Selective detection, isolation and enumeration of Bifidobacterium animalis subsp. lactis BB-12 from Iranian commercial probiotic yoghurts

Document Type : Research(Original) Article

Authors

1 Parmaceutical Sciences Research Center, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz Iran.

2 Parmaceutical Sciences Research Center, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz Iran

3 Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.

4 1. Parmaceutical Sciences Research Center, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz Iran. 2. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran 3. Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.

Abstract

Fermented dairy products are one of the most prevalent vehicles for the delivering of probiotic bacteria to the consumer. A minimal concentration of 106 CFU/g(mL) of a product is required to exert probiotic effects. In this study, we first evaluated the selectivity of WCM 50 and WCM 100 (Wilkins-Chalgren agar supplemented with 50 mg/L and 100 mg/L mupirocin), as well as mMRS (De Man Rogosa Sharpe agar supplemented with 0.1 mg/L Clindamycin plus 10 mg/L Ciprofloxacin) media, using pure cultures of prevalent Bifidobacterial and Lactobacilli probiotic strains. For each strain, the selectivity and cell recovery rate on each medium was compared statistically with that obtained on the non-selective media. Afterwards, one tuf gene-based specific primer set was designed for the detection of Bifidbacterium animalis subsp. lactis BB-12 in commercial probiotic yoghurts. The specificity of designed primer set was evaluated by operation of PCR reactions with extracted DNAs from reference strains and commercial probiotic yoghurts. Finally, strain BB-12 was detected, enumerated and confirmed through tuf gene-based PCR, selective plate count (using WCM 100 medium) and fructose-6-phophate-phosphoketolase assay (F6PPK) respectively, during shelf life and after expiry date of commercial probiotic yoghurts. The results showed that WCM 100 was completely selective for Bifidobacteria, with the recovery about 100%. However, mMRS was not completely selective for Lactobacilli. The PCR assays confirmed the specificity of tuf gene-based primer set for strain BB-12. Although the counts of strain BB-12 had significant decrease during shelf-life, but these counts didn’t fell below CODEX standard (106 CFU/mL), until expiry date of products.

