CURRENT MICROBIOLOGY Vol. 51 (2005), pp.

183–187
DOI: 10.1007/s00284-005-4545-2 Current
Microbiology
An International Journal
ª Springer Science+Business Media, Inc. 2005

Production and Characterization of Nisin-Like Peptide Produced by a

Strain of Lactococcus lactis Isolated from Fermented Milk
Suranjita Mitra,1 Pran Krishna Chakrabartty,2 Swadesh Ranjan Biswas1
1
Department of Botany, Visva-Bharati, Santiniketan 731235, West Bengal, India
2
Department of Microbiology, Bose Institute, P 1/12, CIT Scheme, VII M, Kolkata 700 054, West Bengal, India

Received: 28 December 2004 / Accepted: 29 March 2005

Abstract. An isolate of Lactococcus lactis from fermented milk was found to produce a bacteriocin
peptide. The isolate could grow in a medium with an initial pH of 11.0, in which it produced the
bacteriocin extracellularly at the highest level. The level of the bacteriocin in the medium increased in
parallel to the bacterial growth and reached its peak during the late exponential phase; thereafter it
plateaued. The bacteriocin had a broad antibacterial spectrum similar to that of nisin and inhibited
several related species of lactic acid bacteria and other Gram-positive bacteria. The inhibitory activity of
the bacteriocin was found to be stable over a wide range of pH and temperature. The molecular weight of
the peptide was judged to be 2.5 kDa by SDS-polyacrylamide gel electrophoresis.

Bacteriocins are bactericidal peptides produced by bac-
teria and inhibit the growth of bacteria closely related to
the producer organism. Some strains of lactic acid bac-
teria (LAB) produce extracellular bacteriocin [10]. For
practical applications of bacteriocin the major problems
involved are their low level of production, narrow
antibacterial spectrum, and low stability [6, 8]. The most
extensively studied bacteriocin, nisin, is produced by
certain strains of Lactococcus lactis subsp. lactis. It has
broad-spectrum activity against Gram-positive bacteria.
In the present study, we isolated and characterized a
strain of L. lactis from fermented milk which produces
bacteriocin. We also studied the characteristics of the
bacteriocin production and its antibacterial spectrum.
was used to preserve the cultures at –80
C. Isolates were identified by
their colony morphology, Gram-staining, cell morphology, acid
production using different carbon sources and by 16S rDNA homology.
Bacterial strains used in this study. Bacteriocin production was
identified following development of a zone of growth inhibition of the
indicator bacteria around the colonies of the isolates by a spot-on-
the-lawn method at 37
C [17]. We used four indicator strains-
Lactobacillus plantarum NCDO 955, Pediococcus acidilactici LB42,
Leuconostoc mesenteroides Ly, and Enterococcus faecalis MB1
[18]-to identify bacteriocin-producing LAB. All four strains are
bacteriocin-sensitive and are able to grow at pH 4.0 and above. The
strains were provided by B. Ray, Department of Animal Science,
University of Wyoming, Laramie, Wyoming, USA. L. lactis ATCC
11454 (obtained from Microbial Type Culture Collection (MTCC),
Institute of Microbial Technology, Chandigarh, India), known for its
nisin production, was used as a positive control.

Identification of lactic acid bacteria. The bacteriocin-producing

Materials and Methods
Isolation of lactic acid bacteria. Samples of fermented milk were
collected locally. TGE (tryptone-glucose-yeast extract) medium [3] and
lactic acid bacteria selective Mann Rogosa Sharpe (MRS, Hi Media,
India) medium were used for the isolation of bacteria. Each of the isolates
was streaked on TGE agar plates to obtain pure cultures. From the
isolates one with bac+ (bacteriocin production) phenotype was selected
on the basis of its inhibitory spectrum. TGE broth containing 10% DMSO
isolate was identified by physiological and biochemical tests [5, 15,
21]. The isolate was examined for production of acid from 49 compounds
as sole carbon source using an API 50 CH Test Kit (Bio-merieux,
Crappone, France) according to the manufacturerÕs instructions. The
identification of the isolate was further confirmed by sequencing of a
polymerase chain reaction (PCR) amplified 16S rDNA sequence of the
bacterium [21] with a DNA sequencer (Perkin-Elmer ABI 337). The
homology of the partial sequence was compared with the sequences in
the RDP database employing the BLAST search program [1].

