| β-lactoglobulin（BLG）is a kind of milk protein with high nutritional value and various functional characteristics,but it is also a major allergen in milk,which seriously hinders the application of BLG in food industry.Therefore,how to reduce the allergenicity of BLG is a difficult and hot topic in the field of food safety.In this paper,BLG was covalently modified by ferulic acid and glucose,and its structural changes were characterized by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis,circular dichroism,infrared spectroscopy,intrinsic fluorescence spectrometry and ultraviolet spectrum.The allergenicity of BLG was evaluated by ELISA and cell experiments,and the effects of ferulic acid with glucose modified BLG on allergic reaction,gut flora and serum metabolites in mice were further explored through animal experiments,and the molecular mechanism of ferulic acid combined with glucose to reduce the allergenicity of BLG was explored,and the following conclusions were drawn:（1）After covalent modification of BLG with ferulic acid combined with glucose,SDS-PAGE protein bands migrated upward,but the contents of free amino,tryptophan and sulfhydryl groups decreased significantly,and its secondary structure and conformation structure changed.Compared with single ferulic acid or glucose modification,ferulic acid combined with glucose modification decreased the Ig E binding capacity of BLG,the secretion levels of His and IL-6 in KU812 cells,and the degranulation,IL-4 and TNF-αrelease levels of RBL-2H3 cells.Therefore,ferulic acid combined with glucose can change the epitope structure of BLG and significantly reduce its allergenicity.（2）After the allergic BALB/c mice were stimulated by ferulic acid combined with glucose-modified BLG,the allergic symptom score,rectal temperature fluctuation,liver and thymus indexes of the mice were all decreased,and the contents of specific Ig E,Ig G,Ig G1,histamine and m MCP-1 in serum were also significantly decreased.At the same time,the immune damage degree of the duodenum and spleen of the mice was alleviated,and the antigen re-stimulation ability of spleen and mesenteric lymphocytes in vitro was regulated.The results showed that ferulic acid combined with glucose modified BLG could reduce the allergic risk of mice.（3）BLG can induce allergic reaction in BALB/c mice,destroy the diversity and structural composition of gut flora in mice,and reduce the content of short-chain fatty acids（acetic acid,propionic acid and butyric acid）in gut flora metabolites.However,ferulic acid combined with glucose modified BLG increased the relative abundance of bacterial flora at the family level,such as Lactobacillaceae,Rikenellaceae,Lachnospiraceae,Marinifilaceae and Bacteroidaceae.The relative abundance of Staphylococcaceae and Corynebacteriaceae decreased,while the relative abundance of Lactobacillus,Alistipes,Odoribacter and Bacteroides increased at the genus level.Compared with the control group,the serum non-target metabolism of BLG is rich in metabolites of seven arachidonic acid metabolic pathways,including Prostaglandin D2,12（S）-Hp ETE、PGJ2,12（R）-HETE,Prostaglandin F2a,15-deoxy-delta-12,14-PGJ2,Hepoxilin B3 metabolites were enriched in tryptophan metabolic pathway,while ferulic acid combined with glucose modified BLG only enriched Prostaglandin F2a in arachidonic acid metabolic pathway.Tryptophan metabolic pathway is rich in 3-Hydroxyanthranilic acid、Indoleacetic acid,Kynurenic Acid,4-（2-Amino-3-hydroxyphenyl）-2,4-dioxobutanoic acid,L-Tryptophan、Xanthurenic Acid,showed that BLG modified by ferulic acid combined with glucose had a low degree of activation of arachidonic acid metabolism pathway,which significantly activated tryptophan metabolism pathway.Therefore,ferulic acid combined with glucose modified BLG can alleviate the allergic risk of mice by improving the gut flora and regulating the metabolic pathways of arachidonic acid and tryptophan. |