Keywords

References

  1. Oyetayo V, Oyetayo F. Review-Potential of probiotics as biotherapeutic agents targeting the innate immune system. Afr J Biotechnol. 2005;4(2):123-127.
  2. Ashraf R, Shah NP. Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt—A review. Int J Food Microbiol. 2011;149(3):194-208.
  3. Fotiadis CI, Stoidis CN, Spyropoulos BG, Zografos ED. Role of probiotics, prebiotics and synbiotics in chemoprevention for colorectal cancer. World J Gastroenterol. 2008;14(42):6453-7.
  4. Kadooka Y, Ogawa A, Ikuyama K, Sato M. The probiotic Lactobacillus gasseri SBT2055 inhibits enlargement of visceral adipocytes and upregulation of serum soluble adhesion molecule (sICAM-1) in rats. Int Dairy J. 2011;21(9):623-7.
  5. Quinto EJ, Jimenez P, Caro I, Tejero J, Mateo J, Girbes T. Probiotic Lactic Acid Bacteria: A Review. Food and Nutrition Sciences. 2014;5(18):1765.
  6. Biavati B, Vescovo M, Torriani S, Bottazzi V. Bifidobacteria: history, ecology, physiology and applications. Ann Microbiol. 2000;50(2):117-32.
  7. Darukaradhya J, Phillips M, Kailasapathy K. Selective enumeration of Lactobacillus acidophilus, Bifidobacterium spp., starter lactic acid bacteria and non-starter lactic acid bacteria from Cheddar cheese. Int Dairy J. 2006;16(5):439-45.
  8. Tabasco R, Paarup T, Janer C, Pelaez C, Requena T. Selective enumeration and identification of mixed cultures of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. acidophilus, L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk. Int Dairy J. 2007;17(9):1107-14.
  9. Talwalkar A, Kailasapathy K. Comparison of selective and differential media for the accurate enumeration of strains of Lactobacillus acidophilus, Bifidobacterium spp. and Lactobacillus casei complex from commercial yoghurts. Int Dairy J. 2004;14(2):143-9.
  10. Van de Casteele S, Vanheuverzwijn T, Ruyssen T, Van Assche P, Swings J, Huys G. Evaluation of culture media for selective enumeration of probiotic strains of lactobacilli and bifidobacteria in combination with yoghurt or cheese starters. Int Dairy J. 2006;16(12):1470-6.
  11. Kramer M, Obermajer N, Matijašić BB, Rogelj I, Kmetec V. Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry. Appl Microbiol Biotechnol. 2009;84(6):1137-47.
  12. Garcia-Cayuela T, Tabasco R, Pelaez C, Requena T. Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR. Int Dairy J. 2009;19(6):405-9.
  13. Roy D. Media for the isolation and enumeration of bifidobacteria in dairy products. Int J Food Microbio. 2001;69(3):167-82.
  14. Saccaro DM, Hirota CY, Tamime AY, De Oliveira MN. Evaluation of different selective media for enumeration of probiotic micro-organisms in combination with yogurt starter cultures in fermented milk. Afr J Microbiol Res. 2012;6:2239-45.
  15. Yeung P, Sanders M, Kitts CL, Cano R, Tong PS. Species-specific identification of commercial probiotic strains. J Dairy Sci. 2002;85(5):1039-51.
  16. Raeisi SN, Ouoba LII, Farahmand N, Sutherland J, Ghoddusi HB. Variation, viability and validity of bifidobacteria in fermented milk products. Food control. 2013;34(2):691-7.
  17. Ventura M, Reniero R, Zink R. Specific identification and targeted characterization ofBifidobacterium lactis from different environmental isolates by a combined multiplex-PCR approach. Appl Environ Microbiol. 2001;67(6):2760-5.
  18. Ward P, Roy D. Review of molecular methods for identification, characterization and detection of bifidobacteria. Le Lait. 2005;85(1-2):23-32.
  19. Masco L, Vanhoutte T, Temmerman R, Swings J, Huys G. Evaluation of real-time PCR targeting the 16S rRNA and recA genes for the enumeration of bifidobacteria in probiotic products. Int J Food Microbiol. 2007;113(3):351-7.
  20. Gueimonde M, Debor L, Tölkkö S, Jokisalo E, Salminen S. Quantitative assessment of faecal bifidobacterial populations by real‐time PCR using lanthanide probes. J Appl Microbiol. 2007;102(4):1116-22.
  21. Matsuda K, Tsuji H, Asahara T, Matsumoto K, Takada T, Nomoto K. Establishment of an analytical system for the human fecal microbiota, based on reverse transcription-quantitative PCR targeting of multicopy rRNA molecules. Appl Environ Microbiol. 2009;75(7):1961-9.
  22. Matsuki T, Watanabe K, Tanaka R. Genus-and Species-specific PCR Primers for the Detection and Identification of Bifidobacteria. Curr Issues Intest Microbiol. 2003;4:61-69.
  23. Mullie C, Odou M-F, Singer E, Romond M-B, Izard D. Multiplex PCR using 16S rRNA gene-targeted primers for the identification of bifidobacteria from human origin. FEMS Microbiol Lett. 2003;222(1):129-36.
