Journal of Kerman University of Medical Sciences

16S-Amplified Ribosomal DNA Restriction Analysis Assay for Discriminating Potentially Probiotic Lactobacillus Species Isolated from Traditional Dairy Products

Document Type : Original Article

Authors

Soudeh Ghafouri-Fard 1
Mohammad Amin Hejazi 2
Davoud Afshar 3
Abolfazl Barzegari 4
Solat Eslami 5

1 Associate Professor of Medical Genetics, Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Associate Professor of Food Biotechnology, Department of Food Biotechnology, Branch for Northwest & West region, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education and Extension Organization (AREEO), Tabriz, Iran

3 Assistant Professor of Medical Bacteriology, Department of Microbiology and Virology, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran

4 Ph.D. Candidate of Medical Biotechnology, Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran

5 Assistant Professor of Medical Biotechnology, Dietary Supplements and Probiotic Research Center, Alborz University of Medical Sciences, Karaj, Iran

Abstract
Background: Traditional dairy products are the main sources for probiotic bacteria. This study aimed to isolate and characterize the potentially probiotic Lactobacillus strains isolated from traditional dairy products in Iran.
Methods: Microbial population of each dairy product was enriched and screened for acid- and bile- resistant strains. The isolates were primarily characterized by conventional phenotypic and biochemical tests. The species were finally identified using restriction analysis and 16S rDNA sequencing. In addition, the antibacterial activity of the four most common species was evaluated against seven gastrointestinal pathogens.
Results: From a total of nine yogurt and cheese samples, 17 acid-resistant Lactobacillus strains were isolated (survival>50% in PBS buffer, pH 2.5 for 3 h). Eight of them also showed high tolerance to bile. Also, the superiority of molecular techniques over phenotypic tests for effective identification of species was observed. Biochemical tests were useful in combination with 16S-amplified ribosomal DNA restriction analysis (ARDRA) technique to discriminate the isolates at species level.
Conclusion: 16S–ARDRA in combination with biochemical tests is suggested as a potentially useful method for accurate identification of Lactobacillus species where the 16S-rDNA sequencing could not discriminate between two closely related species. Considering the acid and bile resistance capacity of isolated strains, traditional dairy products may be used as important sources of probiotic strains.

