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Journal of Applied Phycology

, Volume 23, Issue 4, pp 777–787 | Cite as

An investigation of Clostridium species present in nutraceutical preparations of Arthrospira platensis (Spirulina) for human consumption

  • Dirk T. Hoekstra
  • Heinrich Volschenk
  • Michelle Collins
  • Lynn D. McMaster
Article

Abstract

The presence of the anaerobic spore former Clostridium in Arthrospira platensis destined for human consumption is generally not assessed during quality assurance procedures. As this nutraceutical is administered as complementary medicine to the immunocompromised, this study aimed to investigate the presence of these potential pathogens. Anaerobic counts performed on tablets from a single manufacturer indicated an excess of 105 CFU/endospores g−1 tablet for three different A. platensis batches. Tests for coliforms for use as “indicators” of pathogens in the tablets were negative. Using classic culture techniques, five species of Clostridium were isolated. Subsequent use of PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting of tablets showed a divergent microbial population, with a predominance of anaerobic endospore formers, including Clostridium. Sequencing of a 1.5 kb 16S rDNA clone library and phylogenetic analyses of prominent operational taxonomic units confirmed the presence of an additional five Clostridium spp. and other genera in the tablets. A composite molecular ladder, using 16S rRNA DGGE amplicons of 17 representative bacterial species was constructed to assist in identifying anaerobes present in tablets sourced from three different A. platensis manufacturers. Results indicated that commercial A. platensis preparations were contaminated with potentially hazardous clostridia and other anaerobic species. Results suggest that certain commercial A. platensis preparations require stringent microbial quality assurance measures to ensure safe use as a nutraceutical for the immunocompromised and the general public.

Keywords

Arthrospira (Spirulina) platensis Clostridium Anaerobes Health supplements Immunocompromised Nutraceuticals 

Notes

Acknowledgements

Financial assistance from the National Research Foundation and the Cape Peninsula University of Technology Research Fund is gratefully acknowledged. Thanks are due to Monique Fredericks and Diane James for their input.

