Table 1 shows the yields of phycocyanin isolated from Spirulina platensis. The yield using the conventional separation method was 7.2%, and the yield using the hexane separation method without the high-pressure process was 8.4%. This difference in yield was attributed to the lower phycocyanin loss due to the use of fewer chemical components and the simpler separation process. Moreover, we found that the yield of the isolated phycocyanin using both the high-pressure process and the hexane separation method increased up to 10.2%. This increase is attributable to the increased elution of biological active materials from Spirulina platensis as a result of the high pressure destroying the cell membrane. Furthermore, although the cell membrane of Spirulina platensis was destroyed by the high pressure, the temperature-sensitive polypeptide subunits (α and β) that make up phycocyanin were not damaged, thus increasing the yield [12].

We then compared the purities of the isolated phycocyanins obtained using the three different methods, the high-pressure process and hexane separation method, the hexane separation method alone, and the conventional separation method, by measuring the absorbances of these isolated phycocyanins at 620 nm and 280 nm and comparing, as shown in Table 2. The results showed that the purity of the phycocyanin isolated using the conventional separation method (A) was 0.884 and that the purity of the isolated phycocyanin using the hexane separation method without the high-pressure process (C) was 0.882, which was similar to (A). Compared to the results from the two processes of (A) and (C), the data from the combined process (B) showed both a better extraction yield and a higher purity. These data suggest that this high pressure extraction can maintain optimium stability of phycocyanin during the purification process due to less denaturation and broken bonds of the proteins in the complex structure. Also, the purity of the phycocyanin isolated using the hexane separation method was calculated as 0.881 using the absorbance values of 2.125 at 280 nm and 1.873 at 620 nm. The phycocyanin isolated using the high-pressure process and hexane separation method had a purity of 0.909 based on absorbances of 2.104 at 280 nm and 1.912 at 620 nm. Thus, the purity of the phycocyanin isolated using the hexane separation method and high-pressure process was slightly better than that of the phycocyanin standard, 0.904. The higher purity is due to the decrease in the absorbance of the total protein at 280 nm while the absorbance of the specific phycocyanin at 620 nm increased. The phycocyanin isolated using the high-pressure process is thought to be stable because it was isolated under low-temperature and high-pressure conditions; these conditions destroyed the cell membrane of Spirulina platensis without denaturing phycocyanin, which is a chromoprotein that is composed of temperature-sensitive α and β polypeptide subunits [13]. The hexane separation method can be used as a new separation method because it requires fewer chemical components, a simpler separation process, and a shorter separation time. Thus, the high-pressure process with the hexane separation method used in this study is considered to be the best method to isolate under stable coditions a highly purified phycocyanin and is expected to solve the issues associated with the current method, such as the denaturation and destruction of phycocyanin, the long separation time, and the high cost.

Thus, if the phycocyanin is used in the cosmetic industry in the future, the hexane separation method is expected to establish itself as a more effective and stable method due to its use of fewer chemical components and its shorter and simpler process.

The molecular weights of the phycocyanin isolated using the high-pressure process and hexane separation method described in this study and those of the phycocyanin standard are shown in Figure 1. The α and β polypeptide subunits of the phycocyanin standard were detected, and their molecular weights were 18.4 kDa and 21.3 kDa, respectively. These weights are consistent with those of previous reports [14]. Moreover, the phycocyanin isolated using the high-pressure process and hexane separation method had two α and β polypeptide subunits that migrated to the same location on the gel and had the same molecular weights as the phycocyanin standard. Furthermore, the bands were clearer than those of the phycocyanin standard. This finding suggests that the high-pressure process and hexane separation method enabledd isolation of a stable, highly purified phycocyanin with a short treatment time using fewer chemical components and increasing the structural stability of the α and β polypeptide subunits.

