To identify and characterize novel bacteriocins from Weissella hellenica 4-7.
To identify and characterize novel bacteriocins from Weissella hellenica 4-7.
Weissella hellenica 4-7, isolated from the traditional Taiwanese fermented food sian-sianzih (fermented clams), was previously found to produce a bacteriocin active against Listeria monocytogenes and some other Gram-positive bacteria. Bacteriocin activity decreased slightly after autoclaving (121°C for 15 min), but was inactivated by protease K and trypsin. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 3205·6 Da. N-terminal amino acid sequencing yielded a partial sequence, NH2-KGFLSWASKATSWLVGP, by Edman degradation. The obtained partial sequence showed high homology with leucocin B-TA33a; however, at least two different residues were observed. No identical peptide or protein was found, and this peptide was therefore considered to be a novel bacteriocin produced by W. hellenica 4-7 and termed weissellicin L.
The findings obtained in the current study suggest a novel bacteriocin produced by W. hellenica 4-7.
Bacteriocins from Weissella remain rare, and this study is the second report of a bacteriocin produced by W. hellenica.
Lactic acid bacteria (LAB) play an important role in food fermentation and contribute to the organoleptic properties of the final product, as well as its preservation and microbial safety. Antimicrobial activities of LAB, such as the production of lactic acid and ethanol, could also be associated with ribosomally synthesized proteinaceous inhibitors collectively known as ‘bacteriocins’. Bacteriocins are of great interest to the food industry as natural preservatives and possible substitutes for chemical preservation (Cleveland et al. 2001; Ross et al. 2002). Bacteriocins are peptides produced by bacteria that kill or inhibit the growth of closely related bacteria.
Bacteriocins from Lactobacillus, Enterococcus, Lactococcus and Leuconostoc have been frequently reported (Casaus et al. 1997; Ennahar et al. 1999; Foulquié Moreno et al. 2003; Zendo et al. 2003; Settanni and Corsetti 2008; Masuda et al. 2011; Chahad et al. 2012; Nishie et al. 2012; Lin et al. 2013). Several bacteriocins from Weissella, such as weissellicin 110, weissellin A, weissellicin Y and weissellicin M, have been previously reported (Srionnual et al. 2007; Papagianni and Papamichael 2011; Masuda et al. 2012). With the exception of weissellicin Y and weissellicin M, reported by Masuda et al. (2012), the study of bacteriocins from Weissella hellenica remains rare.
In a previous study, we reported that W. hellenica 4-7, isolated from fermented clams (sian-sianzih), is capable of producing a bacteriocin-like inhibitory substance (BLIS) (Chen et al. 2012). However, the primary structure and biochemical characteristics of the BLIS from W. hellenica 4-7 have not yet been discussed. The current study describes the purification and analysis of a bacteriocin from strain W. hellenica 4-7 and discusses its similarities to other known peptides.
Weissella hellenica 4-7 was grown in a modified glucose yeast peptone (GYP) medium containing 2·0% dextrose, 1·5% polypeptone, 0·5% yeast extract, 0·5% sodium acetate and 0·3% (v/v) salt solution (4% MgSO4･7H2O, 0·16% MnSO4･4H2O, 0·2% FeSO4･7H2O and 0·2% NaCl), pH 6·8, at 30°C under aerobic condition without shaking (Chen et al. 2007). Weissella paramesenteroides ATCC 33313T was used as the indicator strain for inhibitory activity assay and was grown in de Man Rogosa and Sharpe (MRS) (De Man et al. 1960) medium (BD, Franklin Lakes, NJ, USA) at 30°C. Inhibitory activity was determined using the agar-well diffusion assay. Briefly, a 2 ml of soft agar (0·5% w/v) was seeded with 100 μl of an overnight culture of the indicator strain and used to overlay an agar plate. Antibacterial activity was determined by pouring the supernatant fluid into the hole made on the agar plates. Plates were incubated overnight at 30 or 37°C and examined for clear zones of inhibition (Table 1).
|Diameter of inhibition zone (mm)a,b|
|Growth temperature (°C)|
|30 min at 80°C||11|
|30 min at 90°C||11|
|15 min at 121°C||10|
To determine the optimum temperature for bacteriocin production, 100 μl of overnight bacterial culture was inoculated into 5 ml MRS broth and then incubated at 25, 30, 37 or 45°C for 24 h. After incubation, bacteriocin activity was determined using the size of the inhibition zone in the agar-well diffusion assay. W. paramesenteroides ATCC 33313T was used as the indicator strain.
