ORIGINAL PAPER

Purification and partial characterization of bacteriocin producedby Lactococcus lactis ssp. lactis LL171

Archana Kumari • Nefise Akkoc • Mustafa Akcelik

Received: 17 August 2011 / Accepted: 29 November 2011 / Published online: 10 December 2011

� Springer Science+Business Media B.V. 2011

Abstract Lactic acid bacteria (LAB) are possessing ability

to synthesize antimicrobial compounds (like bacteriocin)

during their growth. In this regard, novel bacteriocin com-

pound secreting capability of LAB isolated from Tulum

Cheese in Turkey was demonstrated. The synthesized bac-

teriocin was purified by ammonium sulphate precipitation,

dialysis and gel filtration. The molecular weight (&3.4 kDa)

of obtained bacteriocin was confirmed by SDS-PAGE,

which revealed single peptide band. Molecular identification

of LAB strain isolated from Tulum Cheese was conducted

using 16S rDNA gene sequencing as Lactococcus lactis ssp.

lactis LL171. The amino acid sequences (KKIDTRTGKT

MEKTEKKIELSLKNMKTAT) of the bacteriocin from

Lactococcus lactis ssp. lactis LL171 was found unique and

novel than reported bacteriocins. Further, the bacteriocin

was possessed the thermostable property and active at wide

range of pH values from 1 to 11. Thus, bacteriocin reported in

this study has the potential applications property as food

preservative agent.

Keywords Bacteriocin � Cheese � Lactococcus lactis �Lactic acid bacteria � Biopreservation � HPLC

Introduction

The ability of lactic acid bacteria (LAB) to inhibit the

growth of other bacteria has been known for many years

(Rogers 1928), by producing wide variety of compounds

such as low molecular mass antibiotics, metabolic

products, enzymes and bacteriocin. Bacteriocin is one of

the antagonistic compounds found to possess major appli-

cations in food and pharmaceutical industries, as food

preservative and drug, respectively (Drider et al. 2006;

Nagao et al. 2006). Many researchers suggested the useful

criteria for antagonistic activity (bacteriocin) as: (1) narrow

inhibitory spectrum of activity against (closely) related

bacterial species, (2) the presence of an essential, biologi-

cally active protein moiety and (3) a bacteriocins mode of

action (Drider et al. 2006; Nagao et al. 2006).

In an extensive survey of bacteriocin producers, it was

observed that about 43% (out of the 162 strains) lactococcal

strains tested were capable to produce bacteriocin (Kumari

and Garg 2007; Kumari et al. 2008). On the other hand, nisin

is the only bacteriocin from Lactococcus lactis that has been

studied in detail. The inhibitory spectra of the different lac-

tococcal bacteriocins vary but they are generally narrower

than that of nisin (Geis et al. 1983). Schnell et al. (1988)

stated that many of the lactococcal bacteriocins described

are very different from nisin and does not belong to the

lantibiotic family of bacteriocin-like compounds.

The objective of the present study was to describe a

novel bacteriocin produced by L. lactis ssp. lactis LL171

isolated in our laboratory from Turkey Tulum Cheese.

Further, purification and characterization of bacteriocin

was studied in detail for their potential application as food

preservative agent in future.

Materials and methods

Microorganisms

The bacteriocin producing LAB strain was isolated from

Tulum Cheese collected from a local market in Ankara,

A. Kumari (&) � N. Akkoc � M. Akcelik

Department of Biology, Faculty of Science, Ankara University,

Tandogan, 06100 Ankara, Turkey

e-mail: archanamicro@gmail.com

123

World J Microbiol Biotechnol (2012) 28:1647–1655

DOI 10.1007/s11274-011-0971-4

Turkey. Isolated bacterial strain was identified using

microbiological (morphology, Gram staining and motility

tests) and biochemical (catalase, cytochrome oxidase,

arginine hydrolysis and carbohydrate fermentation) as

described in Bergey’s Manual (Holt et al. 1994). Further,

molecular identification of bacterial strain was conducted

using 16S rDNA gene amplification and sequencing.

