Skip to main content
SpringerLink
Log in

Practical identification of human originated Lactobacillus species by amplified ribosomal DNA restriction analysis (ARDRA) for probiotic use

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Probiotics are gaining popularity and increasing the importance of their accurate speciation. Lactobacillus species are commonly used as probiotic strains mostly of clinical importance. Present knowledge indicates that at least 14 Lactobacillus species are associated with the human intestinal tract. Currently, researchers are interested in developing efficient techniques for screening and selecting probiotics bacteria, but unfortunately most of these methods are time-consuming, labor-intensive and costly. The aim of this study is to develop reliable, rapid and accurate method to identify 14 references Lactobacillus species that could have been found in the human alimentary tract by 16S ribosomal DNA restriction analysis. In this study, to develop an effective method for the genotype-based identification of the reference Lactobacillus species, 1.5 kb of 16S rRNA nucleotide sequences of 14 Lactobacillus were collected from the Gene Bank aligned, in silico restricted and analyzed in respect to their 16S-rRNA restriction fragment polymorphism. In silico restriction profiles of 16S-rRNA indicated that FspBI, HinfI and DraI restriction enzymes (RE) are convenient for differentiation of 14 Lactobacillus species in human intestinal tract except Lb. casei and Lb. paracasei. The patterns of our experimental findings obtained from 16S PCR-ARDRA completely confirmed our in silico patterns. The present work demonstrated that 16S PCR-ARDRA method with FspBI, HinfI and DraI RE is a rapid, accurate and reliable method for the identification of Lactobacillus species from human alimentary tract, especially during the identification of large numbers of isolates and any laboratory equipped with a thermo cycler for probiotic use.

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
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Guarner F, Schaafsma G (1998) Probiotics. Int J Food Microbiol 39:237–238

    Article  CAS  PubMed  Google Scholar 

  2. Galdeano CM, de LeBlanc ADM, Vinderola G, Bonet MEB, Perdigón G (2007) Proposed model: mechanisms of immunomodulation induced by probiotic bacteria. Clin Vaccine Immunol 14:485–492

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Boutron-Ruault MC (2007) Probiotiques et cancer colorectal. Nutr Clin Mét 21:85–88

    Article  Google Scholar 

  4. Davis CD, Milner JA (2009) Gastrointestinal microflora, food components and colon cancer prevention. J Nutr Biochem 20:743–752

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Baldwin C, Millette M, Oth D, Ruiz MT, Luquet FM, Lacroix M (2010) Probiotic Lactobacillus acidophilus and L. casei mix sensitize colorectal tumoral cells to 5-fluorouracil-induced apoptosis. Nutr Cancer 623:371–378

    Article  Google Scholar 

  6. Dylewski J, Psaradellis F, Sampalis J (2010) Efficacy of a probiotic formula in the reduction of antibiotic-associated diarrhea, a placebo controlled double-blind randomized, multicenter study. Arch Med Sci 61:56–64

    Article  Google Scholar 

  7. Gao XW, Mubasher M, Fang CY, Reifer C, Miller LE (2010) Dose-response efficacy of a proprietary probiotic formula of Lactobacillus acidophilus CL1285 and Lactobacillus casei LBC80R for antibiotic-associated diarrhea and Clostridium difficile associated diarrhea prophylaxis in adult patients. Am J Gastroenterol 105(7):1636–1641

    Article  PubMed  Google Scholar 

  8. Hirayama K, Rafter J (2000) The role of probiotic bacteria in cancer prevention. Microbes Infect 2:681–686

    Article  CAS  PubMed  Google Scholar 

  9. Songisepp E, Kulisaar T, Hütt P, Elias P, Brilene T, Zilmer M, Mikelsaar M (2004) A new probiotic cheese with antioxidative and antimicrobial. J Dairy Sci 87:2017–2023

    Article  CAS  PubMed  Google Scholar 

  10. Baharav E, Mor F, Halpern M, Weinberger A (2004) Lactobacillus GG bacteria ameliorate arthritis in Lewis Rats. J Nutr 134:1964–1969

