Assessment of the probiotic potential of lactic acid bacteria isolated from kefir grains: evaluation of adhesion and antiproliferative properties in in vitro experimental systems

  • Ioanna Mantzourani
  • Pelagia Chondrou
  • Christos Bontsidis
  • Kyriaki Karolidou
  • Antonia Terpou
  • Athanasios Alexopoulos
  • Eugenia Bezirtzoglou
  • Alex Galanis
  • Stavros PlessasEmail author
Original Article


The main objective of this study was to isolate lactic acid bacteria from kefir grains and investigate their probiotic potential. In this study, 48 bacterial strains were isolated from kefir grains, whereas 39 strains were categorized to the genus Lactobacillus. Evaluation of the probiotic potential of the isolated stains was performed, including resistance to low pH, tolerance to pepsin, pancreatin and bile salts, and antibiotic resistance. In addition, evaluation of adhesion and antiproliferative properties in in vitro experimental systems was also conducted. Strains SP2 and SP5 that displayed the best performance in the conducted in vitro tests were selected for further studies. Firstly, genotypic identification of the two strains was performed by partial 16S rRNA gene sequencing, BLAST analysis, and species-specific multiplex PCR assay. The two strains were confirmed to be Pediococcus pentosaceus SP2 and Lactobacillus paracasei SP5. Then, the adhesion properties of the two strains were examined in vitro. Both strains displayed substantial adherence capacity to HT-29 human colon cancer cells. Moreover, a significant decrease of HT-29 cell growth after treatment with viable P. pentosaceus SP2 or L. paracasei SP5 was recorded. In addition, downregulation of anti-apoptotic genes and over-expression of cell cycle–related genes was recorded by real-time PCR analysis. Treatment with conditioned media of the two strains also caused significant reduction of cancer cell proliferation in a time- and concentration-dependent manner. P. pentosaceus SP2 and L. paracasei SP5 displayed the best probiotic properties that exerted substantial adherence on human colon cancer cells as well as significant anti-proliferative properties.


Probiotics Lactobacillus Kefir Adhesion Anti-proliferation Colon 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

13213_2019_1467_MOESM1_ESM.pptx (507 kb)
Fig S1 Phylogenetic trees based on 16S rRNA gene sequence of Lactobacillus SP2 and Lactobacillus SP5. The partial sequence of the 16S rRNA gene of Lactobacillus SP2 (A) or Lactobacillus SP5 (B) was searched for similarities in GenBank using the BLAST program. The closely related sequences were retrieved, aligned with the Clustal X program and applied for the construction of a phylogenetic tree with the MEGA 6 software, using the neighbor-joining method. The numbers indicate bootstrap values for the branch points. The bar shows the length of branch that represents an amount of 0.005 genetic change. (PPTX 507 kb)
13213_2019_1467_MOESM2_ESM.docx (16 kb)
Supplementary Table S1 (DOCX 15.9 kb)


