Study On Extraction And Purification Of Phycocyanin From Spirulina Platensis

Phycobiliproteins,a class of intracellular colored proteins,were generally produced by algae.Among them,phycocyanin possessed high proportion in phycobiliproteins with wider application area,which was mainly derived from Spirulina platensis.Cell destruction was the first step for the extraction of intracellular proteins.Currently,the methods used to destroy S.platensis cells mainly included water swelling,enzymatic treatment and other assisted extraction techniques.Salt solution extraction and ultrasound-assisted extraction were considered as the low-cost and effective methods for phycobiliproteins extraction.The application field and value of phycocyanin were determined by its purity.Aqueous two-phase system（ATPS）was regarded as a scalable purification method with mild condition,which was beneficial for the purity and stability of phycocyanin.In order to improve the extraction rate of phycobiliprotein from S.platensis powders,the best salt was selected as the extraction solution and its extraction parameters were optimized.Furtherly,ultrasound was used to assist extraction of phycobiliprotein with the salt solution to improve the extraction efficiency.The ultrasound-assisted extraction was scaled up in a 20-L ultrasonic device by external circulation.To improve the purity of phycocyanin,the method of ATPS combined with ultrafiltration was established for the one-step cell separation and phycocyanin purification from the phycobiliprotein extract containing S.platensis cells.The main conclusions of this study were as follows:（1）Using S.platensis as the source of phycobiliprotein,the effects of different sodium salts and chloride salts on the extraction rate of phycobiliprotein were investigated.Na Cl was selected as a promoter for the phycobiliprotein extraction.After the optimization by single factor and response surface method,the optimal extraction conditions were obtained as follows:liquid-solid ratio of 20,rotation speed of 125 rpm,extraction time of 24 h,Na Cl of 40 g/L,15^oC and p H7.Under these conditions,the extraction rate and yield of phycobiliprotein reached 74.8%and 139.4 mg/g,respectively,which were close to the predicted value.In this case,the phycocyanin yield was 92.4 mg/g.The extraction kinetics of phycobiliprotein indicated that a faster and better release of phycobiliprotein was observed during the extraction process with Na Cl solution.The S.platensis cells after extraction with Na Cl solution exhibited severe fragmentation,rougher surface and enhanced pores.The fluorescence of intracellular phycocyanin became weaker after the extraction with Na Cl solution.These results showed that Na Cl promoted the release of intracellular phycocyanin by destroying the integrity of algal cells.The addition of Na Cl didn’t affect the secondary structure of phycobiliprotein,and improve the preservation rate of phycobiliprotein pigment in the extract without loss in the antioxidant activity of phycobiliprotein.（2）Based on the optimal process condition for the extraction of S.platensis phycobiliprotein with Na Cl solution,ultrasound-assisted extraction method was introduced to enhance the extraction efficiency of phycobiliprotein.After the parameter optimization of ultrasound condition,the extraction rate of phycobiliprotein increased to 87.0%under the two 15-min ultrasonic treatments at 8 h and 10 h with the power of60 W and the frequency of 40 k Hz.In this case,the yield of phycobiliprotein was 160.6mg/g and the yield and purity of phycocyanin were 107.5 mg/g and 0.75,respectively.The extraction time was shortened to 16 h with the ultrasonic treatment.According to the process condition of ultrasonic intensification at flask-level,the scaling-up of phycobiliprotein extraction was performed in a 20-L ultrasonic extraction device with external circulation.Under the 150-W ultrasonic treatment with 210-min circulation at8 h,the extraction rate of phycobiliprotein was 90.0%and the yield of phycobiliprotein was 166.1 mg/g,where the yield of phycocyanin reached 111.2 mg/g and no change occurred in its purity.Compared that in the extraction with Na Cl alone,S.platensis filaments were more easily broke into single cells or cell fragments with the ultrasonic treatment,resulting in rougher cell surface with more pores.It facilitated the accelerated release of intracellular phycobiliproteins from algal cells.The fluorescence analysis of intracellular protein further indicate that ultrasonic treatment resulted in more intracellular phycocyanin released into the solution.Compared that obtained by extraction with Na Cl alone,no significant change was observed in the secondary structure and antioxidant activity of phycobiliprotein achieved by ultrasound-assisted extraction with Na Cl.The stability of phycobiliprotein extracted by ultrasound-assisted method was improved due to its more negative Zeta potential in the aqueous system.（3）An ATPS method was established for the one-step cell separation and phycocyanin purification from the phycobiliprotein extract containing algal cells.The ATPS system was determined to be consisted of 20 wt%PEG4000 and 10 wt%C₆H₅K₃O₇ after the selection of phase forming salts and polymers in ATPS,the fitting of phase diagram and the concentration optimization of phase forming salts and polymers.The optimal purification conditions of ATPS were as follows:dilution rate of 2 for the original extract,homogenization time of 3 s,p H 8（without p H adjustment）,and 15^oC.After ATPS purification,the recovery rate of phycocyanin was 102.2%with a purification factor of 1.7,where its purity increased to 1.28.In ATPS,PEG and C₆H₅K₃O₇ promoted the migration of phycocyanin to the top phase by changing the protein microenvironment,resulting in better purification of phycocyanin.The FTIR analysis of the surface of S.platensis cells confirmed the presence ofβ-glucan andα-glucan.After homogenization,the fragments of S.platensis cells with suitable size exhibited the characteristic of polysaccharide micropolymers in ATPS.The possible PEG-glucan system in the ATPS could be one of the reasons for the one-step cell separation.The FTIR spectrum and fluorescence spectrum of phycocyanin demonstrated that the functional integrity of phycocyanin was maintained during its purification by ATPS.Further purification of the ATPS purified phycocyanin was carried out using ultrafiltration with different.The results showed that ultrafiltration with a molecular weight cutoff of 30 k Da could provide the best phycocyanin recovery rate and purification factor.SDS-PAGE and UV spectroscopy analysis also confirmed this conclusion.After the original extract was purified by ATPS and ultrafiltration,the recovery rate of phycocyanin was 92.0%and its purification factor was 3.7,where the purity of phycocyanin was improved 2.78.