The Characterization Of β-lactoglobulin Oligomers Structure And Allergenicity Assessment In Vitro

Whey is a by-product of cheese production, and whey proteins are widely used as ingredients in foods because of it unique functional properties, i.e. emulsification, gelation, thickening, foaming, water-capacity and so on. There are two main allergens in whey, β-lactoglobulin (v-Lg) and a-lactalbumin (α-La),and about50%-80%whey protein is β-Lg. Epidemiological survey showed that about2.5%of the population is allergic to milk, and the number of allergic patients are also increasing as the improvement of the standards of modern living and health conditions,. Current studies show that β-Lg is predominantly dimeric under physiological condition, and there are few reports about allergenicity of BLG dimer at natural state.This work included three parts:1. Purification of β-Lg and its oligomers preparation;2. Characterizationof physical and chemical properties of β-Lg oligomers;3. Digestibility and allergenicity assessment of β-Lg oligomers in vitro. The main methods, results and conclusions are as follows:1. Bovine β-Lg was purifyed by column chromatography, and identified by SDS-PAGE and mass spectrometry, respectively. A single factor experiment was performed, temperature ranging from40℃to75℃, heating time ranging from0.5to2h, metal ions and protein concentration ratio of2:1,1:1,1:2, pH ranging from5.55to8.04.This work aimed to find the best condition for β-Lg oligomers induced by Cu2+, Ca2+, Zn2+and Mg2+. It was shown that the purity of β-Lg reached to99%after two steps chromatography, and the single factor experimental indicated that β-Lg oligomers were mainly dimer at70℃,0.5h heating,1:1of metal ions and protein concentration ratio is and pH7.0can induce, and mass spectrometry identified its existence.2. The particle size of oligomers was analysis by Laser Particle Sizer, appearance shape was observed by environment scanning electron microscope. The free sulfhydryl content was detected by Ellimen’ reagent, and the secondary structure was defined by CD spectrum. It was shown that the average particle size of β-Lg oligomers induced by Cu2+, Ca2+, Zn2+and Mg2+were372nm,1236nm,1167nm and315nm, respectively, and the appearance and secongdary structure also showed significant difference. The free sulfhydryl contents were0.026mM、0.04mM、0.033mM、0.04Mm, respectively. It can concluded that Cu2+mainly induced β-Lg to form oligomerization by free sulfhydryl formation disulfide bond between two molecules, while Ca2+and Mg2+mainly induced β-Lg to form oligomerization by disulfide-exchange between two molecules. The control group and the group of Zn2+induced P-Lg have two types of oligomerization, and Zn2+mainly induced oligomerization by free sulfhydryl formation disulfide bond between two molecules.3. Digestibility of β-Lg oligomers was evaluated by simulation gastrointestinal digestion in vitro, IgG and IgE binding capacity of oligomers were defined by competitive ELISA and western-blotting. It was shown that both P-Lg and it’s oligomers showed strong resistance to pepsin while weak to trypsin. The order of IgG binding capacity of oligomers induced by metal ion was as follow, Zn2+>Cu2+>Mg2+>Ca2+, and IgE binding capacity was as follow Cu2+> Ca2+>Zn2+> Mg2+. Western blot indicated that P-Lg oligomers still keep antigenicity to certain extent.