Identification And Quantitative Study Of Animal-derived Proteins In Honey Based On Liquid Chromatography-tandem Mass Spectrometry

Honey is a natural food produced by honey bees from the nectar of flowers or secretions produced by the botanical sources.In different types of bees will yield differences in the yield,color,taste,flavor and other aspects of honey,which may result in product price differences.A t present,the phenomenon of adulterated honey is frequent,and it is an urgent to establish an accurate and reliable method to identify honey from different bee species.In this study,Apis.ce rana,Apis mellifera,Apis dorsata and Apis florea honey were used as the research object.Usi ng HPLC-MS/MS,the main proteins of honey were identified and detected.The research conte nt is mainly divided into the following three parts:The first part is the identification of animal-derived proteins in honey from Apis.cerana,Apis mellifera and the development of quantitative analysis methods for MRJP1 and 2.After extracting honey protein,it is digested with trypsin and desalted by C18 cartridge.Adjusted peptide concentration,it is analyzed by nano-liquid chromatography tandem mass spectrometry.The mass spectrum data is analyzed by PEAKS 8.5 software.The main proteins are a series of royal jelly major protein family proteins(MRJP1-MRJP7,MRJP9)and enzymes such as α-glucosidase and glucose dehydrogenase.The most abundant proteins in honey are MRJP1 and 2,but the amino acid sequences are significantly different in the Apis.cerana and Apis mellifera honey.After screening,the characteristic peptides of MRJP1 were IMDANVNDLILNTR and FFDYDFGSDER;the characteristic peptides of MRJP2 were IVNNDFNFNDVNFR and IVNDDFNFDDVNFR,respectively.It was established based on LC-MS/MS to accurate quantify the above peptides.And systematic methodological verification was carried out.The sensitivity of the method was determined by limit of quantitation(LOQ)and limit of detection(LOD)of 10 ng/m L and 5 ng/m L in Apis mellifera honey,respectively.The LOD and LOQ were 10ng/m L and 20ng/m L in Apis.cerana honey,respectively.The method was precise(intra-day relative standard RSD <10.01%and inter-day RSD values<10.70%),accurate(84.60 %～111.52%),characteristic and robust.This method can be used to identify adulterations such as Apis.cerana honey mixed with Apis mellifera honey and syrup and provide reference for formulating relevant standards of Apis.cerana honey.In the second part,the identification and analysis of animal-derived proteins in honey from Apis dorsata and Apis florea honey were carried out,and the characteristic peptides of MRJP1 and 2 were screened out.And the quantitative detection method of liquid chromatography tandem mass spectrometry was established.The main animal-derived proteins are the royal jelly major protein family proteins(MRJP1-MRJP7,MRJP9)and enzymes such as α-glucosidase and glucose dehydrogenase.After screening,the characteristic peptides of MRJP1 of the Apis dorsata and Apis florea honey are ENAILSGEYDYTK and EALPHVPILDR;the characteristic peptides of the MRJP2 are IVNNDFNFDDVNFR and ILNNDLNINDINFR,respectively.The sensitivity of the method was determined by its limit of quantitation(LOQ)and limit of detection(LOD)of 20 ng/m L and 10 ng/m L in Apis dorsata honey,respectively.The LOD and LOQ were 15 ng/m L and 20 ng/m L in Apis florea honey,respectively.The method was precise(intra-day relative standard RSD <12.38%and inter-day RSD values<11.25%),accurate(82.78%-110.65%),characteristic and robust.This method can be used to identify adulteration such as Apis dorsata and Apis florea honey mixed with Apis.cerana honey and syrup,and it is conducive to the protection of wild bee species resources.The third part is to analyze the collected 177 honeys based on the honey authenticity evaluation method established.The results show that the content of MRJP1 in Apis.cerana and Apis mellifera honey is relatively stable,and it is not easily degraded under 4 ℃ storage conditions.But there are differences in the content of MRJP1 in honey from different nectar sources and production areas.In the commercial honey testing investigation,the adulteration of Apis.cerana honey,Apis dorsata and Apis florea honey is more serious.The form of adulteration is mainly in blending the Apis mellifera honey in the native honey,and even completely pretending to be the native honey.