    CAS  PubMed  Google Scholar 

  11. Scholz-Ahrens KE, Ade P, Marten B, Weber P, Timm W, Asil Y, Glüer CC, Schrezenmeir J (2007) Prebiotics, probiotics and synbiotics affect mineral absorption, bone mineral content and bone structure. J Nutr 137:838–846

    Google Scholar 

  12. Falagas ME, Betsi GI, Athanasiou S (2006) Probiotics for prevention of recurrent vulvovaginal candidiasis: a review. J Antimicrob Chemother 58:266–272

    Article  CAS  PubMed  Google Scholar 

  13. Ouwehand AC (2007) Antiallergic effects of probiotics. J Nutr 137:794–797

    Google Scholar 

  14. Macfarlane G, Cummings J (2002) Probiotics, infection and immunity. Curr Opin Infect Dis 15:501–506

    Article  CAS  PubMed  Google Scholar 

  15. Lata J, Jurankova J, Kopacova M, Vitek P (2011) Probiotics in hepatology. World J Gastroenterol 17(24):2890–2896

    Article  PubMed Central  PubMed  Google Scholar 

  16. Yıldız GG, Öztürk M, Aslım B (2011) Identification of Lactobacillus strains from breast-fed infant and investigation of their cholesterol reducing effects. World J Microbiol Biotechnol 27(10):2397–2406

    Article  Google Scholar 

  17. Pereira DIA, Gibson GR (2002) Effects of consumption of probiotics and prebiotics on serum lipid levels in human. Crit Rev Biochem Mol Biol 37:259–281

    Article  CAS  PubMed  Google Scholar 

  18. Collins JK, Thornton G, Sullivan GO (1998) Selection of probiotic strains for human application. Int Dairy J 8:487–490

    Article  Google Scholar 

  19. Klaenhammer TR, Kullen MJ (1999) Selection and design of probiotics. Int J Food Microbiol 50:45–57

    Article  CAS  PubMed  Google Scholar 

  20. Tuomola E, Crittenden R, Playne M, Isolauri E, Salminen S (2001) Quality assurance criteria for probiotic bacteria. Am J Clin Nutr 73:393–398

    Google Scholar 

  21. Begley M, Hill C, Gahan CG (2006) Bile salt hydrolase activity in probiotics. Appl Environ Microbiol 72:1729–1738

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Dunne C, O’Mahony L, Murphy L, Thornton G, Morrissey D, O’Halloran S (2001) In vitro selection criteria for probiotic bacteria of human origin, correlation with in vivo findings. Am J Clin Nutr 73:386–392

    Google Scholar 

  23. Reuter G (2001) The Lactobacillus and Bifidobacterium microflora of the human intestine: composition and succession. Curr Issues Intest Microbiol 2:43–53

    CAS  PubMed  Google Scholar 

  24. Walter J (2008) Ecological role of lactobacilli in the gastroin-testinal tract: implications for fundamental and biomedical research. Appl Environ Microbiol 74:4985–4996

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Hammes WP, Hertel C (2006) The genera Lactobacillus and Carnobacterium. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The prokaryotes 4, 3rd edn. Springer, New York, pp 320–403

    Chapter  Google Scholar 

  26. Song YL, Kato N, Liu CX, Matsumiya Y, Kato H, Watanabe K (2000) Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S–23S rRNA intergenic spacer region and its flanking 23S rRNA. FEMS Microbiol Lett 187:167–173

    CAS  PubMed  Google Scholar 

  27. Guan LL, Hagen KE, Tannock GW, Korver DR, Fasenko GM, Allison GE (2003) Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis. Appl Environ Microbiol 69:6750–6757

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Dalezios I, Siebert KJ (2001) Comparison of pattern recognition techniques for the identification of lactic acid bacteria. J Appl Microbiol 91:225–236

