Bacteriocinogenic properties and safety evaluation of Enterococcus faecium YT52 isolated from boza, a traditional cereal based fermented beverage

  • Müge Gök Charyyev
  • Banu Özden Tuncer
  • Didem Akpınar Kankaya
  • Yasin TuncerEmail author
Research Article


The objectives of this study were to characterize the antibacterial substances produced by Enterococcus faecium YT52 isolated from boza, and to evaluate the safety of this strain. E. faecium YT52 inhibits various Gram-positive bacteria, including foodborne pathogens such as Listeria monocytogenes and Bacillus cereus. The manner of action of its antibacterial activity, using proteolytic enzymes, indicate that it produces a bacteriocin. The bacteriocin producing isolate E. faecium YT52 was identified using 16S rRNA gene homology and species-specific polymerase chain reaction (PCR) analysis. The bacteriocin was found to be heat stable and active under both acid and alkaline conditions. The bacteriocin showed primary metabolite kinetics and a bactericidal mode of action against L. monocytogenes. Although enterocin A, B and X genes were detected in E. faecium YT52 by PCR, only one active peptide band (~ 5.5 kDa) with a molecular weight similar to enterocin B was identified by tricine–SDS-PAGE analysis. A safety evaluation of E. faecium YT52 indicated that it is nonhemolytic, gelatinase-negative, and susceptible to clinically relevant antibiotics such as ampicillin, tetracycline and vancomycin. It contains a small number of virulence factors (efaAfm, silent gelE) and antibiotic resistance genes (ermB, tetM, tetL). In conclusion, bacteriocinogenic E. faecium YT52 was found to have a low risk profile, and it may be potentially valuable as a protective culture in the food industry.


Enterococcus faecium Bacteriocin Enterocin gene Tricine–SDS-PAGE Boza Virulence factor 



