Antimicrobial compounds produced by Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian meat product

  • Kátia G. de Carvalho
  • Felipe H. S. Bambirra
  • Monika F. Kruger
  • Matheus S. Barbosa
  • Jamil S. Oliveira
  • Alexandre M. C. Santos
  • Jacques R. Nicoli
  • Marcelo P. Bemquerer
  • Antonio de Miranda
  • Emiliano J. Salvucci
  • Fernando J. M. Sesma
  • Bernadette D. G. M. Franco
Original Paper


Bacteriocins produced by lactic acid bacteria are gaining increased importance due to their activity against undesirable microorganisms in foods. In this study, a concentrated acid extract of a culture of Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian pork product, was purified by cation exchange and reversed-phase chromatographic methods. The amino acid sequences of the active antimicrobial compounds determined by Edman degradation were compared to known protein sequences using the BLAST-P software. Three different antimicrobial compounds were obtained, P1, P2 and P3, and mass spectrometry indicated molecular masses of 4.4, 6.8 and 9.5 kDa, respectively. P1 corresponds to classical sakacin P, P2 is identical to the 30S ribosomal protein S21 of L. sakei subsp. sakei 23 K, and P3 is identical to a histone-like DNA-binding protein HV produced by L. sakei subsp. sakei 23 K. Total genomic DNA was extracted and used as target DNA for PCR amplification of the genes sak, lis and his involved in the synthesis of P1, P2 and P3. The fragments were cloned in pET28b expression vector and the resulting plasmids transformed in E. coli KRX competent cells. The transformants were active against Listeria monocytogenes, indicating that the activity of the classical sakacin P produced by L. sakei 2a can be complemented by other antimicrobial proteins.