To measure the stability of the phycocyanin isolated using the high-pressure process and hexane separation method, the changes of the absorbance of the phycocyanin solution were measured over time, as shown in Figure 2. The phycocyanin from the conventional extraction process had the poorest stability because the absorbance of the phycocyanin from the conventional separation method decreased to 0.808 after 14 days while the phycocyanin standard decreased to 0.841 after 14 days. However, the absorbance of the phycocyanin isolated using the high-pressure process and hexane separation method remained over 0.884 for 14 days. These data suggest that the phycocyanin isolated using the high-pressure process and hexane separation method, compared to two other samples, was more stable because the low temperature and high pressure extraction process effectively destroyed the cell membrane of Spirulina platensis while minimizing the damage to the polypeptide subunits of phycocyanin and increasing the elution of phycobiliprotein. Furthermore, the reduced chemical usage, simple separation process, and short process time of the hexane separation method contributed to the attainment of stable phycocyanin containing two chromophore subunit proteins intact along with a relatively pure form. Phycocyanin with high stability and purity is expected to have an application as a new material in the cosmetic and beauty industry.

To verify the antioxidant activity of phycocyanin, the electron donating ability of the phycocyanin standard and of the phycocyanin isolated using the high-pressure process and hexane separation method were measured. The results are shown in Figure 3. All of the phycocyanins showed increased activity as their concentrations were increased. Phycocyanin isolated using the high-pressure process and hexane separation method showed a high removal rate of oxygen free radicals (83%) at a concentration 50 μM and showed 9% higher activity than that of the phycocyanin standard. These data imply that the purified sample from our process contained more stable chromophore phycocyanin than the samples from conventional processes, which results in better anti oxidant activity than the others.

Reducing power is defined as the ability to donate electrons to free oxygen radicals and other free radicals and is one of the various mechanisms of antioxidant activities; thus, a measurement of reducing power can be used to verify antioxidant activity. All of the phycocyanins showed increased reducing power with increased concentration. Moreover, in the reducing power test, phycocyanin isolated using the described method showed a peak absorbance value of 0.38 at a concentration of 50 μM, which was 0.1 higher than the absorbance of the phycocyanin standard and phycocyanin isolated using the conventional separation method (Table 3). This result, along with the measurement of DPPH scavenging activity, implies that the isolation of stable and highly purified phycocyanin with enhanced antioxidant activity is possible with the high-pressure process and hexane separation method. Furthermore, phycocyanin could be used as a material with antioxidant and anti-aging activities in the cosmetic and beauty industry.

In this study, S. platensis from a culture aquarium was filtered using a filter membrane and washed with water to prepare a dried powder. Washed S. platensis was powdered using a freeze-dryer, and it was used in the experiment after grinding in a grinder. Ten volumes of distilled water were added to 100 g of S. platensis powder and sealed tightly in a special plastic bag, and then hydrostatically pressurized in a 1 L volume high-pressure extraction vessel (Ilshin autoclave, Daejeon, Korea) at 5000 bar and 20 °C for 15 min [15]. After the high-pressure treatment, the sample was immersed in water at 4 °C for 12 h, and filtered by vacuum filtration using filter paper. Then, it was incubated at 4 °C for 12 h, and the water layer was isolated using fractionation. The phycocyanin in the water layer was collected and powdered by freeze-drying as a sample.

To remove beta-carotene and chlorophyll, which have low light stability, and to isolate phycocyanin from the high pressurized sample, the Spirulina platensis extract from the high-pressure treatment was mixed with hexane (Sigma, St. Louis, MO, USA) in a 1:1 volume ratio and shaken using a vortex mixer (VM-96B, JEIO TECH, Daejeon, Korea) for 5 min. Then, it was incubated at 4 °C for 12 h, and the water layer was isolated using fractionation. The phycocyanin in the water layer was collected and powdered by freeze-drying as a sample.