References

  1. Allen SD, Emery CL, Lyerly DM (2003) Clostridium. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH (eds) Manual of Clinical Microbiology. ASM Press, Washington, pp 835–856Google Scholar
  2. Altschul SF, Madden 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. Nucleic Acids Res 25:3389–3444PubMedCrossRefGoogle Scholar
  3. Andrade MR, Costa JAV (2007) Mixotrophic cultivation of microalga Spirulina platensis using molasses as organic substrate. Aquaculture 264:130–134CrossRefGoogle Scholar
  4. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1995) Current Protocols in Molecular Biology. Wiley, ChichesterGoogle Scholar
  5. Ayehunie S, Belay A, Baba TW, Ruprecht RM (1998) Inhibition of HIV-1 replication by an aqueous extract of Spirulina platensis (Arthrospira platensis). J Acq Immun Def Synd 18:7–12Google Scholar
  6. Baicus C, Baicus A (2007) Spirulina did not ameliorate idiopathic chronic fatigue in four N-of-1 randomised controlled trials. Phytother Res 21:570–573PubMedCrossRefGoogle Scholar
  7. Barloy F, Lecadet M, Delecluse M (1998) Cloning and sequencing of three new putative toxin genes from Clostridium bifermentans CH18. Gene 211:293–299PubMedCrossRefGoogle Scholar
  8. Belay A (1997) Mass culture of Spirulina outdoors-the earthrise farms experience. In: Vonshak A (ed) Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology. Taylor and Francis, London, pp 131–158Google Scholar
  9. Belay A (2007) Spirulina (Arthrospira): production and quality assurance. In: Gershwin ME, Belay A (eds) Spirulina in Human Nutrition and Health. CRC, Boca Raton, pp 1–27CrossRefGoogle Scholar
  10. Belay A, Ota Y, Miyakawa K, Shimamatsu H (1993) Current knowledge on potential health benefits of Spirulina. J Appl Phycol 5:235–241CrossRefGoogle Scholar
  11. Blumenthal R, Habig WH (1984) Mechanisim of tetanolysin-induced membrane damage: studies with black lipid membranes. J Bacteriol 157:321–323PubMedGoogle Scholar
  12. Bruggeman H, Gottschalk G (2004) Insights in metabolism and toxin production from the complete genome sequence of Clostridium tetani. Anaerobe 10:53–68CrossRefGoogle Scholar
  13. Chamorro-Cevallos G, Garduno-Siciliano L, Barron BL, Madrigal-Bujaidar E, Cruz-Vega DE, Pages N (2008) Chemoprotective effect of Spirulina (Arthrospira) against cyclophosphamide-induced mutagenicity in mice. Food Chem Toxicol 46:567–574PubMedCrossRefGoogle Scholar
  14. Cohen Z (1997) The chemicals of Spirulina. In: Vonshak A (ed) Spirulina platensis (Arthrospira) Physiology, Cell-biology and Biotechnology. Taylor and Francis, London, pp 175–204Google Scholar
  15. Collins MD, Lawson PA, Willem A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P (1994) The phylogeny of the genus Clostridium: proposal of five genera and eleven new species combinations. Int J Syst Evol Microbiol 44:812–826Google Scholar
  16. Costa JA, Colla LM, Filho PD (2003) Spirulina platensis growth in open raceway ponds using fresh water supplemented with carbon, nitrogen and metal ions. Z Naturforsch 58:76–80Google Scholar
  17. Goksan T, Zekeriyaoglu A (2006) The growth of Spirulina platensis in different culture systems under greenhouse conditions. Turk J Biol 31:47–52Google Scholar
  18. Görs M, Schumann R, Hepperle D, Karsten U (2010) Quality analysis of commercial Chlorella products used as dietary supplement in human nutrition. J Appl Phycol 22:265–276CrossRefGoogle Scholar
  19. Grobbelaar JU (2009) From laboratory to commercial production: a case study of a Spirulina (Arthrospira) facility in Musina, South Africa. J Appl Phycol 21:523–527CrossRefGoogle Scholar
  20. Grzanna R, Polotsky A, Phan PV, Pugh N, Pasco D, Frondoza CG (2006) Immolina, a high-molecular-weight polysaccharide fraction of Spirulina, enhances cytokine expression in human monocytic THP-1 cells. J Altern Complement Med 12:429–435PubMedCrossRefGoogle Scholar
  21. Guis S, Matteia J-P, Cozzoneb P, Bendahan D (2005) Pathophysiology and clinical presentations of rhabdomyolysis. Joint Bone Spine 72:382–391PubMedCrossRefGoogle Scholar
  22. Hang’ombe BM, Kohda T, Mukamoto M, Kozaki S (2006) Purification and sensitivity of Clostridium chauvoei hemolysin to various erythrocytes. Comp Immunol Microbiol 29:263–268CrossRefGoogle Scholar