  1. Argyri A, Zoumpopoulou G, Karatzas KA, Tsakalidou E, Nychas GJ, Panagou E et al (2013) Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests. Food Microbiol 33:282–291. CrossRefGoogle Scholar
  2. Bengoa AA, Llamas MG, Iraporda C, Dueñas MT, Abraham AG, Garrote GL (2018) Impact of growth temperature on exopolysaccharide production and probiotic properties of Lactobacillus paracasei strains isolated from kefir grains. Food Microbiol 69:212–218. CrossRefGoogle Scholar
  3. Bermudez-Brito M, Plaza-Díaz J, Muñoz-Quezada S, Gómez-Llorente C, Gil A (2012) Probiotic mechanisms of action. Ann Nutr Metab 61:160–174. CrossRefGoogle Scholar
  4. Campana R, van Hemert S, Baffone W (2017) Strain-specific probiotic properties of lactic acid bacteria and their interference with human intestinal pathogens invasion. Gut Pathog 9(12).
  5. Chen S, Cao P, Lang F, Wu Z, Pan D, Zeng X et al (2018) Adhesion-related immunomodulatory activity of the screened Lactobacillus plantarum from Sichuan pickle. Curr Microbiol 9.
  6. Cho YJ, Lee HG, Seo KH, Yokoyama W, Kim H (2009) Antiobesity effect of prebiotic polyphenol-rich grape seed flour supplemented with probiotic kefir-derived lactic acid bacteria. J Agric Food Chem 66:12498–12511. CrossRefGoogle Scholar
  7. Chondrou P, Karapetsas A, Kiousi DE, Tsela D, Tiptiri-Kourpeti A, Anestopoulos I et al (2018) Lactobacillus paracasei K5 displays adhesion, anti-proliferative activity and apoptotic effects in human colon cancer cells. Benefic Microbes 0:1–10Google Scholar
  8. Dang F, Jiang Y, Pan R, Zhou Y, Wu S, Wang R et al (2018) Administration of Lactobacillus paracasei ameliorates type 2 diabetes in mice. Food Funct 9:3630–3639. CrossRefGoogle Scholar
  9. Danielsen M, Wind A (2003) Susceptibility of lactobacillus spp. to antimicrobial agents. Int J Food Microbiol 82:1–11. CrossRefGoogle Scholar
  10. Del Carmen S, de LeBlanc AM, Miyoshi A, Rocha CS, Azevedo V, LeBlanc JG (2011) Potential application of probiotics in the prevention and treatment of inflammatory bowel diseases. Ulcers 841651–13.
  11. Deshpande G, Rao S, Patole S, Bulsara M (2010) Updated meta-analysis of probiotics for preventing necrotizing enterocolitis in preterm neonates. Pediatrics 125:921–930. CrossRefGoogle Scholar
  12. Doron S, Snydman DR (2015) Risk and safety of probiotics. Clin Infect Dis 60:S129–S134. CrossRefGoogle Scholar
  13. Drago L, Mattina R, De Vecchi E, Toscano M (2013) Phenotypic and genotypic antibiotic resistance in some probiotics proposed for medical use. Int J Antimicrob Agents 41:396–397. CrossRefGoogle Scholar
  14. EFSA/FEEDAP (2012) Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA J 10:2740–2749Google Scholar
  15. Elisha BG, Courvalin P (1995) Analysis of genes encoding D-alanine: D-alanine ligase-related enzymes in Leuconostoc mesenteroides and lactobacillus spp. Gene 152:79–83. CrossRefGoogle Scholar
  16. Escamilla J, Lane MA, Maitin V (2012) Cell-free supernatants from probiotic Lactobacillus casei and Lactobacillus rhamnosus GG decrease colon cancer cell invasion in vitro. Nutr Cancer 64:871–878. CrossRefGoogle Scholar
  17. 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, Ontario, Canada. . Accessed April 30 and May 1
  18. Garofalo C, Osimani A, Milanović V, Aquilanti L, de Filippis F, Stellato G et al (2015) Bacteria and yeast microbiota in milk kefir grains from different Italian regions. Food Microbiol 49:123–133. CrossRefGoogle Scholar
  19. Granato D, Branco G, Gomes Cruz A, de Assis Fonseca Faria J, Shah N (2010) Probiotic dairy products as functional foods. Compr Rev Food Sci Food Saf 9:455–470.
  20. Howarth GS, Wang H (2013) Role of endogenous microbiota, probiotics and their biological products in human health. Nutrients 5:58–81. CrossRefGoogle Scholar
