
L. acidophilus
and
the
and
suggested
almost
viable
to
However,
products
106
counts
Bifidobacterium
using
by
benefits,
the
L. delbrueckii
and
successfully
(10)
were
broth
thermophilus,
acidophilus
suggested
the
S.
DISCUSSION
of S.
L.
isolated
Although
target
AND
each
bulgaricus,
measured
initial
retentate
stated.
subsp.
by
a 37°C
was
mixture
M, pH
remaining
and
inhibitory
el,
supernatant
(0.001
RESULTS
Similarly
lactic
the
dialysed
and
min.
bacteriocin
10%
(291
min,
10,
with
level
process,
immedi-
produced
incubation
method,
erwise
a control.
crude
sterile
antimicrobial
followed
90
BB-2.
of
4 hr
gel
70,
supernatant
was
using
All
15
liquid
buffer
sample
SDS.
for
the
30
5,
concentrated
fractionated
precipitate
bac-
Ultrasart
and
fractionation
(Bio-Rad
of
80,
the
rpm,
were
Slabcell
Aliquots
were
against
broth
10
(4,500
bath,
using
sample
ST-1
an
proteins
After
(4,800
purified
50 ƒÊl
an
was
saturation
analysis
of
active
using
other
re-
stored
purification.
The
and
partially
supernatant
for
technique.
diffusion
a
to
was
of
The
A 200 ƒÊl
using
filter-sterilised
supernatant
the
pH
superna-
presence
PAGE
pro-
cell-free
material
for
ice
diffusion
bacteriocin
The
supernatant
an
for
removed
4•Ž)
The
bacteriocin.
every
agar
were
min,
Germany)
the
citrate
cells
15
AG,
membrane
and
and
ten-fold
agar
sulfate
culture
membranes.
at 50%
in
assayed
S.
were
to
and
3 hr
After
BB-2
kDa
centrifugation
against
The
centrifuging
adjusted
20
sulfate
broth
containing
assayed
the
the
the
and
The
starting
bacteriocin
nium
monium
against
(BB-2).
of
incubated
purification
of
(Sartorius
using
suspension
organism.
weight
unit
using
6.0
extract
concentrated
were
yogurt
substance
the
Sartorius
against
S. thermophilus
against
activity
diffusion
kDa
for
as
was
precipitate
and
was
bacteriocin)
stability
After
50
20
activity
bacteriocin
screened
of
stability
obtained
1 to
Cell
pH
concentrated
adjusting
rpm,
cells.
neutralized
retentate
obtained
organisms
and
remaining
agar
the
antimicrobial
two
Samples
M NaOH.
2
to
with
by
CS-15R.
adjusted
teriocin
were
This
on
to pH
bath,
con-
isolates
Cell-free
ice
S. thermophilus.
adjusted
32
MRS
of pH
was
The
(final
experiments
activity
treatments,
ately
treated
reacting
determined
preparations
was
5 and
heat
Centrifuge
used
yogurt
on
4•Ž)
for
the
incubator
5.0-5.5
incubation,
(4,500
and
was
of
bacteriocin
were
100•Ž
the
product.
min,
After
from
centrifugation
proteolytic
for
spp.
latter
culture
12
removed
by
at 4°C
and
inhibitory
to
temperature
rpm,
was
remaining
organisms.
S. thermophilus.
overnight
hr
any
bifidobacteria
one
of
studied.
duced
12
move
Louis,
were
of
Only
ther
of
After
tant
St.
The
the
thermophilus
Effect
hr.
zones
37•Ž
proteinaceous
same
6
super-
at
purification
commercial
(ST-1)
every
and
Co.,
The
indicator
determine
bacteria.
maintained
at
incubated
yogurts
eight
probiotic
was
material.
lipase
activity
S. thermophilus.
in
to
pH
containing
and
organisms
adjusted
this
2 hr
Chemical
technique.
commercial
acidophilus
was
and
free
to
and
were
remaining
Characterisation
from
medium
HC1
127
showed
6.0)
sensitivity
(Sigma
samples
the
by
test
Cell
(pH
for
mg/ml)
after
produced
for
that
papain,
hr
and
enzymes.
