of nısın-producıng Lactococcus lactıs subsp. lactıs YB23

Article DOI: 10.2478/v10133-009-0020-8 FB Food BIOTECHNOLOGY PhenotypIc and genotypic characterizatıon of nısın-producıng Lactococcus lactıs subsp. lactıs ısolated from raw mılk ın Turkey Y. Tuncer Süleyman Demirel University, Faculty of Engineering & Architecture, Department of Food Engineering, Isparta, Turkey Correspondence to: Yasin Tuncer E-mail: ytuncer@mmf.sdu.edu.tr ABSTRACT A total of 142 presumptive lactococci were isolated from 40 raw milk samples and screened for bacteriocin production. Four presumptive lactococci isolates (, YB42, YB98 and YB112) exhibited antimicrobial activity against indicator strains. Antimicrobial activity and protease sensitivity assays showed that a strain,, produced bacteriocin that inhibited a broad spectrum of Gram-positive bacteria, and was selected for further analyses. It was identified as Lactococcus lactis subsp. lactis, phenotypically by the API system and genotypically by 16S rdna homology. was tested for determination of antimicrobial activity against indicator strains including known bacteriocin producing lactococci and it showed no activity against nisin producer Lactococcus lactis SIK83. Different ph and heat treatments showed that the bacteriocin produced by L. lactis subsp. lactis exhibited a similar behavior with nisin produced by L. lactis SIK83. The bacteriocin produced by strain was characterized as nisin by PCR analysis of its genomic DNA. Lactic acid production and proteolytic activity in strain were determined as 0.38% and 25.3 μg Tyr/mL, respectively. Keywords: Lactococcus lactis subsp. lactis, nisin, industrial traits Biotechnol. & Biotechnol. Eq. 2009, 23(4), 1504-1508 Introduction Lactic acid bacteria (LAB) are widely used as starter cultures for dairy, meat and vegetable fermentations (16). LAB exert a strong antagonistic activity against many food-contaminating microorganisms as a result of the production of organic acids, hydrogen peroxide, diacetyl, inhibitory enzymes and bacteriocins (24). Many strains of the lactic acid bacteria including lactococci, lactobacilli and pediococci produce bacteriocins (15) which are ribosomally synthesized, extracellularly released, small peptides with bactericidal or bacteriostatic activity usually against closely related strains (7). Nowadays, consumers are increasingly demanding food with reduced amounts of chemical additives, but that are also free from pathogens (28). As a result, bacteriocins produced by lactic acid bacteria have attracted increasing interest for use as natural food preservatives (4). The best characterized bacteriocin to date is nisin that is produced by certain strains of Lactococcus lactis. Nisin displayed antimicrobial activity against Gram-positive bacteria and can appear as five types (nisin A, Z, Q, U or F) that differ in both amino acid composition and biological activity (5, 6, 20, 33, 34). Sensitivity of nisin to α-chymotrypsin, heat stability at low ph and non-toxic nature have promoted its widespread use. Thus far, nisin is the only bacteriocin as bio-preservative that has received acceptance in countries worldwide and widespread application in the food industry. This paper is a report of the phenotypic and genotypic identification of a nisin-producing Lactococcus lactis subsp. lactis strain isolated from raw milk obtained from Turkey. The bacteriocin of L. lactis subsp. lactis strain is described as nisin by the detection of structural gene of this peptide. Industrial traits such as lactic acid production and proteolytic activity of this strain were also determined. Material and Methods Bacterial strains and media L. lactis subsp. lactis strain was grown at 30 0 C in M17 broth (Merck KgaA, Darmstadt, Germany). Glucose at 0.5% (GM17) was added when required. The growth media and incubation temperature of the bacterial cultures used as indicator for the detection of antimicrobial spectrum of L. lactis subsp. lactis are shown in Table 1. A total of 22 