Skip to main content
SpringerLink
Log in

In silico phylogenetic analysis of lactic acid bacteria and new primer set for identification of Lactobacillus plantarum in food samples

  • Original Paper
  • Published:
European Food Research and Technology Aims and scope Submit manuscript

Abstract

Lactic acid bacteria (LAB) include species very closely related both physiologically and genotypically. Therefore, the identification of this bacteria group using conventional phenotypic methods is ambiguous and cumbersome. In this study, we have analyzed a recA gene fragment from 30 bacteria, including LAB and species common in the human gastrointestinal tract, aiming to evaluate the gene conservation among them and the development of primers and PCR conditions able to discriminate Lactobacillus plantarum strains from LAB closely related. The fragment with 995 bp of recA gene has grouped LAB, enterobacteria and bifidobacteria, in different clusters. A novel primer pair, LPrecAF and LPrecAR with 23 and 18 bp, respectively, has allowed the single amplification of a 108 bp fragment of L. plantarum strains contained in culture broth and fermented dairy samples. The observed detection limit for food samples and for cultures broth were 1 × 103 and 7 × 102 CFU mL−1, respectively. This approach proved to be a simple and efficient method for the identification and monitoring of L. plantarum in food, feeds, and culture broth. Moreover, the assay could be used in the studies from human or environmental microbiota.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Borriello SP, Hammes WP, Holzapfel W, Marteau P, Schrezenmeir J, Vaara M, Valtonen V (2003) Safety of probiotics that contain Lactobacilli or Bifidobacteria. Clin Infect Dis 36:775–780

    Article  CAS  Google Scholar 

  2. Heyman M, Ménard S (2002) Probiotic microorganisms: how they affect intestinal athophysiology. Cell Mol Life Sci 59:1151–1165

    Article  CAS  Google Scholar 

  3. Agheyisi R (2008) The probiotics market: Ingredients, supplements, foods—FOD035B. Food & Beverage, June 2008. Available from http://www.bccresearch.com/report/FOD035B.html. Accessed 08 June 2010

  4. Claesson MJ, Sinderen D, O’toole PW (2007) The genus Lactobacillus-a genomic basis for understanding its diversity. FEMS Microbiol Lett 269:22–28

    Article  CAS  Google Scholar 

  5. Kleerebezem M, Boekhorst J, Van Kranenburg R, Molenaar D, Kuipers OP, Leer R, Tarchini R, Peters SA, Sandbrink HM, Fiers MW, Stiekema W, Lankhorst RM, Bron PA, Hoffer SM, Groot MN, Kerkhoven R, de Vries M, Ursing B, de Vos WM, Siezen RJ (2003) Complete genome sequence of Lactobacillus plantarum WCFS1. Proc Natl Acad Sci U S A 100(4):1990–1995

    Article  CAS  Google Scholar 

  6. Paolillo R, Carratelli CR, Sorrentino S, Mazzola N, Rizzo A (2009) Immunomodulatory effects of Lactobacillus plantarum on human colon cancer cells. Int Immunopharmacol 9:1265–1271

    Article  CAS  Google Scholar 

  7. Naruszewicz M, Johansson ML, Zapolska-Downar D, Bukowska H (2002) Effect of Lactobacillus plantarum 299v on cardiovascular disease risk factors in smokers. Am J Clin Nutr 76:1249–1255

    CAS  Google Scholar 

  8. Yen D, Cheung J, Scheerens H, Poulet F, Mcclanahan T, Mckenzie B, Kleinschekl MA, Owyang A, Mattson J, Blumenschein W, Murphy E, Sathe M, Cua DJ, Kastelein RA, Rennick D (2006) IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J Clin Invest 116:1310–1316

    Article  CAS  Google Scholar 

  9. Bukowska H, Pieczul-Mróz J, Jastrzêbska M, Chelstowski K, Naruszewicz M (1997) Decrease in fibrinogen and LDL-cholesterol levels upon supplementation of diet with Lactobacillus plantarum in subjects with moderately elevated cholesterol. Atherosclerosis 137:437–438

    Google Scholar 

  10. Hugenschmidt S, Schwenninger SM, Gnehm N, Lacroix C (2010) Screening of a natural biodiversity of lactic and propionic acid bacteria for folate and vitamin B12 production in supplemented whey permeate. Inter Dairy J 20:852–857

