Study On The Preparation,Enzymatic Hydrolysis And Nano-Selenium Materials Of Rhodomonas Salina Phycoerythrin

Phycoerythrin（PE）is a kind of natural fluorescent pigment protein with various physiological activities such as anti-oxidation,anti-inflammation,anti-tumor and immune regulation.It has broad application prospects in the fields of functional food,medicine,cosmetics and bioengineering.At present,the research on the preparation of phycoerythrin mostly uses Porphyra,Polysiphonia urceolata,and Prophyridium as raw materials.The purification process is complicated and the production cost is high,resulting in the high price of reagent-grade phycoerythrin,which greatly limits its application.Rhodomonas salina belongs to single-celled Cryptomonas,without cell wall.There is only phycoerythrin（PE-545）in the cell,which is rich in phycobiliprotein.The preparation of phycoerythrin by Rhodomonas salina has the advantages of easy cell breakage and easy purification in the later stage,so it can be used as an excellent raw material for the preparation of phycoerythrin.In this paper,the preparation process of PE-545 was optimized by using Rhodomonas salina as raw material,and its physicochemical properties and stability were investigated.At the same time,the enzymatic hydrolysis peptide and selenium nanoparticles（PE-Se NPs）were explored.This study provides an economical and efficient process for preparing reagent grade PE-545,and successfully obtains nanoscale PE-545 selenium complexes,which is of great significance for the large-scale preparation and application of PE-545.The main results are as follows:1.Preparation,physicochemical properties and stability of PE-545The PE-545 spectroscopy purity(A₅₄₅/A₂₈₀)reached 2.45 and the recovery rate was66.51%by repeated freeze-thaw cell fragmentation and two-step ammonium sulfate fractional precipitation.After further purification by Butyl-S-QZT 6FF hydrophobic chromatography,the purity of PE-545(A₅₄₅/A₂₈₀=6.63)reached reagent level,and the recovery rate was 48.9%.The purified PE-545 had the largest characteristic absorption peak at 545 nm,the emission peak was at 587.5 nm.The secondary structure mainly existed in the form ofα-helix,containingαandβsubunits.PE-545 was relatively stable at30～50℃and p H6～8.In the presence of Ca²⁺,Mn²⁺,Zn²⁺,Fe²⁺and Fe³⁺,the spectral properties of PE-545 did not change significantly,while Cu²⁺had a great influence on its spectral properties.2.The study of PE-545 enzymatic hydrolysisPE-545 was hydrolyzed by alkaline protease.On the basis of single factor test,orthogonal test was carried out to determine the optimum enzymatic hydrolysis conditions:p H10,time 3 h,enzyme addition 250 U/g.Under these conditions,the degree of hydrolysis of PE-545 was 45.25%.The enzymatic peptides showed certain antioxidant capacity in vitro.When the degree of hydrolysis was 30%,the reducing capacity(A₇₀₀)reached 0.29.3.Study on PE-545 selenium nanoparticles（PE-Se NPs）The PE-545 dosage and reaction time used for the preparation of PE-Se NPs were optimized,and the particle size,morphology and structure of PE-Se NPs were characterized.The results showed that when 100 mg PE-545,30 mg/m L glutathione solution 1 m L,50 mmol/L Na₂Se O₃solution 250μL and 8.75 m L deionized water were added to the preparation of PE-Se NPs,reacting for 45 min,the obtained PE-Se NPs were about 100 nm in diameter,spherical and well dispersed.Infrared spectroscopy analysis showed that selenium reacted with C=O in the carboxyl group of PE-545 to form C-O=Se bond.In vitro antioxidant test showed that PE-Se NPs exhibited significant Fe²⁺chelating ability,total reducing power and free radical scavenging activity.When the concentration of PE-Se NPs was 0.8 mg/m L,the Fe²⁺chelating ability was 60.29%.When the concentration was 0.2 mg/m L,the hydroxyl radical scavenging rate reached 81.42%.