Research On The Identification Of Allergens In Brassica Campestris Bee Pollen And Its Allergenicity Reduction By Complex Enzymes Treatment

Bee pollen is a functional food rich in nutrients and has an adjunctive therapeutic effect on many diseases.However,due to its origin from plant pollen,it has potential allergenicity,causing food safety risks and threatening human health.At present,the researches on allergens and their allergenic mechanisms in bee pollen are very limited,which seriously limits the development and utilization of bee pollen.In this study,the bee pollen originated from Brassica campestris was used as the research object,and the allergens contained in it were first identified by mass spectrometry combined with bioinformatics.Then,pectinase,cellulase and papain were applied to establish a complex enzyme method to reduce the allergenicity of bee pollen.The two enzymes(pectinase and cellulase)treatment group and three enzymes(pectinase,cellulase and papain)treatment group were set up to study the degradation of allergens in bee pollen.Furthermore,the mouse allergy model was established to evaluate the allergenicity of bee pollen in different treatment groups,aiming to provide a scientific basis for the safety improvement and deep processing and utilization of bee pollen-related products in the future.The main findings are as follows:First,proteomic analysis of bee pollen samples was conducted using Easy-n LC1000-LTQ-Orbitrap Elite mass spectrometry technology,and peptide comparison analysis was performed based on the Brassica protein database.Five new allergens were identified – profilin,cystatin,alcohol dehydrogenase,prolamin and expansin through matching the known allergenic protein sequences from NCBI BLAST and comparing the protein sequences using EMBOSS Needle and All Fam databases.Additionally,the epitopes of T cells and B cells of these five allergenic proteins were predicted by bioinformatics using DNAStar software.Second,the allergenic proteins in natural bee pollen and enzyme-treated bee pollen samples were relatively quantified based on proteomic analysis.Results showed that the content of profilin was decreased significantly in the two enzyme treatment groups;the contents of profilin,cystatin and alcohol dehydrogenase were decreased significantly in the three enzyme treatment groups,compared with the natural bee pollen group.Moreover,the pollen grain of natural bee pollen and enzyme-treated bee pollen samples were observed by scanning electron microscopy,and it was found that the complex enzyme treatment had a wall-breaking effect on bee pollen,and the pollen wall was broken and the contents were released.Dot-blot analysis showed that the protein extracts of bee pollen treated with complex enzymes were significantly reduced in the binding affinity to human specific Ig E antibodies compared with that of the natural bee pollen,indicating that enzymatic treatment could reduce the allergenicity of bee pollen.Metabolomic analysis showed that the contents of amino acids and oligopeptides in complex enzyme treated bee pollen samples were increased significantly compared with those in natural bee pollen,which confirmed the degradation effect of complex enzyme treatment on bee pollen allergens.Additionally,the increases of amino acids and oligopeptides improved the nutritional characteristics of bee pollen.Third,in order to further study the differences in allergenicity of different treatment bee pollen samples,a BALB/c mouse allergy model was established by intraperitoneal injection of protein extract of bee pollen.The histopathological results showed that the mice in ovalbumin treatment group(OVA)and the natural bee pollen treatment group(BP)had an allergic reaction with increased mast cells and infiltration of inflammatory cells in spleen.However,there were no obvious allergic reactions in the two enzyme treatment groups(2E-BP)and three enzyme treatment groups(3E-BP).The detection of allergic mediators(e.g.,Ig E,histamine,etc.)in mice serum showed that the levels of allergic mediators in BP group was significantly increased,while there was no significant difference in 2EBP and 3E-BP groups compared with the CK group,indicating that the enzymatic treatment reduced the allergenicity of bee pollen.Metabolomic analysis showed that the allergic reactions of 2E-BP and 3E-BP groups were reduced,metabolic disorders were alleviated,and the metabolic pathways associated with allergic reactions were regulated compared with BP group.The mice gut microbiota analysis showed that there were significant differences in the mice gut microbiota structure between enzyme-treated bee pollen group and natural bee pollen group.We also found that the allergic reaction and intestinal inflammatory response of mice in enzyme-treated bee pollen group were significantly reduced compared with those in the natural bee pollen group through the analysis of bacteria associated with allergy and inflammatory response.In summary,this study demonstrated that complex enzyme treatment can significantly reduce the allergenicity of bee pollen,and exert positive effects on the nutritional characteristics of bee pollen.Our study could provide a scientific basis for improving the safety of bee pollen products and promoting their wider application in the future.