Extraction and purification of phycocyanin pigment from blue-green algae Spirulina platensis: structural characteristics and antioxidant properties analysis

Document Type : Research Paper

Authors

1 Department of Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, Iran.

2 Evidence-based Phytotherapy & Complementary Medicine Research Center, Alborz University of Medical Sciences, Karaj, Iran.

3 Department of Medicinal Chemistry, Faculty of Pharmacy, Alborz University of Medical Sciences, Karaj, Iran.

10.22059/jfisheries.2025.386883.1447

Abstract

Phycocyanin, a blue pigment with fluorescent and antioxidant properties, is extracted from blue-green algae such as Spirulina. Enhancing the extraction and purification conditions of phycocyanin can broaden its applications as a food and pharmaceutical supplement. This study aimed to extract and purify phycocyanin from dried Spirulina biomass and investigate its structural features, cytotoxicity, and antioxidant activity. Phycocyanin was extracted using an ultrasonic bath technique (100 W power and 120 Hz frequency) and a freeze-thaw method (3 cycles). Purification was performed by ammonium sulfate precipitation (25% and 50% saturation) and dialysis membrane (12-14 kDa). The purity, selectivity, yield, and concentration of the extracted phycocyanin were determined to be 1.37, 14.57, 90.15±0.21 mg/g, and 6.01 mg/mL, respectively. The appearance of characteristic peaks in FTIR (peaks at cm⁻¹ 3443 (NH), cm⁻¹ 1625 (Amide I), cm⁻¹ 1543 (Amide II) (C=O stretching bond), and cm⁻¹ 1456 (CN)) and UV spectra (three distinct peaks at 268, 362, and 620 nanometers), along with α and β bands (with a weight range of 10-17 kDa) in SDS-PAGE gel, confirmed the structure and purity of the extracted phycocyanin. Additionally, the molecular weight of phycocyanin was determined to be 2.6 × 106 g/mol using gel permeation chromatography (GPC). Solubility results indicated a decrease in the solubility of extracted phycocyanin at pH= 4 (near the isoelectric point). Evaluation of cytotoxicity and antioxidant activity revealed a dose-dependent increase in the proliferation and viability of HFF2 cells and DPPH free radical scavenging when exposed to phycocyanin. The findings indicate that the use of various methods in extraction (ultrasonic bath and freeze-thaw) and purification (salt precipitation and dialysis membrane) results in the extraction of phycocyanin with significant potential for applications in various pharmaceutical and food industries.

