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Partial characterization of a class IIa pediocin produced by Pediococcus parvulus 133 strain isolated from meat (Mexican “chorizo”)
Food Control 17 (2006) 909–915 www.elsevier.com/locate/foodcont
Partial characterization of a class IIa pediocin produced by Pediococcus parvulus 133 strain isolated from meat (Mexican “chorizo”) R. Schneider a, F.J. Fernández a, M.B. Aguilar b, I. Guerrero-Legarreta a, A. Alpuche-Solís c, E. Ponce-Alquicira a,¤ a
Departamento de Biotecnología, Bioquímica de Macromoléculas, Universidad Autónoma Metropolitana, Campus Iztapalapa; Av. San Rafael Atlixco No. 186, Col. Vicentina, Iztapalapa, Apdo. Postal 55-534, 09340 México, DF, México b Laboratory of Marine Neuropharmacology and Analytical Biochemistry Unit, Institute for Neurobiology, Universidad Nacional Autónoma de México, Querétaro, México c Molecular Biology Area, Instituto Potosino de Investigación CientíWca y Tecnológica, San Luis Potosí, México Received 21 February 2005; received in revised form 12 June 2005; accepted 14 June 2005
Abstract Pediocin-like bacteriocins are antimicrobial substances produced by some bacteria with high antilisterial activity. Several isolates of Pediococcus acidilactici and two Pediococcus parvulus strains of vegetable origin have been reported to produce this kind of peptide. This work presents the partial characterization of the bacteriocin produced by P. parvulus 133 found in meat and conWrms its identity as a heat resistant, antilisterial bacteriocin. This peptide has a relatively narrow inhibitory spectrum but a high antilisterial activity. Pediocin remained active after heating to 121 °C, but its thermoresistance varied with pH. The pH selective adsorption method resulted in a 150-fold concentration of antimicrobial activity. The Wnal extract was obtained by ultraWltration and resulted in an additional 10-fold concentration of activity. Molecular weight was estimated as 5 kDa and isoelectric point was 8.65. The sequence of the Wrst 17 aminoacids at the N-terminal end of the bacteriocin showed complete coincidence with that previously reported for pediocin A1 (AcH) and with an antilisterial peptide produced by Bacillus coagulans. High sequence similarity was also found with two other antilisterial bacteriocins. © 2005 Elsevier Ltd. All rights reserved. Keywords: Lactic acid bacteria; Pediocin; Meat preservation
1. Introduction Lactic acid bacteria have become attractive as natural food preservatives due to their ability to inhibit other microorganisms by production of high amounts of organic acid and antimicrobial compounds, like bacteriocins. Pediococcus belongs to the homofermentative * Corresponding author. Tel.: +52 55 5804 4726; fax: +52 55 5804 4712. E-mail address: [email protected] (E. Ponce-Alquicira).
0956-7135/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2005.06.010
group of lactic acid bacteria and, although nisin is the only bacteriocin considered to be GRAS (generally recognized as safe) by the US FDA (Food and Drug Administration), a variety of pediocin-producer strains are used as starter cultures in the fermentation of vegetables and meats. Pediocins are bacteriocins produced by some strains of Pediococcus spp., such as P. acidilactici, P. pentosaceus, and P. parvulus. Pediocin AcH or PA-1 was the Wrst and most thoroughly characterized bacteriocin from Class IIa bacteriocins, and it is produced by several P. acidilactici strains found in meat
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(Bhunia, Johnson, & Ray, 1988; Cintas et al., 1995; Nieto Lozano, Nissen Meyer, Sletten, Pelaez, & Nes, 1992; Pucci, Vedamuthu, Kunka, & Vandenbergh, 1988), two vegetable-associated P. parvulus (Bennik, Smid, & Gorris, 1997), and a Lactobacillus plantarum strain isolated from cheese (Ennahar et al., 1996; Loesner, Guenter, SteVan, & Scherer, 2003). One of the most important characteristics of pediocin-like bacteriocins is their high antimicrobial activity against Listeria monocytogenes (Aymerich et al., 1996; Bennik, van Overbeeck, Smid, & Gorris, 1999; Cintas, Casaus, Fernández, & Hernández, 1998; Guyonnet, Fremaux, Cenatiempo, & Berjeaud, 2000). This microorganism is a ubiquitous pathogen in the environment, capable of producing both human and animal infections. Most cases of listeriosis in humans result from food-borne transmission; they aVect mainly immunocompromised patients and have the highest case-fatality rate of foodborne illnesses (Siegman-Igra et al., 2002). In this work we report the partial characterization of a Class IIa bacteriocin produced by a P. parvulus 133 strain isolated from mexican “chorizo” (a traditional, widely consumed meat product), with a view toward its possible application as natural food preservative and/or protective culture.
2. Materials and methods 2.1. Bacterial strain and growth conditions
Table 1 Bacterial strains and pediocin inhibitory spectrum Tested microorganisms
Inhibition
Bacillus subtilis PL1 (1) Brochothrix thermosphacta NCIB-10018 (2) Campylobacter coli NCTC 12143 HG2 (1) Campylobacter fetus NCTC 10842 (1) Campylobacter jejuni dolyei NCTC11987 HG2 (1) Campylobacter jejuni NCTC 12500 HG2 (1) Clostridium sporogenes NCTC 8594 (2) Enterococcus faecium 29 (2) Escherichia coli JM P101 (1) Listeria innocua ATCC33090 (2) Listeria innocua MP 2418 (1) Listeria monocytogenes Scott A (4) Listeria monocytogenes LM 82 (3) Listeria monocytogenes LMB 92000/48 (4) Listeria monocytogenes LMB 911204/47 (4) Staphylococcus carnosus (2) Staphylococcus aureus NCTC 8325 (2)
¡ ¡ ¡ ¡ ¡ ¡ ¡ + ¡ + + + + + + + ¡
(1) Dr. M. Collins, Queen’s University of Belfast, Ireland; (2) Our laboratory; (3) Dr. J. M. Uruburu, Universidad de Valencia, Spain and (4) Dr. F. M. Iniesta, Universidad de Murcia, Spain. (¡) No inhibition halo detected; (+) inhibition halo detected.
2.3. Assay for antibacterial activity Bacteriocin activity was measured by the well diVusion assay as previously described (Schillinger & Lucke, 1989), using L. innocua MP 2418 as sensitive strain. Arbitrary units (AU) per ml were calculated as described by Pucci et al. (1988).
The bacteriocin producer P. parvulus 133 was isolated from Mexican chorizo (fermented raw sausage) by Dr. M. Collin’s research group in the Queen’s University of Belfast. Bacteriocin sensitive strains were obtained from various sources (Table 1). P. parvulus was grown in CGB broth (casein-glucose broth: yeast extract, 0.5%; bacto tryptone, 2%; glucose, 1%; tween 80, 0.1%; magnesium sulphate, 0.01%; manganese sulphate, 0.005%; ammonium citrate, 0.2%; disodium phosphate, 0.2%) at 37 °C. Bacillus subtilis, Brochothrix thermosphacta, Clostridium sporogenes, Enterococcus faecium, Listeria innocua, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus carnosus, and Escherichia coli were grown on trypticase soy broth (Difco Laboratories, Detroit, USA) at 37 °C, with the exception of Brochothrix thermosphacta, which was grown at 30 °C. Clostridium sporogenes was incubated anaerobically with 11% CO2. Campylobacter spp. were grown on Müeller–Hinton broth (Difco Laboratories, Detroit, USA).
2.4. Proteolytic enzyme treatment
2.2. Strain identiWcation
2.6. Antagonistic activity
The producer strain was identiWed by sequencing the 16SrDNA (MIDI LABS, Inc., Newark, USA) followed by a BLAST homology search.
