Probiotics and Antimicrobial Proteins

, Volume 5, Issue 2, pp 99–109 | Cite as

Quantitative Appraisal of the Probiotic Attributes and In Vitro Adhesion Potential of Anti-listerial Bacteriocin-producing Lactic Acid Bacteria

  • Sandipan Mukherjee
  • Atul Kumar Singh
  • Manab Deb Adhikari
  • Aiyagari RameshEmail author


Estimation of bile tolerance, endurance to gastric and intestinal environment and adhesion potential to intestinal cells are significant selection criteria for probiotic lactic acid bacteria (LAB). In this paper, the probiotic potential of native bacteriocin-producing LAB isolated previously from indigenous source has been determined through quantitative approaches. Among fifteen anti-listerial bacteriocin-producing native LAB, ten strains were found to be bile tolerant. The presence of bile salt hydrolase (bsh) gene in native Lactobacillus plantarum strains was detected by PCR and confirmed by nucleic acid sequencing of a representative amplicon. Interestingly, three native LAB strains exhibited significant viability in simulated gastric fluid, analogous to the standard LAB Lactobacillus rhamnosus GG, while an overwhelming majority of the native LAB strains demonstrated the ability to survive and remain viable in simulated intestinal fluid. Quantitative adhesion assays based on conventional plating method and a fluorescence-based method revealed that the LAB isolates obtained from dried fish displayed significant in vitro adhesion potential to human adenocarcinoma HT-29 cells, and the adhesion level was comparable to some of the standard probiotic LAB strains. The present study unravels putative probiotic attributes in certain bacteriocin-producing LAB strains of non-human origin, which on further in vivo characterization could find specific applications in probiotic food formulations targeted for health benefits.


