Spirulina spp, or Arthrospira, is a microscopic and filamentous cyanobacteria with a wide variety of applications including its use as a food source; in fact, Spirulina has been used as food in Mexico since pre-Hispanic times. (Dillon, Phuc, & Dubacq,^[1]; Venkataraman,^[2]).

Nutritional and functional properties of Spirulina
  The popularity of Spirulina as a food supplement is due to its high protein content (about 70% of its dry weight) and high biological value (containing essential amino acids like phenylalanine and methionine). Spirulina also contains vitamins: B₁₂ (cyanocobalamin), B₆ (pyridoxine), B₁ (thiamine) and B₂ (riboflavin), as well as beta carotenes (precursors of vitamin A); minerals (e.g., iron, zinc, selenium, calcium and magnesium), phytochemicals (phenolic acids and tocopherols), and essential fatty acids (such as gamma linoleic acid) (Belay,^[3]; Dillon et al.,^[1]; Habib et al., 2008).

  Spirulina is considered a functional food because of its broad spectrum of biologic effects, which have been demonstrated in vitro and in vivo. Among these, Spirulina has been reported to exhibit anti-inflammatory (Remirez, Ledón, & González,^[4]), anti-hyperlipidemic (Torres-Duran, Ferreira-Hermosillo, & Juarez-Oropeza,^[5]), hypoglycemic (Lima, Facchinetti, & Santos,^[6]), antihypertensive (Torres-Duran et al.,^[5]), antineoplastic (Mittal, Suresh Kumar, Banerjee, Rao, & Kumar,^[7]), antiviral (Lee et al.,^[8]), antianemic (Simsek, Karadeniz, Kalkan, Keles, & Unal,^[9]), and antioxidant activity (Karkos, Leong, Karkos, Sivaji, & Assimakopoulos,^[10]). All of these nutraceutical benefits are attributed to substances in Spirulina known as phycobiliproteins (C-phycocyanin, allophycocyanin, phycoerythrin and phycoerythrocyanin), which constitute their own protein complex in association with their linker polypeptides, called phycobilisome (Gantt, Lipschultz, Grabowski, & Zimmerman,^[11]; Hoseini, Khosravi-Darani, & Mozafari,^[12]). Isolation and testing of these phycobiliproteins has shown that they possess same beneficial effects as the whole microalgae. Hence, they are considered as the actual bioactive agents in this functional food (Eriksen,^[13]; Hoseini et al.,^[12]; Khan et al.,^[14]).

Obtention of C-phycocyanin: brief historical perspective
  Different methods of extraction, isolation and purification (summarized in Figure 1) have been assayed in order to obtain these phycobiliproteins (especially C-phycocyanin) from Spirulina spp (Glazer, Lundell, Yamanaka, & Williams,^[15]; Khan et al.,^[14]). The first attempts involved simple chromatography by using precipitations previously obtained with ammonium sulphate. Subsequently, phycobiliproteins were isolated by crystallization (Carra,^[16]). However, these methods lacked specificity as they extracted the whole phycobilisome without separating each pigment.

Figure 1:

  Years later, a thermal shock-based separation technique allowed pigment separation via density-gradient centrifugation with sucrose (Bekasova, Muslimov, & Krasnovskii,^[17]). Although these processes represented an advance, the overall purity and yield of the process was still low.

  With the use of reversed phase high-performance liquid chromatography (RP-HPLC), it was possible to achieve an isolation of up to 85% of C-phycocyanin and allophycocyanin. (Swanson & Glazer,^[18]) Nevertheless, other compounds apart from the phycobilisome were present in the final products due to the sample pre-treatments needed to perform HPLC. In order to optimize phycobiliprotein isolation, the chromatographic method was modified by adapting resins used in the solid phase, varying the polarity and pH of the eluent solution (Moreno et al.,^[19]), and using magnesium chloride precipitation with further diffusion in polyethylene glycol gel. By enhancing overall specificity, the isolation of pure C-phycocyanin and allophycocyanin was obtained, but the yielding mass was significantly decreased.

  Towards the end of the 20^th century, electrophoresis-based techniques were tested with the addition of laser-induced fluorescence (LIF) detectors. This novel achieved separation with a fairly good yield (≈90 - 100%). Nevertheless, such extractions corresponded to a mixture of both C-phycocyanin and allophycocyanin (Viskari & Colyer,^[20]). Yet another type of electrophoresis was used with polyacrylamide/dodecyl sulphate gel, pre-treating the samples by precipitation with ammonium sulphate followed by further separation in chromatographic columns by Sephadex (Minkova et al., 2003). This technique achieved an isolation of pure C-phycocyanin with a yield of ≈45%.