In order to determine bacteriocin antagonistic activity, the well diVusion assay was used (Schillinger & Lucke, 1989). Pathogenic bacteria and spoilage meat
The eYcacy of proteinase K (Promega, Madison, USA) in bacteriocin inactivation was tested. Aliquots of bacteriocin were incubated with proteinase K at Wnal enzyme concentrations of 0.5 and 1 mg/ml. After samples were incubated 15 min at 37 °C, antibacterial activity was evaluated as described above. 2.5. Bacteriocin production associated with cellular growth Tubes containing 9 ml of CGB broth were inoculated at 1% from an overnight culture of P. parvulus and incubated at 37 °C. Absorbance at 600 nm was registered using a Beckman DU 650 spectrophotometer (Beckman, Fullertown, USA), and antibacterial activity was determined at 2-h intervals during the Wrst 16 h of incubation.
R. Schneider et al. / Food Control 17 (2006) 909–915
microorganisms tested for inhibition were: B. thermosphacta, C. sporogenes, E. faecium, E. coli, C. jejuni, C. coli, C. fetus, B. subtilis, L. innocua, L. monocytogenes, S. carnosus and S. aureus. 2.7. Bacteriocin puriWcation Bacteriocin puriWcation was carried out as previously described (Yang, Johnson, & Ray, 1992) with minor modiWcations. Bacteriocin adsorption to producer cells was carried out for 4 h at pH of 5.5 and desorption was made during 10 h at pH 1. Finally, the extract was ultraWltered through a 10,000 and then through a 5000 kDa cut oV membrane (YM 10/YM 5 Centricon, Millipore, Bedford, USA) following the manufacturer’s instructions. The concentrated extract was stored at ¡80 °C until use. 2.8. Preparative isoelectric focusing The crude extract obtained after centrifugation at 3100 £ g for 20 min of a P. parvulus overnight culture was dialyzed against distilled water for 48 h. Carrier ampholytes (Bio-Lyte® 3/10, Bio-Rad, Hercules, USA) were added to the extract to a Wnal concentration of 1%. The Rotofor cell was run for 3 h at constant power of 12 W. Finally, fractions were collected, and the pH was set to 6. Each fraction was tested for inhibitory activity to determine the pH of the bacteriocin. 2.9. Temperature inactivation at diVerent pH values The bacteriocin-containing supernatant was divided into Wve fractions and adjusted to pH values ranging from 4 to 8. Each fraction was subdivided into 3 aliquots. The Wrst was used as a control, and the second and third were subjected to 95 °C and 121 °C respectively, for 15 min. After thermal treatments, samples were cooled, adjusted to neutral pH (phosphoric acid and NaOH) and assayed for bacteriocin activity. 2.10. Electrophoresis Ten and sixteen (%T) tricine-SDS–PAGE gels were carried out in a Mini Protean II chamber (Bio-Rad, Hercules, USA) as described previously (Bhunia, Johnson, & Ray, 1987; Schägger & von Jagow, 1987). Gels were electrophoresed in parallel at 90 V, 50 W and 5 mA for 3.5 h at 5 °C. Relative molecular weight standards (Polypeptide SDS–PAGE Molecular Weight Standards, Bio-Rad, Hercules, USA) were employed. One of the gels was silver stained (Oakley, Kirsch, & Morris, 1980), and the other was assayed for antimicrobial activity as described by Bhunia et al. (1987). This gel was compared with the silver stained gel to locate the active band. Samples from the Wnal concentrated extract, with and without 2% SDS, were run to test the eVect of SDS on band resolution.
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2.11. Electroblotting to PVDF membranes Proteins in the SDS–PAGE gels were transferred to a PVDF membrane (Sequi-Blot, Bio-Rad, Hercules, USA) in a Mini Trans-Blot Cell (Bio-Rad, Hercules, USA) at 90 V for 40 min at 5 °C, employing 192 mM glycine, 25 mM Tris, and 20% methanol, pH 8.3 as buVer solution. After blotting, the membrane was stained with Coomasie Blue R-250 (0.025% in 40% aqueous methanol) for 30 min at room temperature and destained with 50% aqueous methanol. 2.12. Amino acid sequence determination The PVDF membrane fragment corresponding to the protein band of interest was excised, cut into small pieces, and applied into a Procise 491 Protein Sequencing System (Applied Biosystems, Inc., Foster City CA, USA) using the Blot cartridge. The Pulsed-liquid Blot method was employed. After sequencing, the amino acid sequence was compared with the Protein NCBI database (web site: www.ncbi.nih.gov/BLAST/) to determine homologies with sequences previous reported.
3. Results 3.1. Strain identiWcation A blast search homology of 16s rDNA sequences identiWed the producer strain as Pediococcus parvulus (99.8% of sequence similarity). 3.2. Proteolytic enzyme treatment A total loss of activity was observed with both proteinase K concentrations tested (0.5 and 1 mg/ml), indicating the proteinaceous character of the antimicrobial substance. 3.3. Bacteriocin production associated with cellular growth The aim of this assay was to determine the relationships between the growth rate of P. parvulus and maximal bacteriocin production. Maximal inhibitory activity was found at 12 h of incubation at 37 °C, which corresponded to early stationary phase (Fig. 1). After this, no further increase of inhibitory activity was found. 3.4. Antagonistic activity Results are shown in Table 1. Bacteriocin showed a high inhibitory activity against all of the four L. monocytogenes strains and the two L. innocua strains tested. S. carnosus and E. faecium were also inhibited. No
912
R. Schneider et al. / Food Control 17 (2006) 909–915 35000
U.A./mL
2
30000
O.D
1.8 1.4
20000
1.2 1
15000
0.8
10000
0.6
O.D.
A.U./mL
1.6 25000
0.4
5000
0.2
0
0
0
2
4
6 8 10 Time in Hours
12
14
16
Fig. 1. Bacteriocin production associated with cellular growth. Inhibitory activity was barely detectable at mid log phase, increased until the end of log phase and reached a plateau in early stationary phase.
3.5. Bacteriocin puriWcation Partial bacteriocin puriWcation was carried out by the method described previously (Yang et al., 1992) with minor modiWcations. Maximal and minimal bacteriocin adsorption to bacterial cells were observed at pH values of 5.5 and 1, respectively. The Wnal concentrated extract showed a 150-fold activity increase compared to the antibacterial activity of initial supernatant. UltraWltration through a 10,000 Da cut oV membrane did not result in bacteriocin concentration, but the use of a 5000 Da cut oV membrane yielded a sample retentate with a 10-fold increase of activity compared to the original sample. The 5000 Da Wltrate had no inhibitory activity, suggesting that all bacteriocin remained in the retentate. After 1.5 h of centrifugation (3100 £ g), 2 ml of the original extract were concentrated to approximately 200 l of retentate and 1.8 ml of Wltrate.
Fig. 2. Pediocin tricine-SDS–PAGE (10–16% T). Pediocin sample without extra SDS (lane 2) showed a band with an estimated molecular weight of 5,000 Da. Lane 3 was loaded with a pediocin sample and 2% additional SDS. In this case, band deWnition was not clear, and pediocin activity was more diVuse than in lane 2. Lanes 2a and 3a are the active bands corresponding to lanes 2 and 3, respectively. Lane 1 shows the mobility of molecular weight markers.
7000 pH 8.65 6000 5000 4000 pH 6.83 3000
A.U. /mL
inhibition was observed against B. thermosphacta, B. subtilis, C. sporogenes or S. aureus. Growth of Gram negative bacteria, like Campylobacter spp. and E. coli, was not inhibited.
2000 1000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Fractions Fig. 3. Pediocin isoelectrofocusing. Distribution of bacteriocin activity after preparative isoelectrofocusing. The most active fraction was found at pH 8.65.
3.6. Electrophoresis 3.8. Temperature inactivation at diVerent pH values As shown by SDS–PAGE, the estimated molecular weight for pediocin was around 5 kDa. When SDS (2%) was added to pediocin samples to disrupt any pediocin aggregate, band resolution was not improved; moreover, the band with SDS was more diVuse than the band without extra SDS (Fig. 2). 3.7. Preparative isoelectric focusing Isoelectric focusing of the bacteriocin crude extract yielded twenty fractions, ranging between pH values of 2.5 and 12. Only two of them showed antimicrobial activity. The fraction with pH 8.65 had the highest activity (Fig. 3).
