Production and Characterization of Nisin
Production and Characterization of Nisin
Production and Characterization of Nisin
183–187
DOI: 10.1007/s00284-005-4545-2 Current
Microbiology
An International Journal
ª Springer Science+Business Media, Inc. 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- was used to preserve the cultures at –80C. Isolates were identified by
teria and inhibit the growth of bacteria closely related to their colony morphology, Gram-staining, cell morphology, acid
production using different carbon sources and by 16S rDNA homology.
the producer organism. Some strains of lactic acid bac-
teria (LAB) produce extracellular bacteriocin [10]. For Bacterial strains used in this study. Bacteriocin production was
practical applications of bacteriocin the major problems identified following development of a zone of growth inhibition of the
indicator bacteria around the colonies of the isolates by a spot-on-
involved are their low level of production, narrow
the-lawn method at 37C [17]. We used four indicator strains-
antibacterial spectrum, and low stability [6, 8]. The most Lactobacillus plantarum NCDO 955, Pediococcus acidilactici LB42,
extensively studied bacteriocin, nisin, is produced by Leuconostoc mesenteroides Ly, and Enterococcus faecalis MB1
certain strains of Lactococcus lactis subsp. lactis. It has [18]-to identify bacteriocin-producing LAB. All four strains are
broad-spectrum activity against Gram-positive bacteria. bacteriocin-sensitive and are able to grow at pH 4.0 and above. The
strains were provided by B. Ray, Department of Animal Science,
In the present study, we isolated and characterized a
University of Wyoming, Laramie, Wyoming, USA. L. lactis ATCC
strain of L. lactis from fermented milk which produces 11454 (obtained from Microbial Type Culture Collection (MTCC),
bacteriocin. We also studied the characteristics of the Institute of Microbial Technology, Chandigarh, India), known for its
bacteriocin production and its antibacterial spectrum. nisin production, was used as a positive control.
with pfu DNA polymerase (Fermentas, USA) and a pair of bacteria- against the four bacteriocin-sensitive indicator strains
specific universal primers for 16S rRNA gene (rDNA) sequences [21]. led to identification of an isolate which produced a
The forward primer was 5¢AGAGTTTGATCATGGCTC-3¢ (S-D-Bact-
0027-a-S-18) and the reverse primer was 5¢-CTAGCGATTC
bacteriocin-like substance with an antibacterial activity.
CGACTTCA-3¢ (S-D-Bact-1327-a-A-18); the numbers refer to Both boiled, neutralized and boiled but not neutralized
positions in the 16S rRNA gene of E. coli. PCR was carried out cell-free culture supernatants of the isolate exhibited
with a thermal cycler (Perkin-Elmer) for 35 cycles. Amplified DNA similar inhibitory activity against the indicator strains.
was purified and analyzed by electrophoresis on 0.7% agarose gel and The activity was found to be sensitive to treatment with
compared with a 100 bp DNA ladder as molecular weight marker
(Fermentas, USA).
protease enzymes. The results indicate that the extra-
cellularly released heat-stable antibacterial substance is
Growth and bacteriocin production. Growth and bacteriocin proteinaceous in nature. The isolate fermented milk with
production were studied at 37C in 50 mL tryptone–yeast extract
a concomitant reduction of pH to 4.2.
medium. In a separate experiment carbon source supplement at 1% was
added to the medium to study its effect. A 2% inoculum from an Scanning electron microscopy revealed the isolate
overnight culture was used. Growth was measured turbidimetrically at to be coccoid in structure with a diameter of 1 lm. It is
OD650. An aliquot of culture supernatant was withdrawn at hourly Gram-positive, catalase-negative, VP-positive, and is
intervals and serially diluted. An aliquot from each dilution was able to hydrolyze arginine. The isolate could grow at
spotted on a lawn of L. plantarum NCDO 955 to determine the activity
10C but neither at 45C nor in 6.5% NaCl. The isolate
units (AU) of bacteriocin present per milliliter of culture [3].
did not produce gas from glucose. The data indicate that
Effect of enzymes, heat, pH, and surfactants. Partially purified the strain belongs to the genus Lactococcus. It utilized
bacteriocin preparation (2000 AU/ml) was incubated at 37C with gluconate but not L-arabinose, similar to many species
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
of Lactococcus [4, 11]. The isolate fermented glucose,
residual activity of bacteriocin was determined by a dilution-to- fructose, maltose, lactose, sucrose, trehalose, xylose,
extinction procedure against L. plantarum NCDO 955. To determine ribose, mannitol, amygdalin, and starch, but not raffi-
the effect of heat at different pH, the bacteriocin preparation (2000 AU/ nose. However, the isolate also utilized esculin, in
mL) was adjusted to a desired pH in the range of 2.0 to 12.0 using 1 M HCl contrast to the strain ATCC 19435, the type strain of
or 1 M NaOH. These were then incubated either at 37C for 5 h, at 100C
for 1 h or at 121C for 15 min, readjusted to pH 6.0 for assaying the
L. lactis subsp. lactis [11]. On the basis of acid pro-
residual activity. The effect of SDS, Tween 80, Triton X-100, urea, all at duction from 49 carbohydrates, the isolate was tenta-
1% (v/v), EDTA (10 mM), or mercaptoethanol (50 mM) was determined tively identified as L. lactis having close similarity with
by incubating the partially purified bacteriocin with any of these reagents L. lactis subsp. lactis. In order to study the phylogenetic
at 37C for 5 h. The sample then was diluted 10-fold and assayed for position of L. lactis W8, 1300 nucleotides of the 16S
residual antibacterial activity. Untreated sample was used as control.