PCR amplification of 16S rDNA. Chromosomal DNA from the

Correspondence to: S.R. Biswas; email: swadeshranjan_biswas@ isolate was prepared by the lysozyme–proteinase K procedure [19].
yahoo.com Genei (Bangalore, India) synthesized the primers. PCR was performed
184
with pfu DNA polymerase (Fermentas, USA) and a pair of bacteria-
specific universal primers for 16S rRNA gene (rDNA) sequences [21].
The forward primer was 5¢AGAGTTTGATCATGGCTC-3¢ (S-D-Bact-
0027-a-S-18) and the reverse primer was 5¢-CTAGCGATTC
CGACTTCA-3¢ (S-D-Bact-1327-a-A-18); the numbers refer to
positions in the 16S rRNA gene of E. coli. PCR was carried out
with a thermal cycler (Perkin-Elmer) for 35 cycles. Amplified DNA
was purified and analyzed by electrophoresis on 0.7% agarose gel and
compared with a 100 bp DNA ladder as molecular weight marker
(Fermentas, USA).
Growth and bacteriocin production. Growth and bacteriocin
production were studied at 37
C in 50 mL tryptone–yeast extract
medium. In a separate experiment carbon source supplement at 1% was
added to the medium to study its effect. A 2% inoculum from an
overnight culture was used. Growth was measured turbidimetrically at
OD650. An aliquot of culture supernatant was withdrawn at hourly
intervals and serially diluted. An aliquot from each dilution was
spotted on a lawn of L. plantarum NCDO 955 to determine the activity
units (AU) of bacteriocin present per milliliter of culture [3].
Effect of enzymes, heat, pH, and surfactants. Partially purified
bacteriocin preparation (2000 AU/ml) was incubated at 37
C with
proteinase K (Fermentas, USA), trypsin, papain, catalase, lipase or a-
amylase (Sigma, USA) to a final concentration of 1 mg/mL for 2 h. The
residual activity of bacteriocin was determined by a dilution-to-
extinction procedure against L. plantarum NCDO 955. To determine
the effect of heat at different pH, the bacteriocin preparation (2000 AU/
mL) was adjusted to a desired pH in the range of 2.0 to 12.0 using 1 M HCl
or 1 M NaOH. These were then incubated either at 37
C for 5 h, at 100
C
for 1 h or at 121
C for 15 min, readjusted to pH 6.0 for assaying the
residual activity. The effect of SDS, Tween 80, Triton X-100, urea, all at
1% (v/v), EDTA (10 mM), or mercaptoethanol (50 mM) was determined
by incubating the partially purified bacteriocin with any of these reagents
at 37
C for 5 h. The sample then was diluted 10-fold and assayed for
residual antibacterial activity. Untreated sample was used as control.
Growth inhibition spectrum of bacteriocin. Boiled and neutralized
culture supernatant as well as partially purified bacteriocin obtained by
ammonium sulfate precipitation at 70% saturation were examined for
their property of growth inhibition of Gram-positive and Gram-
negative bacteria by the spot-on-the-lawn method. Bacteriocin was
used at 2000 AU/mL. Partially purified nisin isolated from the culture
of the strain L. lactis ATCC 11454 was used for comparison.
Molecular size approximation by detection of bacteriocin activity
upon SDS-PAGE. Direct detection of bacteriocin on gel was done as
described previously [2]. Activities of ammonium sulfate precipitate of
bacteriocin preparations from boiled and neutralized cell-free culture
supernatant were determined following electrophoresis on SDS-
polyacrylamide (15%) gel. The gel was vertically cut into two halves.
The first half was silver stained to visualize the protein bands, while the
other half was washed in sterile water to remove SDS and overlaid with
TGE soft agar seeded with 106 cells of indicator strain L. plantarum
NCDO 955. It was incubated at 37
C and examined for zone of growth
inhibition caused by bacteriocin within the gel. To determine the size of
the bacteriocin, low-range standard peptide (Amersham Pharmacia
Biotech) was used as the molecular weight marker.
