European Food Research and Technology

, Volume 234, Issue 4, pp 627–638 | Cite as

Molecular diversity among natural populations of Lactobacillus paracasei and Lactobacillus plantarum/paraplantarum strains isolated from autochthonous dairy products

  • Natasa Golic
  • Ivana Strahinic
  • Amarela Terzic-Vidojevic
  • Jelena Begovic
  • Milica Nikolic
  • Maja Tolinacki
  • Ljubisa Topisirovic
Original Paper


The diversity of 87 Lactobacillus paracasei and Lactobacillus plantarum/paraplantarum strains, previously identified from different autochthonous dairy products, was investigated by phenotypic and genotypic approaches. The increased resolution obtained using phenotypic and genotypic characterization allowed the level of strain heterogeneity detection to be widened. Phenotypic diversity was evaluated by studying biochemical characteristics of technological interest, including antimicrobial and proteinase activities, resistance to nisin, aggregation ability, production of exopolysaccharides, acetoin and diacetyl, citrate utilization, and antibiotic susceptibility. Genotypic diversity was generally evaluated by PCR amplification of repetitive bacterial DNA element fingerprinting using the (GTG)5 primer [(GTG)5-PCR]. Moreover, in cases where strains were not discriminated by (GTG)5-PCR combined with phenotypic analysis, pulsed-field gel electrophoresis (PFGE) analysis was performed. The results indicate that L. plantarum/paraplantarum and L. paracasei natural isolates from artisanal dairy products are a gold mine in terms of diversity of strains and could be potentially interesting to dairy companies for the formulation of functional starter cultures in the production of innovative foods.


Lactobacillus (GTG)5-PCR Microbial diversity Artisanal dairy products Natural isolates 



Meters above sea level



This work was funded by the Ministry of Education and Science of the Republic of Serbia, grant No.: 173019. We are grateful to Prof. Dr Vladimir Stevanovic, Faculty of Biology, University of Belgrade, for detailed bio-geographical data of WBC regions from which dairy products were collected, to Dr Natasa Jokovic for clustering fingerprints in Statistica 7.0 for Windows (StatSoft Inc. USA), and to Myra (Macpherson) Poznanovic, official native English editor of the scientific journal Archives of Biological Sciences.


