Identification Of Bovine And Goat Milk Casein Antigen Epitopes And Effects Of Heat Treatment On Their Antigenicity

Bovine is rich in nutritions,alought it is known as one of the eight kinds of foods that may cause allergies in human body.Protein is a major allergen in the dairy products.Goat milk has high nutritional value as well.It is thought that the sensitivity of goat milk is lower than that of bovine milk.However,at present,there are different opinions on whether goat milk can replace bovine as the milk source for the milk allergy patients.Therefore,in this study,the antigenic epitopes of the main allergen of casein in bovine and goat milk were predicted.Standing from the sensitization mechanism,prediction of biological epitopes and validation of cases of main allergen casein of bovine and goat milk were researched using biological software and Caco-2 cell model.Caco-2 cell model was used to investigate whether the antigen epitope peptides can be absorbed in a complete form by the intestinal epithelial cells.And this study can provide a reference for the follow-up study of whether synthetic epitope peptides can cause allergic reactions in the body.Bovine and goat milk casein treating with different heating conditions were compared and analyzed on the aspect of the variation rule between antigenicity and structure,and purposefully to reduce their sensitization.These will lay a certain theoretical foundation for the development of hypoallergenic and non-sensitizing of bovine and goat milk.The main results are as follows:Firstly,prediction of antigen epitopes of bovine and goat milk casein and its verification by immunological detection is investigated.Using three bioinformatics software SOPMA?BepiPred 1.0 and DNASTAR successfully predicted that the antigen epitope peptide of bovine?_s1-CN were 99¹02148¹49 189²08 and the possible antigen epitope peptide of bovine?_s2-CN was72⁷5;the possible antigen epitope peptide of goat milk?_s1-CN were 26³1140¹42 205²08,and the possible antigen peptide of goat milk?_s2-CN was210²15.It is concluded that goat milk casein contains less amount of?_s1-CN but more regions of antigenic epitopes than bovine?_s1-CN,and there is a certain overlapping relationship between them.The bovine and goat?_s2-CN contains less antigenic epitopes.Different epitope peptides were synthesized by solid phase synthesis to be used in the further study.The epitope peptide P-1038?TDAPSFSDIPNPIGSENSEK?derived from bovine?-casein and the epitope peptide P-1037?LSPEVP?derived from the?-casein of goat milk were linked to KLH to prepare complete antigens and followed by immunizing animals to verify the immunogenicity.The results showed that using the synthetic peptide-KLH conjugate as antigen,the titer of?P-1037-KLH?-IgG and the titer of?P-1038-KLH?-IgG all reached 1:204800,indicating that the immunogenicity is very strong;whereas with the synthetic epitope peptides as antigen,the titer of?P-1037-KLH?-Ig G and the titer of?P-1038-KLH?-IgG reached 1:12800 and1:6400 respectively.These results showed that although synthetic epitope peptides are small molecules,they were still immunogenitc to animals.The specificity of?P-1037-KLH?-Ig G is higher than that of?P-1038-KLH?-Ig G detected by crossreaction experiments.Secondly,transportation and absorption of synthetic epitope peptide was studied in caco-2 cells.Caco-2 monolayer cells were logarithmic phase of cell growth for 4-10 days and can be subcultured.After 10 days,the fusion rate reached 90%.The cells began to differentiate and the activity of alkaline phosphatase continued to increase.The activity reached a maximum at 20 days and the TEER value at this time increased to 506?·cm².The fluorescence yellow P_apppp value was 5.0334×10^-8,based on the above indicators,modeling success.The Caco-2 transmembrane transport experiment of synthetic epitope peptides was carried out by by-pass transport.The results showed that the transmission rates of epitope peptides P-1038?TDAPSFSDIPNPIGSENSEK?,P-1039?EDVP?and P-1041?SSEE?derived from bovine?-casein were 1%,14%and 16%;The transmission rates of epitope peptides P-1037?LSPEVP??P-1040?EGN?and P-1042?TQPKTN?derived from goat milk?-casein were 3%,32%and 2%,which displayed that the transmittance rate was related to the fragment size.Six antigenic epitope peptides from bovine and goat milk casein sources were all partially hydrolyzed in intestinal Caco-2 cells.Whether the hydrolyzed small peptide and fragment of transmission causing allergic reaction of bodies needs further investigation.Finally,effects of heat treatment on the antigenicity of bovine and goat milk casein were explored.The purified caseins from bovine and goat milk were subjected to heat treatment under different conditions and then gastrointestinally digested.The immunoreactivity of bovine and goat milk casein was detected by the indirect ELISA.The changes in the structure of bovine and goat milk casein were investigated by circular dichroism,UV spectrum and hydrophobicity changes etc..It is found that the low temperature pasteurization at 63?,high-temperature pasteurization for a long time at 75?,and home boiling at100?did not destroy the spatial structure of bovine and goat milk casein.The reason may be that casein is a heat-resistant protein.When the temperature reached the ultrahigh voltage at 134?,bovine and goat milk casein began to denature,the natural structure was destroyed,and the hidden antigenic epitope was exposed on the surface of the molecule and the antigenicity increases.At the same time,we found that after heat treatment under different conditions,the antigenicity after gastrointestinal digestion decreases and goat milk casein was more easily to digest by gastric protein and trypsin,thereby the antigenicity was lower than that of bovine casein.For cow and goat milk,it is possible to reduce their antigenicity to some extent by reasonably controlling heating conditions.These provide a theoretic basis for the production of low-sensitivity bovine and goat milk.The epitopes of?-casein in bovine and goat milk are mainly located in?_s1-casein,and their epitope peptides are different.These peptides would be partial hydrolysis when transporting the intestinal Caco-2 cells.After heating,the immunoreactivity of two milk decreased,while ultrahigh voltage sterilization may increase the exposure of the antigenic epitopes.however,the antigenic epitopes are hydrolyzed and their antigenicity is reduced after gastrointestinal digestion.Its sensitivities needs to be further studied.