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Purification and characterization of a novel bacteriocin CAMT2 produced by Bacillus amyloliquefaciens isolated from marine fish Epinephelus areolatus
Food Control 51 (2015) 278e282
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Purification and characterization of a novel bacteriocin CAMT2 produced by Bacillus amyloliquefaciens isolated from marine fish Epinephelus areolatus Junying An a, Wenjuan Zhu a, Ying Liu a, *, Xuemei Zhang a, Lijun Sun a, Pengzhi Hong a, Yaling Wang a, Chunhou Xu b, **, Defeng Xu a, Huanming Liu a a
College of Food Science and Technology, Guangdong Ocean University, Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Key Laboratory of Advanced Processing of Aquatic Products of Guangdong Higher Education Institution, Zhanjiang 524088, China College of Agriculture, Guangdong Ocean University, Zhanjiang 524088, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 17 November 2014 Accepted 25 November 2014 Available online 3 December 2014
A novel bacteriocin named CAMT2 was produced by strain ZJHD3-06 which was isolated from the marine fish Epinephelus areolatus and identified as Bacillus amyloliquefaciens, Bacteriocin CAMT2 inhibits important food spoilage and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Vibrio parahaemolyticus. Bacteriocin CAMT2 was purified by ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-50 and reversed phase chromatography on a C18 column. The molecular mass of the purified bacteriocin CAMT2 was about 20.0 kDa and N-terminal sequencing of the peptides revealed low similarity with existing antimicrobial peptides, as determined by an LCeMS/MS system. Bacteriocin CAMT2 was resistant for up to 100 C and pH ranging 2e10, but lost its activity when treated with protease K. The bacteriocin also showed significant antimicrobial activity against L. monocytogenes in a meat model system. These obtained results suggest that bacteriocin CAMT2 has potential for use as a food biopreservative. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Bacteriocin Purification Antimicrobial activity Food biopreservative
1. Introduction The increasing trend of limiting the use of chemical food preservatives has stimulated research in the field of biopreservation to find an attractive and alternative approach to chemical preservatives. Among biopreservatives, bacteriocins are receiving attention due to their GRAS (Generally Recognized as Safe) status and the fact that when applied to foods they have no adverse effects on food (Deegan, Cotter, Hill, & Ross, 2006). Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, and often presenting a bactericidal effect against closely related species (Cotter, Hill, & Ross, 2005). Bacteriocins have the potential for applications in contraception, antimicrobials in personal care formulations, anti-viral formulations and food preservations (Chikindas, 2014; Quintana et al., 2014). But now, the only bacteriocin that is approved for use as a food preservative by the Food
* Corresponding author. Tel.: þ86 15016421906. ** Corresponding author. Tel.: þ86 13828262512. E-mail addresses: [email protected] (Y. Liu), [email protected] (C. Xu). http://dx.doi.org/10.1016/j.foodcont.2014.11.038 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
and Drug Administration (FDA) is nisin from lactic acid bacteria (LAB) (Balciunas et al., 2013). Despite the intensive research on LAB bacteriocins, increasing attention have been addressed to the antimicrobial peptides produced by several other classes of bacteria (Riley & Wertz, 2002). Many species belonging to the genus Bacillus as well as other Gram-positive and Gram-negative bacteria have been shown to produce bacteriocins and/or bacteriocin-like substances (BLS) (Singh et al., 2012). Bacillus sp. presents a great variety of species that produce BLS, which display antimicrobial activity against food spoilage bacteria and food-borne pathogens. These include Bacillus subtilis, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus licheniformis and other (Cherif et al., 2001; Dischinger, Josten, Szekat, Sahl, & Bierbaum, 2009; Sirtori, Cladera, Lorenzini, Tsai, & Brandelli, 2006; Stein, 2005). Some species of Bacillus have a history of safe use in the food industry, including the production of food additives (de Boer & Diderichsen, 1991). Bacillus amyloliquefaciens ZJHD3-06, isolated from marine fish Epinephelus areolatus in the South China Sea, produces a bacteriocin named CAMT2. Preliminary experiments showed that the fermentation supernatant containing bacteriocin CAMT2 exerted a
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broad spectrum inhibitory activity against bacteria and yeast. In the present study, we describe the purification and characterization of bacteriocin CAMT2. The antimicrobial activity against Listeria monocytogenes was tested in a meat model system. 2. Material and methods 2.1. Bacterial strains and culture conditions The bacterium B. amyloliquefaciens ZJHD-06, previously isolated from the intestines of marine fish E. areolatus in the South China Sea, was used for production of bacteriocin CAMT2. Six strains of food spoilage bacteria and food-borne pathogens including the bacteria L. monocytogenes, Staphylococcus aureus, B. subtilis, Escherichia coli, Vibrio parahaemolyticus and yeast C. albicans were used as indicator strains for antimicrobial activity spectrum of bacteriocin CAMT2. The sources, corresponding media, and incubation conditions of the six indicator strains were summarized in Table 1. L. monocytogenes was chosen as the indicator strain for antimicrobial assays various purification steps. All strains were maintained as stock cultures in 20% (v/v) glycerol at 20 C and were transferred twice into the appropriate medium before use and incubated according to the respective growth condition of each strain. 