The other bacterial strains used against the LAB isolated

from Tulum Cheese to test the inhibition spectrum of the

bacteriocin were obtained from the American Type Culture

Collection (ATCC). All indicator strains were grown on

nutrient broth (M002, Himedia) at 37�C for 24 h, with the

exception of Lactococcus lactis ssp. lactis ATCC 19257

and Lactococcus lactis ssp. lactis ATCC 11454 were

grown on MRS broth (De Man, Rogosa and Sharpe med-

ium, Difco Laboratories, Detroit, USA). Clostridium per-

fringens strain ATCC 13124 was cultivated on Clostridium

Agar (RCM Agar, CM0151, Oxoid) in an anaerobic jar

with GasPak. All the strains were stored frozen at -80�C in

appropriate media containing 50% (v/v) glycerol.

Molecular identification of isolated LAB strain

Microbial strain identification was conducted using 16S

rDNA gene sequencing. Molecular identification of LAB is

very precise and accurate to identify up to genus level

(Clarridge 2004; Janda and Abbott 2007).

Genomic DNA isolation from LAB

LAB strain was grown in MRS broth for 8–12 h at 37�C.

DNA isolation was followed as per instruction of bacterial

genomic DNA isolation kit (Fermentas, Finland).

PCR amplification of 16S rRNA gene

Extracted genomic DNA from LAB was used for direct

amplification of 16S rRNA gene portions. Universal

primers were used to amplify the full length 16S rRNA

gene from ribosomal RNA (rrn) operon. The two types of

primers were used for 16S rDNA amplification had the

following sequence: forward primer \50-CCG TCA ATT

CCT TTG AGT TT-30[and reverse primer\50-CTG AGC

CAG GAT CAA ACT CT-30[ as reported by Beasley and

Saris 2004 and The primers for nisin gene were comprised

the following nucleotide sequences; 50-ATG AGT ACA

AAA GAT TTT AAC TTG-30 and 50-ATT TGC TTA CGT

GAA TAA TAC AA-30. PCR amplification of bacterial rrn

operon was performed for 100 ll reaction volume con-

tained 19 PCR amplification buffer, 2 U of Taq DNA

polymerase (Promega, USA), 200 lM of each deoxynu-

cleotides, 100 pmol of each oligonucleotide primers and

template DNA (0.25 lg of DNA from 1 lg/ll of stock

solution) of LAB. Amplification was carried out in a

thermalcycler (Techne-TC.512 England)) with heated lid

(104�C) facility and was run with block temperature con-

trol (thermal regulation by 6�C/Sec). Initial denaturation of

template DNA was done for 2 min at 94�C. PCR was

performed for amplification of 16S rRNA gene under

specific thermal profile as follows; denaturation at 94�C for

45 s, annealing at 55�C for 60 s and polymerization at

72�C for 60 s for 30 cycles followed by final extension at

72�C for 10 min. 5 ll of amplified PCR product was

resolved by electrophoresis on 1.0 % (w/v) agarose gel

(Amresco, Ohio, USA.) and was observed on a UV trans-

illuminator (UVP, 3 UV benchtop transilluminator,

Canada). The remaining PCR product was purified using

the Gel Extraction Kit (QIAquick, Qiagen, GE Healthcare

UK) stored at -20�C for further work.

Agarose gel electrophoresis

Agarose gel (0.7 % w/v) was made in 0.59 Tris Borate

EDTA (TBE) buffer and run at 60 V for 1 h in electro-

phoretic apparatus. The fractionated DNA bands were

visualized under UV Transilluminator (Biometra, Gottin-

gen) and compared with known DNA O’rangerular marker

(fermentas) 1500, 1400, 1300, 1200, 1100, 1000, 900, 800,

700, 600, 500, 400, 300, 200, 100 bp.

DNA sequencing

The purified PCR products were sequenced using BigDye

Terminator Cycle Sequencing Ready Reaction Kit

(Applied Biosystems, Carlsbad, CA, USA) in ABI PRISM

3130 XL Genetic Analyser (Applied Biosystems, Carlsbad,

CA, USA). The quality of gene sequences was analyzed

with the Staden Package software (version 1.5.3). The

obtained 16S rDNA gene sequences were searched for

similarity using the BLAST program (http://www.ncbi.

nlm.nih.gov/BLAST/).