Results and Discussion
A total of 716 bacterial colonies were isolated from
fermented milk samples collected from different sour-
ces. Screening for bacteriocin-producing members
CURRENT MICROBIOLOGY Vol. 51 (2005)
against the four bacteriocin-sensitive indicator strains
led to identification of an isolate which produced a
bacteriocin-like substance with an antibacterial activity.
Both boiled, neutralized and boiled but not neutralized
cell-free culture supernatants of the isolate exhibited
similar inhibitory activity against the indicator strains.
The activity was found to be sensitive to treatment with
protease enzymes. The results indicate that the extra-
cellularly released heat-stable antibacterial substance is
proteinaceous in nature. The isolate fermented milk with
a concomitant reduction of pH to 4.2.
Scanning electron microscopy revealed the isolate
to be coccoid in structure with a diameter of 1 lm. It is
Gram-positive, catalase-negative, VP-positive, and is
able to hydrolyze arginine. The isolate could grow at
10
C but neither at 45
C nor in 6.5% NaCl. The isolate
did not produce gas from glucose. The data indicate that
the strain belongs to the genus Lactococcus. It utilized
gluconate but not L-arabinose, similar to many species
of Lactococcus [4, 11]. The isolate fermented glucose,
fructose, maltose, lactose, sucrose, trehalose, xylose,
ribose, mannitol, amygdalin, and starch, but not raffi-
nose. However, the isolate also utilized esculin, in
contrast to the strain ATCC 19435, the type strain of
L. lactis subsp. lactis [11]. On the basis of acid pro-
duction from 49 carbohydrates, the isolate was tenta-
tively identified as L. lactis having close similarity with
L. lactis subsp. lactis. In order to study the phylogenetic
position of L. lactis W8, 1300 nucleotides of the 16S
rDNA of the bacterium was amplified by PCR, se-
quenced, and subjected to 16S rDNA sequence analysis.
The BLAST result shows 99% nucleotide homology
with the rDNA sequence of L. lactis in the database. The
analyses indicate that the isolate is a strain of L. lactis
having close similarity to subspecies lactis.
The kinetics of growth and bacteriocin production
of the strain L. lactis W8 were studied in TGE medium
(pH 6.5) at temperatures of 30
C, 37
C, and 40
C
(Fig. 1). The strain started producing the bacteriocin at
the early exponential phase. The activity increased
concomitantly with an increase in the cell mass and
reached its peak at the late exponential phase. The
highest level of bacteriocin (2000 AU/mL) coincided
with the highest cell mass production, corresponding to
OD650 of 1.04 = 1011 cells/mL. It was found that pro-
duction of both cell mass and bacteriocin ceased at the
onset of the stationary phase when the pH of the medium
was lowered to 4.2; thereafter the bacteriocin activity
remained constant, indicating that the activity is not
affected by low pH. Previous studies had shown that
although most bacteriocins were produced during the
active growth phase, most often bacteriocin level de-
clined sharply at the end of the exponential phase [7],
S. Mitra et al.: Nisin-Like Peptide Produced by Lactococcus lactis 185

Table 1. Effect of carbon source on growth (OD650) and bacteriocin

production (AU/mL) of L. lactis W8 following 6 h of incubation at
37
C

Carbon sourcea OD650 AU/mL

Glucose 1.04 2000

Galactose 1.26 2000
Mannose 1.0 2000
Cellobiose 1.48 2600
Lactose 1.06 2000
Maltose 0.98 3000
Trehalose 1.32 2400
Sucrose 1.16 2400
Glycerol 0.22 400

a
Each carbohydrate was added at 1% to tryptone-yeast extract medium,
Fig. 1. Growth and bacteriocin production of Lactococcus lactis W8 pH 6.5.
on TGE broth (pH 6.5) at various temperatures. The strain was grown
at 30
C (n), 37
C (d), and 40
C (m), and bacteriocin production at
30
C (h), 37
C (s), and 40
C (n) was measured.