Keywords

Dairy products
probiotics
Lactobacillus
Molecular techniques
16S-ARDRA
  1. Devi SM, Aishwarya S, Halami PM. Discrimination and divergence among Lactobacillus plantarum-group (LPG) isolates with reference to their probiotic functionalities from vegetable origin. Systematic and Applied Microbiology 2016;39(8):562-70.
  2. Zoumpopoulou G, Tzouvanou A, Mavrogonatou E, Alexandraki V, Georgalaki M, Anastasiou R, et al. Probiotic features of lactic acid bacteria isolated from a diverse pool of traditional greek dairy products regarding specific strain-host interactions. Probiotics Antimicrob Proteins 2018;10(2):313-22.
  3. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 2014;11(8):506-14.
  4. Jampaphaeng K, Cocolin L, Maneerat S. Selection and evaluation of functional characteristics of autochthonous lactic acid bacteria isolated from traditional fermented stinky bean (Sataw-Dong). Annals of Microbiology 2017;67(1):25-36.
  5. Barzegari A, Eslami S, Ghabeli E, Omidi Y. Imposition of encapsulated non-indigenous probiotics into intestine may disturb human core microbiome. Front Microbiol 2014; 5: 393.
  6. Rajoka MS, Hayat HF, Sarwar S, Mehwish HM, Ahmad F, Hussain N, et al. Isolation and evaluation of probiotic potential of lactic acid bacteria isolated from poultry intestine. Microbiology 2018;87(1):116-26.
  7. Dutta D, Banerjee S, Mukherjee A, Ghosh K. Selection and probiotic characterization of exoenzyme-producing bacteria isolated from the gut of Catla catla (actinopterygii: cypriniformes: cyprinidae). Acta Ichthyologica et Piscatoria 2015; 45(4): 373-84.
  8. van Loveren H, Sanz Y, Salminen S. Health claims in Europe: probiotics and prebiotics as case examples. Annu Rev Food Sci Technol 2012;3:247-61.
  9. Doron S, Snydman DR. Risk and safety of probiotics. Clin Infect Dis 2015;60 Suppl 2:S129-34.
  10. Jose NM, Bunt CR, Hussain MA. Implications of antibiotic resistance in probiotics. Food Reviews International 2015;31(1):52-62.
  11. Zhang F, Jiang M, Wan C, Chen X, Chen X, Tao X, et al. Screening probiotic strains for safety: Evaluation of virulence and antimicrobial susceptibility of enterococci from healthy Chinese infants. J Dairy Sci 2016;99(6):4282-90.
  12. Eslami S, Hadjati J, Motevaseli E, Mirzaei R, Farashi Bonab S, Ansaripour B, et al. Lactobacillus crispatus strain SJ-3C-US induces human dendritic cells (DCs) maturation and confers an anti-inflammatory phenotype to DCs. APMIS 2016;124(8):697-710.
  13. Motevaseli E, Khorramizadeh MR, Hadjati J, Bonab SF, Eslami S, Ghafouri-Fard S. Investigation of antitumor effects of Lactobacillus crispatus in experimental model of breast cancer in BALB/c mice. Immunotherapy 2018;10(2):119-29.
  14. Eslami S, Barzgari Z, Saliani N, Saeedi N, Barzegari A. Annual fasting; the early calories restriction for cancer prevention. Bioimpacts 2012; 2(4): 213–5.
  15. Ouwehand AC. A review of dose-responses of probiotics in human studies. Benef Microbes 2017;8(2):143-151.
  16. Di Lena M, Quero GM, Santovito E, Verran J, De Angelis M, Fusco V. A selective medium for isolation and accurate enumeration of Lactobacillus casei-group members in probiotic milks and dairy products. International Dairy Journal 2015;47:27-36.
  17. Ruiz P, Seseña S, Palop ML. A comparative study of different PCR-based DNA fingerprinting techniques for typing of lactic acid bacteria. European Food Research and Technology 2014;239(1):87-98.
  18. Donelli G, Vuotto C, Mastromarino P. Phenotyping and genotyping are both essential to identify and classify a probiotic microorganism. Microb Ecol Health Dis 2013;24.
  19. Bagheripoor-Fallah N, Mortazavian A, Hosseini H, Khoshgozaran-Abras S, Rad AH. Comparison of molecular techniques with other methods for identification and enumeration of probiotics in fermented milk products. Crit Rev Food Sci Nutr 2015;55(3):396-413.
  20. Erkkila S, Petaja E. Screening of commercial meat starter cultures at low pH and in the presence of bile salts for potential probiotic use. Meat Sci 2000;55(3):297-300.
  21. Gilliland SE, Staley TE, Bush LJ. Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct. J Dairy Sci 1984;67(12):3045-51.
  22. Garrity GM, Bell JA, Lilburn TG. Taxonomic Outline of the Prokaryotes. Bergey’s Manual of Systematic Bacteriology. 2th ed. New York: Springer; 2004.
  23. Haghshenas B, Haghshenas M, Nami Y, Yari Khosroushahi A, Abdullah N, Barzegari A, et al. Probiotic assessment of lactobacillus plantarum 15HN and enterococcus mundtii 50H isolated from traditional dairies microbiota. Adv Pharm Bull 2016;6(1):37-47.
  24. Bhunia AK, Johnson MC, Ray B. Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici. J Appl Bacteriol 1988;65(4):261-8.
  25. Abedin-Do A, Taherian-Esfahani Z, Ghafouri-Fard S, Ghafouri-Fard S, Motevaseli E. Immunomodulatory effects of Lactobacillus strains: emphasis on their effects on cancer cells. Immunotherapy 2015;7(12):1307-29.
  26. Sharafi H, Derakhshan V, Paknejad M, Alidoust L, Tohidi A, Pornour M, et al. Lactobacillus crustorum KH: novel prospective probiotic strain isolated from Iranian traditional dairy products. Appl Biochem Biotechnol 2015;175(4):2178-94.
  27. Noriega L, Gueimonde M, Sanchez B, Margolles A, de los Reyes-Gavilan CG. Effect of the adaptation to high bile salts concentrations on glycosidic activity, survival at low PH and cross-resistance to bile salts in Bifidobacterium. Int J Food Microbiol 2004;94(1):79-86.
  28. Ozturk M, Meterelliyoz M. Practical identification of human originated Lactobacillus species by amplified ribosomal DNA restriction analysis (ARDRA) for probiotic use. Mol Biol Rep 2015;42(8):1323-32.
  29. Calix-Lara TF, Rajendran M, Talcott ST, Smith SB, Miller RK, Castillo A, et al. Inhibition of Escherichia coli O157:H7 and Salmonella enterica on spinach and identification of antimicrobial substances produced by a commercial Lactic Acid Bacteria food safety intervention. Food Microbiol 2014;38:192-200.
  30. Siroli L, Patrignani F, Serrazanetti DI, Tabanelli G, Montanari C, Gardini F, et al. Lactic acid bacteria and natural antimicrobials to improve the safety and shelf-life of minimally processed sliced apples and lamb's lettuce. Food Microbiol 2015;47:74-84.
  31. Nespolo CR, Brandelli A. Production of bacteriocin-like substances by lactic acid bacteria isolated from regional ovine cheese. Braz J Microbiol 2010;41(4):1009-18.
Journal of Kerman University of Medical Sciences
Volume 25, Issue 2
March and April 2018
Pages 175-186

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