indicator strains used for the antimicrobial activity tests of strain were provided by the Laboratory of Microbial Gene Technology, NLH, Ås, Norway. All strains were kept in 15% glycerol and stored at -20 0 C. Isolation and antimicrobial activity L. lactis subsp. lactis strain was isolated from raw milk sample obtained from Turkey. A total of 40 raw milk samples were diluted in sterile 0.1% (w/v) peptone water (Merck KgaA, Darmstadt, Germany), and 100 µl dilutions were spread on the surface of NRCLA medium plates (11). Plates were incubated at 30 0 C for 48 h. 142 presumptive lactococci isolates were selected for antimicrobial activity tests. Antimicrobial activity of presumptive lactococcal isolates was evaluated as described by van Belkum et al. (32). 1504 Biotechnol. & Biotechnol. Eq. 23/2009/4

TABLE 1 Growth media and incubation temperature of indicator strains and inhibitory spectrum of bacteriocin produced by L. lactis subsp. lactis Indicator strains Media a and temperature ( 0 C) Inhibition zone (ømm) SIK83 Lactobacillus sakei NCDO2714 (nisin sensitive) MRS, 37 15 12 Lactobacillus plantarum LMG 2003 MRS, 37 16 14 Lactococcus lactis SIK83 (nisin producer) GM17, 30 NZ NZ Lactococcus lactis IL1403 (nisin sensitive) GM17, 30 22 13 Lactococcus lactis 105 (lacticin producer) GM17, 30 8 7 Lactococcus lactis LMG2908 GM17, 30 13 8 Lactococcus lactis T1 GM17, 30 9 5 Lactococcus lactis LMG2912 (lacticin 3147 producer) GM17, 30 8 8 Lactococcus lactis JC17 (lacticin 481 producer) GM17, 30 9 10 Lactococcus lactis LMG2132 (lactococcin producer) GM17, 30 3 5 Lactococcus lactis LMG2088 (lactococcin G producer) GM17, 30 10 NZ Lactococcus lactis 731 (lacticin 3147 producer) GM17, 30 4 6 Enterococcus faecalis LMG2708 (nisin sensitive) GM17, 30 6 8 Enterococcus faecalis LMG2602 GM17, 30 10 10 Escherichia coli LMG3083 (ETEC) LB, 37 NZ NZ Salmonella typhimurium SL1344 LB, 37 NZ NZ Pseudomonas fluorescens P1 LB, 37 NZ NZ Listeria innocua LMG2813 (nisin sensitive) LB, 30 6 10 Staphylococcus aureus LMG3022 TSB, 37 14 5 Staphylococcus carnosus LMG2709 TSB, 37 6 5 Pediococcus pentosaceus LMG2001 (pediocin producer) TSB, 37 12 10 Bacillus cereus LMG2732 TSB, 37 5 5 Lactococcus lactis subsp. lactis GM17, 30 NZ NZ a MRS: De Man, Rogosa, Sharpe Broth; GM17: Glucose-M17 Broth (0.5% glucose); LB: Luria Bertani Broth; TSB: Triyptic Soy Broth; NZ: no zone Lactococcal isolates were grown overnight at 30 0 C on M17 agar medium, then the isolates were transferred to M17 plates by using sterile toothpicks. After growth overnight at 30 0 C, an indicator lawn of 3 ml of soft agar (0.7%), containing 100 μl of an overnight culture of indicator strain, was poured on the surface. After 18 h of incubation the colonies were examined for zones of inhibition. Protease sensitivity assay The following enzymes were dissolved in sterile distilled water to final concentrations of 50 mg/ml: proteinase K from Tritirachium album (Cat. No. 0706, ph 7.0, Amresco, Solon, Ohio, USA), trypsin from bovine pancreas (Cat. No. 0785, ph 7.0, Amresco, Solon, Ohio, USA) and α-chymotrypsin from bovine pancreas (Cat. No. C4129, ph 7.0, Sigma Chem Co, USA). Pepsin from porcine gastric mucosa (Cat. No. P6887, ph 3.0, Sigma Chem Co, USA) was dissolved in 0.02 N HCl to a final concentration of 50 mg/ml. All enzyme solutions were filter sterilized with disposable filters (0.45μm, Sartorius, Germany). Twenty-microliter of filtered supernatants of strains, YB42, YB98 and YB112, and 20 μl of each enzyme Biotechnol. & Biotechnol. Eq. 23/2009/4 were spotted 1 cm apart on M17 agar plates and dried for 30 min. Plates were overlaid with Lactococcus lactis IL1403 and incubated at 30 0 C for 24 h. Protease sensitivity was observed as half-moon-shaped zone of inhibition (27). Phenotypic and genotypic identification of L. lactis subsp. lactis Bacteriocin producer L. lactis subsp. lactis strain was identified by the API system (BioMérieux, Marcy I Etaile, France) and 16S rdna homology. Strain was tested for production of acids from carbohydrates and related compounds by use of API 50 CH strips and API 50 CHL medium according to the manufacturer s instructions. Strain was further characterized by 16S rdna homology using universal primers pa 5 -AGA GTT TGA TCC TGG CTC AG-3 and pe 5 -CCG TCA ATT CCT TTG AGT TT-3 (8). Template DNA was isolated from 0.5 ml of overnight culture. Cells were collected by centrifugation in a 1.5 ml sterile Eppendorf tube, resuspended in 0.5 ml of lysis buffer (250 mm NaCl, 10 mm sodium EDTA, 10 mm 1505

