Skip to main content
SpringerLink
Log in

Molecular Identification and Typing of Putative Probiotic Indigenous Lactobacillus plantarum Strain Lp91 of Human Origin by Specific Primed-PCR Assays

  • Published:
Probiotics and Antimicrobial Proteins Aims and scope Submit manuscript

Abstract

In the present scenario, it is now well documented that probiotics confer health benefits to the host and the purported probiotic effects are highly strain specific. Hence, accurate genotypic identification is extremely important to link the strain to the specific health effect. With this aim, specific primed-PCR assays were developed and explored for the molecular identification and typing of a putative indigenous probiotic isolate Lp91 of human faecal origin. PCR with specific primers targeting 23S rRNA gene of genus Lactobacillus and 16S rRNA gene of species L. plantarum resulted positive for Lp91. In addition, BLAST analysis of 16S rRNA gene sequence of Lp91 and multiple sequence alignment of 16S rRNA gene variable (V2-V3) regions along with the reference sequences revealed it as L. plantarum with a sequence identity of more than 99%. Furthermore, resolution of 16S rRNA gene sequences was sufficient to infer a phylogenetic relationship amongst Lactobacillus species. In order to determine strain-level variations, randomly amplified polymorphic DNA (RAPD) banding profiles of Lp91 obtained with OPAA-01, OPAP-01 and OPBB-01 primers were compared with those of reference strains of Lactobacillus spp., and Lp91 could be delineated as a distinct strain. Apart from this, presence of probiotic markers viz. bile salt hydrolase (bsh) and collagen-binding protein (cbp) encoding genes in Lp91 genome could be attributed to its exploitation as a potential probiotic adjunct in the development of indigenous functional foods. Lactobacillus isolates/or strains from the gastrointestinal system, fermented products and other environmental niches could be identified and characterized by employing the PCR methods developed in this study; they are rapid, reproducible and more accurate than the conventional methods based on the fermentation profiles.

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. Adawi D, Ahrne S, Molin G (2001) Effects of different probiotic strains of Lactobacillus and Bifidobacterium on bacterial translocation and liver injury in an acute liver injury model. Int J Food Microbiol 70:213–220

    Article  CAS  Google Scholar 

  2. Ahrne S, Nobaek S, Jeppsson B, Adlerberth I, Wold AE, Molin G (1998) The normal Lactobacillus flora of healthy human rectal and oral mucosa. J Appl Microbiol 85:88–94

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Cunningham-Rundles S, Ahrne S, Bengmark S, Johann-Liang R, Marshall F, Metakis L, Califano C, Dunn AM, Grassey C, Hinds G, Cervia J (2000) Probiotics and immune response. Am J Gastroenterol 95:22–25

    Article  Google Scholar 

  5. Duary RK, Batish VK, Grover S (2010) Expression of atpD gene in putative indigenous 480 probiotic L. plantarum strains under in vitro acidic conditions by RT-qPCR. Res Microbiol 161:399–405

    Article  CAS  Google Scholar 

  6. Dubernet S, Desmasures N, Gueguen M (2002) A PCR based method for identification of lactobacilli at the genus level. FEMS Microbiol Lett 214:217–275

    Article  Google Scholar 

  7. FAO/WHO (2002) Guidelines for the evaluation of probiotics in food. Report of a joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food, London

    Google Scholar 

  8. Guban J, Korver DR, Allison GE, Tannock GW (2006) Relationship of dietary antimicrobial drug administration with broiler performance, decreased population levels of Lactobacillus salivarius, and reduced bile salt deconjugation in the ileum of broiler chickens. Poult Sci 85:2186–2194

    CAS  Google Scholar 

  9. Kumar R, Grover S, Mohanty AK, Batish VK (2010) Molecular cloning and sequence analysis of bile salt hydrolase (bsh) gene from Lactobacillus plantarum MBUL90 strain of human origin. Food Biotech 24:215–226