Keywords

Main Subjects

References

Abalde, J., Betancourt, L., Torres, E., Cid, A., Barwell, C., 1998. Purification and characterization of phycocyanin from the marine cyanobacterium Synechococcus sp. IO9201. Plant Science 136(1), 109-120. DOI: 10.1016/S0168-9452(98)00113
Abdelkhalek, N.K., Ghazy, E.W., Abdel-Daim, M.M., 2015. Pharmacodynamic interaction of Spirulina platensis and deltamethrin in freshwater fish Nile tilapia, Oreochromis niloticus: impact on lipid peroxidation and oxidative stress. Environmental Science and Pollution Research 22(4), 3023-3031. DOI: 10.1007/s11356-014-3578-0.
Bai, Y., Li, X., Xie, Y., Wang, Y., Dong, X., Qi, H., 2023. Ultrasound treatment enhanced the functional properties of phycocyanin with phlorotannin from Ascophyllum nodosum. Frontiers in Nutrition 10(6), 1181262. DOI: 10.3389/fnut.2023.1181262
Cao, J., Zheng, X., Li, Z., Zheng, M., Qian, C., Shen, S., Qi, X., 2024. Exploring marine algae-derived phycocyanin nanoparticles as a safe and effective adjuvant for sunscreen systems. Discover Applied Sciences 6(1), 24. DOI: 10.1007/s42452-024-05665-z.
Chaiklahan, R., Chirasuwan, N., Bunnag, B., 2012. Stability of phycocyanin extracted from Spirulina sp.: Influence of temperature, pH and preservatives. Process Biochemistry 47(4), 659-664. DOI: 10.1016/j.procbio.2012.01.010
Charoensuk, D., Brannan, R. G., Chanasattru, W., Chaiyasit, W., 2018. Physicochemical and emulsifying properties of mung bean protein isolate as influenced by succinylation. International Journal of Food Properties 21(1), 1633-1645. DOI: 10.1080/10942912.2018.1502200.
Chittapun, S., Jonjaroen, V., Khumrangsee, K., Charoenrat, T., 2020. C-phycocyanin extraction from two freshwater cyanobacteria by freeze thaw and pulsed electric field techniques to improve extraction efficiency and purity. Algal Research 46, 101789. DOI: 10.1016/j.algal.2020.101789
Choi, W.Y., Lee, H.Y., 2018. Enhancement of neuroprotective effects of spirulina platensis extract from a high-pressure homogenization process. Applied Sciences 8(4), 634. DOI: 10.3390/app8040634.
Demirel, Z., Sukatar, A., 2019. Purification of phycocyanin from isolated and identified hot spring cyanobacteria. Indian Journal of Experimental Biology 57(5):338-345.
Dierckx, S., Patrizi, M., Merino, M., González, S., Mullor, J.L., Nergiz-Unal, R., 2024. Collagen peptides affect collagen synthesis and the expression of collagen, elastin, and versican genes in cultured human dermal fibroblasts. Frontiers in Medicine 11, 1397517. DOI: 10.3389/fmed.2024.1397517.
Fernandes, F., Campos, J., Serra, M., Fidalgo, J., Almeida, H., Casas, A., Toubarro, D., I.R.N.A. Barros, A., 2023. Exploring the benefits of phycocyanin: From Spirulina cultivation to its widespread applications. Pharmaceutical 16(592), 1-34. DOI: /10.3390/ph16040592.
Gabr, G.A., El-Sayed, S.M., Hikal, M.S., 2020. Antioxidant activities of phycocyanin: A bioactive compound from Spirulina platensis. Journal of Pharmaceutical Research International 32(2), 73-85. DOI: 10.9734/jpri/2020/v32i230407.
Ge, Y., Kang, Y.-K., Dong, L., Liu, L.-H., An, G.-Y. 2019. The efficacy of dietary Spirulina as an adjunct to chemotherapy to improve immune function and reduce myelosuppression in patients with malignant tumors. Translational Cancer Research 8(4), 1065–1073. DOI: 10.21037/tcr.2019.06.13.
Hajiyeva, S., Cankilic, M.Y., Sariozlu, N.Y., 2024. Production, Large-Scale Extraction, and Purification of Phycocyanin by Different Cyanobacteria Isolated from Various Environments. Biointerface Research in Applied Chemistry 14(4), 1-16. DOI: 10.33263/BRIAC144.091.
Hsieh-Lo, M., Castillo, G., Ochoa-Becerra, M.A., Mojica, L., 2019. Phycocyanin and phycoerythrin: Strategies to improve production yield and chemical stability. Algal Research 42, 101600. DOI: https://doi.org/10.1016/j.algal.2019.101600
İlter, I., Akyıl, S., Demirel, Z., Koç, M., Conk-Dalay, M., Kaymak-Ertekin, F., 2018. Optimization of phycocyanin extraction from Spirulina platensis using different techniques. Journal of Food Composition and Analysis 70, 78-88. DOI: 10.1016/j.jfca.2018.04.007
Jaeschke, D.P., Mercali, G.D., Marczak, L.D.F., Müller, G., Frey, W., Gusbeth, C., 2019. Extraction of valuable compounds from Arthrospira platensis using pulsed electric field treatment. Bioresource Technology 283(), 207-212. DOI: 10.1016/j.biortech.2019.03.035
Kaur, S., Khattar, J., Singh, Y., Singh, D., Ahluwalia, A., 2019. Extraction, purification and characterisation of phycocyanin from Anabaena fertilissima PUPCCC 410.5: as a natural and food grade stable pigment. Journal of Applied Phycology 31, 1685-1696. DOI: 10.1007/s10811-018-1722-9.
Kuhnholz, J., Glockow, T., Siebecke, V., Le, A. T., Tran, L.-D., & Noke, A., 2024. Comparison of different methods for extraction of phycocyanin from the cyanobacterium Arthrospira maxima (Spirulina). Journal of Applied Phycology36, 1725–1735, 1-11. DOI: 10.1007/s10811-024-03224-y.