Plasmid isolation and conjugation

Plasmid DNA was isolated by the method of Anderson and

McKay (1983). The plasmid DNA samples were subjected

to electrophoresis in 0.7% agarose gels.

Conjugation procedure was adopted from Gasson and

Davies (1980). Recipient and donor strains were grown in

MRS broth medium at 30�C for 18 h. For the recipient

strain L. lactis LL171, erythromycin (5 ll ml-1) was added

to this medium. 2 ml of the donor and 3 ml of the recipient

culture (both 10-4 diluted) were mixed and the cells were

collected on sterile membrane filters (0.45 lm Sartorius,

Germany). The filters containing the recipient and donor

cells were placed on MRS agar plates and kept at 30�C for

1648 World J Microbiol Biotechnol (2012) 28:1647–1655

123

18 h. Filters were then taken from the MRS agar plates and

washed in 1 ml of sterile Ringer solution to suspend the

cells. Serial dilutions were made (up to 10-8) and from

each dilution the aliquots were spread on to fast slow dif-

ferential agar plates containing the antibiotics and incu-

bated at 30�C for 48 h. Conjugation frequency was

determined according to the ratio of the number of trans-

conjugants per ml to the number of donor per ml. The

stabilities of bacteriocin production phenotype in the

LL171 and its transconjugants were determined after 70

generation according to the method proposed by Picon

et al. (2005).

Purification of bacteriocin

Bacteriocin producing LAB culture strain was grown in

MRS in a 2l fermentor (LAB FORS, Bottmingen, Swit-

zerland) equipped with pH and temperature control, was

operated at constant temperature (35�C) and pH (6.5) for

24 h (Kumari et al. 2008). Culture supernatant was col-

lected by centrifugation at 15,0009g for 30 min at 4�C

(Sigma 2K15, Munich, Germany). The centrifuged culture

supernatant was treated with the gradual addition of

ammonium sulphate to precipitate the bacteriocin as

described below. In 1,000 ml of culture supernatant,

ammonium sulphate was added slowly with constant stir-

ring to achieve 40% saturation and the mixture was kept in

the refrigerator at 4�C for overnight. Stored mixture was

centrifuged at 10,0009g for 30 min at 4�C and the col-

lected precipitate (in the centrifuged pellet) was dissolved

in sodium phosphate buffer 0.05 M (pH 7.0). The super-

natant was subsequently adjusted to 60, 80 and 100% sat-

uration levels by further addition of solid ammonium

sulphate. The precipitates in each case were dissolved in

sodium phosphate buffer as described above. After over-

night incubation at 4�C, the precipitates were again col-

lected by centrifugation (10,0009g at 4�C for 30 min). The

surface pellicles and bottom pellets (containing the bacte-

riocin) were resuspended in a minimal amount of 0.05 M

buffer (pH 7.0).

The bacteriocin solution obtained after each step of

ammonium sulphate fractionation was dialysed with the

same buffer at 4�C for 36 h using a cellulose acetate

membrane (1.0 kDa cut-off, Sigma-Aldrich, Laborchem-

ikalien, Seelze, Germany) and by changing the buffer every

6 h. The bacteriocin preparation obtained after dialysis was

further purified by high performance liquid chromatogra-

phy (HPLC; AKTA prime, Amersham Bioscience, Stock-

holm, Sweden). The dialysate was loaded onto a column of

Superdex 75 (14 ml, fine particle size 13 lm; Amersham

Bioscience) that was previously equilibrated with 0.05 M

sodium phosphate buffer, pH 7.0. Proteins were eluted with

the same buffer at a flow rate of 0.5 ml per min. Fractions

(3 ml) were collected and tested for antibacterial activity

by agar well diffusion on a lawn of indicator strain

(L. lactis ssp. lactis ATCC 11454). Active fractions were

pooled for further analysis.

Molecular mass determination of bacteriocin

The molecular mass of the purified bacteriocin of L. lactis

ssp. lactis LL171 was determined using sodium dodecyl

sulphate–polyacrylamide gel electrophoresis (SDS-PAGE;

15% discontinuous gel) as described in Sambrook et al.