High temperature treatments were applied to bacteriocin extracts at diVerent pH values, ranging from 4 to 8. As shown in Fig. 4, the pH value was crucial for thermoresistance. Bacteriocin activity extracts at low pH values (4–5) showed little or no modiWcation after high temperature treatments although applying the same treatments to higher pH solutions resulted in complete loss of the antimicrobial activity. 3.9. Amino acid sequence determination Automated N-terminal Edman degradation (17 cycles) of intact pediocin showed the following unambig-
inhibition halo (mm)
R. Schneider et al. / Food Control 17 (2006) 909–915 control
14 12 10 8 6 4 2 0
95˚C/15 min 121˚C/15 min
4
5
6
7
8
pH Fig. 4. Pediocin thermoresistance. Samples with pH ranging from 4 to 8 were subjected to 95 °C or 121 °C for 15 min. Total activity loss was observed at pH 7 and 8 when the sample was treated at 121 °C for 15 min. However, minimal activity loss (9%) was observed at pH 4 with the same treatment. When samples were heated to 95 °C for 15 min, activity loss was minimal.
Fig. 5. N-terminal pediocin comparative sequence alignment. For each peptide, the sequence accession number is given. Boxed letters indicate amino acid diVerences with the pediocin sequence.
uous sequence: KYYGNGVTCGKHSCSVD. Similarities found with sequences included in the NCBI database are shown in Fig. 5. As this comparison indicated, the Wrst 17 amino acids at the N-terminus of our pediocin are identical to those of the pediocin A1 (produced by P. acidilactici), the prepediocin (a precursor for pediocin A1), and also to a Class IIa bacteriocin produced by B. coagulans. Prebacteriocin 423, the precursor of a bacteriocin produced by L. plantarum, and the precursor of a sakacin diVer from our pediocin by one and two amino acids, respectively.
4. Discussion Previous characterization of antimicrobial substances produced by Pediococcus spp. determined that most of them have similar characteristics. High antilisterial activity is the most important property they share; relatively small molecular size and high thermostability are others. Pediocin PA-1 from P. acidilactici PAC-1 was the Wrst pediocin described at the molecular level and was demonstrated to be identical to pediocin AcH produced by P. acidilactici strain H (Bhunia et al., 1988; Gonzalez & Kunka, 1987; Henderson, Chopko, & van Wassenaar, 1992; Motlagh et al., 1992). Production of pediocin PA1 by vegetable-associated P. parvulus strains was reported earlier (Bennik et al., 1997). In this study we report production of a bacteriocin belonging to the pediocin family
913
by a meat-associated P. parvulus isolated from Mexican sausage (chorizo). The kinetics of synthesis of our pediocin was in accord with that of others pediocins described (Cintas et al., 1995; Hernández-López, 2002; Solís-Rivera, 2003) in this work, maximal inhibitory activity was also detected at early stationary phase. Eijsink, Skeie, Middelhoven, Brurberg, and Nes (1998), showed that bacteriocins that contained two cystine bridges (PA-1 and enterocin) were more active and have a broader inhibitory spectrum than those with only one cystine bridge. The inhibitory spectrum was very similar to that reported for class IIa bacteriocins (Bennik et al., 1999; Klaenhammer, 1993; Loesner et al., 2003; Meghrous, Lacroix, & Simard, 1999), especially with respect to the high antilisterial activity. This property may suggest the presence of two cystine bridges in the molecule (Eijsink et al., 1998; Fimland et al., 2000). Solís-Rivera (2003) reported 2 log reduction in L. innocua counts when coated frankfurter sausages with calcium alginate gel containing pediocin from P. parvulus 133. In contrast to previous reports that found inhibition of C. sporogenes (Bennik et al., 1997; Cintas et al., 1998), this strain was not inhibited in our study. Unlike pediocin-like bacteriocin, nisin was reported to be an eVective inhibitor of Brochothrix (Cutter & Siragusa, 1994; Tu & Mustapha, 2002). Neither B. thermosphacta nor S. aureus were inhibited by our bacteriocin. In contrast, PA-1 bacteriocin showed antimicrobial activity against this latter microorganism (Ray & Miller, 2000). None of the Gram negative bacteria tested in this study was inhibited. Despite this, addition of chelating agents such EDTA was reported to be eVective to control Gram negative bacteria. This eVect could be mediated, probably, by the binding of magnesium ions by EDTA in the lipopolysaccharides layer of outer membrane of Gram negative bacteria (Nikaido & Vaara, 1987). The heat resistance of bacteriocin was dependent on pH. When bacteriocin was heated at 121 °C for 15 min, a marked loss (84%) of activity was observed at pH 6, and inactivation was total at pH 7 and 8. However, samples at pH 4 showed only moderate activity loss (11%). As the pH increased, bacteriocin became more heat sensitive. The same observation was made previously for substances like pediocin and enterocin (Bhunia et al., 1988; Foulguié Moreno, Callewaert, Devreese, Van Beeumen, & De Vuyst, 2003; Ray & Miller, 2000). Abriouel, Valdivia, Gálvez, and Maqueda (2001) studied the inXuence of physico-chemical factors on the oligomerization and biological activity of a bacteriocin produced by E. faecalis. They found that pH and protein concentration could modify the oligomerization of the bacteriocin. High pH (6–8) and protein concentration (above 0.55 mg/ml) favored dimer formation. Since these pH values induce the highest degree of oligomerization, these results strongly suggest that oligomers are much
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less stable than monomers. Pediocins are reported to form dimers and precipitate when highly puriWed extracts were obtained (Ray & Miller, 2000). Judging by electrophoresis, the P. parvulus bacteriocin also has this characteristic, and this could explain the inXuence of pH on thermostability. These data are important with regard to using bacteriocin in food and should be considered when thermal treatments are included in food processing. The bacteriocin isoelectric point was determined to be 8.65, which agrees with earlier reports for class IIa bacteriocins (Nes & Holo, 2000) but diVers by one pH unit from PA-1, which has a pI of 9.6 (Ray & Miller, 2000). Venema et al. (1997) reported a pI around 9 for pediocin PA-1. These data imply that solubility of the bacteriocin will increase with lower pH values. When the pH is higher aggregates could be formed, resulting in an apparent loss of activity as mentioned by Schved, Lalazar, Henis, and Juven (1993). The amino terminal sequence of the bacteriocin produced by P. parvulus and its physico-chemical properties evaluated in this study suggest that the peptide could be a new member of the Class IIa antilisterial bacteriocins with the YGNGV consensus motif. The diVerence in isoelectric point suggests that the amino acid sequence of our bacteriocin is not 100% identical with PA-1, but diVers at least in some amino acids. As far as we know, this is the Wrst report of a P. parvulus pediocin producer isolated from meat products, but its complete amino acid sequence and/or cloning of the gene coding for the pediocin will be required to determine similarities with other antimicrobial peptides described earlier. Class IIa bacteriocins have great potential to be used as antimicrobial substances in food, either as a puriWed additive or by direct inoculation of the producer strain as a starter-protective culture. With regard to new approaches, genetic modiWcations like the development of overproducing strains are being studied. Also, more research on alternative methods to obtain and purify large amounts of bacteriocins at reduced costs is indispensable to consider industrial scale production.
Acknowledgements We would like to acknowledge the technical assistance of A. Falcón (Laboratory of Marine Neuropharmacology and Analytical Biochemistry Unit, Institute for Neurobiology, Universidad Nacional Autónoma de México) for amino acid sequence determination. We thank Dr. Dorothy D. Pless for revision of the manuscript. Also we would acknowledge SRE (Secretaría de Relaciones Exteriores de México) for supporting the doctoral studies of R. Schneider.