For comparison with the separation method of this study, phycocyanin powder was also extracted by a conventional separation method: In the first, approximately 5.0 g of S. platensis was suspended in 200 mL of 0.1 M Na-phosphate buffer pH 7.0 containing 100 g/mL lyzozyme and 10 mM EDTA. The enzymatic disintegration of the cell-wall was brought about by placing the S. platensis powder in a shaking bath at 30 °C for 24 h. The slurry was then centrifuged for 1 h at 40,000 × g to remove cell debris, yielding a clear supernatant. In the second, the crude phycocyanin was precipitated in 50% ammonium sulfate and was then recovered by centrifugation at 10,000× g for 10 min. The colourless, clear supernatant was discarded and the blue precipitate was dissolved in a small volume of 0.0025 M Na-phosphate buffer pH 7.0 and dialyzed against the same buffer. The dialyzed phycocyanin was then placed in a 2.5 × 30 cm hydroxylapatite column and the two main fractions were pooled following a stepwise elution with phosphate buffers of increasing ionic strength at pH 7.0, as follows: The first fraction was eluted between 2.5 mM and 70 mM and represented c-phycocyanin. The fraction containing c-phycocyanin was purified by chromatography on a 1.5 × 20 cm DEAE Sephadex A-50, according to Binder et al.[16,17].

The extraction yield of the phycocyanin from the three different methods was estimated by the procedure below. The phycocyanin extract in the above method was prepared using three different methods:by the hexane separation method and the high-pressure process used in this study, by the hexane separation method only without the high-pressure process, and by the conventional separation method.

The purity of isolated phycocyanin was checked by the procedure below by the ratio of 280 nm for absorbance of total proteins in phycocyanin to 620 nm of specific protein in the complex [18].

The purity of the standard, a commercially available phycocyanin (Anaspec, Inc., CA, USA) was also measured by the above method [18].

In addition to the measurement of purity, for a closer examination of the phycocyanin isolated using the high-pressure process and the hexane separation method, the molecular weight was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Mini-Protean, Bio-rad Ltd., Hercules, CA, USA) and compared with a phycocyanin standard (Anaspec, Inc., CA, USA). SDS-PAGE was performed using a 12% polyacrylamide slab gel and a 4.5% stacking gel and was confirmed after staining with Coomassie blue R25 (Sigma, St. Louis, MO, USA) and destaining. The molecular weights of the phycocyanin subunits were calculated by comparison with a standard ladder (pre-stained SDS-PAGE standard; Bio-Rad, Seoul, Korea) [14].

To measure the stability of the phycocyanin pigment isolated from Spirulina platensis at a given temperature, an aqueous solution of phycocyanin was incubated at 40 °C under UV exposure. Then, changes in phycocyanin purity were calculated from the absorbance values at 620 nm and 280 nm with an ELISA reader [19].

DPPH scavenging activity was measured using Kang’s method [20]. One milliliter of a 200-μM DPPH solution was added to a series of phycocyanin standards made by solubilizing in ethanol and 2 mL of the phycocyanin isolated using the high-pressure process and hexane separation method. The mixture was vortexed for 10 s to mix completely and incubated at room temperature for 30 min. To measure the remaining DPPH, the absorbance was measured at 517 nm by a UV-vis spectrophotometer (852A Diode Array Spectrophotometer, Agilent Technologies, Seoul, Korea).

The reducing power was measured as follows: 2.5 mL of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide were added to a series of phycocyanin standard concentrations and 2.5 mL of the phycocyanin isolated using the high-pressure process and hexane separation method. The mixture was incubated at 50 °C for 20 min. Then, 2.5 mL of trichloroacetic acid (10%, w/v) was added and centrifuged at 650× g for 10 min. Five milliliters of centrifuged supernatant was mixed with 5 mL of deionized water and 1 mL of 1% ferric chloride, and its absorbance was measured at 700 nm using a UV-vis Spectrophotometer (852A Diode Array Spectrophotometer, Agilent Technologies, Seoul, Korea) [21].

All of the analyzed values were presented as the mean ± S.D., and all of the experimental data were analyzed using the statistics software SPSS 16.0. Tests of the significance of differences were analyzed by ANOVA (analysis of variance) and DMRT (Duncan’s multiple range test) at the level of α = 0.05.