1989. The molecular weight of the fractionated proteins

was compared with standard markers (1.06–40.2 kDa,

Sigma).

The duplicate samples were run on each gel (SDS-

PAGE) to determine the molecular weight and antibacterial

activity of purified bacteriocin compounds as explained

below. The fractionated gel was cut into two half; first half

of the gel was stained with Coomassie brilliant blue R-250

for bacteriocin molecular weight determination. The sec-

ond half of the gel was washed with 0.1% Tween 80 (to

remove SDS) for three times (40 min each) at room tem-

perature. Washed gels were overlaid with 10 ml of soft

nutrient agar medium containing indicator strain (L. lactis

ssp. lactis ATCC 11454) as described by Martinez et al.

1996. Clear zones caused by protein bands with antibac-

terial activity were detected after overnight incubation at

30�C.

Antibacterial activity assay

The antibacterial activity was assayed using modified

protocol of agar well diffusion method (Varadaraj et al.

1993). Antibacterial activity of the purified bacteriocin

against various microbial strains (mentioned in Table 2)

was examined. Test strains (Table 2) were spread (100 ll

volume) on the nutrient agar plates. Wells were made on

the agar plates using a sterile cork borer (4 mm diameter)

and 50 ll of bacteriocin was pipetted aseptically into each

well. The petri plates were incubated at 37�C for 24 h.

Anaerobic test strain (Clostridium perfringens ATCC

13124) was incubated in an anaerobic jar with GasPack.

The zone of inhibition in diameter was measured in mm

using standard scale (Hi Antibiotic Zone Scale-c PW297,

Himedia).

Effect of enzyme, heat, pH and surfactants

on bacteriocin activity

Bacteriocin were treated with the following enzymes at a

final concentration of 1 mg ml-1 trypsin (pH 7, Merck,

Germany), a-chymotrypsin (pH 7, type II, Sigma, USA),

proteinase K (pH 7, Sigma, USA), Pepsin (pH 7, Sigma,

World J Microbiol Biotechnol (2012) 28:1647–1655 1649

123

USA), lipase (pH 7, Sigma, USA), a-amylase (pH 7,

Sigma, USA), Catalase (pH 7, Sigma, USA), and lyzozyme

(pH 7, Sigma, USA). Following incubation at 37�C for 2 h,

enzyme activities were derminated by heating at 100�C for

5 min. Untreated samples were used as the controls (Franz

et al. 1997). Thermal stability of the bacteriocin was con-

ducted at different temperature as follows; (1) 65�C for

30 min, (2) 75�C for 30 min, (3) 85�C for 10 min, (4) 85�C

for 15 min, (5) 90�C for 10 min, (6) 90�C for 15 min, (7)

100�C for 5 min, (8) 100�C for 10 min, (9) 100�C for

15 min, (10) 100�C for 30 min, (11) 100�C for 60 min, and

(12) 121�C for 15 min. After heat or enzyme treatment, the

remaining bacteriocin activity was determined by well-

diffusion assay.

To determine the bacteriocin activity at different pH

values (pH 1–13) was evaluated. The pH of the bacteriocin

in the supernatant was adjusted using specific buffers as

follows; (1) For pH 1 and 2 HCl-KCl buffer were used, (2)

glycine HCl buffer was used for pH 3, (3) acetate buffer

was used for pH 4 and 5, (4) sodium phosphate buffer was

used for pH 6 and 7, (5) Tris–HCl buffer was used for pH 8

and 9, and (6) glycine-NaOH buffer was used for pH 10

and 11.

To determine the effect of different surfactants on bac-

teriocin was investigated, using 2% solutions of (1) Tween

80, (2) Tween 20, (3) TritonX-100 and (4) SDS, with a

final concentration of 1.0 % (V/V) of the surfactants. The

samples were stored at 4�C for 24 h before use.

The treated samples at different temperature, pH and

surfactants were tested for their antimicrobial activity

against L. lactis ssp. lactis ATCC 11454.