Fig. 3. Residual activity of the antibacterial peptide of L. lactis W8

Fig. 2. Effect of initial pH on growth (filled columns) and bacteriocin
production (open columns) of L. lactis W8 grown in TGE medium at
37
C for 10 h.
possibly due to protein degradation, adsorption to the
cell surface, protein aggregation or complex formation
[9, 14, 22]. The optimum temperature for both growth
and bacteriocin production of the strain W8 was a broad
one and ranged from 30
C to 37
C, in contrast to pre-
viously known strains of Lactococcus lactis, which grow
and produce bacteriocin optimally only at 30
C [6, 7].
The strain is capable of growth in a medium with an
initial pH of 11.0 (Fig. 2). No LAB, including the lac-
tococci, are known to grow at this high pH, although
isolates from vegetables have been reported to grow at
pH 9.6 [11]. The initial pH of the growth medium was
found to significantly influence both the cell mass and
bacteriocin production. The production of bacteriocin as
well as cell mass increased with the increase in initial
following incubation at 37
C for 5 h (d), at 100
C for 1 h (m) or at
121
C for 15 min (n) at different pH values.
medium pH. However, the terminal pH of the culture
was consistently lowered to 4.2 regardless of the initial
pH. It was found that the lower the initial pH, the earlier
is the cessation of growth. An initial medium pH of 11.0
had most notable effect on bacteriocin production. Un-
der such a condition the activity of the bacteriocin
produced in TGE medium was 6000 AU/mL compared
with 2000 AU/mL when the initial pH was 6.5 (Fig. 2).
Among the various carbohydrates tested, maltose at
1% stimulated bacteriocin production considerably
(Table 1). A combination of maltose and an initial
medium pH of 11.0 resulted in an increased bacteriocin
production (9000 AU/mL).
The inhibitory activity produced by L. lactis W8 is
lost upon treatment with proteolytic enzymes such as
trypsin, papain or proteinase K. This is in contrast to
nisin, which is resistant to trypsin [9, 16, 20], Nonpro-
186
Table 2. Inhibitory spectrum of bacteriocin produced by Lactococcus lactis strain W8 against lactic acid bacteria and other bacteria
Target strain
Lactobacillius delbrueckii ATCC 4797
Lactobacillus plantarum NCDO 955
Lactobacillus rhamnosus ATCC 7469
Lactococcus lactis ATCC 19257
Pediococcus acidilactici LB 42
Pediococcus acidilactici F+
Bacillus cereus MTCC 1272
Clostridium perfringens ATCC 3624
Enterococcus faecalis MB1
Leuconostoc mesenteroides Ly
Leuconostoc mesenteroides ATCC 10830a
Listeria monocytogenes ATCC 19111
Staphylococcus aureus
E. coli EPEC
E. coli ETEC
Enterobacter aerogenes ATCC 13048
Pseudomonas putida
Salmonella typhimurium ATCC 23565
Vibrio cholerae
L. lactis ATCC 11454 was used as positive control.+, strong inhibition (15 mm and over); –, no inhibition.
MTCC, Microbial Type Culture Collection, Institute of Microbial Technology, India; BR, Bibek Ray, University of Wyoming, USA; LC,
Laboratory Collection; NICED, National Institute of Cholera and Enteric Diseases, Kolkata, India.
teolytic enzymes did not affect the activity. The anti-
bacterial activity is stable at 37
C for 5 h at pH 2.0 to
10.0, at 100
C for 1 h at pH 2.0 to 6.0, and at 121
C for
15 min at pH 2.0 to 4.0 (Fig. 3). The lower the pH the
higher is the stability of antibacterial substance to high
temperature, similar to previous findings for nisin [7, 12,
20]. The inhibitory activity produced by the strain W8
