Ultrafiltration Separation Of MRJPs From Royal Jelly And Utilization Of Recombinant Antibacterial Peptide,Royalisin

Royal jelly (RJ) secreted by hypopharyngeal and mandibular glands of nurse honey bees is the exclusive food for the Western honeybee (Apis mellifera) queen throughout its lifespan and the primary food for the honeybee worker larvae during the first three days. Recently, RJ proteins, especially major royal jelly proteins (MRJPs) are becoming one of research focus. This thesis reported the ongoing studies of MRJPs and royalisin including separation, biological activity assay and application. Major results are as follows:1. Separation and ELISA assay of recombinant royalisinIn order to fully utilize royalisin, fast ELISA assay with MRJP1antibody is established. The technique was used to determine the yield of royalisin produced under different fermentation conditions, and optimal temperature was obtaied.3.01mg/L of royalisn at27℃could be separeted. The yield is much higher than previous yield (1.5mg/L). The results provide basis for further production with fermentation in future.Hemolymph from different honeybee species as antigen was assayed with ELISA. The absorbance of different samples was substituted into created standard curve to calculate the concentration of royalisn. The assayed results showed that the concentration of royalisin (the anitibacterial ability) in Asian honeybee (A cerana) is higher than that in the Western honeybee.2. Fast ELISA assay of RJ freshness with MRJP1antibodyThe fast ELISA assay of RJ freshness with MRJP1antibody was estabilished. The fresh RJ samples stored at40℃from0d to49d (0,7,14,21,28,35,42and49d) were assayed with ELISA, respectively. It was showed the MRJP1in RJ could be degraded greatly under storage, which was proved with SDS-PAGE analysis.3. Ultrafilteration separation of MRJPs from RJWith the MRJPs yield extracted from RJ as index, the effects of pH, ionic strength and water ratio were invstigated, respectively. Then,34orthogonal tests are employed to obtain the optimal conditions to extract MRJPs from RJ. Finally, verification of the optimal condition to extrct MRJPs from RJ and identification of lyophilized MRJPs were implemented. The by-products, total sugar and10-HDA during process were recycled. The results showed that optimal condition are as bellows, water ratio5:1(mL/g), ionic strength0.5mol/L and pH7. Under the optimal condition,82.63%of MRJPs was extracted from fresh RJ. The recycling rate of the two components reached3.6%and2.11%, respectively.