Peptide analysis

Purified bacteriocin of L. lactis ssp. lactis LL171 was

lyophilized under vacuum and the peptide was sequenced

using Edman chemistry, automated sequencer (Shimadzu,

Istanbul, Turkey). The peptide sequence similarity search

was conducted with the existing protein sequence database

at National Centre for Biotechnology Information.

Results and discussion

Strain identification

Microbiological characteristics of LAB strain LL171 was

found as gram-positive, cocci, arginine positive, catalase

and cytocrome oxidase-negative bacterium. Molecular

biology identification of LAB strain by 16S rDNA gene

sequences (1,079 bp) and their similarity search revealed

[98% sequence homology with reported Lactococcus

lactis ssp. lactis (AB510756) and Lactococcus lactis ssp.

lactis (FJ915724). It is known that sequence similarity

C97% is acceptable level for microbial identification and

the microbial strain shall be considered as same species

(Stackebrandt and Goebel 1994; Janda and Abbott 2007).

Thus, the isolated LAB strain was identified as Lactococ-

cus lactis ssp. lactis. Further, based on microbiological,

microscopic, biochemical and molecular biological tech-

niques the LAB strain was named as Lactococcus lactis

ssp. lactis LL171.

Purification and molecular weight determination

of bacteriocin

The highest bacteriocin activity was precipitated at 0–60%

ammonium sulphate concentration, which was further

purified by gel filtration (Fig. 1). Pooled protein fractions

from 60 to 65 revealed the highest bacteriocin activity,

which yielded (at recovery of 10 per cent and a 22-fold

purification as indicated in Table 1) a titre of 2,750 AU/ml

and a specific activity of 16,500 AU/mg protein. In this

study, low recovery rate of bacteriocin were observed.

Notably, other researchers were also obtained low recovery

rates for lactacin B (2.4% recovery) (Barefoot and Klaen-

hammer 1984; Apolonio et al. 2008) and acidocin 8912,

(13.6% recovery) (Tahara et al. 1992). On the other hand,

high recovery rate (41% recovery, 369-fold purification)

was obtained for lactacin F (Muriana and Klaenhammer

1991; Camilla et al. 2008).

SDS-PAGE analysis of the bacteriocin obtained by

HPLC revealed a band (identified based on the clear zone

of inhibition) with a molecular mass of 3.4 kDa (Fig. 2).

The similar approach was used to determine the molecular

masses of mesentericin Y105 and carnosin LA44A, as

2.5–3.0 kDa (Hechard et al. 1992) and 2.5–6.0 kDa (van

Laack et al. 1992), respectively.

Antimicrobial spectrum

The antibacterial activity of bacteriocin was tested against

some pathogenic and nonpathogenic bacteria (Table 2).

The bacteriocin obtained from L. lactis ssp. lactis LL171

displayed a very strong inhibitory activity against Listeria

monocytogenes ATCC 19115, Listeria monocytogenes

ATCC 15813, Bacillus subtilis ATCC 6633, Staphylococ-

cus aureus ATCC 29213, and Clostridium perfringens

ATCC 13124, these all strains where sensitive to the

inhibitory activity of produced bacteriocin. There were no

any inhibitory activity against Shigella sonnei ATCC

25931, Escherichia coli ATCC 25922, Streptococcus fae-

calis ATCC 14508, Streptococcus pneumoniae ATCC

49136, Pseudomonas aeruginosa ATCC 27853, Klebsiella

pneumoniae ATCC 35657, and Proteus vulgaris ATCC

1650 World J Microbiol Biotechnol (2012) 28:1647–1655

123

6380 (Table 2) were resistant for bacteriocin produced

from L. lactis ssp. lactis LL171.

The biggest zone of inhibition (22 mm) was obtained

against Listeria monocytogenes ATCC 19115. Whereas

bacteriocin reported to be active against pathogenic strains

(Batdorj et al. 2006; Todorov and Dicks 2006; Kumari

et al. 2008). The bacteriocin produced by L. lactis ssp.

