Lactobacillus plantarum BM-1 isolated from a traditionally fermented Chinese meat product was found to produce a novel bacteriocin that is active against a wide range of gram-positive and gram-negative bacteria. Production of the bacteriocin BM-1 started early in the exponential phase and its maximum activity (5120 AU/mL) was recorded early during the stationary phase (16 hr). Bacteriocin BM-1 is sensitive to proteolytic enzymes but stable in the pH range of 2.0–10.0 and heat-resistant (15 min at 121°C). This bacteriocin was purified through pH-mediated cell adsorption–desorption and cation-exchange chromatography on an SP Sepharose Fast Flow column. The molecular weight of the purified bacteriocin BM-1 was determined to be 4638.142 Da by electrospray ionization Fourier transform mass spectrometry. Furthermore, the N-terminal amino acid sequence was obtained through automated Edman degradation and found to comprise the following 15 amino acid residues: H2N-Lys-Tyr-Tyr-Gly-Asn-Gly-Val-Tyr-Val-Gly-Lys-His-Ser-Cys-Ser. Comparison of this sequence with that of other bacteriocins revealed that bacteriocin BM-1 contains the consensus YGNGV amino acid motif near the N-terminus. Based on its physicochemical characteristics, molecular weight, and N-terminal amino acid sequence, plantaricin BM-1 is a novel class IIa bacteriocin.
- E. coli
- ESI-FT MS
electrospray ionization Fourier transform mass spectrometry
lactic acid bacteria
- L. monocytogenes
- L. plantarum
deMan Rogosa Sharpe
National Center for Biotechnology Information
- P. acidilactici
- S. aureus
- S. dysenteriae
- S. enteritidis
tryptic soy broth plus 0.5% yeast extract
Because of consumer demand to reduce chemical additives, bacteriocins produced by food-grade lactic acid bacteria have attracted increasing interest in recent years as natural food preservatives. Bacteriocins, ribosomally-synthesized extracellular peptides or proteins, are usually antagonistic to bacteria that are closely related to the producer strain . Some bacteriocins also inhibit various food-spoilage bacteria and food-borne pathogens, such as L. monocytogenes and Staphylococcus aureus. The following four distinct classes of bacteriocins have been identified based on their biochemical and genetic characteristics: (I) lantibiotics, which are small (<5 kDa), membrane-active peptides; (II) small (<10 kDa), heat-stable, non-lanthionine peptides; (III) large (>10 kDa) heat-labile proteins; and (IV) complex bacteriocins. The class II peptides are further divided into three subgroups. In recent years, many studies have investigated class IIa bacteriocins, which contain a consensus YGNGV amino acid motif near the N-terminus and are active against the food-borne pathogen L. monocytogenes [2, 3].
Lactobacillus plantarum is one of the most important LABs used for starter cultures in meat, cereal and vegetable fermentations [4-7]. Many of the bacteriocins produced by this organism have been characterized. Plantaricin C19, an anti-Listeria bacteriocin that contains the consensus sequence YYGNGV on the N-terminal end, has been identified as a pediocin-like bacteriocin . Plantaricin NC8 is a two-peptide bacteriocin (PLNC8α and PLNC8β); these peptides contain 28 and 34 amino acid resides, respectively, and act in a complementary way . The plantaricin produced by L. plantarum LP 31 is a peptide with a molecular weight of 1558.85 Da, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . Plantaricin ASM1, which has a mass of 5045.7 Da, contains a 21-amino acid double glycine-type extension of the N-terminus .
In this study, we describe the physicochemical characteristics of an anti-Listeria bacteriocin produced by L. plantarum BM-1 isolated from a traditionally fermented Chinese meat product. Furthermore, the molecular weight was determined by ESI-FT MS, and the N-terminal amino acid sequence of the bacteriocin BM-1 was sequenced by automated Edman degradation with a protein sequencer.
