Study On The Optimized Purification Of C-PC Based On Deep Eutectic Aqueous Two-phase System And Its Characteristics

Phycocyanin(C-phycocyanin,C-PC)is a natural fluorescent protein that could be eaten directly,which has been widely used in multiple aspects,including food,health care,medical treatment and biological targeted therapy based on its unique biophysical characteristics.Internationally,the purity of C-PC that determines its values is usually divided into three grades,food grade,analytical grade,and reagent grade included.The higher the purity of C-PC is,the greater value of its application will be.Therefore,it is of great theoretical and practical significant to find a more efficient and convenient green method for the separation and purification of C-PC.Deep Eutectic Solvents(DES)is a new type of green solvent following Ionic Liquids(IL),which has similar physical and chemical properties compared with ionic liquids,but could be the raw materials is greener,the production cost is lower and the preparation process is more simple.Deep Eutectic Solvents-Aqueous Two-Phase System(DES-ATPS)is a new green extraction and purification system that combine the advantages of the DES and ATPS,which not display only good biological compatibility,no emulsification,no pollution,low preparation cost and simple operation process,but also has been applied in the purification of various marcomolecules,including nucleic acids,bovine serum albumin,etc.In this study,five kinds of DES-ATPSs were constructed and applied in the purification of C-PC,the purification conditions were optimized using the response surface method,and the stability and antioxidant capability of the purified C-PC were also analyzed.The research contents are as follows:Firstly,five kinds DESs were synthesized using choline chloride as the hydrogen bond acceptor and urea,D-(+)-glucose,D-(-)-fructose,ethylene glycol,and glycerol as the hydrogen bond donors,respectively.DES-ATPSs were then constructed through the DESs and K₂HPO₄salt solution.The difficulty of phase formation of five kinds of DESs was as follows:ChCl-U>D-ChCl-Fru>D-ChCl-Glu>ChCl-EG(?)ChCl-G.The p H values of hydrogen bond donor and their corresponding DESs were measured,and it was found that the type of hydrogen bond donor and its molar ratio had significant influence on its corresponding DES acid-base.The thermodynamic parameters in the extraction process were also determined,and the results showed that the enrichment of C-PC in DES-phase was spontaneous and an endothermic process.Taking Spirulina platensis as research material,the crude extract of C-PC is extracted by repeated freeze-thaw method.DES-ATPS were applied to the subsequent separation and purification of C-PC,and the response surface method were utilized for the optimization of the purification process.The results revealed that purity of C-PC reached the optimal level of 3.376 using the DES synthesized by choline chloride-urea,when the weight of DES was 3.26 g,the salt concentration of K₂HPO₄was 1.0 g m L^-1,the amount of Na Cl added in to the system was 1%,the extraction temperature was25?,and the extraction time was 3.0 h.The purification results was further verified by SDS-PAGE,which proved that the model could be applied to the optimal separation of C-PC.The stability and antioxidant capability of C-PC was also studied by the ABTS and DPPH free radical scavenging methods.C-PC should be processed and applied under the conditions of no higher than 35?and p H environment of 7.0.Certain addition of sugar and salt could improve the stability of C-PC.It was also found that the[choline chloride-urea]DES should not be used for the storage of C-PC for a long time.The IC₅₀of ABTS free radical scavenging rates of the crude extract with purity of 1.211 and the purified sample with purity of 3.365 were 0.116 mg ml^-1and 0.093 mg ml^-1,and the IC₅₀of DPPH scavenging rates were 0.347 mg ml^-1and 0.241 mg ml^-1,respectively,which displayed good antioxidant capability.