2.2. Antimicrobial activity assay Bacteriocin activity was estimated by agar well diffusion method (Batdorj et al., 2006) with some modifications. 50 mL of each indicator bacteria suspension (108 CFU/mL) was added into 10 mL of Nutrient Agar or Potato Dextrose Agar or Trypticase Soyyeast Extract Agar (cooled about 50 C) and mixed by shaking slightly. Then, the mixture was poured into sterile pre-prepared plates with base agar containing 1.5% (w/v) agar, which previously sterile Oxford cups were placed on. After solidification of agar, the Oxford cups were pulled out gently. Then, wells (6 mm in diameter) were filled with 100 mL of the neutralized cell free supernatant (NCFS) containing bacteriocin CAMT2. The plates were left for 2 h at 4 C in sterile conditions before incubating them for 24 h at 37 C. Plates were checked for the presence of 6 mm inhibition zones or larger. To quantify the bacteriocin activity, the bacteriocin titre was determined by the serial 2-fold dilution ^ nio et al., 2008). NCFS was serially diluted twofold method (Apolo with sterile deionized water and 100 mL of each dilution was added into the wells. The titer was defined as 2n, where n is the reciprocal of the highest dilution that resulted in inhibition of the indicator strain. Thus, the arbitrary unit (AU) of antibacterial activity per milliliter was defined as 2n 1000 mL/100 mL. Table 1 Indicator strains, sources, culture conditions, and the inhibitory effects. Indicator strain
Sourcea
Mediumb
L. monocytogenes B. subtilis S. aureus V. parahemolyticus E. coli C. albicans
ATCC ATCC ATCC ATCC ATCC ATCC
TSA-YE, TSB 37 C NA, NB 37 C NA, NB 37 C NA (3%NaCl), NB 37 C NA, NB 37 C PDA, PDB 28 C
a
19111 6633 6538 17802 25922 10231
Incubation conditions Aerobic Aerobic Aerobic Aerobic Aerobic Aerobic
Inhibitionc þþþ þ þþþ þ þþ þþ
ATCC: American Type Culture Collection. TSA-YE: Trypticase Soy-Yeast Extract Agar, TSB: Trypticase Soya Broth, NA: Nutrient Agar, NB: Nutrient Broth, PDA: Potato Dextrose Agar, PDB: Potato Dextrose Broth. c þþþ: Diameter of the inhibition zone is larger than 20 mm; þþ: Diameter of the inhibition zone 15e20 mm; þ: 15e6 mm. b
279
2.3. Purification of bacteriocin CAMT2 2.3.1. Ammonium sulfate precipitation and column chromatography The activated culture of B. amyloliquefaciens ZJHD-06 (108 CFU/ mL) was used to inoculate (3% v/v) 50 mL of nutrient broth and incubated at 30 C with shaking at 120 rpm for 24 h. This culture was used to inoculate (3% v/v) 4 L of modified Tryptone Glucose Extract broth (tryptone 10 g/L, glucose 7.15 g/L, yeast extract 10 g/ L, FeSO4 0.11 mg/L, Vc 0.69 g/L, sea water). The culture was grown for 72 h with shaking at 150 rpm at 30 C and subsequently cells were separated by centrifugation (12,000 g, 15 min at 4 C). The cell free supernatant was submitted to ammonium sulfate precipitation at 60% saturation. After centrifugation at 12,000 g for 20 min at 4 C, the pellet was suspended in pH 7.0, 0.01 M phosphate buffer (PB) and dialyzed using a 1 kDa cut-off membrane against the same buffer at 4 C overnight. Antimicrobial activity was found in the supernatant but not in the re-dissolved precipitate. A 3 mL aliquot of supernatant was applied to a column (d h ¼ 1.6 cm 80 cm) of Sephadex G-50 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with 0.01 M PB pH 7.0. The column was eluted with the same buffer as mobile phase at a flow rate of 1 mL/min, until no absorbance was detected at 275 nm. Fractions of 3 mL were collected and antimicrobial activity was determined. The active fractions were pooled and further purified by reverse phase high-performance liquid chromatography (HPLC) on a C18 column (11 mm 300 mm) (PerkinElmer, Shelton, CT). Fractions of 20 mL were eluted with the following mobile phase at a flow rate of 0.5 mL/min using solvent A (60% methanol) and solvent B (40% water) for 25 min. Fractions were monitored at 220e284 nm and collected manually. Antimicrobial activity was assayed for all fractions with measurable absorbance at 220 nm. 2.3.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The purity of each of the isolated samples was determined using SDS-PAGE (Sch€ agger & von Jagow, 1987). Polyacrylamide concentrations in the stacking and the separating gels were 5.0% and 12.0%, respectively. A 10 mL aliquot of sample was mixed with 10 mL of a twofold concentrated sample buffer and heated for 15 min at € m, Andersson, & Martiasson, 2005). 70 C (Deraz, Karlsson, Hedstro Electrophoresis was conducted at a constant voltage of 80 V for 1 h and 100 V for 2 h. Molecular mass markers were from Sigma (14.4 kDae97.4 kDa). The gel was stained with Coomassie brilliant blue R-250. 2.4. Molecular mass determination and amino acid analysis Molecular mass determination and amino acid analysis were conducted at Beijing Genomics Institute (BGI) located in Shen zhen, China. The single protein gel band with antimicrobial activity was hydrolyzed by trypsin. Peptide fragments were extracted by 50% acetonitrile and 0.1% methanoic acid from the protein hydrolyzate. The peptide fragments were analyzed using an LCeMS/MS system (Shimadzu LC-20AD nanoflow-LC coupled with Thermo Fisher Scientific Q-EXACTIVE ESI-MS). For the MS analysis, the data was acquired in the MS scanning mode using a scan range of 350e2000 (m/z) and MS/MS using a scan range of 100e1800 (m/z). The sequence of the peptide was determined using professional software Mascot 2.3.02. The accuracy of the mass determinations was ±0.002%. The obtained amino acid sequence was compared with deposited sequences using the NCBI BLAST program (http://www. expasy.org/tools/blast).
