Purification and Identification of Astaxanthin and Its Novel Derivative Produced by Radio-tolerant Sphingomonas astaxanthinifaciens

  • Dalal Asker
  • Tarek S. Awad
  • Teruhiko Beppu
  • Kenji Ueda
Part of the Methods in Molecular Biology book series (MIMB, volume 1852)


The red diketocarotenoid, astaxanthin, exhibits extraordinary health-promoting activities such as antioxidant, anti-inflammatory, antitumor, and immune booster, which may potentially protect against many degenerative diseases such as cancers, heart diseases, and exercise-induced fatigue. These numerous health benefits and consumer interest in natural products have therefore increased the market demand of astaxanthin as a nutraceutical and medicinal ingredient in food, aquaculture feed, and pharmaceutical industries. Consequently, many research efforts have been made to discover novel microbial sources with effective biotechnological production of astaxanthin. Using a rapid screening method based on 16S rRNA gene, and effective HPLC-Diode array-MS methods for carotenoids analysis, we isolated a novel astaxanthin-producing bacterium (strain TDMA-17T) that belongs to the family Sphingomonadaceae (Asker et al., FEMS Microbiol Lett 273:140–148, 2007).

In this chapter, we provide a comprehensive description of the methods used for the analysis and identification of carotenoids produced by strain TDMA-17T. We will also describe the methods of isolation and identification for a novel bacterial carotenoid (an astaxanthin derivative), a major carotenoid that is produced by the novel strain. Finally, the identification methods of the novel strain will be summarized.

Key words

Astaxanthin Carotenoids Antioxidant Biotechnology Sphingomonadaceae Sphingomonas astaxanthinifaciens Screening 16S rRNA Phylogenetic HPLC 


  1. 1.
    Johnson EA, An GH (1991) Astaxanthin from microbial sources. Crit Rev Biotechnol 11(4):297–326CrossRefGoogle Scholar
  2. 2.
    Nelis HJ, Deleenheer AP (1991) Microbial sources of carotenoid-pigments used in foods and feeds. J Appl Bacteriol 70(3):181–191CrossRefGoogle Scholar
  3. 3.
    Britton G, Liaaen-Jensen S, Pfander H (1995) Carotenoids today and challenges for the future. Birkhauser, BaselGoogle Scholar
  4. 4.
    Rufer CE et al (2008) Bioavailability of astaxanthin stereoisomers from wild (Oncorhynchus spp.) and aquacultured (Salmo salar) salmon in healthy men: a randomised, double-blind study. Br J Nutr 99(5):1048–1054CrossRefPubMedGoogle Scholar
  5. 5.
    Page GI, Davies SJ (2006) Tissue astaxanthin and canthaxanthin distribution in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Comp Biochem Physiol A Mol Integr Physiol 143(1):125–132CrossRefPubMedGoogle Scholar
  6. 6.
    Fujita T et al (1983) Pigmentation of cultured red sea bream with astaxanthin diester purified from krill oil. Bull Jpn Soc Sci Fish 49:1855–1865CrossRefGoogle Scholar
  7. 7.
    Torrissen OJ (1989) Pigmentation of salmonids—interactions of astaxanthin and canthaxanthin on pigment deposition in rainbow-trout. Aquaculture 79(1–4):363–374CrossRefGoogle Scholar
  8. 8.
    Bjerkeng B, Berge GM (2000) Apparent digestibility coefficients and accumulation of astaxanthin E/Z isomers in Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.). Comp Biochem Phys B 127(3):423–432CrossRefGoogle Scholar
  9. 9.
    An GH, Choi ES (2003) Preparation of the red yeast, Xanthophyllomyces dendrorhous, as feed additive with increased availability of astaxanthin. Biotechnol Lett 25(10):767–771CrossRefPubMedGoogle Scholar
  10. 10.
    Christiansen R et al (1995) Antioxidant status and immunity in Atlantic salmon, Salmo salar L., fed semi-purified diets with and without astaxanthin supplementation. J Fish Dis 18(4):317–328CrossRefGoogle Scholar
  11. 11.
    Christiansen R, Torrissen OJ (1997) Effects of dietary astaxanthin supplementation on fertilization and egg survival in Atlantic salmon (Salmo salar L.). Aquaculture 153(1–2):51–62CrossRefGoogle Scholar
  12. 12.
    Amaya E, Nickell D (2015) Using feed to enhance the color quality of fish and crustaceans. In: Allen Davis D (ed) Feed and feeding practices in aquaculture. Woodhead Publishing, Oxford, pp 269–298Google Scholar
  13. 13.
