| In recent years,with the increasing demands of consumers for food nutrition and health,plant protein,especially soy protein,as a nutritional and functional food component,has become more favored by consumers and producers,and it is widely used in food and animal feed.Soy proteins in plant protein are widely used in the food industry as a food ingredient due to its rich nutritional value and good functional properties.However,the antigenic protein of soy proteins can cause allergic reactions in the human body,which undoubtedly poses a potential threat to people with soy allergies.Among them,?-conglycinin is a major protein allergen in soy proteins,accounting for about 30% of the total protein of soybean seeds,and its three subunits(?,?'and ?)can cause hyperplasia.At present,avoiding ingestion is still the best way to prevent food allergic reactions.In addition,numerous studies have shown that HHP and heat treatment affect the allergenicity of soy protein by changing its protein structure.Therefore,this paper selected ?-conglycinin as the research object to study the effect and mechanism of HHP and heat treatment on its sensitization.The main linear epitopes of ?-conglycinin ? subunit were determined using immunoinformatic combined with serology.The epitope peptide was synthesized by peptide synthesis technology and the corresponding epitope polyclonal antibody was prepared,then the binding ability of epitope antibody with ?-conglycinin was evaluated by dot blotting and Western blotting.The ic-ELISA and Western blotting were used to evaluate the binding ability of ?-conglycinin after HHP and heat treatment with antibody,and spectroscopy was used to characterize the structural characteristics of ?-conglycinin after processing.This explored the changes in the antigenicity,epitope and structure of ?-conglycinin after HHP and heat treatment.By constructing a mouse model allergic,the mechanism of HHP and heat treatment on the sensitization ability of ?-conglycinin was explored.Main contents and conclusions of this paper are as follows:(1)The main linear epitope of ?-conglycinin was identified and the binding ability of the prepared epitope polyclonal antibody was verified.Ten potential antigen epitopes were predicted using immunoinformatic tools combined with epitope database,and 10 epitope antibodies was synthesized.The ic-ELISA results showed that all synthetic epitope peptides had Ig G-binding ability,and synthetic peptides P2 and P6 were also identified as Ig E-binding peptides.The epitope polyclonal antibody corresponding to the epitope peptide was prepared by immunizing rabbits,and the binding ability of the epitope polyclonal antibody with ?-conglycinin was determined by dot blotting and Western blotting.The test results showed that P5 and P8 were possibly located inside the structure of ?-conglycinin;The epitope antibodies PA-1,PA-2,PA-3,PA-4,PA-6,PA-7,PA-9 and PA-10 could effectively bind to ? subunit,while PA-5 and PA-8 bound weakly to proteins,which was related to their recognition sites located inside the structure of ?-conglycinin or its low titer.Moreover,for epitope polypeptides,? subunit and ?' subunit,epitope polyclonal antibody had immunological cross-reaction,because epitope antibodies recognized the same amino acid sequence among the three subunits.The I-TASSER online program was used to simulate the tertiary structure of ?-conglycinin ?subunit and the recognition site of epitope antibodies was shown by Py MOL.The results of this chapter showed that most of the predicted epitopes were located outside the structure of?-conglycinin ? subunit and were easy to bind with antibody.(2)To study the effect of heat treatment on the antigenicity,epitope and structural changes of ?-conglycinin.The ic-ELISA results showed that,compared with untreated ?-conglycinin,the Ig G-binding capacity of heated protein was inhibited as increasing temperature,but its Ig E-binding capacity was enhanced,even at 125 °C.Except for the epitope antibody PA-5,the binding ability of the heated ?-conglycinin with epitope antibodies was not inhibited but was significantly enhanced.The in vitro simulated digestion experiments showed that heat treatment could promote the digestion of ?-conglycinin,and the conformational epitopes were destroyed,but some linear epitopes could resist digestion and existed in small molecule peptides after hydrolyzed.Therefore,the binding ability of heated ?-conglycinin with Ig G,Ig E and epitope antibodies was significantly reduced.In addition,heat treatment significantly changed the structure of ?-conglycinin.The reduced and non-reduced SDS-PAGE showed that thermal denaturation resulted in the formation of protein aggregates and the reduction of ?-conglycinin dimers;The FT-IR analysis results showed that the relative content of the secondary structure of HHP-treated ?-conglycinin changed significantly,the structures of ?-sheet and ?-helix decreased,while the ?-turn and random coil increased;The external fluorescence spectroscopy and UV absorption spectroscopy results showed that the tertiary structure of ?-conglycinin was changed after heat treatment,thereby exposing more hydrophobic groups,and tyrosine and tryptophan residues and other chromophores were buried.(3)To study the effect of HHP treatment on the antigenicity,epitope and structural changes of ?-conglycinin.Compared with the untreated group,HHP treatments of 300 MPa(20 °C,40 °C and 60 °C)and 400 MPa(20 °C and 60 °C)inhibited the Ig G and epitope antibody binding capacity of ?-conglycinin,but its Ig E-binding capacity did not change significantly.The in vitro simulated digestion showed that HHP treatment promoted digestion of ?-conglycinin,and the Ig G-and Ig E-binding capacity of ?-conglycinin after HHP treatment at 400 MPa and 60 °C was lowest.However,the binding capacity of digestion products of HHP-treated ?-conglycinin with epitope antibodies was enhanced.The Western blotting showed that HHP treatment promoted digestion of ?-conglycinin,and a large number of epitopes were destroyed,but new epitopes were exposed and resisted digestion by protease,thus these epitopes were retained.In addition,HHP treatment changed the structure of ?-conglycinin.The SDS-PAGE showed that HHP treatment had no significant effect on the molecular weight distribution of ?-conglycinin protein;The FT-IR analyses indicated that the content of ?-turn and ?-sheet of heated ?-conglycinin decreased,while random coil and ?-helix increased,so the secondary structure of protein becomes disordered;The external fluorescence spectroscopy and UV absorption spectroscopy showed that HHP treatment unfolded the tertiary structure of ?-conglycinin,thereby exposing more hydrophobic groups,tyrosine and tryptophan residues.(4)A sensitized mouse model was constructed to evaluate the effects of HHP and heat treatment on the sensitization of ?-conglycinin.The results of challenge test found that ?-conglycinin would cause severe allergic symptoms in BALB/c mice,while the sensitization symptoms of mice in the HHP treatment and heat treatment groups were effectively alleviated;Compared with the PBS group,the ELISA results showed that the concentrations of Ig E,Ig G and Ig G1 in mice serum of ?-conglycinin group increased significantly,while the Ig G2 a decreased,and the increase/decrease trend was proportional to the protein concentration.The splenic lymphocytes of mouse were isolated and cultured,and their cytokine levels were measured.The experimental results indicated that ?-conglycinin increased the activity of splenic lymphocytes,stimulating cell proliferation and differentiate into Th2 cells.Also,the results showed that the upregulation of IL-4,IL-5,IL-10,IL-12 and IL-13 levels and suppression of IFN-? levels,thus the body's Th1/Th2 immune balance was broken and food allergy was triggered.By changing the protein structure,HHP and heat treatment destroyed the epitope on ?-conglycinin,thereby blocking the occurrence of food allergies and exhibiting low allergenicity. |
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