World J Microbiol Biotechnol DOI 10.1007/s11274-015-1851-0

ORIGINAL PAPER

Purification and characterization of a novel plantaricin, KL-1Y, from Lactobacillus plantarum KL-1 Kittaporn Rumjuankiat1 • Rodney Horanda Perez3 • Komkhae Pilasombut4 • Suttipun Keawsompong1,2 • Takeshi Zendo3 • Kenji Sonomoto3,5 • Sunee Nitisinprasert1,2

Received: 6 November 2014 / Accepted: 3 April 2015  Springer Science+Business Media Dordrecht 2015

Abstract Three bacteriocins from Lactobacillus plantarum KL-1 were successfully purified using ammonium sulfate precipitation, cation-exchange chromatography and reverse-phase HPLC. The bacteriocin peptides KL-1X, -1Y and -1Z had molecular masses of 3053.82, 3498.16 and 3533.16 Da, respectively. All three peptides were stable at pH 2–12 and 25 C and at high temperatures of 80 and 100 C for 30 min and 121 C for 15 min. However, they differed in their susceptibility to proteolytic enzymes and their inhibition spectra. KL-1Y showed broad inhibitory activities against Gram-positive and Gram-negative bacteria, including Salmonella enterica serovar Enteritidis DMST 17368, Pseudomonas aeruginosa ATCC 15442, P. aeruginosa ATCC 9027, Escherichia coli O157:H7 and & Sunee Nitisinprasert [email protected]

E. coli ATCC 8739. KL-1X and -1Z inhibited only Grampositive bacteria. KL-1X, KL-1Y and KL-1Z exhibited synergistic activity. The successful amino acid sequencing of KL-1Y had a hydrophobicity of approximately 30 % and no cysteine residues suggested its novelty, and it was designated ‘‘plantaricin KL-1Y’’. Plantaricin KL-1Y exhibited bactericidal activity against Bacillus cereus JCM 2152T. Compared to nisin, KL-1Y displayed broad inhibitory activities of 200, 800, 1600, 800, 400 and 400 AU/ mL against the growth of Bacillus coagulans JCM 2257T, B. cereus JCM 2152T, Listeria innocua ATCC 33090T, Staphylococcus aureus TISTR 118, E. coli O157:H7 and E. coli ATCC 8739, respectively, whereas nisin had similar activities against only B. coagulans JCM 2257T and B. cereus JCM 2152T. Therefore, the novel plantaricin KL-1Y is a promising antimicrobial substance for food safety uses in the future.

1

Specialized Research Unit: Prebiotics and Probiotics for Health, Department of Biotechnology, Faculty of AgroIndustry, Kasetsart University, Lat Yao, Chatuchak, Bangkok 10900, Thailand

Keywords Bacteriocin  Lactobacillus plantarum  Purification  Characterization  Synergism  Bactericidal activity

2

Center for Advanced Studies for Agriculture and Food, (CASAF) Kasetsart University Institute for Advanced Studies (NRU-KU), Kasetsart University, Chatuchak, Bangkok 10900, Thailand

Introduction

3

Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Higashi-ku, Fukuoka, Japan

4

Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand

5

Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, Higashi-ku, Fukuoka, Japan

Lactic acid bacteria (LAB) produce a variety of antimicrobial substances, including organic acids, diacetyl, hydrogen peroxide, reuterin and bacteriocins (De Vuyst and Vandamme 1994). They are usually classified as ‘‘generally recognized as safe’’ (GRAS), and play an important role in fermented products and food preservation (Chen and Hoover 2003). Bacteriocins produced by food-associated LAB have been identified and characterized, such as nisin (Cleveland et al. 2001), nukacin (Sashihara et al. 2000), enterocin (Wilaipun et al. 2004), lactocyclicin

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(Sawa et al. 2009), leucocin (Sawa et al. 2010), sakacin (Sawa et al. 2013) and plantaricins (Gong et al. 2010). Our laboratory has screened approximately 300 LAB isolates from Nham (fermented pork), fermented meat and fermented vegetables. One active LAB, Lactobacillus plantarum KL-1, isolated from fermented meat, had growth inhibitory activity of 12,800 AU/mL against Lactobacillus sakei subsp. sakei JCM 1157T. A preliminary determination of its antimicrobial substance showed that the strain KL-1 produced a proteinaceous antimicrobial substance defined as a bacteriocin. L. plantarum is one of the most important LAB used in food production (van Reenen et al. 1998). It is commonly associated with plant material (Daeschel et al. 1987) and is used as a starter culture for food fermentation (Kelly et al. 1996). Bacteriocin-producing strains of L. plantarum have been reported from various foods, including meat and meat products (Dicks et al. 2004; Schillinger and Lu¨cke 1989), fermented milk (Todorov et al. 2007), fermented cream (Gong et al. 2010), fermented cereal (Omar et al. 2006) and fermented green olives (Leal-Sa´nchez et al. 2003). A bacteriocin produced by L. plantarum, ‘‘plantaricin’’, has been identified (Atrih et al. 1993; van Reenen et al. 1998). Inhibitory effects of plantaricin S and T, LC74, SA6 and KW30 have been observed against closely related lactic acid bacteria, particularly the mesophilic and thermophilic lactobacilli (Jime´nez-Dı´az et al. 1993; Kelly et al. 1996; Rekhif et al. 1994, 1995). Some plantaricins, such as plantaricin C19, C and F, have shown inhibitory activity against Gram-positive and Gram-negative bacteria (Atrih et al. 1993; Fricourt et al. 1994; Gonza´lez et al. 1994). Although plantaricin can inhibit both Gram-positive and Gram-negative bacteria, it has a narrow inhibitory spectrum, which does not include various foodborne pathogens. This is a major limitation of plantaricin that reduces its possible application in foods (Enan et al. 1996). The strain KL-1 was isolated from fermented meat that is kept for long periods at room temperature without spoilage. This characteristic was of interest to us for further study. This study aimed to purify and characterize the antimicrobial substance from L. plantarum KL-1 with a proteinaceous structure.