Lu, Y., Pan, D., Xia, Q., Cao, J., Zhou, C., He, J., Sun, Y., Xu, S., 2021. Impact of pH-dependent succinylation on the structural features and emulsifying properties of chicken liver protein. Food Chemistry 358, 129868. DOI: 10.1016/j.foodchem.2021.129868.
Li, Y., Zhang, Z., Paciulli, M., Abbaspourrad, A., 2019. Extraction of phycocyanin—A natural blue colorant from dried spirulina biomass: Influence of processing parameters and extraction techniques. Journal of Food Science 85(3):727-735. DOI: 10.1111/1750-3841.14842.
Mao, M., Han, G., Zhao, Y., Xu, X., Zhao, Y., 2024. A review of phycocyanin: Production, extraction, stability and food applications. International Journal of Biological Macromolecules 280(3), 135860. DOI: 10.1016/j.ijbiomac.2024.135860
Moraes, C.C., Sala, L., Cerveira, G. P., & Kalil, S.J., 2011. C-phycocyanin extraction from Spirulina platensis wet biomass. Brazilian Journal of Chemical Engineering 28(1), 45-49. DOI: 10.1590/S0104-66322011000100006.
Munawaroh, H.S.H., Gumilar, G.G., Alifia, C.R., Marthania, M., Stellasary, B., Yuliani, G., Wulandari, A.P., Kurniawan, I., Hidayat, R., Ningrum, A., 2020. Photostabilization of phycocyanin from Spirulina platensis modified by formaldehyde. Process Biochemistry 94, 297-304. DOI: 10.1016/j.procbio.2020.04.021.
Nikolova, K., Petkova, N., Mihaylova, D., Gentscheva, G., Gavrailov, G., Pehlivanov, I., Andonova, V., 2024. Extraction of phycocyanin and chlorophyll from Spirulina by “Green Methods. Separations 11(2), 57. DOI: 10.3390/separations11020057
Paramanya, A., Abiodun, A.O., Ola, M.S., Ali, A., 2024. Enhancing the quality and antioxidant capacity of phycocyanin extracted from Spirulina platensis PCC 7345: A quality-by-design approach. Arabian Journal of Chemistry 17(3), 105653. DOI: 10.1016/j.arabjc.2024.105653.
Patel, A., Mishra, S., Pawar, R., Ghosh, P., 2005. Purification and characterization of C-Phycocyanin from cyanobacterial species of marine and freshwater habitat. Protein Expression and Purification 40(2), 248-255. DOI: 10.1016/j.pep.2004.10.028.
Rame, R., Silvy, D., Novarina, I.H., Agus, P., Ganang, R.D.H., 2018. Cell-wall disruption and characterization of phycocyanin from microalgae: Spirulina platensis using Catalytic ozonation. E3S Web of Conferences.
Rastogi, R. P., Sonani, R. R., & Madamwar, D., 2015. Effects of PAR and UV radiation on the structural and functional integrity of phycocyanin, phycoerythrin and allophycocyanin isolated from the marine cyanobacterium Lyngbya sp. A09DM. Photochemistry and Photobiology 91(4), 837-844. DOI: 10.1111/php.12449.
Renugadevi, K., Valli Nachiyar, C., Sowmiya, P., Sunkar S., 2018. Antioxidant activity of phycocyanin pigment extracted from marine filamentous cyanobacteria Geitlerinema sp TRV57. Biocatalysis and Agricultural Biotechnology 16, 237-242. DOI: 10.1016/j.bcab.2018.08.009.
Ruiz-Domínguez, M.C., Jáuregui, M., Medina, E., Jaime, C., Cerezal, P., 2019. Rapid green extractions of C-phycocyanin from Arthrospira maxima for functional applications. Applied Sciences 9(10), 1987. DOI: 10.3390/app9101987
Sarada, R., Pillai, M.G., Ravishankar, G.A., 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 Biochemistry 34(8), 795-801. DOI: 10.3390/app9101987.
Shao, W., Ebaid, R., El-Sheekh, M., Abomohra, A., Eladel, H., 2019. Pharmaceutical applications and consequent environmental impacts of Spirulina (Arthrospira): An overview. Grasas y Aceites 70(1), e292-e292. DOI: 10.3989/gya.0690181
Soliman, T. N., El-Dein, A. N., Abd Al-Diam, S., Allayeh, A., Awad, H., Flefil, N. S., 2024. Characterization of C-phycocyanin antioxidant, anti-inflammatory, anti-tumour, and anti-HCoV-229E activities and encapsulation for implementation in an innovative functional yogurt. Heliyon 10(11). DOI: 10.3989/gya.0690181.
Stadnichuk, I., Krasilnikov, P., Zlenko, D., 2015. Cyanobacterial phycobilisomes and phycobiliproteins. Microbiology 84, 101-111. DOI: 10.1134/S0026261715020150.
Stanic-Vucinic, D., Minic, S., Nikolic, M.R., Velickovic, T.C., 2018. Spirulina phycobiliproteins as food components and complements. Microalgal Biotechnology 5,129-149. DOI: 10.5772/intechopen.73791.
Sudhakar, M., Jagatheesan, A., Perumal, K., Arunkumar, K., 2015. Methods of phycobiliprotein extraction from Gracilaria crassa and its applications in food colourants. Algal Research 8, 115-120. DOI: 10.1016/j.algal.2015.01.011.
Sun, L., Wang, S., Qiao, Z., 2006. Chemical stabilization of the phycocyanin from cyanobacterium Spirulina platensis. Journal of Biotechnology 121(4), 563-569. DOI: 10.1016/j.jbiotec.2005.08.017.
Tavanandi, H.A., Mittal, R., Chandrasekhar, J., Raghavarao, K., 2018. Simple and efficient method for extraction of C-Phycocyanin from dry biomass of Arthospira platensis. Algal Research 31, 239-251. DOI: 10.1016/j.algal.2018.02.008
Villela, K.S.V., Galindo, R.B., Neira, R.E., Solís, V.E., García-Casillas, P.E., 2024. Stability of Starch-Folic Acid/Polyethylene glycol particles for gastrointestinal drug delivery. Food Hydrocolloids 157, 110318. DOI: 10.1016/j.foodhyd.2024.110318.
Journal of Fisheries
Volume 78, Issue 3
October 2025
Pages 189-203

Files

Share

How to cite

Statistics