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2.5. Antimicrobial activity spectrum of purified bacteriocin CAMT2 The antibacterial activity spectrum of the partially purified bacteriocin (freeze-dried powder of active fractions from Sephadex G-50) with a concentration of 100 mg/L was assessed against six strains of food spoilage bacteria and food-borne pathogens listed in Table 1. Agar well diffusion method was used as described by Batdorj et al. (2006). 2.6. Effect of pH, temperature and proteolytic enzymes on bacteriocin CAMT2 activity The sensitivity of partially purified bacteriocin CAMT2 (100 mg/ L) towards different pH, temperature and proteases was evaluated. To determine pH and temperature resistance, the partially purified peptide was incubated at different pH values between 2.0 and 10.0, and temperatures 60 C, 80 C, 100 C and 121 C for 15 min. Different hydrolytic enzymes including pepsin, trypsin, papain and proteinase K were incubated with bacteriocin for 6 h at 37 C to ensure their effect. The enzyme activity was terminated by heating at 80 C for 10 min before the antimicrobial activity of partially purified bacteriocin CAMT2 was confirmed, pH 7.0 phosphate buffer as control. 2.7. Antimicrobial activity in meat model system Fresh pork (75 g) was mixed with sterile normal saline (175 g). The mixture was chopped for 4 min at 3000 rpm in a cutter and equally placed into three polypropylene tubes labeled I, II and III. 1 mL inoculum of L. monocytogenes (1.0 105 CFU/mL) (Zhang, Kong, & Xiong, 2009) was added to each tube. Partially purified bacteriocin CAMT2 and nisin (purity of 98%, Zhejiang Silver Elephant) were added to tube I and tube II, respectively, until the concentration of bacteriocin CAMT2 and nisin was 75 mg/L, while tube III was kept as control, without the addition of any preservative. These tubes were incubated at 4 C for 15 days. Every three days, samples were homogenized in a blender for 2 min following by decimal serial dilutions before enumerating by plating in Oxford Listeria agar (Acumedia, Lansing, MI, USA). 2.8. Statistical analysis To determine whether there were any differences between the antibacterial activities of samples and control, one-way analysis of variance (ANOVA) was applied by SPSS software and P < 0.05 was considered to indicate statistical significance. All assays were performed in triplicates and the results are the means of three independent experiments.
Fig. 1. Elution profile of purified bacteriocin CAMT2 from B. amyloliquefaciens ZJHD306 on SephadexG-50 column.
G-50 and HPLC were 74,472.7 AU/mg and 134,050.86 AU/mg, respectively. The purified peptide showed a single peak at UV absorption of 220 nm and was positive for antimicrobial activity against L. monocytogenes. SDS-PAGE analysis of this bacteriocin yielded a single band after separation of the active fraction on a HPLC C18 column, while the bacteriocin only purified by ammonium sulfate precipitation showed three bands, as shown in Fig. 3. According to the molecular weight range of protein marker, the molecular mass of bacteriocin was approximately 20 kDa. Most of the bacteriocins produced by Bacillus spp. exhibit broad range molecular mass from the smallest bacteriocin-like substance (800 Da) produced by B. licheniformis (Teixeira, Cladera, Santos, & Brandelli, 2009) to the largest thuricin (950 kDa) produced by B. thuringiensis (Singh et al., 2012). Similarly, a bacteriocin produced by B. subtilis R75 isolated from fermented chunks of mung bean and purified by single step gel exclusion column chromatography had a molecular mass of 12.0 kDa (Sharma, Kapoor, Gautam, & Kumari, 2011). Subtilosin, a ribosomally synthesized cyclical peptide, is a bacteriocin produced by B. amyloliquefaciens (Sutyak et al., 2008) and showed antimicrobial activity against the food-borne pathogen L. monocytogenes Scott A, had a molecular mass of 3.4 KDa,
3. Results and discussion 3.1. Purification of bacteriocin CAMT2 The cell-free fermented broth (CFB) obtained after 72 h growth showed obvious antibacterial activity against L. monocytogenes. The bacteriocin presented in CFB was extracted by ammonium sulfate precipitation at 60% saturation and purified by column chromatography using Sephadex G-50. As shown in Fig. 1, two absorption peaks (F1 and F2) were individually collected and assayed for growth inhibition on L. monocytogenes. The antimicrobial activity was found in fractions 46e48 of peak F2 (Fig. 2). This bacteriocin was finally purified on a semi-preparative reversed phase HPLC. The purified peptide showed a single peak at UV absorption of 220 nm and was positive for antimicrobial activity against L. monocytogenes. Activity titres of active fractions from Sephadex
Fig. 2. Antibacterial activity of elution peaks of bacteriocin CAMT2 from Sephadex G50 against L. monocytogenes. F1: fractions 13e15, F2: fractions 46e48, CK: pH 7.0, 0.01 M phosphate buffer.
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Pseudomonas sp (Scholz et al., 2011). However, antimicrobial activity for bacteriocin CAMT2 observed against Gram-negative organisms such as V. parahaemolyticus and E. coli is particularly rare and has thus far been reported for only a few bacteriocins produced by LAB (Galvez, Abriouel, Lopez, & Ben Omar, 2007). 3.4. Temperature, pH stability and resistance to proteolytic enzymes
Fig. 3. SDS-polyacrylamide gel electrophoresis of purified bacteriocin from B. amyloliquefaciens ZJHD3-06. 1: molecular weight maker, 2: bacteriocin CAMT2 after ammonium sulfate precipitation, 3: bacteriocin CAMT2 after HPLC.
