World J Microbiol Biotechnol (2012) 28:16471655 DOI 10.

1007/s11274-011-0971-4

ORIGINAL PAPER

Purication and partial characterization of bacteriocin produced by Lactococcus lactis ssp. lactis LL171
Archana Kumari Nese 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 compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was puried by ammonium sulphate precipitation, dialysis and gel ltration. The molecular weight (&3.4 kDa) of obtained bacteriocin was conrmed by SDS-PAGE, which revealed single peptide band. Molecular identication 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
A. Kumari (&) N. Akkoc M. Akcelik Department of Biology, Faculty of Science, Ankara University, Tandogan, 06100 Ankara, Turkey e-mail: archanamicro@gmail.com

products, enzymes and bacteriocin. Bacteriocin is one of the antagonistic compounds found to possess major applications 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, biologically 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 lactococcal 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, purication 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,

123

1648

World J Microbiol Biotechnol (2012) 28:16471655

Turkey. Isolated bacterial strain was identied using microbiological (morphology, Gram staining and motility tests) and biochemical (catalase, cytochrome oxidase, arginine hydrolysis and carbohydrate fermentation) as described in Bergeys Manual (Holt et al. 1994). Further, molecular identication of bacterial strain was conducted using 16S rDNA gene amplication 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 37C 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 medium, Difco Laboratories, Detroit, USA). Clostridium perfringens 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 -80C in appropriate media containing 50% (v/v) glycerol. Molecular identication of isolated LAB strain Microbial strain identication was conducted using 16S rDNA gene sequencing. Molecular identication 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 812 h at 37C. DNA isolation was followed as per instruction of bacterial genomic DNA isolation kit (Fermentas, Finland). PCR amplication of 16S rRNA gene Extracted genomic DNA from LAB was used for direct amplication 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 amplication 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 amplication of bacterial rrn operon was performed for 100 ll reaction volume contained 19 PCR amplication buffer, 2 U of Taq DNA polymerase (Promega, USA), 200 lM of each deoxynucleotides, 100 pmol of each oligonucleotide primers and template DNA (0.25 lg of DNA from 1 lg/ll of stock

solution) of LAB. Amplication was carried out in a thermalcycler (Techne-TC.512 England)) with heated lid (104C) facility and was run with block temperature control (thermal regulation by 6C/Sec). Initial denaturation of template DNA was done for 2 min at 94C. PCR was performed for amplication of 16S rRNA gene under specic thermal prole as follows; denaturation at 94C for 45 s, annealing at 55C for 60 s and polymerization at 72C for 60 s for 30 cycles followed by nal extension at 72C for 10 min. 5 ll of amplied PCR product was resolved by electrophoresis on 1.0 % (w/v) agarose gel (Amresco, Ohio, USA.) and was observed on a UV transilluminator (UVP, 3 UV benchtop transilluminator, Canada). The remaining PCR product was puried using the Gel Extraction Kit (QIAquick, Qiagen, GE Healthcare UK) stored at -20C 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 electrophoretic apparatus. The fractionated DNA bands were visualized under UV Transilluminator (Biometra, Gottingen) and compared with known DNA Orangerular marker (fermentas) 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 bp. DNA sequencing The puried 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 30C 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 lters (0.45 lm Sartorius, Germany). The lters containing the recipient and donor cells were placed on MRS agar plates and kept at 30C for