  23. Harley JP, Prescott LM (1993) Laboratory Excercises in Microbiology. McGraw Hill, New YorkGoogle Scholar
  24. Hayashi K, Hayashi T, Kojima I (1996) A natural sulfated polysaccharide, calcium spirulan, isolated from Spirulina platensis; in vitro and ex vivo evaluation of anti-herpes simplex virus and anti-human immunodeficiency virus activities. J Acq Immun Def Synd 12:1463–1471Google Scholar
  25. Hirahashi T, Matsumoto M, Hazeki K, Sacki Y, Ui M, Seya T (2002) Activation of the human innate immune system by Spirulina; augmentation of interferon production and NK cytotoxicity by oral administration of hot water extract on Spirulina platensis. Int Immunopharmacol 2:423–434PubMedCrossRefGoogle Scholar
  26. Hudnell HK (2008) Cyanobacterial Harmful Algal Blooms. State of the Science and Research Needs. Springer LLC, New York, p 893CrossRefGoogle Scholar
  27. Iwasa M, Yamamoto M, Tanaka Y, Kaito M, Adachi Y (2002) Spirulina-associated hepatotoxicity. Am J Gastroenterol 97:3212–3213PubMedCrossRefGoogle Scholar
  28. Jiminez C, Cossio BR, Labella D, Niell FX (2007) The feasibility of industrial production of Spirulina (Arthrospira) in Southern Spain. Aquaculture 217:179–190CrossRefGoogle Scholar
  29. Keijer J, Bunscotena A, Palou A, Franssen-Van Hal N (2005) Beta-carotene and the application of transcriptomics in risk-benefit evaluation of natural dietary components. Biochim Biophys Acta 1740:139–146PubMedGoogle Scholar
  30. Kim C-J, Jung Y-H, Ko S-R, Kim H-I, Park Y-H, Oh H-M (2007) Raceway cultivation of Spirulina platensis using underground water. J Ind Micobiol Biotech 17:853–857Google Scholar
  31. Liu C, Yang Y, Gange SJ, Weber K, Sharp GB, Wilson TE, Levine A, Robison E, Goparaju L, Ganhdi M, Merenstein D (2009) Disclosure of complementary and alternative medicine use to health care providers among HIV-infected women. AIDS Patient Care STDs 23:965–971PubMedCrossRefGoogle Scholar
  32. Lodi A, Soletto D, Solisio C, Converti A (2008) Chromium (III) removal by Spirulina platensis biomass. Chem Eng J 136:151–155CrossRefGoogle Scholar
  33. Lu HK, Hsieh CC, Hsu JJ, Yang YK, Chou HN (2006) Preventative effects of Spirulina platensis on skeletal muscle damage under exercise-induced oxidative stress. Eur J Appl Physiol 98:220–226PubMedCrossRefGoogle Scholar
  34. Majdoub H, Mansour MB, Chaubet F, Roudesli MS, Maaroufi RM (2009) Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis. Biochim Biophys Acta 1790:1377–1381PubMedGoogle Scholar
  35. Maradonna HEM (2009) IMSAMS expansion report. Available at: http://www.imsam.org/. Accessed 30 July 2009.
  36. Martin-Laurent F, Philippot L, Hallet S, Chaussod R, Germon JC, Soulas G, Catroux G (2001) DNA extraction from soils: old bias for new microbial diversity analysis methods. Appl Environ Microbiol 67:2354–2359PubMedCrossRefGoogle Scholar
  37. Matsudo MC, Berreza RP, Sato S, Perego P, Convert A, Carvalho JCM (2009) Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as the nitrogen source. Biochem Eng J 43:52–57CrossRefGoogle Scholar
  38. Mazokopakis EE, Karefilakis CM, Tsartsalis AN, Milkas AN, Ganotakis ES (2008) Acute rhabdomyolysis caused by Spirulina (Arthrospira platensis). Phytomedicine 15:525–527PubMedCrossRefGoogle Scholar
  39. Mitsui K, Mitsui N, Hase J (1982) High molecular weight hemolysin of Clostridium tetani. Infect Immun 35:1086–1090PubMedGoogle Scholar
  40. Oguma K, Yamaguchi T, Sudou K, Yokosawa N, Fujikawa Y (1986) Biochemical classification of Clostridium botulinum type C and D strains and their nontoxigenic derivatives. Appl Environ Microbiol 51:256–260PubMedGoogle Scholar
  41. Rambaut A (2004) Se-Al, a manual sequence alignment editor, v. 2.0 a11. Department of Zoology, University of Oxford, OxfordGoogle Scholar
  42. Rangsayator N, Upatham ES, Kruetrachue M, Pokethitiyook P, Lanza GR (2002) Phytoremediation potential of Spirulina (Arthrospira) platensis: biosorption and toxicity studies of cadmium. Environ Pollut 119:45–53PubMedCrossRefGoogle Scholar
  43. Rechter S, Konig T, Auerochs S, Thulke S, Walter H, Dornenburg H, Walter C, Marshall M (2006) Antiviral activity of Arthrospira-derived spirulan-like substances. Antivir Res 72:197–206PubMedCrossRefGoogle Scholar