References Abriouel, H., Valdivia, E., Gálvez, A., & Maqueda, M. (2001). InXuence of physico-chemical factors on the oligomerization and biological activity of bacteriocin AS-48. Current Microbiology, 42, 89–95. Aymerich, T., Holo, H., Havarstein, L. S., Hugas, M., Garriga, M., & Nes, I. F. (1996). Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocin. Applied and Environmental Microbiology, 62, 1676–1682. Bennik, M. H., Smid, E. J., & Gorris, L. G. M. (1997). Vegetable-associated Pediococcus parvulus produces pediocin PA1. Applied and Environmental Microbiology, 63, 2074–2076. Bennik, M. H. J., van Overbeeck, W., Smid, E. J., & Gorris, L. G. M. (1999). Biopreservation in modiWed atmosphere stored mungbean sprouts: the use of vegetable-associated bacteriocigenic lactic acid bacteria to control the growth of Listeria monocytogenes. Letters in Applied Microbiology, 28, 226–232. Bhunia, A. K., Johnson, M. C., & Ray, B. (1987). Direct detection of an antimicrobial peptide of Pediococcus acidilactici in sodium dodecyl sulfate-polyacrilamide gel electrophoresis. Journal of Industrial Microbiology, 2, 319–322. Bhunia, A. K., Johnson, M. C., & Ray, B. (1988). PuriWcation, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici. Journal of Applied Bacteriology, 65, 261– 268. Cintas, L. M., Casaus, P., Fernández, M. F., & Hernández, P. E. (1998). Comparative antimicrobial activity of enterocin L50, pediocin PA1, nisin A and lactocin S against spoilage and food-borne pathogenic bacteria. Food Microbiology, 15, 289–298. Cintas, L. M., Rodríguez, J. M., Fernández, M. F., Sletten, K., Nes, I. F., Hernández, P. E., et al. (1995). Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum. Applied and Environmental Microbiology, 61, 2643–2648. Cutter, C. N., & Siragusa, G. R. (1994). Decontamination of beef carcass tissue with nisin using a pilot scale mode carcass washer. Food Microbiology, 11, 481–489. Eijsink, V. G. H., Skeie, M., Middelhoven, P. H., Brurberg, M. B., & Nes, I. (1998). Comparative study of class IIa bacteriocin of lactic acid bacteria. Applied and Environmental Microbiology, 64, 3275– 3281. Ennahar, S., Aoude Werner, D., Sorokine, O., Dorseelaer, A. V., Bringel, F., Hubert, J. C., et al. (1996). Production of pediocin AcH by Lactobacillus plantarum WHE92 isolated from cheese. Applied and Environmental Microbiology, 62, 4381–4387. Fimland, G., Johnsen, L., Axelsson, L., Brurberg, B., Nes, I. F., Eijsink, V. G., et al. (2000). A C-terminal disulWde bridge in pediocin-like bateriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum. Journal of Bacteriology, 182, 2643–2648. Foulguié Moreno, M. R., Callewaert, R., Devreese, B., Van Beeumen, J., & De Vuyst, L. (2003). Isolation and biochemical characterisation of enterocins produced by enterococci from diVerent sources. Journal of Applied Microbiology, 94, 214–229. Gonzalez, C. F., & Kunka, B. S. (1987). Plasmid-associated bacteriocin production and sucrose fermentation in Pediococcus acidilactici. Applied and Environmental Microbiology, 46, 81–89. Guyonnet, D., Fremaux, C., Cenatiempo, Y., & Berjeaud, M. J. (2000). Method for the rapid puriWcation of Class IIa bacteriocins and comparison of their activities. Applied and Environmental Microbiology, 66, 1744–1748. Henderson, J. T., Chopko, A. L., & van Wassenaar, P. D. D. (1992). PuriWcation and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC1.0. Archives of Biochemistry and Biophysics, 295, 5–12.
R. Schneider et al. / Food Control 17 (2006) 909–915 Hernández-López, J. C. (2002). Caracterización parcial de la bacteriocina producida por Pediococcus parvulus MXVK133. Biotechnology Master Thesis. Universidad Autónoma Metropolitana. México, DF, Mexico. Klaenhammer, T. R. (1993). Genetics of bacteriocins produced by lactic acid bacteria. FEMS. Microbiology Reviews, 12, 39–86. Loesner, M., Guenter, S., SteVan, S., & Scherer, S. (2003). A pediocinproducing Lactobacillus plantarum strain inhibits Listeria monocytogenes in a multispecies cheese surface microbial ripening consortium. Applied and Environmental Microbiology, 69, 1854–1857. Meghrous, J., Lacroix, C., & Simard, R. E. (1999). The eVects on vegetative cells and spores of three bacteriocins from lactic acid bacteria. Food Microbiology, 16, 105–114. Motlagh, A. M., Bhunia, A. K., Szostek, F., Hansen, T. R., Johnson, M. G., & Ray, B. (1992). Nucleotide and aminoacid sequence of pap-gen (pediocin AcH production) in Pediococcus acidilactici H. Letters in Applied Microbiology, 15, 45–48. Nes, I. F., & Holo, H. (2000). Class II antimicrobial peptides from lactic acid bacteria. Biopolymers (Peptide Science), 55, 50–61. Nikaido, H., & Vaara, M. (1987). Outer membrane. In F. C. Neidhardt (Ed.), Escherichia coli and Salmonella typhimurium: Cellular and molecular biology (92, pp. 379–383). Washington, DC: Am. Soc. Microbiol. Inst. Brewing. Nieto Lozano, J. C., Nissen Meyer, J., Sletten, K., Pelaez, C., & Nes, I. F. (1992). PuriWcation and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici. Journal of General Microbiology, 138, 1985–1990. Oakley, B. A., Kirsch, D. R., & Morris, N. R. (1980). A simpliWed ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry, 105, 361–363. Pucci, M. J., Vedamuthu, E. R., Kunka, B. S., & Vandenbergh, P. A. (1988). Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC1.0. Applied and Environmental Microbiology, 54, 2349–2353.
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Ray, B., & Miller, K. W. (2000). Pediocin. In A. S. Naidu (Ed.), Natural Food Antimicrobial Systems (pp. 525–566). Boca Raton, Florida: CRC Press. Schägger, H., & von Jagow, G. (1987). Trycine-sodium dodecyl sulfatepolyacrilamide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa. Analytical Biochemistry, 166, 368– 379. Schillinger, U., & Lucke, F. K. (1989). Antibacterial activity of Lactobacillus sake isolated from meat. Applied and Environmental Microbiology, 55, 1901–1906. Schved, F., Lalazar, A., Henis, Y., & Juven, B. L. (1993). PuriWcation, partial characterization and plasmid linkage of pediocin SJ-1, a bacteriocin produced by Pediococcus acidilactici. Journal of Applied Bacteriology, 74, 67–77. Siegman-Igra, Y., Levin, R., Weinberger, M., Golan, Y., Schwartz, D., Samra, Z., et al. (2002). Listeria monocytogenes infection in Israel and review of cases worldwide. Emerging Infectious Diseases, 8, 305–310. Solís-Rivera, I., 2003. Evaluación de la estabilidad de pediocina en un recubrimiento comestible. Bsc. Thesis. Universidad Nacional Autónoma de México. México, DF, Mexico. Tu, L., & Mustapha, A. (2002). Reduction of Brochothrix thermosphacta and Salmonella Serotype Typhimurim on vacuum-packaged fresh beef treated with nisin and nisin combined with EDTA. Journal of Food Science, 67, 302–306. Venema, K., Chikindas, M. L., Seegers, J. F. M. L., Haandrikman, A. J., Leenthouts, K. J., Venema, G., et al. (1997). Rapid and eYcient puriWcation method for small, hydrophobic, cationic bacteriocins: puriWcation of lactococcin B and pediocin PA-1. Applied and Environmental Microbiology, 63, 305–309. Yang, R., Johnson, M., & Ray, B. (1992). Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Applied and Environmental Microbiology, 58, 3355–3359.