  1. 1.
    Topisirovic L, Kojic M, Fira D, Golic N, Strahinic I, Lozo J (2006) Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation. Int J Food Microbiol 112:230–235CrossRefGoogle Scholar
  2. 2.
    Cotter PD, Hill C, Ross RP (2005) Bacteriocins: developing innate immunity for food. Nat Rev Microbiol 3:777–788CrossRefGoogle Scholar
  3. 3.
    Kojic M, Vujcic M, Banina A, Cocconcelli P, Cerning J, Topisirovic L (1992) Analysis of the exopolysaccharide production by Lactobacillus casei CG11, isolated from cheese. Appl Environ Microbiol 58:4086–4088Google Scholar
  4. 4.
    Kojic M, Fira D, Bojovic B, Banina A, Topisirovic L (1995) Comparative study on cell envelope-associated proteinases in natural isolates of mesophilic lactobacilli. J Appl Bacteriol 79:61–68CrossRefGoogle Scholar
  5. 5.
    Jokovic N, Nikolic M, Begovic J, Jovcic B, Savic D, Topisirovic L (2008) A survey of the lactic acid bacteria isolated from Serbian artisanal dairy product kajmak. Int J Food Microbiol 127:305–311CrossRefGoogle Scholar
  6. 6.
    Nikolic M, Terzic-Vidojevic A, Jovcic B, Begovic J, Golic N, Topisirovic L (2008) Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat’s milk cheese. Int J Food Microbiol 122:162–170CrossRefGoogle Scholar
  7. 7.
    Veljovic K, Terzic-Vidojevic A, Vukasinovic M, Strahinic I, Begovic J, Lozo J, Ostojic M, Topisirovic L (2007) Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese. J Appl Microbiol 103:2142–2152CrossRefGoogle Scholar
  8. 8.
    Diancourt L, Passet V, Chervaux C, Garault P, Smokvina T, Brisse S (2007) Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination. Appl Environ Microbiol 73:6601–6611CrossRefGoogle Scholar
  9. 9.
    Di Cagno R, Minervini G, Sgarbi E, Lazzi C, Bernini V, Neviani E, Gobbetti M (2010) Comparison of phenotypic (Biolog System) and genotypic (random amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) methods for typing Lactobacillus plantarum isolates from raw vegetables and fruits. Int J Food Microbiol 143:246–253CrossRefGoogle Scholar
  10. 10.
    Siezen RJ, Tzeneva VA, Castioni A, Wels M, Phan HTK, Rademaker JLW, Starrenburg MJC, Kleerebezem M, Molenaar D, van Hylckama Vlieg JET (2010) Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches. Environ Microbiol 12:758–773CrossRefGoogle Scholar
  11. 11.
    Bringel F, Curk MC, Hubert JC (1996) Characterization of lactobacilli by Southern-type hybridization with a Lactobacillus plantarum pyrDFE probe. Int J Syst Bacteriol 46:588–594CrossRefGoogle Scholar
  12. 12.
    Bringel F, Quénée P, Tailliez P (2001) Polyphasic investigation of the diversity within Lactobacillus plantarum related strains revealed two L. plantarum subgroups. Syst Appl Microbiol 24:561–571CrossRefGoogle Scholar
  13. 13.
    Elegado FB, Guerra MA, Macayan RA, Mendoza HA, Lirazan MB (2004) Spectrum of bacteriocin activity of Lactobacillus plantarum BS and fingerprinting by RAPD-PCR. Int J Food Microbiol 15:11–18CrossRefGoogle Scholar
  14. 14.
    Sánchez I, Seseña S, Palop LL (2004) Polyphasic study of the genetic diversity of lactobacilli associated with ‘Almagro’ eggplants spontaneous fermentation, based on combined numerical analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoresis patterns. J Appl Microbiol 97:446–458CrossRefGoogle Scholar
  15. 15.
    Hopwood DA, Bibb JM, Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate KM, Smith CP, Ward JM, Schrempf H (1985) Genetic manipulation of Streptomyces, a laboratory manual. The John Innes Foundation, NorwichGoogle Scholar
  16. 16.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, vol 3, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  17. 17.
    Jovcic B, Begovic J, Lozo J, Topisirovic L, Kojic M (2009) Dynamic of sodium dodecyl sulfate utilization and antibiotic susceptibility of strain Pseudomonas sp. ATCC19151. Arch Biol Sci 61:159–165CrossRefGoogle Scholar
  18. 18.
    Torriani S, Felis GE, Dellaglio F (2001) Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Appl Environ Microbiol 67:3450–3454CrossRefGoogle Scholar
  19. 19.
    Versalovic J, Schneider M, De Bruijn FJ, Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5:25–40Google Scholar
  20. 20.
    Kojic M, Strahinic I, Topisirovic L (2005) Proteinase PI and lactococcin A genes are located on the largest plasmid in Lactococcus lactis subsp. lactis bv. diacetylactis S50. Can J Microbiol 51:305–314CrossRefGoogle Scholar
  21. 21.
    Waterbury PG, Lane MJ (1987) Generation of lambda concatemers for use as pulse-field electrophoresis size markers. Nucleic Acids Res 15:3930CrossRefGoogle Scholar
  22. 22.
    Kojic M, Fira Dj, Banina A, Topisirovic L (1991) Characterization of the cell-wall bound proteinases of Lactobacillus casei HN14. Appl Environ Microbiol 57:1753–1757Google Scholar
  23. 23.
    Tagg JR, McGiven AR (1971) Assay system for bacteriocins. Appl Microbiol 21:943Google Scholar
  24. 24.
    Kojic M, Strahinic I, Fira D, Jovcic B, Topisirovic L (2006) Plasmid content and bacteriocin production by five strains of Lactococcus lactis subsp. lactis isolated from semi-hard homemade cheese. Can J Microbiol 52:1110–1120CrossRefGoogle Scholar