MATERIALS AND METHODS
Bacterial strains and growth conditions
Strain BM-1, the bacteriocin producer strain used in this study, was isolated from a traditionally fermented Chinese meat product and grown in MRS broth  at 37°C for 16 hr. The other strains used to determine the inhibitory spectrum of the investigated bacteriocin and their culture conditions are listed in Table 1. All strains were stored in 15% (v/v) glycerol at −80°C.
|Indicator organism||Medium-incubation, temperature (°C), time (hr)||Sensitivity†|
|Escherichia coli CDC 85933||LB, 37°C, 12 hr||+|
|Enterococcus facealis AS 1.2984||MRS, 37°C, 16 hr||+++|
|Listeria monocytogenes ATCC 54003||TSB-YE, 37°C, 16 hr||+++|
|Lactobacillus plantarum F1, our strain collection||MRS, 37°C, 24 hr||+|
|Lactobacillus acidophilus, our strain collection||MRS, 37°C, 24 hr||−|
|Lactobacillus delbrueckii subsp bulgaricus our strain collection||MRS, 37°C, 24 hr||++|
|Lactobacillus delbrueckii subsp lactis AS 1.2625T||MRS, 37°C, 24 hr||++|
|Lactobacillus acidophilus A1, our strain collection||MRS, 37°C, 24 hr||+|
|Lactobacillus johnsonii J2, our strain collection||MRS, 37°C, 24 hr||−|
|Lactobacillus casei subsp casei ATCC 334||MRS, 37°C, 24 hr||++|
|Lactobacillus pentosus ATCC 8041||MRS, 37°C, 24 hr||+++|
|Lactobacillus curvatus C1, our strain collection||MRS, 37°C, 24 hr||++|
|Salmonella enteritidis CMCC 50041||BPY, 37°C, 16 hr||+|
|Staphylococcus aureus ATCC 6535||LB, 37°C, 16 hr||+|
|Strepyococcus thermophilus T, our strain collection||MRS, 37°C, 16 hr||++|
|Shigella dysenteriae CMCC 51105||GN, 37°C°C, 24 hr||++|
|Pediococcus acidilactici P1, our strain collection||MRS, 37°C, 16 hr||+|
|Bacillus subtilis B1, our strain collection||MRS, 37°C, 16 hr||+|
Identification of Lactobacillus plantarum BM-1
Chromosomal DNA of strain BM-1 was extracted from 1 mL of culture fluid using a DNeasy Blood & Tissue Kit (Qiagen, Gaithersburg, MD, USA). PCR amplifications were performed in GeneAmp PCR System 9700 (Applied Biosystems, Foster, CA, USA) using ExTaq DNA polymerase (TaKaRa, Shiga, Japan). The PCR reaction mix (final volume 25 µL) consisted of 0.25 U ExTaq DNA polymerase, 2.5 µL of 10 × PCR reaction buffer, 1 µL of 25 mM MgCl2, 100 pmol of forward primer (AGA GTT TGA TCC TGG CTC AG) and reverse primer (TAC GGT TAC CTT GTT ACG ACT T), 1 µL of 10 mM dNTP and 0.5 µL of template DNA. The PCR program comprised an initial denaturation step at 94°C for 5 min, followed by 35 cycles of denaturation, annealing and extension steps at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, followed by a final extension step at 72°C for 7 min. The PCR products were purified using a QIAquick PCR purification kit (Qiagen) and PCR products were electrophoresed in a 1.5% agarose gel with 0.5 × Tris/borate/EDTA buffer (45 mM Tris base, 45 mM boric acid, 1 mM EDTA, pH 8.0) and sequenced (Sangong, Shanghai, China). After sequencing, the nucleotide sequence was compared with the NCBI database (http://www.ncbi.nih.gov/BLAST/) to determine homology with previously reported sequences.