This study was supported by the Scientific Research Fund of the Süleyman Demirel University through project no. 3914-YL2-14. Two posters prepared as part of this study were presented at the Food Factor I Conference in Barcelona, Spain, and they were published as abstracts in the Proceedings.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aarestrup FM, Agerso Y, Gerner-Smidt P, Madsen M, Jensen LB (2000) Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark. Diagn Microbiol Infect Dis 37:127–137CrossRefGoogle Scholar
  2. Arıcı M, Daglıoglu O (2002) Boza: a lactic acid fermented cereal beverage as a traditional Turkish food. Food Res Int 18:39–48CrossRefGoogle Scholar
  3. Avci M, Özden Tuncer B (2017) Safety evaluation of enterocin producer Enterococcus sp. strains isolated from traditional Turkish cheeses. Polish J Microbiol 66:223–233CrossRefGoogle Scholar
  4. Aymerich T, Holo H, Havastein LS, Hugas M, Garriga M, Nes IF (1996) Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl Environ Microbiol 62:1676–1682Google Scholar
  5. Ben Barïek O, Cremonesi P, Morandi S, Smaoui S, Hani K, Ghrairi T (2018) Safety characterization and inhibition of fungi and bacteria by a novel multiple enterocin-producing Enterococcus lactis 4CP3 strain. Microb Pathog 118:32–38CrossRefGoogle Scholar
  6. Ben Belgacem Z, Abriouel H, Ben Omar N, Lucas R, Martínez-Canamero M, Gálvez A, Manai M (2010) Antimicrobial activity, safety aspects, and some technological properties of bacteriocinogenic Enterococcus faecium from artisanal Tunisian fermented meat. Food Control 21:462–470CrossRefGoogle Scholar
  7. Cancilla MR, Powell IB, Hillier AJ, Davidson BE (1992) Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels. Appl Environ Microbiol 58:1772–1775Google Scholar
  8. Cariolato D, Andrighetto C, Lombardi A (2008) Occurrence of virulence factors and antibiotic resistances in Enterococcus faecalis and Enterococcus faecium collected from dairy and human samples in North Italy. Food Control 19:886–892CrossRefGoogle Scholar
  9. Casaus P, Nilsen T, Cintas LM, Nes IF, Hernandez PE, Holo H (1997) Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. Microbiology 143:2287–2294CrossRefGoogle Scholar
  10. Cauwerts K, Decostere A, De Graef EM, Haesebrouck F, Pasmans F (2007) High prevalence of tetracycline resistance in Enterococcus isolates from broilers carrying the erm(B) gene. Avian Pathol 36:395–399CrossRefGoogle Scholar
  11. Chajęcka-Wierzchowska W, Zandernowska A, Łaniewska-Trokenheim Ł (2017) Virulence factors of Enterococcus spp. presented in food. LWT-Food Sci Technol 75:670–676CrossRefGoogle Scholar
  12. CLSI Clinical and Laboratory Standards Institute (2012) Performance standards for antimicrobial disk susceptibility tests; approved standard, vol 32(1), 11th edn. CLSI, St. Louis (M02-A11)Google Scholar
  13. De Vuyst L, Vandamme EJ (1994) Bacteriocins of lactic acid bacteria. Microbiology, genetics and applications. Chapman and Hall, New York, p 539CrossRefGoogle Scholar
  14. De Vuyst L, Foulquié Moreno MR, Revets H (2003) Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. Int J Food Microbiol 84:299–318CrossRefGoogle Scholar
  15. Demirgül F, Tuncer Y (2017) Detection of antibiotic resistance and resistance genes in enterococci isolated from sucuk, a traditional Turkish dry-fermented sausage. Korean J Food Sci Anim Resour 37(5):670–681CrossRefGoogle Scholar
  16. Dutka-Malen S, Evers S, Courvalin P (1995) Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clinic Microbiol 33:24–27Google Scholar
  17. Eaton T, Gasson MJ (2001) Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol 67:1628–1635CrossRefGoogle Scholar
  18. Edalatian MR, Najafi MBH, Mortazavi SA, Alegría Á, Delgado S, Bassami MR, Mayo B (2012) Production of bacteriocins by Enterococcus spp. isolated from traditional, Iranian, raw milk cheeses, and detection of their encoding genes. Eur Food Res Technol 234:789–796CrossRefGoogle Scholar
  19. Edwards U, Rogall T, Blocker H, Emde M, Bottger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853CrossRefGoogle Scholar
  20. Favaro L, Basaglia M, Casella S, Hue I, Dousset X, de Melo Franco BDG, Todorov SD (2014) Bacteriocinogenic potential and safety evaluation of non-starter Enterococcus faecium strains isolated from home made white brine cheese. Food Microbiol 38:228–239CrossRefGoogle Scholar
  21. Foulquié Moreno MR, Callewaert R, Devreese B, Van Beeumen J, De Vuyst L (2003) Isolation and biochemical characterisation of enterocins produced by enterococci from different sources. J Appl Microbiol 94:214–229CrossRefGoogle Scholar
  22. Franz CMAP, Du Toit M, von Holy A, Schillinger U, Holzapfel WH (1997) Production of nisin-like bacteriocins by Lactococcus lactis strains isolated from vegetables. J Basic Microbiol 37:187–196CrossRefGoogle Scholar
  23. Garrido AM, Gálvez A, Pulido RP (2014) Antimicrobial resistance in Enterococci. J Infect Dis Ther 2:150Google Scholar