    Lorenz RT, Cysewski GR (2000) Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol 18(4):160–167CrossRefPubMedGoogle Scholar
  14. 14.
    Gorton HL, Williams WE, Vogelmann TC (2001) The light environment and cellular optics of the snow alga Chlamydomonas nivalis (Bauer) Wille. Photochem Photobiol 73(6):611–620CrossRefPubMedGoogle Scholar
  15. 15.
    Gorton HL, Vogelmann TC (2003) Ultraviolet radiation and the snow alga Chlamydomonas nivalis (Bauer) Wille. Photochem Photobiol 77(6):608–615CrossRefPubMedGoogle Scholar
  16. 16.
    Kim JH, Chang HI (2006) High-level production of astaxanthin by Xanthophyllomyces dendrorhous mutant JH1, using chemical and light induction. J Microbiol Biotechnol 16(3):381–385CrossRefGoogle Scholar
  17. 17.
    Fan L et al (1998) Does astaxanthin protect Haematococcus against light damage? Z Naturforsch C 53(1–2):93–100CrossRefPubMedGoogle Scholar
  18. 18.
    Iwamoto T et al (2000) Inhibition of low-density lipoprotein oxidation by astaxanthin. J Atheroscler Thromb 7(4):216–222CrossRefPubMedGoogle Scholar
  19. 19.
    O'connor I, O'brien N (1998) Modulation of UVA light-induced oxidative stress by beta-carotene, lutein and astaxanthin in cultured fibroblasts. J Dermatol Sci 16(3):226–230CrossRefPubMedGoogle Scholar
  20. 20.
    Wang X, Willen R, Wadstrom T (2000) Astaxanthin-rich algal meal and vitamin C inhibit Helicobacter pylori infection in BALB/cA mice. Antimicrob Agents Chemother 44(9):2452–2457CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Jyonouchi H, Sun S, Gross M (1995) Effect of carotenoids on in vitro immunoglobulin production by human peripheral blood mononuclear cells: astaxanthin, a carotenoid without vitamin A activity, enhances in vitro immunoglobulin production in response to a T-dependent stimulant and antigen. Nutr Cancer 23(2):171–183CrossRefPubMedGoogle Scholar
  22. 22.
    Guerin M, Huntley ME, Olaizola M (2003) Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol 21(5):210–216CrossRefPubMedGoogle Scholar
  23. 23.
    Yuan JP et al (2010) Potential health-promoting effects of astaxanthin: a high-value carotenoid mostly from microalgae. Mol Nutr Food Res 55(1):150–165CrossRefPubMedGoogle Scholar
  24. 24.
    Mortensen A (2009) Supplements. In: Britton G, Liaaen-Jensen S, Pfander H (eds) Nutrition and health. Birkhäuser Verlag, Basel, pp 68–82Google Scholar
  25. 25.
    Vaclavik VA, Christian EW (2014) Fat and oil products. In: Vaclavik VA, Christian EW (eds) Essentials of food science. Springer, New York, pp 233–261CrossRefGoogle Scholar
  26. 26.
    März U (2015) FOD025E—the global market for carotenoids. BCC Research, Wellesley, MAGoogle Scholar
  27. 27.
    Solymosi K et al (2015) Food colour additives of natural origin. In: Scotter MJ (ed) Colour additives for foods and beverages. Elsevier Ltd., Amsterdam, pp 3–34Google Scholar
  28. 28.
    Bi W et al (2010) Task-specific ionic liquid-assisted extraction and separation of astaxanthin from shrimp waste. J Chromatogr B Analyt Technol Biomed Life Sci 878(24):2243–2248CrossRefPubMedGoogle Scholar
  29. 29.
    Franco-Zavaleta ME et al (2010) Astaxanthin extraction from shrimp wastes and its stability in 2 model systems. J Food Sci 75(5):C394–C399PubMedGoogle Scholar
  30. 30.
    Pacheco N et al (2009) Effect of temperature on chitin and astaxanthin recoveries from shrimp waste using lactic acid bacteria. Bioresour Technol 100(11):2849–2854CrossRefPubMedGoogle Scholar
  31. 31.
    An GH et al (2004) Pigmentation and delayed oxidation of broiler chickens by the red carotenoid, astaxanthin, from chemical synthesis and the yeast, Xanthophyllomyces dendrorhous. Asian Austral J Anim 17(9):1309–1314CrossRefGoogle Scholar
  32. 32.
    Asker D, Beppu T, Ueda K (2007) Sphingomonas astaxanthinifaciens sp. nov., a novel astaxanthin-producing bacterium of the family Sphingomonadaceae isolated from Misasa, Tottori, Japan. FEMS Microbiol Lett 273(2):140–148CrossRefPubMedGoogle Scholar
  33. 33.