Materials and methods Bacterial strains and growth conditions Lactobacillus plantarum KL-1 was cultivated in MRS medium (de Man, Rogosa and Sharpe, Oxoid, Basingstoke, UK) at 30 C for 18 h without agitation. Bacterial strains used as indicators for the bacteriocin assay were propagated according to the growth conditions shown in Table 1.

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Determination of bacteriocin activity Bacteriocin activity was determined by the spot-on-lawn method (Ennahar et al. 2001) and expressed as either an arbitrary unit (AU) (Parente et al. 1995) or a minimum inhibitory concentration (MIC). In brief, 10 lL of cell free supernatant (CFS) was spotted onto the surface of agar plates that were overlaid with 7 mL of 0.8 % soft agar seeded with 30 lL of freshly grown L. sakei subsp. sakei JCM 1157T or various indicator strains, as shown in Table 1. After an overnight incubation, the inhibition zones on the bacterial lawn were determined. The AU was defined as the reciprocal of the highest dilution producing a clear zone of growth inhibition of the indicator strains, whereas the MIC was defined as the lowest concentration that still showed a clear zone of growth inhibition. Purification procedure The bacteriocin was purified from a 16 h culture of L. plantarum KL-1 grown in 1-L of MRS broth at 30 C. The CFS was obtained by centrifugation for 30 min at 88009g at 4 C and was purified by three steps: (1) preliminary partial purification using adsorption onto Amberlite XAD-16 resin, acetone precipitation or ammonium sulfate precipitation; (2) secondary chromatographic purification using SP-Sepharose resin, Octyl-Sepharose resin or an OASISÒ HLB cartridge; and (3) reverse phase HPLC. Preliminary partial purification steps (PPS) Hydrophobic chromatography with Amberlite XAD-16 resin This method was performed using Amberlite XAD-16 resin according to the modified method of Masuda et al. (2012). One liter of CFS was mixed with 15 g of Amberlite XAD16 (Sigma-Aldrich, St. Louis, MO, USA) activated by 70 % isopropanol and distilled water, and gently shaken at 4 C overnight to allow bacteriocin binding. The unbound substances were removed using distilled water and 150 mL of 45 % ethanol three times. Bacteriocin was eluted with 200 mL of 70 % isopropanol containing 0.1 % trifluoroacetic acid (TFA) that was subsequently removed using a rotary evaporator (Tokyo Rikakikai, Tokyo, Japan). Acetone precipitation Bacteriocin was precipitated by three-fold cold acetone, mixing well and keeping at -30 C overnight. The pellet was obtained by centrifugation at 55009g for 15 min at 4 C and was dissolved in 10 mL of 50 mM phosphate

World J Microbiol Biotechnol Table 1 List of indicator strains and their growth conditions

Indicator microorganisms

Medium

Temperature (C)

Lactobacillus sakei subsp. sakei JCM 1157T

MRS

30

Lactococcus lactis subsp. lactis ATCC 19435T

MRS

30

Pediococcus pentosaceus JCM 5885T

MRS

30

Pediococcus dextrinicus JCM 5887T

MRS

30

T

Lactic acid bacteria

Lactobacillus plantarum JCM 1149

MRS

30

Leuconostoc mesenteroides subsp. mesenteroides JCM 6124T

MRS

30

Enterococcus faecalis JCM 5803T

MRS

30

Enterococcus faecium JCM 5804T

MRS

30

Listeria innocua ATCC 33090T

TSB-YE

37

Kocuria rhizophila NBRC 12708

TSB-YEa

30

Staphylococcus aureus TISTR 118

NB

37

TSB-YEa

37

TSB-YEa NBa

37 37

NBa

37

Salmonella enterica serovar Enteritidis DMST 17368

TSB-YEa

37

Pseudomonas aeruginosa ATCC 15442

NBa

37

Pseudomonas aeruginosa ATCC 9027

NBa

37

Escherichia coli O157:H7

NB

a

37

Escherichia coli ATCC 8739

NBa

37

Other Gram-positive bacteria

Bacillus coagulans JCM 2257T Bacillus subtilis subsp. subtilis JCM 1465 Bacillus cereus JCM 2152T

T

Corynebacterium striatum JCM 1306T Gram-negative bacteria

ATCC American type culture collection, Rockville, Md, USA; JCM Japan collection of microoganisms, Wako, Japan; NBRC NITE biological resource center, Chiba, Japan; TISTR Thailand Institute of Scientific and Technological Research; MRS, De Man, Rogosa and Sharpe broth (Oxoid, UK); TSB-YE, Tryptic Soy Broth (Difco, Md) containing 0.6 % Yeast extract (Nacalai Tesque, Japan); NB, Nutrient broth (Merck, Germany) a