Assay for bacteriocin activity
Bacteriocin activity was determined by a well-diffusion assay using L. monocytogenes ATCC54003 as the indicator strain . The activity was expressed as AU/mL where one AU was defined as the reciprocal of the highest serial twofold dilution with MRS (pH 6.0) broth that shows a clear zone .
Strain BM-1 was used to inoculate sterile MRS broth and the culture incubated at 37°C. Bacteriocin activity (AU/mL) in the cell-free supernatant was measured every 2 hr for 24 hr according to a previously method . Changes in optical density (600 nm) and pH were recorded at the same time intervals. L. monocytogenes ATCC54003 was used as the indicator strain.
Effect of enzymes, pH and temperature on bacteriocin activity
Strain BM-1 was cultured in MRS broth for 20 hr at 37°C. The cells were harvested (12,000 g, 10 min, 4°C) and the cell-free supernatant incubated for 2 hr in the presence of 1 mg/mL catalase (Amresco, Solon, OH, USA), pronase E (Roche, Indianapolis, IN, USA), trypsin (Amresco), proteinase K (Amresco), or α-amylase (Sigma, St Louis, MO, USA). Anti-Listeria activity was determined using a well-diffusion method as described above.
The effect of pH on the activity of bacteriocin BM-1 was determined by adjusting the pH of cell-free supernatants to within the range of 2.0–12.0 (at increments of 2 pH units) with sterile 1 M HCl or 1 M NaOH. After incubation for 2 hr at 37°C, antimicrobial activity was tested using a well-diffusion method.
The effect of temperature on bacteriocin BM-1 activity was tested by incubating cell-free supernatants (adjusted to pH 6.0) at 60 and 100°C for 10, 30 and 90 min. Bacteriocin activity was also assayed at 121°C for 15 and 20 min.
Mode of action of bacteriocin BM-1
To determine the mode of action of bacteriocin BM-1, 10 mL aliquots of filter-sterilized cell-free supernatant (5120 AU/mL) were added to 50 mL cultures of L. monocytogenes ATCC54003 in the exponential phase and the cultures then incubated at 37°C for 10 hr. Cultures of L. monocytogenes ATCC54003 with inactivated bacteriocin (15 min at 121°C) were used as controls. Samples were collected hourly to record the optical density at 600 nm and to determine the number of viable cells (CFU/mL) on TSBYE agar plates.
pH-mediated cell adsorption–desorption
Adsorption of bacteriocin BM-1 to the producer cells was studied using the method described by Yang et al.  with minor modifications. The culture broth was heated for 30 min at 70°C to prevent inactivation of the bacteriocin by proteases. Next, the culture was adjusted to pH 6.0 with 1 M NaOH and stirred for 30 min at room temperature to allow absorption of the bacteriocin to the producer cells. The cells were then collected (16,000 g, 15 min, 4°C) and washed twice with sterile 0.1 M phosphate buffer (pH 6.5). The pellets were resuspended in 100 mM NaCl, adjusted to pH 2.0 with 1 M HCl, and then stirred for 12 hr at 4°C. The cell suspensions were then centrifuged at 16,000 g for 25 min and the supernatants dialyzed against dH2O at 4°C for 24 hr and freeze-dried (fraction І). The bulk volume, protein concentration and bacteriocin activity of this fraction were then determined.
Fraction I was resuspended in 7 mL of 20 mM sodium citrate buffer (pH 3.1) and applied to a SP Sepharose Fast Flow cation-exchange chromatography column (length, 200 mm; internal diameter, 10 mm; GE Healthcare, Uppsala, Sweden) equilibrated with 20 mM sodium citrate buffer (pH 3.1, buffer A) at a flow rate of 1 mL/min. The column was washed with buffer A until no absorbance was detectable at 280 nm. The bacteriocin was then eluted by linear gradient elution (0.1–0.5 M NaCl in buffer A). Fractions of 5 mL were collected and their antimicrobial activity determined. The active fractions were pooled, dialyzed against dH2O, and then freeze-dried (fraction II). The bulk volume, protein concentration and bacteriocin activity of each product were then determined.
Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry
To estimate the molecular mass of fraction II, tricine SDS–PAGE was performed as previously described . This analysis consisted of step gradients of 4% and 16.5% polyacrylamide for the stacking and separating gels, respectively. To determine the position of the active bacteriocin, the gel was cut vertically into two pieces after electrophoresis. One half of the gel was stained with silver stain and the other half (extensively prewashed with sterile distilled water) overlaid with L. monocytogenes ATCC54003 (106 CFU/mL) embedded in TSBYE agar and incubated at 37°C overnight to assay bacteriocin activity. The molecular mass of fraction II was further confirmed by ESI-FT MS using a 9.4T Apex Qe mass spectrometer (Bruker, Bremen, Germany).
N-terminal amino acid sequencing
The partial N-terminal amino acid sequence of fraction II was determined based on automated Edman degradation with a protein sequencer (Procise 491; Applied Biosystems, Foster, CA, USA). After sequencing, the amino acid sequence was compared with the Protein NCBI database (http://www.ncbi.nih.gov/BLAST/) to determine homology with previously reported sequences.
Isolation and identification of bacteriocin-producing strain
In all, 27 LAB strains were originally isolated from a traditionally fermented Chinese meat product. During screening for antibacterial activity, the isolate BM-1 produced the largest inhibition zone against L. monocytogenes 54003. The 16S rDNA gene sequence (GenBank accession number: JX244152) indicated that strain BM-1 has 100% similarity to L. plantarum . Thus, strain BM-1 belongs to the L. plantarum species and was designated L. plantarum BM-1.
Production of bacteriocin BM-1
The cell density of L. plantarum BM-1 increased from 0.1 to 11 (OD600) over 24 hr at 37°C (Fig. 1). The pH decreased from 6.5 to 3.8 during the same period. Production of plantaricin BM-1 started early during the early exponential phase and reached its maximum (5120 AU/mL) early during the stationary phase (16 hr). Over the following 8 hr, the activity remained stable. Therefore, plantaricin BM-1 is produced in a growth-associated manner, as has been demonstrated for all bacteriocins from lactic acid bacteria [17, 18].
Effect of enzymes, pH and temperature on bacteriocin activity
Cell-free supernatants of L. plantarum BM-1 were found to be sensitive to several proteolytic enzymes (pronase E, trypsin and proteinase K; Fig. 2a). These findings indicate that the antibacterial substance is proteinaceous. Moreover, treatment with α-amylase decreased the activity of bacteriocin BM-1, which suggests that the peptide is glycosylated . The antimicrobial activity of bacteriocin BM-1 remained stable in the pH range of 2.0–8.0 (Fig. 2b), demonstrating that it is resistant to acidic conditions. Bacteriocin BM-1 was proven to be relatively stable at moderate temperatures and at 100°C for 30 min. Even after treatment at 121°C for 15 min, some inhibitory activity of bacteriocin BM-1 was detectable (Fig. 2c).
Spectrum of antimicrobial activity of plantaricin BM-1
The inhibitory spectrum of plantaricin BM-1 is presented in Table 1. Plantaricin BM-1 has a relatively wide inhibition spectrum, inhibiting the growth of a number of gram-positive and gram-negative bacteria, including Enterococcus faecalis AS1.2984, L. monocytogenes ATCC54003, Lactobacillus pentosus ATCC 8041, L. plantarum F1, S. aureus ATCC6535, E. coli CDC85933, S. dysenteriae CMCC 51105 and S. enteritidis CMCC 50041.
Mode of action of plantaricin BM-1
Addition of plantaricin BM-1 to a culture of L. monocytogenes ATCC54003 during its exponential growth phase caused a drastic decrease in the number of viable cells (from 7.81 to 5.81 log10 CFU/mL) within 5 hr (Fig. 3). However, prolonged incubation resulted in an increase in the CFU, which may be attributable to proteolytic degradation. During the same period, the optical density of L. monocytogenes ATCC54003 remained relatively constant. These findings suggest that the mode of action of bacteriocin BM-1 is bactericidal rather than bacteriolytic.
