Extraction, purification and characterisation of Phycocyanin from Anabaena fertilissima PUPCCC 410.5: as a natural and food grade stable pigment

  • Sandeep Kaur
  • J. I. S. KhattarEmail author
  • Yadvinder Singh
  • D. P. Singh
  • A. S. Ahluwalia


Diverse phycocyanin (PC) extraction and purification methods for cyanobacteria have been reported in literature. A particular method is not applicable to all cyanobacteria and the best method seems to be genus/species specific. Here, we report PC extraction and purification method for the filamentous cyanobacterium Anabaena fertilissima, a hyperproducer of phycobiliproteins. PC was also characterised in terms of unit assembly and stability. PC was extracted by freeze-thawing in distilled water, concentrated by ammonium sulphate fractionation and purified by size exclusion chromatography. The purification process resulted in 5.85-fold increase in PC purity reaching to 3.28. SDS-PAGE of purified PC demonstrated two bands of 19 and 21 kDa. Based on analysis of non-denaturing PAGE, the native PC from this organism is suggested to be a hexamer (αβ)6 of 240 kDa. Lyophilised PC in powder form was more stable compared to PC in the solution. Half-life of dry PC is 110 days when stored at 4 °C in dark. Storage of PC in light at elevated temperature decreased its half life. When added to milk products, PC was stable up to 9 days when stored at 4 °C. Aqueous solution of PC also exhibited significant free radical scavenging activity. The PC from this organism has potential applications in food as a natural pigment and conservative due to its antioxidant properties.


Anabaena fertilissima Antioxidant Food grade Natural colour Phycocyanin Stability 



The authors are thankful to Head of the Department of Botany, Punjabi University, Patiala and Coordinator, DSA-I of University Grants Commission, New Delhi and Coordinator, FIST of Department of Science and Technology, New Delhi for laboratory facilities.

Funding information

Financial assistance was received from University Grants Commission, New Delhi in the form of research project No. 34-164/2008(SR) and fellowship to Sandeep Kaur under Rajiv Gandhi National Fellowship.

Supplementary material

10811_2018_1722_MOESM1_ESM.doc (580 kb)
ESM 1 (DOC 579 kb)
10811_2018_1722_MOESM2_ESM.doc (50 kb)
ESM 2 (DOC 50 kb)


  1. Abalde J, Betancourt L, Torres E, Cid A, Barwell C (1998) Purification and characterization of phycocyanin from the marine cyanobacterium Synechococcus sp. IO9201. Plant Sci 136:109–120CrossRefGoogle Scholar
  2. Abd El-Baky HH, El-Baroty GS (2012) Characterization and bioactivity of phycocyanin isolated from Spirulina maxima grown under salt stress. Food Funct 3:381–388PubMedCrossRefPubMedCentralGoogle Scholar
  3. Akoglu A, Engin D, Carmakci ML (2015) Some properties of C-phycocyanin from a naïve strain Anabaena affinis isolated from Kovada Lake, Isparta, Turkey. Indian J Mar Sci 44: 538-545Google Scholar
  4. Antelo FS, Costa JAV, Kalil SJ (2008) Thermal degradation kinetics of the phycocyanin from Spirulina platensis. Biochem Eng J 41:43–47CrossRefGoogle Scholar
  5. Apt KE, Collier JL, Grossman AR (1995) Evolution of the phycobiliproteins. J Mol Biol 248:79–96PubMedCrossRefPubMedCentralGoogle Scholar
  6. Bennett A, Bogorad L (1973) Complementary chromatic adaptation in filamentous blue green algae. J Cell Biol 58:419–433PubMedPubMedCentralCrossRefGoogle Scholar