Growth under agitation at 200 rpm

buffer at pH 5.6 (PB) and examined for bacteriocin activity (Zendo et al. 2006). Ammonium sulfate precipitation Ammonium sulfate saturation at 85 % was used to precipitate bacteriocin at 4 C for 24 h with constant stirring. The precipitates were collected by centrifugation at 88009g at 4 C for 20 min and were dissolved in PB. Their inhibitory activities were then determined (Kimura et al. 1998). Secondary purification by chromatography Cation-exchange chromatography using SP-Sepharose This method was performed according to the modified method of Masuda et al. (2012). The active fractions from suitable PPS were applied to the SP-SepharoseTM Fast

Flow cation-exchange column (15-mm internal diameter, 100-mm length; GE Healthcare, Uppsala, Sweden) equilibrated with 100 mL of PB. The fractions were separated at a flow rate of 1 mL/min using a peristaltic pump (Micro Tube Pump MP-3, Tokyo, Japan). One-step elution of 50 mL PB containing 1 M NaCl was used to obtain the bacteriocin solution. Hydrophobic interaction chromatography by Octyl-Sepharose resin The active fractions from suitable PPS containing 1 M ammonium sulfate were applied to an Octyl-SepharoseTM 4 Fast Flow column (length, 50 mm; internal diameter, 10 mm; GE Healthcare, Uppsala, Sweden), which was preequilibrated with 1 M ammonium sulfate in 50 mL of PB and washed with three bed volumes of PB at a constant flow rate of 1 mL/min. The bacteriocin was eluted with 70 % ethanol in PB (Sawa et al. 2009).

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Hydrophobic interaction chromatography by OASIS HLB cartridge The active fractions from suitable PPS were applied to an OASISÒ HLB resin (Waters, Milford, MA, USA), according to the manufacturer’s protocol. Briefly, the column was equilibrated with 50 mL of 100 % methanol followed by 50 mL of MilliQ water. The active fractions were then eluted with 1 mL of 100 % methanol. Reverse phase HPLC The bioactive fractions obtained in the secondary purification step were subjected to reverse phase HPLC (Shimadzu, Kyoto, Japan) using a reverse phase column (Resource RPC 3-mL; GE Healthcare, Uppsala, Sweden) and eluted with a gradient of MilliQ water and acetonitrile containing 0.1 % TFA at a flow rate of 1 mL/min as follows: 0–10 min, 0–30 %; 10–25 min, 30–60 %; 25–30 min, 60–100 %; and 30–35 min, 100 % acetonitrile. The active fractions were subjected to RP-HPLC a second time to obtain the pure bacteriocin. The residual acetonitrile was removed by a Speed-Vac concentrator (Savants, Farmingdale, NY, USA). Determination of protein concentration The protein concentration was determined using a Pierce BCATM protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Protein concentrations were calculated from the standard curve defined by 2 mg/mL bovine albumin serum. Mass spectrometry and amino acid sequence determination The molecular weights of the bacteriocins were analyzed by electrospray ionization time of flight mass spectrometry (ESI-TOF MS) with a JMS-T100LC mass spectrometer (JEOL, Tokyo, Japan). The N-terminal amino acid sequences of the purified bacteriocin were analyzed by Edman degradation on a gas-phase automatic sequence analyzer (PPSQ-31, Shimazu, Kyoto Japan). Computer analysis of amino acid sequence Database searches were performed using National Center for Biotechnology Information (BLAST, http://www.ncbi. nlm.nih.gov/BLAST/). The molecular weights of the N-terminal amino acid sequences were computed by an online program (http://web.expasy.org/compute_pi/). Their hydrophobicities were determined by an online program (https://www.genscript.com/ssl-bin/site2/peptide_calcu lation.cgi).

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Determination of synergistic activity The synergistic activity assay was carried out according to the modified method of Draper et al. (2013). The concentration of each purified peptide was adjusted to 0.1 mM. The combination solutions were prepared using two or three bioactive agents in the ratio of 1:1 or 1:1:1, respectively. The bacteriocin activities both alone (control) and in combination were expressed as AU/mL and were determined by the spot-on-lawn method as described elsewhere. The fractional inhibitory activity (FIA) index (RFIAs) was calculated as follows: RFIA = FIA-X ? FIA-Y or FIAY ? FIA-Z or FIA-Z ? FIA-X or FIA-X ? FIA-Y ? FIAZ, where FIA-X is the bacteriocin activity in the combination/bacteriocin activity of X alone, FIA-Y is the bacteriocin activity in the combination/bacteriocin activity of Y alone and FIA-Z is the bacteriocin activity in the combination/ bacteriocin activity of Z alone. The FIA was interpreted as follows: synergy, FIA [ 2 and 3 for 2 and 3 component interaction. If there was a reduction ratio of \2 and 3 for 2 and 3 component interactions, respectively, as a consequence of the interaction produced by the mixture of bacteriocins, in comparison to the inhibitory effect observed for each bacteriocin alone, it was regarded as an antagonism. No synergy was defined as when the bacteriocin activity of the mixture was equal to the outcome activity of each bacteriocin component. Effect of enzyme, pH and thermal stability The bacteriocin was treated with the proteolytic enzymes trypsin, alpha-chymotrypsin, proteinase K, endoproteinase Glu-C, protease type XIV (Sigma-Aldrich, St. Louis, MO, USA), pepsin (Nacalai Tesque, Kyoto, Japan), and actinase E (Kaken Pharmaceutical, Tokyo, Japan) at a final concentration of 1 mg/mL. The reactions containing the filtersterilized enzyme solution and purified bacteriocin solution at a molar ratio of 1:10 were incubated at 37 C for 3 h and subsequently stopped by heating at 100 C for 5 min (Tagg and McGiven 1971). The residual bacteriocin activities were determined as mentioned elsewhere, using L. sakei subsp. sakei JCM 1157T as an indicator strain. A sample that was not treated with enzyme was used as the control. To determine heat and pH stability, 50 lL of each bacteriocin KL-1X, KL-1Y and KL-1Z at the concentration of 774.2, 7.6 and 50.2 lM, respectively, was added to 50 lL of a buffer composed of glycine/HCl (50 mM), acetic acid/sodium acetate (50 mM), phosphate (50 mM), Tris/HCl (50 mM), Glycine/NaOH (50 mM) or KCl/NaOH (50 mM) at pH of 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0, respectively. Each sample was heated to 80 and 100 C for 30 min, and 121 C for 15 min. The residual bacteriocin activities were determined by the spot-on-lawn method