Partial characterization of a class IIa pediocin produced by Pediococcus parvulus 133 strain isolated from meat (Mexican “chorizo”) R. Schneider a, F.J. Fernández a, M.B. Aguilar b, I. Guerrero-Legarreta a, A. Alpuche-Solís c, E. Ponce-Alquicira a,¤ a
Departamento de Biotecnología, Bioquímica de Macromoléculas, Universidad Autónoma Metropolitana, Campus Iztapalapa; Av. San Rafael Atlixco No. 186, Col. Vicentina, Iztapalapa, Apdo. Postal 55-534, 09340 México, DF, México b Laboratory of Marine Neuropharmacology and Analytical Biochemistry Unit, Institute for Neurobiology, Universidad Nacional Autónoma de México, Querétaro, México c Molecular Biology Area, Instituto Potosino de Investigación CientíWca y Tecnológica, San Luis Potosí, México Received 21 February 2005; received in revised form 12 June 2005; accepted 14 June 2005
Abstract Pediocin-like bacteriocins are antimicrobial substances produced by some bacteria with high antilisterial activity. Several isolates of Pediococcus acidilactici and two Pediococcus parvulus strains of vegetable origin have been reported to produce this kind of peptide. This work presents the partial characterization of the bacteriocin produced by P. parvulus 133 found in meat and conWrms its identity as a heat resistant, antilisterial bacteriocin. This peptide has a relatively narrow inhibitory spectrum but a high antilisterial activity. Pediocin remained active after heating to 121 °C, but its thermoresistance varied with pH. The pH selective adsorption method resulted in a 150-fold concentration of antimicrobial activity. The Wnal extract was obtained by ultraWltration and resulted in an additional 10-fold concentration of activity. Molecular weight was estimated as 5 kDa and isoelectric point was 8.65. The sequence of the Wrst 17 aminoacids at the N-terminal end of the bacteriocin showed complete coincidence with that previously reported for pediocin A1 (AcH) and with an antilisterial peptide produced by Bacillus coagulans. High sequence similarity was also found with two other antilisterial bacteriocins. © 2005 Elsevier Ltd. All rights reserved. Keywords: Lactic acid bacteria; Pediocin; Meat preservation
1. Introduction Lactic acid bacteria have become attractive as natural food preservatives due to their ability to inhibit other microorganisms by production of high amounts of organic acid and antimicrobial compounds, like bacteriocins. Pediococcus belongs to the homofermentative * Corresponding author. Tel.: +52 55 5804 4726; fax: +52 55 5804 4712. E-mail address: [email protected] (E. Ponce-Alquicira).
0956-7135/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2005.06.010
group of lactic acid bacteria and, although nisin is the only bacteriocin considered to be GRAS (generally recognized as safe) by the US FDA (Food and Drug Administration), a variety of pediocin-producer strains are used as starter cultures in the fermentation of vegetables and meats. Pediocins are bacteriocins produced by some strains of Pediococcus spp., such as P. acidilactici, P. pentosaceus, and P. parvulus. Pediocin AcH or PA-1 was the Wrst and most thoroughly characterized bacteriocin from Class IIa bacteriocins, and it is produced by several P. acidilactici strains found in meat
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(Bhunia, Johnson, & Ray, 1988; Cintas et al., 1995; Nieto Lozano, Nissen Meyer, Sletten, Pelaez, & Nes, 1992; Pucci, Vedamuthu, Kunka, & Vandenbergh, 1988), two vegetable-associated P. parvulus (Bennik, Smid, & Gorris, 1997), and a Lactobacillus plantarum strain isolated from cheese (Ennahar et al., 1996; Loesner, Guenter, SteVan, & Scherer, 2003). One of the most important characteristics of pediocin-like bacteriocins is their high antimicrobial activity against Listeria monocytogenes (Aymerich et al., 1996; Bennik, van Overbeeck, Smid, & Gorris, 1999; Cintas, Casaus, Fernández, & Hernández, 1998; Guyonnet, Fremaux, Cenatiempo, & Berjeaud, 2000). This microorganism is a ubiquitous pathogen in the environment, capable of producing both human and animal infections. Most cases of listeriosis in humans result from food-borne transmission; they aVect mainly immunocompromised patients and have the highest case-fatality rate of foodborne illnesses (Siegman-Igra et al., 2002). In this work we report the partial characterization of a Class IIa bacteriocin produced by a P. parvulus 133 strain isolated from mexican “chorizo” (a traditional, widely consumed meat product), with a view toward its possible application as natural food preservative and/or protective culture.
2. Materials and methods 2.1. Bacterial strain and growth conditions
Table 1 Bacterial strains and pediocin inhibitory spectrum Tested microorganisms
Inhibition
Bacillus subtilis PL1 (1) Brochothrix thermosphacta NCIB-10018 (2) Campylobacter coli NCTC 12143 HG2 (1) Campylobacter fetus NCTC 10842 (1) Campylobacter jejuni dolyei NCTC11987 HG2 (1) Campylobacter jejuni NCTC 12500 HG2 (1) Clostridium sporogenes NCTC 8594 (2) Enterococcus faecium 29 (2) Escherichia coli JM P101 (1) Listeria innocua ATCC33090 (2) Listeria innocua MP 2418 (1) Listeria monocytogenes Scott A (4) Listeria monocytogenes LM 82 (3) Listeria monocytogenes LMB 92000/48 (4) Listeria monocytogenes LMB 911204/47 (4) Staphylococcus carnosus (2) Staphylococcus aureus NCTC 8325 (2)
¡ ¡ ¡ ¡ ¡ ¡ ¡ + ¡ + + + + + + + ¡
(1) Dr. M. Collins, Queen’s University of Belfast, Ireland; (2) Our laboratory; (3) Dr. J. M. Uruburu, Universidad de Valencia, Spain and (4) Dr. F. M. Iniesta, Universidad de Murcia, Spain. (¡) No inhibition halo detected; (+) inhibition halo detected.
2.3. Assay for antibacterial activity Bacteriocin activity was measured by the well diVusion assay as previously described (Schillinger & Lucke, 1989), using L. innocua MP 2418 as sensitive strain. Arbitrary units (AU) per ml were calculated as described by Pucci et al. (1988).
The bacteriocin producer P. parvulus 133 was isolated from Mexican chorizo (fermented raw sausage) by Dr. M. Collin’s research group in the Queen’s University of Belfast. Bacteriocin sensitive strains were obtained from various sources (Table 1). P. parvulus was grown in CGB broth (casein-glucose broth: yeast extract, 0.5%; bacto tryptone, 2%; glucose, 1%; tween 80, 0.1%; magnesium sulphate, 0.01%; manganese sulphate, 0.005%; ammonium citrate, 0.2%; disodium phosphate, 0.2%) at 37 °C. Bacillus subtilis, Brochothrix thermosphacta, Clostridium sporogenes, Enterococcus faecium, Listeria innocua, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus carnosus, and Escherichia coli were grown on trypticase soy broth (Difco Laboratories, Detroit, USA) at 37 °C, with the exception of Brochothrix thermosphacta, which was grown at 30 °C. Clostridium sporogenes was incubated anaerobically with 11% CO2. Campylobacter spp. were grown on Müeller–Hinton broth (Difco Laboratories, Detroit, USA).
2.4. Proteolytic enzyme treatment
2.2. Strain identiWcation
2.6. Antagonistic activity
The producer strain was identiWed by sequencing the 16SrDNA (MIDI LABS, Inc., Newark, USA) followed by a BLAST homology search.