Tris hydrochloride (ph 8.0), 10 5 U of lysozyme per ml) and incubated at 37 0 C for 30 min. Sodium dodecyl sulfate (30 μl of a 10% (w/v) solution) was added, and the tube was incubated at 80 0 C for 5 min. The lysate was mixed with 0.7 ml of phenol-chloroform (1:10) by gentle inversion and centrifuged (5 min at 13 000 rpm). Nucleic acids were precipitated from the aqueous phase with propan-2-ol (0.7 ml) and pellet was dissolved in 50 μl of Tris EDTA buffer (ph 8.0) (3). PCR amplification was performed in a total reaction volume of 50 μl. A total of 30 amplification cycles (94 0 C for 30 s, 55 0 C for 60 s, and 72 0 C for 90 s) were performed using a programmable DNA thermocycler (Techne Genius, Cambridge, UK). The PCR product was visualized on 1% (w/v) agarose gel by staining with ethidium bromide using the O GeneRuler TM 100-bp DNA ladder (Fermentas #SM1153, Lithuania). The amplified fragment was purified using DNA gel extraction kit (Fermentas #K0513, Lithuania). Sequencing of the 16S rdna was performed in RefGen Gen Araştırmaları ve Biyoteknoloji Limited Company (Ankara, Turkey) by the use of an automated gene sequencer ABI PRISM 3730XL (Perkin Elmer, USA). Similarity search was conducted using the BLAST program at the NCBI (National Center for Biotechnology Information). Effect of ph and heat on bacteriocin activity To determine the effect of ph on bacteriocin activity, cell free culture supernatant was adjusted to ph levels between ph 2.0 and 11.0 using 6N NaOH or 6N HCl. As a control, samples were treated with proteinase K before adjusting ph and tested against the same indicator. To evaluate the effect of heat on bacteriocin activity, cellfree neutralized culture supernatant (CFNS) was heated at 100 0 C for 5, 10, 15 and 20 min. Untreated sample was used as a control (9). After ph and heat treatments, the remaining bacteriocin activity was assayed by the critical dilution method, using L. lactis subsp. lactis IL1403 as indicator. Briefly, two fold serial dilutions of cell-free culture supernatant were made with fresh GM17 medium, and aliquots (10 µl) of each dilutions were dropped onto the surface of plates which were prepared with GM17 soft agar (0.7% w/v) seeded with approximately 10 6 indicator cells. The activity expressed as arbitrary units (AU) per milliliter was calculated as follows: (1000 x 10-1 ) x (D -1 ), where D was the highest dilution that allowed no growth of the indicator organism after 18 h incubation (9). Each assay was performed in triplicate. Identification of the bacteriocin genes by PCR amplification The PCR analysis was carried out with a volume of 50 μl mixture in a DNA thermocycler (Techne Genius, Cambridge, UK). The primers for the nisin genes comprised the following nucleotide sequences, forward 5 -AAG AAT CTC TCA TGA GT-3 and reverse 5 -CCA TGT CTG AAC TAA CA-3 (26). The PCR conditions were 94 0 C for 40 s, 41 0 C for 1 min, and 72 0 C for 1 min, for 30 cycles. The amplified fragment was visualized on 1% (w/v) agarose gel by staining with ethidium bromide using the O GeneRuler TM 100-bp DNA ladder (Fermentas #SM1153, Lithuania). Isolation of plasmid DNA L. lactis subsp. lactis was grown overnight in GM17 broth at 30 0 C. Plasmid DNA was isolated by the alkali lysis procedure as described by Anderson and McKay (1). Plasmid DNA of was separated by electrophoresis in 0.7% agarose gels using Tris-EDTA buffer (ph 8.0) as electrolyte, stained with ethidium bromide and visualised under UV light. Technological