World J Microbiol Biotechnol

mentioned elsewhere. The bacteriocin activity without heat treatment (control) was defined as 100 % relative activity. Mode of action The bacteriocin was added to the culture at the midlogarithmic growth phase of B. cereus JCM 2152T grown in 10 mL NB on a shaker (Biosan, Riga, Latvia) at 250 rpm and 37 C. The cell density was determined every 2 h for 24 h by absorbance at 600 nm using a microplate reader (Bio Rad Laboratories, Richmond, CA, USA) and the standard plate count method. The culture without the bacteriocin was used as the control. Comparison of antibacterial activities of nisin and bacteriocin KL-1Y The commercial nisin A, Nisaplin (106 International Units (IU)/g, Aplin and Barret, Dorset, UK) was prepared by dissolving in 0.02 N HCl, pH 2, to obtain a final concentration of 0.01 g/mL (104 IU/mL) followed by filtration through a 0.20 lm filter membrane (Sartorius Stedim Biotech GmbH, Go¨ttingen, Germany). Both bacteriocin KL-1Y and nisin A were adjusted to the bacteriocin activity of 102,400 AU/mL against the growth of L. sakei subsp. sakei JCM 1157T and tested for their antagonistic activities against B. cereus JCM 2152T, L. innocua ATCC 33090T, Staphylococcus aureus TISTR118, E. coli O157:H7 and E. coli ATCC 8739, as described elsewhere.

Results Purification of bacteriocin A three-step procedure consisting of a preliminary partial purification step (PPS), secondary purification by chromatography and final purification by HPLC was performed. Three techniques were performed in the first step, Amberlite XAD-16 resin binding, acetone precipitation and ammonium sulfate precipitation, as shown in Table 2. It was found that 85 % ammonium sulfate precipitation gave the highest recovery of 39.2 % partial purified substance (PPS), whereas the yield of Amberlite XAD-16 binding and acetone precipitation were only 10 and 0.5 %, respectively. Therefore, ammonium sulfate precipitation was selected for the first purification step. For the second step of chromatographic purification, purification of PPS by cation exchange chromatography by SP Sepharose and hydrophobic chromatography of Octyl Sepharose and OASIS HLB resulted in recovery yields of 13 and 0.4 %, respectively, as shown in Table 2. The

activity of 1600 AU/mL obtained from the purification using Octyl Sepharose is very low. Moreover, OASIS HLB yielded no bacteriocin. Hence, SP Sepharose resin was chosen for this step. Finally, reverse phase HPLC was used for the final purification. The antimicrobial activities were detected at retention times of approximately 15, 17 and 18 min, indicated as peaks 1, 3 and 5, respectively, in Fig. 1. These three active fractions were separated a second time by reverse-phase HPLC, providing the pure bacteriocins named bacteriocin KL-1X, KL-1Y and KL-1Z with the specific activities of 7.4x102, 2x105 and 1.8x104 AU/mg and yields of 0.1, 15.8 and 2.0 % of SP Sepharose bacteriocin, as shown in Table 3. Therefore, based on three purification steps, the bacteriocins KL-1X, -Y and -Z had yields of 0.004, 0.8 and 0.1 % and purifications of 0.04, 10.5 and 0.9-fold, respectively. Mass spectrometry and amino acid sequencing analysis The molecular masses of the bacteriocins KL-1X, KL-1Y and KL-1Z purified from culture supernatants of L. plantarum KL-1 and analyzed by ESI-TOF MS were 3053.82, 3498.16 and 3533.16 Da, respectively, as shown in Fig. 2. Bacteriocin KL-1Y was successfully amino acid sequenced, which comprised 30 amino acid residues (GRADYNFGYGLGRGTRKFFNGIGRWVR KTF), but the amino acid sequences of KL-1X and -1Z were not obtained. It was presumed that each amino acid sequence was blocked from unknown modifications (Nissen-Meyer et al. 1993) or that the formylmethionine was retained (Cintas et al. 1998). Several pretreatments with alkaline mercaptoethanol, BNPS-skatol and cyanogen bromide (CNBr) were applied; however, sequence analysis was still unsuccessful. A study of the pretreatment techniques for both peptides should be undertaken in the future. Based on the amino acid sequence of bacteriocin KL1Y, it had a molecular weight of 3497.97 Da, close to the value observed by ESI-TOF MS. Protein homology analyses showed no homology to any other plantaricin or bacteriocin. Bacteriocin KL-1Y therefore could be a novel bacteriocin. The amino acid sequence of plantaricin KL-1Y determined in this study has been submitted to The UniProt Knowledgebase (UniProtKB; http://www.uniprot.org/) under accession number C0HJC0. The primary structure of plantaricin KL-1Y had a hydrophobicity of approximately 30 % and no cysteine residues. The primary structures of most mature peptide plantaricins have different hydrophobicities. For example: (1) 20–30 % hydrophobicity of plantaricin Wa (Holo et al. 2001), plantaricin ASM1 (Hata et al. 2010), plantaricin C19 (Atrih et al. 2001) and

