Probiotics and Antimicrobial Proteins

, Volume 7, Issue 3, pp 181–192 | Cite as

Screening, Characterization and In Vitro Evaluation of Probiotic Properties Among Lactic Acid Bacteria Through Comparative Analysis

  • Sundru Manjulata Devi
  • Ann Catherine Archer
  • Prakash M. Halami


The present work aimed to identify probiotic bacteria from healthy human infant faecal and dairy samples. Subsequently, an assay was developed to evaluate the probiotic properties using comparative genetic approach for marker genes involved in adhesion to the intestinal epithelial layer. Several in vitro properties including tolerance to biological barriers (such as acid and bile), antimicrobial spectrum, resistance to simulated digestive fluids and cellular hydrophobicity were assessed. The potential probiotic cultures were rapidly characterized by morphological, physiological and molecular-based methods [such as RFLP, ITS, RAPD and (GTG)5]. Further analysis by 16S rDNA sequencing revealed that the selected isolates belong to Lactobacillus, Pediococcus and Enterococcus species. Two cultures of non-lactic, non-pathogenic Staphylococcus spp. were also isolated. The native isolates were able to survive under acidic, bile and simulated intestinal conditions. In addition, these cultures inhibited the growth of tested bacterial pathogens. Further, no correlation was observed between hydrophobicity and adhesion ability. Sequencing of probiotic marker genes such as bile salt hydrolase (bsh), fibronectin-binding protein (fbp) and mucin-binding protein (mub) for selected isolates revealed nucleotide variation. The probiotic binding domains were detected by several bioinformatic tools. The approach used in the study enabled the identification of potential probiotic domains responsible for adhesion of bacteria to intestinal epithelial layer, which may further assist in screening of novel probiotic bacteria. The rapid detection of binding domains will help in revealing the beneficial properties of the probiotic cultures. Further, studies will be performed to develop a novel probiotic product which will contribute in food and feed industry.


Probiotics Lactic acid bacteria Binding proteins Bile salt hydrolase Phylogeny Genetic comparison 



We extend our gratitude to Prof. Ram Rajasekharan, Director, CSIR-CFTRI, Mysore, for encouragement and support. This work was carried out in the ICMR, New Delhi, funded project on probiotics (No. 5/9/1029/2011-RHN).

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

12602_2015_9195_MOESM1_ESM.tif (1.3 mb)
Supplementary material 1 (TIFF 1343 kb)
12602_2015_9195_MOESM2_ESM.jpg (645 kb)
Supplementary material 2 (JPEG 645 kb)
12602_2015_9195_MOESM3_ESM.doc (27 kb)
Supplementary material 3 (DOC 42 kb)