In order to determine bacteriocin antagonistic activity, the well diVusion assay was used (Schillinger & Lucke, 1989). Pathogenic bacteria and spoilage meat
The eYcacy of proteinase K (Promega, Madison, USA) in bacteriocin inactivation was tested. Aliquots of bacteriocin were incubated with proteinase K at Wnal enzyme concentrations of 0.5 and 1 mg/ml. After samples were incubated 15 min at 37 °C, antibacterial activity was evaluated as described above. 2.5. Bacteriocin production associated with cellular growth Tubes containing 9 ml of CGB broth were inoculated at 1% from an overnight culture of P. parvulus and incubated at 37 °C. Absorbance at 600 nm was registered using a Beckman DU 650 spectrophotometer (Beckman, Fullertown, USA), and antibacterial activity was determined at 2-h intervals during the Wrst 16 h of incubation.
R. Schneider et al. / Food Control 17 (2006) 909–915
microorganisms tested for inhibition were: B. thermosphacta, C. sporogenes, E. faecium, E. coli, C. jejuni, C. coli, C. fetus, B. subtilis, L. innocua, L. monocytogenes, S. carnosus and S. aureus. 2.7. Bacteriocin puriWcation Bacteriocin puriWcation was carried out as previously described (Yang, Johnson, & Ray, 1992) with minor modiWcations. Bacteriocin adsorption to producer cells was carried out for 4 h at pH of 5.5 and desorption was made during 10 h at pH 1. Finally, the extract was ultraWltered through a 10,000 and then through a 5000 kDa cut oV membrane (YM 10/YM 5 Centricon, Millipore, Bedford, USA) following the manufacturer’s instructions. The concentrated extract was stored at ¡80 °C until use. 2.8. Preparative isoelectric focusing The crude extract obtained after centrifugation at 3100 £ g for 20 min of a P. parvulus overnight culture was dialyzed against distilled water for 48 h. Carrier ampholytes (Bio-Lyte® 3/10, Bio-Rad, Hercules, USA) were added to the extract to a Wnal concentration of 1%. The Rotofor cell was run for 3 h at constant power of 12 W. Finally, fractions were collected, and the pH was set to 6. Each fraction was tested for inhibitory activity to determine the pH of the bacteriocin. 2.9. Temperature inactivation at diVerent pH values The bacteriocin-containing supernatant was divided into Wve fractions and adjusted to pH values ranging from 4 to 8. Each fraction was subdivided into 3 aliquots. The Wrst was used as a control, and the second and third were subjected to 95 °C and 121 °C respectively, for 15 min. After thermal treatments, samples were cooled, adjusted to neutral pH (phosphoric acid and NaOH) and assayed for bacteriocin activity. 2.10. Electrophoresis Ten and sixteen (%T) tricine-SDS–PAGE gels were carried out in a Mini Protean II chamber (Bio-Rad, Hercules, USA) as described previously (Bhunia, Johnson, & Ray, 1987; Schägger & von Jagow, 1987). Gels were electrophoresed in parallel at 90 V, 50 W and 5 mA for 3.5 h at 5 °C. Relative molecular weight standards (Polypeptide SDS–PAGE Molecular Weight Standards, Bio-Rad, Hercules, USA) were employed. One of the gels was silver stained (Oakley, Kirsch, & Morris, 1980), and the other was assayed for antimicrobial activity as described by Bhunia et al. (1987). This gel was compared with the silver stained gel to locate the active band. Samples from the Wnal concentrated extract, with and without 2% SDS, were run to test the eVect of SDS on band resolution.
911
2.11. Electroblotting to PVDF membranes Proteins in the SDS–PAGE gels were transferred to a PVDF membrane (Sequi-Blot, Bio-Rad, Hercules, USA) in a Mini Trans-Blot Cell (Bio-Rad, Hercules, USA) at 90 V for 40 min at 5 °C, employing 192 mM glycine, 25 mM Tris, and 20% methanol, pH 8.3 as buVer solution. After blotting, the membrane was stained with Coomasie Blue R-250 (0.025% in 40% aqueous methanol) for 30 min at room temperature and destained with 50% aqueous methanol. 2.12. Amino acid sequence determination The PVDF membrane fragment corresponding to the protein band of interest was excised, cut into small pieces, and applied into a Procise 491 Protein Sequencing System (Applied Biosystems, Inc., Foster City CA, USA) using the Blot cartridge. The Pulsed-liquid Blot method was employed. After sequencing, the amino acid sequence was compared with the Protein NCBI database (web site: www.ncbi.nih.gov/BLAST/) to determine homologies with sequences previous reported.
3. Results 3.1. Strain identiWcation A blast search homology of 16s rDNA sequences identiWed the producer strain as Pediococcus parvulus (99.8% of sequence similarity). 3.2. Proteolytic enzyme treatment A total loss of activity was observed with both proteinase K concentrations tested (0.5 and 1 mg/ml), indicating the proteinaceous character of the antimicrobial substance. 3.3. Bacteriocin production associated with cellular growth The aim of this assay was to determine the relationships between the growth rate of P. parvulus and maximal bacteriocin production. Maximal inhibitory activity was found at 12 h of incubation at 37 °C, which corresponded to early stationary phase (Fig. 1). After this, no further increase of inhibitory activity was found. 3.4. Antagonistic activity Results are shown in Table 1. Bacteriocin showed a high inhibitory activity against all of the four L. monocytogenes strains and the two L. innocua strains tested. S. carnosus and E. faecium were also inhibited. No
912
R. Schneider et al. / Food Control 17 (2006) 909–915 35000
U.A./mL
2
30000
O.D
1.8 1.4
20000
1.2 1
15000
0.8
10000
0.6
O.D.
A.U./mL
1.6 25000
0.4
5000
0.2
0
0
0
2
4
6 8 10 Time in Hours
12
14
16
Fig. 1. Bacteriocin production associated with cellular growth. Inhibitory activity was barely detectable at mid log phase, increased until the end of log phase and reached a plateau in early stationary phase.
3.5. Bacteriocin puriWcation Partial bacteriocin puriWcation was carried out by the method described previously (Yang et al., 1992) with minor modiWcations. Maximal and minimal bacteriocin adsorption to bacterial cells were observed at pH values of 5.5 and 1, respectively. The Wnal concentrated extract showed a 150-fold activity increase compared to the antibacterial activity of initial supernatant. UltraWltration through a 10,000 Da cut oV membrane did not result in bacteriocin concentration, but the use of a 5000 Da cut oV membrane yielded a sample retentate with a 10-fold increase of activity compared to the original sample. The 5000 Da Wltrate had no inhibitory activity, suggesting that all bacteriocin remained in the retentate. After 1.5 h of centrifugation (3100 £ g), 2 ml of the original extract were concentrated to approximately 200 l of retentate and 1.8 ml of Wltrate.
Fig. 2. Pediocin tricine-SDS–PAGE (10–16% T). Pediocin sample without extra SDS (lane 2) showed a band with an estimated molecular weight of 5,000 Da. Lane 3 was loaded with a pediocin sample and 2% additional SDS. In this case, band deWnition was not clear, and pediocin activity was more diVuse than in lane 2. Lanes 2a and 3a are the active bands corresponding to lanes 2 and 3, respectively. Lane 1 shows the mobility of molecular weight markers.
7000 pH 8.65 6000 5000 4000 pH 6.83 3000
A.U. /mL
inhibition was observed against B. thermosphacta, B. subtilis, C. sporogenes or S. aureus. Growth of Gram negative bacteria, like Campylobacter spp. and E. coli, was not inhibited.
2000 1000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Fractions Fig. 3. Pediocin isoelectrofocusing. Distribution of bacteriocin activity after preparative isoelectrofocusing. The most active fraction was found at pH 8.65.
3.6. Electrophoresis 3.8. Temperature inactivation at diVerent pH values As shown by SDS–PAGE, the estimated molecular weight for pediocin was around 5 kDa. When SDS (2%) was added to pediocin samples to disrupt any pediocin aggregate, band resolution was not improved; moreover, the band with SDS was more diVuse than the band without extra SDS (Fig. 2). 3.7. Preparative isoelectric focusing Isoelectric focusing of the bacteriocin crude extract yielded twenty fractions, ranging between pH values of 2.5 and 12. Only two of them showed antimicrobial activity. The fraction with pH 8.65 had the highest activity (Fig. 3).