  7. Bermejo R, Felipe MA, Talavera EM, Alvarez-Pez JM (2006) Expanded bed adsorption chromatography for recovery of phycocyanins from the microalga Spirulina platensis. Chromatographia 63:59–66CrossRefGoogle Scholar
  8. Bernard C, Thomas JC, Mazel D, Mousseau A, Castets AM, Tandeau de Marsac N, Dubacq JP (1992) Characterization of the genes encoding phycoerythrin in the red alga Rhodella violacea: evidence for a splitting of the rpeB gene by an intron. Proc Natl Acad Sci U S A 89:9564–9568PubMedPubMedCentralCrossRefGoogle Scholar
  9. Bharathiraja S, Seo H, Manivasagan P, Moorthy MS, Park S, Oh J (2016) In-vitro photodynamic effect of phycocyanin against breast cancer cells. Molecules 21:1–12CrossRefGoogle Scholar
  10. Bhaskar US, Gopalswamy G, Raghu R (2005) A simple method for differential extraction and purification of C-phycocyanin from Spirulina platensis Geitler. Indian J Exp Biol 43:277–279PubMedPubMedCentralGoogle Scholar
  11. Borowitzka MA (2013) High-value products from microalgae – their development and commercialisation. J Appl Phycol 25:743–756CrossRefGoogle Scholar
  12. Brand-Williams W, Cuvelier ME, Berset C (1995) Use of free radical method to evaluate antioxidant activity. Lebensm Wiss Technol 28:25–30CrossRefGoogle Scholar
  13. Chaiklahan R, Chirasuwan N, Loha V, Tia S, Bunnag B (2011) Separation and purification of phycocyanin from Spirulina sp. using a membrane process. Bioresour Technol 102:7159–7164PubMedCrossRefPubMedCentralGoogle Scholar
  14. Chaneva G, Furnadzhieva S, Minkova K, Lukavsky J (2007) Effect of light and temperature on the cyanobacterium Arthronema africanum - a prospective phycobiliprotein producing strain. J Appl Phycol 19:537–544CrossRefGoogle Scholar
  15. Chen HW, Yang TS, Chen MJ, Chang YC, Wang EIC, Ho CL, Lai YJ, Yu CC, Chao JC, Chao LKP, Liao PC (2014) Purification and immunomodulating activity of C-phycocyanin from Spirulina platensis cultured using power plant flue gas. Process Biochem 49:1337–1344CrossRefGoogle Scholar
  16. Chethana S, Chetan AN, Madhusudan MC, Raghavarao KSMS (2015) Single step aqueous two-phase extraction for downstream processing of C-phycocyanin from Spirulina platensis. J Food Sci Technol 52:2415–2421PubMedCrossRefPubMedCentralGoogle Scholar
  17. de Jesus VC, Gutierrez-Rebolledo GA, Hernandez-Ortega M, Valadez-Carmona L, Mojica-Villegas A, Gutierrez-Salmean G, Chamorro-cevallos G (2016) Methods for extraction, isolation and purification of C-phycocyanin: 50 years of research in review. Int J Food Nutr Sci 3:275–284Google Scholar
  18. de O Moreira I, Passos TS, Chiapinni C, Silveira GK, JCM S, Coca-Vellarde LG, Delizad R, de Lima Araujo KG (2012) Colour evaluation of a phycobiliprotein-rich extract obtained from Nostoc PCC9205 in acidic solutions and yogurt. J Sci Food Agric 92:598–605CrossRefGoogle Scholar
  19. Dikshit R, Tallapragada P (2018) Comparative study of natural and artificial flavoring agents and dyes. In: Holban AM, Grumezescu A (eds) Natural and Artificial Flavoring Agents and Food Dyes. Academic Press, NY, pp 83–111Google Scholar
  20. Edward MR, MacColl R, Eisele LE (1996) Some physical properties of an unusual C-phycocyanin isolated from a photosynthetic thermophile. Biochim Biophys Acta 1276:64–70CrossRefGoogle Scholar
  21. Englard S, Seifter S (1990) Precipitation techniques. Methods Enzymol 182:285–300PubMedCrossRefPubMedCentralGoogle Scholar