The results of pH stability assay for purified bacteriocin CAMT2 confirmed that the bacteriocin was fully pH stable as there was no noticeable reduction in antimicrobial activity after exposing it to a wide range of pH 2.0e10.0. It was found to be stable under 60 C, 80 C and 100 C as there was no obvious reduction in antimicrobial activity. The heat and pH stabilities are a very useful characteristics in the application of bacteriocin as food preservatives, because many food processing procedures involve a heating process and/or acidic or alkaline environment (Lee et al., 1999). However, bacteriocin CAMT2 hardly lost its antimicrobial activity after exposing it to 121 C for 15 min. Likewise, complete inactivation of bacteriocin CAMT2 inhibition activity was observed after treatment with proteinase K. Enzyme treatment with pepsin, trypsin and papain destroyed the antimicrobial activity of bacteriocin CAMT2 in different degrees (Table 2). Similar results of enzyme treatments with proteases have been reported for other bacteriocins or BLS (Batdorj et al., 2006; Cheikhyoussef et al., 2009). 3.5. Antimicrobial activity in meat model system
determined by matrix-assisted laser desorption/ionization time-offlight mass spectrometry (Marx, Stein, Entian, & Glaser. 2001). 3.2. Molecular mass determination and amino acid analysis The molecular mass and amino acid sequence of bacteriocin CAMT2 were analyzed using an LCeMS/MS system. The results showed two different peptides. The partial N-terminal sequences of the two peptides were MKIARTAIGSCLALSLTIPF and MKKWLLFLTTITLILSLGTA. The molecular masses were 19,608.57 Da and 20,265.57 Da, respectively, which was in good agreement with mass obtained by SDS-PAGE (Fig. 3). Upon extensive bioinformatic analysis, no significant similarity was observed to the partial sequences of the two peptides with available bacteriocins in the databases like BACTIBASE or Antimicrobial Peptide database.
The bacterial numbers of L. monocytogenes were tested in the meat model system, which was treated with purified bacteriocin CAMT2 and nisin, respectively. In the meat system containing bacteriocin CAMT2 or nisin, significant lower L. monocytogenes numbers were observed when compared with control. The treatment group with bacteriocin CAMT2 showed almost two orders of magnitude of bacterial counts less than control and one order of magnitude of bacterial counts less than the treatment group with nisin (Fig. 4). Teixeira (2009) reported similar levels of inhibited to the L. Monocytogenes in the meat system, the counts of L. Monocytogenes in the experimental group of their experiment were also about two orders of magnitude lower than the control group. The results indicated that bacteriocin CAMT2 showed significant antimicrobial activity against L. monocytogenes in a meat model system and presents potential for use in as a food biopreservative.
3.3. Antimicrobial activity spectrum of bacteriocin CAMT2 4. Conclusion The spectrum of antimicrobial activity of partially purified bacteriocin CAMT2 (freeze-dried powder of active fractions from Sephadex G-50) was evaluated by the agar diffusion test. As shown in Table 1, the growth of six indicator strains including Grampositive bacteria (L. monocytogenes, B. subtilis, S. aureus), Gramnegative bacteria V. parahaemolyticus, E. coli and yeast C. albicans were all inhibited by bacteriocin CAMT2. The inhibitions of bacteriocin CAMT2 against L. monocytogenes and S. aureus were more obvious (Table 1). Thus, the strong antagonism against a number of food spoilage bacteria and food-borne pathogens advocated the high possibility of using this bacteriocin as a preservatives in food. Similarly, a bacteriocin from B. subtilis R75 can suppress the growth of L. monocytogenes, S. aureus, B. subtilis and E. coli (Sharma et al., 2011). Plantazolicin, a novel microcin B17/streptolysins-like natural product from B. amyloliquefaciens FZB42, was found to be growth inhibitory toward closely related Gram-positive Bacilli, but no activity was observed toward Gram-negative bacteria, such as Erwinia carotovora, E. coli K-12, Klebsiella terrigena and
The purification result of bacteriocin CAMT2 showed two different peptides, which possessed the partial N-terminal sequences of MKIARTAIGSCLALSLTIPF and MKKWLLFLTTITLILSLGTA. The molecular mass of the two peptides were 19,608.57 Da and
Table 2 Effect of proteolytic enzymes and temperature on bacteriocin CAMT2 activity. Enzymes/T emperature
Diameter of inhibition zone ± SD (mm)
Control Trypsin Papain Proteinase K 60 C 80 C 100 C 121 C
20.34 8.91 14.13 0.00 20.23 20.03 14.74 1.80
± ± ± ± ± ± ± ±
0.52 0.14 0.09 0.00 0.11 0.08 0.23 0.00
Decreasing activity (%) 0.00 56.19 30.53 100.0 0.54 1.52 27.53 91.20
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Fig. 4. Changes in Log CFU g1 of L. monocytogenes in meat model system with the different treatments. A: purified bacteriocin CAMT2, -: nisin, :: Control.