To study bacteriocin production, 10 ml of overnight bacterial culture was inoculated into 990 ml of GYP medium. At specific time intervals, 1-ml samples were removed, and the optical density at 600 nm of the culture and the arbitrary activity units (AU) ml−1 (reciprocal of the highest dilution at which activity was still obtained) of the bacteriocin were determined according to the method of Srionnual et al. (2007). W. paramesenteroides ATCC 33313T was used as the indicator strain. The incubation temperature was set based on the results obtained from the optimum growth temperature analysis.
Crude bacteriocin was initially obtained and purified by pH-dependent adsorption and desorption method onto producer cells (Yang et al. 1992). Briefly, W. hellenica 4-7 was grown in 0·8 l modified GYP medium at 30°C for 16 h. The pH was adjusted to 6·0, and the culture pellet was harvested by centrifugation for 20 min at 6100 g. The culture pellet was washed twice in 5 mmol l−1 sodium phosphate buffer (pH 6·0) and resuspended in 40 ml of 0·1 mol l−1 NaCl solution adjusted to pH 2·0 with 5% (v/v) phosphoric acid. The suspension was stirred for 1 h, and cells were removed by centrifugation at 12 400 g for 20 min. The supernatant was filtered through a 0·45-μm-pore-size membrane (Advantec, Tokyo, Japan) and then desalted using a C18 cartridge (Sep-Pak C18, Waters, Milford, MA, USA), prewashed with acetonitrile containing 0·1% trifluoroacetic acid (TFA) and conditioned with 0·1% TFA. The crude bacteriocin was eluted in 30% acetonitrile with 0·1% TFA (Yanagida et al. 2005). Eluted bacteriocin fractions were freeze-dried and stored at −20°C until further study.
Before tests, the crude bacteriocin powder was weighed and then thousand-fold (w/v) diluted by resuspending in 30% acetonitrile solution. To evaluate heat stability, the crude bacteriocin solution was incubated at 80°C for 30 min, 90°C for 30 min or 121°C for 15 min. To analyse sensitivity to various enzymes, the crude bacteriocin solution was treated with proteinase K (Merck, Darmstadt, Germany), trypsin (Wako, Osaka, Japan) or catalase (Wako) (30 IU mg−1, 37°C for 5 h). Next, the inhibitory activity of individual 2 μl crude bacteriocin treatments was determined using the agar-spot test (Schillinger and Lücke 1989). Briefly, a 2 ml of soft agar (0·5% w/v) was seeded with 100 μl of an overnight culture of W. paramesenteroides ATCC 33313T and used to overlay MRS plate. Antibacterial activity was determined by spotting 2 μl of each crude bacteriocin treatment onto the surface of MRS agar plate. Plate was incubated overnight at 30°C, and the size of clear zone was determined.
The purified bacteriocin solution was further tested against the Gram-positive and Gram-negative bacteria listed in Table 2. The inhibitory activity assay was performed using the agar-spot test method.
|Indicator strains||Medium||Incubation temperature (°C)||Weissellicin Lb||Weissellicin 110b||Weissellicin Yc||Weissellicin Mc||Weissellin Ad||Origin|
|Listeria monocytogenes ATCC 19111||BHI||37||+||−||NA||NA||+||a|
|Listeria innocua ATCC 33090T||TSB||37||NA||NA||+||+||NA||Masuda et al. (2012)|
|Lactobacillus sakei subsp. sakei JCM 1157T||MRS||30||+||+||+||+||+||a|
|Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842T||MRS||37||+||−||NA||NA||NA||a|
|Weissella paramesenteroides ATCC 33313T||MRS||30||+||+||+||+||NA||a|
|Weissella hellenica ATCC 51523T||MRS||30||+||+||+||+||NA||a|
|Weissella minor ATCC 35412T||MRS||30||−||−||NA||NA||NA||a|
|Weissella viridescens ATCC 12706T||MRS||30||+||−||NA||NA||NA||a|
|Lactococcus lactis subsp. lactis ATCC 19435T||MRS||30||−||+||+||+||NA||a|
|L. lactis subsp. cremoris ATCC 19257T||MRS||30||−||+||NA||NA||NA||a|
|L. lactis subsp. hordniae ATCC 29071T||MRS||30||−||+||NA||NA||NA||a|
|L. lactis subsp. tructae DSM 21502T||MRS||30||−||−||NA||NA||NA||a|
|Lactococcus taiwanensis 0905C15T||MRS||30||−||+||NA||NA||NA||a|
|Lactococcus garvieae E-9||MRS||30||−||−||NA||NA||NA||Lin et al. (2013)|
|L. garvieae E-10||MRS||30||−||−||NA||NA||NA||Lin et al. (2013)|
|Streptococcus thermophilus ATCC 19258T||MRS||37||+||+||NA||NA||NA||a|
|Escherichia coli ATCC 11775T||LB||37||−||−||−||−||NA||a|
|Vibrio vulnificus ATCC 27562T||BHI||37||−||−||NA||NA||NA||a|
|Bacillus subtilis subsp. subtilis ATCC 6051T||LB||37||−||−||+||+||NA||a|
The molecular size of the crude bacteriocin was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), following the method described by Yanagida et al. (2005). A 4–20% Mini-Protean TGX precast gel (Bio-Rad Laboratories, Hercules, CA, USA) was used, and BLIS molecular size was estimated using Precision Plus Protein Dual Xtea Standards (Bio-Rad). W. paramesenteroides ATCC 33313T was used as the indicator strain.