  22. Gantar M, Simovic D, Djils S, Gonzalez WW, Miksovska J (2012) Isolation, characterization and antioxidative activity of C-phycocyanin from Limnothrix sp. Strain37-2-1. J Biotechnol 159:21–26PubMedPubMedCentralCrossRefGoogle Scholar
  23. Ge X, Tolosa L, Rao G (2004) Dual-labeled glucose binding protein for ratiometric measurement of glucose. Anal Chem 76:1403–1410PubMedCrossRefPubMedCentralGoogle Scholar
  24. Gris EF, Ferreira EA, Falca LD, Bordignon-Luiz MT (2007) Influence of ferulic acid on stability of anthocyanins from cabernet sauvignon grapes in a model system and a yogurt system. Int J Food Sci Technol 42:992–998CrossRefGoogle Scholar
  25. Guan X, Qin S, Zhao F, Zhang X, Tang X (2007) Phycobilisomes linker family in cyanobacterial genomes: divergence and evolution. Int J Biol Sci 3:434–445PubMedPubMedCentralCrossRefGoogle Scholar
  26.  Gupta A, Sainis JK (2010) Isolation of C-phycocyanin from Synechococcus sp., (Anacystis nidulans BD1). J Appl Phycol 22:231–233Google Scholar
  27. Hayashi NR, Terazono K, Hasegawa N, Kodama T, Igarashi Y (1997) Identification and characterization of phycobiliprotein from a thermophilic cyanobacterium, Chroococcidiopsis sp. strain TS-821. J Ferment Bioeng 84:475–477CrossRefGoogle Scholar
  28. Hemlata, Fatma T (2009) Screening of cyanobacteria for phycobilipriteins and effect of different environmental stress on its yield. Bull Environ Contam Toxicol 83:509–515Google Scholar
  29. Hilditch CM, Smith AJ, Balding P, Rogers LJ (1991) C-Phycocyanin from the cyanobacterium Aphanothece halophytica. Phytochemistry 30:3515–3517CrossRefGoogle Scholar
  30. Iwata K, Inayama T, Kato T (1990) Effects of Spirulina platensis on plasma lipoprotein lipase activity in fructose-induced hyperlipidemia in rats. J Nutr Sci Vitaminol 36:165–171PubMedCrossRefPubMedCentralGoogle Scholar
  31. Johnson EM, Kumar K, Das D (2014) Physicochemical parameters optimization, and purification of phycobiliproteins from the isolated Nostoc sp. Bioresour Technol 166:541–547PubMedCrossRefPubMedCentralGoogle Scholar
  32. Kamble SP, Gaikar RB, Padalia RB, Shinde KD (2013) Extraction and purification of C-phycocyanin from dry Spirulina powder and evaluating its antioxidant, anticoagulation and prevention of DNA damage activity. J App Pharm Sci 3:149–153Google Scholar
  33. Kannaujiya VK, Sinha RP (2015) Impacts of varying light regimes on phycobiliproteins of Nostoc sp. HKAR-2 and Nostoc sp. HKAR-11 isolated from diverse habitats. Protoplasma 252:1551–1561PubMedCrossRefGoogle Scholar
  34. Kannaujiya VK, Sinha RP (2016) Thermokinetic stability of phycocyanin and phycoerythrins in food-grade preservatives. J Appl Phycol 28:1063–1070CrossRefGoogle Scholar
  35. Kannaujiya VK, Sundaram S, Sinha RP (2017) Advances in production technology. In: Kannaujiya VK, Sundaram, Sinha RP (eds) Phycobiliproteins: recent developments and future applications. Springer, Cham, pp 83–97CrossRefGoogle Scholar
  36. Khattar JIS, Kaur S, Kaushal S, Singh Y, Singh DP, Rana S, Gulati A (2015) Hyperproduction of phycobiliproteins by the cyanobacterium Anabaena fertilissima PUPCCC 410.5 under optimized culture conditions. Algal Res 12:463–469CrossRefGoogle Scholar
  37. Kronick MN (1986) The use of phycobiliproteins as fluorescent labels in immunoassay. J Immunol Methods 92:1–13PubMedCrossRefGoogle Scholar
  38. Kumar D, Dhar DW, Pabbi S, Kumar N, Walia S (2014) Extraction and purification of C-phycocyanin from Spirulina platensis (CCC540). Indian J Plant Physiol 19:184–188PubMedPubMedCentralCrossRefGoogle Scholar
