Methodological Research About Fast Separation And Purification Of Phycocyanin Via Free Flow Electrophoresis

C-Phycocyanin(C-PC)is a natural blue protein.C-PC can be used as a natural pigment protein additive for food colorant and cosmetic.Moreover,C-PC has some functional characteristics and biological activeness function,such as anti-cancer,promoting cell regeneration,regulating and enhancing immune system,improving resistance to disease.However,the existing methods of separation and purification of C-PC are tedious and time-consuming,both the purity of the products and the recovery rate are low,limiting its extensive application.Free flow electrophoresis(FFE),as a carrier-free and liquid-based electrophoresis separation technique,can be used for analytical and preparative proteins.The mild separation conditions benefit FFE in treating bio-molecule or bio-particle fractionation,which may help maintaining their high bioactivities.The application of FFE on C-PC purification provides possible for producing high purity C-PC(analytical pure)massively.In this work,free flow zone electrophoresis(FFZE)and free-flow isoelectric focusing electrophoresis(FFIEF)were used for separation and purification of C-PC from Spirulina platensis,respectively,improving products purity and recovery rate.The research was consisted of three sections:(1)The sample injection method of FFZE was introduced to improve the resolution of electrophoresis.A simple sheath-flow sample injection method(SFSIM)was developed for FFZE,instead of traditional injection method(TIM)choosing one of the background electrolyte(BGE)inlets as the sample inlet.A needle injector was placed in the center of the separation inlet,into which the background electrolyte(BGE)and sample solution were pumped simultaneously.BGE formed sheath flow outside the sample stream,and cylindrical sample stream was isolated from the upper and lower surface of separation chamber.It was revealed that the resolution of this new technique in separating the two kinds of protein was increased by 3.75 times.(2)FFZE was used for quickly separation and purification of C-PC via SFSIM.It was revealed that C-PC was purified from extracts of Spirulina platensis with more than 4.50 of an analytical pure purity.Especially,the most effective separation was achieved at pH 5.0,and the recovery rate of C-PC could be more than 79%.(3)Recycling free-flow isoelectric focusing electrophoresis(RFFIFF)was used for separation and purification of C-PC.It was revealed that C-PC was purified from extracts of Spirulina platensis with up to 70.31 in carrier ampholytes of pH 3.19-5.8,pH 4-7 and pH 3-10.Especially,the recovery rate of C-PC could reach 82.2% and 90.6% in carrier ampholytes of pH 3.19-5.8,or pH 4-7,respectively.