Download Purification and Characterization of Bacteriocin Produced by Lactococcus lactis

Journal of Applied Sciences Research, 4(11): 1315-1321, 2008
© 2008, INSInet Publication
Purification and Characterization of Bacteriocin Produced by
Isolated Strain of Lactococcus lactis
1
Hanaa A. El-Shafie, 2Nagwa Ibrahim, 2Hanem Abd El-Sabour and 1Yomna A. Mostafa.
1
Microbial Chemistry Department, National Research Centre, Cairo, Egypt.
2
Nutrition Department, Girl College, Ain Shams University, Egypt.
Abstract: A bacteriocin-producing Lactococcus lactis isolated previously from traditional food. The
bacteriocin termed nisin was purified to homogeneity by two-steps purification procedures including 70
% ammonium sulphate precipitation and cation exchange chromatography (DEAE cellulose). Using this
procedure resulted in about 47.2 fold increase in specific activity and a recovery 26.4% of activity.
The specific activity obtained was 9,917IU/mg when compared with the batch fermentation without
recovery (2,105IU/mg). This bacteriocin has a bactericidal mode of action towards the tested spoilage and
pathogenic strains. The Amino acid sequence determined for purified isolated strain exhibited high levels
of similarity to the sequence of the reference strain. Results indicated excess of aspartic acid, valine,
serine, proline and a deficiency of histidine, glycine, alanine, leucine, isoleucine in the isolated strain
compared to the reference. NM R analysis revealed the presence of unsaturated amino acids dehydroalanine
{DHA} and dehydrobutyrine {DHB}.
Key word: Bacteriocins, Lactococcus lactis, nisin, bacteriocin purification
INTRODUCTION
In fermented foods, lactic acid bacteria (LAB)
displays numerous antimicrobial activities. This is
mainly due to the production of antimicrobial
metabolites including organic acids, bacteriocins and
antifungal peptides. Bacteriocin generally exert their
antimicrobial action by interfering with the cell wall or
the membrane of target organisms, either by inhibiting
cell wall biosynthesis or causing pore formation,
subsequently resulting in death [2 1 ]. Several bacteriocins
with industrial potential have been purified and
characterized.The highly promising results of these
studies underline the important role that functional,
bacteriocinogenic LAB strains may play in the food
industry as starter cultures, co-cultures, or bioprotective
cultures, to improve food quality and safety [4 ].
Lactococcus lactis strains are widely distributed
used as starter for several types of cheese, fermented
milk products,and ripened cream butter. T he
fermentation of sugars, leading to a pH decrease which
is important for milk clotting and reduction or
prevention of adventitious microbial growth [1 5 ].
In the last years, the bacteriocins of lactic acid
bacteria have attracted much attention because of their
potential to increase safety and to extend shelf life of
food [5 ]. Currently, only nisin has been Granted
Generally Recognized As Safe (GRAS) status by the
Food and Drug Administration (FDA). In addition,nisin
used in combination with surfactants, chelators and
adjuvants is also effective against both Gram -negative
and resistant Gram-positive bacteria. Many lactic acid
b a c teria p ro d uc e p ro teinaceo us antim icrob ials
bacteriocins, some of which could provide valuable
alternatives to traditional therapeutic antibiotics for the
treatment of infectious diseases [ 1 9 ] . Two such
bacteriocins, nisin and lacticin 3147, which are
produced by strains of Lactococcus lactis are potential
candidates. Nisin Z [1 7 ] and nisin Q [2 5 ] are two natural
variants of the original nisin A, differing in their
propeptide components from nisin A by one and four
amino acids respectively. W e report here the
purification and characterization of nisin , a novel nisin
variant,by Lactococcus lactis.
M ATERIALS AND M ETHODS
M icroorganisms and Culture M edia: Lactic acid
bacterial strain was isolated from yoghurt obtained
from Cairo local market and identified in our
laboratory as Lactococcus lactis[6 ]. Routine cultures
were maintained on MRS media.
Lyophilized reference strain Lactococcus lactis
subsp.lactis NRRL B-1821obtained from the National
Centre for Agricuture Utilization Research USA was
grown and maintained on ATCC17 medium.
Extraction and Purification of Nisin: One liter of
Corresponding Author: Hanaa A. El-Shafie, Microbial Chemistry Department, National Research Centre, Cairo, Egypt.