rDNA of the bacterium was amplified by PCR, se-
Growth inhibition spectrum of bacteriocin. Boiled and neutralized quenced, and subjected to 16S rDNA sequence analysis.
culture supernatant as well as partially purified bacteriocin obtained by The BLAST result shows 99% nucleotide homology
ammonium sulfate precipitation at 70% saturation were examined for
with the rDNA sequence of L. lactis in the database. The
their property of growth inhibition of Gram-positive and Gram-
negative bacteria by the spot-on-the-lawn method. Bacteriocin was analyses indicate that the isolate is a strain of L. lactis
used at 2000 AU/mL. Partially purified nisin isolated from the culture having close similarity to subspecies lactis.
of the strain L. lactis ATCC 11454 was used for comparison. The kinetics of growth and bacteriocin production
Molecular size approximation by detection of bacteriocin activity
of the strain L. lactis W8 were studied in TGE medium
upon SDS-PAGE. Direct detection of bacteriocin on gel was done as (pH 6.5) at temperatures of 30C, 37C, and 40C
described previously [2]. Activities of ammonium sulfate precipitate of (Fig. 1). The strain started producing the bacteriocin at
bacteriocin preparations from boiled and neutralized cell-free culture the early exponential phase. The activity increased
supernatant were determined following electrophoresis on SDS- concomitantly with an increase in the cell mass and
polyacrylamide (15%) gel. The gel was vertically cut into two halves.
The first half was silver stained to visualize the protein bands, while the
reached its peak at the late exponential phase. The
other half was washed in sterile water to remove SDS and overlaid with highest level of bacteriocin (2000 AU/mL) coincided
TGE soft agar seeded with 106 cells of indicator strain L. plantarum with the highest cell mass production, corresponding to
NCDO 955. It was incubated at 37C and examined for zone of growth OD650 of 1.04 = 1011 cells/mL. It was found that pro-
inhibition caused by bacteriocin within the gel. To determine the size of duction of both cell mass and bacteriocin ceased at the
the bacteriocin, low-range standard peptide (Amersham Pharmacia
Biotech) was used as the molecular weight marker.
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
Results and Discussion
affected by low pH. Previous studies had shown that
A total of 716 bacterial colonies were isolated from although most bacteriocins were produced during the
fermented milk samples collected from different sour- active growth phase, most often bacteriocin level de-
ces. Screening for bacteriocin-producing members clined sharply at the end of the exponential phase [7],
S. Mitra et al.: Nisin-Like Peptide Produced by Lactococcus lactis 185
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 30C (n), 37C (d), and 40C (m), and bacteriocin production at
30C (h), 37C (s), and 40C (n) was measured.
Table 2. Inhibitory spectrum of bacteriocin produced by Lactococcus lactis strain W8 against lactic acid bacteria and other bacteria
L. lactis
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- nisin-producing strain L. lactis ATTCC 11454 or vice
bacterial activity is stable at 37C for 5 h at pH 2.0 to versa but inhibited Pediococcus acidilactici F+, which is
10.0, at 100C for 1 h at pH 2.0 to 6.0, and at 121C for resistant to nisin. The results indicate that the strain L.
15 min at pH 2.0 to 4.0 (Fig. 3). The lower the pH the lactis W8 produced a nisin-like peptide which is similar
higher is the stability of antibacterial substance to high but not identical to nisin. The strain W8 was not
temperature, similar to previous findings for nisin [7, 12, inhibitory to the Gram-negative bacteria examined ex-
20]. The inhibitory activity produced by the strain W8 cept Pseudomonas putida, which is resistant to nisin.
differs from that of nisin produced by L. lactis Bacteriocin of LAB is, however, known to be effective
ATCC11454 in having greater heat and pH stability. only against Gram-positive bacteria. The inhibitory
Nisin is unstable at pH greater than 7.0 and 50% of its activity of the bacteriocin produced by the strain
activity is lost when incubated at 121C for 10 min at pH L. lactis W8 in its crude and partially purified form
4.0 [7, 9, 22] and at 100 for 30 min at pH 6.0. The showed similar patterns of spectra.
antibacterial substance produced by L. lactis W8 also SDS-PAGE analysis of partially purified bacterio-
retained its full activity following exposure to SDS, cin of the strain W8 demonstrated that the antibacterial
Tween 80, Triton X-100, urea, EDTA, and mercapto- activity was due to a short peptide of 2.5 kDa (Fig. 4).
ethanol. The peptide produced a clear zone of growth inhibition
Similar to nisin, the bacteriocin produced by the of the overlaid strain L. plantarum NCDO 955 on SDS
strain L. lactis W8 exhibited a broad spectrum of growth gel. The electrophoretic migration of the inhibitory
inhibition of closely related species as well as of other peptide corresponded to the 2.5 kDa peptide band on the
Gram-positive bacteria (Table 2). The bacteriocins stained gel. The molecular weight of the peptide, thus, is
produced by L. lactis ATCC 11454 and L. lactis W8 lower than that of nisin, which has a molecular weight of
both inhibited Bacillus cereus, Listeria monocytogenes, 3.5 kDa.
Staphylococcus aureus, Clostridium perfringens, and In conclusion, the new strain appears to have im-
Leuconostoc mesenteroides. Bacteriocin-producing proved characteristics in respect of production of
strains are known to have self-immunity [5, 7] and a bacteriocin, growth at an extreme alkaline pH, rapid
bacteriocin produced by one strain does not inhibit lowering of medium pH, and high level of bacteriocin
others producing an identical bacteriocin. Bacteriocin production. Stability of the bacteriocin to heat and a
produced by L. lactis W8, however, did not inhibit the wide range of pH are considered to be very important.
S. Mitra et al.: Nisin-Like Peptide Produced by Lactococcus lactis 187