  24. Youn S, Seo J, Ji G. Evaluation of the PCR method for identification of Bifidobacterium species. Lett Appl Microbiol. 2008;46(1):7-13.
  25. Turroni F, Foroni E, Pizzetti P, Giubellini V, Ribbera A, Merusi P, et al. Exploring the diversity of the bifidobacterial population in the human intestinal tract. Appl Environ Microbiol. 2009;75(6):1534-45.
  26. Mathys S, Lacroix C, Mini R, Meile L. PCR and real-time PCR primers developed for detection and identification of Bifidobacterium thermophilum in faeces. BMC Microbiol. 2008;8(1):1.
  27. Matsuki T, Watanabe K, Fujimoto J, Kado Y, Takada T, Matsumoto K, et al. Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria. Appl Environ Microbiol. 2004;70(1):167-73.
  28. Requena T, Burton J, Matsuki T, Munro K, Simon MA, Tanaka R, et al. Identification, detection, and enumeration of human Bifidobacterium species by PCR targeting the transaldolase gene. Appl Environ Microbiol. 2002;68(5):2420-7.
  29. Karimi R, Mortazavian AM, Amiri-Rigi A. Selective enumeration of probiotic microorganisms in cheese. Food Microbiol. 2012;29(1):1-9.
  30. Ferraris L, Aires J, Waligora-Dupriet A-J, Butel M-J. New selective medium for selection of bifidobacteria from human feces. Anaerobe. 2010;16(4):469-71.
  31. Rada V, Petr J. A new selective medium for the isolation of glucose non-fermenting bifidobacteria from hen caeca. J Microbiol Methods. 2000;43(2):127-32.
  32. Rada V, Sirotek K, Petr J. Evaluation of selective media for bifidobacteria in poultry and rabbit caecal samples. J Vet Med B. 1999;46(6):369-73.
  33. Rada V. Detection of Bifidobacteriumspecies by enzymatic methods and antimicrobial susceptibility testing. Biotechnol Tech. 1997;11(12):909-12.
  34. Simpson P, Fitzgerald G, Stanton C, Ross R. The evaluation of a mupirocin-based selective medium for the enumeration of bifidobacteria from probiotic animal feed. J Microbiol Methods. 2004;57(1):9-16.
  35. Serafini F, Bottacini F, Viappiani A, Baruffini E, Turroni F, Foroni E, et al. Insights into physiological and genetic mupirocin susceptibility in bifidobacteria. Appl Environ Microbiol. 2011;77(9):3141-6.
  36. ISO. Milk products-Enumeration of presumptive Lactobacillus acidophilus on a selective medium-Colony-count technique at 37 0C. 2006.
  37. Thitaram S, Siragusa G, Hinton A. Bifidobacterium‐selective isolation and enumeration from chicken caeca by a modified oligosaccharide antibiotic‐selective agar medium. Lett Appl Microbiol. 2005;41(4):355-60.
  38. Orban J, Patterson J. Modification of the phosphoketolase assay for rapid identification of bifidobacteria. J Microbiol Methods. 2000;40(3):221-4.
  39. Ng S, Hart A, Kamm M, Stagg A, Knight S. Mechanisms of action of probiotics: recent advances. Inflamm Bowel Dis. 2009;15(2):300-10.
  40. Corr SC, Hill C, Gahan CG. Understanding the mechanisms by which probiotics inhibit gastrointestinal pathogens. Adv Food Nutr Res. 2009;56:1-15.
  41. Gueimonde M, Tölkkö S, Korpimäki T, Salminen S. New real-time quantitative PCR procedure for quantification of bifidobacteria in human fecal samples. Appl Environ Microbiol. 2004;70(7):4165-9.
  42. Fittipaldi M, Rodriguez NJP, Codony F, Adrados B, Peñuela GA, Morato J. Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR. J Virol Methods. 2010;168(1):228-32.
  43. Nocker A, Camper AK. Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques. FEMS Microbiol Lett. 2009;291(2):137-42.
  44. Fachin L, Moryia J, Gândara ALN, Viotto WH. Evaluation of culture media for counts of Bifidobacterium animalis subsp. lactis Bb 12 in yoghurt after refrigerated storage. Braz J Microbiol. 2008;39(2):357-61.
  45. Pechar R, Rada V, Parafati L, Musilova S, Bunesova V, Vlkova E, et al. Mupirocin-mucin agar for selective enumeration of Bifidobacterium bifidum. Int J Food Microbiol. 2014;191:32-5.
  46. Grand M, Küffer M, Baumgartner A. Quantitative analysis and molecular identification of bifidobacteria strains in probiotic milk products. Eur Food Res Technol. 2003;217(1):90-2.
  47. Solano-Aguilar G, Dawson H, Restrepo M, Andrews K, Vinyard B, Urban JF. Detection of Bifidobacterium animalis subsp. lactis (Bb12) in the intestine after feeding of sows and their piglets. Appl Environ Microbiol. 2008;74(20):6338-47.
  48. Matsuki T, Watanabe K, Tanaka R, Oyaizu H. Rapid identification of human intestinal bifidobacteria by 16S rRNA-targeted species-and group-specific primers. FEMS Microbiol Lett. 1998;167(2):113-21.
Trends in Pharmaceutical Sciences
Volume 2, Issue 4 - Serial Number 4
10
December 2016
Pages 265-276

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