20,265.57 Da, which was in good agreement with mass obtained by SDS-PAGE. It is noteworthy that no significant similarity was observed to the partial sequences of the two peptides with available bacteriocins in the databases. This suggests that bacteriocin CAMT2 might be a novel bacteriocin. The purified bacteriocin CAMT2 showed wider antimicrobial activity spectrum, heat stable for up to 100 C and pH stability in a wide range of pH 2.0e10.0. However, proteolytic enzyme treatment destroyed the antimicrobial activity of bacteriocin CAMT2 by different degrees. The bacteriocin also showed significant antimicrobial activity against L. monocytogenes in a meat model system. These results indicate that Bacteriocin CAMT2 might be used as a potential effective and natural food biopreservative. Acknowledgments This work was financially supported by Guangdong Science and Technology Fund (S2011010000328), National High Technology Research and Development Program of China (863 Program) (No. 2013AA102201), the National Science Fund (No. 30279987) and Higher Educational Cultivation Program for Major Scientific Research Projects of Guangdong Ocean University (No. GDOU2013050205). References ^ nio, A. C. M., Carvalho, M. A. R., Bemquerer, M. P., Santoro, M. M., Pinto, S. Q., Apolo Oliveira, J. S., et al. (2008). Purification and partial characterization of a bacteriocin produced by Eikenella corrodens. Journal of Applied Microbiology, 104, 508e514. Balciunas, E. M., Martinez, F. A. C., Todorov, S. D., de Melo Franco, B. D. G., Converti, A., & de Souza Oliveira, R. P. (2013). Novel biotechnological applications of bacteriocins: a review. Food Control, 32, 134e142. tro, F., & Pre vost, H. Batdorj, B., Dalgalarrondo, M., Choiset, Y., Pedroche, J., Me (2006). Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag. Journal of Applied Microbiology, 101, 837e848.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Purification and characterization of a novel bacteriocin CAMT2 produced by Bacillus amyloliquefaciens isolated from marine fish Epinephelus areolatus Junying An a, Wenjuan Zhu a, Ying Liu a, *, Xuemei Zhang a, Lijun Sun a, Pengzhi Hong a, Yaling Wang a, Chunhou Xu b, **, Defeng Xu a, Huanming Liu a a
College of Food Science and Technology, Guangdong Ocean University, Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Key Laboratory of Advanced Processing of Aquatic Products of Guangdong Higher Education Institution, Zhanjiang 524088, China College of Agriculture, Guangdong Ocean University, Zhanjiang 524088, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 17 November 2014 Accepted 25 November 2014 Available online 3 December 2014
A novel bacteriocin named CAMT2 was produced by strain ZJHD3-06 which was isolated from the marine fish Epinephelus areolatus and identified as Bacillus amyloliquefaciens, Bacteriocin CAMT2 inhibits important food spoilage and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Vibrio parahaemolyticus. Bacteriocin CAMT2 was purified by ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-50 and reversed phase chromatography on a C18 column. The molecular mass of the purified bacteriocin CAMT2 was about 20.0 kDa and N-terminal sequencing of the peptides revealed low similarity with existing antimicrobial peptides, as determined by an LCeMS/MS system. Bacteriocin CAMT2 was resistant for up to 100 C and pH ranging 2e10, but lost its activity when treated with protease K. The bacteriocin also showed significant antimicrobial activity against L. monocytogenes in a meat model system. These obtained results suggest that bacteriocin CAMT2 has potential for use as a food biopreservative. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Bacteriocin Purification Antimicrobial activity Food biopreservative
1. Introduction The increasing trend of limiting the use of chemical food preservatives has stimulated research in the field of biopreservation to find an attractive and alternative approach to chemical preservatives. Among biopreservatives, bacteriocins are receiving attention due to their GRAS (Generally Recognized as Safe) status and the fact that when applied to foods they have no adverse effects on food (Deegan, Cotter, Hill, & Ross, 2006). Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, and often presenting a bactericidal effect against closely related species (Cotter, Hill, & Ross, 2005). Bacteriocins have the potential for applications in contraception, antimicrobials in personal care formulations, anti-viral formulations and food preservations (Chikindas, 2014; Quintana et al., 2014). But now, the only bacteriocin that is approved for use as a food preservative by the Food
* Corresponding author. Tel.: þ86 15016421906. ** Corresponding author. Tel.: þ86 13828262512. E-mail addresses: [email protected] (Y. Liu), [email protected] (C. Xu). http://dx.doi.org/10.1016/j.foodcont.2014.11.038 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
and Drug Administration (FDA) is nisin from lactic acid bacteria (LAB) (Balciunas et al., 2013). Despite the intensive research on LAB bacteriocins, increasing attention have been addressed to the antimicrobial peptides produced by several other classes of bacteria (Riley & Wertz, 2002). Many species belonging to the genus Bacillus as well as other Gram-positive and Gram-negative bacteria have been shown to produce bacteriocins and/or bacteriocin-like substances (BLS) (Singh et al., 2012). Bacillus sp. presents a great variety of species that produce BLS, which display antimicrobial activity against food spoilage bacteria and food-borne pathogens. These include Bacillus subtilis, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus licheniformis and other (Cherif et al., 2001; Dischinger, Josten, Szekat, Sahl, & Bierbaum, 2009; Sirtori, Cladera, Lorenzini, Tsai, & Brandelli, 2006; Stein, 2005). Some species of Bacillus have a history of safe use in the food industry, including the production of food additives (de Boer & Diderichsen, 1991). Bacillus amyloliquefaciens ZJHD3-06, isolated from marine fish Epinephelus areolatus in the South China Sea, produces a bacteriocin named CAMT2. Preliminary experiments showed that the fermentation supernatant containing bacteriocin CAMT2 exerted a