An approximately 2 mg freeze-dried crude bacteriocin sample was resuspended in 0·2 ml of 30% acetonitrile containing 0·1% TFA. The crude bacteriocin solution was subsequently placed on a C18 reverse-phase column (BioBasic-18; Thermo Scientific, Rockford, IL, USA) integrated into a high-performance liquid chromatography (HPLC) system (L-2130, L-2450; Hitachi, Tokyo, Japan). The bacteriocin was initially eluted with a gradient of MilliQ water–acetonitrile containing 0·1% TFA, at a flow rate of 1·0 ml min−1, as follows: 0–10 min, 10–20% (v/v); 10–65 min, 20–100% (v/v); and 65–70 min, 100% (v/v) acetonitrile. The bacteriocin activity of fractions obtained from the first purification procedure was determined against W. paramesenteroides ATCC 33313T. The active eluted solution was freeze-dried on an EYELA FDU-2200 freeze dryer (Tokyo Rikakikai, Tokyo, Japan). The active eluted sample was resuspended and then re-integrated into the HPLC system using the same C18 reverse-phase column (BioBasic-18; Thermo Scientific, Waltham, MA, USA). The bacteriocin was eluted with a gradient of MilliQ water–acetonitrile containing 0·1% TFA, at a flow rate of 0·9 ml min−1, as follows: 0–10 min, 30–45% (v/v); and 10–60 min, 45–70% (v/v) acetonitrile. Detection of peptides was monitored using a UV detector at 220 nm. Antimicrobial activity of detected peaks was tested using the agar-spot diffusion assay.
The molecular mass of the purified bacteriocin was determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) using a mass spectrometer (SCIEX QSTAR Elite; Applied Biosystems, Foster City, CA, USA).
The active peak obtained from the HPLC analysis was further subjected to N-terminal amino acid sequencing using an ABI Procise 494 protein sequencer (Applied Biosystems).
Maximum cell numbers and inhibitory activity against the indicator strain W. paramesenteroides ATCC 33313T were observed at 30°C (Table 1). The highest bacteriocin titres (160 AU ml−1) were obtained after 22 h of incubation at 30°C, and the highest cell density, based on OD at 600 nm, was observed after 18 h of incubation (Fig. 1). Duplicate tests were performed, and similar results were obtained.
The effects of enzymes and heat on the inhibitory agent from Weissella hellenica 4-7 are shown in Table 1. The crude bacteriocin was inactivated by proteinase K or trypsin, but was not affected by catalase treatment. The bacteriocin was considered to be heat stable, as the majority of activity remained after heating at 121°C for 15 min (Table 1).
The purified bacteriocin solution was tested against some Gram-positive and Gram-negative bacteria. The purified bacteriocin solution of W. hellenica 4-7 showed inhibitory activity against Listeria monocytogenes ATCC 19111 and some Gram-positive bacteria, as listed in Table 2, but had no activity against Escherichia coli ATCC 11775T and Vibrio vulnificus ATCC 27562T.
The molecular size of crude bacteriocin from W. hellenica 4-7 was analysed by SDS-PAGE, and the specific band associated with the antibacterial activity was approximately 5–10 kDa (Fig. 2).
In the first purification step of HPLC, a peak with bacteriocin activity was observed at 39·1 min (Fig. 3a). A fraction of this peak was collected and used for the second purification procedure. A peak with bacteriocin activity was observed at 24·3 min in the second purification step (Fig. 3b). This peak was therefore collected and used for further study.
