The Effects Of Output And Harvesting Time On Royal Jelly Quality

Royal jelly(RJ),a natural secretion from the hypopharyngeal and mandibular glands of nurse bees,is a functional food with health-promoting properties.RJ contains a wide cascade of components such as water,proteins,lipids,amino acids,carbohydrates,and vitamins.Bioactive compounds in RJ contribute to the diverse range of pharmaceutical effects of RJ,such as anti-tumor and anti-aging.To augment RJ production from the honeybee colonies,a high RJ-producing honeybee strain(RJBs)in China has been genetically bred from Italian bees(ITBs,Apis mellifera ligustica).To improve the RJ quality,different harvesting time after larval grafting has been adopted.The effects of enhanced output and harvesting time on total protein and 10-HDA content of RJ have been reported.However,contrasting findings still exist,and changes in other small-molecule constituents have not yet elucidated,restricting the overall evaluation of the RJ quality.In this study,we compared metabolic profiles of RJ harvested at 24 h,48 h and 72 h after larval grafting into queen cells from RJBs and ITBs using non-targeted metabolomics based on ultra-performance liquid chromatography–high resolution mass spectrometry(UPLC–HRMS).Water content and antibacterial activity were also compared among these RJ samples.We aimed to comprehensively evaluate the quality of RJ produced by RJBs and to optimize harvesting time,which could provide evidence for RJ production with high quality and quantity.To optimize RJ metabolite extraction,six solvents,including 50% and 80% methanol,ethanol,and acetonitrile,were used separately.The extracted small-molecule compounds were analyzed by UPLC–HRMS.The highest number of metabolite features was obtained with 80% methanol or 80% ethanol,while the 80% acetonitrile had the lowest number of metabolite features and the poorest repeatability.Weakly and medium polar metabolites extracted from 50% solvents were found to less abundant than those from 80% solvents,whereas highly polar metabolites from 80% acetonitrile had the lowest abundance.Thus,80% methanol or 80% ethanol is an optimal solvent for RJ metabolite extraction.A total of 77 high-abundance compounds were identified from RJ harvested at the three harvesting times from RJBs and ITBs.The abundance of most bioactive compounds in RJ from RJBs was not significantly different compared with that of ITBs,although RJBs had a 9-fold higher RJ yield.Notably,the observed 10-HDA(1.98%)and water content(67.26%)in RJBs meet the standard for premium RJ in China.In addition,no significant difference in antibacterial activity was found between the two bee strains.The above results indicate that RJBs not only have a higher RJ yield but also retain an overall high RJ quality.The harvesting at 24 h could lead to a higher RJ quality with lower water content(58.57%)and higher abundance levels of many bioactive compounds,such as 10-HDA,acetylcholine,and taurine.However,grafting labor intensification and RJ yield decrease indicate that the 24-h harvesting is not applicable for commercial RJ production.Although similar metabolic profiles,water content,and RJ output were observed at the two later harvesting times(48 h and 72 h),the 72-h harvesting is superior considering that the 48-h harvesting requires higher labor intensity due to 50% more larval grafting.This study adopted,for the first time,reverse phase liquid chromatography(RPLC)and hydrophilic interaction chromatography(HILIC)each combined with HRMS for non-targeted metabolomics of RJ.We optimized the RJ metabolite extraction protocol and offered technical supports for non-targeted metabolomics analysis of RJ.Our findings reveal the premium quality of RJ from RJBs and the optimal time for RJ harvesting,thus providing evidence for mass production of high-quality RJ.