characterization of L. lactis subsp. lactis The acid production of strain studied was tested by inoculating stationary culture of it (1%, v/v) into skim milk (Fluka, 70166, Germany). Incubation was at 30 0 C for 6 h and the acidity was measured by titration of the cultures at ph 8.2 with 0.1 M NaOH (2). The data were expressed as grams lactic acid per 100 ml milk culture. The determination of the proteolytic activity of the strain was measured according to the International Dairy Federation 149A (14). Stationary culture of strain was inoculated into skim milk and incubated at 30 0 C for 24 h. Afterwards, the concentration of liberated free aromatic amino acids was measured spectrophotometrically at 650 nm (Shimadzu UV-1601 spectrophotometer). Results were expressed as μg tyrosine/ml of skim milk. Results and Discussion A total of 142 presumptive lactococci were isolated from 40 raw milk samples. Four presumptive lactococci isolates (, YB42, YB98 and YB112) exhibited antimicrobial activity against indicator strains. Isolates YB42, YB98 and YB112 were found to show inhibitory activity to only three bacteria (Ent. faecalis LMG2708, Lb. plantarum LMG 2003 and L. lactis IL1403) of 22 indicator strains used in this study (data not shown). Isolate was found to show inhibitory activity against a broad range of bacteria including lactobacilli, lactococci and enterococci. In addition, it showed inhibitory activity against Listeria innocua LMG2813, Staphylococcus aureus LMG3027, Staphylococcus carnosus LMG2709, Pediococcus pentosaceus LMG2001 and Bacillus cereus LMG2732 (Table 1). No inhibitory activity could be detected against nisin-producing L. lactis SIK83 and against tested Gram-negative bacteria. Isolate showed an inhibitory spectrum identical to nisin producing L. lactis SIK83 used as an experimental control. Antimicrobial agents produced by presumptive lactococci isolates YB42, YB98 and YB112 were not inactivated by proteolytic enzymes used in this study. Complete inactivation of antimicrobial agent produced by isolate was observed after treatment with proteinase K and α-chymotrypsin but the activity of was not affected by trypsin and pepsin. These results demonstrated that the antimicrobial agent produced by isolate was bacteriocin because it was completely inactivated by proteolytic enzymes proteinase K 1506 Biotechnol. & Biotechnol. Eq. 23/2009/4

and α-chymotrypsin. Antimicrobial activity of bacteriocins is based on their proteinaceous nature although they might contain some carbohydrate and lipid moieties. Bacteriocins produced by LAB are completely or partilly inactivated after treatment with proteinase K (7, 27, 30, 31). Antimicrobial activity and protease sensitivity assays showed that the strain,, produced a bacteriocin that inhibited Gram-positive bacteria in a broad-spectrum, and was selected for further analyses. The ph and heat stability of CFNS activitiy of compared to that of nisin producing strain L. lactis subsp. lactis SIK83 used as an experimental control is shown in Table 2. Depending on the obtained data, bacteriocin of L. lactis subsp. lactis was similar to nisin (10, 17, 23, 29). TABLE 2 The effects of ph and heat treatments on bacteriocin activity Treatments Bacteriocin activity (AU ml -1 ) SIK-83 Control 12800 6400 ph ph 2.0 25600 10240 ph 3.0 25600 10240 ph 4.0 25600 10240 ph 5.0 12800 6400 ph 6.0 12800 6400 ph 7.0 12800 6400 ph 8.0 12800 6400 ph 9.0 6400 3200 ph 10.0 3200 1600 ph 11.0 1600 800 Heat 100 0 C 5 min 12800 6400 100 0 C 10 min 12800 6400 100 0 C 15 min 12800 6400 100 0 C 20 min 12800 6400 AU: Arbitrary unit : L. lactis subsp. lactis SIK83: L. lactis subsp. lactis SIK83 (nisin producer) Bacteriocin-producing strain was characterized to be a Gram-positive and catalase negative cocci. Strain fermented L-arabinose, ribose, D-xylose, galactose, glucose, fructose, mannose, mannitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, starch, gentibiose and gluconate, but none of the other sugars in the API 50 