  1. 1.
    Patrick OM (2012) Lactic acid bacteria in health and disease. Rwanda J Health Sci 1:39–50Google Scholar
  2. 2.
    Fukao M, Oshima K, Morita H, Toh H, Suda W, Kim SW, Suzuki S, Yakabe T, Hattori M, Yajima N (2013) Genomic analysis by deep sequencing of the probiotic Lactobacillus brevis KB290 harbouring nine plasmids reveals genomic stability. PLoS One 8:1–10CrossRefGoogle Scholar
  3. 3.
    Grosu-Tudor SS, Zamfir M (2012) Probiotic potential of some lactic acid bacteria isolated from Romanian fermented vegetables. Ann Rom Soc Cell Biol 17:234–239Google Scholar
  4. 4.
    Suskovic J, Kos B, Beganovic J, Pauvnc AL, Habjanic K, Matosic S (2010) Antimicrobial activity—the most important property of probiotic and starter lactic acid bacteria. Food Technol Biotechnol 48:296–307Google Scholar
  5. 5.
    Karna BKL, Emata OC, Barraquio VL (2007) Lactic acid bacteria and probiotic bacteria from fermented and probiotic dairy products. Sci Diliman 19:23–34CrossRefGoogle Scholar
  6. 6.
    Gevers D, Huys G, Swings J (2001) Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett 205:31–36CrossRefGoogle Scholar
  7. 7.
    De Angelis MA, Corsetti A, Tosti N, Rossi J, Corbo MR, Gobbetti M (2001) Characterization of non-starter lactic acid bacteria from Italian ewe cheeses based on phenotypic, genotypic and cell wall protein analysis. Appl Environ Microbiol 65:2011–2020CrossRefGoogle Scholar
  8. 8.
    Mohani D, Nagpal R, Kumar M, Bhardwaj A, Mukesh Y, Jain S, Marotta F, Singh V, Parkash O, Yadav H (2008) Molecular approaches for identification and characterization of lactic acid bacteria. J Dig Dis 9:190–198CrossRefGoogle Scholar
  9. 9.
    Kanmani P, Kumar RS, Yuvaraj N, Paari KA, Pattukumar V, Arul V (2013) Probiotics and its functionality valuable products—a review. Crit Rev Food Sci Nut 53:641–658CrossRefGoogle Scholar
  10. 10.
    Naraida L, Susanti Palupi NS, Hana Bostomi RR, Priscilia D, Nurjanah S (2012) Evaluation of probiotic properties of lactic acid bacteria isolated from breast milk and their potency as starter culture for yogurt fermentation. Int J Food Nut Public Health 5:33–60Google Scholar
  11. 11.
    Jacobsen CN, Nielsen VR, Hayford AE, Moller 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
  12. 12.
    Sengupta R, Altermann E, Anderson RC, McNabb WC, Moughan PJ, Roy NC (2013) The role of cell surface architecture of lactobacilli in host-microbe interactions in the gastrointestinal tract. Mediat Inflamm. doi: 10.1155/2013/237921 Google Scholar
  13. 13.
    Van Tassell M, Miller MJ (2011) Lactobacillus adhesion to mucus. Nutrients 3:613–636CrossRefGoogle Scholar
  14. 14.
    Duary RK, Rajput YS, Batish VK, Grover S (2011) Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells. Indian J Med Res 134:664–671CrossRefGoogle Scholar
  15. 15.
    Boonaert CJP, Rouxhet PG (2000) Surface of lactic acid bacteria: relationships between chemical composition and physicochemical properties. Appl Environ Microbiol 66:2548–2554CrossRefGoogle Scholar
  16. 16.
    Turpin W, Humblot C, Guyot JP (2011) Genetic screening of functional properties of lactic acid bacteria in a fermented pearl millet slurry and in the metagenome of fermented starchy foods. Appl Environ Microbiol 77:8722–8734CrossRefGoogle Scholar
  17. 17.
    Devi SM, Halami PM (2011) Detection and characterization of pediocin PA-1/AcH like bacteriocin producing lactic acid bacteria. Curr Microbiol 62:181–185CrossRefGoogle Scholar
  18. 18.
    Barros RR, Carvalho MDS, Peralta JM, Facklam RR, Teixeira LM (2001) Phenotypic and genotypic characterization of Pediococcus strains isolated from human clinical sources. J Clin Microbiol 39:1241–1246CrossRefGoogle Scholar
  19. 19.
    Osmanagaglu O, Kiran F, Ataoglu H (2010) Evaluation of in vitro probiotic potential of Pediococcus pentosaceus OZF isolated from human breast milk. Probiotics Antimicrob Proteins 2:162–174CrossRefGoogle Scholar
  20. 20.
    Sieladie DV, Zombou NF, Kaktcham PM, Cresci A, Fontech F (2011) Probiotic properties of lactobacilli strains isolated from raw cow milk in the western highlands of Cameroon. Innov Rom Food Biotechnol 19:12–28Google Scholar
  21. 21.
    Padmaja RJ, Halami PM (2013) Molecular characterization and toxicity confirmation of LukM/F′-PV producing Staphylococcus aureus isolated from bovine mastitis samples in Mysore, India. Indian J Microbiol 53:276–282CrossRefGoogle Scholar
  22. 22.
    Nithya V, Halami PM (2013) Evaluation of the probiotic characteristics of Bacillus species isolated from different food sources. Ann Microbiol 63:129–137CrossRefGoogle Scholar
  23. 23.
    Grimoud J, Durand H, Courtin C, Monsan P, Ouarnr F, Theodorou V, Roques C (2010) In vitro screening of probiotic lactic acid bacteria and prebiotic glucooligosaccharides to select effective synbiotics. Anaerobe 16:493–500CrossRefGoogle Scholar
  24. 24.
    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
  25. 25.
    Mora D, Fortina MG, Parini C, Daffonchio D, Manachini PL (2000) Genomic sub-populations within the species Pediococcus acidilactici detected multilocus typing analysis: relationships between pediocin AcH/PA-1 producing and non-producing strains. Microbiology 146:2027–2038Google Scholar