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J. Appl. Sci. Res., 4(11): 1315-1321, 2008
MRS
broth
was inoculated with 1% (vol/vol)
inoculum from an overnight MRS broth culture of
Lactococcus lactis and incubated for 24 hr. at 37 o
C. T he supernatant was separated by centrifugation
(7000 *g for 30 min at 4 o C) , adjusted to pH 3.0,
forwardly harvested with 60-80% ammonium sulphate.
The supernatant containing unbound protein was
discarded & the precipitated protein was collected by
centrifugation, resuspend in 0.01 N HCl and dialyzed
against 9 m Urea –HCl (pH 3.0). The bacteriocin was
loaded on DEAE-cellulose sephadex gel filtration
column, pre-equilibrated with the elution
buffer
(20 mM acetate buffer ,pH 3.6) .Finally bound
bacteriocin was eluted with the same buffer at a flow
rate of 1ml/min using a gradient of 0-1.2 NaCl. Active
fractions were pooled ,evaporated, and the antimicrobial
activity was assayed on each fraction.
Nisin Unit: The titre in arbitrary units (AU) per
milliliter is defined as the reciprocal of the highest
dilution of the preparation for which a definite zone of
inhibition is observed.
Spectrum of Inhibitory Activity: The range of
inhibitory activity of the isolated purified and the
reference Lactococcus lactis (NRRL B-1821) strains
were detected and compared with that of commercial
nisin .The well diffusion direct assay described by
Tagg and McGiven [1 1 ] and modified by Benkerroum &
Sandine [1 ] was utilized to detect the inhibition of the
sensitive strains.The GM 17 broth was supplemented
with sodium B-glycerophosphate (2%) and catalase at
final conc. of 100 U.20 ml of M 17 agar inoculated
with 1ml of stationary phase sensitive indicator
cultures( Table 1) was poured in sterile Petri dish and
allowed to harden. Holes were punched out of the agar
,by using a cork bore (4mm of diameter ). The base of
each hole was sealed with 50 ul of GM 17 soft agar
(0.75%agar ) and then filled with 50 ul overnight test
strains. The inoculated plates were incubated at 30 o C
for 18 hr. and checked for the prescence of clear zones
on inhibition as a result of antimicrobial activity.
Solubility of Nisin: Solubility was determined using
concentrated HCl solution of nisin (pH 2.0). Gradually
increasing pH from 2-8, samples were equilibrated for
30 min under continous stirring and the nisin
concentration was determined spectrometrically at
210nm [1 8 ].
Amino Acids Analysis: Mass spectrometry was
conducted using Spectra –Physics Analytical, Inc. A
0099-600 with spectra. Focus optical Scanning detector
and spectra UV 2000 and Ultrasphere C18 Beckman
column. The analysis was carried out using a gradient
of Pico-Tag solvent A&B at 40 o C and flow rate
1ml/min. Detection of the separated Pico-Tag-amino
acids at 254 nm wave length. Before injecting of the
sample, the illustrated was calibrated by two injections
of the standard. Mass spectra were analyzed by using
the manufacture"s software.
NM R Analysis: A Jeol EX-270 NMR Spectrometer,
270 M Hz for H -was used with superconducting magnet
from Oxford and 5mm Dual probehead for H- analysis.
Typical conditions: Spectral width 4000 Hz&15000 Hz,
32 K data points and a flip angle of 450 [1 1 ].
RESULTS AND DISCUSSIONS
Purification of the Inhibitory Peptide from
Lactococcus lactis Strain:
T he highest polypeptide activity produced by
Lactococcus lactis was obtained by subjecting the
supernatant to 70% ammonium sulfate fractionation. It
was decided to proceed with the acidified extract of the
cell pellet for purification purpose, since this comprised
the majority of the inhibitory activity and was also of
higher specific activity .The polypeptide was eluted
effectively with purification fold 47.2 and 26.4% yield.
The activity and purity of the inhibitory substance are
shown in Table (2) and the elution pattern is presented
in Fig. (1).
Table 1: O rigin of Indicator Strains.
Spoilage strains
source
Pathogenic strains
Source
Escherichia coli ATCC 25922
Clinical isolate
Pseudomonas aeruginosa
Local isolate
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus subtilis
Food isolate
Salm onella typhim urium
Local isolate
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus m egaterium
Food isolate
Serratia m arcescens
Clinical isolate
------------------------------------------------------------------------------------------Bacillus cereus
Local isolate
------------------------------------------------------------------------------------------M ycococcus lactis
Food isolate
------------------------------------------------------------------------------------------M icrococcus luteus
Food isolate
------------------------------------------------------------------------------------------Staphylococcus aureus
Food isolate
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Table 2: Activity and purity of antibacterial substance produced by Lactococcus lactis during the fractionation procedure.
