Study On Purification Of Phycoerythrin By Three-Phase Partitioning And Stability Of Phycoerythrin

R-phycoerythrin（R-PE）is a kind of protein as a photosynthetic auxiliary pigment in red algae.This protein is of great significance in many biotechnological applications in food science,immune diagnosis,treatment,cosmetics,protein and cell markers,and analysis.Nowadays,R-PE’s simple and efficient extraction has become a research hotspot.As a simple and efficient non-chromatographic method,three-phase partitioning（TPP）has significant advantages in high throughput,easy scale and cost-effectiveness.It has become an effective method to extract R-PE.In this paper,Porphyra yezoensis was used as raw material to optimize the extraction process of R-PE by three-phase extraction,and the extraction mechanism was deeply studied,Then,by studying the influence of various factors on the stability of R-PE,the optimal conditions that can improve its stability are selected.The main research contents are as follows:The crude extract of R-PE from Porphyra yezoensis was extracted by repeated freeze-thaw method and salted out by ammonium sulfate.A three-phase partitioning system was constructed to extract R-PE and optimized by single factor.Taking the purity as the evaluation index,the optimum extraction conditions were as follows:salt saturation of 70%,temperature of 25℃,p H of 7.0,time of 45 min and volume ratio of upper and lower phases of 1:1.Through optimization,R-PE with purity of 3.90 was successfully purified,and the recovery rate was 39.6%.The experiments of UV,FTIR,fluorescence and SDS-PAGE showed that the purity of R-PE was improved,and its structure was consistent with that before and after purification.In addition,by studying the extraction effect of recovered tert butyl alcohol,it is proved that the three-phase partitioning method is a green and recyclable extraction method.By calculating the distribution of R-PE in the three-phase system,the thermodynamics and kinetics of three-phase partitioning of R-PE were analyzed.The results show that the thermodynamic parameters of the reaction processΔS andΔH are18.90 J/mol^-1K^-1 and-4811.88 J/mol^-1 respectively.In the temperature range of 283.15 K to 303.15 K,theΔG is less than 0,which proves that the reaction can be carried out spontaneously in the range of 10-30℃.The kinetic fitting results showed that the extraction process fitted the second law of the rate model best,and the kinetic equation was Ct=t/（0.7958+0.9836*t）.The physical and chemical factors affecting the stability of R-PE were studied,that is,the effects of temperature,p H,and light on the stability of R-PE were measured.The results showed that temperature,p H,and light had certain effects on the stability of R-PE.The stability of R-PE was the highest when the temperature was 4℃,p H was 6.0,and stored away from light.The effects of additives on the stability of R-PE were studied.The results showed that glucose,sucrose,lactose,and soluble starch could improve the stability of R-PE.Among them,30 mg/m L glucose has the most obvious effect on the stability of R-PE.After adding 30 mg/m L glucose,the C_R value of R-PE is 99.42%,which is 23.75%higher than that of the control group.In this study,high purity R-PE was purified from Porphyra yezoensis by three-phase partitioning,and the extraction mechanism was deeply studied.In addition,the stability of R-PE was studied,and the best storage conditions of R-PE were obtained.This study will provide an important theoretical basis for the separation and purification of phycoerythrin and other algal proteins by TPP.