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World J Microbiol Biotechnol Table 2 Yields of bacteriocins produced by L. plantarum KL-1 in each purification step

Sample

Volume

Activity (AU/mL)

Total activity (AU)

Yield (%)

First step 1000

51,200

5.1 9 107

100

200

25,600

5.1 9 106

10

10

25,600

2.6 9 105

0.5

200

102,400

2.0 9 107

39.2

200

102,400

2.0 9 107

100

SP-Sepharose resin

50

51,200

2.6 9 106

13

Octyl-Sepharose resin

50

1600

8.0 9 104

0.4

1

0

0

0

50

51,200

2.6 9 106

100

Reverse phase HPLC (KL-1X)

5

400

2.0 9 103

0.1

Reverse phase HPLC (KL-1Y)

4

102,400

4.1 9 105

15.8

Reverse phase HPLC (KL-1Z)

4

12,800

5.1 9 104

2

Supernatant Amberlite XAD-16 resin Acetone precipitation Ammonium sulfate precipitation Second step Ammonium sulfate precipitation

OASIS HLB cartridge Third step SP-Sepharose resin

Inhibition spectra of purified plantaricins KL-1X, -1Y and -1Z

Fig. 1 Reverse-phase HPLC profile for the purification of bacteriocin produced by L. plantarum KL-1. The dotted line shows the mobile phase gradient (CH3CN and MilliQ water acidified with 0.1 % trifluoroacetic acid). Three active fractions of purified bacteriocins were determined as 1, 3 and 5, indicated with double-headed arrows

plantaricin J (Anderssen et al. 1998) with cysteine residues of 5,5,3 and 0, respectively; (2) 31–40 % hydrophobicity of plantaricin E and K (Anderssen et al. 1998), plantaricin NC8a and NC8b (Maldonado et al. 2003), plantaricin 149 (Mu¨ller et al. 2007) and plantaricin Wb (Holo et al. 2001) without cysteine residues, except plantaricin Wb, which has 4 cysteines; (3)[40 % hydrophobicity of plantaricin A (Diep et al. 1994) and plantaricin 1.25b (Ehrmann et al. 2000) containing cysteine residues of 0 and 1, respectively. It seemed that the primary structure of plantaricin KL-1Y is similar to plantaricin J (Anderssen et al. 1998), with low hydrophobicity and no cysteine residues.

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The MIC values for plantaricin KL-1X, KL-1Y and KL-1Z at concentrations of 77.42, 194.21 and 160.705 lM, respectively, were determined against Gram-positive and Gram-negative bacteria of 18 and 4 strains, respectively, as shown in Table 4. The inhibitory spectra against L. sakei subsp. sakei JCM 1157T, Lactococcus lactis subsp. lactis ATCC 19435T, Pediococcus pentosaceus JCM 5885T and P. dextrinicus JCM 5887T are shown. Only KL-1Y and -1Z had an MIC value for Leuconostoc mesenteroides subsp. mesenteroides JCM 6124T, of 0.75 and 20.08 lM, respectively. KL-1Y showed a wide inhibitory spectrum of \0.18–97.1 lM against both potential Gram-positive and Gram-negative bacteria pathogens studied, whereas KL-1X and KL-1Z did not. The most sensitive strains to KL-1Y were L. sakei subsp. sakei JCM 1157T and P. dextrinicus JCM. B. coagulans JCM 2257T was the most resistant, with the highest MIC value (97.10 lM). Synergistic activity of three purified bacteriocins Plantaricin KL-1X, KL-1Y and KL-1Z were studied for their synergistic effects in combinations of two and three bioactive agents, as shown in Table 5. All combinations provided higher bacteriocin activities than one alone. The combination of KL-1X, -1Y and -1Z exerted the highest FIA, resulting in a high degree of synergy, although its bacteriocin activities were equivalent to the combination of plantaricins KL-1X and -1Z.