    Article  CAS  Google Scholar 

  11. Quere F, Deschamps A, Urdaci MC (1997) DNA probe and PCR-specific reaction for Lactobacillus plantarum. J Appl Microbiol 82:783–790

    Article  CAS  Google Scholar 

  12. Zhong W, Millsap K, Bialkowska-Hobrzanska H, Reid G (1998) Differentiation of Lactobacillus Species by molecular typing. Appl Environ Microbiol 64(7):2418–2423

    CAS  Google Scholar 

  13. Berger B, Pridmore RD, Barretto C, Delmas-Julien F (2007) Similarity and differences in the Lactobacillus acidophilus group identified by polyphasic analysis and comparative genomics. J Bacteriol 189:1311–1321

    Article  CAS  Google Scholar 

  14. Collins MD, Rodrigues UM, Ash C, Aguire M, Farrow JAE, Martinez-Murcia A, Phillips BA, Williams AM, Wallbanks S (1991) Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16s rRNA. FEMS Microbiol Lett 77:5–12

    Article  CAS  Google Scholar 

  15. O’Flaherty S, Klaenhammer TR (2010) The role and potential of probiotic bacteria in the gut, and the communication between gut microflora and gut/host. Int Dairy J 20:262–268

    Article  Google Scholar 

  16. Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, Nielsen T, Pons N, Levenez F, Yamada T, Mende DR, Li J, Xu JLS, Li D, Cao J, Wang B, Liang H, Zheng H, Xie Y, Tap J, Lepage P, Bertalan M, Batto JM, Hansen T, Le Paslier D, Linneberg A, Nielsen HB, Pelletier E, Renault P, Sicheritz-Ponten T, Turner K, Zhu H, Yu C, Li S, Jian M, Zhou Y, Li Y, Zhang X, Li S, Qin N, Yang H, Wang J, Brunak S, Doré J, Guarner F, Kristiansen K, Pedersen O, Parkhill J, Weissenbach J, Consortium Meta HIT, Bork P, Ehrlich SD, Wang J (2010) A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464:59–66

    Article  CAS  Google Scholar 

  17. Del Piano M, Morell L, Strozzi GP, Allesina S, Barba M, Deida F (2006) Probiotics: from research to consumer. Dig Liver Dis 38(2):248–255

    Article  Google Scholar 

  18. Walker WA, Goulet O, Morelli L, Antoine JM (2006) Progress in the science of probiotics: from cellular microbiology and applied immunology to clinical nutrition. Eur J Clin Nutr 45(1):1–18

    Article  Google Scholar 

  19. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA 4: molecular evolutionary genetics analysis software version 4.0. Mol Biol Evol 24:1596–1599

    Article  CAS  Google Scholar 

  20. Nei M (1986) Definition and estimation of fixation indices. Evolution 40(3):643–645

    Article  Google Scholar 

  21. Gueimonde M, Tolkko S, Korpimki T, Salminen S (2004) New Real-Time Quantitative PCR procedure for quantification of Bifidobacteria in human fecal samples. Appl Environ Microbiol 70:4165–4169

    Article  CAS  Google Scholar 

  22. Singh S, Goswami P, Singh R, Heller KJ (2009) Application of molecular identification tools for Lactobacillus, with a focus on discrimination between closely related species: a review. LWT-Food Sci Technol 42:448–457

    Article  CAS  Google Scholar 

  23. Naser SM, Dawynd P, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerck I, Vancanneyt M, Swings J (2007) Identification of lactobacilli by pheS and rpoA gene sequence analyses. Int System Evol Microbiol 57:2777–2789

    Article  CAS  Google Scholar 

  24. Mathur S, Singh R (2005) Antibiotic resistance in food lactic acid bacteria-A review. Int J Food Microbiol 105:281–295

    Article  CAS  Google Scholar 

  25. Dewangan R, Patel A, Khatri S, Choubey J, Verma MK, Kumar Gupta S, Rishi V (2009) Phylogenetic analysis of Enterococcus, Lactobacillus and Streptococcus strains on the basis of abc (Atp Binding Protein) gene sequences. Curr Res J Biol Sci 1(3):127–130

    CAS  Google Scholar 

  26. Huang CH, Lee FL, Liou JS (2010) Rapid discrimination and classification of the Lactobacillus plantarum group based on a partial dnaK sequence and DNA fingerprinting techniques. Antonie van Leeuwenhoek 97:2892–2896