  21. Klare I, Konstabel C, Werner G, Huys G, Vankerckhoven V, Kahlmeter G et al (2007) Antimicrobial susceptibilities of Lactobacillus, Pediococcus and Lactococcus human isolates and cultures intended for probiotic or nutritional use. J Antimicrob Chemother 59:900–912. CrossRefGoogle Scholar
  22. Klijn N, Weerkamp AH, de Vos WM (1991) Identification of mesophilic lactic acid bacteria by using polymerase chain reaction-amplified variable regions of 16S rRNA and specific DNA probes. Appl Environ Microbiol 57:3390–3393Google Scholar
  23. Koh WY, Utra U, Ahmad R, Rather IA, Park YH (2018) Evaluation of probiotic potential and anti-hyperglycemic properties of a novel Lactobacillus strain isolated from water kefir grains. Food Sci Biotechnol 27:1369–1376. CrossRefGoogle Scholar
  24. Konishi H, Fujiya M, Tanaka H, Ueno N, Moriichi K, Sasajima J et al (2016) Probiotic-derived ferrichrome inhibits colon cancer progression via JNK-mediated apoptosis. Nat Commun 7:12365. CrossRefGoogle Scholar
  25. Mantzourani I, Plessas S, Saxami G, Alexopoulos A, Galanis A, Bezirtzoglou E (2014) Study of kefir grains application in sourdough bread regarding rope spoilage caused by Bacillus spp. Food Chem 143:17–21. CrossRefGoogle Scholar
  26. Marco ML, Tachon S (2013) Environmental factors influencing the efficacy of probiotic bacteria. Curr Opin Biotechnol 24:207–213. CrossRefGoogle Scholar
  27. Orlando A, Refolo MG, Messa C, Amati L, Lavermicocca P, Guerra V (2012) Antiproliferative and proapoptotic effects of viable or heat-killed Lactobacillus paracasei IMPC2.1 and Lactobacillus rhamnosus GG in HGC-27 gastric and DLD-1 colon cell lines. Nutr Cancer 64:1103–1111. CrossRefGoogle Scholar
  28. Orlando A, Linsalata M, Russo F (2016) Antiproliferative effects on colon adenocarcinoma cells induced by co-administration of vitamin K1 and Lactobacillus rhamnosus GG. Int J Oncol 48:2629–2638. CrossRefGoogle Scholar
  29. Ouwehand AC, Tuomola EM, Tölkkö S, Salminen S (2001) Assessment of adhesion properties of novel probiotic strains to human intestinal mucus. Int J Food Microbiol 64:119–126. CrossRefGoogle Scholar
  30. Plessas S, Nouska C, Karapetsas A, Kazakos S, Alexopoulos A, Mantzourani I et al (2017) Isolation, characterization and evaluation of the probiotic potential of a novel Lactobacillus strain isolated from feta-type cheese. Food Chem 226:102–108. CrossRefGoogle Scholar
  31. Raja A, Gajalakshmi P, Mohamed Mahroop Raja M, Mohamed Imran M (2009) Effect of lactobacillus lactis cremoris isolated from kefir against food spoilage bacteria. Am J Food Technol 4:201–209. CrossRefGoogle Scholar
  32. Rivera-Espinoza Y, Gallardo-Navarro Y (2010) Non-dairy probiotic products. Food Microbiol 27:1–11. CrossRefGoogle Scholar
  33. Rosa DD, Dias MMS, Grześkowiak ŁM, Reis SA, Conceição LL, Peluzio MDCG (2017) Milk kefir: nutritional, microbiological and health benefits. Nutr Res Rev 30:82–96. CrossRefGoogle Scholar
  34. Sabokbar N, Khodaiyan F (2015) Characterization of pomegranate juice and whey based novel beverage fermented by kefir grains. J Food Sci Technol 52:3711–3718. Google Scholar
  35. Saxami G, Ypsilantis P, Sidira M, Simopoulos C, Kourkoutas Y, Galanis A (2012) Distinct adhesion of probiotic strain Lactobacillus casei ATCC 393 to rat intestinal mucosa. Anaerobe 18:417–420. CrossRefGoogle Scholar
  36. Saxami G, Karapetsas A, Lamprianidou E, Kotsianidis I, Chlichlia A, Tassou CC et al (2016) Two potential probiotic lactobacillus strains isolated from olive microbiota exhibit adhesion and anti-proliferative effects in cancer cell lines. J Funct Foods 24:461–471. CrossRefGoogle Scholar
  37. Saxami G, Karapetsas A, Chondrou P, Vasiliadis S, Lamprianidou E, Kotsianidis I, Ypsilantis P et al (2017) Potentially probiotic lactobacillus strains with anti-proliferative activity induce cytokine/chemokine production and neutrophil recruitment in mice. Benefic Microbes 8:615–623. CrossRefGoogle Scholar