The purified bacteriocin was further analysed by MALDI-TOF-MS, and the result showed a major peak at 3205·64 Da (Fig. 4). N-terminal amino acid analysis of the purified bacteriocin revealed the following partial sequence: NH2-KGFLSWASKATSWLVGP.
A similar sequence (Accession No.: P81052) was found in the DDBJ database (http://blast.ddbj.nig.ac.jp/blast/blastp?lang=en). It was marked as leucocin B-TA33a from Leuconostoc mesenteroides TA33a. When comparing the bacteriocin sequences, two different amino acid residues were observed between the bacteriocin from W. hellenica 4-7 and leucocin B-TA33a (Fig. 5). In addition, leucocin B-TA33a is the only sequence that shows similarity with our bacteriocin; there is no other sequence with significant similarity found in the database. The results described above strongly suggest that W. hellenica 4-7 naturally produces a novel bacteriocin, which we have termed weissellicin L.
Growth temperature has an influence on the production of bacteriocins (Drosinos et al. 2005; Van den Berghe et al. 2006). The results obtained in the current study indicated that Weissella hellenica 4-7 had higher bacteriocin production when incubated at 30°C for 24 h than when incubated at other temperatures (Table 1). The sensitivity of the substance to proteinase K and trypsin is proof of its proteinaceous nature. In addition, no effect was observed with catalase treatment, evidence that the active agent did not originate from H2O2.
The data obtained in this study indicated that weissellicin L has a completely different protein sequence to other known bacteriocins from Weissella. Additionally, the molecular weight of weissellicin L is smaller than the other four known bacteriocins produced by Weissella sp. (Srionnual et al. 2007; Papagianni and Papamichael 2011; Masuda et al. 2012) (Fig. 5).
As the results showed in the Table 2, bacteriocins from Weissella such as weissellin A, weissellicin Y and weissellicin M were found to have inhibitory abilities against Listeria species except weissellicin 110 (Srionnual et al. 2007; Papagianni and Papamichael 2011; Masuda et al. 2012). Weissellicin L also showed inhibitory ability against Listeria monocytogenes ATCC 19111 (Table 2). The finding here may open future perspective on the use of strain W. hellenica 4-7 to food preservation.
In the previous study of Masuda et al. (2012), it was found that W. hellenica QU 13 could produce two different bacteriocins, termed weissellicin Y and weissellicin M. However, a number of different characteristics were observed among weissellicin L, weissellicin Y and weissellicin M. For example, both weissellicin Y and weissellicin M showed great inhibitory ability against Lactococcus lactis subsp. lactis ATCC 19435T and Bacillus subtilis subsp. subtilis ATCC 6051T. However, no inhibitory activity against these two species was observed with weissellicin L.
When performing BLAST searches of DDBJ databases for partial sequence of weissellicin L, leucocin B-TA33a was the most closely related protein found. It is of great interest that a similar bacteriocin sequence could be observed in two strains belonging to two different genera. Although similar in protein sequence, different inhibitory characteristics were observed. Weissellicin L showed inhibitory activity against Listeria monocytogenes, whereas leucocin B-TA33a did not (Papathanasopoulos et al. 1997). In contrast, leucocin B-TA33a showed inhibitory activity against L. lactis subsp. cremoris, whereas weissellicin L did not (Papathanasopoulos et al. 1997).
The molecular weight of the 17 amino acids was calculated using the Compute pl/Mw tool in the ExPASy Proteomics Server (http://web.expasy.org/compute_pi/), and a result of 1835·14 Da was obtained. However, results from MALDI-TOF-MS indicated that the true molecular weight was approximately 3205·64 Da (Fig. 4). It is therefore considered that more than a dozen of the amino acids remain unknown in this study. To clarify this, an advanced analysis, such as in-gel digestion for mass spectrometric characterization and whole-genome sequencing analysis, will be conducted in the future.
In conclusion, the evidence presented in this report indicates that the W. hellenica isolated from sian-sianzih 4-7 produces a novel bacteriocin, which we have named weissellicin L. Weissellicin L is heat stable and has inhibitory ability against L. monocytogenes. The application of a specific bacteriocin, or the producer, could result in improved food preservation. Future work in our laboratory will also focus on the possibility of applying this novel bacteriocin as a biopreservative.
The authors would like to thank the National Science Council Taiwan for financially supporting this study through a grant to Shwu-fen Pan (Contract No. NSC 101-2313-B-130-001). The authors also thank Mr. Chi-huan Chang for his technical assistance during the amino acid sequence analyses.