Structural Change, Digestibility And Allergenicity Assessment Of The Cross-linked Bovine α-Lactalbumin Catalyzed By Polyphenol Oxidase

Milk is very popular because of its nutritional and delicious.However, it is one of the eight categories allergic food recognized by FAO, and there are8%of young children and2%of adults are allergic to it. Recent findings show that the enzymatic cross-linked milk can reduce its allergenicity, and it may provide a new approach for the development of hypoallergenic milk. Bovine a-lactalbumin can trigger milk allergy, and75%milk allergy patients were allergic to a-lactalbumin specifically. Moreover, the conformational epitopes on a-lactalbumin play a very important role in the sensitization course. Obviously, it is valuable to investigate the relationship between structure and allergenicity of cross-linked bovine a-lactalbumin.In this work, mushroom polyphenol oxidase was used to cross-linke a-lactalbumin, followed by defing the structure change, digestion and allergenicity of cross-linked a-lactalbumin. The main studies include purification of bovine a-lactalbumin and mushroom polyphenol oxidase, processing techniques on cross-linked a-lactalbumin catalyzed by polyphenol oxidase, the defining of structure change of the cross-linked a-lactalbumin and assessment on digestion and allergenicity of the crosslinked protein. The main research methods, results and conclusions are described as follows.1The bovine a-lactalbumin was purified by anion exchange chromatography and gel chromatography with a purity of up to90%and yield rate of21.18%. Then, the polyphenol oxidase enzyme was extracted from agaricus bisporus with enzyme activity up to6,000U/mL.2The effect of a-lactalbumin conformation, the reaction conditions and the substrate on the cross-linked a-lactalbumin were explored, and the optimum enzymatic reaction system were established as follow:1mg/mL of the apo-a-lactalbumin,1mM of caffeic acid is, and1,000U/ml of PPO activity, and cross linking performed at pH7.0,50℃for8h.3The cross-linked a-lactalbumin dimer was identifed by Western-blotting, and the dimer was recoved from the Native-PAGE with a purity of up to80%. The structure of the dimer was analyzed by Far-UV CD spectrum, the fluorescence intensity spectra and the UV absorption spectra, and it was shown that, compare to a-lactalbumin, the dimmer had a higher hydrophobicity and a more loosely structure.4The digestibility of the cross-linked a-lactalbumin was evaluated by simulated gastric and intestinal fluid. It was shown that the native a-lactalbumin was easy to be degraded by gastric and intestinal digestion, but its cross-linked products were relatively resistant to digestion, especially the high polymer.5The potential allergenicity of cross-linked a-lactalbumin product was evaluated. It was shown that, compare to a-lactalbumin, the cross-linked products had a lower binding capacity to IgG and IgE, revealing that the cross-linking catalyzed by polyphenol oxidase enzyme can reduce the allergenicity of the a-lactalbumin.