Effects Of Calcium Ion On Structure And Allergenicity Of Bovine ?-Lactalbumin

Food allergy is an immunological reaction which caused by the ingestion of an allergen in food and refers to a series of clinical symptoms,resulting in tissue or organ damage.It is reported that more than 90% of allergic reaction is caused by the eight kinds of highly allergenic foods,including milk,eggs,peanuts,soybeans,wheat,nuts,fish and aquatic crustaceans.Cow's milk is one of the eight kinds of highly allergenic foods.Bovine ?-lactalbumin is a kind of good nutrition contains many essential amino acids.It accounted for 25% of whey protein,and that have adjusted the physiological function of regulating lactose synthesis.Although bovine ?-LA is similar to human milk ?-LA,it is still considered as one of the major milk allergens.Calcium ion can be combined with ?-LA to change the structure of the protein,therefore affecting the allergenicity of protein.Obviously,it is a great significance to study the effect of calcium ion on the structure and allergenicity of bovine ?-lactalbumin.The results will provide a new idea for the research and development of hypoallergenic formula powder.In this thesis,different concentrations of calcium ions were treated for binding to ?-lactalbumin and evaluation of relationship of structure and allergenicity change.The research contents include: purification of bovine ?-lactalbumin and the calcium removal from native ?-lactalbumin;optimization of the condition of calcium ion binding to bovine ?-lactalbumin;change of the structure,gastrointestinal digestion in vitro and the allergenicity after calcium ion binding to bovine ?-lactalbumin;evaluation of sensitization of holo-?-LA by KU812 cell degranulation model in vitro.The mainmethods,results and conclusion are as follows: 1.Bovine ?-lactalbumin was purified by anion exchange chromatography and gel chromatography with a purity of up to 95%.Through the four parameters of temperature,time,molar ratio and pH of the preliminary study,three factors of optimization were exolored to the optimal conditions by using of response surface method.The results was that the molar ratio apo-?-LA and calcium was 1:4,and the soulution with pH 7.28 should be incubated at 25 °C for 2 hours.2.The structure,digestive stability and antibody binding capacity of calcium binding to bovine ?-LA were evaluated by spectral characterization and in vitro gastrointestinal digestion and indirect competitive ELISA.The results showed that the structure of protein became more compacted calcium ions binding to bovine ?-LA.The surface hydrophobicity of apo-?-LA raised,since fluorescence intensity and UV absorption intensity increased.The surface hydrophobicity of holo-?-LA(1:4)decreased,as the fluorescence intensity and UV absorbance have declined.By Tricine-SDS-PAGE electropherograms,the protein was almost completely disappeared after the stomach digestion for 60 minutes followed by the intestine digestion for 30 minutes.It is noted that calcium binding has almost no effect on the digestive stability.The indirect competitive ELISA results showed that compared to ?-LA in the natural state,and the binding capacity of rabbit and human IgG in apo-?-LA solution increased by 21% and 72%,respectively.while the binding capacity of IgE increased by 30%.It indicated that the removal of calcium ions can significantly enhance the allergenicity of ?-lactalbumin.The rabbit and IgG binding capacity of the holo-?-LA(1:4)solution was reduced by 26% and 400%,respectively.And the IgE binding capacity was reduced by 8%.It was demonstrated that calcium ion-binding bovine ?-LA has reduced the potential allergenicity of bovine ?-LA.3.Based on the degranulation model of KU812 cell line to evaluate the allergenicity of bovine ?-LA,the serum of bovine allergic patients was incubated with KU812 cells,and the intracellular calcium ion after the calcium ion binding to ?-LA stimulated cells before and after calcium bindingand changes in levels and detection of histamine,?-HEX,IL-6,TNF-? was detected.The results showed that compared with bovine ?-LA,?-HEX and IL-6 levels and intracellular calcium concentration of apo-?-LA and holo-?-LA(1:4)did not change obviously,but the content of histamine and TNF-? in apo-?-LA increased,while the content of histamine and TNF-? released in holo-?-LA(1:4)decreased.This showed that calcium ion binding to bovine ?-LA may reduce some of the factors associated with sensitization,therefore reducing the potential allergenicity of bovine ?-LA.