  39. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  40. Liu LN, Su HN, Yan SG, Shao SM, Xie BB, Chen XL (2009) Probing the pH sensitivity of R-phycoerythrin: investigations of active conformational and functional variation. Biochim Biophys Acta 1787:939–946PubMedCrossRefPubMedCentralGoogle Scholar
  41. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  42. MacColl R (1998) Cyanobacterial phycobilisomes. J Struct Biol 124:311–334PubMedCrossRefPubMedCentralGoogle Scholar
  43. Manirafasha E, Murwanashyaka T, Ndikubwimana T, Yue Q, Zeng X, Lu Y, Jing K (2017) Ammonium chloride: a novel effective and inexpensive salt solution for phycocyanin extraction from Arthrospira (Spirulina) platensis. J Appl Phycol 29:1261–1270CrossRefGoogle Scholar
  44. Minkova KM, Tchernov AA, Tcharbadjieva MI, Fournadjieva ST, Antova RE, Busheva MC (2003) Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis. J Biotechnol 102:55–59PubMedCrossRefPubMedCentralGoogle Scholar
  45. Mishra SK, Shrivastav A, Maurya RR, Patidar SK, Haldar S, Mishra S (2010) Effect of light quality on the C-phycoerythrin production in marine cyanobacteria Pseudoanabaena sp. isolated from Gujarat coast, India. Protein Expr Purif 81:5–10CrossRefGoogle Scholar
  46. Mourelle ML, Gómez CP, Legido JL (2017) The potential use of marine microalgae and cyanobacteria in cosmetics and thalassotherapy. Cosmetics 4:46CrossRefGoogle Scholar
  47. Muthulakshmi M, Saranya A, Sudha M, Selvakumar G (2012) Extraction, partial purification, and antibacterial activity of phycocyanin from Spirulina isolated from fresh water body against various human pathogens. J Algal Biomass Utln 3:7–11Google Scholar
  48. Pan M, Jiang TS, Pan JL (2011) Antioxidant activities of rapeseed protein hydrolysates. Food Bioprocess Technol 4:1144–1152CrossRefGoogle Scholar
  49. Pandiraj D, Baldev E, Muhammad Ilyas MH, Thajudin N (2017) Production, extraction and purification of C-phycoerythrin from marine cyanobacterium, Phormidium persicinum NTDP01. Phykos 47:16–22Google Scholar
  50. Pan-utai W, Kahapana W, Lamtham S (2018) Extraction of C-phycocyanin from Arthrospira (Spirulina) and its thermal stability with citric acid. J Appl Phycol 30:231–242CrossRefGoogle Scholar
  51. Parmar A, Singh NK, Kaushal A, Madamwar D (2011) Characterization of an intact phycoerythrin and its cleaved 14 kDa functional subunit from marine cyanobacterium Phormidium sp. A27DM. Process Biochem 46:1793–1799CrossRefGoogle Scholar
  52. Patel A, Mishra S, Pawar R, Ghosh PK (2005) Purification and characterization of C-phycocyanin from cyanobacterial species of marine and fresh water habitat. Protein Expr Purif 40:248–255PubMedCrossRefPubMedCentralGoogle Scholar
  53. Pickup JC, Hussain F, Evans ND, Rolinsk OJ, Birch DJS (2005) Fluorescence-based glucose sensors. Biosens Bioelectron 20:2555–2565PubMedCrossRefPubMedCentralGoogle Scholar
  54. Pleonsil P, Soogarun S, Suwanwong Y (2013) Anti-oxidant activity of holo- and apo-c-phycocyanin and their protective effects on human erythrocytes. Int J Biol Macromol 60:393–398PubMedCrossRefPubMedCentralGoogle Scholar
  55. Pumas C, Peerapornpisal Y, Vacharapiyasophon P, Leelapornpisid P, Boonchum W, Ishii M, Khanongnuch C (2012) Purification and characterization of a thermostable phycoerythrin from hot spring cyanobacterium Leptolyngbya sp. KC45. Int J Agric Biol 14:121–125Google Scholar