123

World J Microbiol Biotechnol (2012) 28:16471655

1649

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 differential agar plates containing the antibiotics and incubated at 30C for 48 h. Conjugation frequency was determined according to the ratio of the number of transconjugants 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). Purication of bacteriocin Bacteriocin producing LAB culture strain was grown in MRS in a 2l fermentor (LAB FORS, Bottmingen, Switzerland) equipped with pH and temperature control, was operated at constant temperature (35C) and pH (6.5) for 24 h (Kumari et al. 2008). Culture supernatant was collected by centrifugation at 15,0009g for 30 min at 4C (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 stirring to achieve 40% saturation and the mixture was kept in the refrigerator at 4C for overnight. Stored mixture was centrifuged at 10,0009g for 30 min at 4C and the collected precipitate (in the centrifuged pellet) was dissolved in sodium phosphate buffer 0.05 M (pH 7.0). The supernatant was subsequently adjusted to 60, 80 and 100% saturation levels by further addition of solid ammonium sulphate. The precipitates in each case were dissolved in sodium phosphate buffer as described above. After overnight incubation at 4C, the precipitates were again collected by centrifugation (10,0009g at 4C for 30 min). The surface pellicles and bottom pellets (containing the bacteriocin) 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 4C for 36 h using a cellulose acetate membrane (1.0 kDa cut-off, Sigma-Aldrich, Laborchemikalien, Seelze, Germany) and by changing the buffer every 6 h. The bacteriocin preparation obtained after dialysis was further puried by high performance liquid chromatography (HPLC; AKTA prime, Amersham Bioscience, Stockholm, Sweden). The dialysate was loaded onto a column of Superdex 75 (14 ml, ne 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 ow 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 puried bacteriocin of L. lactis ssp. lactis LL171 was determined using sodium dodecyl sulphatepolyacrylamide 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 (1.0640.2 kDa, Sigma). The duplicate samples were run on each gel (SDSPAGE) to determine the molecular weight and antibacterial activity of puried bacteriocin compounds as explained below. The fractionated gel was cut into two half; rst half of the gel was stained with Coomassie brilliant blue R-250 for bacteriocin molecular weight determination. The second half of the gel was washed with 0.1% Tween 80 (to remove SDS) for three times (40 min each) at room temperature. 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 antibacterial activity were detected after overnight incubation at 30C. Antibacterial activity assay The antibacterial activity was assayed using modied protocol of agar well diffusion method (Varadaraj et al. 1993). Antibacterial activity of the puried 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 37C 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 nal 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,

123

1650

World J Microbiol Biotechnol (2012) 28:16471655

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 37C for 2 h, enzyme activities were derminated by heating at 100C for 5 min. Untreated samples were used as the controls (Franz et al. 1997). Thermal stability of the bacteriocin was conducted at different temperature as follows; (1) 65C for 30 min, (2) 75C for 30 min, (3) 85C for 10 min, (4) 85C for 15 min, (5) 90C for 10 min, (6) 90C for 15 min, (7) 100C for 5 min, (8) 100C for 10 min, (9) 100C for 15 min, (10) 100C for 30 min, (11) 100C for 60 min, and (12) 121C for 15 min. After heat or enzyme treatment, the remaining bacteriocin activity was determined by welldiffusion assay. To determine the bacteriocin activity at different pH values (pH 113) was evaluated. The pH of the bacteriocin in the supernatant was adjusted using specic 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) TrisHCl 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 bacteriocin was investigated, using 2% solutions of (1) Tween 80, (2) Tween 20, (3) TritonX-100 and (4) SDS, with a nal concentration of 1.0 % (V/V) of the surfactants. The samples were stored at 4C 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 Puried 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.

lactis (FJ915724). It is known that sequence similarity C97% is acceptable level for microbial identication and the microbial strain shall be considered as same species (Stackebrandt and Goebel 1994; Janda and Abbott 2007). Thus, the isolated LAB strain was identied as Lactococcus lactis ssp. lactis. Further, based on microbiological, microscopic, biochemical and molecular biological techniques the LAB strain was named as Lactococcus lactis ssp. lactis LL171. Purication and molecular weight determination of bacteriocin The highest bacteriocin activity was precipitated at 060% ammonium sulphate concentration, which was further puried by gel ltration (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 purication as indicated in Table 1) a titre of 2,750 AU/ml and a specic 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 purication) 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 (identied 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 2.53.0 kDa (Hechard et al. 1992) and 2.56.0 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, Staphylococcus 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 faecalis ATCC 14508, Streptococcus pneumoniae ATCC 49136, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 35657, and Proteus vulgaris ATCC

Results and discussion Strain identication Microbiological characteristics of LAB strain LL171 was found as gram-positive, cocci, arginine positive, catalase and cytocrome oxidase-negative bacterium. Molecular biology identication 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.

123

World J Microbiol Biotechnol (2012) 28:16471655

Activity Unit (AU/ml) (Thousands)

1651
1.4

Fig. 1 High performance liquid chromatography chromatogram of puried bacteriocin produced from Lactococcus lactis ssp. lactis LL171

6 5 4 3

Absorbance (OD 280 nm)

1.2 1.0 0.8 0.6

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

0.4 0.2 0.0

Fraction Number

Table 1 Partial purication of bacteriocin of Lc. lactis subsp. lactis LL171 Purication stage Culture supernatant Ammonium sulphate Gel-ltration superdex-75 Volume (ml) 100 5 18 Activity (AU/ml) 5,280 81,920 2,750 Total activity (AU) 528,000 409,600 49,500 Total protein (mg) 700 45 3 Specic activity (AU/mg) 754 9,102 16,500 Purication (fold) 1 12 22 Recovery (%) 100 77 10