Lactobacillus sakei 2a Bacteriocins Antimicrobial proteins Lactic acid bacteria 


  1. 1.
    Anastasiadou S, Papagianni M, Filiousis G, Ambrosiadis I, Koidis P (2008) Pediocin SA-1, an antimicrobial peptide from Pediococcus acidilactici NRRL B5627: production conditions, purification and characterization. Bioresour Technol 99:5384–5390CrossRefPubMedGoogle Scholar
  2. 2.
    Asaduzzaman SM, Sonomoto K (2009) Lantibiotics: diverse activities and unique modes of action. J Biosci Bioeng 107:475–487CrossRefPubMedGoogle Scholar
  3. 3.
    Atrih A, Rekhif N, Moir AJG, Lebrihi A, Lefebvre G (2001) Mode of action, purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin produced by Lactobacillus plantarum C19. Int J Food Microbiol 68:93–109CrossRefPubMedGoogle Scholar
  4. 4.
    Bambirra FHS, Lima KGC, Franco BDGM, Carmona DCM, Nardi RMD, Barbosa FHF, Nicoli JR (2007) Protective effect of Lactobacillus sakei 2a against experimental challenge with Listeria monocytogenes in gnotobiotic mice. Lett Appl Microbiol 45:663–667CrossRefPubMedGoogle Scholar
  5. 5.
    Bauer R, Chikindas ML, Dicks LMT (2005) Purification, partial amino acid sequence and mode of action of pediocin PD-1, a bacteriocin produced by Pediococcus damnosus NCFB 1832. Int J Food Microbiol 101:17–27CrossRefPubMedGoogle Scholar
  6. 6.
    Ben-Shushan G, Zakin V, Gollop N (2003) Two different propionicins produced by Propionibacterium thoenii P-127. Peptides 24:1733–1740CrossRefPubMedGoogle Scholar
  7. 7.
    Bradford MM (1976) Rapid and sensitive method for quantification of microgram quantities of proteins utilizing principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  8. 8.
    Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria. Nat Rev Microbiol 3:238–250CrossRefPubMedGoogle Scholar
  9. 9.
    Carolissen-Mackay V, Arendse G, Hastings JW (1997) Purification of bacteriocins of lactic acid bacteria: problems and pointers. Int J Food Microbiol 34:1–16CrossRefPubMedGoogle Scholar
  10. 10.
    Chaillou S, Champomier-Verges M, Cornet M, Crutz Lecoq A-M, Dudez A-M, Martin V, Beaufils S, Darbon-Rongere E, Bossy R, Loux V, Zagorec M (2005) The genome sequence of the meat-borne lactic acid bacterium Lactobacillus sakei 23 K. Nat Biotechnol 23:1527–1533CrossRefPubMedGoogle Scholar
  11. 11.
    Chumchalová J, Stiles J, Josephsen J, Plocková M (2004) Characterization and purification of acidocin CH5, a bacteriocin produced by Lactobacillus acidophilus CH5. J Appl Microbiol 96:1082–1089CrossRefPubMedGoogle Scholar
  12. 12.
    Cotter PD, Hill C, Ross RP (2005) Bacteriocins: developing innate immunity for foods. Nat Rev 3:777–788CrossRefGoogle Scholar
  13. 13.
    De Martinis ECP, Franco BDGM (1998) Inhibition of Listeria monocytogenes in a pork product by a Lactobacillus sake strain. Int J Food Microbiol 42:119–126CrossRefPubMedGoogle Scholar
  14. 14.
    Deegan LH, Cotter PD, Hill C, Ross P (2006) Bacteriocins: biological tools for bio-preservation and shelf-life extension. Int Dairy J 16:1058–1071CrossRefGoogle Scholar
  15. 15.
    Drider D, Fimland G, Héchard Y, McMullen LM, Prévost H (2006) The continuing story of class IIa bacteriocins. Microbiol Mol Biol Rev 70:564–582CrossRefPubMedGoogle Scholar
  16. 16.
    Farias LM, Totola AH, Miranda CMS, Carvalho MAR, Damasceno CAV, Tavares CAP, Cisalpino EO, Vieira EC (1994) Extraction, partial purification and characterization of a bacteriocin (fragilicin) produced by a strain of Bacteroides fragilis isolated from Callithrix penicillata. Res Microbiol 145:9–16CrossRefPubMedGoogle Scholar
  17. 17.
    Fimland G, Pirneskoski J, Kaewsrichan J, Jutila A, Kristiansen PE, Kinnunen PKJ, Nissen-Meyer J (2006) Mutational analysis and membrane-interactions of the b-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P. Biochim Biophys Acta 1764:1132–1140PubMedGoogle Scholar
  18. 18.
    Fischer PM (2007) Cellular uptake mechanisms and potential therapeutic utility of peptidic cell delivery vectors: progress 2001–2006. Med Res Rev 27:755–795CrossRefPubMedGoogle Scholar
  19. 19.
    Futaki S (2005) Membrane-permeable arginine-rich peptides and the translocation mechanisms. Adv Drug Deliv Rev 57:547–558CrossRefPubMedGoogle Scholar
  20. 20.
    Gálvez A, Abriouel H, López RL, Ben Omar N (2007) Bacteriocin-based strategies for food biopreservation. Int J Food Microbiol 30:51–70CrossRefGoogle Scholar
  21. 21.
    Ghrairi T, Frere J, Berjeaud JM, Manai M (2008) Purification and characterisation of bacteriocins produced by Enterococcus faecium from Tunisian rigouta cheese. Food Control 19:162–169CrossRefGoogle Scholar
  22. 22.
    Hartnett J, Jill MS, Gracyalny BS, Slater MR (2006) The single step (KRX) competent cells: efficient cloning and high protein yields. Promega Notes 94:27–30Google Scholar
  23. 23.
    Heng NC, Burtenshaw GA, Jack RW, Tagg JR (2007) Ubericin A, a class IIa bacteriocin produced by Streptococcus uberis. Appl Environ Microbiol 73:7763–7766CrossRefPubMedGoogle Scholar
  24. 24.
    Herranz C, Cintas LM, Hernandez PE, Moll GN, Driessen AJ (2001) Enterocin P causes potassium ion efflux from Enterococcus faecium T136 cells. Antimicrob Agents Chemother 45:901–904CrossRefPubMedGoogle Scholar
  25. 25.