Purification of plantaricin BM-1
Plantaricin BM-1 was purified to apparent homogeneity using a two-step purification protocol (Table 2). The first step was to concentrate the plantaricin BM-1 from the growth medium by a method based on the influence of pH on adsorption and release of the bacteriocin; this resulted in an approximately 34.77-fold increase in concentration and 112% recovery. After chromatography on SP Sepharose, one major absorbance peak and a small peak were eluted between 0.1 and 0.5 M NaCl at a flow rate of 1 mL/min (Fig. 4). The fraction with the strongest antimicrobial activity was eluted in 0.3 M NaCl in buffer A (fractions 98 to 124): this provided a 57.6% yield with a specific activity of 7056.67 AU/mg.
|Purification stage†||Volume (mL)||Total protein (mg)||Total activity (AU)||Specific activity (AU/mg)||Purification fold||Recovery (%)|
|Cell culture supernatant||2000||572.17||80,000||139.82||1||100|
Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry
According to tricine SDS–PAGE analysis, fraction II yielded one peptide band in the stained section of the gel, which corresponded to the active bacteriocin BM-1 observed in the other section of the gel, which was overlaid with the indicator strain (Fig. 5). These findings suggest that the molecular weight of fraction II is approximately 4.0 kDa. To further determine the molecular mass of the bacteriocin BM-1, mass spectrometry was performed and revealed that the molecular weight of [M + H+] is 4638.142 Da (Fig. 6).
N-terminal amino acid sequencing
The sequence of residues from 1 to 15 of the purified plantaricin BM-1 was determined to be KYYGNGVTXGKHSXS, where X indicates a blank cycle in which no chromatographic signal was observed. Because this amino acid has to be derivatized before sequencing by Edman degradation, this lack of signal could be attributable to a cysteine residue. The similarities of this sequence to sequences found in the NCBI database are shown in Table 3. The first 15 amino acids at the N-terminus of plantaricin BM-1 were identical to those of the pediocin AcH produced by P. acidilactici. Automated Edman degradation analysis revealed that bacteriocin BM-1 contains the highly conserved YGNGV motif, which has been found in the N-terminal portion of many class IIa bacteriocin .
|Bacteriocin (producer organism)||Sequence and position||Accession number||Molecular weight (MS; Da)||Refs.|
|Bacteriocin BM-1 (L. plantarum)||KYYGNGVTXGKHSXS||4638.142||This study|
|Pediocin AcH (P. acidilactici)||KYYGNGVTCGKHSCS||NP857602.1||4628|||
|Bacteriocin (P. acidilactici UL5)||KYYGNGVTCGKHSCS||2105253A||4624|||
|Pediocin PA-1(P. acidilactici)||KYYGNGVTCGKHSCS||AAB23877.1||4640|||
|Prebacteriocin423 (L. plantarum)||KYYGNGVTCGKHSCS||AAL09346.1||3930.1|||
Antagonistic microorganisms and their antimicrobial metabolites may have some potential as natural preservatives to control the growth of pathogenic or spoilage microbiota in foods. Most bacteriocins produced by LAB, which have been considered to have the status of “generally regarded as safe”, appear to have relatively narrow inhibitory spectra , whereas some bacteriocins, such as plantaricin 423, are active against a wide range of bacteria, including gram-positive and food-borne pathogenic and spoilage bacteria .