    Article  Google Scholar 

  27. 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(8):3450–3454

    Article  CAS  Google Scholar 

  28. Vásquez A, Molin G, Pettersson B, Antonsson M, Ahrne S (2005) DNA-based classification and sequence heterogeneities in the 16S rRNA genes of Lactobacillus casei/paracasei and related species. Syst Appl Microbiol 28(5):430–441

    Article  Google Scholar 

  29. Felis GE, Dellaglio F, Mizzi L, Torriani S (2001) Comparative sequence analysis of a recA gene fragment brings new evidence for a gange in the taxonomy of the Lactobacillus casei group. Int J Syst Evol Microbiol 51(6):2113–2117

    Article  CAS  Google Scholar 

  30. Tiihonen K, Ouwehand AC, Rautonen N (2010) Human intestinal microbiota and healthy ageing. Ageing Res Rev 9:106–117

    Article  Google Scholar 

  31. Akimov VN, Sidarenka AV, Novik GI (2008) Application of molecular methods to classification and identification of bacteria of the genus Bifidobacterium. Microbiology 77:251–260

    Article  Google Scholar 

  32. Gomes AMP, Malcata FX (1999) Agentes probióticos em alimentos: aspectos fisiológicos e terapêuticos e aplicações tecnológicas. Bol Biotecnol 64:12–22

    Google Scholar 

  33. Holzapfel WH, Stile ME (1997) Lactic acid bacteria of foods and their current taxonomy. Int J Food Microbiol 36:1–29

    Article  Google Scholar 

  34. Lee JH, Kim M, Um S (2004) PCR-based Detection and Identification of Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum in Kimchi. Food Sci Biotechnol 13(6):754–775

    CAS  Google Scholar 

  35. Picozzi C, D’Anchise F, Foschino R (2006) PCR detection of Lactobacillus sanfranciscensis in sourdough and panettone baked product. Eur Food Res Technol 222:330–335

    Article  CAS  Google Scholar 

  36. McKillip JL, Jaykus LA, Drakel MA (2000) A comparison of methods for the detection of Escherichia coli O157:H7 from artificially-contaminated dairy products using PCR. J Appl Microbiol 89:49–55

    Article  CAS  Google Scholar 

  37. Zago M, Rosseti L, Reinheimer J, Carminati D, Giraffa G (2008) Detection and identification of Lactobacillus helveticus bacteriophages by PCR. J Dairy Res 75:196–201

    Article  CAS  Google Scholar 

  38. Dickinson J, Kroll RG, Grant KA (1995) The direct application of the polymerase chain reaction to DNA extracted from foods. Lett Appl Microbiol 20:212–216

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge Danisco, C. Hansen, and Sacco Companies by cultures donation, Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq, process: 140352/2008-2 for grants to G.N.C and the financial support from Fundação Araucária. We also thank to Fernando Macedo (UEL), for critically reading the manuscript.

Author information

Authors and Affiliations

  1. Programa de pós graduação em Ciência de Alimentos, DCTA - Universidade Estadual de Londrina, PO Box 6001, Londrina, Paraná, 86051-990, Brazil

    Giselle Nobre Costa

  2. Departamento de Biologia Geral, Centro de Ciências Biológicas, Universidade Estadual de Londrina, PO Box 6001, Londrina, Paraná, 86051-990, Brazil

    Gislayne T. Vilas-Bôas & Laurival A. Vilas-Boas

  3. Departamento de Ciência e Tecnologia de Alimentos, Centro de Ciências Agrárias, Universidade Estadual de Londrina, PO Box 6001, Londrina, Paraná, 86051-970, Brazil

    Lucia H. S. Miglioranza

Authors
  1. Giselle Nobre Costa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gislayne T. Vilas-Bôas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laurival A. Vilas-Boas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lucia H. S. Miglioranza
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Giselle Nobre Costa.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Costa, G.N., Vilas-Bôas, G.T., Vilas-Boas, L.A. et al. In silico phylogenetic analysis of lactic acid bacteria and new primer set for identification of Lactobacillus plantarum in food samples. Eur Food Res Technol 233, 233–241 (2011). https://doi.org/10.1007/s00217-011-1508-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00217-011-1508-7

Keywords

Search

Navigation