    Herranz C, Chen Y, Chung HJ, Cintas LM, Hernandez PE, Montville TJ, Chikindas ML (2001) Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136. Appl Environ Microbiol 67:1689–1692CrossRefPubMedGoogle Scholar
  26. 26.
    Holck AL, Axelsson L, Huhnek K, Krockel L (1994) Purification and cloning of sakacin-674, a bacteriocin from Lactobacillus sake LB674. FEMS Microbiol Lett 115:143–149CrossRefPubMedGoogle Scholar
  27. 27.
    Jamuna M, Jeevaratnam K (2004) Isolation and partial characterization of bacteriocins from Pediococcus species. Appl Microbiol Biotechnol 65:433–439CrossRefPubMedGoogle Scholar
  28. 28.
    Jasniewski J, Cailliez-Grimal C, Millière J-B, Revol-Junelles A-M (2008) Functional differences in Leuconostoc sensitive and resistant strains to mesenterocin 52A, a class IIa bacteriocin. Appl Microbiol Biotechnol 81:339–347CrossRefPubMedGoogle Scholar
  29. 29.
    Kamoun F, Mejdoub H, Aouissaoui H, Reinbolt J, Hammami A, Jaoua S (2005) Purification, amino acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus thuringensis. J Appl Microbiol 98:881–888CrossRefPubMedGoogle Scholar
  30. 30.
    Kemperman R, Jonker M, Nauta A, Kuipers OP, Kok J (2003) Functional analysis of the gene cluster involved in production of the bacteriocin circularin A by Clostridium beijerinckii ATCC 25752. Appl Environ Microbiol 69:5839–5848CrossRefPubMedGoogle Scholar
  31. 31.
    Klaenhammer TR (1993) Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol Rev 12:39–85PubMedGoogle Scholar
  32. 32.
    Knoetze H, Todorov SD, Dicks LM (2008) A class IIa peptide from Enterococcus mundtii inhibits bacteria associated with otitis media. Int J Antimicrob Agents 31:228–234CrossRefPubMedGoogle Scholar
  33. 33.
    Liserre AM, Landgraf M, Destro MT, Franco BDGM (2002) Application of a bacteriocinogenic Lactobacillus sake strain to prevent growth of Listeria monocytogenes in Brazilian sausages (“lingüiça”) packaged under modified atmosphere. Meat Sci 61:449–455CrossRefGoogle Scholar
  34. 34.
    Lopez RS, Garcia MT, Abriouel H, Omar NB, Grande MJ, Martinez-Canamero M, Gálvez A (2007) Semi-preparative scale purification of enterococcal bacteriocin enterocin EJ97, and evaluation of substrates for its production. J Ind Microbiol Biotechnol 34:779–785CrossRefPubMedGoogle Scholar
  35. 35.
    Moll GN, Konings WN, Driessen AJ (1999) Bacteriocins: mechanism of membrane insertion and pore formation. Antonie Van Leeuwenhoek 76:185–198CrossRefPubMedGoogle Scholar
  36. 36.
    Nissen-Meyer J, Rogne P, Oppegård C, Haugen HS, Kristiansen PE (2009) Structure-function relationships of the non-lanthionine-containing peptide (class II) bacteriocins produced by gram-positive bacteria. Curr Pharm Biotechnol 10:19–37CrossRefPubMedGoogle Scholar
  37. 37.
    Park CB, Yi KS, Matsuzaki K, Kim MS, Kim SC (2000) Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: the proline hinge is responsible for the cell-penetrating ability of buforin II. Proc Nat Acad Sci USA 97:3245–3250Google Scholar
  38. 38.
    Prochiantz A (2000) Messenger proteins: homeoproteins, TAT and others. Curr Opin Cell Biol 12:400–406CrossRefPubMedGoogle Scholar
  39. 39.
    Rosa CM, Franco BDGM, Montville TJ, Chikindas M (2002) Purification and mechanistic action of a bacteriocin produced by a Brazilian sausage isolate, Lactobacillus sake 2a. J Food Saf 22:39–54CrossRefGoogle Scholar
  40. 40.
    Saavedra L, Castellano P, Sesma F (2004) Purification of bacteriocins produced by lactic acid bacteria. Methods Mol Biol 268:331–336PubMedGoogle Scholar
  41. 41.
    Schneider R, Fernadez FJ, Aguilar MB, Guerreo-Legaretta I, Alpuche-Solis A, Ponce-Alquicira E (2006) Partial characterization of a class IIa pediocin produced by Pediococcus parvulus 133 strain isolated from meat (Mexican “chorizo”). Food Control 17:909–915CrossRefGoogle Scholar
  42. 42.
    Shai Y (1999) Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1462:55–70CrossRefPubMedGoogle Scholar
  43. 43.
    Suzuki M, Yamamoto T, Kawai Y, Inoue N, Yamazaki K (2005) Mode of action of piscicocin CS526 produced by Carnobacterium piscicola CS526. J Appl Microbiol 98:1146–1151CrossRefPubMedGoogle Scholar
  44. 44.
    Todorov SD, Dicks LMT (2005) Characterization of bacteriocins produced by lactic acid bacteria isolated from spoiled black olives. J Basic Bacteriol 36:318–326Google Scholar
  45. 45.
    Thorén PEG, Persson D, Karlsson M, Nordén B (2000) The antennapedia peptide penetratin translocates across lipid bilayers the first direct observation. FEBS Lett 482:265–268CrossRefPubMedGoogle Scholar
  46. 46.
    Thorén PEG, Persson D, Lincoln P, Nordén B (2005) Membrane destabilizing properties of cell-penetrating peptides. Biophys Chem 114:169–179CrossRefPubMedGoogle Scholar
  47. 47.
    Vera Pingitore E, Hébert EM, Sesma F, Nader-Macias ME (2009) Influence of vitamins and osmolites on growth and bacteriocin production by Lactobacillus salivarius CRL 1328 in a chemically defined medium. Can J Microbiol 55:304–310CrossRefPubMedGoogle Scholar
  48. 48.
    Wool IG (1996) Extraribosomal functions of ribosomal proteins. Trends Biochem Sci 21:164–165PubMedGoogle Scholar
  49. 49.
    Yang R, Johnson MC, Ray B (1992) Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl Environ Microbiol 58:3355–3359PubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2009