World J Microbiol Biotechnol Table 3 Purification of KL-1X, -1Y and -1Z produced by L. plantarum KL-1 Total activity (AU)a

Yield (%)

Total protein (mg)b

Specific activity (AU/mg)

51,200

5.1 9 107

100

2648.5

1.9 9 104

1.0

200

102,400

2.0 9 10

7

39.2

718.5

4

2.8 9 10

1.5

50

51,200

2.6 9 106

5.1

105.7

2.5 9 104

1.3

5

400

2.0 9 103

0.004

2.7

7.4 9 102

5

0.8

2.0

2.0 9 105

0.1

2.8

1.8 9 104

Fraction

Volume (mL)

Culture supernatant

1000

Ammonium sulfate precipitation SP-Sepharose

Activity (AU/mL)

Purification (-fold)

RPC—HPLCc KL-1X KL-1Y

4

102,400

4.1 9 10

KL-1Z

4

12,800

5.1 9 104

a

0.04 10.5 0.9 T

Antimicrobial activity [in arbitrary units (AU)] was assayed by the spot-on-lawn method using Lactobacillus sakei JCM 1157 as an indicator strain

b

The protein concentration (in lg/mL) was estimated by BCATM protein quantification (A540)

c

Reverse phase HPLC

Fig. 2 Electrospray ionization time-of-flight mass spectra of bacteriocins KL-1X, -1Y and -1Z

Effect of enzyme, pH and high temperature on bacteriocin stability The effects of proteolytic enzymes, pH and high temperature on bacteriocin activity were determined using L. sakei JCM 1157T as an indicator strain. The sensitivities of the plantaricins KL-1X, KL-1Y and KL-1Z to 7 proteolytic enzymes are shown in Table 6. All were resistant to endoproteinase Glu-C and sensitive to protease type XIII and pepsin. Both plantaricin KL-1Y and KL-1Z were completely inactivated by trypsin, alpha-chymotrypsin,

protease type XIII, pepsin and actinase E. However, only plantaricin KL-1Z was sensitive to proteinase K. The activities of KL-1X, KL-1Y and KL-1Z were assayed in pHs of 2–10 at 25 C overnight, as shown in Table 7. Both KL-1X and KL-1Z were stable at pH 2–6, whereas KL-1Y was stable at pH 2–10. KL-1X was stable at 80 C at pH 4 and 6, whereas plantaricin KL-1Y was stable at 80, 100 and 121 C at pH 4 and at 80 and 100 C at pH 6. The bacteriocin KL-1Z displayed the greatest thermal stability over a wide pH range, from 2 to 6, and over a temperature range from 80 to 121 C.

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World J Microbiol Biotechnol Table 4 Antimicrobial activity spectrum of KL1-X, -Y and -Z produced by L. plantarum KL-1

MIC (lM)a

Indicator microorganism

KL-1Xb

KL-1Yc

KL-1Zd

Lactobacillus sakei subsp. sakei JCM 1157T

19.35

\0.18

1.25

Lactococcus lactis subsp. lactis ATCC 19435T

19.35

3.03

160.70

Pediococcus pentosaceus JCM 5885T

38.71

0.30

160.70

Pediococcus dextrinicus JCM 5887T

38.71

0.18

20.08 [160.70

T

[77.42

1.51

Leuconostoc mesenteroides subsp. mesenteroides JCM 6124T

[77.42

0.75

20.08

Enterococcus faecalis JCM 5803T

[77.42

24.27

[160.70

Enterococcus faecium JCM 5804T

[77.42

12.13

[160.70

Listeria innocua ATCC 33090T

[77.42

12.13

[160.70

Kocuria rhizophila NBRC 12708

[77.42

6.06

[160.70

Staphylococcus aureus TISTR 118 Bacillus coagulans JCM 2257T

[77.42 [77.42

24.27 97.10

[160.70 [160.70

Bacillus subtilis subsp. subtilis JCM 1465T

[77.42

1.51

[160.70

Bacillus cereus JCM 2152T

[77.42

24.27

[160.70

Corynebacterium striatum JCM 1306T

[77.42

48.55

[160.70

Salmonella enterica serovar Enteritidis DMST 17368

[77.42

48.55

[160.70

Pseudomonas aeruginosa ATCC 15442

[77.42

12.13

[160.70

Pseudomonas aeruginosa ATCC 9027

[77.42

24.27

[160.70

Escherichia coli O157:H7

[77.42

48.55

[160.70

Escherichia coli ATCC 8739

[77.42

48.55

[160.70

Lactobacillus plantarum ATCC 8014

ATCC American type culture collection, Rockville, Md, USA; JCM Japan collection of microorganisms, Wako, Japan; NBRC NITE biological resource center, Chiba, Japan; TISTR Thailand institute of scientific and technological research a

Table 5 The inhibitory activity of KL-1X, -1Y and -1Z alone and in combination

Initial concentration tested (b = 77.42; c = 194.21; d = 160.70 lM)

Combination reaction

KL-1X alone

KL-1Y alone

FIA KL-1Z alone

Combination

KL-1X ? KL-1Y

400

1600



6400

20

KL-1Y ? KL-1Z



1600

3200

12,800

12

KL-1X ? KL-1Z KL-1X ? KL-1Y ? KL-1Z

400 200

– 800

3200 1600

25,600 25,600

72 176

Mode of action of plantaricin KL-1Y To determine the mode of action of plantaricin KL-1Y, B. cereus JCM 2152T, a foodborne pathogen, was used as an indicator strain. In this experiment, the addition of 24.27 lM of plantaricin KL-1Y to the mid log phase (6 h) of a culture solution of B. cereus JCM 2152T resulted in no survival cells detected during 6–24 h, as shown in Fig. 3. However, its optical density still remained with slightly decreasing. In contrast, the cell concentration of the control increased to 12 h with a slow decrease in the optical density. These results suggested that plantaricin KL-1Y has bactericidal activity without cell lysis.

