Separation,Purification And Application Of Phycobiliprotein In Red Algae

This study compared the contents of phycobiliproteins in five red algae species in Fujian Province and selected the highest content of phycoerythrin as the extraction material.In order to fully release the phycoerythrin from the Chinese cabbage,five different extraction methods were compared in this study:freeze-thawing,double enzymatic digestion,single enzymatic hydrolysis(cellulase and pectinase),shaking and ultrasound.The results show that the double enzymatic hydrolysis method can extract more phycoerythrin from the Chinese red cabbage.Single-factor optimized double enzyme extraction method to determine the optimal extraction conditions,the solid-liquid ratio of 1:50,extraction time of 5 h,extraction 2 times,the final obtained phycoerythrin 34.4 mg/g,purity 0.23.The crude phycoerythrin extract was concentrated by ultrafiltration using a hollow fiber membrane having a molecular weight of 10 kDa to obtain a phycoerythrin concentrate with a purity of 1.10.The concentrated solution was precipitated with ammonium sulfate having a saturation of 25%and 45%,respectively,to obtain a phycoerythrin pretreatment solution with a purity of 1.40.To obtain more pure phycoerythrin,four different chromatography columns were compared in this study.DEAE Sepharose FF was used to purify phycoerythrin.A linear elution was performed using 0.01 mol/L phosphate buffer(pH 7.0,containing sodium chloride 0-0.6 mol/L)to obtain phycoerythrin with a purity of 3.11.Purified phycoerythrin was characterized by SDS-PAGE.As a result of SDS-PAGE analysis,R.rubra phycoerythrin contained two different subunits,? and ?,with molecular weights of 15.0 kDa and 19.5 kDa,and no y subunit was found.The spectrum shows three characteristic absorption peaks,500,557,and 620 nm,respectively.The fluorescence spectrum has two emission peaks,574 and 640 nm,respectively.This study evaluated the antioxidative activity of red phycoerythrin,and comprehensively analyzed the ability of phycoerythrin to scavenge DPPH free radicals,superoxide radicals,hydroxyl radicals,and total reducing ability.The results showed that phycoerythrin had good antioxidant activity,among which the IC50 value of DPPH radical scavenging was 0.30 mg/mL,the IC50 value of scavenging superoxide radical was 0.26 mg/mL,and the IC50 value of scavenging hydroxyl radical was 2.60 mg/mL.Studies have found that pH,temperature,light,etc.affect the stability of phycoerythrin.The red yeast phycoerythrin can maintain better stability under the condition of 4-30 ?,pH 4-10 and darkness.This study initially explored the application of phycoerythrin in fluorescent probes and established a rapid detection model.The model uses a diode as the excitation light source,and the light passes through the filter,so that the light at 557 nm wavelength illuminates the erythra of red yam,which is the excitation.Phycoerythrin thus emits fluorescence with a specific wavelength.The fluorescence signal is transmitted through a narrowband filter capable of filtering through a specific wavelength and recorded by a miniature camera.After a series of explorations,a linear relationship between phycoerythrin concentration and fluorescence intensity RGB values was established.R2 reached 0.9962 with a detection limit of 0.04 mg/mL.This lays the foundation for the rapid detection of phycoerythrin as a fluorescent probe.