Purification step
Volum e
Total activity
Total protein
Specific activity
Recovery
Purification
(m l)
(X103 IU )
(m g.)
(IU /m g.)
%
fold
Culture supernatant
900.0
7200.0
3420.0
2105.0
100.0
1.0
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Precipitation with 70%
30.0
3095.0
330.0
9379.0
42.9
4.5
am m onium sulphate
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------D EAE-Cellulose
60.0
1904.0
19.2
9917.0
26.4
47.2
Fig. 1: Elution pattern of nisin produced by isolated strain Lactococcous lactis through DEAE-cellulose measured
as protein concentration and activity.
Antibacterial Spectrum: The isolated purified
strain and the reference strain (Lactococcus lactis
NRRL B-1821) were particularly broadly active in that
they inhibited all the standard indicator strains as did
the commercial nisin (Table 3). However, both strains
are insensitive to commercial nisin.
Solubility of Nisin: W ith respect to practical
application, nisin solubility represents an important
functional property. So solubility was determined
over a wide pH range (2-8) and results are shown in
Fig. (2). At low pH, the solubility of the isolated strain
was 39mg/ml compared to the reference strain
40.8mg/ml. The solubility decreased with the increase
in pH.
Amino Acid Composition: The amino acid analysis of
both strains the isolated purified and the reference
are shown in Table (4). Results indicated excess of
aspartic acid, valine, serine, proline and a deficiency
of histidine, glycine, alanine, leucine, isoleucine in
the
isolated strain compared to the reference.
However, unsaturated amino acids (dehydroalanine
{DHA} and dehydrobutyrine {DHB}) are not
determined (Figs. 3& 4).
NM R Analysis: The chemical structure of the isolated
purified antibiotic and the reference strain was
investigated by H -NM R Spectroscopy. Results
presented in Figs5&6 indicate the presence of two
dehydroalanine (DHA) molecules and one molecule of
dehydrobutyrine (DHB). These unsaturated amino acids
characterized by the presence of vinyl protons which
give resonance signals appeared as a peak. Results
shows that the two vinyl protons of momolecular DHA
give two peaks while the single vinyl proton of DHB
give one peak.
Discussion: Lantibiotics are a diverse group of heavily
modified antimicrobial and/or signalling peptides
produced by a wide range of bacteria, including a
variety of LAB. Based on their diverse structures and
mode of action, at least six separate antibiotic
subgroups are characterized by significant post
translational modifications, which include the formation
of (B-methyl) lanthionines, among other unusual
alterations.
Since their discovery, interest has been fuelled by
their obvious potential as food-grade antimicrobials to
improve safety and quality, a potential which, to date,
has been realized only by the longest characterized
molecule, nisin [2 3 ]. Nisin, a bacteriocin secreted by
Lactococcus lactis, is commercially used as a food
preservative in forty Countries since 1983 [1 0 ].
The production of lantibiotics is widespread among
gram-positive bacteria [2 0 ]. As more lantibiotics are
detected, it is becoming increasingly clear that naturally
occurring variants occur quite commonly,with some
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Table 3: Activity spectrum of isolated and reference Lactococcus lactis strains.
Indicator M icroorganism s
M icroorganism s Sensitivity*
---------------------------------------------------------------------------------------------------------------------------------------------Isolated purified Lactococcus lactis
Reference Lactococcus lactis
Com m ercial nisin
Escherichia coli
+
++
++
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Staphylococcus aureus
++
+++
+++
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus cereus
+++
++++
++++
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus subtilis
++
+++
+++
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus m egaterium
+
+
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------M ycococcus lactis
++
++
+++
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------M icrococcus luteus
++
++
++
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Pseudomonas aeruginosa
+
++
++
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Serratia m arcescens
+
+
++
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Salm onella typhim urium
+
+
+
Experim ental details: (-), not inhibited; (+), (++), (+++): low, high, very high inhibition, respectively.
Fig. 2: Solubility of Nisaplain (commercial nisin), Nisin (Reference strain) and Nisin (isolated strain) in the pH
range 2 to 8 at 25 O C.
Table 4: Am ino acid com position of the purified peptide antibiotic
from Lactococcus lactis (isolated strain and reference
strain).