Authors and Affiliations

  • Kátia G. de Carvalho
    • 1
    • 6
  • Felipe H. S. Bambirra
    • 3
  • Monika F. Kruger
    • 1
  • Matheus S. Barbosa
    • 1
  • Jamil S. Oliveira
    • 2
  • Alexandre M. C. Santos
    • 2
    • 7
  • Jacques R. Nicoli
    • 3
  • Marcelo P. Bemquerer
    • 2
    • 4
  • Antonio de Miranda
    • 5
  • Emiliano J. Salvucci
    • 6
  • Fernando J. M. Sesma
    • 6
  • Bernadette D. G. M. Franco
    • 1
  1. 1.Departamento de Alimentos e Nutrição Experimental, Faculdade de Ciências FarmacêuticasUniversidade de São PauloSão PauloBrazil
  2. 2.Departamento de Bioquímica e Imunologia, Instituto de Ciências BiológicasUniversidade Federal de Minas GeraisBelo HorizonteBrazil
  3. 3.Departamento de Microbiologia, Instituto de Ciências BiológicasUniversidade Federal de Minas GeraisBelo HorizonteBrazil
  4. 4.Embrapa Recursos Genéticos e Biotecnologia Parque Estação BiológicaBrasíliaBrazil
  5. 5.Departamento de BiofisicaUniversidade Federal de São PauloSão PauloBrazil
  6. 6.Centro de Referencia para Lactobacilos (CERELA)TucumánArgentina
  7. 7.Departamento de Ciências FisiológicasUniversidade Federal do Espírito SantoVitóriaBrazil

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