PuriHcation Of Royal Jelly Protein And Storage Changes At Room Temperature

Separation and purification of royal jelly proteins and storage changes at room temperturewere preliminary researched in this subject. The results are as follows:(1) By using ion-exchange chromatography SOURCE15Q and size exclusion chromatographySuperdex75, the major royal jelly proteins can be separated and MRJP1and MRJP2were purified andobtained.(2)70%,80%and90%ethanol were employed to extract royal jelly proteins, alcohol solubleproteins were found in royal jelly, and the content of alcohol soluble proteins in70%ethanol extractwas0.5892±0.01mg/g. Alcohol soluble proteins in the royal jelly were analyzied by gliadin A-PAGEand SDS-PAGE to find alcohol soluble protein-1and alcohol soluble protein-2. These two proteins weresequenced and identified by LC-MS/MSAlcohol soluble protein-2was found to be Royalisin ofhoneybee and alcohol soluble protein-1to be an unknown protein. However, as the content of alcoholsoluble protein-1was too low, alcohol soluble protein-1need to purify and enrich for further precisesequencing.(3) Concanavalin A lectin affinity chromatography was applied to separate glycoproteins in royaljelly.100KDa macromolecular proteins,60~70KDa proteins (MRJP3and MRJP5) and45~58KDaproteins (MRJP1and MRJP2) in royal jelly were found to be glycoproteins by SDS-PAGE,2-DEelectrophoresis analysis and Pro-Q Emerald300glycoprotein staining. N-glycosylated sites ofMRJP1were analyzed and determined by MALDI TOF/TOF and LC-MS/MS. N-glycosylatedsite-177,144of MRJP1was found and site-28was not glycosylated. Furthermore, the unpredictedposition of404and408were foundto glycosylateand need further to verificate by LC-MS.(4) Through CD and SRCD, the variation of secondary structures of royal jelly proteins was testedduring storage at room temperature. The results showed that the CD spectral peaks and SRCD spectralpeaks of the royal jelly proteins were consistent, and there were obvious characteristic peaks of helixand fold. Secondary structures of royal jelly proteins had changed greatly at room temperature duringstorage. Contents of β-fold, β-turn, α-helix, P2and irregular structures in fresh royal jelly wereidentified to make up26.7%,23.7%,22.8%,10.1%and16.7%, respectively. With the length of storagetime, contents of α-helix and β-turn in the royal jelly proteins decreased, while contents of β-sheet andthe secondary structure of royal jelly unfolded. The tertiary structure was studied by the fluorescenceP2increased. The content of the irregular structures had no obvious change. This suggests thatsecondary structures of royal jelly are unfolded during storage at room temperature. The tertiarystructure was studied by the fluorescence spectrum. The results showed that tryptophan residues of royaljelly proteins gradually transferred from internal hydrophobic environment of proteins to externalhydrophilic environment. This meant that tertiary structures of the proteins had changed and royal jellyproteins were unfolded.(5) The browning index of royal jelly can be characterized with the absorption value at420nm and the ratio at520/430nm. It was found that the ratio at520/430nm was better. NBT colorimetric wasused to determine variation of DMFcontents of royal jelly during storage at room temperature. DMFcontents increased from354.873mg/g proteinin fresh royal jelly to666.045mg/g protein after storageof six months. ELISA was employed to determine the CML contents in the royal jelly. CML contentsincreased from50.9ng/g protein in fresh royal jelly to371.08ng/g protein six months later. In addition,changes of the AGEs contents with fluorescence characteristics were measured during storage, includingPentosidine, Pentodilysine, Crossline, Pyrropyridine and Argpyrimidine. Free fluorescent relative valueof these AGEs tended increasing with time length, but the relative value of the total fluorescenceincreased firstly and reached its maximum four months later, then tended to be decreased.