Molecular weight marker (kDa)

Purified bacteriocin

40.2 26.6

Table 2 Antibacterial activity of bacteriocin produced by Lactococcus lactis subsp. lactis LL171 against different test organisms after 24 h at 37C S. no. 1 2 Test organism Zone of inhibition (mean of three trials) (mm) 12.0 16.0 16.0 12.0 0.0 0.0 14.0 13.0 0.0 10.0 0.0 0.0 22.0 18.0 0.0 0.0 12.0

17.0 14.2

Lactococcus lactis subsp. lactis ATCC 19257 Lactococcus lactis subsp. lactis ATCC 11454 Bacillus subtilis ATCC 6633 Salmonella typhi ATCC 19430 Shigella sonnei ATCC 25931 Escherichia coli ATCC 25922 Staphylococcus aureus ATCC 29213 Clostridium perfringens ATCC 13124 Streptococcus pneumoniae ATCC 49136 Enterobacter aerogenes ATCC 13048 Streptococcus faecalis ATCC 14508 Pseudomonas aeruginosa ATCC 27853 Listeria monocytogenes ATCC 19115 Listeria monocytogenes ATCC 15813 Klebsiella pneumoniae ATCC 35657 Proteus vulgaris ATCC 6380 Micrococcus luteus ATCC4698

6.5
3.49

3
Inhibition zone

4 5 6 7 8 9 10 11 12 13 14 15 16 17

2.00
1.06

Fig. 2 Determination of molecular mass of puried bacteriocin (after gel ltration) produced by Lactococcus lactis ssp. lactis LL171. a Gel stained with Coomassie brilliant blue R-250; b gel depicting the bacteriocin activity

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.

123

1652

World J Microbiol Biotechnol (2012) 28:16471655

lactis LL171 is unique because it was extremely antagonistic 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
Table 3 Effect of enzymes, temperature and pH treatment on bacteriocin activity Application Control Enzymes Trypsin (Sigma, No. T-8658) a-Chymotrypsin (Sigma, No. C-7762) Proteinase-K (Sigma, No. P-6556) Pepsin (Sigma, No. P-6887) a-Amylase (Sigma, No. A6380) Lipase (Sigma, No. L-1754) Catalase (Sigma, No. C-3515) Lyzosyme (Sigma, No. L-6876) Temperature 65C/30 min 75C/30 min 85C/10 min 85C/15 min 90C/10 min 90C/15 min 100C/5 min 100C/10 min 100C/15 min 100C/30 min 100C/60 min 121C/15 min pH 2 3 4 5 6 7 8 9 10 11 12 3,200 3,200 3,200 3,200 3,200 3,200 3,200 3,200 1,600 1,600 1,600 3,200 3,200 3,200 3,200 3,200 3,200 3,200 3,200 3,200 3,200 2,400 1,600 3,200 1,600 1,600 3,200 3,200 Activity (AU ml-1) 3,200

Interesting observation: the activity of the sample after autoclaving was repeated 5 times independently and we found same activity

antimicrobial substance produced by LL171 was a bacteriocin-like substance since its inhibitory activity was completely eliminated by treatment with enzyme proteinase K, pepsin and a-chymotrypsin. The activity was, however, not affected by other proteases including trypsin, and non-protease enzymes including catalase and lyzozyme. 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 100C for 30 min, but lost approximately 20% of its initial activity after 60 min. Bacteriocin treated at 121C 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 100C for 120 min and 121C 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 100C. These examples clearly indicates that bacteriocin possess thermostable property. Furthermore, since tolerance of bacteriocin to heat is known to depend on the stage of purication, pH, presence of culture medium, other protective components, etc. that might have inuenced the antimicrobial activity in our ndings too. The heat stability of bacteriocin discussed here indicates that it could be used as biopreservative in combination with thermal processing 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 puried bacteriocin 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 4C. 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, indicating that it can be useful in acidic and non-acidic foods. The stability of bacteriocin to different conditions reects that such compounds can withstand the conditions normally encountered in food processing, so would remain effective during processing.