  56. Rahman DY, Sarian FD, van Wijk A, Martinez-Garcia M, van der Maarel MJEC (2017) Thermostable Phycocyanin from the red microalga Cyanidioschyzon merolae, a new natural blue food colorant. J Appl Phycol 29:1233–1239PubMedCrossRefPubMedCentralGoogle Scholar
  57. Ranjitha K, Kaushik BD (2005) Purification of phycobiliproteins from Nostoc muscorum. J Sci Ind Res 64:372–375Google Scholar
  58. Rastogi RP, Sonani RR, Madamwar D (2015) Physico-chemical factors affecting the in vitro stability of phycobiliproteins from Phormidium rubidium A09DM. Bioresour Technol 190:219–226PubMedCrossRefPubMedCentralGoogle Scholar
  59. Richa KVK, Kesheri M, Singh G, Sinha RP (2011) Biotechnological potentials of phycobiliproteins. Int J Pharm Bio Sci 2:446–454Google Scholar
  60. Rimbau V, Camins A, Romay C, González R, Pallàs M (1999) Protective effects of C-phycocyanin against kainic acid-induced neuronal damage in rat hippocampus. Neurosci Lett 276:75–78PubMedCrossRefGoogle Scholar
  61. Rito-Palomares M, Nuñez L, Amador D (2001) Practical application of aqueous two-phase systems for the development of a prototype process for C-phycocyanin recovery from Spirulina maxima. J Chem Technol Biotechnol 76:1273–1280CrossRefGoogle Scholar
  62. Saini DK, Pabbi S, Shukla P (2018) Cyanobacterial pigments: perspectives and biotechnological approaches. Food Chem Toxicol 120:616–624PubMedCrossRefGoogle Scholar
  63. Salama A, Abdel Ghany A, Osman A, Sitohy M (2015) Maximising phycocyanin extraction from a newly identified Egyptian cyanobacteria strain: Anabaena oryzae SOS13. Int Food Res J 22:517–525Google Scholar
  64. Santiago-Santos M, Ponce Noyola T, Olvera-ramirez ZR, Ortega-López J, Cañizares-Villanueva RO (2004) Extraction and purification of phycocyanin from Calothrix sp. Process Biochem 39:2047–2052CrossRefGoogle Scholar
  65. Sarada R, Pillai MG, Ravishankar GA (1999) Phycocyanin from Spirulina sp.: influence of processing of biomass on phycocyanin yield, analysis of efficacy of extraction methods and stability studies on phycocyanin. Process Biochem 34:795–801CrossRefGoogle Scholar
  66. Sarmadi BH, Ismail A (2010) Antioxidative peptides from food proteins: a review. Peptides 31:1949–1956PubMedCrossRefGoogle Scholar
  67. Sekar S, Chandramohan M (2008) Phycobilproteins as a commodity: trends in applied research, patents and commercialization. J Appl Phycol 20:13–136CrossRefGoogle Scholar
  68. Seo YC, Choi WS, Park JH, Park JO, Jung KH, Lee HY (2013) Stable isolation of phycocyanin from Spirulina platensis associated with high-pressure extraction process. Int J Mol Sci 14:1778–1787PubMedPubMedCentralCrossRefGoogle Scholar
  69. Shalaby EA, Shanab MM (2013) Comparison of DPPH and ABTS assays for determining antioxidant potential of water and methanol extracts of Spirulina platensis. Indian J Mar Sci 42:556–564Google Scholar
  70. Shanab SMM, Mostafa SSM, Shalaby EA, Mahmoud GI (2012) Aqueous extracts of microalgae exhibit antioxidant and anticancer activities. Asian Pac J Trop Biomed 2:608–615PubMedPubMedCentralCrossRefGoogle Scholar
  71. Sidler WA (1994) Phycobilisome and phycobiliprotein structure. In: Bryant DA (ed) The molecular biology of cyanobacteria. Kluwer, Dordrecht, pp 139–216CrossRefGoogle Scholar
  72. Silva LA, Kuhn KR, Moraes CC, Burkert CAV, Kalil SJ (2009) Experimental design as a tool for optimization of C-phycocyanin purification by precipitation from Spirulina platensis. J Braz Chem Soc 20:5–12CrossRefGoogle Scholar