Residue (% )
Reference strain
Isolated strain
Asparagin
H istidine
Glycin
Alanine
Valine
Leucine
Isoleucine
Serin
M ethionine
Lysine
Proline
DHA
DHB
Lanthionine
7.5
3.8
0.3
2.3
2.8
6.1
33.0
4.3
16.0
11.0
6.6
N .D
N .D
N .D
8.5
2.0
0.2
1.3
3.7
5.2
29.0
4.8
15.6
10.0
10.4
N .D
N .D
N .D
Total %
93.72%
90.70%
of these differing in only a single amino acid residue
(nisin A & nisin Z), whereas others involve several
amino acid differences such as between epidermin and
mutacin B-N y 266 [1 6 ].
It has been recommended that "by definition"
natural lantibiotic variants should have only "a few
"amino acid differences, essentially the same ring
pattern, and that cross-immunity should be exhibited
between their respective producing strains [2 6 ].
The bacteriocin was purified from the culture
supernatant fluid of Lactococcus lactis to apparent
homogeneity, as judged by two different protocols,
70% ammonium sulphate precipitation and cation
exchange chromatography. The used protocols resulting
in increase in specific activity and 26.4 % recovery
(Table 1 & Fig 1). In accordance to our results
Coventry et al[3 ] reported that, rude bacteriocin
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Fig. 3: Amino acid composition of the purified peptide antibiotic from isolated strain.
Fig. 4: Amino acid composition of the purified peptide antibiotic from reference strain.
Fig. 5: NMR analysis of the peptide antibiotic purified from isolated strain.
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including nisin prepared by ammonium sulfate
precipitation. In Matsuski et al. [1 4 ] purified a peptide
antibiotic from Lactococcus lactis IO-1 by simple
procedure including acid treatment, ammonium sulfate
precipitation, cation-exchange chromatography and
reversed-phase high liquid chromatography and the
specific activity of the peptide was 122 fold greater
than the starting material and the recovery was 24%
similar to our results. The relative low yield in the last
step of the purification was most probably due to
inactivation of the bacteriocin.
The inhibitory spectrum of bacteriocin against
s e v e r a l G r a m p o s itiv e a nd G r a m n e g a tiv e
microorganisms is determined in order to evaluate the
possibility of using the bacteriocin-producing strains as
an additional barrier against spoilage and /or foodborne microorganisms in foods. The isolated purified
Lactococcus lactis exhibited wide activity spectrum
against pathogenic and spoilage microorganisms tested.
These results are similar to Gupta and Batish [8 ].
The antimicrobial spectrum of the bacteriocin of ITAL
383 was similar to nisin of Lactococcus lactis subsp.
lactis ATCC 11454. It showed a wide spectrum of
inhibitory activity affecting closely related species and
all other Gram-positive microorganisms examined. The
bacteriocin did not inhibit Bacillus megaterium. This
inability of the bacteriocin over some microorganisms
was previously reported [2 ,1 2 ,2 4 ].
Solubility of nisin over a wide range of pH
(2.0-8.0) has been evaluated. It has been noted that the
solubility decease directly with the decrease of pH.
Hurst [1 1 ] and Liu & Hansen [1 3 ] stated that nisin
solubility depends strongly on the pH.
The sequence of the amino acids residues
determined
for the purified Lactococcus lactis
(aspartic-serine-glycine-histedine-alanine-proline-valinemethionine-leucinelysine) exhibited high levels of
similarity to the sequence of the reference strain
(aspartic - serine - glycine - alanine- proline - valine methionine - isoluecine - leucine). Also our results
referred to the presence of an excess of aspartic acid
and the deficiency of histidine in the isolated strain
being similar to nisin Z. However, failure of
appearance of unsaturated amino acids (DHA & DHB)
may be due to acid hydrolysis of the purified peptide
as reported by Gross & Morell[7 ].
NMR analysis of the purified peptide and
the reference revealed that the tested samples may
have
two
DHA
molecules
and
one
DHB
molecule, because these unsaturated amino acids
characterized by the presence of vinyl protons which
give resonance signals appeared as peaks. In case
of DHA two peaks appeared while the single vinyl
proton of DHB give one peak. These results are in
agreement with Jones et al.[1 1 ], Liu & Hensen [1 3 ] and
M atsusaki et al. [1 4 ]. The final results suggest that the
purified peptide belongs to the class of bacteriocins
termed lantibiotics.
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