Lactic acid bacteria Probiotic Adhesion Bacteriocin 



This work was supported by a research grant from Council of Scientific and Industrial Research (CSIR), New Delhi, Government of India [No. 38(1251)/10/EMR-II]. We thank the National Facility of Automated DNA Sequencing, Department of Biochemistry, Delhi University, South Campus for their support in nucleic acid sequencing.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Coolbear T, Crow V, Harnett J, Harvey S, Holland R, Martley F (2008) Developments in cheese microbiology in New Zealand—Use of starter and non-starter lactic acid bacteria and their enzymes in determining flavour. Int Dairy J 18:705–713CrossRefGoogle Scholar
  2. 2.
    Leroy F, De Vuyst L (2004) Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci Technol 15:67–78CrossRefGoogle Scholar
  3. 3.
    Jagannath A, Ramesh A, Ramesh MN, Chandrashekar A, Varadaraj MC (2001) Predictive model for the behavior of Listeria monocytogenes Scott A in Shrikhand, prepared with a biopreservative pediocin K7. Food Microbiol 18:335–343CrossRefGoogle Scholar
  4. 4.
    Jones RJ, Hussein HM, Zagorec M, Brightwell G, Tagg JR (2008) Isolation of lactic acid bacteria with inhibitory activity against pathogens and spoilage organisms associated with fresh meat. Food Microbiol 25:228–234CrossRefGoogle Scholar
  5. 5.
    Singh AK, Ramesh A (2008) Succession of dominant and antagonistic lactic acid bacteria in fermented cucumber: insights from a PCR-based approach. Food Microbiol 25:278–287CrossRefGoogle Scholar
  6. 6.
    Singh AK, Mukherjee S, Adhikari MD, Ramesh A (2012) Fluorescence-based comparative evaluation of bactericidal potency and food application potential of anti-listerial bacteriocin produced by lactic acid bacteria isolated from indigenous samples. Probiotics Antimicrob Proteins 4:122–132CrossRefGoogle Scholar
  7. 7.
    Lebeer S, Vanderleyden J, De Keersmaecker SC (2008) Genes and molecules of lactobacilli supporting probiotic action. Microbiol Mol Biol Rev 72:728–764CrossRefGoogle Scholar
  8. 8.
    Marco ML, Pavan S, Kleerebezem M (2006) Towards understanding molecular modes of probiotic action. Curr Opin Biotechnol 17:204–210CrossRefGoogle Scholar
  9. 9.
    Ouwehand AC, Seppo Salminen, Isolauri E (2002) Probiotics: an overview of beneficial effects. Antonie Van Leeuwenhoek 82:279–289CrossRefGoogle Scholar
  10. 10.
    Servin AL, Coconnier MH (2003) Adhesion of probiotic strains to the intestinal mucosa and interaction with pathogens. Best Pract Res Clin Gastroenterol 17:741–754CrossRefGoogle Scholar
  11. 11.
    Yan F, Polk DB (2010) Probiotics: progress toward novel therapies for intestinal diseases. Curr Opin Gastroenterol 26:95–101CrossRefGoogle Scholar
  12. 12.
    Alander M, Satokari R, Korpela R, Saxelin M, Vilpponen-Salmela T, Mattila-Sandholm T (1999) Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption. Appl Environ Microbiol 65:351–354Google Scholar
  13. 13.
    Elliott SN, Buret MW, Miller MJS, Wallace JL (1998) Bacteria rapidly colonize and modulate healing of gastric ulcers in rats. Am J Physiol Gastrointest Liver Physiol 275:425–432Google Scholar
  14. 14.
    Collado MC, Meriluoto J, Salminen S (2007) Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus. Lett Appl Microbiol 45:454–460CrossRefGoogle Scholar
  15. 15.
    Reid G, Burton J (2002) Use of Lactobacillus to prevent infection by pathogenic bacteria. Microbes Infect 4:319–324CrossRefGoogle Scholar
  16. 16.
    Pagnini C, Saeed R, Bamias G, Aresneau KO, Pizarro TT, Cominelli F (2010) Probiotics promote gut health through stimulation of epithelial innate immunity. Proc Natl Acad Sci 107:454–459CrossRefGoogle Scholar
  17. 17.
    Zhou JS, Gill HS (2005) Immunostimulatory probiotic Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 do not induce pathological inflammation in mouse model of experimental autoimmune thyroiditis. Int J Food Microbiol 103:97–104CrossRefGoogle Scholar
  18. 18.
    Nueno-Palop C, Narbad A (2011) Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut. Int J Food Microbiol 145:390–394CrossRefGoogle Scholar
  19. 19.
    Charteris WP, Kelly PM, Morelli L, Collins JK (1998) Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. J Appl Microbiol 84:759–768CrossRefGoogle Scholar
  20. 20.
    Huang Y, Adams MC (2004) In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria. Int J Food Microbiol 91:253–260CrossRefGoogle Scholar
  21. 21.
    Gopal PK, Prasad J, Smart J, Gill HS (2001) In vitro adherence properties of Lactobacillus rhamnosus DR20 and Bifidobactirium lactis DR10 strains and their antagonistic activity against an enterotoxigenic Escherichia coli. Int J Food Microbiol 67:207–216CrossRefGoogle Scholar
  22. 22.
    Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A, Zucco F (2005) The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol 21:1–26CrossRefGoogle Scholar
  23. 23.
    Tuomola EM, Salminen SJ (1998) Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures. Int J Food Microbiol 5:45–51CrossRefGoogle Scholar
  24. 24.
    Forestier C, De Champs C, Vatoux C, Joly B (2001) Probiotic activities of Lactobacillus casei rhamnosus: in vitro adherence to intestinal cells and antimicrobial properties. Res Microbiol 152:167–173CrossRefGoogle Scholar
  25. 25.
    Binachi MA, Del Rio D, Pellegrini N, Sansebastiano G, Neviani E, Brighenti F (2004) A fluorescence-based method for the detection of adhesive properties of lactic acid bacteria to Caco-2 cells. Lett Appl Microbiol 39:301–305CrossRefGoogle Scholar