  26. 26.
    Jeyaram K, Romi W, Anand STH, Devi AR, Devi SS (2010) Bacterial species associated with traditional starter cultures used for fermented bamboo shoot production in Manipur state of India. Int J Food Microbiol 143:1–8CrossRefGoogle Scholar
  27. 27.
    Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680CrossRefGoogle Scholar
  28. 28.
    Altschul SF, Maddan TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25:3389–3402CrossRefGoogle Scholar
  29. 29.
    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefGoogle Scholar
  30. 30.
    Albright SC, Winston WL, Zappe C (1999) Data analysis and decision making with microsoft excel. Brooks/Cole Publishing Company, Pacific Grove, CAGoogle Scholar
  31. 31.
    Thumu SCR, Halami PM (2013) Phenotypic expression, molecular characterization and transferability of erythromycin resistance genes in Enterococcus spp. isolated from naturally fermented food. J Appl Microbiol 116:689–699CrossRefGoogle Scholar
  32. 32.
    Adimpong DB, Nielsen DS, Sorensen KI, Derkx PMF, Jespersen L (2012) Genotypic characterization and safety assessment of lactic acid bacteria from indigenous African fermented food products. BMC Microbiol 12:1–12CrossRefGoogle Scholar
  33. 33.
    Svec P, Vancanneyt M, Seman M, Snauwaert C, Lefebvre K, Sedlacek I, Swings J (2005) Evaluation of (GTG)5-PCR for identification of Enterococcus spp. FEMS Microbiol Lett 247:59–63CrossRefGoogle Scholar
  34. 34.
    Tuo Y, Yu H, Ai L, Wu Z, Guo B, Chen W (2013) Aggregation and adhesion properties of 22 Lactobacillus strains. J Dairy Sci 96:4252–4257CrossRefGoogle Scholar
  35. 35.
    Gusils C, González S, Oliver G (1999) Some probiotic properties of chicken lactobacilli. Can J Microbiol 45:981–987CrossRefGoogle Scholar
  36. 36.
    Kos B, Suskovic J, Vukovic S, Simpraga M, Frece J, Matosic S (2003) Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Appl Microbiol 94:981–987CrossRefGoogle Scholar
  37. 37.
    Turpin W, Humblot C, Noordine ML, Thomas M, Guyot JP (2012) Lactobacillaceae and cell adhesion: genomic and functional screening. PLoS One 7:1–14CrossRefGoogle Scholar
  38. 38.
    Mc Auliffe 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
  39. 39.
    Pridmore RD, Berger B, Desiere F, Vilanova D, Barretto C, Pittet AC, Zwahlen MC, Rouvet M, Altermann E, Barrangou R, Mollet B, Mercenier A, Klaenhammer T, Arigoni F, Schell MA (2004) The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc Natl Acad Sci USA 101:2512–2517CrossRefGoogle Scholar
  40. 40.
    Macias-Rodriguez ME, Zogorac M, Ascencio F, Vazquez-Juarez R, Rojas M (2009) Lactobacillus fermentum BCS87 expresses mucus- and mucin-binding proteins on the cell surface. J Appl Microbiol 107:1866–1874CrossRefGoogle Scholar
  41. 41.
    Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, Pavlova N, Karamychev V, Polouchine N, Shakhova V, Grigoriev I, Lou Y, Rohksar D, Lucas S, Huang K, Goodstein DM, Hawkins T, Plengvidhya V, Welker D, Hughes J, Goh Y, Benson A, Baldwin K, Lee JH, Muniz ID, Dosti B, Smeianov V, Wechter W, Barabote R, Lorca G, Altermann E, Barrangou Ganesan B, Xie Y, Rawsthorne H, Tamir D, Parker C, Breidt F, Broadbent J, Hutkins R, O’Sullivan D, Steele J, Unlu G, Saier M, Klaenhammer T, Richardson P, Kozyavkin S, Weimer B, Mills D (2006) Comparative genomics of lactic acid bacteria. Proc Natl Acad Sci USA 103:15611–15616CrossRefGoogle Scholar
  42. 42.
    Ross S, Karner F, Axelsson L, Jonsson H (2000) Lactobacillus mucosae sp. nov., a new species with in vitro mucus-binding activity isolated from pig intestine. Int J Syst Evol Microbiol 50:251–258CrossRefGoogle Scholar
  43. 43.
    Boekhorst J, Helmer Q, Kleerebezem M, Siezen RJ (2006) Comparative analysis of proteins with a mucus-binding domain found exclusively in lactic acid bacteria. Microbiology 152:273–280CrossRefGoogle Scholar
  44. 44.
    Venton D (2012) Highlight: tiny bacterial genome opens a huge mystery: AT mutational bias in Hodgkinia. Genome Biol Evol 4:28–29CrossRefGoogle Scholar
  45. 45.
    Zhou M, Theunissen D, Wels M, Siezen RJ (2010) LAB: secretome: a genome-scale comparative analysis of the predicted extracellular and surface-associated proteins of lactic acid bacteria. BMC Genomics 11:1–16CrossRefGoogle Scholar
  46. 46.
    Buck BL, Altermann E, Svingerud T, Klaenhammer TR (2005) Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM. Appl Environ Microbiol 71:8344–8351CrossRefGoogle Scholar
  47. 47.
    Schillinger U, Yousif NMK, Sesar L, Franz CMAP (2003) Use of group-specific and RAPD-PCR analyses for rapid differentiation of Lactobacillus strains from probiotic yogurts. Curr Microbiol 47:453–456CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sundru Manjulata Devi
    • 1
  • Ann Catherine Archer
    • 1
  • Prakash M. Halami
    • 1
  1. 1.Microbiology and Fermentation Technology DepartmentCSIR- Central Food Technological Research InstituteMysoreIndia

Personalised recommendations