    Asker D et al (2018) Screening and profiling of natural ketocarotenoids from environmental aquatic bacterial isolates. Food Chem 253:247–254CrossRefPubMedGoogle Scholar
  34. 34.
    Asker D (2017) Isolation and characterization of a novel, highly selective astaxanthin-producing marine bacterium. J Agric Food Chem 65(41):9101–9109CrossRefPubMedGoogle Scholar
  35. 35.
    Asker D et al (2012) A novel radio-tolerant astaxanthin-producing bacterium reveals a new astaxanthin derivative: astaxanthin dirhamnoside. In: Barredo J-L (ed) Microbial carotenoids from bacteria and microalgae: methods and protocols. Humana Press, New York, pp 61–97CrossRefGoogle Scholar
  36. 36.
    Asker D et al (2012) Isolation, characterization, and diversity of novel radiotolerant carotenoid-producing bacteria. In: Barredo J-L (ed) Microbial carotenoids from bacteria and microalgae: methods and protocols. Humana Press, New York, pp 21–60CrossRefGoogle Scholar
  37. 37.
    Asker D et al (2009) Astaxanthin dirhamnoside, a new astaxanthin derivative produced by a radio-tolerant bacterium, Sphingomonas astaxanthinifaciens. J Antibiot (Tokyo) 62(7):397–399CrossRefGoogle Scholar
  38. 38.
    Asker D, Beppu T, Ueda K (2007) Unique diversity of carotenoid-producing bacteria isolated from Misasa, a radioactive site in Japan. Appl Microbiol Biotechnol 77(2):383–392CrossRefPubMedGoogle Scholar
  39. 39.
    Asker D, Isaka K (2006) Production of astaxanthin by microorganisms. Office TP, Japan. Patent JP340676AGoogle Scholar
  40. 40.
    Del Rio E et al (2008) Efficiency assessment of the one-step production of astaxanthin by the microalga Haematococcus pluvialis. Biotechnol Bioeng 100(2):397–402CrossRefPubMedGoogle Scholar
  41. 41.
    Lubián LM et al (2000) Nannochloropsis (Eustigmatophyceae) as source of commercially valuable pigments. J Appl Phycol 12(3):249–255CrossRefGoogle Scholar
  42. 42.
    De La Fuente JL et al (2010) High-titer production of astaxanthin by the semi-industrial fermentation of Xanthophyllomyces dendrorhous. J Biotechnol 148(2–3):144–146CrossRefPubMedGoogle Scholar
  43. 43.
    Jacobson GK et al. (2003) Astaxanthin over-producing strains of Phaffia rhodozyma, methods for their cultivation, and their use in animal feeds. US patent 20030049241Google Scholar
  44. 44.
    Liu YS, Wu JY (2006) Hydrogen peroxide-induced astaxanthin biosynthesis and catalase activity in Xanthophyllomyces dendrorhous. Appl Microbiol Biotechnol 73(3):663–668CrossRefPubMedGoogle Scholar
  45. 45.
    Calo P et al (1995) Ketocarotenoids in halobacteria: 3-hydroxy-echinenone and trans-astaxanthin. J Appl Bacteriol 79:282CrossRefGoogle Scholar
  46. 46.
    Yokoyama A, Izumide H, Miki W (1994) Production of astaxanthin and 4-ketozeaxanthin by the marine bacterium, Agrobacterium aurantiacum. Biosci Biotechnol Biochem 58:1842–1844CrossRefGoogle Scholar
  47. 47.
    Tsubokura A, Yoneda H, Mizuta H (1999) Paracoccus carotinifaciens sp. nov., a new aerobic gram-negative astaxanthin-producing bacterium. Int J Syst Bacteriol 49(Pt 1):277–282CrossRefPubMedGoogle Scholar
  48. 48.
    Yokoyama A et al (1996) New trihydroxy-keto-carotenoids isolated from an astaxanthin-producing marine bacterium. Biosci Biotechnol Biochem 60:200–203CrossRefPubMedGoogle Scholar
  49. 49.
    Osanjo GO et al (2009) A salt lake extremophile, Paracoccus bogoriensis sp. nov., efficiently produces xanthophyll carotenoids. Afr J Microbiol Res 3(8):426–433Google Scholar
  50. 50.
    Hirasawa K, Tsuborkura A (2014). Method for separating carotenoid. US Patent 8853460B2Google Scholar
  51. 51.