  73. Sonani RR, Singh NK, Kumar J, Thakar D, Madamwar D (2014) Concurrent purification and antioxidant activity of phycobiliproteins from Lyngbya sp. A09DM: an antioxidant and anti-aging potential of phycoerythrin in Caenorhabditis elegans. Process Biochem 49:1757–1766CrossRefGoogle Scholar
  74. Sonani RR, Rastogi RP, Patel R, Madamwar D (2016) Recent advances in production, purification and applications of phycobiliproteins. World J Biol Chem 7:100–109PubMedPubMedCentralCrossRefGoogle Scholar
  75. Sonani RR, Patel S, Bhastana B, Jakharia K, Chaubey MG, Singh NK, Madamwar D (2017) Purification and antioxidant activity of phycocyanin from Synechococcus sp. R42DM isolated from industrially polluted site. Bioresour Technol 31:325–331CrossRefGoogle Scholar
  76. Song W, Zhao C, Wang S (2013) A large-scale preparation method of high purity C-Phycocyanin. Int J Biosci Biochem Bioinformatics 3:293–297Google Scholar
  77. Soni B, Kalavadia B, Trivedi U (2006) Extraction, purification and characterization of phycocyanin from Oscillatoria quadripunctulata isolated from the rocky shores of bet-Dwarka, Gujarat, India. Process Biochem 41:2017–2023CrossRefGoogle Scholar
  78. Soni B, Trivedi U, Madamwar D (2008) A novel method of single step hydrophobic interaction chromatography for the purification of phycocyanin from Phormidium fragile and its characterization for antioxidant property. Bioresour Technol 99:188–194PubMedCrossRefGoogle Scholar
  79. Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101:87–96PubMedCrossRefGoogle Scholar
  80. Stadnichuk IN, Tropin IV (2017) Phycobiliproteins: structure, function and biotechnological applications. Appl Biochem Microbiol 53:1–10CrossRefGoogle Scholar
  81. Tandeau de Marsac N (2003) Phycobiliproteins and phycobilisomes: the early observations. Photosynth Res 76:197–205CrossRefGoogle Scholar
  82. Tavanandi HA, Mittal R, Chandrasekhar J, Raghavarao KSMS (2018) Simple and efficient method for C-phycocyanin from dry biomass of Arthospira platensis. Algal Res 31:239–251CrossRefGoogle Scholar
  83. Thangam R, Suresh V, AsenathPrincy W, Rajkumar M, SenthilKumar N, Gunasekaran P, Rengasamy R, Anbazhagan C, Kaveri K, Kannan S (2013) C-Phycocyanin from Oscillatoria tenuis exhibited an antioxidant and in vitro antiproliferative activity through induction of apoptosis and G0/G1 cell cycle arrest. Food Chem 140:262–272PubMedCrossRefPubMedCentralGoogle Scholar
  84. Thomas JC, Passaquet C (1999) Characterization of a phycoerythrin without α-subunits from a unicellular red alga. J Biol Chem 274:2472–2482PubMedCrossRefPubMedCentralGoogle Scholar
  85. Yuan YV, Walsh NA (2006) Antioxidant and antiproliferative activities of extracts from a variety of edible seaweeds. Food Chem Toxicol 44:1144–1150PubMedCrossRefPubMedCentralGoogle Scholar
  86. Zhang YM, Chen F (1999) A simple method for efficient separation and purification of c-phycocyanin and allophycocyanin from Spirulina platensis. Biotechnol Tech 13:601–603CrossRefGoogle Scholar
  87. Zhang XF, Wang X, Luo GH (2017) Ultrasound-assisted three phase partitioning of phycocyanin from Spirulina platensis. Eur J Pure Appl Chem 4:1–15Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of BotanyPunjabi UniversityPatialaIndia
  2. 2.Department of BotanyKanya Maha VidyalayaJalandharIndia
  3. 3.Department of Botany and Environmental ScienceSri Guru Granth Sahib World UniversityFatehgarh SahibIndia
  4. 4.Department of BotanyPanjab UniversityChandigarhIndia

Personalised recommendations