neutralising
which
stand
previously
organisms
tested
The
the
filled
Beckman
was
1.0
to
bath
then
the
BY BACTERIA
measured.
chymotrypsin,
USA).
an
of
described
after
MO,
water
were
to
as
producer
centrations
left
diffusion
to proteolytic
catalase
a 37•Ž
and
incubated
the
inhibition
in
Wells
sample
inhibition
from
with
2 hr
reaction.
for
then
Sensitivity
natant
for
PRODUCED
pro-
lower
than
ssp.
bulgaricus,
the
those
products
105-106
in one
was
threshof
the
128
N.P. SHAH and L. Dr
Table
1.
Preliminary
inhibitory
screening
activity
of yogurt
against
11mm, (+++) = 11-13mm, (++++) = >13mm,
and probiotic
probiotic
bacteria
isolates
for
organisms.
(-) = no zone. Zone size is inclusive of well
diameter.
Inhibitory activity was not observed by LA-1, LA-7, and LA-8. Inhibition of LA-6 to LA-8 was
not observed.
The 32 isolates were screened for the presence of
inhibitory activity against the eight strains each of L.
acidophilus and Bifidobacterium spp. Table 1 summarizes the results from the preliminary screening of sensitive organisms to antagonistic activity from the producer organisms using the spot on the lawn method.
The results showed that bifidobacteria strains were more
vulnerable to inhibitory activity as evidenced by much
larger zones of inhibition compared to L. acidophilus
strains. Inhibition of several bifidobacterial strains was
observed by a majority of producer strains of L. acidophilus, two strains of S. thermophilus and three strains
of L. delbrueckii subsp. bulgaricus. However, only
minimal inhibition zones were observed by two L. acidophilus strains, three L delbrueckii subsp. bulgaricus,
and two strains of Bifidobacterium spp. against five of
the indicator L. acidophilus strains.
L. acidophilus
hibitory
effect
thermophilus,
bifidobacteria
producer
strains
on bifidobacteria
posed a greater inisolates
than S.
L. delbrueckii
subsp. bulgaricus
and
producer strains. An exception
was the
ST-1 producer
strain, which distinctively
targeted the
BB-2 and BB-3 strains, producing
zones of inhibition
greater than 13 mm.
As the inhibitory
substance
may be due to organic
acid, hydrogen peroxide
or bacteriocin,
the agar diffusion assay was performed
for the confirmation
type of antagonistic
compound.
Table 2 shows
of the
the in-
hibitory activity of the producer
organisms
after neutralization
and treatment with catalase of the cell-free
supernatant
in order to eliminate the effects of acid and
hydrogen
peroxide.
S. thermophilus-1
was the only
strain found to be active against its target organisms,
while all the other strains lost their inhibitory
activity.
BACTERIOCIN
Table
Zones
ST1
and
2.
Agar diffusion
of inhibition:
producer
BB3
+ = zones
organism
was
screening
PRODUCED
BY BACTERIA
of organisms
possessing
<9mm,
++
the
organism
only
= 10-13mm,
found
- = no
129
antimicrobial
activity.
zone.
to have ƒÕantibacterial
activity
against
BB2
organisms.
Treatment of the supernatant to eliminate possible inhibitory effects of organic acids (neutral pH) and hydrogen peroxide (reaction with catalase) confirms the
inhibitory compound of ST-1 to be a bacteriocin (referred to as ST-1 bacteriocin), as shown in Table 2.
From the plate count results, the viable counts of
bifidobacteria in yogurts were found to be lower, especially in the commercial product from which ST-1 was
isolated (data not shown). This may suggest that the
survival of bifidobacteria may be related to the inhibitory compound produced by ST-1. Dave and Shah (3)
reported that a strain of bifidobacteria lost its viability
in yogurts made from commercial starter cultures that
contained yogurt and probiotic bacteria. Their investigation could be related to the production of antimicrobial substances by yogurt organisms present in yogurt.
The inhibitory activity of the ST-1 broth was lost
after treatment with proteolytic enzymes including chy-
motrypsin and papain, while the activity was unaffected
after treatment with lipase. These results agree with the
study of Aktypis et al. (I) which noted the same effect
of thermophilin T after the treatments with chymotrypsin, papain, and lipase.