    Bubrick P (1991) Production of astaxanthin from Haematococcus. Bioresour Technol 38:237–239CrossRefGoogle Scholar
  52. 52.
    Miller MW, Yoneyama M, Soneda M (1976) Phaffia, a new yeast genus in the Deuteromycotina (Blastomycetes). Int J Syst Bacteriol 26:286–291CrossRefGoogle Scholar
  53. 53.
    Harker M, Hirschberg J, Oren A (1998) Paracoccus marcusii sp. nov., an orange gram-negative coccus. Int J Syst Bacteriol 48(Pt 2):543–548CrossRefPubMedGoogle Scholar
  54. 54.
    Lee JH et al (2004) Paracoccus haeundaensis sp. nov., a Gram-negative, halophilic, astaxanthin-producing bacterium. Int J Syst Evol Microbiol 54(Pt 5):1699–1702CrossRefPubMedGoogle Scholar
  55. 55.
    Kametani K, Matsumura T (1983) Determination of 238U, 234U, 226Ra and 228Ra in spring waters of sanin district. Radioisotopes 32(1):18–21CrossRefPubMedGoogle Scholar
  56. 56.
    Nelis HJ, De Leenheer AP (1989) Profiling and quantitation of bacterial carotenoids by liquid chromatography and photodiode array detection. Appl Environ Microbiol 55(12):3065–3071PubMedPubMedCentralGoogle Scholar
  57. 57.
    Britton G, Liaaen-Jensen S, Pfander H (2004) Carotenoids handbook. Birkhäuser, BaselCrossRefGoogle Scholar
  58. 58.
    Takaichi S et al (2003) Fatty acids of astaxanthin esters in krill determined by mild mass spectrometry. Comp Biochem Physiol B Biochem Mol Biol 136(2):317–322CrossRefPubMedGoogle Scholar
  59. 59.
    Breithaupt DE (2004) Identification and quantification of astaxanthin esters in shrimp (Pandalus borealis) and in a microalga (Haematococcus pluvialis) by liquid chromatography-mass spectrometry using negative ion atmospheric pressure chemical ionization. J Agric Food Chem 52(12):3870–3875CrossRefPubMedGoogle Scholar
  60. 60.
    Matsuno T et al (1984) The occurence of enantiomeric and meso-astaxanthin in aquatic animals. Bull Jpn Soc Sci Fish 50:1589–1592CrossRefGoogle Scholar
  61. 61.
    Johnson EA, Schroeder W (1995) Astaxanthin from the yeast Phaffia rhodozyma. Stud Mycol 38:81–90Google Scholar
  62. 62.
    Rønneberg H et al (1980) Natural occurrence of enatiomeric and meso-astaxanthin 1. Ex lobster eggs (Homarus gammarus). Helv Chim Acta 63:711–715CrossRefGoogle Scholar
  63. 63.
    Bernhard K et al (1982) Carotenoids of the carotenoprotein asteriarubin. Optical purity of asterinic acid. Helv Chim Acta 65:2224–2229CrossRefGoogle Scholar
  64. 64.
    Yokoyama A, Adachi K, Shizuri Y (1995) New carotenoid glucosides, astaxanthin glucoside and adonixanthin glucoside, isolated from the astaxanthin-producing marine bacterium, Agrobacterium aurantiacum. J Nat Prod 58:1929–1933CrossRefGoogle Scholar
  65. 65.
    Misawa N, Shimada H (1998) Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts. J Biotechnol 59(3):169–181CrossRefGoogle Scholar
  66. 66.
    Maoka T, Tsushima M, Matsuno T (1989) New acetylenic carotenoids from the starfishes Asterina pectinifera and Asterias amurensis. Comp Biochem Physiol 93:829–834Google Scholar
  67. 67.
    Maoka T et al (1985) Stereochemical investigation of the carotenoids in the antarctic krill Euphausia superba. Bull Jpn Soc Sci Fish 51:1671–1673CrossRefGoogle Scholar
  68. 68.
    Hertzberg S et al (1983) Carotenoid sulfates: 2. Structural elucidation of bastaxanthin. Acta Chem Scand 37:267–280CrossRefGoogle Scholar
  69. 69.
    Cowan ST (1968) A dictionary of microbial taxonomic usage. Oliver & Boyd, EdinburghGoogle Scholar
  70. 70.
    Staley JT, Krieg NJ (1984) Classification of prokaryotic organisms: an overview. The Williams & Wilkins Co., BaltimoreGoogle Scholar
  71. 71.
    Vandamme P et al (1996) Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60(2):407–438PubMedPubMedCentralGoogle Scholar
  72. 72.