Act

ivit

y U

nit

(A

U/m

l) (

Th

ou

san

ds)

Ab

sorb

ance (O

D 280 n

m)

Fraction Number

6

5

4

3

2

1

0 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 72 75 79 82 85 88

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Fig. 1 High performance liquid

chromatography chromatogram of

purified bacteriocin produced from

Lactococcus lactis ssp. lactis LL171

Table 1 Partial purification of bacteriocin of Lc. lactis subsp. lactis LL171

Purification stage Volume

(ml)

Activity

(AU/ml)

Total activity

(AU)

Total protein

(mg)

Specific activity

(AU/mg)

Purification

(fold)

Recovery

(%)

Culture supernatant 100 5,280 528,000 700 754 1 100

Ammonium sulphate 5 81,920 409,600 45 9,102 12 77

Gel-filtration

superdex-75

18 2,750 49,500 3 16,500 22 10

6.5

Molecular weight Purified bacteriocin marker (kDa)

3.49

1.06

Inhibition zone

17.0

26.6

14.2

2.00

40.2 A B

Fig. 2 Determination of molecular mass of purified bacteriocin (after

gel filtration) produced by Lactococcus lactis ssp. lactis LL171. a Gel

stained with Coomassie brilliant blue R-250; b gel depicting the

bacteriocin activity

Table 2 Antibacterial activity of bacteriocin produced by Lacto-coccus lactis subsp. lactis LL171 against different test organisms after

24 h at 37�C

S.

no.

Test organism Zone of inhibition

(mean of three trials)

(mm)

1 Lactococcus lactis subsp. lactisATCC 19257

12.0

2 Lactococcus lactis subsp. lactisATCC 11454

16.0

3 Bacillus subtilis ATCC 6633 16.0

4 Salmonella typhi ATCC 19430 12.0

5 Shigella sonnei ATCC 25931 0.0

6 Escherichia coli ATCC 25922 0.0

7 Staphylococcus aureus ATCC 29213 14.0

8 Clostridium perfringens ATCC 13124 13.0

9 Streptococcus pneumoniae ATCC

49136

0.0

10 Enterobacter aerogenes ATCC 13048 10.0

11 Streptococcus faecalis ATCC 14508 0.0

12 Pseudomonas aeruginosa ATCC

27853

0.0

13 Listeria monocytogenes ATCC 19115 22.0

14 Listeria monocytogenes ATCC 15813 18.0

15 Klebsiella pneumoniae ATCC 35657 0.0

16 Proteus vulgaris ATCC 6380 0.0

17 Micrococcus luteus ATCC4698 12.0

World J Microbiol Biotechnol (2012) 28:1647–1655 1651

123

lactis LL171 is unique because it was extremely antago-

nistic to Gram-positive food spoilage bacteria as well as to

pathogenic organisms such as Listeria monocytogenes.

Effect of enzymes, heat, pH and surfactants

on bacteriocin activity

The effect of enzymes, pH and heat treatments on the

activity of the bacteriocin produced by LL171 is presented

in Table 3. Protease sensitivity assay demonstrated that the

antimicrobial substance produced by LL171 was a bacte-

riocin-like substance since its inhibitory activity was

completely eliminated by treatment with enzyme protein-

ase K, pepsin and a-chymotrypsin. The activity was,

however, not affected by other proteases including trypsin,

and non-protease enzymes including catalase and lyzo-

zyme. When lipase and a-amylase were applied, both

enzymes were lost 50% activity (Fig. 3). The L. lactis ssp.

lactis LL171 bacteriocin retained full activity at 100�C for

30 min, but lost approximately 20% of its initial activity

after 60 min. Bacteriocin treated at 121�C for 15 min

(sterilization temperature) revealed &40% of loss from the

initial activity. This study clearly demonstrated that the

bacteriocin obtained from strain LL171 is thermostable

(Table 3). The reason for the bacteriocins heat stablility

could be due due to its complex nature. Several studies

have been reported that the bacteriocin treated at 100�C for

120 min and 121�C for 15 min were stable at this high

temperature (Do et al. 2001; Pilar et al. 2008). Pediocin

SJ-1 (Schved et al. 1993) was not affected by heat treatment

for 30 min at 100�C. These examples clearly indicates that

bacteriocin possess thermostable property. Furthermore,

since tolerance of bacteriocin to heat is known to depend on

the stage of purification, pH, presence of culture medium,

other protective components, etc. that might have influenced

the antimicrobial activity in our findings too. The heat sta-

bility of bacteriocin discussed here indicates that it could be

used as biopreservative in combination with thermal pro-

cessing to preserve the food products. Furthermore, when

comparatively low temperature is employed for processing

compared to high temperature being used at present, the

retention of nutrients would be higher. However, more

studies on these aspects are needed.

