Effect Of C18 Unsaturated Fatty Acid On The Allergenicity Of Bovine α-lactalbumin And β-lactoglobulin

Cow’s milk allergy has become one of the food safety issues of wide concern to governments and the public.Although more than 30 potentially allergic proteins have been found in cow’s milk,α-lactalbumin（BLA）andβ-lactoglobulin（BLG）are considered the dominant whey protein allergens.It is well known that BLA and BLG are not exist in the pure forms when human ingestion or inhalation of cow’s milk,and generally associated with other nutrition compounds which are concomitantly ingested or inhaled.According to previous studies,BLA and BLG can bind the oleic acid（OA）and other C18 unsaturated fatty acids（UFAs）in food matrix to form a complexes,which can induce apoptosis in tumor and immature cells but not in healthy differentiated cells.However,in the view of cow’s milk allergy,it is unclear whether the C18 UFAs affect the allergenicity of BLA and BLG.In this study,BLA and BLG will be treated with 5 different C18 UFAs（FA:OA,linoleic acid（LA）,conjugated linoleic acid（CLA）,α-linolenic acid（ALA）,andγ-linolenic acid（GLA））to form FA-protein complexes,respectively.The interaction of FA with protein will be defined well,and IgE binding ability of the FA-protein complexes will be tested.Moreover,human basophil KU812 degranulation model will be used to assess the allergenicity of the complexes in vitro.The digestibility of the FA-protein complexes was tested in the simulating gastric and intestinal fluids,followed by analyzing the specific IgE binding capacity of the digestion products.Afterwards,a BALB/c mouse model will be used to evaluate the allergenicity of the FA-protein complexes in vivo by testing the relevant biological substancesrelated to the food allergy.Finally,focusing on the intestinal immune cells,the antigen-presenting capacity by dendritic cells and T cell differentiation from sensitized BALB/c mice will be carried out to test the allergenicity of the complexes.This study aims to illustrate comprehensively the immunological function of FA-protein complexes in each steps of allergic reaction.Accordingly,the effect mechanism of C18 UFAs on the allergenicity of BLA and BLG will be stated clearly,and it helps to understand the allergenicity of milk protein in food matrix,and also guid the allergenicity control of milk protein by food processing.The main methods,results and conclusions were described as follows.1.To further study the C18 UFAs in food matrix how to effect the allergenicity of milk protein,here firstly used BLA as a proof protein to construct the in vitro human basophils KU812 degranulation model and BALB/c mice sensitization model,provide the methodology foundation of allergenicity evaluation in milk protein.The results of flow cytometry showed that the FcεRI,which is a high-affinity IgE receptor considered as a key molecule in inducing basophils degranulation,was significantly expressed（30%）on the cell surface.In addition,in BALB/c mouse model,FcεRI⁺c-kit⁺as the surface markers to identify the peritoneal mast cell,IgE⁺CD200R⁺CD4^-CD45R^-as the surface markers to identify the peripheral blood basophils.2.Based on BLA and BLG could bind C18 UFAs in food matrix to form bioactive complexes,the FA-protein complexes were prepared and the molar ratio of FA to protein was tested as 6-10.Electrophoresis and spectrum technology identified that C18 UFAs could induce the structure changes of BLA and BLG,and the change of the tertiary structure was the most significant.With the increase in the degree of unsaturation,the protein structure gradually expanded and the surface hydrophobicity gradually enhanced.In addition,through the IgG/IgE binding capacity and mouse basophil activation test,the C18 UFAs could enhance the antigenicity and IgE binding ability of BLA and BLG.3.Non-reducing SDS-PAGE and indirect ELISA were used to test the electrophoresis behavior and IgG/IgE binding ability of FA-treated BLA/BLG under different physical and chemical conditions.Among the different temperature,different time,and different mole ratio,only temperature had a siginificant influence on the protein patterns and IgG/IgE combining ability after FA-treated BLA/BLG,while the time and mole ratio did not have a obviously effects.The optimum physicochemical conditions for the preparation of FA-protein complexes was 60^oC,30min and 1:50.Scanning electron microscopy,particle size and zata potential showed that FA induced the granule and particle size becoming larger and the stability becoming stronger,which indirectly indicated that the FA induced the aggregation and formed a high polymer.Additionaly,indirect competitive ELISA test verified that FA under the optimum physicochemical conditions could also increase the IgE binding ablity of BLA and BLG.4.In vitro KU812 cell degranulation test indicated that C18 UFAs could enhance the ability of BLA and BLG to stimulate the degranulation of the KU812 cells,releasing the increased levels of histamine,β-HEX and IL-6.Moreover,flow cytometry was used to detect the Lyn,Syk,NF-κB and MAPK family proteins’expressions after KU812 cells activated degranulation,and it was found that the FA-protein complexes could promote the activity of original kinase Lyn and Syk proteins,strengthen the signal transmission of the entire pathway,and further promote the degranulation capacity of KU812.The test in KU812 cell degranulation model verified that C18 UFAs could increased the allergenicity of BLA and BLG by inducing the IgE/FcεRI mediated signal pathway.5.Tricine-SDS-PAGE and IgE binding capacity showed that pH 3.0 and gastrointestinal digestion time of 60min were determined as the optimum digestion conditions for simulated infants’digestion of BLA and BLG;as well as pH 2.0 and gastrointestinal digestion time of 60min were the optimum digestive conditions for simulated adults’digestion of BLA and BLG.Under optimal digestive conditions for infants and adults,the FA-proteins complexes resistance to gastrointestinal digestion,and the digests of FA-proteins complexes had a higher IgE binding ability than proteins,especially the increased allergies were prominently in infants than adults.6.ALA as a proof model was used in the sensitizated and challenged phase in BALB/c mice to investigate the effects of allergenicity of BLA and BLG through humoral immunity,cellular immunity and effector cell expression.The results showed that the LA-protein complexes activated the peripheral blood B cells（B220⁺）expression,increased the Th2-related specific antibody IgG,IgG₁ and IgG_2a levels,and decreased the Th1-related specific antibody IgG_2aa levels.Moreover,CD4⁺T lymphocytes efficient expression,Th2 cytokines efficient secretion,Th1 cytokines effective inhibition,and regulatory T cells significant reduction were also observed,which indicated the allergic reactions towards Th2 direction resulting in increased allergenicity.Additionally,LA also promoted the mast cells expression and the release of mMCP-1 and histamine levels of BLA and BLG.Finally,histological analysis showed that LA could promote the allergic reactions of BLA and BLG in the lung and intestine tissues.7.The intestinal PP and MLN were isolated in mice,and the effect of FA-protein complexes on intestinal mucosal immune system was further identified by flow cytometry.Results showed that PP and MLN in FA-protein complexes sensitizated and challenged mice reduced the dendritic cells（DC）expression,decreased the Th1cells and Tregs expression,but increased the Th2 cells expression,which reflected that ALA could induced the BLA/BLG disrupted the Th1/Th2 balance towards a Th2-immune allergic reaction,leading to enhanced allergenicity.