  38. Sharifi M, Moridnia A, Mortazavi D, Salehi M, Bagheri M, Sheikhi A (2017) Kefιr: a powerful probiotic with anticancer properties. Med Oncol 34(183).
  39. Shi Y, Cui X, Gu S, Yan X, Li R, Xia S et al (2018) Antioxidative and probiotic activities of lactic acid bacteria isolated from traditional artisanal milk cheese from Northeast China. Probiotics Antimicrob Proteins.
  40. Sidira M, Kourkoutas Y, Kanellaki M, Charalampopoulos D (2015) In vitro study on the cell adhesion ability of immobilized lactobacilli on natural supports. Food Res Int 76:532–539. CrossRefGoogle Scholar
  41. Szajewska H, Kołodziej M (2015) Systematic review with meta-analysis: Lactobacillus rhamnosus GG in the prevention of antibiotic-associated diarrhoea in children and adults. Aliment Pharmacol Ther 42:1149–1157. CrossRefGoogle Scholar
  42. Tane S, Chibazakura T (2009) Cyclin a overexpression induces chromosomal double-strand breaks in mammalian cells. Cell Cycle (Georgetown, Tex) 8(23):3900–3903.
  43. Tiptiri-Kourpeti A, Spyridopoulou K, Santarmaki V, Aindelis G, Tompoulidou E, Lamprianidou E et al (2016) Lactobacillus casei exerts anti-proliferative effects accompanied by apoptotic cell death and up-regulation of TRAIL in colon carcinoma cells. PLoS One 11:e0147960. CrossRefGoogle Scholar
  44. Vardjan T, Mohar Lorbeg P, Rogelj I, Čanžek Majhenič A (2013) Characterization and stability of lactobacilli and yeast microbiota in kefir grains. J Dairy Sci 96:2729–2736. CrossRefGoogle Scholar
  45. Ventura M, Canchaya C, Meylan V, Klaenhammer TR, Zink R (2003) Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification. Appl Environ Microbiol 69:6908–6922. CrossRefGoogle Scholar
  46. Wang P, Wu Z, Wu J, Pan D, Zeng X, Cheng K (2016) Effects of salt stress on carbohydrate metabolism of Lactobacillus plantarum ATCC 14917. Curr Microbiol 73:491–497. CrossRefGoogle Scholar
  47. Weese JS, Martin H (2011) Assessment of commercial probiotic bacterial contents and label accuracy. Can Vet J 52:43–46Google Scholar
  48. Xu Q, Gu S, Chen Y, Quan J, Lv L, Chen D et al (2018) Protective effect of Pediococcus pentosaceus LI05 against Clostridium difficile infection in a mouse model. Front Microbiol 9(2396).
  49. Zhao X, Higashikawa F, Noda M, Kawamura Y, Matoba Y, Kumagai T et al (2012) The obesity and fatty liver are reduced by plant-derived Pediococcus pentosaceus LP28 in high fat diet-induced obese mice. PLoS One 7:e30696. CrossRefGoogle Scholar
  50. Zheng Y, Lu Y, Wang J, Yang L, Pan C, Huang Y (2013) Probiotic properties of lactobacillus strains isolated from Tibetan kefir grains. PLoS One (7):e69868.
  51. Zmora N, Zilberman-Schapira G, Suez J, Mor U, Dori-Bachash M, Bashiardes S et al (2018) Personalized gut mucosal colonization resistance to empiric probiotics is associated with unique host and microbiome features. Cell 174:1388–1405. CrossRefGoogle Scholar

Copyright information

© Università degli studi di Milano 2019

Authors and Affiliations

  • Ioanna Mantzourani
    • 1
  • Pelagia Chondrou
    • 2
  • Christos Bontsidis
    • 1
  • Kyriaki Karolidou
    • 2
  • Antonia Terpou
    • 3
  • Athanasios Alexopoulos
    • 1
  • Eugenia Bezirtzoglou
    • 1
  • Alex Galanis
    • 2
  • Stavros Plessas
    • 1
    Email author
  1. 1.Laboratory of Microbiology, Biotechnology and Hygiene, Faculty of Agricultural DevelopmentDemocritus University of ThraceOrestiadaGreece
  2. 2.Department of Molecular Biology and GeneticsDemocritus University of ThraceAlexandroupolisGreece
  3. 3.Food Biotechnology Group, Section of Analytical Environmental and Applied Chemistry, Department of ChemistryUniversity of PatrasPatrasGreece

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