The residual activities of the partially purified bacte-

riocin from L. lactis ssp. lactis LL171 revealed that the

bacteriocin retained its total activity in the pH range of 1-9

even after 15 days storage at 4�C. However, 66% of loss in

bacteriocin activity at pH 10 and 11 after 24 h storage was

observed and 75% loss after 15 days of storage. At pH 12,

no detectable activity was found after 7 days, whereas

there was 10 and 3.9% activity retained after 8 and 24 h of

storage, respectively. At pH 13, all the bacteriocin activity

was lost after 8 h of storage, this loss in activity at high pH

was irreversible. These facts were also reported by Millette

et al. (2007) for a bacteriocin produced by L. lactis isolated

from human. Bacteriocin from L. lactis ssp. lactis LL171

was not only active and stable over a wide pH range but it

was also extremely heat stable at neutral pH values, indi-

cating that it can be useful in acidic and non-acidic foods.

The stability of bacteriocin to different conditions reflects

that such compounds can withstand the conditions nor-

mally encountered in food processing, so would remain

effective during processing.

Table 3 Effect of enzymes, temperature and pH treatment on bac-

teriocin activity

Application Activity (AU ml-1)

Control 3,200

Enzymes

Trypsin (Sigma, No. T-8658) 3,200

a-Chymotrypsin (Sigma, No. C-7762) –

Proteinase-K (Sigma, No. P-6556) –

Pepsin (Sigma, No. P-6887) –

a-Amylase (Sigma, No. A6380) 1,600

Lipase (Sigma, No. L-1754) 1,600

Catalase (Sigma, No. C-3515) 3,200

Lyzosyme (Sigma, No. L-6876) 3,200

Temperature

65�C/30 min 3,200

75�C/30 min 3,200

85�C/10 min 3,200

85�C/15 min 3,200

90�C/10 min 3,200

90�C/15 min 3,200

100�C/5 min 3,200

100�C/10 min 3,200

100�C/15 min 3,200

100�C/30 min 3,200

100�C/60 min 2,400

121�C/15 min 1,600

pH

2 3,200

3 3,200

4 3,200

5 3,200

6 3,200

7 3,200

8 3,200

9 3,200

10 1,600

11 1,600

12 1,600

Interesting observation: the activity of the sample after autoclaving

was repeated 5 times independently and we found same activity

1652 World J Microbiol Biotechnol (2012) 28:1647–1655

123

Non-ionic detergents such as Tween 20, Tween 80 and

Triton X-100 (1% final concentration used) did not

revealed any significant increase in bacteriocin activity

(Table 4). This clearly indicated that either these agents are

not capable of dissociating bacteriocin aggregates or no

aggregates are causing loss of activity. However, the

anionic detergent SDS caused 200 and 300% increase in

bacteriocin activity when used at 0.5 and 1%, respectively

(Table 4). The increase in bacteriocin activity could be due

to attributable to dispersion of the bacteriocin complex

thereby releasing more units for the activity (Diop et al.

2007). Further, SDS itself an antibacterial agent thus

increased bacteriocin activity shall be obtained (Giesova

et al. 2004; Albano et al. 2007).

The genetic nature of bacteriocin production at strain

LL171

In order to determine whether the bacteriocin production

ability of the strain LL171 is chromosomally or plasmid

DNA encoded, a PCR assay was applied by using the

primers specific to the nisin A structural gene. By ana-

lyzing the extracts of genomic and plasmid DNAs of

LL171 separately, a 174 bp product was obtained from

genomic DNA indicating that the bacteriocin production

genes were located on the chromosomal DNA (Fig. 4a).