  24. Gharairi T, Frere J, Berjeaud J, Manai M (2008) Purification and chacterisation of bacteriocins produced by Enterococcus faecium from Tunisian rigouta cheese. Food Control 19:162–169CrossRefGoogle Scholar
  25. Hadji-Sfaxi I, El-Ghaish S, Ahmadova A, Batdorj B, Le Blay-Laliberté G, Barbier G, Haertlé T, Chobert J-M (2011) Antimicrobial activity and safety of use of Enterococcus faecium PC4.1 isolated from Mongol yogurt. Food Control 22:2020–2027CrossRefGoogle Scholar
  26. Hernández D, Cardell E, Zárate V (2005) Antimicrobial activity of lactic acid bacteria isolated from Tenerife cheese: initial characterization of plantaricin TF711, a bacteriocin-like substance produced by Lactobacillus plantarum TF711. J Appl Microbiol 99:77–84CrossRefGoogle Scholar
  27. Hu C-B, Malaphan W, Zendo T, Nakayama J, Sonomoto K (2010) Enterocin X, a novel two-peptide bacteriocin from Enterococcus faecium KU-B5, has an antibacterial spectrum entirely different from those of its component peptides. Appl Environ Microbiol 76:4542–4545CrossRefGoogle Scholar
  28. Inoğlu ZN, Tuncer Y (2013) Safety assessment of Enterococcus faecium and Enterococcus facalis strains isolated from Turkish tulum cheese. J Food Safety 33:369–377CrossRefGoogle Scholar
  29. Iranmanesh M, Ezzatpanah H, Mojgani N, Mak T (2015) Characterization and kinetics of growth of bacteriocin like substance produced by lactic acid bacteria isolated from ewe milk and traditional sour buttermilk in Iran. J Food Process Technol 6:529CrossRefGoogle Scholar
  30. Isleroglu H, Yıldırım Z, Tokatlı M, Oncul N, Yıldırım M (2012) Partial characterization of enterocin KP produced by Enterococcus faecalis KP, a cheese isolate. Int J Dairy Technol 65:90–96CrossRefGoogle Scholar
  31. Ivanova I, Kabadjova P, Pantev A, Danova S, Dousset X (2000) Detection, purification and partial characterization of a novel bacteriocin substance produced by Lactococcus lactis subsp. lactis B14 isolated from boza—Bulgarian traditional cereal beverage. Biocatalysis 41:47–53Google Scholar
  32. Jackson CR, Fedorka-Cray PJ, Barrett JB (2004) Use of a genus- and species-specific multiplex PCR for identification of enterococci. J Clin Microbiol 42(8):3558–3565CrossRefGoogle Scholar
  33. Jahan M, Krause DO, Holley RA (2013) Antimicrobial resistance of Enterococcus species from meat and fermented meat products isolated by a PCR-based rapid screening method. Int Food Microbiol 163:89–95CrossRefGoogle Scholar
  34. Kabak B, Dobson ADW (2011) An introduction to the traditional fermented foods and beverages of Turkey. Crit Rev Food Sci Nutr 51:248–260CrossRefGoogle Scholar
  35. Ke D, Picard FJ, Martineau F, Ménard C, Roy PH, Ouellette M, Bergeron MG (1999) Development of a PCR assay for rapid detection of enterococci. J Clin Microbiol 37:3497–3503Google Scholar
  36. Klaenhammer T (1988) Bacteriocins of lactic acid bacteria. Biochimie 70:337–349CrossRefGoogle Scholar
  37. Koral G, Tuncer Y (2014) Nisin Z-producing Lactococcus lactis subsp. lactis GYL32 isolated from Boza. J Food Process Preserv 38:1044–1053CrossRefGoogle Scholar
  38. Lee H-J, Kim WJ (2010) Isolation and characterization of anti-listerial and amylase sensitive enterocin producing Enterococcus faecium DB1 from Gajami-sikhae, a fermented flat fish in Korea. Food Sci Biotechnol 19:373–381CrossRefGoogle Scholar
  39. Marco ML, Heeney D, Binda S, Cifelli CJ, Cotter PD, Foligné B, Gänzle M, Kort R, Pasin G, Pihlato A, Smid EJ, Hutkins R (2017) Health benefits of fermented foods: microbiota and beyond. Curr Opin Biotechnol 44:94–102CrossRefGoogle Scholar
  40. Mojsova S, Krstevski K, Dzadzovski I, Popova Z, Sekulovski P (2015) Phenotypi and genotypic characteristics of enterocin producing enterococci against pathogenic bacteria. Mac Vet Rev 38(2):209–216CrossRefGoogle Scholar
  41. Morandi S, Brasca M, Andrighetto C, Lombardi A, Lodi R (2006) Technological and molecular characterisation of enterococci isolated from North-West Italian dairy products. Int Dairy J 16:867–875CrossRefGoogle Scholar
  42. Ndlovu B, Schoeman H, Franz CMAP, du Toit M (2015) Screening, identification and characterization of bacteriocins produced by wine-isolated LAB strains. J Appl Microbiol 118:1007–1022CrossRefGoogle Scholar
  43. Ogier JC, Serror P (2008) Safety assessment of dairy microorganisms: the Enterococcus genus. Int J Food Microbiol 126:291–301CrossRefGoogle Scholar
  44. Ouoba LII, Lei V, Jensen LB (2008) Resistance of potential probiotic lactic acid bacteria and bifidobacteria of African and European origin to antimicrobials: determination and transferability of the resistance genes to other bacteria. Int J Food Microbiol 121:217–224CrossRefGoogle Scholar
  45. Özden Tuncer B, Ay Z, Tuncer Y (2013) Occurrence of enterocin genes, virulence factors and antibiotic resistance in three bacteriocin producer Enterococcus faecium strains isolated from Turkish tulum cheese. Turkish J Biol 37:443–449CrossRefGoogle Scholar