Bacteriocins possess a number of properties that allow them to be identified according to their molecular
weight, susceptibility to enzymes, heat tolerance, and
resistance to low pH. Some characteristics of ST-1 bacteriocin was studied including sensitivity to various temperatures and pH, and the molecular weight of the bacteriocin was determined using sodium SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) analysis.
Table 3 shows the effect of temperature on the crude
ST-1 bacteriocin and Table 4 the effect of pH on stability of ST-1 bacteriocin. The bacteriocin retained full
activity after various heat treatments including auto-
130
N.P. SHAH and L. LY
Table
3.
Effect of temperature
on the stability
Table
4.
Effect of pH on stability
of ST-1 bacteriocin.
of the ST-1 bacteriocin.
Fig. 1.
SDS-PAGE
of purified
claving at 121°C for 15 min and was active over a wide
range of pH. The bacteriocin was found to be optimally
active at pH between 6 to 10, and some inactivation
occurred at pH between 3 to 5. However, total loss of
activity was observed below pH 2.0. These findings
agree with the results of Dave and Shah (3) and Aktypis
et al. (I). `Thermophilin T,' a bacteriocin produced by
S. thermophilus ACA-DC 0040 was found to be stable
at pH between 1 to 9, and inactivation of the bacteriocin occurred only at pH 10 to 12. The bacteriocin was
heat stable at 121°C for 30 min without any loss of
activity.
A series of purification steps were employed to partially purify the bacteriocin and to determine the molecular weight of the putative bacteriocin compound.
The compound was concentrated by passing the supernatant through 5, 10, and 20 kDa membrane filters (Sartorius). After screening for inhibitory activity of the
retentate and permeate fractions, the retentate fraction
ST-1 bacteriocin.
only
displayed
weight
of
20
kDa.
tio
10)
activity.
Thus
bacteriocin
appeared
to
concentrated
retentate
(concentration
the
The
was
precipitated
saturation).
Some
tionation
say.
inhibitory
stage,
lost
activity
as
However,
using
to
a considerable
After
dialysis,
the
mined
using
modification
tein
remaining
in
approximately
purified
still
point,
80
obtained
bacteriocin
concentration
not
of Lowry
last
kDa.
ra(50%
this
frac-
diffusion
as-
activity
was
was
(3).
was
deter-
The
to
be
of
the
weight
of
ap-
band
of
the
yogurts
sold
in
the
the
inhibitory
a molecular
1 shows
the
found
pro-
analysis
SDS-PAGE
Figure
from
than
shown).
assay
fractionate
greater
after
agar
(data
showed
bacteriocin
the
the
extent
the
bacteriocin
proximately
remained
by
molecular
sulfate
protein
3.6 ƒÊg/ƒÊl.
be
ammonium
determined
at this
the
SDS-PAGE.
CONCLUSIONS
In
this
Australian
study,
market
eight
were
commercial
assessed
for
ac-
BACTERIOCIN
PRODUCED
tion of bacteriocins released by yogurt bacteria against
probiotic bacteria. Of the 32 isolates obtained, 12 strains
(consisting of L. acidophilus 2-6, S. thermophilus 1
and 5, L. delbrueckii ssp. bulgaricus 1,4, and 5, and
bifidobacteria 4 and 6) were identified to release inhibitory activity against the eight isolates of Bifidobacterium spp. and five strains of L. acidophilus. However, only one strain of yogurt bacteria, S. thermophilus
(ST-1) was found to be a bacteriocin-producing organism. The bacteriocin compound was proteinaceous in
nature as confirmed by treatment to various proteolytic
enzymes.
The ST-1 strain was found to target the Bifidobacterium sp.-2 (BB-2) and Bifidobacterium sp.-3 (BB-3).
The bacteriocin was heat stable, even after autoclaving
for 121°C for 15 min. Further, the inhibitory compound
was found to be active at a pH range of 6 to 10.
Purification of the bacteriocin was carried out by
ultrafiltration, precipitation with ammonium sulphate
and dialysis. SDS-PAGE of the bacteriocin showed that
it had a molecular mass of approximately 80 kDa.
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