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broad spectrum inhibitory activity against bacteria and yeast. In the present study, we describe the purification and characterization of bacteriocin CAMT2. The antimicrobial activity against Listeria monocytogenes was tested in a meat model system. 2. Material and methods 2.1. Bacterial strains and culture conditions The bacterium B. amyloliquefaciens ZJHD-06, previously isolated from the intestines of marine fish E. areolatus in the South China Sea, was used for production of bacteriocin CAMT2. Six strains of food spoilage bacteria and food-borne pathogens including the bacteria L. monocytogenes, Staphylococcus aureus, B. subtilis, Escherichia coli, Vibrio parahaemolyticus and yeast C. albicans were used as indicator strains for antimicrobial activity spectrum of bacteriocin CAMT2. The sources, corresponding media, and incubation conditions of the six indicator strains were summarized in Table 1. L. monocytogenes was chosen as the indicator strain for antimicrobial assays various purification steps. All strains were maintained as stock cultures in 20% (v/v) glycerol at 20 C and were transferred twice into the appropriate medium before use and incubated according to the respective growth condition of each strain. 2.2. Antimicrobial activity assay Bacteriocin activity was estimated by agar well diffusion method (Batdorj et al., 2006) with some modifications. 50 mL of each indicator bacteria suspension (108 CFU/mL) was added into 10 mL of Nutrient Agar or Potato Dextrose Agar or Trypticase Soyyeast Extract Agar (cooled about 50 C) and mixed by shaking slightly. Then, the mixture was poured into sterile pre-prepared plates with base agar containing 1.5% (w/v) agar, which previously sterile Oxford cups were placed on. After solidification of agar, the Oxford cups were pulled out gently. Then, wells (6 mm in diameter) were filled with 100 mL of the neutralized cell free supernatant (NCFS) containing bacteriocin CAMT2. The plates were left for 2 h at 4 C in sterile conditions before incubating them for 24 h at 37 C. Plates were checked for the presence of 6 mm inhibition zones or larger. To quantify the bacteriocin activity, the bacteriocin titre was determined by the serial 2-fold dilution ^ nio et al., 2008). NCFS was serially diluted twofold method (Apolo with sterile deionized water and 100 mL of each dilution was added into the wells. The titer was defined as 2n, where n is the reciprocal of the highest dilution that resulted in inhibition of the indicator strain. Thus, the arbitrary unit (AU) of antibacterial activity per milliliter was defined as 2n 1000 mL/100 mL. Table 1 Indicator strains, sources, culture conditions, and the inhibitory effects. Indicator strain
Sourcea
Mediumb
L. monocytogenes B. subtilis S. aureus V. parahemolyticus E. coli C. albicans
ATCC ATCC ATCC ATCC ATCC ATCC
TSA-YE, TSB 37 C NA, NB 37 C NA, NB 37 C NA (3%NaCl), NB 37 C NA, NB 37 C PDA, PDB 28 C
a
19111 6633 6538 17802 25922 10231
Incubation conditions Aerobic Aerobic Aerobic Aerobic Aerobic Aerobic
Inhibitionc þþþ þ þþþ þ þþ þþ
ATCC: American Type Culture Collection. TSA-YE: Trypticase Soy-Yeast Extract Agar, TSB: Trypticase Soya Broth, NA: Nutrient Agar, NB: Nutrient Broth, PDA: Potato Dextrose Agar, PDB: Potato Dextrose Broth. c þþþ: Diameter of the inhibition zone is larger than 20 mm; þþ: Diameter of the inhibition zone 15e20 mm; þ: 15e6 mm. b
279
2.3. Purification of bacteriocin CAMT2 2.3.1. Ammonium sulfate precipitation and column chromatography The activated culture of B. amyloliquefaciens ZJHD-06 (108 CFU/ mL) was used to inoculate (3% v/v) 50 mL of nutrient broth and incubated at 30 C with shaking at 120 rpm for 24 h. This culture was used to inoculate (3% v/v) 4 L of modified Tryptone Glucose Extract broth (tryptone 10 g/L, glucose 7.15 g/L, yeast extract 10 g/ L, FeSO4 0.11 mg/L, Vc 0.69 g/L, sea water). The culture was grown for 72 h with shaking at 150 rpm at 30 C and subsequently cells were separated by centrifugation (12,000 g, 15 min at 4 C). The cell free supernatant was submitted to ammonium sulfate precipitation at 60% saturation. After centrifugation at 12,000 g for 20 min at 4 C, the pellet was suspended in pH 7.0, 0.01 M phosphate buffer (PB) and dialyzed using a 1 kDa cut-off membrane against the same buffer at 4 C overnight. Antimicrobial activity was found in the supernatant but not in the re-dissolved precipitate. A 3 mL aliquot of supernatant was applied to a column (d h ¼ 1.6 cm 80 cm) of Sephadex G-50 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with 0.01 M PB pH 7.0. The column was eluted with the same buffer as mobile phase at a flow rate of 1 mL/min, until no absorbance was detected at 275 nm. Fractions of 3 mL were collected and antimicrobial activity was determined. The active fractions were pooled and further purified by reverse phase high-performance liquid chromatography (HPLC) on a C18 column (11 mm 300 mm) (PerkinElmer, Shelton, CT). Fractions of 20 mL were eluted with the following mobile phase at a flow rate of 0.5 mL/min using solvent A (60% methanol) and solvent B (40% water) for 25 min. Fractions were monitored at 220e284 nm and collected manually. Antimicrobial activity was assayed for all fractions with measurable absorbance at 220 nm. 2.3.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The purity of each of the isolated samples was determined using SDS-PAGE (Sch€ agger & von Jagow, 1987). Polyacrylamide concentrations in the stacking and the separating gels were 5.0% and 12.0%, respectively. A 10 mL aliquot of sample was mixed with 10 mL of a twofold concentrated sample buffer and heated for 15 min at € m, Andersson, & Martiasson, 2005). 70 C (Deraz, Karlsson, Hedstro Electrophoresis was conducted at a constant voltage of 80 V for 1 h and 100 V for 2 h. Molecular mass markers were from Sigma (14.4 kDae97.4 kDa). The gel was stained with Coomassie brilliant blue R-250. 2.4. Molecular mass determination and amino acid analysis Molecular mass determination and amino acid analysis were conducted at Beijing Genomics Institute (BGI) located in Shen zhen, China. The single protein gel band with antimicrobial activity was hydrolyzed by trypsin. Peptide fragments were extracted by 50% acetonitrile and 0.1% methanoic acid from the protein hydrolyzate. The peptide fragments were analyzed using an LCeMS/MS system (Shimadzu LC-20AD nanoflow-LC coupled with Thermo Fisher Scientific Q-EXACTIVE ESI-MS). For the MS analysis, the data was acquired in the MS scanning mode using a scan range of 350e2000 (m/z) and MS/MS using a scan range of 100e1800 (m/z). The sequence of the peptide was determined using professional software Mascot 2.3.02. The accuracy of the mass determinations was ±0.002%. The obtained amino acid sequence was compared with deposited sequences using the NCBI BLAST program (http://www. expasy.org/tools/blast).