High temperature treatments were applied to bacteriocin extracts at diVerent pH values, ranging from 4 to 8. As shown in Fig. 4, the pH value was crucial for thermoresistance. Bacteriocin activity extracts at low pH values (4–5) showed little or no modiWcation after high temperature treatments although applying the same treatments to higher pH solutions resulted in complete loss of the antimicrobial activity. 3.9. Amino acid sequence determination Automated N-terminal Edman degradation (17 cycles) of intact pediocin showed the following unambig-
inhibition halo (mm)
R. Schneider et al. / Food Control 17 (2006) 909–915 control
14 12 10 8 6 4 2 0
95˚C/15 min 121˚C/15 min
4
5
6
7
8
pH Fig. 4. Pediocin thermoresistance. Samples with pH ranging from 4 to 8 were subjected to 95 °C or 121 °C for 15 min. Total activity loss was observed at pH 7 and 8 when the sample was treated at 121 °C for 15 min. However, minimal activity loss (9%) was observed at pH 4 with the same treatment. When samples were heated to 95 °C for 15 min, activity loss was minimal.
Fig. 5. N-terminal pediocin comparative sequence alignment. For each peptide, the sequence accession number is given. Boxed letters indicate amino acid diVerences with the pediocin sequence.
uous sequence: KYYGNGVTCGKHSCSVD. Similarities found with sequences included in the NCBI database are shown in Fig. 5. As this comparison indicated, the Wrst 17 amino acids at the N-terminus of our pediocin are identical to those of the pediocin A1 (produced by P. acidilactici), the prepediocin (a precursor for pediocin A1), and also to a Class IIa bacteriocin produced by B. coagulans. Prebacteriocin 423, the precursor of a bacteriocin produced by L. plantarum, and the precursor of a sakacin diVer from our pediocin by one and two amino acids, respectively.
4. Discussion Previous characterization of antimicrobial substances produced by Pediococcus spp. determined that most of them have similar characteristics. High antilisterial activity is the most important property they share; relatively small molecular size and high thermostability are others. Pediocin PA-1 from P. acidilactici PAC-1 was the Wrst pediocin described at the molecular level and was demonstrated to be identical to pediocin AcH produced by P. acidilactici strain H (Bhunia et al., 1988; Gonzalez & Kunka, 1987; Henderson, Chopko, & van Wassenaar, 1992; Motlagh et al., 1992). Production of pediocin PA1 by vegetable-associated P. parvulus strains was reported earlier (Bennik et al., 1997). In this study we report production of a bacteriocin belonging to the pediocin family
913
by a meat-associated P. parvulus isolated from Mexican sausage (chorizo). The kinetics of synthesis of our pediocin was in accord with that of others pediocins described (Cintas et al., 1995; Hernández-López, 2002; Solís-Rivera, 2003) in this work, maximal inhibitory activity was also detected at early stationary phase. Eijsink, Skeie, Middelhoven, Brurberg, and Nes (1998), showed that bacteriocins that contained two cystine bridges (PA-1 and enterocin) were more active and have a broader inhibitory spectrum than those with only one cystine bridge. The inhibitory spectrum was very similar to that reported for class IIa bacteriocins (Bennik et al., 1999; Klaenhammer, 1993; Loesner et al., 2003; Meghrous, Lacroix, & Simard, 1999), especially with respect to the high antilisterial activity. This property may suggest the presence of two cystine bridges in the molecule (Eijsink et al., 1998; Fimland et al., 2000). Solís-Rivera (2003) reported 2 log reduction in L. innocua counts when coated frankfurter sausages with calcium alginate gel containing pediocin from P. parvulus 133. In contrast to previous reports that found inhibition of C. sporogenes (Bennik et al., 1997; Cintas et al., 1998), this strain was not inhibited in our study. Unlike pediocin-like bacteriocin, nisin was reported to be an eVective inhibitor of Brochothrix (Cutter & Siragusa, 1994; Tu & Mustapha, 2002). Neither B. thermosphacta nor S. aureus were inhibited by our bacteriocin. In contrast, PA-1 bacteriocin showed antimicrobial activity against this latter microorganism (Ray & Miller, 2000). None of the Gram negative bacteria tested in this study was inhibited. Despite this, addition of chelating agents such EDTA was reported to be eVective to control Gram negative bacteria. This eVect could be mediated, probably, by the binding of magnesium ions by EDTA in the lipopolysaccharides layer of outer membrane of Gram negative bacteria (Nikaido & Vaara, 1987). The heat resistance of bacteriocin was dependent on pH. When bacteriocin was heated at 121 °C for 15 min, a marked loss (84%) of activity was observed at pH 6, and inactivation was total at pH 7 and 8. However, samples at pH 4 showed only moderate activity loss (11%). As the pH increased, bacteriocin became more heat sensitive. The same observation was made previously for substances like pediocin and enterocin (Bhunia et al., 1988; Foulguié Moreno, Callewaert, Devreese, Van Beeumen, & De Vuyst, 2003; Ray & Miller, 2000). Abriouel, Valdivia, Gálvez, and Maqueda (2001) studied the inXuence of physico-chemical factors on the oligomerization and biological activity of a bacteriocin produced by E. faecalis. They found that pH and protein concentration could modify the oligomerization of the bacteriocin. High pH (6–8) and protein concentration (above 0.55 mg/ml) favored dimer formation. Since these pH values induce the highest degree of oligomerization, these results strongly suggest that oligomers are much
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less stable than monomers. Pediocins are reported to form dimers and precipitate when highly puriWed extracts were obtained (Ray & Miller, 2000). Judging by electrophoresis, the P. parvulus bacteriocin also has this characteristic, and this could explain the inXuence of pH on thermostability. These data are important with regard to using bacteriocin in food and should be considered when thermal treatments are included in food processing. The bacteriocin isoelectric point was determined to be 8.65, which agrees with earlier reports for class IIa bacteriocins (Nes & Holo, 2000) but diVers by one pH unit from PA-1, which has a pI of 9.6 (Ray & Miller, 2000). Venema et al. (1997) reported a pI around 9 for pediocin PA-1. These data imply that solubility of the bacteriocin will increase with lower pH values. When the pH is higher aggregates could be formed, resulting in an apparent loss of activity as mentioned by Schved, Lalazar, Henis, and Juven (1993). The amino terminal sequence of the bacteriocin produced by P. parvulus and its physico-chemical properties evaluated in this study suggest that the peptide could be a new member of the Class IIa antilisterial bacteriocins with the YGNGV consensus motif. The diVerence in isoelectric point suggests that the amino acid sequence of our bacteriocin is not 100% identical with PA-1, but diVers at least in some amino acids. As far as we know, this is the Wrst report of a P. parvulus pediocin producer isolated from meat products, but its complete amino acid sequence and/or cloning of the gene coding for the pediocin will be required to determine similarities with other antimicrobial peptides described earlier. Class IIa bacteriocins have great potential to be used as antimicrobial substances in food, either as a puriWed additive or by direct inoculation of the producer strain as a starter-protective culture. With regard to new approaches, genetic modiWcations like the development of overproducing strains are being studied. Also, more research on alternative methods to obtain and purify large amounts of bacteriocins at reduced costs is indispensable to consider industrial scale production.
Acknowledgements We would like to acknowledge the technical assistance of A. Falcón (Laboratory of Marine Neuropharmacology and Analytical Biochemistry Unit, Institute for Neurobiology, Universidad Nacional Autónoma de México) for amino acid sequence determination. We thank Dr. Dorothy D. Pless for revision of the manuscript. Also we would acknowledge SRE (Secretaría de Relaciones Exteriores de México) for supporting the doctoral studies of R. Schneider.
