Prediction Of Soybean And Milk Allergen Peptides And Effects Of Processing Treatment On Their Antigenicity And Conformational Structure

Soybean and milk allergy has a high incidence in children and adults, and epitopes ofmilk allergen were known clearly,while the allergen mechanism of the α subunit of β-conglycinin is not entirely clear, mainly for the lack of awareness of its allergen epitopes. Inthis paper, three immunoinformatics tools, DNAStar, the BepiPred and SOPMA, were used tobuild the method of predicting the potential epitopes and the predicted epitopes wereconfirmed by dot-blot inhibition. In addition, the effects of the heat treatment of β-conglycinin and pulsed electric fields processing of α-casein were discussed. Influencingfactors of structural modification and antigenicity were assessed. These work providedtheoretical and technical basis for the further development of low-allergic or non-allergicsoybean and milk processing, the main findings and conclusions are as follows:1. DNAStar,the BepiPred1.0Server and SOPMA network server successfully predicted15peptides of α subunit of β-conglycinin. Sera from soybean allergic subjects were used asantibody for dot blot validation,11peptides were comfired as the IgE linear epitope of αsubunit of β-conglycinin. The peptide sequence HEQREEQEWPRKEEKRGEKGSEEEDEwas the main IgE epitope of α subunit of the β-conglycinin.The accuracy using three softwarepredicting epitopes was high, which providing a new method for epitope prediction.2. In order to further validate the accuracy of the above-described method, this methodwas applied to predict allergen epitopes which was known.Three kinds of bioinformaticssoftware, DNAStar, SOPMA and the BepiPred,were used to predict linear epitopes ofsoybean Gly m Bd30K and alpha-casein, combined with SWISS-MODEL to analyze thetertiary structure of these two proteins. Four peptides of Gly m Bd30K and Five peptides ofalpha-casein were predicted. The predicted epitopes were compared with known epitopes. Thecorrect rate of prediction epitope of soybean Gly m Bd30K was75%, and alpha-casein was60%, which build the foundation for the prediction of soybean allergen epitope.3.The purified α subunit of β-conglycinin with biological activity was processed by aseries of heat treatment and immunized New Zealand white rabbits to obtain polyclonalantibody ofα subunit of β-conglycinin. Polyclonal antibody were applied to ELISA to detectantigenic changes of α subunit of β-conglycinin between untreated and heat treatmentαsubunit of β-conglycinin; Circular dichroism, fluorescence spectroscopy and ultravioletspectrum were used to detected structural changes. The results showed that the antigenicityofα subunit of β-conglycinin was elevated with low temperature (50℃-70℃) within a shorttime (35min) heating treatment, this may be due to that protein internal structure were unfoldwere a low temperature and exposed epitopes; When the heating temperature was over80℃,antigenicity ofα subunit of β-conglycinin was reduced with the increase of the temperatureand heating time,as the disulfide of the protein breaking and rearrangement. The heating temperature is90℃and the treatment time was35min, which was the key point of theαsubunit of β-conglycinin secondary structure destroyed.4. Purified α-casein which was treated by pulsed electric field, were researched underthe conditions of different processing time, pulse strength and treatment temperature. TheELISA, circular dichroism, fluorescence spectroscopy and UV spectroscopy methods wereused to study the relationship between the antigenic and structural changes. The resultsshowed that when the pulse strength was35kV/cm, the treatment temperature was10℃andthe treatment time were not over200μs, the antigenicity of α-casein decreased, while in thelow intensity of the pulse processing and high environment temperature (30℃and50℃), theantigenicity of α-casein increased. This may be that low-intensity pulsed induced the structureof the protein open, and exposing the hidden peptide. High temperature can be suppressed tothe destruction of the pulse on the protein structure, but high intensity pulses (35kV/cm)directly damaged the secondary structure of α-casein.