| Phycocyanin (PC) is the important component of phycobiliprotein, mainly existed in blue-green algae, red algae, cryptophyta and some species of pyrrophyta. Arthrospira (Spirulina) platensis has a high content of phycocyanin. As the natural blue colorant, phycocyanin has a variety of bioactive properties, such as antioxidant, free radicals scanvenging, immunology improving, anti-inflammatory, hepatocyte protecting, as well as the high efficient fluorescent characteristic. So it has been widely applied to the fields of food, cosmetics, pharmaceuticals, molecular probes. Nowadays, the main problem of commercial production of phycocyanin is due to be absence of the stable-efficient-economic preparation technology. This study focused on the extraction and purification of phycocyanin and how to produce food grade phycocyanin in pilot scale, aiming at offerring some basal data for the large scale production of phycocyanin.Different extraction methods have different impacts on the yield. Firstly, response surface methodology was used to optimize the extraction assisted with ultrasonic wave. Then, comparison was conducted among different extraction methods. Secondly, hydrophobic interaction chromatography (HIC) was made use of to purify phycocyanin after the treatment of ammonium sulfate. It has many advantages, such as reducing steps, improving efficiency. Analytical phycocyanin was obtained with the single step chromatography of Macro-Prep Methyl HIC. Phycocyanin is only produced in laboratory in China. The pilot scale production of phycocyanin was studied in this study, achieving the production capability of 1 kg. The technological data was helpful for the large scale production.Ultrasound technology was used to extract phycocyanin from Spirulina platensis. The effects of processing parameters were optimized by the response surface methodology (RSM). The high determination coefficient(R2=99.95%) indicated that the mathematical model was quite accurate to be applied to optimize phycocyanin extraction. The results of the optimization were described as follows:ratio of PBS to raw material 21 mL/g, ultrasonic power 640 W, and extraction time 14 min. The yield of phycocyanin was 10.76% under these conditions, which perfectly matched the predicted value (10.77%). Comparison was made among three different methods including ultrasonic wave extraction, freezing and thawing extraction and air incubating extraction. The phycocyanin yields were 10.76%, 7.89%, and 6.57%, respectively. Ultrasonic wave extraction method achieved a great reduction in time and got the highest yield.Macro-Prep Methyl HIC was used to purify phycocyanin. Ammonium sulfate was gradually resolved into phycocyanin crude extract to make its concentration 1.25 mol/L. The mixture was kept static for 12 h and then centrifuged. After centrifugation, supernatant was degassed and injected into the HIC. Only by using the single column chromatography could the purity be improved to 4.017 and the recovery was 19.38%. UV-vis absorption spectrum and fluorescence excitation-emission spectra of the purified protein was quite fit for the characteristics of phycocyanin. Native-PAGE showed only one band, which indicated that the purified phycocyanin was homogeneous. SDS-PAGE had two bands. Molecular weight of the two bands were 15.4 kDa and 17.3 kDa, respectively. They are a subunit and β subuint of phycocyanin. The whole purification consists of two major operations:ammonium sulfate precipitation and HIC. After precipitation, the mixture was centrifuged. Supernatant was degassed and then directly purified by HIC with no special treatments. The former operation unit was easily linked to the latter. Generally, the purification procedure was economic and fit for commercial production of phycocyanin with high purity.Spirulina powder was mixed with water in the ratio of 1:20, and cell walls were disrupted by 3 times freezing and thawing. After that, centrifugation was conducted to obtain the phycocyanin crude extract to which ammonium sulphate was gradually added to achieve 30% saturation. Resulting solution was kept under static condition for 12 h and then filtrated by the system composed of 2μm～1 um～0.65 um～0.22 urn membranes. The solution gained from flitration was desalted and concentrated by untrafiltration. Finally, concentrated solution was dried by spray dryer.1 kg procution capability in medium scale was successfully achieved according to the procedure. |
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