Isolation, Identification And Biological Activities Of Water-soluble Proteins From Queen Bee Larvae

Honeybee queen larvae (QL), the byproduct of royal jelly (RJ) production, are one of excellent and rich protein reservoir as a food resource in China. Long term therapeutic practice evidence indicates that QL have many beneficial effects and showed development potential in the future. However, the information about the basis of bioactive compounds has not clearly been defined yet, which lead to low utilization. The present work was therefore undertaken to explore the characterization of the water-soluble proteins in QL and investigate the relationship between the composition and the bioactivities to provide scientific basis for the utilization and development of QL.(1) Purification and identification of water-soluble proteins from QL. High abundant proteins and low molecular weight (LMW) proteins in QL were characterized and identified by mass spectrometry. Major proteins were isolated by ion exchange, gel filtration and reverse phase chromatography. Two proteins, queen larvae protein-1(QLP-1) and queen larvae protein-2(QLP-2), were obtained, which was composed by major royal jelly protein1(MRJP1) and MRJP2as well as their degradation proteins. gel filtration chromatography (GPC) results showed that the molecular distribution of LMW proteins was between5and20kDa. The LMW proteins were dominated by their degradation proteins of MRJPs, antioxidant proteins, antibacterial proteins and proteins associated with the development and metabolism of larvae.(2) Structure characterization of water-soluble proteins. The QLP-1and QLP-2were examined by native PAGE to verify the homogeneity of the protein. Both proteins migrated as a single diffuse band on the gel. The second structure of these proteins was dominated by β-fold (>33%), with lower content of a-helix in the relatively stable conformation. The content of β-fold structure increased compared to the MRJP1and MRJP2isolated from RJ. Results showed that little difference was observed in the sterol profiles of QL and RJ. Results showed that sterols were found to be integrated with MRJP1oligomer and QLP-1through the determination of sterols in QLP-1and QLP-2.(3) The bioactivities of QL proteins. The major bee larvae protein was identified as QLP-2. The profile of the hydrolysis was characterized by gel filtration chromatography and tricine-SDS-PAGE. The proteins were more digestible into peptides with molecular weights lower than3kDa. Hydrolysis capacity in intestinal digestion was considerably greater than that in gastric digestion. The angiotensin I converting enzyme (ACE) inhibitory activity of the hydrolysate increased during the hydrolysis and the IC50reached0.21mg/mL after3h digestion. An ACE inhibitory peptide was purified sequentially by gel filtration and RP-HPLC and was identified to be Leu-Leu-Lys-Pro-Tyr (632.40Da) with highest ACE inhibitory activity (IC50=54.9μM). Two antioxidant peptides were also purified and their amino acid sequences were identified as Glu-Trp (333.14Da) and Asn-Tyr-Pro-Phe (539.24Da). The median effect concentration (EC50) values of Glu-Trp and Asn-Tyr-Pro-Phe for1,1-diphenyl-2-pycrylhydrazyl (DPPH) radical scavenging were0.19and1.47mg/mL, respectively. Two peptides also showed strong ABTS and hydroxyl radical scavenging abilities as well as reducing power. Proteins with immune enhancing activity were screened based on the mice model. After intragastric administration of QLP-1, the spleen index and thymus index significantly increased (P<0.05), while the remaining samples had no significant effect on the normal mice thymus index (P>0.05). QLP-1(0.24g· kg-1dose) significantly enhanced the immune suppressed mice carbon clearance ability, and the mice ear swelling degree increased significantly (P<0.05). The concentration of TNF-a and IFN-y in the serum of DTH mice was significantly increased (P<0.05). The results showed that QLP-1in the larvae was the main responsible component enhancing immune function, which can improve cell immune of mouse, increase the activity of macrophages and NK cells and induce the production of TNF-a and IFN-y.