  Afterwards, different combinations of methods were tested in order to increase the ease of separation and isolation as well as the grade of purity and final yield. One such method, HPLC coupled with a flame ionization detector (Zolla & Bianchetti,^[21]), was able to separate C-phycocyanin from allophycocyanin, yet it destroyed the original sample. In order to resolve this problem, an integral procedure was devised that extracted the phycobiliproteins with sodium phosphate neutral buffer, and then further purified them via dialysis and gel filtration chromatography (Bhaskar, Gopalaswamy, & Raghu,^[22]). This procedure yielded C-phycocyanin with a purity of 4.98. Another method, pre-treated the sample in the same manner, but the purification process consisted of ion-exchange chromatography (Patel, Mishra, Pawar, & Ghosh,^[23]), yielding C-phycocyanin with a purity of 4.42.

  Another widely studied method for isolating C-phycocyanin from Spirulina platensis combined chromatography with expanded bed adsorption, anion interchange, and hydroxyapatite columns (Niu, Wang, Lin, & Zhou,^[24]). These techniques yielded 4.45 mg of C-phycocyanin per gram of dried S. platensis with a purity of 3.2. This method offers several advantages, such as the possibility of using different resins with special charge characteristics (i.e., anionic or cationic). For instance, by using Q-Sepharose (Silveira, de Menezes Quines, Burkert, & Kalil, 2008) it was possible to isolate C-phycocyanin from Spirulina platensis with ~75% yield and a purity of 3.4. The problem with these kinds of techniques is that they are often strongly dependent on the pH and temperature of the eluent solutions.

  On the other hand, hydrophobic interaction chromatography with ammonium sulphate and liquid nitrogen precipitation pretreatments (Soni, Trivedi, & Madamwar,^[25]) are capable of isolating C-phycocyanin with a purity of 4.5 but with poor yields. This may be due to the original cyanobacterium (Phormidium fragile) from which the phycobilin was isolated. To improve the yield, C-phycocyanin was extracted from Spirulina platensis with high-speed counter-current chromatography (HSCCC) (Yin et al.,^[26]), obtaining 78.7 mg per 200 mg of crude extract with a purity of 4.25. Nowadays, one of the most widely used methods is ionic exchange chromatography, which involves pre treating vegetable samples of Spirulina platensis with two aqueous phases(Patil, Chethana, Sridevi, & Raghavarao,^[27]), leading to a purity of 6.69.

  The aim of the present review was to describe different methods for C-phycocyanin extraction and purification and compare the results in order to determine the method with the best cost-benefit ratio.

Methods

  We performed an exhaustive search (using Scopus and PubMed databases) to find methods for the separation, isolation and purification of C-phycocyanin from different microorganisms. Search words were: extraction, separation, isolation and purification of C-phycocyanin.

Results

Summaries and general characteristics are herein presented (see Table) for the 86 reports found.

Table :

Discussion

  Novel extraction, isolation, and purification processes for C-phycocyanin have been sought and developed since the 1980’s.(Khan et al.,^[73]) Throughout this process, it has been demonstrated that the original biomass is of critical importance in order to reach the best cost-benefit ratio when isolating phycobiliproteins. Another feature that must be considered is the freshness of the biomass and the subsequent pretreatment processes. In this series, C-phycocyanin was obtained from fresh biomass and dried at room temperature (as opposed to using lyophilized powders)(Chaiklahan et al.,^[15] 2011; Niu et al.,^[50]).

  Regarding extraction and purification methods, previous studies have shown that multiple cycles improved the purity grade of the C-phycocyanin extract, although yields significantly decreased. For instance, an aqueous two-phase system with polyethylene glycol 4000 (Patil et al.,^[4]) managed to increase purity, but yields were importantly reduced. Similarly, a multiple extraction process for obtaining C-phycocyanin by using a Sephadex column (Minkova et al., 2003) achieved a yield of 46% with acceptable purity (established by Rito-Palomares et al.,^[5]).

  Based on the information herein gathered, a protocol was proposed with a one-step extraction processin order to obtain both a good yield and a high grade of purity. This was accomplished by using an aqueous two-phase system with a posterior ultrafiltration, giving C-phycocyaninin a yield of 57% and a purity of 3.9, thus surpassing the results of previous methodologies. It can be clearlyseen that the preferred method should not be based on adsorption or elucidation, thus ruling out chromatography, because these processes diminish the yield of the extract (Cruz de Jesus,^[74]).

Conclusion

  To maximize the health benefits that may be obtained from nutraceuticals, such as C-phycocyanin, it is essential to seek innovative methods for their isolation and purification,and thus preserve the valuable properties of these bioactive substances from natural sources. The current review makes it evident that to obtain nutraceuticals from extractsand achieve good yield and high purity;it is convenient to use aqueoustwo–phase systems for extraction together with ultrafiltration for purification.It is also essential to consider the freshness and species of the primary biomass, as these factors heavily influencethe concentration and viability of the desired phycobiliproteins and therefore affect the yield and purity.

References