    Colwell RR (1970) Polyphasic taxonomy of the genus Vibrio: numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus, and related Vibrio species. J Bacteriol 104:410–433PubMedPubMedCentralGoogle Scholar
  73. 73.
    Murray RGE et al (1990) Report of the Ad-Hoc committee on approaches to taxonomy within the Proteobacteria. Int J Syst Bacteriol 40(2):213–215CrossRefGoogle Scholar
  74. 74.
    Tindall BJ et al (2010) Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 60(Pt 1):249–266CrossRefPubMedGoogle Scholar
  75. 75.
    Egan S, Thomas T, Kjelleberg S (2008) Unlocking the diversity and biotechnological potential of marine surface associated microbial communities. Curr Opin Microbiol 11(3):219–225CrossRefPubMedGoogle Scholar
  76. 76.
    Schleifer KH, Ludwig W (1989) Phylogenetic relationships of bacteria. Elsevier Science Publishers B.V., AmsterdamGoogle Scholar
  77. 77.
    Stackebrandt E et al (2002) Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52:1043–1047PubMedGoogle Scholar
  78. 78.
    Stackebrandt E, Goebel BM (1994) A place for DNA–DNA reassociation and 16s ribosomal-RNA sequence-analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44(4):846–849CrossRefGoogle Scholar
  79. 79.
    Woese CR (1987) Bacterial evolution. Microbiol Rev 51(2):221–271PubMedPubMedCentralGoogle Scholar
  80. 80.
    Fox GE, Wisotzkey JD, Jurtshuk P Jr (1992) How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 42(1):166–170CrossRefPubMedGoogle Scholar
  81. 81.
    Martinez-Murcia AJ, Collins MD (1990) A phylogenetic analysis of the genus Leuconostoc based on reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol Lett 70(1):73–83CrossRefGoogle Scholar
  82. 82.
    Wayne LG et al (1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37(4):463–464CrossRefGoogle Scholar
  83. 83.
    Johnson JL (1984) Nucleic acids in bacterial classification. Williams & Wilkins, Baltimore, MDGoogle Scholar
  84. 84.
    Yarza P et al (2008) The All-Species Living Tree project: a 16S rRNA-based phylogenetic tree of all sequenced type strains. Syst Appl Microbiol 31(4):241–250CrossRefPubMedGoogle Scholar
  85. 85.
    Takeuchi M, Hamana K, Hiraishi A (2001) Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 51(Pt 4):1405–1417CrossRefPubMedGoogle Scholar
  86. 86.
    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425PubMedGoogle Scholar
  87. 87.
    Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, pp 607–654Google Scholar
  88. 88.
    Beveridge TJ, Popkin TJ, Cole RM (1994) Electron microscopy. In P. Gerhardt (ed.), Methods for general molecular bacteriology. American Society for Microbiology, Washington, D.C., pp 42–71Google Scholar
  89. 89.
    Norris JR, Ribbons DW, Varma AK (1985) Methods in microbiology. Academic Press, LondonGoogle Scholar
  90. 90.
    Cowan ST, Steel KJ (1993) Manual for the identification of medical bacteria. Cambridge University Press, LondonGoogle Scholar
  91. 91.
    Collins MD (1994) Isoprenoid quinones. In: O’Donnell MGAG (ed) Chemical methods in prokaryotic systematics. Wiley, Chichester, pp 265–310Google Scholar
  92. 92.
    Kawahara K et al (1991) Chemical structure of glycosphingolipids isolated from Sphingomonas paucimobilis. FEBS Lett 292(1–2):107–110PubMedGoogle Scholar
  93. 93.
    Tindall BJ (1990) Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66:199–202CrossRefGoogle Scholar
  94. 94.
    Mesbah M, Whitman WB (1989) Measurement of deoxyguanosine/thymidine ratios in complex mixtures by high-performance liquid chromatography for determination of the mole percentage guanine + cytosine of DNA. J Chromatogr 479(2):297–306CrossRefPubMedGoogle Scholar
  95. 95.
    Meyers SP, Bligh D (1981) Characterization of astaxanthin pigments from heat-processed crawfish waste. J Agric Food Chem 29(3):505–508CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Dalal Asker
    • 1
    • 2
  • Tarek S. Awad
    • 2
  • Teruhiko Beppu
    • 3
  • Kenji Ueda
    • 3
  1. 1.Food Science and Technology Department, Faculty of AgricultureAlexandria UniversityAlexandriaEgypt
  2. 2.Department of Materials Science and EngineeringUniversity of TorontoTorontoCanada
  3. 3.Life Science Research Center, College of Bioresource SciencesNihon UniversityFujisawaJapan

Personalised recommendations