References Abriouel, H., Valdivia, E., Gálvez, A., & Maqueda, M. (2001). InXuence of physico-chemical factors on the oligomerization and biological activity of bacteriocin AS-48. Current Microbiology, 42, 89–95. Aymerich, T., Holo, H., Havarstein, L. S., Hugas, M., Garriga, M., & Nes, I. F. (1996). Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocin. Applied and Environmental Microbiology, 62, 1676–1682. Bennik, M. H., Smid, E. J., & Gorris, L. G. M. (1997). Vegetable-associated Pediococcus parvulus produces pediocin PA1. Applied and Environmental Microbiology, 63, 2074–2076. Bennik, M. H. J., van Overbeeck, W., Smid, E. J., & Gorris, L. G. M. (1999). Biopreservation in modiWed atmosphere stored mungbean sprouts: the use of vegetable-associated bacteriocigenic lactic acid bacteria to control the growth of Listeria monocytogenes. Letters in Applied Microbiology, 28, 226–232. Bhunia, A. K., Johnson, M. C., & Ray, B. (1987). Direct detection of an antimicrobial peptide of Pediococcus acidilactici in sodium dodecyl sulfate-polyacrilamide gel electrophoresis. Journal of Industrial Microbiology, 2, 319–322. Bhunia, A. K., Johnson, M. C., & Ray, B. (1988). PuriWcation, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici. Journal of Applied Bacteriology, 65, 261– 268. Cintas, L. M., Casaus, P., Fernández, M. F., & Hernández, P. E. (1998). Comparative antimicrobial activity of enterocin L50, pediocin PA1, nisin A and lactocin S against spoilage and food-borne pathogenic bacteria. Food Microbiology, 15, 289–298. Cintas, L. M., Rodríguez, J. M., Fernández, M. F., Sletten, K., Nes, I. F., Hernández, P. E., et al. (1995). Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum. Applied and Environmental Microbiology, 61, 2643–2648. Cutter, C. N., & Siragusa, G. R. (1994). Decontamination of beef carcass tissue with nisin using a pilot scale mode carcass washer. Food Microbiology, 11, 481–489. Eijsink, V. G. H., Skeie, M., Middelhoven, P. H., Brurberg, M. B., & Nes, I. (1998). Comparative study of class IIa bacteriocin of lactic acid bacteria. Applied and Environmental Microbiology, 64, 3275– 3281. Ennahar, S., Aoude Werner, D., Sorokine, O., Dorseelaer, A. V., Bringel, F., Hubert, J. C., et al. (1996). Production of pediocin AcH by Lactobacillus plantarum WHE92 isolated from cheese. Applied and Environmental Microbiology, 62, 4381–4387. Fimland, G., Johnsen, L., Axelsson, L., Brurberg, B., Nes, I. F., Eijsink, V. G., et al. (2000). A C-terminal disulWde bridge in pediocin-like bateriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum. Journal of Bacteriology, 182, 2643–2648. Foulguié Moreno, M. R., Callewaert, R., Devreese, B., Van Beeumen, J., & De Vuyst, L. (2003). Isolation and biochemical characterisation of enterocins produced by enterococci from diVerent sources. Journal of Applied Microbiology, 94, 214–229. Gonzalez, C. F., & Kunka, B. S. (1987). Plasmid-associated bacteriocin production and sucrose fermentation in Pediococcus acidilactici. Applied and Environmental Microbiology, 46, 81–89. Guyonnet, D., Fremaux, C., Cenatiempo, Y., & Berjeaud, M. J. (2000). Method for the rapid puriWcation of Class IIa bacteriocins and comparison of their activities. Applied and Environmental Microbiology, 66, 1744–1748. Henderson, J. T., Chopko, A. L., & van Wassenaar, P. D. D. (1992). PuriWcation and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC1.0. Archives of Biochemistry and Biophysics, 295, 5–12.
R. Schneider et al. / Food Control 17 (2006) 909–915 Hernández-López, J. C. (2002). Caracterización parcial de la bacteriocina producida por Pediococcus parvulus MXVK133. Biotechnology Master Thesis. Universidad Autónoma Metropolitana. México, DF, Mexico. Klaenhammer, T. R. (1993). Genetics of bacteriocins produced by lactic acid bacteria. FEMS. Microbiology Reviews, 12, 39–86. Loesner, M., Guenter, S., SteVan, S., & Scherer, S. (2003). A pediocinproducing Lactobacillus plantarum strain inhibits Listeria monocytogenes in a multispecies cheese surface microbial ripening consortium. Applied and Environmental Microbiology, 69, 1854–1857. Meghrous, J., Lacroix, C., & Simard, R. E. (1999). The eVects on vegetative cells and spores of three bacteriocins from lactic acid bacteria. Food Microbiology, 16, 105–114. Motlagh, A. M., Bhunia, A. K., Szostek, F., Hansen, T. R., Johnson, M. G., & Ray, B. (1992). Nucleotide and aminoacid sequence of pap-gen (pediocin AcH production) in Pediococcus acidilactici H. Letters in Applied Microbiology, 15, 45–48. Nes, I. F., & Holo, H. (2000). Class II antimicrobial peptides from lactic acid bacteria. Biopolymers (Peptide Science), 55, 50–61. Nikaido, H., & Vaara, M. (1987). Outer membrane. In F. C. Neidhardt (Ed.), Escherichia coli and Salmonella typhimurium: Cellular and molecular biology (92, pp. 379–383). Washington, DC: Am. Soc. Microbiol. Inst. Brewing. Nieto Lozano, J. C., Nissen Meyer, J., Sletten, K., Pelaez, C., & Nes, I. F. (1992). PuriWcation and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici. Journal of General Microbiology, 138, 1985–1990. Oakley, B. A., Kirsch, D. R., & Morris, N. R. (1980). A simpliWed ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry, 105, 361–363. Pucci, M. J., Vedamuthu, E. R., Kunka, B. S., & Vandenbergh, P. A. (1988). Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC1.0. Applied and Environmental Microbiology, 54, 2349–2353.
915
Ray, B., & Miller, K. W. (2000). Pediocin. In A. S. Naidu (Ed.), Natural Food Antimicrobial Systems (pp. 525–566). Boca Raton, Florida: CRC Press. Schägger, H., & von Jagow, G. (1987). Trycine-sodium dodecyl sulfatepolyacrilamide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa. Analytical Biochemistry, 166, 368– 379. Schillinger, U., & Lucke, F. K. (1989). Antibacterial activity of Lactobacillus sake isolated from meat. Applied and Environmental Microbiology, 55, 1901–1906. Schved, F., Lalazar, A., Henis, Y., & Juven, B. L. (1993). PuriWcation, partial characterization and plasmid linkage of pediocin SJ-1, a bacteriocin produced by Pediococcus acidilactici. Journal of Applied Bacteriology, 74, 67–77. Siegman-Igra, Y., Levin, R., Weinberger, M., Golan, Y., Schwartz, D., Samra, Z., et al. (2002). Listeria monocytogenes infection in Israel and review of cases worldwide. Emerging Infectious Diseases, 8, 305–310. Solís-Rivera, I., 2003. Evaluación de la estabilidad de pediocina en un recubrimiento comestible. Bsc. Thesis. Universidad Nacional Autónoma de México. México, DF, Mexico. Tu, L., & Mustapha, A. (2002). Reduction of Brochothrix thermosphacta and Salmonella Serotype Typhimurim on vacuum-packaged fresh beef treated with nisin and nisin combined with EDTA. Journal of Food Science, 67, 302–306. Venema, K., Chikindas, M. L., Seegers, J. F. M. L., Haandrikman, A. J., Leenthouts, K. J., Venema, G., et al. (1997). Rapid and eYcient puriWcation method for small, hydrophobic, cationic bacteriocins: puriWcation of lactococcin B and pediocin PA-1. Applied and Environmental Microbiology, 63, 305–309. Yang, R., Johnson, M., & Ray, B. (1992). Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Applied and Environmental Microbiology, 58, 3355–3359.