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2.5. Antimicrobial activity spectrum of purified bacteriocin CAMT2 The antibacterial activity spectrum of the partially purified bacteriocin (freeze-dried powder of active fractions from Sephadex G-50) with a concentration of 100 mg/L was assessed against six strains of food spoilage bacteria and food-borne pathogens listed in Table 1. Agar well diffusion method was used as described by Batdorj et al. (2006). 2.6. Effect of pH, temperature and proteolytic enzymes on bacteriocin CAMT2 activity The sensitivity of partially purified bacteriocin CAMT2 (100 mg/ L) towards different pH, temperature and proteases was evaluated. To determine pH and temperature resistance, the partially purified peptide was incubated at different pH values between 2.0 and 10.0, and temperatures 60 C, 80 C, 100 C and 121 C for 15 min. Different hydrolytic enzymes including pepsin, trypsin, papain and proteinase K were incubated with bacteriocin for 6 h at 37 C to ensure their effect. The enzyme activity was terminated by heating at 80 C for 10 min before the antimicrobial activity of partially purified bacteriocin CAMT2 was confirmed, pH 7.0 phosphate buffer as control. 2.7. Antimicrobial activity in meat model system Fresh pork (75 g) was mixed with sterile normal saline (175 g). The mixture was chopped for 4 min at 3000 rpm in a cutter and equally placed into three polypropylene tubes labeled I, II and III. 1 mL inoculum of L. monocytogenes (1.0 105 CFU/mL) (Zhang, Kong, & Xiong, 2009) was added to each tube. Partially purified bacteriocin CAMT2 and nisin (purity of 98%, Zhejiang Silver Elephant) were added to tube I and tube II, respectively, until the concentration of bacteriocin CAMT2 and nisin was 75 mg/L, while tube III was kept as control, without the addition of any preservative. These tubes were incubated at 4 C for 15 days. Every three days, samples were homogenized in a blender for 2 min following by decimal serial dilutions before enumerating by plating in Oxford Listeria agar (Acumedia, Lansing, MI, USA). 2.8. Statistical analysis To determine whether there were any differences between the antibacterial activities of samples and control, one-way analysis of variance (ANOVA) was applied by SPSS software and P < 0.05 was considered to indicate statistical significance. All assays were performed in triplicates and the results are the means of three independent experiments.
Fig. 1. Elution profile of purified bacteriocin CAMT2 from B. amyloliquefaciens ZJHD306 on SephadexG-50 column.
G-50 and HPLC were 74,472.7 AU/mg and 134,050.86 AU/mg, respectively. The purified peptide showed a single peak at UV absorption of 220 nm and was positive for antimicrobial activity against L. monocytogenes. SDS-PAGE analysis of this bacteriocin yielded a single band after separation of the active fraction on a HPLC C18 column, while the bacteriocin only purified by ammonium sulfate precipitation showed three bands, as shown in Fig. 3. According to the molecular weight range of protein marker, the molecular mass of bacteriocin was approximately 20 kDa. Most of the bacteriocins produced by Bacillus spp. exhibit broad range molecular mass from the smallest bacteriocin-like substance (800 Da) produced by B. licheniformis (Teixeira, Cladera, Santos, & Brandelli, 2009) to the largest thuricin (950 kDa) produced by B. thuringiensis (Singh et al., 2012). Similarly, a bacteriocin produced by B. subtilis R75 isolated from fermented chunks of mung bean and purified by single step gel exclusion column chromatography had a molecular mass of 12.0 kDa (Sharma, Kapoor, Gautam, & Kumari, 2011). Subtilosin, a ribosomally synthesized cyclical peptide, is a bacteriocin produced by B. amyloliquefaciens (Sutyak et al., 2008) and showed antimicrobial activity against the food-borne pathogen L. monocytogenes Scott A, had a molecular mass of 3.4 KDa,
3. Results and discussion 3.1. Purification of bacteriocin CAMT2 The cell-free fermented broth (CFB) obtained after 72 h growth showed obvious antibacterial activity against L. monocytogenes. The bacteriocin presented in CFB was extracted by ammonium sulfate precipitation at 60% saturation and purified by column chromatography using Sephadex G-50. As shown in Fig. 1, two absorption peaks (F1 and F2) were individually collected and assayed for growth inhibition on L. monocytogenes. The antimicrobial activity was found in fractions 46e48 of peak F2 (Fig. 2). This bacteriocin was finally purified on a semi-preparative reversed phase HPLC. The purified peptide showed a single peak at UV absorption of 220 nm and was positive for antimicrobial activity against L. monocytogenes. Activity titres of active fractions from Sephadex
Fig. 2. Antibacterial activity of elution peaks of bacteriocin CAMT2 from Sephadex G50 against L. monocytogenes. F1: fractions 13e15, F2: fractions 46e48, CK: pH 7.0, 0.01 M phosphate buffer.
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Pseudomonas sp (Scholz et al., 2011). However, antimicrobial activity for bacteriocin CAMT2 observed against Gram-negative organisms such as V. parahaemolyticus and E. coli is particularly rare and has thus far been reported for only a few bacteriocins produced by LAB (Galvez, Abriouel, Lopez, & Ben Omar, 2007). 3.4. Temperature, pH stability and resistance to proteolytic enzymes
Fig. 3. SDS-polyacrylamide gel electrophoresis of purified bacteriocin from B. amyloliquefaciens ZJHD3-06. 1: molecular weight maker, 2: bacteriocin CAMT2 after ammonium sulfate precipitation, 3: bacteriocin CAMT2 after HPLC.
