Study On Isolation, Purification, Bio-activity And Preliminary Structure Identification Of Royal Jelly Active Protein

The isolation,purification,bio-activity and preliminary structure identification of royal jelly active proteins were studied in this paper,the results showed as follows:Royal jelly protein was separated and protein content and antioxidation of the fractions were determined.The protein fraction,which was of high protein content and antioxidation,was selected to be the target fraction.Single-factor and orthogonal tests were taken to optimized extraction process of target fraction.Analysis of variance showed that pH and ratio of solid to liquid influenced protein extraction significantly,but temperature,extraction time,NaC1 concentration did not affect significantly.The optimum extraction process was pH 8.0,temperature 0℃,ratio of solid to liquid 1:20,time 2h. Under these conditions,the relative extraction rate was up to 87.21%.Royal jelly albumin extract was prepared under the optimal conditions.And chemiluminescence was applied to study antioxidation of albumin extract.The results indicated that royal jelly albumin extract performed higher scavenging effect on hydroxyl free radical,superoxide anionic radical,hydrogen peroxide and protective effect on DNA damage in vitro with IC₅₀ 3.81mg/mL,5.16mg/mL,7.43μg/mL and 4.34mg/mL, respectively.Besides,it also showed ability on scavenging DPPH·free radical with IC₅₀ 0.315mg/mL and some inhibition to mice liver lipid peroxidation.Immunomodulatory effect of royal jelly albumin extract on normal mice was also investigated.The results displayed that royal jelly albumin extract could improve mice cellular immune function, humoral immune function,non-specific immune function and NK cell activity, significantly.In order to isolate and purify royal jelly albumin,it was applied to Sephadex G-100 gel filtration chromatography,DEAE-52 ion-exchange chromatography,respectively,or combination of these two methods.SDS-PAGE analysed the purity of elution peak.After the albumin was applied to Sephadex G-100 chromatography,and the elution peak then applied to DEAE-52 ion-exchange chromatography,a single protein component（royal jelly albumin-57kDa,RJA-57） could be obtained.SDS-PAGE,Native-PAGE,Sephadex G-100 gel filtration chromatography and UV absorption spectrum were used to identify the purity of RJA-57.All of the results demonstrated that RJA-57 was composed of only one protein,And the antioxidation activity of RJA-57 was preliminarily studied in vitro. The results showed that RJA-57 could scavenge DPPH radical and protective effect on DNA damage effectively.The IC₅₀ of them were 0.139mg/mL,2.47mg/mL respectively. RJA-57 also exhibited some inhibition of lipid peroxidation of rat liver.Amino acid composition analysis of RJA-57 indicated that RJA-57 contained all of the 8 essential amino acids in human body.It was rich in tryptophan and sulfur-containing amino acid especially.Thus,RJA-57 was a high quality protein.FT-IR infrared spectrum of RJA-57 exhibited protein characteristic absorption peaks.And preliminarily judged that the content ofβ-sheet in RJA-57 was much higher than that of a-helix.Circular dichroism spectrum analyzed the secondary structure of RJA-57 at 25℃,70℃, respectively.The results showed that the main secondary structure of RJA-57 wasβ-sheet and random coil at both temperature.And with temperature increased,the content of a-helix,β-sheet would decrease,whereas random coil increase.Intrinsic fluorescence and Fluorescence quenching were applied to study conformation of tryptophan residues.The results showed that some tryptophan residues was exposed to the surface of RJA-57 molecule,but the others were embedded in the hydrophobic region.