CHL reaction test (data not shown). Comparison of these carbohydrate fermentation reactions to the API 50 CHL databank revealed 99.8% homology to L. lactis subsp. lactis 1, confirming its identification. Further confirmation was achieved by PCR amplifying an approximately 930 bp fragment of 16S rdna sequence using universal primers pa 5 -AGA GTT TGA TCC TGG CTC AG-3 and pe 5 -CCG TCA ATT CCT TTG AGT TT-3. The PCR product was sequenced and the sequence Biotechnol. & Biotechnol. Eq. 23/2009/4 pattern of this fragment (930 bp) showed 99% homology with L. lactis subsp. lactis genome using the BLAST program at the NCBI. Fig. 1. PCR amplification of nisin gene fragments from genomic DNA of L. lactis subsp. lactis (lane 2); lane 1 - negative control; lane 3 - amplification of nisin gene fragment from genomic DNA of L. lactis subsp. lactis SIK83 (positive control); lane 4-100 bp DNA ladder (Fermentas #SM1153, Lithuania); lane 5 - amplification from plasmid extracts of the Fig. 2. Plasmid profile of L. lactis subsp. lactis (lane 1 and 2) The primers used for the nisin genes comprised the following nucleotide sequences, forward 5 -AAG AAT CTC TCA TGA GT-3 and reverse 5 -CCA TGT CTG AAC TAA CA-3. The results showed that about 900 bp fragment was amplified from the genomic DNA of L. lactis subsp. lactis (Fig. 1, lane 2), which was identical to that amplified from a nisin-producing strain of L. lactis subsp. lactis SIK83 (Fig. 1, lane 3). No DNA fragment was amplified using total plasmid DNA extracted from L. lactis subsp. lactis (Fig. 1, lane 5). The amplified fragment from the genomic DNA of L. lactis subsp. lactis was the same size like the one described by other researches (13, 26). In other studies, nisinproducing Lactococcus lactis strains were detected by PCR with nisin gene-specific primers in various fermented food products such as dry fermented sausage (26), Nigerian cheese product wara (22), Thai fermented sausage nham (21) and from raw milk samples (13), as confirmed in this study. Plasmid analyses showed that L. lactis subsp. lactis contains three distinct plasmids with sizes of 32.6, 21.8 and 16.8 kb (Fig. 2). Most L. lactis strains contain multiple 1507

plasmids ranging in size from 3 to 130 kb (19). PCR analysis suggests that the genes encoding nisin production are located on the genome. Nisin production genes might be located on a conjugative transposon on the chromosome (12, 13, 25). Finally, after incubation in skim milk at 30 0 C for 6 h, lactic acid production in the nisin-producing L. lactis subsp. lactis was determined as 0.38%. Proteolytic activity of was measured as 25.3 μg Tyr/mL, after 24 h in skim milk at 30 0 C. L. lactis strains may be considered useful when they are able to produce at least 0.4% lactic acid after incubation at 30 0 C for 6 h at 1% inoculation rates in milk. Proteolytic activity was 25 μg/ml tyrosine equivalents after 24 h of incubation in milk, indicating that the strain can be useful as proteolytic starter (18). Lactic acid production and proteolytic activity suggest that nisin-producing L. lactis subsp. lactis have a potential to be used as commercial starter cultures for manufacturing dairy products. Conclusions Bacteriocin producer presumptive lactococci strain was isolated from raw milk in Turkey. was identified as Lactococcus lactis subsp. lactis, both phenotypically and genotypically. Antibacterial spectrum, ph and heat stability of CFNS activitiy of exhibited a similar behavior with nisin. The bacteriocin produced by was characterized as nisin by PCR analysis. According to the results of this study, L. lactis subsp. lactis may be used as commercial starter cultures for manufacturing dairy products. REFERENCES 1. Anderson D.G., McKay L.L. (1983) Appl. Environ. Microbiol., 46, 549-552. 2. Bradley R.L., Arnold E., Barbano D.M., Semerad R.G., Smith D.E., Vines B.K. (1992) In: Standard Methods For The Examination Of Dairy Product (R.T. Marshall, Ed.) 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