123

World J Microbiol Biotechnol (2012) 28:16471655 Table 4 Effect of surfactant treatment on bacteriocin of Lactococcus lactis subsp. lactis LL171 Application Concentration (%) Activity (AU ml-1) Surfactant Control Surfactant Tween 80 Tween 20 TritonX-100 SDS SDS SDS 1.0 1.0 1.0 0.1 0.5 1.0 0 0 0 0 200 300 3,200 3,200 3,200 3,200 6,336 9,536 Bacteriocin ? surfactant 3,200

1653

Non-ionic detergents such as Tween 20, Tween 80 and Triton X-100 (1% nal concentration used) did not revealed any signicant 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 specic to the nisin A structural gene. By analyzing 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 consequence 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 determinants were transferred by a chromosomally located conjugative transposon. Additionally, the bacteriocin production level of donor strain LL171 cannot be exceeded by the three different transconjugants, which were able to produce 8001,600 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 production 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 efcient 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

Fig. 3 Inhibition zones of bacteriocin produced by LL171 after treatment with different enzymes

123

1654

World J Microbiol Biotechnol (2012) 28:16471655

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 monocytogenes 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 biopreservative 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 identication 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 Tas kale, Seyit Nesimi Bulut, brahim Erdogan, Mine Gunes and Meral Kaya. I

Fig. 4 a Nisin gene amplication using specic primers lane 1: Negative control. lane 2: Amplication from plasmid extracts of LL171 lane 3: marker (fermentas) 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 bp. lane 4: Amplication from genomic DNA of LL171. b The plasmid prole of LL171. lane 1: Supercoiled marker (sigma) 16.2, 14.1, 12.2, 10.2, 8.0, 7.2, 6.0, 5.0, 4.0, 2.9, 2.1 kb, lane 2: Plasmids of LL171

References
Akcelik O, Tukel C, Ozcengiz G, Akcelik M (2006) Characterization of bacteriocins from two Lactococcus lactis subsp. lactis isolates. Mol Nutr Food Res 50:306313 Albano H, Todorov SD, van Reenen CA, Hogg T, Dicks LMT, Teixeira P (2007) Characterization of two bacteriocins produced by Pediococcus acidilactici isolated from Alheira, a fermented sausage traditionally produced in Portugal. Int J Food Microbiol 116:239247 Anderson DG, Mckay LL (1983) A simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol 46:549552 Apolonio ACM, Carvalho MAR, Bemquerer MP, Santoro MM, Pinto SQ, Oliveira JS, Santos KV, Farias LM (2008) Purication and partial characterization of a bacteriocin produced by Eikenella corrodens. J Appl Microbiol 104:508514 Barefoot SF, Klaenhammer TR (1984) Purication and characterization of Lactobacillus acidophilus bacteriocin lactacin B. Antimicrob Agents Chemother 26:328334 Batdorj B, Dalgalarrondo M, Choiset Y, Pedroche J, Metro F, Prevost H, Chobert JM, Haertle T (2006) Purication and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag. J Appl Microbiol 101:837848 Beasley SS, Saris PEJ (2004) Nisin-producing Lactococcus lactis strains isolated from human milk. Appl Environ Microbiol 70:50515053 Camilla O, Rogne P, Emanuelsen L, Kristiansen PE, Fimland G, Nissen-Meyer J (2008) The two-peptide class II bacteriocins: structure, production and mode of action. J Mol Microbiol Biotechnol 13:210219 Clarridge JE (2004) Impact of 16S rRNA gene sequence analysis for identication of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev 17:840862 Diop MB, Robin DD, Emmanuel T, Abib N, Jacqueline D, Philippe T (2007) Bacteriocin producers from traditional food products. Biotechnol Agron Soc Environ 11:13706233

as a potential donor to improve the starter culture properties 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. Calculation 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 Information 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.