In this study, L. plantarum BM-1, which produces a bacteriocin, was isolated from a traditionally fermented Chinese meat product. The plantaricin BM-1 produced by L. plantarum BM-1 was purified and partially characterized. According to our results, plantaricin BM-1 is produced in a growth-associated manner (Fig. 1) and its maximal activity occurs during the early stationary growth phase at 37°C in MRS broth. Treatment of plantaricin BM-1 with proteolytic enzymes (pronase E, trypsin, proteinase K and α-amylase) resulted in loss of activity (Fig. 2a), which suggest it is proteinaceous. Moreover, treatment with catalase did not affect the activity of plantaricin BM-1, which indicates that hydrogen peroxide is not responsible for the inhibition. Similar characteristics have been reported for plantaricin C , plantaricin D  and plantaricin MG . Plantaricin BM-1 exhibits good acid resistance and heat stability (Fig. 2b,c respectively), similarly to some of the other bacteriocins produced by L. plantarum . The observed acid resistance and heat stability would be very useful characteristics for an additive for the processing and conservation of various foods.
According to our results, plantaricin BM-1 is capable of inhibiting a wide range of LAB and some gram-positive pathogens, including L. plantarum (our strain collection), L. monocytogenes, and S. aureus. Similar findings have been reported for plantaricins S, C, C19 and BN, as summarized by Olasupo . Stevens et al. theorized that the bacteriocins of LAB inhibit gram-negative bacteria inefficiently because the outer membrane of these bacteria obstructs the site used by this bacteriocin . However, plantaricin BM-1 also has activity against gram-negative bacteria (E. coli, S. dysenteriae and S. enteritidis). Similar findings have been reported by Messi et al.  and Todorov and Dicks .
In this study, plantaricin BM-1 was purified using a two-step purification protocol (Table 2). According to tricine SDS–PAGE and mass spectrometry analyses, the molecular weight of plantaricin BM-1 is 4638.142 Da (Figs. 5 and 6). In addition, the composition of the 15 amino acids at the N-terminus of plantaricin BM-1 was determined to be KYYGNGVTXGKHSXS (Table 3), where X indicates a blank cycle in which no chromatographic signal was observed. The amino acid sequence and molecular weight of plantaricin BM-1 produced by L. plantarum indicate that it is a novel class IIa bacteriocin. The first 15 amino acids at the N-terminus of bacteriocin BM-1 are highly similar to those of pediocin AcH (produced by P. acidilactici), bacteriocin UL5 (produced by P. acidilactici UL5), CoaA (produced by Bacillus coagulans), pediocin PA-1 (produced by P. acidilactici) and prebacteriocin 423, which is the precursor of a bacteriocin produced by L. plantarum. However, the molecular weight of plantaricin BM-1 (4638.142 Da) is definitely larger than that of plantaricin 423 [19, 27], and the inhibitory spectrum of plantaricin BM-1 clearly differs from that of plantaricin 423. Thus, we postulate that these two bacteriocins are not identical and that plantaricin BM-1 is a novel class IIa bacteriocin produced by L. plantarum.
In conclusion, an anti-Listeria bacteriocin produced by L. plantarum BM-1 was isolated from a traditional Chinese meat product. This bacteriocin showed a broad inhibition spectrum, which includes some food-spoiling and pathogenic organisms in food, such as L. monocytogenes, S. aureus, E. coli, and Salmonella. Mass spectrometry analysis showed that the molecular weight of plantaricin BM-1 is 4638.142 Da. The NH2-terminal amino acid sequence demonstrated that plantaricin BM-1 contains the consensus YGNGV amino acid motif. Therefore, plantaricin BM-1 was classified as a class IIa bacteriocin. Because plantaricin BM-1 is heat-resistant and stable over a pH range of 2.0–10.0, our findings will lay a theoretical foundation for the application of this bacteriocin as a natural biopreservative in the food industry.
This study was supported by the National Natural Sciences Foundation of China (Contract No. 21076223), a National “863” Program Grant (No. 2012AA101606-05) and the Program on the Detection and Control of Spoilage Organisms and Pesticide Residues in Agricultural Products (PXM2012_014207_000011).
The authors declare they have no conflicts of interest.