    Article  CAS  Google Scholar 

  10. Kumar R, Grover S, Batish VK (2011) Hypocholesterolemic effect of dietary inclusion of two putative probiotic bile salt hydrolase (Bsh) producing Lactobacillus plantarum strains in Sprague-Dawley rats. Brit J Nutr 105:561–573

    Article  CAS  Google Scholar 

  11. Lambert JM, Bongers RS, de Vos WM, Kleerebezem M (2008) Functional analysis of four bile salt hydrolase and penicillin acylase family members in Lactobacillus plantarum WCFS1. Appl Environ Microbiol 74:4719–4726

    Article  CAS  Google Scholar 

  12. Pidiyar VJ, Jangid K, Patole MS, Shouche YS (2004) Studies on cultured and uncultured microbiota of wild Culex quinquefasciatus mosquito midgut based on 16S ribosomal RNA gene analysis. Am J Trop Med Hyg 70:597–603

    CAS  Google Scholar 

  13. Rodas AM, Ferrer S, Pardo I (2005) Polyphasic study of wine Lactobacillus strains: taxonomic implications. Int J Syst Evol Microbiol 55:197–207

    Article  CAS  Google Scholar 

  14. Sanchez B, Bressollier P, Urdaci MC (2008) Exported proteins in probiotic bacteria: adhesion to intestinal surfaces, host immunomodulation and molecular cross-talking with the host. FEMS Immunol Med Microbiol 54:1–17

    Article  CAS  Google Scholar 

  15. 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  Google Scholar 

  16. Spano G, Beneduce L, Tarantino D, Zapparoli G, Massa S (2002) Characterization of Lactobacillus plantarum from wine must by PCR species-specific and RAPD-PCR. Lett Appl Microbiol 35:370–374

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Tannock GW, Dashkevicz MP, Feighner SD (1989) Lactobacilli and bile salt hydrolase in the murine intestinal tract. Appl Environ Microbiol 55:1848–1851

    CAS  Google Scholar 

  19. Velez MP, De Keersmaecker SC, Vanderleyden J (2007) Adherence factors of Lactobacillus in the human gastrointestinal tract. FEMS Microbiol Lett 276:140–148

    Article  CAS  Google Scholar 

  20. Wang Y, Qian P-Y (2009) Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS ONE 4(10):e7401

    Article  Google Scholar 

  21. Weiss A, Lettner HP, Kramer W, Mayer HK, Kneifel W (2005) Molecular methods used for the identification of potentially probiotic Lactobacillus reuteri strains. Food Technol Biotechnol 43:295–300

    CAS  Google Scholar 

  22. www.operon.com/products/downloads/OperonsRAPD10merSequences.xls

Download references

Acknowledgments

R.K. greatly appreciates the financial support received from Indian Council of Agricultural Research (ICAR, India) in terms of fellowship to work on his doctoral thesis.

Conceived, conceptualized and supervised the present study: R.K., S.G. and V.K.B; performed the experiments: R.K.; analysis and interpretation of data: R.K., S.G. and V.K.B.; contributed reagents/materials/analysis tools: S.G. and V.K.B.; preparation of the manuscript: R.K., S.G. and V.K.B. and have no conflict of interest.

Author information

Authors and Affiliations

  1. Dairy Microbiology Division, Molecular Biology Unit, National Dairy Research Institute, Karnal, 132001, India

    Rajesh Kumar, Sunita Grover & Virender Kumar Batish

  2. Department of Microbiology and Immunology, National Institute of Nutrition, Hyderabad, 500007, India

    Rajesh Kumar

Authors
  1. Rajesh Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sunita Grover
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Virender Kumar Batish
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Virender Kumar Batish.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kumar, R., Grover, S. & Batish, V.K. Molecular Identification and Typing of Putative Probiotic Indigenous Lactobacillus plantarum Strain Lp91 of Human Origin by Specific Primed-PCR Assays. Probiotics & Antimicro. Prot. 3, 186–193 (2011). https://doi.org/10.1007/s12602-011-9083-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12602-011-9083-6

Keywords

Search

Navigation