  44. Rellán S, Osswald J, Saker M, Gago-Martinez A, Vasconcelos V (2009) First detection of anatoxin-a in human and animal dietary supplements containing cyanobacteria. Food Chem Toxicol 47:2189–2195PubMedCrossRefGoogle Scholar
  45. Rose PD, Maart BA, Dunn KM, Rowswell RA, Britz P (1996) High algal rate oxidation ponding for the treatment of tannery effluents. Water Sci Technol 33:219–227Google Scholar
  46. Roy KR, Arunasee KM, Reddy NP, Dheeraj B, Reddy VG, Redanna P (2007) Alteration of mitochondrial membrane potential by Spirulina platensis C-phycocyanin induces apoptosis in the doxorubicinresistant human hepatocellular-carcinoma cell line HepG2. Biotechnol Appl Biochem 47:159–167PubMedCrossRefGoogle Scholar
  47. Schilter B, Andersson C, Anton R, Constable A, Kleiner J, O’ Brien J, Renwick AG, Korver O, Smit F, Walker R (2003) Guidance for the safety assessment of botanical and botanical prepaprations for use in food and food supplements. Food Chem Toxicol 41:1625–1649PubMedCrossRefGoogle Scholar
  48. Shannon CE, Weaver W (1963) The Mathematical Theory of Communication. University of Illinois Press, UrbanaGoogle Scholar
  49. Simpore J, Zongo F, Kabore F, Dansou D, Bere A, Nikiema J-P, Pignatelli S, Biondi DM, Ruberto G, Musumeci S (2005) Nutrition rehabilitation of HIV-infected and HIV-negative undernourished children utilizing Spirulina. Ann Nutr Metab 49:373–380PubMedCrossRefGoogle Scholar
  50. Song Z, Williams CJ, Edyvean RGT (2004) Treatment of tannery wastewater by chemical co-agulation. Desalination 164:249–259CrossRefGoogle Scholar
  51. Swofford DL (2000) PAUP*: phylogenetic analysis by parsimony, version 4. Sinauer, MassachusettsGoogle Scholar
  52. Teas J, Hebert JR, Fitton JH, Zimba PV (2004) Algae—a poor man’s HAART? Med Hypotheses 62:507–510PubMedCrossRefGoogle Scholar
  53. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882PubMedCrossRefGoogle Scholar
  54. Torres-Duran PV, Ferreira-Hermosillo A, Juarez-Oropeza MA (2007) Antihyperlipemic and antihypertensive effects of Spirulina maxima in an open sample of the Mexican population: a preliminary report. Lipids Health Dis 6:33PubMedCrossRefGoogle Scholar
  55. Vaneechoutte M, Cartwright CP, Williams EC, Jager B, Tichy H-V, De Baere T, de Rouck A, Verschraegen G (1996) Evaluation of 16SrRNA gene restriction analysis for the identification of cultured organisms of clinically important Clostridium species. Anaerobe 2:249–256CrossRefGoogle Scholar
  56. Vanella R, Verma SK (2006) Co2+, Cu2+ and Zn2+ accumulation by cyanobacterium Spirulina platensis. Biotechnol Prog 22:1282–1293CrossRefGoogle Scholar
  57. Vonshak A (ed.) (1997) Spirulina platensis (Arthrospira) physiology, cell biology and biotechnology. Taylor and Francis, LondonGoogle Scholar
  58. Wadowsky RM, Laus S, Libert T, States SJ, Ehrlich GD (1994) Inhibition of PCR-based assay for Bordetella pertussis by using calcium alginate fiber and aluminum shaft components of a nasopharyngeal swab. J Clin Microbiol 32:1054–1057PubMedGoogle Scholar
  59. Walker R (2004) Criteria for risk assessments of food supplements. Toxicol Lett 149:187–195PubMedCrossRefGoogle Scholar
  60. Watanabe K, Kodama Y, Harayama S (2001) Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting. J Microbiol Methods 44:253–262PubMedCrossRefGoogle Scholar
  61. Yamani E, Kaba-Mebri J, Mouala C, Gresenquet G, Rev JL (2009) Use of Spirulina supplement for nutritional management of HIV-infected patients: study in Bangui, Central African Republic. Med Trop 69:66–70Google Scholar
  62. Yeates C, Gillings MR, Davison AD, Altavilla N, Veal DA (1998) Methods for microbial DNA extraction from soil for PCR amplification. Biol Proced Online 1:40–47PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Dirk T. Hoekstra
    • 1
  • Heinrich Volschenk
    • 2
  • Michelle Collins
    • 1
  • Lynn D. McMaster
    • 1
  1. 1.Department of Agricultural and Food Sciences, Faculty of Applied SciencesCape Peninsula University of TechnologyCape TownSouth Africa
  2. 2.Department of MicrobiologyStellenbosch UniversityStellenboschSouth Africa

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