Examination of the plasmid contents of the strain LL171

revealed that it has 10 distinct plasmids with molecular

weights varying from 2.1 to 33.1 kb (Fig. 4b). As a con-

sequence of conjugation trials, the bacteriocin production

genes were transferred to the erythromycin resistant strain

L. lactis MG1361 with a frequency of 2 9 10-3 per donor

cell. All bacteriocin producing transconjugants were found

to be plasmid free, indicating that the bacteriocin deter-

minants were transferred by a chromosomally located

conjugative transposon. Additionally, the bacteriocin pro-

duction level of donor strain LL171 cannot be exceeded by

the three different transconjugants, which were able to

produce 800–1,600 AU bacteriocin per ml. The stability of

bacteriocin production of LL171 was determined as 90%

where the transconjugants as 50% in average.

Studies have shown that bacteriocin production genes

are located either on the conjugative plasmids (Horn et al.

1991; Akcelik et al. 2006) or linked with conjugative

transposons on the chromosome (Rauch and de Vos 1992).

In this study, the production of bacteriocin in strain LL171

was found to be located on a conjugative transposon

residing in the chromosome. Conjugative nature of pro-

duction facilitates relation with genetic manipulations,

providing developments in industrial starter cultures and

bringing an economical gain in the fermentation industry.

However, Picon et al. (2005) claimed that at least 50%

stability is required for any traits of starter cultures to be

efficient at the industrial level after 70 generations. In this

circumstance, the stability of nisin production at the

transconjugants has indicated that LL171 strain can be used

Table 4 Effect of surfactant treatment on bacteriocin of Lactococcuslactis subsp. lactis LL171

Application Concentration (%) Activity (AU ml-1)

Surfactant Bacteriocin ?

surfactant

Control – – 3,200

Surfactant

Tween 80 1.0 0 3,200

Tween 20 1.0 0 3,200

TritonX-100 1.0 0 3,200

SDS 0.1 0 3,200

SDS 0.5 200 6,336

SDS 1.0 300 9,536

Fig. 3 Inhibition zones of

bacteriocin produced by LL171

after treatment with different

enzymes

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as a potential donor to improve the starter culture proper-

ties even though the low production of transcojugants

compared to producer LL171.

Amino acid sequence of bacteriocin

Amino acid sequencing of the active peptide indicated

the presence of 29 amino acids in the sequence

KKIDTRTGKTMEKTEKKIELSLKNMKTAT. Calcula-

tion of the molecular mass of the peptide from the sequence

yielded an approximate molecular weight of 3.344 kDa,

which was consistent with the molecular mass obtained by

SDS-PAGE. The peptide sequence does not show any

characteristic suggestive of a cyclic molecule.

On comparison with PIR/PDB database (Protein Infor-

mation Resource/Protein Data Bank), no homology with

any previously reported bacteriocin or other proteins

sequences was found. Therefore, the obtained bacteriocin

molecule is novel. The protein sequence reported in this

work was deposited in the UniProt Knowledgebase under

the accession number P85833.

Conclusions

The study revealed that bacteriocin from L. lactis ssp. lactis

LL171 isolated from Tulum Cheese possesses a wide

spectrum of inhibitory activity against Listeria monocyt-

ogenes ATCC 19115, Bacillus subtilis ATCC 6633,

Staphylococcus aureus ATCC 29213, and Clostridium

perfringens ATCC 13124. Bacteriocin obtained from strain

LL171 was extremely thermostable, and pH stable.

Therefore, it has a potential for application as a biopre-

servative in different thermally processed food products as

such or in combination with other preservation methods.

Acknowledgments This work was supported by the grant from

Tubitak, Turkey under 2216-Research Fellowships for Foreign Citi-

zens program with the project entitled ‘‘Isolation and identification of

new bacteriocin producing lactic acid bacteria, antibacterial activity

against food spoilage and human pathogenic bacteria and its potential

as biopreservatives’’. I am happy to acknowledge the company I

enjoyed by working along with my colleagues and friends Deniz

Yuksel, Duygu Abbasoglu, Neslihan Taskale, Seyit Nesimi Bulut,

Ibrahim Erdogan, Mine Gunes and Meral Kaya.

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