  46. Özmen Toğay S, Ay M, Güneşer O, Karagül Yüceer Y (2016) Investigation of antimicrobial activity and entA and entB genes in Enterococcus faecium and Enterococcus faecalis strains isolated from naturally fermented Turkish white cheeses. Food Sci Biotechnol 25:1633–1637CrossRefGoogle Scholar
  47. Poeta P, Igrejas G, Costa D, Sargo R, Rodrigues J, Torres C (2008) Virulence factors and bacteriocin in faecal enterococci of wild boars. J Basic Microbiol 48:385–392CrossRefGoogle Scholar
  48. Reviriego C, Eaton T, Martín R, Jiménez E, Fernández L, Gasson MJ, Rodríguez JM (2005) Screening of virulence determinants in Enterococcus faecium strains isolated from breast milk. J Hum Lac 21:131–137CrossRefGoogle Scholar
  49. Rice LB (1998) Tn916 family conjugative transposons and dissemination of antimicrobial resistance determinants. Antimicrob Agents Chemother 42:1871–1877CrossRefGoogle Scholar
  50. Ryan MP, Rea MC, Hill C, Ross RP (1996) An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad spectrum bacteriocin lacticin 3147. Appl Environ Microbiol 62:612–619Google Scholar
  51. Şahingil D, İşleroğlu H, Yildirim Z, Akçelik M, Yildirim M (2011) Characterization of lactococcin BZ produced by Lactococcus lactis subsp. lactis BZ isolated from boza. Turkish J Biol 35:21–33Google Scholar
  52. Schägger H, von Jagow G (1987) Tricine–SDS-PAGE for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379CrossRefGoogle Scholar
  53. Semedo T, Almeida M, Silva Lopes MF, Figueiredo Marques JJ, Barreto Crespo MT, Tenreiro R (2003) Virulence factors in food, clinical and reference enterococci: a common trait in the genus? Sys Appl Microbiol 26:13–22CrossRefGoogle Scholar
  54. Tamang JP, Watanabe K, Holzapfel WH (2016) Review: diversity of microorganisms in global fermented foods and beverages. Front Microbiol 7:1–28Google Scholar
  55. Todorov SD (2010) Diversity of bacteriocinogenic lactic acid bacteria isolated from boza, a cereal-based fermented beverage from Bulgaria. Food Control 21:1011–1021CrossRefGoogle Scholar
  56. Todorov SD, Dicks LMT (2004) Characterization of mesentericin ST99, a bacteriocin produced by Leuconostoc mesenteroides subsp. dextranicum ST99 isolated from Boza. J Indian Microbiol Biotechnol 31:323–329CrossRefGoogle Scholar
  57. Todorov SD, Dicks LMT (2005a) Production of bacteriocin ST33LD by Leuconostoc mesenteroides subsp. mesenteroides, as recorded in the presence of different medium components. World J Microbiol Biotechnol 21:1585–1590CrossRefGoogle Scholar
  58. Todorov SD, Dicks LMT (2005b) Pediocin ST18, an anti-listerial bacteriocin produced by Pediococcus pentosaceus ST18 isolated from Boza, a traditional cereal beverage from Bulgaria. Process Biochem 40:365–370CrossRefGoogle Scholar
  59. Todorov SD, Dicks LMT (2006) Screening for bacteriocin-producing lactic acid bacteria from Boza, a traditional cereal beverage from Bulgaria: comparison of the bacteriocins. Process Biochem 41:11–19CrossRefGoogle Scholar
  60. Tuncer Y (2009) Some technological properties of phenotypically identified enterococci strains isolated from Turkish tulum cheese. Afr J Biotechnol 8:7008–7016Google Scholar
  61. Tuncer Y, Özden B (2010) Partial biochemical characterization of nisin-like bacteriocin produced by Lactococcus lactis subsp. lactis YBD11 isolated from boza, a traditional fermented Turkish beverage. Rom Biotechnol Lett 15:4940–4948Google Scholar
  62. Tuncer M, Özden Tuncer B, Tuncer Y (2014) Safety evaluation of enterocin B producer Enterococcus faecalis MYE58 strain isolated from raw milk. Gıda 39(5):275–282 (in Turkish)Google Scholar
  63. van Belkum MJ, Hayema BJ, Geis A, Kok J, Venema G (1989) Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid. Appl Environ Microbiol 55:1187–1191Google Scholar
  64. Von Mollendorff JW, Todorov SD, Dicks LMT (2006) Comparison of bacteriocins produced by lactic acid bacteria isolated from boza, a cereal-based fermented beverage from the Balkan Peninsula. Curr Microbiol 53:209–216CrossRefGoogle Scholar
  65. Yogurtcu NN, Tuncer Y (2013) Antibiotic susceptibility patterns of Enterococcus strains isolated from Turkish Tulum cheese. Int J Dairy Technol 66(2):236–242CrossRefGoogle Scholar
  66. Yousif NMK, Dawyndt P, Abriouel H, Wijaya A, Schillinger U, Vancanneyt M, Swings J, Dirar HA, Holzapfel WH, Franz CMAP (2005) Molecular characterization, technological properties and safety aspects of enterococci from “Hussuwa”, an African fermented sorghum product. J Appl Microbiol 96:216–228CrossRefGoogle Scholar
  67. Zorba M, Hancioglu O, Genc M, Karapinar M, Ova G (2003) The use of starter cultures in the fermentation of boza, a traditional Turkish beverage. Process Biochem 38:1405–1411CrossRefGoogle Scholar

Copyright information

© Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL) 2019

Authors and Affiliations

  1. 1.Department of Food Engineering, Faculty of EngineeringSüleyman Demirel UniversityIspartaTurkey

Personalised recommendations