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Bacteriocin activity (AU/mL)

Comparison of the inhibitory activity of plantaricin KL-1Y and nisin Nisin is a known bacteriocin that is available in the market. The efficiency of plantaricin KL-1Y and nisin were investigated. Equivalent bacteriocin activities of 102,400 AU/mL for plantaricin KL-1Y and nisin against 6 potential pathogens were determined, as shown in Table 8. Plantaricin KL-1Y exhibited inhibitory activity to all strains of Gram-positive and Gram-negative bacteria tested, where nisin showed similar activities to only the Gram-positive bacteria of B. coagulans JCM 2257T and B. cereus JCM 2152T.

World J Microbiol Biotechnol Table 6 Effect of proteolytic enzymes on the plantaricins KL-1X, 1Y and -1Z produced by L. plantarum KL-1 against L. sakei subsp. sakei JCM 1157T Diameter (mm)a

Proteolytic enzyme

KL-1X

KL-1Y

KL-1Z

pH 3 buffer (untreated with enzyme)

16

16

13

pH 5.5 buffer (untreated with enzyme)

16

16

13

pH 8 buffer (untreated with enzyme)

16

16

13

Trypsin

16

0

0

Alpha-chymotrypsin

16

0

0

Protease type XIII

0

0

0

Pepsin

0

0

0

Proteinase K

16

16

0

Endoproteinase Glu-C

16

15

13

Actinase E

16

0

0

a

Clear zones of inhibition indicated as diameter in mm

Discussion Three bacteriocins, KL-1X, -1Y and -1Z, were successfully purified using the three steps of ammonium sulfate precipitation, cation exchange chromatography and reverse phase HPLC, indicating their hydrophobic cationic structures, which corresponded to previously reported bacteriocin structures (Todorov et al. 2004). KL-1Y and KL-1Z were thermally stable at pH 2–6, similar to other plantaricins (Gong et al. 2010). Because many food processing procedures involve a heating step, the plantaricin group including plantaricin KL-1Y and KL-1Z would be a choice as preservatives for the food industry. These three peptides were different in their inhibition spectrum and enzyme susceptibility. Both KL-1Y and KL1Z were sensitive to the same proteolytic enzymes, except proteinase K. KL-1X was more resistant to all enzymes, except protease type XIII and pepsin. Based on these results, plantaricin KL-1Z was the most sensitive and KL-1X Table 7 pH and thermal stability of KL-1X, -1Y and -1Z at pH 2–12

pH

was more resistant to proteolytic enzymes. Both KL-1Y and KL-1Z were sensitive to trypsin, alpha-chymotrypsin and pepsin, which are active in the gastrointestinal tract (Dallas et al. 2012). The proteinaceous nature of bacteriocin can be degraded by proteolytic enzymes and later be found in an inactive form in the human body (Perez et al. 2014). Therefore, bacteriocin from L. plantarum KL-1 would not cause the emergence of antibiotic resistance. Plantaricin KL-1Y showed a broad inhibitory activity against both Gram-positive and Gram-negative bacteria, where bacteriocin KL-1X and -1Z could inhibit only Grampositive bacteria. Based on their differences in proteolytic enzyme sensitivity, broad inhibitory activity and molecular weight, it was concluded that L. plantarum KL-1 produced three different bacteriocins with different characteristics. The combination of both KL-1X and KL-1Y, KL-1X and KL-1Z or KL-1Y and KL-1Z showed higher synergistic activities than alone. The two peptides, plantaricin Wa and plantaricin Wb alone had low antimicrobial activity but they showed the synergism when all of them were mixed in 1:1 ratio (Holo et al. 2001). However, in this study a synergistic effect had also occurred by the combination of three bioactive agents with the highest bacteriocin activities equal to that of KL-1X and KL-1Z. This was the first report where it was quantitatively shown that a positive interaction occurs between three bacteriocins from L. plantarum KL-1. The amino acid sequence of KL-1Y contains no unusual amino acids, such as lanthionine and beta-methylanthionine, and is tolerant to high temperature, indicating that it belongs to bacteriocin class II. The cysteine content is an important characteristic of class II bacteriocin. Bacteriocins of class IIa have at least two cysteines with disulfide bridges (Ennahar et al. 2000). Although the primary structure of other plantaricins contained 0–5 cysteine residues, KL-1Y did not contain any cysteine and showed inhibition activity against E. coli O157:H7 which was not reported by the previous studies. These structural differences may affect its inhibitory spectrum and need for further study.

Relative activity of KL-1X (%)

Relative activity of KL-1Y (%)

Relative activity of KL-1Z (%)

C

80 C

100 C

121 C

C

80 C

100 C

121 C

C

80 C

100 C

121 C

2 4

100 100

50 100

50 50

25 50

100 100

50 100

50 100

12.5 100

100 100

100 100

100 100

100 100

6

100

100

25

6.25

100

100

100

50

100

100

100

100

8

50

50

25

6.25

100

50

50

50

50

50

50

25

10

50

50

25

6.25

100

50

50

6.25

50

50

50

6.25

12

50

25

25

6.25

50

50

50

0

25

25

6.25

0

The control was incubated at 25 C overnight. The relative activity of the control at pH 2, 4 and 6 without any treatment was defined as 100 % C control

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World J Microbiol Biotechnol Fig. 3 Mode of action of plantaricin KL-1Y produced by L. plantarum KL-1. Growth of B. cereus JCM 2152T determined by the optical density at 600 nm without bacteriocin (–O–) and with bacteriocin (•••O•••); cell concentration of B. cereus JCM 2152T without bacteriocin (–X– ) and with bacteriocin (•••X•••). The arrow indicates the addition of KL-1Y at 6 h cultivation