differs from that of nisin produced by L. lactis
ATCC11454 in having greater heat and pH stability.
Nisin is unstable at pH greater than 7.0 and 50% of its
activity is lost when incubated at 121
C for 10 min at pH
4.0 [7, 9, 22] and at 100
for 30 min at pH 6.0. The
antibacterial substance produced by L. lactis W8 also
retained its full activity following exposure to SDS,
Tween 80, Triton X-100, urea, EDTA, and mercapto-
ethanol.
Similar to nisin, the bacteriocin produced by the
strain L. lactis W8 exhibited a broad spectrum of growth
inhibition of closely related species as well as of other
Gram-positive bacteria (Table 2). The bacteriocins
produced by L. lactis ATCC 11454 and L. lactis W8
both inhibited Bacillus cereus, Listeria monocytogenes,
Staphylococcus aureus, Clostridium perfringens, and
Leuconostoc mesenteroides. Bacteriocin-producing
strains are known to have self-immunity [5, 7] and a
bacteriocin produced by one strain does not inhibit
others producing an identical bacteriocin. Bacteriocin
produced by L. lactis W8, however, did not inhibit the
CURRENT MICROBIOLOGY Vol. 51 (2005)
L. lactis
Source W8 ATCC 11454
MTCC + +
BR + +
MTCC + +
MTCC + +
BR + +
BR + )
MTCC + +
MTCC + +
MTCC + +
BR + +
MTCC + +
MTCC + +
LC + +
NICED ) )
NICED ) )
MTCC ) )
LC + )
MTCC ) )
NICED ) )
nisin-producing strain L. lactis ATTCC 11454 or vice
versa but inhibited Pediococcus acidilactici F+, which is
resistant to nisin. The results indicate that the strain L.
lactis W8 produced a nisin-like peptide which is similar
but not identical to nisin. The strain W8 was not
inhibitory to the Gram-negative bacteria examined ex-
cept Pseudomonas putida, which is resistant to nisin.
Bacteriocin of LAB is, however, known to be effective
only against Gram-positive bacteria. The inhibitory
activity of the bacteriocin produced by the strain
L. lactis W8 in its crude and partially purified form
showed similar patterns of spectra.
SDS-PAGE analysis of partially purified bacterio-
cin of the strain W8 demonstrated that the antibacterial
activity was due to a short peptide of 2.5 kDa (Fig. 4).
The peptide produced a clear zone of growth inhibition
of the overlaid strain L. plantarum NCDO 955 on SDS
gel. The electrophoretic migration of the inhibitory
peptide corresponded to the 2.5 kDa peptide band on the
stained gel. The molecular weight of the peptide, thus, is
lower than that of nisin, which has a molecular weight of
3.5 kDa.
In conclusion, the new strain appears to have im-
proved characteristics in respect of production of
bacteriocin, growth at an extreme alkaline pH, rapid
lowering of medium pH, and high level of bacteriocin
production. Stability of the bacteriocin to heat and a
wide range of pH are considered to be very important.
S. Mitra et al.: Nisin-Like Peptide Produced by Lactococcus lactis
Fig. 4. SDS-PAGE and antimicrobial activity of partially purified
bacteriocin. Lane 1, low-molecular-weight protein markers (the values
are numbers in kDa); lane 2, partially purified bacteriocin of Lacto-
coccus lactis W8 (silver stained); lane 3, zone of Inhibition of Lac-
tobacillus plantarum NCDO955 by the bacteriocin of Lactococcus
lactis W8 on SDS gel.
L. lactis is a food-grade microorganism, widely used in
food industries and has GRAS status [13]. As such, the
bacteriocin produced by the strain W8 has a potential to
be used as a natural food preservative.
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