The results of pH stability assay for purified bacteriocin CAMT2 confirmed that the bacteriocin was fully pH stable as there was no noticeable reduction in antimicrobial activity after exposing it to a wide range of pH 2.0e10.0. It was found to be stable under 60 C, 80 C and 100 C as there was no obvious reduction in antimicrobial activity. The heat and pH stabilities are a very useful characteristics in the application of bacteriocin as food preservatives, because many food processing procedures involve a heating process and/or acidic or alkaline environment (Lee et al., 1999). However, bacteriocin CAMT2 hardly lost its antimicrobial activity after exposing it to 121 C for 15 min. Likewise, complete inactivation of bacteriocin CAMT2 inhibition activity was observed after treatment with proteinase K. Enzyme treatment with pepsin, trypsin and papain destroyed the antimicrobial activity of bacteriocin CAMT2 in different degrees (Table 2). Similar results of enzyme treatments with proteases have been reported for other bacteriocins or BLS (Batdorj et al., 2006; Cheikhyoussef et al., 2009). 3.5. Antimicrobial activity in meat model system
determined by matrix-assisted laser desorption/ionization time-offlight mass spectrometry (Marx, Stein, Entian, & Glaser. 2001). 3.2. Molecular mass determination and amino acid analysis The molecular mass and amino acid sequence of bacteriocin CAMT2 were analyzed using an LCeMS/MS system. The results showed two different peptides. The partial N-terminal sequences of the two peptides were MKIARTAIGSCLALSLTIPF and MKKWLLFLTTITLILSLGTA. The molecular masses were 19,608.57 Da and 20,265.57 Da, respectively, which was in good agreement with mass obtained by SDS-PAGE (Fig. 3). Upon extensive bioinformatic analysis, no significant similarity was observed to the partial sequences of the two peptides with available bacteriocins in the databases like BACTIBASE or Antimicrobial Peptide database.
The bacterial numbers of L. monocytogenes were tested in the meat model system, which was treated with purified bacteriocin CAMT2 and nisin, respectively. In the meat system containing bacteriocin CAMT2 or nisin, significant lower L. monocytogenes numbers were observed when compared with control. The treatment group with bacteriocin CAMT2 showed almost two orders of magnitude of bacterial counts less than control and one order of magnitude of bacterial counts less than the treatment group with nisin (Fig. 4). Teixeira (2009) reported similar levels of inhibited to the L. Monocytogenes in the meat system, the counts of L. Monocytogenes in the experimental group of their experiment were also about two orders of magnitude lower than the control group. The results indicated that bacteriocin CAMT2 showed significant antimicrobial activity against L. monocytogenes in a meat model system and presents potential for use in as a food biopreservative.
3.3. Antimicrobial activity spectrum of bacteriocin CAMT2 4. Conclusion The spectrum of antimicrobial activity of partially purified bacteriocin CAMT2 (freeze-dried powder of active fractions from Sephadex G-50) was evaluated by the agar diffusion test. As shown in Table 1, the growth of six indicator strains including Grampositive bacteria (L. monocytogenes, B. subtilis, S. aureus), Gramnegative bacteria V. parahaemolyticus, E. coli and yeast C. albicans were all inhibited by bacteriocin CAMT2. The inhibitions of bacteriocin CAMT2 against L. monocytogenes and S. aureus were more obvious (Table 1). Thus, the strong antagonism against a number of food spoilage bacteria and food-borne pathogens advocated the high possibility of using this bacteriocin as a preservatives in food. Similarly, a bacteriocin from B. subtilis R75 can suppress the growth of L. monocytogenes, S. aureus, B. subtilis and E. coli (Sharma et al., 2011). Plantazolicin, a novel microcin B17/streptolysins-like natural product from B. amyloliquefaciens FZB42, was found to be growth inhibitory toward closely related Gram-positive Bacilli, but no activity was observed toward Gram-negative bacteria, such as Erwinia carotovora, E. coli K-12, Klebsiella terrigena and
The purification result of bacteriocin CAMT2 showed two different peptides, which possessed the partial N-terminal sequences of MKIARTAIGSCLALSLTIPF and MKKWLLFLTTITLILSLGTA. The molecular mass of the two peptides were 19,608.57 Da and
Table 2 Effect of proteolytic enzymes and temperature on bacteriocin CAMT2 activity. Enzymes/T emperature
Diameter of inhibition zone ± SD (mm)
Control Trypsin Papain Proteinase K 60 C 80 C 100 C 121 C
20.34 8.91 14.13 0.00 20.23 20.03 14.74 1.80
± ± ± ± ± ± ± ±
0.52 0.14 0.09 0.00 0.11 0.08 0.23 0.00
Decreasing activity (%) 0.00 56.19 30.53 100.0 0.54 1.52 27.53 91.20
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Fig. 4. Changes in Log CFU g1 of L. monocytogenes in meat model system with the different treatments. A: purified bacteriocin CAMT2, -: nisin, :: Control.
20,265.57 Da, which was in good agreement with mass obtained by SDS-PAGE. It is noteworthy that no significant similarity was observed to the partial sequences of the two peptides with available bacteriocins in the databases. This suggests that bacteriocin CAMT2 might be a novel bacteriocin. The purified bacteriocin CAMT2 showed wider antimicrobial activity spectrum, heat stable for up to 100 C and pH stability in a wide range of pH 2.0e10.0. However, proteolytic enzyme treatment destroyed the antimicrobial activity of bacteriocin CAMT2 by different degrees. The bacteriocin also showed significant antimicrobial activity against L. monocytogenes in a meat model system. These results indicate that Bacteriocin CAMT2 might be used as a potential effective and natural food biopreservative. Acknowledgments This work was financially supported by Guangdong Science and Technology Fund (S2011010000328), National High Technology Research and Development Program of China (863 Program) (No. 2013AA102201), the National Science Fund (No. 30279987) and Higher Educational Cultivation Program for Major Scientific Research Projects of Guangdong Ocean University (No. GDOU2013050205). References ^ nio, A. C. M., Carvalho, M. A. R., Bemquerer, M. P., Santoro, M. M., Pinto, S. Q., Apolo Oliveira, J. S., et al. (2008). Purification and partial characterization of a bacteriocin produced by Eikenella corrodens. Journal of Applied Microbiology, 104, 508e514. Balciunas, E. M., Martinez, F. A. C., Todorov, S. D., de Melo Franco, B. D. G., Converti, A., & de Souza Oliveira, R. P. (2013). Novel biotechnological applications of bacteriocins: a review. Food Control, 32, 134e142. tro, F., & Pre vost, H. Batdorj, B., Dalgalarrondo, M., Choiset, Y., Pedroche, J., Me (2006). Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag. Journal of Applied Microbiology, 101, 837e848.
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