  26. 26.
    Blay GL, Fliss I, Lacroix C (2004) Comparative detection of bacterial adhesion to Caco-2 cells with ELISA, radioactivity and plate count method. J Microbiol Methods 59:211–221CrossRefGoogle Scholar
  27. 27.
    Lee YK, Ho PS, Low CS, Arvilommi H, Salminen S (2004) Permanent colonization by Lactobacillus casei is hindered by the low rate of cell division in mouse gut. Appl Environ Microbiol 70:670–674CrossRefGoogle Scholar
  28. 28.
    Chang JH, Shim YY, Cha SK, Chee KM (2010) Probiotic characteristics of lactic acid bacteria isolated from kimchi. J Appl Microbiol 109:220–230CrossRefGoogle Scholar
  29. 29.
    Vitali B, Minervini G, Rizzello CG, Spisni E, Maccaferri S, Brigidi P, Gobbetti M, Di Cagno R (2012) Novel probiotic candidates for humans isolated from raw fruits and vegetables. Food Microbiol 31:116–125CrossRefGoogle Scholar
  30. 30.
    Singh AK, Ramesh A (2009) Evaluation of a facile method of template DNA preparation for PCR-based detection and typing of lactic acid bacteria. Food Microbiol 26:513–540CrossRefGoogle Scholar
  31. 31.
    Jacobsen CN, Rosenfeldt NV, Hayford AE, Møller PL, Michaelsen KF, Paerregaard A, Sandstrom B, Tvede M, Jakobsen M (1999) Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Appl Environ Microbiol 65:4949–4956Google Scholar
  32. 32.
    Ohland CL, MacNaughton WK (2010) Probiotic bacteria and intestinal epithelial barrier function. Am J Physiol Gastrointest Liver Physiol 298:807–810CrossRefGoogle Scholar
  33. 33.
    Begley M, Gahan CGM, Hill C (2005) The interaction between bacteria and bile. FEMS Microbiol Rev 29:625–651CrossRefGoogle Scholar
  34. 34.
    Chateau N, Deschamps AM, Hadj SA (1994) Heterogeneity of bile salts resistance in the Lactobacillus isolates of a probiotic consortium. Lett Appl Microbiol 18:42–44CrossRefGoogle Scholar
  35. 35.
    Burns P, Vinderola G, Binetti A, Quiberoni A, de los Reyes-Gavilan C, Reinheimer J (2008) Bile-resistant derivatives obtained from non-intestinal dairy lactobacilli. Int Dairy J 18:377–385CrossRefGoogle Scholar
  36. 36.
    Grill JP, Cayuela C, Antoine JM, Schneider F (2000) Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistance. J Appl Microbiol 89:553–563CrossRefGoogle Scholar
  37. 37.
    Kaushik JK, Kumar A, Duary RK, Mohanty AK, Grover S, Batish VK (2009) Functional and probiotic attributes of an indigenous isolate of Lactobacillus plantarum. PLoS ONE 4:1–11CrossRefGoogle Scholar
  38. 38.
    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 74:4719–4726CrossRefGoogle Scholar
  39. 39.
    Jones BV, Begley M, Hill C, Gahan CGM, Marchesi JR (2008) Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome. Proc Natl Acad Sci 105:13580–13585CrossRefGoogle Scholar
  40. 40.
    McAuliffe O, Cano RJ, Klaenhammer TR (2005) Genetic analysis of two bile salt hydrolase activities in Lactobacillus acidophilus NCFM. Appl Environ Microbiol 71:4925–4929CrossRefGoogle Scholar
  41. 41.
    Patel AK, Singhania RR, Pandey A, Chincholkar SB (2010) Probiotic bile salt hydrolase: current developments and perspectives. Appl Biochem Biotechnol 162:166–180CrossRefGoogle Scholar
  42. 42.
    Liong MT, Shah NP (2005) Acid and bile tolerance and cholesterol removal ability of lactobacilli strains. J Dairy Sci 88:55–66CrossRefGoogle Scholar
  43. 43.
    Blum S, Haller D, Pfeifer A, Schiffrin EJ (2002) Probiotics and immune response. Clin Rev Allergy Immunol 22:287–309CrossRefGoogle Scholar
  44. 44.
    Langerholc T, Maragkoudakis PA, Wollgast J, Gradisnik L, Cencic A (2011) Novel and established cell line models—An indispensable tool in food science and nutrition. Trends Food Sci Technol 22:S11–S20CrossRefGoogle Scholar
  45. 45.
    Ossowski I, Reunanen J, Satokari R, Vesterlund S, Kankainen M, Huhtinen H, Tynkkynen S, Salminen S, De Vos WM, Palva A (2010) Mucosal adhesion properties of the probiotic Lactobacillus rhamnosus GG SpaCBA and SpaFED pilin subunits. Appl Environ Microbiol 76:2049–2057CrossRefGoogle Scholar
  46. 46.
    Jensen H, Grimmer S, Naterstad K, Axelsson L (2012) In vitro testing of commercial and potential probiotic lactic acid bacteria. Int J Food Microbiol 153:216–222CrossRefGoogle Scholar
  47. 47.
    Tallon R, Arias S, Bressollier P, Urdaci MC (2007) Strain- and matrix-dependent adhesion of Lactobacillus plantarum is mediated by proteinaceous bacterial compounds. J Appl Microbiol 102:442–451CrossRefGoogle Scholar
  48. 48.
    Archimbaud C, Shankar N, Forestier C, Baghdayan A, Gilmore MS, Charbonne F, Joly B (2002) In vitro adhesive properties and virulence factors of Enterococcus faecalis strains. Res Microbiol 153:75–80CrossRefGoogle Scholar
  49. 49.
    Hoefel D, Groobya WL, Monisa PT, Andrews S, Saint CP (2003) A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity. J Microbiol Methods 52:379–388CrossRefGoogle Scholar
  50. 50.
    Ramiah K, van Reenen CA, Dicks LMT (2008) Surface-bound proteins of Lactobacillus plantarum 423 that contribute to adhesion of Caco-2 cells and their role in competitive exclusion and displacement of Clostridium sporogenes and Enterococcus faecalis. Res Microbiol 159:470–475CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Sandipan Mukherjee
    • 1
  • Atul Kumar Singh
    • 1
  • Manab Deb Adhikari
    • 1
  • Aiyagari Ramesh
    • 1
    Email author
  1. 1.Department of BiotechnologyIndian Institute of Technology GuwahatiGuwahatiIndia

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