  25. 25.
    Kojic M, Svircevic J, Banina A, Topisirovic L (1991) A bacteriocin-producing strain of Lactococcus lactis subsp. diacetylactis S50. Appl Environ Microbiol 57:1835–1837Google Scholar
  26. 26.
    Lozo J, Vukasinovic M, Strahinic I, Topisirovic L (2004) Characterization and antimicrobial activity of bacteriocin 217 produced by natural isolate Lactobacillus paracasei subsp. paracasei BGBUK2-16. J Food Prot 67:2727–2734Google Scholar
  27. 27.
    Strahinic I, Begovic J, Fira D, Ostojic M, Topisirovic L (2005) Analysis of natural isolates of lactobacilli resistant to bacteriocin nisin. Genetika 37:77–87CrossRefGoogle Scholar
  28. 28.
    Klare I, Konstabel C, Muller-Bertling S, Reissbrodt R, Huys G, Vancanneyt M, Swings J, Goossens H, Witte W (2005) Evaluation of new broth media for microdilution antibiotic susceptibility testing of lactobacilli, pediococci, lactococci, and bifidobacteria. Appl Environ Microbiol 71:8982–8986CrossRefGoogle Scholar
  29. 29.
    Methods for antimicrobial dilution and disc susceptibility testing of infrequently isolated or Fastidious Bacteria; Approved guideline (2006) CLSI document M45-A. Clinical and Laboratory Standards Institute, Wayne, PAGoogle Scholar
  30. 30.
    International standard ISO10932:2010. Milk and milk products—determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB)Google Scholar
  31. 31.
    European Committee on Antimicrobial Susceptibility Testing (EUCAST). Clinical breakpoints and epidemiological cut-off values: definitions of clinical breakpoints and epidemiological cut-off values. 28 Feb 2007, date last accessed
  32. 32.
    Giraffa G, Gatti M, Rossetti L, Senini L, Neviani E (2000) Molecular diversity within Lactobacillus helveticus as revealed by genotypic characterization. Appl Environ Microbiol 66:1259–1265CrossRefGoogle Scholar
  33. 33.
    Giraffa G, Andrighetto C, Antonello C, Gatti M, Lazzi C, Marcazzan G, Lombardi A, Neviani E (2004) Genotypic and phenotypic diversity of Lactobacillus delbrueckii subsp. lactis strains of dairy origin. Int J Food Microbiol 91:129–139CrossRefGoogle Scholar
  34. 34.
    Terzic-Vidojevic A, Tolinacki M, Nikolic M, Lozo J, Begovic J, Gurban oglu Gulahmadov S, Alekperovich Kuliev A, Dalgalarrondo M, Chobert J-M, Haertlé T, Topisirovic L (2009) Phenotypic and genotypic characterization of lactic acid bacteria isolated from Azerbaijani traditional dairy products. Afr J Biotechnol 8:2576–2588Google Scholar
  35. 35.
    Beresford TP, Fitzsimons NA, Brennan NL, Cogan T (2001) Recent advances in cheese microbiology. Int Dairy J 11:259–274CrossRefGoogle Scholar
  36. 36.
    Nikolic M, Jovcic B, Kojic M, Topisirovic L (2010) Surface properties of Lactobacillus and Leuconostoc isolates from homemade cheeses showing auto-aggregation ability. Eur Food Res Tech 231:925–931CrossRefGoogle Scholar
  37. 37.
    Begovic J, Brandsma JB, Jovcic B, Tolinacki M, Veljovic K, Meijer WC, Topisirovic L (2011) Analysis of dominant lactic acid bacteria from artisanal raw milk cheeses produced on the mountain Stara Planina, Serbia. Arch Biol Sci 63:11–20CrossRefGoogle Scholar
  38. 38.
    Lozo J, Jovcic B, Kojic M, Dalgalarrondo M, Chobert J, Haertlé T, Topisirovic L (2007) Molecular characterization of a novel bacteriocin and an unusually large aggregation factor of Lactobacillus paracasei subsp. paracasei BGSJ2-8, a natural isolate from home-made cheese. Curr Microbiol 55:266–271CrossRefGoogle Scholar
  39. 39.
    Strahinic I, Kojic M, Tolinacki M, Fira D, Topisirovic L (2010) The presence of prtP proteinase gene in natural isolate Lactobacillus plantarum BGSJ3-18. Lett Appl Microbiol 50:43–49CrossRefGoogle Scholar
  40. 40.
    Tolinacki M, Kojic M, Lozo J, Terzic-Vidojevic A, Topisirovic L, Fira D (2010) Characterization of bacteriocin-producing strain Lactobacillus paracasei subsp. paracasei BGUB9. Arch Biol Sci 62:889–899CrossRefGoogle Scholar
  41. 41.
    Terzic-Vidojevic A, Nikolic M, Veljovic K, Tolinacki M, Busarcevic M, Topisirovic L (2009) Analysis of lactic acid bacteria microflora in traditional Caucasus cow’s milk cheeses. Arch Biol Sci 61:395–407CrossRefGoogle Scholar
  42. 42.
    Reniero R, Cocconcelli PS, Bottazzi V, Morelli L (1992) High frequency of conjugation in Lactobacillus mediated by an aggregation-promoting factor. J Gen Microbiol 138:763–768Google Scholar
  43. 43.
    Collado MC, Meriluoto J, Salminen S (2007) Measurement of aggregation properties between probiotics and pathogens: in vitro evaluation of different methods. J Microbiol Methods 71:71–74CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Natasa Golic
    • 1
  • Ivana Strahinic
    • 1
  • Amarela Terzic-Vidojevic
    • 1
  • Jelena Begovic
    • 1
  • Milica Nikolic
    • 1
  • Maja Tolinacki
    • 1
  • Ljubisa Topisirovic
    • 1
  1. 1.Institute of Molecular Genetics and Genetic EngineeringUniversity of BelgradeBelgradeSerbia

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