123

World J Microbiol Biotechnol (2012) 28:16471655 Do TM, Plockova M, Chumchalova J (2001) Lactococcus lactis ssp. lactis LTM 32, a new bacteriocin-producing strain isolated from Vietnamese fermented milk. Czech J Food Sci 19:171176 Drider D, Fimland G, Hechard Y, McMullen LM, Prevost H (2006) The continuing story of class IIA bacteriocins. Microbiol Mol Biol Rev 70:564582 Franz CMAP, Du Toit M, Von Holy A, Schillinger U, Holzapfel WH (1997) Production of nisin-like bacteriocins by Lactococcus lactis strains isolated from vegetables. J Basic Microbiol 3: 187196 Gasson MJ, Davies FL (1980) High frequency conjugation associated with streptococcus lactis donor cell aggregation. J Bacteriol 143:12601264 Geis A, Singh J, Teuber M (1983) Potential of lactic Streptococci to produce bacteriocin. Appl Environ Microbiol 45:205211 Giesova M, Chumchalova J, Plockova M (2004) Effect of food preservatives on the inhibitory activity of acidocin CH5 and bacteriocin D10. Eur Food Res Technol 218:194197 Hechard Y, Derijard B, Letellier F, Cenatiempo Y (1992) Characterization and purication of mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides. J Gen Microbiol 138:27252731 Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994) Bergeyss manual of determinative bacteriology, 9th edn. Williams and Wilkins, Baltimore Horn N, Swindell S, Dodd H, Gasson M (1991) Nisin biosynthesis genes are encoded by a novel conjugative transposon. Mol Gen Genet 228:129135 Janda JM, Abbott SL (2007) 16S rRNA gene sequencing for bacterial identication in the diagnostic laboratory: pluses, perils, and pitfalls. J Clin Microbiol 45(9):27612764 Kumari A, Garg AP (2007) A bacteriocin from Lactococcus lactis CCSUB94 isolated from milk and milk products. Res J Microbiol 2:375380 Kumari A, Garg AP, Makeen K, Lal M, Gupta C, Chandra S (2008) Bacteriocin production on soya nutri nuggets extract medium (SNNEM) by Lactococcus lactis ssp. lactis CCSUB202. Int J Dairy Sci 3:4954 Martinez B, Suarez JE, Rodriguez A (1996) Lactococcin 972: a homodimeric lactococcal bacteriocin whose primary target is not the plasma membrane. Microbiology 142:23932398 Millette M, Dupont C, Archambault D, Lacroix M (2007) Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococcus acidilactici isolates. J Appl Microbiol 102:274282 Muriana PM, Klaenhammer TR (1991) Purication and partial characterization of lactacin F, a bacteriocin produced by

1655 Lactobacillus acidophilus 11088. Appl Environ Microbiol 57:114121 Nagao J, Asaduzzaman SM, Aso Y, Okuda K, Nakayama J, Sonomoto K (2006) Lantibiotics: insight and foresight for new paradigm. J Biosci Bioeng 102:139149 Picon A, De Torres B, Gaya P, Nunez M (2005) Cheese making with Lactococcus lactis strain expressing a mutant oligopeptide binding protein as a starter results in a different peptide prole. Int J Food Microbiol 104:299307 Pilar C-M, Arlindo S, Boehme K, de Miguel T, Pascoal A, Velazquez JB (2008) Current applications and future trends of lactic acid bacteria and their bacteriocins for the biopreservation of aquatic food. Food Bioprocess Technol 1:4363 Rauch PJG, De Vos MW (1992) Characterization of the novel nisinsucrose conjugative transposon Tn5276 and its insertion in Lactococcus lactis. J Bacteriol 174:12801287 Rogers LA (1928) The inhibitory effect of Streptococcus lactis on Lactobacillus bulgaricus. J Bacteriol 16:321325 Sambrook J, Fitsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York Schnell N, Entian KD, Schneider U, Gotz F, Zahner H, Kellner R, Jung G (1988) Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature (London) 333:276566 Schved F, Lalazar A, Henis Y, Juven BJ (1993) Purication, partial characterization and plasmid-linkage of pediocin SJ-1, a bacteriocin produced by Pediococcus acidilactici. J Appl Bacteriol 74:6777 Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species denition in bacteriology. Int J Syst Bacteriol 44:846849 Tahara T, Kanatani K, Yashida K, Miura H, Sakamato M, Oshimura M (1992) Purication and some properties of acidocin 8912, a novel bacteriocin produced by Lactobacillus acidophilus TK 8912. Biosci Biotech Biochem 56:12121215 Todorov SD, Dicks LMT (2006) Medium components effecting bacteriocin production by two strains of Lactobacillus plantarum ST414BZ and ST664BZ isolated from boza. Biologia (Bratislava) 61:269274 Van-Laack RLJM, Schillinger U, Halzaptel WH (1992) Characterization of a bacteriocin produced by Leuconostoc carnosum LA 44A. Int J Food Microbiol 16:183195 Varadaraj MC, Nirmala D, Keshava N, Manjrekar SP (1993) Antimicrobial activity of neutralized extracellular culture ltrates of lactic acid bacteria isolated from a cultured Indian milk product (dahi). Int. J Food Microbiol 20:259267

123