    Article  CAS  PubMed  Google Scholar 

  29. Collins MD, Rodrigues U, Ash C, Aguirre M, Farrow JAE, Martinez-Murcia A et al (1991) Phylogenic 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 

  30. Hammes WP, Vogel RF (1995) The genus Lactobacillus. In: Wood BJB, Holzapfel W (eds) The genera of lactic acid bacteria. Blackie Academic & Professional, Glasgow, pp 19–54

    Chapter  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  32. 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. Food Sci Technol 42(2):448–457

    CAS  Google Scholar 

  33. Hammes WP, Hertel C (2003) The Genera Lactobacillus and Cronobacterium. In: The prokaryotes: an evolving electronic resource for the microbiological community, 3rd edn. Springer, New York. http://link.springer.ny.com/link/service/books/10125/

  34. Holzapfel WH, Haberer P, Geisen R, Bjorkroth J, Schillinger U (2001) Taxonomy and important features of probiotic microorganisms in food and nutrition. Am J Clin Nutr 73:365–373

    Google Scholar 

  35. Cato EP, Moore WEC, Johnson JL (1983) Synonymy of strains of ‘‘Lactobacillus acidophilus’’ group A2 (Johnson et al., 1980) with the type strain of Lactobacillus crispatus (Brygoo and Aladame, 1953) Moore and Holdeman 1970. Int J Syst Bacteriol 33:426–428

    Article  Google Scholar 

  36. Fujisawa T, Benno Y, Yaeshima T (1992) Taxonomic study of the Lactobacillus acidophilus group, with recognization of Lactobacillus gallinarum sp. nov. Lactobacillus johnsonii sp. nov. and synonymy of Lactobacillus acidophilus group A3 (Johnson et al. 1980) with the type strain of Lactobacillus amylovorus (Nakamura 1981). Int J Syst Bacteriol 42:487–491

    Article  CAS  PubMed  Google Scholar 

  37. Song YL, Kato N, Matsumiya Y, Liu CX, Kato H, Watanabe K (1999) Identification of Lactobacillus species of human origin by a commercial kit, API 50CHL. J Assoc Rapid Methods Autom Microbiol 10:77–82

    CAS  Google Scholar 

  38. Yeung PSM, Sanders ME, Kitts CL, Cano R, Tong PS (2002) Species specific identification of commercial probiotic strains. J Dairy Sci 85:1039–1051

    Article  CAS  PubMed  Google Scholar 

  39. Walter J, Tannock GW, Tilsala-Timisjarvi A, Rodtong S, Loach DM, Munro K et al (2000) Detection and identification of gastrointestinal Lactobacillus species by DGGE and species-specific PCR primers. Appl Environ Microbiol 66:297–303

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Chavagnat F, Haueter M, Jimeno J, Casey MG (2002) Comparison of partial tuf gene sequences for the identification of lactobacilli. FEMS Microbiol Lett 217:177–183

    Article  CAS  PubMed  Google Scholar 

  41. Vasquez A, Ahrne S, Pettersson B, Molin G (2001) Temporal temperature gradient gel electrophoresis (TTGE) as a tool for identification of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus. Lett Appl Microbiol 32:215–219

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This research was supported by Scientific Research Projects of Abant Izzet Baysal University (2011.03.01.406). We would like to acknowledge to İ. ÇAKIR, Abant İzzet Baysal University, for Lb. johnsonii and Lb. reuteri reference strains.

Author information

Authors and Affiliations

  1. Department of Biology, Faculty of Literature and Science, Abant İzzet Baysal University, 14280, Bolu, Turkey

    Mehmet Öztürk & Merve Meterelliyöz

Authors
  1. Mehmet Öztürk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Merve Meterelliyöz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mehmet Öztürk.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Öztürk, M., Meterelliyöz, M. Practical identification of human originated Lactobacillus species by amplified ribosomal DNA restriction analysis (ARDRA) for probiotic use. Mol Biol Rep 42, 1323–1332 (2015). https://doi.org/10.1007/s11033-015-3877-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-015-3877-7

Keywords

Search

Navigation