Table 8 Inhibitory activity of Nisaplin and plantaricin KL1-Y Indicator strain T

Lactobacillus sakei subsp. sakei JCM 1157 (control) Bacillus coagulans JCM 2257T Bacillus cereus JCM 2152

T

Listeria innocua ATCC 33090T

Nisaplin (104 IU; AU/mL)

Plantaricin KL-1Y (AU/mL)

102,400

102,400

200

200

800

800

0

1600

Staphylococcus aureus TISTR 118

0

800

Escherichia coli O157:H7

0

400

Escherichia coli ATCC 8739

0

400

ATCC American type culture collection, Rockville, Md, USA; JCM Japan collection of microoganisms, Wako, Japan; TISTR Thailand institute of scientific and technological research

Plantaricin KL-1Y showed effective wide inhibitory activities against Gram-positive bacteria, especially Leuc. mesenteroides, and Gram-negative bacteria, which cause foodborne illness. Some LAB can also cause food spoilage, such as Leuc. mesenteroides subsp. mesenteroides, which has been reported as a food contaminant (Bo¨hme et al. 2010; Elizaquı´vel et al. 2008; Schirmer et al. 2009). KL-1Y had strong inhibitory activity against Leuc. mesenteroides subsp. mesenteroides JCM 6124T, with a low MIC level of 0.75 lM, whereas the other plantaricins did not (van Reenen et al. 1998). However, other LAB species could also produce other bacteriocins, including weissellicin Y, weissellicin M (Masuda et al. 2012) and lactocyclicin Q (Sawa et al. 2009), which inhibit Leuc. mesenteroides subsp. mesenteroides JCM 6124T with MICs of 3.26, 1.41, and 1.03 lM, respectively. Plantaricin KL-1Y inhibited not only LAB but also a wide range of Gram-positive bacteria, including L. innocua ATCC 33090T, K. rhizophila NBRC

123

12708, S. aureus TISTR 118, B. coagulans JCM 2257T, B. subtilis subsp. subtilis JCM 1465T, B. cereus JCM 2152T and C. striatum JCM 1306T, which cause foodborne disease and food spoilage (Chen and Hoover 2003). In addition, plantaricin KL-1Y also inhibited the growth of S. enterica serovar Enteritidis DMST 17368, P. aeruginosa ATCC 15442, Ps. aeruginosa ATCC 9027, E. coli O157:H7 and E. coli ATCC 8739. To date, a few bacteriocins, such as plantaricin MG, have been reported to inhibit some Gram-negative bacteria, including E. coli, P. fluorescens, P. putida and S. Typhimurium (Gong et al. 2010). However, no plantaricin has been reported to inhibit the growth of E. coli O157:H7. Therefore, plantaricin KL1Y could serve as antimicrobial substance with a wide inhibitory spectrum against those foodborne pathogens. Currently, nisin is used in a commercial preservative named Nisaplin (Danisco, UK) in many products. Nisaplin contains 2.5 % nisin and 77.5 % NaCl, with the

World J Microbiol Biotechnol

remainder made up of non-fat dried milk containing 12 % protein and 6 % carbohydrate (Cotter et al. 2005). It displays antimicrobial activity against a wide range of important Gram-positive foodborne pathogens and spoilage agents, such as vegetative pathogens, including Listeria, Staphylococcus and Mycobacterium (Chen and Hoover 2003) and the spore-forming bacteria, Bacillus or Clostridium (Delves-Broughton et al. 1996). Nisin shows little or no activity against Gram-negative bacteria except in combination with EDTA, citrate or lactate against S. enterica serovar Typhimurium and E. coli O157:H7 (Chen and Hoover 2003). Nisin can be used as a preservative in low pH foods that are not heat processed (DelvesBroughton et al. 1996), which is a limitation. On the other hand, the novel plantaricin KL-1Y exhibited inhibitory activities against E. coli, Pseudomonas, Salmonella and retained its activity and stability at pH 2–10 and at high temperatures, which would be very useful characteristics for food-processing procedures involving a neutral pH and heating steps. In conclusion, three bacteriocins from L. plantarum KL1 were purified using ammonium sulfate precipitation, cation-exchange chromatography (SP Sepharose) and reverse-phase HPLC. Bacteriocin KL-1Y comprised 30 amino acid residues and showed no homology to any other known bacteriocins, suggesting its novelty. It was named plantaricin KL-1Y. It displayed a broad antibacterial activity spectrum against both Gram-positive and Gramnegative bacteria and was stable at acidic and alkaline pHs as well as at high temperatures. Plantaricin KL-1Y is a promising antimicrobial substance for use as a food biopreservative. Acknowledgments We express our gratitude to The Royal Golden Jubilee Ph.D. program for a Ph.D. scholarship. Our thanks also go to S&P Syndicate Public Company Limited and the Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan. Conflict of Interests

We have no conflict of interest.

Funding This study was funded by The Royal Golden Jubilee Ph.D. program, Thailand. Ethical approval

Not required.

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Purification and characterization of a novel plantaricin, KL-1Y, from Lactobacillus plantarum KL-1.

Three bacteriocins from Lactobacillus plantarum KL-1 were successfully purified using ammonium sulfate precipitation, cation-exchange chromatography a...
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