| ?-lactoglobulin(?-Lg)is a high-quality protein in milk, and it has the best proportion of amino acids, high branched-chain amino acid content, and a strong ability to combine with fatty acids. But ?-Lg is also the main allergens in milk, which seriously impact on the consumer uptake of nutrients in milk, and greatly threaten the health and growth of the allergic people, especially infants and young children. Therefore, how to reduce and eliminate the allergenicity of ?-Lg became a research hotspot at home and abroad. In this paper, ?-Lg as the raw material, the anti-?-Lg polyclonal antibody were prepared and the indirect competitive enzyme-linked immunosorbent assay for detecting the antigenicity of ?-Lg were established. ?-Lg were modified by ultrasound treatment and ultrasound combined with Maillard reaction treatment, meanwhile the structure, antigenicity, allergenicity and functional properties of ?-Lg were researched after modification. Results are as follows.1. ?-Lg was used as the antigen to intravenously immunize the Japanese big ear rabbit, then anti-?-Lg polyclonal antibody were obtained. The indirect enzyme-linked immunosorbent assay were used to detect serum titer and western blotting experiment were used to detect the specificity of serum, we found that the serum we obtained has high effective serum and good specificity.The operating condition of the indirect competitive enzyme-linked immunosorbent assay were determined by chessboard titration: the optimal concentration of coating antigen was 2?g/m L, the optimal dilution of anti-?-Lg and the optimal dilution of HRP-IgG was1:102400 and 1:20,000 respectively. And then we got the regression equation: y=-42.82+79.62, R2=0.9938 and the most suitable detect range is 0.5 ~64?g/m L.2. Ultrasonic treatment could not change the molecular weight of ?-Lg; The content of ?-sheet increased and the content of random decreased after ultrasonic treatment. The content of ?-sheet firstly increased and then decreased with the increasing of ultrasonic treatment time and power, while the content of random firstly decreased and then increased. The free sulfhydryl groups of ?-Lg decreased and surface hydrophobicity firstly increased and then decreased with the increasing of ultrasonic treatment time and power. The free sulfhydryl groups reached the maximum(H0=1945) at 400 W for 15 min. Antigenicity firstly increased and then decreased with the increasing of ultrasonic treatment time and power, and the difference of allergenicity was not significant.3. The free amino content of ?-Lg decreased significantly, molecular weight shifted up and dimer generated after ultrasound combined with Maillard reaction modification. The UV absorbance increased significantly. The endogenous fluorescence intensity decreased significantly, and firstly increased and then decreased with the increasing of ultrasonic power, more than 10 nm redshift. The endogenous fluorescence intensity reached the minimum at 400 W for 25 min. The free sulfhydryl groups, antigenicity and allergenicity were all decreased.4. The solubility, emulsifying activity and emulsifying stability, oxidation resistance(reducing power, DPPH radical scavenging ability and Trolox equivalent antioxidant capacity) of ultrasonic pretreatment of samples of Maillard reaction were all significantly increased. |
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